WO2008075713A1 - Marqueur pour le diagnostic du cancer et molécule cible pour une thérapie - Google Patents

Marqueur pour le diagnostic du cancer et molécule cible pour une thérapie Download PDF

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Publication number
WO2008075713A1
WO2008075713A1 PCT/JP2007/074408 JP2007074408W WO2008075713A1 WO 2008075713 A1 WO2008075713 A1 WO 2008075713A1 JP 2007074408 W JP2007074408 W JP 2007074408W WO 2008075713 A1 WO2008075713 A1 WO 2008075713A1
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Prior art keywords
gene
small cell
cancer
trim33
polynucleotide
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PCT/JP2007/074408
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English (en)
Japanese (ja)
Inventor
Johji Inazawa
Sana Yokoi
Issei Imoto
Hitoshi Tsuda
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Tokyo Medical And Dental University
Daiichi Sankyo Co., Ltd.
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Publication of WO2008075713A1 publication Critical patent/WO2008075713A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to a means for diagnosing small cell cancer such as small cell lung cancer or breast cancer, and preferably means for utilizing amplification or expression enhancement of a specific gene as a cancer diagnostic marker.
  • the present invention also relates to a means for treating small cell cancer such as small cell lung cancer or breast cancer by suppressing amplification or expression enhancement of the specific gene.
  • TRIM33 also called tripartite motif 33, sometimes called “Ectodermin” has been identified as a factor involved in ectoderm formation in Xenopus embryogenesis, and by ubiquitinating Smad4, a mesoderm-inducing factor It is known to inhibit (Non-Patent Document 1: Cell, 121, pp.87-99, 2005). Regarding the relationship between TRIM33 gene and cancer, TRIM3 3 gene has been shown to increase expression in colorectal cancer! /, And its ability to be demonstrated S /! (Non-patent document 1, Fig. 7D And 7E).
  • RNA interference suppression of TRIM33 gene expression in the melanoma C32 cell line or breast cancer MB231 cell line by RNA interference has been shown to suppress the growth of these cells (Figs. 7F and 7G). Proliferation inhibition has been shown to be Smad4-dependent (Fig. 7G).
  • TRM33 gene is amplified or increased in breast cancer cells. Relationship between TRM33 gene and other cancers (eg small cell lung cancer) There is no report about. Further, TRM33 gene is a gene present in the genetic child seat 1Ro13.1, the force is also reported that the human genome 1 ⁇ 13 area you are amplified in breast cancer s (Non-Patent Document 2: Cancer Research, 60, pp.4519- 4525, 2000; Non-Patent Document 3: Cancer Genet Cytogenet, 117, pp.153-158, 2000), these publications show that specific TRIM33 genes are amplified or upregulated in breast cancer! / No suggestion! /, And no teaching.
  • the gene expression pattern in cancer is the gene expression pattern in the tissue of the origin.
  • the gene expression pattern differs for each tissue that often inherits the turn. Therefore, while there are genes that change in common across cancer types, in general, gene expression patterns in cancer differ depending on the tissue of origin (for example, classification such as colon cancer and lung cancer). (Molecular biology of the cell (3rd edition) .1256
  • Non-Patent Document 1 suggests the relationship between TRIM33 gene and other cancers (for example, small cell lung cancer)! / I think.
  • Non-Patent Document 1 Cell, 121, pp.87-99, 2005
  • Non-patent document 2 Cancer Research, 60, pp.4519-4525, 2000
  • Non-patent document 3 Cancer Genet Cytogenet, 117, pp.153-158, 2000
  • An object of the present invention is to provide means for using amplification or increased expression of a specific gene as a marker for cancer diagnosis, preferably means for diagnosing small cell cancer such as small cell lung cancer or breast cancer. is there.
  • Another object of the present invention is to provide a means for treating small cell cancer such as small cell lung cancer or breast cancer by suppressing the amplification or increased expression of the specific gene. Means to do
  • TRIM33 gene is amplified in small cell carcinoma such as small cell lung cancer and breast cancer cells, and its expression is enhanced. I found out. Furthermore, we found that the expression level of TRIM33 gene is a prognostic factor for breast cancer. Further, the gene can be used as a marker for small cell carcinoma such as small cell lung cancer and breast cancer, and by detecting the amplification or expression enhancement using, for example, a probe that can specifically bind to the gene, It was found that small cell carcinoma such as small cell lung cancer and breast cancer can be diagnosed easily and reliably.
  • RNA interference by inhibiting the expression of the TRIM33 gene by means such as RNA interference, it is possible to remarkably suppress the growth of small cell carcinomas such as small cell lung cancer and breast cancer cells, thereby producing small cell lung cancer. It has been found that it can treat small cell cancer and breast cancer.
  • the present invention is based on the above findings. Based on this, it was completed.
  • the present inventors have doubled a specific region on 1 ⁇ 13.2 (hereinafter also referred to as an amplicon according to the present invention) in a cell line derived from human small cell lung cancer (LU-140). It was found that amplification was performed on a minute chromosome (dmin) (Fig. 1 (C), (D)). Furthermore, the present inventors found that the TRIM33 gene was amplified in a homogeneous chromosome region (HSR) in a human breast cancer-derived cell line (MCF7) (Fig. 2, (A)). .
  • HSR homogeneous chromosome region
  • MCF7 human breast cancer-derived cell line
  • the amplicon according to the present invention has, as its original locus, 1 ⁇ 13 ⁇ 2 substantially represented by four types of BAC clones (RP1 1-1115211, RP11-1129E18, RP11-625J15, RP11-254K1). Present in the chromosomal region.
  • These BAC clones are clones of the BAC library (RPCI human BAC library 11) of human chromosome fragments created from human blood-derived chromosomes.
  • Each clone in the BAC library represents a certain fragment (+ strand, strand) of the chromosome.
  • the terminal portion of each clone (+ strand, strand) was sequenced, and its base sequence became clear.
  • the full-length nucleotide sequence of each clone (+ strand, single strand) of the BAC library has been revealed!
  • the partial base sequence of the end of RP11-1152I1 (+ strand) is the base sequence described in SEQ ID NO: 1.
  • the partial base sequence at the end of the RP11— 1129E18 (+ strand) region is Shiki Iki (GenBank accession number: AQ697738) described in SEQ ID NO: 3.
  • the partial base sequence at the end of the RP11-1129E18 (-strand) region is the base sequence described in SEQ ID NO: 4 (GenBank accession number: AQ720255).
  • the partial base sequence at the end of the RP11-625J15 (+ strand) region is the base sequence described in SEQ ID NO: 5 (GenBank accession number: AQ405632).
  • the partial base sequence at the end of the RP11-625J15 (strand) region is the base sequence described in SEQ ID NO: 6 (GenBank Accession No: AQ455093).
  • the partial base sequence at the end of the RP11-254K1 (+ strand) region is the base set forth in SEQ ID NO: 7. Sequence (GenBank accession number: AQ478938). The partial base sequence at the end of the RP11—254K1 (—strand) region is the base sequence described in SEQ ID NO: 8 (GenBank Accession No: AQ478940). Since these 4 clones partially overlap, it is possible to create a contig sequence formed from them.
  • nucleotide sequence described in GenBank accession number NT-0192713.18 is Contig's IJ of the genomic fragment of the first human chromosome.
  • RP11—115211, RP11—1129E18, RP1 1—625J15 and RP11—254K1 are the 10597582th power and 10752202, respectively, in the base banking IJ described in GenBank accession number NT—019273 ⁇ 18.
  • the Contig sequence consisting of these 4 clones is the base sequence from the 1097582nd position of IJ No. 10597582, described in GenBank accession number NT _019273-18 (about 0 ⁇ It is considered to correspond to an area of 5 Mbps.
  • the amplicon according to the present invention is a base sequence consisting of a base sequence from GenBank accession number NT-0119273.18, IJ No. 10597582, No. 11 to No. 121,868.
  • the force S is considered to be a chromosomal region substantially represented by the polynucleotide represented by.
