Mechanisms Of Castration-resistant Prostate Cancer Research Articles

Abstract A029: Whole genome and transcriptome analysis of paired metastatic biopsies identified resistance mechanisms in castration-resistant prostate cancer

Abstract Background: Androgen receptor signaling inhibitors (ARSIs), including abiraterone and enzalutamide, are widely used to treat prostate cancer. However, resistance inevitably develops, and subsequent treatment options remain limited. Prior studies of resistant tumors identified genetic mechanisms of ARSI resistance converging on AR, including its gene and enhancer amplifications, as well as mutations and structural variants involving the ligand-binding domain. Additionally, a phenotypic switch from AR-driven adenocarcinoma to AR-independent neuroendocrine carcinoma is well recognized and thought to be mediated by epigenetic reprogramming. Genomic analysis of paired metastatic biopsies before and after ARSIs offers a unique opportunity to systematically define resistance mechanisms, but such data are limited, in part due to difficulty obtaining serial biopsies. Methods: We obtained paired metastatic biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) before the initiation of an ARSI and at disease progression while on the ARSI. We performed whole-genome sequencing (WGS) and RNA-seq on these samples and conducted bioinformatic and statistical analyses contrasting pairs pre- vs. post-ARSI to search for genetic and transcriptomic alternations associated with ARSI resistance. Results: We generated WGS data for 45 paired biopsies and concurrent RNA-seq data for 35 pairs. WGS analysis identified further copy number gain in the genomic region spanning AR and its known upstream enhancer in multiple patients, driven by tandem duplications, and associated with increased AR expression. Overlaying publicly available chromatin immunoprecipitation sequencing data of transcription factors known to be important in prostate cancer (AR, FOXA1, and HOXB13) and the enhancer marker H3K27ac, we identified putative novel enhancers both upstream and downstream of AR and plan to further assess their effects on AR expression in cell lines. Paired WGS analysis also identified genes with mutations newly arising post-ARSI, yet with only limited recurrence suggesting intertumor heterogeneity. Interestingly, our paired RNA-seq analysis identified SSTR1, whose expression was consistently and significantly decreased in most ARSI-resistant samples. In a separate retrospective analysis of a cohort of 123 patients, we found that low SSTR1 expression was associated with worse overall survival, and patient survival was improved with post-biopsy ARSIs only when SSTR1 expression was high. SSTR1 encodes somatostatin receptor type 1, and its activation by endogenous somatostatin or exogenous somatostatin analogues exerts an anti-proliferative effect in cells expressing SSTR1, consistent with our observation of its downregulation in resistant tumors. Conclusions: Whole genome and transcriptome analysis of paired metastatic biopsies identified amplification of putative novel AR enhancers and decreased SSTR1 expression involved in ARSI resistance that warrant further investigation in additional patients and experimental systems. Citation Format: Xiaolin Zhu, Daniël Vis, Martin Sjöström, Raunak Shrestha, Jeroen Kneppers, Tesa Severson, Yanyun Zhu, Meng Zhang, Arian Lundberg, Thaidy Moreno Rodriguez, Alana Weinstein, Haolong Li, Adam Foye, Rahul Aggarwal, Andries M Bergman, Eric Small, Wilbert Zwart, David Quigley, Michiel van der Heijden, Felix Feng. Whole genome and transcriptome analysis of paired metastatic biopsies identified resistance mechanisms in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A029.

Molecular Mechanisms of Noncoding RNA in the Occurrence of Castration-Resistant Prostate Cancer.

Although several therapeutic options have been shown to improve survival of most patients with prostate cancer, progression to castration-refractory state continues to present challenges in clinics and scientific research. As a highly heterogeneous disease entity, the mechanisms of castration-resistant prostate cancer (CRPC) are complicated and arise from multiple factors. Among them, noncoding RNAs (ncRNAs), the untranslated part of the human transcriptome, are closely related to almost all biological regulation, including tumor metabolisms, epigenetic modifications and immune escape, which has encouraged scientists to investigate their role in CRPC. In clinical practice, ncRNAs, especially miRNAs and lncRNAs, may function as potential biomarkers for diagnosis and prognosis of CRPC. Therefore, understanding the molecular biology of CRPC will help boost a shift in the treatment of CRPC patients. In this review, we summarize the recent findings of miRNAs and lncRNAs, discuss their potential functional mechanisms and highlight their clinical application prospects in CRPC.

