WO2014188775A1 - Method for preparing androgen receptor activity-dependent breast cancer cell line, screening method using cell line, and method, kit and marker for determining acquisition of androgen receptor activity dependence in breast cancer patient - Google Patents

Method for preparing androgen receptor activity-dependent breast cancer cell line, screening method using cell line, and method, kit and marker for determining acquisition of androgen receptor activity dependence in breast cancer patient Download PDF

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WO2014188775A1
WO2014188775A1 PCT/JP2014/058262 JP2014058262W WO2014188775A1 WO 2014188775 A1 WO2014188775 A1 WO 2014188775A1 JP 2014058262 W JP2014058262 W JP 2014058262W WO 2014188775 A1 WO2014188775 A1 WO 2014188775A1
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breast cancer
androgen receptor
receptor activity
cells
estrogen
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French (fr)
Japanese (ja)
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慎一 林
里圭 藤井
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国立大学法人東北大学
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor

Definitions

  • the present invention relates to a method for producing a hormone therapy-resistant breast cancer cell line, a screening method for a therapeutic agent for breast cancer using the cell line, a method for determining acquisition of hormone therapy resistance in breast cancer patients, a kit, and a marker.
  • Estrogen is a female hormone closely related to the development and proliferation of breast cancer, and most breast cancer cells are hormone-dependent tumors that proliferate in an estrogen-dependent manner.
  • breast cancer cells receive estrogen, a signal transduction system related to cell proliferation is activated via the estrogen receptor (ER), cell proliferation is promoted, and cancer progresses.
  • ER estrogen receptor
  • ER estrogen receptor
  • Hormonal agents as breast cancer treatments are large, drugs that exhibit an inhibitory action on estrogen receptors such as tamoxifen, toremifene, fulvestrant, and raloxifene (antiestrogens); and estrogen synthases such as anastrozole, letrozole, and exemestane It is classified into two types of drugs (aromatase inhibitor (AI)) that inhibit aromatase.
  • AI aromatase inhibitor
  • a cell line (type 1) that is estrogen-independent and estrogen receptor (ER) activity-dependent A cell line that is estrogen-independent and ER-independent (type 2), A cell line (type 3) that is estrogen independent, ER activity independent, and growth factor (GF) dependent and expresses HER2 and epidermal growth factor receptor (EGFR), Cell lines that are dependent on androgen metabolites (type 4) and / or cell lines that are insensitive to aromatase inhibitor (AI) and are dependent on ER activity (type 5) (Patent Document 1).
  • the present invention provides a cell line having a novel resistance mechanism that is completely different from the conventional hormone therapy resistance mechanism, and provides a screening method for a therapeutic agent for hormone therapy resistant breast cancer cells using the cell line. It is another object of the present invention to provide a method for determining whether or not a breast cancer patient has acquired such a resistance mechanism.
  • the present inventors tried to establish a hormone therapy-resistant breast cancer cell line under various culture conditions.
  • T47D-TE8 cells were used, and this was used in an estrogen-depleted medium supplemented with androgen.
  • AR androgen receptor
  • the cell line unexpectedly acquired androgen receptor (AR) activity dependency when cultured.
  • the gene expression of the AR-dependent acquired strain was analyzed, it was found that the expression levels of PSA (KLK3), DDC, and AR were significantly increased in the cell line that acquired the AR-dependent.
  • the present invention is based on these new findings.
  • Item 1 A method for determining the presence or absence of androgen receptor activity-dependent acquisition in a breast cancer patient, using as an index at least one selected from the group consisting of KLK3, DDC and AR.
  • Item 2 The method of claim 1, wherein the breast cancer patient is a female patient.
  • Item 3 A kit for determining the presence or absence of androgen receptor activity-dependent acquisition in a breast cancer patient, comprising means for measuring at least one expression level selected from the group consisting of KLK3, DDC and AR.
  • Item 4 The kit according to claim 3, wherein the breast cancer patient is a female patient.
  • Item 5 A marker for determining the presence or absence of acquisition of androgen receptor activity dependency in a breast cancer patient, comprising at least one selected from the group consisting of KLK3, DDC and AR.
  • Item 6. The marker according to Item 5, wherein the breast cancer patient is a female patient.
  • Item 7 A method for screening a therapeutic agent for breast cancer that is resistant to hormone therapy, comprising contacting a test substance with hormone therapy-resistant breast cancer cells, culturing the cells, and measuring cell proliferation and / or androgen receptor activity Compared to the results of culturing and measuring the hormone therapy resistant breast cancer cells not contacted with the test substance under the same conditions, and whether the test substance caused cell proliferation and / or decreased AR activity Determining whether or not,
  • the method wherein the hormone therapy resistant breast cancer cells are cell lines that are estrogen independent, estrogen receptor activity independent, and androgen receptor activity dependent.
  • Item 8 A method for producing androgen receptor activity-dependent breast cancer cells, comprising culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
  • Item 9 An androgen receptor activity-dependent breast cancer cell line obtained by a method comprising culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
  • a cell line having a novel resistance mechanism that is completely different from the conventional hormone therapy resistance mechanism and a method for screening a therapeutic agent for hormone therapy resistant breast cancer cells using the cell line.
  • a method for screening a therapeutic agent for hormone therapy resistant breast cancer cells using the cell line can be provided.
  • the result of the cell proliferation test of T47D-TE8, A3T and B4T in the presence of estrogen in the Examples is shown.
  • the ER activity measurement result of T47D-TE8, A3T, and B4T in an Example is shown.
  • the result (ER) of the western blotting in an Example is shown.
  • the measurement result of the ER expression level by real-time RT-PCR in an Example is shown.
  • the result of the cell proliferation test of T47D-TE8, A3T, and B4T in the presence of androgen in Examples is shown.
  • the AR activity measurement result of T47D-TE8, A3T, and B4T in an Example is shown.
  • the ER activity measurement result of T47D-TE8, A3T, and B4T in an Example is shown.
  • the result (AR and PSA) of the Western blotting in an Example is shown.
  • the measurement result of AR and KLK3 expression level by real-time RT-PCR in an Example is shown.
  • the measurement result of the DDC expression level by real-time RT-PCR in an Example is shown.
  • the result of the cell proliferation test of T47D-TE8, A3T and B4T in the presence of DHT in the Examples is shown.
  • the base sequence of KLK3 gene is shown.
  • the base sequence of the DDC gene is shown.
  • the base sequence of the AR gene is shown.
  • the present invention relates to an androgen receptor-dependent (androgen receptor activity-dependent) method comprising the step of culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
  • a method for producing a breast cancer cell is provided.
  • the T47D strain used as a parent cell in the present invention is a human breast cancer cell strain and has been deposited with ATCC No .: HTB-133.
  • “T47D cells” include those transformed with the T47D strain as long as the effects of the present invention are not impaired.
  • a vector containing a GFP gene as a reporter gene downstream of an estrogen response element (ERE) is used for T47D cells, and a known method (Yamaguchi Y. et al., Cancer Res, 2005, 65: 4653) is used. -4662) and T47D-TE8 cells transformed with the above T47D strain.
  • the medium depleted of estrogen used in the culturing step in the present invention is a medium obtained by adding estrogen in advance to the medium as serum such as fetal calf serum (FCS).
  • FCS fetal calf serum
  • culturing in a medium depleted of estrogen means adding cells to the medium from which estrogen has been removed as described above and culturing. If the medium as described above is used, even if a trace amount of estrogen is originally contained in the cell or a trace amount of estrogen is produced by the cell during the culture, the medium in which the estrogen is depleted in the present invention is used.
  • T47D cells are cultured in the presence of androgen.
  • Examples of the androgen include, but are not limited to, testosterone, dihydrotestosterone, and dehydroepiandrosterone, and testosterone is preferable.
  • the concentration of androgen is not particularly limited, and can be appropriately set within a range of, for example, 5 to 500 nM, preferably 10 to 300 nM, more preferably 50 to 150 nM.
  • the type of the medium is not particularly limited as long as estrogen is depleted and androgen is present, and those that can be used for culturing breast cancer cells can be widely used.
  • RPMI1640 medium and the like can be mentioned.
  • the culture temperature is not particularly limited and can be appropriately set in the range of 36.5 to 38.0 ° C, more preferably 37.0 to 37.5 ° C.
  • An inert gas containing air or an appropriate concentration of oxygen as the gas phase and containing an appropriate concentration of CO 2 in order to keep the concentration of the medium in a predetermined range can be used.
  • the culture time is not particularly limited, and can be appropriately set within a range of, for example, 720 to 3000 hours, more preferably 2000 to 2500 hours.
  • an androgen receptor activity-dependent breast cancer cell line can be obtained.
  • the method of the present invention makes it possible to produce a breast cancer cell line that is not only dependent on androgen receptor activity but also estrogen-independent and estrogen receptor activity-independent.
  • the androgen receptor may be referred to as an androgen receptor or AR.
  • an androgen metabolite-dependent cell line can be obtained by using MCF7-E10 cells as a parent cell and culturing in the presence of estrogen and in the presence of androgen (Patent Document 1, Type 4).
  • the type 4 cell line derived from MCF7-E10 cells is dependent on dihydrotestosterone (DHT) which is a metabolite of testosterone which is an androgen, and 3 ⁇ -hydroxysteroid dehydrogenase type 1 (3 ⁇ which is a DHT metabolizing enzyme.
  • DHT dihydrotestosterone
  • HSD3B1 converts DHT to 3 ⁇ -diol.
  • type 4 cell lines have acquired estrogen depletion resistance by a mechanism in which ER activity is induced by 3 ⁇ -diol obtained from androgen via DHT. It is considered (Patent Document 1). However, the type 4 cell line shows a decrease in androgen receptor expression and does not depend on androgen receptor activity.
  • the breast cancer cell line obtained by the method of the present invention is dependent on androgen receptor activity, and is estrogen-independent and estrogen receptor-independent, and thus has a completely different mechanism from that of the type 4 cell line. It is clear that you have earned. Further, the breast cancer cell line obtained by the method of the present invention is independent of estrogen and estrogen receptor activity which are female hormones, and has acquired androgen receptor activity dependency which is a receptor activity of male hormones. Therefore, in the method of the present invention, it can be said that the breast cancer cell line is masculinized. There has been no report that breast cancer cells are masculinized like this.
  • Breast cancer cell line The present invention provides a breast cancer cell line having androgen receptor dependency (androgen receptor activity dependency) obtained by the above method.
  • the characteristics of the breast cancer cell line of the present invention and the production method thereof are as described above.
  • the present invention is also a method for screening a therapeutic agent for breast cancer that is resistant to hormone therapy, comprising contacting a test substance with hormone therapy-resistant breast cancer cells, culturing the cells, Or the step of measuring androgen receptor activity, and the test substance is a cell proliferation and / or compared with the results of culturing and measuring the hormone therapy resistant breast cancer cells not contacted with the test substance under the same conditions Determining whether the AR activity has been reduced, Provided is a method wherein the hormone therapy resistant breast cancer cells are cell lines that are estrogen independent, estrogen receptor activity independent, and androgen receptor dependent (androgen receptor activity dependent).
  • an androgen receptor-activity-dependent breast cancer cell prepared by the above method may be used. it can.
  • the contact between the cells and the test substance can be easily performed by adding the test substance to the medium. Contact with the test substance may be performed by a short pulse or may be present in the medium throughout the culture period. Therefore, contact and culture can be performed simultaneously.
  • As the medium a normal medium, an estrogen-depleted medium described below, or a medium with a predetermined additive added thereto can be used as necessary.
  • Cell proliferation can be carried out by counting the number of cells by a conventional method. Androgen receptor activity can be measured according to a method known per se.
  • cell proliferation and androgen receptor activity may be measured and evaluated, or both of them may be measured and evaluated.
  • cell growth is reduced (suppressed) in hormonal therapy-resistant breast cancer cells contacted with the test substance compared to hormonal therapy-resistant breast cancer cells not contacted with the test substance, and AR activity is reduced. When it falls, it can be judged that the said to-be-tested substance can become an effective therapeutic agent with respect to the breast cancer cell which acquired androgen receptor activity dependence.
  • Cell growth was reduced in hormone therapy-resistant breast cancer cells in contact with the test substance, and if AR activity did not decrease, androgen-androgen receptor system was acquired, but it did not inhibit the androgen receptor system.
  • the cells of the present invention are transplanted into animals to create model animals of the respective mechanisms, and are closer to individuals. Can be used to evaluate drug effects in experimental systems.
  • the present invention relates to the presence or absence of acquisition of androgen receptor dependency (androgen receptor activity dependency) in a breast cancer patient using at least one selected from the group consisting of KLK3, DDC and AR as an index.
  • a method for determining at least one gene product selected from the group consisting of KLK3, DDC and AR is used as an index.
  • the gene product includes mRNA that is a transcription product of the gene and a protein that is a translation product of the mRNA.
  • KLK3, DDC and AR or their gene products mean those derived from endogenous genes (on the genome).
  • the determination method of the present invention includes a step of measuring at least one expression level selected from the group consisting of KLK3, DDC and AR in a sample collected from a subject, and an expression level and control of the subject sample measured in the above step A step of comparing the expression level.
  • PSA prote specific antigen
  • KLK3 kallikrein 3
  • DDC L-Dopa decarboxylase
  • the base sequence of the DDC gene is known per se, and examples thereof include the one shown by GenBank accession number M76180.1 (SEQ ID NO: 2).