  • the chromosomal region substantially represented by the polynucleotide represented by the nucleotide sequence consisting of nucleotide sequence 1059758 from the 2nd to 11121868 of the nucleotide sequence described in GenBank accession number NT-0192773.18 is TRIM33
  • BCAS2 breast carcino ma amplified sequence—2
  • DENND2C DENN / MADD dom ain containing 2C
  • the transcription region of the TRIM33 gene is GenBank accession number NT-019273.
  • the transcription region of the BCAS2 gene is substantially indicated by a polynucleotide represented by the nucleotide sequence up to the 11031950th position of the 11017863th nucleotide of the nucleotide sequence described in GenBank accession number NT-019273.18. It is thought to be conformed to the chromosomal region. Therefore, the transcription region of the BCAS2 gene is considered to be conformed to the chromosomal region substantially indicated by the polynucleotide represented by RP11-254K1.
  • the transcription region of the DENND2C gene is a polynucleotide represented by the nucleotide sequence from the 11033155th force of IJ's 11033155th to 11120417th described in GenBank accession number NT-0192 73.18. It is thought that it is actually conformed to the chromosomal region shown. Therefore, the transcription region of the DENND2C gene is considered to be coordinated with a chromosomal region substantially corresponding to the polynucleotide represented by RP11-254K1.
  • the present invention provides a diagnostic marker for small cell cancer or breast cancer, preferably lung small cell cancer or breast cancer, comprising the amplicon according to the present invention.
  • the disease site of a patient who develops small cell cancer such as small cell lung cancer or breast cancer is amplified by the amplicon according to the present invention as compared to the site of a healthy subject as a control. It is thought that there is. Therefore, when the amplicon according to the present invention is amplified in a biological sample derived from lung or milk tissue isolated from a certain person, compared to that isolated from a healthy person, the person is Diagnosed as having or having a high probability of suffering from small cell cancer or breast cancer, and / or having a poor or high prognosis.
  • Amplification of the amplicon according to the present invention means that the portion included in the amplicon is also amplified. Therefore, it is clear that the portion contained in the amplicon according to the present invention also serves as a diagnostic marker for small cell cancer such as small cell lung cancer or breast cancer.
  • a diagnostic marker for small cell cancer or breast cancer preferably small cell lung cancer or breast cancer comprising a portion contained in the amplicon according to the present invention.
  • the chromosome region where the TRIM33 gene is conformed Region chromosomal region where BCAS2 gene is conformed, chromosomal region where DENND2C gene is conformed, chromosomal region where TRIM33 gene transcription region is conformed, chromosomal region where BCAS2 gene transcription region is conformed, transcription region of DENND2C gene
  • examples include the chromosomal region to be conformed, TRIM33 gene, BCAS2 gene, and DENND2C gene.
  • the present inventors As a result of the expression analysis of the TRIM33 gene, the BCAS2 gene, and the DENND2C gene in 20 cell lines derived from human small cell lung cancer, the present inventors have found that the expression levels of the respective genes ( The amount of each gene transcript) was found to be increased compared to the expression level of each gene in healthy lungs (Fig. 1 (E)). Furthermore, the present inventor measured the amount of TRIM33 protein in cells using a cell line derived from small cell lung cancer (4 types) and a cell line derived from breast cancer (MCF7). It was found to be increased compared to the TRIM33 protein mass (Fig. 2 (B)).
  • the present inventors use an immunochemical technique to increase the expression of the TRIM33 gene only in the small cell carcinoma part in a mixed small cell lung cancer (mixed small cell cancer and linear cancer) tissue.
  • Figure 3 Furthermore, the present inventors have found that expression of the TRIM3 3 gene is increased in breast cancer tissue using an immunochemical technique (FIG. 3).
  • the prognosis of patients whose TRI M33 gene expression is strongly detected in breast cancer tissue is poor compared to the prognosis of patients whose expression of TRIM33 gene is not detected or weakly detected in breast cancer tissue.
  • FIG. 6 (A) (B) the prognosis of patients whose TRI M33 gene expression is strongly detected in breast cancer tissue.
  • the transcription product of the gene conforming to the amplicon according to the present invention and the protein encoded by the gene also serve as a diagnostic marker for small cell cancer such as small cell lung cancer or breast cancer. It is done. That is, when the expression of the gene is increased in a biological sample derived from lung or milk tissue isolated from a certain person, the person is suffering from small cell cancer such as small cell lung cancer or breast cancer. Diagnosed as having a strong or likely likelihood and / or having a poor or likely prognosis.
  • diagnosis of small cell cancer or breast cancer preferably small cell lung cancer or breast cancer comprising a transcription product of a gene conforming to the amplicon according to the present invention or a protein encoded by the gene, is preferable.
  • Markers are provided.
  • genes conforming to the amplicon according to the present invention include TRIM33 gene, BCAS2 gene, and DENND2C gene.
  • amplification of the amplicon according to the present invention in mammals including humans which is a diagnostic agent for small cell cancer or breast cancer, preferably small cell lung cancer or breast cancer, and A diagnostic agent capable of detecting an increased expression of a gene conforming to the amplicon is provided.
  • the presence of the above-mentioned diagnostic agent for diagnosis of human small cell cancer or breast cancer preferably human small cell cancer or breast cancer, small cell cancer or breast cancer, preferably small cell lung cancer or breast cancer is suspected.
  • the above diagnostic agent for prognosis of patients with small cell cancer or breast cancer preferably small cell lung cancer or breast cancer.
  • amplification of the amplicon according to the present invention and / or increased expression of a gene conforming to the amplicon is detected in a biological sample isolated from a human.
  • the above diagnostic agent comprising a nucleic acid probe that hybridizes with a region under stringent conditions; the base sequence described in GenBank Accession Number NT-0192713.18 and the base sequence consisting of up to 11 121868, or its complementary base
  • a polynucleotide comprising at least 15 consecutive nucleotides contained in the sequence or a condition that is stringent with the polynucleotide
  • the above diagnostic agent comprising a polynucleotide consisting of at least 15 bases that hybridizes underneath; a chromosomal region to
  • a method for diagnosing small cell cancer or breast cancer, preferably small cell lung cancer or breast cancer wherein the amplicon according to the present invention is used in a biological sample isolated from a human. And / or an increase in the expression of a gene conforming to the amplicon, and preferably a step of detecting an increase in the TRIM33 gene and / or an increase in the expression of the TRIM33 gene is provided.
  • a method for prognosing small cell cancer or breast cancer, preferably small cell lung cancer or breast cancer which comprises a step of detecting an increase in expression of the gene to be detected, preferably amplification of TRIM33 gene and / or detection of increased expression of TRIM33 gene .
  • the above diagnostic method characterized by using any one of the above diagnostic agents.
  • the present invention provides a method for screening a substance having a growth inhibitory action on small cell cancer cells or breast cancer cells, preferably lung small cell cancer cells or breast cancer cells, which is conformed to the amplicon according to the present invention.
  • a screening method comprising a step of selecting a substance having an action of suppressing amplification and / or expression of a gene, preferably TRIM33 gene, is provided.
  • a substance having an action of suppressing amplification and / or expression of a gene conforming to the amplicon according to the present invention preferably an action of suppressing amplification and / or expression of a TRIM33 gene.
  • a medicament for treating small cell cancer or breast cancer preferably small cell lung cancer or breast cancer, which contains, as an active ingredient, a substance having the above, for example, a substance screened by the screening method described above.
  • a double-stranded polynucleotide comprising a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 17 of the Sequence Listing and a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 18 of the Sequence Listing;
  • Examples include a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 19 in the sequence listing and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 20 in the sequence listing.
  • a method for treating small cell cancer or breast cancer preferably small cell cancer such as small cell lung cancer, more preferably small cell lung cancer, wherein a therapeutically effective amount of the above substance is applied to small cell lung cancer or the like.
