Castration-resistant Prostate Cancer Models Research Articles

Abstract 4615: Development of an antibody-drug conjugate with broad anticancer activity

Abstract Treatment for patients with advanced solid tumors that include triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and castrate resistant prostate cancer (CRPC), as well as Mantle Cell Lymphoma (MCL), while increasing survival is not curative. We have identified a membrane bound protease, “activated” matriptase, as an attractive target antigen for highly selective antibody delivery of cytotoxins as activated matriptase expression is restricted to epithelial tumors and some B-cell lymphomas. We generated a novel ADC by linking M69, a mouse antibody specific to activated matriptase, to monomethyl auristatin E (MMAE) via a PEGylated, releasable di-peptide linker as a proof-of-principle prototype. Both in cell lines and in human xenograft models of TNBC, NSCLC, CRPC and MCL, the conjugate was found to exhibit potent anticancer activity against all of these tumor types without toxicity. Encouraged by these results, we are also exploring the use of this ADC against gastric and pancreatic cancer, and in combination with chemotherapy and immunotherapy. As this is a mouse antibody, we are also generating a chimeric antibody for toxicity studies in primates. Citation Format: Siang-Yo Lin, Zoltan Szekely, Chen-Yong Lin, Joseph R. Bertino, Gulam Mohmad Rather. Development of an antibody-drug conjugate with broad anticancer activity [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 4615. doi:10.1158/1538-7445.AM2017-4615

Abstract LB-320: Reconstituting B55/protein phosphatase 2A activity in B55/PP2A-defective prostate cancer cells suppresses proliferation and tumorigenicity through pleiotropic mechanisms

Abstract Prostate cancer accounts for close to 30,000 deaths annually in the United States. The most frequent alteration in the prostate oncogenome landscape is loss of chromosome 8p, which is also observed in other epithelial tumors. This region harbors NKX3.1, a prostate specific tumor suppressor, but 8p copy number loss does not correlate with its mRNA expression. The PPP2R2A gene, which encodes a regulatory subunit of protein phosphatase 2 (PP2A) designated B55, is located in 8p21.2 and is hemizygously lost in more that 50% of prostate adenocarcinomas. TCGA data analysis shows that hemizygous loss of PPP2R2A correlates with its reduced expression, poorer prognosis and that loss of PPP2R2A increases with metastasis to more than 80%. Of note, while homozygous loss is less common (4-10%), deletion of other subunits of the B55, but not other PP2A regulatory families, increases dramatically with metastasis despite their independent chromosomal locations. However, functional evidence that PPP2R2A acts as an haploinsufficient tumor suppressor and the potential mechanisms implicated are lacking. We have identified prostate cancer (PrCa) cell lines with reduced expression of B55 and shown that reconstitution of B55 expression reduces proliferation and induces senescence and loss of viability. B55 reconstitution in PC3 cells, an AR-negative model of human castration-resistant prostate cancer (CRPC), also blocks transformation in vitro and tumorigenicity in SCID mice. Reconstitution of B55 in PCa cells with reduced expression (PC3 and DU145 cells) induces cell cycle defects in G1, G2/M and mitosis diminishing and delaying phosphorylation of pRB pocket proteins and mitotic substrates and altering associated kinase networks. Mitogenic signaling is also attenuated. Importantly, the effects of B55 reconstitution are at least partially mimicked via treatment of these cells with PP2A activating drugs, which have anti-tumorigenic activity, indicating that development of specific B55/PP2A activating drugs targeting the unaltered B55 alleles remaining in PCa cells may have therapeutic potential for a high proportion of prostate tumors and perhaps other epithelial tumors such as luminal B breast and ovarian cancers which share this alteration. Citation Format: Ziran Zhao, Alison Kurimchak, Petr Makhov, Katherine Johnson, Vladimir Kolenko, James Duncan, Xavier Graña. Reconstituting B55/protein phosphatase 2A activity in B55/PP2A-defective prostate cancer cells suppresses proliferation and tumorigenicity through pleiotropic mechanisms [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 LB-320. doi:10.1158/1538-7445.AM2017-LB-320

