Resistance In Castration-resistant Prostate Cancer Research Articles

Inhibition of enhancer of zeste homolog 2 (EZH2) overcomes enzalutamide resistance in castration-resistant prostate cancer

Enzalutamide, approved by the United States Food and Drug Administration in 2018 for the management of metastatic castration-resistant prostate cancer (CRPC), is an androgen receptor (AR) inhibitor. It blocks androgen binding to the AR, AR nuclear translocation, and AR-mediated DNA binding. Unfortunately, a considerable proportion of tumors eventually develop resistance during the treatment. The molecular mechanisms underlying enzalutamide resistance are not completely understood. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressor complex 2, has been proposed as a prognostic marker for prostate cancer (PCa). With the goal to test whether EZH2 also plays a critical role in acquisition of enzalutamide resistance in CRPC, here we examined whether EZH2 inhibition/depletion enhances the efficacy of enzalutamide in enzalutamide-resistant PCa cells. We show that combining the EZH2 inhibitor GSK126 with enzalutamide synergistically inhibits cell proliferation and colony formation and promotes apoptosis in enzalutamide-resistant PCa cells. EZH2 depletion also overcomes enzalutamide resistance in both cultured cells and xenograft tumors. Mechanistically, we found that EZH2 directly binds to the promoter of prostate-specific antigen and inhibits its expression in enzalutamide-resistant PCa cells. In agreement, bioinformatics analysis of clinical RNA sequencing data involving GSEA indicated a strong correlation between AR and EZH2 gene expression during PCa progression. Our study provides critical insights into the mechanisms underlying enzalutamide resistance, which may offer new approaches to enhance the efficacy of enzalutamide in CRPC.

Results from a phase Ib/II study of enzalutamide and metformin in men with castration resistant prostate cancer (CRPC).

5054 Background: Preclinical models have shown that autophagy is a cell survival mechanism to overcome treatment-induced stress and facilitate progression and resistance in CRPC. Metformin (met) demonstrates autophagy inhibition and resensitizes CRPC tumors to enzalutamide (enza) as a combination in in vitro models. A Phase Ib/II trial was conducted to evaluate the combination. Methods: Eligible patients (pts) had established CRPC, ECOG 0-2, adequate hematologic and organ function, and ≤ 2 prior treatments for CRPC. Major exclusion criteria included prior enza or met treatment, presence of brain metastases, history of DM2 or seizures. An initial cohort of 3 pts were treated with enza 160 mg PO daily and met 500 mg PO bid (DL1), with met dose escalated to 1000 mg PO bid (DL2) in subsequent 3 pts if dose-limiting toxicity (DLT) was observed in ≤1 pt in DL 1, and cycles of 28 days. Once primary objective of maximum tolerated dose (MTD) was established, additional pts were enrolled at MTD for a total of 24 evaluable pts. Secondary objectives of the study included PSA response and safety. Results: From 8/24/16-12/31/18, a total of 3 pts were enrolled to DL1, with no DLTs observed in that cohort, with an additional 21 enrolled to DL2; 12 remain on trial. Median age was 71, with 8/24 (33%) and 3/24 (12.5%) with prior docetaxel and abiraterone treatment, respectively. One DLT due to Grade 3 abdominal pain was observed at DL2 prior to establishing MTD. Grade 3 AEs observed in 1/24 (4%) of pts were diarrhea, fatigue, hypertension, lower GI bleed, myalgia; 2/24 (8%) had abdominal pain. One pt had Grade 4 hallucinations. Among pts who discontinued, 8 were discontinued for progression, 3 withdrew consent, and 1 experienced an SAE (seizure). Pts received median of 11 cycles (1-31) of treatment. PSA response ≥ 50% was observed in 19/24 (79%) pts. Conclusions: The combination of enzalutamide 160 mg PO daily and metformin 1000 mg PO bid is well-tolerated, and clinically active. Clinical trial information: NCT02339168.

Genetic Alterations Detected in Cell-Free DNA Are Associated With Enzalutamide and Abiraterone Resistance in Castration-Resistant Prostate Cancer.

PURPOSEAndrogen receptor (AR) gene alterations, including ligand-binding domain mutations and copy number (CN) gain, have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration-resistant prostate cancer (mCRPC). The goal of this study was to validate AR gene alterations detected in cell-free DNA (cfDNA) as markers of enzalutamide and abiraterone resistance in patients with mCRPC.METHODSPatients with mCRPC (N = 62) were prospectively enrolled between 2014 and 2018. Blood was collected before therapies—enzalutamide (n = 25), abiraterone (n = 35), or enzalutamide and abiraterone (n = 2) —and at disease progression. We used deep next-generation sequencing to analyze cfDNA for sequence variants and CN status in AR and 45 additional cancer-associated genes. Primary end points were prostate-specific antigen response, progression-free survival (PFS), and overall survival (OS).RESULTSElevated tumor-specific cfDNA (circulating tumor DNA) was associated with a worse prostate-specific antigen response (hazard ratio [HR], 3.17; 95% CI, 1.11 to 9.05; P = .031), PFS (HR, 1.76; 95% CI, 1.03 to 3.01; P = .039), and OS (HR, 2.92; 95% CI, 1.40 to 6.11; P = .004). AR ligand-binding domain missense mutations (HR, 2.51; 95% CI, 1.15 to 5.72; P = .020) were associated with a shorter PFS in multivariable models. AR CN gain was associated with a shorter PFS; however, significance was lost in multivariable modeling. Genetic alterations in tumor protein p53 (HR, 2.70; 95% CI, 1.27 to 5.72; P = .009) and phosphoinositide 3-kinase pathway defects (HR, 2.62; 95% CI, 1.12 to 6.10; P = .026) were associated with a worse OS in multivariable models.CONCLUSIONThese findings support the conclusion that high circulating tumor DNA burden is associated with worse outcomes to enzalutamide and abiraterone in men with mCRPC. Tumor protein p53 loss and phosphoinositide 3-kinase pathway defects were associated with worse OS in men with mCRPC. AR status associations with outcomes were not robust, and additional validation is needed.

