Expression Of Androgen Receptor Variants Research Articles

Caffeic acid phenethyl ester suppresses the expression of androgen receptor variant 7 via inhibition of CDK1 and AKT.

Androgen receptor (AR) splice variant 7 (AR-V7) is capable to enter nucleus and activate downstream signaling without ligand. AR-V7 assists the tumor growth, cancer metastasis, cancer stemness, and the evolvement of therapy-resistant prostate cancer (PCa). We discovered that caffeic acid phenethyl ester (CAPE) can repress the expression and downstream signaling of AR-V7 in PCa cells. CAPE blocked the gene transcription, nuclear localization, and protein abundance of AR-V7. CAPE inhibited the expression of U2AF65, SF2 and hnRNPF, which were splicing factors for AR-V7 intron. Additionally, CAPE decreased protein stability of AR-V7 and enhanced the proteosome-degradation of AR-V7. We observed that CDK1 and AKT regulated the expression and stability of AR-V7 via phosphorylation of Ser81 and Ser213, respectively. CAPE decreased the expression of CDK1 and AKT. Overexpression of CDK1 restored the abundance of AR-V7 in CAPE-treated PCa cells. Overexpression of AR-V7, AKT or CDK1 rescued the proliferation of PCa cells under CAPE treatment. Intraperitoneal injection of 10 mg/kg CAPE retarded the growth of 22Rv1 xenografts in nude mice and suppressed the protein levels of AR-V7, CDK1 and AKT in 22Rv1 xenografts. Our study provided the rationale of applying CAPE for inhibition of AR-V7 in prostate tumors.

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest

BackgroundTherapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported.MethodsTo characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay.ResultsAR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA.ConclusionVTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

OR26-1 Alternative Polyadenylation as a Therapeutic Vulnerability in Prostate Cancer

Abstract Prostate cancer is the second leading cause of male cancer death in the United States. While localized disease can be cured by radiation or surgery, metastatic prostate cancer presents a clinical challenge. Metastatic prostate cancer can initially be controlled by endocrine therapies that target the androgen receptor (AR), however, these tumors will inevitably develop resistance. This stage of the disease, termed castration-resistant prostate cancer (CRPC), is responsible for the majority of prostate cancer-specific deaths. Truncated AR variant (AR-V) proteins are broadly enriched in CRPC cell lines and clinical samples, and can function as ligand-independent, constitutively active transcription factors. We found that blocking an alternative poly(A) site located in AR intron 3 reduced expression of multiple AR-V mRNA and protein species and increased expression of full-length (FL) AR mRNA and protein in 22Rv1 and LNCaP95 CRPC cells. We found the cleavage and polyadenylation specificity factor (CPSF) component, CPSF1, regulates selection of this alternative poly(A) site based on the finding that knockdown of CPSF1 also reduced expression of AR-Vs and increased expression of FL-AR in 22Rv1 and LNCaP95 cells. Further, knockdown of CPSF1 inhibited growth of these CRPC cell lines, as well as androgen-dependent LNCaP cells. To define how CPSF1 regulates alternative polyadenylation of AR and to determine novel pathways that are manipulated by CPSF1 in prostate cancer, we identified CPSF1-dependent gene expression using RNA-seq and CPSF1-dependent poly(A) site usage using poly(A)-ClickSeq (PAC-Seq). Gene set enrichment analysis of RNA-seq data revealed the Glycolysis Hallmark gene was positively regulated by CPSF1, and nominated multiple differentially-expressed genes encoding regulators of glycolysis as CPSF1 targets. These results highlight AR independent pathways that are also regulated by CPSF1 in prostate cancer. We are currently integrating RNA-Seq and PAC-seq datasets to define novel CPSF1 regulated pathways that can be targeted therapeutically in prostate cancer. This research will determine trans-acting factors and cis-regulatory elements that drive alternative polyadenylation of AR mRNA transcripts to promote CRPC progression. This research will also identify AR independent targets of CPSF1 that could be exploited for development of new therapies for CRPC. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