  • the AR gene is known per se, and for example, the gene indicated by GenBank accession number M34233.1 (SEQ ID NO: 3) can be mentioned.
  • the subject is not particularly limited, but examples include mammals including humans. Examples of non-human mammals include mice, rats, dogs, cats, cows, sheep and horses.
  • a preferred test subject of the determination method of the present invention is a human.
  • the subject may be female (female) or male (male), but is particularly useful for testing female subjects.
  • a method for measuring KLK3, DDC and / or AR a method known per se can be appropriately employed, and is not particularly limited.
  • cancer tissue is removed from a breast cancer patient, antigen detection by immunostaining, RT- It can be performed by detecting mRNA by PCR.
  • the measured value of the expression level of KLK3, DDC and / or AR obtained by the above method is statistically significantly larger than the numerical value of the control (breast cancer patient who has not acquired androgen receptor activity dependency) Can be determined to have acquired androgen receptor activity dependency.
  • the control breast cancer patient who has not acquired androgen receptor activity dependency
  • a method of determining that the androgen receptor activity dependency is acquired can be mentioned.
  • a method for measuring all of KLK3, DDC, and AR can be mentioned. In that case, if all three values are significantly larger than the control, it is determined that the androgen receptor activity dependency is acquired.
  • both KLK3 and DDC are measured, and both KLK3 and DDC values are significantly larger than the control, it is determined that the androgen receptor activity dependence has been acquired, and both KLK3 and DDC are As a result of the measurement, when only one of KLK3 and DDC is significantly larger than the control, there is a method for determining that the androgen receptor activity dependency is acquired.
  • the subject determined to have acquired androgen receptor activity dependency by the above method can be treated appropriately for breast cancer that has acquired androgen receptor activity dependency.
  • treatment methods such as bicalutamide, flutamide, enzalutamide and the like can be mentioned.
  • the present invention includes a step of determining whether or not androgen receptor activity is acquired in a breast cancer patient using at least one selected from the group consisting of KLK3, DDC and AR as an index, and androgen receptor activity in the determination step.
  • a method for treating a breast cancer patient comprising a step of treating a breast cancer patient determined to have acquired dependence suitable for breast cancer that has acquired androgen receptor activity dependence.
  • the present invention relates to androgen receptor dependency (androgen receptor activity dependency) in breast cancer patients, comprising means for measuring at least one expression level selected from the group consisting of KLK3, DDC and AR. Gender)
  • a kit for determining the presence or absence of acquisition is provided.
  • the means for measuring at least one expression level selected from the group consisting of KLK3, DDC, and AR is not particularly limited.
  • PCR immunostaining, Northern blotting, Western blotting, ELISA, Means used for the microarray method and the like can be mentioned.
  • KLK3, DDC and / or AR as primers or probes for PCR
  • examples include reverse transcriptase, DNA polymerase, and the like; antibodies that specifically bind to PSA, DDC, and / or AR used for immunostaining, and microarrays to which probes used for microarray methods are immobilized.
  • Examples of a primer for detecting KLK3 by RT-PCR include F: 5′-TGTCCGTGACGTGGATT-3 ′ (SEQ ID NO: 4) R: 5′-ACGAGAGGCCACAAGCA-3 ′ (SEQ ID NO: 5).
  • primers for detecting DDC by RT-PCR include F: 5′-GAAGTCGGTCCTATCTGCAAC-3 ′ (SEQ ID NO: 6), R: 5′-ACTCCACTCCATTCAGAAGGT-3 ′ (SEQ ID NO: 7), and the like. it can.
  • primers for detecting AR by RT-PCR include F: 5′-ATGTGGAAGCTGCAAGGTCT-3′CT (SEQ ID NO: 8) R: 5′-CGAAGACGACAAGATGGACA-3 ′ (SEQ ID NO: 9).
  • the kit of the present invention may contain other components as necessary. Examples of other components include, but are not limited to, a tool for collecting a specimen, a positive control sample, and a negative control sample. It can also include a document in which a procedure for performing the above determination method is written.
  • the present invention relates to the presence or absence of acquisition of androgen receptor dependency (androgen receptor activity dependency) in breast cancer patients, comprising at least one selected from the group consisting of KLK3, DDC and AR.
  • a marker for determination means an index for evaluating whether or not a subject or a sample collected from the subject has a specific property, whether it is in a specific state, or the like.
  • KLK3, DDC, and AR included in the determination marker are not limited to these genes per se as long as they can be used as markers, and are mRNA and translation products of gene transcripts. Protein etc. are also included.
  • a marker containing at least one selected from the group consisting of KLK3, DDC and AR examples include proteins.
  • double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof are included.
  • gene refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.
  • mRNA, cDNA, protein derived from at least one gene selected from the group consisting of KLK3, DDC, and AR is not limited to the whole mRNA, the whole cDNA, and the whole protein. As long as it can be specified that they are derived from, all (partial) or all of these mRNAs, part (fragment) or all of cDNA, and part (fragment) or all of each of proteins are included. It is.
  • T47D-TE8 cells used for the establishment were derived from the human breast cancer cell line T47D (ATCC number: HTB-133) from the estrogen response element (ERE) upstream of the short half-life GFP cDNA in the pd2EGFP-1 vector (Clontech). ) was introduced according to a known method (Yamaguchi Y. et al. Cancer Res, 2005, 65: 4653-4662). T47D-TE8 cells were cultured in a normal medium.
  • I-2 Cell culture method
  • RPMI 1640 medium SIGMA (sometimes called “normal medium") supplemented with 10% FCS (Tissue Culture Biologicals) and 1% penicillin / streptomycin (GIBCO) was used.
  • FCS Tissue Culture Biologicals
  • GIBCO penicillin / streptomycin
  • DCC-FCS dextran-coated charcoal-treated FCS
  • GIBCO penicillin / streptomycin
  • estradiol, testosterone, and DHT were purchased from Sigma-Aldrich Corp. Fulvestrant was obtained from AstraZeneca. Letrozole was obtained from Novartis Pharma (Tokyo, Japan). Baicartamide (androgen receptor inhibitor) was obtained from LKT Laboratories (St. Paul, MN, USA).
  • RNA PCR kit RNA PCR kit
  • Luciferase assay ERE activity was measured using the trade name “Dual-Luciferase Reporter System” (Promega) basically according to the method described in Biochem Biophys Res Commun 2001, 285: 340-347. After culturing in a depletion medium for 3 days, 3 ⁇ 10 5 cells were seeded in a 6 cm dish and cultured in the same medium for 48 hours.
  • T47D-TE8 cells were cultured for about 3 months in a medium prepared by adding 10% DCC-FCS (charcoal-treated fetal bovine serum) and 1% Penicillin / Streptomycin to Phenol red free RPMI medium to give 100 nM testosterone. did.
  • DCC-FCS charcoal-treated fetal bovine serum
  • Penicillin / Streptomycin to Phenol red free RPMI medium
  • the proportion of cells that fluoresce gradually decreased.
  • those that fluoresced were cloned, and two cell lines A3T and B4T that became fluorescent when testosterone was added were designated as type 6 cells.
  • A3T and B4T gradually lost fluorescence as the culture was continued.
  • the parent strain T47D-TE8 was controlled when 100 pM of estradiol was added (the number of cells on the fourth day when ethanol was added) The number of cells increased by a factor of about 2, but A3T and B4T showed no change in the number of cells.
  • an ERE luciferase reporter and a pRL luciferase reporter as an internal control were temporarily introduced to carry out a luciferase assay.
  • a luciferase assay was performed using estradiol (100 pM (E2)) as the drug, 100 pM estradiol and the antiestrogenic drug fulvestrant 1 ⁇ M (E2 + Ful) or ethanol as the control. It was. The results are shown in FIG.
  • T47D-TE8 increased ER activity about 8-fold by adding 100 pM estradiol, but A3T and B4T showed almost no change in activity. From these results, it was considered that A3T and B4T do not function the cell growth mechanism via ER possessed by T47D-TE8.
  • the expression level of mRNA was measured by real-time RT-PCR using RPL13A as an internal control (FIG. 4).
  • the expression of ER and its response gene, PgR was markedly decreased in A3T and B4T. From the above, it was shown that not only ER function but also ER expression was almost lost in A3T and B4T.
  • Testosterone was added and cultured for 4 days to conduct a cell proliferation test. Specifically, a cell proliferation test was performed in the same manner as II-2 except that testosterone was used instead of estrogen (upper part of FIG. 5). T47D-TE8 showed no change in cell number compared to control, but A3T and B4T showed about 1.4-fold increase in testosterone at 10 nM.
  • an ARE luciferase reporter including a PSA promoter upstream region and a pRL luciferase reporter as an internal control were temporarily introduced, and a luciferase assay was performed.
  • the ARE luciferase reporter was prepared by using an androgen reporter plasmid (including the PSA promoter region, 5800 bases upstream from the PSA transcription start site instead of the estrogen reporter plasmid (Tk-ERE-Luci) -TK-luci (introducing ARE instead of ERE) Except that the above-mentioned I-6. In accordance with the method described in.
  • T47D-TE8, A3T and B4T cell lines were added with testosterone 10 nM (TS), testosterone 10 nM and baicaltamide 10 ⁇ M (TS + Bic) or ethanol added as a control and cultured for 2 days.
  • the gene expression ratio between samples was analyzed using an Agilent oligo microarray and data analysis software GeneSpringGX. Among the genes whose expression was more than doubled in A3T compared to T47D-TE8, genes related to both “Androgen” and “Cancer” were picked up by pathway analysis software IPA. The gene with the highest ratio after PSA was DDC (L-Dopa decarboxylase). DDC is a coactivator of AR and has been reported to promote cell growth by overexpression in prostate cancer cells (Wafa et al. Int.J.Cancer 2012, 130: 2835-2844).
  • FIG. 10 shows the relative value of the expression level with T47D-TE8 as 1.
  • increased DDC expression was predicted to be a major factor in increasing AR activity.
  • a cell proliferation test was performed by adding an androgen, a DDC inhibitor, and NSD-1015.
  • Fig. 11 In T47D-TE8, A3T, and B4T, the number of cells increased compared to the control by adding DHT1nM. This growth promoting reaction was suppressed by A3T / B4T by addition of 200 ⁇ M NSD-1015 but not by T47D-TE8.
  • T47D-TE8 originally has an AR-mediated proliferation pathway that is enhanced in A3T and B4T, and that DDC contributes greatly to its activation. .
  • breast cancer resistance to hormone therapy is that breast cancer, like prostate cancer, shows growth dependent on signals through androgen and androgen receptor. There seems to be a possibility. At that time, DDC seems to play an important role. Anti-androgen treatment may be effective for advanced / recurrent breast cancer that has acquired such a resistance mechanism.
  • Primary antibodies include anti-ER ⁇ antibody (ER1D5, Immunotech, Marseille, France), anti-PR (progesterone receptor) antibody (MAB429, Chemicon, Temecula, CA, USA), anti-HER2 antibody (A0485, DAKO, Carpinteria, CA, USA) )), Anti-Ki-67 antibody (MIB1, DAKO, Carpinteria, CA, USA) and anti-AR antibody (AR441, DAKO) were used.
  • antigen activation of ER ⁇ , PR, Ki-67, and AR heat treatment was performed at 120 ° C. for 5 minutes in an autoclave using a citrate buffer.
  • the dilution factor is ER ⁇ ; 1/50, PR; 1/30, HER2; 1/200, Ki-67; 1/50, AR; 1/50, 3.3'-diaminobenzidine ( DAB) solution was used and nuclear staining was performed with hematoxylin.
  • ER ⁇ , PgR, AR, and Ki67 measure the number of positive cells in more than 1000 cancer cells and evaluate the ratio by calculating LI [labeling Index: percentage of positive cells (%)].
  • ER ⁇ and PgR are 1% As described above, AR was positive for LI 10% or more.
  • HER2 immunostaining was evaluated on a 4-stage (score) basis for the expression of HER2 protein on the cell membrane according to the staining kinetics. All immunohistochemical staining results were determined only by cancer invasion. The results are shown in Table 2 below.
  • SEQ ID NO: 4 is a primer.
  • SEQ ID NO: 5 is a primer.
  • SEQ ID NO: 6 is a primer.
  • SEQ ID NO: 7 is a primer.
  • SEQ ID NO: 8 is a primer.
  • SEQ ID NO: 9 is a primer.

Abstract

The present invention addresses the problem of providing a cell line having a novel resistance mechanism that is completely different from the resistance mechanisms in conventional hormone therapy, a screening method using the cell line, and a method for determining whether or not a breast cancer patient has acquired the aforesaid resistance mechanism. Provided are: a method for preparing androgen receptor activity-dependent breast cancer cells, said method comprising a step for culturing T47D-TE8 cells in an estrogen-depleted medium in the presence of androgen; a screening method using the breast cancer cells; and a method and a kit for determining the presence or absence of the acquisition of androgen receptor activity dependence in a breast cancer patient with the use of at least one member selected from the group consisting of KLK3, DDC and AR as an indicator.

Description

アンドロゲンレセプター活性依存性乳癌細胞株の作製方法、当該細胞株を用いたスクリーニング方法、ならびに乳癌患者におけるアンドロゲンレセプター活性依存性獲得の判定方法、キット及びマーカーMethod for producing androgen receptor activity-dependent breast cancer cell line, screening method using the cell line, and method for determining androgen receptor activity-dependent acquisition in breast cancer patients, kit and marker
　［関連出願の相互参照］
　本出願は、２０１３年５月２３日に出願された、日本国特許出願第２０１３－１０８７７４号明細書（その開示全体が参照により本明細書中に援用される）に基づく優先権を主張する。 [Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2013-108774 filed on May 23, 2013, the entire disclosure of which is incorporated herein by reference.