Suppressive Role of Androgen/Androgen Receptor Signaling via Chemokines on Prostate Cancer Cells.

Androgen/androgen receptor (AR) signaling is a significant driver of prostate cancer progression, therefore androgen-deprivation therapy (ADT) is often used as a standard form of treatment for advanced and metastatic prostate cancer patients. However, after several years of ADT, prostate cancer progresses to castration-resistant prostate cancer (CRPC). Androgen/AR signaling is still considered an important factor for prostate cancer cell survival following CRPC progression, while recent studies have reported dichotomic roles for androgen/AR signaling. Androgen/AR signaling increases prostate cancer cell proliferation, while simultaneously inhibiting migration. As a result, ADT can induce prostate cancer metastasis. Several C-C motif ligand (CCL)-receptor (CCR) axes are involved in cancer cell migration related to blockade of androgen/AR signaling. The CCL2-CCR2 axis is negatively regulated by androgen/AR signaling, with the CCL22-CCR4 axis acting as a further downstream mediator, both of which promote prostate cancer cell migration. Furthermore, the CCL5-CCR5 axis inhibits androgen/AR signaling as an upstream mediator. CCL4 is involved in prostate carcinogenesis through macrophage AR signaling, while the CCL21-CCR7 axis in prostate cancer cells is activated by tumor necrotic factor, which is secreted when androgen/AR signaling is inhibited. Finally, the CCL2-CCR2 axis has recently been demonstrated to be a key contributor to cabazitaxel resistance in CRPC.

The influence of treatment sequence in the prognostic value of TMPRSS2-ERG as a biomarker of taxane resistance in castration-resistant prostate cancer.

235 Background: TMPRSS2-ERG expression at peripheral blood has been correlated with lower docetaxel benefit. This multicenter study prospectively assessed the role of TMPRSS2-ERG mRNA as a taxane-resistance biomarker in blood and retrospectively in tumors, and explored the impact of prior abiraterone/enzalutamide (A/E) in castration-resistant prostate cancer (CRPC) patients and in vitro. Methods: TMPRSS2-ERG was tested by quantitative reverse-transcription PCR. We included 204 patients (137 blood and 124 tumor samples) treated with taxanes. TMPRSS2-ERG expression was correlated with PSA-progression-free survival (PFS), radiological-PFS (RX-PFS), and overall survival (OS). Independent association with survival was evaluated by multivariate Cox modeling. ERG knockdown, combinatorial and sequential experiments with enzalutamide and docetaxel were performed in VCaP cells. Results: In the multivariate analysis prior A/E (HR: 1.833; P = 0.005) and blood TMPRSS2-ERG (HR: 2, 95%CI; 1.1-3.67; P = 0.018) , were independently associated to lower PSA-PFS. In patients without prior A/E, both blood and tumor TMPRSS2-ERG independently predicted lower PSA-PFS (HR 2.92; P = 0.014 and HR 1.82; P = 0.045, respectively) to taxanes. This was not observed in patients with prior A/E. There was a significant interaction between blood TMPRSS2-ERG detection and prior A/E related to PSA-PFS (P = 0.032) and RX-PFS (P = 0.009). In vitro stable ERG inhibition did not sensitize VCaP cells to docetaxel. Concomitant enzalutamide and taxanes were synergistic, but prior enzalutamide reduced docetaxel cytotoxicity in VCaP cells. Enzalutamide induced neuroendocrine markers and reduced E-cadherin expression. Conclusions: Prior hormone-therapy may influence taxanes response and TMPRSS2-ERG prognostic value. Thus, multiple and sequential biomarkers are needed in CRPC follow-up evaluation.

Preclinical study using circular RNA 17 and micro RNA 181c-5p to suppress the enzalutamide-resistant prostate cancer progression