Spindle pole body component 25 in the androgen-induced regression of castration-resistant prostate cancer.

BackgroundAndrogen plays a critical role in the development and growth of prostate cancer (PCa) by binding to the androgen receptor, a steroid receptor for testosterone and dihydrotestosterone (DHT). Androgen deprivation therapy, a clinical endocrine therapy, has resulted in increases in the occurrence of castration-resistant prostate cancer (CRPC); however, the mechanisms of CRPC have not yet fully been determined. We previously showed that spindle pole body component 25 (SPC25), a component of the NDC80 complex that is critical in kinetochore formation and chromosome segregation during the cell cycle, plays a critical role in PCa tumorigenesis and cancer stemness. However, it is not yet known whether SPC25 plays a role in CRPC; thus, we sought to address this question in the current study.MethodsSPC25 levels were detected in androgen-insensitive PCa cells using the public database and bioinformatics tools. In vitro, SPC25 levels were determined in androgen-sensitive and androgen-insensitive PCa cells treated with or without DHT. The growth of the PCa cells was assessed by the Cell Counting Kit-8 assay. The invasiveness and migratory potential of the PCa cells were assessed by the transwell cell invasive assay and migratory assay, respectively. Gain-of-function and loss-of-function experiments examined the transfection of androgen-sensitive and androgen-insensitive PCa cells by plasmids carrying small-interfering ribonucleic acids for SPC25 or SPC25, respectively.ResultsSPC25 levels were significantly reduced in the androgen-insensitive PCa cells treated with DHT in the Public database. In vitro, PCa cell growth, invasion, and metastasis was reduced in androgen-insensitive PCa cells but increased in androgen-sensitive PCa cells treated with DHT, partially through DHT-regulated expression of SPC25 at transcriptional but not at translational levels.ConclusionsAndrogen treatment reduces CRPC growth, invasion, and metastasis partially through its regulation of SPC25. SPC25 represents a promising target in the treatment of CRPC.

CYP1B1-catalyzed 4-OHE2 promotes the castration resistance of prostate cancer stem cells by estrogen receptor \u03b1-mediated IL6 activation

BackgroundResistance to androgen deprivation therapy remains a major challenge for the clinical treatment of patients with castration-resistant prostate cancer (CRPC). CYP1B1, a critical enzyme that catalyzes the conversion of estradiol to 4-Hydroxy-17β-estradiol (4-OHE2), has been reported to promote the development and progression of hormone-related cancer, but its role in CRPC is unclear.MethodsTo explore the underlying mechanism which CYP1B1 promotes the prostate cancer stem cells (PCSCs) characteristics, bioinformatics analyses of human clinical prostate cancer (PCa) datasets were performed. CYP1B1, IL6, and estrogen receptor-α (ERα) expression levels were evaluated in PCa and CRPC tissues via immunohistochemistry. The high-performance liquid chromatography-mass spectrometry assay was carried out to examine intracellular 4-OHE2 levels. Serum-free suspension culture and flow cytometry assays were performed to evaluate PCSCs. Chromatin immunoprecipitation was used to validate that 4-OHE2 recruited ERα to the IL6 promoter.ResultsCYP1B1 expression was significantly increased in CRPC tissues and androgen-independent PCa cell lines. CYP1B1+ PCa cells were significantly enriched in bicalutamide-treated LNCaP cells, and CYP1B1 knockdown reduced the cell viability under bicalutamide treatment. In addition, CYP1B1 knockdown decreased the intracellular 4-OHE2 concentration, accompanied by reduced PCSC characteristics. In PCa cells, 4-OHE2 stimulated ERα transcriptional activity and upregulated the expression of IL6 and downstream genes of the IL6-STAT3 signaling. 4-OHE2 increased cell viability under bicalutamide treatment and promoted PCSC characteristics, while IL6 neutralizing antibody reversed these effects. Mechanistically, siERα and the ER antagonist ICI182780 significantly attenuated 4-OHE2-induced IL6 expression, and 4-OHE2 promoted the binding of ERα to the estrogen response element of the IL6 promoter.ConclusionsOur findings indicate that CYP1B1-catalyzed 4-OHE2 enhanced PCSC characteristics and attenuated bicalutamide sensitivity by ERα-mediated the IL6-STAT3 pathway activation. Our study further emphasizes the role of CYP1B1 in castration resistance and illustrates a novel mechanism of CRPC development.Graphical 54dyAth8NatsYXqV4_5FMeVideo .

