Castration-resistant Prostate Cancer Specimens Research Articles

Abstract 2266: Detection of androgen receptor splice variants from clinical sequencing

Abstract Background: Androgen receptor splice variant-7 (AR-v7) has been widely studied as a biomarker of resistance to AR-targeted therapy in castration-resistant prostate cancer (CRPC). Various methods have been used to detect AR-v7 in CRPC specimens, including next generation sequencing (NGS) technology. However, NGS technologies applied in clinical sequencing have focused on using blood or fresh-frozen CRPC tissue specimens. There is not yet an AR-v7 detection system tailored to formalin-fixed paraffin-embedded (FFPE) tissue specimens commonly used for clinical sequencing in hospitals. Methods: In this respect, we propose a novel approach to identify AR-v7 in targeted RNA sequencing (RNA-seq) data derived from FFPE specimens. This approach can identify, in addition to AR-v7, all constitutively active AR splice variants (AR-Vs) that are co-expressed with AR-v7. In short, our two-step approach first gathers soft-clipped or divided reads that are adjacent to the splicing sites of AR-Vs and then yields the number of splitting reads that support the existence of AR-Vs. Next, the algorithm selects the paired-end reads whose one side and the other side are mapped to the preceding and following exons, respectively. The final number of spanning reads are calculated following to the removal of low-quality reads. Results: We validated the proposed approach using two large-scale, independent RNA-seq datasets of prostate cancer (PC) samples: 111 samples from Seoul National University Hospital (SNUH) and 558 samples from The Cancer Genome Atlas (TCGA) dataset. The majority of both datasets were localized PC tumors, especially in the SNUH dataset, where localized PC accounted for 91% of the total. Overall, our approach successfully identified samples with putative AR-Vs, including AR-v7, AR-v3, and AR-v9. Statistical analyses of 111 SNUH PC patients suggested potential detection threshold in clinical sequencing settings (at least 200 split reads). In the SNUH dataset, the AR-v7 positive group, which included 5 patients (5%) with values above the threshold, shared the following characteristics: higher AR-v7/full length AR expression levels, AR amplification and co-expression with AR-v3/AR-v9. AR-v7 positivity was experimentally validated by Sanger sequencing. The similar pattern was also found in the analysis of TCGA dataset. Conclusions: These results demonstrate that the detection system can successfully identify AR-v7 positivity not only for metastatic PC but also for patients with localized PC through clinical sequencing. We expect that the further in-depth analyses including larger samples and clinical outcomes can discover clinical applicability of AR-v7. Citation Format: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun. Detection of androgen receptor splice variants from clinical sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2266.

Abstract 5863: Double-negative prostate cancer emerges through a mixed basal, club, and hillock cell identity driven by KLF5

Abstract The purpose of this study was to characterize the phenotypes and drivers of therapy-induced lineage plasticity during the development of castration resistant prostate cancer (CRPC). While the majority of localized prostate cancer can be cured with surgery or radiation, metastatic disease is lethal. Therapies targeting the androgen receptor (AR) are initially effective, but eventually men will develop CRPC. About 70-75% of CRPC displays restored AR signaling (ARPC), while the remaining 25-30% display lineage plasticity evidenced by the emergence of AR-negative subtypes including those that display neuroendocrine (NE) markers, or double negative CRPC (DNPC) lacking both AR and NE markers. AR independent manifestations of CRPC represent a clinical challenge because no effective therapies are available and therapeutic targets are largely unknown. We analyzed publicly available bulk and single-cell RNA-seq datasets derived from clinical CRPC specimens to characterize the cellular identity of AR-negative tumors. Using a database of all known transcriptional regulators, we identified potential drivers of these phenotypes. We performed siRNA-mediated knockdown experiments in a model of DNPC to explore the extent to which the candidate regulators maintain AR-negative cell identity and sustain growth of DNPC prostate cancer cells. We identified a subset of CRPC tumors that co-express gene sets defining basal, club, and hillock cells in the benign prostate. This data supports the existence of epithelial cell lineage plasticity manifesting within CRPC tumors, as well as individual CRPC tumor cells, wherein AR-positive tumor cells transform to a phenotype encompassing basal, club, and hillock cell identities. We nominated the stem cell transcription factor KLF5 as a regulator of basal, club, and hillock cell identities in CRPC. In cell line models of DNPC, knock down of KLF5 inhibited cell growth and reduced the expression of genes defining basal, club, and hillock cell identities. This work links a prevalent and poorly-defined subtype of AR-negative CRPC to AR-negative epithelial cell types in benign prostate tissue. Inhibition of KLF5 and its downstream effectors could represent a therapeutic strategy to prevent or delay lineage plasticity and thereby extend patient response to AR-targeted therapies. Citation Format: Samuel Pitzen, Yinjie Qiu, Sarah Munro, Scott Dehm. Double-negative prostate cancer emerges through a mixed basal, club, and hillock cell identity driven by KLF5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5863.