Androgen-deprivation therapy (ADT) with newly developed antiandrogen enzalutamide (Enz) may increase the castration-resistant prostate cancer (CRPC) patients survival an extra 4.8 months. Yet eventually most patients may fail with development of Enz resistance. While recent clinical studies indicated that the increased expression of the androgen receptor (AR) splicing variant ARv7 might have key roles for the development of Enz resistance in CRPC, its detailed mechanism, especially its linkage to the circular RNAs (circRNAs), a form of non-coding RNA, however, remains unclear. Here we found from human clinical sample survey that circRNA17 (hsa_circ_0001427) has a lower expression in higher Gleason score PCa, and results from in vitro cell lines studies also revealed the lower expression in CRPC C4–2 Enz-resistant (EnzR-C4–2) cells compared to their parental Enz-sensitive (EnzS-C4–2) cells. Mechanism dissection indicated that suppressing circRNA17 in EnzS-C4–2 cells increased ARv7 expression that might then lead to increase the Enz resistance and cell invasion. Mechanism dissection demonstrated that Enz could suppress the circRNA17 expression at the transcriptional level via suppressing transcription of its host gene PDLIM5, and circRNA17 could regulate ARv7 expression via altering the expression of miR-181c-5p that involved the direct binding of miR-181c-5p to the 3′UTR of ARv7. Preclinical study using in vivo mouse model with xenografted EnzR-CWR22Rv1 cells revealed that adding circRNA17 or miRNA-181c-5p could suppress the EnzR-CWR22Rv1 cells growth. Together, results from these preclinical studies suggest that circRNA17 may function as suppressor to alter the Enz sensitivity and cell invasion in CRPC cells via altering the miR-181c-5p/ARv7 signaling and targeting this newly identified signaling may help in the development of a better therapy to further suppress the EnzR cell growth.

The Response of Prostate Cancer to Androgen Deprivation and Irradiation Due to Immune Modulation.

This study investigated changes in the immune system and the biological consequences of androgen deprivation therapy (ADT) and radiotherapy (RT) for augmenting the treatment response in prostate cancer, particularly for castration-resistant prostate cancer (CRPC). Human and murine prostate cancer cell lines were used to examine the response to ADT and RT in vitro and in vivo. Biological changes following treatment and related immune modulation in the tumor microenvironment were examined. Our results showed that CRPC cells were demonstrated to be more resistant to the RT and ADT treatments. ADT increased tumor inhibition following irradiation. The underlying changes included increased cell death, attenuated myeloid-derived suppressor cell recruitment, and an increase in the number of tumor-infiltrating T cells (TILs). Furthermore, when high-dose fractionated RT was given to the primary CRPC tumor, a smaller size of secondary non-irradiated tumor associated with increased TILs was noted in ADT-treated mice. In conclusion, treatment resistance in CRPC was associated with a more immunosuppressive microenvironment. Enhanced antitumor immunity was responsible for the augmented RT-induced tumoricidal effect induced by ADT. Immune modulation could be a promising strategy for prostate cancer, especially for metastatic CRPC.

Inhibition of cholesterol biosynthesis overcomes enzalutamide resistance in castration-resistant prostate cancer (CRPC)

Enzalutamide, a nonsteroidal second-generation antiandrogen, has been recently approved for the management of castration-resistant prostate cancer (CRPC). Although patients can benefit from enzalutamide at the beginning of this therapy, acquired enzalutamide resistance usually occurs within a short period. This motivated us to investigate the mechanism involved and possible approaches for overcoming enzalutamide resistance in CRPC. In the present study, we found that 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), a crucial enzyme in the mevalonate pathway for sterol biosynthesis, is elevated in enzalutamide-resistant prostate cancer cell lines. HMGCR knockdown could resensitize these cells to the drug, and HMGCR overexpression conferred resistance to it, suggesting that aberrant HMGCR expression is an important enzalutamide-resistance mechanism in prostate cancer cells. Furthermore, enzalutamide-resistant prostate cancer cells were more sensitive to statins, which are HMGCR inhibitors. Of note, a combination of simvastatin and enzalutamide significantly inhibited the growth of enzalutamide-resistant prostate cancer cells in vitro and tumors in vivo Mechanistically, simvastatin decreased protein levels of the androgen receptor (AR), which was further reduced in combination with enzalutamide. We observed that the decrease in AR may occur through simvastatin-mediated inhibition of the mTOR pathway, whose activation was associated with increased HMGCR and AR expression. These results indicate that simvastatin enhances the efficacy of enzalutamide-based therapy, highlighting the therapeutic potential of statins to overcome enzalutamide resistance in CRPC.