Abstract 836: ATR inhibitor BAY 1895344 shows potent anti-tumor efficacy in monotherapy and strong combination potential with the targeted alpha therapy Radium-223 dichloride in preclinical tumor models

Abstract The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, induction of DNA repair, or cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first and only approved targeted alpha therapy so far. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It exhibits strong cytotoxic effects on adjacent cells via the induction of DNA DSB. Here, we disclose for the first time the structure and functional characterization of the novel ATR kinase inhibitor BAY 1895344. In vitro, BAY 1895344 is a selective low-nanomolar inhibitor of ATR kinase activity, potently inhibiting proliferation of a broad spectrum of human tumor cell lines (median IC50 of 78 nM). A clear separation between highly sensitive (IC50 <10 nM) and less sensitive cell lines was observed. The majority of the sensitive cell lines are characterized by mutations affecting the ATM (ataxia telangiectasia mutated) pathway. In cellular mechanistic assays BAY 1895344 inhibited hydroxyurea-induced H2AX phosphorylation demonstrating the anticipated mode of action. BAY 1895344 is an ATR inhibitor that exhibits strong in vivo anti-tumor efficacy in monotherapy in a variety of xenograft models of different indications that are characterized by DDR deficiencies, inducing stable disease in ovarian and colorectal cancer or even complete tumor remission in mantle cell lymphoma models. In addition, we could demonstrate that combination treatment with BAY 1895344 and Radium-223 exhibits clear synergistic anti-tumor activity in a bone metastases xenograft model of CRPC. Our findings validate the concept of synthetic lethality of genetically determined DNA repair deficiency and ATR blockade by demonstrating strong monotherapy efficacy of the highly potent ATR inhibitor BAY 1895344 in a variety of tumor indications. Furthermore, the mechanism-based combination potential of DNA damage induction by Radium-223 with BAY 1895344 creates a powerful new treatment option for CRPC patients with bone metastases. The start of clinical investigation of BAY 1895344 is planned early 2017. Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Philip Lienau, Gesa Deeg, Eleni Lagkadinou, Li Liu, Sven Golfier, Christoph Schatz, Arne Scholz, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. ATR inhibitor BAY 1895344 shows potent anti-tumor efficacy in monotherapy and strong combination potential with the targeted alpha therapy Radium-223 dichloride in preclinical tumor models [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 836. doi:10.1158/1538-7445.AM2017-836

Abstract 5143: Fusogenic targeted liposomes: novel nanotherapy for specific treatment of prostate cancer

Abstract Metastatic or castrate-resistant prostate cancer is the second-leading cause of cancer mortality in males. In the past 3 years, chemotherapies have extended survival, but efficacy is limited by dose-limiting toxicities due to suboptimal biodistribution. Here, we have addressed the lack of specificity and accumulation in Castrate Resistant Prostate Cancer (CRPC) by developing a unique nano-carrier for drug delivery: molecular targeted fusogenic liposomes. Previously, we have developed a platform whereby lipid nanoparticles are formulated with fusion associated small transmembrane (FAST) protein p14. The p14 protein catalyzes mixing of the nanoparticle lipids with target plasma membrane to deliver the cargo directly into the cytoplasm. In this study, we have engineered the p14 protein to incorporate the prostate cancer targeting ligand bombesin, which binds specifically to the Gastrin-releasing Peptide (GRPR) that is overexpressed in prostate cancer. We hypothesized that this novel targeted fusogenic liposome formulation would significantly improve the biodistribution and efficacy of chemotherapy. A clinical liposomal doxorubicin formulation (DOXIL) was modified to incorporate targeted fusogenic p14 protein, and the efficacy and biodistribution of these new formulations was evaluated using in vitro and in vivo models of CRPC. In PC3 cells, compared to conventional liposomes, intracellular levels of doxorubicin are increased by 15 and 25 times when p14 or p14-bombesin liposomes are used. Additionally, the IC50 is reduced from 85mM to 2mM. In mice bearing PC3 tumors treated with targeted fusogenic liposomal doxorubicin, we observed improved tumor growth inhibition by 57% (vs control) and 1.5 fold increased accumulation in tumors within 24h after administration. These proof of concept experiments indicate that molecular targeted fusogenic nanomedicines can improve both the intracellular delivery and tumor accumulation of chemotherapy, improving outcomes in this preclinical model. This highlights the potential for improved outcomes in patients with CRPC through the enhancement of approved drugs. Note: This abstract was not presented at the meeting. Citation Format: Jihane Mriouah, Rae Lynn Nesbitt, Susan Richter, Melinda Wuest, Desmond Pink, Deborah Sosnowski, Roy Duncan, Frank Wuest, Andries Zijlstra, John Lewis. Fusogenic targeted liposomes: novel nanotherapy for specific treatment of 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 5143. doi:10.1158/1538-7445.AM2017-5143

