WO2012023285A1 - Ehmt2 as a target gene for cancer therapy and diagnosis - Google Patents

Ehmt2 as a target gene for cancer therapy and diagnosis Download PDF

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WO2012023285A1
WO2012023285A1 PCT/JP2011/004608 JP2011004608W WO2012023285A1 WO 2012023285 A1 WO2012023285 A1 WO 2012023285A1 JP 2011004608 W JP2011004608 W JP 2011004608W WO 2012023285 A1 WO2012023285 A1 WO 2012023285A1
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ehmt2
cancer
double
gene
stranded molecule
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Ryuji Hamamoto
Yusuke Nakamura
Takuya Tsunoda
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Oncotherapy Science, Inc.
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Definitions

  • the present invention relates to methods of detecting and diagnosing cancer as well as methods of treating and/or preventing cancer, including cancers associated with the overexpression of EHMT2 such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • the present invention also relates to methods of screening for a candidate substance for treating and/or preventing an EHMT2-associated cancer.
  • the present invention relates to double-stranded molecules that reduce EHMT2 gene expression and uses thereof.
  • Histone methylation plays dynamic and crucial roles in regulating chromatin structure. Precise coordination and organization of open and closed chromatin regions control normal cellular processes such as DNA replication, repair, recombination and transcription. Histone lysine methylation regulates transcription positively or negatively depending on the methylation state of various methylation sites (NPL1). For instance, methylation of histone H3 at lysine 9 (H3K9) has served as the proto-type for studying the regulation of histone function by lysine methylation.
  • H3K9 Di- or tri-methylation of H3K9 creates a binding site for chromodomain (CD)-containing proteins of the heterochromatin protein 1 (HP1) family (NPL2, 3), which is thought to lead to gene repression through changes in higher-order chromatin structure. Methylation-dependent HP1 recruitment can be antagonized by adjacent H3 serine 10 phosphorylation.
  • Histones are subject to a system of combinatorially acting posttranslational modifications, referred to as the "histone code" (NPL4-6).
  • NPL4-6 a system of combinatorially acting posttranslational modifications
  • EHMT2 also known as G9a, is mainly responsible for mono-methylation and di-methylation of H3K9 in euchromatin (NPL12).
  • EHMT2 is essential for early embryonic development and is involved in the transcriptional silencing of developmentally regulated genes. Knockout of EHMT2 causes embryonic lethality in mice, indicating a major role for epigenetic repression in early mammalian development (NPL13).
  • EHMT2 also appears to function as a coactivator for nuclear receptors, acting synergistically with CARM1 and other nuclear receptor (NR) coactivators (NPL17). Additionally, the complex of EHMT2 and DNMT1 enhances DNA and histone methylation of in vitro assembled chromatin substrates, indicating that direct cooperation between EHMT2 and DNMT1 provides a mechanism of coordinated H3K9 and DNA methylation during cell division (NPL18).
  • SIAH single in absentia homolog proteins are members of the RING-finger-containing E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina) protein (NPL19, 20). It has been suggested that the SIAH1 protein plays a key role in biological processes such as the cell cycle, cell apoptosis and oncogenesis (NPL21-23).
  • NPL1 Kouzarides T. Curr Opin Genet Dev 2002;12:198-209.
  • NPL2 Bannister AJ. et al. Nature 2001;410:120-4.
  • NPL3 Lachner M et al. Nature 2001;410:116-20.
  • NPL4 Fischle W et al. Nature 2005;438:1116-22.
  • NPL5 Hirota T et al. Nature 2005;438:1176-80.
  • NPL6 Strahl BD and Allis CD. Nature 2000;403:41-5.
  • NPL7 Hamamoto R et al. Nat Cell Biol 2004;6:731-40.
  • NPL8 Hamamoto R et al. Cancer Sci 2006;97:113-8.
  • NPL9 Kunizaki M et al. Cancer Res 2007;67:10759-65.
  • NPL10 Silva FP. Oncogene 2008;27:2686-92.
  • NPL11 Tsuge M et al. Nat Genet 2005;37:1104-7.
  • NPL12 Tachibana M et al. J Biol Chem 2001;276:25309-17.
  • NPL13 Tachibana M et al. Genes Dev 2002;16:1779-91.
  • NPL14 Gyory I et al. Nat Immunol 2004;5:299-308.
  • NPL15 Nishio H and Walsh MJ. Proc Natl Acad Sci U S A 2004;101:11257-62.
  • NPL16 Roopra A et al. Mol Cell 2004;14:727-38.
  • NPL17 Lee DY et al. J Biol Chem 2006;281:8476-85.
  • NPL18 Esteve PO et al. Genes Dev 2006;20:3089-103.
  • NPL19 Carthew RW and Rubin GM. Cell 1990;63:561-77.
  • NPL20 Hu G et al Genomics 1997;46:103-11.
  • NPL21 Okabe H et al. Cancer Res 2003;63:3043-8.
  • NPL22 Wen YY et al. Mol Carcinog;49:440-9.
  • NPL23 Wen YY et al. Cancer Sci;101:73-9.
  • the present invention relates to the discovery, through microarray analysis and RT-PCR, that EHMT2 is overexpressed in clinical bladder cancer, lung cancer (including squamous cell carcinoma (SCC), adenocarcinoma (ADC), alveolus cell carcinoma (ACC), small cell lung cancer (SCLC) and large cell carcinoma (LCC)), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), esophageal cancer, breast cancer, cervical cancer and osteosarcoma tissues.
  • SCC squamous cell carcinoma
  • ADC adenocarcinoma
  • ACC alveolus cell carcinoma
  • SCLC small cell lung cancer
  • LCC large cell carcinoma
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • esophageal cancer breast cancer, cervical cancer and osteosarcoma tissues.
  • functional knockdown of endogenous EHMT2 by siRNA in cancer cell lines results in drastic suppression of cancer cell growth
  • EHMT2 Since it is only scarcely expressed in normal adult organs, EHMT2 is an appropriate molecular target for a therapeutic approach with minimal adverse effects. For example, EHMT2 can suppress transcription of the SIAH1 gene by binding to its promoter region (-293 to +51) and methylating lysine 9 of histone H3. Furthermore, an EHMT2 specific inhibitor BIX-01294 significantly suppresses the growth of cancer cells. These results demonstrate that dysregulation of EHMT2 plays an important role in the growth regulation of cancer cells, and EHMT2 is a valid therapeutic target for various types of cancer.
  • NSCLC non-small cell lung cancer
  • AML non-small cell lung cancer
  • CML esophageal cancer
  • breast cancer cervical cancer
  • osteosarcoma esophageal cancer
  • EHMT2 an increase in the level of expression of EHMT2 as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, particularly bladder cancer, lung cancer (NSCLC(SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • NSCLC lung cancer
  • SCLC SCLC
  • AML CML
  • esophageal cancer breast cancer
  • cervical cancer and osteosarcoma.
  • the EHMT2 polynucleotide can be detected by appropriate probes or, alternatively, the EHMT2 polypeptide can be detected by an anti-EHMT2 antibody.
  • the methods of the present invention can be carried out using as an index the binding activity to an EHMT2 polypeptide, an expression level of an EHMT2 gene, a biological activity of an EHMT2 polypeptide, or an expression level and/or activity of a reporter gene controlled under a transcriptional regulatory region of EHMT2 gene.
  • Substances that bind to an EHMT2 polypeptide, or suppress an EHMT2 expression or activity, or a reporter gene expression or activity can be identified as candidate substances for treating and/or preventing cancer, or inhibiting cancer cell growth.
  • the biological activity of the EHMT2 polypeptide to be detected includes cell proliferative activity (cell proliferation enhancing activity), methyltransferase activity or the activity of suppressing transcription of the SIAH1 gene.
  • a decrease in the biological activity of the EHMT2 polypeptide as compared to a control level in the absence of the test substance may indicate that the test substance may be used to reduce symptoms of cancer, or treating and/or preventing cancer.
  • the agent is a EHMT2 inhibitor (e.g., BIX-01294).
  • an EHMT2 inhibitor inhibits the histone methyltransferase activity.
  • the cancer being treated and/or prevented is bladder cancer, lung cancer (NSCLC (SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • the agent is an inhibitory nucleic acid (e.g., an antisense, ribozyme, double stranded molecule, aptamer).
  • the agent may be a nucleic acid molecule or vector for providing nucleic acid molecules including double-stranded molecules.
  • expression of EHMT2 may be inhibited by introduction of a double-stranded molecule into a target cell in an amount sufficient to inhibit expression of the EHMT2 gene.
  • the method includes the step of administering to a subject a pharmaceutically effective amount of double-stranded molecule against an EHMT2 gene or a vector encoding such a molecule, wherein the double-stranded molecule inhibits expression of an EHMT2 gene as well as cell proliferation when introduced into a cell that expresses an EHMT2 gene.
  • a double-stranded molecule against EHMT2 inhibits the expression of an EHMT2 gene as well as inhibiting the cell proliferation induced thereby when introduced into a cell expressing an EHMT2 gene.
  • the cancer being treated and/or prevented is bladder cancer, lung cancer (NSCLC (SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • NSCLC lung cancer
  • ADC ADC
  • ACC ACC
  • LCC LCC
  • SCLC SCLC
  • AML AML
  • CML CML
  • esophageal cancer breast cancer
  • cervical cancer or osteosarcoma.
  • the double-stranded molecules of the present invention may be composed of a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 34 and 35 and the antisense strand includes a sequence which is complementary to the target sequence.
  • the sense and the antisense strands of the molecule hybridize to each other to form a double-stranded molecule.
  • the double-stranded molecule of the present invention inhibits expression of the EHMT2 gene and inhibit cell proliferation.
  • the present invention provides following [1] to [27]: [1] A method of detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of an EHMT2 gene in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer, wherein the expression level is determined by a method selected from the group consisting of: (a) detecting an mRNA of the EHMT2 gene; (b) detecting a protein encoded by the EHMT2 gene; and (c) detecting a biological activity of a protein encoded by the EHMT2 gene; [2] The method of [1], wherein said increase is at least 10% greater than said normal control level; [3] The method of [1], wherein the subject-derived biological sample is a biopsy specimen; [4] A kit for diagnosing cancer, which comprises a reagent selected from the group consisting of: (a rea
  • the methods and materials of the present invention are capable of identifying cancer prior to detection of overt clinical symptoms and may be used in cancer therapy. In some embodiments, the methods and compositions provide cancer therapy without adverse effect. It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention.
  • Figure 1 demonstrates the elevated EHMT2 expression in bladder cancer.
  • Part A depicts the EHMT2 gene expression in normal and tumor bladder tissues in British cases. Expression levels of EHMT2 were analyzed by quantitative real-time PCR, and the result is shown by box-whisker plot. The Mann-Whitney U-test was used for statistical analysis.
  • Part B depicts the statistical analysis of EHMT2 expression categorized by histological grade of bladder tumors. The P value was calculated using the Kruskal-Wallis test.
  • Part C depicts the statistical analysis of EHMT2 expression categorized by pathological stages of bladder tumors. The P value was calculated using the Kruskal-Wallis test.
  • Part D depicts the expression ratio between bladder normal and tumor tissues in Japanese populations. The signal intensity of each sample was analyzed by cDNA microarray, and expression ratio, the signal intensity in the tumor sample divided by that in normal tissue, is shown.
  • Part E depicts the tissue microarray images of bladder tumors stained by standard immunohistochemistry for protein expression of EHMT2. Clinical information for each section is represented above the histological pictures. Counterstaining was done with hematoxylin and eosin. Original magnification, x200.
  • Figure 2 demonstrates the elevated EHMT2 expression levels in lung cancer.
  • Part A depicts the expression ratio between normal lung and small cell lung cancer (SCLC) tissues. Signal intensity of each sample was analyzed by cDNA microarray (right), and the result is shown by box-whisker plot (median 50% boxed). Mann-Whitney U-test was used for the statistical analysis. Expression ratio is the signal intensity in the tumor sample divided by that in the normal sample (left).
  • Part B depicts the immunohistochemical staining of EHMT2 in lung tissues. Clinical information for each section is represented above histological pictures. Counterstaining was done with hematoxylin and eosin. Original magnification, x200.
  • Figure 3 demonstrates the involvement of EHMT2 in the growth of bladder and lung cancer cells.
  • Part A depicts the expression levels of EHMT2 in various cell lines. A western blot was performed to measure the protein level of EHMT2, and an anti-ACTB antibody was used as an internal control.
  • Part B depicts the effects of EHMT2 knockdown on the protein expression. Lysates from SBC-5 cells after siRNA treatment were immunoblotted with anti-EHMT2 and tubulin antibodies. Expression of tubulin served as an internal control.
  • Part C depicts the results of a colony formation assay after siRNA treatment. Giemsa staining was performed 7 days after treatment with siRNAs.
  • Part D depicts the effect of siRNA knockdown of EHMT2 on the viability of a bladder cancer cell line (SW780, RT4) and lung cancer cell lines (LC319, SBC5, A549).
  • Relative cell number is the cell number normalized to siEGFP-treated cells. The numbers are a mean value +/-SD of three independent experiments. P values were calculated using Student's t-test (*, P ⁇ 0.05;**, P ⁇ 0.01; ***, P ⁇ 0.001).
  • Part E depicts the results of FACS analysis after treatment with EHMT2 siRNAs. SBC5 cells were treated with siRNAs and analyzed by FACS 72 h after siRNA treatment.
  • FIG. 4 demonstrates that SIAH1 expression is directly regulated by EHMT2.
  • Part A depicts a two-dimensional, unsupervised hierarchical cluster analysis of SW780 and A549 cells after knockdown of EHMT2 expression. Differentially expressed genes were selected for this analysis. Darker color indicates up-regulated genes and lighter color indicates down-regulated genes.
  • Part B depicts the results of microarray analysis of A549 cells after treatment with siRNAs targeting EGFP(control; siEGFP) and EHMT2 (siEHMT2#2). P values were calculated using Student's t-test (**, P ⁇ 0.01).
  • Part C depicts the results of validation of the microarray data using quantitative real-time PCR and western blot analyses in A549 cells after treatment with siRNAs targeting EGFP (control; siEGFP) and EHMT2 (siEHMT2). P values were calculated using Student's t-test (**, P ⁇ 0.01). Samples for western blot analysis were fractionated by SDS-PAGE and immunoblotted with anti-EHMT2 (NB100-40825, Novus Biologicals) and anti-SIAH1 (sc-5506, Santa Cruz) antibodies. Anti-ACTB was used as an internal control. Part D depicts the results of a ChIP assay using anti-FLAG and anti-H3K9me2 antibodies.
  • the ChIP assay was performed 48 hours after transfection with pCAGGSn-3FC (Mock) and pCAGGSn-3FC-EHMT2 (3xFLAG-EHMT2) into 293T cells.
  • Left top panel depicts a schematic diagram of the SIAH1 promoter region.
  • Left bottom panel depicts the result of real-time PCR analysis using a primer pair shown in Table 1.
  • Cross-linked and sheared chromatin was immunoprecipitated with anti-FLAG antibody (M2, Sigma). The results are shown as percentage of the input chromatin.
  • the right top panels depict the results of immunoblot analysis using anti-FLAG antibody.
  • the input samples were fractionated by SDS-PAGE and immunoblotted with anti-FLAG antibody. Expression of ACTB was detected as an internal control.
  • the right bottom panel depicts the result of quantification of H3K9diMe ChIP at the SIAH1 promoter region using real-time PCR.
  • Cross-linked and sheared chromatin was immunoprecipitated with anti-diMeH3K9 antibody (ab1220, abcam).
  • P values were calculated using Student's t-test (***, P ⁇ 0.001).
  • Figure 5 demonstrates elevated EHMT2 expression levels in AML, CML and esophageal cancer in Japanese populations.
  • the signal intensity of each sample was analyzed by cDNA microarray, and the results are shown by box-whisker plot (median 50% boxed). The Mann-Whitney U-test was used for the statistical analysis.
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia.
  • Figure 6 demonstrates expression levels of EHMT2 in normal tissue, 14 bladder cancer cell lines and five lung cancer cell lines. Expression levels of EHMT2 were analyzed by quantitative real-time PCR. Data were normalized by GAPDH and SDH expressions.
  • Figure 7 demonstrates the results of quantitative real-time PCR analyses after knockdown of EHMT2 by siEHMT2.
  • Quantitative real-time PCR analyses show suppression of endogenous expression of EHMT2 by EHMT2-specific siRNAs (siEHMT2#1 and #2) in A549 and SBC5 cells.
  • siEGFP and siNC were used as controls.
  • Relative mRNA expression is shown as the expression level normalized by expression levels of siEGFP-treated cells. The results are presented as mean expression level +/-SD of three independent experiments. P values were calculated using Student's t-test (**, P ⁇ 0.01; ***, P ⁇ 0.001).
  • Figure 8 demonstrates that the treatment with siEHMT2 may decrease cancer cells in the S phase and increase cancer cells in the sub-G1 phase.
  • the SBC5 cells were treated with siRNAs and analyzed by FACS 72 h after siRNA treatment. Numerical analysis of the FACS result classifying cells by cell cycle status are shown. The proportion of cancer cells in sub-G 1 phase is significantly high after treatment with siEHMT2#1 compared to control siRNAs-treated cancer cells. The results are presented as the mean +/-SD of three independent experiments. P values were calculated using Student's t-test.
  • Figure 9 demonstrates reduction of the growth rate of several types of cancer cell lines by treatment with BIX-01294.
  • Part A depicts expression levels of EHMT2 in various types of cancer cells analyzed by quantitative real-time PCR.
  • Part B depicts the effect of BIX-01294 on the viability of cancer cell lines. Cancer cell lines were treated for 2 days with the inhibitor BIX-01294 at 2, 4 and 6 micromolar. This result was normalized against the results of treatment with pure water as the negative control (N.C).
  • N.C negative control
  • Statistical analysis was performed based on three independent experiments. P values were calculated using Student's t-test.
  • Part C depicts the results of cell cycle distribution analyzed by flow cytometry. Cell cycle distribution was analyzed by flow cytometry after coupled staining with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU and 7-amino-actinomycin D (7-AAD) as described below.
  • FITC fluorescein iso
  • Figure 10 demonstrates the expression levels of EHMT2 in 78 normal tissues.
  • GAPDH expression is shown as a control for signal intensity.
  • Figure 11 demonstrates the elevated EHMT2 expression levels in lung cancer.
  • Part A depicts expression of EHMT2 in normal lung, 17 non-small cell lung cancer (NSCLC) and 6 SCLC tissues. Expression levels were analyzed by quantitative real-time PCR, and data were normalized against EHMT2 expression levels in normal lung tissue.
  • FIG 12 demonstrates that SIAH1 expression may be directly regulated by EHMT2.
  • the ChIP assay was performed using anti-EHMT2 (Middle) and anti-H3K9me2 (Right) antibodies after treatment of SBC5 cells with siEGFP or siEHMT2#2 for 48 h. The results are shown as a percentage of the input chromatin. Left panels depict the result of the immunoblot analysis using anti-EHMT2 antibody. The input samples were fractionated by SDS-PAGE and immunoblotted with anti-EHMT2 antibody. Expression of ACTB was detected as an internal control.
  • FIG. 13 demonstrates that SIAH1 regulated by EHMT2 regulates cancer cell growth and apoptosis.
  • Part A depicts the results of western blot analyses in SBC5 cells after treatment with siRNAs targeting EGFP (control; siEGFP), EHMT2 (siEHMT2#2) and SIAH1 (siSIAH1) for 72 h.
  • Anti-PARP1 sc-8007, Santa Cruz
  • anti-cleaved caspase 3 #9661S, Cell Signaling
  • Part B depicts the result of colony formation assay of SBC5 cells.
  • siRNAs were transfected 24 h after preparation of cells and Giemsa staining was performed 96 h after treatment with siRNAs.
  • Part C depicts the results of cell growth assay of SBC5 cells treated with indicated siRNAs.
  • siEHMT2#2 and either siEGFP or siSIAH1 were transfected 24 h after preparation of cells, and subsequently, cell viability was measured 48 h and 96 h after siRNA treatment. The results are presented as the mean +/-SD of three independent experiments. P values were calculated using Student's t-test (**, P ⁇ 0.01).
  • Figure 14 demonstrates gene ontology pathway analysis based on the Affymetrix's microarray data.
  • Figure 14 demonstrates gene ontology pathway analysis based on the Affymetrix's microarray data.
  • Figure 14 demonstrates gene ontology pathway analysis based on the Affymetrix's microarray data.
  • an isolated or purified antibody refers to an antibody that is substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • polypeptide When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In one embodiment, antibodies of the present invention are isolated or purified.
  • SDS sodium dodecyl sulfate
  • EHMT2 gene encompasses polynucleotides that encode the human EHMT2 or any of the functional equivalents of the human EHMT2 gene.
  • the EHMT2 gene can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids. Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleic acid molecules are used interchangeably unless otherwise specifically indicated and, similarly to the amino acids, are referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
  • the polynucleotide, oligonucleotide, nucleotides, nucleic acids, or nucleic acid molecules may be composed of DNA, RNA or a combination thereof.
  • cancer refers to cancer over-expressing the EHMT2 gene.
  • examples of cancers over-expressing EHMT2 include, but are not limited to, bladder cancer, lung cancer (including squamous cell carcinoma (SCC), adenocarcinoma (ADC), alveolus cell carcinoma (ACC), small cell lung cancer (SCLC) and large cell carcinoma (LCC)), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • SCC squamous cell carcinoma
  • ADC adenocarcinoma
  • ACC alveolus cell carcinoma
  • SCLC small cell lung cancer
  • LCC large cell carcinoma
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • esophageal cancer breast cancer, cervical cancer and osteosarcoma.
  • double-stranded molecule refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • double-stranded molecule is also referred to as “double-stranded nucleic acid”, “double-stranded nucleic acid molecule”, “double-stranded polynucleotide” and “double-stranded polynucleotide molecule”.
  • the Gene and Protein The present invention is based in part on the discovery that the gene encoding EHMT2 is over-expressed in cancer tissues as compared to non-cancerous tissues.
  • the nucleic acid and polypeptide sequences of EHMT2 are provided, for example, in the following SEQ ID numbers: EHMT2 polynucleotide: SEQ ID NOs:1 or 3; EHMT2 polypeptide: SEQ ID NOs:2 or 4.
  • the present invention is also based in part on the discovery that EHMT2 may directly bind to the promoter region of SIAH1 and may regulate the transcription of SIAH1.
  • the nucleic acid and polypeptide sequences of SIAH1 in the present invention are provided, for example, in the following SEQ ID numbers: SIAH1 polynucleotide: SEQ ID NOs: 5 or 7; SIAH1 polypeptide: SEQ ID NOs: 6 or 8.
  • the above sequence data is also available via the following GenBank accession numbers: SIAH1: NM_001006610 or NM_003031.
  • the promoter region of SIAH1 may be obtained by amplifying the 5' upstream region of the SIAH1 gene with a primer set, for example, a primer set of SEQ ID NO: 18 and 19, which is designed based on genomic sequence information.
  • a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptide that retains the biological ability may be used as such a functional equivalent in the present invention.
  • Such functional equivalents include those wherein one or more amino acids are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the protein.
  • the polypeptide may be composed of an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the respective protein, at least about 90% to 95% homology, or about 96%, 97%, 98% or 99% homology.
  • the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the natural occurring nucleotide sequence of the genes.
  • a polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a function equivalent to that of the above proteins of the present invention, it is within the scope of the present invention.
  • stringent (hybridization) conditions refers to conditions under which a nucleic acid molecule will hybridize, typically in a complex mixture of nucleic acids, to its target sequence but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times higher than background level, or at least 10 times higher than the background level.
  • Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
  • Hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the above human proteins can be routinely selected by a person skilled in the art.
  • hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, or 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions may alternatively be used.
  • An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min.
  • factors such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
  • modifications of one, two or more amino acids in a protein will not influence the function of the protein.
  • mutated or modified proteins i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition
  • mutated or modified proteins have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).
  • the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in the sequence.
  • the number of amino acid mutations is not particularly limited. However, in general, 5% or less of the amino acid sequence is altered. Accordingly, in a typical embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or fewer, 20 amino acids or fewer, 10 amino acids or fewer, 5 or 6 amino acids or fewer, or 3 or 4 amino acids or fewer.
  • An amino acid residue to be mutated may be mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution).
  • properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
  • A, I, L, M, F, P, W, Y, V hydrophilic amino
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cystein (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
  • Such conservatively modified polypeptides are included in the present EHMT2 protein.
  • the present invention is not restricted thereto and the EHMT2 protein includes non-conservative modifications, so long as at least one biological activity of the EHMT2 protein is retained.
  • the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
  • the EHMT2 gene of the present invention encompasses polynucleotides that encode such functional equivalents of the EHMT2 protein.
  • a gene amplification method for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the EHMT2 protein, using a primer synthesized based on the sequence information of the protein encoding DNA (e.g. SEQ ID NO: 1 or 3).
  • PCR polymerase chain reaction
  • Polynucleotides and polypeptides that are functionally equivalent to the human EHMT2 gene and protein, respectively, may have a high homology to the originating nucleotide or amino acid sequence thereof .
  • High homology typically refers to a homology of 40% or higher, 60% or higher, 80% or higher, 90% to 95% or higher, or 96%, 97%, 98%, 99% or higher.
  • the homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".
  • a method for Diagnosing Cancer The expression of EHMT2 gene was found to be elevated in bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma (Fig. 1, 2, 5, 6, Table 4) and was not expressed in normal tissues. Accordingly, the EHMT2 genes identified herein as well as their transcription and translation products are useful as diagnostic markers for cancers such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. For example, cancer can be diagnosed or detected by comparing the expression level of EHMT2 gene between a subject-derived sample with a normal sample. In one aspect, the present invention provides methods for detecting or diagnosing cancer, determining the presence of cancer, or determining a predisposition for developing cancer, more particularly EHMT2-associated cancer, by determining the expression level of EHMT2 in the subject.
  • the present invention provides a method for detecting or diagnosing cancer in a subject, such method including the step of determining an expression level of an EHMT2 gene in a subject-derived biological sample, wherein an increase of the level as compared to a normal control level of the gene indicates the presence or suspicion of cancer cells in the sample, which, in turn, suggests that the subject suffers from or is at risk of developing cancer.
  • the expression level of the EHMT2 gene may be determined by any known method, examples of which include: (a) detecting the mRNA of an EHMT2 gene; (b) detecting the protein encoded by an EHMT2 gene; and (c) detecting the biological activity of the protein encoded by an EHMT2 gene.
  • cancer to be diagnosed by the present method includes bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose whether a subject suffers from the disease.
  • the present invention provides a method for the use of EHMT2 as a diagnostic marker for cancer.
  • the present invention may be used to detect or identify cancerous cells in a subject-derived tissue, such cells being characterized by an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the tissue.
  • EHMT2 expression results may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease.
  • the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease.
  • the present invention when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the EHMT2 gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc.
  • some well-known diagnostic tumor markers in blood include, but are not limited to, IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA.
  • the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.
  • the present invention also provides the following methods [1] to [10]: [1] A method of detecting or diagnosing cancer in a subject, including determining an expression level of an EHMT2 gene in a subject derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer; [2] The method of [1], wherein the expression level is at least 10% greater than the normal control level; [3] The method of [1], wherein the expression level is detected by a method selected from the group consisting of: (a) detecting an mRNA of the EHMT2 gene, (b) detecting a protein encoded by the EHMT2 gene, and (c) detecting a biological activity of a protein encoded by the EHMT2 gene; [4] The method of [1], wherein the cancer is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma; [5] The method of [3
  • a subject to be diagnosed by the present method may be a mammal.
  • Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the methods of the present invention may include a step of collecting a biological sample from the subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it includes, or is capable of including, a transcription or translation product of EHMT2.
  • Exemplary biological samples include, but are not limited to, bodily tissues which are desired for diagnosing or are suspected of suffering from cancer; and fluids, such as biopsy specimen, blood, sputum or urine.
  • the biological sample may contain a cell population including an epithelial cell, a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, the cell may be purified from the obtained bodily tissues and fluids, and then used as the biological sample.
  • the present invention provides methods for determining the expression level of EHMT2 in a subject-derived biological sample.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of EHMT2 may be quantified using probes and hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a chip or an array, for example.
  • the use of an array may allow detection of the expression level of a plurality of genes (e.g., various cancer specific genes) including EHMT2.
  • the cDNA of EHMT2, or fragments thereof may be used as the probes.
  • the probe may be labeled with a suitable label, such as dyes, fluorescent and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of EHMT2 may be quantified by amplification-based detection methods (e.g., RT-PCR) using primers.
  • primers may be prepared based on the available sequence information of the gene.
  • the primers used in the Example (SEQ ID NO: 14 and 15) may be employed for the detection by RT-PCR or Northern blot.
  • the present invention is not restricted to the use of these specific primer or probe sequences.
  • a probe or primer used for the present method may hybridize under stringent, moderately stringent, or low stringent conditions to the mRNA of EHMT2 or a fragment thereof.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the diagnosis of the present invention.
  • the quantity of EHMT2 protein may be determined.
  • Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody that specifically recognizes the protein or a fragment thereof.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment retains the binding ability to EHMT2 protein or fragment thereof.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be observed via immunohistochemical analysis using an antibody against EHMT2 protein or a fragment thereof. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of EHMT2 gene.
  • methods for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begin with a determination of EHMT2 gene expression level. Once determined, using any of the aforementioned techniques, this value is as compared to a control level.
  • control level refers to the expression level of a test gene detected in a control sample and encompasses both a normal control level and a cancer control level.
  • normal control level refers to a level of gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer.
  • a normal individual is one with no clinical symptom of bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma.
  • a normal control level can be determined using a normal cell obtained from a non-cancerous tissue.
  • a "normal control level” may also be the expression level of a test gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma.
  • cancer control level refers to an expression level of a test gene detected in the cancerous tissue or cell of an individual or population suffering from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma.
  • an increase in the expression level of EHMT2 detected in a subject-derived sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma.
  • the subject-derived sample may be any tissues obtained from test subjects, e.g., patients suspected of having cancer.
  • tissues may include epithelial cells. More particularly, tissues may be epithelial cells collected from a suspected cancerous area.
  • the expression level of EHMT2 in a sample can be compared to a cancer control level of EHMT2 gene.
  • a similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer.
  • the expression levels of other cancer-related genes are also measured and compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
  • the control level may be determined at the same time with the test biological sample by using a sample or samples previously collected and stored from a subject or subjects whose disease state (cancerous or non-cancerous) is or are known.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression levels of EHMT2 gene in samples from subjects whose disease state are known.
  • the control level can be from a database of expression patterns from previously tested cells.
  • the expression level of EHMT2 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • a control level may be utilized that is determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample.
  • the methods of the present invention may use a standard value of the expression levels of the EHMT2 gene in a population with a known disease state.
  • the standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 times the S.D. or mean +/- 3 times the S.D. may be used as a standard value.
  • the expression level of other cancer-associated genes for example, genes known to be differentially expressed in bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma may also be determined, in addition to the expression level of the EHMT2 gene. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference which is cancerous indicates that the subject is suffering from or at a risk of developing bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • gene expression levels are deemed to be "altered” or “increased” when the gene expression changes or increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level.
  • the expression level of bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma marker genes including EHMT2 gene in a biological sample can be considered to be increased if it increases from a control level of the corresponding bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma marker gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • Differences between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell.
  • control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
  • the present invention also provides EHMT2 as a suitable target for cancer therapy. Therefore, cancer treatment targeting EHMT2 is provided by the present invention.
  • the cancer treatment targeting EHMT2 refers to suppression or inhibition of EHMT2 activity and/or expression in a cancer cell or tissue, or in a subject having cancer.
  • any anti-EHMT2 agents may be used for the cancer treatment targeting EHMT2.
  • the anti-EHMT2 agents may include following substances or active ingredients: (a) a double-stranded molecule of the present invention, (b) DNA encoding the double-stranded molecule, or (c) a vector encoding the double-stranded molecule.
  • the present invention provides a method of (i) diagnosing whether a subject has cancer suitable for treatment with an anti- EHMT2 agent, and/or (ii) selecting a subject for cancer treatment targeting EHMT2, which method includes the steps of: a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of EHMT2 with a normal control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is increased as compared to the normal control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • such a method includes the steps of: a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of EHMT2 with a cancerous control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is similar or equivalent to the cancerous control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • kits for Diagnosing Cancer The present invention also provides a kit for diagnosing cancer, which may also be useful in monitoring the efficacy of a cancer therapy.
  • the present invention also provides a kit for determining if a subject suffering from cancer may be treated with a double-stranded molecule or inhibitor of the present invention or vector encoding thereof.
  • the kit of the present invention may also be useful in assessing and/or monitoring the efficacy of a cancer treatment.