　本発明は、ホルモン療法耐性乳癌細胞株の作製方法、当該細胞株を用いた乳癌治療剤のスクリーニング方法及び乳癌患者におけるホルモン療法耐性獲得の判定方法、キット及びマーカーに関する。 The present invention relates to a method for producing a hormone therapy-resistant breast cancer cell line, a screening method for a therapeutic agent for breast cancer using the cell line, a method for determining acquisition of hormone therapy resistance in breast cancer patients, a kit, and a marker.
　エストロゲンは、乳癌の発生及び増殖に深く関係する女性ホルモンであり、乳癌細胞の多くはエストロゲン依存性に増殖するホルモン依存性腫瘍である。乳癌細胞がエストロゲンを受容すると、エストロゲンレセプター（ＥＲ）を介して細胞増殖に関わるシグナル伝達系が活性化され、細胞増殖が促進されて癌が進展する。 Estrogen is a female hormone closely related to the development and proliferation of breast cancer, and most breast cancer cells are hormone-dependent tumors that proliferate in an estrogen-dependent manner. When breast cancer cells receive estrogen, a signal transduction system related to cell proliferation is activated via the estrogen receptor (ER), cell proliferation is promoted, and cancer progresses.
　乳癌患者の７０％以上の乳癌はエストロゲンレセプター（ＥＲ)陽性であり、ホルモン療法の治療対象である。 Breast cancer in more than 70% of breast cancer patients is estrogen receptor (ER) positive and is a subject of treatment for hormone therapy.
　乳癌治療薬としてのホルモン剤は、大きく、タモキシフェン、トレミフェン、フルベストラント、ラロキシフェン等のエストロゲンレセプターの阻害作用を示す薬剤（抗エストロゲン剤）；及びアナストロゾール、レトロゾール、エグゼメスタン等のエストロゲン合成酵素であるアロマターゼを阻害する薬剤（アロマターゼ阻害剤（ＡＩ））の２種類に分類される。 Hormonal agents as breast cancer treatments are large, drugs that exhibit an inhibitory action on estrogen receptors such as tamoxifen, toremifene, fulvestrant, and raloxifene (antiestrogens); and estrogen synthases such as anastrozole, letrozole, and exemestane It is classified into two types of drugs (aromatase inhibitor (AI)) that inhibit aromatase.
　しかし、これらの薬剤はいずれも、やがて耐性を獲得されてしまうという問題がある。ホルモン療法を受けた乳癌患者のうち１／３は、癌がホルモン治療に耐性を獲得し、再発する。再発後は、化学療法が行われるが、予後が不良であり、治療が困難である。したがって、ホルモン療法耐性の獲得が、臨床上、重大な問題となっており、耐性機序の解明と新たな治療方法が望まれている。 However, all of these drugs have a problem that resistance is eventually acquired. One third of breast cancer patients who have received hormonal therapy relapse because the cancer has acquired resistance to hormonal therapy. Chemotherapy is given after recurrence, but the prognosis is poor and treatment is difficult. Therefore, acquisition of resistance to hormonal therapy is a serious problem clinically, and elucidation of the resistance mechanism and a new treatment method are desired.
　かかる状況の下、本発明者は、既に、下記５種類のホルモン療法耐性乳癌細胞株の樹立している：
エストロゲン非依存性、かつ、エストロゲンレセプター（ＥＲ)活性依存性である細胞株（タイプ１）、
エストロゲン非依存性、かつ、ＥＲ活性非依存性である細胞株（タイプ２）、
エストロゲン非依存性、ＥＲ活性非依存性、かつ、成長因子（ＧＦ）依存性であり、ＨＥＲ２及び上皮増殖因子レセプター（ＥＧＦＲ）を発現する細胞株（タイプ３）、
アンドロゲン代謝産物依存性である細胞株（タイプ４）、及び／又は
アロマターゼ阻害剤（ＡＩ）非感受性、かつ、ＥＲ活性依存性である細胞株（タイプ５）
（特許文献１）。 Under such circumstances, the present inventor has already established the following five types of hormone therapy resistant breast cancer cell lines:
A cell line (type 1) that is estrogen-independent and estrogen receptor (ER) activity-dependent,
A cell line that is estrogen-independent and ER-independent (type 2),
A cell line (type 3) that is estrogen independent, ER activity independent, and growth factor (GF) dependent and expresses HER2 and epidermal growth factor receptor (EGFR),
Cell lines that are dependent on androgen metabolites (type 4) and / or cell lines that are insensitive to aromatase inhibitor (AI) and are dependent on ER activity (type 5)
(Patent Document 1).
特開２０１３－１７４１４JP2013-17414
　本発明は、従来のホルモン療法耐性機序とは全く異なる新規の耐性機序を有する細胞株を提供すること、当該細胞株を用いたホルモン療法耐性乳癌細胞に対する治療剤のスクリーニング方法を提供すること、及び乳癌患者がかかる耐性機序を獲得してしまったか否かを判定する方法を提供することを課題とする。 The present invention provides a cell line having a novel resistance mechanism that is completely different from the conventional hormone therapy resistance mechanism, and provides a screening method for a therapeutic agent for hormone therapy resistant breast cancer cells using the cell line. It is another object of the present invention to provide a method for determining whether or not a breast cancer patient has acquired such a resistance mechanism.
　上記の状況の下、本発明者らは、種々の培養条件でホルモン療法耐性乳癌細胞株の樹立を試みたところ、親細胞としてＴ４７Ｄ－ＴＥ８細胞を用い、これをアンドロゲンを添加したエストロゲン枯渇培地で培養すると、意外にも当該細胞株がアンドロゲンレセプター（ＡＲ）活性依存性を獲得することを見出した。また、当該ＡＲ依存性獲得株の遺伝子発現を解析したところ、ＡＲ依存性を獲得した上記細胞株においてＰＳＡ（ＫＬＫ３）、ＤＤＣ及びＡＲの発現量が有意に増加していることを見出した。本発明はこれらの新規の知見に基づくものである。 Under the circumstances described above, the present inventors tried to establish a hormone therapy-resistant breast cancer cell line under various culture conditions. As a parent cell, T47D-TE8 cells were used, and this was used in an estrogen-depleted medium supplemented with androgen. It was found that the cell line unexpectedly acquired androgen receptor (AR) activity dependency when cultured. Moreover, when the gene expression of the AR-dependent acquired strain was analyzed, it was found that the expression levels of PSA (KLK3), DDC, and AR were significantly increased in the cell line that acquired the AR-dependent. The present invention is based on these new findings.
　従って、本発明は以下の項を提供する：
　項１．ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを指標とする、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定する方法。 Accordingly, the present invention provides the following sections:
Item 1. A method for determining the presence or absence of androgen receptor activity-dependent acquisition in a breast cancer patient, using as an index at least one selected from the group consisting of KLK3, DDC and AR.
　項２．乳癌患者が女性患者である、請求項１に記載の方法。 Item 2. 2. The method of claim 1, wherein the breast cancer patient is a female patient.
　項３．ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの発現量を測定する手段を含む、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定するためのキット。 Item 3. A kit for determining the presence or absence of androgen receptor activity-dependent acquisition in a breast cancer patient, comprising means for measuring at least one expression level selected from the group consisting of KLK3, DDC and AR.
　項４．乳癌患者が女性患者である、請求項３に記載のキット。 Item 4. The kit according to claim 3, wherein the breast cancer patient is a female patient.
　項５．ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを含む、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定するためのマーカー。 Item 5. A marker for determining the presence or absence of acquisition of androgen receptor activity dependency in a breast cancer patient, comprising at least one selected from the group consisting of KLK3, DDC and AR.
　項６．乳癌患者が女性患者である、項５に記載のマーカー。 Item 6. Item 6. The marker according to Item 5, wherein the breast cancer patient is a female patient.
　項７．ホルモン療法に耐性を示す乳癌に対する治療剤をスクリーニングする方法であって、ホルモン療法耐性乳癌細胞に被験物質を接触させる工程と、この細胞を培養し、細胞の増殖及び／又はアンドロゲンレセプター活性を測定する工程と、前記被検物質と接触させていない前記ホルモン療法耐性乳癌細胞を同じ条件下で培養及び測定した結果と比較して前記被検物質が細胞の増殖及び／又はＡＲ活性の低下をもたらしたか否かを判定する工程とを含み、
　前記ホルモン療法耐性乳癌細胞が、エストロゲン非依存性、エストロゲンレセプター活性非依存性、かつアンドロゲンレセプター活性依存性である細胞株である、方法。 Item 7. A method for screening a therapeutic agent for breast cancer that is resistant to hormone therapy, comprising contacting a test substance with hormone therapy-resistant breast cancer cells, culturing the cells, and measuring cell proliferation and / or androgen receptor activity Compared to the results of culturing and measuring the hormone therapy resistant breast cancer cells not contacted with the test substance under the same conditions, and whether the test substance caused cell proliferation and / or decreased AR activity Determining whether or not,
The method wherein the hormone therapy resistant breast cancer cells are cell lines that are estrogen independent, estrogen receptor activity independent, and androgen receptor activity dependent.
　項８．Ｔ４７Ｄ細胞を、エストロゲンを枯渇させた培地で、かつアンドロゲンの存在下で培養する工程を含む、アンドロゲンレセプター活性依存性の乳癌細胞の作製方法。 Item 8. A method for producing androgen receptor activity-dependent breast cancer cells, comprising culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
　項９．Ｔ４７Ｄ細胞を、エストロゲンを枯渇させた培地で、かつアンドロゲンの存在下で培養する工程を含む方法により得られる、アンドロゲンレセプター活性依存性の乳癌細胞株。 Item 9. An androgen receptor activity-dependent breast cancer cell line obtained by a method comprising culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
　本発明によれば、従来のホルモン療法耐性機序とは全く異なる新規の耐性機序を有する細胞株を提供すること、及び当該細胞株を用いたホルモン療法耐性乳癌細胞に対する治療剤のスクリーニング方法を提供することができる。また、本発明によれば、ホルモン療法により乳癌患者がアンドロゲンレセプター活性依存性を獲得たか否かを判定することができる。 According to the present invention, there is provided a cell line having a novel resistance mechanism that is completely different from the conventional hormone therapy resistance mechanism, and a method for screening a therapeutic agent for hormone therapy resistant breast cancer cells using the cell line. Can be provided. In addition, according to the present invention, it is possible to determine whether or not a breast cancer patient has acquired androgen receptor activity dependency by hormone therapy.
実施例におけるエストロゲン存在下でのT47D-TE8、A3T及びB4Tの細胞増殖試験の結果を示す。The result of the cell proliferation test of T47D-TE8, A3T and B4T in the presence of estrogen in the Examples is shown. 実施例におけるT47D-TE8、A3T及びB4TのER活性測定結果を示す。The ER activity measurement result of T47D-TE8, A3T, and B4T in an Example is shown. 実施例でのウエスタンブロッティングの結果（ＥＲ）を示す。The result (ER) of the western blotting in an Example is shown. 実施例におけるリアルタイムRT-PCRによるER発現量の測定結果を示す。The measurement result of the ER expression level by real-time RT-PCR in an Example is shown. 実施例におけるアンドロゲン存在下でのT47D-TE8、A3T及びB4Tの細胞増殖試験の結果を示す。The result of the cell proliferation test of T47D-TE8, A3T, and B4T in the presence of androgen in Examples is shown. 実施例におけるT47D-TE8、A3T及びB4TのAR活性測定結果を示す。The AR activity measurement result of T47D-TE8, A3T, and B4T in an Example is shown. 実施例におけるT47D-TE8、A3T及びB4TのER活性測定結果を示す。The ER activity measurement result of T47D-TE8, A3T, and B4T in an Example is shown. 実施例でのウエスタンブロッティングの結果（AR及びPSA）を示す。The result (AR and PSA) of the Western blotting in an Example is shown. 実施例におけるリアルタイムRT-PCRによるAR及びKLK3発現量の測定結果を示す。The measurement result of AR and KLK3 expression level by real-time RT-PCR in an Example is shown. 実施例におけるリアルタイムRT-PCRによるDDC発現量の測定結果を示す。The measurement result of the DDC expression level by real-time RT-PCR in an Example is shown. 実施例におけるDHT存在下でのT47D-TE8、A3T及びB4Tの細胞増殖試験の結果を示す。The result of the cell proliferation test of T47D-TE8, A3T and B4T in the presence of DHT in the Examples is shown. ＫＬＫ３遺伝子の塩基配列を示す。The base sequence of KLK3 gene is shown. ＤＤＣ遺伝子の塩基配列を示す。The base sequence of the DDC gene is shown. ＡＲ遺伝子の塩基配列を示す。The base sequence of the AR gene is shown.
　アンドロゲンレセプター活性依存性の乳癌細胞の作製方法
　本発明は、Ｔ４７Ｄ細胞を、エストロゲンを枯渇させた培地で、かつアンドロゲンの存在下で培養する工程を含む、アンドロゲンレセプター依存性（アンドロゲンレセプター活性依存性）の乳癌細胞の作製方法を提供する。 The present invention relates to an androgen receptor-dependent (androgen receptor activity-dependent) method comprising the step of culturing T47D cells in a medium depleted of estrogen and in the presence of androgen. A method for producing a breast cancer cell is provided.
