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WO2022250465A1 - Nouveau biomarqueur pour la prédiction de la résistance à l'enzalutamide dans le cas du cancer de la prostate et son utilisation - Google Patents

Nouveau biomarqueur pour la prédiction de la résistance à l'enzalutamide dans le cas du cancer de la prostate et son utilisation Download PDF

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WO2022250465A1
WO2022250465A1 PCT/KR2022/007465 KR2022007465W WO2022250465A1 WO 2022250465 A1 WO2022250465 A1 WO 2022250465A1 KR 2022007465 W KR2022007465 W KR 2022007465W WO 2022250465 A1 WO2022250465 A1 WO 2022250465A1
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taz
yap1
slc22a3
prostate cancer
mdvr
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PCT/KR2022/007465
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English (en)
Korean (ko)
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강민용
전성수
서은정
지별아
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사회복지법인 삼성생명공익재단
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Publication of WO2022250465A1 publication Critical patent/WO2022250465A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a novel biomarker for predicting resistance to enzalutamide, a treatment for castration-resistant prostate cancer, in prostate cancer patients and its use, and more specifically, to a composition for predicting enzalutamide resistance in prostate cancer patients, and A kit and a method for providing information for predicting enzalutamide resistance in prostate cancer patients.
  • Prostate cancer is a disease with the fastest increasing incidence among male cancers in Korea.
  • Localized prostate cancer in which cancer is confined to the prostate, has a relatively high survival rate because radical surgery or radiation therapy can be applied.
  • the rate of survival without recurrence for 10 years after radical prostatectomy is about 70 to 85%.
  • metastatic prostate cancer is a terminal stage 4 cancer, and the average survival time is rapidly reduced to two and a half years because the cancer progresses faster than stage 1 or 2 prostate cancer, which is operable.
  • Prostate cancer characteristically has an androgen receptor (AR), and when the level of androgen hormone in the blood is lowered, such as in castration treatment, the cell signal transmission system through the receptor is blocked, thereby killing cancer cells.
  • AR androgen receptor
  • CRPC castration resistant prostate cancer
  • next-generation androgen receptor inhibitors such as enzalutamide are being used along with an anticancer drug called Docetaxel.
  • Enzalutamide was approved for the treatment of metastatic castration-resistant prostate cancer patients who had previously been treated with docetaxel in 2013, and asymptomatic or mildly symptomatic metastatic castration-resistant prostate cancer in 2015 and 2019, respectively. It was approved for cancer and high-risk non-metastatic castration-resistant prostate cancer, making it the first targeted treatment that can be used for castration-resistant prostate cancer regardless of metastasis. However, 10 to 25% of castration-resistant prostate cancer patients show resistance to enzalutamide, so they do not see the therapeutic effect of enzalutamide, and timely prostate cancer treatment is delayed, resulting in poor prognosis.
  • An object of the present invention is to provide a composition for predicting enzalutamide resistance in prostate cancer patients capable of detecting novel biomarkers predicting enzalutamide resistance.
  • an object of the present invention is to provide a kit for predicting enzalutamide resistance in prostate cancer patients comprising the above composition.
  • an object of the present invention is to provide a method for providing information for predicting enzalutamide resistance in prostate cancer patients by measuring the protein expression level of a novel biomarker predicting enzalutamide resistance.
  • the present inventors confirmed that the castration-resistant prostate cancer cell line having enzalutamide-resistance expressed the SLC22A3 gene at a significantly lower level than the castration-resistant prostate cancer cell line without drug resistance, and thus the expression of the SLC22A3 gene in the two castration-resistant prostate cancer cell lines.
  • the expression levels of proteins involved in were compared and analyzed.
  • YAP1/TAZ acts on the upstream region of SLC22A3 gene to reduce the expression of SLC22A3 gene, and it was first discovered that AR-V7 protein is also involved in this YAP1/TAZ action.
  • a combination of AR-V7 proteins is presented as a biomarker for predicting or discriminating enzalutamide resistance in prostate cancer, and a composition for predicting enzalutamide resistance in prostate cancer patients, including an agent capable of detecting such a biomarker, and
  • the present invention was completed by providing a kit and a method of providing information for predicting enzalutamide resistance of prostate cancer patients using the kit.
  • One aspect of the present invention provides a composition for predicting enzalutamide resistance in prostate cancer patients, including an agent for measuring the expression levels of biomarkers SLC22A3 and YAP1/TAZ.
