KR20220160452A - Pharmaceutical composition for inhibiting of resistance against enzalutamide of prostate cancer and pharmaceutical composition for treating enzalutamide resistance-prostate cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for suppressing enzalutamide resistance in prostate cancer, and a pharmaceutical composition for treating prostate cancer with enzalutamide resistance. The pharmaceutical composition for suppressing enzalutamide resistance in prostate cancer contains a YAP1/TAZ inhibitor as an active ingredient, thereby sensitizing prostate cancer to enzalutamide to reduce the resistance of prostate cancer to enzalutamide and increase the therapeutic effect of enzalutamide. In addition, enzalutamide can be used to treat prostate cancer in patients with prostate cancer whose sensitivity to enzalutamide has recovered.

Description

Pharmaceutical composition for inhibiting of resistance against enzalutamide of prostate cancer and pharmaceutical composition for treating enzalutamide resistance-prostate cancer}

The present invention relates to a pharmaceutical composition for inhibiting enzalutamide resistance in prostate cancer and a pharmaceutical composition for treating prostate cancer having enzalutamide resistance.

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, it is required to develop a drug that suppresses resistance to enzalutamide so that a therapeutic effect of enzalutamide can be expected by enhancing sensitivity to enzalutamide in prostate cancer having resistance to enzalutamide. Keytruda, currently marketed as an immuno-anticancer drug, entered phase 3 clinical trials in 2020 in the hope of increasing the enzalutamide sensitivity of cells by blocking the PD-1 receptor of enzalutamide-resistant cells. Studies on drugs for inhibiting mead resistance are lacking.

Julie N. Graff, et al., Oncotarget. 2016 Aug 16; 7(33): 52810-52817. Jennifer L. Bishop, et al., Oncotarget. 2015 Jan; 6(1): 234-242.

An object of the present invention is to provide a pharmaceutical composition for suppressing resistance of prostate cancer capable of enhancing the sensitivity of prostate cancer patients to enzalutamide.

In addition, an object of the present invention is to provide a pharmaceutical composition for the treatment of prostate cancer having resistance to enalutamide.

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 discovered for the first time that AR-V7 protein is also involved in this YAP1/TAZ action, thereby activating or expressing YAP1/TAZ. Regarding a pharmaceutical composition for inhibiting enzalutamide resistance in prostate cancer and a pharmaceutical composition for treating prostate cancer having enzalutamide resistance, which confirms that inhibition enhances sensitivity to enzalutamide and contains a YAP1/TAZ inhibitor as an active ingredient completed the present invention.

One aspect of the present invention provides a pharmaceutical composition for inhibiting enzalutamide resistance of prostate cancer, comprising a YAP1/TAZ inhibitor as an active ingredient.

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.

As used in the present invention, "inhibition of resistance to Enzalutamide" refers to a case in which a prostate cancer patient does not die of cancer cells even if he or she takes Enzalutamide and thus does not show a therapeutic effect by Enzalutamide. Resistance or resistance to Enzalutamide ), which means that a therapeutic effect by Enzalutamide can be expected by enhancing the sensitivity to Enzalutamide in order to overcome such resistance to Enzalutamide.

According to one embodiment of the present invention, the YAP1/TAZ inhibitor may include both YAP1 and TAZ expression inhibitors and activity inhibitors.

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 is known to enter the nucleus and regulate gene expression.

More specifically, the YAP1/TAZ inhibitor may be at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, ribozymes, antibodies, antigen-binding fragments thereof, compounds, peptides, peptide mimetics, and aptamers.

According to one embodiment of the present invention, the expression inhibitor of YAP1/TAZ is an antisense oligonucleotide, siRNA, shRNA, and ribonucleotide that complementarily bind to YAP1 and TAZ genes, or the mRNA of a gene promoting the expression of YAP1/TAZ, respectively. It may be one or more selected from the group consisting of zymes.

The antisense oligonucleotide is a DNA or RNA sequence capable of binding to YAP1 and TAZ mRNA, and has essential activities for translation, cytoplasmic translocation, maturation, or all other overall biological functions of YAP1 and TAZ mRNA. can hinder The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. Such antisense oligonucleotides may be modified at one or more bases, sugars or backbone positions to enhance efficacy.