PD07-02 TARGETING WNT5A/FZD2 SIGNALING OVERCOMES RESISTANCE TO ENZALUTAMIDE IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022PD07-02 TARGETING WNT5A/FZD2 SIGNALING OVERCOMES RESISTANCE TO ENZALUTAMIDE IN ADVANCED PROSTATE CANCER Shu Ning, Chengfei Liu, Wei Lou, Alan Lombard, Leandro D'Abronzo, Aiming Yu, Christopher Evans, and Allen Gao Shu NingShu Ning More articles by this author , Chengfei LiuChengfei Liu More articles by this author , Wei LouWei Lou More articles by this author , Alan LombardAlan Lombard More articles by this author , Leandro D'AbronzoLeandro D'Abronzo More articles by this author , Aiming YuAiming Yu More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration resistant prostate cancer (CRPC). Unfortunately, drug resistance occurs frequently due to mechanisms that are not completely understood. Wnt5a, a representative ligand of non-canonical Wnt signaling, is expressed in circulating tumor cells from CRPC patients treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt receptor in prostate cancer. Here we determine the contribution of non-canonical Wnt5a/FZD2 to enzalutamide treatment resistance, and explore the potential of targeting Wnt5a/FZD2 to overcome antiandrogen resistance in castration resistant prostate cancer. METHODS: Wnt5a/FZD2 expression was examined in enzalutamide resistant C4-2B MDVR cells. Wnt5a and FZD2 expression were modulated using specific siRNAs. Cell growth, colony formation, migration and invasion were determined in vitro. RNA sequencing was analyzed on C4-2B MDVR cells with WNT5a/FZD2 knocked down; gene expression of non-canonical Wnt signaling, AR and AR-V7 signature genes were analyzed. A novel RNA bioengineered Wnt5a siRNA (tRNA-siWnt5A) was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth and sensitivity to enzalutamide was evaluated in vitro and in vivo. RESULTS: Wnt5a and FZD2 are highly co-upregulated in CRPC patients and enzalutamide resistant C4-2B MDVR cells compared to parental C4-2B cells. Knocking down Wnt5a and FZD2 abrogates the increase of full-length AR and AR variant expression and diminishes the enrichment of genes involved in the non-canonical Wnt signaling pathway. Blocking Wnt5a and FZD2 using specific siRNAs suppresses prostate cancer cell growth, colony formation, migration and invasion. Wnt5a and FZD2 knockdown with siRNA resensitized C4-2B MDVR cells to enzalutamide treatment. Targeting Wnt5a using the bioengineered tRNA-siWnt5A inhibited the growth of enzalutamide resistant prostate cancer cells and resensitized cells to enzalutamide in vitro, and resistant CRPC LuCaP35CR PDX tumor growth in vivo. CONCLUSIONS: Our studies suggest that Wnt5a/FZD2 confers enzalutamide resistance and prostate cancer survival and proliferation. Targeting the non-canonical Wnt5a/FZD2 pathway suppresses tumors expressing high level of Wnt5a and FZD2, not only overcoming resistance but potentiating anti-tumor effects of enzalutamide in CRPC patients. Source of Funding: CA250082, CA 225836, DOD PC180180 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e98 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shu Ning More articles by this author Chengfei Liu More articles by this author Wei Lou More articles by this author Alan Lombard More articles by this author Leandro D'Abronzo More articles by this author Aiming Yu More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement PDF DownloadLoading ...

Urinary exosome-based androgen receptor-variant 7 detection in metastatic castration-resistant prostate cancer patients.

BackgroundAndrogen receptor variant 7 (AR-V7) detection provides important information for the clinical management of abiraterone in metastatic castration-resistant prostate cancer (mCRPC). We performed a non-invasive urine-derived exosomal AR-V7 analysis of mCRPC patients.MethodsA total of 34 mCRPC patients were recruited including 16 patients treated with abiraterone (ABI) with stable prostate-specific antigen (PSA)/radiograph response (the ABI-Sta group) and 18 were resistant to abiraterone (the ABI-Res group). Urine was collected from patients and healthy control patients for the analysis. Exosomal ribonucleic acid was isolated from urine. Urinary exosome-based androgen receptor-variant 7 was detected by quantitative real-time polymerase chain reaction assay. Characteristics of patients and survival data were collected. The correlation between AR-V7 expression and the therapeutic effect/survival outcomes of abiraterone was analyzed.ResultsUrine is the ideal biological sample for exosome separation and AR full-length analysis. Positive urine-derived exosomal AR-V7 was detected in 32.4% (11 of 34) of the mCRPC patients’ urine samples. Positive AR-V7 was more common in the ABI-Res patients than the ABI-Sta patients (50.0% vs. 12.5%, respectively; P=0.009), and was associated with a higher PSA progression rate and poorer overall survival (OS) (P=0.0031, and P=0.0012, respectively).ConclusionsThe present study showed that the detection of urine-derived exosomal AR-V7 provides a sensitive and feasible clinical workflow. The predicting role of urine-derived exosomal AR-V7 in mCRPC patients should be further verified using studies with greater sample sizes.