Abstract A083: Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3

Abstract Pten loss leading to constitutive PI3K/Akt/mTor activation is a well-characterized PCa survival pathway frequently activated in castration-resistant prostate cancer (CRPC). Unfortunately, targeted mono-therapies to PI3K/Akt/mTor with or without anti-androgen therapy failed to achieve significant response in clinical trials with CRPC patients. The mechanisms that drive PI3K resistance in CRPC are unknown. The androgen receptor (AR) is also a major driver of prostate cancer (PCa) survival, even in CRPC. However, surprisingly very little is known about how AR promotes PCa survival. We previously identified an AR-dependent survival pathway in androgen-dependent tumor cells whereby AR induction of integrin α6β1 and adhesion to laminin in the tumor microenvironment activates NF-κB to induce Bcl-xL. This pathway acts in parallel to the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin α6β1 is required to effectively kill tumor cells and suppress tumor growth. While this drug combination was effective in androgen-responsive tumors in vivo, it poorly suppressed CRPC. Thus, we hypothesized there are additional survival mechanisms operating in CRPC. To identify CRPC survival pathways, we mined the gene expression data comparing androgen-sensitive to CRPC tumors in a series of human PDX LuCaP models. We found the hypoxia-inducible protein, BNIP3, to be preferentially elevated in CRPC tumors, and this correlated with increased integrin α6β1 expression and enhanced AR signaling. Furthermore, elevated BNIP3 predicts for poor outcome in PCa patients. We discovered that androgen induces BNIP3 expression specifically in CRPC C4-2 cell lines, but not the androgen-dependent parental LNCaP cells. BNIP3 expression is dependent on AR induction of integrin α6β1, adhesion to laminin, and nuclear translocation of HIF-1α. The CRPC C4-2 cells are highly resistant to PI3K inhibition by PX866 compared to LNCaP when adherent to laminin, and siRNA-mediated loss of integrin α6 or BNIP3 sensitizes these cells to PI3K inhibition. Blocking Bnip3 expression in C4-2 cells injected orthotopically into mice reduced tumor growth and induced apoptosis. BNIP3 can promote survival by activating autophagy and/or mitophagy. Even though autophagic flux was specifically enhanced in laminin-adherent CRPC C4-2 cells, but not in LNCaP cells, this was not dependent on BNIP3. WT BNIP3 can restore PI3K drug resistance in shBNIP3-expressing cells, but the BNIP3 ΔLIR mutant, which is unable to target mitochondria to autophagosomes, cannot restore drug resistance. We have identified an AR-dependent survival pathway in CRPC involving BNIP3-induced mitophagy that is dependent on and mediated by integrin α6β1 adhesion to laminin that promotes resistance to PI3K inhibition. Laminin is abundant in prostate tissue, lymph nodes, and bone—the primary sites for PCa growth. Thus, laminin within the tumor microenvironment of both primary and metastatic PCa drives drug resistance in CRPC. Citation Format: Eric Nollet, Sourik Ganguly, Veronique Schulz, Eva Corey, Cindy K. Miranti. Cell adhesion to laminin, by integrin α6β1, mediates drug resistance to PI3K in castration-resistant prostate cancer through AR-dependent induction of BNIP3 [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A083.

Abstract A080: Caveolin-1 detected in tumor-derived exosomes induces cancer stem cell phenotypes in castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is by far the most common male malignancy and the third leading cause of cancer-related death in the US. Regardless of a great response of hormonal therapy for metastatic PCa patients, eventually PCa relapses to an end-stage castration-resistant prostate cancer (CRPC) leading to mortality. Although introducing newer androgen receptor (AR) pathway inhibitors is encouraging, treatment resistance in CRPC continues to remain a clinical problem. Clinical evidence of treatment resistant CRPC indicates carcinoma cell undergoing transdifferentiation. However, the mechanism as well as cell lineage are not fully characterized, which highlights an unmet clinical need to uncover potential therapeutic targets for targeted therapy to improve the overall survival of CRPC patients. Exosome is characterized as nano-grade small extracellular vesicle derived from the endosomal system from many kinds of cells. Therefore, exosome can carry many kinds of message molecules as a delivery vehicle in a paracrine or endocrine manner. Overall, tumor-derived exosomes (TDE) has been shown to play a critical role in promoting cancer progression or drug resistance. Our recent data have shown that TDE from PCa cells is able to induce neuroendocrine differentiation of CRPC, which leads us to further elucidate the role of PCa-derived exosome. Previously by using xenograft model, we have reported that elevated caveolin-1 (Cav-1) in LNCaP cell can accelerate its tumor growth but also increase the tumor incidence of low tumorigenic cells in a paracrine manner, implying that Cav-1 can be secreted extracellualrly. However, the functional role of secreted Cav-1 in PCa is still being debated. The expression of Cav-1 was considered to function as a suppressor of cell transformation and as preventing the development and metastases of mammary tumor; on the other hand, Cav-1 served as an oncogene that correlates with PCa progression. Furthermore, recent studies have shown that Cav-1 expression is associated with chemoresistance in multiple cancers as well as cancer stem cell (CSC) property in lung cancer. CSC theory has raised significant attention in recent years because of its self-renewal and quiescent nature, which underlies its role as a cancer-initiating cell population responsible for recurrent tumor with treatment resistance. In this study, we purified TDE from various PCa cell lines including either Cav-1 positive (Cav-1High) or negative (Cav-1low) cells using membrane-based affinity chromatography, and further characterized its size and number using Zetaview (Particle Matrix, Mebane, NC) and Exocet quantitation kit (SBI, Palo Alto, CA) separately. We found that TDE derived from metastatic PCa cells without AR expression is capable of inducing CSC phenotypes in AR-expressing recipient cells. Further analyses demonstrated that the high accumulation of Cav-1 in TDE appears to be a potential candidate. Indeed, Cav-1High cells or exogenous Cav-1-treated PCa cell lines can increase CSC phenotypes in vitro and in vivo. Mechanistically, the presence of Cav-1 is able to increase stem cell transcription Yamanaka factors, including KLF4, SOX2, OCT4, and Nanog. Furthermore, from microarray screening, we found RPC32 as another potential Cav-1 downstream effector, which is characterized as a subunit of RNA polymerase III (Pol III). RPC32 has two different isoforms (α and β), and only RPC32α is known to be highly regulated and expressed in stem cell and transformed cells. Therefore, we hypothesize that RPC32α is responsible for the elevated Yamanaka factors protein expression leading to CSC development. Nevertheless, we conclude that the presence of Cav-1 in PCa TDE plays an inductive role in the appearance of treatment-resistant CRPC in a paracrine manner, which provides a potential prognostic marker and therapeutic target. Citation Format: Chun-Jung Lin, Andrew Dang, Elizabeth Hernandez, Jer-Tsong Hsieh. Caveolin-1 detected in tumor-derived exosomes induces cancer stem cell phenotypes in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A080.