Abstract 3205: A nanomolar potency small-molecule compound against castration-resistant and bone metastatic prostate cancer

Abstract A standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that suppresses androgen receptor (AR) signaling axis. Although initially responsive, most patients receiving ADT eventually develop metastatic castration-resistant prostate cancer (CRPC), with more than 90% of them exhibiting bone metastases. A mechanism by which CRPC cells evade ADT is the expression of constitutively active AR variants (AR-Vs), such as the well-characterized AR-V7. Recently we developed GH501, a flurbiprofen-modified small-molecule compound, and investigated its anti-cancer activity and mechanism of action in pre-clinical models of CRPC. At nanomolar range, GH501 effectively induces cell cycle arrest and apoptosis in CRPC cells regardless of their resistance status to enzalutamide treatment. RNA-seq analysis of GH501 combined with Western blotting analysis identified key targets implicated in CRPC progression, including the full-length AR, AR-V7 variant, and other important genes implicated in CRPC progression. Importantly, low doses of GH501 effectively inhibit the skeletal growth of CRPC in a xenograft model without obvious in vivo toxicities. These preclinical results indicate that GH501 is a promising small-molecule compound that can be further developed for the treatment of lethal prostate cancer. Citation Format: Kenza Mamouni, Lajos Gera, Xin Li, Daqing Wu. A nanomolar potency small-molecule compound against castration-resistant and bone metastatic 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 3205. doi:10.1158/1538-7445.AM2017-3205

CDK4/6 Therapeutic Intervention and Viable Alternative to Taxanes in CRPC.

Resistance to second-generation androgen receptor (AR) antagonists and CYP17 inhibitors in patients with castration-resistant prostate cancer (CRPC) develops rapidly through reactivation of the androgen signaling axis and has been attributed to AR overexpression, production of constitutively active AR splice variants, or the selection for AR mutants with altered ligand-binding specificity. It has been established that androgens induce cell-cycle progression, in part, through upregulation of cyclin D1 (CCND1) expression and subsequent activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Thus, the efficacy of the newly described CDK4/6 inhibitors (G1T28 and G1T38), docetaxel and enzalutamide, was evaluated as single agents in clinically relevant in vitro and in vivo models of hormone-sensitive and treatment-resistant prostate cancer. CDK4/6 inhibition (CDK4/6i) was as effective as docetaxel in animal models of treatment-resistant CRPC but exhibited significantly less toxicity. The in vivo effects were durable and importantly were observed in prostate cancer cells expressing wild-type AR, AR mutants, and those that have lost AR expression. CDK4/6i was also effective in prostate tumor models expressing the AR-V7 variant or the AR F876L mutation, both of which are associated with treatment resistance. Furthermore, CDK4/6i was effective in prostate cancer models where AR expression was lost. It is concluded that CDK4/6 inhibitors are a viable alternative to taxanes as therapeutic interventions in endocrine therapy-refractory CRPC.Implications: The preclinical efficacy of CDK4/6 monotherapy observed here suggests the need for near-term clinical studies of these agents in advanced prostate cancer. Mol Cancer Res; 15(6); 660-9. ©2017 AACR.