  • a therapeutic method comprising the steps of administering to a small cell cancer patient or a breast cancer patient, preferably a small cell cancer patient such as a small cell lung cancer, more preferably a small cell lung cancer patient, and TRIM33 for the manufacture of the medicament
  • Use of a substance having an action of suppressing gene amplification and / or expression is provided by the present invention.
  • the present invention also provides a growth inhibitor for small cell carcinoma cells such as small cell lung cancer or breast cancer cells, comprising a double-stranded polynucleotide selected from the following group. .
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 17 of the sequence listing and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 18 of the sequence listing
  • (2 ) A double-stranded polynucleotide comprising a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 19 in the Sequence Listing and a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 20 in the Sequence Listing.
  • FIG. 1 shows the TRM33 amplification ((A) to (D)) and expression level ((E)) in SCLC cell lines.
  • A A representative CGH array image of LU-140 cells is shown.
  • B shows the replication number profile of chromosome 1 in LU-140 cells.
  • C Representative FISH images of RP11-625J15 (TRIM33, green signal) and control RP11-315D10 (RET, red signal) hybridized to the metaphase chromosome of SCLC cell line LU-140.
  • D Amplicon map of 1 ⁇ 13.2 site in LU-140 cell line.
  • E shows TRIM33 expression in SCLC cell lines and normal lung as measured by RT-PCR analysis.
  • FIG. 2 shows TRM33 amplification and overexpression in breast cancer cell lines.
  • A BAC RP11-625J15 (TRIM33, green signal) and RP 11 _315D 10 (RET, red signal) were used. The results of a two-color FISH test on metaphase chromosomes derived from the breast cancer cell line MCF7 are shown.
  • B Immunoblotting of protein extracts in 4 SCLC, 2 NSCLC, and 1 breast cancer cell lines
  • FIG. 3 is a photograph showing the immunohistochemical staining of TRIM33 protein in lung primary small cell carcinoma and breast cancer. Shows strong nuclear staining (A-D) in small cell carcinoma and low expression in adenocarcinoma (D, tip of arrow) and low expression in normal lung (stars in A and C). Intensive nuclear staining in invasive ductal cell carcinoma (F), invasive lobular carcinoma (H, arrow tip), and intraepithelial ductal carcinoma (G), and low expression in normal mammary gland (H, arrow) Show.
  • A-D strong nuclear staining
  • F invasive ductal cell carcinoma
  • H, arrow tip invasive lobular carcinoma
  • G intraepithelial ductal carcinoma
  • H, arrow normal mammary gland
  • FIG. 4 shows the effect of enhanced TRIM33 expression on cancer cell proliferation.
  • A Western blotting results using protein extract and anti-Myc antibody are shown.
  • B Number of colonies formed by TRIM33 transform HeLa and SBC-3 cells.
  • C Growth-promoting action of TRIM33 on SBC-3 cells established as a clone that stably expresses TRI M33, and SBC-3 cells transfected with an empty vector (empty) as a control The results are shown.
  • FIG. 5 is a graph showing the growth inhibitory effect of siRNA targeting TRM33 mRNA.
  • A Shows the protein level of TRIM33 protein determined by Western blotting.
  • B shows the effect of siRNA oligonucleotides on the survival of SBC_5 and MCF7 cells.
  • C Control and TRM33_siRNA-treated The results of measuring the growth inhibitory action of TGF- / 3 on MCF7 cells by BrdU incorporation are shown.
  • FIG. 6 shows the correlation between the expression level of TRIM33 and the survival rate in 134 cases of breast cancer.
  • A The overall survival of the TRIM33 high group is significantly shorter than that of the TRIM33 low group.
  • B The progression-free survival of the TRIM33 high group is also significantly shorter than that of the TRIM33 low group.
  • the diagnostic marker provided by the present invention is a marker for cancer diagnosis detected in patients with small cell cancer such as small cell lung cancer or breast cancer, and the amplification of the amplicon according to the present invention and / or the present invention. Increased expression of genes conforming to such amplicons It is a marker for cancer diagnosis.
  • diagnosis marker refers to a biologically-derived substance used to determine the presence or absence of a certain disease, the degree thereof, or the prognosis after surgery, or the substance. Means the substance. Therefore, the marker for cancer diagnosis means a biological substance used for determining the presence or absence of cancer, its degree, or the prognosis after the operation, or a substance originating from the substance.
  • a marker for diagnosing cancer according to the present invention is preferably a marker for diagnosing small cell cancer such as small cell lung cancer or breast cancer.
  • the amplicon according to the present invention or a portion thereof, or a gene conforming to the amplicon, a transcription product of the gene, or the gene It is characterized by consisting of gene products.
  • the amplicon according to the present invention or a portion thereof is amplified and / or the expression of the gene is increased, the individual to be diagnosed has small cell cancer such as small cell lung cancer or breast cancer. Diagnosed as having or likely to be affected.
  • the prognosis of a patient with small cell cancer or breast cancer, such as small cell lung cancer, in which the amplicon according to the present invention or a portion thereof is amplified and / or the expression of the gene is increased is poor. It is diagnosed that the possibility is high.
  • genes that are conformed to the amplicon according to the present invention include TRIM33 gene, BC AS2 gene, and DENND2C gene.
  • the TRIM33 gene (sometimes simply referred to as “TRIM33”) is a gene involved in ectoderm formation in Xenopus embryogenesis and is inhibited by ubiquitination of the mesoderm-inducing factor Smad4 Responsible for the process. Information on this gene is available to those skilled in the art (Non-Patent Document l: Cell, 121, pp. 87-99, 2005). Since the TRIM33 gene is present in the chromosome of any mammalian animal and the TRI M33 protein encoded thereby is expressed in any mammalian animal, the cancer marker of the present invention is Forces available in mammals The most preferred subject is a human. For example, the full length of human-derived TRIM33 gene is exemplified in SEQ ID NO: 22 in the sequence listing.
  • the BCAS2 gene is a gene that conforms to the region of chromosome 1 ⁇ 13.3 to 21 and was found to be amplified in breast cancer (Cancer Lett., 185, pp.219-223, 2002). This Genetic information is available to those skilled in the art.
  • GenBank accession number NM-005872 discloses the full-length ORF base sequence of the BCAS2 gene.
  • DENND2C gene Information on the DENND2C gene is available to those skilled in the art.
  • the full-length ORF base sequence of the DENND2C gene is disclosed in GenBank accession number NM-198459.
  • the amplicon according to the present invention can be obtained from mammals including humans by a method known per se. Specifically, for example, the cell nucleus is separated and purified from mammals including humans, the genomic DNA contained in the cell nucleus is extracted, the genomic DNA is fragmented with a restriction enzyme, etc.
  • a genomic library is created by incorporating DNA into a vector such as BAC. From this genomic library, the probe for detecting the amplicon according to the present invention (for example, RP11-115211, RP11-1129E18, RP11-625J15, RP11-254K1, etc.) is used to simply amplify the amplicon according to the present invention. Can be separated.
  • the amplicon according to the present invention comprises a polynucleotide represented by a nucleotide sequence consisting of a base sequence of GenBank accession number NT-0192713.18, IJ No. 10597582, No. 11121868, and the like. Since it is considered to be a substantially chromosomal region, for example, the probe can be designed based on this base sequence.
  • a part or all of the amplicon according to the present invention can be used.
  • a part of the amplicon according to the present invention is used, two or more of the amplicons according to the present invention are used. May be used.
  • a chromosomal region in which the TRIM33 gene is conformed As part of the amplicon according to the present invention, a chromosomal region in which the TRIM33 gene is conformed, a chromosomal region in which the BCAS 2 gene is conformed, a chromosomal region in which the DENND2C gene is conformed, and a transcription region of the TRIM33 gene.
  • a chromosomal region to which the transcription region of the DENND2C gene conforms examples include a chromosomal region to which the transcription region of the DENND2C gene conforms, and a chromosomal region to which the transcription region of the BCAS2 gene conforms.
  • the chromosomal region in which a certain gene is conformed means a part of the genome in which the gene is located, and the genome including the sense strand of the gene and the corresponding antisense strand! / Means part.