Analysis of cabazitaxel-resistant mechanism in human castration-resistant prostate cancer.

Cabazitaxel (CBZ) is approved for docetaxel‐resistant castration‐resistant prostate cancer (CRPC). However, efficacy of CBZ for CRPC is limited and there are no effective treatments for CBZ‐resistant CRPC. In order to investigate the CBZ‐resistant mechanism, the establishment of a CBZ‐resistant cell line is urgently needed. We established CBZ‐resistant CRPC cell lines DU145CR and PC3CR by incubating DU145 and PC3 cells with gradually increasing concentrations of CBZ for approximately 2 years. We analyzed the gene expression profiles and cell cycle changes using microarray and flow cytometry. Pathway analysis revealed DU145CR cells had enhanced gene clusters of cell division and mitotic nuclear division. Enhancement of ERK signaling was detected in DU145CR cells. DU145CR cells had resistance to G2/M arrest induced by CBZ through ERK signaling activation. The MEK inhibitor PD184352 significantly inhibited cell proliferation of DU145CR. In contrast to DU145CR, PC3CR cells had enhancement of PI3K/AKT signaling. The PI3K/mTOR inhibitor NVP‐BEZ 235 had a significant antitumor effect in PC3CR cells. Cabazitaxel ‐resistant CRPC cells established in our laboratory had enhancement of cell cycle progression signals and resistance to G2/M arrest induced by CBZ. Enhancement of ERK signaling or PI3K/AKT signaling were detected in the cell lines, so ERK or PI3K/AKT could be therapeutic targets for CBZ‐resistant CRPC.

Abstract 5904: Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression

Abstract Purpose Prostate cancer (CaP) driven by androgen receptor (AR) can be targeted therapeutically by androgen deprivation therapy (ADT); however, in 10-20% of cases, ADT fails, allowing disease recurrence. Mechanisms for recurrence include dysregulation of AR at the gene, RNA, and protein level. Recently, AR negative cell growth has been implicated as a mechanism of resistance. This study aims to identify mechanisms of drug resistance in castration-resistant prostate cancer (CRPC) that can be exploited therapeutically to reduce disease recurrence. Experimental Procedures Using the BCaP model, a novel series of cell lines derived from a single human prostate epithelial cell, we can study the progression of CaP from benign to metastasis. Additionally, using the LNCaP-C4 series we can study progression to CRPC. To determine the RNA binding capacity of DDX3, we utilized 1) RIP-qPRC for identification of mRNA targets and 2) RNAscope for visual colocalization. Changes in RNA and protein expression were determined using qPCR, Western blot, and IF. Results DDX3 is an ATP-dependent RNA helicase that can aid translation of target mRNAs, or in stress conditions, form ribonucleoprotein granules that prevent translation. While DDX3 is implicated in several cancers, its role as a translational regulator in CaP remains unstudied. In BCaP and LNCaP-C4, DDX3 protein expression increases through progression, concurrent with localization to cytoplasmic puncta. RIP-qPCR identified AR as an mRNA target of DDX3 in the metastatic and CRPC cell lines. Because of the dual role of DDX3 in translational control, AR expression in these models was investigated; while AR mRNA expression increased through progression, AR protein expression decreased. Treatments in vitro with RK33, a commercially available inhibitor of DDX3, restored the protein expression of AR and downstream AR signaling (PSA, Nkx3.1). These data suggest DDX3 acts as a translational repressor of AR in metastasis and CRPC. To confirm that increased DDX3 expression is sufficient to reduce AR protein expression, we overexpressed DDX3 using two models: induced cellular stress and a DDX3 expression vector. Here, we saw DDX3 localized to puncta, and subsequent reduction in AR and PSA protein. Because DDX3 is sufficient to reduce AR protein expression in metastases and CRPC, co-treatment of DDX3 inhibitors with anti-androgen therapy may prevent AR negative cell growth underlying recurrence. in vitro co-treatments with RK33 and bicalutamide, an AR antagonist, show decreased proliferation compared to either treatment alone, suggesting increased efficacy of bicalutamide with DDX3 inhibition. Conclusions DDX3 as a repressor of AR translation could have clinical implications as a mechanism of resistance to ADT. Based on preliminary data, DDX3 could be contributing to the regulation of AR post-transcriptionally, and targeting DDX3 could reduce resistance and disease recurrence. Citation Format: Jordan E. Vellky, William Ricke. Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression [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 5904.