The shRNA (small hairpin RNA or short hairpin RNA) represents an RNA sequence that makes a strong hairpin turn, which can be used to suppress or silence gene expression through RNA interference.

The siRNA (small interference RNA) refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing, and since it can suppress the expression of a target gene, it is provided as an efficient gene knockdown method or gene therapy method. The siRNA molecule may have a double-stranded structure in which the sense strand and the antisense strand are located opposite to each other, or may have a single-stranded structure having self-complementary sense and antisense strands. As used herein, the term complementary is meant to encompass incomplete complementarity as well as 100% complementarity.

The ribozyme refers to RNA having an enzyme function that catalyzes biochemical reactions such as RNA splicing, tRNA synthesis, and protein synthesis, and has a unique secondary structure such as a hairpin structure.

According to one embodiment of the present invention, the YAP1/TAZ activity inhibitor specifically binds to YAP1 and TAZ proteins, respectively, or an antibody, antigen-binding fragment thereof, compound, or peptide that specifically binds to the YAP1/TAZ complex. , It may be one or more selected from the group consisting of peptide mimetics and aptamers.

As used in the present invention, "specifically binding" means that the binding ability to plagiarism material is superior to other materials.

The antibody refers to a specific protein molecule directed against an antigenic site. In the present invention, it means an antibody that specifically binds to YAP1 and TAZ as a target substance, and includes all 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.

Peptide mimetics are small protein molecules with a chain structure designed to imitate peptides, which means that molecular properties such as stability and biological activity are more advantageously controlled by partially modifying the chemical structure of commonly existing peptides. do.

An aptamer is a nucleic acid or peptide capable of binding specifically and friendly to a target substance. In the present invention, it refers to a peptide that binds specifically and strongly to YAP1/TAZ, and has higher specificity and affinity than antibodies has the advantage of having

According to one embodiment of the present invention, the YAP1/TAZ inhibitor may be one or more compounds selected from the group consisting of verteporfin, YAP/TAZ inhibitor-1, YAP/TEAD inhibitor-1, and CA3.

The verteporfin is a benzoporphyrin derivative that is used as a photosensitizer in photodynamic therapy to remove abnormal blood vessels in the eye caused by diseases such as wet macular degeneration. Recently, YAP1 It is known to inhibit the formation of the /TEAD complex.

The YAP/TAZ inhibitor-1 (YAP/TAZ inhibitor-1) serves to inhibit the formation of Tead and YAP/TAZ complexes.

The YAP/TEAD inhibitor 1 (YAP/TEAD inhibitor 1) is also referred to as peptide 17, and serves to inhibit the YAP-TEAD protein-protein interaction.

The CA3 (CIL56) acts to strongly suppress the transcriptional activity of YAP1/TEAD.

According to one embodiment of the present invention, when verteporfin is treated alone in enzalutamide-resistant castration-resistant prostate cancer cell lines, the YAP1/TAZ protein expression is significantly reduced, similar to castration-resistant prostate cancer without drug resistance. It was confirmed that the expression of SLC22A3 appeared. In addition, it was confirmed that the castration-resistant prostate cancer cell line having enzalutamide resistance was restored in sensitivity to enzalutamide by verteporfin, and cell proliferation was inhibited by enzalutamide.

In addition, according to one embodiment of the present invention, when YAP/TAZ inhibitor-1 is treated alone in a castration-resistant prostate cancer cell line having enzalutamide resistance, YAP1/TAZ protein expression is partially reduced, and castration-resistant prostate cancer cell lines having no drug resistance are reduced. As with resistant prostate cancer, it was confirmed that the expression of SLC22A3 appeared, suggesting that sensitivity to ensalutamide would be restored by YAP/TAZ inhibitor-1.

A pharmaceutical composition for inhibiting enzalutamide resistance of prostate cancer containing such a YAP1 inhibitor as an active ingredient inhibits enzalutamide resistance and thereby sensitizes prostate cancer to enzalutamide, thereby reducing resistance of prostate cancer to enzalutamide and enzalutamide. May enhance the effectiveness of lutamide.