  • the cancer to be diagnosed by the present kit includes, but is not limited to, bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • the kit may include at least one reagent for detecting the expression level of the EHMT2 gene in a subject-derived biological sample, which reagent may be selected from the group consisting of: (a) a reagent for detecting mRNA of the EHMT2 gene or a fragment thereof; (b) a reagent for detecting the EHMT2 protein or a fragment thereof; and (c) a reagent for detecting the biological activity of the EHMT2 protein.
  • Suitable reagents for detecting mRNA of the EHMT2 gene include nucleic acids that specifically bind to or identify the EHMT2 mRNA, such as oligonucleotides which have a complementary sequence to a part of the EHMT2 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the EHMT2 mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art.
  • the reagent for detecting the EHMT2 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the EHMT2 mRNA may be included in the kit.
  • a probe or primer of the present invention is typically a substantially purified oligonucleotide.
  • the oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about a 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bases of consecutive sense strand nucleotide sequence of a nucleic acid encoding an EHMT2 sequence, or an anti sense strand nucleotide sequence of a nucleic acid encoding an EHMT2 sequence, or of a naturally occurring mutant of these sequences.
  • an oligonucleotide having a 5-50 nucleotide length can be used as a primer for amplifying the genes, to be detected.
  • mRNA or cDNA of a EHMT2 gene can be detected with an oligonucleotide probe or primer of a specific size, generally 15- 30 bases in length.
  • the length of the oligonucleotide probe or primer can be selected from 15-25 nucleotides.
  • Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR).
  • probes or primers can also include tags, labels, or linker sequences. Further, probes or primers can be modified with a detectable label or affinity ligand to be captured. Alternatively, in hybridization based detection procedures, a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).
  • Suitable reagents for detecting the EHMT2 protein may include antibodies to the EHMT2 protein or a fragment thereof.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used as the reagent, so long as the fragment retains the binding ability to the EHMT2 protein or a fragment thereof.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the antibody may be labeled with signal generating molecules or other detectable labels via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods may be employed for the present invention.
  • more than one reagent for detecting the EHMT2 protein may be included in the kit.
  • the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed EHMT2 protein in the biological sample.
  • the cell may be cultured in the presence of a subject-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability, the cell proliferating activity of the biological sample can be determined.
  • the reagent for detecting the EHMT2 mRNA may be immobilized on a solid matrix.
  • more than one reagent for detecting the biological activity of the EHMT2 protein may be included in the kit.
  • the kit may contain more than one of the aforementioned reagents.
  • the kit may include a solid matrix and reagent for binding a probe against the EHMT2 gene or antibody against the EHMT2 protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the EHMT2 protein.
  • tissue samples obtained from subject suffering from cancer or a normal subject may serve as useful control reagents.
  • a kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, URL etc.) with instructions for use.
  • These reagents and such may be provided in a container with a label.
  • Suitable containers include bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials, such as glass or plastic.
  • the reagent when the reagent is a probe against the EHMT2 mRNA, the reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one detection site.
  • the measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid (probe).
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a different strip separated from the test strip.
  • the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal may provide a quantitative indication of the amount of EHMT2 mRNA present in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot. In some embodiments, the bar or dot may span the width of a test strip.
  • the kit of the present invention may further include a positive and/or negative controls sample, and/or an EHMT2 standard sample.
  • the positive control sample of the present invention may be prepared by collecting EHMT2 positive samples.
  • EHMT2 positive samples may be obtained, for example, from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma cell lines, including lung adenocarcinoma cell (ADC) lines such as A427, NCI-H1781, A549, LC319 and the like; lung squamous cell carcinoma (SCC) cell lines such as NCI-H26, EBC-1, NCI-H520, NCI-H2170 and the like; SCLC cell lines such as DMS114, DMS273, SBC-3, SBC-5, H196, H446 and the like; and bladder cancer cell lines such as 5637, 253J, 253JBV, EJ28, HT1197, HT1376, HT1576, J
  • the EHMT2 positive samples may be obtained from clinical bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma tissues.
  • positive control samples may be prepared by determining a cut-off value and preparing a sample containing an amount of an EHMT2 mRNA or protein more than the cut-off value.
  • the phrase "cut-off value" refers to a threshold value dividing between a normal range and a cancerous range.
  • ROC receiver operating characteristic
  • the present kit may include an EHMT2 standard sample containing a cut-off value amount of an EHMT2 mRNA or polypeptide.
  • negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal tissues, or may be prepared by preparing a sample containing an EHMT2 mRNA or protein less than the cut-off value.
  • the present invention provides use of a reagent for preparing a diagnostic reagent for diagnosing cancer.
  • the reagent can be selected from the group consisting of: (a) a reagent for detecting mRNA of the EHMT2 gene; (b) a reagent for detecting the EHMT2 protein; and (c) a reagent for detecting the biological activity of the EHMT2 protein.
  • such reagent is an oligonucleotide that hybridizes to the EHMT2 polynucleotide, or an antibody that binds to the EHMT2 polypeptide.
  • EHMT2 is involved in cancer cell growth. Accordingly, substances that suppress an expression level of EHMT2 gene and/or a biological activity of EHMT2 polypeptide are useful for treating and/or preventing cancer. Such substances can be screened using an EHMT2 gene, polypeptides encoded by the gene, or a transcriptional regulatory region of the gene. Thus, the present invention also provides a method of screening for a candidate substance for treating and/or preventing cancer using EHMT2 gene, EHMT2 polypeptide, or transcriptional regulatory region of the gene.
  • substances to be identified through the present screening methods may be any compound or composition including several compounds.
  • the test substance exposed to a cell or protein according to the screening methods of the present invention may be a single substance or a combination of substances.
  • the substances may be contacted sequentially or simultaneously.
  • the substances screened by the present screening method may be suitable candidate substances for treating and/or preventing cancer, and/or inhibiting cancer cell growth.
  • the cancer may be characterized by an association with EHMT2 overexpression.
  • the screened substances may be applied to the cancers correlated or associated with EHMT2 overexpression.
  • the cancers correlated or associated with EHMT2 overexpression include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • test substance for example, cell extracts, cell culture supernatant, products of fermenting microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural compounds can be used in the screening methods of the present invention.
  • test substance for example, cell extracts, cell culture supernatant, products of fermenting microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural compounds can be used in the screening methods of the present invention.
  • test substance of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • Methods of the present invention utilizing libraries are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67).
  • a candidate substance obtained by the present screening method is a protein
  • for obtaining a DNA encoding the protein either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein.
  • an amino acid sequence or partial sequence may be used to screen a database of DNA sequences such as Genebank to obtain a DNA encoding the protein. The usefulness in preparing the candidate substance for treating or preventing cancer of the obtained DNA may be confirmed.
  • Test substances used in the screenings described herein may also be antibodies that specifically bind to an EHMT2 protein or partial peptides thereof, including partial peptides that lack the biological activity of the original proteins in vivo.
  • test substance libraries are well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.
  • suppression of the expression level and/or biological activity of EHMT2 may lead to suppression of the growth of cancer cells. Therefore, when a substance suppresses the expression and/or activity of EHMT2, such suppression is indicative of a therapeutic effect in a subject.
  • a therapeutic effect refers to a clinical benefit. Examples of such clinical benefit include but are not limited to; (a) reduction in expression of the EHMT2 gene, (b) a decrease in size, prevalence, or metastatic potential of the cancer in the subject, (c) preventing cancers from forming, or (d) preventing or alleviating a clinical symptom of cancer.
  • test substance libraries Construction of test substance libraries is facilitated by knowledge of the molecular structure of substances known to have the properties sought, and/or the molecular structure of EHMT2 protein.
  • One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
  • Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new substances that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new substance will link to the target molecule and allow experimental manipulation of the structures of the substance and target molecule to perfect binding specificity. Prediction of what the molecule-substance interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive analysis, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • CHARMM performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • test substances may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, including bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • Combinatorial chemical synthesis Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening.
  • simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library.
  • An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
  • Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6).
  • peptide libraries see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No.
  • WO 91/19735 encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J.
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples.
  • Furka et al. 14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93
  • Houghten US Patent 4,631,211
  • Rutter et al. US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
  • Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target.
  • Tuerk and Gold discloses the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • a large library of nucleic acid molecules ⁇ e.g., 10 15 different molecules) can be used for screening.
  • the present invention provides methods of screening for a candidate substance applicable to the treatment and/or prevention of cancer using an EHMT2 polypeptide.
  • the EHMT2 polypeptide to be used may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • the EHMT2 polypeptide may be a recombinant polypeptide, a protein derived from the nature or a partial peptide thereof.
  • EHMT2 polypeptides may be included in EHMT2 polypeptides used for the present screening so long as the modified peptide retains at least one biological activity of the original polypeptide. Examples of such functional equivalents are described above in the section entitled "The Gene and Protein".
  • the polypeptides may be further linked to other substances.
  • a linking process and linker are chosen so that does not interfere with the biological activity of the original polypeptide and/or fragment.
  • Usable substances include, for example: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.
  • the polypeptides used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on a selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include those described in: 1) Peptide Synthesis, Interscience, New York, 1966; 2) The Proteins, Vol.
  • the polypeptides may be obtained by adapting any known genetic engineering methods to the production of the instant polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
  • a suitable vector including a polynucleotide encoding the objective protein in an expressible form e.g., downstream of a regulatory sequence including a promoter
  • the host cell may be cultured to produce the protein.
  • a gene encoding an EHMT2 polypeptide may be expressed in host (e.g., animal, bacteria, or fungal) cells by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
  • a promoter may be used for the expression. Any commonly used promoters may be employed, including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3.
  • the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SR-alpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such.
  • EHMT2 polypeptides may be performed according to any conventional methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such. EHMT2 polypeptides may also be produced in vitro using a conventional in vitro translation system.
  • the over-expression of EHMT2 gene may be detected in bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma, in spite of no expression in normal organs (Fig. 1, 2, 5, 6, Table 4). Accordingly, using the EHMT2 gene and proteins encoded thereby, the present invention provides a method of screening for a substance that binds to EHMT2 polypeptide. Due to the expression of EHMT2 in cancer, a substance that binds to EHMT2 polypeptide may suppress the proliferation of cancer cells, and is thus useful for treating and/or preventing cancer.
  • the present invention also provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer using the EHMT2 polypeptide.
  • an embodiment of this screening method includes the steps of: (a) contacting a test substance with an EHMT2 polypeptide ; (b) detecting the binding activity between the polypeptide and the test substance; and (c) selecting the test substance that binds to the polypeptide.
  • the potential therapeutic effect of a test substance for treating and/or preventing cancer can also be evaluated or estimated.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance for treating and/or preventing cancer and/or inhibiting cancer associated with over-expression of EHMT2, the method including steps of: (a) contacting a test substance with a polypeptide encoded by a polynucleotide of EHMT2; (b) detecting the binding activity between the polypeptide and the test substance; and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when a substance binds to the polypeptide.
  • the therapeutic effect may be correlated with the binding level of the test substance and EHMT2 protein(s).
  • the test substance when the test substance binds to an EHMT2 protein, the test substance may identified or selected as a candidate substance having the requisite therapeutic effect.
  • the test substance when the test substance does not bind to an EHMT2 protein, the test substance may characterized as having no significant therapeutic effect.
  • the EHMT2 polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from nature or a partial peptide thereof.
  • the polypeptide to be contacted with a test substance may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • the polypeptide is isolated from cells expressing EHMT2, or chemically synthesized to be contacted with a test substance in vitro.
  • test substances used by the present invention may be proteins such as antibodies or synthetic chemical compounds.
  • screening substances that bind to an EHMT2 polypeptide many methods well known by a person skilled in the art may be used. Such a screening may be conducted by, for example, the immunoprecipitation method.
  • an EHMT2 polypeptide may contain an antibody recognition site.
  • EHMT2 polypeptides to be used for the present screening method may be prepared as described above.
  • the polypeptide encoded by EHMT2 gene can be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity is known, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)).
  • Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available.
  • a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the EHMT2 polypeptide by the fusion may be used.
  • Epitopes such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the EHMT2 polypeptide (Experimental Medicine 13: 85-90 (1995)).
  • an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent.
  • the immune complex consists of the EHMT2 polypeptide, a polypeptide including the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the EHMT2 polypeptide itself rather than against an added epitope, which antibodies can be prepared as described above.
  • An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody or any other antibody that binds to Protein A, Protein G or Protein L.
  • polypeptide encoded by EHMT2 gene is prepared as a fusion protein with an epitope, such as GST, a complex can be formed in the same manner as in the use of the antibody against the EHMT2 polypeptide, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.
  • Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the EHMT2 polypeptide is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • a protein binding to the EHMT2 polypeptide can be obtained by preparing a cDNA library from cultured cells expected to express a protein binding to the EHMT2 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled EHMT2 polypeptide with the above filter, and detecting the plaques expressing proteins that bind to the EHMT2 polypeptide according to the label.
  • a phage vector e.g., ZAP
  • the polypeptide of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the EHMT2, or a peptide or polypeptide (for example, GST) that is fused to the EHMT2 polypeptide. Methods using radioisotopes or fluorescence and such may be also used.
  • a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”).
  • the polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
  • a reporter gene for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • a substance binding to an EHMT2 polypeptide may also be screened using affinity chromatography.
  • an EHMT2 polypeptide may be immobilized on a carrier of an affinity column, and a composition containing test substances is applied to the column.
  • a composition herein may be, for example, cell extracts, cell lysates, antibody libraries etc.
  • the column is washed, and substances bound to the EHMT2 polypeptide can be collected.
  • the test substance is a protein
  • the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention.
  • a biosensor When such a biosensor is used, the interaction between an EHMT2 polypeptide and a test substance can be observed in real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between an EHMT2 polypeptide and a test substance using a biosensor such as BIAcore.
  • the EHMT2 protein may promote cell proliferation of cancer cells (Fig. 3). Moreover, the EHMT2 protein may also be a histone methyltransferase(Tachibana M et al. J Biol Chem 2001;276:25309-17.).
  • the present invention provides a method for screening for a substance that suppresses the proliferation of cancer cells expressing EHMT2, and a method of screening for a candidate substance useful for treating and/or preventing cancer, in particular EHMT2 associated cancers including bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • EHMT2 associated cancers including bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • the present invention provides a method of screening for a substance for treating and/or preventing cancer using the polypeptide encoded by the EHMT2 gene including the steps as follows: (a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the EHMT2 gene (i.e., EHMT2 polypeptide); (b) detecting the biological activity of the polypeptide of step (a); and (c) selecting the test substance that suppresses the biological activity of the polypeptide as compared to the biological activity of said polypeptide detected in the absence of the test substance.
  • the therapeutic effect of the test substance in suppressing the biological activity (e.g., the cell-proliferating activity) of EHMT2 polypeptide, or a candidate substance for treating and/or preventing cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance that suppresses the biological activity of EHMT2 polypeptide, or a candidate substance for treating and/or preventing cancer, using the EHMT2 polypeptide or fragments thereof, including the following steps: a) contacting a test substance with the EHMT2 polypeptide or a functional fragment thereof; and b) detecting the biological activity of the polypeptide or fragment of step (a), and c) correlating the biological activity of b) with the therapeutic effect of the test substance.
  • a method of screening for a candidate substance that suppresses the biological activity of EHMT2 polypeptide, or a candidate substance for treating and/or preventing cancer using the EHMT2 polypeptide or fragments thereof, including the following steps: a) contacting
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance in the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer associated with over-expression of EHMT2, the method including steps of: (a) contacting a test substance with the EHMT2 polypeptide or a functional fragment thereof; (b) detecting the biological activity of the polypeptide or fragment of step (a); and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when a substance suppresses the biological activity of the polypeptide encoded by the polynucleotide corresponding to the EHMT2 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.
  • cancers associated with over-expression of the EHMT2 gene include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • the therapeutic effect may be correlated with the biological activity of the EHMT2 polypeptide or a functional fragment thereof.
  • the test substance when the test substance suppresses or inhibits the biological activity of the EHMT2 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not suppress or inhibit the biological activity of the EHMT2 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • any polypeptides can be used for screening so long as they suppress a biological activity of the EHMT2 protein.
  • the biological activities of the EHMT2 protein include cell-proliferating activity and methyltransferase activity of the EHMT2 protein.
  • EHMT2 protein can be used and polypeptides functionally equivalent to the EHMT2 protein can also be used. Details of polypeptides functionally equivalent to the EHMT2 protein (i.e., functional equivalent of the EHMT2 protein) have been described above under the item " The Gene and Protein". Such polypeptides may be expressed endogenously or exogenously by cells.
  • the present invention also provides a screening method following the method described in the above section "(i) Screening for an EHMT2 Binding Substance", such method including the steps of: a) contacting a test substance with the EHMT2 polypeptide or a fragment thereof; b) detecting the binding between the polypeptide or fragment and the test substance; c) selecting the test substance that binds to the polypeptide; d) contacting the test substance selected in step c) with the EHMT2 polypeptide or a fragment thereof; e) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the substance; and f) selecting the substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.
  • the substance isolated by this screening is a candidate for antagonists of the polypeptide encoded by EHMT2 gene.
  • the term "antagonist” refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding EHMT2.
  • a substance isolated by this screening is a candidate for substance which inhibits the in vivo interaction of the EHMT2 polypeptide with molecules (including DNAs and proteins).
  • the biological activity to be detected in the present method is cell proliferation, it can be detected, for example, by preparing cells which express the EHMT2 polypeptide, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring survival cells or the colony forming activity assay, for example, as shown in Fig. 3.
  • the substances that reduce the speed of proliferation of the cells expressed EHMT2 are selected as a candidate substance for treating or preventing cancer.
  • cells expressing EHMT2 gene are isolated and cultured cells exogenously or endogenously expressing EHMT2 gene in vitro.
  • the method includes the step of: (a) contacting a test substance with cells overexpressing EHMT2; (b) measuring cell-proliferating activity; and (c) selecting the test substance that reduces the cell-proliferating activity in the comparison with the cell-proliferating activity in the absence of the test substance.
  • the method of the present invention may further include the steps of: (d) selecting the test substance that have no effect to the cells no or little expressing EHMT2.
  • the methyltransferase activity can be determined by contacting a EHMT2 polypeptide with a substrate (e.g., histone H3 or fragments thereof comprising lysine 9) and a co-factor (e.g., S-adenosyl-L-methionine) under conditions suitable for methylation of the substrate and detecting the methylation level of the substrate.
  • a substrate e.g., histone H3 or fragments thereof comprising lysine 9
  • a co-factor e.g., S-adenosyl-L-methionine
  • the method includes the step of: [1] A method of screening for a candidate substance for treating or preventing cancer associated with EHMT2 overexpression, said method comprising the steps of: (a) contacting a polypeptide encoded by a polynucleotide corresponding to the EHMT2 gene with a substrate and a cofactor in the presence of the test substance; (b) detecting the methylation level of the substrate; (c) determining the methyltransferase activity by correlating the methylation level of the step (b) with the methyltransferase activity; and (d) selecting the test substance that reduces the methyltransferase activity as compared to the methyltransferase activity detected in the absence of the test substance; [2] The method of [1], wherein the substrate is a histone or a fragment thereof comprising at least one methylation region.
  • SAHH S-adenosyl homocysteine hydrolase
  • methyltransferase activity of a EHMT2 polypeptide can be determined by methods known in the art.
  • the EHMT2 and a substrate can be incubated with a labeled methyl donor, under suitable assay conditions.
  • Histone H3 peptides, and S-adenosyl-[methyl- 14 C]-L-methionine, or S-adenosyl-[methyl- 3 H]-L-methionine can be used as the substrate and labeled methyl donor, respectively.
  • Transfer of the radiolabel to a histone H3 peptide can be detected, for example, by SDS-PAGE electrophoresis and fluorography.
  • histone H3 peptide can be separated from the methyl donor by filtration, and the amount of radiolabel retained on the filter quantitated by scintillation counting.
  • suitable labels that can be attached to methyl donors such as chromogenic and fluorescent labels, and methods of detecting transfer of these labels to histone peptides, are known in the art.
  • histone peptide and histone H3 peptide refer to a full length of histone or a fragment thereof and a full length of histone H3 or fragment thereof, respectively.
  • the methyltransferase activity of EHMT2 can be determined using an unlabeled methyl donor (e.g. S-adenosyl-L-methionine) and reagents that selectively recognize methylated histone peptides.
  • methylated substrate e.g. S-adenosyl-L-methionine
  • substrate to be methylated and methyl donor under the condition capable of methylation of the substrate, methylated substrate can be detected by immunological method. Any immunological techniques using an antibody recognizing methylated substrate can be used for the detection.
  • an antibody against methylated histone is commercially available (e.g. ab1220, abcam).
  • ELISA or immunoblotting with antibodies recognizing methylated histone can be used for the present invention.
  • the histone H3 fragment to be used as a substrate typically retains lysine 9.
  • Such histone H3 fragment may be composed of at least 10 amino acid residues, at least 15 amino acid residues, or at least 20 amino acid residues.
  • a modified peptide of the histone H3 or fragment thereof may be used for which the methyltransferase has increased affinity/activity.
  • Such peptides can be designed by exchanging and/or adding and/or deleting amino acids and testing the substrate in serial experiments for methyltransferase assay using the EHMT2 polypeptide.
  • any functional equivalent of the EHMT2 polypeptide can be used so long as such functional equivalents retain methyltransferase activity of the EHMT2 polypeptide.
  • the functional equivalent of the EHMT2 polypeptide retains a SET-domain (e.g., amino acid position 1039-1154 SEQ ID NO: 2 or amino acid position 1005-1120 SEQ ID NO: 4) of the EHMT2 polypeptide.
  • an agent enhancing the methylation of the substrate can be used.
  • SAHH or a functional equivalent thereof is one known enhancing agent for the methylation. In the presence of the agent enhancing the methylation of the substrate, the methyltransferase activity can thereby be determined with higher sensitivity.
  • EHMT2 may be contacted with a substrate and a cofactor in the presence of an enhancing agent.
  • detection of methyltransferase activity can be performed by preparing cells which express the EHMT2 polypeptide, culturing the cells in the presence of a test substance, and determining methylation level of a histone, for example, by using the antibody specific binding to a methylation region of the histone for EHMT2 (i.e., histone H3 lysine 9). More specifically, the method includes the step of: [1] contacting a test substance with cells expressing EHMT2; [2] detecting a methylation level of histone H3 lysine 9; and [3] selecting the test substance that reduces the methylation level in the comparison with the methylation level in the absence of the test substance.
  • suppress the biological activity are preferably at least 10% suppression of the biological activity of EHMT2 in comparison with in absence of the substance, at least 25%, 50% or 75% suppression, or at least 90% suppression.
  • control cells that do not express EHMT2 polypeptide are used.
  • the present invention also provides a method of screening for a candidate substance that inhibits cell growth or a candidate substance for treating and/or preventing an EHMT2- associated disease, using the EHMT2 polypeptide or fragments thereof including the steps as follows: a) culturing cells which express an EHMT2 polypeptide or a functional fragment thereof, and control cells that do not express an EHMT2 polypeptide or a functional fragment thereof in the presence of the test substance; b) detecting the biological activity of the cells which express the protein and control cells; and c) selecting the test substance that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance.
  • suppressing the biological activity of EHMT2 reduces cell growth.
  • candidate substances that have the potential to, or are useful to, treat and/or prevent cancers can be identified.
  • the potential, or utility, of these candidate substances to treat or prevent cancers may be evaluated by secondary and/or further screening to identify therapeutic substances, compounds or agents for cancers.
  • a substance that inhibits the biological activity of an EHMT2 protein also inhibits the activity of a cancer, such a substance has an EHMT2-specific therapeutic effect.
  • a decrease in the expression of EHMT2 by siRNA results in the inhibition of cancer cell proliferation (Fig. 3). Accordingly, the present invention provides a method of screening for a substance that inhibits the expression of EHMT2.
  • a substance that inhibits the expression of EHMT2 may suppress the proliferation of cancer cells, and thus is useful for treating or preventing cancer, particularly EHMT2-associated cancers such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
  • screening may include, for example, the following steps: (a) contacting a test substance with a cell expressing EHMT2; and (b) selecting the test substance that reduces the expression level of EHMT2 as compared to a control.
  • the screening method of the present invention may include, following steps: (a) contacting a test substance with a cell expressing an EHMT2 gene; (b) detecting an expression level of the EHMT2 gene in the cell of the step (a); and (c) selecting the test substance that reduces the expression level detected in the step (b) in comparison with the expression level of an EHMT2 gene detected in the absence of the test substance.
  • such screening may include, for example, the following steps: a) contacting a test substance with a cell expressing the EHMT2 gene; b) detecting the expression level of the EHMT2 gene; and c) correlating the expression level of b) with the therapeutic effect of the test substance.
  • the therapeutic effect may be correlated with the expression level of the EHMT2 gene.
  • the test substance when the test substance reduces the expression level of the EHMT2 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level of the EHMT2 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • Cells expressing the EHMT2 include, for example, cell lines established from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma or cell lines transfected with EHMT2 expression vectors; any of such cells can be used for the above screening of the present invention.
  • the expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining and flow cytometry analysis.
  • the phrase "reduce the expression level" as defined herein includes at least 10% reduction of expression level of EHMT2 in comparison to the expression level in absence of the substance, at least 25%, 50% or 75% reduced level, or at least 95% reduced level.
  • the substance herein includes chemical compounds, double-strand nucleotides, proteins, peptides, polynucleotides, aptamers, and so on. The preparation of the double-strand nucleotides will be described bellow.
  • a substance that reduces the expression level of EHMT2 can be selected as candidate substances to be used for the treatment or prevention of cancer.
  • cells expressing EHMT2 gene are isolated and cultured cells exogenously or endogenously expressing EHMT2 gene in vitro.
  • the screening method of the present invention may include the following steps: (a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of EHMT2 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression or activity of said reporter gene; and (c) selecting the test substance that reduces the expression or activity of said reporter gene.
  • reporter genes and host cells are well known in the art.
  • Illustrative reporter genes include, but are not limited to, luciferase, green fluorescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of EHMT2 gene.
  • the transcriptional regulatory region of EHMT2 gene herein is the region from transcription start site to at least 500bp upstream, preferably 1000bp, more preferably 5000 or 10000bp upstream.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of the gene. Methods for identifying a transcriptional regulatory region, and assay protocols are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
  • the vector containing the said reporter construct may be introduced into host cells and the expression or activity of the reporter gene may be detected by methods well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on).
  • "Reduces the expression or activity” as defined herein includes at least 10% reduction of the expression or activity of the reporter gene in comparison with expression or activity in absence of the test substance, at least 25%, 50% or 75% reduction, or at least 95% reduction.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of EHMT2, the method including steps of: (a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of EHMT2 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression level or activity of said reporter gene; and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression level or activity of said reporter gene.
  • the therapeutic effect of the test substance on inhibiting the cell growth or the therapeutic effect of a candidate substance for treating or preventing EHMT2 associating disease may be evaluated. Therefore, the present invention also provides a method for screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening for a candidate substance for treating or preventing EHMT2 associating disease.
  • the present invention provides a method which includes the following steps of: (a) contacting a test substance with a cell into which a vector, composed of the transcriptional regulatory region of the EHMT2 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) detecting the expression level or activity of said reporter gene; and (c) correlating the expression level of (b) with the therapeutic effect of the test substance.
  • the therapeutic effect may be correlated with the expression level or activity of said reporter gene.
  • the test substance when the test substance reduces the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified or selected as the substance or as a candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified as a substance having no significant therapeutic effect.
  • the present invention provides a method for screening for a substance for inhibiting the binding between the EHMT2 polypeptide and the promoter region of SIAH1 using such a binding of EHMT2 polypeptide and the promoter region of SIAH1 as an index. Furthermore, the present invention also provides a method for screening a candidate substance that inhibits or reduces the growth of cancer cells expressing EHMT2 gene, e.g. bladder cancer cells, lung cancer cells, AML cells, CML cells, esophageal cancer cells, breast cancer cells, cervical cancer cells or osteosarcoma cells, and a method for screening for a candidate substance for treating or preventing cancers, e.g. bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • cancer cells expressing EHMT2 gene e.g. bladder cancer cells, lung cancer cells, AML cells, CML cells, esophageal cancer cells, breast cancer cells, cervical cancer cells or osteosarcoma.
  • the present invention provides the following methods of [1] to [4]: [1] A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of: (a) contacting a EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to a promoter region of SIAH1 in the presence of a test substance; (b) detecting a binding between the polypeptide and the polynucleotide; (c) comparing the binding level detected in the step (b) with the binding level between the polypeptide and the polynucleotide detected in the absence of the test substance; and (d) selecting the test substance that reduces the binding level; [2] The method of [1], wherein the functional equivalent of the EHMT2 polypeptide comprises the DNA-binding domain; [3] The method of [2], wherein the functional equivalent of EHMT2 comprises the amino acid sequence of SEQ ID NO: 9; and [4] The method of any one of [1] to [3
  • the therapeutic effect of the test substance in inhibiting the cell growth or a candidate substance for treating or preventing EHMT2 associating disease may be evaluated. Therefore, the present invention also provides a method for screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening for a substance or a candidate substance for treating or preventing cancer.
  • the method includes the steps of: (a) contacting a EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to the promoter region of SIAH1 in the presence of a test substance; (b) detecting the level of binding between the polypeptide and the polynucleotide; (c) comparing the binding level detected in the step (b) with the binding level between the polypeptide and the polynucleotide detected in the absence of the test substance; and (d) correlating the binding level of c) with the therapeutic effect of the test substance.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance for treating or preventing cancer or inhibiting cancer, the method including steps of: (a) contacting an EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to the promoter region of SIAH1 in the presence of a test substance; (b) detecting a binding level between the polypeptide and the polynucleotide; (c) comparing the binding level detected in the step (b) with the binding level between the polypeptide and the polynucleotide detected in the absence of the test substance; and (d) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when a test substance reduces the binding level.
  • the therapeutic effect may be correlated with the binding level of the EHMT2 polypeptide and a polynucleotide corresponding to the promoter region of SIAH1.
  • the test substance when the test substance reduces the binding level of the EHMT2 polypeptide and a polynucleotide corresponding to the promoter region of SIAH1 as compared to a level detected in the absence of the test substance, the test substance may identified or selected as a substance or a candidate substance having the therapeutic effect.
  • test substance when the test substance does not reduce the binding level of EHMT2 polypeptide and a polynucleotide corresponding to the promoter region of SIAH1 as compared to a level detected in the absence of the test substance, the test substance may identified as a substance having no significant therapeutic effect.
  • a functional equivalent of an EHMT2 polypeptide is a polypeptide that has a biological activity equivalent to an EHMT2 polypeptide (SEQ ID NO: 2, 4) (see, (1) Genes and Polypeptides).
  • the functional equivalent of EHMT2 polypeptide may be a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 2 or 4 comprising the DNA region-binding domain.
  • An example of such functional equivalent includes, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 9.
  • Many methods well known by one skilled in the art can be used in screening for a substance that inhibits the binding of EHMT2 polypeptide to the polynucleotide.
  • Such a screening can be conducted using, for example, ChIP assay, gel shift assay, affinity chromatography or a biosensor using the surface plasmon resonance phenomenon.
  • some protein-DNA binding assay kits are commercially available (e.g., Protein-DNA Binding Assay, Clontech).
  • a polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof.
  • the polypeptide is isolated from cells expressing EHMT2, or chemically synthesized to be contacted with a polynucleotide corresponding to a promoter region of SIAH1 in vitro.
  • a polynucleotide to be used for screening can be a synthesized polynucleotide or a DNA derived from natural sources, or a partial oligonucleotide thereof. Any test substances aforementioned can be used for screening.
  • the present screening method can be performed using cells that express the EHMT2 polypeptide and have the SIAH1 gene.
  • Cells that expresses the EHMT2 polypeptide and possess the SIAH1 gene include, for example, cell lines established from cancer, e.g. bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • cells can be prepared by introducing an expression vector of the EHMT2 gene into a cell that possesses the SIAH gene.
  • the binding of EHMT2 polypeptide to the SIAH1 promoter region can be detected by, for example, ChIP assay using an anti-EHMT2 antibody or antibody against a tag protein fused with the EHMT2 polypeptide and a primer set for amplification of the SIAH1 promoter region (see, "Example 1, Chromatin Immunoprecipitation Assay").
  • the EHMT2 polypeptide suppresses the expression of the SIAH1 gene by binding to its promoter region, and consequently, inhibits apoptotic cell death in cancer cells .
  • substances that are useful for treating or preventing cancers or substances that have the potential to treat or prevent cancers can be identified.
  • the potential of these candidate substance to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance that inhibits the binding of the EHMT2 polypeptide to the SIAH1 promoter region inhibits activities of cancer such as cell growth or proliferation, such a substance may have an EHMT2-specific therapeutic effect.
  • candidate substances that (i) bind to the EHMT2 polypeptide; (ii) suppress/reduce the biological activity (e.g., the cell-proliferating activity, the methyltransferase activity) of the EHMT2 polypeptide; (iii) reduce the expression level of EHMT2 gene; or (iv) suppress/reduce the binding of the EHMT2 polypeptide to the SIAH1 promoter region candidate substances that are useful for treating or preventing cancers or have the potential to treat or prevent cancers (e.g., bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma) can be identified.