Abstract B013: KLF5 drives basal cell identity to promote prostate cancer lineage plasticity

Abstract The purpose of this study was to characterize the phenotypes and drivers of therapy-induced lineage plastic castration resistant prostate cancer (CRPC). Since the majority of primary prostate cancer presents with a luminal cell identity, which relies on androgen receptor (AR) signaling for growth and survival, AR-targeted therapies have become an effective method of slowing progression. However, CRPC cells can develop resistance through reduced or lost dependence on AR. These cells can transition their identity to neuroendocrine CRPC (NEPC), which lacks AR and expresses neuroendocrine lineage specific markers like chromogranin A. More frequently, these cells display transition to a cell identity that lacks AR and neuroendocrine markers and has been termed double-negative CRPC (DNPC). Identifying effective therapeutic targets to treat or prevent DNPC remains a significant clinical challenge. By utilizing prostate cancer cell lines, patient derived xenografts (PDX), and publicly available data from clinical CRPC specimens, we have identified an upregulation in basal cell identity in a subset of CRPC in response to AR-targeted therapies. Additionally, we show that the stem cell transcription factor KLF5, which is highly expressed in healthy prostate basal cells, is uniquely upregulated in CRPC subtypes displaying basal cell identity. In a cell line model of DNPC, we found that knockdown of KLF5 decreased the expression of genes that define basal cell identity. From these results, we conclude that AR-targeted therapies, while effective at targeting luminal-like adenocarcinoma, promote upregulation of KLF5 which can drive a basal cell identity and AR independence. These studies nominate KLF5 signaling as a potential therapeutic target to block the emergence of basal identity, inhibit prostate cancer lineage plasticity, and increase the durability of AR-targeted therapies. Citation Format: Samuel P. Pitzen, Yinjie Qiu, Sarah Munro, Scott M. Dehm. KLF5 drives basal cell identity to promote prostate cancer lineage plasticity [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 B013.

Abstract LB048: AR-targeted linked-read sequencing reveals complex AR structural variants in CRPC tumors

Abstract Inhibition of the androgen receptor (AR) transcription factor is the mainstay therapeutic strategy for systemic prostate cancer. However, development of a resistance phenotype termed castration resistant prostate cancer (CRPC) enables disease progression that underlies virtually all prostate cancer mortality. In the majority of CRPC, AR is transcriptionally re-activated in part due to accumulation of numerous alterations in the AR gene. These genomic alterations include missense mutations in exons that encode the ligand binding domain of AR, amplification of the AR gene body and/or an upstream transcriptional enhancer, and AR structural variants (SVs) resulting from inaccurate repair of DNA double strand breaks. In CRPC patient samples, these AR genomic alterations co-occur within tumors, which makes elucidating the fitness contribution of each alteration difficult. While short-read DNA sequencing has allowed for characterization of these events, they are underpowered to preserve linkage information. To address this challenge, we performed AR-targeted linked-read DNA-sequencing of CRPC cell line and patient derived xenograft (PDX) models with the goal of resolving sub-clonal and complex AR SVs. This approach confirmed the heterogeneity of AR SVs in these PDXs, including phased AR SVs that can be traced back to a single high molecular weight DNA molecule. These phased AR SVs were consistently found to co-occur in CRPC specimens with amplification of AR. Our results define a new class of AR SVs where multiple rearrangements co-occur on amplified DNA molecules to yield a complex array of AR gene structures in CRPC. Citation Format: Andrej Zivanovic, Sarah A. Munro, Jeffrey T. Miller, Yingming Li, Courtney N. Passow, Scott M. Dehm. AR-targeted linked-read sequencing reveals complex AR structural variants in CRPC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB048.

