WO2011096196A1

WO2011096196A1 - Lsd1 for target genes of cancer therapy and diagnosis

Info

Publication number: WO2011096196A1
Application number: PCT/JP2011/000551
Authority: WO
Inventors: Ryuji Hamamoto; Yusuke Nakamura; Takuya Tsunoda
Original assignee: Oncotherapy Science, Inc.
Priority date: 2010-02-02
Filing date: 2011-02-01
Publication date: 2011-08-11

Abstract

The present invention relates to the roles played by a LSD1 gene in carcinogenesis and features a method for treating or preventing cancer by administering a double-stranded molecule against the LSD1 gene or a composition, vector or cell containing such a double- stranded molecule. The present invention also features methods and kits for detecting or diagnosing cancer in a subject, using the over-expressed LSD1 gene. Also, disclosed are methods of screening for candidate substances for treating and preventing cancer or inhibiting cancer cell growth, using as an index their effect on the expression or activity of LSD1.

Description

LSD1 FOR TARGET GENES OF CANCER THERAPY AND DIAGNOSIS

The present invention relates to the field of biological science, more specifically to the field of cancer research, cancer diagnosis and cancer therapy. In particular, the present invention relates to methods for detecting and diagnosing cancer, particularly bladder cancer, colorectal cancer and lung cancer as well as methods for treating and preventing of a subject with cancer, particularly bladder cancer, colorectal cancer and lung cancer. Moreover, the present invention relates to methods for screening a candidate substance for treating and/or preventing cancer, particularly bladder cancer, colorectal cancer and lung cancer.
Priority
The present application claims priority to US Serial No. 61/300,683, filed February 2, 2010, and US Serial No. 61/363,887, filed July 13, 2010, the disclosures of which are incorporated herein by reference in their entireties.

Histone methylation plays important dynamic roles in regulating chromatin structure. Precise conformational regulation of chromatins is crucial for normal cellular processes such as DNA replication, DNA repair, chromosome recombination and mRNA transcription. Although histone methylation was considered to be a static modification until recently, the discovery of lysine-specific demethylase 1 (LSD1), which specifically demethylates mono- and di-methylated histone H3 at lysine 4 (H3-K4), indicated the histone methylation was reversible (NPL1). Subsequently, a JmjC domain-containing protein was identified to possess histone demethylase activity, and the JmjC domain was shown to be a demethylase signature motif (NPL2). JmjC-domain-containing enzymes catalyze the removal of methyl groups using a hydroxylation reaction, requiring iron and alpha-ketoglutarate cofactors. Several additional proteins were identified as histone lysine demethylases on the basis of the presence of the JmjC motif (NPL3-9). Although information of histone demethylases in their physiological function has been accumulated, their involvement in human disease remains unclear.

LSD1, also known as AOF2, is a histone demethylase that does not belong to the JmjC family, that catalyzes the demethylation of histone H3-K4 and K9. LSD1 is composed of several domains, including a SWIRM domain, a conserved motif shared by many chromatin regulatory complexes, an amine oxidase domain (AOD) and a Tower domain found in BRCA2 (NPL18-20). LSD1 cooperates with CoREST, a CtBP co-repressor complex, and demethylates histone H3-K4 through this interaction (NPL21, 22). LSD1 also demethylates histone H3-K9 and regulates transcription, in the presence of the human androgen receptor (AR) 23-25. In addition to histone proteins, LSD1 was reported to demethylate p53 lysine 370, and repress p53-mediated transcriptional activation and apoptosis (NPL26-28). LSD1 was also shown to form a stable complex with RB and decrease H3-K4 methylation levels, causing reduction of transcription from the Epstein Barr Virus-C promoter 29.

On the other hand, MYPT1(myosin phosphatase target subunit 1) is known as a regulator of RB1 phosphorylation (NPL29), and relates to myosin phosphatase activity (NPL30,31). Additionaly, LSD1 has been implicated in demethylating a lysine residue on DNMT1, which is methylated by SETD7, a histone lysine methyltransferase (NPL32).
Previously, it was reported that SMYD3, a histone methyltransferase, stimulates cell proliferation through its methyltransferase activity and plays a crucial role in human carcinogenesis (PL1, NPL10-14). Dysfunction of histone methylation, particularly dysfunction of lysine methylation or demethylation was also shown to contribute to human carcinogenesis (NPL15-17). However, the mechanism for how abnormal lysine methylation of non-histone proteins causes carcinogenesis has not been clarified. Though the role of LSD1 in transcriptional regulation has been described (NPL1,18,26), the relationship between abnormal histone demethylation and human carcinogenesis, and the biological importance of LSD1 dysregulation in human cancer is unclear.

[PTL1] WO2005/071102

[NPL1] Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 2004;119:941-53.
[NPL2] Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins. Nature 2006;439:811-6.
[NPL3] Christensen J, Agger K, Cloos PA, Pasini D, Rose S, Sennels L, Rappsilber J, Hansen KH, Salcini AE, Helin K. RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell 2007;128:1063-76.
[NPL4] Iwase S, Lan F, Bayliss P, de la Torre-Ubieta L, Huarte M, Qi HH, Whetstine JR, Bonni A, Roberts TM, Shi Y. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell 2007;128:1077-88.
[NPL5] Klose RJ, Yan Q, Tothova Z, Yamane K, Erdjument-Bromage H, Tempst P, Gilliland DG, Zhang Y, Kaelin WG, Jr. The retinoblastoma binding protein RBP2 is an H3K4 demethylase. Cell 2007;128:889-900.
[NPL6] Lee MG, Norman J, Shilatifard A, Shiekhattar R. Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein. Cell 2007;128:877-87.
[NPL7] Whetstine JR, Nottke A, Lan F, Huarte M, Smolikov S, Chen Z, Spooner E, Li E, Zhang G, Colaiacovo M, Shi Y. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 2006;125:467-81.
[NPL8] Yamane K, Tateishi K, Klose RJ, Fang J, Fabrizio LA, Erdjument-Bromage H, Taylor-Papadimitriou J, Tempst P, Zhang Y. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. Mol Cell 2007;25:801-12.
[NPL9] Yamane K, Toumazou C, Tsukada Y, Erdjument-Bromage H, Tempst P, Wong J, Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 2006;125:483-95.
[NPL10] Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva FP, Li M, Yagyu R, Nakamura Y. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol 2004;6:731-40.
[NPL11] Hamamoto R, Silva FP, Tsuge M, Nishidate T, Katagiri T, Nakamura Y, Furukawa Y. Enhanced SMYD3 expression is essential for the growth of breast cancer cells. Cancer Sci 2006;97:113-8.
[NPL12] Kunizaki M, Hamamoto R, Silva FP, Yamaguchi K, Nagayasu T, Shibuya M, Nakamura Y, Furukawa Y. The lysine 831 of vascular endothelial growth factor receptor 1 is a novel target of methylation by SMYD3. Cancer Res 2007;67:10759-65.
[NPL13] Silva FP, Hamamoto R, Kunizaki M, Tsuge M, Nakamura Y, Furukawa Y. Enhanced methyltransferase activity of SMYD3 by the cleavage of its N-terminal region in human cancer cells. Oncogene 2008;27:2686-92.
[NPL14] Tsuge M, Hamamoto R, Silva FP, Ohnishi Y, Chayama K, Kamatani N, Furukawa Y, Nakamura Y. A variable number of tandem repeats polymorphism in an E2F-1 binding element in the 5' flanking region of SMYD3 is a risk factor for human cancers. Nat Genet 2005;37:1104-7.
[NPL15] Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 2006;6:846-56.
[NPL16] Takeshita F, Minakuchi Y, Nagahara S, Honma K, Sasaki H, Hirai K, Teratani T, Namatame N, Yamamoto Y, Hanai K, Kato T, Sano A, et al. Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo. Proceedings of the National Academy of Sciences of the United States of America 2005;102:12177-82.
[NPL17] Schneider R, Bannister AJ, Kouzarides T. Unsafe SETs: histone lysine methyltransferases and cancer. Trends in biochemical sciences 2002;27:396-402.
[NPL18] Shi Y, Whetstine JR. Dynamic regulation of histone lysine methylation by demethylases. Mol Cell 2007;25:1-14.
[NPL19] Mimasu S, Sengoku T, Fukuzawa S, Umehara T, Yokoyama S. Crystal structure of histone demethylase LSD1 and tranylcypromine at 2.25 A. Biochem Biophys Res Commun 2008;366:15-22.
[NPL20] Yang M, Culhane JC, Szewczuk LM, Gocke CB, Brautigam CA, Tomchick DR, Machius M, Cole PA, Yu H. Structural basis of histone demethylation by LSD1 revealed by suicide inactivation. Nat Struct Mol Biol 2007;14:535-9.
[NPL21] Lee MG, Wynder C, Cooch N, Shiekhattar R. An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature 2005;437:432-5.
[NPL22] Gatta R, Mantovani R. NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations. Nucleic Acids Res 2008;36:6592-607.
[NPL23] Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, Peters AH, Gunther T, Buettner R, Schule R. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 2005;437:436-9.
[NPL24] Kahl P, Gullotti L, Heukamp LC, Wolf S, Friedrichs N, Vorreuther R, Solleder G, Bastian PJ, Ellinger J, Metzger E, Schule R, Buettner R. Androgen receptor coactivators lysine-specific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res 2006;66:11341-7.
[NPL25] Wissmann M, Yin N, Muller JM, Greschik H, Fodor BD, Jenuwein T, Vogler C, Schneider R, Gunther T, Buettner R, Metzger E, Schule R. Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat Cell Biol 2007;9:347-53.
[NPL26] Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, Berger SL. p53 is regulated by the lysine demethylase LSD1. Nature 2007;449:105-8.
[NPL27] Scoumanne A, Chen X. The lysine-specific demethylase 1 is required for cell proliferation in both p53-dependent and -independent manners. J Biol Chem 2007;282:15471-5.
[NPL28] Tsai WW, Nguyen TT, Shi Y, Barton MC. p53-targeted LSD1 functions in repression of chromatin structure and transcription in vivo. Mol Cell Biol 2008;28:5139-46.
[NPL29] Wu Y, Muranyi A, Erdodi F, Hartshorne DJ. Localization of myosin phosphatase target subunit and its mutants. J Muscle Res Cell Motil. 2005;26:123-134.
[NPL30] Ito M, Nakano T, Erdodi F, Hartshorne DJ. Myosin phosphatase: structure, regulation and function. Mol Cell Biochem.2004;259:197-209.
[NPL31] Matsumura F, Hartshorne DJ. Myosin phosphatase target subunit: Many roles in cell function. Biochem Biophys Res Commun.2008;369:149-156.
[NPL32] Wang J, Hevi S, Kurash JK, Lei H, Gay F, Bajko J, Su H, Sun W, Chang H, Xu G, Gaudet F, Li E, Chen T. The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation. Nat Genet.2009;41:125-129.

The present invention relates to LSD1, and the role it plays in cancer carcinogenesis. As such, the present invention relates to novel compositions and methods for detecting, diagnosing, treating and/or preventing cancer, e.g. bladder cancer, colorectal cancer and lung cancer, as well as methods for screening for useful substances for treating and/or preventing cancer.
In particular, the present invention arises from the discovery that LSD1 gene is overexpressed in cancer cells and double-stranded molecules composed of specific sequences (e.g., SEQ ID NOs: 17 and 18, or SEQ ID NOs: 19 and 20), which inhibit LSD1 expression, are effective for inhibiting cellular growth of cancer cells.

Moreover, the present invention is based, at least in part, on the discovery that LSD1 polypeptide interacts with MYPT1 polypeptide. In the present invention, we identified MYPT1 polypeptide as a binding partner of LSD1 polypeptide, and confirmed that MYPT1 polypeptide, methylated by SETD7 polypeptide, is demethylated by LSD1 polypeptide. Further, lysine 442 of the MYPT1 polypeptide is identified as a target residue of methylation/demethylation regulated by the SETD7 polypeptide and the LSD1 polypeptide. The knockdown of LSD1 by siRNA directed against LSD1 increased MYPT1 protein levels and decreased the amount of phosphorylated RB1 (serine 807/811). Conversely, overexpression of LSD1 resulted in more rapid degradation of MYPT1 protein, and increased ubiquitination of MYPT1. Mutant MYPT1 containing a substitution of lysine 442 to alanine degraded more rapidly compared with the wild-type. These results demonstrate that overexpressed LSD1 enhances MYPT1 ubiquitination through demethylation of lysine 442, and results in decreased MYPT1 protein and increased phosphorylated RB1. Thus, we show that LSD1 can enhance cell-cycle progression and carcinogenesis.

Therefore, in one aspect, the present invention provides an isolated double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, the molecule including a sense strand and an antisense strand complementary thereto, the sense and antisense strands hybridized to each other to form the double-stranded molecule. These double-stranded molecules may be utilized in an isolated state or encoded in vectors and expressed from the vectors. Accordingly, in another aspect, the present invention provides such double-stranded molecules as well as vectors and host cells expressing the double-stranded molecules.

In another aspect, the present invention provides methods for inhibiting cell growth and treating cancer, particularly cancers including bladder cancer, colorectal cancer and lung cancer, by administering the double-stranded molecules or vectors of the present invention to a subject in need thereof. Such methods encompass administering to a subject a composition composed of one or more of the double-stranded molecules or vectors.
In another aspect, the present invention provides compositions for treating cancers including bladder cancer, colorectal cancer and lung cancer containing at least one of the double-stranded molecules or vectors of the present invention.

In yet another aspect, the present invention provides a method of detecting or diagnosing cancer in a subject by determining an expression level of LSD1 in a subject- derived biological sample. An increase in the expression level of LSD1 as compared to a normal control level of LSD1 indicates the presence of cancer in the subject, e.g., bladder cancer, colorectal cancer or lung cancer.
In a further aspect, the present invention provides a method of screening for a candidate
substance for treating and/or preventing cancer. Such a substance binds LSD1 polypeptide , reduces the biological activity of the LSD1 polypeptide, reduces the expression of LSD1 gene, reduces the expression of a reporter gene under the control of LSD1 gene regulatory regions,or reduces or increases the expression of downstream genes of LSD1. Genes downstream of LSD1 are involved in various chromatin-modifying pathways such as chromatin remodeling,, centromeric heterochromatin formation, and chromatin assembly.

In a further aspect, the present invention provides a method of screening for a candidate substance for treating and/or preventing cancer using (i) the binding activity between LSD1 polypeptide and MYPT1 polypeptide, (ii) the demethylation activity of MYPT1 polypeptide by LSD1 polypeptide, (iii) the phosphorylation level of the RB1 polypeptide, (iv) the MYPT1 polypeptide level, or (v) the ubiquitination level of the MYPT1 polypeptide in a cell expressing LSD1 as an index.
In a further aspect, the present invention provides a method of identifying a substance that modulates the demethylation level of a MYPT1 polypeptide by LSD1 polypeptide.
In a further aspect, the present invention provides a kit for detecting or diagnosing cancer, which comprises a reagent for detecting a mRNA, protein, or biological activity of LSD1.

In a further aspect, the present invention provides a kit for measuring a demethylase activity of LSD1 polypeptide, said kit comprising a methylated MYPT1 polypeptide and a reagent for detecting the demethylation level of the MYPT1 polypeptide.
In a further aspect, the present invention provides a kit for detecting for the ability of a test substance to inhibit demethylation of MYPT1 polypeptide by LSD1 polypeptide, said kit comprising LSD1 polypeptide, methylated MYPT1 polypeptide, and a reagent for detecting the demethylation level of the MYTP1 polypeptide.

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 of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention.

Fig.1 depicts elevated LSD1 expression in bladder cancer in British and Japanese patients. A LSD1 gene expression in normal and tumor bladder tissues in British cases. Expression levels of LSD1 were analyzed by quantitative real-time PCR, and the result is shown by box-whisker plot (median 50% boxed). Mann-Whitney U-test was used for statistical analysis. B Expression ratio between normal and tumor bladder tissues from Japanese patients. Signal intensity for each sample was analyzed by cDNA microarray, and the expression ratio is the signal intensity in tumor divided by that in normal (1 is normal). C Immunohistochemical staining of LSD1 in bladder tissues. Nonimmunized rabbit IgG was used as a negative control. Original magnification, x40 (left panels) and x400 (right panels).

Fig.2 depicts LSD1 protein overexpression in stable T-REx 293 cell lines. A Western blotting using anti-V5 antibody. B Immunocytochemical analysis of the T-REx LSD1 cell lines. Cells were stained with anti-V5 monoclonal antibody (Alexa488), beta-tubulin (Alexa594) and 4', 6'-diamidine-2'-phenylindole dihydrochloride (DAPI). LSD1 proteins were localized in the nucleus.

Fig.3 depicts elevated LSD1 expression in lung and colorectal cancer in Japanese patients. A Expression ratio of LSD1 between small cell lung cancer (SCLC) tissue samples and corresponding normal lung tissues. cDNA microarray signal intensity for each sample was analyzed, and tumor/normal expression ratio is shown (1 is normal). B Expression ratio (tumor/normal) for colorectal cancer. Signal intensity for each sample was analyzed by cDNA microarray, and the expression ratio is the signal intensity in tumor divided by that in normal (1 is normal). C Comparison of LSD1 expression in normal and tumor lung tissues in Japanese patients. Signal intensity of each sample was analyzed by cDNA microarray, and the result is shown by box-whisker plot. Mann-Whitney U-test was used for the statistical analysis. D Comparison of LSD1 expression between normal and colorectal tumor tissues in Japanese patients. Signal intensity of each sample was analyzed by cDNA microarray, and the result is shown by box-whisker plot (median 50% boxed). Mann-Whitney U-test was used for the statistical analysis.

Fig.4 depicts involvement of LSD1 in the growth of bladder and lung cancer cells. A Expression of LSD1 in normal bladder tissue, normal lung tissue, 12 bladder cancer cells and five lung cancer cells. Expression levels of LSD1 were analyzed by quantitative real-time PCR. Data were normalized by GAPDH and SDH expressions, and relative LSD1 expression shows the ratio compared to the value in normal bladder tissue (1 = normal bladder tissue). B Quantitative real-time PCR showing suppression of endogenous LSD1 expression by two independent LSD1-specific siRNAs (siLSD1#1, #2) in A549 and SBC5 cells. siRNA targeting EGFP (siEGFP) and siNegative Control (B-Bridge International) were used as controls. mRNA expression levels were normalized by GAPDH and SDH expressions, and values are relative to siEGFP (siEGFP = 1). Results are the mean +/-SD of three independent experiments. P values were calculated using Student's t-test. C Validation of LSD1 expression at the protein level. Lysates from A549 and SBC5 cells after siRNA treatment were immunoblotted with anti-LSD1 and ACTB antibodies. Expression of ACTB was served as a control. D Effect of LSD1 siRNA knockdown on the viability of bladder cancer cell lines (SW780 and RT4) and lung cancer cell lines (A549, LC319 and SBC5). Relative cell numbers are normalized to the number of siEGFP-treated cells (siEGFP = 1): results are the mean +/-SD of three independent experiments. P values were calculated using Student's t-test. E Numerical analysis of the FACS result, classifying cells by cell cycle status. The proportion of T-REx-LSD1 cells in S and G₂/M phases is slightly higher than control cells (T-REx-Mock and T-REx-CAT). Results are the mean +/-SD of three independent experiments. Fisher's PLSD Post-Hoc test was used to calculate P values.

Fig.5 depicts microarray competitive expression data for different candidate downstream genes were sorted by two-dimensional, unsupervised hierarchical cluster analysis after knockdown of LSD1 expression in SW780 and A549 cell lines. Two different control siRNAs (siEGFP and siFFLuc) were used and the signal intensities between control and siLSD1-treated samples were compared. Four different experiments were performed for each control, and genes were clustered based on the normalization of totally 16 results of A549 (8) and SW780 (8) as shown in this figure. Randomly selected five genes for the validation using qRT-PCR were underlined. Red (bottom panel), Up-regulated; Green (top and middle panel), Down-regulated.

Fig.6 depicts confirmation of the microarray data using quantitative real-time PCR. Five genes (HIF1AN, HECTD2, HELLS, RNF146 and BUF2), which were down-regulated by siLSD1 in the microarray result, were randomly selected, and the expression levels of those genes were measured based on three independent experiments. P-values were calculated using Student's t-test.