  • a “gene” can be defined as a unit of genetic function defined by a base sequence of a certain region in high molecular DNA or RNA.
  • Eukaryotic genes can include exons and introns.
  • a gene can include a region on a nucleic acid having a specific control function such as a promoter or an operator.
  • a sense strand of a chromosomal region in which a gene is coordinated is also included in the gene by reverse transcription of the transcription product of the gene.
  • a single-stranded polynucleotide or a partial polynucleotide thereof forming the amplicon according to the present invention can be exemplified.
  • a cDNA library is prepared from an appropriate source in which the expression of the gene has been confirmed according to a conventional method, and the gene is obtained from the library. It can be obtained by selecting a desired clone using an appropriate probe or primer peculiar to the gene. Examples of gene origin include various cells and tissues in which the gene expression has been confirmed, or cultured cells derived from these cells (such as cell lines derived from small cell carcinoma such as breast cancer or small cell lung cancer). it can.
  • Isolation of total RNA from these sources, isolation and purification of mRNA, acquisition of cDNA and its cloning, etc. can all be performed according to conventional methods.
  • Selection of a desired clone from the cDNA library can be performed, for example, by confirming the expressed protein for each clone using a known protein expression system and further using the function of the protein as an index.
  • An example of the function of TRI M33 is the ubiquitination activity of Smad4.
  • the full-length ORF of the TRIM33 gene is disclosed in, for example, GenBank accession number NM-015906.3 or SEQ ID NO: 22 in the sequence listing. Based on these sequences, an appropriate probe or a specific probe specific to the TRIM33 gene Primers can be designed.
  • An example of such a primer is a primer consisting of a base sequence shown in SEQ ID NOs: 9 and 10 in the sequence listing.
  • the full-length ORF of the BCAS2 gene is disclosed in, for example, NM-005872.2, and it is possible to design appropriate probes and primers specific to the BCAS2 gene based on this sequence.
  • the ORF full length of the DENND2C gene is disclosed in, for example, NM 198459.2, and based on this sequence, DENND2C c It is possible to design appropriate probes and primers specific to the gene.
  • the desired gene can be obtained from various cells and tissues in which the expression of the gene has been confirmed by general means such as PCR using the above primers.
  • Reaction conditions for PCR are not particularly limited and can be selected by those skilled in the art. For example, 94 ° C for 30 seconds (denaturation), 55 ° C for 30 seconds, 1 minute (annealing), 72 ° C
  • a reaction step consisting of 2 minutes (elongation) is defined as one cycle. For example, after 30 cycles, the reaction can be performed at 72 ° C for 7 minutes.
  • the amplified DNA fragment can be cloned into an appropriate vector that can be amplified in a host such as Escherichia coli.
  • Techniques such as primer preparation and cloning of target genes are well known and commonly used by those skilled in the art. For example, Molecular Cloning 2nd Edition, Current Protocols in Molecular Biogy. Supplementl-38, John Wiley & Sons (1987—1997). ) Etc. can be performed according to the method described in S).
  • DNA consisting of a base sequence having a deletion, substitution and / or addition of one to several bases in the base sequence of the human TRIM33 gene is suitable for chemical synthesis, genetic engineering, or mutagenesis.
  • a genetic engineering technique can be used in addition to a method of contacting a drug that becomes a mutagen with the TRIM33 gene or a method of irradiating ultraviolet rays.
  • Site-specific mutagenesis which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position, and follows the method described in Molecular Cloning, 2nd edition, above. It can be carried out.
  • the BCAS2 gene and DENND2C gene can also be obtained in the same manner as the TRIM33 gene.
  • a gene conforming to the amplicon according to the present invention a transcription product of the gene, or a gene product of the gene may be used.
  • the transcript of the gene conforming to the amplicon according to the present invention can be obtained in the same manner as the acquisition of the gene conforming to the amplicon according to the present invention.
  • a method for obtaining a gene product (eg, TRIM33 protein, BCAS2 protein, DENND2C protein, etc.) of a gene conforming to the amplicon according to the present invention is not particularly limited. Obtain the gene according to the method and apply this DNA
  • the gene product may be expressed by introducing it into an appropriate expression system, and the protein expressed by an appropriate method may be separated and purified.
  • the type of vector is not particularly limited.
  • the vector should replicate autonomously (for example, a plasmid), or be integrated into the genome of the host cell when introduced into the host cell and replicated together with the integrated chromosome. You can use a cluster.
  • an expression vector can be used, and it is desirable that elements necessary for transcription (eg, promoter, terminator, selection marker, etc.) are functionally linked.
  • a promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected by those skilled in the art depending on the type of host.
  • a transformant can be prepared by introducing a recombinant vector containing the gene into an appropriate host (eg, bacteria, yeast, fungi, and higher eukaryotic cells). The gene product is cultured in an appropriate nutrient medium under conditions that allow gene expression, and the expressed gene product can be isolated and purified by an appropriate means available to those skilled in the art.
  • the diagnostic agent for small cell cancer such as small cell lung cancer or breast cancer provided by the present invention is amplifying and / or conforming to the amplicon according to the present invention in mammals including humans.
  • a diagnostic agent capable of detecting increased expression of a gene preferably a diagnostic agent for diagnosis of small cell cancer or breast cancer such as human small cell lung cancer, small cell cancer such as small cell lung cancer or Diagnostic agents for diagnosis of patients suspected of having breast cancer, amplification of amplicons according to the present invention and / or increased expression of genes conforming to the amplicons in biological samples isolated from humans It is a diagnostic agent that can detect.
  • Amplification of the amplicon according to the present invention means that the portion included in the amplicon is also amplified. Therefore, a diagnostic agent that can detect a portion contained in the amplicon according to the present invention is also included in the diagnostic agent according to the present invention.
  • amplification of the amplicon according to the present invention means that the number of copies of the amplicon according to the present invention increases.
  • the amplification of the amplicon according to the present invention is not limited to the amplification at its original locus (1 ⁇ 13.2), but also includes amplification in a homogeneously stained region or a double microchromosome.
  • the degree of amplification of the amplicon according to the present invention is increased as long as the amount of the amplicon contained in the test sample is increased compared with the amount of the amplicon in the healthy subject as a control! / , And les, not particularly limited.
  • the gene conformed to the amplicon according to the present invention means a gene included in the amplicon according to the present invention, for example, TRIM33 gene, BCAS2 gene, DENND2C gene Is exemplified.
  • TRIM33 gene a gene included in the amplicon according to the present invention
  • BCAS2 gene a gene included in the amplicon according to the present invention
  • DENND2C gene a gene included in the amplicon according to the present invention.
  • the diagnostic agent for small cell cancer such as small cell lung cancer or breast cancer provided by the present invention is suitably used for diagnosis of humans suspected of having small cell cancer such as small cell lung cancer or breast cancer.
  • the biological sample include collected blood, body fluid, sputum, milk, biopsy tissue, or tissue separated by surgery.
  • Examples of the diagnostic agent capable of detecting amplification of the amplicon according to the present invention include diagnostic agents capable of detecting amplification of a gene conforming to the amplicon according to the present invention.
  • diagnostic agents examples include diagnostic agents that can detect TRIM33 gene amplification, diagnostic agents that can detect BCAS2 gene amplification, and diagnostic agents that can detect DENND2C gene amplification. it can.
  • a diagnostic agent capable of detecting the amplification of the TRIM33 gene for example, a diagnostic agent containing a nucleic acid probe that hybridizes under stringent conditions with a chromosomal region in which the TRIM33 gene is conformed; Diagnostic agent containing a nucleic acid probe that hybridizes under stringent conditions with the chromosomal region to which the transcription region conforms; Diagnostic agent containing a nucleic acid probe that hybridizes under stringent conditions with the TRIM33 gene; GenBan kcession number NT— A nucleotide sequence consisting of at least 15 bases contained in the nucleotide sequence consisting of the 10843084th to 10961466th nucleotides of the IJ base described in 019273.18 or its complementary nucleotide sequence, or a stringent condition with the polynucleotide Polynucleoside consisting of at least 15 bases that hybridize below It can be exemplified diagnostic agent comprising a de
  • a diagnostic agent comprising a nucleic acid probe represented by any one of the base sequences described in SEQ ID NOs: 5 to 8 in the sequence listing, SEQ ID NO: 9 to 10 in the sequence listing
  • a diagnostic agent containing a primer consisting of one nucleotide sequence can be exemplified.