　本発明において親細胞として用いるＴ４７Ｄ株は、ヒト乳癌細胞株であり、ＡＴＣＣ番号：ＨＴＢ－１３３で寄託されている。本発明において、「Ｔ４７Ｄ細胞」には、本発明の効果を棄損しない限りにおいて、Ｔ４７Ｄ株を形質転換したものも含まれる。例えば、本発明において、Ｔ４７Ｄ細胞には、エストロゲン応答配列（ＥＲＥ）の下流にレポーター遺伝子としてＧＦＰ遺伝子を含むベクターを用い、公知の方法（Yamaguchi Y. et al., Cancer Res, 2005, 65: 4653-4662）で上記Ｔ４７Ｄ株を形質転換したＴ４７Ｄ－ＴＥ８細胞等も含まれる。 The T47D strain used as a parent cell in the present invention is a human breast cancer cell strain and has been deposited with ATCC No .: HTB-133. In the present invention, “T47D cells” include those transformed with the T47D strain as long as the effects of the present invention are not impaired. For example, in the present invention, a vector containing a GFP gene as a reporter gene downstream of an estrogen response element (ERE) is used for T47D cells, and a known method (Yamaguchi Y. et al., Cancer Res, 2005, 65: 4653) is used. -4662) and T47D-TE8 cells transformed with the above T47D strain.
　本発明における培養工程で用いるエストロゲンを枯渇させた培地としては、ウシ胎児血清（ＦＣＳ）等の血清として事前にエストロゲンを除去したものを培地に添加したものを示す。本発明において、エストロゲンを枯渇させた培地で培養するとは、上記のようにエストロゲンを除去した培地に細胞を添加して培養することを意味する。上記のような培地を用いていれば、細胞中に元々微量のエストロゲンが含まれていたり、または培養中に細胞により微量のエストロゲンが生成されても、本発明におけるエストロゲンを枯渇させた培地での培養に該当する。本発明においては、Ｔ４７Ｄ細胞はアンドロゲンの存在下で培養することを特徴とする。アンドロゲンとしては、特に限定されないが、テストステロン、ジヒドロテストステロン、デヒドロエピアンドロステロン等を挙げることができ、テストステロンが好ましい。アンドロゲンの濃度としては特に限定されないが、例えば、5～500ｎＭ、好ましくは10～300ｎＭ、より好ましくは50～150ｎＭの範囲で適宜設定できる。 The medium depleted of estrogen used in the culturing step in the present invention is a medium obtained by adding estrogen in advance to the medium as serum such as fetal calf serum (FCS). In the present invention, culturing in a medium depleted of estrogen means adding cells to the medium from which estrogen has been removed as described above and culturing. If the medium as described above is used, even if a trace amount of estrogen is originally contained in the cell or a trace amount of estrogen is produced by the cell during the culture, the medium in which the estrogen is depleted in the present invention is used. Corresponds to culture. In the present invention, T47D cells are cultured in the presence of androgen. Examples of the androgen include, but are not limited to, testosterone, dihydrotestosterone, and dehydroepiandrosterone, and testosterone is preferable. The concentration of androgen is not particularly limited, and can be appropriately set within a range of, for example, 5 to 500 nM, preferably 10 to 300 nM, more preferably 50 to 150 nM.
　培地の種類は、エストロゲンが枯渇しており、かつアンドロゲンが存在している限り特に限定されず、乳癌細胞の培養に用いることができるものを広く使用することができる。例えば、RPMI1640培地等が挙げられる。また、上記培地には、抗生物質等の所定の添加物を加えてもよい。培養温度は特に限定されず、36.5～38.0℃、より好ましくは37.0～37.5℃の範囲で適宜設定できる。気相として空気もしくは適当な濃度の酸素を含み培地の濃度を所定の範囲に保つために適当な濃度のＣＯ2を含む不活性ガスを用いることができる。培養時間は特に限定されないが、例えば、720～3000時間、より好ましくは2000～2500時間の範囲で適宜設定できる。 The type of the medium is not particularly limited as long as estrogen is depleted and androgen is present, and those that can be used for culturing breast cancer cells can be widely used. For example, RPMI1640 medium and the like can be mentioned. Moreover, you may add predetermined additives, such as antibiotics, to the said culture medium. The culture temperature is not particularly limited and can be appropriately set in the range of 36.5 to 38.0 ° C, more preferably 37.0 to 37.5 ° C. An inert gas containing air or an appropriate concentration of oxygen as the gas phase and containing an appropriate concentration of CO 2 in order to keep the concentration of the medium in a predetermined range can be used. The culture time is not particularly limited, and can be appropriately set within a range of, for example, 720 to 3000 hours, more preferably 2000 to 2500 hours.
　本発明の方法により、アンドロゲンレセプター活性依存性の乳癌細胞株を得ることができる。また、本発明の方法により、アンドロゲンレセプター活性依存性だけでなくエストロゲン非依存性、エストロゲンレセプター活性非依存性も有する乳癌細胞株を作製することができる。本明細書において、アンドロゲンレセプターは、アンドロゲン受容体又はＡＲと表記することもある。 By the method of the present invention, an androgen receptor activity-dependent breast cancer cell line can be obtained. In addition, the method of the present invention makes it possible to produce a breast cancer cell line that is not only dependent on androgen receptor activity but also estrogen-independent and estrogen receptor activity-independent. In the present specification, the androgen receptor may be referred to as an androgen receptor or AR.
　一方、ＭＣＦ７－Ｅ１０細胞を親細胞とし、エストロゲン枯渇、かつアンドロゲン存在下で培養することにより、アンドロゲン代謝産物依存性細胞株を得ることができる（特許文献１、タイプ４）。具体的には、ＭＣＦ７－Ｅ１０細胞由来のタイプ４細胞株は、アンドロゲンであるテストステロンの代謝物であるジヒドロテストステロン（ＤＨＴ）依存性であり、ＤＨＴ代謝酵素である３β－ヒドロキシステロイドデヒドロゲナーゼタイプ１（３β－ＨＳＤタイプ１：ＨＳＤ３Ｂ１）の発現の増大が見られる。ＨＳＤ３Ｂ１によりＤＨＴは３β－ジオールに変換される。３β－ジオールはエストロゲン様の作用をするものであるため、タイプ４細胞株においては、アンドロゲンからＤＨＴを介して得られる３β－ジオールによってＥＲ活性が誘導される機構でエストロゲン枯渇耐性を獲得しているものと考えられる（特許文献１）。しかし、当該タイプ４細胞株では、アンドロゲンレセプター発現の低下が見られ、アンドロゲンレセプター活性依存性を有しない。 On the other hand, an androgen metabolite-dependent cell line can be obtained by using MCF7-E10 cells as a parent cell and culturing in the presence of estrogen and in the presence of androgen (Patent Document 1, Type 4). Specifically, the type 4 cell line derived from MCF7-E10 cells is dependent on dihydrotestosterone (DHT) which is a metabolite of testosterone which is an androgen, and 3β-hydroxysteroid dehydrogenase type 1 (3β which is a DHT metabolizing enzyme. -Increased expression of HSD type 1: HSD3B1). HSD3B1 converts DHT to 3β-diol. Since 3β-diol acts like an estrogen, type 4 cell lines have acquired estrogen depletion resistance by a mechanism in which ER activity is induced by 3β-diol obtained from androgen via DHT. It is considered (Patent Document 1). However, the type 4 cell line shows a decrease in androgen receptor expression and does not depend on androgen receptor activity.
　本発明の方法により得られる乳癌細胞株は、アンドロゲンレセプター活性依存性を有し、かつエストロゲン非依存性及びエストロゲンレセプター活性非依存性であるため、タイプ４細胞株とは全く異なる機構によりエストロゲン枯渇耐性を獲得していることが明らかである。また、本発明の方法により得られる乳癌細胞株は女性ホルモンであるエストロゲン非依存性及びエストロゲンレセプター活性非依存性であり、かつ男性ホルモンの受容体活性であるアンドロゲンレセプター活性依存性を獲得していることから、本発明の方法においては、乳癌細胞株が男性化しているということができる。乳癌細胞がこのような男性化をするということは、これまでに報告がない。 The breast cancer cell line obtained by the method of the present invention is dependent on androgen receptor activity, and is estrogen-independent and estrogen receptor-independent, and thus has a completely different mechanism from that of the type 4 cell line. It is clear that you have earned. Further, the breast cancer cell line obtained by the method of the present invention is independent of estrogen and estrogen receptor activity which are female hormones, and has acquired androgen receptor activity dependency which is a receptor activity of male hormones. Therefore, in the method of the present invention, it can be said that the breast cancer cell line is masculinized. There has been no report that breast cancer cells are masculinized like this.
　乳癌細胞株
　本発明は、上記方法により得られるアンドロゲンレセプター依存性（アンドロゲンレセプター活性依存性）を有する乳癌細胞株を提供する。本発明の乳癌細胞株の特徴及びその製造方法は、前述の通りである。 Breast cancer cell line The present invention provides a breast cancer cell line having androgen receptor dependency (androgen receptor activity dependency) obtained by the above method. The characteristics of the breast cancer cell line of the present invention and the production method thereof are as described above.
　スクリーニング方法
　本発明は、またホルモン療法に耐性を示す乳癌に対する治療剤をスクリーニングする方法であって、ホルモン療法耐性乳癌細胞に被験物質を接触させる工程と、この細胞を培養し、細胞の増殖及び／又はアンドロゲンレセプター活性を測定する工程と、前記被検物質と接触させていない前記ホルモン療法耐性乳癌細胞を同じ条件下で培養及び測定した結果と比較して前記被検物質が細胞の増殖及び／又はＡＲ活性の低下をもたらしたか否かを判定する工程とを含み、
　前記ホルモン療法耐性乳癌細胞が、エストロゲン非依存性、エストロゲンレセプター活性非依存性、かつアンドロゲンレセプター依存性（アンドロゲンレセプター活性依存性）である細胞株である、方法を提供する。 Screening method The present invention is also a method for screening a therapeutic agent for breast cancer that is resistant to hormone therapy, comprising contacting a test substance with hormone therapy-resistant breast cancer cells, culturing the cells, Or the step of measuring androgen receptor activity, and the test substance is a cell proliferation and / or compared with the results of culturing and measuring the hormone therapy resistant breast cancer cells not contacted with the test substance under the same conditions Determining whether the AR activity has been reduced,
Provided is a method wherein the hormone therapy resistant breast cancer cells are cell lines that are estrogen independent, estrogen receptor activity independent, and androgen receptor dependent (androgen receptor activity dependent).
　本発明のスクリーニング方法に用いるエストロゲン非依存性、エストロゲンレセプター活性非依存性、かつアンドロゲンレセプター活性依存性である細胞株としては、前記の方法により作製したアンドロゲンレセプター活性依存性の乳癌細胞を用いることができる。 As an estrogen-independent, estrogen receptor-independent, and androgen receptor-activity-dependent cell line used in the screening method of the present invention, an androgen receptor-activity-dependent breast cancer cell prepared by the above method may be used. it can.
　細胞と被検物質との接触は、培地に被検物質を添加することにより容易に行うことができる。被検物質との接触は、短時間のパルスで行ってもよく、培養期間中を通じて培地に存在させてもよい。したがって、接触と培養とは同時に行うことができる。培地としては、必要に応じて以下に説明する普通培地、エストロゲン枯渇培地、又はこれらに所定の添加物を添加した培地等を使用することができる。細胞の増殖は、常法により細胞数をカウントすること等により、行うことができる。アンドロゲンレセプター活性の測定は、自体公知の方法に従い行うことができる。 The contact between the cells and the test substance can be easily performed by adding the test substance to the medium. Contact with the test substance may be performed by a short pulse or may be present in the medium throughout the culture period. Therefore, contact and culture can be performed simultaneously. As the medium, a normal medium, an estrogen-depleted medium described below, or a medium with a predetermined additive added thereto can be used as necessary. Cell proliferation can be carried out by counting the number of cells by a conventional method. Androgen receptor activity can be measured according to a method known per se.
　本発明の方法において、細胞の増殖及びアンドロゲンレセプター活性の一方のみを測定し評価しても、これらの両方を測定し評価してもよい。本発明の方法において、被検物質に接触させていないホルモン療法耐性乳癌細胞と比較して被検物質に接触させたホルモン療法耐性乳癌細胞において細胞の増殖が低下（抑制）され、かつＡＲ活性が低下した場合、当該被検物質は、アンドロゲンレセプター活性依存性を獲得した乳癌細胞に対して有効な治療薬となり得ると判断できる。被検物質に接触させたホルモン療法耐性乳癌細胞において細胞の増殖は低下したが、かつＡＲ活性が低下しない場合、アンドロゲン－アンドロゲンレセプターの系を阻害する機構ではないもののアンドロゲンレセプター活性依存性を獲得した乳癌細胞に対して有効な治療薬となり得る余地があると判断できる。また、被検物質に接触させたホルモン療法耐性乳癌細胞において細胞の増殖は低下しないが、かつＡＲ活性が低下した場合、当該被検物質単独ではアンドロゲンレセプター活性依存性を獲得した乳癌細胞に対して有効ではない可能性があるが、他の治療薬と組合わせることで有効な治療を構築できる余地があると判断できる。 In the method of the present invention, only one of cell proliferation and androgen receptor activity may be measured and evaluated, or both of them may be measured and evaluated. In the method of the present invention, cell growth is reduced (suppressed) in hormonal therapy-resistant breast cancer cells contacted with the test substance compared to hormonal therapy-resistant breast cancer cells not contacted with the test substance, and AR activity is reduced. When it falls, it can be judged that the said to-be-tested substance can become an effective therapeutic agent with respect to the breast cancer cell which acquired androgen receptor activity dependence. Cell growth was reduced in hormone therapy-resistant breast cancer cells in contact with the test substance, and if AR activity did not decrease, androgen-androgen receptor system was acquired, but it did not inhibit the androgen receptor system. It can be judged that there is room for effective treatment for breast cancer cells. In addition, in the hormone therapy-resistant breast cancer cells brought into contact with the test substance, cell proliferation does not decrease, but when the AR activity decreases, the test substance alone can prevent androgen receptor activity-dependent breast cancer cells. Although it may not be effective, it can be determined that there is room for effective treatment by combining with other therapeutic agents.