Fig.7 depicts the interaction between LSD1 and MYPT1. A, Interacting protein partners in SBC5 cell lysates were enriched by anti-LSD1 immunoprecipitation, separated by SDS-PAGE and silver stained. The strong bands were cut out and identified by mass spectrometry. The strongest binding partner was MYPT1. Normal rabbit IgG (NRIgG) was used for negative control. B, Immunoprecipitates from lysates of SBC5 cells using anti-LSD1 and -MYPT1 antibodies were immunoblotted with anti-LSD1 and -MYPT1 antibodies, respectively. Normal rabbit IgG (NRIgG) was used for negative control. C, Immunocytochemical analysis of 293T cells after transfection with FLAG-tagged LSD1 and HA-tagged MYPT1 expression vectors. Cells were stained with anti-FLAG antibody (Alexa Fluor(registered trademark)488 ), anti-HA antibody (Alexa Fluor(registered trademark)594) and 4', 6'-diamidine-2'-phenylindole dihydrochloride (DAPI). Scale bar; 20 micro m. D, LSD1 directly interacts with N-terminal region of MYPT1 (1-500), but not BSA. GST-N-MYPT1 (residues 1-500) and His-LSD1 were purified by Glutathione Sepharose 4B and TALON Metal affinity Resin, respectively, and mixed them in TBS buffer (pH 7.6). Samples were purified with TALON beads and separated by SDS-PAGE. Proteins were detected by CBB staining. E, FLAG-tagged LSD1 deletion mutants were co-immunoprecipitated with HA tagged MYPT1. After co-transfection with HA-tagged MYPT1 and FLAG-tagged LSD1 deletion mutants in 293T cells, immunoprecipitaion was performed with FLAG antibody. The FLAG peptide was used for the elution of FLAG-tagged proteins. After SDS-PAGE, Western blot was conducted using anti-HA and anti-FLAG antibodies.

Fig.8 depicts the methylation/demethylation of MYPT1 by SETD7 and LSD1. A, In vitro methyltransferase assay of SETD7 using immunoprecipitated FLAG-tagged MYPT1 as a substrate. Purified FLAG-MYPT1 was incubated with [³H]-labeled S-adenosylmethionine (SAM) as a methyl donor, in the presence of recombinant SETD7. BSA was used as a negative control. B, Dose-dependent methylation of N-MYPT1 by increasing amounts of SETD7. His-tagged N-terminal fragment (1-500) of MYPT1 (N-MYPT1) was used as a substrate and incubated with indicated concentration of SETD7. The reaction products were analyzed by SDS-PAGE followed by fluorography. Ponceau S staining was performed for the loading control. C, In vitro demethylation of N-MYPT1 by LSD1. The N-MYPT1 recombinant proteins were methylated by SETD7. After performing dialysis to remove SAM, they were incubated with recombinant LSD1 or BSA at 37 degrees C for 4 hours. The reaction products were analyzed by SDS-PAGE followed by fluorography. The signal intensity corresponding to MYPT1 methylation was quantified by image J. Mean +/- SD of three independent experiments. P values were calculated using Student's t-test (***, P < 0.001). D, In vivo methylation/demethylation of MYPT1. 293T cells were transfected with FLAG-tagged MYPT1 and FLAG-tagged SETD7 expression vectors together with a mock or a FLAG-tagged LSD1 vector. After methionine starvation in methionine-free medium for 1 hr, cells were treated with cycloheximide (CHX) and labeled with_L-[methyl-³H] methionine for 5 hours. MYPT1, LSD1 and SETD7 were immunoprecipitated using anti-FLAG M2 agarose and analyzed by SDS-PAGE followed by fluorography. The signal intensity corresponding MYPT1 methylation was quantified by image J. E, MYPT1 can be demethylated by LSD1. N-MYPT1 recombinant proteins methylated by SETD7 were incubated with recombinant LSD1 in a dose dependent manner or BSA at 37 degrees C for 4 hours. The signal intensity corresponding MYPT1 methylation was quantified by image J. Mean +/- SD of three independent experiments. P values were calculated using Student's t-test (*, P < 0.05).

Fig.9 depicts that lysine 442 of MYPT1 can be an essential target of methylation/demethylation dynamics regulated by SETD7 and LSD1. A, Typical mass chromatograms of methylated and unmethylated MYPT1 peptides. MYPT1 samples were digested with bovine trypsin, and an aliquot of digest was analyzed by nano LC- MS/MS using LCQ Deca XP plus.

B, Quantitative analysis of methylated and unmethylated MYPT1 peptides analyzed by iCarta (KYA Technologies). Mean +/- SD of five independent experiments. P values were calculated using Student's t-test (**, P < 0.01)). C, In vitro methyltransferase assay of wild type (WT) N-MYPT1 and N-MYPT1(K442A) mutant.

D, MS/MS spectra of MYPT1 peptide (KTGSYGALAEITASK). Typical MS/MS spectra of unmethylated (upper panel) and methylated (bottom panel) MYPT1 peptides. His-N-MYPT1 (1-500) was incubated with SETD7 and digested with bovine trypsin. An aliquot of digest was analyzed by nano LC-MS/MS using LCQ Deca XP plus. The peptides were separated using nano ESI spray column packed with a reversed-phase material. The mass spectrometer was operated in the positive-ion mode and the spectra were acquired in a data-dependent MS/MS mode. The MS/MS spectra were searched against in-house database using local MASCOT server (version: 2.2.1).

Fig.10 depicts the regulation of RB1 phosphorylation by LSD1 through demethylation of MYPT1. A, Overexpression of MYPT1 in 293T cells. After transfection with a FLAG-tagged MYPT1 or a mock vector, western blot analysis was performed with antibodies against FLAG and RB1 (serine 807/ 811). The amount of ACTB in each sample was used as an internal control. B, Depletion of MYPT1 enhanced RB1 phosphorylation. 293T cells were transfected with control siRNA (siEGFP) or two independent MYPT1 siRNAs. After 48 hours, western blot analysis was performed with antibodies against MYPT1, RB1 (serine 807/ 811) and CDK4. The amount of ACTB in each sample was used as an internal control. C, Depletion of LSD1 enhanced MYPT1 expression and decreased RB1 phosphorylation (serine 807/ 811). D, E2F reporter assay after depletion of LSD1 in A549 cells. Depletion of LSD1 decreased E2F luciferase activity comparing with that of control siRNA (siEGFP). Mean +/- SD of three independent experiments. P values were calculated using Student's t-test (***, P < 0.001). E, E2F reporter assay after overexpression of MYPT1 in 293T cells. Exogenous expression of MYPT1 decreased E2F luciferase activity compared to control (mock transfection). Mean +/- SD of three independent experiments. P values were calculated using Student's t-test (***, P < 0.001).

F,G, Quantitative real-time PCR analysis. mRNA levels of MYPT1 (F) and RB1 (G) after treatment with two independent LSD1 siRNAs. siEGFP was used for a negative control. H, mRNA levels of MYPT1 in Setd7 WT and Setd7 knockout MEF cells.

Fig.11 depicts protein stability of MYPT1 regulated by SETD7 and LSD1. A, LSD1 shortens the half life of MYPT1. 293T cells were transfected with a mock or a FLAG-LSD1 expression vector. Cells were treated with cycloheximide (CHX) and immunoblotted to examine endogenous MYPT1 protein levels. B, LSD1 induced ubiquitination of MYPT1. After transfection with a FLAG-tagged MYPT1 expression vector together with a mock, a FLAG-tagged full-lenrth LSD1 or a FLAG-tagged inactive LSD1 (1-500) expression vector, western blot analysis was performed with an antibody against ubiquitin. C, LSD1 induced polyubiquitination of MYPT1. After transfection with a FLAG-tagged MYPT1 expression vector together with a mock, a FLAG-tagged full-length LSD1 or a FLAG-tagged inactive LSD1 (1-500), cells were treated with 25 micro M of MG132 for 6 hours. FLAG-MYPT1 immunoprecipitates were prepared and immunoblotted for HA-ubiquitin. D, 293T cells were transfected with a HA-tagged wild-type MYPT1 or mutant MYPT1 containing a substitution of lysine 442 to alanine expression vector. Cells were treated with cycloheximide (CHX) and immunoblotted to examine exogenous MYPT1 protein levels. E, F, Western blot analysis of lysates from WT/Setd7^-/- MEF cells and WT (Aof2^2lox/+)/Lsd1-deficient ES cells (Aof2^1lox/1lox) using antibodies against SETD7, MYPT1 and LSD1. Expression of Actb was used as an internal control.

G, Schematic model for the dynamic regulation of RB1 phosphorylation through methylation and demethylation of MYPT1.

Fig.12 depicts tissue images of lung tumors stained by standard immunohistochemistry for protein expression of MYPT1. Clinical information for each section is represented above histological pictures. Counterstaining was done with hematoxylin and eosin.

Definition
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or polynucleotides.

The term "gene", "polynucleotide", "oligonucleotide" "nucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleic acid residues and, unless otherwise specifically indicated are referred to by their commonly accepted single-letter codes. The terms apply to nucleic acid (nucleotide) polymers in which one or more nucleic acids are linked by ester bonding. The nucleic acid polymers may be composed of DNA, RNA or a combination thereof and encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
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.

Unless otherwise defined, the terms "cancer" refers to cancers over-expressing the LSD1 gene. Examples of cancers over-expressing LSD1 gene include, but are not limited to, bladder cancer, lung cancer and colorectal cancer.
The terms "isolated" 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 else be included in the natural source. Thus, an isolated antibody, polypeptide or polynucleotide refer to an antibody, polypeptide or polynucleotide that are substantially free of cellular material from the cell or tissue source from which they are derived, substantially free of other celluar material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Genes and proteins
The LSD1 (lysine (K)-specific demethylase 1A: also referred as to KDM1A or AOF2) encodes a nuclear protein containing a SWIRM domain, a FAD-binding motif, and an amine oxidase domain. LSD1 polypeptide is a component of several histone deacetylase complexes, though it silences genes by functioning as a histone demethylase. The MYPT1 (myosin phosphatase target subunit 1:also referred as to MBS, MGC133042 or PPP1R12A), which is also called the myosin-binding subunit of myosin phosphatase, is known as a regulator of RB1 phosphorylation. MYPT1 is one of the subunits of myosin phosphatase and relates to myosin phosphatase activity.

The SETD7 (SET domain containing lysine methyltransferase 7: also refered as to SET7, SET9 or SET7/9) is a histone lysine methyltransferase responsible for the methylation of histone H3 K4 (the lysine residue at amino acid 4). The SETD7 is also known to methylate Lys189 of the TAF10, a member of the TFIID transcription factor complex, and Lys372 of the p53 tumor suppressor. The RB1 (retinoblastoma 1) is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The RB1 is well-known to be a key regulator in cell-cycle progression in cancer cells through its phosphorylation and dephosphorylation.

The nucleic acid sequences of the above mentioned genes and the amino acid sequences of the corresponding encoded polypeptides are known in the art. For example, the exemplary amino acid sequences of LSD1, MYPT1, SETD7 and RB1 polypeptide include, but not limited to, the amino acid sequences shown in SEQ ID NOs: 22 and 24 for LSD1, SEQ ID NOs: 26 and 29 for MYPT1, SEQ ID NO:31 for SETD7 and SEQ ID NO: 33 for RB1. The sequences are also available via GeneBank accession numbers, NP_001009999 and NP_055828 for LSD1, NP_002471 and NP_001137358 for MYPT1, NP_085151 for SETD7 and NP_000312 for RB1, respectively. Thus, the exemplary nucleic acid sequences of the LSD1 gene, the MYPT1 gene , the SETD7 gene and the RB1 gene may contain nucleic acid sequences encoding amino acid sequences shown in SEQ ID NOs: 22 and 24 for LSD1, SEQ ID NOs: 26 and 29 for MYPT1, SEQ ID NO: 31 for SETD7 and SEQ ID NO: 33 for RB1, respectively. The examples of such nucleic acid sequences include nucleic acid sequnces shown in SEQ ID NOs: 21 and 23 for LSD1, SEQ ID NOs: 25, 27 and 28 for MYPT1, SEQ ID NO: 30 for SETD7 and SEQ ID NO: 32 for RB1, but are not limited to. These nucleic acid sequence data are also available via GeneBank accession numbers, NM_001009999.2 and NM_015013.3 for LSD1, NM_002480, NM_001143885 and NM_001143886 for MYPT1, NM_030648 for SETD7 and NM_000321 for RB1, respectively.

According to an aspect of the present invention, functional equivalents of a polypeptide are also considered to be the "polypeptide". Herein, a "functional equivalent" of a polypeptide is a polypeptide that has a biological activity equivalent to the polypeptide. Namely, any polypeptide that retains a biological ability of the polypeptide 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 polypeptide. Alternatively, functional equivalents may be composed an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the amino acid sequence of the polypetide, more preferably at least about 90% to 95% homology, even more preferably 96%, 97%, 98% or 99%. 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)". In other embodiments, a functional equivalent may be a polypeptide encoded by a polynucleotide that hybridizes to the polynucleotide having the natural occurring nucleotide sequence of the gene under a stringent condition.
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 functional equivalent to that of the polypeptide, 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 to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will be different 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 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 of background, preferably 10 times of background hybridization. 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.

In the context of the present invention, a hybridization condition for isolating a polynucleotide encoding a functional equivalent of a polypeptide 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, preferably 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions are often preferably 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. However, 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 addition to hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a functional equivalent of a polypeptide, using primers synthesized based on the sequence information of the polynucleotide or polypeptide.
Generally, it is known that modifications of one or more amino acids in a polypeptide do not influence the function of the polypeptide. In fact, mutated or modified polypeptides, having amino acid sequences modified by substituting, deleting, inserting, and/or adding one or more amino acid residues of a certain amino acid sequence, can 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 polypeptide results in a polypeptide with similar functions, are acceptable in the context of the instant invention.

So long as the biological activity of the polypeptide is maintained, the number of amino acid mutations is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 6 amino acids or less, and even more preferably 3 amino acids or less.

An amino acid residue to be mutated is preferably 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) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified polypeptides are included in functional equivalents of a polypepitde. However, the present invention is not restricted thereto and functional equivalents of a polypeptide may include non-conservative modifications, so long as at least one biological activity of the polypeptide is retained. Furthermore, the modified polypeptides do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these polypeptides.
Also, in the context of the present invention, a gene encompasses polynucleotides that encode such functional equivalents of the polypeptide.

Double stranded molecule
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, 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", "double-stranded polynucleotide molecule", "double-stranded oligonucleotide" and "double-stranded oligonucleotide molecule".

As used herein, the term "target sequence" refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the
whole mRNA of the target gene if a double-stranded nucleic acid molecule containing the sequence is introduced into a cell expressing the gene. A nucleotide sequence within a mRNA or cDNA sequence of a target gene can be determined to be a target sequence when a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. When a target sequence is shown by a 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 to the sense strand of a double-stranded molecule. For example, when a target sequence is shown in a 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 an 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. For example, when a target sequence is the RNA sequence shown in SEQ ID NO: 17 or 19 and the 3' side half region of the sense strand of the double-stranded molecule is composed of DNA, "a sequence corresponding to a target sequence" is "CUAUGUAGCUGATCTTGGA" (for SEQ ID NO: 17) or "GUGAUACUGUGCTTGTCCA" (for SEQ ID NO: 19). 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 nucleotides that constitute the antisense strand. For example, when a target sequence is the RNA sequence shown in SEQ ID NO: 17 or 19 and the 5' end of the antisense strand of the double-stranded molecule is composed of DNA, " a complementary sequence to a target sequence " is "TCCAAGATCAGCUACAUAG" (for SEQ ID NO: 17) or "TGGACAAGCACAGUAUCAC" (for SEQ ID NO: 19). On the otherhand, when a double-stranded molecule is composed of RNA, the sequence corresponding to a target sequence shown in SEQ ID NO: 17 or SEQ ID NO: 19 is the ribonucleotide sequence shown in SEQ ID NO:17 or SEQ ID NO: 19 and the complementary sequence to the target sequence is the ribonucleotide sequence shown in SEQ ID NO: 18 or SEQ ID NO: 20.

A double-stranded molecule may have one or two 3'overhang(s) 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 complementary sequence thereto.

As used herein, the term "siRNA" refers to a double-stranded RNA molecule which 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. Alternatively, siRNA may also be directly introduced in cells to be treated. Methods of introducing siRNA in a subject are well known in the art. For example, administration of siRNA in conjunction with a delivery substance is preferable for the introduction of siRNA.
The siRNA includes a part of sense nucleic acid sequence of the target gene (also referred to as "sense strand"), a part of antisense nucleic acid sequence of the target gene (also referred to as "antisense strand") or both (nucleotide "t" is replaced with "u" in a siRNA). 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 RNA molecule having 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 the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions is joined by a loop region, and 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 used 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 a part of sense nucleic acid sequence of the target gene (also referred to as "sense strand"), a part of antisense nucleic acid sequence of the target gene (also referred to as "antisense strand") or both (nucleotide "t" is replaced with "u" in RNA). 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 polynucleotide 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 are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is 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 the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, and 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".

A double-stranded molecule directed against LSD1, of which the antisense strand hybridizes to the LSD1 mRNA, induces degradation of the LSD1 mRNA by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and inhibiting expression of the protein. As demonstrated herein, the expression of LSD1 in cancer cell lines, which overexpress LSD1 gene, is inhibited by dsRNA directed against LSD1 gene, and consequently, the growth of the cancer cell lines is suppressed (Fig. 4). Therefore the present invention provides isolated double-stranded molecules that are capable of inhibiting the expression of the LSD1 gene and cell growth when introduced into a cell expressing the gene. The double-stranded molecules of the present invention are useful for inhibiting cancer cell growth relating to the overexpression of LSD1 gene, and can provide new methods for treating cancers. For example, the double-stranded molecules of the present invention are suitable for treating cancers such as bladder cancer, colorectal cancer, and lung cancer, in which the overexpression of LSD1 gene was observed.
The target sequence of the double-stranded molecule against LSD1 gene includes, for example, a nucleotide sequence selected from SEQ ID NOs: 17 and 19.

Specifically, the present invention provides the following double-stranded molecules [1] to [18]:
[1] An isolated double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, wherein the double-stranded molecule contains a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule, wherein the sense strand contains a nucleotide sequence corresponding to a part of LSD1 gene sequence;
[2] The double-stranded molecule of [1], wherein the double-stranded molecule acts on mRNA of LSD1 gene, matching a target sequence selected from SEQ ID NOs: 17 and 19;
[3] The double-stranded molecule of [1], wherein the sense strand contains a nucleotide sequence corresponding to a target sequence selected from SEQ ID NOs: 17 and 19;
[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 length of less than about 100 nucleotides pairs in length (i.e., the double-stranded portion is less than about 100 nucleotide base pairs);
[5] The double-stranded molecule of any one of [1] to [4], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having a length of 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 a length of 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 a length of less than about 25 nucleotide pairs in length;
[8] The double-stranded molecule of [7], wherein the sense strand hybridize with 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;
[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 selected from SEQ ID NOs: 17 and 19, [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 the sense strand and a region flanking the 3'-end of the 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 contains one or two 3' overhang(s).

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 (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. recommend to avoid 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/, can 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.

Any other algorithms developed for designing siRNA may be also used for designing target sequences of the double-stranded molecules of the present invention.
In the present invention, two nucleotide sequences shown in SEQ ID NOs: 17 and 19 are demonstrated to be suitable for target sequences of the double-stranded molecules of the present invention.
Double-stranded molecules targeting the above-mentioned target sequences were respectively examined and it was confirmed that they possessed ability to suppress the growth of cells expressing LSD1 gene. Therefore, the present invention provides double-stranded molecules targeting the nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and 19 for a LSD1 gene.

The double-stranded molecule of the present invention may be directed to a single target LSD1 gene sequence or may be directed to a plurality of target LSD1 gene sequences.

A double-stranded molecule of the present invention targeting the LSD1 gene includes isolated polynucleotides that contain any of the target sequence selected from the LSD1 gene sequence and/or complementary sequences to the target sequence. Examples of polynucleotides targeting a LSD1 gene include those containing the sequence corresponding to SEQ ID NO: 17 or 19 and/or complementary sequences to these nucleotide sequences.

In an 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 another polypeptide 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 the 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 a 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 preferred embodiments, double-stranded molecules targeting the LSD1 gene may have a sequence selected from among SEQ ID NOs: 17 and 19 as a target sequence. Accordingly, preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 17 or 19 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 17 or 19 and a complementary sequence thereto.

In other words, a double-stranded molecule of the present invention comprises 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 comprising 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 and complementary sequence thereto is replaced with "u". Alternatively, a part of the polynucleotide of either or both of the strands may be DNA, and when the target sequence is defined with a RNA sequence, the nucleotide "u" within the target sequence and complementary sequence thereto is replaced with "t".
In one embodiment of the present invention, such a double-stranded molecule of the present invention comprises 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 comprising a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.