Differential Expression of the Androgen Receptor, Splice Variants and Relaxin 2 in Renal Cancer

Background: The role of the androgen receptor (AR) in renal cell carcinoma (RCC) is unclear. We aimed to analyze the expression of AR and its splice variants (SVs) and their correlation with relaxin 2 (RLN2) and cytokines in RCC. Methods: We investigated the expression of RLN2 and AR variants in 25 clear cell RCC (ccRCC) and 9 papillary (pRCC) tumor tissues and the corresponding controls using quantitative PCR and serum RLN2, testosterone and cytokine levels in matched samples using ELISA and chemiluminescent immunometric assay, respectively. Results: ccRCC tissues but not pRCC tissues more frequently expressed AR and the SVs than did normal tissues. All pRCC samples expressed more AR than did ccRCC samples. The highest expression of all AR variants except AR-V12 was found in low-stage tumors, with dominant expression of AR-V7. In males in the ccRCC cohort, the expression of AR-FL, AR-V1 and AR-V3 was significantly correlated with that of RLN2. The secretion pattern of proinflammatory IL-6 was higher in ccRCC than in pRCC. Conclusions: The results highlight additional molecular differences between ccRCC and pRCC, suggesting the influence of external factors on the whole kidney or genetic predispositions to developing certain types of renal cancer, and may support further pathological analysis and studies of targeted hormone therapy.

Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage

Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for treatment customization. However, the absence of a uniform CTC isolation platform and consensus on an analytical assay has prevented the incorporation of these measurements in routine clinical practice. Here, we present a single-CTC sensitive digital droplet PCR (ddPCR) assay for the quantitation of the two most common AR-Vs, AR-V7, and AR-v567es, using antigen agnostic CTC enrichment. In a cohort of 29 mCRPC patients, we identify AR-V7 in 66% and AR-v567es in 52% of patients. These results are corroborated using another gene expression platform (NanoStringTM) and by analysis of RNA-Seq data from patients with mCRPC (SU2C- PCF Dream Team). We next quantify AR-V expression in matching EpCAM-positive vs EpCAM-negative CTCs, as EpCAM-based CTC enrichment is commonly used. We identify lower AR-V prevalence in the EpCAM-positive fraction, suggesting that EpCAM-based CTC enrichment likely underestimates AR-V prevalence. Lastly, using single CTC analysis we identify enrichment for AR-v567es in patients with neuroendocrine prostate cancer (NEPC) indicating that AR-v567es may be involved in lineage plasticity, which warrants further mechanistic interrogation.

TROP-2 co-expression with androgen receptor splice variants as a new therapeutic target in prostate cancer.

5060 Background: Tumor-associated calcium signal transducer 2 (TROP-2, TACSTD2) is a transmembrane glycoprotein that is highly expressed in many epithelial cancers. Overexpression of TROP-2 is postulated to mediate cancer cell growth, invasion, and is associated with more aggressive disease. TROP-2 has emerged as a therapeutic target for antibody-drug conjugates in clinical trials including sacituzumab govitecan and DS-1062. Here, we evaluated the expression of TROP-2 in tumor biopsies and circulating tumor cells (CTCs) in men with metastatic castration resistant prostate cancer (mCRPC) to evaluate TROP-2 as a clinically relevant target. Methods: RNA-seq data from the SU2C-PCF database and PROMOTE clinical trial (NCT#01953640) was assessed for TACSTD2 and androgen receptor (AR) splice variant ( AR_V7/AR_V9) expression. Prostate cancer ChIP-seq data was analyzed to identify binding of the AR to the TROP-2 promoter. EpCAM and TROP-2 captured CTCs were isolated from patients with mCRPC using the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform and assessed for splice variant, neuroendocrine (NE), and AR-regulated gene signatures, in addition to CTC enumeration and TROP-2 protein expression. Results: TROP-2 expression was detectable in 90% of patients, in both bone and visceral metastatic biopsies (SUC2-PCF). Although TROP-2 low biopsies were infrequent (10%), 58% of these samples showed high levels of NE markers, as compared with 5% in all other patients. In the PROMOTE study, elevated TROP-2 gene expression was significantly higher in biopsies with high AR_V7 expression than in those with low (p = 0.04) or negative (p <.01) AR_V7 expression. ChIP-seq data demonstrated binding of AR at the TROP-2 promoter as well as at a potential enhancer site upstream, suggesting that TROP-2 expression can be regulated by AR activity. Splice variants and NE gene signatures were expressed in CTCs captured with both EpCAM and TROP-2, although markedly different gene expression profiles between EpCAM and TROP-2 CTCs were observed in a subset of patients with neuroendocrine prostate cancer. Detection of AR_V7 from TROP-2 CTCs corresponded to shorter overall survival in 20 patients with mCRPC. TROP-2 protein expression was identified on EpCAM captured CTCs, although patients exhibited a wide degree of both intra- and inter-patient heterogeneity. Conclusions: Our findings demonstrate that TROP-2 is highly expressed in mCRPC, and is reduced in a subset of patient tumors expressing neuroendocrine markers. In the PROMOTE clinical trial with abiraterone acetate, TROP-2 AR variant expression correlated with increased TROP-2 expression. Binding of the AR to the TROP-2 promoter and potential enhancer was observed in prostate cancer cell lines and biopsies. These results indicate TROP-2 is a high value a biomarker and therapeutic target mCRPC.