  • the therapeutic potential of these candidate substances may be evaluated by secondary and/or further screening to identify therapeutic substances for cancers. For example, when a substance that binds to the EHMT2 polypeptide inhibits the above-described activities of cancer, it may be concluded that such a substance has the EHMT2-specific therapeutic effect.
  • the term "isolated double-stranded molecule” refers to a nucleic acid molecule that inhibits expression of a target gene and includes, for example, a short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)), a short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA), or a small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • dsD/R-NA double-stranded chimera of DNA and RNA
  • shD/R-NA small hairpin chimera of DNA and RNA
  • a target sequence is a nucleotide sequence within the mRNA or cDNA sequence of a gene, which will result in suppression of translation of the whole mRNA if a double-stranded nucleic acid molecule of the present invention is introduced within a cell expressing the gene.
  • a nucleotide sequence within the mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded polynucleotide including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene.
  • the double-stranded polynucleotide which suppresses the gene expression may comprise the target sequence and may further comprise a 3'overhang having 2 to 5 nucleotides in length (e.g., uu) on one or both strands of the double-stranded polynucleotide.
  • a sense strand sequence of a double-stranded cDNA i.e., a sequence that mRNA sequence is converted into DNA sequence
  • a double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
  • the phrase "corresponding to” means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule.
  • a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region
  • base "t”s within the RNA region are replaced with base "u”s.
  • base "u"s within the DNA region are replaced with "t”s.
  • the target sequences are mainly shown in DNA.
  • the present invention also provides a double-stranded molecule whose target sequence includes or is limited to SEQ ID NO: 34 or SEQ ID NO: 35 which is shown in DNA but can be replaced with RNA.
  • a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
  • a double-stranded molecule may have one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and a complementary sequence.
  • siRNA refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA includes an EHMT2 sense nucleic acid sequence (also referred to as “sense strand”), an EHMT2 antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin.
  • the siRNA may either be a dsRNA or shRNA.
  • dsRNA refers to a construct of two RNA molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or “antisense” RNAs selected from a protein coding sequence of target gene sequence, but also or alternatively, may comprise a nucleotide sequence selected from non-coding region of the target gene.
  • shRNA refers to an siRNA having a stem-loop structure, composed of first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • siD/R-NA refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA.
  • a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used.
  • the siD/R-NA includes an EHMT2 sense nucleic acid sequence (also referred to as "sense strand"), an EHMT2 antisense nucleic acid sequence (also referred to as "antisense strand”) or both.
  • the siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin.
  • the siD/R-NA may either be a dsD/R-NA or shD/R-NA.
  • the term "dsD/R-NA” refers to a construct of two molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or "antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotides having a nucleotide sequence selected from non-coding region of the target gene.
  • One or both of the two molecules constructing the dsD/R-NA may be composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules may be composed of RNA and the other composed of DNA (hybrid double-strand).
  • shD/R-NA refers to an siD/R-NA having a stem-loop structure, composed of a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • an "isolated nucleic acid” is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state.
  • examples of isolated nucleic acid includes DNA, RNA, and derivatives thereof.
  • a double-stranded molecule against EHMT2 that hybridizes to target mRNA may decrease or inhibit production of EHMT2 protein encoded by EHMT2 gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the protein.
  • the expression of EHMT2 in several cancer cell lines may be inhibited by dsRNA (Fig. 3).
  • the present invention provides isolated double-stranded molecules that are capable of inhibiting the expression of an EHMT2 gene when introduced into a cell expressing the gene.
  • the target sequence of double-stranded molecule may be designed by an siRNA design algorithm such as that mentioned below. Examples of EHMT2 target sequences include the nucleotide sequences of SEQ ID NOs: 34 and 35.
  • [1] to [19] are the following double-stranded molecules [1] to [19]: [1] An isolated double-stranded molecule that, when introduced into a cell, inhibits expression of EHMT2 and cell proliferation, such molecules composed of a sense strand and a complementary antisense strand , hybridized to each other to form the double-stranded molecule; [2] The double-stranded molecule of [1], wherein said double-stranded molecule inhibits expression, translation, or stability of mRNA, matching a target sequence of SEQ ID NO: 34 or 35; [3] The double-stranded molecule of [1] or [2], wherein the sense strand contains a nucleotide sequence corresponding to a target sequence of SEQ ID NO: 34 or 35; [4] The double-stranded molecule of any one of [1] to [3], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a less than about
  • the double-stranded molecule of the present invention will be described in more detail below.
  • Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Patent No. 6,506,559, herein incorporated by reference in its entirety).
  • a computer program for designing siRNAs is available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). The computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.
  • Target Sites 1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex. 2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.
  • BLAST which can be found on the NCBI server at:ncbi.nlm.nih.gov/BLAST/, may be used (Altschul SF et al., Nucleic Acids Res 1997 Sep 1, 25(17): 3389-402). 3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
  • the target sequence of the double-stranded molecules of the present invention may be designed as SEQ ID NO: 34 and 35 for EHMT2 gene.
  • the present invention provides double-stranded molecule targeting the sequences of SEQ ID NO: 34 and 35 for EHMT2 gene
  • the double-stranded molecule of the present invention may be directed to a single target EHMT2 gene sequence or may be directed to a plurality of target EHMT2 gene sequences.
  • the siRNA may be directed to multiple targets of the EHMT2 gene sequence.
  • the composition may contain siRNAs of EHMT2 directed to two, three, four, or five or more target sequences of EHMT2, respectively.
  • EHMT2 target sequence is meant a nucleotide sequence that is identical or substantially identical to a portion of the EHMT2 gene.
  • the target sequence can include the 5' untranslated (UT) region, the open reading frame (ORF) or the 3' untranslated region of the human EHMT2 gene.
  • siRNA molecules of the invention can be defined by their ability to hybridize specifically to mRNA or cDNA from a EHMT2 gene under stringent conditions.
  • a double-stranded molecule of the present invention targeting the above-mentioned targeting sequence of EHMT2 gene includes isolated polynucleotides that contain the nucleic acid sequences of target sequences and/or complementary sequences to the target sequence.
  • Examples of polynucleotides targeting EHMT2 gene include those containing the sequence of SEQ ID NO: 34 or 35 and/or complementary sequences thereof;
  • the present invention is not limited to these examples, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of EHMT2 gene.
  • the phrase "minor modification" as used in connection with a nucleic acid sequence indicates one, two or several substitutions, deletions, additions or insertions of nucleic acids to the sequence.
  • a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and the other polynucleotide has a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule. Examples of such double-stranded molecules include dsRNA and dsD/R-NA.
  • a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand and the antisense strand are linked by an intervening strand, and hybridize to each other to form a hairpin loop structure.
  • Examples of such double-stranded molecule include shRNA and shD/R-NA.
  • a double-stranded molecule of the present invention is composed a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule.
  • a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence or complementary sequence is replaced with "u".
  • such a double-stranded molecule of the present invention includes a stem-loop structure, composed of the sense and antisense strands.
  • the sense and antisense strands may be joined by a loop.
  • the present invention also provides the double-stranded molecule composed of a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.
  • double-stranded molecules targeting the EHMT2 gene may have a sequence selected from among SEQ ID NOs: 34 and 35 as a target sequence.
  • examples of the double-stranded molecule of the present invention include a polynucleotide and a complementary sequence thereto, such as a polynucleotide that has a sequence corresponding to SEQ ID NO: 34 or 35 and a complementary sequence thereto.
  • the term "several" as applies to nucleic acid substitutions, deletions, additions and/or insertions may mean 3-7, preferably 3-5, more preferably 3-4, even more preferably 3 nucleic acid residues.
  • a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples.
  • double-stranded molecules composed of sense strands of various portions of mRNA of EHMT2 genes or antisense strands complementary thereto were tested in vitro for their ability to decrease production of EHMT2 gene product in cancer cell lines according to standard methods.
  • reduction in EHMT2 gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g. RT-PCR using primers for the EHMT2 mRNA mentioned under Example 1 item "Quantitative real time PCR".
  • Sequences that decrease the production of an EHMT2 gene product in in vitro cell-based assays can then be tested for their inhibitory effects on cell growth. Sequences that inhibit cell growth in in vitro cell-based assays can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of an EHMT2 gene product and decreased cancer cell growth.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide
  • binding means the physical or chemical interaction between two polynucleotides.
  • the polynucleotide includes modified nucleotides and/or non-phosphodiester linkages, these polynucleotides may also bind each other as same manner.
  • complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches.
  • the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by hybridization.
  • such duplexes contain no more than 1 mismatch for every 10 matches.
  • such duplexes contain no mismatches.
  • the polynucleotide may be less than 3982 nucleotides in length for EHMT2.
  • the polynucleotide may be less than 500, 200, 100, 75, 50, or 25 nucleotides in length for EHMT2.
  • the isolated polynucleotides of the present invention are useful for forming double-stranded molecules against EHMT2 gene or preparing template DNAs encoding the double-stranded molecules.
  • the polynucleotides may be longer than 19 nucleotides, longer than 21 nucleotides, or have a length of between about 19 and 25 nucleotides.
  • the present invention provides a double-stranded molecule composed of a sense strand and an antisense strand, wherein the sense strand is a nucleotide sequence corresponding to a target sequence.
  • the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  • the double-stranded molecule may serve as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells.
  • the identified target RNA may be cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA.
  • a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the EHMT2 gene under stringent conditions.
  • target sequence or “target nucleic acid” or “target nucleotide”.
  • target nucleic acid or “target nucleotide”.
  • nucleotide sequence of the "target sequence” can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
  • the double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages.
  • Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule.
  • Chemical modifications which may be incorporated into the present molecules include those described in WO03/070744, and WO2005/045037. In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include, but are not limited to, phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base” nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
  • modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule.
  • modifications include, but are not limited to, chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
  • modifications can be used to increase or decrease affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine.
  • the double-stranded molecule is a double-stranded molecule with a 3' overhang
  • the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200).
  • published documents such as US20060234970 are available.
  • the present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene, such as EHMT2.
  • the double-stranded molecules of the present invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
  • RNA e.g., dsD/R-NA or shD/R-NA.
  • a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability.
  • DNA and RNA i.e., a hybrid type double-stranded molecule composed of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule containing both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule.
  • the hybrid of a DNA strand and an RNA strand may be either where the sense strand is DNA and the antisense strand is RNA, or vice versa so long as it can inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.
  • the chimera type double-stranded molecule may be either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the molecule may contain as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule may be required to be RNA within a range to induce sufficient inhibition of the expression.
  • an upstream partial region i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands
  • the upstream partial region indicates the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand.
  • regions flanking to 5'-end of sense strand and/or 3'-end of antisense strand are referred to upstream partial region.
  • a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand are composed of RNA.
  • the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
  • sense strand 5'-[-----DNA-----]-3' 3'-(RNA)-[DNA]-5' : antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-[DNA]-5' : antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(-----RNA-----)-5' : antisense strand
  • the upstream partial region may be a domain composed of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules.
  • examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA.
  • the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
  • the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA).
  • shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence.
  • the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • a loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure.
  • Such loop sequence may be joined to 5' or 3' end of a sense strand and 3' or 5' end of an antisense strand to form the hairpin loop structure.
  • the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a nucleotide sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A].
  • the target sequence may be selected from among, for example, the nucleotide sequences of SEQ ID NO: 34 and 35.
  • the present invention is not limited to these examples, and the target sequence in [A] may be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted EHMT2 gene.
  • the region [A] hybridizes to [A'] to form a loop composed of the region [B].
  • the intervening single-stranded portion [B], i.e., loop sequence may be 3 to 23 nucleotides in length.
  • the loop sequence for example, can be selected from among the following sequences (http://www.ambion.com/techlib/tb/tb_506.html).
  • loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26): CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26; UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.
  • the loop sequence can be selected from among AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto: GAGUUUGGCUAUGAGGCUA -[B]- UAGCCUCAUAGCCAAACUC (for target sequence SEQ ID NO: 34). GCAAAUAUUUCACCUGCCA -[B]- UGGCAGGUGAAAUAUUUGC (for target sequence SEQ ID NO: 35).
  • nucleotides can be added to 3'end of the sense strand and/or antisense strand of the target sequence, as 3' overhangs.
  • nucleotides constituting a 3' overhang include "t" and "u", but are not limited thereto.
  • the number of nucleotides to be added is at least 2, generally 2 to 10, or 2 to 5.
  • the added nucleotides may be single stranded at the 3'end of the antisense strand or sense strand of the double-stranded molecule.
  • a 3' overhang sequence may be added to the 3' end of the single polynucleotide.
  • the method for preparing the double-stranded molecule is not particularly limited and includes chemical synthetic methods known in the art.
  • chemical synthesis method sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule.
  • Specific example for the annealing includes wherein the synthesized single-stranded polynucleotides are mixed in a molar ratio of preferably at least about 3:7, about 4:6, or in a substantially equimolar amount (i.e., a molar ratio of about 5:5).
  • the mixture is heated to a temperature at which double-stranded molecules dissociate and then gradually cooled down.
  • the annealed double-stranded polynucleotide can be purified by methods known in the art. Examples of purification methods include methods utilizing agarose gel electrophoresis or methods in which remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
  • the regulatory sequences flanking EHMT2 sequences may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner.
  • the double-stranded molecules can be transcribed intracellularly by cloning EHMT2 gene templates into a vector containing, e.g., an RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • snRNA small nuclear RNA
  • the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence.
  • a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter
  • the regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules are described below.
  • Vector containing a double-stranded molecule of the present invention contemplates vectors containing one or more of the double-stranded molecules described herein, and a cell containing such a vector.
  • a vector encoding a double-stranded molecule that, when introduced into a cell, inhibits expression of EHMT2 and cell proliferation, the double-stranded molecule composed of a sense strand and an antisense strand complementary thereto and hybridized to each other;
  • a vector of the present invention may encode a double-stranded molecule of the present invention in an expressible form.
  • the phrase "in an expressible form” indicates that the vector, when introduced into a cell, will express the molecule.
  • the vector includes regulatory elements necessary for expression of the double-stranded molecule.
  • the expression vector encodes the nucleic acid sequence of the double-stranded molecule of the present invention and is adapted for expression of said double-stranded molecule.
  • Such vectors of the present invention may be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.
  • Vectors of the present invention can be produced, for example, by cloning EHMT2 sequence into an expression vector so that regulatory sequences are operatively-linked to the EHMT2 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5).
  • RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • a first promoter e.g., a promoter sequence flanking to the 3' end of the cloned DNA
  • RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene.
  • two vector constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then form a double-stranded molecule.
  • the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector that contains both the sense and complementary antisense sequences of the target gene.
  • the vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
  • the vectors of the present invention include, for example, viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell.
  • Another example of useable vector includes Bacille Calmette Guerin (BCG) vectors.
  • BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
  • dsRNAs for EHMT2 were tested for their ability to inhibit cell growth.
  • the dsRNA for EHMT2 (Fig. 3, 7) effectively knocked down the expression of the gene in several cancer cell lines, which coincided with suppression of cell proliferation.
  • the present invention provides methods for inhibiting cancer cell growth by inducing dysfunction of the EHMT2 gene via inhibiting the expression of EHMT2.
  • EHMT2 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention that specifically target the EHMT2 gene.
  • the present double-stranded molecules and vectors to inhibit cell growth of cancerous cell indicates that they can be used for methods for treating cancer such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • cancer such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • the present invention provides methods to treat patients with cancer by administering a double-stranded molecule against EHMT2 gene or a vector expressing the molecule.
  • the treating methods of the present invention may be carried out without adverse effect because EHMT2 gene was minimally detected in normal organs (Fig. 1, 2, 5, 6, 10).
  • [1] A method for inhibiting a growth of cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses the EHMT2 gene, such method including the step of administering at least one isolated double-stranded molecule or vector encoding the double-stranded molecule that inhibits the expression of EHMT2 and cell proliferation in a cell over-expressing the gene, wherein the double-stranded molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule; [2] The method of [1], wherein the double-stranded molecule inhibits the expression of mRNA which matches a target sequence of SEQ ID NO: 34 or 35; [3] The method of [1], wherein the sense strand contains the sequence corresponding to a target sequence of SEQ ID NO: 34 or 35; [4] The method of any one of [1] to [3], wherein the cancer
  • the growth of cells expressing an EHMT2 gene may be inhibited by contacting the cells with a double-stranded molecule against an EHMT2 gene, a vector expressing the molecule or a composition containing the same.
  • the cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art.
  • the phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule.
  • Cell growth may be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
  • the term "specifically inhibit” in the context of inhibitory polynucleotides and polypeptides refers to the ability of an agent or ligand to inhibit the expression or the biological function of EHMT2. Specific inhibition typically results in at least about a 2-fold inhibition over background, greater than about 10-fold or greater than 100-fold inhibition of EHMT2 expression (e.g., transcription or translation) or measured biological function (e.g., cell growth or proliferation, inhibition of apoptosis, intracellular signaling from EHMT2). Expression levels and/or biological function can be measured in the context of comparing treated and untreated cells, or a cell population before and after treatment. In some embodiments, the expression or biological function of EHMT2 is completely inhibited, or inhibited to below detectable levels.
  • the growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention.
  • Exemplary cells include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • patients suffering from or at risk of developing a disease related to EHMT2 may be treated with the administration of a double-stranded molecule of the present invention, at least one vector expressing the molecule or a composition containing the molecule.
  • patients suffering from cancer may be treated according to the present methods.
  • the type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed.
  • patients treated by the methods of the present invention may be selected by detecting the expression of EHMT2 in a biopsy from the patient by RT-PCR or immunoassay.
  • the biopsy specimen from the subject may be confirmed for EHMT2 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.
  • each of the molecules may have different structures but act on mRNA that matches the same target sequence of EHMT2.
  • multiple types of the double-stranded molecules may act on mRNA that matches a different target sequence of same gene.
  • the method may utilize double-stranded molecules directed to one, two or more target sequences of EHMT2.
  • a double-stranded molecule of present invention may be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts.
  • a DNA encoding the double-stranded molecule may be introduced into cells as a vector.
  • transfection-enhancing agent such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), may be employed.
  • a treatment is deemed “efficacious” if it leads to clinical benefit such as, reduction in expression of EHMT2 gene, or a decrease in size, prevalence, proliferation, or metastatic potential of the cancer in the subject.
  • “efficacious” means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating any particular tumor type.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis includes 10%, 20%, 30% or more reduction, or inducing disease stability.
  • a double-stranded molecule of the present invention degrades EHMT2 mRNA in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, as compared to standard cancer therapies, the present invention requires the delivery of significantly less double-stranded molecule at or near the site of cancer in order to exert therapeutic effect.
  • an effective amount of the double-stranded molecule of the present invention can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is local or systemic.
  • an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, including from about 2 nM to about 50 nM, and about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered.
  • the precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
  • the present methods can be used to inhibit the growth or metastasis of cancer expressing EHMT2 gene; for example bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • a double-stranded molecule containing a target sequence of EHMT2 gene e.g., SEQ ID NO: 34 and 35
  • SEQ ID NO: 34 and 35 is useful for the treatment of cancer.
  • the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical composition different from the double-stranded molecule.
  • the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
  • the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
  • Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma tissue, and can also increase the blood half-life of the double-stranded molecule.
  • Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream.
  • the liposomes encapsulating the double-stranded molecule of the present invention may include a ligand molecule that can deliver the liposome to the cancer site.
  • exemplary ligands include those ligands which bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens.
  • the liposomes encapsulating the present double-stranded molecule may be modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure.
  • a liposome of the invention can include both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization-inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No.
  • Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
  • target tissue characterized by such microvasculature defects for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen.
  • liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
  • Opsonization-inhibiting moieties suitable for modifying liposomes include water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, or from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM.sub.1.
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization-inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • natural polysaccharides containing amino acids or carboxylic acids e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan
  • aminated polysaccharides or oligosaccharides linear or branched
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
  • Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH 3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
  • Vectors expressing a double-stranded molecule of the present invention are discussed above. Such vectors expressing at least one double-stranded molecule of the present invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • a suitable delivery reagent including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • the double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites.
  • the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation.
  • injections or infusions of the double-stranded molecule or vector are given at or near the site of the cancer.
  • the double-stranded molecule of the present invention can be administered in a single dose or in multiple doses.
  • the infusion can be a single sustained dose or can be delivered by multiple infusions.
  • administration can include injection of the double-stranded molecule directly into the tissue at or near the site of cancer. In some cases, administration includes multiple injections of the double-stranded molecule into the tissue at or near the site of cancer.
  • the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, or from about seven to about ten days.
  • the double-stranded molecule may be injected at or near the site of cancer once a day for seven days.
  • the effective amount of a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.
  • a cancer overexpressing EHMT2 can be treated with at least one active ingredient selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of: a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of EHMT2 with a normal control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is increased as compared to the normal control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • such a method includes the steps of: a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of EHMT2 with a cancerous control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is similar or equivalent to the cancerous control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • the cancer includes, but is not limited to, bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is an aspect of the present invention to confirm whether the expression level of EHMT2 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing EHMT2, such method including the steps of: i) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of EHMT2 with normal control; and iii) administrating at least one component selected from the group consisting of (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, to a subject with a cancer overexpressing EHMT2 compared with normal control.
  • the present invention also provides a pharmaceutical composition containing at least one component selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, for use in administrating to a subject having a cancer overexpressing EHMT2.
  • the present invention further provides a method for identifying a subject to be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof, which method may include the step of determining an expression level of EHMT2 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with.
  • a subject to be treated by the present method may be a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of EHMT2 in cancer cells or tissues obtained from a subject may be determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of EHMT2 may be quantified using probes by hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a chip or an array.
  • an array may be used for detecting the expression level of EHMT2.
  • the cDNA of EHMT2 may be used as the probes.
  • the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of EHMT2 e.g., SEQ ID NO: 1 or 3
  • primers may be prepared based on the available sequence information of the gene.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of EHMT2.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the diagnosis of the present invention.
  • the quantity of observed protein SEQ ID NO: 2 or 4
  • Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be measured via immunohistochemical analysis using an antibody against the EHMT2 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of EHMT2 gene.
  • the expression level of a target gene, e.g., the EHMT2 gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level may be determined at the same time with the cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known.
  • normal cells obtained from non-cancerous regions of an organ that has the cancer to be treated may be used as normal control.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of EHMT2 gene in samples from subjects whose disease states are known.
  • the control level can be derived from a database of expression patterns from previously tested cells.
  • the expression level of EHMT2 gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples.
  • a control level is determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample.
  • a standard value of the expression levels of EHMT2 gene in a population with a known disease state may be used. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.
  • a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • cancerous control level When the expression level of EHMT2 gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.
  • compositions containing a double-stranded molecule of the present invention In addition to the above, the present invention also provides pharmaceutical compositions that include the present double-stranded molecule or the vector coding for double-stranded molecules.
  • composition is used to refer to a product that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutical when used in relation to the modifier "pharmaceutical” (as in “pharmaceutical composition”), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, in the context of the present invention, the term “pharmaceutical composition” refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier”, as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • active ingredient herein refers to a substance in composition that is biologically or physiologically active. Particularly, in the context of pharmaceutical composition, the term “active ingredient” refers to a substance that shows an objective pharmacological effect.
  • active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly.
  • such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on.
  • active ingredient may also be referred to as “bulk”, “drug substance” or “technical product”.
  • compositions [1] to [32] [1] A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer and the cancer cell express at least one EHMT2 gene, including an isolated double-stranded molecule that inhibits the expression of EHMT2 and the cell proliferation or a vector encoding the double-stranded molecule, wherein the molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule; [2] The composition of [1], wherein the double-stranded molecule inhibits the expression of mRNA which matches a target sequence of SEQ ID NO: 34 or 35; [3] The composition of [1], wherein the double-stranded molecule, wherein the sense strand contains a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35; [4] The composition of any one of [1] to [3], wherein the cancer to be treated is bladder cancer,
  • compositions of the present invention are described in additional detail below.
  • the double-stranded molecule of the present invention may be formulated as a pharmaceutical composition prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention may be characterized as being at least sterile and pyrogen-free.
  • pharmaceutical composition includes formulations for human and veterinary use.
  • the pharmaceutical compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
  • suitable pharmaceutical formulations of the present invention include those suitable for oral, rectal, nasal, topical (including buccal, sub-lingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation.
  • Other formulations include implantable devices and adhesive patches that release a therapeutic agent.
  • the above-described formulations may be adapted to give sustained release of the active ingredient.
  • the present pharmaceutical composition contains the double-stranded molecule or vector encoding that of the present invention (e.g., 0.1 to 90% by weight), or a pharmaceutically acceptable salt of the molecule, mixed with a pharmaceutically acceptable carrier medium.
  • exemplary physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the composition may contain multiple types of the double-stranded molecules, each of the molecules may be directed to the same target sequence, or different target sequences of EHMT2.
  • the composition may contain double-stranded molecules directed to EHMT2.
  • the composition may contain double-stranded molecules directed to one, two or more target sequences EHMT2.
  • the present composition may contain a vector coding for one or plural double-stranded molecules.
  • the vector may encode one, two or several kinds of the present double-stranded molecules.
  • the present composition may contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
  • compositions of the present invention can also include conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers e.g., tromethamine hydrochloride
  • additions of chelants such as, for example, DTPA or DTPA-bisamide
  • calcium chelate complexes for example calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • Pharmaceutical compositions of the present invention can be packaged for use in liquid form, or can be lyophilized.
  • conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, or 25-75%, of one or more double-stranded molecule of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, or 1-10% by weight, of one or more double-stranded molecule of the present invention encapsulated in a liposome as described above, and propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • the present composition may contain other pharmaceutically active ingredients so long as they do not inhibit the in vivo function of the double-stranded molecules of the present invention.
  • the composition may contain chemotherapeutic agents conventionally used for treating cancers.
  • compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
  • active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
  • the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the EHMT2 gene.
  • the present invention provides for the use of the double-stranded nucleic acid molecule of the present invention in manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene.
  • the present invention relates to a use of double-stranded nucleic acid molecule inhibiting the expression of an EHMT2 gene in a cell, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and target to a sequence of SEQ ID NO: 34 or 35, for manufacturing a pharmaceutical composition for treating cancer expressing EHMT2 gene.
  • the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the EHMT2 gene.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of EHMT2 gene in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and target to a sequence of SEQ ID NO: 34 or 35 as active ingredients.
  • the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of EHMT2 gene in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NO: 34 or 35.
  • a EHMT2 inhibitor was tested for their ability to inhibit cell growth.
  • the EHMT2 inhibitor (Fig. 9) effectively reduces growth rate of the several type of cancer cell lines.
  • the present invention provides methods for inhibiting cancer cell growth by an EHMT2 inhibitor.
  • Such ability of EHMT2 inhibitors to inhibit cell growth of cancerous cell indicates that they can be used for methods for treating cancer such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • the present invention provides methods to treat patients with cancer by administering an EHMT2 inhibitor.
  • an "EHMT2 inhibitor” refers to a substance that inhibits the function of the EHMT2 polypeptide.
  • an EHMT2 inhibitor inhibits histone methyltransferase activity of the EHMT2 polypeptide.
  • EHMT2 inhibitors include, for example, BIX-01294.
  • [1] A method for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses the EHMT2 gene, such method including the step of administering at least one EHMT2 inhibitor to a subject; [2] The method of [1], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma; [3] The method of [1] or [2], wherein the EHMT2 inhibitor is the BIX-01294.
  • the growth of cells expressing an EHMT2 gene may be inhibited by contacting the cells with an EHMT2 inhibitor.
  • the phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule.
  • Cell growth may be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
  • the growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the EHMT2 gene.
  • Exemplary cells include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
  • patients suffering from or at risk of developing disease related to EHMT2 may be treated with the administration of an EHMT2 inhibitor.
  • patients suffering from cancer may be treated according to the present methods.
  • the type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed.
  • patients treated by the methods of the present invention may be selected by detecting the expression of EHMT2 in a biopsy specimen from the patient by RT-PCR or immunoassay.
  • the biopsy specimen from the subject may be confirmed for EHMT2 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.
  • a treatment is deemed “efficacious” if it leads to clinical benefit such as, a decrease in size, prevalence, growth rate, or metastatic potential of the cancer in the subject.
  • “efficacious” means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer as described in " Method of Inhibiting or Reducing Growth of a Cancer Cell or Treating Cancer using a Double-Stranded Molecule of the Present Invention ".
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof includes any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effectively treating and/or providing prophylaxis of cancer may decrease mortality and improve the prognosis of individuals having cancer, decrease the levels of tumor markers in the blood, and alleviate detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or prophylaxis may include 10%, 20%, 30% or more reduction, or induction of disease stability.
  • an effective amount of an EHMT2 inhibitor to be administered to a given subject by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of an EHMT2 inhibitor is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, from about 2 nM to about 50 nM, or from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of an EHMT2 inhibitor can be administered.
  • the precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
  • the present methods can be used to inhibit the growth or metastasis of cancer expressing EHMT2 gene; for example bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
  • an EHMT2 inhibitor can also be administered to a subject in combination with a pharmaceutical composition different from the inhibitor compound of the present invention.
  • an EHMT2 inhibitor can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • an EHMT2 inhibitor can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
  • an EHMT2 inhibitor can be administered to the subject either as a naked, or in conjunction with a delivery reagent.
  • Suitable delivery reagents for administration in conjunction with the present a inhibitor compound include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation.
  • administration includes injections or infusions of the inhibitor compound at or near the site of the cancer.
  • An EHMT2 inhibitor can be administered in a single dose or in multiple doses. Where the administration of an EHMT2 inhibitor is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions.
  • the inhibitor compound is directly injected into the tissue is at or near the site of cancer. In another embodiment, the inhibitor compound is direction injected into the tissue at or near the site of cancer multiple times.
  • the inhibitor compound can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • an EHMT2 inhibitor can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, or from about seven to about ten days.
  • the inhibitor compound is injected at or near the site of cancer once a day for seven days.
  • the effective amount of a inhibitor compound administered to the subject can include the total amount of a inhibitor compound administered over the entire dosage regimen.
  • a cancer overexpressing EHMT2 can be treated with EHMT2 inhibitor.
  • the cancers to be treated by the present method include, but are not limited to, bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Accordingly, prior to the administration of an EHMT2 inhibitor as active ingredient, it is one aspect of the present invention to confirm whether the expression level of EHMT2 in the cancer cells or tissues to be treated is elevated as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing EHMT2, such method including the steps of: i) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of EHMT2 with normal control; and iii) administrating an EHMT2 inhibitor, to a subject with a cancer overexpressing EHMT2 compared with normal control.
  • the present invention also provides a pharmaceutical composition containing an EHMT2 inhibitor, for use in administrating to a subject having a cancer overexpressing EHMT2.
  • the present invention further provides a method for identifying a subject to be treated with an EHMT2 inhibitor, which method may include the step of determining an expression level of EHMT2 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with.
  • compositions containing an EHMT2 inhibitor include an EHMT2 inhibitor.
  • an EHMT2 inhibitor include an EHMT2 inhibitor.
  • compositions [1] to [4] [1] A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer or the cancer cell express the EHMT2 gene, including an EHMT2 inhibitor and a pharmaceutically acceptable carrier; [2] The composition of [1], wherein the EHMT2 inhibitor is BIX-01294.; [3] The composition of [1] or [2], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma; Suitable compositions of the present invention are described in additional detail below.
  • An EHMT2 inhibitor may be formulated as a pharmaceutical composition prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention may be characterized as being at least sterile and pyrogen-free. Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the present pharmaceutical composition may contain an EHMT2 inhibitor (e.g., 0.1 to 90% by weight), or a pharmaceutically acceptable salt of the molecule, mixed with a pharmaceutically acceptable carrier medium.
  • exemplary physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the composition may contain plural kinds of EHMT2 inhibitors, each of the molecules may be directed by the same mechanism, or different mechanism.
  • compositions of the present invention can also include conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the present invention can be packaged for use in liquid form, or can be lyophilized.
  • conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, preferably 25-75%, of one or more EHMT2 inhibitors.
  • a pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, preferably 1-10% by weight, of one or more EHMT2 inhibitors encapsulated in a liposome, and propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • the present composition may contain other pharmaceutically active ingredients so long as they do not inhibit the in vivo function of the histone methyltransferase inhibitor of the present invention.
  • the composition may contain chemotherapeutic agents conventionally used for treating cancers.
  • the present invention provides the use of an EHMT2 inhibitor in manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene.
  • the present invention further provides an EHMT2 inhibitor for use in treating a cancer expressing the EHMT2 gene.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with an EHMT2 inhibitor.
  • the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is an EHMT2 inhibitor.
