WO2008038832A1 - Agent thérapeutique, procédé de détection et kit de détection pour cancer du sein et cancer ovarien - Google Patents

Agent thérapeutique, procédé de détection et kit de détection pour cancer du sein et cancer ovarien Download PDF

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Publication number
WO2008038832A1
WO2008038832A1 PCT/JP2007/069413 JP2007069413W WO2008038832A1 WO 2008038832 A1 WO2008038832 A1 WO 2008038832A1 JP 2007069413 W JP2007069413 W JP 2007069413W WO 2008038832 A1 WO2008038832 A1 WO 2008038832A1
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prdm14
gene
cancer
breast cancer
expression level
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PCT/JP2007/069413
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English (en)
French (fr)
Japanese (ja)
Inventor
Minoru Toyota
Takashi Tokino
Kouichi Hirata
Noriko Nishikawa
Tousei Ohmura
Kohzoh Imai
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Sapporo Medical University
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Priority to JP2008536470A priority Critical patent/JP5219818B2/ja
Publication of WO2008038832A1 publication Critical patent/WO2008038832A1/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a therapeutic agent for breast cancer and ovarian cancer, a method for detecting breast cancer and ovarian cancer, and a kit for detecting breast cancer and ovarian cancer.
  • Histone modification is important as a regulatory mechanism of gene expression.
  • histone methyl ⁇ plays an important role in gene activation and inactivation (see Fig. 1).
  • Proteins with histone methylhi activity are SET (Suvar3-9, Enhancer-of-zeste, Tri thorax) domain, or PR (PRDI-BF1 and RIZ) with 20-30% homology with this SET domain. It is suggested to have a domain (see Figure 2).
  • SET Sud3-9, Enhancer-of-zeste, Tri thorax
  • PR PRDI-BF1 and RIZ
  • SMYD3 (SET and MYND domain-containing protein 3) has been shown to have a histone methyl enzyme activity, and the expression of the SMYD3 gene has increased in large intestine cancer and liver cancer. This has been shown to be involved in canceration and cancer cell proliferation (see Non-Patent Document 1). Furthermore, the polymorphism of the CCGCC sequence in the SMYD3 promoter region is also associated with carcinogenesis in colorectal cancer, liver cancer and breast cancer. It has been shown to be involved in risk (see Non-Patent Document 2).
  • EZH2 (enhaner of zeste homolog 2) with SET domain, which is known as histone methylase involved in methylone of histone H3 lysine 27, is clearly expressed in prostate cancer.
  • SET domain which is known as histone methylase involved in methylone of histone H3 lysine 27.
  • histone methylase in cancer is a molecular mechanism of carcinogenesis. It has been thought to be important not only for elucidating the structure, but also for obtaining basic knowledge on the development of new cancer diagnosis and treatment methods.
  • PRDM PR-domain containing protein
  • PRDM2 gene and PRDM5 gene function as a tumor suppressor gene. It is speculated that the functions of all other proteins belonging to the PRDM family have not been elucidated yet, and the role in cancer is not known at present. Reference list
  • Non-patent literature 1 Hatnamoto R et al., Nature Cell Biology, 6: 731-40, 2004
  • Non-patent literature 2 Tsuge M et al., Nature Genetics, 37: 1104-7, 2005
  • Non-patent literature 3 Vararabally S et al., ature, 419: 624-9, 2002
  • Non-Patent Document 4 Vire E et al., Nature, 439: 871-4, 2006
  • Non-patent literature 5 Keller A. D. et al., Genes & Development, 5: 868, 1991 Non-patent literature 6. Turner, C. A. et al., Cell, 77: 297-306, 1994
  • Non-patent literature 7 Du Y et al., Cancer Research, 61: 8094-9, 2001
  • Non-patent literature 8 Tokumaru Y et al., Oncogene, 22: 6954-8, 2003
  • Non-Patent Document 9 Deng Q et al., Oncogene, 23: 4903-10, 2004 Disclosure of Invention
  • the task of the present invention is to elucidate the role of the PRDM family in cancer Means for Solving the Problems in Providing Novel Cancer Therapeutic Agents and Methods for Detecting Cancer
  • the present inventors have conducted intensive studies to solve the above problems, and among the PRDM families, the expression of the gene encoding PRDM14 is more specific in breast cancer and ovarian cancer than in normal tissues.
  • the increase in the specific expression of the PRDM14 gene is due to gene amplification, and the expression of PRDM14 is involved in the proliferation of cancer cells and the decreased sensitivity to anticancer drugs. I also found out.
  • PRDM14 gene expression inhibitors and PRDM inhibitors are effective in the treatment of breast cancer or ovarian cancer, and the expression level of PRDM gene is also increased. It was discovered that breast cancer and ovarian cancer can be detected by quantification, and the present invention has been completed.
  • the present invention relates to a therapeutic agent for breast cancer or ovarian cancer comprising a PRDM14 gene expression inhibitor.
  • the present invention also relates to the therapeutic agent selected from antisense, siRA or shRA that inhibits expression of the PRDM14 gene. Furthermore, the present invention provides an antisense ability to inhibit the expression of the PRDM14 gene.
  • the present invention also relates to the therapeutic agent, which is an antisense polynucleotide containing a base sequence complementary to the base sequence of the polynucleotide to be coded or a part thereof.
  • the present invention still further provides an siRNA against a peptide whose siRNA or shRNA that inhibits the expression of the PRDM14 gene has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a peptide that is a part thereof. Or it is shRNA, It is related with the said therapeutic agent.
  • the present invention also relates to a therapeutic agent for breast cancer or ovarian cancer comprising an inhibitor of PRDM14.
  • the present invention also relates to the above therapeutic agent, wherein the inhibitor of PRDM14 is an anti-PRDM14 antibody or a fragment thereof. Furthermore, the present invention also relates to a method for detecting breast cancer or ovarian cancer in a subject based on the expression level of PRDM14 gene in a biological sample obtained from the subject. Further, the present invention provides an internal standard in a biological sample obtained from a healthy subject of the same kind as the subject, wherein the ratio of the expression level of the PRDM14 gene to the expression level of the internal standard gene in the biological sample obtained from the subject is It relates to the method, wherein breast cancer or ovarian cancer is detected on the basis that the expression level of the PRDM14 gene is higher than the expression level of the gene by at least 5 times.
  • the present invention further relates to the aforementioned method, wherein the expression level of the PRDM14 gene is quantified based on the expression level of mRNA encoding PRDM14.
  • the present invention also relates to the above method, wherein the expression level of mRNA encoding PRDM14 is quantified by reverse transcription polymerase chain reaction.
  • the present invention provides a sense primer wherein the reverse transcription polymerase chain reaction includes a part of a base sequence that is the same or substantially the same as the base sequence represented by SEQ ID NO: 2, and the base sequence represented by SEQ ID NO: 2
  • the method is carried out using an antisense primer containing a part of a complementary base sequence in the same or substantially the same base sequence.
  • the present invention relates to a sense primer containing a part of the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 2, and the same or substantially identical to the base sequence represented by SEQ ID NO: 2.
  • the present invention also relates to a breast cancer or ovarian cancer detection kit for performing the above method, comprising an antisense primer containing a part of the base sequence complementary to the base sequence.