RF27 | LBSUN342 Ar-targeted Linked-read Sequencing Reveals Complex Ar Structural Variants In Castration Resistant Prostate Cancer Models

Abstract Androgen deprivation therapies are employed for the treatment of advanced prostate cancer to suppress the transcriptional activity of the androgen receptor (AR). However, over the course of therapy, AR transcriptional activity re-emerges, and the disease progresses to castration resistant prostate cancer (CRPC). During CRPC progression, AR becomes heavily altered by genomic aberrations that include amplification, missense mutations, and structural variants (SVs). AR SVs are heterogeneous across CRPC specimens, with variability noted in SV class, breakpoint locations, and co-occurrence with amplification. Further variability has been observed for sub-clonal enrichment of AR SVs within tumors. A barrier to understanding the clinical significance of AR SVs is the challenge of deciphering this high degree of variability from short-read DNA-sequencing data. To address this challenge, we performed AR-targeted linked-read DNA-sequencing of CRPC cell line and patient-derived xenograft (PDX) models with the goal of resolving sub-clonal and complex AR SVs. We benchmarked this approach using prostate cancer cell lines and LuCaP86.2 PDX tissue harboring known AR SVs. Using this benchmarking approach, we identified linked AR SVs that co-occurred on single DNA molecules in LuCaP35CR and LuCaP105CR PDX models. We also identified a novel AR SV in the LuCaP77 PDX model and a castration-resistant derivative, LuCaP77CR. These results have revealed heterogeneous trajectories of AR SVs in CRPC and define a new class of complex AR SVs where multiple rearrangements co-occur on single DNA molecules to yield highly rearranged AR gene structures. Presentation: Monday, June 13, 2022 1:05 p.m. - 1:10 p.m.

TP53 loss-of-function causes vulnerability to autophagy inhibition in aggressive prostate cancer.

TP53 loss-of-function is commonly found in aggressive prostate cancer. However, a highly-efficient therapy for this tumor subtype is still lacking. In this study, we investigated the relationship between TP53 mutation status and autophagy in prostate cancer and assessed the efficacy of autophagy inhibitors on TP53-deficient tumors. We first evaluated the expression patterns of p53 and autophagy-related proteins, namely LC3B, ULK1 and BECLIN1, as well as their relationship in treatment-naïve and castration-resistant prostate cancer specimens through immunohistochemistry. Subsequently, we generated a Trp53-deleted genetically-engineered mouse model, established prostate tumor organoid lines from the mice and assessed the efficacy of autophagy inhibitors in overcoming Enzalutamide resistance in the tumor organoid model. We also investigated the impact of TP53 re-expression in modulating responses to autophagy inhibitors using LNCaP cell line, which harbored a TP53 missense mutation. Lastly, we attempted to identify potential autophagy-related genes that were crucial for TP53-deficient tumor maintenance. TP53 loss-of-function was associated with increased levels of autophagy-related proteins in aggressive prostate cancers and Trp53-deleted genetically-engineered mouse-derived tumors. Moreover, the generated androgen receptor-independent tumor organoids were highly vulnerable to autophagy inhibition. Upon TP53 re-expression, not only did the surviving LNCaP cells demonstrate resistance, but they also showed growth advantage in response to autophagy inhibition. Lastly, PEX14, an important peroxisomal regulator was differentially upregulated in aggressive tumors with TP53 loss-of-function mutations, thus implying the importance of peroxisome turnover in this tumor subtype. Our results support the potential use of autophagy inhibitors in prostate cancers that contain TP53 loss-of-function mutations.

Pathological Roles of Prostaglandin E2-specific E-type Prostanoid Receptors in Hormone-sensitive and Castration-resistant Prostate Cancer.

Prostaglandin (PG) E2 mediates malignant aggressiveness by binding to four specific E-type prostanoid receptors (EP1R - 4R). This study aimed to clarify the pathological significance of EPRs in hormone-sensitive prostate cancer (HSPC) and castration-resistant prostate cancer (CRPC). EP1R - 4R expression was examined in 102 HSPC and 27 CRPC specimens. The relationships between their expression and proliferation index (PI), apoptotic index (AI), and vascular endothelial growth factor (VEGF)-A expression were analyzed. EP4R expression in CRPC was significantly higher compared to that in HSPC, even in advanced disease (T3/4, N1, and/or M1). EP4R expression was significantly correlated with PI, AI, and VEGF-A expression in CRPC. Such significant relationships were not detected between EP1R - 3R and CRPC. EP4R expression in CRPC was significantly higher than that in HSPC and was associated with cancer cell proliferation, apoptosis, and pro-angiogenetic potential.