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 a LSD1 gene. Herein, the phrase "minor modification" as used in connection with a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleotide(s) to the sequence. In the context of the present invention, the term "several" as applied to nucleotide substitutions, deletions, additions and/or insertions may mean 3 to 7, preferably 3 to 5, more preferably 3 or 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 some portions of mRNA of the LSD1 gene and antisense strands complementary thereto were tested in vitro for their ability to decrease production of a LSD1 gene product in bladder cancer and lung cancer cell lines (e.g., using SW780, 1637, A549 and SBC-5) according to standard methods. For example, reduction in the LSD1 gene product in cells transfected with the candidate double-stranded molecule compared to that in cells transfected no oligonucleotide or control siRNA (e.g., siRNA against EGFP) can be detected by, e.g., RT-PCR using primers for a LSD1 mRNA mentioned under Example: "Quantitative Real-time PCR". Candidate target sequences which decrease the production of the LSD1 gene product in vitro cell-based assays can then be tested for their inhibitory effects on cell growth. Target sequences which inhibit cell growth in vitro cell-based assay may then be tested for their in vivo ability using animals with cancer, e.g., nude mouse xenograft models, to confirm decreased production of the LSD1 product and decreased cancer cell growth.

When the polynucleotide contained in double-stranded molecule is RNA or derivatives thereof, base "t" should be replaced with "u" in the nucleotide sequences. Thus, as used herein, the phrase " a sequence corresponding to a target sequence" refers to a nucleotide sequence in which base "t"s of the target sequence are replaced with "u"s in RNA or derivatives thereof, or a nucleotide sequence in which base "u"s of the target sequence are replaced with "t"s in DNA or derivatives thereof. 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 isolated double-stranded molecule of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization of the sense strand and antisense strand. In a preferred embodiment, such double-stranded molecules contain no more than 1 mismatch for every 10 matches. In an especially preferred embodiment, where the strands of the duplex are fully complementary, such double-stranded molecules contain no mismatches.

The polynucleotide is preferably less than 3125 or 3053 nucleotides in length for LSD1. For example, the polynucleotide can be less than 500, 200, 100, 75, 50, or 25 nucleotides in length. The isolated polynucleotides of the present invention are useful for forming double-stranded molecules against the LSD1 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, preferably longer than 21 nucleotides, and more preferably has a length of between about 19 and 25 nucleotides. Accordingly, the present invention provides the double-stranded molecules comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence. In preferable embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pairs in length (i.e. the double-stranded portion is between 19 and 25 base pairs in length).

The double-stranded molecule serves 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 is 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 present invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the LSD1 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 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 present 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. The skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; 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 with deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, see, e.g., US20060234970. 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.

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 either 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 the opposite 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 preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.

As a preferred 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. Preferably, 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 the 5'-end of the sense strand and/or 3'-end of antisense strand are referred to as the upstream partial region. That is, in preferable embodiments, a region flanking the 3'-end of the antisense strand, or the regions flanking the 5'-end of sense strand and 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 preferably is 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, preferred 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 double-stranded molecule is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the inhibition of target gene expression is much higher when the entire antisense strand is RNA (US20050004064).

In the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) or 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 a sense strand containing a sequence corresponding to the target sequence and an antisense containing a complementary sequence corresponding to the 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 the 5' or 3' end of a sense 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 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 NOs: 17 and 19 for LSD1.

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 LSD1 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 preferably 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from among the sequences disclose on the Ambion website (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 preferred 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:
CUAUGUAGCUGAUCUUGGA-[B]-UCCAAGAUCAGCUACAUAG
(for target sequence of SEQ ID NO: 17);
GUGAUACUGUGCUUGUCCA-[B]- UGGACAAGCACAGUAUCAC
(for target sequence of SEQ ID NO: 19).

Furthermore, in order to enhance the inhibition activity of the double-stranded molecules, several nucleotide can be added to 3 'end of the sense strand and/or antisense strand of the target sequence, as 3' overhangs. The preferred examples of nucleotides for a 3' overhang include but are not limited to "t" and "u". The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotides form (a) single strand(s) at the 3 'end of the sense strand and/or antisense strand of the double-stranded molecule. In cases where a double-stranded molecule 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 though it is preferable to use a chemical synthetic method 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, more preferably about 4:6, and most preferably 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 is gradually cooled down. The annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Examples of purification methods include methods utilizing agarose gel electrophoresis. Remaining single-stranded polynucleotides may be optionally removed by, e.g., degradation with appropriate enzyme.

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 will be described below.

Vectors containing a double-stranded molecule of the present invention:
Also included in the present invention are vectors encoding one or more of the double-stranded molecules described herein, and a cell containing such a vector.
Specifically, the present invention provides the following vector of [1] to [11].
[1] A vector, encoding a double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of LSD1 and cell proliferation, such double-stranded molecules composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule.
[2] The vector of [1], encoding the double-stranded molecule directed against mRNA of LSD1, corresponding to a target sequence selected from among SEQ ID NOs: 17 and 19;
[3] The vector of [1], wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from among SEQ ID NOs: 17 and 19;
[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 to the antisense strand at the target sequence to form the double-stranded molecule having a length of 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 to the antisense strand at the target sequence to form the double-stranded molecule having a length of 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 to the antisense strand at the target sequence to form the double-stranded molecule having a length of 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 to 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;
[8] The vector of [7], encoding the double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pair 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;
[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 selected from SEQ ID NOs: 17 and 19, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[11] The vector of any one of [1] to [10], wherein the double-stranded molecule contains one or two 3' overhang(s).

A vector of the present invention preferably encodes 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 a preferred embodiment, the vector includes regulatory elements necessary for expression of the 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 the sequence encoding the double-stranded molecule into an expression vector so that regulatory sequences are operatively-linked to the coding sequences of the double-stranded molecule 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, an RNA molecule that is the antisense strand 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). After transcribed, the sense and antisense strands hybridize to each other in vivo to generate a double-stranded molecule 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 antisense strands which then form a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector 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 double-stranded molecule and thereby suppresses the proliferation of the cell. Another example of a useable vector includes Bacille Calmette Guerin (BCG). 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.

Methods of inhibiting cancer cell growth and treating cancer using double-stranded molecules:
The present invention provides methods for inhibiting cancer cell growth, e.g.,, bladder cancer, colorectal cancer and lung cancer cell growth, by inducing dysfunction of a LSD1 gene via inhibiting the expression of LSD1. The LSD1 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention which specifically target of the LSD1 gene or the vectors of the present invention that can express any of the double-stranded molecules.
The present double-stranded molecules and vectors encoding said double-stranded molecules inhibit cell growth of cancerous cells, and can be used for methods for treating cancer. Thus, the present invention provides methods to treat patients with cancer associated with overexpression of LSD1, for example, bladder cancer, colorectal cancer or lung cancer, by administering a double-stranded molecule against a LSD1 gene or a vector expressing the molecule without adverse effect because that the gene is hardly expressed in normal organs.

Specifically, the present invention provides the following methods of [1] to [34]:
[1] A method of treating and/or preventing cancer, or inhibiting cancer cell growth in a subject comprising administering to a subject a pharmaceutically effective amount of a double-stranded molecule against a LSD1 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, the double-stranded molecule comprising a sense strand and an antisense strand complementary thereto, the strands hybridized to each other to form the double-stranded molecule;
[2] The method of [1], wherein the double-stranded molecule is directed against mRNA which matches a target sequence selected from SEQ ID NOs: 17 and 19;
[3] The method of [1], wherein the sense strand contains the nucleotide sequence corresponding to a target sequence selected from SEQ ID NOs: 17 and 19;
[4] The method of any one of [1] to [3], wherein the cancer is selected from the group consisting of bladder cancer, colorectal cancer and lung cancer;
[5] The method of any one of [1] to [4], wherein plural or multiple kinds of the double-stranded molecules are administered;
[6] The method of any one of [1] to [3], 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 lengths;
[7] The method of [6], 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 lengths;
[8] The method of [7], 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 lengths;
[9] The method of [8], wherein the sense strand of the double-stranded moleculehybridizes with antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotides pairs in lengths;
[10] The method of [9], 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;
[11] The method of any one of [1] to [10], 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;
[12] The method of [11], 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 selected from SEQ ID NOs: 17 and 19, [B] is the intervening single strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A];
[13] The method of [1] to [12], wherein the double-stranded molecule is an RNA;
[14] The method of [1] to [12], wherein the double-stranded molecule contains both DNA and RNA;
[15] The method of [14], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[16] The method of [15] wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[17] The method of [14], wherein the double-stranded molecule is a chimera of DNA and RNA;
[18] The method of [17], wherein 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;
[19] The method of [18], wherein the flanking region is composed of 9 to 13 nucleotides;
[20] The method of any one of [1] to [19], wherein the double-stranded molecule contains one or two 3' overhang(s);
[21] The method of any one of [1] to [20], 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.

[22] The method of [1], wherein the double-stranded molecule is encoded by a vector;
[23] The method of [22], wherein the double-stranded molecule encoded by the vector is directed against mRNA which matches a target sequence selected from SEQ ID NO: 17 and 19.

[24] The method of [22], 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 NOs: 17 and 19.
[25] The method of any one of [22] to [24], wherein the cancer to be treated is selected from the group consisting of bladder cancer, colorectal cancer and lung cancer;
[26] The method of any one of [22] to [25], wherein plural or multiple kinds of the double-stranded molecules are administered;
[27] The method of any one of [22] to [26], wherein the sense strand of the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotides in length;
[28] The method of [27], 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;
[29] The method of [28], 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;
[30] The method of [29], wherein the sense strand of the double-stranded molecule hybridizes with antisense strand at the target sequence to form the double-stranded molecule having has a length of less than about 25 nucleotide pairs in length;
[31] The method of [30], 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;
[32] The method of any one of [22] to [31], 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;
[33] The method of [32], 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 selected from SEQ ID NOs: 17 and 19, [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
[34] The method of any one of [22] to [33], 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.

In another embodiments, [1] a method of either of treating or preventing cancer, or both, or inhibiting cancer cell growth in a subject comprising administering to a subject a pharmaceutically effective amount of a double-stranded molecule against a LSD1 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, the double-stranded molecule comprising a sense strand and an antisense strand complementary thereto, the strands hybridized to each other to form the double-stranded molecule is provided.

The method of the present invention will be described in more detail below.
The growth of cells expressing a LSD1 gene may be inhibited by contacting the cells with a double-stranded molecule directed against the LSD1 gene, a vector expressing the double-stranded molecule or a composition containing the double-stranded molecule or vector. 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 double-stranded 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 target gene of the double-stranded molecule of the present invention. Exemplary cells include bladder cancer cells, colorectal cancer cells and lung cancer cells.

Thus, patients suffering from or at risk of developing a disease related to LSD1 may be treated by administering at least one of the present double-stranded molecules, at least one vector expressing at least one of the molecules or at least one composition containing at least one of the molecules. For example, patients of bladder cancer, colorectal cancer or lung 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. Bladder cancer, colorectal cancer and lung cancer may be diagnosed, for example, with tumor markers, such as CEA for colorectal cancer and lung cancer, CYFRA and pro-GRP for lung cancer, and TPA for bladder cancer. Diagnosis can also be aided using Chest X-Ray and/or Sputum Cytology. More preferably, patients treated by the methods of the present invention are selected by detecting the expression of LSD1 gene in a biological sample from the patient by conventional methods such as RT-PCR or immunoassay. Preferably, before the treatment of the present invention, the biopsy specimen from the subject is confirmed for a LSD1 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.

According to the present method to inhibit cell growth and thereby to treat cancer, when administering plural or multiple kinds of the double-stranded molecules (or vectors or compositions carrying said double-stranded molecules), each of the molecules may have different structures but target mRNA which matches the same target sequence. Alternatively, multiple or plural kinds of the double-stranded molecules may act on mRNA which matches a different target sequence of the same gene or acts on mRNA which matches a different target sequence of a different gene. For example, the method may utilize double-stranded molecules directed against one, two, or more target sequences of LSD1 gene. Alternatively, the method may utilize the double-stranded molecules directed against target sequences of the LSD1 gene and other genes.

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 on a vector. For introducing the double-stranded molecules and vectors into the cells, a 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 a LSD1 gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that the treatment 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 the particular tumor type.

It is understood that the double-stranded molecule of the present invention degrades LSD1 mRNA in substoichiometric amounts. Without wishing to be bound by any theory, the double-stranded molecule of the present invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less double-stranded molecule needs to be delivered at or near the site of cancer to exert a 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 present invention is an concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from 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 LSD1; for example bladder cancer, colorectal cancer and lung cancer. In particular, a double-stranded molecule containing a target sequence of LSD1 (i.e., SEQ ID NO: 17 or 19) is particularly preferred for the treatment of bladder cancer, colorectal cancer and lung cancer.

For treating cancer, the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical agent 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 the present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, with a delivery substance (e.g., delivery vehicle), or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
Suitable delivery substances for administration of the double-stranded molecule include the Mirus Transit TKO lipophilic substance; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. Preferred examples of delivery substances are liposomes.

Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as lung tumor tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the present invention are 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. A variety 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.

Preferably, the liposomes encapsulating the double-stranded molecule of the present invention includes a ligand molecule (e.g., targeting moiety) that can deliver the liposome to the cancer site. 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, are preferred.

Particularly preferably, the liposomes encapsulating the double-stranded molecule of the present invention are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibiting moieties bound to the surface of the structure. In one embodiment, a liposome may include both opsonization-inhibiting moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing liposomes 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 modified with opsonization-inhibiting moieties can deliver the double-stranded molecule of the present invention to tumor cells.

Opsonization-inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably 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 GM1. 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.
Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or a derivative 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 substance, including the Mirus Transit LT1 lipophilic substance; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for local delivery of recombinant viral vectors, which express a double-stranded molecule of the present invention, 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 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. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of 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. Preferably, the double-stranded molecule is injected directly into the tissue at or near the site of cancer. Multiple injections of the double-stranded molecule into the tissue at or near the site of cancer are particularly preferred.

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, more preferably from about seven to about ten days. In a preferred dosage regimen, the double-stranded molecule is injected at or near the site of cancer once a day for seven days. Where a dosage regimen includes 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.

Compositions containing double-stranded molecules:
In addition to the above, the present invention also provides pharmaceutical compositions that include at least one of the double-stranded molecules of the present invention or the vectors coding for the molecules. Specifically, the present invention provides the following compositions [1] to [34]:
[1] A composition for treating and/or preventing cancer, and inhibiting cancer cell growth, wherein the cancer cell and the cancer expresses a LSD1 gene, including a pharmaceutically effective amount of an isolated double-stranded molecule directed against LSD1 gene or pharmaceutically acceptable salt thereof, or a vector encoding the double-stranded molecule, which molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits in vivo expression of a LSD1 gene as well as cell proliferation, and pharmaceutically acceptable carrier;
[2] The composition of [1], wherein the double-stranded molecule acts on (is directed against) mRNA which matches a target sequence selected from among SEQ ID NOs: 17 and 19;
[3] The composition of [1], wherein the double-stranded molecule, wherein the sense strand contains a nucleotide sequence corresponding to a target sequence selected from SEQ ID NOs: 17 and 19;
[4] The composition of any one of [1] to [3], wherein the cancer is selected from the group consisting of bladder cancer, colorectal cancer and lung cancer;
[5] The composition of any one of [1] to [4], wherein the composition contains plural or multiple kinds of double-stranded molecules;
[6] The composition of [3], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[7] The composition of [6], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[8] The composition of [7], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[9] The composition of [8], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[10] The composition of [9], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[11] The composition of [1], 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;
[12] The composition of [11], 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 containing a nucleotide sequence corresponding to a target sequence selected from SEQ ID NOs: 17 and 19, [B] is the intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is the antisense strand containing a nucleotide sequence complementary to [A];
[13] The composition of any one of [1] to [12], wherein the double-stranded molecule is an RNA;
[14] The composition of any one of [1] to [12], wherein the double-stranded molecule is DNA and/or RNA (Alternatively, The composition of any one of [1] to [12], wherein the double-stranded molecule is either of DNA or RNA, or both);
[15] The composition of [14], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[16] The composition of [15], wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[17] The composition of [14], wherein the double-stranded molecule is a chimera of DNA and RNA;
[18] The composition of [17], wherein a region flanking the 3'-end of the antisense strand, or both of a region flanking the 5'-end of the sense strand and a region flanking the 3'-end of the antisense strand are composed of RNA;
[19] The composition of [18], wherein the flanking region is composed of 9 to 13 nucleotides;
[20] The composition of any one of [1] to [19], wherein the double-stranded molecule contains one or two 3' overhang(s);
[21] The composition of any one of [1] to [20], wherein the composition includes a transfection-enhancing agent;
[22] The composition of [1], wherein the double-stranded molecule is encoded by a vector;
[23] The composition of [22], wherein the double-stranded molecule encoded by the vector acts on mRNA which matches a target sequence selected from SEQ ID NOs: 17 and 19;
[24] The composition of [23], wherein the sense strand of the double-stranded molecule encoded by the vector contains the nucleotide sequence corresponding to a target sequence selected from SEQ ID NOs: 17 and 19;
[25] The composition of any one of [22] to [24], wherein the cancer is selected from the group consisting of bladder cancer, colorectal cancer and lung cancer;
[26] The composition of any one of [22] to [25], wherein the composition contains the vector encodes plural or multiple kinds of double-stranded molecules or plural or multiple kinds of vectors, each of the vectors encoding a different double-stranded molecule;
[27] The composition of any one of [22] to [26], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 100 nucleotide pairs in length;
[28] The composition of [27], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 75 nucleotide pairs in length;
[29] The composition of [28], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 50 nucleotide pairs in length;
[30] The composition of [29], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having less than about 25 nucleotide pairs in length;
[31] The composition of [30], wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form the double-stranded molecule having between about 19 and about 25 nucleotide pairs in length;
[32] The composition of any one of [22] to [31], 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;
[33] The composition of [32], 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 nucleotide sequence corresponding to a target sequence selected from SEQ ID NOs: 17 and 19, [B] is a intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a nucleotide sequence complementary to [A]; and
[34] The composition of any one of [22] to [33], wherein the composition includes a transfection-enhancing agent.

In another embodiments, [1] a composition, including a pharmaceutically effective amount of an isolated double-stranded molecule directed against LSD1 gene or pharmaceutically acceptable salt thereof, or a vector encoding the double-stranded molecule, which molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits in vivo expression of a LSD1 gene as well as cell proliferation, and pharmaceutically acceptable carrier for a purpose selected from the group consisting of:
(a) treating cancer,
(b) preventing cancer,
(c) inhibiting cancer cell growth, and
(d) combinations thereof, wherein the cancer cell and the cancer expresses a LSD1 gene is provided.

Additional details of the compositions of the present invention are described below.
Compositions of the present invention preferably formulated as pharmaceutical compositions, according to techniques known in the art. Compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical compositions" include formulations for human and veterinary use. Methods for preparing the 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 compositions of the present invention contain at least one of the double-stranded molecules of the present invention or vectors encoding them (e.g., 0.1 to 90% by weight), or pharmaceutically acceptable salts of the molecules, mixed with a pharmaceutically acceptable carrier. Preferred pharmaceutically acceptable carrier 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 or multiple kinds of the double-stranded molecules, each of the molecules may be directed to different target sequences of LSD1, or target sequences of LSD1 and other genes. For example, the composition may contain double-stranded molecules directed to one, two or more target sequences of LSD1. Alternatively, for example, the composition may contain double-stranded molecules directed to a target sequence of LSD1 and double-stranded molecules directed to target sequences of other genes.

Furthermore, the present composition may contain a vector coding for one or plural or multiple double-stranded molecules. For example, the vector may encode one, two or several kinds of the double-stranded molecules of the present invention. Alternatively, the present composition may contain plural or multiple kinds of vectors, each of the vectors coding for a different double-stranded molecule.

Moreover, the double-stranded molecules of the present invention may be contained as liposomes encapsulating the molecules in the present composition. See under the item of "Methods of inhibiting cancer cell growth and treating cancer using double-stranded molecules " for details of liposomes.

Compositions of the present invention may 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). 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, solid compositions for oral administration can include any of carriers and excipients listed above and 10-95%, preferably 25-75%, of one or more double-stranded molecule of the present invention. Compositions for aerosol (inhalational) administration can include 0.01-20% by weight, preferably 1-10% by weight, of one or more double-stranded molecule of the present invention encapsulated in a liposome as described above, and propellant. For intranasal delivery, compositions may include carriers such as lecithin..

In addition to the above, the present composition may contain other pharmaceutical 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.

In another embodiment, the present invention also provides the use of the double-stranded molecules of the present invention in manufacturing a pharmaceutical composition for treating a cancer characterized by the expression of LSD1. For example, the present invention relates to a use of double-stranded molecule inhibiting the expression of a LSD1 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 molecule and targets to a nucleotide sequence selected from among SEQ ID NOs: 17 and 19, for manufacturing a pharmaceutical composition for treating cancer expressing LSD1, such as colorectal cancer, bladder cancer and lung cancer.

Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating cancer characterized by the expression of LSD1, 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 LSD1 in a cell which over-expresses the gene, wherein the double-stranded molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule to target a nucleotide sequence selected from SEQ ID NOs: 17 and 19 as active ingredients.

In another embodiment, the present invention also provides a method or process for manufacturing a pharmaceutical composition for treating cancer characterized by the expression of LSD1, 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 LSD1 in a cell which over-expresses the gene, wherein the double-stranded molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule to target a nucleotide sequence selected from SEQ ID NOs: 17 and 19.

Method of detecting or diagnosing cancer
The expression of LSD1 was found to be specifically elevated in bladder cancer (Fig.1A-C), colorectal cancer(Fig.3B,D), lung cancer (Fig.3A,C), bladder cancer cell lines, and lung cancer cell lines (Fig.4A). The LSD1 gene and its transcription and translation products find diagnostic utility as markers for cancer, e.g. bladder cancer, colorectal cancer and lung cancer. Also, by measuring the expression of LSD1 in a subject-derived biological sample, cancer, e.g. bladder cancer, colorectal cancer and lung cancer, can be diagnosed. Specifically, the present invention provides a method for detecting or diagnosing cancer, e.g. bladder cancer, colorectal cancer and lung cancer by determining the expression level of LSD1 in a subject-derived biological sample. Lung cancers that can be diagnosed by the present method include NSCLC and SCLC. Furthermore, NSCLC, including lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) and Large cell carcinoma (LCC), can also be diagnosed or detected by the present invention.

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 that a subject suffers from the disease. That is, the present invention provides a diagnostic marker LSD1 for examining cancer. Alternatively, the present invention provides a method for detecting or identifying cancer cells in a subject-derived tissue, for example bladder tissue sample, colorectal tissue sample and lung tissue sample, said method including the step of determining the expression level of the LSD1 gene in a subject-derived biological tissue, wherein 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. Such result 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 disease 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 LSD1 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 lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA, bladder tumor markers in blood are BTA, IAP and so on, colorectal tumor markers in blood are IAP, SLX, STN, CA19-9, CEA and so on. Namely, in this particular 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.

Specifically, the present invention provides the following methods [1] to [10]:
[1] A method of detecting or diagnosing cancer in a subject, comprising determining an expression level of LSD1 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 the mRNA of LSD1;
(b) detecting the protein encoded by LSD1 gene; and
(c) detecting the biological activity of the protein encoded by LSD1 gene.
[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 cancer is selected from the group consisting of bladder cancer, colorectal cancer and lung cancer;
[4] The method of [1], wherein the expression level is determined by detecting hybridization of a probe to a gene transcript of the gene;
[5] The method of [1], wherein the expression level is determined by detecting the binding of an antibody against the protein encoded by a gene as the expression level of the gene;
[6] The method of [1], wherein the biological sample is selected from biopsy, sputum, blood, pleural effusion and urine.;
[7] The method of [1], wherein the subject-derived biological sample includes an epithelial cell;
[8] The method of [1], wherein the subject-derived biological sample includes a cancer cell; and
[9] The method of [1], wherein the subject-derived biological sample includes a cancerous epithelial cell.
[10] The method of [3], wherein when the cancer is bladder cancer, the subject-derived biological sample is a bladder tissue derived from the subject; when the cancer is lung cancer, the subject-derived biological sample is a lung tissue; and when the cancer is colorectal cancer, the subject-derived biological sample is a colorectal tissue derived from the subject.

The method of diagnosing cancer (e.g., bladder cancer, colorectal cancer and lung cancer) will be described in more detail below.
A subject to be diagnosed by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.

It is preferred to collect 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 the objective transcription or translation product of LSD1. The biological samples include, but are not limited to, bodily tissues and fluids which are desired for diagnosing or are suspected of harboring cancer cells, such as biopsy, blood, sputum, pleural effusion and urine. Preferably, the biological sample contains a cell population including an epithelial cell, more preferably a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell may be purified from the obtained bodily tissues and fluids, and then used as the biological sample.

For example, according to the present invention, suitable cancers for diagnosis or detection include bladder cancer , lung cancer and colorectal cancer. In order to diagnose or detect theses cancers, a subject-derived biological sample is preferably collected from following organs:
bladder: for bladder cancer,
lung: for lung cancer, and
colorectum: for colorectal cancer.

According to the present invention, the expression level of LSD1 in the subject-derived biological sample is 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 LSD1 may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including LSD1. Those skilled in the art can prepare such probes utilizing the sequence information of the LSD1 (e.g., SEQ ID NO: 21 and 23; GenBank accession number: NM_001009999.2 and NM_015013.3). For example, the cDNA of LSD1 may be used as a probe. If necessary, the probe may be labeled with a suitable label, such as a dye, fluorescent molecule, or isotope, and the expression level of the gene may be detected as the intensity of the labels.

Furthermore, the transcription product of LSD1 may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers or probes used in the Example (SEQ ID NOs: 5, 6, 7 and 8) may be employed for the detection by RT-PCR or Northern blot, but the present invention is not restricted thereto.

Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of LSD1. As also defined above, 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 C 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 C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C 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 a LSD1 protein may be determined. A method for determining the quantity of the protein as the translation product includes 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 retains the binding ability to a LSD1 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.

The intensity of staining may be observed via immunohistochemical analysis using an antibody against a LSD1 protein as another method to detect the expression level of a LSD1 gene based on its translation product.. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of a LSD1 gene.

Furthermore, the quantity of LSD1 protein can be determined by measuring the biological activity of LSD1 protein, such as histone demethylation or MYPT1 demethylation. As described above, LSD1 is a component of several histone deacetylase complexes, and it silences genes by functioning as a histone demethylase. Further, in the present invention, MYPT1 protein was found to be demethylated by LSD1 protein, which influences the affinity of MYPT1 in the ubiquitin-proteasome pathway. Therefore, demethylase activity to histone or MYPT1 protein is useful for quantification of LSD1 protein based on its biological activity. The demethylation level of histone or MYPT1 protein can be determined by the methods well known in the art.

Alternatively, cell proliferation enhancing activity may be used as a biological activity of LSD1 protein. According to the present invention, inhibiting the expression of LSD1 gene led to suppress the cell growth in bladder cancer and lung cancer cells, therefore, LSD1 protein is presumed to promote cell proliferation. For determining the cell proliferation enhancing activity of LSD1 protein, the cell is cultured in the presence of a biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability, the cell proliferation enhancing activity of the biological sample can be determined.

Moreover, in addition to the expression level of a LSD1 gene, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in bladder cancer, colorectal cancer and lung cancer may also be determined to confirm the accuracy of the diagnosis.
The expression level of cancer marker gene including a LSD1 gene in a biological sample can be considered to be increased if it increases from the control level of the corresponding cancer 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.

The control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of a LSD1 gene in samples from subjects whose disease state are known. Furthermore, the control level can be a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of the LSD1 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample. Moreover, it is preferred, to use the standard value of the expression levels of the LSD1 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 S.D. or mean +/- 3 S.D. may be used as standard value.

In the context of the present invention, a control level determined from a biological sample that is known not to be 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 a LSD1 gene is increased as compared to the normal control level or is similar to the cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer. 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 cancer.

Difference 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.

A kit for diagnosing or detecting cancer:
The present invention provides a kit for diagnosing or detecting cancer or predisposition for developing cancer. In the context of the present invention, preferred examples of cancer to be diagnosed or detected are bladder cancer, colorectal cancer and lung cancer. Specifically, the kit includes at least one reagent for detecting the expression of the LSD1 gene in a patient-derived biological sample, which reagent may be selected from the group of:
(a) a reagent for detecting mRNA of the LSD1 gene;
(b) a reagent for detecting an LSD1 protein; and
(c) a reagent for detecting a biological activity of an LSD1 protein.

Suitable reagents for detecting mRNA of the LSD1 gene include nucleic acids that specifically bind to or identify the LSD1 mRNA, such as oligonucleotides which have a complementary sequence to a part of the LSD1 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the LSD1 mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art. If needed, the reagent for detecting the LSD1 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the LSD1 mRNA may be included in the kit.

A probe or primer of the present invention typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25, consecutive sense strand nucleotide sequence of a nucleic acid comprising a LSD1 sequence, or an anti sense strand nucleotide sequence of a nucleic acid comprising a LSD1 sequence, or of a naturally occurring mutant of these sequences. In particular, for example, in a preferred embodiment, an oligonucleotide having 5-50 in length can be used as a primer for amplifying the genes, to be detected. 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 DNA microarray analysis).

On the other hand, suitable reagents for detecting the LSD1 protein include antibodies to the LSD1 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 as the reagent, so long as the fragment retains the ability to specifically bind to the LSD1 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. Furthermore, the antibody may be labeled with signal generating molecules 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 LSD1 protein may be included in the kit.

Furthermore, the biological activity can be determined by, for example, measuring demethylase activity, or the cell proliferation enhancing activity due to the expressed LSD1 protein in the biological sample.

For example, the demethylase activity in a biological sample can be determined by incubating the biological sample with a methylated substrate such as methylated histone and methylated MYPT1 protein, and then, detecting residual methylated histone or MYPT1 protein using antibody against methylated histone or MYPT1 protein. Thus, the present kit may include methylated histone, or MYPT1 protein and anti-methylated histone antibody or anti-methylated MYPT1 protein. Otherwise, the present kit may include methylated histone or methylated MYPT1 protein with labeled methyl group for detecting formaldehyde released by histone or MYPT1 protein demethylation. The labeled methyl group may be prepared by incubated histone or MYPT1protein with an appropriate labeled methyl donor, e.g. S- adenosyl [methyl-³H] methionine (SAM) in the presence of an appropriate methyltransferase such as SETD7.
On the other hand, cell proliferation enhancing activity can be determined by cultivating cells in the presence of the biological sample and then detecting the speed of proliferation, or measuring the cell cycle or the colony forming ability. Thus, the present kit can include medium and container for cultivation of cells.

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 LSD1 gene or antibody against the LSD1 protein, a medium and container for culturing cells, positive and negative control samples, and a secondary antibody for detecting an antibody against the LSD1 protein. 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, etc.) with instructions for use. These reagents and such may be included 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.

As an embodiment of the present invention, when the reagent is a probe against the LSD1 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 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 provides a quantitative indication of the amount of LSD1 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 spanning the width of a test strip.

The kit of the present invention may further include a positive control sample, negative control sample and/or LSD1 standard sample. The positive control sample of the present invention may be prepared by collecting LSD1 positive tissue samples, confirmed by assaying the level of LSD1. In one embodiment, the LSD1 positive tissue samples may be composed of cancer cells expressing LSD1. Such cancer cells include, but are not limited to, bladder cancer cells, colorectal cancer cells or lung cancer cells. Alternatively, a purified LSD1 protein or polynucleotide may be added to LSD1 free sample to form the positive sample or the LSD1 standard.

Screening for an anti- cancer substance
In the context of the present invention, substances to be identified through the present screening methods may be any substance or composition including several substances. 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.
Alternatively, the present invention provides a method of evaluating therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer cell growth.

Any test substances, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide substances, synthetic micromolecular substances (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural substances 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-substance" library method and (5) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of substances (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 substances 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 substance in which a part of the structure of the substance screened by any 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 the screened test substance 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. The obtained DNA can be used to prepare the test substance which is a candidate for treating or preventing cancer.

Test substances useful in the screenings described herein can also be antibodies that specifically bind to a LSD1 protein or partial peptides of LSD1 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.

(i) Molecular modeling:
Construction of test substance libraries is facilitated by knowledge of the properties sought, and/or the molecular structure of LSD1 protein. One approach to preliminary screening of test substances suitable for further evaluation is computer modeling of the interaction between the test substance and LSD1 protein.

Computer modeling technology allows 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 computers, 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 review computer modeling of drugs interactive with specific proteins, such as 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 for treating or preventing cancer, such as bladder cancer, colorectal cancer and lung cancer.

(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 exemplified. 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 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.

Screening for a substance that binds to LSD1 polypeptide
In present invention, over-expression of LSD1 was detected in bladder cancer, colorectal cancer and lung cancer, and low expression of LSD1 was detected in the corresponding normal (non-cancerous) organs (Figs. 1, 3 and 4). Therefore, using the LSD1 gene and proteins encoded by the gene, the present invention provides a method of screening for a substance that binds to LSD1 polypeptide. Due to the expression of LSD1 in cancer such as bladder cancer, colorectal cancer and lung cancer, a substance that binds to LSD1 polypeptide is expected to suppress the proliferation of cancer cells overexpressing LSD1 (e.g., bladder cancer cells, colorectal cancer cells or lung cancer cells), and thus be useful for treating or preventing cancer associated with LSD1 overexpression (e.g., bladder cancer, colorectal cancer or lung cancer). Therefore, the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells overexpressing LSD1 (e.g.,bladder cancer cells, colorectal cancer cells and lung cancer cells), and a method for screening a substance for treating or preventing cancer associated with LSD1 overexpression (e.g., bladder cancer, colorectal cancer and lung cancer) using the LSD1 polypeptide. In an embodiment of the present method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, the method includes the steps of:
(a) contacting a test substance with an LSD1 polypeptide or a fragment thereof;
(b) detecting the binding activity between the LSD1 polypeptide or fragment thereof and the test substance; and
(c) selecting the test substance that binds to the LSD1 polypeptide or fragment thereof as a candidate substance for treating or preventing cancer.

In another embodiment, the present invention also provides a method of evaluating therapeutic effect of a test substance on treating or preventing cancer, or inhibiting cancer cell growth, the method includes the steps;
(a) contacting a test substance with an LSD1 polypeptide or a fragment thereof;
(b) detecting the binding activity between the LSD1 polypeptide or fragment thereof and the test substance; and
(c) correlating the potential therapeutic effect of the test substance with the binding activity detected in the step (b), wherein the potential therapeutic effect is shown when the test substance binds to the polypeptide or fragment thereof as a candidate substance for treating or preventing cancer.

In the present invention, the therapeutic effect may be correlated with the binding activity to LSD1 polypeptide or a functional fragment thereof. For example, when the test substance binds to LSD1 polypeptide or a functional fragment thereof, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not bind to LSD1 polypeptide or a functional fragment thereof, the test agent or compound may identified as the substance having no significant therapeutic effect.

The method of the present invention will be described in more detail below.
The LSD1 polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof. The polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.

As a method of screening for proteins, for example, that bind to the LSD1 polypeptide, many methods well known by a person skilled in the art can be used. Such a screening can be conducted by, for example, immunoprecipitation method, specifically, in the following manner. The gene encoding the LSD1 polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.

The promoter to be used for the expression may be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141 (1982)), the EF-alpha promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa et al., Gene 108: 193 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)) the SR alpha promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), the CMV immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946 (1989)), the HSV TK promoter and so on.

The introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B., Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)) and so on.

The polypeptide encoded by the LSD1 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 has been revealed, 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 florescence 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 LSD1 polypeptide by the fusion is also reported. 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 LSD1 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 LSD1 polypeptide, a polypeptide including the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the LSD1 polypeptide, besides using antibodies against the above epitopes, 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. If the polypeptide encoded by the LSD1 gene is prepared as a fusion protein with an epitope, such as GST, an immune complex can be formed in the same manner as in the use of the antibody against the LSD1 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 LSD1 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.

As a method of screening for proteins binding to the LSD1 polypeptide, for example, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used. Specifically, a protein binding to the LSD1 polypeptide can be obtained by preparing a cDNA library from cultured cells (e.g., SW780, RT4, A549, LC319 and SBC5) expected to express a protein binding to the LSD1 polypeptide using a phage vector (e.g., ZAP), being expressed the protein on LB-agarose, fixing the protein expressed on a filter, contacting the purified and labeled LSD1 polypeptide with the above filter, and detecting the plaques expressing proteins bound to the LSD1 polypeptide according to the label. The LSD1 polypeptide may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the LSD1, or a peptide or polypeptide (for example, GST) that is fused to the LSD1 polypeptide. Methods using radioisotope 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, LSD1 polypeptide 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 LSD1 polypeptide, 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 LSD1 polypeptide can also be screened using affinity chromatography. For example, LSD1 polypeptide may be immobilized on a carrier of an affinity column, and a test substance is applied to the column. Test substances herein may be, for example, cell extracts, cell lysates, etc. After loading the test substance, the column is washed, and substances bound to LSD1 polypeptide can be prepared. 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 LSD1 polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between LSD1 polypeptide and a test substance using a biosensor such as BIAcore.

The methods of screening for substances that bind to LSD1 polypeptide when the immobilized LSD1 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 LSD1 polypeptide (including agonist and antagonist) are well known to one skilled in the art.

In addition to the full length of LSD1 polypeptide, fragments of the polypeptide may be used for the present screening, so long as the fragment retains at least one biological activity of the naturally occurring LSD1 polypeptide. Such biological activities include cell proliferation activity and histone demethylase activity, and so on.
LSD1 polypeptides or fragments thereof may be further linked to other substances, so long as the polypeptide or fragment retains at least one biological activity. Usable substances include: 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 or fragment.

LSD1 polypeptides or fragments used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
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, LSD1 polypeptides may be obtained through any known genetic engineering methods for producing 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) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the LSD1 polypeptide is expressed in host (e.g., animal) cells and such 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 the LSD1 gene can be performed according to any 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.

The LSD1 polypeptide may also be produced in vitro adopting an in vitro translation system.
The LSD1 polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.
Test substances screened by the present method as substances that bind to LSD1 polypeptide can be candidate substances that have the potential to treat or prevent cancers. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to further identify or confirm the therapeutic efficacy of the substance for cancers. For example, these candidate substances may be further examined for suppression of cancer cell proliferation by contacting the substance with a cancer cell overexpressing the LSD1 gene.

Screening for a substance suppressing the biological activity of LSD1 polypeptide
The present invention provides a method for screening a substance that suppresses a biological activity of LSD1 polypeptide (e.g., cancer cell proliferation enhancing activity, MYPT1-binding activity or demethylase activity), and a method for screening a substance for treating or preventing cancer associating with LSD1 overexpression, including bladder cancer, colorectal cancer and lung cancer. Thus, the present invention provides a method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, including the steps as follows:
(a) contacting a test substance with an LSD1 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 the polypeptide detected in the absence of the test substance.

In another embodiment, the present invention provides a method of evaluating the therapeutic effect of a test substance on treating or preventing cancer, or inhibiting cancer cell growth, the method including the steps as follows:
(a) contacting a test substance with a LSD1 polypeptide;
(b) detecting the biological activity of the polypeptide of step (a); and
(c) correlating the potential therapeutic effect of the test substance with the biological activity detected in step (b), wherein the potential therapeutic effect is shown when the test substance suppresses the biological activity of the polypeptide as compared to the biological activity of the polypeptide detected in the absence of the test substance.

In the present invention, the therapeutic effect may be correlated with the biological activity of LSD1 polypeptide. For example, when the test substance suppresses or inhibits the biological activity of LSD1 polypeptide as compared to a level detected in the absence of the substance, the test substance may identified or selected as a candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity LSD1 polypeptide as compared to a level detected in the absence of the substance, the test substance may identified as a 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 retain a biological activity of the LSD1 polypeptide. For example, LSD1 polypeptide and functional equivalents thereof can be used in the present screening method. Examples of a biological activity of LSD1 polypeptide includes cell proliferation enhancing activity, MYPT1-binding activity and demethylase activity. Demethylation is defined as the catalysis of the transfer of a methyl group to an another compound, e.g., acceptor molecule. The LSD1 polypeptide can demethylate a methylated substrate. The exemplified substrates of LSD1 polypeptide include methylated histone and methylated MYPT1 polypeptide. The fragments of such methylated polypeptides containing at least one methylation site may also be used as substrates of LSD1 polypeptide. LSD1 polypeptide is known to demethylate histone H3 lysine 4 and lysine 9. We demonstrate that LSD1 polypeptide also demethylates a lysine 442 of MYPT1 polypeptide. Therefore, histone H3, or a fragment thereof containing

lysine

4 or 9, or MYPT1 polypeptide, or a fragment thereof containing lysine 442 of MYPT1 polypeptide (SEQ ID NO: 26) is useful as a substrate to be demethylated by LSD1 polypeptide.

The substance isolated by this screening is a candidate for antagonists of the LSD1 polypeptide. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. The term also refers to molecules that reduce or inhibit expression of the gene encoding LSD1. Moreover, a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the LSD1 polypeptide with molecules (including DNAs and proteins).

In the present invention, we have revealed that suppressing the expression of LSD1 gene reduces cell growth. Thus, by screening for a candidate substance that reduces the biological activity of LSD1 polypeptide, a candidate substance for treating or preventing cancers can be identified. The ability of the candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to further identify or confirm the therapeutic efficacy of the substance for treating cancers.