　本発明の細胞は、上述のようにしてインビトロでの薬剤スクリーニングに用いることができるのに加えて、これらの細胞を動物に移植してそれぞれの機序のモデル動物を作製し、より個体に近い実験系で薬剤の効果を評価するために使用することができる。 In addition to being able to be used for in vitro drug screening as described above, the cells of the present invention are transplanted into animals to create model animals of the respective mechanisms, and are closer to individuals. Can be used to evaluate drug effects in experimental systems.
　また、新規な薬剤を見出すことだけでなく、既存薬剤の投与順序や併用の組合せ等に関しても、最適な方法を見出すために、上記と同様のインビトロ又はインビボ実験を行うことができる。 In addition to finding new drugs, in vitro or in vivo experiments similar to those described above can be performed in order to find an optimal method regarding the order of administration of existing drugs, combinations of combinations, and the like.
　アンドロゲンレセプター活性依存性獲得の判定方法
　本発明は、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを指標とする、乳癌患者におけるアンドロゲンレセプター依存性（アンドロゲンレセプター活性依存性）獲得の有無を判定する方法を提供する。本発明においては、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの遺伝子産物を指標とすることを特徴とする。ここで、遺伝子産物とは、上記遺伝子の転写産物であるｍＲＮＡ及び当該ｍＲＮＡの翻訳産物であるタンパク質を含む。尚、本発明において、ＫＬＫ３、ＤＤＣ及びＡＲまたはこれらの遺伝子産物は、内在性の（ゲノム上の）遺伝子由来のものを意味する。 Method for determining acquisition of androgen receptor activity dependency The present invention relates to the presence or absence of acquisition of androgen receptor dependency (androgen receptor activity dependency) in a breast cancer patient using at least one selected from the group consisting of KLK3, DDC and AR as an index. A method for determining In the present invention, at least one gene product selected from the group consisting of KLK3, DDC and AR is used as an index. Here, the gene product includes mRNA that is a transcription product of the gene and a protein that is a translation product of the mRNA. In the present invention, KLK3, DDC and AR or their gene products mean those derived from endogenous genes (on the genome).
　本発明の判定方法は、被験者から採取した検体中のＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの発現量を測定する工程、及び上記工程において測定した被験者検体の発現量とコントロールにおける発現量とを比較する工程を含む。 The determination method of the present invention includes a step of measuring at least one expression level selected from the group consisting of KLK3, DDC and AR in a sample collected from a subject, and an expression level and control of the subject sample measured in the above step A step of comparing the expression level.
　ＰＳＡ（前立腺特異抗原(prostate specific antigen)）は、前立腺腫瘍マーカーとして知られている糖蛋白である。ＫＬＫ３（kallikrein 3）は、このＰＳＡをコードする遺伝子である。ＫＬＫ３遺伝子の塩基配列としては、例えば、GenBankアクセッション番号NM_001648.2で示されるもの（配列番号１）が挙げられる。ＤＤＣ（L-Dopa decarboxylase）は、ＡＲコファクターであり、前立腺癌における予後不良因子である。ＤＤＣ遺伝子の塩基配列は自体公知であり、例えば、GenBankアクセッション番号M76180.1で示されるもの（配列番号２）が挙げられる。ＡＲ遺伝子は自体公知であり、例えば、GenBankアクセッション番号M34233.1で示されるもの（配列番号３）が挙げられる。 PSA (prostate specific antigen) is a glycoprotein known as a prostate tumor marker. KLK3 (kallikrein 3) is a gene encoding this PSA. Examples of the base sequence of the KLK3 gene include the one shown by GenBank accession number NM_001648.2 (SEQ ID NO: 1). DDC (L-Dopa decarboxylase) is an AR cofactor and a poor prognosis factor in prostate cancer. The base sequence of the DDC gene is known per se, and examples thereof include the one shown by GenBank accession number M76180.1 (SEQ ID NO: 2). The AR gene is known per se, and for example, the gene indicated by GenBank accession number M34233.1 (SEQ ID NO: 3) can be mentioned.
　被験者としては、特に限定されるものではないが、ヒトを含む哺乳類が例示される。非ヒト哺乳類としては、マウス、ラット、イヌ、ネコ、ウシ、ヒツジ、ウマ等が挙げられる。本発明の判定方法の好ましい被験対象は、ヒトである。被験者は、女性（雌）でも男性（雄）でもよいが、特に女性被験者の検査に有用である。 The subject is not particularly limited, but examples include mammals including humans. Examples of non-human mammals include mice, rats, dogs, cats, cows, sheep and horses. A preferred test subject of the determination method of the present invention is a human. The subject may be female (female) or male (male), but is particularly useful for testing female subjects.
　ＫＬＫ３、ＤＤＣ及び／又はＡＲの測定方法としては、自体公知の方法を適宜採用することができ、特に限定されないが、例えば、乳癌患者から癌組織を摘出し、免疫染色による抗原の検出、ＲＴ－ＰＣＲによるｍＲＮＡの検出等により行うことができる。 As a method for measuring KLK3, DDC and / or AR, a method known per se can be appropriately employed, and is not particularly limited. For example, cancer tissue is removed from a breast cancer patient, antigen detection by immunostaining, RT- It can be performed by detecting mRNA by PCR.
　上記の方法により得られた被験者のＫＬＫ３、ＤＤＣ及び／又はＡＲの発現量の測定値が、コントロール（アンドロゲンレセプター活性依存性を獲得していない乳癌患者）の数値よりも統計的に有意に大きい場合にアンドロゲンレセプター活性依存性を獲得していると判定することができる。例えば、ＫＬＫ３、ＤＤＣ及びＡＲのうち１つのみを測定した場合、被験者の数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法が挙げられる。また、例えば、ＫＬＫ３、ＤＤＣ及びＡＲの全てを測定する方法が挙げられる。その場合、３つ全ての数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法、少なくとも２つの数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法、及び少なくとも１つの数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法が挙げられる。また、ＫＬＫ３、ＤＤＣ及びＡＲのうち２つを測定する方法が挙げられる。その場合、測定した２つ両方の数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法、及び少なくとも１つの数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法が挙げられる。例えば、ＫＬＫ３及びＤＤＣの両方を測定した結果、ＫＬＫ３及びＤＤＣの両方の数値がコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法、及びＫＬＫ３及びＤＤＣの両方を測定した結果、ＫＬＫ３及びＤＤＣの一方のみがコントロールよりも有意に大きい場合、アンドロゲンレセプター活性依存性を獲得していると判定する方法が挙げられる。 When the measured value of the expression level of KLK3, DDC and / or AR obtained by the above method is statistically significantly larger than the numerical value of the control (breast cancer patient who has not acquired androgen receptor activity dependency) Can be determined to have acquired androgen receptor activity dependency. For example, when only one of KLK3, DDC, and AR is measured, and when the numerical value of the subject is significantly larger than the control, a method of determining that the androgen receptor activity dependency is acquired can be mentioned. Further, for example, a method for measuring all of KLK3, DDC, and AR can be mentioned. In that case, if all three values are significantly larger than the control, it is determined that the androgen receptor activity dependency is acquired. If at least two values are significantly larger than the control, androgen receptor activity dependency is determined. And a method of determining that the androgen receptor activity dependency is acquired when at least one numerical value is significantly larger than that of the control. Moreover, the method of measuring two of KLK3, DDC, and AR is mentioned. In that case, if both of the two measured values are significantly greater than the control, a method for determining that the androgen receptor activity dependence has been acquired, and if at least one value is significantly greater than the control, the androgen receptor A method for determining that the activity dependence is acquired is given. For example, when both KLK3 and DDC are measured, and both KLK3 and DDC values are significantly larger than the control, it is determined that the androgen receptor activity dependence has been acquired, and both KLK3 and DDC are As a result of the measurement, when only one of KLK3 and DDC is significantly larger than the control, there is a method for determining that the androgen receptor activity dependency is acquired.
　上記方法により、アンドロゲンレセプター活性依存性を獲得していると判定された被験者には、アンドロゲンレセプター活性依存性を獲得した乳癌に適する治療を施すことができる。例えば、ビカルタミド、フルタミド、エンザルタミド等の治療方法が挙げられる。 The subject determined to have acquired androgen receptor activity dependency by the above method can be treated appropriately for breast cancer that has acquired androgen receptor activity dependency. For example, treatment methods such as bicalutamide, flutamide, enzalutamide and the like can be mentioned.
　従って、本発明は、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを指標とする、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定する工程、及び
上記判定工程においてアンドロゲンレセプター活性依存性を獲得したと判定された乳癌患者に、アンドロゲンレセプター活性依存性を獲得した乳癌に適する治療を施す工程
を含む、乳癌患者の治療方法も提供する。 Therefore, the present invention includes a step of determining whether or not androgen receptor activity is acquired in a breast cancer patient using at least one selected from the group consisting of KLK3, DDC and AR as an index, and androgen receptor activity in the determination step. There is also provided a method for treating a breast cancer patient, comprising a step of treating a breast cancer patient determined to have acquired dependence suitable for breast cancer that has acquired androgen receptor activity dependence.
　アンドロゲンレセプター活性依存性獲得の判定用キット
　本発明は、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの発現量を測定する手段を含む、乳癌患者におけるアンドロゲンレセプター依存性（アンドロゲンレセプター活性依存性）獲得の有無を判定するためのキットを提供する。 KIT FOR DETERMINING ANDROGEN RECEPTOR ACTIVITY Dependence Acquisition The present invention relates to androgen receptor dependency (androgen receptor activity dependency) in breast cancer patients, comprising means for measuring at least one expression level selected from the group consisting of KLK3, DDC and AR. Gender) A kit for determining the presence or absence of acquisition is provided.
　ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの発現量を測定するための手段としては、特に限定されないが、例えば、ＰＣＲ、免疫染色法、ノーザンブロット法、ウエスタンブロット法、ＥＬＩＳＡ法、マイクロアレイ法等に用いる手段を挙げることができる。ＫＬＫ３、ＤＤＣ及び／又はＡＲの発現量を測定するための手段としては、例えば、ＰＣＲ用のプライマー又はプローブとしてのＫＬＫ３、ＤＤＣ及び／又はＡＲのｍＲＮＡ、ｃＤＮＡ等に特異的に結合するオリゴヌクレオチド、逆転写酵素、ＤＮＡポリメラーゼ等；免疫染色法に用いるＰＳＡ、ＤＤＣ及び／又はＡＲに特異的に結合する抗体等；マイクロアレイ法に用いるプローブを固定したマイクロアレイ等が挙げられる。ＲＴ－ＰＣＲによりＫＬＫ３を検出するためのプライマーとしては、例えば、F:5'-TGTCCGTGACGTGGATT-3'（配列番号４） R:5'-ACGAGAGGCCACAAGCA-3' （配列番号５）等を挙げることができる。ＲＴ－ＰＣＲによりＤＤＣを検出するためのプライマーとしては、例えば、F:5'-GAAGTCGGTCCTATCTGCAAC-3' （配列番号６）、R:5'-ACTCCACTCCATTCAGAAGGT-3' （配列番号７）等を挙げることができる。ＲＴ－ＰＣＲによりＡＲを検出するためのプライマーとしては、例えば、F:5'-ATGTGGAAGCTGCAAGGTCT-3' （配列番号８） R:5'-CGAAGACGACAAGATGGACA-3' （配列番号９）等を挙げることができる。また、本発明のキットには、必要に応じて他の成分を含めることができる。他の成分としては、例えば検体を採取するための道具、ポジティブコントロール試料及びネガティブコントロール試料などが挙げられるが、これに限定されない。上記判定方法を行うための手順を書き記した書面等を含むこともできる。 The means for measuring at least one expression level selected from the group consisting of KLK3, DDC, and AR is not particularly limited. For example, PCR, immunostaining, Northern blotting, Western blotting, ELISA, Means used for the microarray method and the like can be mentioned. As a means for measuring the expression level of KLK3, DDC and / or AR, for example, oligonucleotides that specifically bind to mRNA, cDNA, etc. of KLK3, DDC and / or AR as primers or probes for PCR, Examples include reverse transcriptase, DNA polymerase, and the like; antibodies that specifically bind to PSA, DDC, and / or AR used for immunostaining, and microarrays to which probes used for microarray methods are immobilized. Examples of a primer for detecting KLK3 by RT-PCR include F: 5′-TGTCCGTGACGTGGATT-3 ′ (SEQ ID NO: 4) R: 5′-ACGAGAGGCCACAAGCA-3 ′ (SEQ ID NO: 5). . Examples of primers for detecting DDC by RT-PCR include F: 5′-GAAGTCGGTCCTATCTGCAAC-3 ′ (SEQ ID NO: 6), R: 5′-ACTCCACTCCATTCAGAAGGT-3 ′ (SEQ ID NO: 7), and the like. it can. Examples of primers for detecting AR by RT-PCR include F: 5′-ATGTGGAAGCTGCAAGGTCT-3′CT (SEQ ID NO: 8) R: 5′-CGAAGACGACAAGATGGACA-3 ′ (SEQ ID NO: 9). . The kit of the present invention may contain other components as necessary. Examples of other components include, but are not limited to, a tool for collecting a specimen, a positive control sample, and a negative control sample. It can also include a document in which a procedure for performing the above determination method is written.