P300-Mediated Acetylation of Histone Demethylase JMJD1A Prevents Its Degradation by Ubiquitin Ligase STUB1 and Enhances Its Activity in Prostate Cancer.

The androgen receptor (AR) pathway plays a central role in the development of castration-resistant prostate cancer (CRPC). The histone demethylase JMJD1A has been shown to regulate activities of AR and c-Myc transcription factors and promote prostate cancer progression. Here, we report that JMJD1A protein stability is controlled by the ubiquitin ligase STUB1. High levels of JMJD1A were strongly correlated with low STUB1 levels in human CRPC specimens. STUB1 inhibited AR activity, AR-V7 levels, and prostate cancer cell growth partly through degradation of JMJD1A. Furthermore, the acetyltransferase p300 acetylated JMJD1A at lysine (K) 421, a modification that recruits the BET family member BRD4 to block JMJD1A degradation and promote JMJD1A recruitment to AR targets. Increased levels of both total and K421-acetylated JMJD1A were observed in prostate cancer cells as they developed resistance to the AR antagonist enzalutamide. Treatment of prostate cancer cells with either p300 or BET inhibitors destabilized JMJD1A, and enzalutamide-resistant prostate cancer cells were more sensitive than parental cells to these inhibitors. Together, our findings identify a critical role for acetylation of JMJD1A in regulating JMJD1A stability and AR activity in CRPC. These newly identified mechanisms controlling JMJD1A protein stability provide potential druggable targets to encourage the development of additional therapies for advanced prostate cancer. SIGNIFICANCE: Identification of mechanisms regulating JMJD1A protein stability reveals new strategies to destabilize JMJD1A and concomitantly inhibit AR activities as potential prostate cancer therapy.

Determining the prognostic significance of IKKα in prostate cancer.

As the survival of castration-resistant prostate cancer (CRPC) remains poor, and the nuclear factor-κB (NF-κB) pathways playkey roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF-κB pathway through generating inhibitory κB kinase subunitα (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p-IKKα S180) and threonine 23 (p-IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome. A cohort of 115 patients with hormone-naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'Isurvival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth. High levels of cytoplasmic IKKα were associated with a higher cancer-specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log-rank, 0.11-0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27-5.33; P = .01) and cytoplasmic p-IKKα S180 (HR, 2.10; 95% CI, 1.17-3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T-cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate-specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF-κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF-κB and Akt pathways correlated with members of the canonical NF-κB pathway in CRPC. The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p-IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.

Combined targeting of EGFR and HER2 against prostate cancer stem cells

ABSTRACTProgression of prostate cancer has been associated with EGFR and HER2 activation and to tumor-initiating cells contribution toward chemotherapy resistance. We investigated the efficacy of a dual intervention against EGFR and HER2 to deplete the tumor-initiating cells, optimize the chemotherapy management and prevent the progression of castration-resistant prostate cancer (CRPC) cells. Using DU145, PC3, and 22Rv1 CRPC cell lines, biochemical analysis revealed activation of EGFR, HER2, MAPK, and STAT3 in DU145 and 22Rv1, and AKT and SRC in DU145 and PC-3. pSTAT3 nuclear staining was observed in DU145 xenografts and in 12 out of 14 CRPC specimens. The in vivo dual targeting of ErbB receptors with Cetuximab and Trastuzumab combined with chemotherapy caused an effective antitumor response in DU145 xenografted mice displaying STAT3 activation; conversely PC-3 bearing mice experienced tumor relapse. The potentiating of in vivo cytotoxic effect in DU145 model was accompanied by a significant decrease of prostatosphere-forming capacity assessed in vitro on residual tumor cells. Additionally, combined treatment in vitro with Cetuximab, Trastuzumab and chemotherapy negatively affected DU145 and 22Rv1 sphere formation, suggesting the critical function of ErbB receptors for tumor-initiating cells proliferation; no effect on PC-3 clonogenic potential was observed, indicating that other receptors than EGFR and HER2 may sustain PC3 tumor-initiating cells. These findings provided the preclinical evidence that the dual inhibition of EGFR and HER2 by targeting tumor-initiating cells may improve the efficacy of the current chemotherapy regimen, bringing benefits especially to castration-resistant patients with activated STAT3, and preventing disease progression.

