KR20230092844A - Novel biomarker for predicting of resistance against enzalutamide of prostate cancer and uses thereof - Google Patents

Abstract

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 provides a novel biomarker capable of predicting enzalutamide resistance in prostate cancer patients. By presenting the present invention, it is possible to provide a composition and kit for predicting enzalutamide resistance in prostate cancer patients, promptly and accurately determine individuals with enzalutamide resistance at an early stage, and provide appropriate treatment.

Description

Novel biomarker for predicting of resistance against enzalutamide of prostate cancer and uses thereof {Novel biomarker for predicting of resistance against enzalutamide of prostate cancer and uses thereof}

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 (PC) 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. In particular, the rate of survival without recurrence for 10 years after radical prostatectomy is about 70 to 85%. However, 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. However, if the cancer progresses despite blocking testosterone in the blood, it is called castration resistant prostate cancer (CRPC). ), 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. Therefore, if it is possible to predict or determine the patient's resistance to Enzalutamide before treating castration-resistant prostate cancer, a more positive treatment effect can be expected by selecting an appropriate treatment or treatment drug. However, some studies on biomarkers for diagnosing the onset of prostate cancer or predicting treatment prognosis of prostate cancer are currently being conducted, but studies on biomarkers for predicting enzalutamide resistance in prostate cancer patients are still insufficient.

Korean Patent Registration No. 10-2229255 Korean Patent Registration No. 10-2208140

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.

In addition, an object of the present invention is to provide a kit for predicting enzalutamide resistance in prostate cancer patients comprising the above composition.

In addition, 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. As a result, 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. Alternatively, 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.

According to one embodiment of the present invention, the prostate cancer may be castration-resistant prostate cancer.

"Prediction of Enzalutamide resistance" used in the present invention 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.

According to one embodiment of the present invention, the expression level of the biomarker may be measured in prostate cancer cells or tissues of a prostate cancer patient.

As used in the present invention, "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. In the present invention, 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.

In a recent study, it was confirmed that the expression of genes XLOC_l2_002352 , FADS2 , SLC22A3 and MALAT1 was reduced in prostate cancers resistant to enalutamide compared to prostate cancers without drug resistance (John Greene, et al., Scientific Reports volume 9, Article number: 10739, 2019), the mechanism of expression of the SLC22A3 gene or the correlation between the SLC22A3 gene and YAP1/TAZ has not been elucidated.

According to one embodiment of the present invention, 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. When 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. Therefore, 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.

According to one embodiment of the present invention, 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.

More specifically, 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.

According to one embodiment of the present invention, the agent may be a primer or probe that specifically binds to the gene of the biomarker SLC22A3 and YAP1/TAZ.

As used herein, “specifically 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.

As used herein, "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. 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.

As used herein, “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.

According to one embodiment of the present invention, 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.

As used herein, "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. For example, 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, and cows.

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.

Meanwhile, the 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. Recently, a study has been published showing that AR-V7 RNA is resistant to enzalutamide treatment in castration-resistant prostate cancer patients (C Thoma, N Engl J Med 2014; 371:1028-1038). However, in this study, 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.

According to one embodiment of the present invention, when YAP1/TAZ and AR-V7 proteins are knocked down in a castration-resistant prostate cancer cell line (MDVR) having enzalutamide resistance When YAP1/TAZ or AR-V7 protein is knocked down alone In comparison, it was confirmed that the protein expression level of the SLC22A3 gene significantly increased. In addition, it was confirmed that YAP1/TAZ and AR-V7 proteins were located in the nucleus of castration-resistant prostate cancer cell lines having enzalutamide resistance. Therefore, since 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.

In addition, one aspect of the present invention provides a kit for predicting enzalutamide resistance in prostate cancer patients comprising the composition.

As used in the present invention, 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.

According to one embodiment of the present invention, 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.

According to one embodiment of the present invention, 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.

According to one embodiment of the present invention, the kit may include antibodies that bind specifically to SLC22A3 and YAP1/TAZ proteins, respectively.