　アンドロゲンレセプター活性依存性獲得の判定用マーカー
　本発明は、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを含む、乳癌患者におけるアンドロゲンレセプター依存性（アンドロゲンレセプター活性依存性）獲得の有無を判定するためのマーカーを提供する。本発明において、「マーカー」とは、被験者又は被験者から採取した検体が特定の性質を有するか否か、特定の状態にあるか否か等を評価するための指標となるものを意味する。本発明において、判定用マーカーに含まれる「ＫＬＫ３、ＤＤＣ及びＡＲ」は、マーカーとして用いられ得るものである限りにおいて、これらの遺伝子自体に限られず、遺伝子の転写物であるｍＲＮＡ、翻訳産物であるタンパク質等も含まれる。従って、本発明において、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを含むマーカーとしては、ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの遺伝子に由来するｍＲＮＡ、ｃＤＮＡ、タンパク質等が挙げられる。尚、本発明において、特に言及しない限り、２本鎖DNA、１本鎖DNA(センス鎖又はアンチセンス鎖)、及びそれらの断片が含まれる。また、本発明において「遺伝子」とは、特に言及しない限り、調節領域、コード領域、エクソン、及びイントロンを区別することなく示すものとする。また、本発明において、「ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの遺伝子に由来するｍＲＮＡ、ｃＤＮＡ、タンパク質」は、ｍＲＮＡ全体、ｃＤＮＡ全体、タンパク質全体に限られず、これらの遺伝子に由来するものであることが特定できる限りにおいて、これらのｍＲＮＡの一部（断片）又は全部、ｃＤＮＡの一部（断片）又は全部、及びタンパク質の各々の一部（断片）又は全部がいずれも含まれる。 Marker for determining androgen receptor activity-dependent acquisition The present invention relates to the presence or absence of acquisition of androgen receptor dependency (androgen receptor activity dependency) in breast cancer patients, comprising at least one selected from the group consisting of KLK3, DDC and AR. Provide a marker for determination. In the present invention, the “marker” means an index for evaluating whether or not a subject or a sample collected from the subject has a specific property, whether it is in a specific state, or the like. In the present invention, “KLK3, DDC, and AR” included in the determination marker are not limited to these genes per se as long as they can be used as markers, and are mRNA and translation products of gene transcripts. Protein etc. are also included. Therefore, in the present invention, as a marker containing at least one selected from the group consisting of KLK3, DDC and AR, mRNA, cDNA, derived from at least one gene selected from the group consisting of KLK3, DDC and AR, Examples include proteins. In the present invention, unless otherwise specified, double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof are included. In the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified. In the present invention, “mRNA, cDNA, protein derived from at least one gene selected from the group consisting of KLK3, DDC, and AR” is not limited to the whole mRNA, the whole cDNA, and the whole protein. As long as it can be specified that they are derived from, all (partial) or all of these mRNAs, part (fragment) or all of cDNA, and part (fragment) or all of each of proteins are included. It is.
　Ｉ．材料及び測定方法
　Ｉ－１．樹立に使用した親細胞
　T47D-TE8細胞は、ヒト乳癌細胞株T47D(ATCC番号：HTB-133)から、ｐｄ２ＥＧＦＰ－１ベクター（Clontech）において半減期の短いＧＦＰのｃＤＮＡの上流にエストロゲン応答配列（ＥＲＥ）を配したレポータープラスミドを公知の方法にしたがって導入することにより樹立した（Yamaguchi Y.et al. Cancer Res,2005,65:4653-4662）。T47D-TE8細胞は、普通培地で培養した。 I. Material and Measuring Method I-1. The parental T47D-TE8 cells used for the establishment were derived from the human breast cancer cell line T47D (ATCC number: HTB-133) from the estrogen response element (ERE) upstream of the short half-life GFP cDNA in the pd2EGFP-1 vector (Clontech). ) Was introduced according to a known method (Yamaguchi Y. et al. Cancer Res, 2005, 65: 4653-4662). T47D-TE8 cells were cultured in a normal medium.
　Ｉ－２．細胞培養法
　細胞の通常の培養には、１０％ＦＣＳ（Tissue Culture Biologicals）及び１％ペニシリン／ストレプトマイシン（GIBCO）を添加したＲＰＭＩ１６４０培地（SIGMA）（「普通培地」と呼ぶことがある）を用いた。耐性株のエストロゲン枯渇培養には、１０％デキストラン被覆チャコール処理ＦＣＳ（ＤＣＣ－ＦＣＳ；Tissue Culture Biologicals）および１％ペニシリン／ストレプトマイシン（GIBCO）を添加したフェノールレッド無含有ＲＰＭＩ１６４０培地（GIBCO）（「枯渇培地」又は「エストロゲン枯渇培地」と呼ぶことがある）を用いた。細胞は、すべて５％ＣＯ2、３７℃に調整したＣＯ２インキュベーターで培養した。 I-2. Cell culture method For normal culture of cells, RPMI 1640 medium (SIGMA) (sometimes called "normal medium") supplemented with 10% FCS (Tissue Culture Biologicals) and 1% penicillin / streptomycin (GIBCO) was used. . For the estrogen-depleted culture of resistant strains, phenol red-free RPMI 1640 medium (GIBCO) supplemented with 10% dextran-coated charcoal-treated FCS (DCC-FCS; Tissue Culture Biologicals) and 1% penicillin / streptomycin (GIBCO) ("depletion medium" Or “estrogen-depleted medium”). All cells were cultured in a CO 2 incubator adjusted to 5% CO 2 and 37 ° C.
　特に記載がない場合、実験結果は、平均±ＳＤ（ｎ＝３）で表す。 Unless otherwise stated, the experimental results are expressed as mean ± SD (n = 3).
　Ｉ－３．試薬等
エストラジオール、テストステロン、ＤＨＴは、Sigma-Aldrich Corpから購入した。フルベストラントはアストラゼネカから入手した。レトロゾールはノバルティスファーマ（Tokyo, Japan）から入手した。バイカルタミド（アンドロゲンレセプター阻害剤）はLKT Laboratories (St. Paul, MN,USA)から入手した。 I-3. Reagents, etc. Estradiol, testosterone, and DHT were purchased from Sigma-Aldrich Corp. Fulvestrant was obtained from AstraZeneca. Letrozole was obtained from Novartis Pharma (Tokyo, Japan). Baicartamide (androgen receptor inhibitor) was obtained from LKT Laboratories (St. Paul, MN, USA).
　Ｉ－４．リアルタイムＲＴ－ＰＣＲ
　それぞれの条件下で培養した細胞から商品名「Isogen」（Nippon Gene）を用いて総ＲＮＡを抽出し、商品名「RNA PCR kit」（Takara Shuzo）を用いて第１鎖ｃＤＮＡを合成した。リアルタイムＲＴ－ＰＣＲは、商品名「StepOne real-time PCR system」（Applied Biosystems）を用いて標準プロトコールにしたがって行った。目的の遺伝子の発現を、内部標準としてのＧＡＰＤＨ又はＲＰＬ１３Ａに対する相対値として算出した。データは、平均±ＳＤ（ｎ＝２）で示した。 I-4. Real-time RT-PCR
Total RNA was extracted from the cells cultured under each condition using the trade name “Isogen” (Nippon Gene), and the first strand cDNA was synthesized using the trade name “RNA PCR kit” (Takara Shuzo). Real-time RT-PCR was performed according to a standard protocol using the trade name “StepOne real-time PCR system” (Applied Biosystems). The expression of the target gene was calculated as a relative value to GAPDH or RPL13A as an internal standard. Data are shown as mean ± SD (n = 2).
　Ｉ－５．ウェスタンブロッティング
１００mmディッシュで増殖させた細胞を氷冷生理食塩水で洗浄し、ホスファターゼ阻害剤カクテル(「Phos STOP」、Roche)及びプロテアーゼ阻害剤カクテル（Roche）を含む１mLのリシスバッファー（「Complete Lysis-M」、Roche Diagnotics GmbH）で溶解させた。５分後、ライセートをパルスソニケーションに供し、１４，０００rpmで１０分間遠心分離した後、１０％ポリアクリルアミドゲル（Wako）でＳＤＳ－ＰＡＧＥを行い、タンパク質をＰＶＤＦ膜（GE Healthcare Bioscience）に転写した。この膜を、一次抗体（５％ＢＳＡ、０．０５％ Ｔｗｅｅｎ－２０含有溶液中）と反応させた。次に、西洋ワサビペルオキシダーゼ結合二次抗体を適用した。化学発光基質（「Immunsutar AP substrate」、 BIO-RAD）と反応させた後、目的のタンパクのバンドを、Ｘ線フィルムを感光させることにより可視化した。 I-5. Cells grown in Western blotting 100 mm dishes were washed with ice-cold saline and 1 mL of lysis buffer (“Complete Lysis-” containing phosphatase inhibitor cocktail (“Phos STOP”, Roche) and protease inhibitor cocktail (Roche). M ", Roche Diagnotics GmbH). After 5 minutes, the lysate was subjected to pulse sonication, centrifuged at 14,000 rpm for 10 minutes, then subjected to SDS-PAGE with 10% polyacrylamide gel (Wako), and the protein was transferred to a PVDF membrane (GE Healthcare Bioscience). . This membrane was reacted with a primary antibody (in a solution containing 5% BSA, 0.05% Tween-20). Next, a horseradish peroxidase-conjugated secondary antibody was applied. After reacting with a chemiluminescent substrate (“Immunsutar AP substrate”, BIO-RAD), the band of the protein of interest was visualized by exposing the X-ray film.
　Ｉ－６．ルシフェラーゼアッセイ
　ＥＲＥ活性を、商品名「Dual-Luciferase Reporter System」（Promega）を用いて、基本的にBiochem Biophys Res Commun 2001, 285:340-347に記載された方法に従って測定した。枯渇培地で３日間培養後、３×１０5個の細胞を６cmディッシュに播き、同じ培地中で４８時間培養した。０．５μgのエストロゲンレポータープラスミド（Ｔｋ－ＥＲＥ－Ｌｕｃi）及び０．０５μgのコントロールベクター（ｐＲＬ―ＴＫ, Promega）を３００μLの無血清培地中で５μLの商品名「TransIt」LT-1試薬(Takara Bio)と混合し、製造業者の指示書に従ってトランスフェクションを行った。薬剤の存在下又は非存在下で40時間細胞を培養した後、商品名「Dual-Luciferase Reporter System」（Promega）を製造業者の指示書に従って用いてルシフェラーゼ活性を測定し、コントロールに対する相対値を算出した。 I-6. Luciferase assay ERE activity was measured using the trade name “Dual-Luciferase Reporter System” (Promega) basically according to the method described in Biochem Biophys Res Commun 2001, 285: 340-347. After culturing in a depletion medium for 3 days, 3 × 10 5 cells were seeded in a 6 cm dish and cultured in the same medium for 48 hours. 0.5 μg of estrogen reporter plasmid (Tk-ERE-Luci) and 0.05 μg of control vector (pRL-TK, Promega) in 300 μL of serum-free medium, 5 μL of the trade name “TransIt” LT-1 reagent (Takara Bio ) And transfection was performed according to the manufacturer's instructions. After culturing the cells for 40 hours in the presence or absence of the drug, measure the luciferase activity using the trade name “Dual-Luciferase Reporter System” (Promega) according to the manufacturer's instructions and calculate the relative value to the control did.
　ＩＩ．タイプ６細胞株の樹立及び酵素活性の測定
　ＩＩ－１．タイプ６細胞株の樹立
　エストロゲン枯渇・アンドロゲン添加の条件下での長期培養によりT47D-TE8細胞から取得した細胞はアロマターゼ阻害薬で治療された乳癌のモデルを表す。具体的には、Phenol red free RPMI培地に10%DCC-FCS(チャコール処理ウシ胎児血清),1%Penicillin/Streptomycinを添加しテストステロン100nMとなるよう調製した培地でT47D-TE8細胞を約３カ月培養した。培養中、蛍光を発する細胞の割合は徐々に少なくなっていった。コロニーのうち蛍光を発するものをクローニングし、テストステロン添加にて蛍光が強くなる２つの細胞株A3T・B4Tを、タイプ６細胞とした。A3T,B4Tは培養を続けるうちに、徐々に蛍光を失っていった。 II. Establishment of type 6 cell line and measurement of enzyme activity II-1. Cells obtained from T47D-TE8 cells by long-term culture under the conditions of establishment of estrogen depletion and androgen addition of type 6 cell line represent a model of breast cancer treated with an aromatase inhibitor. Specifically, T47D-TE8 cells were cultured for about 3 months in a medium prepared by adding 10% DCC-FCS (charcoal-treated fetal bovine serum) and 1% Penicillin / Streptomycin to Phenol red free RPMI medium to give 100 nM testosterone. did. During culture, the proportion of cells that fluoresce gradually decreased. Among the colonies, those that fluoresced were cloned, and two cell lines A3T and B4T that became fluorescent when testosterone was added were designated as type 6 cells. A3T and B4T gradually lost fluorescence as the culture was continued.