The pharmaceutical composition may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions. In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier is commonly used in preparation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, and polyvinylpyrrolidone. , cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

The dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type and degree of response to be achieved by administration of the pharmaceutical composition, the subject of administration Various factors, including the type of subject, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used simultaneously or concurrently with the subject, and other components of the composition, and well known in the medical field It may vary according to similar factors, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment. For example, it may be 0.001 to 1000 mg/kg, 0.01 mg/kg to 100 mg/kg, or 0.1 mg/kg to 10 mg/kg per day, but is not limited thereto.

In addition, the pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans through various routes. The route and method of administration of the pharmaceutical composition may be independent, and are not particularly limited in the method, and may follow any route and method of administration as long as the pharmaceutical composition can reach the target site. . The pharmaceutical composition may be administered orally or parenterally. The parenteral administration method includes, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, etc., and a method of applying, spraying, or inhaling the pharmaceutical composition to a diseased area may also be used. but not limited thereto.

Another aspect of the invention is a YAP1/TAZ inhibitor; And it provides a pharmaceutical composition for the treatment of prostate cancer having enalutamide resistance containing enalutamide.

The pharmaceutical composition for the treatment of prostate cancer having resistance to enzalutamide according to the present invention can exhibit the effect of enzalutamide on prostate cancer treatment by recovering sensitivity to enzalutamide due to the YAP1/TAZ inhibitor.

Since the description of the YAP1/TAZ inhibitor and Enzalutamide is in common with the above description, the description thereof is omitted to avoid excessive complexity.

The pharmaceutical composition for inhibiting resistance of prostate cancer to Enzalutamide according to the present invention includes a YAP1/TAZ inhibitor as an active ingredient to sensitize prostate cancer to Enzalutamide, thereby reducing resistance of prostate cancer to Enzalutamide and It is possible to increase the therapeutic effect of enzalutamide, and furthermore, prostate cancer can be treated using enzalutamide for prostate cancer patients whose sensitivity to enzalutamide has been restored.

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 showed 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
9 shows the regulation of SLC22A3 gene expression by YAP1/TAZ inhibitors, (A) and (B) are YAP1/TAZ inhibitors Y/T in MDVR. This is the result confirmed by the protein and mRNA expression levels of SLC22A3 depending on whether or not the treatment was performed, and (C) is Y/T in, a YAP1/TAZ inhibitor in MDVR. This is the result of confirming the colony formation rate according to the treatment.

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. As YAP/TAZ inhibitors, verteporfin (VER (Sigma-Aldrich) and YAP/TAZ inhibitor-1 (Y/T in.) (MedchemExpress) were used at concentrations of 5 μM and 20 μM, respectively. was used as

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, YAP1/TAZ inhibitors verteporfin (VER) or YAP/TAZ inhibitor-1 (Y/T in.) were used.

In the case of verteporfin treatment, MDVR cells were treated with VER (5 μM), ENZ (20 μM) or both for 36 hours, and protein and nucleic acid expression levels were confirmed from each cell together with control cells, and colony generation experiments were performed. . As a result, verteporfin decreased the protein expression of YAP1/TAZ and increased the protein and transcript expression levels 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.

In the case of YAP/TAZ inhibitor-1 treatment, MDVR cells were Y/T in. (30 μM), ENZ (20 mM) or both of them were treated for 24 hours, and protein and nucleic acid expression levels were confirmed from each cell together with the control. Then, each drug was treated for 10 hours to perform a colony formation experiment. As a result, YAP/TAZ inhibitor-1 decreased the protein expression of YAP1/TAZ and increased the protein and transcript expression levels of SLC22A3 regardless of whether or not Enzalutamide was treated (FIG. 9A and B). In addition, YAP/TAZ inhibitor-1 reduced the colony forming ability of MDVR (FIG. 9C).