In addition, according to one embodiment of the present invention, the kit may further include an antibody specifically binding to the AR-V7 protein.

Since the antibody is the same as described above, it is omitted here.

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.

According to one embodiment of the present invention, the prostate cancer patient in step a) may be a castration-resistant prostate cancer patient.

According to one embodiment of the present invention, 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. In the present invention, it is preferable to use the above-described composition or kit for predicting enzalutamide resistance in prostate cancer patients.

According to one embodiment of the present invention, the biomarker in step b) may be measured at the level of nucleic acid or protein expression.

According to one embodiment of the present invention, 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.

In addition, according to one embodiment of the present invention, 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.

According to one embodiment of the present invention, the control group may be a normal person or a prostate cancer patient without Enzalutamide resistance.

According to one embodiment of the present invention, in 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

Meanwhile, 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.

According to one embodiment of the present invention, cancer cells from castration-resistant prostate cancer patients, of which enzalutamide resistance has not been confirmed, are collected and stained for SLC22A3, TAZ, and AR-V7 proteins by immunohistochemical analysis. As a result, normal prostate cells are stained. Compared to , 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.

In 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, and (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 (F) 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, (F) and (G) are immunofluorescence staining images confirming the co-localization of Flag-YAP1 and AR-V7 in the cell nucleus (accumulation Bar = 20 μM), (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, and (F) to (G) are the protein and mRNA expression levels of SLC22A3 according to the concentration or treatment of DAC. These are the confirmed results, (H) shows the change in SLC22A3 expression by Enzalutamide and DAC, (I) shows that the increase in SLC22A3 protein expression by DAC is reduced by TAZ overexpression, and (J) shows the SLC22A3 The image shows the increased expression when DNMT1 was removed.
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 And, (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, and (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, and "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, and (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, and (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 , and (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, and (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

Hereinafter, the present invention will be described in more detail. However, these descriptions are merely presented as examples to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

1. Materials and Methods

1-1. RNA sequencing

Total RNA was extracted with an RNA extraction kit and RNA integrity was checked with a Bioanalyzer (Agilent). 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).

1-2. Gene Set Enrichment Analysis (GSEA) & ssGSEA

In the GSEA analysis, the Hallmark gene set of the Molecular Signatures Database (MSIGDB 7.0) was used. To perform the GSEA of YAP activation, a previously published data set (JJ Alumkal et al., PNAS. September 29, 2009 106 (39) 16663-16668) was used by applying the CORDENOSI_YAP_CONNSERED gene set of MSigDB. ssGSEA (single sample GSEA) was calculated using the GSVA package.

1-3. Validation set

For validation analysis, 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).

1-4. Cell culture and reagents

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 μM of enzalutamide (ENZ) was added to RPMI medium. Decitabine (DAC) (Sigma, A3656) 1 μM 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 μM.

1-5. Gene knockdown, expression constructs and transfection

Cells were treated with Lipofectamine RNAiMAX (Invitrogen) using scrambled siRNA (siScr) (Santa Cruz) as a control or YAP1-targeting siRNA (Sata Cruz, sc-108080) and TAZ-targeting siRNA (Santa Cruz, sc-38568) as test groups. ), DNMT1 targeting siRNA (Thermo Fisher, 110914), AR-V7 targeting siRNA (Ambion, Life Technologies, GUAGUUGUGAGUAUCAUGA) and SLC22A3 targeting siRNA (Sigma-Aldrich, EHU002291) were transfected. HA-TAZ, Flag-MST1, Flag-YAP1, and retroviral vector shRNA targeting YAP1 or TAZ were provided by KAIST. For ectopic expression, transfection was performed using Lipofectamine 2000. Retroviruses were produced through techniques known in the art.

1-6. Western blot and immunoprecipitation

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).

1-7. qRT-PCR.

RNA was extracted from cells and cDNA was synthesized by reverse transcription with M-MLV reverse transcriptase (Promega) according to the manufacturer's protocol. cDNA was mixed with SYBR green and primers for each target gene in Table 1 below.

