Lethal Castration-resistant Prostate Cancer Research Articles

Abstract 6059: Discovery of ETS-007, a first-in-class degrader targeting N-terminal domain of androgen receptor derived from AR liquid-liquid phase separation inhibitor

Abstract Patients with advanced prostate cancer inevitably acquire resistance to anti-androgen therapies, and progress to lethal castration-resistant prostate cancer (CRPC). The resistance mechanisms involve AR gene amplification and overexpression, constitutively active ligand-independent splice variants such as AR-V7, and the mutations in the AR ligand-binding domain (LBD). Our previous study has shown that the AR N-terminal domain (NTD) is responsible for AR liquid-liquid phase separation (LLPS) capability, which is essential for AR/AR-V7 transcriptional activity. We conducted a compound library screening using an AR LLPS phenotypic assay and identified ET0516 as a hit to specifically inhibit AR LLPS and its downstream gene expression, indicating that inhibition of AR. LLPS through targeting its NTD is a feasible approach to overcome the resistance to current anti-androgens therapy. Here we report the identification of a novel AR degrader, ETS-007 which is originated and evolved from internal LLPS phenotypic screening, that effectively degrades both AR and AR-V7 proteins with DC50 of single-digit nM by inducing a protein-protein interaction between AR NTD and an E3 ligase complex. ETS-007 induced AR and AR-V7 polyubiquitination, followed by degradation which could be blocked by the protease inhibitor MG132. ETS-007 significantly inhibited cell proliferation in AR and/or AR-V7 positive cell lines, e.g. LNCaP and 22RV1, and downregulated the androgen responsive genes such as PSA and TMPRSS2. Compared with enzalutamide and ARV-110, ETS-007 could block transcriptional activities of AR-V7, and AR mutants in luciferase report assay. Oral administration of ETS-007 in an enzalutamide-resistant 22RV1 xenograft model led to intratumoral AR-V7 degradation and concomitant decrease in PSA protein levels, resulting in a remarkable tumor regression. Taken together, our findings provide preclinical evidence that ETS-007 as a potent AR-NTD degrader derived from our LLPS platform can effectively block the transcriptional activities of both AR and AR-V7 for the treatment of metastatic CRPC. Citation Format: Yingjie Li, Mingyue Fei, Yingke Miao, Lijian Feng, Xiaofei Fan, Jinping Li, Yaozhong Chen, Ming Xu, Wenqi Cui, Qiangang Zheng, Jidong Zhu. Discovery of ETS-007, a first-in-class degrader targeting N-terminal domain of androgen receptor derived from AR liquid-liquid phase separation inhibitor [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 6059.

Abstract 3588: Androgen synthesis disruption, ferroptosis induction and prostate cancer inhibition by androgen and vitamin D combination