Genome-wide Expression Analysis Identifies the Association between SEC14L2 and Castration-resistant Prostate Cancer Survival.

Background: Mechanism of castration-resistant prostate cancer (CRPC) is still unclear. Our objective is to investigate the association between genes expression and CRPC through the genome-wide approach and functional researches.Methods: Differentially expressed genes (DEGs) between PCa and CRPC tissues were identified using expression profile obtained from Gene Expression Omnibus database (GEO). Survival analysis was performed using online database Gene Expression Profiling Interactive Analysis (GEPIA). Oncomine database was further used to explore the relationship between DEGs expression levels with clinical parameters. After in silico study, SEC14L2-knockdown CRPC cells and normal prostatic epithelial cells were used for in vitro study to verify its biological functions.Results: A total of 3 consistently changed DEGs (SEC14L2, DMD, SEL1L) were identified correlating with CRPC after cross validation in three independent datasets. Low expression of SEC14L2 was associated with poorer disease-free survival and higher Gleason score than normal/high expression of SEC14L2. SEC14L2 knockdown promoted cell proliferation, migration, invasion as well as cell cycle progression in CRPC cells (all P<0.05) while no significant effects were observed in normal prostatic epithelial cells.Conclusions: Low expression of SEC14L2 was significantly associated with CRPC, and correlated with PCa aggressiveness and poorer prognosis. SEC14L2 might be a potential biomarker or drug target for CRPC.

Exosomes Promote the Transition of Androgen-Dependent Prostate Cancer Cells into Androgen-Independent Manner Through Up-Regulating the Heme Oxygenase-1.

BackgroundCastration-resistant prostate cancer (CRPC) is still considered incurable, even though the mechanisms of CRPC had been extensively researched. Studies have demonstrated that exosomes in the tumor microenvironment contribute to prostate cancer development and progression. However, the role of exosomes in the process of CRPC progression has not yet been determined.MethodsCo-culturing and exosome treatment assays combined with in vitro and in vivo assays were performed to determine the function of exosomes in the transformation of androgen-dependent prostate cancer (ADPC) cells into androgen-independent cells. Then, the mRNA expression profiles of ADPC cells and ADPC cells co-cultured with androgen-independent prostate cancer (AIPC) cell-derived exosomes were studied using microarrays. After silencing the expression of heme oxygenase-1 (HMOX1), Western blotting, quantitative real-time PCR, immunohistochemistry (IHC) studies, and MTS assay were used to confirm the mechanisms of exosome participation in CRPC progression.ResultsThe results showed that ADPC cells acquired tolerance for androgen deprivation due to the exosome-mediated communication between cells. AIPC cell-derived exosomes promoted the transformation of ADPC cells into androgen-independent cells in vivo and in vitro. Microarray analysis revealed that HMOX1 in ADPC cells was up-regulated after treatment with AIPC cell-derived exosomes. Further results showed that HMOX1 is overexpressed in human AIPC specimens and protects ADPC cells from androgen deprivation.ConclusionsOur findings revealed that exosomes contribute to CRPC progression via promoting the transition of prostate cancer cells into an androgen-independent growth stage by activating HMOX1.

Novel functions of the luteinizing hormone/chorionic gonadotropin receptor in prostate cancer cells and patients.

Prostate cancer (PCa) cells become castrate-resistant after initial tumor regression following castration-based lowering of testosterone (T). De-novo intra-tumoral steroid synthesis is a suggested biological mechanism of castration resistant PCa, but the regulators are unknown. Testicular T production is controlled by the luteinizing hormone/choriogonadotropin receptor (LHCGR). To elucidate the influence of LHCGR on PCa development the presence and effects of LHCGR in PCa and whether LHCGR in serum holds prognostic information in PCa patients is investigated. LHCGR expression was investigated by RT-PCR, WB, IHC, qPCR in PCa cell lines and prostatic tissue. Steroid production was measured in media from cell lines with LC-MS/MS and expression of steroidogenic enzymes with qPCR. Serum LHCGR (sLHCGR) was measured with ELISA in PCa patients (N = 157). Presence of LHCGR was established in prostatic tissue and PCa cell lines. Cell proliferation increased by 1.29-fold in LNCaP (P = 0.007) and 1.33-fold in PC-3 cells (P = 0.0007), when stimulated by luteinizing hormone. Choriogonadotropin stimulation decreased proliferation 0.93-fold in DU145 cells (P = 0.05), but none of the treatments altered steroid metabolite secretion. Low sLHCGR concentration was associated with a higher risk of biochemical failure after radical prostatectomy (HR = 3.05, P = 0.06) and castration resistance (HR = 6.92, P = 0.004) compared to high sLHCGR concentration. LHCGR is expressed in PCa and may exert a growth regulatory role in PCa derived cell lines. A potential prognostic role of sLHCGR for determining recurrence risk in PCa patients is found in this pilot study but needs verification in larger cohorts.