  • the enzalutamide is an androgen receptor or androgen receptor inhibitor, which binds to the androgen receptor to prevent male hormone from binding to the androgen receptor, or blocks the delivery of androgen receptor to which male hormone binds into the cell nucleus. Or, by blocking the androgen receptor entering the cell from binding to the DNA of the target gene, signal transduction by male hormones is blocked to inhibit the growth of cancer cells.
  • Enzalutamide is an oral drug used for the treatment of castration-resistant prostate cancer with or without metastasis.
  • the castration-resistant prostate cancer is prostate cancer that continues to grow even when the amount of testosterone in the blood is reduced to a very low level (50 ng/dL) by androgen deprivation therapy.
  • Early-stage prostate cancer requires testosterone for growth, but castration-resistant prostate cancer that is resistant to testosterone blockade treatment continues to grow without testosterone and the cancer progresses, so the prognosis is very poor.
  • the prostate cancer may be castration-resistant prostate cancer.
  • Prediction of Enzalutamide resistance refers to a case in which a prostate cancer patient does not die of cancer cells even if he or she takes Enzalutamide and does not show a therapeutic effect by Enzalutamide. ), which means that before prescribing treatment drugs for prostate cancer patients, it is necessary to determine whether or not the patient is resistant to enzalutamide.
  • the expression level of the biomarker may be measured in prostate cancer cells or tissues of a prostate cancer patient.
  • biomarker is a substance that is generally detectable in biological samples, and organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, sugars, etc. All inclusive.
  • nucleic acids or proteins of SLC22A3 and YAP1/TAZ can be used as biomarkers.
  • the solute carrier family 22 member 3 (SLC22A3) is known as organic cation transporter 3 (OCT3), and is expressed by the SLC22A3 gene in various tissues to remove endogenous organic cations, drugs, toxins, etc. .
  • YAP1 (yes-associated protein 1) is a transcriptional regulator that activates the transcription of genes involved in cell proliferation and cell death inhibition, and is a protein molecule that acts in the final step of Hippo signal transduction. YAP1 works with various functional partners. Among them, the YAP1/TAZ complex combined with TAZ (WWTR1) is phosphorylated when Hippo signaling is activated, and both YAP1 and TAZ are phosphorylated and present in the cytoplasm, while Hippo signaling is inhibited. When not activated, YAP1/TAZ enters the nucleus and regulates gene expression.
  • the castration-resistant prostate cancer cell line (MDVR) having enzalutamide resistance has the SLC22A3 gene among genes involved in various signal transductions compared to the castration-resistant prostate cancer cell line (C4-2B) without resistance.
  • the expression level of was markedly decreased.
  • the YAP1/TAZ protein was expressed at a high level, the expression of the SLC22A3 gene was remarkably reduced. In particular, it was confirmed that the YAP1/TAZ protein was locally located in the cell nucleus.
  • the YAP1/TAZ protein which regulates the transcription level of the SLC22A3 gene, is a biomarker to predict enzalutamide resistance in prostate cancer, and the expression levels of SLC22A3 and YAP1/TAZ proteins to predict enzalutamide resistance in prostate cancer. It is preferable to observe in the nucleus of prostate cancer cells or the nucleic acid fraction of cancer cell lysates.
  • the agent may be capable of measuring the presence and expression level of each gene or protein by specifically binding to each gene or protein of the biomarker.
  • the agent may be one or more selected from the group consisting of primers, probes, antibodies, aptamers, oligopeptides, and PNAs that specifically bind to genes or proteins of the biomarker SLC22A3 and YAP1/TAZ, respectively.
  • the agent may be a primer or probe that specifically binds to the gene of the biomarker SLC22A3 and YAP1/TAZ.
  • binding means that the binding ability to a target substance is superior to other substances to the extent that the presence or absence of the target substance can be detected by binding.
  • primer refers to a primer capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerases) at a suitable temperature and buffer. It refers to a single stranded oligonucleotide.
  • suitable conditions i.e., four different nucleoside triphosphates and polymerases
  • the suitable length of a primer varies depending on various factors, such as temperature and use of the primer, but typically consists of 15-30 nucleotides.
  • probe refers to a linear oligomer of natural or modified monomers or linkages, comprising deoxyribonucleotides and ribonucleotides, and specifically hybridizing to a target nucleotide sequence It can be naturally occurring or artificially synthesized.