Abstract Background: Metastatic castration-resistant prostate cancer (CRPC) is a fatal condition that progresses to end-of-life within two years, and it is the fifth leading cause of cancer death in men globally. New avenues for preventing or delaying lethal CRPC progression are a critical unmet need. Hormonal vitamin D3 (calcitriol) inhibits prostate cancer - both castration sensitive and castration resistant - in preclinical models, as documented widely. However, vitamin D as an anti-cancer therapeutic is clinically untenable since prostate tumor suppression in patients requires hormone dosing at a supraphysiologic level which induces hypercalcemia and other toxicities. Results: Herein we provide the first evidence that a subnanomolar, non-calcemic calcitriol dose can inhibit CRPC cell proliferation, G1 to S phase transition, clonal growth, migration, and invasion in vitro, and xenograft growth in vivo, only upon concurrent dosing with 5α-DHT at a physiologic or subphysiologic level. Calcitriol as a single treatment is non-inhibitory at a subnanomolar dose. HSD3β1, a hydroxysteroid dehydrogenase, was markedly induced by calcitriol, while DHT cotreatment reduced the induction. HSD3β1 catalyzes conversion of dehydroepiandrosterone, an adrenal androgen, to androstenedione (AED) - a rate-limiting step in the androgen biosynthesis pathway. AKR1C3, an aldo-keto reductase which promotes androgen biosynthesis, was also reduced by calcitriol/DHT cotreatment. In contrast, AKR1D1, an androgen-inactivating 5β-reductase, was robustly induced by a non-calcemic calcitriol dose in the presence of DHT, not by calcitriol singly. AKR1D1 converts AED and testosterone to 5β-diols. HSD3β1 and AKR1C3 are elevated in CRPC. Disruption of androgen biosynthesis and net reduction of the intratumor androgen pool are expected outcomes of reduced HSD3β1 and AKR1C3, and elevated AKR1D1. The hormone pair led to LC3B induction, ferritin degradation, and induction of ALOX-5 and NCoA4 - the genes which regulate ferroptosis, indicating autophagy mediated ferroptosis (ferritinophagy). Apoptosis and necroptosis were absent. We conclude that vulnerabilities created by combined androgen and non-calcemic vitamin D dosing can lead to cell death by ferroptosis, and this hormone pairing may be a new avenue for arresting CRPC clinical progression. Significance: The translational potential of our results is bolstered by the precedent that supraphysiologic testosterone improved serum PSA scores and other clinical parameters for a subset of therapy-inert CRPC patients who received repeated cycles of once-a-month single testosterone dosing- a protocol known as bipolar androgen therapy (BAT)1. Our findings may offer a strategic framework for enhancing the efficacy of BAT by including oral dosing of a non-calcemic level of vitamin D3. W81XWH-21-1-0307 (DoD); IK6 BX004207 (VA). Ref 1: Nat Rev Urol 2023; PMID 36543976 Citation Format: Bobae Park, Jacqueline Vaquiz, Okunola Igbekoyi, Bandana Chatterjee. Androgen synthesis disruption, ferroptosis induction and prostate cancer inhibition by androgen and vitamin D combination [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 3588.

Agent-based modeling of the prostate tumor microenvironment uncovers spatial tumor growth constraints and immunomodulatory properties

Inhibiting androgen receptor (AR) signaling through androgen deprivation therapy (ADT) reduces prostate cancer (PCa) growth in virtually all patients, but response may be temporary, in which case resistance develops, ultimately leading to lethal castration-resistant prostate cancer (CRPC). The tumor microenvironment (TME) plays an important role in the development and progression of PCa. In addition to tumor cells, TME-resident macrophages and fibroblasts express AR and are therefore also affected by ADT. However, the interplay of different TME cell types in the development of CRPC remains largely unexplored. To understand the complex stochastic nature of cell-cell interactions, we created a PCa-specific agent-based model (PCABM) based on in vitro cell proliferation data. PCa cells, fibroblasts, “pro-inflammatory” M1-like and “pro-tumor” M2-like polarized macrophages are modeled as agents from a simple set of validated base assumptions. PCABM allows us to simulate the effect of ADT on the interplay between various prostate TME cell types. The resulting in vitro growth patterns mimic human PCa. Our PCABM can effectively model hormonal perturbations by ADT, in which PCABM suggests that CRPC arises in clusters of resistant cells, as is observed in multifocal PCa. In addition, fibroblasts compete for cellular space in the TME while simultaneously creating niches for tumor cells to proliferate in. Finally, PCABM predicts that ADT has immunomodulatory effects on macrophages that may enhance tumor survival. Taken together, these results suggest that AR plays a critical role in the cellular interplay and stochastic interactions in the TME that influence tumor cell behavior and CRPC development.