  • Example 1 General Methods Tissue samples and RNA preparation
  • Bladder tissue samples and RNA preparation were described previously (Wallard MJ et al. Br J Cancer 2006;94:569-77.). Briefly, 126 surgical specimens of primary urothelial carcinoma were collected, either at cystectomy or transurethral resection of bladder tumor (TUR-Bt), and snap-frozen in liquid nitrogen. 26 normal bladder tissues were collected from areas of macroscopically-normal regions in patients with no evidence of malignancy. Five sequential sections of 7 micron thickness were cut from each tissue and stained using Histogene(trademark) staining solution (Arcturus, California, USA) following the manufacturer's protocol, and assessed for cellularity and tumor grade by an independent consultant urohistopathologist.
  • Vimentin and Uroplakin were sourced and qRT-PCR performed according to the manufacturer's instructions (Assays on demand, Applied Biosystems, Warrington, UK). Vimentin is primarily expressed in messengerchymally derived cells, and was used as a stromal marker. Uroplakin is a marker of urothelial differentiation and is preserved in up to 90% of epithelially derived tumors (Olsburgh J et al. The Journal of pathology 2003;199:41-9.). Use of tissues for this study was approved by Cambridge shire Local Research Ethics Committee (Ref 03/018).
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • SCLC small cell lung cancer SBC-5
  • the Normal human lung fibroblast HFL-1 and the normal human colon fibroblast CCD-18Co were used as normal control cells.
  • D-MEM Dulbecco's modified Eagle's medium
  • E-MEM Eagle's minimal essential medium
  • F-12K medium for HFL-1 cells
  • Leibovitz's L-15 for SW780 cells
  • McCoy's 5A medium for RT4 and T24 cells
  • RPMI1640 medium for 5637, A549 and LC319 cells supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma).
  • the cDNAs were amplified by RT-PCR using poly (A) + RNAs isolated from various human organs as templates; the lengths of the amplicons ranged from 200 to 1,100 bp, without any repetitive or poly (A) sequences.
  • Many types of tumors and corresponding non-neoplastic tissues were prepared in 8-micrometer sections, as described previously ( Kitahara O et al. Cancer Res 2001;61:3544-9.). A total of 30,000-40,000 cancer or noncancerous cells were collected selectively using the EZ cut system (SL Microtest GmbH, Germany) according to the manufacturer's protocol. Extraction of total RNA, T7-based amplification, and labeling of probes were performed as described previously ( Kitahara O et al.
  • Nonspecific binding was blocked by incubating sections with 3% BSA in a humidified chamber for 30 min at ambient temperature, then a 1:1000 dilution of rabbit polyclonal anti-EHMT2 antibody (NB100-40825, Novus Biologicals) overnight at 4 degrees C. Sections were washed twice with PBS, incubated with 1 micro-g/micro-L goat anti-rabbit biotinylated IgG in PBS containing 1% BSA for 30 min at ambient temperature, and then incubated with ABC reagent for 30 min. Immunostaining was visualized using 3,3'-diaminobenzidine. Slides were dehydrated through graded alcohol to sylene washing and mounted on cover slips. Hematoxylin was used for nuclear counterstaining.
  • EnVision+ kit/horseradish peroxidase (Dako, Glostrup, Denmark) was applied. Briefly, slides of paraffin-embedded lung tumor specimens were processed under high pressure (125 degrees C, 30 s) in antigen-retrieval solution, high pH 9 (S2367, Dako Cytomation, Carpinteria, CA, USA), treated with peroxidase blocking regent, and then treated with protein blocking regent (K130, X0909, Dako Cytomation). Tissue sections were incubated with a rabbit anti-EHMT2 polyclonal antibody followed by HRP-conjugated secondary antibody (Dako Cytomation).
  • Antigen was visualized with substrate chromogen (Dako liquid DAB chromogen; Dako Cytomation). Finally, tissue specimens were stained with Mayer's haematoxylin (Muto pure chemicals Ltd, Tokyo, Japan) for 20 s to discriminate the nucleus from the cytoplasm.
  • substrate chromogen Dako liquid DAB chromogen; Dako Cytomation.
  • Quantitative real-time PCR As described above, the inventors obtained 126 bladder cancer tissues and 26 normal bladder tissues in Cambridge Addenbrooke's Hospital. For quantitative RT-PCR reactions, specific primers for all human GAPDH (housekeeping gene), SDH (housekeeping gene) and EHMT2 were designed (primer sequences in Table 1). PCR reactions were performed using the ABI prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK) following the manufacture's protocol. 50% SYBR GREEN universal PCR Master Mix without UNG (Applied Biosystems, Warrington, UK), 50 nM each of the forward and reverse primers and 2 microliter of reversely-transcribed cDNA were applied.
  • Amplification conditions were 5 min at 95 degrees C and then 45 cycles each consisting of 10 sec at 95 degrees C, 1 min at 55 degrees C and 10 sec at 72 degrees C. Then, reactions were heated for 15 sec at 95 degrees C, 1 min at 65 degrees C to draw the melting curve, and cooled to 50 degrees C for 10 sec.
  • Reaction conditions for target gene amplification were as described above and the equivalent of 5 ng of reverse transcribed RNA was used in each reaction. mRNA levels were normalized to GAPDH and SDH expression.
  • siRNA Transfection siRNA oligonucleotide duplexes were purchased from SIGMA Genosys for targeting the human EHMT2 transcript or the EGFP and FFLuc transcripts as control siRNAs. siRNA sequences are described in Table 2. siRNA duplexes (100 nM final concentration) were transfected in lung cancer cell lines with lipofectamine 2000 (Invitrogen) for 72 h, and cell viability was examined using Cell Counting Kit 8 (DOJINDO).
  • Flow cytometry assays for cell cycle analysis
  • the present inventors collected the cells after trypsin treatment, washed them twice with 1,000 micro-L of Assay Buffer and centrifuged for 5 min at 5,000 rpm. Cells were resuspended in 200 micro-L of Assay Buffer. 1,000 micro-L of fixative buffer was added, and the samples incubated at room temperature for 1 h. Finally, the present inventors added the propidium iodide reagent and analyzed cell cycle profiles by flow cytometry (LSR II, BD Biosciences). The proportion of each cell division was calculated and analyzed using Student's T test for significance.
  • ChIP assays were performed using ChIP Assay kit (Millipore, Billerica, MA) according to the manufacture's protocol. Briefly, the fragment of EHMT2 (SEQ ID NO: 9) and chromatin complexes was immnoprecipitated with anti-FLAG antibody 48 h after transfection with pCAGGS-n3FC (Mock) and pCAGGS-n3FC-EHMT2 (3xFLAG-EHMT2) vectors. After the bound DNA fragments to EHMT2 were eluted, and the amount was subjected to quantitative real-time PCR reactions. Primer sequences are shown in Table 1.
  • Coupled cell cycle and cell proliferation assay A 5'-bromo-2'-deoxyuridine (BrdU) flow kit (BD Pharmingen, San Diego, CA) was used to determine the cell cycle kinetics and to measure the incorporation of BrdU into DNA of proliferating cells.
  • the assay was performed according to the manufacturer's protocol. Briefly, SBC5 cells (2 x 10 5 per well) were seeded overnight in 6-well tissue culture plates and treated with an optimized concentration of siRNAs in medium containing 10% FBS for 72 hours, followed by addition of 10 micromolar BrdU, and incubations continued for an additional 30 min.
  • Both floating and adherent cells were pooled from triplicates wells per treatment point, fixed in a solution containing paraformaldehyde and the detergent saponin, and incubated for 1 hour with DNAase at 37 degrees C (30 micro g per sample).
  • FITC-conjugated anti-BrdU antibody (1:50 dilution in Wash buffer; BD Pharmingen, San Diego, CA) was added and incubation continued for 20 minutes at room temperature. Cells were washed in Wash buffer and total DNA was stained with 7-amino-actinomycin D (7-AAD; 20 microliter per sample), followed by flow cytometric analysis using FACScan (BECKMAN COULTER) and total DNA content (7-AAD) was determined CXP Analysis Software Ver. 2.2 (BECKMAN COULTER).
  • Example 2 EHMT2 expression is up-regulated in clinical cancer tissues
  • expression profiles were examined for several histone methyltransferase genes using a small number of clinical bladder samples, and a significant difference of EHMT2 gene expression was found between cancer and normal tissues (data not shown). Consequently, the present inventors analyzed 126 bladder cancer samples and 26 normal control samples (British), and found significant elevation of EHMT2 expression in tumor cells compared with in normal cells (P ⁇ 0.0001, Mann-Whitney U test) (Fig. 1A, Table 1). No significant difference was observed in expression levels among cancer samples of different stages and grades (Fig. 1B, Table 3).
  • EHMT2 expression is up-regulated at an early stage in bladder carcinogenesis, and remains high in the advanced stages of the disease.
  • Subclassification of tumors according to metastasis status, gender, smoking history and recurrence status identified no significant differences of EHMT2 expression levels (Table 3).
  • the present inventors then analyzed the expression patterns of EHMT2 in a number of Japanese clinical bladder cancer samples by cDNA microarray (Fig. 1C, Table 4), and confirmed significant overexpression in bladder cancers of Japanese patients (P ⁇ 0.0001, Mann-Whitney U-test). Consisting with this, expression levels of EHMT2 in bladder cancer cell lines were significantly higher than those in two normal human fibroblast cell lines (Fig. 6).
  • the present inventors then examined EHMT2 protein expression levels in lung tissue by immunohistochemistry (Fig. 2B) and observed strong EHMT2 staining in the nucleus of cancer tissues and weak staining in non-neoplastic tissues. Additionally, the present inventors examined microarray expression analysis of a large number of clinical samples derived from Japanese subjects and found that EHMT2 expression was also significantly up-regulated in various types of cancer compared with corresponding non-neoplastic tissues (Table 4; Fig. 5). These data indicate that EHMT2 is involved in many types of human cancer.
  • Example 3 Growth regulation of cancer cells by EHMT2
  • siRNAs against EHMT2 siEHMT2#1 and #2
  • siEGFP and siNC siRNAs against siRNAs
  • the present inventors transfected these siRNAs into A549 and SBC5 cells, in which EHMT2 was highly expressed (Fig.3A; Fig. 6).
  • EHMT2 expression in the cells transfected with two independent EHMT2 siRNAs was significantly suppressed in comparison with those transfected with control siRNAs at the mRNA and protein levels (Fig. 3B; Fig.7).
  • Fig. 3C colony formation assay and cell growth assay was performed.
  • the present inventors observed inhibition of colony formation (Fig. 3C) and significant growth suppression of two bladder cancer cell lines (SW780 and RT4) and three lung cancer cell lines (LC319, A549 and SBC5) after treatment with two EHMT2 siRNAs though no effect was observed for control siRNAs (Fig.3D).
  • Fig.3D control siRNAs
  • the cell cycle status of cancer cells were analyzed after treatment with siRNAs using flow cytometry stained with a FITC-conjugated anti-BrdU antibody and 7-AAD.
  • Example 4 SIAH1 directly regulated by EHMT2 is a key regulation of cancer cell growth and apoptosis
  • the present inventors identified target genes regulated by EHMT2 using microarray expression analysis.
  • the inventors isolated total RNA from SW780 and A549 cells 24 h after treatment with siEHMT2. The expression profile of these cells was analyzed by Affymetrix's HG-U133 Plus 2.0 Array in comparison with those treated with control siRNAs (siEGFP and siFFLuc), and the inventors identified a set of genes that were significantly up/down-regulated.
  • the sub-G1 population of cancer cells was significantly increased by knockdown of EHMT2, according to FACS analysis ( Figure 3E), illustrating that EHMT2 is associated with regulation of apoptosis in cancer cells.
  • the present inventors identified a down-stream gene for EHMT2 which was known to be involved in apoptotic regulation in cancer cells, by microarray analysis.
  • candidate genes the present inventors observed significant up-regulation of SIAH1, a tumor suppressor gene, after treatment with siEHMT2 (Fig. 4A, Fig. 4B).
  • Quantitative real-time PCR and Western blot also confirmed the up-regulation of SIAH1 after treatment with siEHMT2 (Fig. 4C).
  • a chromatin immunoprecipitation (ChIP) assay was performed. EHMT2 protein was highly enriched at the promoter region of SIAH1 after transfection with the 3xFLAG-EHMT2 vector together with increased levels of di-methylation on histone H3K9 (Fig. 4D). These results show that EHMT2 can directly suppress SIAH1 expression at the transcriptional level through the enhancement of histone H3K9 methylation status. Additionally, to validate the function of endogenous EHMT2 protein in cancer cells, the present inventors performed ChIP analysis of cells after treatment 12 with EHMT2 siRNA, using anti-EHMT2 and -H3K9me2 antibodies.
  • the present inventors then tried to clarify the significance of SIAH1 suppressed by EHMT2 in cancer cells. Since knockdown of EHMT2 significantly increased the sub-G1 population of cancer cells, the present inventors performed detailed apoptosis analysis using the SIAH1 siRNA whose effects were already validated. Cleaved PARP1 and caspase 3 were observed in SBC5 cells after treatment with siEHMT2, demonstrating that apoptosis may be induced by knockdown of EHMT2. Subsequently, the present inventors examined effects of SIAH1 knockdown on EHMT2 siRNA-induced apoptosis.
  • BIX-01294 reduced the growth rate of several cancer cell lines
  • a small molecule compound, BIX-01294 a diazepin-quinazolin-amine derivative
  • BIX-01294 specifically inhibits EHMT2 enzymatic activity and reduces H3K9me2 levels at the chromatin regions of several EHMT2 target genes ( Chang Y, et al. Nat Struct Mol Biol 2009;16:312-7., Kubicek S, et al. Mol Cell 2007;25:473-81.).
  • EHMT2 is involved in the proliferation of cancer cells, the inventors evaluated effects of BIX-01294 on the growth of cancer cell lines.
  • Fig. 9A the inventors chose cancer cells that showed a wide variety of EHMT2 expression levels.
  • Fig. 9B the growth of cancer cells was significantly suppressed by BIX-01294 treatment in a dose dependent manner, and the effect was correlated with EHMT2 expression levels.
  • the cell cycle status of SBC-5 cells after treatment with BIX-01294 was examined, and the proportion of cells in S phase significantly decreased and that in sub-G 1 phase increased in a dose dependent manner (Fig. 9C).
  • Histone modifications including methylation, acetylation, phosphorylation and ubiquitination, are considered to play critical roles in transcriptional activation and repression through the regulation of chromatin structure.
  • Histone methylation was once thought to be a stable modification, but is more recently recognized as being dynamically regulated by both histone methyltransferases and demethylases.
  • EHMT2 is mainly responsible for mono-methylation and di-methylation of H3K9 in euchromatin, and these play a unique role in transcriptional regulation and chromatin remodeling (12-14, 35, 36).
  • the present inventors demonstrated the significant up-regulation of EHMT2 in bladder and lung cancers by quantitative RT-PCR and immunohistochemistry at RNA and protein levels.
  • EHMT2 expression is shown to be dysregulated in a great majority of human tumors (Table 4).
  • the inventors demonstrate that EHMT2 may serve an important role in the growth regulation of cancer cells and confirmed that knockdown of EHMT2 suppresses the growth of various bladder and lung cancer cells, with the number of cells in the sub-G 1 phase increasing (Fig. 3D and E).
  • SIAH1 is a tumor suppressor gene located in chromosomal band 16q12-q13, a frequently deleted region in human tumors arising from various tissues ( Medhioub M et al. Int J Cancer 2000;87:794-7., Okabe H et al. Hepatology 2000;31:1073-9.). It was also reported that E3 ubiquitin ligases, including SIAH1, played an important role in regulating breast carcinogenesis ( Chen C et al.
  • the expression profile data show that expression levels of SIAH in tumor tissues are significantly low compared with corresponding non-neoplastic tissues in various types of cancer, including bladder and lung cancers (Table 5). These data reveal that SIAH1 is one of the key regulators in human carcinogenesis.
  • the microarray data of the present inventors showed that SIAH1 was up-regulated by EHMT2 knockdown, and the elevation was confirmed using quantitative real-time PCR and western blot analyses (Fig. 4C). Additionally, the inventors found that EHMT2 directly binds to the promoter region of SIAH1 and regulated the transcription of SIAH1 through the histone methylation analyzed by ChIP assay (Fig. 4D, Fig. 12).
  • EHMT2 was overexpressed in various types of cancer, including bladder and lung cancers, and play a crucial role in the proliferation of cancer cells.
  • the BioGPS database revealed that expression of EHMT2 in many types of normal tissues is very low (Fig. 10), indicating that EHMT2 is a good target for cancer therapy.
  • the inventors evaluated effects of BIX-01294, a highly specific inhibitor of EHMT2 (Kubicek S et al. Mol Cell. 2007 Feb 9;25(3):473-81), treatment and found that this chemical compound effectively suppressed the growth of cancer cells (Fig. 9).
  • This result shows that EHMT2 inhibitors can work as anti-cancer drugs. Further validation with functional analyses of this protein in the context of human carcinogenesis and optimization of EHMT2 inhibitors as anti-cancer drugs may assist to development of novel therapeutic strategies for human cancer.
  • the data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
  • EHMT2 can be conveniently used as a molecular diagnostic marker for identifying and detecting cancer, in particular, bladder cancer, lung cancer (SCC, ADC, ACC, SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Accordingly, the EHMT2 gene and the proteins encoded thereby find utility in diagnostic kits and assays of cancer.
  • the present invention further demonstrates that the cell growth may be suppressed by a double-stranded nucleic acid molecule that specifically targets the EHMT2 gene.
  • the double-stranded nucleic acid molecule is useful for the development of anti-cancer pharmaceuticals.
  • EHMT2 polypeptide is a useful target for the development of anti-cancer pharmaceuticals.
  • substances that block the expression of EHMT2 protein or prevent its activity may find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of bladder cancer, lung cancer (SCC, ADC, ACC, SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.

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Abstract

Objective methods for diagnosing a predisposition to developing cancer, including bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma caner is described herein. The present invention provides a diagnostic method that utilizes the expression level of EHMT2 as an index of cancer. The present invention further provides methods of screening for therapeutic substances for the treatment of EHMT2-associated disease, such as a cancer, e.g. bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. The invention further provides methods of inhibiting cell growth and treating or alleviating one or more symptoms of EHMT2-associated diseases. The invention also features double-stranded molecules that inhibit EHMT2 expression, vectors encoding the double-stranded molecule and compositions containing them.

Description

EHMT2 AS A TARGET GENE FOR CANCER THERAPY AND DIAGNOSIS
The present invention relates to methods of detecting and diagnosing cancer as well as methods of treating and/or preventing cancer, including cancers associated with the overexpression of EHMT2 such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. The present invention also relates to methods of screening for a candidate substance for treating and/or preventing an EHMT2-associated cancer. Moreover, the present invention relates to double-stranded molecules that reduce EHMT2 gene expression and uses thereof.
Priority
The present application claims the benefit of U.S. Provisional Application No. US 61/375,462 filed on August 20, 2010, and Japanese Patent Application No. 2011-154301 filed on June 25, 2011, the entire contents of which are herein incorporated by reference.
Histone methylation plays dynamic and crucial roles in regulating chromatin structure. Precise coordination and organization of open and closed chromatin regions control normal cellular processes such as DNA replication, repair, recombination and transcription. Histone lysine methylation regulates transcription positively or negatively depending on the methylation state of various methylation sites (NPL1). For instance, methylation of histone H3 at lysine 9 (H3K9) has served as the proto-type for studying the regulation of histone function by lysine methylation. Di- or tri-methylation of H3K9 creates a binding site for chromodomain (CD)-containing proteins of the heterochromatin protein 1 (HP1) family (NPL2, 3), which is thought to lead to gene repression through changes in higher-order chromatin structure. Methylation-dependent HP1 recruitment can be antagonized by adjacent H3 serine 10 phosphorylation. Thus, histones are subject to a system of combinatorially acting posttranslational modifications, referred to as the "histone code" (NPL4-6). Despite a large body of information for the prominent role of histone methylation in transcriptional regulation, their physiological function and their involvement in human disease is still not well understood. The present inventors previously reported that SMYD3, a histone methyltransferase, stimulates cell proliferation through its methyltransferase activity and plays a crucial role in human carcinogenesis (NPL 7-11, PTL 1-3).
EHMT2, also known as G9a, is mainly responsible for mono-methylation and di-methylation of H3K9 in euchromatin (NPL12). EHMT2 is essential for early embryonic development and is involved in the transcriptional silencing of developmentally regulated genes. Knockout of EHMT2 causes embryonic lethality in mice, indicating a major role for epigenetic repression in early mammalian development (NPL13). Previous studies found that EHMT2 functions as a corepressor, targeted to specific genes by associating with various transcriptional repressors and corepressors including CDP/Cut, Blimp-1/PRDI-BF1, and REST/NRSF (NPL14-16). Meanwhile, EHMT2 also appears to function as a coactivator for nuclear receptors, acting synergistically with CARM1 and other nuclear receptor (NR) coactivators (NPL17). Additionally, the complex of EHMT2 and DNMT1 enhances DNA and histone methylation of in vitro assembled chromatin substrates, indicating that direct cooperation between EHMT2 and DNMT1 provides a mechanism of coordinated H3K9 and DNA methylation during cell division (NPL18).
SIAH (seven in absentia homolog) proteins are members of the RING-finger-containing E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina) protein (NPL19, 20). It has been suggested that the SIAH1 protein plays a key role in biological processes such as the cell cycle, cell apoptosis and oncogenesis (NPL21-23).
[PTL1] WO2005/071102
[PTL2] WO2007/004526
[PTL3] WO2008/152816
Non-Patent Literature
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The present invention relates to the discovery, through microarray analysis and RT-PCR, that EHMT2 is overexpressed in clinical bladder cancer, lung cancer (including squamous cell carcinoma (SCC), adenocarcinoma (ADC), alveolus cell carcinoma (ACC), small cell lung cancer (SCLC) and large cell carcinoma (LCC)), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), esophageal cancer, breast cancer, cervical cancer and osteosarcoma tissues. As demonstrated herein, functional knockdown of endogenous EHMT2 by siRNA in cancer cell lines results in drastic suppression of cancer cell growth, suggesting the role of EHMT2 in maintaining the viability of cancer cells. Since it is only scarcely expressed in normal adult organs, EHMT2 is an appropriate molecular target for a therapeutic approach with minimal adverse effects. For example, EHMT2 can suppress transcription of the SIAH1 gene by binding to its promoter region (-293 to +51) and methylating lysine 9 of histone H3. Furthermore, an EHMT2 specific inhibitor BIX-01294 significantly suppresses the growth of cancer cells. These results demonstrate that dysregulation of EHMT2 plays an important role in the growth regulation of cancer cells, and EHMT2 is a valid therapeutic target for various types of cancer.
Accordingly, it is an object of the present invention to provide a method of diagnosing or determining a predisposition to cancer, particularly bladder cancer, lung cancer (non-small cell lung cancer; NSCLC (including SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma in a subject by determining an expression level of EHMT2 in a subject-derived biological sample. An increase in the level of expression of EHMT2 as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, particularly bladder cancer, lung cancer (NSCLC(SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. In some methods of the present invention, the EHMT2 polynucleotide can be detected by appropriate probes or, alternatively, the EHMT2 polypeptide can be detected by an anti-EHMT2 antibody.
It is another object of the present invention to provide a kit that includes a reagent for detecting a transcription or translation product of the EHMT2 gene.
It is yet another object of the present invention to provide a reagent for diagnosis or detection of cancer, which may comprise a nucleic acid that binds to a transcriptional product of the EHMT2 gene, or an antibody that binds to a translational product of the EHMT2 gene.
It is yet another object of the present invention to provide use of a nucleic acid that binds to a transcriptional product of the EHMT2 gene, or an antibody that binds to a translational product of the EHMT2 gene for the manufacture of a reagent for diagnosis or detection of cancer.
It is a further object of the present invention to provide methods for identifying a candidate substance that inhibits the growth of cells over-expressing the EHMT2 gene. Such substances are useful for the treatment or prevention of EHMT2- associated diseases, including cancer. The methods of the present invention can be carried out using as an index the binding activity to an EHMT2 polypeptide, an expression level of an EHMT2 gene, a biological activity of an EHMT2 polypeptide, or an expression level and/or activity of a reporter gene controlled under a transcriptional regulatory region of EHMT2 gene. Substances that bind to an EHMT2 polypeptide, or suppress an EHMT2 expression or activity, or a reporter gene expression or activity can be identified as candidate substances for treating and/or preventing cancer, or inhibiting cancer cell growth. The biological activity of the EHMT2 polypeptide to be detected includes cell proliferative activity (cell proliferation enhancing activity), methyltransferase activity or the activity of suppressing transcription of the SIAH1 gene. A decrease in the biological activity of the EHMT2 polypeptide as compared to a control level in the absence of the test substance may indicate that the test substance may be used to reduce symptoms of cancer, or treating and/or preventing cancer.
It is a further object of the present invention to provide methods for identifying a candidate substance that inhibits the growth of cells over-expressing the EHMT2 gene. Such substances are useful in the treatment and/or prevention of EHMT2-associated diseases, such as cancer.
It is yet a further object of the present invention to provide a method for treating and/or preventing cancer, or inhibiting the growth of a cancerous cell over-expressing EHMT2, by administering an agent that inhibits a function of the EHMT2 protein. In some embodiments, the agent is a EHMT2 inhibitor (e.g., BIX-01294).
It is yet a further object of the present invention to provide a pharmaceutical composition suitable for the treatment and/or prevention of an EHMT2-associated cancer that includes a pharmaceutically acceptable carrier and an EHMT2 inhibitor. In one aspect of the present invention, an EHMT2 inhibitor inhibits the histone methyltransferase activity. In one embodiment, the cancer being treated and/or prevented is bladder cancer, lung cancer (NSCLC (SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
It is yet a further object of the present invention to provide a method for treating and/or preventing cancer, or inhibiting the growth of a cancerous cell over-expressing EHMT2, by administering an agent that inhibits the expression of an EHMT2 gene and/or a function of the EHMT2 protein. In some embodiments of the present invention, the agent is an inhibitory nucleic acid (e.g., an antisense, ribozyme, double stranded molecule, aptamer). The agent may be a nucleic acid molecule or vector for providing nucleic acid molecules including double-stranded molecules. For example, expression of EHMT2 may be inhibited by introduction of a double-stranded molecule into a target cell in an amount sufficient to inhibit expression of the EHMT2 gene. Thus, in one embodiment of the present invention, the method includes the step of administering to a subject a pharmaceutically effective amount of double-stranded molecule against an EHMT2 gene or a vector encoding such a molecule, wherein the double-stranded molecule inhibits expression of an EHMT2 gene as well as cell proliferation when introduced into a cell that expresses an EHMT2 gene.
It is yet a further object of the present invention to provide a pharmaceutical composition suitable for the treatment and/or prevention of an EHMT2-associated cancer that includes a pharmaceutically acceptable carrier and an active agent including one or more double-stranded molecules against an EHMT2 gene or a vector encoding such a molecule. In one aspect of the present invention, a double-stranded molecule against EHMT2 inhibits the expression of an EHMT2 gene as well as inhibiting the cell proliferation induced thereby when introduced into a cell expressing an EHMT2 gene. In one embodiment, the cancer being treated and/or prevented is bladder cancer, lung cancer (NSCLC (SCC, ADC, ACC, LCC), SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
It is yet a further object of the present invention to provide double-stranded molecules against the EHMT2 gene, or a vector encoding such a molecule for use in the aforementioned method for treating and/or preventing cancer or in the aforementioned composition for treating and/or preventing cancer. The double-stranded molecules of the present invention may be composed of a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 34 and 35 and the antisense strand includes a sequence which is complementary to the target sequence. The sense and the antisense strands of the molecule hybridize to each other to form a double-stranded molecule. When introduced into a cell expressing an EHMT2 gene, the double-stranded molecule of the present invention inhibits expression of the EHMT2 gene and inhibit cell proliferation.
More specifically, the present invention provides following [1] to [27]:
[1] A method of detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of an EHMT2 gene in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer, wherein the expression level is determined by a method selected from the group consisting of:
(a) detecting an mRNA of the EHMT2 gene;
(b) detecting a protein encoded by the EHMT2 gene; and
(c) detecting a biological activity of a protein encoded by the EHMT2 gene;
[2] The method of [1], wherein said increase is at least 10% greater than said normal control level;
[3] The method of [1], wherein the subject-derived biological sample is a biopsy specimen;
[4] A kit for diagnosing cancer, which comprises a reagent selected from the group consisting of:
(a) a reagent for detecting an mRNA of an EHMT2 gene;
(b) a reagent for detecting a protein encoded by an EHMT2 gene; and
(c) a reagent for detecting a biological activity of a protein encoded by an EHMT2 gene;
[5] The kit of [4], wherein the reagent is a probe or a primer set to the mRNA of the EHMT2 gene, or an antibody against the protein encoded by the EHMT2 gene;
[6] A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with an EHMT2 polypeptide or a fragment thereof;
(b) detecting the binding activity between the polypeptide or fragment and the test substance; and
(c) selecting the test substance that binds to the polypeptide or the fragment as a candidate substance for treating and/or preventing cancer;
[7] A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing an EHMT2 gene;
(b) detecting an expression level of the EHMT2 gene in the cell of the step (a); and
(c) selecting the test substance that reduces the expression level detected in the step (b) in comparison with the expression level of the EHMT2 gene detected in the absence of the test substance;
[8] A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with an EHMT2 polypeptide or a fragment thereof;
(b) detecting a biological activity of the polypeptide or the fragment of step (a); and
(c) selecting the test substance that suppresses the biological activity of the polypeptide or the fragment detected in the step (b) in comparison with the biological activity detected in the absence of the test substance;
[9] The method of [8], wherein the biological activity is a cell proliferative activity or histone methyltransferase activity;
[10] A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, the method comprising the steps of:
(a) contacting a test substance with a cell into which a vector comprising the transcriptional regulatory region of an EHMT2 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region has been introduced,
(b) measuring the expression or activity level of the reporter gene; and
(c) selecting a substance that reduces the expression and/or activity level of the reporter gene detected in the step (b) in comparison with the expression or activity level in the absence of the test substance;
[11] A method of screening for a candidate substance that inhibits binding between an EHMT2 polypeptide and a SIAH1 promoter region, the method comprising steps of:
(a) contacting the EHMT2 polypeptide or a functional equivalent thereof with a polynucleotide corresponding to the SIAH1 promoter region in the presence of a test substance;
(b) detecting binding between the polypeptide and the polynucleotide;
(c) comparing the binding level detected in the step (b) with the level detected in the absence of the test substance; and
(d) selecting the test substance that reduces or inhibits the binding level in comparison with the level detected in the absence of the test substance;
[12] An isolated double-stranded molecule comprising a sense strand and an antisense strand complementary thereto, wherein the strands hybridize to each other to form the double-stranded molecule, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 34 and 35, and wherein the double-stranded molecule, when introduced into a cell expressing an EHMT2 gene, inhibits expression of the EHMT2 gene and cell proliferation;
[13] The double-stranded molecule of [12], wherein the sense strand hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pairs in length;
[14] The double-stranded molecule of [12] or [13], wherein the double-stranded molecule has one or two 3' overhangs at the 3' ends of the sense strand and/or the antisense strand;
[15] The double-stranded molecule of any one of [12] to [14], wherein said double-stranded molecule is a single polynucleotide comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence;
[16] The double-stranded molecule of [15], wherein said polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 34 and 35; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is an antisense strand comprising a complementary sequence to the target sequence of [A] ;
[17] A vector encoding the double-stranded molecule of any one of [12] to [16] ;
[18] A method of treating and/or preventing a cancer in a subject, the method comprising the step of administering to the subject a pharmaceutically effective amount of a double-stranded molecule that inhibits expression of an EHMT2 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule inhibits expression of the EHMT2 gene and proliferation when introduced into a cell expressing the EHMT2 gene;
[19] The method of [18], wherein the double-stranded molecule is that of any one of claims 11 to 15;
[20] The method of [18], wherein the vector is that of [17] ;
[21] A composition for treating and/or preventing a cancer, the composition comprising a pharmaceutically effective amount of a double-stranded molecule that inhibits expression of an EHMT2 gene or a vector encoding the double-stranded molecule, and a pharmaceutically acceptable carrier, wherein the double-stranded molecule inhibits expression of an EHMT2 gene and cell proliferation when introduced into a cell expressing the EHMT2 gene;
[22] The composition of [21], wherein the double-stranded molecule is that of any one of [12] to [16];
[23] The composition of [22], wherein the vector is that of [17];
[24] A method for inhibiting a growth of cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses an EHMT2 gene, the method comprising the step of administering at least one EHMT2 inhibitor to a subject;
[25] The method of [24], wherein the EHMT2 inhibitor is BIX-01294;
[26] A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses the EHMT2 gene, comprising at least one EHMT2 inhibitor; and
[27] The composition of [26], wherein the EHMT2 inhibitor is BIX-01294.
The methods and materials of the present invention are capable of identifying cancer prior to detection of overt clinical symptoms and may be used in cancer therapy. In some embodiments, the methods and compositions provide cancer therapy without adverse effect.