  • the agent is selected from the group consisting of antibodies, aptamers, oligopeptides and PNA (peptide nucleic acid) that bind specifically to proteins of the biomarker SLC22A3 and YAP1/TAZ, respectively. It may contain one or more. In the present invention, it is preferably an antibody so that an antigen-antibody reaction can be used for specific detection of a target protein.
  • antibody refers to a specific protein molecule directed against an antigenic site. In the present invention, it means an antibody that specifically binds to the target protein SLC22A3 or YAP1/TAZ, and includes both monoclonal antibodies, polyclonal antibodies and recombinant antibodies. In addition, the antibody includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains.
  • a functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, or the like.
  • the antibody can be readily prepared through techniques known in the art.
  • monoclonal antibodies can be prepared by the hybridoma method well known in the art (Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or by phage antibody libraries (Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991) technique.
  • Polyclonal antibodies can be produced by a method of injecting a target protein antigen into an animal and collecting blood from the animal to obtain antibody-containing serum.
  • Such polyclonal antibodies can be prepared from animals such as goats, rabbits, sheep, monkeys, horses, pigs, cows, and dogs.
  • the antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
  • composition for predicting enzalutamide resistance of prostate cancer patients according to the present invention may further include an agent for measuring the expression level of AR-V7.
  • AR-V7 (androgen receptor splice variant 7) is one of various variants of androgen receptor (AR) mRNA, and has a C-terminus of the androgen-binding domain compared to full-length AR (androgen receptor full length, AR (FL)). It is in partially truncated form.
  • AR-V7 RNA is resistant to enzalutamide treatment in castration-resistant prostate cancer patients (C Thoma, N Engl J Med 2014; 371:1028-1038).
  • the interaction between AR-V7 and YAP1/TAZ and the possibility of regulating the expression of the SLC22A3 gene by AR-V7 were not studied.
  • YAP1/TAZ and AR-V7 proteins are knocked down in a castration-resistant prostate cancer cell line (MDVR) having enzalutamide resistance
  • MDVR castration-resistant prostate cancer cell line
  • YAP1/TAZ or AR-V7 protein is knocked down alone
  • the protein expression level of the SLC22A3 gene significantly increased.
  • YAP1/TAZ and AR-V7 proteins were located in the nucleus of castration-resistant prostate cancer cell lines having enzalutamide resistance.
  • the AR-V7 protein together with YAP1/TAZ regulates the transcription level of the SLC22A3 gene, it can be used as a biomarker to predict enzalutamide resistance in prostate cancer, and the expression of SLC22A3, YAP1/TAZ and AR-V7 proteins By measuring all the levels, it will be able to be used to more accurately predict enzalutamide resistance in prostate cancer.
  • the preparation for measuring the expression level of AR-V7 is the same as the description of the preparation of the above-described biomarker SLC22A3 and YAP1/TAZ, so it is omitted here.
  • one aspect of the present invention provides a kit for predicting enzalutamide resistance in prostate cancer patients comprising the composition.
  • the "kit for predicting enzalutamide resistance in prostate cancer patients” refers to a substance capable of predicting whether or not enzalutamide resistance is present through a biological sample collected from a test subject or a prostate cancer patient, and through this, prostate cancer Enzalutamide resistance can be quickly and easily diagnosed.
  • the kit may use an immunoassay.
  • the immunoassay is an analysis method for confirming the presence and expression level of a target protein using an antigen-antibody reaction, and examples thereof include immunoblotting, radioimmunoassay, and radioactive immunoprecipitation. (radio-immunoprecipitation), immunoprecipitation, immunodiffusion, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunophile It may be an immunoaffinity purification or the like, but is not limited thereto. In the present invention, it can be performed according to previously developed protein quantitative or qualitative immunoassay protocols.
  • the kit is an ELISA kit, a Western blot kit, a radioimmunoassay kit, an immunodiffusion kit, an immunoprecipitation kit, an immunohistochemistry kit, an immunofluorescence staining kit, and a protein microarray kit. It may be one or more selected from the group consisting of.
  • the kit may further include one or more other component compositions, solutions or devices suitable for the assay method.
  • the kit may include antibodies that bind specifically to SLC22A3 and YAP1/TAZ proteins, respectively.
  • the kit may further include an antibody specifically binding to the AR-V7 protein.
  • Another aspect of the present invention is a) collecting a biological sample from a prostate cancer patient; b) measuring the expression levels of the biomarkers SLC22A3 and YAP1/TAZ in the biological sample; and c) providing information for predicting enzalutamide resistance in prostate cancer patients, comprising comparing the measured expression level of the biomarker with a control group.