PRPF6 promotes androgen receptor/androgen receptor-variant 7 actions in castration-resistant prostate cancer cells.

Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to cis-regulatory elements in AR target genes and associates with JMJD1A to enhance AR-induced transactivation. PRPF6 also promotes expression of AR-FL and AR-V7. Moreover, PRPF6 depletion reduces tumor growth in prostate cancer-derived cell lines and results in significant suppression of xenograft tumors even under castration condition in mouse model. Furthermore, PRPF6 is obviously highly expressed in human prostate cancer samples. Collectively, our results suggest PRPF6 is involved in enhancement of oncogenic AR signaling, which support a previously unknown role of PRPF6 during progression of prostate cancer and castration-resistant prostate cancers.

Expression of AR-V7 (Androgen Receptor Variant 7) Protein in Granular Cytoplasmic Structures Is an Independent Prognostic Factor in Prostate Cancer Patients.

Simple SummaryThe expression of the androgen receptor (AR) and its splice variant AR-V7 is crucial for prostate cancer (PCa) biology. An immunohistochemical staining was performed on a tissue microarray with specimens from 410 PCa patients. AR staining, neither in the nucleus nor in the cytoplasm was associated with prognosis. AR-V7 staining of the general cytoplasm was associated with a shorter relapse free survival (RFS), whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. Further subgroup stratification for AR-V7 granular staining revealed it as an independent prognostic factor in younger patients (age ≤ 65), patients with negative CK20 staining and patients with perineural invasion. Altogether, AR-V7 protein detected in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox’s regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox’s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

Identification of long non-coding RNAs in advanced prostate cancer associated with androgen receptor splicing factors

The molecular and cellular mechanisms of development of castration-resistant prostate cancer (CRPC) remain elusive. Here, we analyzed the comprehensive and unbiased expression profiles of both protein-coding and long non-coding RNAs (lncRNAs) using RNA-sequencing to reveal the clinically relevant molecular signatures in CRPC tissues. For protein-coding genes upregulated in CRPC, we found that mitochondria-associated pathway, androgen receptor (AR), and spliceosome associated genes were enriched. Moreover, we discovered AR-regulated lncRNAs, CRPC-Lncs, that are highly expressed in CRPC tissues. Notably, silencing of two lncRNAs (CRPC-Lnc #6: PRKAG2-AS1 and #9: HOXC-AS1) alleviated CRPC tumor growth, showing repression of AR and AR variant expression. Mechanistically, subcellular localization of the splicing factor, U2AF2, with an essential role in AR splicing machinery was modulated dependent on the expression level of CRPC-Lnc #6. Thus, our investigation highlights a cluster of lncRNAs which could serve as AR regulators as well as potential biomarkers in CRPC.

Androgen receptor variants: RNA-based mechanisms and therapeutic targets.