It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are not exclusive or restrictive of other alternate embodiments of the invention. Other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.
Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and the embodiments that follow:
Figure 1 demonstrates the elevated EHMT2 expression in bladder cancer. Part A depicts the EHMT2 gene expression in normal and tumor bladder tissues in British cases. Expression levels of EHMT2 were analyzed by quantitative real-time PCR, and the result is shown by box-whisker plot. The Mann-Whitney U-test was used for statistical analysis. Part B depicts the statistical analysis of EHMT2 expression categorized by histological grade of bladder tumors. The P value was calculated using the Kruskal-Wallis test. Part C depicts the statistical analysis of EHMT2 expression categorized by pathological stages of bladder tumors. The P value was calculated using the Kruskal-Wallis test. Part D depicts the expression ratio between bladder normal and tumor tissues in Japanese populations. The signal intensity of each sample was analyzed by cDNA microarray, and expression ratio, the signal intensity in the tumor sample divided by that in normal tissue, is shown.
Part E depicts the tissue microarray images of bladder tumors stained by standard immunohistochemistry for protein expression of EHMT2. Clinical information for each section is represented above the histological pictures. Counterstaining was done with hematoxylin and eosin. Original magnification, x200.
Figure 2 demonstrates the elevated EHMT2 expression levels in lung cancer. Part A depicts the expression ratio between normal lung and small cell lung cancer (SCLC) tissues. Signal intensity of each sample was analyzed by cDNA microarray (right), and the result is shown by box-whisker plot (median 50% boxed). Mann-Whitney U-test was used for the statistical analysis. Expression ratio is the signal intensity in the tumor sample divided by that in the normal sample (left). Part B depicts the immunohistochemical staining of EHMT2 in lung tissues. Clinical information for each section is represented above histological pictures. Counterstaining was done with hematoxylin and eosin. Original magnification, x200.
Figure 3 demonstrates the involvement of EHMT2 in the growth of bladder and lung cancer cells. Part A depicts the expression levels of EHMT2 in various cell lines. A western blot was performed to measure the protein level of EHMT2, and an anti-ACTB antibody was used as an internal control. Part B depicts the effects of EHMT2 knockdown on the protein expression. Lysates from SBC-5 cells after siRNA treatment were immunoblotted with anti-EHMT2 and tubulin antibodies. Expression of tubulin served as an internal control. Part C depicts the results of a colony formation assay after siRNA treatment. Giemsa staining was performed 7 days after treatment with siRNAs. Part D depicts the effect of siRNA knockdown of EHMT2 on the viability of a bladder cancer cell line (SW780, RT4) and lung cancer cell lines (LC319, SBC5, A549). Relative cell number is the cell number normalized to siEGFP-treated cells. The numbers are a mean value +/-SD of three independent experiments. P values were calculated using Student's t-test (*, P < 0.05;**, P < 0.01; ***, P < 0.001). Part E depicts the results of FACS analysis after treatment with EHMT2 siRNAs. SBC5 cells were treated with siRNAs and analyzed by FACS 72 h after siRNA treatment. Representative histograms of this experiment are shown along with numerical analysis of the FACS result, classifying cells by cell cycle status. The proportion of cancer cells in sub-G1 phase is significantly higher after treatment with siEHMT2#1 compared to control siRNAs-treated cancer cells. The results are presented as mean cell number +/-SD of three independent experiments. P values were calculated using Student's t-test.
Figure 4 demonstrates that SIAH1 expression is directly regulated by EHMT2. Part A depicts a two-dimensional, unsupervised hierarchical cluster analysis of SW780 and A549 cells after knockdown of EHMT2 expression. Differentially expressed genes were selected for this analysis. Darker color indicates up-regulated genes and lighter color indicates down-regulated genes. Part B depicts the results of microarray analysis of A549 cells after treatment with siRNAs targeting EGFP(control; siEGFP) and EHMT2 (siEHMT2#2). P values were calculated using Student's t-test (**, P < 0.01). Part C depicts the results of validation of the microarray data using quantitative real-time PCR and western blot analyses in A549 cells after treatment with siRNAs targeting EGFP (control; siEGFP) and EHMT2 (siEHMT2). P values were calculated using Student's t-test (**, P < 0.01). Samples for western blot analysis were fractionated by SDS-PAGE and immunoblotted with anti-EHMT2 (NB100-40825, Novus Biologicals) and anti-SIAH1 (sc-5506, Santa Cruz) antibodies. Anti-ACTB was used as an internal control. Part D depicts the results of a ChIP assay using anti-FLAG and anti-H3K9me2 antibodies. The ChIP assay was performed 48 hours after transfection with pCAGGSn-3FC (Mock) and pCAGGSn-3FC-EHMT2 (3xFLAG-EHMT2) into 293T cells. Left top panel depicts a schematic diagram of the SIAH1 promoter region. Left bottom panel depicts the result of real-time PCR analysis using a primer pair shown in Table 1. Cross-linked and sheared chromatin was immunoprecipitated with anti-FLAG antibody (M2, Sigma). The results are shown as percentage of the input chromatin. The right top panels depict the results of immunoblot analysis using anti-FLAG antibody. The input samples were fractionated by SDS-PAGE and immunoblotted with anti-FLAG antibody. Expression of ACTB was detected as an internal control. The right bottom panel depicts the result of quantification of H3K9diMe ChIP at the SIAH1 promoter region using real-time PCR. Cross-linked and sheared chromatin was immunoprecipitated with anti-diMeH3K9 antibody (ab1220, abcam). P values were calculated using Student's t-test (***, P < 0.001).
Figure 5 demonstrates elevated EHMT2 expression levels in AML, CML and esophageal cancer in Japanese populations. The signal intensity of each sample was analyzed by cDNA microarray, and the results are shown by box-whisker plot (median 50% boxed). The Mann-Whitney U-test was used for the statistical analysis. AML, acute myeloid leukemia; CML, chronic myelogenous leukemia.
Figure 6 demonstrates expression levels of EHMT2 in normal tissue, 14 bladder cancer cell lines and five lung cancer cell lines. Expression levels of EHMT2 were analyzed by quantitative real-time PCR. Data were normalized by GAPDH and SDH expressions.
Figure 7 demonstrates the results of quantitative real-time PCR analyses after knockdown of EHMT2 by siEHMT2. Quantitative real-time PCR analyses show suppression of endogenous expression of EHMT2 by EHMT2-specific siRNAs (siEHMT2#1 and #2) in A549 and SBC5 cells. siEGFP and siNC were used as controls. Relative mRNA expression is shown as the expression level normalized by expression levels of siEGFP-treated cells. The results are presented as mean expression level +/-SD of three independent experiments. P values were calculated using Student's t-test (**, P < 0.01; ***, P < 0.001).
Figure 8 demonstrates that the treatment with siEHMT2 may decrease cancer cells in the S phase and increase cancer cells in the sub-G1 phase. The SBC5 cells were treated with siRNAs and analyzed by FACS 72 h after siRNA treatment. Numerical analysis of the FACS result classifying cells by cell cycle status are shown. The proportion of cancer cells in sub-G1 phase is significantly high after treatment with siEHMT2#1 compared to control siRNAs-treated cancer cells. The results are presented as the mean +/-SD of three independent experiments. P values were calculated using Student's t-test.
Figure 9 demonstrates reduction of the growth rate of several types of cancer cell lines by treatment with BIX-01294. Part A depicts expression levels of EHMT2 in various types of cancer cells analyzed by quantitative real-time PCR. Part B depicts the effect of BIX-01294 on the viability of cancer cell lines. Cancer cell lines were treated for 2 days with the inhibitor BIX-01294 at 2, 4 and 6 micromolar. This result was normalized against the results of treatment with pure water as the negative control (N.C). Statistical analysis was performed based on three independent experiments. P values were calculated using Student's t-test. Part C depicts the results of cell cycle distribution analyzed by flow cytometry. Cell cycle distribution was analyzed by flow cytometry after coupled staining with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU and 7-amino-actinomycin D (7-AAD) as described below.
Figure 10 demonstrates the expression levels of EHMT2 in 78 normal tissues. The data were derived from BioGPS (http://biogps.gnf.org/#goto=genereport&id=54904). GAPDH expression is shown as a control for signal intensity.
Figure 11 demonstrates the elevated EHMT2 expression levels in lung cancer. Part A depicts expression of EHMT2 in normal lung, 17 non-small cell lung cancer (NSCLC) and 6 SCLC tissues. Expression levels were analyzed by quantitative real-time PCR, and data were normalized against EHMT2 expression levels in normal lung tissue. Part B depicts mRNA expression levels of EHMT2 in 2 normal human cell lines and 4 lung cancer cell lines examined by quantitative real-time PCR. Expression levels were normalized by GAPDH and SDH expression, and values are relative to CCD-18Co (CCD-18Co=1).
Figure 12 demonstrates that SIAH1 expression may be directly regulated by EHMT2. The ChIP assay was performed using anti-EHMT2 (Middle) and anti-H3K9me2 (Right) antibodies after treatment of SBC5 cells with siEGFP or siEHMT2#2 for 48 h. The results are shown as a percentage of the input chromatin. Left panels depict the result of the immunoblot analysis using anti-EHMT2 antibody. The input samples were fractionated by SDS-PAGE and immunoblotted with anti-EHMT2 antibody. Expression of ACTB was detected as an internal control.
Figure 13 demonstrates that SIAH1 regulated by EHMT2 regulates cancer cell growth and apoptosis. Part A depicts the results of western blot analyses in SBC5 cells after treatment with siRNAs targeting EGFP (control; siEGFP), EHMT2 (siEHMT2#2) and SIAH1 (siSIAH1) for 72 h. Anti-PARP1 (sc-8007, Santa Cruz) and anti-cleaved caspase 3 (#9661S, Cell Signaling) antibodies were used as apoptosis markers, and anti-GAPDH antibody was used as an internal control. Part B depicts the result of colony formation assay of SBC5 cells. Indicated siRNAs were transfected 24 h after preparation of cells and Giemsa staining was performed 96 h after treatment with siRNAs. Part C depicts the results of cell growth assay of SBC5 cells treated with indicated siRNAs. siEHMT2#2 and either siEGFP or siSIAH1 were transfected 24 h after preparation of cells, and subsequently, cell viability was measured 48 h and 96 h after siRNA treatment. The results are presented as the mean +/-SD of three independent experiments. P values were calculated using Student's t-test (**, P < 0.01).
Figure 14 demonstrates gene ontology pathway analysis based on the Affymetrix's microarray data.
Figure 14 demonstrates gene ontology pathway analysis based on the Affymetrix's microarray data.
Figure 14 demonstrates gene ontology pathway analysis based on the Affymetrix's microarray data.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods, devices, and materials of the present invention are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
The disclosure of each publication, GenBank Accession or other sequence, patent or patent application mentioned in this specification is specifically incorporated by reference herein in its entirety. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Definition:
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
The terms "isolated" and "purified" when used herein in relation to a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicate that the substance is substantially free from at least one substance that may also be included in the natural source. Thus, an isolated or purified antibody refers to an antibody that is substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In one embodiment, antibodies of the present invention are isolated or purified.
In the context of the present invention, the phrase "EHMT2 gene" encompasses polynucleotides that encode the human EHMT2 or any of the functional equivalents of the human EHMT2 gene. The EHMT2 gene can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.
Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
The terms "gene", "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably unless otherwise specifically indicated and, similarly to the amino acids, are referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers. The polynucleotide, oligonucleotide, nucleotides, nucleic acids, or nucleic acid molecules may be composed of DNA, RNA or a combination thereof.
Unless otherwise defined, the term "cancer" refers to cancer over-expressing the EHMT2 gene. Examples of cancers over-expressing EHMT2 include, but are not limited to, bladder cancer, lung cancer (including squamous cell carcinoma (SCC), adenocarcinoma (ADC), alveolus cell carcinoma (ACC), small cell lung cancer (SCLC) and large cell carcinoma (LCC)), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
As used herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)). Herein, "double-stranded molecule" is also referred to as "double-stranded nucleic acid", "double-stranded nucleic acid molecule", "double-stranded polynucleotide" and "double-stranded polynucleotide molecule".
The Gene and Protein:
The present invention is based in part on the discovery that the gene encoding EHMT2 is over-expressed in cancer tissues as compared to non-cancerous tissues. The nucleic acid and polypeptide sequences of EHMT2 are provided, for example, in the following SEQ ID numbers:
EHMT2 polynucleotide: SEQ ID NOs:1 or 3;
EHMT2 polypeptide: SEQ ID NOs:2 or 4.
The above sequence data is also available via the following GenBank accession numbers:
EHMT2: NM_006709 or NM_025256.
Moreover, the present invention is also based in part on the discovery that EHMT2 may directly bind to the promoter region of SIAH1 and may regulate the transcription of SIAH1. The nucleic acid and polypeptide sequences of SIAH1 in the present invention are provided, for example, in the following SEQ ID numbers:
SIAH1 polynucleotide: SEQ ID NOs: 5 or 7;
SIAH1 polypeptide: SEQ ID NOs: 6 or 8.
The above sequence data is also available via the following GenBank accession numbers:
SIAH1: NM_001006610 or NM_003031.
In addition, the promoter region of SIAH1 may be obtained by amplifying the 5' upstream region of the SIAH1 gene with a primer set, for example, a primer set of SEQ ID NO: 18 and 19, which is designed based on genomic sequence information.
According to an aspect of the present invention, functional equivalents are also considered to be above "polypeptides". Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptide that retains the biological ability may be used as such a functional equivalent in the present invention. Such functional equivalents include those wherein one or more amino acids are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the protein. Alternatively, the polypeptide may be composed of an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the respective protein, at least about 90% to 95% homology, or about 96%, 97%, 98% or 99% homology. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the natural occurring nucleotide sequence of the genes.
A polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a function equivalent to that of the above proteins of the present invention, it is within the scope of the present invention.
The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize, typically in a complex mixture of nucleic acids, to its target sequence but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times higher than background level, or at least 10 times higher than the background level. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
Hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the above human proteins can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, or 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions may alternatively be used. An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. Several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
In general, modifications of one, two or more amino acids in a protein will not influence the function of the protein. In fact, mutated or modified proteins (i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition)have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)). Accordingly, one of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence which alter a single amino acid or a small percentage of amino acids or those considered to be a "conservative modifications", wherein the alteration of a protein results in a protein with similar functions, provide functionally equivalent proteins and polypeptides. Thus, in one embodiment, the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in the sequence.
So long as the activity of the protein is maintained, the number of amino acid mutations is not particularly limited. However, in general, 5% or less of the amino acid sequence is altered. Accordingly, in a typical embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or fewer, 20 amino acids or fewer, 10 amino acids or fewer, 5 or 6 amino acids or fewer, or 3 or 4 amino acids or fewer.
An amino acid residue to be mutated may be mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Aspargine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cystein (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
Such conservatively modified polypeptides are included in the present EHMT2 protein. However, the present invention is not restricted thereto and the EHMT2 protein includes non-conservative modifications, so long as at least one biological activity of the EHMT2 protein is retained. Furthermore, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
Moreover, the EHMT2 gene of the present invention encompasses polynucleotides that encode such functional equivalents of the EHMT2 protein. In addition to hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the EHMT2 protein, using a primer synthesized based on the sequence information of the protein encoding DNA (e.g. SEQ ID NO: 1 or 3). Polynucleotides and polypeptides that are functionally equivalent to the human EHMT2 gene and protein, respectively, may have a high homology to the originating nucleotide or amino acid sequence thereof . "High homology" typically refers to a homology of 40% or higher, 60% or higher, 80% or higher, 90% to 95% or higher, or 96%, 97%, 98%, 99% or higher. The homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".
A method for Diagnosing Cancer:
The expression of EHMT2 gene was found to be elevated in bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma (Fig. 1, 2, 5, 6, Table 4) and was not expressed in normal tissues. Accordingly, the EHMT2 genes identified herein as well as their transcription and translation products are useful as diagnostic markers for cancers such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. For example, cancer can be diagnosed or detected by comparing the expression level of EHMT2 gene between a subject-derived sample with a normal sample. In one aspect, the present invention provides methods for detecting or diagnosing cancer, determining the presence of cancer, or determining a predisposition for developing cancer, more particularly EHMT2-associated cancer, by determining the expression level of EHMT2 in the subject.
Accordingly, the present invention provides a method for detecting or diagnosing cancer in a subject, such method including the step of determining an expression level of an EHMT2 gene in a subject-derived biological sample, wherein an increase of the level as compared to a normal control level of the gene indicates the presence or suspicion of cancer cells in the sample, which, in turn, suggests that the subject suffers from or is at risk of developing cancer.
The expression level of the EHMT2 gene may be determined by any known method, examples of which include:
(a) detecting the mRNA of an EHMT2 gene;
(b) detecting the protein encoded by an EHMT2 gene; and
(c) detecting the biological activity of the protein encoded by an EHMT2 gene.
In one embodiment, cancer to be diagnosed by the present method includes bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose whether a subject suffers from the disease. Thus, the present invention provides a method for the use of EHMT2 as a diagnostic marker for cancer.
Alternatively, the present invention may be used to detect or identify cancerous cells in a subject-derived tissue, such cells being characterized by an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the tissue. EHMT2 expression results may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease. In other words, the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease. For example, according to the present invention, when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the EHMT2 gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc. For example, some well-known diagnostic tumor markers in blood include, but are not limited to, IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA. In one embodiment of the present invention, the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.
The present invention also provides the following methods [1] to [10]:
[1] A method of detecting or diagnosing cancer in a subject, including determining an expression level of an EHMT2 gene in a subject derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer;
[2] The method of [1], wherein the expression level is at least 10% greater than the normal control level;
[3] The method of [1], wherein the expression level is detected by a method selected from the group consisting of:
(a) detecting an mRNA of the EHMT2 gene,
(b) detecting a protein encoded by the EHMT2 gene, and
(c) detecting a biological activity of a protein encoded by the EHMT2 gene;
[4] The method of [1], wherein the cancer is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
[5] The method of [3], wherein the expression level is determined by detecting hybridization of a probe to a gene transcript of the gene;
[6] The method of [3], wherein the expression level is determined by detecting binding of an antibody against the protein encoded by the EHMT2 gene;
[7] The method of [1], wherein the biological sample includes a biopsy specimen, sputum or blood;
[8] The method of [1], wherein the subject-derived biological sample includes an epithelial cell;
[9] The method of [1], wherein the subject-derived biological sample includes a cancer cell;
[10] The method of [1], wherein the subject-derived biological sample includes a cancerous epithelial cell.
The method of diagnosing cancer of the present invention is described in more detail below.
A subject to be diagnosed by the present method may be a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
The methods of the present invention may include a step of collecting a biological sample from the subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it includes, or is capable of including, a transcription or translation product of EHMT2. Exemplary biological samples include, but are not limited to, bodily tissues which are desired for diagnosing or are suspected of suffering from cancer; and fluids, such as biopsy specimen, blood, sputum or urine. The biological sample may contain a cell population including an epithelial cell, a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, the cell may be purified from the obtained bodily tissues and fluids, and then used as the biological sample.
The present invention provides methods for determining the expression level of EHMT2 in a subject-derived biological sample. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of EHMT2 may be quantified using probes and hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array, for example. In one embodiment, the use of an array may allow detection of the expression level of a plurality of genes (e.g., various cancer specific genes) including EHMT2. Those skilled in the art can prepare such probes utilizing the sequence information of EHMT2. For example, the cDNA of EHMT2, or fragments thereof, may be used as the probes. If necessary, the probe may be labeled with a suitable label, such as dyes, fluorescent and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
Furthermore, the transcription product of EHMT2 may be quantified by amplification-based detection methods (e.g., RT-PCR) using primers. Such primers may be prepared based on the available sequence information of the gene. For example, the primers used in the Example (SEQ ID NO: 14 and 15) may be employed for the detection by RT-PCR or Northern blot. However the present invention is not restricted to the use of these specific primer or probe sequences.
In some embodiments, a probe or primer used for the present method may hybridize under stringent, moderately stringent, or low stringent conditions to the mRNA of EHMT2 or a fragment thereof. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the quantity of EHMT2 protein may be determined. Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody that specifically recognizes the protein or a fragment thereof. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment retains the binding ability to EHMT2 protein or fragment thereof. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of EHMT2 gene based on its translation product, the intensity of staining may be observed via immunohistochemical analysis using an antibody against EHMT2 protein or a fragment thereof. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of EHMT2 gene.
In the context of the present invention, methods for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begin with a determination of EHMT2 gene expression level. Once determined, using any of the aforementioned techniques, this value is as compared to a control level.
In the context of the present invention, the phrase "control level" refers to the expression level of a test gene detected in a control sample and encompasses both a normal control level and a cancer control level. The phrase "normal control level" refers to a level of gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer. A normal individual is one with no clinical symptom of bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma. A normal control level can be determined using a normal cell obtained from a non-cancerous tissue. A "normal control level" may also be the expression level of a test gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma. On the other hand, the phrase "cancer control level" refers to an expression level of a test gene detected in the cancerous tissue or cell of an individual or population suffering from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma. An increase in the expression level of EHMT2 detected in a subject-derived sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma. In the context of the present invention, the subject-derived sample may be any tissues obtained from test subjects, e.g., patients suspected of having cancer. For example, tissues may include epithelial cells. More particularly, tissues may be epithelial cells collected from a suspected cancerous area. Alternatively, the expression level of EHMT2 in a sample can be compared to a cancer control level of EHMT2 gene. A similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer. When the expression levels of other cancer-related genes are also measured and compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
The control level may be determined at the same time with the test biological sample by using a sample or samples previously collected and stored from a subject or subjects whose disease state (cancerous or non-cancerous) is or are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression levels of EHMT2 gene in samples from subjects whose disease state are known. Furthermore, the control level can be from a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of EHMT2 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. In some embodiments, a control level may be utilized that is determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample. Moreover, the methods of the present invention may use a standard value of the expression levels of the EHMT2 gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 times the S.D. or mean +/- 3 times the S.D. may be used as a standard value.
To improve the accuracy of the diagnosis, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma may also be determined, in addition to the expression level of the EHMT2 gene. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference which is cancerous indicates that the subject is suffering from or at a risk of developing bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
In the context of the present invention, gene expression levels are deemed to be "altered" or "increased" when the gene expression changes or increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level. Accordingly, the expression level of bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma marker genes including EHMT2 gene in a biological sample can be considered to be increased if it increases from a control level of the corresponding bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and/or osteosarcoma marker gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
Differences between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
The present invention also provides EHMT2 as a suitable target for cancer therapy. Therefore, cancer treatment targeting EHMT2 is provided by the present invention. In some embodiments, the cancer treatment targeting EHMT2 refers to suppression or inhibition of EHMT2 activity and/or expression in a cancer cell or tissue, or in a subject having cancer. Any anti-EHMT2 agents may be used for the cancer treatment targeting EHMT2. In the present invention, the anti-EHMT2 agents may include following substances or active ingredients:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding the double-stranded molecule, or
(c) a vector encoding the double-stranded molecule.
Accordingly, in a one embodiment, the present invention provides a method of (i) diagnosing whether a subject has cancer suitable for treatment with an anti- EHMT2 agent, and/or (ii) selecting a subject for cancer treatment targeting EHMT2, which method includes the steps of:
a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of EHMT2 with a normal control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is increased as compared to the normal control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
Alternatively, such a method includes the steps of:
a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of EHMT2 with a cancerous control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is similar or equivalent to the cancerous control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
A kit for Diagnosing Cancer:
The present invention also provides a kit for diagnosing cancer, which may also be useful in monitoring the efficacy of a cancer therapy. The present invention also provides a kit for determining if a subject suffering from cancer may be treated with a double-stranded molecule or inhibitor of the present invention or vector encoding thereof. The kit of the present invention may also be useful in assessing and/or monitoring the efficacy of a cancer treatment. The cancer to be diagnosed by the present kit includes, but is not limited to, bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma. Further, the kit may include at least one reagent for detecting the expression level of the EHMT2 gene in a subject-derived biological sample, which reagent may be selected from the group consisting of:
(a) a reagent for detecting mRNA of the EHMT2 gene or a fragment thereof;
(b) a reagent for detecting the EHMT2 protein or a fragment thereof; and
(c) a reagent for detecting the biological activity of the EHMT2 protein.
Suitable reagents for detecting mRNA of the EHMT2 gene include nucleic acids that specifically bind to or identify the EHMT2 mRNA, such as oligonucleotides which have a complementary sequence to a part of the EHMT2 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the EHMT2 mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art. In some embodiments, the reagent for detecting the EHMT2 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the EHMT2 mRNA may be included in the kit.
A probe or primer of the present invention is typically a substantially purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about a 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bases of consecutive sense strand nucleotide sequence of a nucleic acid encoding an EHMT2 sequence, or an anti sense strand nucleotide sequence of a nucleic acid encoding an EHMT2 sequence, or of a naturally occurring mutant of these sequences. For example, in one embodiment, an oligonucleotide having a 5-50 nucleotide length can be used as a primer for amplifying the genes, to be detected. Alternatively, mRNA or cDNA of a EHMT2 gene can be detected with an oligonucleotide probe or primer of a specific size, generally 15- 30 bases in length. In other embodiments, the length of the oligonucleotide probe or primer can be selected from 15-25 nucleotides. Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR). In these assays, probes or primers can also include tags, labels, or linker sequences. Further, probes or primers can be modified with a detectable label or affinity ligand to be captured. Alternatively, in hybridization based detection procedures, a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).
Suitable reagents for detecting the EHMT2 protein may include antibodies to the EHMT2 protein or a fragment thereof. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used as the reagent, so long as the fragment retains the binding ability to the EHMT2 protein or a fragment thereof. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof. Furthermore, the antibody may be labeled with signal generating molecules or other detectable labels via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods may be employed for the present invention. Moreover, more than one reagent for detecting the EHMT2 protein may be included in the kit.
Furthermore, the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed EHMT2 protein in the biological sample. For example, the cell may be cultured in the presence of a subject-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability, the cell proliferating activity of the biological sample can be determined. In some embodiments, the reagent for detecting the EHMT2 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the biological activity of the EHMT2 protein may be included in the kit.
The kit may contain more than one of the aforementioned reagents. Furthermore, the kit may include a solid matrix and reagent for binding a probe against the EHMT2 gene or antibody against the EHMT2 protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the EHMT2 protein. For example, tissue samples obtained from subject suffering from cancer or a normal subject may serve as useful control reagents. A kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, URL etc.) with instructions for use. These reagents and such may be provided in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.
In one embodiment of the present invention, when the reagent is a probe against the EHMT2 mRNA, the reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one detection site. The measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid (probe). A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a different strip separated from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal may provide a quantitative indication of the amount of EHMT2 mRNA present in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot. In some embodiments, the bar or dot may span the width of a test strip.
The kit of the present invention may further include a positive and/or negative controls sample, and/or an EHMT2 standard sample. The positive control sample of the present invention may be prepared by collecting EHMT2 positive samples. Such EHMT2 positive samples may be obtained, for example, from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma cell lines, including lung adenocarcinoma cell (ADC) lines such as A427, NCI-H1781, A549, LC319 and the like; lung squamous cell carcinoma (SCC) cell lines such as NCI-H26, EBC-1, NCI-H520, NCI-H2170 and the like; SCLC cell lines such as DMS114, DMS273, SBC-3, SBC-5, H196, H446 and the like; and bladder cancer cell lines such as 5637, 253J, 253JBV, EJ28, HT1197, HT1376, HT1576, J82, MT197, RT4, SCaBER, SW780, T24, UMUC3 and the like. Alternatively, the EHMT2 positive samples may be obtained from clinical bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma tissues. Alternatively, positive control samples may be prepared by determining a cut-off value and preparing a sample containing an amount of an EHMT2 mRNA or protein more than the cut-off value. Herein, the phrase "cut-off value" refers to a threshold value dividing between a normal range and a cancerous range. For example, one skilled in the art may determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include an EHMT2 standard sample containing a cut-off value amount of an EHMT2 mRNA or polypeptide. On the contrary, negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal tissues, or may be prepared by preparing a sample containing an EHMT2 mRNA or protein less than the cut-off value.
Alternatively, the present invention provides use of a reagent for preparing a diagnostic reagent for diagnosing cancer. In some embodiments, the reagent can be selected from the group consisting of:
(a) a reagent for detecting mRNA of the EHMT2 gene;
(b) a reagent for detecting the EHMT2 protein; and
(c) a reagent for detecting the biological activity of the EHMT2 protein.
In some embodiments, such reagent is an oligonucleotide that hybridizes to the EHMT2 polynucleotide, or an antibody that binds to the EHMT2 polypeptide.
Screening for an Anti-cancer Substance:
Through the present invention, it has been demonstrated that EHMT2 is involved in cancer cell growth. Accordingly, substances that suppress an expression level of EHMT2 gene and/or a biological activity of EHMT2 polypeptide are useful for treating and/or preventing cancer. Such substances can be screened using an EHMT2 gene, polypeptides encoded by the gene, or a transcriptional regulatory region of the gene. Thus, the present invention also provides a method of screening for a candidate substance for treating and/or preventing cancer using EHMT2 gene, EHMT2 polypeptide, or transcriptional regulatory region of the gene.
In the context of the present invention, substances to be identified through the present screening methods may be any compound or composition including several compounds. Furthermore, the test substance exposed to a cell or protein according to the screening methods of the present invention may be a single substance or a combination of substances. When a combination of substances is used in the methods, the substances may be contacted sequentially or simultaneously.
The substances screened by the present screening method may be suitable candidate substances for treating and/or preventing cancer, and/or inhibiting cancer cell growth. In the present invention, the cancer may be characterized by an association with EHMT2 overexpression. Accordingly, the screened substances may be applied to the cancers correlated or associated with EHMT2 overexpression. In some embodiments, the cancers correlated or associated with EHMT2 overexpression include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
Any test substance, for example, cell extracts, cell culture supernatant, products of fermenting microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural compounds can be used in the screening methods of the present invention. The test substance of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound" library method and (5) synthetic library methods using affinity chromatography selection. Methods of the present invention utilizing libraries are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of compounds may be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698; 5,403,484, and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Pat. Application 2002103360).
A compound in which a part of the structure of the substance screened by any one of the present screening methods is converted by addition, deletion and/or replacement, is included in the substances obtained by the screening methods of the present invention.
Furthermore, when a candidate substance obtained by the present screening method is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. Alternatively, an amino acid sequence or partial sequence may be used to screen a database of DNA sequences such as Genebank to obtain a DNA encoding the protein. The usefulness in preparing the candidate substance for treating or preventing cancer of the obtained DNA may be confirmed.
Test substances used in the screenings described herein may also be antibodies that specifically bind to an EHMT2 protein or partial peptides thereof, including partial peptides that lack the biological activity of the original proteins in vivo.
Although the construction of test substance libraries is well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.
In some embodiments of the present invention suppression of the expression level and/or biological activity of EHMT2 may lead to suppression of the growth of cancer cells. Therefore, when a substance suppresses the expression and/or activity of EHMT2, such suppression is indicative of a therapeutic effect in a subject. In the context of the present invention, a therapeutic effect refers to a clinical benefit. Examples of such clinical benefit include but are not limited to;
(a) reduction in expression of the EHMT2 gene,
(b) a decrease in size, prevalence, or metastatic potential of the cancer in the subject,
(c) preventing cancers from forming, or
(d) preventing or alleviating a clinical symptom of cancer.
(i) Molecular modeling:
Construction of test substance libraries is facilitated by knowledge of the molecular structure of substances known to have the properties sought, and/or the molecular structure of EHMT2 protein. One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new substances that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new substance will link to the target molecule and allow experimental manipulation of the structures of the substance and target molecule to perfect binding specificity. Prediction of what the molecule-substance interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive analysis, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
An example of the molecular modeling system described generally above includes the CHARMM and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMM performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
A number of articles have been published on the subject of computer modeling of drugs interactive with specific proteins, examples of which include Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect to a model receptor for nucleic acid components, Askew et al., J Am Chem Soc 1989, 111: 1082-90.
Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17: 266-78; Shoichet et al., Science 1993, 259: 1445-50.
Once a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors, or "test substances" may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, including bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
(ii) Combinatorial chemical synthesis:
Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology 1995 supplement; Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino compounds, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
(iii) Other candidates:
Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 106 -108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples. Furka et al. (14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Patent 4,631,211) and Rutter et al. (US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target. Tuerk and Gold (Science. 249:505-510 (1990)) discloses the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers. In the SELEX method, a large library of nucleic acid molecules {e.g., 1015 different molecules) can be used for screening.
I. Protein based screening methods
The present invention provides methods of screening for a candidate substance applicable to the treatment and/or prevention of cancer using an EHMT2 polypeptide.
In the context of the present screening method, the EHMT2 polypeptide to be used may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides. Further, the EHMT2 polypeptide may be a recombinant polypeptide, a protein derived from the nature or a partial peptide thereof.