  • Steps a) to c) will be examined in detail in the following, and the description of the contents common to the above will be omitted to avoid excessive complexity.
  • Step a) is a process of collecting a biological sample from a prostate cancer patient who needs to be tested for enalutamide resistance.
  • the prostate cancer patient in step a) may be a castration-resistant prostate cancer patient.
  • the biological sample in step a) may be prostate cancer cells or tissues containing the same.
  • Step b) is a process of measuring the expression levels of the biomarkers SLC22A3 and YAP1/TAZ in the biological sample, and an antigen-antibody reaction may be used to measure the biomarkers at the protein level.
  • an antigen-antibody reaction may be used to measure the biomarkers at the protein level.
  • the biomarker in step b) may be measured at the level of nucleic acid or protein expression.
  • the nucleic acid expression levels of the biomarkers SLC22A3 and YAP1/TAZ are determined by polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, and real-time RT-PCR. , It may be measured by one or more methods selected from the group consisting of nuclease protection assay, in situ hybridization, DNA microarray, and Northern blot.
  • the protein expression levels of the biomarkers SLC22A3 and YAP1/TAZ can be measured by ELISA, Western blot, radioimmunoassay, immunodiffusion, immunoprecipitation, immunohistochemistry, immunofluorescence, and protein microarray. It may be measured by one or more methods selected from the group consisting of
  • Step c) is a process of determining whether resistance to Enzalutamide is present by comparing the expression levels of SLC22A3 and YAP1/TAZ measured in the biological sample with a control group that does not exhibit resistance to Enzalutamide.
  • control group may be a normal person or a prostate cancer patient without Enzalutamide resistance.
  • step c) when the measured expression level of the biomarker SLC22A3 is lower than that of the control group and the expression level of YAP1/TAZ is higher than that of the control group, determining that the person has resistance to Enzalutamide it could be
  • the method for providing information for predicting enzalutamide resistance of prostate cancer patients according to the present invention may further measure the expression level of AR-V7 in a biological sample in step b). Since the expression level of AR-V7 is measured in the same way as the expression level of the biomarkers SLC22A3 and YAP1/TAZ described above, it is omitted here. Thereafter, when the measured expression level of the biomarker SLC22A3 is lower than that of the control group, and the expression level of YAP1/TAZ and AR-V7 is higher than that of the control group, it can be determined as having resistance to enalutamide.
  • cancer cells from castration-resistant prostate cancer patients are collected and stained for SLC22A3, TAZ, and AR-V7 proteins by immunohistochemical analysis.
  • SLC22A3, TAZ, and AR-V7 proteins are stained.
  • low level of SLC22A3 protein in cell nucleus and high level of TAZ and AR-V7 proteins were expressed, indicating that the test subject had castration-resistant prostate cancer showing resistance to Enzalutamide.
  • the present invention by presenting a new biomarker capable of predicting enzalutamide resistance in prostate cancer patients, by providing a composition and kit for predicting enzalutamide resistance in prostate cancer patients, individuals with enzalutamide resistance at an early stage can be rapidly and We can accurately diagnose and provide appropriate treatment.
  • Figure 1 shows the characteristics of transcriptome changes of Enzalutamide-resistant (EnzR) cell lines
  • A is the GSEA analysis result of EnzR cells compared to C4-2B cells
  • B is using the DAVID website GO analysis results of significantly up- or down-regulated genes
  • C is a Venn diagram showing commonly up- or down-regulated genes using data sets including GSE110802, GSE130534, GSE137833 and GSE136129
  • D is a heatmap of commonly up- or down-regulated major genes
  • E is the result of confirming the expression of the SLC22A3 and GULP1 genes in the C4-2B cell line and the MDVR cell line.
  • Figure 2 shows the prevention of phosphorylation of YAP1/TAZ from MST1 by abundant AR (FL),
  • A is the result of comparing the protein levels of YAP1 and TAZ, and
  • B is the YAP1/TAZ target gene in MDVR cells.
  • C is the result of confirming the transcription level of CTGF and CYR61
  • C is the result of confirming the mRNA expression of YAP1 and TAZ
  • D is the result of confirming the protein level of YAP1 and TAZ in MDVR
  • E is Flag-MST1
  • This is the result of confirming the protein levels of YAP1 and TAZ in AR(FL)-deleted MDVR.