Prostate cancer is the second leading cause of male cancer death in the United States. The androgen receptor (AR) transcription factor is a master regulator of normal glandular homeostasis in the prostate, as well as growth and survival of prostate cancer cells. Therefore, AR-targeted therapies are effective for improving overall survival of patients with advanced prostate cancer that is incurable by surgery or radiation. However, prostate cancer will inevitably progress on AR-targeted therapies to a castration-resistant prostate cancer (CRPC) phenotype that accounts for virtually all prostate cancer-specific death. mRNA transcript variants of the AR gene are expressed in CRPC cells and can be translated to produce AR variant (AR-V) proteins that function as ligand-independent, constitutively active transcription factors. AR-Vs are able to support growth of CRPC cells by promoting expression of AR target genes that are normally suppressed by AR-targeted therapies. Knowledge of mechanisms that govern expression of AR-Vs is incomplete. Studies have shown genomic rearrangements of the AR gene underlie expression of diverse AR-Vs in certain CRPC tumors, but post-transcriptional processes represent a broader regulatory mechanism for expression of AR-Vs in CRPC. This review focuses on alternative splicing, 3' end processing, miRNA-mediated mRNA repression, of AR and AR-V expression and the potential these mechanisms hold as therapeutic targets for CRPC.

Diverse AR Gene Rearrangements Mediate Resistance to Androgen Receptor Inhibitors in Metastatic Prostate Cancer.

Prostate cancer is the second leading cause of male cancer deaths. Castration-resistant prostate cancer (CRPC) is a lethal stage of the disease that emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. The purpose of this study was to identify genomic mechanisms that contribute to the development and progression of CRPC. We used whole-genome and targeted DNA-sequencing approaches to identify mechanisms underlying CRPC in an aggregate cohort of 272 prostate cancer patients. We analyzed structural rearrangements at the genome-wide level and carried out a detailed structural rearrangement analysis of the AR locus. We used genome engineering to perform experimental modeling of AR gene rearrangements and long-read RNA sequencing to analyze effects on expression of AR and truncated AR variants (AR-V). AR was among the most frequently rearranged genes in CRPC tumors. AR gene rearrangements promoted expression of diverse AR-V species. AR gene rearrangements occurring in the context of AR amplification correlated with AR overexpression. Cell lines with experimentally derived AR gene rearrangements displayed high expression of tumor-specific AR-Vs and were resistant to endocrine therapies, including the AR antagonist enzalutamide. AR gene rearrangements are an important mechanism of resistance to endocrine therapies in CRPC.

Abstract P6-03-12: Characterization of anti-androgen resistant androgen receptor-positive triple negative breast cancer cells

Abstract Triple negative breast cancer (TNBC) has a poor prognosis in part due to a lack of targeted therapeutics that have improved outcome for other breast cancer subtypes. Gene expression analysis has identified several subtypes of TNBC, including subtypes that express androgen receptor (AR). However, to date the use of AR inhibitors (ARI) in AR+ TNBC has produced disappointing results, likely due to inherent resistance. Additionally, patients that initially benefit from ARIs ultimately develop acquired resistance with resultant progressive disease. In order to better understand the mechanisms of resistance to ARIs in AR+ TNBC, we developed cell lines with acquired resistance to standard ARI therapy. Methods: Bicalutamide (Bica) and enzalutamide (Enza)-resistant cell lines (Bica-R and Enza-R) were generated via continuous passaging of the wild type MDA-MB- 231 (Wt-231) cells in drug-containing media. Cell viability assays were conducted over a 72-hour period using respective anti androgen drugs at varying concentrations. IC-50 for respective drugs was determined via linear regression on normalized control values. Immuno-histochemical analysis and Western blotting for AR and other proteins were compared between the sensitive and resistant lines. Real time PCR was used to detect AR variants (AR-Vs). Additionally we evaluated the efficacy of a CDK inhibitor abemaciclib (Abe) in the resistant cell lines in comparison to the Wt-231 cells. Results: The ARI-resistant cell lines showed higher IC-50 values compared to the Wt cells. For the Bica-R and Enza-R cells the values were at 729 μM and 496μM with Bica and Enza respectively, whereas the values are at 134 μM and 91μM with Bica and Enza respectively for the Wt-231 cells. The resistant lines also exhibited a different morphology, compared to the sensitive cell lines with a more mesenchymal appearance. Expression of AR-Vs differed between the Wt and resistant AR+ TNBC cells. Confocal microscopy revealed different AR localization in the resistant cells along with changes in cell size: compared to Wt cells, Bica-R cells show more diffuse distribution of AR while still maintaining high nuclear levels; Enza-R cells are 3-4 fold size larger than Wt cells and show predominantly nuclear AR localization. Chromogranin A, a protein known for its protective role in regulating endothelial barrier function against increased production of tumor necrosis factor during cancer progression, was found to be down regulated in the resistant cells compared to the sensitive cells. The CDK inhibitor Abe inhibited the growth of the Bica-R cells (IC50 5.74 μM) and was equally effective in inhibiting growth of Wt cells (IC50 5.4 μM). Enza treatment inhibited growth of the Bica-R cells with IC-50 values of 173 μM compared to 91 μM in Wt cells. Interestingly, synergistic effects on cytotoxicity were observed between Abe and Enza in the Bica-R cells, where combination of Enza and 0.1 μM Abe inhibited the growth of the bicalutamide-resistant cells at even lower Enza concentrations (71 μM) than when used as single agents (173 μM). Conclusion: ARI-resistant AR+ TNBC is distinct from AI-sensitive AR+ TNBC at the protein and genomic level. CDK inhibition appears effective in AR+ TNBC, even in cells with acquired-resistance to ARIs and the combination of CDK inhibition and AR inhibition is currently being evaluated in several ongoing clinical trials for patients with AR+ TNBC. Citation Format: Saswati Bhattacharya, Noah Siegel, Ruth O'Regan. Characterization of anti-androgen resistant androgen receptor-positive triple negative breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-12.