Primer name primer sequence sequence number CTGF F: 5'-CCA ATG ACA ACG CCT CCT G-3' One R: 5'-TGG TGC AGC CAG AAA GCT C-3' 2 CYR61 F: 5'-GGT CAA AGT TAC CGG GCA GT-3' 3 R: 5'-GGA GGC ATC GAA TCC CAG C-3' 4 ANKRD1 F: 5'-CAC TTC TAG CCC ACC CTG TGA-3' 5 R: 5'-CCA CAG GTT CCG TAA TGA TTT-3' 6 YAP1 F: 5'-TAG CCC TGC GTA GCC AGT TA-3' 7 R: 5'-TCA TGC TTA GTC CAC TGT CTG T-3' 8 TAZ F: 5'-GAT CCT GCC GGA GTC TTT CTT-3' 9 R: 5'-CAC GTC GTA GGA CTG CTG G-3' 10 SLC22A3 F: 5'-ATC GTC AGC GAG TTT GAC CTT-3' 11 R: 5'-ACC TGT CTG CTG CAT AGC CTA-3' 12 DNMT1 F: 5'-AGG CGG CTC AAA GAT TTG GAA-3' 13 R: 5'-GCA GAA ATT CGT GCA AGA GAT TC-3' 14 Cyclin A1 F: 5'-GAG GTC CCG ATG CTT GTC AG-3' 15 R: 5'-GTT AGC AGC CCT AGC ACT GTC-3' 16 Cyclin D1 F: 5'-GCT GCG AAG TGG AAA CCA TC-3' 17 R: 5'-CCT CCT TCT GCA CAC ATT TGA A-3' 18 GSTP1 F: 5'-CCC TAC ACC GTG GTC TAT TTC C-3' 19 R: 5'-CAG GAG GCT TTG AGT GAG C-3' 20

1-8. Chromatin immunoprecipitation (Chip) analysis

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

1-9. Luciferase assay

The 8xGTIIc-luciferase reporter construct and pTk-Renilla (transfection control) 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.

1-10. Immunofluorescence analysis

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.

1-11. Colony formation assay

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.

1-12. Tissue and immunofluorescence analysis

Tissues were prepared as slides by embedding them in paraffin, and stained with hematoxylin and eosin (H&E) for histological analysis. To monitor the expression of each target protein in tissue slides, TAZ antibody (Abcam), AR-V7 antibody (ab198394), SLC22A3 antibody (ab75402) or DBA antibody (Vector, FL-1031) were used as probes. .

2. Results

2-1. Characteristics of changes in EnzR cell lines through transcriptome analysis

To characterize transcriptome changes in EnzR cells, RNA sequencing was performed in both C4-2B cells (Ctrl, control) and MDVR cells (EnzR) followed by GSEA using the hallmark gene set. As a result, in 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). . In addition, to evaluate transcriptome changes during resistance to Enzalutamide, 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). To confirm the biological role of these genes in EnzR, GO (Gene Ontology) was analyzed using the DAVID website. As a result, genes up-regulated in EnzR are related to the regulation of cell proliferation, system development, and cell differentiation, and genes down-regulated in EnzR are related to cell-cell signaling. -cell signaling), cell adhesion, and ion transport (FIG. 1B). Taken together, these transcriptome changes may be associated with the development of enzalutamide resistance. To identify driver genes associated with enalutamide resistance, the DEG lists of four independent data sets, including GSE110802, GSE137833, GSE130534 and GSE136129, were compared with our data set for analysis. As a result, it was confirmed that SLC22A3, GULP1, and TFPI, except for AKR1C3, were downregulated by ENZ treatment (Fig. 1 C and D). In particular, a clear difference in expression of SLC22A3 was confirmed in both the control group and EnzR, indicating that the decrease in SLC22A3 expression was highly correlated with enzalutamide resistance (FIG. 1E).