Identification of Core Genes and Potential Drugs for Castration-Resistant Prostate Cancer Based on Bioinformatics Analysis.

Prostate cancer (PCa) is a common malignant tumor in elderly men worldwide. Most primary PCas inevitably progress into castration-resistant prostate cancer (CRPC) after androgen deprivation therapy. The mechanisms contributing to this progression are still controversial. In this study, functional module genes, DNA methylations, core regulators, and potential drugs in primary PCa and CRPC were explored by integrating a series of bioinformatics analyses. First, 588 differentially expressed genes (DEGs) were identified. Combined with related genes, protein-protein interaction networks were constructed, and 22 and 14 significant modules were identified in primary PCa and CRPC, respectively. More DEGs were identified in differentially methylated genes in CRPC modules. The hub genes in CRPC included CDC20 and CDK1. Moreover, core noncoding RNAs and transcription factors that significantly regulate CRPC modules were identified, including TUG1, MALAT1, E2F3, and MED1. Finally, the prediction of potential drugs for primary PCa and CRPC was also performed. Exisulind and phosphodiesterase-4 inhibitors were predicted as potential drugs for CRPC. The results of this study provide a new way for biologists and pharmacists to understand the potential molecular mechanisms of CRPC and also provide valuable references for drug redirection and new drug development for PCa.

Integrative Exome Sequencing Analysis in Castration-Resistant Prostate Cancer in Chinese Population.

Castration-resistant Prostate Cancer (CRPC) is a fatal disease with rapid growth. The malignancy usually presents with metastasis and poor prognosis, and causes 100% mortality. Therefore, the treatment of CRPC is extremely challenging, and its pathogenesis need to be elucidated urgently. The high throughput sequencing technology was used to sequence the whole exome associated with CRPC, to explore the molecular mechanism of CRPC, and to find the potential therapeutic targets. We performed whole-exome sequencing of FFPE tissue from 11 Chinese adult male patients. Genomic DNA was fragmented and enriched for whole-exome sequencing using the QiAamp DNA FFPE Tissue KIT, sequenced on an Illumina HiSeq2000 platform, and the relevant genes were analyzed using biological information. Finally, immunohistochemistry method was used to detect the phosphorylation level of LATS1 in CRPC tissues of MST1 mutant and non-mutant patients. We have screened 85 significant mutant genes with relatively high mutation rates of TP53, AR, KMT2, DMAPK1, PIK3R1, SH2B3, WHSC1, KMT2D, MST1 and MAPK1. We first found that MST1 has multiple mutations in CRPC patients, and the MST1 plays an important role in the Hippo pathway. Immunohistochemistry results showed that the phosphorylation level of LATS1 in the mutant patients was significantly lower than that in the non-mutant patients. We speculate that MST1 would be a new potential target for the treatment of CRPC by regulating Hippo signaling pathway. The results provided an important clue to the molecular mechanism of CRPC.

Androgen receptor: what we know and what we expect in castration-resistant prostate cancer

Androgen deprivation therapy is an important therapy for prostate cancer (PCa) in aging men. Under the background of castration, it is inevitable that prostate cancer will develop into castration-resistant prostate cancer (CRPC), which has a high mortality rate, after 2-3years. Androgen receptor (AR) plays a key role in PCa development and is essential to CRPC. More recent research studies have reported that the development of CRPC is largely due to altered mechanisms related to AR, so it is important for us to understand the roles of AR and detailed AR-related mechanisms in CRPC. The multiple AR-related mechanisms promoting the development of CRPC are as follows: (1) enhanced transformation and increased synthesis of intratumoral androgen; (2) AR overexpression, which enables CRPC to be hypersensitive to low levels of androgen; (3) AR cofactors, which enhanced AR transactivation; (4) AR-spliced variants, which mediated downstream gene expression without androgen; (5) the interaction between the AR pathway and classic tumor-related pathways; and» (6) AR mutations, which reduced AR specificity and enhanced AR transcription.