Insight into Recent Advances in Degrading Androgen Receptor for Castration-Resistant Prostate Cancer

Induced protein degradation has emerged as an innovative drug discovery approach, complementary to the classical method of suppressing protein function. The androgen receptor signaling pathway has been identified as the primary driving force in the development and progression of lethal castration-resistant prostate cancer. Since androgen receptor degraders function differently from androgen receptor antagonists, they hold the promise to overcome the drug resistance challenges faced by current therapeutics. Proteolysis-targeting chimeras (PROTACs), monomeric degraders, hydrophobic tagging, molecular glues, and autophagic degradation have demonstrated their capability in downregulating intracellular androgen receptor concentrations. The potential of these androgen receptor degraders to treat castration-resistant prostate cancer is substantiated by the advancement of six PROTACs and two monomeric androgen receptor degraders into phase I or II clinical trials. Although the chemical structures, in vitro and in vivo data, and degradation mechanisms of androgen receptor degraders have been reviewed, it is crucial to stay updated on recent advances in this field as novel androgen receptor degraders and new strategies continue to emerge. This review thus provides insight into recent advancements in this paradigm, offering an overview of the progress made since 2020.

Androgen receptor nucleocytoplasmic trafficking – A one-way journey

The androgen receptor (AR) is a key regulator of the growth and proliferation of prostate cancer. The majority of lethal castration-resistant prostate cancer (CRPC) growth is still dependent on AR activity. The AR need to be in the nucleus to exert its biological action as a transcription factor. As such, defining the mechanisms that regulate the subcellular localization of AR are important. Previously it was believed that AR was imported into the nucleus in a ligand-dependent manner and subsequently exported out of the nucleus upon ligand withdrawal. Recent evidence has challenged this decades-old paradigm and showed that the AR is degraded, not exported, in the nucleus. This review discusses the current understanding of how AR nucleocytoplasmic localization is regulated by import and through nuclear degradation.

Identifying Patients With Rapid Progression From Hormone-Sensitive to Castration-Resistant Prostate Cancer: A Retrospective Study

Prostate cancer (PCa) is the second most prevalent malignancy and the fifth cause of cancer-related deaths in men. A crucial challenge is identifying the population at risk of rapid progression from hormone-sensitive prostate cancer (HSPC) to lethal castration-resistant prostate cancer (CRPC). We collected 78 HSPC biopsies and measured their proteomes using pressure cycling technology and a pulsed data-independent acquisition pipeline. We quantified 7355 proteins using these HSPC biopsies. A total of 251 proteins showed differential expression between patients with a long- or short-term progression to CRPC. Using a random forest model, we identified seven proteins that significantly discriminated long- from short-term progression patients, which were used to classify PCa patients with an area under the curve of 0.873. Next, one clinical feature (Gleason sum) and two proteins (BGN and MAPK11) were found to be significantly associated with rapid disease progression. A nomogram model using these three features was generated for stratifying patients into groups with significant progression differences (p-value = 1.3×10−4). To conclude, we identified proteins associated with a fast progression to CRPC and an unfavorable prognosis. Based on these proteins, our machine learning and nomogram models stratified HSPC into high- and low-risk groups and predicted their prognoses. These models may aid clinicians in predicting the progression of patients, guiding individualized clinical management and decisions.

An unbiased seed-based RNAi selection screen identifies small RNAs that inhibit androgen signaling and prostate cancer cell growth

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration-resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here, we explore a therapeutic strategy for PCa based on the ability of shRNAs/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3' UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell-cycle genes. When tested in mice, the toxic shRNAs also inhibited castration-resistant PCa and exhibited therapeutic efficacy in pre-established tumors. Our findings highlight RNAi of androgen signaling networks as a promising therapeutic strategy for PCa.

Tricyclic Diterpenoids Selectively Suppress Androgen Receptor-Positive Prostate Cancer Cells.