When the biological activity to be detected in the present method is cell proliferation enhancing activity, it can be detected, for example, by preparing cells which express the LSD1 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, for example, shown in Fig.4. The substances that reduce the speed of proliferation of the cells expressing LSD1 are selected as candidate substances for treating or preventing cancer, particularly cancers including bladder cancer, colorectal cancer and lung cancer.

More specifically, the method includes the step of:
(a) contacting a test substance with cells overexpressing LSD1;
(b) measuring cell proliferation enhancing activity in the cells of step (a); and
(c) selecting the test substance that reduces the cell proliferation enhancing activity in the comparison with the cell proliferation enhancing 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 LSD1.

Cells expressing LSD1 polypeptides include, for example, cell lines established from cancer, e.g. bladder cancer, colorectal cancer or lung cancer. Such cells can be used for the above screening of the present invention so long as the cells express the gene. Alternatively cells can be transfected with an expression vector encoding the LSD1 polypeptide, so as to express the gene.

In the present invention, the present inventors have screened a protein interacting with LSD1 through mass spectrometric analysis coupled with immunoprecipitation using anti-LSD1 antibody, and identified MYPT1 as a binding partner (Fig. 7A). MYPT1 is myosin phosphatase target subunit 1 and relates to myosin phosphatase activity (Ito M, et al., Mol Cell Biolchem, 2004, 259, 197-209). To verify binding between endogenous LSD1 and MYPT1 proteins, the immunoprecipitation with anti-LSD1 and anti-MYPT1 antibodies was conducted , and confirmed the endogenous interaction (Fig. 7B). To identify the region of LSD1 which interacts with MYPT1, plasmid clones that were designed to express parts of LSD1 protein were constructed. A co-immunoprecipitation assay was performed and revealed that the central region of LSD1 was required for binding to MYPT1(271-500) (Fig. 7E). Moreover, an in vitro binding assay indicated that MYPT1 is directly associated with LSD1 through its N-terminal region (Fig. 7D). Together, these data show that N-terminal region of MYPT1 directly binds to the central portion of LSD1. Furthermore, in the present invention, MYPT1 was demonstrated to be demethylated through the interaction with LSD1 (Fig. 8C, 8D), and then ubiquitinated by the ubiquitin-proteasome pathway (Fig. 11B, 11C). As a result of ubiquitination, MYPT1 polypeptide is destabilized, resulting in and increased amount of phosphorylated RB1 to induce carcinogenesis (Fig. 10B).

Thus, a substance that inhibits the binding between an LSD1 polypeptide and an MYPT1 polypeptide may be a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth. Therefore, candidate substances for treating or preventing cancer, or inhibiting cancer cell growth can be screened using such binding of the LSD1 polypeptide and the MYPT1 polypeptide as an index. Therefore, the present invention provides a method for screening a substance for inhibiting the binding between the LSD1 polypeptide and the MYPT1 polypeptide, which includes the step of contacting LSD1 polypeptide with MYPT1 polypeptide in the presence of a test substrate and the step of selecting the test substrate that inhibits the binding between LSD1 polypeptide and MYPT1 polypeptide as compared to that detected in absence of the test substrate . Furthermore, the present invention also provides a method for screening a candidate substance for inhibiting or reducing a cancer cell growth, which cancer cell expresses LSD1 and MYPT1 genes, e.g. bladder cancer cell, colorectal cancer cell and lung cancer cell, and a candidate substance for treating or preventing cancers, e.g. bladder cancer, colorectal cancer or lung cancer, using LSD1-MYPT1 binding as an index.

Specifically, the present invention provides the following methods of [1] to [5]:
[1] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, the method comprising the steps of:
(a) contacting a polypeptide comprising an MYPT1-binding domain of an LSD1 polypeptide with a polypeptide comprising an LSD1-binding domain of an MYPT1 polypeptide in the presence of a test substance;
(b) detecting binding between the polypeptides;
(c) comparing the binding level detected in the step (b) with that detected in the absence of the test substance; and
(d) selecting the test substance that inhibits the binding between the polypeptides as a candidate substance for treating or preventing cancer;
[2] The method of [1], wherein the polypeptide comprising the MYPT1-binding domain comprises residues 290-524 of SEQ ID NO: 22 or 271-500 of SEQ ID NO: 24;
[3] The method of [1] or [2], wherein the polypeptide comprising the LSD1-binding domain
comprises residues 1-413 of SEQ ID NO: 29;
[4] The method of [3], wherein the polypeptide comprising the LSD1-binding domain
comprises residues 1-500 of SEQ ID NO: 26; and
[5] The method of any one of [1] to [4], wherein the cancer is selected from the group consisting of bladder cancer, colorectal cancer and lung cancer.

Polypeptides to be used for the screening can be recombinant polypeptides or proteins derived from natural sources, or a partial peptide thereof. Preferably, the LSD1 polypeptide to be used for the screening is a polypeptide comprising an MYPT1-binding domain. In preferred embodiments, the MYPT1-binding domain may contain residues 290-524 of SEQ ID NO: 22 or 271-500 of SEQ ID NO: 24. In more preferred embodiments, the LSD1 polypeptide to be used in the screening is a polypeptide comprising an amino acid sequence of SEQ ID NOs: 22 or 24, and more preferably a polypeptide consisting an amino acid sequence of SEQ ID NOs: 22 or 24. On the other hand, the MYPT1 polypeptide to be used for the screening is preferably a polypeptide comprising an LSD1-binding domain. In preferred embodiments, the LSD1-binding domain may contain residues 1-413 of SEQ ID NO: 29. In more preferred embodiments, the LSD1-binding domain may contain residues 1-500 of SEQ ID NO:26. In more preferred embodiments, the MYPT1 polypeptide to be used in the screening is a polypeptide comprising an amino acid sequence of SEQ ID NOs: 26 or 29, and more preferably a polypeptide consisting an amino acid sequence of SEQ ID NOs: 26 or 29.

As a method of screening for substances that inhibits the binding of LSD1 polypeptide and MYPT1 polypeptide, many methods well known by one skilled in the art can be used. Such a screening can be conducted using, for example, an immunoprecipitation, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)), a two-hybrid system utilizing cells ("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)"), affinity chromatography and A biosensor using the surface plasmon resonance phenomenon. For example, the binding between LSD1 polypeptide and MYPT1 polypeptide may be detected by immunoprecipitation assay using anti-LSD1 antibody and/or MYPT1 antibody, as described in "[Example 1] Materials and methods". Immunoprecipitation may be conducted using either purified LSD1 polypeptide and MYPT1 polypeptide, or a cell lysate, which cell expresses LSD1 polypeptide and MYPT1 polypeptide.

Any aforementioned test substances can be used. In some embodiments, this method further comprises the step of detecting the binding of the candidate substance to LSD1or MYPT1 polypeptide, or detecting the level of binding LSD1 or MYPT1 polypeptide.
When the biological activity to be detected in the present method is the demethylase activity, it can be determined by contacting a LSD1 polypeptide with a substrate (e.g., a histone H3 comprising tri- or di-methylated lysine 4 and/or lysine 9, or a methylated MYPT1 polypeptide) under a suitable condition for demethylation of the substrate and detecting the demethylation level of the substrate.

More specifically, the method includes the steps of:
(a) contacting a LSD1 polypeptide with a substrate to be demethylated in the presence of the test substance under the condition capable of demethylation of substrate.
(b) detecting the demethylation level of the substrate; and
(c) selecting the test substance that decreases the demethylation level of the substrate as compared to the demethylation level detected in the absence of the test substance.
Alternatively, the method may include the steps of:
(a) contacting a LSD1 polypeptide with a substrate to be demethylated in the presence of the test substance under the condition capable of demethylation of substrate.
(b) detecting the methylation level of the substrate; and
(c) selecting the test substance that increases the methylation level of the substrate as compared to the methylation level detected in the absence of the test substance.

Preferably, a substrate to be demethylated by LSD1 polypeptide is a histone H3 or a fragment thereof comprising tri-or di-methylated lysine 4 and/or lysine 9 of histone H3, or a MYPT1 polypeptide or a fragment thereof comprising one or more methylation site(s) of MYPT1 polypeptide.

In the present invention, the demethylase activity of LSD1 polypeptide can be determined by methods known in the art. For example, LSD1 polypeptide may be incubated with a substrate with a labeled methylation site, under a suitable condition for demethylation. For example, a histone H3 peptide having tri- or di-[methyl-¹⁴C]-lysine , or tri- or di-[methyl-³H]-lysine in 4th and/or 9th amino acid residue, or a MYPT1 polypeptide having tri-, di- or mono- [methyl-¹⁴C]-lysine, or tri-, di- or mono- [methyl-3H]-lysine may be preferably used as a substrate for demethylation. The labeled methylated substrate may be prepared by labeled methyl donor, e.g. S- adenosyl-L-[methyl-³H]-methionine (SAM) or [methyl-¹⁴C]-SAM.

The demethylase activity can be determined based on the radioactivity in the substrate after incubation (i.e., the higher radioactivity in the substrate indicates the lower demethylase activity of LSD1 polypeptide). The radioactivity in the substrate may be detected, for example, by SDS-polyacrylamide gel electrophoresis and autoradiography. Alternatively, following the incubation the substrate may be separated from LSD1 polypeptide by conventional methods such as gel filtration and immunoprecipitation, and the radioactivity in the substrate may be measured by methods well-known in the art. Other suitable labels that can be attached to methyl group in a substrate, such as chromogenic and fluorescent labels, and methods of detecting these labels, are known in the art.

Alternatively, demethylase activity of LSD1 polypeptide may be determined using a mass spectrometry or reagents that selectively recognize a methylated substrate. For example, antibodies against the methylated substrate may be preferably used as such reagents. Any immunological techniques using such antibodies can be used for the detection of methylation level of the substrate. For example, when the substrate is a methylated histone, antibodies against a methylated histone (e.g., a histone H3 comprising tri- or di-methylated lysine 4 and/or lysine 9) may be preferably used. When the substrate is a methylated MYPT1 polypeptide, antibodies against a methylated MYPT1 polypeptide, in particular antibodies against a methylated lysine 442 of MYPT1 polypeptide having the amino acid sequence of SEQ ID NO: 26 (lysine 355 of SEQ ID NO: 29) may be preferably used. For example, ELISA or Immunoblotting with antibodies recognizing a methylated substrate may be used for the present invention.

Alternatively, demethylase activity of LSD1 polypeptide may be determined by detecting formaldehyde or hydrogen peroxide released from a methylated substrate due to demethylation using conventional methods.
Furthermore, the method for detecting demethylase activity can be performed by preparing cells which express the LSD1 gene, culturing the cells in the presence of a test substance, and determining the demethylation level (methylation level) of histones or MYPT1 polypeptide in the cells, for example, by using the antibody specific binding to the methylation site of the histone or MYPT1 polypeptide.

More specifically, the method includes the step of:
(a) contacting a test substance with a cell expressing LSD1 gene;
(b) detecting the demethylation level (methylation level) of the histone H3 lysine 4 and/or lysine 9, or MYPT1 polypeptide (Alternatively, detecting the demethylation level (methylation level) of either of the histone H3 lysine 4 and lysine 9, or both, or MYPT1 polypeptide); and
(c) selecting the test substance that decreases the demethylation level (increases the methylation level) as compared to the demethylation level (methylation level) detected in the absence of the test substance.

"Suppress the biological activity (e.g., cell proliferation enhancing activity , MYPT1-binding activity and demethylase activity)", as defined herein, means preferably at least 10% suppression in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression.

In the preferred embodiments, control cells which do not express LSD1
polypeptide are used. Accordingly, the present invention also provides a method of
screening for a candidate substance for inhibiting the cell growth or a candidate substance
for treating or preventing LSD1 associating disease, using the LSD1 polypeptide or
fragments thereof including the steps as follows:
a) culturing cells which express a LSD1 polypeptide or a functional
fragment thereof, and control cells that do not express a LSD1
polypeptide or a functional fragment thereof in the presence of a 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 biological activity detected in the control cells and in the absence of said test substance.

According to the present invention, MYPT1 polypeptide was identified as a novel substrate for demethylase activity of LSD1 polypeptide, and lysine 442 of MYPT1 polypeptide (SEQ ID NO: 26) was identified as an important demethylation site for LSD1 polypeptide. In the MYPT1 polypeptide, an isoform having an amino acid sequence of SEQ ID NO: 29 is known, in addition to the isoform having an amino acid sequence of SEQ ID NO: 26. When the MYPT1 polypeptide having an amino acid sequence of SEQ ID NO: 29 is used as a substrate for LSD1 polypeptide, lysine 355, corresponding to lysine 442 of SEQ ID NO: 26, may be demethylated by LSD1 polypeptide. Thus, in another embodiments, the present invention provides a method for screening a substance that modulates demethylation level of a MYPT1 polypeptide by a LSD1 polypeptide. Among substances identified in the screening, substances that inhibit demethylation of MYPT1 polypeptide are candidate substances for treating or preventing cancer, or inhibiting cancer cell growth.

Specifically, the present invention provides the following methods of [1] to [11]:
[1] A method for screening a substance that modulates demethylation level of a MYPT1 polypeptide by a LSD1 polypeptide, wherein the method comprises the steps of:
(a) contacting an LSD1 polypeptide or a functional equivalent thereof with a methylated MYPT1 polypeptide or a functional equivalent thereof in the presence of a test substance under a condition suitable for demethylation of the methylated MYPT1 polypeptide or the functional equivalent;
(b) detecting the demethylation level of the MYPT1 polypeptide or the functional equivalent thereof; and
(c) selecting the test substance that modulates the demethylation level of the MYPT1 polypeptide or the functional equivalent thereof in comparison with the demethylation level in the absence of the test substance;
[2] The method of [1], wherein the functional equivalent of the methylated MYPT1 polypeptide comprises a fragment of an MYPT1 polypeptide retaining a methylated lysine residue corresponding to the lysine 442 of the amino acid sequence of SEQ ID NO: 26;
[3] The method of [2], wherein the fragment of the MYPT1 polypeptide is a polypeptide containing residues 1-413 of SEQ ID NO: 29;
[4] The method of [3], wherein the fragment of the MYPT1 polypeptide is a polypeptide containing residues 1-500 of SEQ ID NO: 26;
[5] The method of any one of [1] to [4], wherein the functional equivalent of the LSD1 polypeptide comprises an amino oxidase domain of LSD1 polypeptide;
[6] The method of [5], wherein the amino oxidase domain comprises residues 308-850 of SEQ ID NO: 22 or residues 288-826 of SEQ ID NO: 24;
[7] The method of [6], wherein the amino oxidase domain comprises residues 290-850 of SEQ ID NO: 22 or residues 270-826 of SEQ ID NO: 24;
[8] The method of any one of [1] to [7], wherein the methylated MYPT1 polypeptide or the functional equivalent thereof is prepared by methylating a MYPT1 polypeptide or a functional equivalent thereof using a SETD7 polypeptide;
[9] The method of [8], wherein the step (a) comprises incubating a MYPT1 polypeptide or a functional equivalent thereof and a SETD7 polypeptide in the presence of a methyl donor, and simultaneously or subsequently adding a LSD1 polypeptide in the incubation mixture;
[10] The method of [9], wherein the methyl donor is S-adenosyl methionine; and
[11] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, wherein the method comprises the steps of:
(a) identifying a test substance that modulates the demethylation level of an MYPT1 polypeptide by an LSD1 polypeptide using the method of any one of [1] to [10]; and
(b) selecting the test substance that decreases the demethylation level of the MYPT1 polypeptide in comparison with the demethylation level in the absence of the test substance.

In the present screening methods, detection of demethylation level of MYPT1 polypeptide can be conducted by aforementioned methods.
LSD1 polypeptide to be used for the screening may either the full length of LSD1 polypeptide such as a polypeptide containing an amino acid sequence of SEQ ID NOs: 22 or 24, or a functional equivalent thereof such as a fragment of the full length of LSD1 polypeptide. Preferably, the functional equivalents of LSD1 polypeptide retain demethylase activity for MYPT1 polypeptide. Such functional equivalents may include a polypeptide containing an amino oxidase domain of LSD1 polypeptide. The amino oxidase domain is considered to be responsible for demethylase activity via an oxidation reaction that removes methyl groups from lysine. The examples of amino oxidase domain of LSD1 polypeptides include regions having residues 308-850 of SEQ ID NO: 22 and regions having residues 288-826 of SEQ ID NO: 24. Furthermore, in the present invention, the MYPT1-binding region was found to be located in a region having residues 270-500 of SEQ ID NO: 24. Therefore, in preferred embodiments, the functional equivalent of LSD1 polypeptide is a polypeptide containing residues 290-850 of SEQ ID NO: 22 or residues 270-826 of SEQ ID NO: 24.

MYPT1 polypeptide to be used for the screening may either the full length of MYPT1 polypeptide such as a polypeptide containing an amino acid sequence of SEQ ID NOs: 26 or 29, or a functional equivalent thereof such as a fragment of the full length of MYPT1 polypeptide. Preferably, the functional equivalents of MYPT1 polypeptide retains at least one demethylation site capable to be demethylated by LSD1 polypeptide. Such demethylation site includes lysine 442 of SEQ ID NO: 26 and lysine 355 of SEQ ID NO: 29 (corresponding to lysine 442 of SEQ ID NO: 26). Thus, preferred examples of the functional equivalent of MYPT1 polypeptide include a fragment of a MYPT1 polypeptide retaining a methylated lysine residue corresponding to the lysine 442 of the amino acid sequence of SEQ ID NOs: 26. Preferably, such fragments may contain a contiguous sequence of the amino acid sequence of SEQ ID NO: 26 including the methylated lysine 442, having more than 10 amino acid residues. More preferably, the fragments may have more than 15, 20, 25, 30, 50, 75, 100, 150, 200, 250, 300, 350 or 400 amino acid residues. Further more preferably, the fragments may contain residues 1-413 of SEQ ID NO: 29, more preferably, residues 1-500 of SEQ ID NO: 26.

According to the present invention, methylation/demethylation of MYPT1 polypeptide is regulated by SETD7 polypeptide and LSD1 polypeptide. Namely, methylated lysine of MYPT1 polypeptide by SETD7 polypeptide can be demethylated by LSD1 polypeptide. Thus, methylated MYPT1 polypeptides or functional equivalents thereof to be used in the screening may be prepared by methylating a MYPT1 polypeptides or the functional equivalents using a SETD7 polypeptide. The typical examples of SETD7 polypeptide include a polypeptide containing an amino acid sequence of SEQ ID NO: 31. Alternatively, instead of contacting a LSD1 polypeptide with a methylated MYPT1 polypeptide, a MYPT1 polypeptide may be incubated with a SETD7 polypeptide in the presence of a methyl donor to form an incubation mixture, and LSD1 polypeptide can be added simultaneously or subsequently to the incubation mixture. In this case, S-adenosyl methionine may be preferably used as a methyl donor.

Furthermore, the present invention also provides a method for screening a candidate substance for inhibiting or reducing a cancer cell growth, which cancer cell expresses LSD1, e.g. bladder cancer cell, colorectal cancer cell and lung cancer cell, and screening a candidate substance for treating or preventing cancer associated with LSD1 overexpression, e.g. bladder cancer, colorectal cancer or lung cancer. Substances identified as inhibitors of demethylation of MYPT1 polypeptide by LSD1 polypeptide in the above screening are good candidates for cancer therapeutic agent.

Screening for a substance altering the expression of LSD1
The present invention provides a method of screening for a substance that inhibits the expression of LSD1. A substance that inhibits the expression of LSD1 is expected to suppress the proliferation of cancer cells (e.g.,bladder cancer, colorectal cancer or lung cancer cells), and thus may be useful for treating or preventing cancer (e.g., bladder cancer, colorectal cancer or lung cancer). Therefore, the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells overexpressing LSD1, such as bladder cancer, colorectal cancer and lung cancer cells, and a method for screening a candidate substance for treating or preventing cancer associating with LSD1 overexpression such as bladder cancer, colorectal cancer or lung 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 LSD1 gene;
(b) detecting the expression level of LSD1 gene in the cell; and
(c) selecting the test substance that reduces the expression level of LSD1gene in comparison with the expression level detected in absence of the test substance.

Furthermore, the present invention provides a method of evaluating the ability of a test substance to suppress the proliferation of cancer cells, or treat or prevent cancer, the method comprising the steps of:
(a) contacting a test substance with a cell expressing the LSD1 gene;
(b) detecting the expression level of the LSD1 gene ; and
(c) correlating the potential therapeutic effect of the test substance with the expression level detected in step (b), wherein the potential therapeutic effect is shown when the test substance reduces the expression level of LSD1gene in comparison with the expression level detected in absence of the test substance.

In the present invention, the therapeutic effect may be correlated with the expression level of the LSD1 gene. For example, when the test substance reduces the expression level of the LSD1 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as a candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level of the LSD1 gene 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.