  • Figure 3 shows the retention of YAP1/TAZ in the nucleus by AR-V7
  • (A) and (B) are YAP1 and TAZ proteins and mRNAs depending on whether AR-V7-targeting siRNA (siv7) was transfected or not.
  • (C) is the result of confirming the mRNA expression levels of YAP1/TAZ target genes CTGF, Cyr61, and AnkrD1, and
  • (D) is YAP1/TAZ transcriptional activity by siRNA targeting AR-V7 in MDVR.
  • (E) is the result of confirming the nuclear localization of YAP1 and TAZ
  • (H) is the result confirming the decrease in nuclear localization of YAP1/TAZ by AR-V7 deletion.
  • Figure 4 shows the transcriptional induction of DNMT1 and the inhibition of SLC22A3 expression by TAZ.
  • A) to (C) are the results of confirming the protein and mRNA expression levels of DNMT1 and SLC22A3 according to YAP1 and TAZ deficiency in MDVR
  • D) and (E) are the results of confirming the protein and mRNA expression levels of SLC22A3 by TAZ and DNMT1 in MDVR
  • (F) to (G) are the protein and mRNA expression levels of SLC22A3 according to the concentration or treatment of DAC.
  • Figure 5 shows the regulation of SLC22A3 gene expression by AR (FL) / AR-V7-YAP1 / TAZ-DNMT1 in MDVR cells.
  • (A) and (B) show AR (FL) and TAZ or YAP1 by DTH.
  • (C) is the result of confirming the interaction of AR (FL) and DNMT1 through TAZ
  • (D) is the result of confirming the interaction of YAP1 / TAZ-DNMT1 mediated by AR-V7
  • (E) and (F) are the results of confirming the protein and mRNA expression levels of SLC22A3 according to whether or not siRNA targeting YAP1 / TAZ, DNMT1 and / or AR-V7 was treated in MDVR cells
  • (G) is normal ( This is an image confirming the expression of TAZ, AR-V7, and SLC22A3 in normal) and CRPC tissues.
  • Figure 6 shows the promoter regulation of the SLC22A3 gene by the YAP1/TAZ-DNMT1 axis (Axis) in MDVR cells
  • A is a schematic diagram of the SLC22A3 promoter
  • "A" and "B” are YAP1/TEAD-binding It means a gene fragment containing or not including a motif
  • B is the result of confirming the abundant Flag-YAP1 of the "B” gene fragment
  • C) is the result of confirming the abundant YAP1 or TAZ of the "B” gene fragment
  • (D) is the result of confirming the abundance of YAP1 through AR-V7 of the "B” gene fragment
  • E is the result of confirming the abundant DNMT1 of the "B” gene fragment.
  • Figure 7 shows the enzalutamit resistance requirement of SLC22A3 regulated by TAZ-DNMT1
  • A is the result of confirming cell proliferation by joint treatment of ENZ and DAC
  • B is the joint treatment of ENZ and DAC.
  • This is the result of confirming the mRNA levels of the cell proliferation marker genes Cyclin A , Cyclin D1 and GSTP1
  • C) and (D) are siScr or SLC22A3 targeting siRNA transfected MDVR cells transfected with ENZ and DAC co-treatment
  • E) and (F) show the protein expression levels and colony formation rate of YAP1, TAZ and SLC22A3 by introduction of shYAP1 or shTAZ retroviral vectors and SLC22A3-targeting siRNA in MDVR cells. This is the result of checking
  • Figure 8 shows the regulation of SLC22A3 gene expression by the YAP1/TAZ inhibitor
  • (A) and (B) are the results confirmed by the protein and mRNA expression levels of SLC22A3 according to whether or not the YAP1/TAZ inhibitor VER was treated in MDVR
  • (C) is the result of confirming the colony formation rate according to the YAP1/TAZ inhibitor VER treatment in MDVR
  • (D) is the synergistic effect of the combination of MET and ENZ in MDVR and the ectopic expression of SLC22A3 when cell proliferation is inhibited. is the result of checking
  • a library for sequencing was generated using the QuantSeq 3' Library Prep Kit (Lexogen, Inc.) according to the manufacturer's instructions, and the sequence was read using the HiSeq 2000 system (Illumina).
  • the read reads were mapped to the hg19 human reference genome by applying default parameter values using TopHat (version 2.1.1). Abundance values of mapped reads for each gene were calculated using Cufflinks (version 2.2.1). Data processing and analysis were performed using R/Bioconductor libraries (version 1.4.1106).