  • BCAS2 residue Diagnostic agent containing a nucleic acid probe that hybridizes under stringent conditions with the chromosomal region to which the gene is conformed; Nucleic acid probe that hybridizes under stringent conditions with the chromosomal region to which the transcription region of the BCAS2 gene is conformed Diagnostic agent; Diagnostic agent comprising a nucleic acid probe that hybridizes with the BCAS2 gene under stringent conditions; GenBank accession number NT-0119273.18 Examples thereof include a diagnostic agent comprising a polynucleotide comprising at least 15 consecutive bases contained in the complementary base sequence or a polynucleotide comprising at least 15 bases that hybridizes with the polynucleotide under stringent conditions.
  • a diagnostic agent containing a nucleic acid probe represented by any one of the nucleotide sequences set forth in SEQ ID NOs: 7 to 8 in the sequence listing, SEQ ID NOS: 11 to 12 in the sequence listing A diagnostic agent containing a primer consisting of one base sequence can be exemplified.
  • a diagnostic agent capable of detecting the amplification of the DENND2C gene for example, a diagnostic agent containing a nucleic acid probe that hybridizes under stringent conditions with a chromosomal region in which the DENN D2C gene conforms; a transcription region of the DENND2C gene; Diagnostic agent containing nucleic acid probe that hybridizes under stringent conditions with the chromosomal region to which it conforms; Diagnostic agent containing nucleic acid probe that hybridizes with DEN ND2C gene under stringent conditions; GenBank Accession Number NT— 019273.18 Or a polynucleotide comprising at least 15 contiguous bases contained in the base sequence consisting of the 11033155th to the 11120417th base sequence or the complementary base sequence thereof, or hybridizing with the polynucleotide under stringent conditions Polynuclear consisting of at least 15 bases It can be exemplified diagnostic agent comprising a plastid.
  • a diagnostic agent comprising a nucleic acid probe represented by any one of the nucleotide sequences set forth in SEQ ID NOs: 7 to 8 in the sequence listing, SEQ ID NOS: 13 to 14 in the sequence listing Examples thereof include a diagnostic agent containing a primer consisting of any one of the described base sequences.
  • examples of the diagnostic agent capable of detecting amplification of the amplicon according to the present invention include diagnostic agents including a nucleic acid probe that hybridizes with the amplicon according to the present invention under stringent conditions.
  • diagnostic agents include GenBank succession A base sequence consisting of at least 15 bases included in the base sequence consisting of up to the 10597582th power of IJ, No. 111 to 21868, or its complementary base sequence described in the number ⁇ _019273 ⁇ 18
  • a diagnostic agent containing a polynucleotide comprising at least 15 bases that hybridizes with a nucleotide under stringent conditions can be preferably exemplified. More specifically, a diagnostic agent containing a nucleic acid probe represented by any one of the base sequences described in SEQ ID NOs:! To 4 in the sequence listing can be preferably exemplified.
  • the length of the polynucleotide (including a nucleic acid probe or primer) contained in the diagnostic agent according to the present invention is preferably at least 15 bases (Molecular Cloning: A laboratory Manual, 2nd). Ed., Old Spring Harbor Laboratory, old Spring Harbor. N., 1989; sometimes referred to as Molecular Cloning 2nd edition).
  • the diagnostic marker according to the present invention is specifically detected by Southern hybridization or Northern hybridization, the length of the polynucleotide may be about 19 to 40 bp (Molecular Cloning No. 1). 2 edition).
  • the length of the polynucleotide can be preferably exemplified by about 300 to 3000 bp (the first clinical FISH protocol). Chromosome ⁇ Gene diagnosis method 1st edition, Joji Inazawa et al., 1997; sometimes referred to as clinical FISH protocol).
  • the detection of the amplification of the amplicon according to the present invention using the diagnostic agent capable of detecting the amplification of the amplicon according to the present invention includes, for example, conventional methods such as FISH method, Southern blot method, CGH array method, etc. Can be implemented.
  • CGH array method first, DNA from the test sample and normal tissue DNA as a control are labeled with FITC (green) and Texas red (red), respectively, by nick translation, and equal amounts of both labeled DNAs.
  • This solution is prepared by adding the solution to a slide glass labeled with the diagnostic agent according to the present invention after the preparation of the mixed solution and performing competitive hybridization and detecting fluorescence. The power to do S.
  • stringent conditions refers to, for example, 6 X SSC, 0.5% SDS and 50% honremamide in the night. This refers to the condition of washing at 68 ° C in a solution of 0.1 X SSC, 0.5% S DS after warming at C.
  • the polynucleotide to be hybridized may not be a polynucleotide having a base sequence complementary to the base sequence of the other polynucleotide to be hybridized. Hybridization can be performed, for example, according to the method described in the Molecular Cloning Second Edition or the Clinical FISH Protocol.
  • the "chromosomal region substantially represented by a certain polynucleotide” means a portion of a chromosome corresponding to a double-stranded polynucleotide formed from the polynucleotide and its complementary polynucleotide.
  • corresponding includes complete matching, and high homology (for example, 80% or more, preferably 90% or more, more preferably 93% or more, particularly preferably 95%). More preferably 98% or more).
  • a hybridized under stringent conditions with a chromosomal region in which the gene conformed to the amplicon according to the present invention is conformed is as follows. Examples of such genes include TRIM33 gene, BCAS2 gene, and DENND2C gene.
  • any one of SEQ ID NOS: 5 to 8 in the sequence listing, a nucleic acid probe represented by the base sequence set forth in 1, and a primer represented by the base sequence set forth in SEQ ID NO: 9 or 10 in the sequence listing Can be preferably exemplified.
  • a polynucleotide that hybridizes under stringent conditions with the chromosomal region to which the BCAS2 gene is conformed a polynucleotide that hybridizes under stringent conditions with the chromosomal region to which the transcription region of the BCAS 2 gene is conformed, and the BCAS2 gene and string.
  • the BCAS2 gene sequence the base sequence IJ described in SEQ ID NO: 7 in the sequence listing, the base sequence IJ described in SEQ ID NO: 8 in the sequence listing, the base sequence described in SEQ ID NO: 11 in the sequence listing,
  • the nucleotide sequence according to SEQ ID NO: 12 any force selected from the group consisting of: a polynucleotide having at least 15 bases contiguous in the nucleotide sequence of 1 or a complementary nucleotide sequence thereof, or under stringent conditions with the polynucleotide Examples of polynucleotides that hybridize with each other.
  • nucleic acid probe represented by the base sequence described in SEQ ID NO: 7 or 8 in the sequence listing, and a primer represented by the base sequence described in SEQ ID NO: 11 or 12 in the sequence listing may be preferably exemplified. it can.
  • a polynucleotide that hybridizes under stringent conditions with a chromosomal region in which the DENND2C gene is conformed and a polynucleotide that hybridizes under stringent conditions with a chromosomal region in which the transcription region of the DENND2C gene is conformed, DENND2C
  • a polynucleotide that hybridizes under stringent conditions with the gene included in the nucleotide sequence consisting of the 11033155th to 11120417th of the nucleotide sequence described in GenBank Session Number NT-019273 or its complementary nucleotide sequence Examples thereof include a polynucleotide comprising at least 15 consecutive nucleotides, or a polynucleotide that hybridizes with the polynucleotide under stringent conditions.