  • the present invention relates to the above method, wherein the expression level of PRDM14 gene is quantified based on the expression level of PRDM14.
  • the present invention also relates to the above method, wherein the expression level of PRDM14 is quantified by an antigen antibody reaction using an anti-PRDM14 antibody or a fragment thereof.
  • the present invention also relates to a kit for detecting breast cancer or ovarian cancer for performing the above-described method, which comprises an anti-PRDM14 antibody or a fragment thereof.
  • the invention's effect The therapeutic agent of the present invention comprises a PRDM14 gene expression inhibitor or a PRM14 inhibitor. Therefore, this therapeutic agent is induced by an anticancer agent by suppressing the growth of cancer cells and increasing the sensitivity to the anticancer agent through PR 14 gene expression inhibitory action or PRDM14 inhibitory action. It is possible to enhance apoptosis of cancer cells and obtain an excellent therapeutic effect. Further, by using the therapeutic agent of the present invention together with an anticancer agent having a cell growth inhibitory effect, a synergistic effect can be obtained in the therapeutic effect of cancer, and a good prognosis can be expected.
  • the PRDM14 gene or PRDM14 which is the target of the therapeutic agent of the present invention, is specifically highly expressed in breast cancer tissues and ovarian cancer tissues, while the expression level in normal tissues is extremely small.
  • the therapeutic agent can exert its effect specifically in the target cancer tissue that requires treatment, is safe with few side effects, and has a high life.
  • the PRDM14 gene When using anti-sense, siRNA or shRNA against PRDM14 gene as an expression inhibitor, or when using PRDM14 antibody as a PRDM14 inhibitor, the specificity to PRDM14 gene or PRDM14 is extremely high. The possibility of undesired effects on proteins and proteins is extremely low, and side effects are reduced, resulting in high safety.
  • the detection method and detection kit for breast cancer or ovarian cancer according to the present invention are specifically elevated in the PRDM14 gene expression level S, breast cancer tissue and ovarian cancer tissue, which are detection targets, but low in normal tissues Therefore, it is possible to detect breast cancer and ovarian cancer with good accuracy, and contribute to early detection of these cancers.
  • FIG. 1 is a schematic diagram showing the function of histone methylase.
  • FIG. 2 is a schematic diagram showing the structures of SMYD 3 having a SET domain, and PRDM1 and PRDM2 having a PR domain.
  • FIG. 3A is a view showing expression profiles of PRDM1-15 genes in cancer tissues of breast cancer patients case 8, 9, 87, and 96.
  • FIG. 3B is a diagram showing expression profiles of PRDM1-15 genes in cancer tissues of breast cancer cases 7-9, 19, 20, 87, 96, and 11 12.
  • Figure 4A shows a number of clinical cases (Normal breast, Non-invasive ductal carcinoma (DCIS), Breast cancer positive), (Normal stomach; and Gastric 3 ⁇ 4 (Gastric cancer)). It is a figure which shows the expression level of PRDM14 gene.
  • Figure 4B shows various ovarian cancer cell lines (SK0V-3, 0VCA-3, PA-1, Caov-3, MH, KUM, AM0C-2, MCAS, KF, KFr, HTB0A, TOV-21G, SW626 N T0V112D. And OV-90), normal ovary, and teratocarcinoma cell line (NCC-IT-A3) showing PRDM14 gene expression levels.
  • FIG. 5 is a diagram showing immunostaining of PRDM14 in cancer tissues of breast cancer patients case 86, 92, 96, and 105.
  • Figure 6 shows the relationship between the presence or absence of gene amplification of genes located on chromosome 8ql3.3 (SLC05A1, PRDM14, LACTB2, EYA1, and TPD52) in each breast cancer patient and the expression level (mRNA expression level) of the PRDM14 gene. is there.
  • Figure 7 shows the gene expression levels (mRNA expression levels) of the genes located on chromosome 8ql3.3 (SLC05A1, SUL Fl, PRDM14, NC0A2, T-fraction 1, LACTB2, XKR9, EYAl) in each breast cancer patient and the PRDM14 gene. It is a figure which shows the relationship with amplification.
  • FIG. 8 shows the intracellular localization of PRDM14.
  • FIG. 9A is a diagram showing colon formation by expression of PRDM14 in breast cancer cell lines MCF7 and SKBr-3.
  • FIG. 9B is a graph showing an increase in the number of colodies by PRDM14 expression in the random cancer cell lines MCF7 and SKBr-3.
  • FIG. 10 is a graph showing a decrease in sensitivity to an anticancer agent (CDDP, VP-16, adriamycin or docetaxel) in a breast cancer cell line SKBr-3 stably expressing PRDM14.
  • FIG. 11 shows the gene transfer efficiency in the ovarian cancer cell line PA-1.
  • FIG. 12 shows suppression of PRDM14 gene expression in PA-1 cells into which siRNA for PRDM14 has been introduced.
  • FIG. 14 is a graph showing the enhancement of apoptosis induced by treatment with an anticancer agent (CDDP or docetaxenore) in PA-1 cells into which siRNA for PRDM14 has been introduced.
  • subject in the present invention means any individual organism, preferably It is a vertebrate, more preferably a mammal, and more preferably a human individual.
  • the ability of a subject to be healthy or to be affected by any disease When treatment for breast cancer or ovarian cancer is intended, the subject or experiment suffering from the disease
  • Afflicted subjects such as rodents such as mice, rats, gerbils, guinea pigs, felines such as cats, pumas and tigers, deer such as deer and deer, etc.
  • Mink, Hidge, Goat, Ussi, Uma, Monkey, Human etc. are preferred! /.
  • PRDM14J simply means a protein that is PRDM14 (PR domain-containing protein 14) itself
  • PRDM14 gene means that PRDM14 It means a gene to be coded
  • rpRDMHj also means a gene encoding PRDM14.
  • PRDM14 used in the present invention is a protein having a PR domain and a zinc finger domain called PR domain-containing protein 14 (also known as PFMll or MGC59730) .For example, it is identical to the amino acid sequence represented by SEQ ID NO: 1.
  • examples include proteins containing one or substantially the same amino acid sequence.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more, of the amino acid sequence represented by SEQ ID NO: 1, More preferred examples include amino acid sequences having a homology of about 90% or more, more preferably about 95% or more, and most preferably about 98% or more.
  • the PRDM14 gene used in the present invention specifically includes, for example, DNA containing the base sequence represented by SEQ ID NO: 2 or the base sequence represented by SEQ ID NO: 2 under stringent conditions.
  • DNA that encodes a protein that contains a base sequence to be hyperlysed and that has substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is included.
  • sequence number The DNA containing the base sequence represented by No. 2 and the base sequence that specifically hybridizes under stringent conditions includes, for example, about 60% or more, preferably about 70% of the base sequence represented by SEQ ID NO: 2.
  • stringent conditions are usually conditions of 42 ° C, 2 XSSC and 0.1% SDS, preferably, conditions of 65 ° C, 0.1 XSSC and 0.1% SDS. It is.
  • a therapeutic agent for breast cancer or ovarian cancer comprising an PRDM14 gene expression inhibitor or an PRDM14 inhibitor is provided.