2-2. Prevention of phosphorylation of YAP1/TAZ from MST1 by abundant AR(FL)

Although it has been previously reported that there is an association between YAP1 and AR(FL) in the nucleus of CRPC cells, the expression of YAP1 and TAZ has not been reported in CRPC resistant to Enzalutamide. Therefore, we investigated the distinct expression patterns of YAP1/TAZ and AR(FL) in CRPC cells (C4-2B) and enzalutamide-resistant CRPC cells (MDVR). C4-2B and MDVR cells were treated with 10 nM DHT for 6 hours before treatment with control or 20 μM ENZ for 36 hours. As a result, AR(FL), which was increased by DHT exposure in C4-2B, was decreased by ENZ treatment, whereas AR(FL) was dominantly expressed in MDVR regardless of ENZ treatment. The protein expression of AR-V7 and YAP1/TAZ also showed a pattern similar to that of AR (FL) (Fig. 2A). These results indicate that YAP1/TAZ expression correlates with the amount of total AR (FL) in C4-2B and MDVR. In addition, we investigated whether the expression of target genes (CTGF and CYR61) reflecting YAP1/TAZ activity in C4-2B or MDVR was affected by Enzalutamide in the presence of DHT (10 nM). As a result, ENZ treatment decreased the expression level of two genes in C4-2B, but both genes were highly expressed in MDVR regardless of ENZ treatment, indicating that YAP1/TAZ is continuously activated in MDVR (Fig. 2). B). In addition, as a result of evaluating the expression of YAP1/TAZ at the mRNA level, the transcription of YAP1/TAZ in MDVR was slightly increased compared to C4-2B, but the difference in expression of YAP1/TAZ mRNA in C4-2B or MDVR was not different from DHT and ENZ treatment. There was no significant difference by whether or not (Fig. 2 C). These results indicate that increased mRNA levels of YAP1/TAZ are not sufficient to enrich YAP1/TAZ protein in MDVR. In addition, to test whether 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. As a result, DHT treatment increased the amount of Flag-YAP1 or HA-TAZ protein in C4-2B, and decreased in the presence of ENZ (Fig. 2D). In addition, the stability of YAP1/TAZ was measured in AR(FL)-silenced MDVR cultured in the presence of cycloheximide (CHX), which inhibits protein synthesis. After transfecting C4-2B and MDVR cells with control vector or Flag-MST1, they were treated with control or 20 μM ENZ for 36 hours in the presence of 10 nM DHT. As a result, the half-life of YAP1 or TAZ in MDVR delivered with siRNA AR(FL) was significantly decreased compared to the control group (Fig. 2E). These results suggest that the presence of AR(FL) is required for YAP1/TAZ stability, as YAP1/TAZ was not stabilized in AR(FL)-knockdown MDVR.

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). In comparison, strong expression of MST1 markedly increased YAP1 phosphorylation while reducing total YAP1 protein, regardless of the amount of AR(FL) protein (Fig. 2 F, Lines 3 and 4). In addition, MDVR in which AR (FL) is saturated showed a decrease in YAP1 phosphorylation level as total YAP1 protein increased, which was not changed by ENZ treatment or MST1 overexpression (FIG. 2 F, Line 5 to 8). These results indicate that degradation of YAP1/TAZ via MST1 is likely to be inhibited by high expression of AR(FL). To confirm this, we analyzed the phosphorylation level of YAP1 in MDVR silenced by siRNA against AR(FL), MST1 or both. As a result, depletion of AR(FL) reduced the YAP1/TAZ protein level, which was restored to a level similar to that of siScr-expressing MDVR by knockdown of MST1 (Fig. 2G). Taken together, these results suggest that the abundant AR (FL) protein in ENZ-resistant MDVR contributes to increasing YAP1/TAZ protein levels through inhibition of phosphorylation of YAP1 by MST1.