Abstract 764: MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer

Abstract Although death rates for prostate cancer are down about 50% as a result of improvements in early detection and treatment, it is still the second leading cause of death (26,120 cases) for American men. However, the genetic mechanism underlying the development and progression of prostate cancer remains poorly understood. It is therefore important to identify the genetic risk factors for prostate cancer. Genome-wide association studies (GWAS) showed evidence for an association of prostate cancer risk with independent genetic variants on chromosome 8q24, conferring prostate cancer susceptibility at this locus. Nevertheless, at the locus with these genetic variants, no specific gene has been identified as responsible for the risk of prostate cancer. At 8q24.21, there is a long-range interaction of these genetic variants with c-MYC or long non-coding RNAs (lncRNAs) in a tissue-specific manner, including prostate, suggesting master genetic factors at 8q24.21 that contribute to this genetic risk. The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. Here, the copy numbers and expressions of miRs-1204~1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with expression of miR-1205. Furthermore, functional analysis with a miR-1205 mimic, a miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression but not apoptosis. In these cells, miR-1205 downregulated expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) gene and targeted a site in its 3’-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Wen Wen, Yicun Wang, Yue Zhang, Xuelian Cui, Xiaoguang M. Liu, Runhua Liu. MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer [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 764.

MicroRNA-1205, encoded on chromosome 8q24, targets EGLN3 to induce cell growth and contributes to risk of castration-resistant prostate cancer.

The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. A long-range interaction of genetic variants with c-MYC or long non-coding PVT1 at this locus contributes to the genetic risk of prostate cancer. At this locus is a cluster of genes for six miRs (miR-1204, −1205, −1206, −1207–3p, −1207–5p, and −1208), but their functional role remains elusive. Here, the copy numbers and expressions of miRs-1204~1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and in primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with expression of miR-1205. Furthermore, functional analysis with an miR-1205 mimic, an miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression and inhibited hydrogen peroxide-induced apoptosis. In these cells, miR-1205 downregulated expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) gene and targeted a site in its 3’-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer.

DHX15 is up-regulated in castration-resistant prostate cancer and required for androgen receptor sensitivity to low DHT concentrations.

DHX15 is a member of the DEAH-box (DHX) RNA helicase family. Our previous study identified it as an AR coactivator which contributes to prostate cancer progression. We investigated DHX15 expression in castration resistant prostate cancer specimens and the influence of DHX15 on the responsiveness of prostate cancer cells to DHT stimulation. We also explored the role DHX15 played in enzalutamide resistance and the interacting domain in DHX15 with AR. DHX15 expression level in human CRPC specimens and prostate cancer specimens was detected by tissue microarray (TMA) immunostaining analysis. Colony formation assay was performed to determine the proliferation of cells treated with enzalutamide or DHT. siRNAs were used to knockdown DHX15. The interactions between DHX15 and AR were detected using co-immunoprecipitation assay. The expression level of DHX15 was upregulated in human CRPC specimens compared with hormone naïve prostate cancer specimens. DHX15 knockdown reduced AR sensitivity to low DHT concentrations in C4-2 cells. Inactivation of DHX15 sensitizes the enzalutamide treatment in C4-2 cells. Deletion mutagenesis indicated that DHX1 5 interacts with AR through its N terminal domain. These findings suggest that DHX15 contributes to prostate cancer progression. DHX15 is required for androgen receptor sensitivity to low DHT concentrations and contributes to enzalutamide resistance in C4-2 cells. Targeting DHX15 may improve the ADT treatment.

Abstract A063: Preclinical evaluation of an arginine vasopressin receptor 1A (AVPR1A) antagonist in castration-resistant prostate cancer