　ＩＩ－２．エストロゲンに対する反応（図１、２）
　親株であるT47D-TE8（枯渇培地で３日間培養し、ステロイドを枯渇させたもの）及び上記ＩＩ－１で樹立したA3T及びB4Tを、２万細胞／ウェルの密度で２４ウェル培養プレートに播き、種々の濃度のエストロゲンの存在下及び非存在下で培養した。４日後、細胞をＰＢＳで１回洗浄し、トリプシン／ＥＤＴＡ処理によって回収した。細胞をパーティクルカウンター（「CDA-500 Sysmex automated cell counter」(Sysmex Corporation)でカウントした。結果を図１に示す。親株T47D-TE8はエストラジオール100pM添加時にコントロール（エタノールを添加した４日目の細胞数）に比べ細胞数は約2倍に増加したが、A3T・B4Tは細胞数の変化はみられなかった。 II-2. Response to estrogen (Figures 1 and 2)
The parent strain T47D-TE8 (cultured in a depletion medium for 3 days and depleted of steroid) and A3T and B4T established in II-1 above were seeded in a 24-well culture plate at a density of 20,000 cells / well, Cultures were performed in the presence and absence of various concentrations of estrogen. After 4 days, cells were washed once with PBS and harvested by trypsin / EDTA treatment. The cells were counted with a particle counter (“CDA-500 Sysmex automated cell counter” (Sysmex Corporation). The results are shown in FIG. 1. The parent strain T47D-TE8 was controlled when 100 pM of estradiol was added (the number of cells on the fourth day when ethanol was added) The number of cells increased by a factor of about 2, but A3T and B4T showed no change in the number of cells.
　次に、ER(エストロゲン受容体)活性を測定するため、EREルシフェラーゼレポーター、内部コントロールとしてpRLルシフェラーゼレポーターを一時導入し、ルシフェラーゼアッセイを行った。具体的には、前述のＩ－６に記載の方法に従い、薬剤としてエストラジオール（100pM（Ｅ２））、エストラジオール100pM及び抗エストロゲン薬　フルベストラント1μＭ（Ｅ２＋Ｆｕｌ）又はコントロールとしてエタノールを用いてルシフェラーゼアッセイを行った。結果を図２に示す。T47D-TE8はコントロールに比べて、エストラジオール100pM添加により約8倍にER活性は上昇したが、A3T・B4Tはほとんど活性の変化はなかった。これらの結果から、A3T・B4TはT47D-TE8が有しているERを介した細胞増殖機構が機能していないと考えられた。 Next, in order to measure ER (estrogen receptor) activity, an ERE luciferase reporter and a pRL luciferase reporter as an internal control were temporarily introduced to carry out a luciferase assay. Specifically, according to the method described in I-6 above, a luciferase assay was performed using estradiol (100 pM (E2)) as the drug, 100 pM estradiol and the antiestrogenic drug fulvestrant 1 μM (E2 + Ful) or ethanol as the control. It was. The results are shown in FIG. Compared with the control, T47D-TE8 increased ER activity about 8-fold by adding 100 pM estradiol, but A3T and B4T showed almost no change in activity. From these results, it was considered that A3T and B4T do not function the cell growth mechanism via ER possessed by T47D-TE8.
　ＩＩ－３．エストロゲン受容体とその応答遺伝子の発現（図３、４）
　親株であるT47D-TE8（枯渇培地で３日間培養し、ステロイドを枯渇させたもの）及び上記ＩＩ－１で樹立したA3T・B4Tについてウエスタンブロッティングを行ったところ、A3T・B4TのERの発現蛋白はほぼ消失していた（図３）。尚、ウエスタンブロッティングはテストステロン（10nM）を添加し、2日間培養したもの及びテストステロンを添加していないものの両方について行った。 II-3. Expression of estrogen receptor and its response gene (Figs. 3 and 4)
Western blotting was performed on the parent strain T47D-TE8 (cultured in a depletion medium for 3 days and depleted of steroid) and A3T / B4T established in II-1 above. The protein expressed in the ER of A3T / B4T was Almost disappeared (FIG. 3). Western blotting was performed for both those with testosterone (10 nM) added and cultured for 2 days and those without testosterone.
　また、リアルタイムRT-PCRにてmRNAの発現量を、RPL13Aを内部コントロールとして測定した（図４）。T47D-TE8に比べ、A3T及びB4TではERとその応答遺伝子であるPgRの発現が著明に低下していた。以上から、A3T及びB4TではERの機能のみならずERの発現もほぼ喪失していることが示された。 In addition, the expression level of mRNA was measured by real-time RT-PCR using RPL13A as an internal control (FIG. 4). Compared with T47D-TE8, the expression of ER and its response gene, PgR, was markedly decreased in A3T and B4T. From the above, it was shown that not only ER function but also ER expression was almost lost in A3T and B4T.
　ＩＩ－４．アンドロゲン（テストステロン）に対する反応（図５～７）
　テストステロンを添加し4日間培養し細胞増殖試験を行った。具体的には、エストロゲンの代わりにテストステロンを用いる以外、前記ＩＩ－２と同様にして、細胞増殖試験を行った（図５上）。T47D-TE8はコントロールに比べて細胞数の変化はみられなかったが、A3T・B4Tではテストステロン10nMにて約1.4倍の増加を示した。 II-4. Response to androgen (testosterone) (Figures 5-7)
Testosterone was added and cultured for 4 days to conduct a cell proliferation test. Specifically, a cell proliferation test was performed in the same manner as II-2 except that testosterone was used instead of estrogen (upper part of FIG. 5). T47D-TE8 showed no change in cell number compared to control, but A3T and B4T showed about 1.4-fold increase in testosterone at 10 nM.
　また、Ａ３Ｔ及びＢ４Ｔのそれぞれについて、アンドロゲンレセプターブロッカーであるバイカルタミド（Ｂｉｃ）、エストロゲンレセプターブロッカーであるフルベストラン（Ｆｕｌ）１００ｎＭ、又はアロマターゼ阻害剤（ＡＩ）であるレトロゾール（Ｌｅｔ）１００ｎＭを添加し、4日間培養したものについても同様に細胞増殖試験を行った（図５、中央、下）。増殖促進反応は、フルベストラントやレトロゾールを添加しても抑制されず、ビカルタミドにて抑制されたことから、この反応はエストロゲン受容体ではなくアンドロゲン受容体を介していることが示唆された。 For each of A3T and B4T, add androgen receptor blocker bicalutamide (Bic), estrogen receptor blocker fulvestrane (Ful) 100 nM, or aromatase inhibitor (AI) letrozole (Let) 100 nM. A cell proliferation test was also conducted on the cells cultured for 4 days (FIG. 5, center, bottom). The growth-promoting reaction was not inhibited by the addition of fulvestrant or letrozole, but was inhibited by bicalutamide, suggesting that this reaction is mediated by the androgen receptor, not the estrogen receptor.
　次に、アンドロゲン受容体(AR)活性を測定するため、PSAプロモーター上流領域を含むAREルシフェラーゼレポーター、内部コントロールとしてpRLルシフェラーゼレポーターを一時導入し、ルシフェラーゼアッセイを行った。AREルシフェラーゼレポーターの調製は、エストロゲンレポータープラスミド（Ｔｋ－ＥＲＥ－Ｌｕｃi）の代わりにアンドロゲンレポータープラスミド（PSAプロモーター領域を含む、PSA転写開始点より上流5800塩基―TK-luci（EREの代わりにAREを導入したもの））を用いる以外、前記Ｉ－６．に記載の方法に従って行った。当該試験には、T47D-TE8、Ａ３Ｔ及びＢ４Ｔ細胞株に、テストステロン10nM添加（ＴＳ）、テストステロン10nM及びバイカルタミド10μM添加（ＴＳ＋Ｂｉｃ）またはコントロールとしてエタノールを添加し、２日間培養したものを使用した。 Next, in order to measure the androgen receptor (AR) activity, an ARE luciferase reporter including a PSA promoter upstream region and a pRL luciferase reporter as an internal control were temporarily introduced, and a luciferase assay was performed. The ARE luciferase reporter was prepared by using an androgen reporter plasmid (including the PSA promoter region, 5800 bases upstream from the PSA transcription start site instead of the estrogen reporter plasmid (Tk-ERE-Luci) -TK-luci (introducing ARE instead of ERE) Except that the above-mentioned I-6. In accordance with the method described in. In this test, T47D-TE8, A3T and B4T cell lines were added with testosterone 10 nM (TS), testosterone 10 nM and baicaltamide 10 μM (TS + Bic) or ethanol added as a control and cultured for 2 days.
　結果を図６に示す。T47D-TE8ではテストステロン10nM添加（ＴＳ）によりAR活性の変化はなかったが、A3T・B4Tではコントロールに比べ2～3倍の活性上昇が認められた。 The results are shown in FIG. In T47D-TE8, there was no change in AR activity due to the addition of 10 nM testosterone (TS), but in A3T and B4T, an increase in activity was observed 2 to 3 times that in the control.
　AREとEREの配列が類似していることにより、ARがEREに作用するという報告があるため（Peters et al. Cancer Res.2009,69:6131-6140）、EREルシフェラーゼレポーター、内部コントロールとしてpRLルシフェラーゼレポーターを一時導入し、ルシフェラーゼアッセイを行った（図７）。A3T・B4Tではテストステロン添加によるEREルシフェラーゼ値の上昇はなく、テストステロンによる細胞増殖促進反応はEREを介していないことが証明された。以上から、A3T・B4Tはアンドロゲン(テストステロン)により細胞増殖が促進し、その反応はARを介していることが示された。 Because ARE and ERE sequences are similar, AR has been reported to act on ERE (Peters et al. Cancer Res.2009,69: 6131-6140), so the ERE luciferase reporter and pRL luciferase as an internal control A reporter was temporarily introduced and a luciferase assay was performed (FIG. 7). In A3T and B4T, there was no increase in ERE luciferase level due to the addition of testosterone, and it was proved that the cell growth promotion reaction by testosterone was not mediated by ERE. From the above, it was shown that cell growth of A3T / B4T was promoted by androgen (testosterone), and the reaction was mediated by AR.
　ＩＩ－５．ARとその応答遺伝子の発現　(図８～図９)
　親株であるT47D-TE8及び上記ＩＩ－１で樹立したA3T・B4Tについて、前記Ｉ－２の培養法と同じ培地（TE8:RPMI+FCS+P/S   A3T・B4T：フェノールレッドフリーRPMI+DCC-FCS+P/S）を用いてウエスタンブロッティングを行いT47D-TE8・A3T・B4Tの蛋白発現量を調べた。結果を図８に示す。ARとその応答遺伝子であるPSAの発現が、A3T・B4TではT47D-TE8に比べて著明に上昇していた。 II-5. Expression of AR and its response gene (Figs. 8-9)
For the parent strain T47D-TE8 and A3T / B4T established in II-1 above, the same medium as the culture method of I-2 (TE8: RPMI + FCS + P / S A3T • B4T: phenol red-free RPMI + DCC− Western blotting was performed using FCS + P / S) to examine the protein expression levels of T47D-TE8, A3T, and B4T. The results are shown in FIG. The expression of AR and its response gene, PSA, was significantly increased in A3T and B4T compared to T47D-TE8.
　また、ウエスタンブロッティングと同様に前記Ｉ－２の培地中のT47D-TE8・A3T・B4TについてリアルタイムRT-PCRによりAR及びPSAのmRNA発現量を調べたところ（図９）、A3T及びB4Tでは発現上昇がみられた。とりわけPSAの発現量上昇は顕著であった。A3T及びB4TではARの発現量の増加がARを介した増殖経路の活性化に寄与していると考えられた。 Similarly to Western blotting, the expression levels of AR and PSA mRNA were examined by real-time RT-PCR for T47D-TE8 / A3T / B4T in the medium of I-2 (FIG. 9). The expression increased in A3T and B4T. Was seen. In particular, the increase in the expression level of PSA was remarkable. In A3T and B4T, an increase in the expression level of AR was thought to contribute to activation of the growth pathway via AR.