2-3. Retention of YAP1/TAZ in the nucleus by AR-V7

Through the above experiments, it was confirmed that YAP1/TAZ plays a role as a transcriptional activator of SLC22A3 in MDVR and performs epigenetic regulation. In this experiment, we hypothesized that the positive correlation between YAP1/TAZ and AR-V7 in MDVR offsets the expression of the SLC22A3 gene via DNMT1 for DNA methylation, suggesting that AR-V7 deficiency does not affect YAP1/TAZ, DNMT1 or SLC22A3 proteins. 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. As a result, although knockdown of AR-V7 did not change the YAP1/TAZ protein level in MDVR, DNMT1 was significantly reduced to a level similar to that of C4-2B, and SLC22A3 was expressed in MDVR (Fig. 3A). And even in the absence of AR-V7, the YAP1/TAZ transcript, which is expressed at a high level in MDVR compared to C4-2B, did not change (Fig. 3B). However, depletion of 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 expression 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 nucleic acid fractions. As a result, 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). In addition, we investigated whether Flag-YAP1 colocalizes with AR-V7 or DNMT1 in DHT-treated C4-2B or MDVR. As a result, C4-2B ectopically expressed Flag-YAP1 distributed in the cytoplasm and nucleus without AR-V7 expression, whereas Flag-YAP1 in MDVR was mostly located in the nucleus and overlapped with AR-V7 expression (Fig. 3 F). In addition, DNMT1 was expressed at a high level in MDVR expressing Flag-YAP1, and DNMT1 and Flag-YAP1 were equally located in the nucleus in MDVR compared to C4-2B (Fig. 3G). These results indicate that the positive correlation between AR-V7 and YAP1/TAZ is related to nuclear localization of YAP1/TAZ and regulates their activity. In addition, nuclear localization of YAP1/TAZ and changes in DNMT1 and SLC22A3 protein levels by AR-V7 deficiency in MDVR were investigated. As a result, AR-V7 silencing decreased DNMT1 protein level in the nucleus (N), but increased SLC22A3 protein level in the cytoplasm (C), accompanied by loss of nuclear localization of YAP1/TAZ in MDVR (Fig. 3H ). Taken together, it can be seen that AR-V7 associated with enzalutamide resistance activates YAP1/TAZ by being preserved in the nucleus co-localized with DNMT1, and as a result, the expression level of SLC22A3 protein is reduced, especially in the cytoplasm.

2-4. Inhibition of SLC22A3 expression in MDVR of DNMT1 induced by TAZ

We hypothesized that 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. As a result, in the knockdown of YAP1/TAZ, the amount of DNMT1 protein was decreased, whereas the level of SLC22A3 protein was markedly increased (Fig. 4A). In addition, as a result of confirming whether YAP1, TAZ or both deficiency increased the mRNA expression of SLC22A3, YAP1 and TAZ silencing increased the mRNA expression level of SLC22A3 compared to YAP1 or TAZ deficiency alone (Fig. 4B) . In addition, as a result of observing changes in DNMT1 mRNA expression in the absence of YAP1 or TAZ, DNMT1 mRNA expression was significantly reduced in TAZ knockdown, but not in YAP1 knockdown (FIG. 4C). These results indicate that ectopic expression of TAZ upregulates the mRNA expression of DNMT1. In addition, to determine the epistatic relationship between TAZ and DNMT1 in the regulation of SLC22A3 expression, the effect of TAZ or DNMT1 alone or joint deficiency on SLC22A3 expression was investigated. DNMT1 silencing induced an increase in SLC22A3 levels, slightly reducing DNMT1 protein, more than deprivation of TAZ. Co-deficiency of TAZ and DNMT1 increased the level of SLC22A3 protein in MDVR compared to lack of TAZ or DNMT1 alone, suggesting that TAZ and DNMT1 function as upstream regulators of SLC22A3 (Fig. 4D). . In addition, 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). These results indicate that DNMT1 may be a downstream mediator of TAZ that suppresses SLC22A3 expression in MDVR. In addition, to further investigate the role of DNMT1 in suppressing 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. As a result, while the high level of SLC22A3 in C4-2B was not significantly changed by DAC treatment, DNMT1 protein was reduced in DAC-treated MDVR and SLC22A3 was recovered to a level similar to that of C4-2B (FIG. 4F ). Also, in MDVR treated with DAC, the mRNA of SLC22A3 increased compared to untreated MDVR (Fig. 4G). These results indicate that proper expression of DNMT1 suppresses the mRNA expression of SLC22A3 in MDVR. In addition, DNMT1 lowered the protein level of SLC22A3 in MDVR, but when treated with DAC, the protein level of DNMT1 was reduced and the expression of SLC22A3 protein was increased (FIG. 4H). In addition, MDVR exposed to ENZ exhibited a low level of DNMT1 expression by DAC treatment, and significantly increased the expression of SLC22A3 (FIG. 4I). This result also supports the fact that DNMT1 suppresses the expression of SLC22A3 in MDVR. In addition, as a result of observing SLC22A3 expression by DNMT1 deficiency using immunofluorescence analysis, SLC22A3 was expressed in the plasma membrane and cytoplasm of DNMT1-deficient MDVR, whereas it was not expressed in siScr-transfected MDVR (J in FIG. 4 ).