Molecular mechanisms of castration-resistant prostate cancer progressed from prostate cancer by androgen deprivation treatment

Androgen deprivation treatment (ADT) is the gold standard of care for patients with locally advanced or metastatic hormone-sensitive prostate cancer. However, most of these patients have developed to the castration-resistant prostate cancer (CRPC) after months or years through a series of different molecular mechanisms during ADT. So far, CRPC is still incurable and deadly, therefore, it is very important for researchers to study different molecular mechanism in the occurrence and development of CRPC, which may provide some new directions for targeted therapy. Key words: Prostatic neoplasms; Receptors, androgen; Carcinoma, neuroendocrine; Neoplastic stem cells; Transformation

Abstract 503: MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer and third deadliest cancer in American men, yet the only established risk factors are familial genetics, age and race. Men of African Ancestry (moAA) have higher PCa incidence rates when compared to Caucasian men in the United States, which are due to differences in genetic susceptibility variants. Moreover, high mortality rates of PCa are associated with castration-resistant prostate cancer (CRPC) due to maintenance of androgen receptor (AR) signaling in PCa cells following androgen ablation therapy. The 8q24 chromosomal locus is a highly susceptible PCa region that has frequent amplifications of the c-MYC oncogene and downstream PVT1 gene. PVT1 is a long non-protein coding gene that encodes six annotated microRNAs (miRNAs), including microRNA-1205 (miR-1205), yet the function of these miRNAs are poorly understood. To elucidate the role of miR-1205 in PCa, we examined miR-1205 mRNA expression in a cohort of normal (n=22), benign (n=42), and malignant (n=26) histologically confirmed prostatic tissues obtained from prostatectomy or transrectal biopsies of moAA men in Ibadan, Nigeria. One-way ANOVA analysis determined changes in the relative expression of miR-1205 between groups (F(2,87) = 1.153) and a Tukey post hoc test revealed decreased miR-1205 expression in benign (4.61 ± 7.5) and malignant tumors (3.39 ± 3.53) when compared to normal tissues (6.55 ± 9.5). These data suggest that miR-1205 may function as a miRNA tumor suppressor and is characteristic to moAA-associated PCa. To elucidate the molecular mechanism of miR-1205, we examined AR and c-MYC mRNA expression using androgen-dependent LNCaP and CRPC C4-2B and 22RV1 cells. We observed a two-fold decrease of miR-1205 expression and overexpression of AR and c-MYC in C4-2B and 22RV1 cells when compared to LNCaP, suggesting that miR-1205 may regulate AR and c-MYC signaling in CRPC. Next, we identified Fry-like (FRYL) as a putative target using a miSVR computer algorithm and subsequently performed whole transcriptome analysis on prostate tumors and adjacent normal tissue from fourteen PCa patients using the Galaxy web platform. FRYL and AR overexpression was observed in diseased patients suggesting that FRYL may function as an oncogene. Moreover, FRYL was overexpressed in C4-2B and 22RV1 cells when compared to LNCaP, further suggesting a role in CRPC development. C4-2B cells transfected with miR-1205 and the 3'UTR of FRYL in a luciferase expressing vector revealed a significant decrease in luciferase activity when compared to control cells, indicating direct binding of miR-1205 to the 3'UTR of FRYL. These observations strongly suggest that miR-1205 acts as a tumor suppressor that may regulate AR and c-MYC expression, and directly targets the 3'UTR of FRYL in PCa cells. Further understanding the role of miR-1205 regulation of FRYL, AR, and c-MYC signaling may provide novel insights into the molecular mechanisms of CRPC. Citation Format: Michelle K. Naidoo, Dibash K. Das, Adeodat Ilboudo, Akintunde Orunmuyi, Gabriel O. Ogun, S. A. Adebayo, E. O. Olapade-Olaopa, Olorunseun Ogunwobi. MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 503.

Recent advances on the progressive mechanism and therapy in castration-resistant prostate cancer.