Abstract Background: Advanced prostate cancer is treated by androgen deprivation, but despite initial responses most tumors inevitably recur. The recurrent disease is termed castration-resistant prostate cancer (CRPC) and is characterized by active androgen receptor (AR) signaling despite decreased androgen levels. AR activity in CRPC occurs through a variety of mechanisms including upregulation of AR coactivators, such as Vav3, and expression of constitutively active AR variants, which lack the ligand binding domain and are linked to poor prognosis. We previously demonstrated that PC cells are growth inhibited by depletion of either Vav3 or the clinically prevalent AR variant, AR-V7 (1). Gene expression profiling in CRPC cells revealed that arginine vasopressin receptor 1a (AVPR1a) was significantly reduced by depletion of either Vav3 or AR-V7. AVPR1a is a G protein-coupled receptor that has not been previously linked to PC. Analysis of publicly available datasets showed that the AVPR1a gene exhibits a significant increase in copy number in human CRPC specimens and AVPR1a mRNA is upregulated in aggressive PC (2-4). Methods: To explore the role of AVPR1a in PC cells, we selectively depleted AVPR1a in a panel of prostate cancer and prostate epithelial cell lines and examined cell survival and proliferation in culture and soft agar, as well as cell migration and invasion. Conversely, we overexpressed AVPR1a in an androgen-dependent PC cell line, LNCaP, and examined growth in vitro and in vivo in androgen-depleted conditions. We investigated AVPR1a antagonism in two distinct in vivo models of CRPC progression and growth using an effective and safe-in-humans AVPR1a antagonist, relcovaptan. Results: We showed that depletion of AVPR1a greatly inhibited the proliferation of multiple CRPC cell lines but had less to no effect on androgen-dependent PC cells or nontumorigenic prostate epithelial cells. Depletion of AVPR1a also decreased CRPC cell anchorage-independent growth. Conversely, overexpression of AVPR1a in androgen-dependent LNCaP cells conferred castration resistance in vitro and in vivo. Similar to depletion of AVPR1a, treatment of PC cells with the AVPR1a antagonist relcovaptan decreased CRPC anchorage-independent growth. AVPR1a depletion or antagonism was also effective at decreasing CRPC metastatic properties: migration and invasion. In a preclinical xenograft model of progression to castration resistance, we found that relcovaptan halted tumor growth and stabilized circulating levels of a clinical tumor progression marker, prostate specific antigen (PSA). We evaluated relcovaptan efficacy on end-stage metastatic CRPC growth. C4-2B CRPC cells were injected into the tibias of castrated mice to model tumor growth and bone remodeling. Relcovaptan significantly inhibited tumor growth and prevented cancer cell-induced bone remodeling. Conclusion: AVPR1a is a promising new therapeutic target against CRPC. We propose that AVPR1a antagonists may be repurposed for PC therapy.

Identification of an oncogenic network with prognostic and therapeutic value in prostate cancer.

Identifying critical pathways governing disease progression is essential for accurate prognosis and effective therapy. We developed a broadly applicable and novel systems‐level gene discovery strategy. This approach focused on constitutively active androgen receptor (AR) splice variant‐driven pathways as representative of an intractable mechanism of prostate cancer (PC) therapeutic resistance. We performed a meta‐analysis of human prostate samples using weighted gene co‐expression network analysis combined with experimental AR variant transcriptome analyses. An AR variant‐driven gene module that is upregulated during human PC progression was identified. We filtered this module by identifying genes that functionally interacted with AR variants using a high‐throughput synthetic genetic array screen in Schizosaccharomyces pombe. This strategy identified seven AR variant‐regulated genes that also enhance AR activity and drive cancer progression. Expression of the seven genes predicted poor disease‐free survival in large independent PC patient cohorts. Pharmacologic inhibition of interacting members of the gene set potently and synergistically decreased PC cell proliferation. This unbiased and novel gene discovery strategy identified a clinically relevant, oncogenic, interacting gene hub with strong prognostic and therapeutic potential in PC.

Interleukin-6/signal transducer and activator of transcription 3 promotes prostate cancer resistance to androgen deprivation therapy via regulating pituitary tumor transforming gene 1 expression.

Prostate cancer can progress from androgen dependence to androgen deprivation resistance with some unknown mechanisms. The current study aims to explore the possible role of pituitary tumor transforming gene1 (PTTG1) in castration‐resistant prostate cancer (CRPC). Initially, we found that PTTG1 expression was significantly increased in androgen‐independent prostate cancer cell lines PC3, DU145 and CRPC specimens compared with that in androgen‐dependent prostate cancer cell line LNCaP and initial prostate cancer specimens. PTTG1 overexpression significantly enhanced the cell survival rate, clonality and tumorigenicity in LNCaP cells upon androgen‐deprivation therapy (ADT). While knockdown of PTTG1 expression significantly elevated the sensitivity of DU145 cells to ADT. The effects of PTTG1 overexpression on LNCaP cells may be ascribed to the induced EMT and increased CD44+ CD24‐ cancer stem cell population. Furthermore, we detected that PTTG1 expression was regulated by interleukin‐6 via activated signal transducer and activator of transcription 3 (STAT3) directly binding to the region −500 to +1 of PTTG1 promoter in LNCaP cells. In conclusion, our results elucidate that interleukin‐6/STAT3 activation can increase PTTG1 expression and, consequently, promote the resistance to ADT in CRPC by inducing EMT and increasing the cancer stem cell population, suggesting that PTTG1 may be a novel therapeutic target for CRPC.