2-5. Regulation of SLC22A3 gene expression by AR(FL)/AR-V7-YAP1/TAZ-DNMT1

As described above, it was shown that excessive AR(FL) and AR-V7 localize in the nucleus for two reasons, activating YAP1/TAZ and inducing the expression of DNMT1, which suppresses the expression of SLC22A3. In this experiment, it was expected that the interaction between YAP1/TAZ and AR(FL)/AR-V7 would regulate SLC22A3 expression by bringing DNMT1 to the promoter of the upper region of SLC22A3. First, it was investigated whether AR(FL)/AR-V7 form a YAP1/TAZ complex depending on DHT treatment. MDVR cells were treated with 1nM DHT for 36 hours, and immunoprecipitation was performed using each antibody. As a result, in AR(FL) immunoprecipitation, YAP1 or TAZ showed a weak interaction with AR(FL) when DHT was not treated, whereas AR(FL) precipitated well with TAZ or YAP1 when DHT was treated. , and the interaction between AR(FL) and AR-V7 did not change (Fig. 5A). These results indicate that AR(FL) activated by DHT treatment is likely to interact with YAP1 or TAZ. In addition, TAZ immunoprecipitation showed an interaction with DNMT1 or AR(FL) without DHT, whereas TAZ showed a strong association with DNMT1 or AR(FL) due to DHT treatment (FIG. 5B). These results explain the strong interaction between DNMT1 and TAZ in MDVR's response to DHT, suggesting that AR(FL) can interact with TAZ to expand its interaction with DNMT1 to a wider range. To confirm that TAZ is required for the formation of AR (FL) complexes with DNMT1, AR ( FL) was precipitated with AR (FL) antibody. As a result, in AR(FL) immunoprecipitation, DNMT1 and AR(FL) were precipitated together in the control group, whereas DNMT1 and AR(FL) were less precipitated in MDVR with low TAZ expression (FIG. 5C). These results indicate that AR(FL) interacts with DNMT1 and that TAZ is required to form this complex. To determine whether nuclear localization of YAP1 or TAZ by AR-V7 contributes to the interaction of YAP1/TAZ with DNMT1, nucleic acid fractions of scrambled or DHT-treated MDVR transfected with siRNA for AR-V7 were used to detect DNMT1 Immunoprecipitation was performed. As a result, as expected, AR-V7 knockdown of MDVR showed that the amount of YAP1 or TAZ binding to DNMT1 was reduced (Fig. 5D). These results suggest that DNMT1 immunoprecipitation of AR-V7-deficient MDVR interacts weakly with YAP1, TAZ or AR-V7, DNMT1 forms a complex containing AR-V7, and YAP1 or TAZ is nucleated by AR-V7. indicates that it is conserved in