BackgroundAlthough there have been great advances in mechanisms and therapeutic methods of prostate cancer, the mortality rate of prostate cancer remains high. The castration-resistant prostate cancer (CRPC), which develops from hormone-sensitive prostate cancer, foreshadows a more dismal outcome. Concomitant with the researches in the mechanism of CRPC and therapy for CRPC, more and more landmark progress has been made in recent years.MethodsA number of clinical and experimental studies were reviewed to indicate the novel advancement in the progressive mechanism and therapy of CRPC.ResultsThe androgen receptor (AR) is still a vital driver in the progression of CRPC, while other multiple mechanisms also contribute to this progression, such as tumor immunity, cancer stem cells, epithelial–mesenchymal transition and DNA repair disorder. In terms of the therapeutic methods of CRPC, chemotherapy with drugs, such as docetaxel, has been the first-line therapy for CRPC for many years. Besides, newer agents, which target some of the above mechanisms, show additional overall survival benefits for CRPC patients. These therapies include drugs targeting the androgen axis pathway (androgen synthesis, androgen receptor splice variants, coactivators of AR and so on), PI3K-AKT pathway, WNT pathway, DNA repair, rearrangement of ETS gene, novel chemotherapy and immunotherapy, bone metastasis therapy and so on. Understanding these novel findings on the mechanisms of CRPC and the latest potential CRPC therapies will direct us for further exploration of CRPC.ConclusionThrough comprehensive consideration, the predominant mechanism of CRPC might be the AR signal axis concomitant with tumor microenvironment, stress, immunity, tumor microenvironment and so on. For CRPC therapy, targeting the AR axis pathway and chemotherapy are the first-line treatments at present. However, with the advancements in CRPC therapy made by the researchers, other novel potential methods will occupy more and more important position in the treatment of CRPC, especially the therapies targeting the tumor microenviroment, tumor immunity and DNA repair and so on.

Research advances in prostate cancer stem cells and radiotherapy resistance of castration-resistant prostate cancer

Prostate cancer tends to progress to castration-resistant prostate cancer within 24 months after castration. For the management of castration-resistant prostate cancer, the biggest challenge is therapeutic resistance which includes radiotherapy resistance. Relevant studies suggest that the radiotherapy resistance of castration-resistant prostate cancer is associated with prostate cancer stem cells and miRNA related to prostate cancer stem cells. This article reviews the research advances in the radiotherapy resistance of castration-resistant prostate cancer in terms of the possible mechanisms of castration-resistant prostate cancer, prostate cancer stem cells, and miRNA related to prostate cancer stem cells. Key words: Castration Resistant Prostate Cancer; Cancer stem cell; Prostate cancer stem cell; Micro-ribonucleic acid

Transcriptomic features of primary prostate cancer and their prognostic relevance to castration-resistant prostate cancer.

Although various mechanisms of castration-resistant prostate cancer (CRPC) have been discovered, reliable biomarkers for monitoring CRPC progression are lacking. We sought to identify molecules that predict the progression of advanced prostate cancer (AdvPC) into CRPC. The study used primary-site samples (N=45 for next-generation sequencing (NGS); N=243 for real-time polymerase chain reaction) from patients with prostate cancer (PC). Five public databases containing microarray data of AdvPC and CRPC samples were analyzed. The NGS data showed that each progression step in PC associated with distinct gene expression profiles. Androgen receptor (AR) associated with tumorigenesis, advanced progression, and progression into CRPC. Analysis of the paired and unpaired AdvPC and CRPC samples in the NGS cohort showed that 15 genes associated with progression into CRPC. This was validated by cohort-1 and public database analyses. Analysis of the third cohort with AdvPC showed that higher serine peptidase inhibitor, Kazal type 1 (SPINK1) and lower Sp8 transcription factor (SP8) expression associated with progression into CRPC (log-rank test, both P<0.05). Multivariate regression analysis showed that higher SPINK1 (Hazard Ratio (HR)=4.506, 95% confidence intervals (CI)=1.175–17.29, P=0.028) and lower SP8 (HR=0.199, 95% CI=0.063–0.632, P=0.006) expression independently predicted progression into CRPC. Gene network analysis showed that CRPC progression may be mediated through the AR-SPINK1 pathway by a HNF1A-based gene network. Taken together, our results suggest thatSPINK1 and SP8 may be useful for classifying patients with AdvPC who have a higher risk of progressing to CRPC.

Direct Metabolic Interrogation of Dihydrotestosterone Biosynthesis from Adrenal Precursors in Primary Prostatectomy Tissues.