Taken together, treatment with YAP1/TAZ inhibitors 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.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Pharmaceutical composition for inhibiting of resistance against enzalutamide of prostate cancer and pharmaceutical composition for treating enzalutamide resistance-prostate cancer <130> BPN210104P1 <150> KR 10-2021-0068227 <151> 2021-05-27 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> CTGF-F <400> 1 ccaatgacaa cgcctcctg 19 <210> 2 <211> 19 <212> DNA <213> artificial sequence <220> <223> CTGF-R <400> 2 tggtgcagcc agaaagctc 19 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> CYR61-F <400> 3 ggtcaaagtt accgggcagt 20 <210> 4 <211> 19 <212> DNA <213> artificial sequence <220> <223> CYR61-R <400> 4 ggaggcatcg aatcccagc 19 <210> 5 <211> 21 <212> DNA <213> artificial sequence <220> <223> ANKRD1-F <400> 5 cacttctagc ccaccctgtg a 21 <210> 6 <211> 21 <212> DNA <213> artificial sequence <220> <223> ANKRD1-R <400> 6 ccacaggttc cgtaatgatt t 21 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> YAP1-F <400> 7 tagccctgcg tagccagtta 20 <210> 8 <211> 22 <212> DNA <213> artificial sequence <220> <223> YAP1-R <400> 8 tcatgcttag tccactgtct gt 22 <210> 9 <211> 21 <212> DNA <213> artificial sequence <220> <223> TAZ-F <400> 9 gatcctgccg gagtctttct t 21 <210> 10 <211> 19 <212> DNA <213> artificial sequence <220> <223> TAZ-R <400> 10 cacgtcgtag gactgctgg 19 <210> 11 <211> 21 <212> DNA <213> artificial sequence <220> <223> SLC22A3-F <400> 11 atcgtcagcg agtttgacct t 21 <210> 12 <211> 21 <212> DNA <213> artificial sequence <220> <223> SLC22A3-R <400> 12 acctgtctgc tgcatagcct a 21 <210> 13 <211> 21 <212> DNA <213> artificial sequence <220> <223> DNMT1-F <400> 13 aggcggctca aagatttgga a 21 <210> 14 <211> 23 <212> DNA <213> artificial sequence <220> <223> DNMT1-R <400> 14 gcagaaattc gtgcaagaga ttc 23 <210> 15 <211> 20 <212> DNA <213> artificial sequence <220> <223> Cyclin A1-F <400> 15 gaggtcccga tgcttgtcag 20 <210> 16 <211> 21 <212> DNA <213> artificial sequence <220> <223> Cyclin A1-R <400> 16 gttagcagcc ctagcactgt c 21 <210> 17 <211> 20 <212> DNA <213> artificial sequence <220> <223> Cyclin D1-F <400> 17 gctgcgaagt ggaaaccatc 20 <210> 18 <211> 22 <212> DNA <213> artificial sequence <220> <223> Cyclin D1-R <400> 18 cctccttctg cacacatttg aa 22 <210> 19 <211> 22 <212> DNA <213> artificial sequence <220> <223> GSTP1-F <400> 19 ccctacaccg tggtctattt cc 22 <210> 20 <211> 19 <212> DNA <213> artificial sequence <220> <223> GSTP1-R <400> 20 caggaggctt tgagtgagc 19 <210> 21 <211> 21 <212> DNA <213> artificial sequence <220> <223> A-F <400> 21 ctagcactta tgtttcaagg c 21 <210> 22 <211> 21 <212> DNA <213> artificial sequence <220> <223> A-R <400> 22 gtgctcctaa catatctaag a 21 <210> 23 <211> 21 <212> DNA <213> artificial sequence <220> <223> B-F <400> 23 tggagagccc tgtcgaaata g 21 <210> 24 <211> 21 <212> DNA <213> artificial sequence <220> <223> B-R <400> 24 tgtgtcatct tctgccctcc t 21

Claims (5)

A pharmaceutical composition for inhibiting resistance to enzalutamide in prostate cancer, comprising a YAP1/TAZ inhibitor as an active ingredient. The method of claim 1,
The pharmaceutical composition of which the prostate cancer is castration-resistant prostate cancer. The method of claim 1,
The YAP1/TAZ inhibitor is a group consisting of antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), ribozymes, antibodies, antigen-binding fragments thereof, compounds, peptides, peptide mimetics and aptamers A pharmaceutical composition that is one or more selected from. The method of claim 1,
The pharmaceutical composition of claim 1 , wherein the YAP1/TAZ inhibitor is at least one compound selected from the group consisting of verteporfin, YAP/TAZ inhibitor-1, YAP/TEAD inhibitor-1, and CA3. YAP1/TAZ inhibitors; And Enzalutamide-resistant pharmaceutical composition for the treatment of prostate cancer comprising Enzalutamide.

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