2-6. Biomarkers Predicting Enzalutamide Resistance

The effect of knockdown of AR-V7 or DNMT1 in YAP1/TAZ-silenced MDVR on the mRNA or protein expression of SLC22A3 was investigated. As a result, YAP1/TAZ deletion further increased the protein or transcript levels of SLC22A3 compared to DNMT1 or AR-V7 silencing, and AR-V7 and YAP1/TAZ co-silencing greatly increased the mRNA or protein levels of SLC22A3. Co-silencing of YAP1/TAZ and DNMT1 did not significantly affect the mRNA or protein levels of SLC22A3 (Fig. 5E and F). These results suggest that AR-V7, YAP1 or TAZ can be specific markers for patients with enzalutamide resistance. To confirm this, immunohistochemical analysis of TAZ, SLC22A3 and AR-V7 was performed in tissues of normal subjects and CRPC patients. As a result, while normal tissue did not express TAZ and AR-V7 and expressed SLC22A3 highly, CRPC tissue strongly expressed TAZ or AR-V7 while expressing SLC22A3 at a low level (G in FIG. 5 ). Therefore, it can be inferred that the CRPC tissue of this experiment has enalutamide resistance. These results suggest that non-expression of SLC22A3 together with strong expression of TAZ and AR-V7 can be a significant predictive marker for CRPC with enzalutamide resistance.

2-7. Promoter regulation of SLC22A3 gene by YAP1/TAZ-DNMT1 axis

In the above experiments, it was confirmed that 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. It is hypothesized that 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. As a result, YAP was abundant in the -1.5 kb region indicated by "B", whereas Flag-YAP was abundant in the negative control region indicated by "A". YAP was not enriched (Fig. 6B). In MDVR expressing HA-TAZ, HA-TAZ was abundant in the B region (FIG. 6C). To test whether the presence of AR-V7 affects YAP1 abundance in the upper region of SLC22A3, MDVR cells were transfected with siScr or scAR-V7 and incubated with 1 nM DHT for 36 hours. As a result, as expected, when AR-V7 was deficient, Flag-YAP1 was decreased in the B region of the SLC22A3 promoter (Fig. 6D). 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).

2-8. Enzalutamide resistance requiring SLC22A3 modulated by TAZ-DNMT1

To investigate the effect of the YAP1/TAZ-DNMT1 axis on colony forming of enzalutamide-resistant MDVR, 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). To confirm the inhibitory effect of the co-treatment of ENZ and DAC in MDVR, mRNA expression of genes related to cell proliferation was measured. As a result, ENZ and DAC treatment decreased the expression of Cyclin A and Cyclin D , and GSTP1 , which is expressed at a low level in CRPC, increased in both C4-2B and MDVR (FIG. 7B). These results indicate that inhibition of DNMT1 by DAC causes a decrease in the colony forming ability of MDVR.

Given that DNMT1 directly inhibited SLC22A3 expression and that DNMT1 inhibition attenuated colony formation, we hypothesized that inhibition of SLC22A3 by DNMT1 confers the ability to form colonies in MDVR resistant to enalutamide, and then investigated SLC22A3 SLC22A3-knockdown MDVRs were generated using siRNA for SLC22A3, and control cells transfected with siScr were used to examine the effect of inhibition of DNMT1 through SLC22A3 induction on the decrease in colony formation ability in the presence of ENZ. As a result, in Western blot analysis, the increase in SLC22A3 protein level by treatment with DAC and ENZ was efficiently reduced in SLC22A3-deficient MDVR (FIG. 7C). In addition, the effect of SLC22A3 knockdown on the loss of colony forming ability of DNMT1-inhibiting MDVR response to enalutamide was investigated. As a result, compared to the untreated control group, the siScr-transfected MDVR showed a decrease in colony formation ability by about 51% in the presence of ENZ and DAC, whereas the SLC22A3-silenced MDVR increased the colony formation ability by about 75% (Fig. 7D ). These results suggest that MDVR can obtain colony-forming ability through the suppressive function of DNMT1 on SLC22A3. From this, YAP1/TAZ acts on top of DNMT1 to inhibit SLC22A3, and as a result, the effect of YAP1/TAZ on the MDVR proliferation response to enzalutamide is expected to be dependent on SLC22A3, so shRNA-mediated YAP1 or TAZ knockdown MDVR was transfected with siRNA against SLC22A3. As a result, the expression level of YAP1 or TAZ in the YAP1-/TAZ-knockdown MDVR was significantly lower than that of the control MDVR (FIG. 7E). These results suggest that YAP1-/TAZ-deleted MDVRs show loss of colony formation ability in the absence of ENZ, regardless of SLC22A3 expression, suggesting that YAP1/TAZ is involved in MDVR proliferation. In addition, the decrease in colony formation in YAP1/TAZ-deficient MDVR was restored by 70% by SLC22A3 deficiency in the presence of ENZ (FIG. 7F). These results suggest that YAP1/TAZ-mediated inhibition of SLC22A3 contributes to enzalutamide resistance in MDVR, and the cause of enzalutamide resistance can be explained by inhibition of SLC22A3 expression.