  • breast cancer refers to a malignant tumor that occurs in the mammary gland, fat layer, and skin constituting breast tissue, and is classified into invasive breast cancer, non-invasive breast cancer, and Paget's disease. Including.
  • ovarian cancer refers to a malignant tumor that occurs in the surface epithelium, sex stroma, and embryonic cells constituting ovarian tissue, and epithelial ovarian cancer, sex cord stromal oocyte cancer Including those classified as germ cell ovarian cancer.
  • the PRDM14 gene expression inhibitor used in the therapeutic agent of the present invention is not particularly limited as long as it can inhibit the expression of the PRDM14 gene, and transcription of PRDM14 gene into mRNA or from mRNA to protein.
  • examples thereof include compounds that inhibit the expression of polynucleotides encoding such peptides or salts thereof.
  • the “polynucleotide that codes for a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a peptide that is a part thereof” may be DNA. It ’s RNA.
  • PRDM14 gene expression inhibitors include antisense that specifically inhibits PRDM14 gene expression, siRNA (small interfering RNA) or shRNA (short hairpin RNA), and anti-blocking PRDM14 gene expression.
  • a preferred example of the sense is a nucleotide sequence of a polynucleotide that codes for a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a peptide that is a part thereof.
  • antisense polynucleotides may be DNA or RNA.
  • siRNA or shRNA that specifically inhibits the expression of the PRDM14 gene is a protein or a part thereof having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. SiRNA or shRNA against the peptide.
  • the siRNA used in the present invention is a polynucleotide that encodes a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a peptide that is a part thereof (for example, , Preferably comprising a sense strand sequence of 18 to 28 nucleotides in succession from the mRNA of PRDM14 and an antisense strand sequence which is a complementary sequence thereof. It is particularly preferred that it contains a sense strand sequence of ⁇ 22 nucleotide nucleotides and an antisense strand sequence that is a complementary sequence thereof.
  • suitable siRNA sequences eg, Dykxhoorn DM et al., Nature Rev. Mol. Cell Biol. 4; 457-67 (2003); Khvorova A et al , Cell, 115: 209-16 (2003), etc.).
  • siRNA sequences (combination of sense strand and antisense strand) in the present invention are shown below.
  • Antisense 5, -UAGGUCUUCACUUCACUGGTT-3 '(SEQ ID NO: 4)
  • Antisense 5, -GAUCUGAGAAGAUUUUCCCTT-3 '(SEQ ID NO: 8)
  • the selected siRNA sequence further comprises deletion, substitution or attachment of 1 to 3, preferably 1 to 2 nucleotides in the sense strand sequence, so long as the target mRNA can be cleaved. It may be mutated to include cocoons.
  • the mutation is preferably located on the 5th side, since a mutation on the 3 'side from the center tends to cause inactivation.
  • the selected siRNA does not show a so-called off-target effect (effect of suppressing the expression of a gene other than the target gene partially homologous to the used siRNA) in clinical use. It is preferable. Therefore, in order to avoid the off-target effect, it is desirable to confirm that there is no cross-reaction for candidate siRNA in advance using a gene chip or the like.
  • the shRNA that can be used in the present invention includes a single-stranded loop sequence that covalently binds between the sense strand sequence and the antisense strand sequence thereto, and is an intracellular RNase D icer. Is an RNA that is processed to form siRNA.
  • a poly-T sequence consisting of 1 to 6, preferably 1 to 5, T at the end of the hairpin type DNA encoding siRNA, as a transcription termination signal sequence or for overhang.
  • TTTT or TTTTT consisting of 4 or 5 Ts is connected.
  • the shRNA as a siRNA precursor transcribed from the vector DNA preferably has an overhang consisting of 2 to 4 U at the 3 'end of its antisense strand.
  • RNA and antisense RNA can be more stable against degradation by nucleases.
  • a known loop sequence can be appropriately used as the single-stranded loop sequence.
  • Another embodiment of the siRNA in the present invention is formed from tandem DNA, which comprises a DNA sequence encoding the sense strand and a DNA sequence encoding the antisense strand, 5 ′ ⁇ 3 ′.
  • Sense RNA that is generated in the same direction after transcription in the cell, consisting of a sequence that includes a promoter at the 5 'end of each strand and a poly T sequence at the 3' end of each strand.
  • the poly-T sequence is preferably composed of 1 to 5, particularly 4 to 5 T as a transcription stop signal sequence.
  • the generated siRNA may have an overhang consisting of 2 to 4 U at the 3, terminal ends of the sense and Z or antisense strands.
  • antisense RNA or siRNA that specifically inhibits the expression of the PRDM14 gene can be formulated by a known method and administered to the subject.
  • antisense RNA or siRNA that specifically inhibits the expression of the PDM14 gene can be directly introduced into the target tissue, breast cancer tissue or ovarian cancer tissue.
  • Antisense DNA that specifically inhibits expression of the PRDM14 gene can be incorporated into the downstream of an appropriate promoter sequence and administered to a subject as an antisense RNA expression vector, for example.
  • antisense RNA expression vectors, siRNA expression vectors, and shRNA expression vectors that specifically inhibit the expression of the PRDM14 gene can be prepared and administered to the subject. That is, in the present invention, an antisense RNA, siRNA or DNA encoding shRNA that specifically inhibits expression of the PRDM14 gene is incorporated into an expression vector, and the expression of the PRDM14 gene is controlled under the control of an appropriate promoter.
  • Is transcribed into RNA that specifically inhibits Expression vectors used in the present invention include plasmid vectors and viral vectors.
  • Plasmid vectors can be prepared using known methods, and are also commercially available vectors such as pi GENE (registered trademark) U6 vector, pi GENE (registered trademark) HI vector, pi GENE (registered trademark).
  • tRNA vector Tekarabio Co., Ltd.
  • a plasmid vector contains a drug resistance gene, a transcription termination sequence, a restriction enzyme cleavage site, a replication origin, etc., in addition to the DNA sequence encoding the antisense RNA of the present invention, siRNA, and shRNA. Can be included.
  • the vi / less vector for example, an adenovirus vector, an adeno-associated winores betater, a lentiwinores vector, a retrowinole vector, a herpesvirus vector, and the like can be used. It is preferable that the viral vector has been modified so as to lack self-replication ability. Methods for producing viral vectors are known to those skilled in the art.
  • the virus vector can be introduced into cells by directly injecting betater into the affected area and infecting the cells.
  • the PRDM14 inhibitor used in the therapeutic agent of the present invention is not particularly limited as long as it can specifically inhibit PRDM14.
  • the amino acid sequence represented by SEQ ID NO: 1 examples thereof include compounds containing the same or substantially the same amino acid sequence, or compounds that inhibit the function of peptides that are parts thereof, or salts thereof.
  • a preferred example of an inhibitor of PRDM14 is an anti-PRDM14 antibody or a fragment thereof.
  • the anti-PRDM14 antibody is not limited as long as it specifically recognizes PRDM14, and even a monoclonal antibody is a polyclonal antibody. It may be a cell-cell-expressing antibody (intrabody), a humanized antibody, or the like.
  • fragment includes any fragment as long as it specifically recognizes the target PRDM14.