Abiraterone + prednisone (Abi) +/- veliparib (Vel) for patients (pts) with metastatic castration-resistant prostate cancer (CRPC): NCI 9012 updated clinical and genomics data.

5001 Background: In preclinical CRPC models, PARP1 inhibition synergizes with AR targeted therapy, especially in ETS fusion-positive tumors. We hypothesized: 1. Co-targeting PARP-1 + AR is superior to AR inhibition and 2. ETS +ve predicts response. Methods: Pts had metastatic (mets) disease biopsy (bx), stratified by IHC-ETS status and randomized to Abi (Arm A) or Abi + Vel (Arm B). Primary endpoint: PSA response rate (RR > = 50% decline). Secondary endpoints: safety, objective RR (ORR), progression free survival (PFS), and molecular analysis including if DNA repair gene deficiency (DRD: BRCA 1, BRCA 2, ATM, FANCA, PALB2, RAD51B, RAD51C) predicts response. 148 pts stratified by IHC-ETS status were randomized to detect a 20% PSA RR improvement assuming a 5% 1-sided type I error and 80% power. An elastic net multivariable Cox model was used to analyze PFS. Mets bx underwent targeted exon sequencing and capture transcriptome analysis. Results: 72 pts were randomly assigned to Arm A and 76 to Arm B. PSA RR: Arm A 63.9%, Arm B 72.4% (p = 0.27). ORR: Arm A 45%, Arm B 52.2%, p = 0.51. Median PFS: Arm A 10.1 months (m), Arm B 11.3 m, p = 0.95. More Arm-B pts were on therapy for 12+ (45% vs 38%) and 18+ cycles (22% vs 17%). ETS status had no impact. Mets tissue sequencing (N = 80): 42 pts (53%) were ETS +ve, 19 (25%) had DRD, 47 (59%) had AR amplification/copy gain, 32 (40%) had PTEN mutation (mut), 33 (41%) had TP53 mut, 37 (46%) had PIK3CA activation (a) and 12 (15%) had WNT-a. Irrespective of arm pts with DRD had a higher PSA and ORR ( > = 87%) vs wild type (58%, 39%; p = 0.013, p = 0.002, respectively), higher PSA decline rate of > = 90% (74% vs 26%, p = 0.0004) and longer median PFS (95% CI): DRD 16.6 m (11 - NR) vs wild type: 8 m (5.4 – 13.3); p = 0.02. PFS was longer in pts with normal PTEN (13.5 vs 6.2 m, p = 0.02), TP53 (13.3 vs 7.8 m, p = 0.04) and PIK3CA (10.3 vs 8.3 m, p = 0.03). Controlling for clinical factors, DRD, PTEN, TP53 and PIK3CA are associated with PFS in this order of importance. Conclusions: There was a modest trend in favor of Abi + Vel but no difference by ETS. Pts with DRD, normal PTEN,TP53 and PIK3CA had better PFS raising new hypotheses regarding the importance of integrating molecular analysis in therapeutic trials. Clinical trial information: NCT01576172.

BIRC6 Targeting as Potential Therapy for Advanced, Enzalutamide-Resistant Prostate Cancer.