Further, in the present invention, the downstream genes of LSD1 gene affected by the knockdown of LSD1 were examined. Table2 indicates the list of genes down-regulated in A549 and SW480 cells transfected with LSD1 siRNA. Table 3 indicates the list of genes up-regulated in A549 and SW480 cells transfected with LSD1 siRNA. The expression level of these downstream genes can be used as indexes of the expression level of LSD1 gene.

Therefore, the present invention also provides the method of screening for a candidate substance for treating or preventing cancer associated with LSD1 overexpression (e.g., bladder cancer, lung cancer or colorectal cancer) or preventing proliferation of cancer cells overexpressing LSD1 (e.g., bladder cancer cells, lung cancer cells or colorectal cancer cells), the method including the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene and a downstream gene of LSD1 gene selected from the genes shown in Table 2 and Table 3;
(b) detecting the expression level of the downstream gene; and
(c) selecting the test substance that alters the expression level of the downstream gene in comparison with the expression level detected in absence of the test substance.

More specifically, when a downstream gene is a gene selected from the genes shown in Table 2, a test substance that reduces the expression level is selected as a candidate substance. On the other hand, when a downstream gene is a gene selected from the genes shown in Table 3, a test substance that increases the expression level is selected as a candidate substance. Therefore, in preferred embodiments, the present screening method includes the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene and a downstream gene of LSD1 gene selected from the genes shown in Table 2;
(b) detecting the expression level of the downstream gene; and
(c) selecting the test substance that reduces the expression level of the downstream gene in comparison with the expression level detected in absence of the test substance.

Alternatively, the present screening method may include the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene and a downstream gene of LSD1 gene selected from the genes shown in Table 3;
(b) detecting the expression level of the downstream gene; and
(c) selecting the test substance that increases the expression level of the downstream gene in comparison with the expression level detected in absence of the test substance.

The method of the present invention will be described in more detail below.
Cells expressing the LSD1 and downstream genes shown in Table 2 and Table 3 include, for example, cell lines established from bladder cancer, lung cancer or colorectal cancer; such cells can be used for the above screening methods of the present invention (e.g., SW780, RT4, A549, LC319, SBC5). 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. "Reduce the expression level" as defined herein are preferably at least 10% reduction of expression level of LSD1 or the downstream genes in comparison to the expression level in absence of the test substance, more preferably at least 25%, 50% or 75% reduced level and most preferably at 95% reduced level. Test substances herein include, for example, chemical substances, double-strand molecules, and so on. Methods for preparation of chemical substances and the double-strand molecules are described in the above description. In the method of screening, test substances that reduces the expression level of LSD1 or the downstream genes can be selected as candidate substances to be used for the treatment or prevention of cancer associating LSD1 overexpression, such as bladder cancer, colorectal cancer and lung cancer. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance for cancers.

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 LSD1 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 the reporter gene; and
(c) selecting the test substance that reduces the expression level or activity of the reporter gene in comparison with the expression level or activity detected in absence of the test substance.

Furthermore, the present invention provides a method of evaluating therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer cell growth, the method including steps of:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of LSD1 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 the reporter gene; and
(c) correlating the potential therapeutic effect of the test substance with the expression level or activity detected in step (b), wherein the potential therapeutic effect is shown when the test substance reduces the expression level or activity of the reporter gene in comparison with the expression level or activity detected in absence of the test substance.

In the present invention, the therapeutic effect may be correlated with the expression level or activity of the reporter gene. For example, when the test substance reduces the expression level or activity of the reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected 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 identified as the substance having no significant therapeutic effect.

Suitable reporter genes and host cells are well known in the art. For example, reporter genes are luciferase, green florescence 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 LSD1. The transcriptional regulatory region of LSD1 herein is the region from the start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5000 or 10,000 bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated or amplified by PCR. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of any one of these genes. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).

The vector containing the reporter construct is transfected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on). "Reduces the expression or activity" as defined herein are preferably at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.

In the present invention, we have revealed that suppressing the expression of LSD1 gene reduces cell growth. Thus, by screening for a candidate substance that reduces the expression or activity of the reporter gene, a candidate substance for treating or preventing cancers can be identified. The ability of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to further identify and confirm the therapeutic efficacy of the substance.

Screening for a substance using the phosphorylation level of a RB1 polypeptide, the MYPT1 polypeptide level or the ubiquitination level of a MYPT1 polypeptide as index
In the present invention, it was confirmed that overexpression of MYPT1 diminished RB1 phosphorylation (serine 807/811) in 293T cells (Fig. 10A). On the other hand, the amount of phosphorylated RB1 (serine 807/811) was increased dramatically after treatment with MYPT1 siRNAs, although the level of CDK4 protein was not influenced (Fig. 10B). Furthermore, after knockdown of LSD1, an increase of MYPT1 protein was observed and the amount of phosphorylated RB1 (serine 807/811) was decreased (Fig. 10C). In contrast, real-time PCR analysis showed that transcriptional levels of MYPT1 and RB1 expressions were unchanged (Fig. 10F, 10G). These data reveal that when MYPT1 is demethylated by LSD1, MYPT1 protein level is decreased and in turn RB1 phosphorylation level is increased.

Furthermore, in the present invention, the amount of ubiquitinated MYPT1 protein in 293T cells that overexpressed full-length LSD1 was much higher than that in the cells transfected with a mock vector or in the cells with the partial LSD1 protein without the enzymatic activity (residues 1-500) (Fig. 11B). In addition, after treatment with MG132, which suppresses degradation of ubiquitinated proteins, poly-ubiuquitination of MYPT1 was observed only in the cells having the full-length LSD1(Fig. 11C). These data show that overexpression of LSD1 can promote poly-ubiquitination of MYPT1 and destabilize MYPT1 protein in cancer cells, resulting in an increased amount of phosphorylated RB1.
Thus, the phosphorylation level of a RB1 polypeptide, the MYPT1 polypeptide level or the ubiquitination level of the MYPT1 polypeptide can be used as indexes of the expression level and/or activity of LSD1 polypeptide in cells.
Therefore, the present invention provides a method for screening a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth using at least one such level as an index.

Specifically, the present invention provides the following methods of [1] to [2]:
[1] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, wherein the method comprises the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene, MYPT1 gene, and RB1 gene;
(b) detecting at least one level selected from the group consisting of:
i) the phosphorylation level of the RB1polypeptide;
ii) the MYPT1 polypeptide level; and
iii) the ubiquitination level of the MYPT1polypeptide.
(c) selecting the test substance that has at least one property selected from the group consisting of:
i) decreasing the phosphorylation level of the RB1polypeptide,
ii) increasing the MYPT1 polypeptide level,
iii) decreasing the ubiquitination level of the MYPT1 polypeptide,
in comparison with those of control level,detected in absence of the test substance; and
[2] The method of [1], wherein the phosphorylation level of the RB1 polypeptide is detected by an antibody against phospholylated RB1 (Ser807/811).

Furthermore, the present invention provides the following methods of [1] to [2]:
[1] A method evaluating therapeutic effect of a test substance on treating or preventing cancer, or inhibiting cancer cell growth, wherein the method comprises the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene, MYPT1, gene and RB1 gene;
(b) detecting at least one level selected from the group consisting of:
i) the phosphorylation level of the RB1polypeptide;
ii) the MYPT1 polypeptide level; and
iii) the ubiquitination level of the MYPT1polypeptide.
(c) Correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown when the test substance has at least one property selected from the group consisting of:
i) decreasing the phosphorylation level of the RB1polypeptide,
ii) increasing the MYPT1 polypeptide level,
iii) decreasing the ubiquitination level of the MYPT1 polypeptide,
in comparison with the control level detected in absence of the test substance; and
[2] The method of [1], wherein the phosphorylation level of the RB1 polypeptide is detected by an antibody against phospholylated RB1 (Ser807/811).

Additional details of the present screening method will describe below.
Any cells can be used for the present screening methods, as log as the cells express LSD1 gene, MYPT1 gene and RB1 gene. Such cells may be established cell lines, known to express LSD1 gene, MYPT1 gene and RB1 gene, for example, cell lines established from bladder cancer, colorectal cancer or lung cancer. Alternatively, the cells may be cells transformed with any of LSD1 gene, MYPT1 gene and RB1 gene. One skilled in the art can prepare expression vectors for these genes and conduct cellular transformation by those vectors using conventional methods. Details of those genes was described in the item " Genes and proteins ".

Phosphorylation level of a RB1 polypeptide can be detected by methods known in the art. For example, antibodies against a phosphorylated RB1 polypeptide may be used as suitable reagents for the detection. Examples of such antibodies include antibodies against phospho-RB1 (Ser 807), phoshpo-RB1 (Ser 811) or phoshpo-RB1 (Ser807/ 811). These antibodies are commercially available. Any immunological techniques using such antibodies can be used for the detection of phosphorylation level of a RB1 polypeptide. For example, ELISA or immunoblotting may be used for the present invention.

Alternatively, the cell may be contacted with a test substance in the presence of radio-labeled ATP (e.g., ³²P-ATP) and then the radioactivity incorporated in RB1 polypeptide may be detected as an index of phosphorylation level of RB1 polypeptide. The radioactivity in the RB1 polypeptide may be detected, for example, by SDS-polyacrylamide gel electrophoresis and autoradiography. Alternatively, following the incubation, the RB1 polypeptide may be separated from other components by conventional methods such as gel filtration and immunoprecipitation, and then the radioactivity in the RB1 polypeptide may be measured by methods well-known in the art. Other suitable labels that can be attached to a phosphate group in a RB1 polypeptide, such as chromogenic and fluorescent labels, and methods of detecting these labels, are known in the art.

MYPT1 polypeptide level can be detected by methods well-known in the art. For example, antibodies against a MYPT1 polypeptide may be used as suitable reagents for the detection. Alternatively, cells to be used in the screening may be transformed with a fusion gene containing MYPT1 gene and a gene of commercially available epitope, and after contacting with a test substance, MYPT1 polypeptide level may be detected using an antibodies against the epitope. Examples of commercially available epitopes include 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. Antibodies against those epitopes are also commercially available. Any immunological techniques, for example, ELISA, immunoblotting and such can be used for the detection of MYPT1 polypeptide level. To clarify MYPT1 polypeptide level destabilized by LSD1 polypeptide before the screening, protein synthesis in the cell to be used for the screening may be inhibited by addition of protein synthesis inhibitor such as cycloheximide.

Ubiquitination level of the MYPT1 polypeptide can be detected by methods well-known in the art. For example, antibodies against ubiquitin may be used as suitable reagents for the detection. Such antibodies are commercially available. Any immunological techniques, for example, ELISA, immunoblotting and such can be used for the detection of ubiquitination level of the MYPT1 polypeptide. During the screening, a proteasome inhibitor such as MG132 or Lactacystin may be added in order to prevent degradation of ubiquitinated MYPT1 polypeptide.

A kit for measuring a demethylase activity or detecting for the ability of a test substance to inhibit demethylase activity
The present invention further provides a kit for measuring a demethylase activity of a LSD1 polypeptide. In the present invention, methylated MYPT1 polypeptide was identified as a novel substrate of LSD1 polypeptide. Thus, the present invention provides a kit for measuring a demethylase activity of a LSD1 polypeptide, containing a methylated MYPT1 polypeptide or a functional equivalent thereof as a substrate of LSD1 polypeptide. Such kit can be used for measuring LSD1-mediated demethylase activity in a sample containing a LSD1 polypeptide or a LSD1 polypeptide purified or isolated from a sample.
Furthermore, the present invention provides a kit for detecting for the ability of a test substance to inhibit demethylation of MYPT1polypeptide by a LSD1 polypeptide, containing a LSD1 polypeptide and a methylated MYPT1 polypeptide as a substrate for LSD1 polypeptide.

The above kits of the present invention find a use for identifying a substance that modulate a demethylation level of a MYPT1 polypeptide by a LSD1 polypeptide. Furthermore, the kits of the present invention are useful for screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth.

Specifically, the present invention provides the following kits of [1] to [4]:
[1] A kit for measuring a demethylase activity of a LSD1 polypeptide, wherein the kit comprises the following components (a) and (b):
(a) a component selected from (i) and (ii):
(i) a methylated MYPT1 polypeptide or a functional equivalent thereof;
(ii) a MYPT1 polypeptide or a functional equivalent thereof, a SETD7 polypeptide and a methyl donor;
(b) a reagent for detecting the demethylation level of the MYPT1 polypeptide or functional equivalent thereof;
[2] A kit for detecting for the ability of a test substance to inhibit demethylation of MYPT1polypeptide by a LSD1 polypeptide, wherein the kit comprises the following components of (a) to (c):
(a) an LSD1 polypeptide or a functional equivalent thereof;
(b) a component selected from (i) and (ii):
(i) a methylated MYPT1 polypeptide or a functional equivalent thereof:
(ii) a MYPT1 polypeptide or a functional equivalent thereof, a SETD7 polypeptide and a methyl donor;
(c) a reagent for detecting the demethylation level of the MYPT1 polypeptide or functional equivalent thereof;
[3] The kit of [1] or [2], wherein the functional equivalent of the methylated MYPT1 polypeptide comprises a fragment of the MYPT1 polypeptide having a methylated lysine residue corresponding to the lysine 442 of the amino acid sequence of SEQ ID NO: 34; and
[4] The kit of any one of [1] to [4], wherein the methyl donor is S-adenosyl methionine.

Details of the kits of the present invention will be described below.
MYPT1 polypeptide contained in the kits of the present invention may either the full length of MYPT1 polypeptide such as a polypeptide containing an amino acid sequence of SEQ ID NOs: 26 or 29, or a functional equivalent thereof such as a fragment of the full length of MYPT1 polypeptide. Herein, the functional equivalent of MYPT1 polypeptide refers to a modified polypeptide, a fragment or a modified fragment of the full length of MYPT1 polypeptide, capable of being demethylated by a LSD1 polypeptide. Preferably, the functional equivalents of MYPT1 polypeptide retains at least one demethylation site capable to be demethylated by LSD1 polypeptide. Such demethylation site includes lysine 442 of SEQ ID NO: 26 and lysine 355 of SEQ ID NO: 29 (corresponding to lysine 442 of SEQ ID NO: 26). Thus, preferred examples of the functional equivalent of MYPT1 polypeptide include a fragment of a MYPT1 polypeptide retaining a methylated lysine residue corresponding to the lysine 442 of the amino acid sequence of SEQ ID NOs: 26. Preferably, such fragments may contain a contiguous sequence of the amino acid sequence of SEQ ID NO: 26 including the methylated lysine 442, having more than 10 amino acid residues. More preferably, the fragments may have more than 15, 20, 25, 30, 50, 75, 100, 150, 200, 250, 300, 350 or 400 amino acid residues. Further more preferably, the fragments may contain residues 1-413 of SEQ ID NO: 29, yet more preferably, residues 1-500 of SEQ ID NO: 26.

According to the present invention, methylation/demethylation of MYPT1 polypeptide is regulated by SETD7 polypeptide and LSD1 polypeptide. Namely, MYPT1 polypeptide methylated by SETD7 polypeptide can be demethylated by LSD1 polypeptide. Thus, in stead of methylated MYPT1 polypeptides or functional equivalents thereof, the kit of the present invention may contain a MYPT1 polypeptide or a functional equivalent thereof, a SETD7 polypeptide and a methyl donor. In this case, a user can prepare a methylated MYPT1 polypeptide or functional equivalent thereof by incubating MYPT1 polypeptide or functional equivalent thereof with SETD7 polypeptide in the presence of a methyl donor such as S-adenosyl methionine. Examples of the SETD7 polypeptide includes a polypeptide having an amino acid sequence of SEQ ID NO: 33.

MYPT1 polypeptide or functional equivalent thereof may have one or more labeled methyl group(s) such as radiolabeled methyl group(s). Examples of other suitable labels that can be attached to the methyl group(s) includes chromogenic labels, fluorescent labels and such. MYPT1 polypeptide with labeled methyl group(s) can be prepared by methods well-known in the art.

LSD1 polypeptide contained in the kits of the present invention may be either the full length of LSD1 polypeptide such as a polypeptide containing an amino acid sequence of SEQ ID NOs: 22 or 24, or a functional equivalent thereof such as a fragment of the full length of LSD1 polypeptide. Herein, the functional equivalent of LSD1 polypeptide refers to a modified polypeptide, a fragment or a modified fragment of the full length of LSD1 polypeptide, having demethylase activity for MYPT1 polypeptide. Such functional equivalents may include a polypeptide containing an amino oxidase domain of LSD1 polypeptide. The examples of amino oxidase domain of LSD1 polypeptides include regions having residues 308-850 of SEQ ID NO: 22 and regions having residues 288-826 of SEQ ID NO: 24. Furthermore, in the present invention, MYPT1-binding region was found to be located in a region having residues 270-500 of SEQ ID NO: 24. Therefore, in preferred embodiments, the functional equivalents of LSD1 polypeptide may be a polypeptide containing residues 290-850 of SEQ ID NO: 22 or residues 270-826 of SEQ ID NO: 24.

Reagents for detecting the demethylation level of the MYPT1 polypeptide may be any reagents that is able to be used for detection of demethylation level of the MYPT1 polypeptide. For example, antibodies against a methylated MYPT1 polypeptide, in particular antibodies against a methylated lysine 442 of the amino acid sequence of SEQ ID NO: 26 (lysine 355 of SEQ ID NO: 29) may be preferably used as a such reagent. The anti-methylated MYPT1 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 methylated MYPT1 polypeptide. Methods to prepare these kinds of antibodies 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 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. For example, radiolabels, chromogenic labels, fluorescent labels and such may be preferably used for labeling the antibody. When the kit contains an anti-methylated MYPT1 antibody with label, the kit may further contain reagent(s) for detecting a signal generated by the label. Alternatively, the antibodies may be conjugated with such enzyme that catalyses a chromogenic reaction, for example, peroxidase, alkaline phosphatase and such. When the kit contains an anti-methylated MYPT1antibody conjugated with the enzyme, the kit may further contain a chromogenic substrate for the enzyme. Alternatively, a secondary antibody labeled or conjugated with an enzyme that catalyses a chromogenic reaction may be contained in the kit of the present invention.

When the kit contains a labeled methylated MYPT1 polypeptide or functional equivalent thereof, the reagents for detecting the demethylation level of the MYPT1 polypeptide may be reagents for detecting signal generated by the label. For example, when the MYPT1 polypeptide is labeled with a radiolabel, the reagents for the detection of demethylation level may be liquid scintillators, reagents for autoradiography and the like.

Alternatively, the reagents for detecting the demethylation level of the MYPT1 polypeptide may be reagents for detecting hydrogen peroxide or formaldehyde released by MYPT1 polypeptide demethylation. Such reagents are well-known in the art.

The kit may contain more than one of the aforementioned reagents. Furthermore, the kit may include a solid matrix for binding an anti-methylated MYPT1 antibody, a medium or buffer and container for incubating the polypeptides under suitable condition for demethylation, a cofactor for demethylation such as FAD (flavin adenine dinucleotide), positive and negative control samples. The 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, etc.) with instructions for use. These substances and such may be included 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.
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.

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] Materials and methods
Tissue samples and RNA preparation
121 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 specimens of normal bladder urothelial tissue were collected from areas of macroscopically normal bladder urothelium in patients with no evidence of malignancy. Five sequential sections of 7 micrometers thickness were cut from each tissue and stained using Histogene^TM staining solution (Arcturus, CA, USA) following the manufacturer's protocol, and assessed for cellularity and tumor grade by an independent consultant urohistopathologist. Slides were then transferred for microdissection using a Pix Cell II laser capture microscope (Arcturus). This technique employs a low-power infrared laser to melt a thermoplastic film over the cells of interest, to which the cells become attached. Additionally, the sections were graded according to the degree of inflammatory cell infiltration (low, moderate and severe). Samples showing significant inflammatory cell infiltration were excluded.

Approximately 10,000 cells were microdissected from both stromal and epithelial/tumor compartments in each tissue. RNA was extracted using an RNeasy Micro Kit (QIAGEN, Crawley, UK). Areas of tumor or stroma containing significant inflammatory cell infiltration were avoided to prevent contamination. Total RNA was reverse transcribed, and qRT-PCR performed as below. Given the low yield of RNA from such small samples, NanoDrop^TM quantification was not performed, but correction for the endogenous 18S CT value was used as an accurate measure of the amount of intact starting RNA. 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 mesenchymally 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. Use of tissues for this study was approved by Cambridgeshire Local Research Ethics Committee (Ref 03/018).