2-9. Inhibition of MDVR proliferation by YAP1/TAZ inhibitors

To investigate the effect of YAP1/TAZ inhibitors on colony formation and SLC22A3 expression in MDVR, the YAP1/TAZ inhibitor verteporfin (VER) was used. MDVR cells were treated with only 5 μM VER or 20 μM ENZ for 36 hours, or treated with VER (5 μM) and ENZ (20 μM) together, and the cells were crushed together with a control or used for colony generation experiments. As a result, 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). In addition, verteporfin reduced the colony forming ability of MDVR (FIG. 8C).

In addition, as a result of treating MDVR transfected with the control vector or SLC22A3 vector with Enzalutamide, metformin (MET), which has known anticancer efficacy, or both, MET alone or the combination of MET and ENZ slightly reduced the colony-forming ability of MDVR. In MDVR expressing SLC22A3, colony formation by ENZ or MET treatment alone was reduced by 22% and 40%, respectively, compared to the vector-treated control group. And the combination of ENZ and MET markedly reduced colony formation by ectopic expression of SLC22A3 in MDVR (Fig. 8D). These results suggest that overexpression of SLC22A3 promotes metformin uptake and synergistically inhibits MDVR proliferation in the presence of ENZ. Taken together, treatment with metformin can restore SLC22A3 expression as a novel therapeutic strategy to overcome enzalutamide resistance in CRPC.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (7)

Biomarker AR-V7 in castration-resistant prostate cancer cells or tissues containing them; And an agent for measuring the protein expression level of SLC22A3 or YAP1 / TAZ complex,
The YAP1 / TAZ complex is measured in the nucleus or nucleic acid fraction of cancer cells.
A composition for predicting enzalutamide resistance in castration-resistant prostate cancer patients. The method of claim 1,
The agent is a composition comprising at least one selected from the group consisting of antibodies, aptamers, oligopeptides and PNAs that bind specifically to proteins of the biomarker. A kit for predicting enzalutamide resistance in castration-resistant prostate cancer patients comprising the composition of claim 1 or 2. The method of claim 3,
The kit is a kit using an immunoassay. a) biomarker AR-V7 in prostate cancer cells or tissues containing castration-resistant prostate cancer patients; and measuring the protein expression level of SLC22A3 or YAP1/TAZ complex; and
b) comparing the protein expression level of the measured biomarker with a control group;
The YAP1 / TAZ complex is measured in the nucleus or nucleic acid fraction of cancer cells.
A method for providing information to predict enzalutamide resistance in castration-resistant prostate cancer patients. The method of claim 5,
The biomarker in step a) is one in which the protein expression level is measured by at least one method selected from the group consisting of ELISA, Western blot, radioimmunoassay, immunodiffusion, immunoprecipitation, immunohistochemistry, immunofluorescence, and protein microarray. way of being. The method of claim 5,
Step b) is performed when the protein expression level of the measured biomarker AR-V7 is higher than that of the control group and the protein expression level of SLC22A3 is lower than that of the control group or the protein expression level of the YAP1/TAZ complex is higher than that of the control group, Enzalutamide A method of determining that a person has resistance.

Images