Androgen receptor (AR) is a viable therapeutic target for lethal castration-resistant prostate cancer (CRPC), because the continued progression of CRPC is mainly driven by the reactivation of AR transcriptional activity. The current FDA-approved AR antagonists binding to ligand binding domain (LBD) become ineffective in CRPC with AR gene amplification, LBD mutation, and the evolution of LBD-truncated AR splice variants. Encouraged by the fact that tricyclic aromatic diterpenoid QW07 has recently been established as a potential N-terminal AR antagonist, this study aims to explore the structure-activity relationship of tricyclic diterpenoids and their potential to suppress AR-positive cell proliferation. Dehydroabietylamine, abietic acid, dehydroabietic acid, and their derivatives were selected, since they have a similar core structure as QW07. Twenty diterpenoids were prepared for the evaluation of their antiproliferative potency on AR-positive prostate cancer cell models (LNCaP and 22Rv1) using AR-null cell models (PC-3 and DU145) as comparisons. Our data indicated that six tricyclic diterpenoids possess greater potency than enzalutamide (FDA-approved AR antagonist) towards LNCaP and 22Rv1 AR-positive cells, and four diterpenoids are more potent than enzalutamide against 22Rv1 AR-positive cells. The optimal derivative possesses greater potency (IC50 = 0.27 µM) and selectivity than QW07 towards AR-positive 22Rv1 cells.

Abstract A037: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2

Abstract Background: Treatment-related neuroendocrine prostate cancer (NEPC) is an aggressive subset of castration-resistant prostate cancer (CRPC), found in ~20% of lethal CRPC. The mechanisms underlying the progression of prostate cancer to NEPC are largely unclear, and new drug targets are desperately needed. NEPC is known to be highly vascularized. Elevated expression of NE markers and increased angiogenesis are two prominent phenotypes of NEPC, and thus are expected to be linked. However, direct molecular links between these two phenotypes are still elusive, whose elucidation will substantially expand our knowledge in NEPC and enable the development of effective treatments for NEPC. Through RNAi & cDNA screening and functional validations, we previously discovered that GPCR-kinase 3 (GRK3) is essential preferentially for highly metastatic cancer cells as compared to lowly metastatic cancer cells. The mechanisms of GRK3 in prostate cancer progression were mostly unknown. Methods: We assessed GRK3’s expression in patient samples through IHC staining on TMA and data mining on public RNA-seq datasets with large cohorts. Using molecular and cell biology methods, we determined the impacts of GRK3 and HDAC2 overexpression and silencing on NE marker expression and angiogenesis in prostate cancer cells. Through biochemistry, we investigated the nature of GRK3-HDAC2 relation. By ChIP-PCR, we measured the impacts of GRK3 genetic and pharmacological modulations on HDAC2’s epigenetic activity. Through compound library screening and biochemical characterization, we searched for novel GRK3 inhibitors to study GRK3 biology and to evaluate whether GRK3 is a suitable drug target for prostate cancer. Results: GRK3 is significantly overexpressed in metastatic prostate tumors from patients, especially in NEPC. GRK3 promotes both angiogenesis and neuroendocrine differentiation in prostate cancer cells, indicating that it is a key missing link for these two phenotypes. Mechanistically, GRK3 enhances the epigenetic repressor activity of histone deacetylase 2 (HDAC2) to suppress key repressors of angiogenesis or NE phenotype. We have identified several compounds that block kinase activity of GRK3 much more potently than that of GRK2, the closest-related kinase to GRK3. Of note, our GRK3 inhibitors could substantially reduce angiogenesis and NE marker expression, as well as significantly inhibit NEPC cell growth in culture and in mouse xenografts. Conclusion: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression. Its mechanism of actions is at least in part through enhancing HDAC2’s epigenetic activity. Results based on our novel GRK3 inhibitors suggest that GRK3 is a valuable new drug target for aggressive prostate cancer. Citation Format: Samira Naderinezhad, Zheng Wang, Guoliang Zhang, Michael Ittmann, Martin Gleave, Wenliang Li. Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2 [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 A037.