Cell culture
All cell lines were grown in monolayers in appropriate media: Eagle's minimal essential medium (EMEM) for 253J, 253JBV, HT1197, HT1376, J82, SCaBER, UMUC3 bladder cancer cells and SBC5 small cell lung cancer cells; RPMI1640 medium for 5637 bladder cancer cells and A549, H2170 and LC319 non-small cell lung cancer cells; Dulbecco's modified Eagle's medium (DMEM) for EJ28 bladder cancer cells, RERF-LC-AI non-small cell lung cancer cells and 293T cells; McCoy's 5A medium for RT4 and T24 bladder cancer cells; Leibovitz's L-15 for SW780 cells supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma); MEF-medium (High glucose DMEM) for wild and Setd7^-/- MEF cells supplemented with 10% fetal bovine serum, 1x penicillin-streptomycin, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 1x non-essential amino acids and 1 mM Na-pyruvate); ES cell medium (High glucose DMEM) for wild and Lsd1-deficient ES cells supplemented with 15% fetal bovine serum, 100 U/ml penicillin, 100 micro g/ml streptomycin, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 1x non-essential amino acids and Leukemia Inhibitory Factor. ES cells were grown on a feeder layer. All cells were maintained at 37 degrees C in humid air with 5% CO₂, (253J, 253J-BV, HT1197, HT1376, J82, SCaBER, UMUC3, SBC5, 5637, A549 H2170, LC319, EJ28, RERF-LC-AI, RT4, T24, 293T, MEF cells and ES cells) or without CO₂(SW780). Cells were transfected with FuGENE6^TM (ROCHE, Basel, Switzerland) according to manufacturer's protocols.

Expression profiling in cancers using cDNA microarrays
A genome-wide cDNA microarray with 36,864 cDNAs or ESTs selected from the UniGene database of the National Center for Biotechnology Information (NCBI) was established. 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 labelling of probes were performed as described previously (Kitahara O et al. Cancer Res 2001;61:3544-9.). 2.5-microgram aliquots of twice-amplified RNA (aRNA) from each cancerous and noncancerous tissue were then labelled, respectively, with Cy3-dCTP or Cy5-dCTP. Detailed expression profiling data of bladder, lung, and colorectal cancers in this study were based on the data reported previously by Drs.Takefumi Kikuchi ,Yu-Min Lin and Ryo Takata respectively (Kikuchi T et al. Oncogene 2003;22:2192-205., Takata R et al. Clin Cancer Res 2005;11:2625-36., Lin YM et al. Oncogene 2002;21:4120-8.).

Quantitative Real-time PCR
As described above, 121 bladder cancer and 26 normal bladder tissues were obtained from Cambridge Addenbrooke's Hospital. For quantitative RT-PCR reactions, specific primers for all human GAPDH (housekeeping gene), SDH (housekeeping gene), LSD1, MYPT1 and RB1 were designed.

PCR reactions were performed using the ABI prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK) following the manufacturer's protocol. 50% SYBR Green universal PCR Master Mix without UNG (Applied Biosystems, Warrington, UK) or LightCycler(registered trademark)480 SYBR Green I Master(Roche), 50 nM each of the forward and reverse primers and 2 microlitters of reverse transcriptional cDNA were applied. Amplification conditions were firstly at 95 degrees C for 5 min and then 45 cycles, each at 95 degrees C for 10 sec, at 55 degrees C for 1 min and at 72 degrees C for 10 sec. After this, samples were treated at 95 degrees C for 15 sec, then at 65 degrees C for 1 min to draw the melting curve, then 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.

To determine relative RNA levels within the samples, standard curves for the PCR reactions were prepared from a series of two-fold dilutions of cDNA covering the range 2-0.625 ng of RNA for the 18S reaction and 20-0.5 ng of RNA for all target genes. The ABI prism 7700 measured changes in fluorescence levels throughout the 45 cycles PCR reaction and generated a cycle threshold (C_t) value for each sample correlating to the point at which amplification entered the exponential phase. This value was used as an indicator of the amount of starting template; hence a lower C_t values indicated a higher amount of initial intact cDNA.

Transfection with small interfering RNAs
The small interfering RNA (siRNA) oligonucleotide duplexes were purchased from SIGMA Genosys for targeting the human LSD1 and MYPT1 transcript. siEGFP and siNegative control, which is a mixture of three different oligonucleotide duplexes, were used as control siRNAs. The siRNA sequences are described below.

siRNA duplexes (100 nM final concentration) were transfected to bladder cancer cell lines, lung cancer cell lines, and 293T with Lipofectamine 2000 (Invitrogen). After 72 hours, cell viability was examined using cell counting kit 8 (DOJINDO).

Construction of stable cell lines, constitutively expressing LSD1
V5-tagged LSD1 expression vectors (pcDNA5/FRT/V5-His-LSD1) were prepared and were transfected those into Flp-In T-REx 293 cells (Invitrogen), which contains a Flp recombination target (FRT) site in its genome to express LSD1 conditionally and stably. V5-tagged chloramphenicol acetyltransferase (CAT) expression vectors (pcDNA5/FRT/V5-His-CAT) were used as a negative control for the experiments. LSD1 expression at the protein level was evaluated by Western blot and immunocytochemistry (Fig. 2).

Flow cytometry assays (FACS) for cell cycle analysis
Three stable T-REx 293 cell lines were prepared: mock-transfected with unmodified vector (pcDNA5/FRT/V5-His), vector expressing CAT (pcDNA5/FRT/V5-His-CAT) and vector expressing LSD1 (pcDNA5/FRT/V5-His-LSD1). Then, the cells after trypsin treatment were collected, washed twice with 1,000 microlitters of Assay Buffer and centrifuged for 5 min at 5,000 rpm. Cells were resuspended in 200 microlitters of Assay Buffer. 1,000 microlitters of fixative buffer was added, and the samples incubated at room temperature for 1 hr. Finally, the propidium iodide reagent was added 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.

Immunohistochemical staining
Sections of human bladder cancer were stained by VECTASTAIN^TM ABC KIT (VECTOR LABORATORIES, CA, USA). 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 1:100 dilution of rabbit polyclonal anti-LSD1 antibody (abcam) overnight at 4 degrees C. Sections were washed twice with PBS (-), incubated with 5 microgram/microlitter goat anti-mouse 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 xylene washing and mounted on cover slips. Hematoxylin was used for nuclear counterstaining.

Microarray hybridization and statistical analysis for the clarification of down-stream genes
Purified total RNA was labelled and hybridized onto Affymetrix GeneChip U133 Plus 2.0 oligonucleotide arrays (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Probe signal intensities were normalized by RMA and Quantile normalization methods (using R and Bioconductor). Next, signal intensity fluctuation due to inter-experimental variation was estimated. Each experiment was replicated (1 and 2), and the standard deviation (stdev) of log₂(intensity₂/intensity₁) was calculated for each of a set of intensity ranges with the midpoints being at log₂((intensity₁+intensity₂) / 2) = 5, 7, 9, 11, 13, and 15. The preset inventors modeled intensity variation using the formula stdev (log₂(intensity₂/intensity₁)) = a * (log₂((intensity₁+intensity₂) / 2)) + b and estimated parameters a and b using the method of least squares. Using these values, the standard deviation of intensity fluctuation was calculated. The signal intensities of each probe were then compared between siLSD1 (EXP) and controls (EGFP/FFLuc) (CONT) and tested for up/down-regulation by calculating the z-score: log₂(intensity_EXP/intensity_CONT) / (a * (log₂((intensity_EXP+intensity_CONT) / 2)) + b). Resultant P-values for the replication sets were multiplied to calculate the final P-value of each probe. These procedures were applied to each comparison: siEGFP vs. siLSD1, siFFLuc vs. siLSD1, and siEGFP vs. siFFLuc, respectively. The present inventors determined up/down-regulated gene sets as those that simultaneously satisfied the following criteria: (1) The Benjamini-Hochberg false discovery rate (FDR) <=0.05 for siEGFP vs. siLSD1, (2) FDR<=0.05 for siFFLuc vs. siLSD1 and the regulation direction is the same as (1), and (3) siEGFP vs. siFFLuc has the direction opposite to (1) and (2) or P > 0.05 for siEGFP vs. siFFLuc. Finally, the present inventors performed a pathway analysis 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.

Immunoprecipitation
Transfected 293T cells were washed with PBS and lysed in CelLytic^TMM Cell Lysis Reagent (Sigma) containing complete protease inhibitor cocktail (Roche). In a typical immunoprecipitation reaction, whole-cell extract was incubated with an optimum concentration of the following antibodies: anti-FLAG (M2; Sigma), anti-HA (Y-11; Santa Cruz, Santa Cruz, CA), anti-MYPT1 (H-130; Santa Cruz), anti-LSD1 (Sigma), anti-SETD7 (2967C2a-1; Cosmo Bio), anti-Ubiquitin (FL-76; Santa Cruz), anti-pRb (serine 807/811; Santa Cruz), anti-CDK4 (B-10; Santa Cruz) or anti-Actin (I-19; Santa Cruz), and 30 microliters of Protein A/ G Plus-Agarose beads (Santa Cruz) at 4 degrees C for 1 hr. After the beads were washed 3 times with 1 ml of TBS buffer (pH 7.6), the proteins bound to the beads were eluted by boiling in Lane Marker Reducing Sample Buffer (Thermo Scientific).

In vitro binding assay
For the in vitro binding assay, GST-N-MYPT1 (residues 1-500) and His-LSD1 were purified by Glutathione Sepharose 4B (Amersham, GE Healthcare) and TALON Metal affinity Resin (Clontech), respectively, and mixed them in TBS buffer (pH 7.6). After binding with TALON beads for 1hour, samples were washed three times with TBS buffer, and boiled in sample buffer for subjecting to SDS-PAGE.

Immunostaining
Cultured cells were fixed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 30 min and permeabilized with 0.5% Triton X-100 in PBS (Sigma). Fixed cells were blocked with 5% BSA or 5% skim milk in PBS for 30 min and incubated with primary antibodies overnight at 4 degrees C. Then they were incubated with Alexa Fluor conjugated second antibody (Molecular Probes, invitrogen) and observed using a Leica confocal microscopy.

Mass spectrometry
A protein band of SDS-polyacrylamide gel electrophoresis was excised and reduced with dithiothreitol and propionamide by acrylamide. After washing the gel, the band was digested with bovine trypsin (TPCK treated, Worthington Biochemical Corp., Lakewood NJ) at 37 degrees C overnight. An aliquot of digest was analyzed by nano LC-MS/MS using LCQ Deca XP plus (Thermo Fisher Scientific, San Jose, CA). The peptides were separated using nano ESI spray column (100 micro m i.d.x 50 mm L) packed with a reversed-phase material (Inertsil ODS-3, 3 micrometers, GL Science, Japan) at a flow rate 200 nl/min. The mass spectrometer was operated in the positive-ion mode and the spectra were acquired in a data-dependent MS/MS mode. The MS/MS spectra were searched against the in-house database using local MASCOT server (version: 2.2.1, Matrix Sciences, UK). A peak area of selected mass chromatogram was calculated using Qual Browser V1.3 (Thermo Fisher Scientific, San Jose, CA) or iCarta (KYA Technologies, Japan).

In vitro methylation and demethylation assays
To perform in vitro methylation assay, SETD7 (Upstate) was incubated with recombinant His-N-MYPT1 (residues 1-500) as a substrate and 2 micro-Ci S-adenosyl-L- [methyl-³H] methionine (SAM; Amersham Biosciences) as a methyl donor in a mixture of 10 microlitters of methylase activity buffer (50 mM Tris-HCl at pH 8.5), for 1 hr at 30 degrees C. Samples were resolved on a 5-20% SDS-PAGE gel (Ready Gel; Bio-Rad) and visualized by fluorography and ponceau S (MP biomedical) staining. To test the ability of LSD1 to demethylate MYPT1, firstly MYPT1 was methylated with SETD7, and then purified methylated MYPT1 with TALON beads. After dialysis, methylated SETD7 was incubated with recombinant LSD1 or BSA in demethylation buffer (50 mM Tris-HCl [pH 8.5], 50 mM KCl, 5 mM MgCl₂, and 5% glycerol) at 37 degrees C for 4 hours. For MS/MS analysis of LSD1 demethylation, firstly His-N- MYPT1 was methylated with SETD7 for 2 hours at 30 degrees C, sequential adding recombinant LSD1 and incubating for 12 hours at 30 degrees C.

In vivo labeling experiments
Cells were starved for 1 hr in methionine-free medium, including cycloheximide (100 microgram/ml) and chloramphenicol (40 microgram/ml). They were then labeled with L-[methyl-³H] methionine (10 micro-Ci/ml, Perkin Elmer) for 5 hours. FLAG-MYPT1 was immunoprecipitated with FLAG-M2 agarose (Sigma) and methylated MYPT1 was visualized by fluorography.

Luciferase assay
The transcriptional activity of E2F was analyzed by the Cignal^TM E2F Reporter Assay Kit (SuperArray Bioscience Corporation). Cells were transfected with an E2F-responsive luciferase construct, which encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the E2F transcriptional response element [TRE], negative control or positive control. After 24 hours of transfection, dual luciferase assay was performed using Dual-Luciferase Reporter Assay System (Promega), and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicate, and Student's t-test was used for statistical analysis.

[Example2] Up-regulation of LSD1 expression in clinical cancer tissues
The present inventors first examined expression levels of various histone demethylase genes involved demethylase candidate genes, which possess JmjC domain, in 121 bladder cancer samples and 26 normal control samples obtained in the UK, and found significant elevation of LSD1 expression in tumors compared with in normal tissues (P < 0.0001, Mann-Whitney U-test) (Fig. 1A). As shown in Table 1, there was no significant correlation of LSD1 expression with clinical and pathological features (tumor grade, metastasis status, gender, smoking history and recurrence status), suggesting the involvement of LSD1 transactivation from an early stage of bladder carcinogenesis. Then, the expression patterns of LSD1 in a number of Japanese clinical bladder cancer samples were analyzed by cDNA microarray (Fig. 1B), and confirmed significant overexpression in bladder cancers of Japanese patients (P < 0.0001, Mann-Whitney U-test). To evaluate protein expression levels of LSD1 in bladder tissues, immunohistochemical analysis was performed using an anti-LSD1 antibody and detected strong LSD1 staining, mainly in the nucleus of malignant cells while no staining was observed in any of non-neoplastic tissues (Fig. 1C). In addition, microarray expression analysis of a large number of clinical samples derived from Japanese subjects was examined and found that LSD1 expression was also significantly up-regulated in lung and colorectal cancer tissues compared with corresponding non-neoplastic tissues (P < 0.0001, P = 0.0009, respectively, Fig. 3), indicating its possible involvement in many types of human cancer.

[Example3] Growth regulation of cancer cells by LSD1
To investigate roles of LSD1 in human carcinogenesis, a knockdown experiment was performed using two independent siRNAs targeting LSD1 (siLSD#1 and #2). Firstly, LSD1 expression was examined in various bladder and lung cancer cell lines, and compared to that in normal bladder and lung tissues (Fig. 4A). Expression levels of LSD1 in bladder and lung cancer cell lines were significantly higher than those in normal bladder and lung tissues. In addition, LSD1 expression in A549 and SBC5 cells transfected with siLSD1#1 and #2 was significantly suppressed, compared to that transfected with control siRNAs, siEGFP or siNegative control (Fig. 4B). The knockdown effect at the protein level was also confirmed (Fig. 4C). Using the same siRNAs, cell growth assays were perfomed in two bladder cancer cell lines (SW780, RT4) and three lung cancer cell lines (A549, LC319 and SBC5), and significant growth suppression by the siLSD1 was found. No growth-suppressive effect was observed when two control siRNAs were used (Fig. 4D).

To elucidate the mechanism how LSD1 up-regulation influences to the growth of cancer cells, the effect of LSD1 overexpression was examined using human embryonic kidney fibroblast (HEK293) cells containing the Flp-In T-REx system (T-REx-293, Invitrogen). The V5 tagged LSD1 expression vector, empty vector (mock) or V5 tagged CAT expression vector (control) were transfected into the T-REx-293 cells to establish stable cell lines expressing LSD1. LSD1 proteins in the T-REx-LSD1 cells were mainly located in the nucleus (Fig. 2B). The cell cycle status were analyzed by FACS analysis (Fig. 4E) and it was found that the proportions at the S phase and G₂/M phases were slightly but significantly increased in the T-REx-LSD1 cells compared with those in the control cells (S: P = 0.0055 [Mock, LSD1] and P = 0.0026 [CAT, LSD1], respectively; G₂/M: P = 0.0027 [Mock, LSD1] and P = 0.0020 [CAT, LSD1], respectively). Conversely, the proportion at the G₀/G₁ phase in the T-REx-LSD1 cells was slightly lower than that in the control cells (P = 0.0006 [Mock, LSD1] and P = 0.0003 [CAT, LSD1], respectively). These results show that LSD1 overexpression plays an important role in cell growth through enhancement of the cell cycle progression.

[Example 4] Identification of the downstream genes by microarray expression analysis
To identify signal pathways downstream to LSD1, microarray expression analysis was performed. After knocking down of LSD1 in SW780 and A549 cancer cells, total RNA was isolated from SW780 and A549 at 24 hour after the treatment with siLSD1#1. The expression profiles of these cells were compared to the cells treated with control siRNAs (siEGFP and siFFLuc) using Affymetrix's HG-U133 Plus 2.0 Array. Expression of 198 genes decreased and 72 genes increased statistically by the knockdown of LSD1, so these 270 genes were suggested to be the downstream genes affected by knockdown of LSD1 (Fig. 5). Table 2 indicates the list of genes down-regulated in A549 and SW480 cells transfected with LSD1 siRNA . Table 3 indicates the list of genes up-regulated in A549 and SW480 cells transfected with LSD1 siRNA. Because it was confirmed that the several genes randomly selected among these down-regulated downstream gene candidates were down-regulated by siLSD1 using quantitative RT-PCR (Fig. 6), this microarray data must be reproducible.

Signal pathway analysis for determining the downstream candidates using the Gene Ontology database (Table 4) indicated that LSD1 could regulate a wide variety of chromatin functions, including chromatin remodelling at centromere, centromeric heterochromatin formation, chromatin assembly or disassembly and methylation-dependent chromatin silencing. Therefore, dysfunction of LSD1 expression likely contributes to human carcinogenesis partially through these chromatin regulations.

[Example5] Interaction of LSD1 with MYPT1
To further investigate biological activity of LSD1, a protein(s) interacting with LSD1 was screened through mass spectrometric analysis coupled with immunoprecipitation using anti-LSD1 antibody. The results identified MYPT1 as the LSD1 binding partner (Fig. 7A). MYPT1 is myosin phosphatase target subunit 1 and relates to myosin phosphatase activity (Ito, M. et al., Mol Cell Biochem 259, 197-209 (2004), Matsumura, F. & Hartshorne, D.J. Biochem Biophys Res Commun 369, 149-156 (2008)). To verify binding between endogenous LSD1 and MYPT1 proteins, immunoprecipitations with anti-LSD1 and -MYPT1 antibodies were conducted, and the endogenous interaction was confirmed (Fig. 7B). Co-localization in the nucleus was also found (Fig. 7C). To identify the region of LSD1 which interacts with MYPT1, plasmid clones that were designed to express parts of LSD1 protein were constructed and co-immunoprecipitaion assay was performed, and the MYPT1-binding activity was located to the central region of LSD1 (Fig. 7E). Moreover, an in vitro binding assay indicated that MYPT1 is directly associated with LSD1 through its N-terminal region (Fig. 7D). Together, these data imply that N-terminal region of MYPT1 directly binds to the central portion of LSD1.

[Example 6] Methylation of MYPT1 by SETD7 and demethylation of MYPT1 by LSD1
LSD1 was reported to remove the methyl group of histone H3 lysine 4 (H3K4) (Shi, Y. et al. Cell 119, 941-953 (2004), Shi, Y.J. et al. Mol Cell 19, 857-864 (2005)) for which SETD7 was shown to methylate. LSD1 was also reported to demethylate a lysine residue on DNMT1 methylated by SETD7 (Wang, J. et al. Nat Genet 41, 125-129 (2009)). The present inventors investigated whether LSD1 demethylates one or more lysine residues on MYPT1 methylated by SETD7.

An in vitro methylation assay revealed that SETD7 is able to methylate full -length MYPT1 (Fig. 8A), and the N-terminal portion of MYPT1 (residues 1-500) (Fig. 8B), but not the C-terminal region of MYPT1 (data not shown). Subsequently an LSD1-dependent demethylase assay was performed using an N-terminal portion of MYPT1 protein after it was methylated by SETD7. The amount of methylated MYPT1 was decreased by addition of LSD1 in a dose-dependent manner (Fig. 8C and Fig. 8E), indicating demethylase activity on MYPT1. To further confirm the metylation and demethylation of MYPT1 in vivo, an in vivo labeling experiment was performed (Fig. 8D), and confirmed that the level of MYPT1 methylation was decreased after treatment with LSD1. These results show that MYPT1 can be methylated by SETD7 and demethylated by LSD1 both in vitro and in vivo.