  • the antibody fragment include an Fab fragment, an F (ab) ′ 2 fragment, and a single chain.
  • An antibody (scFv) etc. are mentioned.
  • anti-PRDM14 antibody or a fragment thereof a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a peptide that is a part thereof is used. Antibodies or fragments thereof that specifically bind are included.
  • the anti-PRDM14 antibody or fragment thereof used in the present invention can be formulated by a known method and administered to a subject.
  • target PRDM14 When producing an antibody that specifically recognizes the target PRDM14, Part or all of the subject PRDM14 can be used. Specifically, (1) target cells, tissues or organs expressing PRDM14, or a purified product thereof, or (2) the target PRDM14 gene or a part thereof using insect recombination technology. Recombinant proteins or peptides introduced and expressed in host cells such as animal cells, (3) peptides that are chemically synthesized in full length or in part according to the amino acid sequence of PRDM14 of interest can be used. .
  • a protein other than the target PRDM14 can be used as an immunizing antigen.
  • a protein for example, PRDM14 derived from an animal species different from the target
  • a protein into which an amino acid sequence of this protein has been artificially introduced with mutations such as deletion, substitution, and calorie as an antigen for immunization. it can.
  • the position and number of mutations such as deletions, substitutions and additions introduced into PRDM14 are not particularly limited, but are preferably positions and numbers that retain the three-dimensional structure of PRDM14.
  • immunization antigens are used to immunize mammals such as rats, mice, guinea pigs, rabbits, hidges, horses, tusks and the like.
  • immunization in order to induce antibody production, it is preferable to immunize multiple times after emulsification using an immune assistant.
  • an immunity assistant Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide gel, and the like can be used.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • the dose of antigen per mammal can be set as appropriate according to the type of B
  • the administration site is, for example, intravenous, subcutaneous, intraperitoneal, or the like.
  • Immunization is usually performed at intervals of several 13 to several weeks, preferably at intervals of 4 days to 3 weeks, for a total of 2 to 8 times, preferably 2 to 5 times. Then, 5 to 10 days after the final immunization, the antibody titer against PRDM14 is measured, and after the antibody titer has increased, blood is collected to obtain an antiserum.
  • the antibody titer can be measured by ELISA (enzyme linked immunosorbent assay) radioimmunoassay or the like.
  • a known method such as salting out with ammonium sulfate, gel / chromatography, ion exchange chromatography, affinity chromatography, etc. is appropriately selected or combined. Use can do.
  • antibody-producing cells When preparing monoclonal antibodies, immunize mammals with immunizing antigens as in polyclonal antibodies, and collect antibody-producing cells 2-5 days after the final immunization.
  • antibody-producing cells include spleen cells, lymph node cells, thymocytes, and peripheral blood cells. Generally, spleen cells are used.
  • cell fusion between antibody-producing cells and myeloma cells is performed in order to obtain a hyperidoma.
  • myeloma cells to be fused with antibody-producing cells cell lines derived from mammals such as humans and mice and generally available can be used.
  • the cell line used has drug selectivity and cannot survive in a selective medium (for example, HAT medium) in a non-fused state, but can survive when fused with antibody-producing cells. Those having properties are preferred.
  • antibody-producing cells and myeloma cells are mixed at a predetermined ratio (for example, 1: 1 to 1:10 in a cell culture medium such as serum-free DM EM, R PM I-1640 medium, etc. ), And a fusion reaction is carried out in the presence of a cell fusion promoter such as polyethylene glycol or by electric pulse treatment (for example, electroporation).
  • a cell fusion promoter such as polyethylene glycol or by electric pulse treatment (for example, electroporation).
  • the cells are cultured in a selective medium and the target hyperpridoma is selected.
  • the culture supernatant of the grown hyperidoma is screened for the presence of the antibody of interest.
  • Hypridoma screening may be carried out in accordance with ordinary methods, and is not particularly limited. For example, a part of the culture supernatant of hyperidoma is collected, and ELISA or radioimmunoassay is used. Can be screened.
  • cloning of a hyperidoma can be performed by, for example, a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, or the like, and finally a hyperidoma that produces a monoclonal antibody is obtained.
  • Monoclonal antibodies can be collected from the resulting hyperidoma according to a conventional method. For example, hybridomas containing 10-20% fetal bovine serum RPM I-1640 medium, MEM medium, etc. in normal culture conditions (eg 37 ° C, 5% CO 2 concentration) By culturing for 3 to 10 days, a monoclonal antibody can be obtained from the culture supernatant. Hypridoma Can be transplanted into the abdominal cavity of a mouse or the like, and ascites can be collected 10 to 14 days later, and a monoclonal antibody can be obtained from the ascites.
  • PRDM14 gene expression inhibitor or PRDM14 inhibitor is a pharmaceutically acceptable carrier (eg, excipient, binder, disintegrating agent, lubricant, stabilizer, preservative, pH adjustment).
  • a pharmaceutically acceptable carrier eg, excipient, binder, disintegrating agent, lubricant, stabilizer, preservative, pH adjustment.
  • the therapeutic agent of the present invention may contain a label or nanocapsule that can be specifically delivered to a target tissue.
  • the therapeutic agent of the present invention may contain both an PRDM14 gene expression inhibitor and a PRDM14 inhibitor.
  • the therapeutic agent of the present invention is a known anticancer agent effective for the treatment of breast cancer or ovarian cancer (e.g., fluorouracil, tamoxifen, anastomasol, aclarubicin, doxorubicin, tegafur, cyclophosphamide, irinotecan, cytarabine, paclitaxel, docetaxel, May further contain other medicinal ingredients such as epilubicin, carbobratin, cisplatin, thiotepa, or pharmaceutically acceptable salts thereof, and can also be used in combination with these active ingredients.
  • a known anticancer agent effective for the treatment of breast cancer or ovarian cancer e.g., fluorouracil, tamoxifen, anastomasol, aclarubicin, doxorubicin, tegafur, cyclophosphamide, irinotecan, cytarabine, paclitaxel
  • Examples of the administration route of the therapeutic agent of the present invention include oral administration and parenteral administration (for example, intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, local administration, etc.).
  • Dosage forms include sprays, capsules, tablets, granules, syrups,?
  • Examples include L agents, suppositories, injections, and suspensions.
  • the affected area can be exposed by surgical operation, and the therapeutic agent of the present invention can be directly administered to the cancer tissue by means of a syringe or the like. Can be done by administration.
  • a preferred route of administration is topical administration.
  • the dose and the number of doses vary depending on the intended effect, administration method, treatment period, age, weight, sex, etc. of the subject
  • the dose is Adults are usually 1 0 0 / Z g ⁇ l 0 per day O mg, preferably 1 to 20 mg, can be appropriately selected, and the dosage when using antisense DNA, antisense RNA, siRNA, shRA or a vector expressing these to the PRDM14 gene is
  • the subject is a human, it can be appropriately selected from the range of 0.0 lmg / kg to: L g / kg, preferably 0.1 lm gZk g to 50 O mg Gkg per adult.
  • the number of times can be appropriately selected from a range of once to several times a day. However, since the dose may vary depending on various conditions, it is not limited to the above range.