Abstract 3438: Dual-strands pre-microRNA-150 (miR-150-5p and miR-150-3p) act as tumor suppressor in prostate cancer

Abstract Background: Prostate cancer (PCa) is the second most common cause of cancer and the sixth leading cause of cancer death among men in the world. Androgen signaling through the androgen receptor (AR) is an important oncogenic pathway for PCa progression. The initial response rate of PCa to androgen deprivation therapy (ADT) can be up to 80%, but most patients experience disease relapse and progress to castration-resistant prostate cancer (CRPC). Although several clinical trials, such as molecularly-targeted therapies for CRPC have been carried out, these treatments provide limited benefits and, are not curative. Therefore, identification of effective biomarkers for detection of CRPC and understanding the molecular mechanisms of androgen-independent signaling and metastatic signaling pathways underlying PCa using current genomic approaches would help to improve therapies for and prevention of the disease. Currently, numerous studies have indicated that miRNAs are aberrantly expressed in several cancers, including CRPC. In this study, we constructed a miRNA expression signature to identify miRNA regulated RNA networks in CRPC using autopsy specimens from patients with ADT. Based on the signature, dual-strands of pre-miR-150 (miR-150-5p and miR-150-3p) were significantly reduced in CRPC specimens. The aim of this study was to investigate the functional significance of both strands of miR-150-5p and miR-150-3p and these miRNAs regulated RNA networks in CRPC. Results: Downregulation of miR-150-5p and miR-150-3p were validated in hormone naive PCa and CRPC specimens compared to non-cancerous prostate tissues (p < 0.0001). Restoration of miR-150-5p and miR-150-3p significantly suppressed cancer cell migration and invasion in PCa cell lines (P < 0.0001). Gene expression data and in silico database analysis showed that Sparc/Osteonectin, Cwcv AND Kazal-Like Domains Proteoglycan 1 (SPOCK1) was regulated by both miRNAs. Knockdown of SPOCK1 inhibited cancer cell aggressiveness. Overexpression of SPOCK1 was observed in PCa clinical specimens. Conclusions: In miRNA biogenesis, it is the general consensus that processing of the pre-miRNA through Dicer1 generates a miRNA duplex (a passenger strand and a guide strand), and that the passenger strand has degradation and no regulatory activity and disintegrates in cells. Our present data showed that both strands of pre-miR-150 (miR-150-5p and miR-150-3p) inhibited cancer cell aggressiveness, suggesting these miRNAs as tumor-suppressors. Identification of miRNA-mediated cancer networks may provide novel molecular pathogenesis of the disease. Citation Format: Atsushi Okato, Takayuki Arai, Akira Kurozumi, Mayuko Kato, Yusuke Goto, Keiichi Koshizuka, Satoko Kojima, Tomohiko Ichikawa, Naohiko Seki. Dual-strands pre-microRNA-150 (miR-150-5p and miR-150-3p) act as tumor suppressor 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 3438. doi:10.1158/1538-7445.AM2017-3438

Transcript Levels of Androgen Receptor Variant 7 and Ubiquitin‐Conjugating Enzyme 2C in Hormone Sensitive Prostate Cancer and Castration‐Resistant Prostate Cancer

This study is designed to identify the androgen receptor variant 7 (AR-V7) status, clinical significance of AR-V7 in hormone sensitive prostate cancer (HSPC). Then, we evaluated AR-V7 and changes of its target gene, ubiquitin-conjugating enzyme E2C (UBE2C) which is an anaphase-promoting complex/cyclosome (APC/C)-specific ubiquitin-conjugating enzyme, in castration-resistant prostate cancer (CRPC) in serial tumor biopsies from patients receiving androgen deprivation therapy. We used RT-PCR and Q-PCR assay to evaluate AR-V7, androgen receptor full length (AR-FL), and UBE2C in tumor biopsies from patients with HSPC and CRPC. We examined associations between mRNA expression of AR-V7 and clinicopathologic factors. Furthermore, to identify other potential genes involved in the development of CRPC, RNA sequencing was conducted, using paired prostate cancer (PCa) tissues obtained immediately prior to treatment and at the time of therapeutic resistance. A total of 13 HSPC patients and three CRPC patients were enrolled. Neither a high Gleason score (score of 8 and 9) nor a high risk of PCa (a high risk of locally advanced PCa according to NCCN guidelines) was correlated with mRNA expression of AR-V7 in HSPC (P = 0.153 and P = 0.215). The mRNA expression of AR-FL, but not AR-V7, was significantly associated with the mRNA expression of UBE2C level in HSPC (P = 0.007). However, increased expression of AR-V7, not AR-FL, paralleled increased expression of UBE2C in the CRPC specimens (P = 0.03). AR-V7 expression status before ADT was likely related to shorter CRPC development in patients treating ADT. The result of the RNA-sequencing analysis using serial samples from the same patient before and after castration demonstrated an increased level of the PI3K regulatory subunit 1 (P = 0.018). Our study revealed the role of UBE2C as a marker of the androgen signaling pathway in PCa. Differential gene expression analysis using serial samples from the same patient before and after castration revealed potential genes and pathways involved in development of CRPC. Further studies are needed to determine whether these genes and pathways are potential therapeutic target for CRPC. Prostate 77:60-71, 2017. © 2016 Wiley Periodicals, Inc.