Abstract 334: Proteostasis by HSP70/STUB1 complex regulates androgen receptor variants expression and confers resistance to enzalutamide and abiraterone

Abstract INTRODUCTION AND OBJECTIVES: Proteostasis is a complementary process by which cells control the protein biosynthesis, folding, trafficking and degradation. Evidence suggests that proteomic instability, such as protein misfolding and aggregation plays pivotal roles in cancer cell survival and progression. AR-V7 lacking the ligand binding domain confers resistance to enzalutamide and abiraterone in prostate cancer, targeting AR-V7 is an logical strategy to overcome the resistance. The mechanisms of AR-V7 proteostasis haven’t been fully studied so far. The objectives of this project are to investigate the roles of proteostasis in regulating AR-V7 expression and sensitivity to enzalutamide and abiraterone treatment. METHODS: Expression of HSP70 and STUB1 was determined by qRT-PCR and western blot. Expression of HSP70 and STUB1 was downregulated using specific siRNA. HSP70/STUB1 and AR-V7 interaction was determined by co-immunoprecipitation and dual immunofluorescence. The gene regulating mechanisms underlying the HSP70 inhibition in drug resistant prostate cancer cells was determined by RNA sequencing analyses. The effects of HSP70 inhibition on enzalutamide sensitivity were examined in vitro and in vivo. The correlation between HSP70 and AR-V7 in high Gleason score prostate tumors was determined by qRT-PCR. RESULTS: In the present study, we analyzed enzalutamide and abiraterone resistant prostate cancer cells and found ubiquitin mediated proteolysis pathway was suppressed, and E3 ubiquitin ligases STUB1 is downregulated in enzalutamide and abiraterone resistant prostate cancer cells. STUB1 binds to AR-V7, degrades AR-V7 expression and suppresses its activity. HSP70, the STUB1 binding protein, also binds to AR-V7 and enhances AR-V7 transcriptional activity. Mechanistically, STUB1 disassociates HSP70 from AR-V7 binding and increases AR-V7 degradation. Targeting HSP70 by siRNA or small molecular inhibitors (Apoptozole and Ver155008) significantly suppressed prostate cancer growth and improved enzalutamide and abiraterone treatment through AR-V7 inhibition in vitro and in vivo. Additionally, HSP70 expression is upregulated in mCRPC tumors and correlates with AR-V7 levels in high Gleason score and metastatic prostate tumor specimens. CONCLUSION: Enzalutamide and abiraterone treatment induces the imbalance of AR-V7 proteostasis through the ubiquitin-proteolysis alteration. STUB1/HSP70 complex controls AR and AR variants proteostasis. Targeting HSP70 could be a valuable strategy to overcome the next generation anti-androgen resistance and improve their therapy. Citation Format: Chengfei Liu, Wei Lou, Joy C. Yang, Liangren Liu, Cameron M. Armstrong, Alan P. Lombard, Ruining Zhao, Onika DV Noel, Clifford G. Tepper, Hong-Wu Chen, Marc Dall’Era, Marc Dall’Era, Christopher P. Evans, Allen C. Gao. Proteostasis by HSP70/STUB1 complex regulates androgen receptor variants expression and confers resistance to enzalutamide and abiraterone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 334.