Abstract 1813: The role of androgen receptor variant AR-V9 in prostate cancer

Abstract The expression of androgen receptor (AR) variants is a frequent, yet poorly-understood mechanism of clinical resistance to AR-targeted therapy for castration-resistant prostate cancer (CRPC). Among the multiple AR variants expressed in CRPC, AR-V7 is considered the most clinically-relevant AR variant due to broad expression in CRPC, correlations of AR-V7 expression with clinical resistance, and growth inhibition when AR-V7 is knocked down in CRPC models. Therefore, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The aim of this study was to understand whether other AR variants are co-expressed with AR-V7 and promote resistance to AR-targeted therapies. To test this, we utilized RNA-seq to characterize AR expression in CRPC models. RNA-seq revealed the frequent coexpression of AR-V9 and AR-V7 in multiple CRPC models and metastases. Furthermore, long-read single-molecule real-time (SMRT) sequencing of AR isoforms revealed that AR-V7 and AR-V9 shared a common 3'terminal cryptic exon. To test this, we knocked down AR-V7 in prostate cancer cell lines and confirmed that AR-V9 mRNA and protein expression were also impacted. In reporter assays with AR-responsive promoters, AR-V9 functioned as a constitutive activator of androgen/AR signaling. Similarly, infection of AR-V9 lentiviral construct in LNCaP cells induced androgen-independent cell proliferation. In conclusion, these data implicate co-expression of AR-V9 with AR-V7 as an important component of constitutive AR signaling and therapeutic resistance in CRPC. Citation Format: Yeung Ho, Jamie L. Van Etten, Christine Henzler, Yingming Li, Rendong Yang, Elizabeth Tseng, Ting Hon, Tyson Clark, Scott M. Dehm. The role of androgen receptor variant AR-V9 in 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 1813.

Abstract 1394: Resistance to BET inhibitor leads to new therapeutic vulnerabilities in castration resistant prostate cancer

Abstract BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration resistant prostate cancer (CRPC) cells. Bromodomain and extraterminal protein (BET) inhibitors displace BRD4 protein from chromatin, resulting in the inhibition of oncogenic transcriptional programs. Several BET inhibitors (BETi) are in phase I clinical trials for a variety of malignancies including CRPC. Here we describe a general mechanism of acquired resistance to BETi due to modulation of cellular pathways that are amenable to targeted therapies in CRPC cells. BETi resistant CRPC cells displayed cross resistance to a variety of BETi in the absence of gatekeeper mutations or persistent drug pump activation. Resistant cells exhibited reduced chromatin bound BRD4, and were less sensitive to Proteolysis Targeting Chimeric (PROTAC) -mediated degradation or genetic knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR-signaling due to CDK9-mediated serine-81 phosphorylation of AR, with a consequent increase in sensitivity to CDK9 inhibitors and anti-androgens in BETi resistant cells. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DNA damage response (DDR) genes was observed due to the loss of BRD4 from their proximal promoter regions in the resistant cells, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy in CRPC. However, data showing the reactivation of AR-signaling and increased DNA damage in the BETi resistant cells provide unique opportunities for combination therapies in managing CRPC. Citation Format: Aishwarya S. Pawar, Irfan A. Asangani. Resistance to BET inhibitor leads to new therapeutic vulnerabilities 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 1394.

Abstract 5834: Epigenetic re-programming sensitizes prostate cancer cells to anti-androgen therapy

Abstract Prostate cancer (Pca) remains the second leading cause of cancer-related deaths in men in the United States. Although advanced Pca usually responds to therapies that suppress androgen-axis signaling, resistance inevitably develops, leading to the emergence of castration-resistant prostate cancer (CRPC). Importantly, the clinical efficacy of novel therapies targeting androgen receptor (AR) signaling, such as abiraterone and enzalutamide, has confirmed that most CRPC cases demonstrate intact AR signaling. Since resistance to these therapies inevitably develops, approaches that improve the response duration and address the key pathways of resistance are warranted. Mechanisms underlying androgen deprivation therapy (ADT) resistance remain poorly defined, but may depend on epigenetic reprogramming. Our group previously showed that EZH2 is overexpressed and associates with Pca progression and poor prognosis. We substantiated this finding by screening a library of epigenetic inhibitors for their ability to render ADT-resistant CRPC cells sensitive to enzalutamide. Of these compounds, the EZH2 inhibitors facilitated a substantial increase in enzalutamide-mediated growth inhibition. Given these data, we hypothesized that EZH2-mediated epigenetic reprogramming might be crucial for the development and maintenance of ADT resistance, and targeting EZH2 might sensitize resistant CRPC cells to ADT. To test this hypothesis, we performed loss-of-function of EZH2 experiments on multiple CRPC cell lines, and determined that loss-of-function of EZH2 significantly sensitized Pca cells to ADT. Interestingly, by employing RNA-seq and ChIP-seq approaches to dissect the regulatory role of EZH2, we found that EZH2 inhibition significantly up-regulates AR signaling by changing the AR cistrome. Further experimentation confirmed that the loss-of-function of EZH2-mediated deregulation of AR signaling strengthens the AR-dependency of Pca, thus increasing the sensitivity of Pca to ADT. To co-target EZH2 and AR with greater efficiency, we employed antisense oligonucleotides (ASOs) directed against EZH2 and AR at the mRNA level. We determined that co-targeting EZH2 and AR by ASOs constitutes a novel, viable therapy for CRPC in vitro and in vivo. In conclusion, we have identified EZH2 as a critical epigenetic regulator for ADT resistance and have shown that co-targeting EZH2 and AR by ASOs to be a promising, potential strategy for preventing and treating CRPC. Citation Format: Lanbo Xiao, Jean Ching-Yi Tien, Josh Vo, Abhijit Parolia, Lisha Wang, Mengyao Tan, Yuanyuan Qiao, Sudhanshu Shukla, Xiaoju Wang, Heng Zheng, Fengyun Su, Xuhong Cao, Arul Chinnaiyan. Epigenetic re-programming sensitizes prostate cancer cells to anti-androgen therapy [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 5834.