Abstract 3161: Development and implementation of a non-invasive NGS-based diagnostic test for precision medicine in prostate cancer

Abstract The growth and survival of prostate cancer tumors relies primarily on the functioning of the androgen receptor (AR) signaling pathway. Recent studies suggested that the presence of AR-V7, a c-terminal truncated form of AR, in circulating tumor cells (CTCs) of patients with castration-resistant prostate cancer (CRPC) is associated with inherent and/or acquired resistance to enzalutamide and abiraterone, the stand of care androgen deprivation therapies in prostate cancer. A series of data also indicate that AR-Vs (eg, including AR-V7, ARv567es, AR-T878A, AR-F876L, and Arv567es) may drive resistance in CRPC. Expression of AR-Vs has been shown to correlate with disease progression and shortened survival and AR-V7 is most abundant in CRPC specimens. Considering that truncated ARs with C-terminal loss (splice variants) lack a functional LBD and are constitutively active, C-terminal AR-directed therapies may not be effective, and novels agents are needed that target mutated ARs including AR-Vs. Circulating tumor cell (CTC)-based AR-V7 tests are currently being tested in the clinic. However, nearly half of the CRPC patients do not have enough CTCs for AR-V7 test, raising the request for a complementary, non-CTC platform to detect AR variants as well as other resistance markers in circulation. Here we report the successful development of a non-invasive, next generation sequencing-based diagnostics platform that offers comprehensive profiling of genomic alterations in prostate cancer, including splicing variants (such as AR-V7), point mutation, copy number, and translocation using biofluid samples (blood and urine) from CRPC patients. To the best of our knowledge, this is the first non-invasive diagnostic test that measures genetic alterations especially AR-V7 in all patients with prostate cancer, regardless of their status of CTC enumeration. The successful development and clinical validation of this test has potential to enable precision medicine in prostate cancer. Citation Format: Pan Du, Amy Wang, Tak Cheng, Winston Patrick Kuo. Development and implementation of a non-invasive NGS-based diagnostic test for precision medicine in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3161.

Direct regulation of LAMP1 by tumor-suppressive microRNA-320a in prostate cancer

Advanced prostate cancer (PCa) metastasizes to bone and lymph nodes, and currently available treatments cannot prevent the progression and metastasis of the disease. Therefore, an improved understanding of the molecular mechanisms of the progression and metastasis of advanced PCa using current genomic approaches is needed. Our miRNA expression signature in castration-resistant prostate cancer (CRPC) revealed that microRNA-320a (miR-320a) was significantly reduced in cancer tissues, suggesting that miR-320a may be a promising anticancer miRNA. The aim of this study was to investigate the functional roles of miR-320a in naïve PCa and CRPC cells and to identify miR-320a-regulated genes involved in PCa metastasis. The expression levels of miR-320a were significantly reduced in naïve PCa, CRPC specimens, and PCa cell lines. Restoration of mature miR-320a in PCa cell lines showed that miR-320a significantly inhibited cancer cell migration and invasion. Moreover, we found that lysosomal-associated membrane protein 1 (LAMP1) was a direct target of miR-320a in PCa cells. Silencing of LAMP1 using siRNA significantly inhibited cell proliferation, migration, and invasion in PCa cells. Overexpression of LAMP1 was observed in PCa and CRPC clinical specimens. Moreover, downstream pathways were identified using si-LAMP1-transfected cells. The discovery of tumor-suppressive miR-320a-mediated pathways may provide important insights into the potential mechanisms of PCa metastasis.