  • the present invention further provides a method for detecting breast cancer or ovarian cancer in a subject based on the expression level of PRDM14 gene in a biological sample obtained from the subject.
  • the “biological sample obtained from the subject” in the present invention is not particularly limited, for example, blood, serum, ascites, or mammary gland, fat layer or skin constituting the breast, secreted from the mammary gland Secretions produced from the mammary gland (ductal fluid), or the surface epithelium, sex stroma, or embryonic cells constituting the ovary can be used.
  • the expression level of PRDM14 gene is generally low in any normal tissue, and the expression level is increased in breast cancer tissue or ovarian cancer tissue.
  • the biological sample obtained from the subject constitutes the breast.
  • any biological sample collected by any method can be used in the method of the present invention.
  • the biological sample may be collected by surgery, and may be collected by a puncture needle for tissue collection. It may be collected, or it may be collected as a secretion secreted from the mammary gland or a lavage fluid secreted from the mammary gland (ductal fluid).
  • M NA encoding PRDM14 Includes a polynucleotide RNA encoding a protein having the amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a peptide that is a part thereof.
  • the expression level of the PRDM14 gene is expressed using a gene whose expression level does not vary greatly between tissues or individuals (for example, housekeeping genes such as] 3-actin gene and GAPDH gene) as an internal standard gene. It is preferable to correct based on the amount.
  • detecting breast cancer or ovarian cancer on the basis of the expression level of the PRDM14 gene means, for example, the ratio of the expression level of the PRDM14 gene to the expression level of the internal standard gene in the biological sample obtained from the subject.
  • a subject suffers from breast cancer or ovarian cancer if the expression level of the PRDM14 gene relative to the expression level of the internal standard gene in a biological sample obtained from a healthy subject of the same type as Is included.
  • the ratio of the expression level of the PRDM14 gene to the expression level of the internal standard gene in the biological sample obtained from the subject is the same as that in the biological sample obtained from the same healthy subject as the subject.
  • the standard is preferably at least 5 times more than the ratio of the expression level of the PRDM14 gene to the expression level of the internal standard gene, more preferably at least 10 times higher, and particularly preferably at least 10 times higher.
  • the standard is 0 times more.
  • a known technique such as a hybridization technique (a Northern hybridization method, a dot hybridization method, a dot hybridization method, RNase protection assay, cDNA microarray, etc.), gene amplification technology (reverse transcription polymerase chain reaction (RT-PCR) (including competitive RT-PCR, real-time PCR, etc.)) .
  • a hybridization technique a Northern hybridization method, a dot hybridization method, a dot hybridization method, RNase protection assay, cDNA microarray, etc.
  • gene amplification technology reverse transcription polymerase chain reaction (RT-PCR) (including competitive RT-PCR, real-time PCR, etc.)
  • a polynucleotide encoding PRDM14 or an oligonucleotide or a polynucleotide that can hybridize to a part thereof can be used as a probe.
  • the oligonucleotide is used.
  • Nucleotides or polynucleotides can be used as primers.
  • the “polynucleotide encoding PRDM14 or a part thereof” includes both DNA and RNA, and includes, for example, mRNA, cDNA, cRNA and the like.
  • the nucleotide constituting the oligonucleotide or polynucleotide may be any of deoxyliponucleotide and ribonucleotide.
  • the base length of the oligonucleotide to be used is not particularly limited, but is usually 15 to 100 bases, preferably 17 to 35 bases.
  • the base length of the oxide is not particularly limited, but is usually 50 to 1000 bases, preferably 150 to 500 bases.
  • the oligonucleotide or polynucleotide capable of hybridizing to the polynucleotide encoding PRDM14 or a part thereof can specifically hybridize under stringent conditions to the polynucleotide encoding PRDM14 or a part thereof.
  • the “stringent conditions” here are usually conditions of 4 2 ° C, 2xSSC and 0.1% SDS, preferably 65 ° C, conditions of 0.1xSSC and 0.1% SDS. It is. ⁇
  • nucleotide sequence of the oligonucleotide or polynucleotide that can hybridize to the polynucleotide encoding PRDM14 or a part thereof can be appropriately designed based on the known knowledge from the nucleotide sequence of the target PRDM14 gene. Labels such as fluorescent dyes and radioisotopes may be added to the primer and probe.
  • RT-PCR in the quantification of the expression level of mRNA encoding PRDM14 in a biological sample obtained from a subject, RT-PCR is among the above-mentioned various techniques from the viewpoint of accuracy and ease of operation. It is particularly preferable to use
  • a method for measuring the expression level of mRNA encoding PRDM14 in a biological sample obtained from a subject will be specifically described below using RT-PCR as an example.
  • RNA is extracted from a biological sample collected from the subject, and cDNA is synthesized from the extracted total RNA.
  • PCR is performed using primers that can hybridize to cDNA that encodes PRDM14 using the synthesized cDNA as a saddle. So Then, the amount of mRNA encoding PRDM14 can be measured by quantifying the PCR-amplified fragment amplified in PCR. At this time, it is desirable to perform PCR under conditions such that the amount of PCR amplified fragments produced reflects the amount of initial truncated cDNA (for example, the number of PCR cycles in which PCR amplified fragments increase exponentially).
  • a pair of primers specific for the gene encoding PRDM14 for example, a sense primer containing a part of a base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 2, and An antisense primer containing a part of a base sequence complementary to the base sequence identical or substantially the same as the base sequence represented by SEQ ID NO: 2 can be mentioned, and the base length of the primer is, for example, 17 to 3 5 bases, preferably 20 to 28 bases.
  • the method for quantifying the PCR amplified fragment is not particularly limited, and for example, a quantification method using a radioisotope (RI), a quantification method using a fluorescent dye, and the like can be appropriately used.
  • RI radioisotope
  • fluorescent dye a quantification method using a fluorescent dye
  • Examples of quantification methods using RI include: (1) RI-labeled nucleotide (eg, 32 P-labeled dCTP) is added to the reaction solution as a substrate and incorporated into the PCR-amplified fragment. After RI labeling and separating the PCR amplified fragment by electrophoresis, etc., the radioactivity is measured and the PCR amplified fragment is quantified. (2) The RI amplified primer is used to label the PCR amplified fragment.
  • RI-labeled nucleotide eg, 32 P-labeled dCTP
  • a method of quantifying the PCR amplified fragments by measuring the radioactivity (3) After electrophoresis of the PCR amplified fragments, plotting on a membrane and using a RI-labeled probe Examples include a method of quantifying PCR amplified fragments by hybridization and measuring radioactivity. Radioactivity can be measured using, for example, a liquid scintillation counter, an X-ray film, or an imaging plate.
  • Fluorescent dyes that interfer with double-stranded DNA for example, ethidium promide (EtBr), SYBR (registered trademark) Gr eenl
  • Fluorescent dyes staining the PCR amplified fragment using PicoGreen (registered trademark), etc., and measuring the fluorescence intensity emitted by excitation light irradiation and measuring the PCR amplified fragment
  • a primer labeled with Examples include a method in which the CR amplified fragment is labeled with a fluorescent dye, the PCR amplified fragment is separated by electrophoresis, and then the fluorescence intensity is measured to quantify the PCR amplified fragment.