  • transcriptome data of an enzalutamide resistant (EnzR) prostate cancer cell line was downloaded from the Gene Expression Omnibus (GEO) database and used. Data sets for verifying the results of this experiment were obtained from the GEO website (Accession numbers: GSE110802, GSE130534, GSE136129, and GSE137833).
  • C4-2B cells were purchased from ATCC (American Type Culture Collection) as a human castration-resistant prostate cancer cell line, and MDVR cells were a human castration-resistant prostate cancer cell line with enzalutamide resistance, University of California, USA (Research Director: Christopher P. .Evans). Both C4-2B cells and MDVR cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS) and penicillin/streptomycin. To maintain MDVR cells, 20 ⁇ M of enzalutamide (ENZ) was added to RPMI medium.
  • Decitabine (DAC) (Sigma, A3656) 1 ⁇ M was dissolved in an aqueous solution and stored at -20°C.
  • the DAC-containing medium was replaced with fresh DAC-containing medium daily.
  • Dihydoxytestosterone (DHT) was added to the cells at 10 nM each.
  • Vertepofin (VERTeporfin, VER) was used at a concentration of 5 ⁇ M.
  • RNAiMAX Lipofectamine RNAiMAX
  • siScr scrambled siRNA
  • YAP1-targeting siRNA Sata Cruz, sc-108080
  • TAZ-targeting siRNA Santa Cruz, sc-385678
  • DNMT1 targeting siRNA Thermo Fisher, 110914
  • AR-V7 targeting siRNA Ambion, Life Technologies, GUAGUUGUGAGUAUCAUGA
  • SLC22A3 targeting siRNA Sigma-Aldrich, EHU002291
  • HA-TAZ, Flag-MST1, Flag-YAP1, and retroviral vector shRNA targeting YAP1 or TAZ were provided by KAIST.
  • transfection was performed using Lipofectamine 2000.
  • Retroviruses were produced through techniques known in the art.
  • Cell protein lysates were obtained using a lysis buffer containing 0.5% Triton X-100.
  • Cell fractionation (Subcellular fractionation) was performed using a homogenizer (homogenizer) as a method known in the art from the protein lysate. Briefly, cells were disrupted using a pestle in 200 ul of cold hypotonic buffer. The lysate was centrifuged at 1000 rpm, and the precipitate was used as a nuclear fraction and the supernatant was used as a cytoplasmic fraction.
  • Western blot and immunoprecipitation were performed in at least two independent experiments. The supernatant was pretreated with IP buffer containing normal rabbit IgG and A/G agarose beads and used for immunoprecipitation.
  • the antibody used here was AR-V7 (ab8394).
  • the binding region of Flag-YAP1, HA-TAZ or DNMT1 in the chromatin of MDVR cells for immunoprecipitation was prepared by a method known in the art. Briefly, MDVR cells were transfected with Flag-YAP1, HA-TAZ, siScr or siAR-V7 cross-linked with 1% formaldehyde, which was then chilled with 1M glycine. Cells were lysed with lysis buffer (containing 1% SDS, 10 mM EDTA, 50 mM Tris-Hcl (pH 8.0) and protease inhibitors) and then subjected to sonication. Subsequent experimental procedures were performed according to methods known in the art.
  • Fragment A (negative control) was amplified using a primer that binds upstream of SLC22A3, and fragment B (including the TEAD-binding motif TGGAATG) was amplified using a primer that specifically binds to SLC22A3. Amplified (see Figure 5). The primers used here are shown in Table 2 below.
  • Primer name primer sequence sequence number A fragment F: 5'-CTA GCA CTT ATG TTT CAA GGC-3' 21 R: 5'-GTG CTC CTA ACA TAT CTA AGA-3' 22 B fragment F: 5'-TGG AGA GCC CTG TCG AAA TAG-3' 23 R: 5'-TGT GTC ATC TTC TGC CCT CCT-3' 24
  • the 8xGTIIc-luciferase reporter construct and pTk-Renilla were co-transfected into MDVR cells.
  • the activity of luciferase was measured using a luciferase assay reagent (Promega luciferase assay reagent) according to the manufacturer's instructions.
  • C4-2B or MDVR cells were washed with PBS, fixed with 4% paraformaldehyde at 4°C, permeabilized with PBS containing 0.1% Triton X-100, blocked with 2% BSA-PBS, and then stained with primary antibody, rabbit Incubation was performed for 1 hour with anti-AR-V7 antibody (ab198394), goat anti-Flag antibody (ab1257), rabbit anti-DNMT1 antibody (ab188453) or mouse anti-SLC22A3 antibody (ab75402).