In addition to naturally-occurring EHMT2 polypeptides, functional equivalents of the polypeptides may be included in EHMT2 polypeptides used for the present screening so long as the modified peptide retains at least one biological activity of the original polypeptide. Examples of such functional equivalents are described above in the section entitled "The Gene and Protein".
The polypeptides may be further linked to other substances. In some embodiments, a linking process and linker are chosen so that does not interfere with the biological activity of the original polypeptide and/or fragment. Usable substances include, for example: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments. The polypeptides used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on a selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include those described in:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; and
7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
Alternatively, the polypeptides may be obtained by adapting any known genetic engineering methods to the production of the instant polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector including a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence including a promoter) may be prepared, transformed into a suitable host cell, and then the host cell may be cultured to produce the protein. More specifically, a gene encoding an EHMT2 polypeptide may be expressed in host (e.g., animal, bacteria, or fungal) cells by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. A promoter may be used for the expression. Any commonly used promoters may be employed, including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SR-alpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such. The introduction of the vector into host cells to express an EHMT2 polypeptide may be performed according to any conventional methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.
EHMT2 polypeptides may also be produced in vitro using a conventional in vitro translation system.
(i) Screening for an EHMT2 Binding Substance:
In one aspect of the present invention, the over-expression of EHMT2 gene may be detected in bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma, in spite of no expression in normal organs (Fig. 1, 2, 5, 6, Table 4). Accordingly, using the EHMT2 gene and proteins encoded thereby, the present invention provides a method of screening for a substance that binds to EHMT2 polypeptide. Due to the expression of EHMT2 in cancer, a substance that binds to EHMT2 polypeptide may suppress the proliferation of cancer cells, and is thus useful for treating and/or preventing cancer. Therefore, the present invention also provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer using the EHMT2 polypeptide. In particular, an embodiment of this screening method includes the steps of:
(a) contacting a test substance with an EHMT2 polypeptide ;
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) selecting the test substance that binds to the polypeptide.
Alternatively, according to the present invention, the potential therapeutic effect of a test substance for treating and/or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance for treating and/or preventing cancer and/or inhibiting cancer associated with over-expression of EHMT2, the method including steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of EHMT2;
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when a substance binds to the polypeptide.
In one aspect of the present invention, the therapeutic effect may be correlated with the binding level of the test substance and EHMT2 protein(s). For example, when the test substance binds to an EHMT2 protein, the test substance may identified or selected as a candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not bind to an EHMT2 protein, the test substance may characterized as having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
The EHMT2 polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from nature or a partial peptide thereof. The polypeptide to be contacted with a test substance may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides. In preferred embodiments, the polypeptide is isolated from cells expressing EHMT2, or chemically synthesized to be contacted with a test substance in vitro.
In one embodiment, test substances used by the present invention may be proteins such as antibodies or synthetic chemical compounds. As a method of screening substances that bind to an EHMT2 polypeptide, many methods well known by a person skilled in the art may be used. Such a screening may be conducted by, for example, the immunoprecipitation method.
When the immunoprecipitation method is used, an EHMT2 polypeptide may contain an antibody recognition site. EHMT2 polypeptides to be used for the present screening method may be prepared as described above.
Alternatively, the polypeptide encoded by EHMT2 gene can be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity is known, to the N- or C- terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available. Also, a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the EHMT2 polypeptide by the fusion may be used. Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the EHMT2 polypeptide (Experimental Medicine 13: 85-90 (1995)).
In immunoprecipitation, an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent. The immune complex consists of the EHMT2 polypeptide, a polypeptide including the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the EHMT2 polypeptide itself rather than against an added epitope, which antibodies can be prepared as described above. An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody or any other antibody that binds to Protein A, Protein G or Protein L. If the polypeptide encoded by EHMT2 gene is prepared as a fusion protein with an epitope, such as GST, a complex can be formed in the same manner as in the use of the antibody against the EHMT2 polypeptide, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.
Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the EHMT2 polypeptide is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35S-methionine or 35S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used as a method of screening for proteins binding to the EHMT2 polypeptide using the polypeptide. In particular, a protein binding to the EHMT2 polypeptide can be obtained by preparing a cDNA library from cultured cells expected to express a protein binding to the EHMT2 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled EHMT2 polypeptide with the above filter, and detecting the plaques expressing proteins that bind to the EHMT2 polypeptide according to the label. The polypeptide of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the EHMT2, or a peptide or polypeptide (for example, GST) that is fused to the EHMT2 polypeptide. Methods using radioisotopes or fluorescence and such may be also used.
Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)").
In the two-hybrid system, the polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein. As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
A substance binding to an EHMT2 polypeptide may also be screened using affinity chromatography. For example, an EHMT2 polypeptide may be immobilized on a carrier of an affinity column, and a composition containing test substances is applied to the column. A composition herein may be, for example, cell extracts, cell lysates, antibody libraries etc. After loading test substances, the column is washed, and substances bound to the EHMT2 polypeptide can be collected. When the test substance is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention. When such a biosensor is used, the interaction between an EHMT2 polypeptide and a test substance can be observed in real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between an EHMT2 polypeptide and a test substance using a biosensor such as BIAcore.
The methods of screening for molecules that bind when the immobilized EHMT2 polypeptide is exposed to synthetic chemical substances, or natural substance banks or a random phage peptide display library, and the methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9 (1996)) to isolate not only proteins but chemical substances that bind to the EHMT2 protein (including agonist and antagonist) are well known to one skilled in the art.
(ii) Screening for a Substance that Suppresses the Biological Activity of EHMT2:
In one aspect of the present invention, the EHMT2 protein may promote cell proliferation of cancer cells (Fig. 3). Moreover, the EHMT2 protein may also be a histone methyltransferase(Tachibana M et al. J Biol Chem 2001;276:25309-17.). Using these biological activities as an index, the present invention provides a method for screening for a substance that suppresses the proliferation of cancer cells expressing EHMT2, and a method of screening for a candidate substance useful for treating and/or preventing cancer, in particular EHMT2 associated cancers including bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Thus, the present invention provides a method of screening for a substance for treating and/or preventing cancer using the polypeptide encoded by the EHMT2 gene including the steps as follows:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the EHMT2 gene (i.e., EHMT2 polypeptide);
(b) detecting the biological activity of the polypeptide of step (a); and
(c) selecting the test substance that suppresses the biological activity of the polypeptide as compared to the biological activity of said polypeptide detected in the absence of the test substance.
According to the present invention, the therapeutic effect of the test substance in suppressing the biological activity (e.g., the cell-proliferating activity) of EHMT2 polypeptide, or a candidate substance for treating and/or preventing cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance that suppresses the biological activity of EHMT2 polypeptide, or a candidate substance for treating and/or preventing cancer, using the EHMT2 polypeptide or fragments thereof, including the following steps:
a) contacting a test substance with the EHMT2 polypeptide or a functional fragment thereof; and
b) detecting the biological activity of the polypeptide or fragment of step (a), and
c) correlating the biological activity of b) with the therapeutic effect of the test substance.
Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance in the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer associated with over-expression of EHMT2, the method including steps of:
(a) contacting a test substance with the EHMT2 polypeptide or a functional fragment thereof;
(b) detecting the biological activity of the polypeptide or fragment of step (a); and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when a substance suppresses the biological activity of the polypeptide encoded by the polynucleotide corresponding to the EHMT2 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.
Examples of cancers associated with over-expression of the EHMT2 gene include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
In the context of the present invention, the therapeutic effect may be correlated with the biological activity of the EHMT2 polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of the EHMT2 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity of the EHMT2 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
Any polypeptides can be used for screening so long as they suppress a biological activity of the EHMT2 protein. Examples of the biological activities of the EHMT2 protein include cell-proliferating activity and methyltransferase activity of the EHMT2 protein. For example, EHMT2 protein can be used and polypeptides functionally equivalent to the EHMT2 protein can also be used. Details of polypeptides functionally equivalent to the EHMT2 protein (i.e., functional equivalent of the EHMT2 protein) have been described above under the item " The Gene and Protein". Such polypeptides may be expressed endogenously or exogenously by cells.
In another aspect, the present invention also provides a screening method following the method described in the above section "(i) Screening for an EHMT2 Binding Substance", such method including the steps of:
a) contacting a test substance with the EHMT2 polypeptide or a fragment thereof;
b) detecting the binding between the polypeptide or fragment and the test substance;
c) selecting the test substance that binds to the polypeptide;
d) contacting the test substance selected in step c) with the EHMT2 polypeptide or a fragment thereof;
e) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the substance; and
f) selecting the substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.
The substance isolated by this screening is a candidate for antagonists of the polypeptide encoded by EHMT2 gene. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding EHMT2. Moreover, a substance isolated by this screening is a candidate for substance which inhibits the in vivo interaction of the EHMT2 polypeptide with molecules (including DNAs and proteins).
When the biological activity to be detected in the present method is cell proliferation, it can be detected, for example, by preparing cells which express the EHMT2 polypeptide, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring survival cells or the colony forming activity assay, for example, as shown in Fig. 3. The substances that reduce the speed of proliferation of the cells expressed EHMT2 are selected as a candidate substance for treating or preventing cancer. In some embodiments, cells expressing EHMT2 gene are isolated and cultured cells exogenously or endogenously expressing EHMT2 gene in vitro.
More specifically, the method includes the step of:
(a) contacting a test substance with cells overexpressing EHMT2;
(b) measuring cell-proliferating activity; and
(c) selecting the test substance that reduces the cell-proliferating activity in the comparison with the cell-proliferating activity in the absence of the test substance.
In preferable embodiments, the method of the present invention may further include the steps of:
(d) selecting the test substance that have no effect to the cells no or little expressing EHMT2.
When the biological activity to be detected in the present method is methyltransferase activity, the methyltransferase activity can be determined by contacting a EHMT2 polypeptide with a substrate (e.g., histone H3 or fragments thereof comprising lysine 9) and a co-factor (e.g., S-adenosyl-L-methionine) under conditions suitable for methylation of the substrate and detecting the methylation level of the substrate.
More specifically, the method includes the step of:
[1] A method of screening for a candidate substance for treating or preventing cancer associated with EHMT2 overexpression, said method comprising the steps of:
(a) contacting a polypeptide encoded by a polynucleotide corresponding to the EHMT2 gene with a substrate and a cofactor in the presence of the test substance;
(b) detecting the methylation level of the substrate;
(c) determining the methyltransferase activity by correlating the methylation level of the step (b) with the methyltransferase activity; and
(d) selecting the test substance that reduces the methyltransferase activity as compared to the methyltransferase activity detected in the absence of the test substance;
[2] The method of [1], wherein the substrate is a histone or a fragment thereof comprising at least one methylation region.
[3] The method of [2], wherein the substrate is a histone H3 or a fragment thereof comprising at least one methylation region.
[4] The method of [3], wherein .the methylation region is lysine 9 of histone H3.
[4] The method of [1], wherein the cofactor is an S-adenosylmethionine.
[6] The method of [1], wherein the polypeptide is contacted with the substrate and cofactor in the presence of an enhancing agent for the methylation.
[7] The method of [1], wherein the enhancing agent for the methylation is S-adenosyl homocysteine hydrolase (SAHH).
In the present invention, methyltransferase activity of a EHMT2 polypeptide can be determined by methods known in the art. For example, the EHMT2 and a substrate can be incubated with a labeled methyl donor, under suitable assay conditions. Histone H3 peptides, and S-adenosyl-[methyl-14C]-L-methionine, or S-adenosyl-[methyl-3H]-L-methionine can be used as the substrate and labeled methyl donor, respectively. Transfer of the radiolabel to a histone H3 peptide can be detected, for example, by SDS-PAGE electrophoresis and fluorography. Alternatively, following the reaction, the histone H3 peptide can be separated from the methyl donor by filtration, and the amount of radiolabel retained on the filter quantitated by scintillation counting. Other suitable labels that can be attached to methyl donors, such as chromogenic and fluorescent labels, and methods of detecting transfer of these labels to histone peptides, are known in the art. Herein, "histone peptide" and "histone H3 peptide" refer to a full length of histone or a fragment thereof and a full length of histone H3 or fragment thereof, respectively.
Alternatively, the methyltransferase activity of EHMT2 can be determined using an unlabeled methyl donor (e.g. S-adenosyl-L-methionine) and reagents that selectively recognize methylated histone peptides. For example, after incubation of the EHMT2, substrate to be methylated and methyl donor, under the condition capable of methylation of the substrate, methylated substrate can be detected by immunological method. Any immunological techniques using an antibody recognizing methylated substrate can be used for the detection. For example, an antibody against methylated histone is commercially available (e.g. ab1220, abcam). ELISA or immunoblotting with antibodies recognizing methylated histone can be used for the present invention.
In the present invention, the histone H3 fragment to be used as a substrate typically retains lysine 9. Such histone H3 fragment may be composed of at least 10 amino acid residues, at least 15 amino acid residues, or at least 20 amino acid residues. Alternatively, a modified peptide of the histone H3 or fragment thereof may be used for which the methyltransferase has increased affinity/activity. Such peptides can be designed by exchanging and/or adding and/or deleting amino acids and testing the substrate in serial experiments for methyltransferase assay using the EHMT2 polypeptide.
In the present invention, any functional equivalent of the EHMT2 polypeptide can be used so long as such functional equivalents retain methyltransferase activity of the EHMT2 polypeptide. Generally, the functional equivalent of the EHMT2 polypeptide retains a SET-domain (e.g., amino acid position 1039-1154 SEQ ID NO: 2 or amino acid position 1005-1120 SEQ ID NO: 4) of the EHMT2 polypeptide.
In the present invention, an agent enhancing the methylation of the substrate can be used. SAHH or a functional equivalent thereof is one known enhancing agent for the methylation. In the presence of the agent enhancing the methylation of the substrate, the methyltransferase activity can thereby be determined with higher sensitivity. EHMT2 may be contacted with a substrate and a cofactor in the presence of an enhancing agent.
Furthermore, detection of methyltransferase activity can be performed by preparing cells which express the EHMT2 polypeptide, culturing the cells in the presence of a test substance, and determining methylation level of a histone, for example, by using the antibody specific binding to a methylation region of the histone for EHMT2 (i.e., histone H3 lysine 9).
More specifically, the method includes the step of:
[1] contacting a test substance with cells expressing EHMT2;
[2] detecting a methylation level of histone H3 lysine 9; and
[3] selecting the test substance that reduces the methylation level in the comparison with the methylation level in the absence of the test substance.
The phrase "suppress the biological activity" as defined herein are preferably at least 10% suppression of the biological activity of EHMT2 in comparison with in absence of the substance, at least 25%, 50% or 75% suppression, or at least 90% suppression.
In some embodiments, control cells that do not express EHMT2 polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance that inhibits cell growth or a candidate substance for treating and/or preventing an EHMT2- associated disease, using the EHMT2 polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express an EHMT2 polypeptide or a functional fragment thereof, and control cells that do not express an EHMT2 polypeptide or a functional fragment thereof in the presence of the test substance;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test substance that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance. As one embodiment of the present invention, suppressing the biological activity of EHMT2 reduces cell growth. Thus, by screening for a candidate substance that inhibits the biological activity of EHMT2, candidate substances that have the potential to, or are useful to, treat and/or prevent cancers can be identified. The potential, or utility, of these candidate substances to treat or prevent cancers may be evaluated by secondary and/or further screening to identify therapeutic substances, compounds or agents for cancers. For example, when a substance that inhibits the biological activity of an EHMT2 protein also inhibits the activity of a cancer, such a substance has an EHMT2-specific therapeutic effect.
II. Screening for a Substance that Alters the Expression of EHMT2:
In one aspect of the present invention, a decrease in the expression of EHMT2 by siRNA results in the inhibition of cancer cell proliferation (Fig. 3). Accordingly, the present invention provides a method of screening for a substance that inhibits the expression of EHMT2. A substance that inhibits the expression of EHMT2 may suppress the proliferation of cancer cells, and thus is useful for treating or preventing cancer, particularly EHMT2-associated cancers such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Therefore, the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer. In the context of the present invention, such screening may include, for example, the following steps:
(a) contacting a test substance with a cell expressing EHMT2; and
(b) selecting the test substance that reduces the expression level of EHMT2 as compared to a control.
Alternatively, the screening method of the present invention may include, following steps:
(a) contacting a test substance with a cell expressing an EHMT2 gene;
(b) detecting an expression level of the EHMT2 gene in the cell of the step (a); and
(c) selecting the test substance that reduces the expression level detected in the step (b) in comparison with the expression level of an EHMT2 gene detected in the absence of the test substance.
In the context of the present invention, such screening may include, for example, the following steps:
a) contacting a test substance with a cell expressing the EHMT2 gene;
b) detecting the expression level of the EHMT2 gene; and
c) correlating the expression level of b) with the therapeutic effect of the test substance.
In the context of present invention, the therapeutic effect may be correlated with the expression level of the EHMT2 gene. For example, when the test substance reduces the expression level of the EHMT2 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level of the EHMT2 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
Cells expressing the EHMT2 include, for example, cell lines established from bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma or cell lines transfected with EHMT2 expression vectors; any of such cells can be used for the above screening of the present invention. The expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining and flow cytometry analysis. The phrase "reduce the expression level" as defined herein includes at least 10% reduction of expression level of EHMT2 in comparison to the expression level in absence of the substance, at least 25%, 50% or 75% reduced level, or at least 95% reduced level. The substance herein includes chemical compounds, double-strand nucleotides, proteins, peptides, polynucleotides, aptamers, and so on. The preparation of the double-strand nucleotides will be described bellow. In the method of screening, a substance that reduces the expression level of EHMT2 can be selected as candidate substances to be used for the treatment or prevention of cancer. In some embodiments, cells expressing EHMT2 gene are isolated and cultured cells exogenously or endogenously expressing EHMT2 gene in vitro.
Alternatively, the screening method of the present invention may include the following steps:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of EHMT2 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) selecting the test substance that reduces the expression or activity of said reporter gene.
Suitable reporter genes and host cells are well known in the art. Illustrative reporter genes include, but are not limited to, luciferase, green fluorescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of EHMT2 gene. The transcriptional regulatory region of EHMT2 gene herein is the region from transcription start site to at least 500bp upstream, preferably 1000bp, more preferably 5000 or 10000bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of the gene. Methods for identifying a transcriptional regulatory region, and assay protocols are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
The vector containing the said reporter construct may be introduced into host cells and the expression or activity of the reporter gene may be detected by methods well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on). "Reduces the expression or activity" as defined herein includes at least 10% reduction of the expression or activity of the reporter gene in comparison with expression or activity in absence of the test substance, at least 25%, 50% or 75% reduction, or at least 95% reduction.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of EHMT2, the method including steps of:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of EHMT2 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression level or activity of said reporter gene; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression level or activity of said reporter gene.
According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or the therapeutic effect of a candidate substance for treating or preventing EHMT2 associating disease may be evaluated. Therefore, the present invention also provides a method for screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening for a candidate substance for treating or preventing EHMT2 associating disease.
According to another aspect, the present invention provides a method which includes the following steps of:
(a) contacting a test substance with a cell into which a vector, composed of the transcriptional regulatory region of the EHMT2 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) detecting the expression level or activity of said reporter gene; and
(c) correlating the expression level of (b) with the therapeutic effect of the test substance.
In the present invention, the therapeutic effect may be correlated with the expression level or activity of said reporter gene. For example, when the test substance reduces the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified or selected as the substance or as a candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified as a substance having no significant therapeutic effect.
III. Screening for a Substance that Inhibits the binding of EHMT2 to a promoter region of SIAH1:
In the present invention, it was confirmed that the EHMT2 polypeptide interacts with the promoter region of SIAH1 (Fig. 4D). Thus, a substance that inhibits the binding between EHMT2 polypeptide and the promoter region of SIAH1 can be screened using such a binding of EHMT2 polypeptide and promoter region of SIAH1 as an index. Such substances may have potential therapeutic effect on cancer treatment as those proteins are involved in cancer cell growth. Therefore, the present invention provides a method for screening for a substance for inhibiting the binding between the EHMT2 polypeptide and the promoter region of SIAH1 using such a binding of EHMT2 polypeptide and the promoter region of SIAH1 as an index. Furthermore, the present invention also provides a method for screening a candidate substance that inhibits or reduces the growth of cancer cells expressing EHMT2 gene, e.g. bladder cancer cells, lung cancer cells, AML cells, CML cells, esophageal cancer cells, breast cancer cells, cervical cancer cells or osteosarcoma cells, and a method for screening for a candidate substance for treating or preventing cancers, e.g. bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
Specifically, the present invention provides the following methods of [1] to [4]:
[1] A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to a promoter region of SIAH1 in the presence of a test substance;
(b) detecting a binding between the polypeptide and the polynucleotide;
(c) comparing the binding level detected in the step (b) with the binding level between the polypeptide and the polynucleotide detected in the absence of the test substance; and
(d) selecting the test substance that reduces the binding level;
[2] The method of [1], wherein the functional equivalent of the EHMT2 polypeptide comprises the DNA-binding domain;
[3] The method of [2], wherein the functional equivalent of EHMT2 comprises the amino acid sequence of SEQ ID NO: 9; and
[4] The method of any one of [1] to [3], wherein the cancer is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
According to the present invention, the therapeutic effect of the test substance in inhibiting the cell growth or a candidate substance for treating or preventing EHMT2 associating disease may be evaluated. Therefore, the present invention also provides a method for screening for a candidate substance that suppresses the proliferation of cancer cells, and a method for screening for a substance or a candidate substance for treating or preventing cancer.
More specifically, the method includes the steps of:
(a) contacting a EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to the promoter region of SIAH1 in the presence of a test substance;
(b) detecting the level of binding between the polypeptide and the polynucleotide;
(c) comparing the binding level detected in the step (b) with the binding level between the polypeptide and the polynucleotide detected in the absence of the test substance; and
(d) correlating the binding level of c) with the therapeutic effect of the test substance.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance for treating or preventing cancer or inhibiting cancer, the method including steps of:
(a) contacting an EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to the promoter region of SIAH1 in the presence of a test substance;
(b) detecting a binding level between the polypeptide and the polynucleotide;
(c) comparing the binding level detected in the step (b) with the binding level between the polypeptide and the polynucleotide detected in the absence of the test substance; and
(d) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when a test substance reduces the binding level.
In the present invention, the therapeutic effect may be correlated with the binding level of the EHMT2 polypeptide and a polynucleotide corresponding to the promoter region of SIAH1. For example, when the test substance reduces the binding level of the EHMT2 polypeptide and a polynucleotide corresponding to the promoter region of SIAH1 as compared to a level detected in the absence of the test substance, the test substance may identified or selected as a substance or a candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the binding level of EHMT2 polypeptide and a polynucleotide corresponding to the promoter region of SIAH1 as compared to a level detected in the absence of the test substance, the test substance may identified as a substance having no significant therapeutic effect.
In one aspect of the present invention, a functional equivalent of an EHMT2 polypeptide is a polypeptide that has a biological activity equivalent to an EHMT2 polypeptide (SEQ ID NO: 2, 4) (see, (1) Genes and Polypeptides). For example, the functional equivalent of EHMT2 polypeptide may be a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 2 or 4 comprising the DNA region-binding domain. An example of such functional equivalent includes, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 9.
Many methods well known by one skilled in the art can be used in screening for a substance that inhibits the binding of EHMT2 polypeptide to the polynucleotide. Such a screening can be conducted using, for example, ChIP assay, gel shift assay, affinity chromatography or a biosensor using the surface plasmon resonance phenomenon. Also, some protein-DNA binding assay kits are commercially available (e.g., Protein-DNA Binding Assay, Clontech).
A polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof. In preferred embodiments, the polypeptide is isolated from cells expressing EHMT2, or chemically synthesized to be contacted with a polynucleotide corresponding to a promoter region of SIAH1 in vitro. In addition, a polynucleotide to be used for screening can be a synthesized polynucleotide or a DNA derived from natural sources, or a partial oligonucleotide thereof. Any test substances aforementioned can be used for screening.
In some embodiments, the present screening method can be performed using cells that express the EHMT2 polypeptide and have the SIAH1 gene. Cells that expresses the EHMT2 polypeptide and possess the SIAH1 gene include, for example, cell lines established from cancer, e.g. bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma. Alternatively cells can be prepared by introducing an expression vector of the EHMT2 gene into a cell that possesses the SIAH gene. The binding of EHMT2 polypeptide to the SIAH1 promoter region can be detected by, for example, ChIP assay using an anti-EHMT2 antibody or antibody against a tag protein fused with the EHMT2 polypeptide and a primer set for amplification of the SIAH1 promoter region (see, "Example 1, Chromatin Immunoprecipitation Assay").
In one aspect of the present invention, the EHMT2 polypeptide suppresses the expression of the SIAH1 gene by binding to its promoter region, and consequently, inhibits apoptotic cell death in cancer cells . Thus, by screening for candidate compounds that inhibits the binding of EHMT2 polypeptide to the SIAH1 promoter region, substances that are useful for treating or preventing cancers or substances that have the potential to treat or prevent cancers can be identified. The potential of these candidate substance to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance that inhibits the binding of the EHMT2 polypeptide to the SIAH1 promoter region inhibits activities of cancer such as cell growth or proliferation, such a substance may have an EHMT2-specific therapeutic effect.
By screening for candidate substances that (i) bind to the EHMT2 polypeptide; (ii) suppress/reduce the biological activity (e.g., the cell-proliferating activity, the methyltransferase activity) of the EHMT2 polypeptide; (iii) reduce the expression level of EHMT2 gene; or (iv) suppress/reduce the binding of the EHMT2 polypeptide to the SIAH1 promoter region, candidate substances that are useful for treating or preventing cancers or have the potential to treat or prevent cancers (e.g., bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma) can be identified. The therapeutic potential of these candidate substances may be evaluated by secondary and/or further screening to identify therapeutic substances for cancers. For example, when a substance that binds to the EHMT2 polypeptide inhibits the above-described activities of cancer, it may be concluded that such a substance has the EHMT2-specific therapeutic effect.
Double Stranded Molecules:
As used herein, the term "isolated double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene and includes, for example, a short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)), a short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA), or a small hairpin chimera of DNA and RNA (shD/R-NA)).
As used herein, a target sequence is a nucleotide sequence within the mRNA or cDNA sequence of a gene, which will result in suppression of translation of the whole mRNA if a double-stranded nucleic acid molecule of the present invention is introduced within a cell expressing the gene. A nucleotide sequence within the mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded polynucleotide including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. The double-stranded polynucleotide which suppresses the gene expression may comprise the target sequence and may further comprise a 3'overhang having 2 to 5 nucleotides in length (e.g., uu) on one or both strands of the double-stranded polynucleotide.
When a target sequence is shown by cDNA sequence, a sense strand sequence of a double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence. A double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region are replaced with base "u"s. On the other hand, when a target sequence is shown in RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region are replaced with "t"s. In the context of the present invention, the target sequences are mainly shown in DNA. In other words, the present invention also provides a double-stranded molecule whose target sequence includes or is limited to SEQ ID NO: 34 or SEQ ID NO: 35 which is shown in DNA but can be replaced with RNA.
Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
A double-stranded molecule may have one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and a complementary sequence.
As use herein, the term "siRNA" refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes an EHMT2 sense nucleic acid sequence (also referred to as "sense strand"), an EHMT2 antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.
As used herein, the term "dsRNA" refers to a construct of two RNA molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" RNAs selected from a protein coding sequence of target gene sequence, but also or alternatively, may comprise a nucleotide sequence selected from non-coding region of the target gene.
The term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, composed of first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
As use herein, the term "siD/R-NA" refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes an EHMT2 sense nucleic acid sequence (also referred to as "sense strand"), an EHMT2 antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.
As used herein, the term "dsD/R-NA" refers to a construct of two molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotides having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA may be composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules may be composed of RNA and the other composed of DNA (hybrid double-strand).
The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop structure, composed of a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
As used herein, an "isolated nucleic acid" is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In one aspect of the present invention, examples of isolated nucleic acid includes DNA, RNA, and derivatives thereof.
A double-stranded molecule against EHMT2 that hybridizes to target mRNA may decrease or inhibit production of EHMT2 protein encoded by EHMT2 gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the protein. As demonstrated herein, the expression of EHMT2 in several cancer cell lines may be inhibited by dsRNA (Fig. 3). Accordingly, the present invention provides isolated double-stranded molecules that are capable of inhibiting the expression of an EHMT2 gene when introduced into a cell expressing the gene. The target sequence of double-stranded molecule may be designed by an siRNA design algorithm such as that mentioned below.
Examples of EHMT2 target sequences include the nucleotide sequences of SEQ ID NOs: 34 and 35.
Of particular interest in the present invention are the following double-stranded molecules [1] to [19]:
[1] An isolated double-stranded molecule that, when introduced into a cell, inhibits expression of EHMT2 and cell proliferation, such molecules composed of a sense strand and a complementary antisense strand , hybridized to each other to form the double-stranded molecule;
[2] The double-stranded molecule of [1], wherein said double-stranded molecule inhibits expression, translation, or stability of mRNA, matching a target sequence of SEQ ID NO: 34 or 35;
[3] The double-stranded molecule of [1] or [2], wherein the sense strand contains a nucleotide sequence corresponding to a target sequence of SEQ ID NO: 34 or 35;
[4] The double-stranded molecule of any one of [1] to [3], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a less than about 100 nucleotide pairs in length;
[5] The double-stranded molecule of [4], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[6] The double-stranded molecule of [5], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[7] The double-stranded molecule of [6], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[8] The double-stranded molecule of [7], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[9] The double-stranded molecule of any one of [1] to [8], composed of a single polynucleotide having both the sense and antisense strands linked by an intervening single-strand;
[10] The double-stranded molecule of [9], having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a nucleotide sequence corresponding to a target sequence of SEQ ID NO: 34 or 35, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A];
[11] The double-stranded molecule of any one of [1] to [10], composed of RNA;
[12] The double-stranded molecule of any one of [1] to [10], composed of both DNA and RNA;
[13] The double-stranded molecule of [12], wherein the molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[14] The double-stranded molecule of [13] wherein the sense and the antisense strands are composed of DNA and RNA, respectively;
[15] The double-stranded molecule of [12], wherein the molecule is a chimera of DNA and RNA;
[16] The double-stranded molecule of [15], wherein a region flanking the 3'-end of the antisense strand, or both of a region flanking the 5'-end of sense strand and a region flanking the 3'-end of antisense strand are RNA;
[17] The double-stranded molecule of [16], wherein the flanking region is composed of 9 to 13 nucleotides; and
[18] The double-stranded molecule of any one of [1] to [17], wherein the molecule has one or two 3' overhang(s).
[19] The double-stranded molecule of any one of [1] to [8] or [11]-[17], wherein the molecule has two 3' overhangs.
The double-stranded molecule of the present invention will be described in more detail below.
Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Patent No. 6,506,559, herein incorporated by reference in its entirety). For example, a computer program for designing siRNAs is available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html).
The computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.
Selection of Target Sites:
1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. For example, BLAST, which can be found on the NCBI server at:ncbi.nlm.nih.gov/BLAST/, may be used (Altschul SF et al., Nucleic Acids Res 1997 Sep 1, 25(17): 3389-402).
3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
Using the above protocol, the target sequence of the double-stranded molecules of the present invention may be designed as
SEQ ID NO: 34 and 35 for EHMT2 gene.
Double-stranded molecules targeting the above-mentioned target sequences were respectively examined for their ability to suppress the growth of cells expressing the target genes. Therefore, the present invention provides double-stranded molecule targeting the sequences of SEQ ID NO: 34 and 35 for EHMT2 gene,
The double-stranded molecule of the present invention may be directed to a single target EHMT2 gene sequence or may be directed to a plurality of target EHMT2 gene sequences. Alternatively, the siRNA may be directed to multiple targets of the EHMT2 gene sequence. For example, the composition may contain siRNAs of EHMT2 directed to two, three, four, or five or more target sequences of EHMT2, respectively. EHMT2 target sequence is meant a nucleotide sequence that is identical or substantially identical to a portion of the EHMT2 gene. The target sequence can include the 5' untranslated (UT) region, the open reading frame (ORF) or the 3' untranslated region of the human EHMT2 gene.
An siRNA of EHMT2 which hybridize to target mRNA and decreases or inhibits production of the EHMT2 polypeptide product encoded by the EHMT2 gene by associating with the normally single-stranded mRNA transcript, may thereby interfere with translation of EHMT2 and thus, suppress the expression of the protein. Thus, in some embodiments of the present invention, the siRNA molecules of the invention can be defined by their ability to hybridize specifically to mRNA or cDNA from a EHMT2 gene under stringent conditions.
A double-stranded molecule of the present invention targeting the above-mentioned targeting sequence of EHMT2 gene includes isolated polynucleotides that contain the nucleic acid sequences of target sequences and/or complementary sequences to the target sequence. Examples of polynucleotides targeting EHMT2 gene include those containing the sequence of SEQ ID NO: 34 or 35 and/or complementary sequences thereof; However, the present invention is not limited to these examples, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of EHMT2 gene. Herein, the phrase "minor modification" as used in connection with a nucleic acid sequence indicates one, two or several substitutions, deletions, additions or insertions of nucleic acids to the sequence.