Purpose: A major mechanism of castration-resistant prostate cancer (CRPC) involves intratumoral biosynthesis of dihydrotestosterone (DHT) from adrenal precursors. We have previously shown that adrenal-derived androstenedione (AD) is the preferred substrate over testosterone (T) for 5α-reductase expressed in metastatic CRPC, bypassing T as an obligate precursor to DHT. However, the metabolic pathway of adrenal-derived DHT biosynthesis has not been rigorously investigated in the setting of primary disease in the prostate.Experimental Design: Seventeen patients with clinically localized prostate cancer were consented for fresh tissues after radical prostatectomy. Prostate tissues were cultured ex vivo in media spiked with an equimolar mixture of AD and T, and stable isotopic tracing was employed to simultaneously follow the enzymatic conversion of both precursor steroids into nascent metabolites, detected by liquid chromatography-tandem mass spectrometry. CRPC cell line models and xenograft tissues were similarly assayed for comparative analysis. A tritium-labeled steroid radiotracing approach was used to validate our findings.Results: Prostatectomy tissues readily 5α-reduced both T and AD. Furthermore, 5α-reduction of AD was the major directionality of metabolic flux to DHT. However, AD and T were comparably metabolized by 5α-reductase in primary prostate tissues, contrasting the preference exhibited by CRPC in which AD was favored over T. 5α-reductase inhibitors effectively blocked the conversion of AD to DHT.Conclusions: Both AD and T are substrates of 5α-reductase in prostatectomy tissues, resulting in two distinctly nonredundant metabolic pathways to DHT. Furthermore, the transition to CRPC may coincide with a metabolic switch toward AD as the favored substrate. Clin Cancer Res; 23(20); 6351-62. ©2017 AACR.

STAT3 and STAT5A are potential therapeutic targets in castration-resistant prostate cancer.

Mechanisms of castration-resistant prostate cancer (CRPC) are not well understood, thus hindering rational-based drug design. Activation of STAT3/5A, key components of the JAK/STAT pathway, is implicated in aggressive PC, yet their clinical relevance in CRPC remains elusive. Here, we evaluated the possible role of STAT3/5A in CRPC using immunological, quantitative mRNA expression profiling, and pharmacological methods. We observed a strong nuclear immunoreactivity for STAT3 and STAT5A in 93% (n=14/15) and 80% (n=12/15) of CRPC cases, respectively, compared with benign prostatic hyperplasia (BPH). We demonstrated that PC cells express varying levels of STAT3 and STAT5A transcripts. In addition, we demonstrate that pimozide, a psychotropic drug and an indirect inhibitor of STAT5, attenuated PC cells growth, and induced apoptosis in a dose-dependent manner. Furthermore, our analysis of the PC public data revealed that the STAT3/5A genes were frequently amplified in metastatic CRPC. These findings suggest that STAT3/5A potentially serves as a predictive biomarker to evaluate the therapeutic efficacy of a cancer drug targeting the JAK/STAT pathway. Since the JAK/STAT and AR pathways are suggested to be functionally synergistic, inhibition of the JAK/STAT signaling alone or together with AR may lead to a novel treatment modality for patients with advanced PC.

Abstract 3522: Structural characterization of androgen receptor variant 7 in prostate cancer

Abstract Prostate cancer is an extremely common disease that affects one in every seven men in their lifetime. The standard care for late-stage cancer is designed to inhibit the activation of androgen receptor (AR). While this therapeutic approach is initially efficient, the cancer almost always develops resistance and gives rise to castration-resistant prostate cancer (CRPC). When therapy fails, the median survival of patients who suffer from CRPC is 12-16 months. It is critical to understand the mechanism of CRPC better to ameliorate the outcome of late-stage prostate cancer patients. AR is a transcription factor that consists of an N-terminal domain (NTD), a DNA binding domain (DBD) and a ligand binding domain (LBD). Upon activation by androgen, an intramolecular interaction between NTD and LBD occurs. The AR translocates into the nucleus where it forms a homodimer. It binds to the promoter and enhancer elements of the AR target genes and triggers gene transcription. It has been demonstrated in many clinical studies that AR is important for the progression of prostate cancer including CRPC. In CRPC, although androgen production and binding is inhibited, how the AR can still be activated is a fundamental question to be answered. One of the several different mechanisms proposed is the alternative splicing of the AR generating truncated AR variants that lack LBD. These variants do not require androgens to be activated and they are intrinsically resistant to clinically approved therapeutics. Despite the fact that the AR variants have been shown to be critical in late-stage prostate cancer, the mechanism of gene transcription initiation by the AR variants is not well-known. We propose to characterize the mechanism of the AR variant activation. Since ARV7 is the most commonly observed variant, we utilize ARV7. We develop a FRET based methodology to identify how ARV7 dimers form and to test if full-length AR is necessary for transcriptional activation mediated by ARV7. We are able to demonstrate possible intermolecular interaction between the full length AR and ARV7 measuring the FRET intensity between the fluorophores attached to the N and C terminus of two proteins. FRET measurements are analyzed by tracking single cells and recording the FRET intensities during the process. Our preliminary data suggest that the full-length AR and ARV7 may interact in the presence of androgen. ARV7 is always localized in the nucleus whereas the AR requires androgen to be activated and translocates into the nucleus. Being in close proximity, putative interaction between the AR and ARV7 occurs in the nucleus. Additionally, we use NTD and DBD point mutants to determine critical positions of intermolecular interaction between them. Supporting the significance of ARV7 in prostate cancer, our results clarify the dimerization process. It gives an opportunity to test clinical drugs currently in development to effectively inhibit ARV7 activation. Citation Format: Fatma Özgün, Zeynep Kaya, Halil Bayraktar, Selen Manioğlu, Doğancan Özturan, Nathan Lack. Structural characterization of androgen receptor variant 7 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3522. doi:10.1158/1538-7445.AM2017-3522