  • the fluorescence intensity can be measured using, for example, a CCD camera, a fluorescence scanner, or a spectrofluorometer.
  • the anti-PRDM14 antibody is not limited as long as it specifically recognizes PRDM14, and may be a monoclonal antibody or a polyclonal antibody, an intracellular expression antibody (intrabody), human It may be an antibody. Also,
  • the “fragment” includes any fragment as long as it specifically recognizes the target PRDM14.
  • Examples of the antibody fragment include F ab fragment, F (ab) 2 fragment, single chain antibody (sc FV) and the like.
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • CLIA chemiluminescence immunoassay
  • FIA fluorescence immunoassay Law
  • a specific method for quantifying the expression level of PRDM14 in a biological sample obtained from a subject using the anti-PRDM14 antibody or a fragment thereof is, for example, an organism obtained from the subject using the anti-PRDM14 antibody or a fragment thereof.
  • the captured PRDM14 binds to the anti-PRDM14 antibody or fragment thereof having a binding site different from that of PRDM14 for the anti-PRDM14 antibody or fragment thereof.
  • It can be quantified using labeling substances that can be used (for example, enzymes such as peroxidase and alkaline phosphatase, fluorescent substances such as FITC and rhodamine, or antibodies (secondary antibodies) or substances labeled with RI). .
  • the expression level of PRDM14 in a biological sample obtained from a subject can also be quantified by measuring the activity of PRDM14 contained in the biological sample obtained from the subject.
  • the activity of PRDM14 can be measured according to a known method for hishistylase activity, transcription activation ability or transcription repression ability.
  • the expression level of PRDM14 gene increases with the progression of canceration or progression of breast tissue or ovarian tissue (ie, expression of PRDM14 gene in breast cancer cells or ovarian cancer cells). The level is higher than the expression level of the PRDM14 gene in normal cells), and the presence or absence of cancer cells in the sample is detected using the expression level of the PRDM14 gene in the biological sample collected from the subject as an index.
  • the expression level of PRDM14 gene in a biological sample collected from a subject is higher than the expression level of PRDM14 gene in a biological sample collected from a healthy subject of the same type as the subject, It can be determined that cancer cells are present in the collected biological sample.
  • the expression level of PRDM14 gene is higher than that in cancer cells, it can be determined that cancer cells are present in a biological sample collected from the subject's breast tissue or ovarian tissue.
  • the expression level of the PRDM14 gene between a subject and a healthy subject of the same kind as the subject it is preferable to use cells of the same tissue or organ as the biological sample.
  • the amount of PRDM14 gene expression in healthy subjects (healthy group) is quantified, the normal range is set from the distribution of the values, and the PRDM14 expression level in the subject is above the normal range and below the normal range It is preferable to determine whether or not.
  • the expression level of the PRDM14 gene in the subject biological sample is above the normal range, it is possible to detect cancer in the subject by determining that cancer cells are present in the subject sample.
  • the expression level of PRDM14 gene in a biological sample obtained from a subject is preferably at least 5 times greater than the expression level of PRDM14 gene in a biological sample obtained from a healthy subject of the same type as the subject. It is possible to detect cancer in a subject on the basis of the above, more preferably on the basis of at least 10 times more, particularly preferably on the basis of at least 10 times more.
  • the expression level of the PRDM14 gene is determined by the gene whose expression level does not vary greatly between tissues or individuals (for example, a gene such as actin gene, GA PDH gene, etc.).
  • the ratio of the expression level of the PRDM14 gene to the expression level of the internal standard gene in the biological sample obtained from the subject is The ratio is preferably at least 5 times greater than the ratio of the expression level of the PRDM14 gene to the expression level of the internal standard gene in a biological sample obtained from a healthy subject of the same type as the subject, and more preferably at least It can be based on the fact that it is 10 times larger, particularly preferably at least 10 times larger.
  • the kit for detecting cancer according to the present invention is a biological sample collected from a subject.
  • Reagents for quantifying the expression level of PRDM14 gene include oligonucleotides or polynucleotides that can be hybridized to the polynucleotide encoding PRDM14 of interest or fragments thereof, or anti-PRDM14 antibodies or fragments thereof.
  • the cancer detection kit of the present invention may be in any form as long as it contains the above-described oligonucleotide or polynucleotide, or the above-described antibody or fragment thereof, and can appropriately include reagents, instruments and the like.
  • the cancer detection kit of the present invention contains the above oligonucleotide or polynucleotide, a solution used for extraction of DNA or RNA from tissues or cells (for example, a tissue or cell lysis buffer, phenol) / Chloform, ethanol, etc.), equipment, reagents required for reverse transcription (eg H 2 0, buffer, Mg)
  • a solution used for extraction of DNA or RNA from tissues or cells for example, a tissue or cell lysis buffer, phenol) / Chloform, ethanol, etc.
  • equipment reagents required for reverse transcription (eg H 2 0, buffer, Mg)
  • reagents required for PCR e.g., H 2 0, buffer, M g C 1 2, d NTP mix,
  • PCR amplified fragments eg., RI, fluorescent dyes, etc.
  • DNA microarray DNA chip, etc.
  • the oligonucleotide or polynucleotide is a base represented by SEQ ID NO: 2.
  • a sense primer containing a part of the base sequence identical or substantially identical to the sequence, and a part of the base sequence complementary to the base sequence identical or substantially identical to the base sequence represented by SEQ ID NO: 2 It preferably contains an antisense primer.
  • the base length is, for example, 17 to 35 bases, preferably 20 to 28 bases.
  • kits for diagnosing cancer of the present invention contains the anti-PRDM14 antibody or fragment thereof, a solid phase carrier (for example, immunoplate, latex particle, etc.) for immobilizing the antibody or fragment thereof , there anti V- globulin antibody (second antibody) anti PRDM1 4 antibody or agent that binds to a fragment thereof, such as, an antibody (secondary antibody containing) or substance for labeling a fragment thereof (e.g., an enzyme, a fluorescent substance, etc.
  • an antibody secondary antibody containing
  • substance for labeling a fragment thereof e.g., an enzyme, a fluorescent substance, etc.
  • various reagents eg, enzyme substrate, buffer, diluent, etc.
  • PRDM1 ABI: Hs00153357
  • PRDM2 ⁇ : Hs00210612
  • PRDM3 ABI: Hs00602795
  • PRDM4 ⁇ : Hs00183764
  • PRDM5 ABI : Hs00218855 s
  • PRDM6 ABI: Hs01373000
  • PRDM7 ABI: Hs00364862
  • PRDM8 ABI: Hs00220274
  • PRDM9 ABI: Hs00360639
  • PRDM10 ABI: Hs00360651, PRDMU: ABI: Hs00220293
  • PRDM12 ABI: Hs0022: 80
  • PRDM14 ABI: Hs00225842
  • PRDM15 ABI: Hs00411330
  • GAPDH ABI: Hs99999905 were used.
  • the PCR reaction was performed at 96 ° C for 5 seconds at 6 ° C with a total of 20 ⁇ 1 solution containing cDNAl / i 1, ABI TaqMan Fast Master mix 10 ⁇ 1, primer (50 ⁇ ) ⁇ ⁇ 1 and water 8 ⁇ 1. Performed in 1 5 seconds, 40 cycles,
  • DCIS Noninvasive ductal carcinoma
  • PRDM14 mRNA was significantly higher than the expression levels of other PRDM mRNAs.