[Example 7] Lysine 442 of MYPT1 as an essential target of methylation/demethylation dynamics regulated by SETD7 and LSD1.

To identify a site(s) of methylation/demethylation, mass spectrometric analysis was performed after in vitro methylation and demethylation assays. Detailed MS/MS analysis indicated that lysine 442 of MYPT1 is mono-methylated after treatment with SETD7 (Fig. 9D). MS data was also analyzed to quantify the amount of methylated peptides (meKTGSYGALAEITASK) and unmethylated peptides (KTGSYGALAEITASK) using MYPT1 samples incubated with SETD7, and subsequent LSD1-dependent demethylation assay (Fig. 9A). The mass chromatogram data showed that methylated MYPT1 peptides were notably increased after treatment with SETD7, and sequential LSD1 treatment significantly diminished its amount. Consistently, the amount of unmethylated MYPT1 peptides was decreased after SETD7 treatment and was restored by following LSD1 treatment (Fig. 9A, B). Furthermore, it was found that the methylation signal of mutant MYPT1 containing a substitution of lysine 442 to alanine was much weaker than that of wild-type MYPT1 after incubation with SETD7 (Fig. 9C), indicating that lysine 442 of MYPT1 is a target of methylation/demethylation by SETD7 and LSD1.

[Example 8] Regulation of RB1 phosphorylation through demethylation of MYPT1 by LSD1
Phosphorylation and dephosphorylation of RB1 is well-known to be a key regulator in cell-cycle progression in cancer cells (Classon, M. & Harlow, E. Nat Rev Cancer 2, 910-917 (2002), Cobrinik, D. Oncogene 24, 2796-2809 (2005), Knudsen, E.S. & Knudsen, K.E. Nat Rev Cancer 8, 714-724 (2008), Liu, H. et al, Curr Opin Genet Dev 14, 55-64 (2004)). Myosin phosphatase (MP) was recently reported to regulate dephosphorylation of RB1 in a MYPT1-dependent manner (Wu, Y. et al. J Muscle Res Cell Motil 26, 123-134 (2005), Matsumura, F. & Hartshorne, D.J. Biochem Biophys Res Commun 369, 149-156 (2008), Kiss, A. et al. Exp Cell Res 316, 68-77.). Hence, the relationship between MYPT1 expression and RB1 phosphorylation status was examined.

Overexpression of MYPT1 diminished RB1 phosphorylation (serine 807/811) in 293T cells (Fig. 10A). Conversely, the amount of phosphorylated RB1 (serine 807/811) was increased dramatically after treatment with MYPT1 siRNAs, although the level of CDK4 protein was not influenced (Fig. 10B). The results indicate that the increased MYPT1 expression induces the dephosphorylation of RB1 (serine 807/ 811).

The inventors subsequently examined protein expression levels of LSD1 and MYPT1, and RB1 as well as the RB1 phosphorylation status. After knockdown of LSD1, an increase in MYPT1 protein and decreased phosphorylated RB1 (serine 807/811) were observed (Fig. 10C). In contrast, real-time PCR analysis showed that transcriptional levels of MYPT1 and RB1 expressions were unchanged (Fig. 10F, G). These data revealed that MYPT1 becomes unstable when it is demethylated by LSD1, and decreased MYPT1 protein level might in turn increase the amount of phosphorylated RB1. MYPT1 stability may thus be regulated by methylation/ demethylation dynamics, and the amount of MYPT1 is likely to be a key factor in the regulation of the phosphorylation status of RB1. Hence, an E2F reporter assay was performed to investigate the effect of LSD1 demethylation on the cell-cycle progression. After treatment with LSD1 siRNA, E2F-luciferase activity was significantly decreased compared with that after treatment with control siRNA (Fig. 10D). Consistent with this result, overexpression of MYPT1 impaired E2F luciferase activity (Fig. 10E), indicating that E2F activity can be regulated by MYPT1 protein expression levels.

[Example 9] Reguration of protein stability of MYPT1 by SETD7 and LSD1
Endogenous MYPT1 stability with/without exogenous LSD1 proteins was examined after inhibiting protein synthesis by cycloheximide. Endogenous MYPT1 protein in 293T cells transfected with exogenous LSD1 degraded more rapidly compared with that in mock-transfected cells (Fig. 11A). In order to clarify the mechanism for regulation of MYPT1 protein stability in more detail, a ubiquitination assay was performed. It was recently reported that MYPT1 stability can be regulated by the E3 ubiquitin ligase SIAH2 (Twomey, E. et al. Exp Cell Res 316, 68-77). The amount of ubiquitinated MYPT1 protein in 293T cells that overexpressed full-length LSD1 was much higher than that in the cells transfected with a mock vector or in the cells with the partial LSD1 protein without the enzymatic activity (residues 1-500) (Fig. 11B). In addition, after treatment with MG132, poly-ubiquitination of MYPT1 was observed only in the cells having the full-length LSD1 (Fig. 11C). These results show that overexpression of LSD1 can promote poly-ubiquitination of MYPT1 and destabilize MYPT1 protein in cancer cells. In addition, mutant MYPT1 containing a substitution of lysine 442 to alanine degraded more rapidly compared with wild-type MYPT1 in 293T cells (Fig. 11D), indicating that lysine 442 plays a crucial role in the stability of MYPT1 regulated by the ubiquitin-proteasome pathway. Finally, MYPT1 expression was examined using Setd7^-/- MEF and Lsd1-deficient ES cells (Aof2^1lox/1lox) to evaluate the methylation/demethylation dynamics of MYPT1, as regulated by SETD7 and LSD1, and related to MYPT1 stability. Western blot analysis showed that MYPT1 protein expression was substantially reduced in Setd7^-/- MEF, but increased in Aof2^1lox/1loxES cells (Fig. 11E, F). However, MYPT1 expression at the RNA level was not changed in Setd7^-/- MEF cells (Fig. 10H). The data also show that the stability of MYPT1 protein is physiologically regulated by SETD7 and LSD1 in vivo.

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 had been thought to be a stable modification, but is at present considered to be dynamically regulated by both histone methyltransferases and demethylases. LSD1, the first reported histone lysine demethylase, uses an amine oxidase reaction to catalyze the removal of methyl groups (Shi Y et al. Cell 2004;119:941-53.). According to the present invention, LSD1 was confirmed to be significantly up-regulated in bladder cancer, by real-time PCR, microarray data and immunohistochemistry. Microarray analysis was also indicated LSD1 to be aberrantly over-expressed in lung and colorectal cancers.

Northern blot analysis revealed that LSD1 expression was barely detectable in 16 normal tissues except the testis (data not shown). The aberrant overexpression of LSD1 in many tumor types makes it a good candidate therapeutic molecular target with minimum side effects. To date, synthetic inhibitors of classical histone deacetylases (HDACs) have been widely used as biological tools for epigenetic studies, and some have advanced to clinical studies. In addition, development of histone methyltransferase and demethylase inhibitors has recently been reported (Greiner D et al. Nat Chem Biol 2005;1:143-5., Kubicek S et al. Mol Cell 2007;25:473-81.). Bisguanidine and biguanide, polyamine analogues, may be potential inhibitors for LSD1-dependent demethylation (Forneris F, et al. Trends Biochem Sci 2008;33:181-9., Huang Y et al. Proc Natl Acad Sci U S A 2007;104:8023-8., Culhane JC et al. Curr Opin Chem Biol 2007;11:561-8.).

Unlike JmjC family demethylases, LSD1 mediates an amine oxidase reaction, which reduces the oxidation state and generates hydrogen peroxide. The non-selective mono-amine oxidase inhibitor, tranylcypromine, which is currently used for the treatment of mental disorders, has been studied as an LSD1 inhibitor. Tranylcypromine generally functions by covalently modifying the flavin cofactor (Yang M et al. Nat Struct Mol Biol 2007;14:535-9., Culhane JC et al. Curr Opin Chem Biol 2007;11:561-8., Schmidt DM et al. Biochemistry 2007;46:4408-16., Yang M et al. Biochemistry 2007;46:8058-65.).
LSD1 inhibitors have great potential as anti-cancer reagents.

The pathway analysis using the LSD1 knock down by siRNA indicated that LSD1 is involved in regulation of a variety of chromatin functions such as chromatin remodelling, heterochromatin formation and DNA dynamics (Table.4). The results reveal that abnormally high levels of LSD1 expression cause dysregulation of chromatin structure and gene transcription, and contribution to malignant transformation of the cells. These data are consistent with evidence that the disruption of chromatin remodelling is tightly associated with human cancer (Wang GG, Allis CD et al. Trends Mol Med 2007;13:373-80.).

Furthermore, in the present invention, pathway analysis using the KEGG database also confirmed that LSD1 regulates the p53 and TGF-beta pathways (Table 5). LSD1 was known to regulate the function of p53 through the demethylation of mono- and di-methyl groups at K370, a site which is mono-methylated by SMYD2 (Huang J et al. Nature 2007;449:105-8., Scoumanne A et al. J Biol Chem 2007;282:15471-5., Tsai WW et al. Mol Cell Biol 2008;28:5139-46.).

LSD1 may prevent the accumulation of the di-methyl groups of p53 by demethylating p53K370Me2 and then inhibit the binding of 53BP1 to p53 (Huang J, et al. Nature 2007;449:105-8.). This is another example of how LSD1 can repress p53-mediated transcriptional up-regulation and prevent apoptosis, and contribute to human carcinogenesis in addition to chromatin modification.
In conclusion, the inventors found that LSD1 is overexpressed in bladder, lung and colorectal cancers, through early to late stages in carcinogenesis. LSD1 is present in the nucleus and promotes cancer cell proliferation at least in part through regulation of a wide variety of chromatin functions.

Additionally, the results disclosed herein propose a dynamic model for the regulation of MYPT1 protein stability through lysine methylation and demethylation in cancer cells (Fig. 11). The overexpressed LSD1 may enhance MYPT1 ubiquitination through its demethylase activity and result in the increase of the amount of phosphorylated RB1. Subsequently, released E2F activates transcription of genes required for S phase, and cell-cycle progression is enhanced. Consistent with these results, MYPT1 protein expression is significantly reduced in lung cancer, which constitutively overexpresses LSD1 (Fig.12).
The results disclosed herein show that histone methyltransferases and demethylases regulate non-histone protein as well, and their dysregulation can also play very critical roles in carcinogenesis.

The present inventors have shown that the cell growth is suppressed by a double-stranded nucleic acid molecule that specifically targets and inhibits expression of the LSD1 gene. Thus, the double-stranded nucleic acid molecule is useful for the development of anti-cancer pharmaceuticals.
The expression of human genes LSD1 are markedly elevated in bladder, colorectal or lung cancer. Accordingly, this gene can be conveniently used as diagnostic markers of cancers and the proteins encoded thereby may be used in diagnostic assays of cancers.

Furthermore, LSD1 polypeptide is a useful target for the development of anti-cancer pharmaceuticals. For example, substances that bind LSD1or block the expression of LSD1, or prevent biological activity of LSD1 may find therapeutic utility as anti-cancer or diagnostic agents, particularly anti-cancer agents for the treatment of bladder, colorectal or lung cancer.
Furthermore, methylated MYPT1 polypeptide was identified as a novel substrate for LSD1 polypeptide. Accordingly, the kit that contains methylated MYPT1 polypeptide as a substrate for LSD1 polypeptide is useful for screening for candidate of anti-cancer agents.

While the invention has been described in detail and with reference to specific
embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. 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.

Claims

A method of detecting or diagnosing cancer in a subject, comprising determining an expression level of LSD1 gene in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates the presence of cancer in said subject or that said subject suffers from cancer, wherein the expression level is determined by a method selected from the group consisting of:
(a) detecting a mRNA of LSD1 gene;
(b) detecting a protein encoded by LSD1 gene; and
(c) detecting a biological activity of a protein encoded by LSD1 gene.
The method of claim 1, wherein said increase is at least 10% greater than said normal control level.
The method of claim 1, wherein the subject-derived biological sample is selected from the group consisting of biopsy, sputum, blood, pleural effusion and urine.
A kit for detecting or diagnosing cancer, which comprises a reagent selected from the group consisting of:
(a) a reagent for detecting a mRNA of LSD1 gene;
(b) a reagent for detecting a protein encoded by LSD1 gene; and
(c) a reagent for detecting a biological activity of a protein encoded by LSD1 gene.
The kit of claim 4, wherein the reagent is a probe or primers to a gene transcript of LSD1 gene.
The kit of claim 4, wherein the reagent is an antibody against a protein encoded by LSD1 gene.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a LSD1 polypeptide;
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) selecting the test substance that binds to the polypeptide.
A method of screening for a candidate substance for treating or preventing cancer or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene;
(b) detecting the expression level of LSD1 gene in the cell; and
(c) selecting the test substance that reduces the expression level of LSD1 gene in comparison with the expression level in the absence of the test substance.
A method of screening for a candidate substance for treating or preventing cancer or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a test substance with a LSD1 polypeptide;
(b) detecting a biological activity of the polypeptide of step (a); and
(c) selecting the test substance that suppresses the biological activity of the polypeptide in comparison with the biological activity detected in the absence of the test substance.
The method of claim 9, wherein the biological activity is cell proliferation enhancing activity, MYPT1-binding activity or demethylase activity.
A method of screening for a candidate substance for treating or preventing cancer or inhibiting 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 LSD1 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 of said reporter gene; and
(c) selecting the test substance that reduces the expression or activity level of said reporter gene, in comparison with the level in the absence of the test substance.
A method of screening for a candidate substance for treating or preventing cancer or inhibiting cancer cell growth, the method including steps of:
(a) contacting a test substance with a cell expressing LSD1 gene and a downstream gene selected from the genes shown in Table 2 and Table 3;
(b) detecting the expression level of the downstream gene; and
(c) selecting the test substance that alters the expression level of the downstream gene in comparison with the expression level detected in absence of the test substance.
The method of claim 12, wherein the downstream gene of LSD1 gene is selected from the genes shown in Table 2 and step (c) comprises selecting the test substance that reduces the expression level of the downstream gene in comparison with the expression level detected in absence of the test substance.
The method of claim 12, wherein the downstream gene of LSD1 gene is selected from the genes shown in Table 3 and step (c) comprises selecting the test substance that increases the expression level of the downstream gene in comparison with the expression level detected in absence of the test substance.
An isolated double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, the molecule comprising a sense strand and an antisense strand complementary thereto, the strands hybridized to each other to form the double-stranded molecule, wherein the sense strand comprises the nucleotide sequence corresponding to a target sequenceof SEQ ID NO: 17 or 19.
The double-stranded molecule of claim 15, wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule, wherein said double-stranded molecule is between 19 and 25 nucleotide pair in length.
The double-stranded molecule of claim 15, which consists of a single polynucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
The double-stranded molecule of claim 17, having the general formula 5'-[A]-[B]-[A']-3', or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence of SEQ ID NO: 17 or 19, [B] is an intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is an antisense strand including a complementary sequence to [A].
A vector encoding the double-stranded molecule of any one of claims 15 to 18.
Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 17 or 19 and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vector, when introduced into a cell expressing LSD1 gene, inhibits cell proliferation.
A method of either of treating or preventing cancer, or both, or inhibiting cancer cell growth in a subject, comprising administering to a subject a pharmaceutically effective amount of a double-stranded molecule directed against a LSD1 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, the molecule comprising a sense strand and an antisense strand complementary thereto, the strands hybridized to each other to form the double-stranded molecule.
The method of claim 21, wherein the double-stranded molecule is that of any one of claims 15 to 18.
A composition, which comprises a pharmaceutically effective amount of a double-stranded molecule directed against a LSD1 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits in vivo expression of a LSD1 gene and cell proliferation, the molecule comprising a sense strand and an antisense strand complementary thereto, the strands hybridized to each other to form the double-stranded molecule, and a pharmaceutically acceptable carrier for a purpose selected from the group consisting of:
(a) treating cancer,
(b) preventing cancer,
(c) inhibiting cancer cell growth, and
(d) combinations thereof.
The composition of claim 23, wherein the double-stranded molecule is that of any one of claims 15 to 18.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a polypeptide comprising a MYPT1-binding domain of a LSD1 polypeptide with a polypeptide comprising a LSD1-binding domain of a MYPT1 polypeptide in the presence of a test substance;
(b) detecting a binding between the polypeptides;
(c) comparing the binding level detected in the step (b) with that detected in the absence of the test substance; and
(d) selecting the test substance that inhibits the binding between the polypeptides as a candidate substance for treating or preventing cancer.
The method of claim 25, wherein the polypeptide comprising the MYPT1-binding domain comprises residues 290-524 of SEQ ID NO: 22 or residues 271-500 of SEQ ID NO: 24.
The method of claim 25, wherein the polypeptide comprising the LSD1-binding domain
comprises residues 1-413 of SEQ ID NO: 29.
A method for identifying a substance that modulates the demethylation level of a MYPT1 polypeptide by a LSD1 polypeptide, said method comprising the steps of:
(a) contacting a LSD1 polypeptide or a functional equivalent thereof with a methylated MYPT1 polypeptide or a functional equivalent thereof in the presence of a test substance under a condition suitable for demethylation of the methylated MYPT1 polypeptide or the functional equivalent;
(b) detecting the demethylation level of the MYPT1 peptide; and
(c) selecting the test substrate that modulates the demethylation level of the MYPT1 polypeptide or the functional equivalent thereof in comparison with the demethylation level in the absence of the test substance.
The method of claim 28, wherein the functional equivalent of the methylated MYPT1 polypeptide comprises a fragment of the MYPT1 polypeptide having a methylated lysine residue corresponding to lysine 442 of the amino acid sequence of SEQ ID NO: 26.
The method of claim 28 or 29, wherein the methylated MYPT1 polypeptide or the functional equivalent thereof is prepared by methylating a MYPT1 polypeptide or a functional equivalent thereof by a SETD7 polypeptide.
The method of claim 28, wherein step (a) comprises incubating a MYPT1 polypeptide or a functional equivalent thereof and a SETD7 polypeptide in the presence of a methyl donor to form an incubation mixture, and simultaneously or subsequently adding a LSD1 polypeptide to the incubation mixture.
The method of claim 31, wherein the methyl donor is S-adenosyl methionine.
A kit for measuring a demethylase activity of a LSD1 polypeptide, wherein the kit comprises the following components (a) and (b):
(a) a component selected from the group consisting of (i) and (ii):
(i) a methylated MYPT1 polypeptide or a functional equivalent thereof;
(ii) a MYPT1 polypeptide or a functional equivalent thereof capable of being methylated by a SETD7 polypeptide, a SETD7 polypeptide and a methyl donor; and
(b) a reagent for detecting the demethylation level of the MYPT1 polypeptide or the functional equivalent thereof.
A kit for detecting for the ability of a test substance to inhibit demethylation of MYPT1polypeptide by a LSD1 polypeptide, wherein the kit comprises the following components (a) (b) and (c):
(a) a LSD1 polypeptide or a functional equivalent thereof;
(b) a component selected from the group consisting of (i) and (ii):
(i) a methylated MYPT1 polypeptide or a functional equivalent thereof;
(ii) a MYPT1 polypeptide or a functional equivalent thereof, a SETD7 polypeptide and a methyl donor; and
(c) a reagent for detecting the demethylation level of the MYPT1 polypeptide or the
functional equivalent thereof.
The kit of claim 33 or 34, wherein the functional equivalent of the methylated MYPT1 polypeptide comprises a fragment of the MYPT1 polypeptide having a methylated lysine residue corresponding to the lysine 442 of the amino acid sequence of SEQ ID NO: 26.
The kit of any one of claims 33 to 35, wherein the methyl donor is S-adenosyl methionine.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) identifying a test substance that modulates demethylation level of a MYPT1 polypeptide by a LSD1 polypeptide using the method of claim 28; and
(b) selecting the test substance that decreases the demethylation level of the MYPT1 polypeptide in comparison with the demethylation level in the absence of the test substance.
A method of screening for a candidate substance for treating or preventing cancer, or
inhibiting cancer cell proliferation, said method comprising the steps of:
(a) contacting a test substance with a cell expressing LSD1 gene, MYPT1 gene and RB1 gene;
(b) detecting at least one level selected from the group consisting of:
i) the phosphorylation level of a RB1polypeptide;
ii) the MYPT1 polypeptide level; and
iii) the ubiquitination level of a MYPT1polypeptide.
(c) selecting the test substance that has at least one property selected from the group consisting of:
i) decreasing the phosphorylation level of the RB1polypeptide;
ii) increasing the MYPT1 polypeptide level; and
iii) decreasing the ubiquitination level of the MYPT1 polypeptide,
in comparison with the control level detected in absence of the test substance.
The method of claim 38, wherein the phosphorylation level of the RB1 polypeptide is
detected by an antibody against phospholylated RB1 (Ser807/811).