In one embodiment, a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and the other polynucleotide has a complementary sequence to the target sequence, i.e., antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule. Examples of such double-stranded molecules include dsRNA and dsD/R-NA.
In an another embodiment, a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand. Generally, the sense strand and the antisense strand are linked by an intervening strand, and hybridize to each other to form a hairpin loop structure. Examples of such double-stranded molecule include shRNA and shD/R-NA.
In other words, a double-stranded molecule of the present invention is composed a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule. In the double-stranded molecule including the polynucleotides, a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence or complementary sequence is replaced with "u".
In one embodiment of the present invention, such a double-stranded molecule of the present invention includes a stem-loop structure, composed of the sense and antisense strands. The sense and antisense strands may be joined by a loop. Accordingly, the present invention also provides the double-stranded molecule composed of a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.
In the present invention, double-stranded molecules targeting the EHMT2 gene may have a sequence selected from among SEQ ID NOs: 34 and 35 as a target sequence. Accordingly, examples of the double-stranded molecule of the present invention include a polynucleotide and a complementary sequence thereto, such as a polynucleotide that has a sequence corresponding to SEQ ID NO: 34 or 35 and a complementary sequence thereto.
In one aspect of the present invention, the term "several" as applies to nucleic acid substitutions, deletions, additions and/or insertions may mean 3-7, preferably 3-5, more preferably 3-4, even more preferably 3 nucleic acid residues.
According to the present invention, a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples. In the Examples herein below, double-stranded molecules composed of sense strands of various portions of mRNA of EHMT2 genes or antisense strands complementary thereto were tested in vitro for their ability to decrease production of EHMT2 gene product in cancer cell lines according to standard methods. Furthermore, for example, reduction in EHMT2 gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g. RT-PCR using primers for the EHMT2 mRNA mentioned under Example 1 item "Quantitative real time PCR". Sequences that decrease the production of an EHMT2 gene product in in vitro cell-based assays can then be tested for their inhibitory effects on cell growth. Sequences that inhibit cell growth in in vitro cell-based assays can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of an EHMT2 gene product and decreased cancer cell growth.
When the isolated polynucleotide is RNA or derivatives thereof, base "t" should be replaced with "u" in the nucleotide sequences. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide, and the term "binding" means the physical or chemical interaction between two polynucleotides. When the polynucleotide includes modified nucleotides and/or non-phosphodiester linkages, these polynucleotides may also bind each other as same manner. Generally, complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. Furthermore, the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by hybridization. In one embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In another embodiment, where the strands of the duplex are fully complementary, such duplexes contain no mismatches.
The polynucleotide may be less than 3982 nucleotides in length for EHMT2. For example, the polynucleotide may be less than 500, 200, 100, 75, 50, or 25 nucleotides in length for EHMT2. The isolated polynucleotides of the present invention are useful for forming double-stranded molecules against EHMT2 gene or preparing template DNAs encoding the double-stranded molecules. When the polynucleotides are used for forming double-stranded molecules, the polynucleotide may be longer than 19 nucleotides, longer than 21 nucleotides, or have a length of between about 19 and 25 nucleotides.
Accordingly, the present invention provides a double-stranded molecule composed of a sense strand and an antisense strand, wherein the sense strand is a nucleotide sequence corresponding to a target sequence. In some embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
The double-stranded molecule may serve as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells. The identified target RNA may be cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the EHMT2 gene under stringent conditions. Herein, the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence" or "target nucleic acid" or "target nucleotide". In the context of the present invention, the nucleotide sequence of the "target sequence" can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
The double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. Chemical modifications which may be incorporated into the present molecules include those described in WO03/070744, and WO2005/045037. In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include, but are not limited to, phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Examples of such modifications include, but are not limited to, chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212). In another embodiment, modifications can be used to increase or decrease affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. The present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene, such as EHMT2.
Furthermore, the double-stranded molecules of the present invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule composed of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule containing both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule.
The hybrid of a DNA strand and an RNA strand may be either where the sense strand is DNA and the antisense strand is RNA, or vice versa so long as it can inhibit expression of the target gene when introduced into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may be either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. In order to enhance stability of the double-stranded molecule, the molecule may contain as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule may be required to be RNA within a range to induce sufficient inhibition of the expression.
As one example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA. In this example, the upstream partial region indicates the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. Alternatively, regions flanking to 5'-end of sense strand and/or 3'-end of antisense strand are referred to upstream partial region. That is, in some embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand are composed of RNA. For instance, the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
sense strand:
5'-[-----DNA-----]-3'
3'-(RNA)-[DNA]-5'
: antisense strand,
sense strand:
5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5'
: antisense strand, and
sense strand:
5'-(RNA)-[DNA]-3'
3'-(-----RNA-----)-5'
: antisense strand.
The upstream partial region may be a domain composed of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
In the context of the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
A loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Such loop sequence may be joined to 5' or 3' end of a sense strand and 3' or 5' end of an antisense strand to form the hairpin loop structure. Thus, the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a nucleotide sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A]. The target sequence may be selected from among, for example, the nucleotide sequences of SEQ ID NO: 34 and 35.
The present invention is not limited to these examples, and the target sequence in [A] may be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted EHMT2 gene. The region [A] hybridizes to [A'] to form a loop composed of the region [B]. The intervening single-stranded portion [B], i.e., loop sequence may be 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from among the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26;
UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and
UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.
Examples of double-stranded molecules of the present invention having hairpin loop structure are shown below. In the following structure, the loop sequence can be selected from among AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto:
GAGUUUGGCUAUGAGGCUA -[B]- UAGCCUCAUAGCCAAACUC
(for target sequence SEQ ID NO: 34).
GCAAAUAUUUCACCUGCCA -[B]- UGGCAGGUGAAAUAUUUGC
(for target sequence SEQ ID NO: 35).
Furthermore, in order to enhance the inhibition activity of the double-stranded molecules, several nucleotides can be added to 3'end of the sense strand and/or antisense strand of the target sequence, as 3' overhangs. Examples of nucleotides constituting a 3' overhang include "t" and "u", but are not limited thereto. The number of nucleotides to be added is at least 2, generally 2 to 10, or 2 to 5. The added nucleotides may be single stranded at the 3'end of the antisense strand or sense strand of the double-stranded molecule. In cases where double-stranded molecules consists of a single polynucleotide to form a hairpin loop structure, a 3' overhang sequence may be added to the 3' end of the single polynucleotide.
The method for preparing the double-stranded molecule is not particularly limited and includes chemical synthetic methods known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Specific example for the annealing includes wherein the synthesized single-stranded polynucleotides are mixed in a molar ratio of preferably at least about 3:7, about 4:6, or in a substantially equimolar amount (i.e., a molar ratio of about 5:5). Next, the mixture is heated to a temperature at which double-stranded molecules dissociate and then gradually cooled down. The annealed double-stranded polynucleotide can be purified by methods known in the art. Examples of purification methods include methods utilizing agarose gel electrophoresis or methods in which remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
The regulatory sequences flanking EHMT2 sequences may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. The double-stranded molecules can be transcribed intracellularly by cloning EHMT2 gene templates into a vector containing, e.g., an RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
Alternatively, the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence. The regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules are described below.
Vector containing a double-stranded molecule of the present invention:
As noted above, the present invention contemplates vectors containing one or more of the double-stranded molecules described herein, and a cell containing such a vector.
Of particular interest to the present invention are the following vector of [1] to [10].
[1] A vector, encoding a double-stranded molecule that, when introduced into a cell, inhibits expression of EHMT2 and cell proliferation, the double-stranded molecule composed of a sense strand and an antisense strand complementary thereto and hybridized to each other;
[2] The vector of [1], encoding the double-stranded molecule, wherein the double-stranded molecule inhibits expression of EHMT2, and wherein the double-stranded molecule comprises nucleotide sequence of SEQ ID NO: 34 or 35 as a target sequence;
[3] The vector of [1], wherein the sense strand contains a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35;
[4] The vector of any one of [1] to [3], encoding the double-stranded molecule ,wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[5] The vector of [4], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[6] The vector of [5], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[7] The vector of [6] encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[8] The vector of [7], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[9] The vector of any one of [1] to [8], wherein the double-stranded molecule is composed of a single polynucleotide having both the sense and antisense strands linked by an intervening single-strand; and
[10] The vector of [9], encoding the double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3', or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A].
A vector of the present invention may encode a double-stranded molecule of the present invention in an expressible form. Herein, the phrase "in an expressible form" indicates that the vector, when introduced into a cell, will express the molecule. In one embodiment, the vector includes regulatory elements necessary for expression of the double-stranded molecule. Accordingly, in one embodiment, the expression vector encodes the nucleic acid sequence of the double-stranded molecule of the present invention and is adapted for expression of said double-stranded molecule. Such vectors of the present invention may be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.
Vectors of the present invention can be produced, for example, by cloning EHMT2 sequence into an expression vector so that regulatory sequences are operatively-linked to the EHMT2 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5). For example, RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene. Alternatively, two vector constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then form a double-stranded molecule. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector that contains both the sense and complementary antisense sequences of the target gene.
The vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
The vectors of the present invention include, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell. Another example of useable vector includes Bacille Calmette Guerin (BCG) vectors. BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
Method of Inhibiting or Reducing Growth of a Cancer Cell or Treating Cancer using a Double-Stranded Molecule of the Present Invention:
In present invention, dsRNAs for EHMT2 were tested for their ability to inhibit cell growth. The dsRNA for EHMT2 (Fig. 3, 7) effectively knocked down the expression of the gene in several cancer cell lines, which coincided with suppression of cell proliferation.
Accordingly, the present invention provides methods for inhibiting cancer cell growth by inducing dysfunction of the EHMT2 gene via inhibiting the expression of EHMT2. EHMT2 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention that specifically target the EHMT2 gene.
Such ability of the present double-stranded molecules and vectors to inhibit cell growth of cancerous cell indicates that they can be used for methods for treating cancer such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Thus, the present invention provides methods to treat patients with cancer by administering a double-stranded molecule against EHMT2 gene or a vector expressing the molecule. In one aspect, the treating methods of the present invention may be carried out without adverse effect because EHMT2 gene was minimally detected in normal organs (Fig. 1, 2, 5, 6, 10).
Of particular interest to the present invention are the following methods [1] to [32]:
[1] A method for inhibiting a growth of cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses the EHMT2 gene, such method including the step of administering at least one isolated double-stranded molecule or vector encoding the double-stranded molecule that inhibits the expression of EHMT2 and cell proliferation in a cell over-expressing the gene, wherein the double-stranded molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
[2] The method of [1], wherein the double-stranded molecule inhibits the expression of mRNA which matches a target sequence of SEQ ID NO: 34 or 35;
[3] The method of [1], wherein the sense strand contains the sequence corresponding to a target sequence of SEQ ID NO: 34 or 35;
[4] The method of any one of [1] to [3], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
[5] The method of [3], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[6] The method of [5], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[7] The method of [6], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[8] The method of [7], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having a length of less than about 25 nucleotide pairs in length;
[9] The method of [8], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having a length of between about 19 and about 25 nucleotide pairs in length;
[10] The method of any one of [1] to [9], wherein the double-stranded molecule is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[11] The method of [10], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35, [B] is the intervening single strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A];
[12] The method of any one of [1] to [11], wherein the double-stranded molecule is an RNA;
[13] The method of any one of [1] to [11], wherein the double-stranded molecule contains both DNA and RNA;
[14] The method of [13], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[15] The method of [14] wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[16] The method of [13], wherein the double-stranded molecule is a chimera of DNA and RNA;
[17] The method of [16], wherein a region flanking the 3'-end of the antisense strand, or both of a region flanking the 5'-end of sense strand and a region flanking the 3'-end of antisense strand are composed of RNA;
[18] The method of [17], wherein the flanking region is composed of 9 to 13 nucleotides;
[19] The method of any one of [1] to [18], wherein the double-stranded molecule contains one or two 3' overhang(s);
[20] The method of any one of [1] to [19], wherein the double-stranded molecule is contained in a composition which includes, in addition to the molecule, a transfection-enhancing agent and pharmaceutically acceptable carrier;
[21] The method of [1], wherein the double-stranded molecule is encoded by a vector;
[22] The method of [21], wherein the double-stranded molecule encoded by the vector inhibits the expression of mRNA which matches a target sequence of SEQ ID NO: 34 or 35;
[23] The method of [21], wherein the sense strand of the double-stranded molecule encoded by the vector contains the sequence corresponding to a target sequence selected from among SEQ ID NO: 34 and 35;
[24] The method of any one of [21] to [23], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
[25] The method of [23], wherein the double-stranded molecule encoded by the vector has less than about 100 nucleotide pairs in length;
[26] The method of [25], wherein the double-stranded molecule encoded by the vector has a length of less than about 75 nucleotide pairs in length;
[27] The method of [26], wherein the double-stranded molecule encoded by the vector has less than about 50 nucleotide pairs in length;
[28] The method of [27], wherein the double-stranded molecule encoded by the vector has less than about 25 nucleotide pairs in length;
[29] The method of [28], wherein the double-stranded molecule encoded by the vector has between about 19 and about 25 nucleotide pairs in length;
[30] The method of any one of [21] to [29], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[31] The method of [30], wherein the double-stranded molecule encoded by the vector has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35, [B] is a intervening single-strand is composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[32] The method of any one of [21] to [31], wherein the double-stranded molecule encoded by the vector is contained in a composition which includes, in addition to the molecule, a transfection-enhancing agent and pharmaceutically acceptable carrier.
The method of the present invention will be described in more detail below.
The growth of cells expressing an EHMT2 gene may be inhibited by contacting the cells with a double-stranded molecule against an EHMT2 gene, a vector expressing the molecule or a composition containing the same. The cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule. Cell growth may be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
The term "specifically inhibit" in the context of inhibitory polynucleotides and polypeptides refers to the ability of an agent or ligand to inhibit the expression or the biological function of EHMT2. Specific inhibition typically results in at least about a 2-fold inhibition over background, greater than about 10-fold or greater than 100-fold inhibition of EHMT2 expression (e.g., transcription or translation) or measured biological function (e.g., cell growth or proliferation, inhibition of apoptosis, intracellular signaling from EHMT2). Expression levels and/or biological function can be measured in the context of comparing treated and untreated cells, or a cell population before and after treatment. In some embodiments, the expression or biological function of EHMT2 is completely inhibited, or inhibited to below detectable levels. Typically, specific inhibition is a statistically meaningful reduction in EHMT2 expression or biological function (e.g., p <= 0.05) using an appropriate statistical test.
The growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention. Exemplary cells include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
Thus, patients suffering from or at risk of developing a disease related to EHMT2 may be treated with the administration of a double-stranded molecule of the present invention, at least one vector expressing the molecule or a composition containing the molecule. For example, patients suffering from cancer may be treated according to the present methods. The type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed. For example, patients treated by the methods of the present invention may be selected by detecting the expression of EHMT2 in a biopsy from the patient by RT-PCR or immunoassay. In some cases, before the treatment of the present invention, the biopsy specimen from the subject may be confirmed for EHMT2 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.
According to one aspect of the present method to inhibit cell growth and thereby treat cancer, through the administration of multiple types of the double-stranded molecules (or vectors expressing or compositions containing the same), each of the molecules may have different structures but act on mRNA that matches the same target sequence of EHMT2. Alternatively, multiple types of the double-stranded molecules may act on mRNA that matches a different target sequence of same gene. Alternatively, for example, the method may utilize double-stranded molecules directed to one, two or more target sequences of EHMT2.
For inhibiting cell growth, a double-stranded molecule of present invention may be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts. Alternatively, as described above, a DNA encoding the double-stranded molecule may be introduced into cells as a vector. For introducing the double-stranded molecules and vectors into the cells, transfection-enhancing agent, such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), may be employed.
A treatment is deemed "efficacious" if it leads to clinical benefit such as, reduction in expression of EHMT2 gene, or a decrease in size, prevalence, proliferation, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating any particular tumor type.
To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels." While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
The treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. For example, reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis includes 10%, 20%, 30% or more reduction, or inducing disease stability.
It is understood that a double-stranded molecule of the present invention degrades EHMT2 mRNA in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, as compared to standard cancer therapies, the present invention requires the delivery of significantly less double-stranded molecule at or near the site of cancer in order to exert therapeutic effect.
One skilled in the art can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is local or systemic. Generally, an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, including from about 2 nM to about 50 nM, and about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
The present methods can be used to inhibit the growth or metastasis of cancer expressing EHMT2 gene; for example bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma. In particular, a double-stranded molecule containing a target sequence of EHMT2 gene (e.g., SEQ ID NO: 34 and 35) is useful for the treatment of cancer.
For treating cancer, the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical composition different from the double-stranded molecule. Alternatively, the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
In one aspect of the present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. Varieties of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
The liposomes encapsulating the double-stranded molecule of the present invention may include a ligand molecule that can deliver the liposome to the cancer site. Exemplary ligands include those ligands which bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens.
The liposomes encapsulating the present double-stranded molecule may be modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure. In one embodiment, a liposome of the invention can include both opsonization-inhibition moieties and a ligand.
Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization-inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, target tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
Opsonization-inhibiting moieties suitable for modifying liposomes include water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, or from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM.sub.1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization-inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
In some embodiments, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
The opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
Vectors expressing a double-stranded molecule of the present invention are discussed above. Such vectors expressing at least one double-stranded molecule of the present invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express a double-stranded molecule of the present invention, to an area of cancer in a patient are within the skill of the art.
The double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites. For example, the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. In some embodiments, injections or infusions of the double-stranded molecule or vector are given at or near the site of the cancer.
The double-stranded molecule of the present invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the present invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. For example, administration can include injection of the double-stranded molecule directly into the tissue at or near the site of cancer. In some cases, administration includes multiple injections of the double-stranded molecule into the tissue at or near the site of cancer.
One skilled in the art can also readily determine an appropriate dosage regimen for administering the double-stranded molecule of the present invention to a given subject. For example, the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, or from about seven to about ten days. In one embodiment, the double-stranded molecule may be injected at or near the site of cancer once a day for seven days. Where a dosage regimen requires multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.
In the present invention, a cancer overexpressing EHMT2 can be treated with at least one active ingredient selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof.
Accordingly, in a preferred embodiment, the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of:
a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of EHMT2 with a normal control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is increased as compared to the normal control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
Alternatively, such a method includes the steps of:
a) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of EHMT2 with a cancerous control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of EHMT2 is similar or equivalent to the cancerous control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
The cancer includes, but is not limited to, bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is an aspect of the present invention to confirm whether the expression level of EHMT2 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ. Thus, in one embodiment, the present invention provides a method for treating a cancer (over)expressing EHMT2, such method including the steps of:
i) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated;
ii) comparing the expression level of EHMT2 with normal control; and
iii) administrating at least one component selected from the group consisting of
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
to a subject with a cancer overexpressing EHMT2 compared with normal control. Alternatively, the present invention also provides a pharmaceutical composition containing at least one component selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
for use in administrating to a subject having a cancer overexpressing EHMT2. In other words, the present invention further provides a method for identifying a subject to be treated with:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof,
which method may include the step of determining an expression level of EHMT2 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with.
The method of treating a cancer of the present invention will be described in more detail below.
A subject to be treated by the present method may be a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
According to the present invention, the expression level of EHMT2 in cancer cells or tissues obtained from a subject may be determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of EHMT2 may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array. For example, an array may be used for detecting the expression level of EHMT2. Those skilled in the art can prepare such probes utilizing the sequence information of EHMT2. For example, the cDNA of EHMT2 may be used as the probes. If necessary, the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
Furthermore, the transcription product of EHMT2 (e.g., SEQ ID NO: 1 or 3) may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers may be prepared based on the available sequence information of the gene.
In some embodiments, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of EHMT2. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the quantity of observed protein (SEQ ID NO: 2 or 4) may be determined. Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody specifically recognizing the protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment or modified antibody retains the binding ability to the EHMT2 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of EHMT2 gene based on its translation product, the intensity of staining may be measured via immunohistochemical analysis using an antibody against the EHMT2 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of EHMT2 gene.
The expression level of a target gene, e.g., the EHMT2 gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
The control level may be determined at the same time with the cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known. In addition, normal cells obtained from non-cancerous regions of an organ that has the cancer to be treated may be used as normal control. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of EHMT2 gene in samples from subjects whose disease states are known. Furthermore, the control level can be derived from a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of EHMT2 gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples. In some embodiments, a control level is determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample. Moreover, a standard value of the expression levels of EHMT2 gene in a population with a known disease state may be used. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.
In the context of the present invention, a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level".
When the expression level of EHMT2 gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.
Compositions containing a double-stranded molecule of the present invention:
In addition to the above, the present invention also provides pharmaceutical compositions that include the present double-stranded molecule or the vector coding for double-stranded molecules.
In the context of the present invention, the term "composition" is used to refer to a product that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such terms, when used in relation to the modifier "pharmaceutical" (as in "pharmaceutical composition"), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, in the context of the present invention, the term "pharmaceutical composition" refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
The phrase "pharmaceutically acceptable carrier" or "physiologically acceptable carrier", as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
The term "active ingredient" herein refers to a substance in composition that is biologically or physiologically active. Particularly, in the context of pharmaceutical composition, the term "active ingredient" refers to a substance that shows an objective pharmacological effect. For example, in case of pharmaceutical compositions for use in the treatment or prevention of cancer, active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly. Preferably, such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on. Before being formulated, the "active ingredient" may also be referred to as "bulk", "drug substance" or "technical product".
Of particular interest to the present invention are the following compositions [1] to [32]:
[1] A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer and the cancer cell express at least one EHMT2 gene, including an isolated double-stranded molecule that inhibits the expression of EHMT2 and the cell proliferation or a vector encoding the double-stranded molecule, wherein the molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
[2] The composition of [1], wherein the double-stranded molecule inhibits the expression of mRNA which matches a target sequence of SEQ ID NO: 34 or 35;
[3] The composition of [1], wherein the double-stranded molecule, wherein the sense strand contains a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35;
[4] The composition of any one of [1] to [3], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
[5] The composition of any one of [1] to [4], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[6] The composition of [5], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[7] The composition of [6], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[8] The composition of [7], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[9] The composition of [8], wherein the sense strand of the double-stranded molecule hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[10] The composition of any one of [1] to [9], wherein the double-stranded molecule is composed of a single polynucleotide containing the sense strand and the antisense strand linked by an intervening single-strand;
[11] The composition of [10], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand sequence contains a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35, [B] is the intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is the antisense strand contains a sequence complementary to [A];
[12] The composition of any one of [1] to [11], wherein the double-stranded molecule is an RNA;
[13] The composition of any one of [1] to [11], wherein the double-stranded molecule contains both of DNA and RNA;
[14] The composition of [13], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[15] The composition of [14], wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[16] The composition of [13], wherein the double-stranded molecule is a chimera of DNA and RNA;
[17] The composition of [14], wherein a region flanking the 3'-end of the antisense strand, or both of a region flanking the 5'-end of sense strand and a region flanking the 3'-end of antisense strand are composed of RNA;
[18] The composition of [17], wherein the flanking region is composed of 9 to 13 nucleotides;
[19] The composition of any one of [1] to [18], wherein the double-stranded molecule contains one or two 3' overhang(s);
[20] The composition of any one of [1] to [19], wherein the composition includes a transfection-enhancing agent and pharmaceutically acceptable carrier;
[21] The composition of [1], wherein the double-stranded molecule is encoded by a vector and contained in the composition;
[22] The composition of [21], wherein the double-stranded molecule encoded by the vector acts at mRNA which matches a target sequence of SEQ ID NO: 34 or 35;
[23] The composition of [21], wherein the sense strand of the double-stranded molecule encoded by the vector contains the sequence corresponding to a target sequence of SEQ ID NO: 34 or 35;
[24] The composition of any one of [21] to [23], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
[25] The composition of any one of [21] to [24], wherein the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotides;
[26] The composition of [25], wherein the double-stranded molecule encoded by the vector has less than about 75 nucleotide pairs in length;
[27] The composition of [26], wherein the double-stranded molecule encoded by the vector has less than about 50 nucleotide pairs in length;
[28] The composition of [27], wherein the double-stranded molecule encoded by the vector has less than about 25 nucleotide pairs in length;
[29] The composition of [28], wherein the double-stranded molecule encoded by the vector has between about 19 and about 25 nucleotide pairs in length;
[30] The composition of [21], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[31] The composition of [30], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence of SEQ ID NO: 34 or 35, [B] is a intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[32] The composition of any one of [21] to [31], wherein the composition includes a transfection-enhancing agent and pharmaceutically acceptable carrier.
Suitable compositions of the present invention are described in additional detail below.
The double-stranded molecule of the present invention may be formulated as a pharmaceutical composition prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention may be characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical composition" includes formulations for human and veterinary use. Thus, the pharmaceutical compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
In the context of the present invention, suitable pharmaceutical formulations of the present invention include those suitable for oral, rectal, nasal, topical (including buccal, sub-lingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Other formulations include implantable devices and adhesive patches that release a therapeutic agent. When desired, the above-described formulations may be adapted to give sustained release of the active ingredient.
Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
The present pharmaceutical composition contains the double-stranded molecule or vector encoding that of the present invention (e.g., 0.1 to 90% by weight), or a pharmaceutically acceptable salt of the molecule, mixed with a pharmaceutically acceptable carrier medium. Exemplary physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
According to the present invention, the composition may contain multiple types of the double-stranded molecules, each of the molecules may be directed to the same target sequence, or different target sequences of EHMT2. For example, the composition may contain double-stranded molecules directed to EHMT2. Alternatively, for example, the composition may contain double-stranded molecules directed to one, two or more target sequences EHMT2.
Furthermore, the present composition may contain a vector coding for one or plural double-stranded molecules. For example, the vector may encode one, two or several kinds of the present double-stranded molecules. Alternatively, the present composition may contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
Moreover, the present double-stranded molecule may be contained as liposomes in the present composition. See the section entitled "Method of Inhibiting or Reducing Growth of a Cancer Cell or Treating Cancer using a Double-Stranded Molecule of the Present Invention" for details of liposomes.
Pharmaceutical compositions of the present invention can also include conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the present invention can be packaged for use in liquid form, or can be lyophilized.
For solid compositions, conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
For example, a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, or 25-75%, of one or more double-stranded molecule of the invention. A pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, or 1-10% by weight, of one or more double-stranded molecule of the present invention encapsulated in a liposome as described above, and propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.
In addition to the above, the present composition may contain other pharmaceutically active ingredients so long as they do not inhibit the in vivo function of the double-stranded molecules of the present invention. For example, the composition may contain chemotherapeutic agents conventionally used for treating cancers.
The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives. Furthermore, it should be understood that, in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
Alternatively, the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the EHMT2 gene.
In another embodiment, the present invention provides for the use of the double-stranded nucleic acid molecule of the present invention in manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene. For example, the present invention relates to a use of double-stranded nucleic acid molecule inhibiting the expression of an EHMT2 gene in a cell, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and target to a sequence of SEQ ID NO: 34 or 35, for manufacturing a pharmaceutical composition for treating cancer expressing EHMT2 gene.
The present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the EHMT2 gene.
Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of EHMT2 gene in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and target to a sequence of SEQ ID NO: 34 or 35 as active ingredients.
In another embodiment, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of EHMT2 gene in a cell, which over-expresses the gene, which molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NO: 34 or 35.
Method of Inhibiting or Reducing Growth of a Cancer Cell or Treating Cancer using an EHMT2 inhibitor:
In present invention, a EHMT2 inhibitor was tested for their ability to inhibit cell growth. The EHMT2 inhibitor (Fig. 9) effectively reduces growth rate of the several type of cancer cell lines.
Accordingly, the present invention provides methods for inhibiting cancer cell growth by an EHMT2 inhibitor. Such ability of EHMT2 inhibitors to inhibit cell growth of cancerous cell indicates that they can be used for methods for treating cancer such as bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Thus, the present invention provides methods to treat patients with cancer by administering an EHMT2 inhibitor.
Herein, an "EHMT2 inhibitor" refers to a substance that inhibits the function of the EHMT2 polypeptide. In some embodiments, an EHMT2 inhibitor inhibits histone methyltransferase activity of the EHMT2 polypeptide. Such EHMT2 inhibitors include, for example, BIX-01294.
Of particular interest to the present invention are the following methods [1] to [4]:
[1] A method for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses the EHMT2 gene, such method including the step of administering at least one EHMT2 inhibitor to a subject;
[2] The method of [1], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
[3] The method of [1] or [2], wherein the EHMT2 inhibitor is the BIX-01294.
The method of the present invention will be described in more detail below.
The growth of cells expressing an EHMT2 gene may be inhibited by contacting the cells with an EHMT2 inhibitor. The phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule. Cell growth may be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
The growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the EHMT2 gene. Exemplary cells include bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
Thus, patients suffering from or at risk of developing disease related to EHMT2 may be treated with the administration of an EHMT2 inhibitor. For example, patients suffering from cancer may be treated according to the present methods. The type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed. For example, patients treated by the methods of the present invention may be selected by detecting the expression of EHMT2 in a biopsy specimen from the patient by RT-PCR or immunoassay. In some cases, before the treatment of the present invention, the biopsy specimen from the subject may be confirmed for EHMT2 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.
A treatment is deemed "efficacious" if it leads to clinical benefit such as, a decrease in size, prevalence, growth rate, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer as described in " Method of Inhibiting or Reducing Growth of a Cancer Cell or Treating Cancer using a Double-Stranded Molecule of the Present Invention ".
The treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof includes any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effectively treating and/or providing prophylaxis of cancer may decrease mortality and improve the prognosis of individuals having cancer, decrease the levels of tumor markers in the blood, and alleviate detectable symptoms accompanying cancer. For example, reduction or improvement of symptoms constitutes effectively treating and/or prophylaxis may include 10%, 20%, 30% or more reduction, or induction of disease stability.
One skilled in the art can readily determine an effective amount of an EHMT2 inhibitor to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of an EHMT2 inhibitor is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, from about 2 nM to about 50 nM, or from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of an EHMT2 inhibitor can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
The present methods can be used to inhibit the growth or metastasis of cancer expressing EHMT2 gene; for example bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma.
For treating cancer, an EHMT2 inhibitor can also be administered to a subject in combination with a pharmaceutical composition different from the inhibitor compound of the present invention. Alternatively, an EHMT2 inhibitor can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, an EHMT2 inhibitor can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
In the present methods, an EHMT2 inhibitor can be administered to the subject either as a naked, or in conjunction with a delivery reagent.
Suitable delivery reagents for administration in conjunction with the present a inhibitor compound include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. In one aspect of the present invention, administration includes injections or infusions of the inhibitor compound at or near the site of the cancer.
An EHMT2 inhibitor can be administered in a single dose or in multiple doses. Where the administration of an EHMT2 inhibitor is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. In one embodiment, the inhibitor compound is directly injected into the tissue is at or near the site of cancer. In another embodiment, the inhibitor compound is direction injected into the tissue at or near the site of cancer multiple times.
One skilled in the art can also readily determine an appropriate dosage regimen for administering inhibitor compound of the present invention to a given subject. For example, the inhibitor compound can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, an EHMT2 inhibitor can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, or from about seven to about ten days. In one dosage regimen of the present invention, the inhibitor compound is injected at or near the site of cancer once a day for seven days. Where a dosage regimen requires multiple administrations, it is understood that the effective amount of a inhibitor compound administered to the subject can include the total amount of a inhibitor compound administered over the entire dosage regimen.
In the present invention, a cancer overexpressing EHMT2 can be treated with EHMT2 inhibitor.
The cancers to be treated by the present method include, but are not limited to, bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Accordingly, prior to the administration of an EHMT2 inhibitor as active ingredient, it is one aspect of the present invention to confirm whether the expression level of EHMT2 in the cancer cells or tissues to be treated is elevated as compared with normal cells of the same organ. Thus, in one embodiment, the present invention provides a method for treating a cancer (over)expressing EHMT2, such method including the steps of:
i) determining the expression level of EHMT2 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated;
ii) comparing the expression level of EHMT2 with normal control; and
iii) administrating an EHMT2 inhibitor,
to a subject with a cancer overexpressing EHMT2 compared with normal control. Alternatively, the present invention also provides a pharmaceutical composition containing an EHMT2 inhibitor, for use in administrating to a subject having a cancer overexpressing EHMT2. In other words, the present invention further provides a method for identifying a subject to be treated with an EHMT2 inhibitor, which method may include the step of determining an expression level of EHMT2 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with.