Evolution of androgen receptor variant (ARV) profiles in serial metastatic solid and liquid biopsies in metastatic castrate resistant prostate cancer (mCRPC) treated with abiraterone acetate/ prednisone (AA/P).

e16559 Background: ARVs that develop early during treatment with abiraterone acetate/ prednisone (AA/P) may play a role in treatment resistance. We evaluated metastatic site and whole blood mRNA expression of full-length AR (AR-FL) and ARVs to characterize treatment-emergent ARVs (TE-ARVs) from men with mCRPC pre- and post- 12 weeks of AA/P collected in a prospective clinical trial (NCT#01953640). Methods: RNAseq performed on paired metastatic site biopsy (n = 40) and whole blood (n = 25) passed quality control. Reads were aligned to the GRCh38 reference genome with the spliced-alignment TopHat2 package. AR-FL or ARVs were detected if ≥ 2 splice reads aligned to unique splice junctions for AR-FL, AR-8, AR-45, AR-23, AR-V3, AR-V5V6, AR-V7, AR-V8, AR-V9, AR-V10, AR-V12, AR-V13, and AR-V14, and normalized to splice reads per million (SRPM). Cox proportional hazard regression analysis was performed on AR-FL and AR-Vs with ≥1 SRPM for association with time to treatment change (TTTC). Results: In metastatic site biopsies post-AA/P, the average number of splice reads was 27,376,541 (range 7,753,998 to 62,456,773). The median number of ARVs detected was 2 (range 0-8), with a total of total of 110 ARVs identified (Table). Dynamic shifts in ARV profiles were observed post-AAP, with 41 TE-ARVs identified in 17 unique patients. The most common TE-ARV was AR-8 (n = 8), followed by AR-45 (n = 5), AR-23 (n = 5), and AR-V7 (n = 4). The presence of AR-V7 post-AAP was adversely associated with TTTC (hazard ratio 2.46, p = 0.013). The identification of early TE- ARV was not associated with TTTC. In whole blood samples, post-AA/P detection of AR-FL was low (n = 3) and no ARVs were detected. Conclusions: No specific patterns were observed in ARV profiles obtained in the metastatic biopsies after 12 weeks of treatment with AA/P. Clinical trial information: 01953640. [Table: see text]

Cigarette smoking augments androgen receptor activity and promotes resistance to antiandrogen therapy.

Cigarette smoking is associated with worse outcomes in prostate cancer, whose growth is dependent on androgen receptor (AR) signaling. We aimed to elucidate the biological effect of cigarette smoking on AR signaling and its clinical influence on oncological outcome. Gene expression levels after exposure to tobacco smoke condensate (TSC) were evaluated by quantitative real-time polymerase chain reaction and Western blot analysis in prostate cancer cells. Cellular sensitivities to enzalutamide and docetaxel after TSC exposure were evaluated using a prostate cancer cell proliferation assay. Prognosis was compared between current smokers and nonsmokers when treated with AR-axis-targeting (ARAT) agent enzalutamide and docetaxel. Expression of AR variants as well as prostate-specific antigen was augmented after TSC exposure, which occurred after Akt phosphorylation. These inductions were suppressed by Akt inhibitor LY294002 as well as antioxidant N-acetylcysteine. Consistently, TSC exposure augmented cellular resistance to enzalutamide. In clinical data, cigarette smoking was associated with worse progression-free survival and cancer-specific survival when patients with prostate cancer were treated with ARAT agents but not docetaxel. It was suggested that cigarette smoking leads to detrimental oncological outcome when prostate cancer patients are treated with ARAT agents through induction of aberrant AR signaling. Accordingly, we recommend that patients with advanced prostate cancer should refrain from cigarette smoking.

Androgen receptor variant 7 (AR-V7) in sequencing therapeutic agents for castratrion resistant prostate cancer: A critical review.