Purpose: Enzalutamide resistance has emerged as a major problem in the management of castration-resistant prostate cancer (CRPC). Research on therapy resistance of CRPCs has primarily focused on the androgen receptor pathway. In contrast, there is limited information on antiapoptotic mechanisms that may facilitate the treatment resistance. The inhibitor of apoptosis proteins (IAP) family is well recognized for its role in promoting treatment resistance of cancers by inhibiting drug-induced apoptosis. Here, we examined whether BIRC6, an IAP family member, has a role in enzalutamide resistance of CRPCs and could provide a therapeutic target for enzalutamide-resistant CRPC.Experimental Design: Use of enzalutamide-resistant CRPC models: (i) the transplantable, first high-fidelity LTL-313BR patient-derived enzalutamide-resistant CRPC tissue xenograft line showing primary enzalutamide resistance, (ii) MR42D and MR49F CRPC cells/xenografts showing acquired enzalutamide resistance. Specific BIRC6 downregulation in these models was produced using a BIRC6-targeting antisense oligonucleotide (ASO-6w2). Gene expression was determined by qRT-PCR and gene expression profiling. Molecular pathways associated with growth inhibition were assessed via gene enrichment analysis.Results: Of eight IAPs examined, BIRC6 was the only one showing elevated expression in both enzalutamide-resistant CRPC models. Treatment with ASO-6w2 markedly suppressed growth of LTL-313BR xenografts and increased tumor apoptosis without inducing major host toxicity. Pathway enrichment analysis indicated that GPCR and matrisome signaling were the most significantly altered pathways. Furthermore, ASO-6w2 inhibited expression of prosurvival genes that were upregulated in the LTL-313BR line.Conclusions:BIRC6 targeting inhibited the growth of enzalutamide-resistant CRPC models and may represent a new option for clinical treatment of advanced, enzalutamide-resistant prostate cancer. Clin Cancer Res; 23(6); 1542-51. ©2016 AACR.

Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer

Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC.

Combination of sorafenib and enzalutamide as a potential new approach for the treatment of castration-resistant prostate cancer

Enzalutamide, a novel androgen receptor (AR) antagonist, prolongs overall survival of patients with castration-resistant prostate cancer (CRPC); however, patients eventually progress with enzalutamide resistance. We studied the efficacy of sorafenib combined with enzalutamide in a CRPC model and explored a potential strategy to improve enzalutamide efficacy in vitro and in LNCaP xenografts. The results indicated that enzalutamide combined with sorafenib potently decreased cell proliferation and induced apoptosis in the prostate cancer cell lineLNCaP. In castrate-resistant LNCaP xenografts, the combination of enzalutamide with sorafenib significantly suppressed tumor growth compared with eachsingle agent. Western blots and immunohistochemical staining assay showed that the expression of AR was down-regulated, and the extracellular signal-regulated kinase (ERK) signaling pathway was inhibited after combination treatment, suggesting a synergistic inhibitory effect on the AR and ERK pathways. These results demonstrated that sorafenib therapy improved the efficacy of enzalutamide in the CRPC model, indicating a promising therapeutic strategy for clinical CRPC patients.

Abstract B11: Re-wired E2F function in response to RB loss as a potential driver of castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy, and second leading cause of cancer death in males in the United States, causing over 30,000 deaths per year. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). AR promotes proliferation and is required for PCa cell growth and survival. As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease, castration-resistant prostate cancer (CRPC). It has been shown that an underlying mechanism of this resistance revolves around the retinoblastoma (RB) tumor suppressor protein. RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors thus, resulting in a decrease in AR levels. RB additionally controls cell cycle progression through its ability to bind and inhibit E2F1 mediated transcription of genes required for G1/S progression. Surprisingly, genome-wide assessment of E2F activity in the absence of RB has been understudied. To better understand the molecular pathways that are altered upon RB loss and the roles they play in CRPC, RNA-Seq and ChIP-Seq analyses were performed in isogenic RB cell lines in both hormone sensitive and CRPC models. Data to be discussed reveal new insight into the differential effects of RB loss on AR and E2F1 function and role as potential drivers of late stage disease. Citation Format: Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Kexin Xu, Fugen Li, Brown Myles, Karen E. Knudsen. Re-wired E2F function in response to RB loss as a potential driver of castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B11.

Estrogen related receptor alpha in castration-resistant prostate cancer cells promotes tumor progression in bone.

Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFβ1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated with ERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events.

C-MYC drives histone demethylase PHF8 during neuroendocrine differentiation and in castration-resistant prostate cancer.