Compositions containing an EHMT2 inhibitor:
In addition to the above, the present invention also provides a pharmaceutical composition that includes an EHMT2 inhibitor. Of particular interest to the present invention are the following compositions [1] to [4]:
[1] A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer or the cancer cell express the EHMT2 gene, including an EHMT2 inhibitor and a pharmaceutically acceptable carrier;
[2] The composition of [1], wherein the EHMT2 inhibitor is BIX-01294.;
[3] The composition of [1] or [2], wherein the cancer to be treated is bladder cancer, lung cancer, AML, CML, esophageal cancer, breast cancer, cervical cancer or osteosarcoma;
Suitable compositions of the present invention are described in additional detail below.
An EHMT2 inhibitor may be formulated as a pharmaceutical composition prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention may be characterized as being at least sterile and pyrogen-free. Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
The present pharmaceutical composition may contain an EHMT2 inhibitor (e.g., 0.1 to 90% by weight), or a pharmaceutically acceptable salt of the molecule, mixed with a pharmaceutically acceptable carrier medium. Exemplary physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
According to the present invention, the composition may contain plural kinds of EHMT2 inhibitors, each of the molecules may be directed by the same mechanism, or different mechanism.
Pharmaceutical compositions of the present invention can also include conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the present invention can be packaged for use in liquid form, or can be lyophilized.
For solid compositions, conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
For example, a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, preferably 25-75%, of one or more EHMT2 inhibitors. A pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, preferably 1-10% by weight, of one or more EHMT2 inhibitors encapsulated in a liposome, and propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.
In addition to the above, the present composition may contain other pharmaceutically active ingredients so long as they do not inhibit the in vivo function of the histone methyltransferase inhibitor of the present invention. For example, the composition may contain chemotherapeutic agents conventionally used for treating cancers.
In another embodiment, the present invention provides the use of an EHMT2 inhibitor in manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene.
The present invention further provides an EHMT2 inhibitor for use in treating a cancer expressing the EHMT2 gene.
Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with an EHMT2 inhibitor.
In another embodiment, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of EHMT2 gene, wherein the method or process includes a step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is an EHMT2 inhibitor.
Hereinafter, the present invention is described in more detail with reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: General Methods
Tissue samples and RNA preparation
Bladder tissue samples and RNA preparation were described previously (Wallard MJ et al. Br J Cancer 2006;94:569-77.). Briefly, 126 surgical specimens of primary urothelial carcinoma were collected, either at cystectomy or transurethral resection of bladder tumor (TUR-Bt), and snap-frozen in liquid nitrogen. 26 normal bladder tissues were collected from areas of macroscopically-normal regions in patients with no evidence of malignancy. Five sequential sections of 7 micron thickness were cut from each tissue and stained using Histogene(trademark) staining solution (Arcturus, California, USA) following the manufacturer's protocol, and assessed for cellularity and tumor grade by an independent consultant urohistopathologist. Approximately 10,000 cells were microdissected from both stromal and epithelial/tumor compartments in each tissue. To validate the accuracy of microdissection, primers and probes for Vimentin and Uroplakin were sourced and qRT-PCR performed according to the manufacturer's instructions (Assays on demand, Applied Biosystems, Warrington, UK). Vimentin is primarily expressed in messenchymally derived cells, and was used as a stromal marker. Uroplakin is a marker of urothelial differentiation and is preserved in up to 90% of epithelially derived tumors (Olsburgh J et al. The Journal of pathology 2003;199:41-9.). Use of tissues for this study was approved by Cambridge shire Local Research Ethics Committee (Ref 03/018).
Cell culture
Cancer cell lines used in this study were as follows: lung adenocarcinoma (ADC) LC319 and A549; lung squamous cell carcinoma (SCC) RERF-LC-AI; small cell lung cancer (SCLC) SBC-5; bladder cancer 5637, 253J, 253JBV, EJ28, HT1197, HT1376, J82, RT4, SCaBER, SW780, T24 and UMUC3. The Normal human lung fibroblast HFL-1 and the normal human colon fibroblast CCD-18Co were used as normal control cells. All cell lines were grown in monolayers in appropriate media: Dulbecco's modified Eagle's medium (D-MEM) for EJ28, RERF-LC-AI and 293T cells; Eagle's minimal essential medium (E-MEM) for CCD-18Co, 253J, 253J-BV, HT1197, HT1376, J82, SCaBER, UMUC3 and SBC5 cells; F-12K medium for HFL-1 cells; Leibovitz's L-15 for SW780 cells; McCoy's 5A medium for RT4 and T24 cells; RPMI1640 medium for 5637, A549 and LC319 cells supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma). All cells were maintained at 37 degrees C in humid air with 5% CO2 condition (CCD-18Co, HFL-1, 5637, 253J, 253J-BV, EJ28, HT1197, HT1376, J82, RT4, SCaBER, T24, UMUC3, A549, LC319, RERF-LC-AI, SBC5 and 293T) or without CO2 (SW780). Cells were transfected with FuGENE6TM (Roche Applied Science, Basel, Switzerland) according to the manufacturer's protocols.
Expression profiling in cancers using cDNA microarrays
The inventors established a genome-wide cDNA microarray with 36,864 cDNAs or ESTs selected from the UniGene database of the National Center for Biotechnology Information (NCBI). This microarray system was constructed essentially as described previously ( Kikuchi T et al. Oncogene 2003;22:2192-205., Kitahara O et al. Cancer Res 2001;61:3544-9., Nakamura T et al. Oncogene 2004;23:2385-400.). Briefly, the cDNAs were amplified by RT-PCR using poly (A)+ RNAs isolated from various human organs as templates; the lengths of the amplicons ranged from 200 to 1,100 bp, without any repetitive or poly (A) sequences. Many types of tumors and corresponding non-neoplastic tissues were prepared in 8-micrometer sections, as described previously ( Kitahara O et al. Cancer Res 2001;61:3544-9.). A total of 30,000-40,000 cancer or noncancerous cells were collected selectively using the EZ cut system (SL Microtest GmbH, Germany) according to the manufacturer's protocol. Extraction of total RNA, T7-based amplification, and labeling of probes were performed as described previously ( Kitahara O et al. Cancer Res 2001;61:3544-9.). 2.5-micro-g aliquots of twice-amplified RNA (aRNA) from each cancerous and noncancerous tissue were then labeled, respectively, with Cy3-dCTP or Cy5-dCTP. Detailed expression profiling data of bladder and lung cancers, shown in this study, were based on the data reported previously by Drs. Ryo Takata and Takefumi Kikuchi, respectively (Kikuchi T et al. Oncogene 2003;22:2192-205., Takata R, et al. Clin Cancer Res 2005;11:2625-36.).
Immunohistochemical Staining and Tissue Microarray
Immunohistochemical analysis was performed using a specific polyclonal rabbit-EHMT2 antibody as described previously (Cho HS, , et al. (2011). Cancer Res 71, 1-6., Unoki M, et al. (2009). Br J Cancer 101, 98-105.). For clinical bladder tissue microarray, VECTASTAINTM ABC KIT (Vector Laboratories, Burlingame, CA) was applied. Briefly, endogenous peroxidase activity of xylene-deparaffinized and dehydrated sections was inhibited by treatment with 0.3% H2O2/methanol. Nonspecific binding was blocked by incubating sections with 3% BSA in a humidified chamber for 30 min at ambient temperature, then a 1:1000 dilution of rabbit polyclonal anti-EHMT2 antibody (NB100-40825, Novus Biologicals) overnight at 4 degrees C. Sections were washed twice with PBS, incubated with 1 micro-g/micro-L goat anti-rabbit biotinylated IgG in PBS containing 1% BSA for 30 min at ambient temperature, and then incubated with ABC reagent for 30 min. Immunostaining was visualized using 3,3'-diaminobenzidine. Slides were dehydrated through graded alcohol to sylene washing and mounted on cover slips. Hematoxylin was used for nuclear counterstaining. For clinical lung cancer tissue microarray, EnVision+ kit/horseradish peroxidase (Dako, Glostrup, Denmark) was applied. Briefly, slides of paraffin-embedded lung tumor specimens were processed under high pressure (125 degrees C, 30 s) in antigen-retrieval solution, high pH 9 (S2367, Dako Cytomation, Carpinteria, CA, USA), treated with peroxidase blocking regent, and then treated with protein blocking regent (K130, X0909, Dako Cytomation). Tissue sections were incubated with a rabbit anti-EHMT2 polyclonal antibody followed by HRP-conjugated secondary antibody (Dako Cytomation). Antigen was visualized with substrate chromogen (Dako liquid DAB chromogen; Dako Cytomation). Finally, tissue specimens were stained with Mayer's haematoxylin (Muto pure chemicals Ltd, Tokyo, Japan) for 20 s to discriminate the nucleus from the cytoplasm.
Quantitative real-time PCR.
As described above, the inventors obtained 126 bladder cancer tissues and 26 normal bladder tissues in Cambridge Addenbrooke's Hospital. For quantitative RT-PCR reactions, specific primers for all human GAPDH (housekeeping gene), SDH (housekeeping gene) and EHMT2 were designed (primer sequences in Table 1). PCR reactions were performed using the ABI prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK) following the manufacture's protocol. 50% SYBR GREEN universal PCR Master Mix without UNG (Applied Biosystems, Warrington, UK), 50 nM each of the forward and reverse primers and 2 microliter of reversely-transcribed cDNA were applied. Amplification conditions were 5 min at 95 degrees C and then 45 cycles each consisting of 10 sec at 95 degrees C, 1 min at 55 degrees C and 10 sec at 72 degrees C. Then, reactions were heated for 15 sec at 95 degrees C, 1 min at 65 degrees C to draw the melting curve, and cooled to 50 degrees C for 10 sec. Reaction conditions for target gene amplification were as described above and the equivalent of 5 ng of reverse transcribed RNA was used in each reaction. mRNA levels were normalized to GAPDH and SDH expression.
Figure JPOXMLDOC01-appb-T000001
siRNA Transfection
siRNA oligonucleotide duplexes were purchased from SIGMA Genosys for targeting the human EHMT2 transcript or the EGFP and FFLuc transcripts as control siRNAs. siRNA sequences are described in Table 2. siRNA duplexes (100 nM final concentration) were transfected in lung cancer cell lines with lipofectamine 2000 (Invitrogen) for 72 h, and cell viability was examined using Cell Counting Kit 8 (DOJINDO).
Figure JPOXMLDOC01-appb-T000002
Flow cytometry assays (FACS) for cell cycle analysis
The present inventors collected the cells after trypsin treatment, washed them twice with 1,000 micro-L of Assay Buffer and centrifuged for 5 min at 5,000 rpm. Cells were resuspended in 200 micro-L of Assay Buffer. 1,000 micro-L of fixative buffer was added, and the samples incubated at room temperature for 1 h. Finally, the present inventors added the propidium iodide reagent and analyzed cell cycle profiles by flow cytometry (LSR II, BD Biosciences). The proportion of each cell division was calculated and analyzed using Student's T test for significance.
Chromatin immunoprecipitation assay (ChIP)
ChIP assays were performed using ChIP Assay kit (Millipore, Billerica, MA) according to the manufacture's protocol. Briefly, the fragment of EHMT2 (SEQ ID NO: 9) and chromatin complexes was immnoprecipitated with anti-FLAG antibody 48 h after transfection with pCAGGS-n3FC (Mock) and pCAGGS-n3FC-EHMT2 (3xFLAG-EHMT2) vectors. After the bound DNA fragments to EHMT2 were eluted, and the amount was subjected to quantitative real-time PCR reactions. Primer sequences are shown in Table 1.
Microarray hybridization and statistical analysis for the clarification of down-stream genes
Microarray analysis to identify down-stream genes were done described previously ( Hayami S, et al. Int J Cancer 2010., Hayami S, et al. Mol Cancer 2010;9:59., Yoshimatsu M, et al. Int J Cancer 2010;in press.). Briefly, purified total RNA was labeled and hybridized onto Affymetrix GeneChip U133 Plus 2.0 oligonucleotide arrays (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. A pathway analysis was performed using the hyper-geometric distribution test, which calculates the probability of overlap between the up/down-regulated gene set and each GO category compared against another gene list that is randomly sampled. The present inventors applied the test to the identified up/down-regulated genes to test whether or not they are significantly enriched (FDR<=0.05) in each category of "Biological processes" (857 categories) as defined by the Gene Ontology database.
Coupled cell cycle and cell proliferation assay
A 5'-bromo-2'-deoxyuridine (BrdU) flow kit (BD Pharmingen, San Diego, CA) was used to determine the cell cycle kinetics and to measure the incorporation of BrdU into DNA of proliferating cells. The assay was performed according to the manufacturer's protocol. Briefly, SBC5 cells (2 x 105 per well) were seeded overnight in 6-well tissue culture plates and treated with an optimized concentration of siRNAs in medium containing 10% FBS for 72 hours, followed by addition of 10 micromolar BrdU, and incubations continued for an additional 30 min. Both floating and adherent cells were pooled from triplicates wells per treatment point, fixed in a solution containing paraformaldehyde and the detergent saponin, and incubated for 1 hour with DNAase at 37 degrees C (30 micro g per sample). FITC-conjugated anti-BrdU antibody (1:50 dilution in Wash buffer; BD Pharmingen, San Diego, CA) was added and incubation continued for 20 minutes at room temperature. Cells were washed in Wash buffer and total DNA was stained with 7-amino-actinomycin D (7-AAD; 20 microliter per sample), followed by flow cytometric analysis using FACScan (BECKMAN COULTER) and total DNA content (7-AAD) was determined CXP Analysis Software Ver. 2.2 (BECKMAN COULTER).
Example 2: EHMT2 expression is up-regulated in clinical cancer tissues
In order to identify the histone methyltransferase involved in human carcinogenesis, expression profiles were examined for several histone methyltransferase genes using a small number of clinical bladder samples, and a significant difference of EHMT2 gene expression was found between cancer and normal tissues (data not shown). Consequently, the present inventors analyzed 126 bladder cancer samples and 26 normal control samples (British), and found significant elevation of EHMT2 expression in tumor cells compared with in normal cells (P < 0.0001, Mann-Whitney U test) (Fig. 1A, Table 1). No significant difference was observed in expression levels among cancer samples of different stages and grades (Fig. 1B, Table 3). This shows that EHMT2 expression is up-regulated at an early stage in bladder carcinogenesis, and remains high in the advanced stages of the disease. Subclassification of tumors according to metastasis status, gender, smoking history and recurrence status identified no significant differences of EHMT2 expression levels (Table 3). The present inventors then analyzed the expression patterns of EHMT2 in a number of Japanese clinical bladder cancer samples by cDNA microarray (Fig. 1C, Table 4), and confirmed significant overexpression in bladder cancers of Japanese patients (P < 0.0001, Mann-Whitney U-test). Consisting with this, expression levels of EHMT2 in bladder cancer cell lines were significantly higher than those in two normal human fibroblast cell lines (Fig. 6). To evaluate protein expression levels of EHMT2 in clinical tissues, immunohistochemical analysis using was performed anti-EHMT2 specific antibody. This antibody strongly stained the nucleus of various types of bladder cancer tissues, while signals in normal bladder tissue were weak (Fig. 1E). In addition to bladder tissues, expression levels of EHMT2 in lung tissues were measured. cDNA microarray experiments showed that EHMT2 expression was also highly elevated in lung tumor tissues compared with corresponding non-neoplastic tissues (Fig. 2A, Table 4), and the over expression of EHMT2 in lung cancer was also validated by quantitative real-time PCR (Fig. 11A). In addition, expression levels of EHMT2 in four lung cancer cell lines were significantly higher than in two normal human fibroblast cell lines (Fig. 11B). The present inventors then examined EHMT2 protein expression levels in lung tissue by immunohistochemistry (Fig. 2B) and observed strong EHMT2 staining in the nucleus of cancer tissues and weak staining in non-neoplastic tissues. Additionally, the present inventors examined microarray expression analysis of a large number of clinical samples derived from Japanese subjects and found that EHMT2 expression was also significantly up-regulated in various types of cancer compared with corresponding non-neoplastic tissues (Table 4; Fig. 5). These data indicate that EHMT2 is involved in many types of human cancer.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Example 3: Growth regulation of cancer cells by EHMT2
To investigate the role of EHMT2 in human carcinogenesis, the present inventors performed knockdown experiments using siRNAs against EHMT2 (siEHMT2#1 and #2) and two control siRNAs (siEGFP and siNC) (Table 2). The present inventors transfected these siRNAs into A549 and SBC5 cells, in which EHMT2 was highly expressed (Fig.3A; Fig. 6). EHMT2 expression in the cells transfected with two independent EHMT2 siRNAs was significantly suppressed in comparison with those transfected with control siRNAs at the mRNA and protein levels (Fig. 3B; Fig.7). Using the siRNAs, colony formation assay and cell growth assay was performed. The present inventors observed inhibition of colony formation (Fig. 3C) and significant growth suppression of two bladder cancer cell lines (SW780 and RT4) and three lung cancer cell lines (LC319, A549 and SBC5) after treatment with two EHMT2 siRNAs though no effect was observed for control siRNAs (Fig.3D). To further assess the mechanism of growth suppression induced by the siRNA, the cell cycle status of cancer cells were analyzed after treatment with siRNAs using flow cytometry stained with a FITC-conjugated anti-BrdU antibody and 7-AAD. The proportion of cancer cells in the S phase decreased and that in the sub-G1 phase increased in a significant manner after treatment with siEHMT2 (Fig. 3E). The result was confirmed by PI staining and FACS analysis (Fig. 8). These results suggest that EHMT2 plays a crucial role in G1/S transition. It also suggests that apoptosis can be induced after EHMT2 knockdown.
Example 4: SIAH1 directly regulated by EHMT2 is a key regulation of cancer cell growth and apoptosis
To define the mechanism by which EHMT2 regulates the cell cycle and apoptosis, the present inventors identified target genes regulated by EHMT2 using microarray expression analysis. In order to clarify early responding genes after knockdown of EHMT2, the inventors isolated total RNA from SW780 and A549 cells 24 h after treatment with siEHMT2. The expression profile of these cells was analyzed by Affymetrix's HG-U133 Plus 2.0 Array in comparison with those treated with control siRNAs (siEGFP and siFFLuc), and the inventors identified a set of genes that were significantly up/down-regulated. The sub-G1 population of cancer cells was significantly increased by knockdown of EHMT2, according to FACS analysis (Figure 3E), illustrating that EHMT2 is associated with regulation of apoptosis in cancer cells. The present inventors identified a down-stream gene for EHMT2 which was known to be involved in apoptotic regulation in cancer cells, by microarray analysis. Among candidate genes, the present inventors observed significant up-regulation of SIAH1, a tumor suppressor gene, after treatment with siEHMT2 (Fig. 4A, Fig. 4B). Quantitative real-time PCR and Western blot also confirmed the up-regulation of SIAH1 after treatment with siEHMT2 (Fig. 4C). To evaluate the possibility that EHMT2 transcriptionally regulates the SIAH1 expression, a chromatin immunoprecipitation (ChIP) assay was performed. EHMT2 protein was highly enriched at the promoter region of SIAH1 after transfection with the 3xFLAG-EHMT2 vector together with increased levels of di-methylation on histone H3K9 (Fig. 4D). These results show that EHMT2 can directly suppress SIAH1 expression at the transcriptional level through the enhancement of histone H3K9 methylation status. Additionally, to validate the function of endogenous EHMT2 protein in cancer cells, the present inventors performed ChIP analysis of cells after treatment 12 with EHMT2 siRNA, using anti-EHMT2 and -H3K9me2 antibodies. The data showed that the siRNA treatment clearly diminished the binding of endogenous EHMT2 to the promoter region of SIAH1, and reduced H3K9 di-methylation in the region (Figure 12). Therefore, endogenous EHMT2 protein can bind to the promoter region of SIAH1 and, through di-methylation of histone H3-K9, affects regulation of gene expression.
The present inventors then tried to clarify the significance of SIAH1 suppressed by EHMT2 in cancer cells. Since knockdown of EHMT2 significantly increased the sub-G1 population of cancer cells, the present inventors performed detailed apoptosis analysis using the SIAH1 siRNA whose effects were already validated. Cleaved PARP1 and caspase 3 were observed in SBC5 cells after treatment with siEHMT2, demonstrating that apoptosis may be induced by knockdown of EHMT2. Subsequently, the present inventors examined effects of SIAH1 knockdown on EHMT2 siRNA-induced apoptosis. Importantly, the cleavage of PARP1 and caspase 3 was not observed in SBC5 cells treated with both EHMT2 and SIAH1 siRNAs (Figure 13A). The data reveal that SIAH1 is an essential factor for EHMT2 siRNA-induced apoptosis. Furthermore, the present inventors conducted growth assay after treatment with siEHMT2 and either siEGFP or siSIAH1. The growth of SBC5 cells was significantly suppressed treatment with siEHMT2, but the growth suppression was recovered by knockdown of SIAH1 (Figure 13C). This result was confirmed by a colony formation assay (Figure 5B). Taken together, those findings show that SIAH is directly regulated by EHMT2, and it plays a key role in regulating growth and apoptosis of cancer cells overexpressing EHMT2.
Example 5: BIX-01294 reduced the growth rate of several cancer cell lines
A small molecule compound, BIX-01294 (a diazepin-quinazolin-amine derivative), specifically inhibits EHMT2 enzymatic activity and reduces H3K9me2 levels at the chromatin regions of several EHMT2 target genes ( Chang Y, et al. Nat Struct Mol Biol 2009;16:312-7., Kubicek S, et al. Mol Cell 2007;25:473-81.). Since EHMT2 is involved in the proliferation of cancer cells, the inventors evaluated effects of BIX-01294 on the growth of cancer cell lines. To examine the relationship between EHMT2 expression and BIX-01294 effects, the inventors chose cancer cells that showed a wide variety of EHMT2 expression levels (Fig. 9A). As shown in Fig. 9B, the growth of cancer cells was significantly suppressed by BIX-01294 treatment in a dose dependent manner, and the effect was correlated with EHMT2 expression levels. The cell cycle status of SBC-5 cells after treatment with BIX-01294 was examined, and the proportion of cells in S phase significantly decreased and that in sub-G1 phase increased in a dose dependent manner (Fig. 9C). These results indicate that the enzymatic activity of EHMT2 can be closely related to the growth of cancer cells, and that inhibition of EHMT2 may induce growth suppression and apoptosis of cancer cells.
Discussion
Histone modifications, including methylation, acetylation, phosphorylation and ubiquitination, are considered to play critical roles in transcriptional activation and repression through the regulation of chromatin structure. Histone methylation was once thought to be a stable modification, but is more recently recognized as being dynamically regulated by both histone methyltransferases and demethylases. EHMT2 is mainly responsible for mono-methylation and di-methylation of H3K9 in euchromatin, and these play a unique role in transcriptional regulation and chromatin remodeling (12-14, 35, 36). In this study, the present inventors demonstrated the significant up-regulation of EHMT2 in bladder and lung cancers by quantitative RT-PCR and immunohistochemistry at RNA and protein levels. Together with microarray-based expression profiles of a large number of clinical tissues, EHMT2 expression is shown to be dysregulated in a great majority of human tumors (Table 4). The inventors demonstrate that EHMT2 may serve an important role in the growth regulation of cancer cells and confirmed that knockdown of EHMT2 suppresses the growth of various bladder and lung cancer cells, with the number of cells in the sub-G1 phase increasing (Fig. 3D and E). The pathway analysis using the cells in which EHMT2 expression was knocked down by siRNA, shows that EHMT2 is involved in regulation of cell apoptosis and a variety of chromatin functions such as chromatin remodeling and transcriptional regulation (Fig. 14).
It has been suggested that SIAH1 is a tumor suppressor gene located in chromosomal band 16q12-q13, a frequently deleted region in human tumors arising from various tissues ( Medhioub M et al. Int J Cancer 2000;87:794-7., Okabe H et al. Hepatology 2000;31:1073-9.). It was also reported that E3 ubiquitin ligases, including SIAH1, played an important role in regulating breast carcinogenesis ( Chen C et al. Mol Cancer Res 2006;4:695-707.), and a recent study indicated that SIAH1 induces apoptosis by activation of the JNK pathway and inhibits invasion by inactivation of the ERK pathway in breast cancer cells ( Wen YY et al. Cancer Sci;101:73-9.). The present inventors previously reported that the paternally expressed gene 10 (PEG10), which was highly expressed in hepatocellular carcinomas (HCCs), associated with SIAH1, and PEG10 overexpression decreased the cell death mediated by SIAH1 ( Okabe H, et al., (2003). Cancer Res 63, 3043-3048.). Moreover, the expression profile data show that expression levels of SIAH in tumor tissues are significantly low compared with corresponding non-neoplastic tissues in various types of cancer, including bladder and lung cancers (Table 5). These data reveal that SIAH1 is one of the key regulators in human carcinogenesis. The microarray data of the present inventors showed that SIAH1 was up-regulated by EHMT2 knockdown, and the elevation was confirmed using quantitative real-time PCR and western blot analyses (Fig. 4C). Additionally, the inventors found that EHMT2 directly binds to the promoter region of SIAH1 and regulated the transcription of SIAH1 through the histone methylation analyzed by ChIP assay (Fig. 4D, Fig. 12). Consistently, the apoptosis induction and growth suppression after treatment with EHMT2 siRNA was recovered by SIAH1 knockdown (Fig. 13). According to the series of experiments, EHMT2 directly regulates SIAH1 expression through methylation of histone H3-K9. This negative regulation of SIAH1 expression in cancer cells may be a mechanism how EHMT2 contributes to human carcinogenesis.
Figure JPOXMLDOC01-appb-T000005
In the present study, it was found that EHMT2 was overexpressed in various types of cancer, including bladder and lung cancers, and play a crucial role in the proliferation of cancer cells. Importantly, the BioGPS database revealed that expression of EHMT2 in many types of normal tissues is very low (Fig. 10), indicating that EHMT2 is a good target for cancer therapy. Indeed, the inventors evaluated effects of BIX-01294, a highly specific inhibitor of EHMT2 (Kubicek S et al. Mol Cell. 2007 Feb 9;25(3):473-81), treatment and found that this chemical compound effectively suppressed the growth of cancer cells (Fig. 9). This result shows that EHMT2 inhibitors can work as anti-cancer drugs. Further validation with functional analyses of this protein in the context of human carcinogenesis and optimization of EHMT2 inhibitors as anti-cancer drugs may assist to development of novel therapeutic strategies for human cancer.
The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
In particular, the gene-expression analysis of cancers described herein using the combination of laser-capture dissection and genome-wide cDNA microarray identify EHMT2 as a gene that is markedly elevated in cancer as compared to normal organs. As such, it finds utility in the context of cancer prevention and therapy. For example, given its differential expression, EHMT2 can be conveniently used as a molecular diagnostic marker for identifying and detecting cancer, in particular, bladder cancer, lung cancer (SCC, ADC, ACC, SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma. Accordingly, the EHMT2 gene and the proteins encoded thereby find utility in diagnostic kits and assays of cancer.
The present invention further demonstrates that the cell growth may be suppressed by a double-stranded nucleic acid molecule that specifically targets the EHMT2 gene. Thus, the double-stranded nucleic acid molecule is useful for the development of anti-cancer pharmaceuticals. Furthermore, EHMT2 polypeptide is a useful target for the development of anti-cancer pharmaceuticals. For example, substances that block the expression of EHMT2 protein or prevent its activity may find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of bladder cancer, lung cancer (SCC, ADC, ACC, SCLC), AML, CML, esophageal cancer, breast cancer, cervical cancer and osteosarcoma.
All publications, databases, sequences, patents, and patent applications cited herein are herby incorporated by reference.
While the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims (27)

  1. A method of detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of an EHMT2 gene in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer, wherein the expression level is determined by any one of method selected from the group consisting of:
    (a) detecting an mRNA of an EHMT2 gene;
    (b) detecting a protein encoded by an EHMT2 gene; and
    (c) detecting a biological activity of a protein encoded by an EHMT2 gene.
  2. The method of claim 1, wherein said increase is at least 10% greater than said normal control level.
  3. The method of claim 1, wherein the subject-derived biological sample is a biopsy specimen.
  4. A kit for diagnosing cancer, which comprises a reagent selected from the group consisting of:
    (a) a reagent for detecting an mRNA of an EHMT2 gene;
    (b) a reagent for detecting a protein encoded by an EHMT2 gene; and
    (c) a reagent for detecting a biological activity of a protein encoded by an EHMT2 gene.
  5. The kit of claim 4, wherein the reagent is a probe or a primer set that binds to the mRNA of the EHMT2 gene, or an antibody against the protein encoded by the EHMT2 gene.
  6. A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, said method comprising the steps of:
    (a) contacting a test substance with an EHMT2 polypeptide or a fragment thereof;
    (b) detecting the binding activity between the polypeptide or the fragment and the test substance; and
    (c) selecting the test substance that binds to the polypeptide or the fragment as a candidate substance for treating and/or preventing cancer.
  7. A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, said method comprising the steps of:
    (a) contacting a test substance with a cell expressing an EHMT2 gene;
    (b) detecting an expression level of the EHMT2 gene in the cell of the step (a); and
    (c) selecting the test substance that reduces the expression level detected in the step (b) in comparison with the expression level of an EHMT2 gene detected in the absence of the test substance.
  8. A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, said method comprising the steps of:
    (a) contacting a test substance with an EHMT2 polypeptide or a fragment thereof;
    (b) detecting a biological activity of the polypeptide or the fragment of the step (a); and
    (c) selecting the test substance that suppresses the biological activity of the polypeptide or the fragment detected in the step (b) in comparison with the biological activity detected in the absence of the test substance.
  9. The method of claim 8, wherein the biological activity is a cell proliferative activity or histone methyltransferase activity.
  10. A method of screening for a candidate substance for treating and/or preventing a cancer, or inhibiting a cancer cell growth, said method comprising the steps of:
    (a) contacting a test substance with a cell into which a vector comprising the transcriptional regulatory region of EHMT2 genes and a reporter gene that is expressed under the control of the transcriptional regulatory region has been introduced,
    (b) measuring the expression or activity of said reporter gene in the step (a); and
    (c) selecting a substance that reduces the expression and/or activity levels of said reporter gene detected in the step (b) in comparison with the expression and/or activity levels in the absence of the test substance.
  11. A method of screening for a candidate substance that inhibits binding between a EHMT2 polypeptide and a SIAH1 promoter region, said method comprising the steps of:
    (a) contacting an EHMT2 polypeptide or functional equivalent thereof with a polynucleotide corresponding to an SIAH1 promoter region in the presence of a test substance;
    (b) detecting binding between the polypeptide and the polynucleotide;
    (c) comparing the binding level detected in the step (b) with the level detected in the absence of the test substance; and
    (d) selecting the test substance that reduces or inhibits the binding level in comparison with the level detected in the absence of the test substance.
  12. An isolated double-stranded molecule comprising a sense strand and an antisense strand complementary thereto, wherein the strands hybridize to each other to form the double-stranded molecule, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 34 and 35, and wherein said double-stranded molecule, when introduced into a cell expressing an EHMT2 gene, inhibits expression of said gene as well as cell proliferation.
  13. The double-stranded molecule of claim 12, wherein the sense strand hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pairs in length.
  14. The double-stranded molecule of claim 12 or 13, wherein the double-stranded molecule has one or two 3' overhangs at the 3' ends of the sense strand and/or the antisense strand.
  15. The double-stranded molecule of any one of claims 12 to 14, wherein said double-stranded molecule is a single polynucleotide comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  16. The double-stranded molecule of claim 15, wherein said polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 34 and 35; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is an antisense strand comprising a complementary sequence to the target sequence of [A].
  17. A vector encoding the double-stranded molecule of any one of claims 12 to 16.
  18. A method of treating and/or preventing a cancer in a subject, said method comprising the step of administering to said subject a pharmaceutically effective amount of a double-stranded molecule against an EHMT2 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule inhibits expression of an EHMT2 gene and cell proliferation when introduced into a cell expressing the EHMT2 gene.
  19. The method of claim 18, wherein the double-stranded molecule is that of any one of claims 12 to 16.
  20. The method of claim 18, wherein the vector is that of claim 17.
  21. A composition for treating and/or preventing a cancer, said composition comprising a pharmaceutically effective amount of a double-stranded molecule against an EHMT2 gene or a vector encoding the double-stranded molecule, and a pharmaceutically acceptable carrier, wherein the double-stranded molecule inhibits expression of an EHMT2 gene and cell proliferation when introduced into a cell expressing the EHMT2 gene.
  22. The composition of claim 21, wherein the double-stranded molecule is that of any one of claims 12 to 16.
  23. The composition of claim 22, wherein the vector is that of claim 17.
  24. A method for inhibiting a growth of a cancer cell or treating a cancer, wherein the cancer cell or the cancer expresses the EHMT2 gene, said method comprising the step of administering at least one EHMT2 inhibitor to a subject.
  25. The method of claim 24, wherein the EHMT2 inhibitor is BIX-01294.
  26. A composition for inhibiting growth of a cancer cell or treating a cancer, wherein the cancer cell or the cancer express the EHMT2 gene, comprising at least one EHMT2 inhibitor;
  27. The composition of claim 26, wherein the EHMT2 inhibitor is BIX-01294.
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