Abstract LB032: Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2

Abstract Treatment-related neuroendocrine prostate cancer (t-NEPC, here as NEPC) is an aggressive subset of castration-resistant prostate cancer (CRPC), found in ~20% of lethal CRPC. The mechanisms underlying the progression of prostate cancer to NEPC are largely unclear, and new drug targets are desperately needed. NEPC is known to be highly vascularized. Elevated expression of NE markers and increased angiogenesis are two prominent phenotypes of NEPC, and thus are expected to be linked. However, direct molecular links between these two phenotypes are still elusive, whose elucidation will substantially expand our knowledge in NEPC and enable the development of effective treatments for NEPC. Through RNAi & cDNA screening and functional validations, we previously discovered that GPCR-kinase 3 (GRK3) is essential preferentially for highly metastatic cancer cells as compared to lowly metastatic cancer cells. The mechanisms of GRK3 in prostate cancer progression were mostly unknown. Our new data indicate that GRK3 is significantly overexpressed in metastatic prostate tumors from patients, especially in NEPC. GRK3 promotes both angiogenesis and neuroendocrine differentiation in prostate cancer cells, indicating that it is a key missing link for these two phenotypes. Mechanistically, GRK3 enhances the epigenetic repressor activity of histone deacetylase 2 (HDAC2) to suppress key repressors of angiogenesis or NE phenotype. Through compound library screening, we have identified several compounds that block kinase activity of GRK3 much more potently than that of GRK2, the closest-related kinase to GRK3. Of note, our GRK3 inhibitors could substantially reduce angiogenesis and NE marker expression, as well as significantly inhibit NEPC cell growth in culture and in mouse xenografts. In summary, kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression. Its mechanism of actions is at least in part through enhancing HDAC2’s epigenetic activity. Results based on our novel GRK3 inhibitors suggest that GRK3 is a valuable new drug target for aggressive prostate cancer. Citation Format: Samira Naderinezhad, Guoliang Zhang, Zheng Wang, Dayong Zheng, Junwei Lian, Wenliang Li. Kinase GRK3 connects angiogenesis and neuroendocrine differentiation in prostate cancer progression by enhancing epigenetic activity of HDAC2 [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 LB032.

The Transmembrane Protein TM4SF3 Interacts With AR and AR-V7 and is Recruited to AR Target Genes.

Prostate cancer transitions from an early treatable form to the lethal castration-resistant prostate cancer (CRPC). Androgen receptor (AR) and constitutively active AR splice variants, such as AR-V7, may be major drivers of CRPC. Our laboratory recently identified a novel mechanism of AR regulation via the transmembrane protein transmembrane 4 superfamily 3 (TM4SF3), which exhibits a physical interaction, nuclear colocalization, and mutual stabilization with AR. Here, we have mapped the interaction domains within AR and TM4SF3 and discovered that TM4SF3 also physically interacts with AR-V7, regulating its protein stability and the viability of CRPC cells expressing AR-V7. Ubiquitination of TM4SF3 and AR-V7 was detected for the first time and TM4SF3 interaction with either AR or AR-V7 resulted in mutual deubiquitination of both proteins, showing that mutual stabilization results from deubiquitination. Interestingly, nuclear TM4SF3 was co-recruited to the promoters of AR- and AR-V7-regulated genes and required for their expression, showing that TM4SF3 interaction is critical for their transcriptional functions. The results collectively show the multiple critical regulatory functions of TM4SF3 on AR or AR-V7 in prostate cancer cells.

Druggable Metabolic Vulnerabilities Are Exposed and Masked during Progression to Castration Resistant Prostate Cancer.

There is an urgent need for exploring new actionable targets other than androgen receptor to improve outcome from lethal castration-resistant prostate cancer. Tumor metabolism has reemerged as a hallmark of cancer that drives and supports oncogenesis. In this regard, it is important to understand the relationship between distinctive metabolic features, androgen receptor signaling, genetic drivers in prostate cancer, and the tumor microenvironment (symbiotic and competitive metabolic interactions) to identify metabolic vulnerabilities. We explore the links between metabolism and gene regulation, and thus the unique metabolic signatures that define the malignant phenotypes at given stages of prostate tumor progression. We also provide an overview of current metabolism-based pharmacological strategies to be developed or repurposed for metabolism-based therapeutics for castration-resistant prostate cancer.