Epigenetic factors play critical roles in prostate cancer (PCa) development. However, how they contribute to neuroendocrine differentiation (NED) and castration-resistant PCa (CRPC) is not fully understood. Using bioinformatics and biochemical approaches to analyze cell-based models of NED and CRPC, we found a cluster of epigenetic factors whose expression is downregulated during NED and upregulated in CRPC (i.e. follow a Down-Up pattern). Two histone demethylases within this cluster, PHF8 and KDM3A, are post-transcriptionally regulated by c-MYC through miR-22, which targets both PHF8 and KDM3A. We also found that the c-MYC/miR-22/PHF8 axis is downstream of androgen receptor (AR) signaling in CRPC cells. The co-expression of PHF8 with AR in clinical CRPC samples, normal mouse prostate, and adenocarcinomas of the prostate during PCa progression in a transgenic (TRAMP) mouse model supports the connection between PHF8 and AR. Knockdown of PHF8 impedes cell cycle progression in CRPC cells and has more profound effects on their growth than on the parental LNCaP cell line. Furthermore, PHF8 knockdown sensitizes LNCaP-Abl cells to the AR antagonist enzalutamide. Our data reveal novel mechanisms that underlie the regulation of PHF8 and KDM3A during NED and in CRPC, and support the candidacy of PHF8 as a therapeutic target in CRPC.

WOMEN IN CANCER THEMATIC REVIEW: New roles for nuclear receptors in prostate cancer.

Prostate cancer has, for decades, been treated by inhibiting androgen signalling. This is effective in the majority of patients, but inevitably resistance develops and patients progress to life-threatening metastatic disease - hence the quest for new effective therapies for 'castrate-resistant' prostate cancer (CRPC). Studies into what pathways can drive tumour recurrence under these conditions has identified several other nuclear receptor signalling pathways as potential drivers or modulators of CRPC.The nuclear receptors constitute a large (48 members) superfamily of transcription factors sharing a common modular functional structure. Many of them are activated by the binding of small lipophilic molecules, making them potentially druggable. Even those for which no ligand exists or has yet been identified may be tractable to activity modulation by small molecules. Moreover, genomic studies have shown that in models of CRPC, other nuclear receptors can potentially drive similar transcriptional responses to the androgen receptor, while analysis of expression and sequencing databases shows disproportionately high mutation and copy number variation rates among the superfamily. Hence, the nuclear receptor superfamily is of intense interest in the drive to understand how prostate cancer recurs and how we may best treat such recurrent disease. This review aims to provide a snapshot of the current knowledge of the roles of different nuclear receptors in prostate cancer - a rapidly evolving field of research.

Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer.

Clinical resistance to the second-generation antiandrogen enzalutamide in castration resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights the unmet medical need for next generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide), and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR:CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm bound to heat shock proteins. Thus, we have identified third generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC.

Epigenetic modulation of AR gene expression in prostate cancer DU145 cells with the combination of sodium butyrate and 5'-Aza-2'-deoxycytidine.

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. Castration-resistant prostate cancer (CRPC) is a consequence of androgen deprivation therapy. Unchecked CRPC followed by metastasis is lethal. Some CRPCs show decreased AR gene expression due to epigenetic mechanisms such as DNA methylation and histone deacetylation. The aim of this study was to epigenetically modulate the methylated state of the AR gene leading to targeted demethylation and AR gene expression in androgen-independent human prostate cancer DU145 cell line, representing the CRPC model with very low or undetectable AR levels. The cell treatment was based on single and combined applications of two epigenetic inhibitors, sodium butyrate (NaB) as histone deacetylases inhibitor and 5'-Aza-2'-deoxycytidine (Aza-dC) as DNA methyltransferases inhibitor. We found that the Aza-dC in combination with NaB may help reduce the toxicity of higher NaB concentrations in cancer cells. In normal RWPE-1 cells and even stronger in cancer DU145 cells, the combined treatment induced both AR gene expression on the mRNA level and increased histone H4 acetylation in AR gene promoter. Also activation and maintenance of G2/M cell cycle arrest and better survival in normal RWPE-1 cells compared to cancer DU145 cells were observed after the treatments. These results imply the selective toxicity effect of both inhibitors used and their potentially more effective combined use in the epigenetic therapy of prostate cancer patients.