LncRNA HOXD-AS1 Regulates Proliferation and Chemo-Resistance of Castration-Resistant Prostate Cancer via Recruiting WDR5

Castration-resistant prostate cancer (CRPC) that occurs after the failure of androgen deprivation therapy is the leading cause of deaths in prostate cancer patients. Thus, there is an obvious and urgent need to fully understand the mechanism of CRPC and discover novel therapeutic targets. Long noncoding RNAs (lncRNAs) are crucial regulators in many human cancers, yet their potential roles and molecular mechanisms in CRPC are poorly understood. In this study, we discovered that an lncRNA HOXD-AS1 is highly expressed in CRPC cells and correlated closely with Gleason score, T stage, lymph nodes metastasis, and progression-free survival. Knockdown of HOXD-AS1 inhibited the proliferation and chemo-resistance of CRPC cells invitro and invivo. Furthermore, we identified several cell cycle, chemo-resistance, and castration-resistance-related genes, including PLK1, AURKA, CDC25C, FOXM1, and UBE2C, that were activated transcriptionally by HOXD-AS1. Further investigation revealed that HOXD-AS1 recruited WDR5 to directly regulate the expression of target genes by mediating histone H3 lysine 4 tri-methylation (H3K4me3). In conclusion, our findings indicate that HOXD-AS1 promotes proliferation, castration resistance, and chemo-resistance in prostate cancer by recruiting WDR5. This sheds a new insight into the regulation of CRPC by lncRNA and provides a potential approach for the treatment of CRPC.

Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer.

Estrogens are considered to potentially play some roles in the development and progression of prostate cancer through estrogen receptor beta (ERβ). However, additional factors which could influence the clinical outcome of the patients through modulating these steroid signalings have also been proposed. Among these, increased expression of serotonin receptor especially that of 5-hydroxytryptamine receptor Type 4 (5-HTR4) has been recently proposed to be involved in autocrine/paracrine mechanisms of castration-resistant prostate cancer, but the presence and clinical significance of 5-HTR4 in hormone-naive prostate cancer (HNPC) and its interaction with hormonal signaling pathways have remained virtually unknown. We evaluated the status of 5-HTR4 in 112 human HNPC cases (acinar adenocarcinoma) using immunohistochemistry and correlated the findings with clinicopathological features of individual patients and the status of androgen receptor (AR) and ERβ. To further elucidate its underlying mechanisms, androgen-dependent human prostate carcinoma cell line, LNCaP, expressing 5-HTR4, was treated by 5-HTR4 agonist. 5-HTR4 immunoreactivity was detected in 34% of prostate cancer cases examined (38/112) and was significantly correlated with the status of ERβ but not with that of AR and other clinicopathological factors of the patients. Results of in vitro studies demonstrated that 24 h incubation with 5-HTR4 agonist (10 nM) increased the expression level of ERβ messenger RNA compared to controls. 5-HTR4 agonist (100 nM) significantly inhibited LNCaP carcinoma cell migration (P < 0.05). Results of our present study indicated that 5-HTR4 signaling upregulated ERβ expression in HNPCs and could impact on biological processes in HNPC.