  • nucleic acid probe represented by the base sequence described in SEQ ID NO: 7 or 8 in the sequence listing, and a primer represented by the base sequence described in SEQ ID NO: 13 or 14 in the sequence listing are preferably exemplified. Can do.
  • a diagnostic agent for small cell cancer such as small cell lung cancer or breast cancer provided by the present invention
  • Drugs can be exemplified.
  • Such a diagnostic agent can detect increased expression of the gene conforming to the amplicon according to the present invention.
  • a diagnostic agent comprising a polynucleotide comprising at least 15 bases that hybridizes under stringent conditions with a transcription product of a gene conforming to the amplicon according to the present invention
  • a polynucleotide comprising at least 15 bases that hybridizes with a transcription product of TRIM33 gene under stringent conditions and a polynucleotide comprising at least 15 bases that hybridizes with a transcription product of BCAS2 gene under stringent conditions.
  • DENND2C gene transcript and string Or at least one of these forces can be exemplified.
  • the polynucleotide (including the nucleic acid probe or primer) contained in the diagnostic agent according to the present invention includes the base sequence of the amplicon according to the present invention, the base sequence of the gene conformed to the amplicon according to the present invention, etc. And can be produced by a conventional method, for example, a chemical synthesis method.
  • a diagnostic agent capable of detecting the gene product of the gene conforming to the amplicon according to the present invention can be exemplified.
  • the gene product of the gene conforming to the amplicon according to the present invention is recognized, preferably specifically recognized. Diagnostic agents including antibodies can be exemplified. Examples of the gene product of the gene conforming to the amplicon according to the present invention include TRIM33 protein, BCAS2 protein or DENND2C protein.
  • a diagnostic agent containing an antibody that recognizes the TRIM33 protein preferably a polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 21 in the Sequence Listing can be exemplified.
  • the antibody herein may be either a polyclonal antibody or a monoclonal antibody, or may be a chimeric antibody in which antibodies derived from two or more species are combined.
  • An antibody can be produced using the gene product or a fragment thereof as an antigen.
  • the antigen is composed of at least 8, preferably at least 10, more preferably at least 12, even more preferably 15 or more amino acids.
  • a region represented by an amino acid sequence unique to the gene product is preferable to use as an antigen.
  • per se known antibody production methods can be used.
  • the antigen is administered to the animal by a method known per se to induce immunity, and the antibody is recovered from the serum of the immunized animal by a known antibody recovery method, or the antibody-producing cells are recovered and known per se Can be produced by introducing means for transformation into permanent proliferating cells.
  • the polynucleotide (including the case of being a nucleic acid probe or primer) or antibody contained in the diagnostic agent according to the present invention is the amplification of the amplicon according to the present invention and / or the expression of the gene conforming to the amplicon.
  • the renucleotide, the nucleic acid probe, the primer or the antibody can be labeled with an appropriate label such as a fluorescent label or a radioisotope.
  • an appropriate label such as a fluorescent label or a radioisotope.
  • labeling means are well known and commonly used by those skilled in the art.
  • the method for diagnosing small cell cancer such as small cell lung cancer or breast cancer comprises amplifying the amplicon according to the present invention and / or the amplicon in a biological sample isolated from human force. It includes a step of detecting increased expression of a gene conforming to.
  • the method includes a step of detecting amplification of the amplicon according to the present invention using the polynucleotide, nucleic acid probe, or primer described above, or conforms to the amplicon using the antibody described above.
  • a step of detecting increased expression of the gene It is also preferable to detect the amplification of the amplicon according to the present invention and the increased expression of the gene conforming to the amplicon by combining the above two steps. More specifically, the above diagnostic method including the step of detecting amplification of TRIM33 gene and / or enhanced expression of TRIM33 gene can be exemplified.
  • the amplification of the amplicon according to the present invention and / or amplification of the amplicon according to the present invention is performed on a biological sample isolated from a human. It is possible to detect an increase in the expression of a gene conforming to the.
  • the diagnostic method including the step of detecting the amplification of TRIM33 gene and / or the increased expression of TRIM33 gene, typically, (1) the amount of TRIM33 gene and / or TRIM33 contained in a biological sample isolated from human A step of measuring the expression level of the gene product of the gene, and (2) the amount of the TRIM33 gene and / or the expression level of the gene product of the TRIM33 gene measured in (1) above and included in a healthy human-derived biological sample It is preferable to include a step of comparing the amount of the TRIM33 gene and / or the expression level of the gene product of the TRIM33 gene. Prepare in advance the amount of TRIM33 gene and / or the expression level of the gene product of TRIM33 gene contained in a healthy human-derived biological sample as a standard value, and perform the above step (2) using this standard value. You can also.
  • the screening method provided by the present invention is a screening method for a substance having a growth inhibitory action on small cell cancer cells such as small cell lung cancer or breast cancer cells, and is conformed to the amplicon according to the present invention. Suppresses the amplification and / or expression of genes It includes the step of selecting a substance to have!
  • the substance selected by the above method can be used as an active ingredient of a medicament for the treatment of small cell cancer such as small cell lung cancer or breast cancer, and the substance selected by the above method can be used as a pulmonary substance. It can be used as a growth inhibitor for small cell cancer cells such as small cell cancer or breast cancer cells.
  • the types of substances subject to screening are not particularly limited, and include, for example, organic low molecular weight compounds, high molecular weight compounds, inorganic compounds, nucleic acids (including small molecule RNA and non-natural bases used for RNAi, etc. Any substance is included, including nucleic acids and the like, sugars, lipids, or peptides (including oligopeptides or polypeptides).
  • a screening method including the step of detecting an inhibitory effect on the amplification and / or expression of the TRIM33 gene by a test substance, typically, (1) a small cell, such as small cell lung cancer, TRIM33 in a cancer cell or a breast cancer cell It is preferable to include a step of measuring the number of gene copies and / or the expression level of the gene product of the TRIM33 gene in the presence or absence of the test substance.
  • Small cell carcinoma cells such as small cell lung cancer or breast cancer cells are isolated from humans including patients.
  • Small cell cancer cells or breast cancer cells may be used, but for example, small cell cancer cells or breast cancer cells such as small cell lung cancer that stably express the TRIM33 protein may be prepared and used as established cell lines. Good.
  • a small cell carcinoma cell line for example, LU-140
  • MCF7 breast cancer cell line
  • the number of copies of the TRIM33 gene can be measured by a conventional method using the above-described nucleic acid probe or primer, for example, the Northern blot method or the CGH method.
  • the expression level of the gene product of TRIM33 gene can be detected, for example, by Western plotting.
  • the medicament provided by the present invention is a medicament for the treatment of small cell cancer such as small cell lung cancer or breast cancer, and amplification and / or amplification of a gene conforming to the amplicon according to the present invention. It contains a substance having an action of suppressing the expression of the gene as an active ingredient.
  • the medicament include a substance having an action of amplifying the TRIM33 gene and / or suppressing the expression of the gene as an active ingredient. Or, for example, on It may be a medicine containing a substance screened by the screening method described above as an active ingredient.
  • the medicament of the present invention can be administered to mammals including humans.
  • the administration form of the medicament of the present invention is not particularly limited and can be administered orally or parenterally.
  • the medicament of the present invention may be a substance that is an active ingredient, but a pharmaceutical composition containing a substance that is an active ingredient and a pharmacologically and pharmaceutically acceptable additive for pharmaceutical preparation is prepared and administered. I hope to do it.
  • the medicament of the present invention containing a protein as an active ingredient can be formulated according to a conventional method for preparing a protein preparation, and the medicament of the present invention containing a polynucleotide as an active ingredient can also be used in the art. It can be formulated and used by various means.
  • the medicament of the present invention containing a nucleic acid can also be used, for example, in the form of a recombinant vector containing a nucleic acid which is an active ingredient.
  • the above recombinant vector contains various sequences necessary for gene expression or promoting gene expression so that the gene product is efficiently expressed in vivo from the nucleic acid which is an active ingredient. Incorporate them in the proper order!
  • the total length is not particularly limited.