A COP1-GATA2 axis suppresses AR signaling and prostate cancer

Androgen receptor (AR) signaling is crucial for driving prostate cancer (PCa), the most diagnosed and the second leading cause of death in male patients with cancer in the United States. Androgen deprivation therapy is initially effective in most instances of AR-positive advanced or metastatic PCa. However, patients inevitably develop lethal castration-resistant PCa (CRPC), which is also resistant to the next-generation AR signaling inhibitors. Most CRPCs maintain AR expression, and blocking AR signaling remains a main therapeutic approach. GATA2 is a pioneer transcription factor emerging as a key therapeutic target for PCa because it promotes AR expression and activation. While directly inhibiting GATA2 transcriptional activity remains challenging, enhancing GATA2 degradation is a plausible therapeutic strategy. How GATA2 protein stability is regulated in PCa remains unknown. Here, we show that constitutive photomorphogenesis protein 1 (COP1), an E3 ubiquitin ligase, drives GATA2 ubiquitination at K419/K424 for degradation. GATA2 lacks a conserved [D/E](x)xxVP[D/E] degron but uses alternate BR1/BR2 motifs to bind COP1. By promoting GATA2 degradation, COP1 inhibits AR expression and activation and represses PCa cell and xenograft growth and castration resistance. Accordingly, GATA2 overexpression or COP1 mutations that disrupt COP1-GATA2 binding block COP1 tumor-suppressing activities. We conclude that GATA2 is a major COP1 substrate in PCa and that COP1 promotion of GATA2 degradation is a direct mechanism for regulating AR expression and activation, PCa growth, and castration resistance.

Spatio-temporal analysis of prostate tumors in situ suggests pre-existence of treatment-resistant clones

The molecular mechanisms underlying lethal castration-resistant prostate cancer remain poorly understood, with intratumoral heterogeneity a likely contributing factor. To examine the temporal aspects of resistance, we analyze tumor heterogeneity in needle biopsies collected before and after treatment with androgen deprivation therapy. By doing so, we are able to couple clinical responsiveness and morphological information such as Gleason score to transcriptome-wide data. Our data-driven analysis of transcriptomes identifies several distinct intratumoral cell populations, characterized by their unique gene expression profiles. Certain cell populations present before treatment exhibit gene expression profiles that match those of resistant tumor cell clusters, present after treatment. We confirm that these clusters are resistant by the localization of active androgen receptors to the nuclei in cancer cells post-treatment. Our data also demonstrates that most stromal cells adjacent to resistant clusters do not express the androgen receptor, and we identify differentially expressed genes for these cells. Altogether, this study shows the potential to increase the power in predicting resistant tumors.

Androgen receptor variant-7 regulation by tenascin-c induced src activation

BackgroundBone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC.MethodsProstate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation.ResultsUtilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells.ConclusionOverall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC.9BxMEZaJbqgytBg6QvviEaVideo

Squalene Epoxidase Metabolic Dependency Is a Targetable Vulnerability in Castration-Resistant Prostate Cancer.

This study reveals PTEN and p53 deficiency confers a dependence on SQLE-mediated cholesterol metabolism, providing insights for new therapeutic strategies for treating castration-resistant prostate cancer.