Cdc20/p55 mediates the resistance to docetaxel in castration-resistant prostate cancer in a Bim-dependent manner.

At least to date, no effective treatment for advanced castration-resistant prostate cancer (CRPC) has been established. Recent studies indicated that cell division cycle 20 homolog (Cdc20) overexpression is associated with poor prognosis in patients with castration-resistant prostate cancer. However, the mechanism of Cdc20 in the development of docetaxel resistance in CRPC remains elusive. In this study, the transcription of Cdc20 was confirmed in three independent CRPC cell lines derived from different tissues, including LNCaP, PC3, and DU145. Docetaxel resistant (DR) cell lines were generated within the background of DU145 and PC3. The protein levels of Cdc20 and the biological phenotype were detected in both wild-type and DR cell lines. To further explore the mechanism of Cdc20 overexpression, stable cell lines with Cdc20 or Bcl-2 interacting mediator of cell death (Bim) deprivation were generated and examined for biological parameters. In addition, a specific Cdc20 inhibitor was used in DR cell lines to explore the potential solution for docetaxel resistant CRPC. Here, we identified Cdc20 is overexpressed in docetaxel resistant CRPC cell lines, including LNCaP, PC3, and DU145. We also reported that DR cell lines, which mimic the recurrent prostate cancer cells after docetaxel treatment, have higher levels of Cdc20 protein compared with the CRPC cell lines. Interestingly, the protein levels of Bim, an E3 ligase substrate of Cdc20, were decreased in DR cell lines compared with the wild-type, while the mRNA levels were similar. More importantly, in DR cell lines, the biological phenotype induced by Cdc20 deletion could be significantly reversed by the additional knockdown of Bim. As a result, docetaxel resistant prostate cancer cells treated with the pharmacological Cdc20 inhibitor became sensitive to docetaxel treatment. In conclusion, our data collectively demonstrated that Cdc20 overexpression facilitates the docetaxel resistant of the CRPC cell lines in a Bim-dependent manner. Furthermore, additionally targeting Cdc20 might be a promising solution for the treatment of the CRPC with docetaxel resistance.

Niclosamide in combination with abiraterone and prednisone in men with castration-resistant prostate cancer (CRPC): initial results from a phase Ib/II trial.

192 Background: Prostate cancer is driven by androgen receptor (AR) signaling. The variant AR-V7 lacks the ligand binding domain, constitutively activates the AR pathway, and confers resistance to Abiraterone (Abi) and enzalutamide (Enza). We discovered that the anti-helminthic drug niclosamide targets AR-V7 and sensitizes resistant CRPC to Enza and Abi. We hypothesize that niclosamide/PDMX1001 potentiates the efficacy of Abi against CRPC. Here we report the initial results of this ongoing investigator-initiated phase Ib/II trial. Methods: Eligible patients (pts) have progressive CRPC with serum testosterone < 50 ng/dl. No prior Abi was allowed. In the Phase Ib cohort, pts received Abi 1000 mg PO qd, prednisone 5 mg PO bid, with intrapatient dose-escalation of niclosamide/PDMX1001 from 400 mg PO bid to 1600 mg PO tid. Trough niclosamide/PDMX1001 levels were measured. The Phase II cohort will enroll 27 patients with detectable AR-V7 in the peripheral blood. Co-primary endpoints include toxicity and response as determined by the Prostate Cancer Working Group 2 criteria. Results: Of 6 pts (age 74-83) in the Phase Ib cohort, five pts tolerated a niclosamide/PDMX1001 dose of 1,600 mg po tid without dose limiting toxicity; per protocol, this is the recommended Phase II dose. Niclosamide/PDMX1001 trough level was 0.305 and 0.496 µM in the two pts analyzed thus far, higher than the target level of 0.1µM required for anti-cancer activity. Of 6 pts, two pts achieved complete PSA response ( < 0.01 ng/ml), compared to historical control 0/30 pts treated with Abi alone; two with partial PSA response (≥50% decrease). Of the remaining two pts, one was prematurely taken off from the study after one cycle because of rising PSA, and the other had PSA decrease of 17.1%, but biopsy of the only enlarged lymph node showed all necrotic tissue. The only toxicity was Grade 1 nausea and diarrhea. The Phase II cohort will now enroll. Molecular correlative studies will be presented. Conclusions: The combination of niclosamide/PDMX1001, Abi and prednisone is well tolerated with promising safety and efficacy data. Targeted serum trough levels of niclosamide are clinically achievable. Clinical trial information: NCT02807805.

A phase Ib/II trial of indomethacin and enzalutamide to treat castration-resistant prostate cancer (CRPC).