  • PRDM14 mRNA was highly expressed.
  • PRDM3, 6 and 7 mRNA were also highly expressed.
  • RA Replacement 3 ⁇ 4 ⁇ m> All RA were extracted.
  • breast cancer cell lines MCF7, MDA-MB-231, MDA-MB-435S, MDA-MB-436, MDA_MB-468, SKBr-3 and T-47D gastric cancer cell lines MKN7, KatoIII, JRST, SNU638, JRST, AZ521, MKN28 and MKN74, ovarian cancer cell lines SKOV-3, OVCA-3, PA-1, Caov-3, thigh, KURA, AM0C2, MCAS, KF, KFr, HTB0A, TOV-21G, Total RNA was also extracted from SW626, T0V112D and 0V-90, normal ovary, and NCC-IT-A3, a teratocarcinoma cell line.
  • PRDM14 gene expression was analyzed by real-time PCR in the same manner as in (1) above.
  • GAPDH gene as an internal standard, the ratio of PRDM14 gene expression to GAPDH gene expression (ie, PRDM14 gene expression level ZGAPDH gene expression level) indicates the expression of PRDM14 gene in each sample.
  • PRDM14 gene expression was frequently increased in breast cancer tissues.
  • PRDM14 gene expression was only slightly increased in gastric cancer tissues.
  • the ovarian cancer cell line PA-1 expressed the PRDM14 gene at a very high level. Since PA-1 showed a teratocarcinoma tissue type, we also examined its expression in the teratocarcinoma cell line NCC-IT-A3 and found that the PRDM14 gene was highly expressed.
  • Noninvasive breast cancer (DCIS) patients 4
  • DCIS Noninvasive ductal carcinoma
  • DCIS Noninvasive ductal carcinoma
  • DCIS Non-invasive ductal carcinoma
  • tubl 319 ⁇ Male 72 Gastric cancer Well differentiated tubular adenocarcinoma (tubl)
  • Example 4 Four patients with breast cancer whose PRDM14 gene expression level was increased in Example 1 (cases 8, 6, 2, 96, 10 5: sex, age, and disease classification are shown in Table 4) The obtained cancer tissue was fixed with formalin, and a paraffin section was prepared by a conventional method. After anti-PRDM14 antibody (ABGENT, AP1214A, 50 times) was used as the primary antibody at 4 ° C, after incubation at 2 ° C, 2 Incubation was performed using streptavidin-labeled goat anti-rabbit antibody (DAK0) as the next antibody, and immunostaining was performed using EnVision-Plus (DAK0). Moreover, nuclear staining was performed using hematoxylin. The results are shown in FIG.
  • P ⁇ 14 Sense primer: 5, -CCTGACGGACACCCTCTCTGA-3, (SEQ ID NO: 9); Antisense primer: 5, -GTGGGTACCTGCATCCTAACACAT-3 '(SEQ ID NO: 10), Albumin: Sense primer: 5' -AAGCTGAGTTTGCAGAAGTTTCCAAG- 3 '(sequence number
  • SLC05A1 Sense primer: 5 '-CTACTTTGGAGCAGTCATTGACAC-3, (SEQ ID NO: 1 3); Antisense primer: 5' -CACGTTGTACTCCCAGCAAGA-3, (SEQ ID NO: 14), LACTB2: Sense primer: 5,-TCCTTACCCACCAGMTGGCMTC-3 '( ⁇ Self-sense number 1 5); end antisense primer: 5, -TGAAATTCTTCGTGGTTTTTGGGTC-3 '(SEQ ID NO: 1 6): sense primer: 5' -GAAGTTCTGGTGTGCGTGAGTG-3-3 (SEQ ID NO: 1 7); antisense primer: 5, -AATCCCCTAAAGTTAACCTAATGATG-3, (Self column number 1 8), TPD52: Sense primer: 5 '-CCTCTAACTCCTGGGGTTGATTTC-3, (Self column number 1 9); Antisense primer: 5, -CCTTCAGTTGAGTGCCGCTTTG-3' (SEQ ID NO:
  • the PCR reaction was performed using TaqMan PCR (Applied Biosystems) method using a total solution of 20 ⁇ 1 containing cDNA 1, ABI TaqMan Fast Master mix 10 / i 1, primer (50 ⁇ ) ⁇ 1 and water 8 ⁇ 1. The test was performed at 6 ° C for 5 seconds, 60 ° C for 15 seconds, and 40 cycles.
  • the expression level of PRDM14 mRNA was quantified by real-time PCR using the total RNA obtained from each sample in the same manner as in Example 1.
  • the GAPDH gene was used as an internal standard. Primers were purchased from Applied Biosystems as in Example 1, and PRDM14: ABI: Hs00225842 and GAPDH: ABI: Hs99999905 were used, respectively.
  • the PCR reaction was performed using TaqMan PCR (Applied Biosystems) method with a total solution of 20 ⁇ 1 containing cDNAl / z 1, ABI TaqMan Fast Master mi 10 ⁇ 1, primer (50 ⁇ ) 1 ⁇ 1 and water 8 ⁇ 1. The test was performed at 6 ° C for 5 seconds, 60 ° C for 15 seconds, and 40 cycles.
  • Example 3 (1) 17 breast cancer patients (C asel, 1 1, 17 ⁇ 20 , 81, 85, 94, 96 ⁇ 98, 105, 106, 108, 1 12, 114: gender,
  • genes that are present in the chromosome 8ql3.3 region, SLC05A1, SULF1, NC0A2, TRAM1, LACTB2, XKR9 The mRNA expression level of EYA1 was also examined.
  • PRDM14 gene (Genbank Accession number: BC052311) (SEQ ID NO: 2)
  • the expression vector pCMV-Tag2A (Stratagene) was inserted into the expression vector pCMV-Tag2A / PRDM14 and introduced into SKBr-3 cells, a cell line that does not express PRDM14, by electroporation. did. Cells were fixed with 100% methanol 24 hours after introduction, and anti-PRDM14 antibody (ABGENT, AP1214A, used at 100-fold dilution) as the primary antibody, and anti-rabbit antibody labeled with Alexa488 as the secondary antibody (Molecular Probe) , Alexa Flour 488, goat anti rabbit IgG, used at 500-fold dilution).
  • the PRDM14 gene (Genbank Accession number: BC052311) (SEQ ID NO: 2) was inserted into the expression vector pCMV-Tag2A (Stratagene) to produce the expression construct pCMV-Tag2A / PRDM14.
  • pCMV- Tag2A / PRDM14 was introduced into breast cancer cell lines MCF7 and SKBr-3 cells by electroporation, and the cells into which the construct was introduced were selected using the drug marker G418 and analyzed did. Fourteen days after introduction of the construct by electroporation, the cells were fixed with methanol, stained with Giemsa solution, and the number of colonies was counted. Using cells into which only the expression vector (pCMV-Tag2A) was introduced as a control line, the ability to form a coloie was compared (an experiment was performed three times for each sample).