  • the antisense polynucleotide may be, for example, an oligonucleotide having 10 bases or more, preferably 15 bases or more, so that complementary binding is possible.
  • an antibody preferably a monoclonal antibody
  • the antibody can be produced by a usual method, and is preferably a gene product of a gene conforming to the amplicon according to the present invention, preferably Monoclonal antibodies capable of specifically binding to the TRIM33 protein can also be produced by a general method of those skilled in the art.
  • a cell growth inhibitor for small cell carcinoma cells such as small cell lung cancer or breast cancer cells
  • a preferred polynucleotide as an active ingredient of the medicament of the present invention for example, the following (1) or (2) duplex Polynucleotide: (1) a double-stranded polynucleotide comprising a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 17 of the Sequence Listing and a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 18 of the Sequence Listing; And (2) a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 19 of the sequence listing and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 20 of the sequence listing
  • the force S that can be, is not limited to these.
  • Examples of pharmaceutical additives that are pharmacologically and pharmaceutically acceptable include excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, dyes, diluents, The ability to use bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, adhesives, etc.
  • Examples of pharmaceutical compositions suitable for oral administration include, for example, tablets, capsules, powders, fine granules, granules, liquids, syrups and the like.
  • compositions suitable for parenteral administration include injections, drops, suppositories, inhalants, transdermal absorption agents, eye drops, ear drops, ointments, creams, or patches. be able to.
  • the dosage of the medicament of the present invention is not particularly limited, and an appropriate dosage may be selected according to various conditions such as the type of substance as an active ingredient, the purpose of treatment or prevention, the age and symptoms of the patient, the route of administration and the like.
  • the power that can be selected In general, an adult can be selected from a dose range of about 0.001 mg per day; about 100 Omg.
  • an appropriate medium RPMI-1640 or Dulbecco with 10% urine fetal serum and 100 units / ml penicillin / 100 g / ml streptomycin. Maintained in modified Eagle medium).
  • a CGH array (MCG Cancer Array -800) was prepared using 800 BAC / PAC clones with genes and STS (sequence_tagged site) markers that may be important for cancer development and progression (Cancer Sci., 95 , Pp.559-563, 2004), and hybridization was performed according to the method described in the literature (Cancer Sci., 96, pp.100-110, 2005; Cancer Sci., 96, pp. 676-683, 2005). Specifically, the test DNA from each GBM cell line and the control DNA obtained from healthy male volunteers were labeled with Cy3_dCTP or Cy5_dCTP, respectively, precipitated with ethanol in the presence of Cot-1 DNA, and then the hybridization mixture.
  • Metaphase chromosomes were prepared from each cell line. FISH analysis was performed by using the BAC located near the region to be analyzed as a probe by the method reported previously (Clin. Cancer Res., 15, pp.4705-4713, 2003).
  • Single-stranded cDNA was generated using total RNA strength, Superscript First-Strand Synthesis System (Invitrogen), and amplified using specific primers for each gene (TRM33 gene, BCAS2 gene, or FLJ37099 gene).
  • TRM33 gene Superscript First-Strand Synthesis System
  • BCAS2 gene BCAS2 gene
  • FLJ37099 gene specific primers for each gene
  • GPDH glyceraldehyde-3-phosphate dehydrogenase gene
  • pCMV-Tag3_TRM33 The plasmid that expresses Myc-tagged TRIM33 (pCMV-Tag3_TRM33) is cloned in frame into the eukaryotic expression vector pCMV-Tag3 (Stratagene) together with the above RT-PCR product, which is the entire coding region of TRIM33.
  • pCMV-Tag3_TRIM33 or empty vector pCMV-Tag3_mock
  • FuGENE6 Roche
  • the expression of TRIM33 protein in transiently transfected cells was detected 48 hours after transfection. This was confirmed by Western blotting analysis using Signaling Technology). Cells were incubated for 2 weeks in 6-well plates with appropriate concentrations of G418, fixed with 70% ethanol and stained with crystal violet.
  • Stable TRIM33 transfectants and controls were obtained by transfecting pCMV-Tag3_TRM33 or pCMV-Tag3_mock, respectively, into cells with low expression of TRIM33. 2 ⁇ 10 3 cells were seeded in 96-well plates for measurement of cell proliferation. The number of viable cells was measured by colorimetric quantitative analysis (cell counting kit_8, Dojindo Laboratories) using a water-soluble tetrazolium salt.
  • tissue microarray (TMA) blocks of tissue core samples taken from the center of each tumor were prepared.
  • TMA tissue microarray
  • tissue cores were collected from each representative tissue piece, and these cores were placed on the receiving block using a Tissue Microarrayer (Beecher Instruments).
  • TMAs containing 138 core samples were constructed using a 2.0 mm diameter core, placed 7 to 0.8 mm apart in the receiving block. The paraffin in the sample was removed with various concentrations of ethanol and rehydrated.
  • a tumor was TRM33 negative when all malignant cells were not nuclear-stained, and a tumor was TRM33-positive when nuclear staining was observed in more than 50% of the tumor cell cells.
  • Small lymphocytes (not showing pl6 nuclear staining) were used as a negative internal control and the following criteria were applied.
  • SiRNA treatment was performed according to the previously reported method (Cell, 121, pp. 87-99, 2005). Using Lipofectamine 2000 protocol (Invitrogen), double-stranded RNA oligomers (Sigma genosys, 75 ng m 2 ) were transfected into each cell. As a control-siRNA, an unrelated sequence of RNA was used.
  • the dmin pattern was not shown in the strain, indicating that it was located away from the 1 pl3 amplicon (FIG. 1D).
  • the affected sites that were the least common among the 13 amplicons in BAC RP 11-115211 and 1 ⁇ 11-25411 were identified, that is, the sites having the TRIM33, BCAS2, and FLJ37099 genes.
  • the amplicon size was only 500kb.
  • Figure 1 shows the amplification ((A) to (D)) and expression level ((E) of TRIM33 in SCLC cell lines.
  • A Representative CGH array of LU-140 cells Image shows a marked increase in TRIM33 replication as a clear green signal (arrow)
  • B shows the replication profile of chromosome 1 in LU-140 cells.
  • C RP11-625J15 (TRIM33, green signal) hybridized to metaphase chromosome of SCLC cell line LU-140 and control RP11-3
  • a typical FISH image of 15D10 shows that TRIM33 (green) is amplified on a double microchromosome BAC RP11-315D10 shows two signals in the same cell line
  • D Amplicon map of 1 ⁇ 13 ⁇ 2 site in LU-140 cell line 7 types used as probes in FIS sputum test Shows the position of BAC (vertical bar) and three genes (TRIM33, BCAS2, and FLJ37099), the number of replicates per cell and LU -Range of dmin determined by FISH test of metaphase chromosomes derived from -140 cells.
  • TRIM33, BCAS2, and FLJ37099 are individual targets for amplification. These genes were detected in all SCLC cell lines except Lu-141 and S-1 by semiquantitative RT-PCR. The expression level of was examined. As shown in Figure 1E, TRIM33, BCAS2, and FL J37099 expression levels are normal in 17 (85%), 4 (20%), and 4 (20%) of 20 cell lines, respectively. Of these three genes, TR IM33 was most frequently overexpressed compared to lung cDNA.
  • Figure 1 (E) shows TRIM33 expression in SCLC cell lines and normal lung as measured by RT-PCR analysis.
  • ACC-LC_80 1: ACC-LC_80, 2: ACC_LC_173, 3 ⁇ u_130, 4: Lul34A, 5: Lul34B, 6: Lu_143, 7: 87-5, 8: S_2, 9: ACC_LC_5, 10: ACC_LC_172, 11: ACC_LC_170, 12 : PC_6, 13: SBC_5, 14: Lu_24, 15: Lu_135, 16: Lu_13 8, 17: Lu-139, 18: Lu-165, 19: SBC-3, 20: Lu-140, 21: Normal lung. Overexpression of TRIM33 was observed in 17 out of 20 cell lines (85.0%).