　マイクロアレイによる遺伝子解析（図１０、１１）
　アンドロゲン添加時の、T47D-TE8に対するA3Tの遺伝子発現量の比を検出することで、A3Tのアンドロゲン依存性増殖経路に関与する遺伝子を同定することができると考え、サンプルとして、T47D-TE8及びA3TにDHTをそれぞれ１nM添加し24時間後に採取したRNAを用いてマイクロアレイを行った。各遺伝子について、T47D-TE8での発現量に対するA3Tでの発現量の比（A3T-DHT/TE8-DHT）を下記表１に示す。 Gene analysis by microarray (Figs. 10 and 11)
By detecting the ratio of A3T gene expression level to T47D-TE8 at the time of androgen addition, we think that it is possible to identify genes involved in the androgen-dependent growth pathway of A3T, and as a sample, T47D-TE8 and A3T Microarrays were performed using RNA collected at 24 hours after adding 1 nM each of DHT. The ratio of the expression level in A3T to the expression level in T47D-TE8 (A3T-DHT / TE8-DHT) is shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　アジレント社オリゴマイクロアレイ、データ解析ソフトGeneSpringGXを用いて、サンプル間の遺伝子の発現比を解析した。T47D-TE8に比べA3Tで2倍以上の発現上昇がみられた遺伝子の中から、パスウェイ解析ソフトIPAにて「Androgen」と「Cancer」両者に関連する遺伝子をピックアップした。PSAに次いで高い比の遺伝子が、DDC(L-Dopa decarboxylase)であった。DDCはARのcoactivatorであり、前立腺癌細胞では過剰発現により細胞増殖促進効果が報告されている(Wafa et al. Int.J.Cancer 2012,130:2835-2844)。 The gene expression ratio between samples was analyzed using an Agilent oligo microarray and data analysis software GeneSpringGX. Among the genes whose expression was more than doubled in A3T compared to T47D-TE8, genes related to both “Androgen” and “Cancer” were picked up by pathway analysis software IPA. The gene with the highest ratio after PSA was DDC (L-Dopa decarboxylase). DDC is a coactivator of AR and has been reported to promote cell growth by overexpression in prostate cancer cells (Wafa et al. Int.J.Cancer 2012, 130: 2835-2844).
　実際にリアルタイムRT-PCRを行いmRNAの発現量を測定したところ、A3T及びB4TではT47D-TE8に比べ著明な発現上昇を認めた（図１０）。尚、図１０は、T47D-TE8を１とした発現量の相対値を示す。A3TではDDCの発現増加が、AR活性上昇の大きな要因であると予測された。 Actually, real-time RT-PCR was performed and the mRNA expression level was measured. A3T and B4T showed a marked increase in expression compared to T47D-TE8 (FIG. 10). FIG. 10 shows the relative value of the expression level with T47D-TE8 as 1. In A3T, increased DDC expression was predicted to be a major factor in increasing AR activity.
　さらに検証するため、アンドロゲン及びDDC阻害剤、NSD-1015を添加し細胞増殖試験を行った。（図１１）
T47D-TE8、A3T、B4TはDHT1nM添加によりコントロールに比べ細胞数は増加した。この増殖促進反応はNSD-1015 200μM添加にて、A3T・B4Tでは抑制されたが、T47D-TE8では抑制されなかった。以上の結果から、T47D-TE8が元来保持しているARを介した増殖経路が、A3T・B4Tでは活性が高められており、その活性化にDDCが大きく寄与していることが示唆された。これらの結果から推察されることは、乳癌のホルモン療法耐性の機序のひとつとして、乳癌が、前立腺癌のように、アンドロゲンとアンドロゲン受容体を介したシグナルに依存した増殖を示すようになることがあり得ると考えられる。その際、ＤＤＣが重要な役割を果たしていると思われる。このような耐性機序を獲得した進行・再発乳癌には、抗アンドロゲン剤治療が有効である可能性がある。 For further verification, a cell proliferation test was performed by adding an androgen, a DDC inhibitor, and NSD-1015. (Fig. 11)
In T47D-TE8, A3T, and B4T, the number of cells increased compared to the control by adding DHT1nM. This growth promoting reaction was suppressed by A3T / B4T by addition of 200 μM NSD-1015 but not by T47D-TE8. The above results suggest that T47D-TE8 originally has an AR-mediated proliferation pathway that is enhanced in A3T and B4T, and that DDC contributes greatly to its activation. . From these results, it can be inferred that one of the mechanisms of breast cancer resistance to hormone therapy is that breast cancer, like prostate cancer, shows growth dependent on signals through androgen and androgen receptor. There seems to be a possibility. At that time, DDC seems to play an important role. Anti-androgen treatment may be effective for advanced / recurrent breast cancer that has acquired such a resistance mechanism.
　ＩＩＩ．AR依存性AI耐性メカニズムの実在性の検証
　初発乳癌手術後に術後補助療法としてAIを投与中に再発し、再発病変の切除手術が行われ、初発・再発病変組織が使用可能であった症例21例を対象とした。内訳は東北大学病院7例、東北公済病院5例、宮城県がんセンター5例、岩手県立中央病院4例。なお以上の検体を用いた研究計画は各病院の倫理委員会にて承認を受けた。
10%ホルマリン固定パラフィン包埋標本より未染色標本を作製し、ヒストファインキット（ニチレイバイオサイエンス）を使用しstreptavidin-biotin 増幅法にて行った。
1次抗体には抗ERα抗体(ER1D5, Immunotech, Marseille, France)、 抗PR（プロゲステロンレセプター）抗体(MAB429, Chemicon, Temecula, CA, USA)、抗HER2抗体(A0485, DAKO, Carpinteria, CA, USA))、抗Ki-67抗体(MIB1, DAKO, Carpinteria, CA, USA)、抗AR抗体(AR441, DAKO)を使用した。ERα、PR、Ki-67、ARの抗原賦活化には、クエン酸緩衝液を用いてオートクレーブで120℃, 5分の熱処理を行った。希釈倍率は各々ERα；1/50、PR；1/30、HER2；1/200、Ki-67；1/50、AR；1/50、抗原・抗体複合体の発色には3.3'-diaminobenzidine(DAB)溶液を使用し、ヘマトキシリンで核染色を施した。ERα、PgR、AR、Ki67は1000個以上の癌細胞における陽性細胞数を計測し、その割合を算出するLI［labeling Index:陽性細胞の割合(%)］で評価し、ERα、PgRは1%以上、ARはLI 10%以上を陽性とした。HER2の免疫染色は細胞膜上のHER2蛋白の発現に対し、染色動態に従って4段階(スコア)に評価した。全ての免疫組織化学染色の結果は、癌の浸潤巣のみで判定した。結果を下記表２に示す。 III. Verification of the reality of the AR-dependent AI resistance mechanism 21 cases of recurrence during the administration of AI as a postoperative adjuvant therapy after initial breast cancer surgery, resection of the recurrent lesion, and use of the first / recurrent diseased tissue Example was targeted. The breakdown is 7 Tohoku University Hospitals, 5 Tohoku Kosai Hospitals, 5 Miyagi Cancer Centers, and 4 Iwate Prefectural Central Hospitals. The research plan using the above samples was approved by the ethics committee of each hospital.
Unstained specimens were prepared from 10% formalin-fixed paraffin-embedded specimens and subjected to the streptavidin-biotin amplification method using a histofine kit (Nichirei Bioscience).
Primary antibodies include anti-ERα antibody (ER1D5, Immunotech, Marseille, France), anti-PR (progesterone receptor) antibody (MAB429, Chemicon, Temecula, CA, USA), anti-HER2 antibody (A0485, DAKO, Carpinteria, CA, USA) )), Anti-Ki-67 antibody (MIB1, DAKO, Carpinteria, CA, USA) and anti-AR antibody (AR441, DAKO) were used. For antigen activation of ERα, PR, Ki-67, and AR, heat treatment was performed at 120 ° C. for 5 minutes in an autoclave using a citrate buffer. The dilution factor is ERα; 1/50, PR; 1/30, HER2; 1/200, Ki-67; 1/50, AR; 1/50, 3.3'-diaminobenzidine ( DAB) solution was used and nuclear staining was performed with hematoxylin. ERα, PgR, AR, and Ki67 measure the number of positive cells in more than 1000 cancer cells and evaluate the ratio by calculating LI [labeling Index: percentage of positive cells (%)]. ERα and PgR are 1% As described above, AR was positive for LI 10% or more. HER2 immunostaining was evaluated on a 4-stage (score) basis for the expression of HER2 protein on the cell membrane according to the staining kinetics. All immunohistochemical staining results were determined only by cancer invasion. The results are shown in Table 2 below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　前述したように、耐性株においてARの発現が亢進していたことから、ARはAR依存性増殖を示す乳癌の指標となる可能性があると考えられる。耐性株が持つ性質と同様に、ERα陰転化・AR発現亢進の特徴を示すAI耐性乳癌の存在を探索するため、AI耐性乳癌症例21例におけるホルモン受容体の発現を免疫組織化学的に検討した。表２に示すように、再発時にERαが陽性から陰転化した症例は6例であった。初発時にARが陰性であった症例は9例であり、再発時に陽転化した症例は3例であった。初発時にARが陽性であった症例は12例であり、うち2例は再発時に陰転化した。以上の結果から、ARの転写活性が亢進している可能性のある症例もあることが示唆された。 As described above, since the expression of AR was increased in resistant strains, AR is considered to be an indicator of breast cancer exhibiting AR-dependent growth. To investigate the presence of AI-resistant breast cancer, which has the characteristics of ERα negative conversion and AR expression, as well as the properties of resistant strains, we examined the expression of hormone receptors in 21 AI-resistant breast cancer cases immunohistochemically. . As shown in Table 2, there were 6 cases in which ERα was negatively converted from positive at the time of recurrence. Nine cases were negative for AR at the first occurrence, and 3 cases were positively converted at the time of recurrence. Twelve cases were positive for AR at the first onset, of which 2 were negatively converted at the time of recurrence. These results suggest that there may be cases where AR transcriptional activity may be enhanced.
配列番号４はプライマーである。
配列番号５はプライマーである。
配列番号６はプライマーである。
配列番号７はプライマーである。
配列番号８はプライマーである。
配列番号９はプライマーである。 SEQ ID NO: 4 is a primer.
SEQ ID NO: 5 is a primer.
SEQ ID NO: 6 is a primer.
SEQ ID NO: 7 is a primer.
SEQ ID NO: 8 is a primer.
SEQ ID NO: 9 is a primer.

Claims (10)

  1. 　ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを指標とする、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定する方法。 A method for determining the presence or absence of acquisition of androgen receptor activity dependency in a breast cancer patient using at least one selected from the group consisting of KLK3, DDC and AR as an index.
  2. 　乳癌患者が女性患者である、請求項１に記載の方法。 The method according to claim 1, wherein the breast cancer patient is a female patient.
  3. 　ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つの発現量を測定する手段を含む、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定するためのキット。 A kit for determining whether or not androgen receptor activity is acquired in breast cancer patients, comprising means for measuring at least one expression level selected from the group consisting of KLK3, DDC and AR.
  4. 　乳癌患者が女性患者である、請求項３に記載のキット。 The kit according to claim 3, wherein the breast cancer patient is a female patient.
  5. 　ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを含む、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定するためのマーカー。 A marker for determining the presence or absence of acquisition of androgen receptor activity dependency in breast cancer patients, comprising at least one selected from the group consisting of KLK3, DDC and AR.
  6. 　乳癌患者が女性患者である、請求項５に記載のマーカー。 The marker according to claim 5, wherein the breast cancer patient is a female patient.
  7. 　ホルモン療法に耐性を示す乳癌に対する治療剤をスクリーニングする方法であって、ホルモン療法耐性乳癌細胞に被験物質を接触させる工程と、この細胞を培養し、細胞の増殖及び／又はアンドロゲンレセプター活性を測定する工程と、前記被検物質と接触させていない前記ホルモン療法耐性乳癌細胞を同じ条件下で培養及び測定した結果と比較して前記被検物質が細胞の増殖及び／又はＡＲ活性の低下をもたらしたか否かを判定する工程とを含み、
    　前記ホルモン療法耐性乳癌細胞が、エストロゲン非依存性、エストロゲンレセプター活性非依存性、かつアンドロゲンレセプター活性依存性である細胞株である、方法。     A method for screening a therapeutic agent for breast cancer that is resistant to hormone therapy, comprising contacting a test substance with hormone therapy-resistant breast cancer cells, culturing the cells, and measuring cell proliferation and / or androgen receptor activity Compared to the results of culturing and measuring the hormone therapy resistant breast cancer cells not contacted with the test substance under the same conditions, and whether the test substance caused cell proliferation and / or decreased AR activity Determining whether or not,
    The method wherein the hormone therapy resistant breast cancer cells are cell lines that are estrogen independent, estrogen receptor activity independent, and androgen receptor activity dependent.
  8. 　Ｔ４７Ｄ細胞を、エストロゲンを枯渇させた培地で、かつアンドロゲンの存在下で培養する工程を含む、アンドロゲンレセプター活性依存性の乳癌細胞の作製方法。 A method for producing androgen receptor activity-dependent breast cancer cells, comprising culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
  9. 　Ｔ４７Ｄ細胞を、エストロゲンを枯渇させた培地で、かつアンドロゲンの存在下で培養する工程を含む方法により得られる、アンドロゲンレセプター活性依存性の乳癌細胞株。 An androgen receptor activity-dependent breast cancer cell line obtained by a method comprising a step of culturing T47D cells in a medium depleted of estrogen and in the presence of androgen.
  10. ＫＬＫ３、ＤＤＣ及びＡＲからなる群より選択される少なくとも一つを指標とする、乳癌患者におけるアンドロゲンレセプター活性依存性獲得の有無を判定する工程、及び
    上記判定工程においてアンドロゲンレセプター活性依存性を獲得したと判定された乳癌患者に、アンドロゲンレセプター活性依存性を獲得した乳癌に適する治療を施す工程
    を含む、乳癌患者の治療方法。
          With the step of determining the presence or absence of acquisition of androgen receptor activity dependency in breast cancer patients, using at least one selected from the group consisting of KLK3, DDC and AR as an index, and acquiring androgen receptor activity dependency in the above determination step A method for treating a breast cancer patient, comprising the step of applying a treatment suitable for breast cancer that has acquired dependence on androgen receptor activity to the determined breast cancer patient.
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