Abstract 1483: RNA interference seed-based approach to inhibit androgen signaling and induce prostate cancer cell death

Abstract The goal of this study is to identify RNA interference (RNAi) seed sequences that inhibit androgen signaling and induce toxicity in prostate cancer (PCa) cells. The high prevalence of acquired resistance to androgen deprivation therapy, as well as first and second generation androgen receptor (AR) antagonists, drives the development of lethal castration resistant prostate cancer (CRPC). As a result, PCa remains one of the leading causes of cancer related deaths in men. AR amplification, gain of function mutations and constitutively active splice isoforms of the AR, and the overexpression of AR coregulators, play considerable roles in promoting CRPC by sustaining AR activity in the presence of the low androgen environment and/or AR antagonists. The development of specific small molecule inhibitors of AR splice isoforms has been largely unsuccessful, and inhibitors of individual AR coregulators have not translated well into the clinic, likely due to their large number and functional redundancies. Therefore, novel therapeutic strategies for targeting androgen signaling in CRPC are necessary. Previous studies in our lab have demonstrated RNAi-mediated PCa cell death through seed region (nucleotides 2-8) complementarity of shRNA/siRNA guide strands to the 3’UTR of the AR and multiple AR coregulators and essential genes. In the current study, we used an unbiased novel seed-based shRNA screen to identify seed sequences that are toxic to different PCa cells, including CRPC cells expressing the clinically relevant constitutively active AR variant 7 (AR-V7). Common motifs were identified among toxic seed sequences, and the miRDB target prediction database was used to select those seeds that were predicted to target the AR and AR coregulators. Using RNA-seq analyses we confirmed that selected seed sequences inhibited global androgen signaling and identified potential direct targets known to be relevant to PCa. Conclusions: These studies have identified androgen signaling inhibitory sequences that have the potential to be used in RNAi-seed based therapies for PCa. The ability to inhibit the expression of the AR and multiple essential AR coregulators simultaneously with one agent, and the reduced likelihood of developed resistance, are considerable benefits of this approach. Citation Format: Joshua M. Corbin, Maria J. Ruiz-Echevarria. RNA interference seed-based approach to inhibit androgen signaling and induce prostate cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1483.

Abstract 1070: Dual inhibition of AR and β-catenin induces cell death in abiraterone-resistant prostate cancer

Abstract Abiraterone acetate has been clinically approved for treatment of patients with advanced-stage prostate cancer. It reduces testosterone production by blocking the enzyme cytochrome P450 17 alpha-hydroxylase. Despite exhibiting improved survival outcomes, almost all patients with advance-stage disease relapse, progressing to a more aggressive and lethal castration-resistant prostate cancer. Our pathway-related analysis and characterization of gene expression identified activation of the β-catenin pathway in abiraterone-resistant prostate cancer. Increased expression of androgen receptor (AR) and β-catenin and their crosstalk between distinct signaling pathways causes activation of AR target genes and regulatory networks for which overcoming innate or acquired resistance remains a major challenge. Here we show that co-targeting AR and β-catenin induces cell death in abiraterone-resistant prostate cancer. For these studies, we generated abiraterone-resistant C4-2B cells (C4-2B-Abi) by growing them in progressive concentration of abiraterone 5-20µM to develop resistance and maintained in 5µM abiraterone in the culture medium for >20 generations. Treatment of C4-2B-Abi cells individually with abiraterone (20µM) and ICG001 (2µM) for 72 h exhibited a partial suppressive effect in cell growth compared to C4-2B parental cell line. A combination of abiraterone and ICG001 (20µM + 2µM) exhibited marked cell growth inhibition with significant increase in G1-phase cell cycle arrest, inhibition of migration and invasion in C4-2B-Abi cells, compared to the parental counterpart. Furthermore, combination treatment led to significant reduction in cell proliferation as assessed by PCNA along with reduction in the protein expression of AR, AR-v7, CDC20, PSA, β-catenin and its downstream targets, cyclinD1 and c-Myc, which were more prominent in C4-2B-Abi cells, compared to parental C4-2B cells. Co-targeting AR and β-catenin potentiated cell death in C4-2B-Abi cells as evident by increased PARP cleavage. Together, our data identify activation of the β-catenin pathway as a major mechanism contributing to abiraterone resistance and demonstrate that combined treatment with abiraterone and ICG001 appears to be an effective regimen for treatment of abiraterone-resistant cancer cells. This opens new therapeutic modality for advance-stage castration-resistant prostate cancer patients. Citation Format: Ibrahim M. Atawia, Prem P. Kushwaha, Shiv S. Verma, Spencer Lin, Eswar Shankar, Osama Abdel-Gawad, Sanjay Gupta. Dual inhibition of AR and β-catenin induces cell death in abiraterone-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1070.