Background:androgen receptor variant 7 (AR-V7) has been suggested as potential marker for treatment selection in men with metastatic castration-resistant prostate cancer (mCRPC). The aim of the present review is to critically analyze: frequency of the AR-V7 expression in mCRPC cases—impact of AR-V7 expression on abiraterone, enzalutamide, and taxane therapy.Methods:we searched in the Medline and Cochrane Library database from the literature of the past 10 years. We critically evaluated the level of evidence according to the European Association of Urology (EAU) guidelines.Results:12 clinical trials were selected. The determination of AR-V7 in peripheral blood using circulating tumor cells mRNA seems to be the preferred method. At baseline, the mean percentage of cases with AR-V7 positivity was 18.3% (range 17.8%–28.8%). All data on mCRPC submitted to enzalutamide or abiraterone reported a significantly (P <.05) lower clinical progression-free survival (CPFS) and overall survival (OS) in AR-V7+ than AR-V7− cases (CPFS hazard ratio [HR]: 2.3; 95% CI 1.1–4.9; OS HR: 3.0; 95% CI 1.4–6.3). In mCRPC cases submitted to chemotherapies data are not homogeneous and some studies showed no association between CPFS or OS and AR-V7 status (OS HR 1.6; 95% CI 0.6–4.4; P = .40).Conclusions:the suggestion is that taxane therapy is more efficacious than abiraterone or enzalutamide for men with AR-V7+ CRPC. On the contrary, clinical outcomes did not seem to differ significantly on the basis of the type of therapy used among AR-V7− cases.

Differential expression of androgen receptor variants in hormone-sensitive prostate cancer xenografts, castration-resistant sublines, and patient specimens according to the treatment sequence.

Androgen receptor variants (AR-vs), especially AR-v7 and AR-v 5, 6, and 7 exon-skipped (AR-v567es), are reportedly key players in the development of castration-resistant prostate cancer (CRPC). We previously established a mouse xenograft model (JDCaP) from a metastatic skin lesion from a Japanese patient with CRPC and that was revealed to exhibit androgen sensitivity. In the present study, we established multiple castration-resistant xenograft models from JDCaP mice to investigate the biological features of CRPC. Tissue from JDCaP mice was transplanted into male and female nude mice, and after serial passaging, castration-resistant sublines (JDCaP-CR2M and JDCaP-CR4M in male mice, JDCaP-CR2F and JDCaP-CR4F in female mice) were established. We investigated anti-androgen and testosterone sensitivity and the messenger RNA expression pattern of full-length AR and AR-vs. In addition, we compared AR protein levels of patient specimens among primary, local-recurrent, and two skin-metastatic tumors. All JDCaP-CR sublines showed continuous growth following the administration of bicalutamide, although the effects of testosterone varied among sublines. Parental JDCaP and JDCaP-CR2M, JDCaP-CR4M, and JDCaP-CR4F sublines expressed AR-v7, whereas JDCaP-CR2F exhibited elevated AR-v567es expression resulting from genomic deletion, which was confirmed by DNA sequencing. Moreover, we confirmed AR-v7 expression in the tumor of the original patient after androgen-deprivation therapy. Each JDCaP-CR subline showed different AR-v-expression patterns, with JDCaP-CR2F expressing AR-v567es due to genomic deletion. Our results indicated that AR-vs emerged after androgen-deprivation therapy and appeared essential for acquisition of castration resistance.

MiR-103a-2-5p/miR-30c-1-3p inhibits the progression of prostate cancer resistance to androgen ablation therapy via targeting androgen receptor variant 7.

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly used to treat advanced prostate cancer. However, the acquisition of androgen ablation therapy resistance remains a challenge. Recently, androgen receptor splicing variants lacking the ligand-binding domain have been reported to play a critical role in the acquisition of androgen ablation therapy resistance. In the present study, we revealed that the messenger RNA expression and the protein levels of an androgen receptor variant 7 (AR-V7) were higher in prostate cancer tissue samples and in the AR-positive prostate cancer cell line, VCaP. In contrast, microRNA (miR)-30c-1-3p/miR-103a-2-5p expression was significantly downregulated in tumor tissues and cells. miR-30c-1-3p/miR-103a-2-5p overexpression could inhibit AR-V7 expression, suppress VCaP cell growth, and inhibit AR-V7 downstream factor expression by directly targeting the 3'-untranslated region of AR-V7. Under enzalutamide (Enza) treatment, the effects of AR-V7 overexpression were the opposite of those of miR-103a-2-5p/miR-30c-1-3p overexpression; more importantly, the effects of miR-103a-2-5p/miR-30c-1-3p overexpression could be significantly reversed by AR-V7 overexpression under Enza. In summary, we demonstrated a novel mechanism of the miR-30c-1-3p/miR-103a-2-5p/AR-V7 axis modulating the cell proliferation of AR-positive prostate cancer cells via AR downstream targets. The clinical application of miR-30c-1-3p/miR-103a-2-5p needs further in vivo validation.