  • MCF7 breast cancer cell line (Cancer Genet. Cytogenet., 117, pp.153-158, 2000) that gave high level expression in this region by CGH reported previously was subjected to FIS H test, and TRIM33 replication in MCF7 I checked the number.
  • TRIM33 protein Expression levels of TRIM33 protein in four SCLC cell lines (LU-24, LU_139, LU_165, and LU_140), two NSCLC cell lines (PC-10 and A549), and one breast cancer cell line (MCF7) Compared. As shown in FIG. 2B, the maximum amount of TRIM33 was observed in LU-140 and MCF7, and the minimum amount in the NSCLC cell line. The expression level of TRIM33 protein was consistent with the amplification level of this gene.
  • Fig. 2 shows the amplification and overexpression of TRIM33 in breast cancer cell lines.
  • A shows the results of a two-color FISH test for metaphase chromosomes derived from breast cancer cell line MCF7 using BA C RP11-625J15 (TRIM33, green signal) and RP11_315D10 (RET, red signal).
  • B AC RP11-625J15 gave a strong signal as HSR in MCF7.
  • BAC RP11-31 5D10 gave two signals in the same cell line.
  • B shows immunoblotting of protein extracts in 4 types of SCLC, 2 types of NSCLC, and 1 type of breast cancer cell line.
  • TRIM33 protein coincided with the amplification level of the gene in LU-140 and MCF7.
  • FIG. 3 is a photograph showing immunohistochemical staining of TRM33 protein in primary small cell lung cancer and breast cancer.
  • FIG. 4 shows the effect of enhanced TRIM33 expression on cancer cell proliferation.
  • a construct containing the full-length sequence of TRIM33 and a Myc tag or an empty vector (pCMV-Tag3_mock, controller) was transfected into HeLa and SCLC cell line SBC-3.
  • A The results of Western blotting using 50 g of protein extract and anti-Myc antibody are shown. This indicates that Myc-tagged TRIM33 protein is expressed in the cells transiently tranfected with pCMV-Tag3_TRIM33!
  • the upper row shows the results of Western blot analysis of 2 clones (clone 1 and 2) transfected with pCMV_Tag3B-TRIM33 using 50 ⁇ g of protein extract and anti-Myc-tag antibody. Both clones expressed Myc-tagged TRM33 protein!
  • the lower graph shows the effect of stable TRIM3 3 expression on the proliferation of SBC-3 cells. Cell viability was measured on a given day with water-soluble tetrazolium salt assembly (each point on the graph is the average of three independent test results and the bar represents SE).
  • TRM33 suppresses the anti-proliferative effect of TGF- ⁇ by reducing the Smad response
  • control and TRIM33_siRNA-treated MCF7 cell DNA synthesis / cell cycle progression were assessed by BrdU incorporation.
  • Inhibition of TRIM33 inhibited the basal rate of BrdU incorporation and markedly enhanced the growth-suppressing action induced by exogenous TGF-3 (Fig. 5C). This result indicates that TRM33 exerts a growth promoting effect on SCLC and breast cancer cells by attenuating the cell growth inhibitory effect of TGF- / 3 by TRM33.
  • Fig. 5 shows the growth inhibitory action of siRNA targeting TRIM33 mRNA.
  • MCF7 cells showing amplification and overexpression of TRIM33 and SBC-5 cells showing overexpression of TRIM33 were treated with siRNA targeting TRIM33 or control siRNA using Lipofectamine 2000.
  • A Protein level of TRIM33 protein determined by Western blotting. Cells were treated with 75 ng m 2 oligonucleotide (TRIM33 or controller) or Lipofectamine 2000 alone, and cells were collected 48 and 120 hours after transfer. Untreated cells were maintained under the same conditions. 50 g of whole cell lysate was separated by polyacrylamide gel electrophoresis and analyzed by immunoblotting.
  • TRM33 is a prognostic factor for breast cancer
  • a prognostic factor analysis was performed on 134 cases with a clear prognosis among the 138 breast cancer cases stained in (g) above.
  • the TRIM33 high group is significant (p value: 0.0285 or 0 ⁇ 011) for both overall survival and progression-free survival. ) Had a poor prognosis. From these results, TRIM33 was considered useful as a prognostic marker.
  • nucleotide sequence set forth in SEQ ID NO: 2 RP11-115211 probe, -strand
  • nucleotide sequence set forth in SEQ ID NO: 4 RP11-1129E18 probe, -strand Base sequence described in SEQ ID NO: 5: RP11-625J15 probe, + strand, NT— 019273 (10389 3839, 120498679): 10785181-10949822 (Length: 164642) Start: 114679019-114 843660 (Length: 164642)
  • nucleotide sequence set forth in SEQ ID NO: 8 RP11-254KI probe, -strand
  • SEQ ID NO: 9 nucleotide sequence: F primer used for TRM33 gene expression analysis
  • SEQ ID NO: 10 nucleotide sequence: TRM33 gene expression analysis
  • R primer SEQ ID NO: 11 nucleotide sequence: BCAS2 gene
  • F primer used for expression analysis Base sequence described in SEQ ID NO: 12: used for expression analysis of BCAS2 gene
  • Base sequence described in SEQ ID NO: 13 F primer used for expression analysis of DENND2C gene
  • Base sequence described in 14 R primer used for expression analysis of DENND2C gene
  • Base sequence described in SEQ ID NO: 17 TRM33 siRNA (l) sense strand
  • Amino acid sequence set forth in SEQ ID NO: 21 antigen of TRIM33 antibody
  • Nucleotide sequence described in SEQ ID NO: 25 Nucleotide sequence consisting of Nos. 10597582 to 11121868 of the nucleotide sequence described in GenBank accession number NT-0192713.18 Industrial applicability
  • the present invention provides a means for easily and reliably diagnosing small cell cancer such as small cell lung cancer and breast cancer. According to the present invention, a medicament for the treatment of small cell cancer such as small cell lung cancer and breast cancer is provided.

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Abstract

L'invention concerne un marqueur destiné à être utilisé dans le diagnostic d'un carcinome à petites cellules ou d'un cancer du sein. Ce marqueur comprend une région chromosomique, un gène ou une protéine choisi dans le groupe constitué par les élément suivants : (1) une région chromosomique comprenant sensiblement un polynucléotide ayant une séquence nucléotidique se situant entre la position -10597582 et la position -11121868 dans la séquence nucléotidique décrite par le Numéro de Matricule de GenBank NT_019273.18 ; (2) le gène TRIM33 (motif tripartite 33) ; (3) le gène BCAS2 (séquence 2 amplifiée du carcinome du sein) ; (4) le gène DENND2C (domaine DENN/MADD contenant 2C) ; (5) la protéine TRIM33 ; (6) la protéine BCAS2 ; et (7) la protéine DENND2C.
PCT/JP2007/074408 2006-12-20 2007-12-19 Marqueur pour le diagnostic du cancer et molécule cible pour une thérapie WO2008075713A1 (fr)

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CN110387424A (zh) * 2019-07-12 2019-10-29 安徽农业大学 Bcas2基因用于判断圩公猪初情期启动时间的应用
WO2020069661A1 (fr) * 2018-10-04 2020-04-09 Cdi Laboratories, Inc. Biomarqueurs sérologiques de diagnostic précoce du cancer du poumon

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069661A1 (fr) * 2018-10-04 2020-04-09 Cdi Laboratories, Inc. Biomarqueurs sérologiques de diagnostic précoce du cancer du poumon
WO2020069637A1 (fr) * 2018-10-04 2020-04-09 Cdi Laboratories, Inc. Biomarqueurs sérologiques pour le diagnostic précoce du cancer du poumon
US20220003768A1 (en) * 2018-10-04 2022-01-06 Cdi Laboratories, Inc. Serological biomarkers for early diagnosis of lung cancer
CN110387424A (zh) * 2019-07-12 2019-10-29 安徽农业大学 Bcas2基因用于判断圩公猪初情期启动时间的应用

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