Abstract 5637: Transformation of the gut microbiome in response to androgen deprivation and the transition to castration-resistant prostate cancer

Abstract Androgen deprivation therapy (ADT) remains the standard care for men with advanced prostate cancer. Although initially effective, many patients fail to maintain durable responses and progress to lethal castration-resistant prostate cancer. Commensal bacteria play important role in maintaining homeostasis and proper immune function. However, ADT alters gut bacterial compositions and the ramifications being investigated. In this study, we used a mouse model of Pten-deficient prostate cancer to characterize differences in bacterial composition of gut bacteria in mice with castration naïve-prostate cancer (CNPC) and gut microbiota from mice three and six weeks after treatment ADT via surgical castration representing castration-sensitive prostate cancer (CSPC) and CRPC, respectively. Fecal samples were collected and processed for 16s rRNA amplicon sequencing. ADT resulted in significant changes to biological diversity and local diversity was highest in CSPC (Shannon index, P=0.04). Community composition also differed significantly different between the groups (Adonis OTU Bray-Curtis, P=0.001, NMDS stress=0.07). Despite dissimilarities between bacterial communities among the groups, CNPC and CRPC shared a greater similarity. Taxa associated with CNPC included Unclassified S247, Prevotella L. reuteri, unclassified Rikenellaceae and unclassified Bacteroidales; taxa associated with CSPC included R. gnavus, Dorea, unclassified Desulfovibrionaceae, Dehalobacterium, A. muciniphilia, Bifidobacterium, and B. producta; and taxa associated with CRPC included Turicibacter, R. flavefaciens, L. garvieae, unclassified Peptostreptococcaceae and unclassified Bacilli. Mice with CSPC and CRPC had an increased abundance genes associated with of catabolic metabolic pathways associated with aromatic compound, steroid Benzoate, and toluene degradation as well as genes associated to pathways corresponding to methane metabolism, phosphonate and phosphinate metabolism, and nitrogen metabolism. Genes enriched in fecal taxa from CNPC and CRPC mice were associated with folate biosynthesis, galactose metabolism, amino sugar and nucleotide sugar metabolism, and carbon fixation pathways in prokaryotes. This study provides preclinical evidence to support the association between the gut microbiome and prostate cancer phenotypes associated with sensitivity to ADT. Citation Format: Chisato Wakamori, Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Kazutoshi Fujita, Eri Banno, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuto Nishio, Hirotsugu Uemura. Transformation of the gut microbiome in response to androgen deprivation and the transition to castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5637.

Gremlin1 is a therapeutically targetable FGFR1 ligand that regulates lineage plasticity and castration resistance in prostate cancer.

Among the greatest hurdles in clinical management of prostate cancer (PCa) are the progression to lethal castration-resistant prostate cancer (CRPC) and the lack of suitable targeted therapies for advanced disease. Here we identify Gremlin1 as a ligand for fibroblast growth factor receptor 1 (FGFR1), which promotes lineage plasticity and drives castration resistance. Importantly, we generate a specific anti-Gremlin1 therapeutic antibody and demonstrate synergistic effect with androgen deprivation therapy (ADT) in CRPC. GREM1 transcription is suppressed by androgen receptor (AR) and released following ADT. We show that Gremlin1 binds to FGFR1 and activates downstream MAPK signaling. Gremlin1 interacts with FGFR1 differently to its canonical ligand FGF1, as revealed through protein structure docking and mutagenesis experiments. Altogether, our data indicate Gremlin1 as a promising candidate therapeutic target for CRPC.