  • Cells were washed with 1X PBS and secondary antibodies Alexa Fluor 488 donkey anti-goat (molecular probes in LifeTechnologies A-11055), Cy3-conjugated AffiniPure F (ab')2' Fragment Donkey, anti-rabbit IgG (711-166 -152) or Alexa Fluor 488 donkey anti-mouse IgG (molecular probes in life technologies, A 21202) for 1 hour. Cells were then washed 4 times with 1X PBS. Nuclei were stained with DAPI and cells were embedded with DAKO mounting medium.
  • C4-2B or MDVR cells were cultured for 2 weeks at 500 to 1,000 cells per well in a 6-well plate, fixed with 4% paraformaldehyde for 15 minutes, and 0.5% (w/v) crystal violet (crystal violet). violet) (Sigma-Aldrich) for 15 minutes.
  • Tissues were prepared as slides by embedding them in paraffin, and stained with hematoxylin and eosin (H&E) for histological analysis.
  • H&E hematoxylin and eosin
  • TAZ antibody Abcam
  • AR-V7 antibody ab198394
  • SLC22A3 antibody ab75402
  • DBA antibody Vector, FL-1031
  • RNA sequencing was performed in both C4-2B cells (Ctrl, control) and MDVR cells (EnzR) followed by GSEA using the hallmark gene set.
  • EnzR cell cycle-related pathways, including the G2M checkpoint and E2F target gene sets, were significantly activated, while androgen response-related gene sets were significantly inactivated (Fig. 1A).
  • a t-test was performed between the control group and EnzR and 923 differentially expressed genes (DEGs) were identified (p ⁇ 0.05 , fold difference > 1).
  • YAP1/TAZ is increased at the protein level
  • the protein level of exogenous YAP1/TAZ in C4-2B transfected with Flag-YAP1 or HA-TAZ was examined under DHT or ENZ+DHT conditions.
  • DHT treatment increased the amount of Flag-YAP1 or HA-TAZ protein in C4-2B, and decreased in the presence of ENZ (Fig. 2D).
  • the stability of YAP1/TAZ was measured in AR(FL)-silenced MDVR cultured in the presence of cycloheximide (CHX), which inhibits protein synthesis.
  • CHX cycloheximide
  • MST1 is known to induce degradation of YAP1 by direct phosphorylation, and androgen receptor (AR) plays a role in regulating YAP1 by androgen. Therefore, assuming that enhanced AR (FL) expression protects phosphorylation of YAP1 from MST1, whether ectopic expression of MST1 increases the phosphorylation level of YAP1 protein in C4-2B and MDVR with or without ENZ treatment investigated. As a result, ENZ treatment slightly increased YAP1 phosphorylation while decreasing total YAP1 protein, resulting in the expression of AR protein at a level similar to that of C4-2B (F, Lines 1 and 2 in Fig. 2).
  • YAP1/TAZ plays a role as a transcriptional activator of SLC22A3 in MDVR and performs epigenetic regulation.
  • the effect on the level was investigated.
  • C4-2B and MDVR cells were transfected with siScr or siRNA for AR-V7, treated with DHT at 32 hours, harvested at 36 hours after transfection, and subjected to Western blotting.
  • AR-V7 reduced the mRNA expression of YAP1/TAZ target genes such as Ctgf , Cyr61 , and AnkrD1 and the transcriptional activity of YAP1/TAZ in MDVR, suggesting that AR-V7 inhibits YAP1 without altering AP1/TAZ protein levels. / indicates that it positively regulates the transcriptional activity of TAZ (Fig. 3 C and D). Considering the nuclear localization of AR-V7, it was confirmed whether YAP1/TAZ, DNMT1, and AR-V7 were expressed by DHT exposure in the nucleus of C4-2B or MDVR.
  • C4-2B and MDVR cells were treated with DHT 10 nM for 24 hours, and Western blotting was performed using the nucleic acid fraction.
  • YAP1 and TAZ proteins slightly increased in the nucleus of C4-2B treated with DHT, whereas YAP1/TAZ and DNMT1 were elevated in the nucleus of MDVR regardless of DHT treatment (Fig. 3E).
  • Flag-YAP1 colocalizes with AR-V7 or DNMT1 in DHT-treated C4-2B or MDVR.