TPS394 Background: Enzalutamide (Enza) and abiraterone (Abi) are commonly used to treat CRPC. Resistance is the most common cause of treatment failure. We discovered that a critical steroidogenic enzyme AKR1C3 was significantly elevated and contributed to intratumoral androgen synthesis in Enza-resistant prostate cancer cells and tumors. Overexpression of AKR1C3 induced androgen receptor variant 7 (AR-V7) expression, while inhibition of AKR1C3 downregulated AR-V7. We then discovered that indomethacin (Indo) inhibited AKR1C3 activation and sensitized resistant CRPC cells to Enza and Abi. One patient accidentally took Indo and achieved biochemical as well as radiological response of his prostate cancer. These findings prompted us to design a clinical trial to test the combination of Indo with Enza for the treatment of CRPC and to study the underlying mechanisms of action and resistance. Methods: This investigator-initiated single-arm Phase Ib/II trial enrolls patients with progressive CRPC after Abi, adequate vital organ function, ECOG performance status 0-2, and serum testosterone < 50 ng/dl. Major exclusion criteria include prior Enza treatment, brain metastasis and history of seizure. In the Phase Ib cohort, patients receive Enza 160 mg po qd and Indo 50 mg po tid to determine toxicity. The Phase II expansion will enroll 26 patients with 21 evaluable patients. This sample size provides 90% power to detect, at the 0.05 level (1-sided), the difference between a PSA response rate of 50% expected with the study treatment and a historical control of 20% with Enza alone. Co-primary endpoints are safety and PSA response of ≥50% decrease. Secondary endpoints include overall response rate as determined by the Prostate Cancer Working Group 2 criteria (PCWG2), progression-free survival and overall survival. Molecular correlative studies are exploratory endpoints. Serum and intratumoral androgen levels, full-length AR, AR-V7 and AKR1C3 will be measured to assess the effect of the combination therapy. To date, 4 patients have been enrolled to the trial (clinicaltrials.gov Identifier No: NCT02935205; this trial is funded by DoD Prostate Cancer Research Program IMPACT award). Clinical trial information: NCT02935205.

PTEN Loss Promotes Intratumoral Androgen Synthesis and Tumor Microenvironment Remodeling via Aberrant Activation of RUNX2 in Castration-Resistant Prostate Cancer.

Purpose: Intratumoral androgen synthesis (IAS) is a key mechanism promoting androgen receptor (AR) reactivation and antiandrogen resistance in castration-resistant prostate cancer (CRPC). However, signaling pathways driving aberrant IAS remain poorly understood.Experimental Design: The effect of components of the AKT-RUNX2-osteocalcin (OCN)-GPRC6A-CREB signaling axis on expression of steroidogenesis genes CYP11A1 and CYP17A1 and testosterone level were examined in PTEN-null human prostate cancer cell lines. Pten knockout mice were used to examine the effect of Runx2 heterozygous deletion or abiraterone acetate (ABA), a prodrug of the CYP17A1 inhibitor abiraterone on Cyp11a1 and Cyp17a1 expression, testosterone level and tumor microenvironment (TME) remodeling in vivoResults: We uncovered that activation of the AKT-RUNX2-OCN-GPRC6A-CREB signaling axis induced expression of CYP11A1 and CYP17A1 and testosterone production in PTEN-null prostate cancer cell lines in culture. Deletion of Runx2 in Pten homozygous knockout prostate tumors decreased Cyp11a1 and Cyp17a1 expression, testosterone level, and tumor growth in castrated mice. ABA treatment also inhibited testosterone synthesis and alleviated Pten loss-induced tumorigenesis in vivoPten deletion induced TME remodeling, but Runx2 heterozygous deletion or ABA treatment reversed the effect of Pten loss by decreasing expression of the collagenase Mmp9.Conclusions: Abnormal RUNX2 activation plays a pivotal role in PTEN loss-induced IAS and TME remodeling, suggesting that the identified signaling cascade represents a viable target for effective treatment of PTEN-null prostate cancer, including CRPC. Clin Cancer Res; 24(4); 834-46. ©2017 AACR.

Genomic Resistance Patterns to Second-Generation Androgen Blockade in Paired Tumor Biopsies of Metastatic Castration-Resistant Prostate Cancer

PurposePatients with castration-resistant prostate cancer (CRPC) receive second-generation androgen-deprivation therapy, but frequently experience relapse or do not respond. Understanding the genetic mechanisms of resistance will help to identify strategies and biomarkers that are essential for the next line of therapy.Patients and MethodsWe analyzed whole exomes of patient-matched pre- and post-treatment tumors from patients with CRPC. These patients had received the secondary androgen-deprivation therapy agent, abiraterone, which suppresses androgens to below castration levels, or enzalutamide, which competitively inhibits the key androgen signaling effector, androgen receptor.ResultsWe observed that abiraterone-resistant tumors harbored alterations in AR and MYC, whereas enzalutamide-resistant tumors gained alterations in cell-cycle pathway genes, such as mutation in cyclin-dependent kinase N2A (CDKN2A) or amplification of CDK6. Experimentally, overexpressing cell-cycle kinases promoted enzalutamide resistance in androgen-sensitive LnCAP cells that was mitigated via CDK4/6 blockade—palbociclib and ribociclib.ConclusionCDK4/6-mediated resistance observed in preclinical experiments suggests that CDK4/6 amplifications may sufficiently promote enzalutamide resistance in CRPC, and that these patients may respond to palbociclib or ribociclib. The overall observations suggest that, in genomically selected advanced CRPC, clinical strategies against abiraterone- or enzalutamide-resistant tumors may require treatment strategies that are tailored to the resistance mechanisms that are specific to those patient subpopulations.