  • PRDM14 In order to analyze the function of PRDM14 in more detail, we introduced the PRDM14 gene into the breast cancer cell line SKBr-3 and examined the effect of PRDM14 on resistance to anticancer drugs.
  • the PRDM14 gene (Genbank Accession number: BC052311) (self-column number 2) was inserted into the expression vector pCMV_Tag2A (Stratagene) to construct a construct pCMV-Tag2A / PRDM14 for stable expression.
  • This construct was introduced into SKBr-3 cells, a cancer cell line, by electroporation using Nucleofector (Amaxa), and the cells into which the construct was introduced were selected with 0.7 mg / ml G418, and PRDM14 residue was obtained. A cell line that stably expressed the gene was established.
  • As a control we were prepared expression vector P CMV-Tag2A only the introduced cell lines.
  • Cisplatin an anticancer agent, for cell lines that do not overexpress PRDM14 (cell lines into which only the expression vector has been introduced) and cell lines that stably express PRDM14 (2 clones: P-8 and P-9) (CDDP) (50 M), etoposide (VP-16) (50 ⁇ M), adriamycin (200 ng / ml), docetaxel ( ⁇ / M) for 48 hours each, and cell survival by MTT method The rate was measured.
  • PRDM14 gene was knocked down in ovarian cancer cell line PA-1 overexpressing PRDM14 using siRNA against PRDM14.
  • a vector expressing GFP was introduced into PA-1 cells, and the gene transfer efficiency was examined and optimized.
  • Microscopic image of PA-1 cells into which GFP expression vector has been introduced magnification is 400 times) (cell image by phase contrast microscope (left: Phase), cell image by fluorescence microscope (middle: GFP), and overlay of the above two cell images
  • Figure 11 shows the combination (right: Merge). As is clear from Fig. 11, it is understood that gene transfer has become possible in about 80% of cells.
  • siRNA A-PRDM14-1 siRNA-PRDM14-2
  • siRNA_PRDM14-3 siRNA-PRDM14-3
  • siRNA-PRDM14-l Sense: 5, -CCAGUGAAGUGAAGACCUATT-3, (SEQ ID NO: 3), Antisense: 5'-UAGGUCUUCACUUCACUGGTT-3 '(SEQ ID NO: 4);
  • siRNA_PRDM14-2 Sense: 5, -GGACAAGGGCGAUAGGAAATT-3 '(SEQ ID NO: 5), Antisense: 5' -UUUCCUAUCGCCCUUGUCCTT-3, (SEQ ID NO: 6);
  • siRNA-PRDM14-3 Sense: 5, -GGGAAAAUCUUCUCAGAUCTT-3 '(SEQ ID NO: 7), Antisense: 5' -GAUCUGAGAAGAUUUUCCCTT-3 '(SEQ ID NO: 8)
  • siRNAs As controls, siRNAs (siRNA-control-1, siRNA-control-2, and siRNA-control-3) having the following three types of sequences were used.
  • siRNA-control-1 5,-ATCCGCGCGATAGTACGTA-3 '(SEQ ID NO: 2 1)
  • siRNA-control-2 5,-TTACGCGTAGCGTMTACG-3, (SEQ ID NO: 2 2)
  • siRNA-control-3 5, -TATTCGCGCGTATAGCGGT— 3 '(SEQ ID NO: 2 3)
  • PRDM14 For the expression level of PRDM14 gene 48 hours after siRNA introduction, using ABI7900 real-time PCR analyzer (Applied Biosystems) and using GAPDH gene as an internal standard in the same manner as in Example 1, PRDM14 relative to the expression level of GAPDH gene Analysis by gene expression ratio (ie, PRDM14 gene expression level ZGAPDH gene expression level) revealed that the PRDM14 expression level in cells transfected with control siRNA was 100% as shown in Figure 12 In some cases, the expression level of the PRDM14 gene was suppressed to 14.3% in the cells into which the siRNA-PRDM14-1 to 3 force vector was introduced.
  • -PADM cells introduced with PRDM14-1 to 3 cocktail are approximately 40% or 96 hours after 48 hours compared to cells transfected with control siRNA (siRNA-control-1 to 3). Later, it decreased by about 70%, and a marked suppression of cell proliferation was observed. Therefore, it was shown that the growth of cancer cells can be suppressed by suppressing the expression of the PRDM14 gene in cancer cells or by inhibiting PRDM14 in cancer cells.
  • the present inventors analyzed the PRDM family-first gene expression profile by using a combination of the real-time PCR method and the delta / delta CT method, and the PRDM14 gene in breast cancer and ovarian cancer. It was revealed for the first time that the expression of is specifically increased. In addition, this increase in the expression of the PRDM14 gene is due to chromosome amplification, and the increase in the expression of the PRDM14 gene has been found to increase the expression in cancer tissues even at the protein level, and PRDM14 promotes cell proliferation. Thus, it was suggested for the first time that the PRDM14 gene has an action as an oncogene. Furthermore, based on these findings, suppression of PRDM14 gene expression in cancer cells significantly suppressed cancer cell growth and It was clarified that the anticancer drug sensitivity of cancer cells was enhanced.
  • the PRDM14 gene expression inhibitor or PRDM14 inhibitor used as the therapeutic agent of the present invention exerts an effect of suppressing the growth of breast cancer or ovarian cancer cells, and in cancer cells treated with anticancer agents, It was shown that the sensitivity to anticancer drugs can be increased.
  • detecting breast cancer or ovarian cancer in a subject based on the expression level of PRDM14 gene in a biological sample obtained from the subject. It was shown that this is possible.
  • the expression of the PRDM14 gene used in the present invention is specifically increased in breast cancer or ovarian cancer lesions. Therefore, the therapeutic agent of the present invention comprising a PRDM14 gene expression inhibitor or PRDM14 inhibitor can be safely used as a therapeutic agent for breast cancer or ovarian cancer, and a high therapeutic effect is obtained. It is expected to be applied to pharmaceutical products. Furthermore, according to the present invention, since a detection method and a detection kit for breast cancer and ovarian cancer based on the expression of the PRDM14 gene can also be provided, the present invention is not limited to treatment of breast cancer or ovarian cancer. This will greatly contribute to the diagnosis and research.

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PCT/JP2007/069413 2006-09-27 2007-09-27 Agent thérapeutique, procédé de détection et kit de détection pour cancer du sein et cancer ovarien WO2008038832A1 (fr)

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Cited By (4)

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JP2015033381A (ja) * 2013-07-10 2015-02-19 国立大学法人 東京大学 がん幹細胞分子マーカー
EP3690444A4 (en) * 2017-09-26 2021-05-26 The University Of Tokyo PROCEDURE FOR CONFIRMING EXPRESSION OF PRDM14
EP3996684A4 (en) * 2019-07-09 2024-06-19 Ariz Precision Medicine, Inc. CANCER TREATMENT USING TARGETED PHARMACEUTICAL SIRNA FORMULATIONS TO DECREASE THE EXPRESSION OF THE PRDM14 PROTEIN
US12012597B2 (en) 2020-05-14 2024-06-18 Ariz Precision Medicine, Inc. Cancer treatment using siRNA to modulate expression of PRDM2/RIZ protein

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