  • YAP1/TAZ-induced DNMT1 in MDVR offsets the expression of the SLC22A3 gene through methylation of the CpG island in the promoter of SLC22A3, so we investigated the effect of YAP1/TAZ deficiency on DNMT1 or SLC22A3 expression in MDVR. investigated.
  • the knockdown of YAP1/TAZ the amount of DNMT1 protein was decreased, whereas the level of SLC22A3 protein was markedly increased (Fig. 4A).
  • DNMT1 silencing increased the amount of SLC22A3 transcript and overexpression of TAZ reduced the expression of SLC22A3 transcript, which was restored by knockdown of DNMT1 in TAZ-expressing C4-2B (Fig. 4E).
  • DNMT1 may be a downstream mediator of TAZ that suppresses SLC22A3 expression in MDVR.
  • changes in SLC22A3 protein expression in response to decitabine (DAC), a DNMT1 inhibitor were observed.
  • C4-2B and MDVR cells were treated with 1 nM DAC for 36 hours.
  • nucleic acid fractions of scrambled or DHT-treated MDVR transfected with siRNA for AR-V7 were used to detect DNMT1 Immunoprecipitation was performed.
  • AR-V7 knockdown of MDVR showed that the amount of YAP1 or TAZ binding to DNMT1 was reduced (Fig. 5D).
  • TAZ-induced DNMT1 suppresses the mRNA level of SLC22A3 in MDVR, and that AR(FL)/AR-V7 and YAP1/TAZ together with DNMT1 are involved in MDVR.
  • the protein complex containing DNMT1 binds to genomic DNA and directly inhibits the expression of SLC22A3, and after analyzing the genomic sequence of the SLC22A3 gene, the transcription start site presumed to be the TEAD-binding sequence (TGGAATG) , TSS) was confirmed to have a sub-1.5 kb (Fig. 6A). Chip analysis was performed using an anti-Flag antibody in MDVR expressing Flag-YAP.
  • DNMT1 To confirm whether TAZ-induced DNMT1 forms a complex containing TAZ, a Chip assay was performed using an anti-DNMT1 antibody in MDVR expressing HA-TAZ. As a result, DNMT1 was markedly increased in the "B" region by the ectopic expression of HA-TAZ compared to vector expression, indicating that YAP1/TAZ or DNMT1 constituting the complex of AR(FL) and AR-V7 is the promoter of SLC22A3. It indicates that it directly regulates (E in FIG. 6).
  • C4-2B and MDVR cells were treated with 50 mM ENZ and/or 1 nM DAC for 14 days, followed by colony formation. formation was observed. As expected, the clonogenicity was reduced by 50% in C4-2B treated with ENZ alone or ENZ and DAC, while the DAC-treated group showed no change in colony formation, indicating a low level of DNMT1 in C-2B. This suggests that it does not affect colony formation (Fig. 7A). However, in MDVR treated with DAC alone or with ENZ and DAC, the ability to form colonies by ENZ was suppressed (Fig. 7A).
  • VER YAP1/TAZ inhibitor verteporfin
  • MDVR cells were treated with only 5 ⁇ M VER or 20 ⁇ M ENZ for 36 hours, or treated with VER (5 ⁇ M) and ENZ (20 ⁇ M) together, and the cells were crushed together with a control or used for colony generation experiments.
  • verteporfin decreased the protein expression of YAP1/TAZ and increased the expression level of SLC22A3 regardless of whether or not enzalutamide was treated (Fig. 8A and B).
  • verteporfin reduced the colony forming ability of MDVR (FIG. 8C).

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Abstract

La présente invention concerne un nouveau biomarqueur pour la prédiction de la résistance à l'enzalutamide, qui est un agent de traitement contre le cancer de la prostate hormono-résistant, chez un patient atteint d'un cancer de la prostate, et une utilisation de celui-ci. La présente invention présente le nouveau biomarqueur qui peut prédire la résistance à l'enzalutamide chez un patient atteint d'un cancer de la prostate, et fournit une composition et un kit pour la prédiction de la résistance à l'enzalutamide chez un patient atteint d'un cancer de la prostate, ce qui permet d'identifier rapidement et avec précision un individu ayant une résistance à l'enzalutamide à un stade précoce et de fournir un traitement approprié.
PCT/KR2022/007465 2021-05-27 2022-05-26 Nouveau biomarqueur pour la prédiction de la résistance à l'enzalutamide dans le cas du cancer de la prostate et son utilisation WO2022250465A1 (fr)

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