Optimized design and analysis of preclinical intervention studies in vivo.

Recent reports have called into question the reproducibility, validity and translatability of the preclinical animal studies due to limitations in their experimental design and statistical analysis. To this end, we implemented a matching-based modelling approach for optimal intervention group allocation, randomization and power calculations, which takes full account of the complex animal characteristics at baseline prior to interventions. In prostate cancer xenograft studies, the method effectively normalized the confounding baseline variability, and resulted in animal allocations which were supported by RNA-seq profiling of the individual tumours. The matching information increased the statistical power to detect true treatment effects at smaller sample sizes in two castration-resistant prostate cancer models, thereby leading to saving of both animal lives and research costs. The novel modelling approach and its open-source and web-based software implementations enable the researchers to conduct adequately-powered and fully-blinded preclinical intervention studies, with the aim to accelerate the discovery of new therapeutic interventions.

Androgen-induced Long Noncoding RNA (lncRNA) SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells

Long noncoding RNAs (lncRNA) have been associated with the development of cancer. However, the interplay between lncRNAs and androgen receptor (AR) signaling in prostate cancer is still unclear. Here, we identified lncRNAs induced by androgen in AR-positive prostate cancer cells, where induction was abolished by AR knockdown as well as an anti-androgen, bicalutamide. By combining these data, we identified an androgen-regulated lncRNA, suppressor of cytokine signaling 2-antisense transcript 1 (SOCS2-AS1), the expression of which was higher in castration-resistant prostate cancer model cells, i.e long-term androgen-deprived (LTAD) cells, than in parental androgen-dependent LNCaP cells. SOCS2-AS1 promoted castration-resistant and androgen-dependent cell growth. We found that SOCS2-AS1 knockdown up-regulated genes related to the apoptosis pathway, including tumor necrosis factor superfamily 10 (TNFSF10), and sensitized prostate cancer cells to docetaxel treatment. Moreover, we also demonstrated that SOCS2-AS1 promotes androgen signaling by modulating the epigenetic control for AR target genes including TNFSF10 These findings suggest that SOCS2-AS1 plays an important role in the development of castration-resistant prostate cancer by repressing apoptosis.

Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant-Driven Prostate Cancer Model

Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the treatment of AR-overexpressed CRPC driven by AR splice variants that are not clinically actionable at present. Prostate 76:1536-1545, 2016. © 2016 Wiley Periodicals, Inc.

Abstract 334: Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer

Abstract (A) Introduction: Because of the central role of androgen receptor (AR) signaling in prostate cancer (PC), AR inhibition is the primary modality of initial PC treatment. While AR-targeted therapies are often initially effective in castration-resistant prostate cancer (CRPC), disease progression is common. AR and glucocorticoid receptor (GR) are structurally similar nuclear receptors, bind the same DNA response elements, and regulate common genes. Previous work from our lab demonstrated that GR expression is upregulated following AR antagonism, GR expression decreased the effectiveness of AR blockade, and inhibiting GR activity reversed these effects. However, the only FDA-approved GR antagonist, mifepristone, is not GR-specific and has significant drug-drug interactions; therefore, we report our initial data with a new generation of selective GR-antagonists (SGRMs) for PC treatment using models of CRPC. (B) Experimental Procedures: The LAPC4, CWR-22Rv1 and VCAP PC cell lines were utilized. GR target gene expression analysis was performed at various time-points by qRT-PCR and protein immunoblots subsequent to treatment with AR agonist (R1881, 1nM), AR antagonist (enzalutamide, 10 μM), GR agonist (dexamethasone, 100nM) and the new SGRMs CORT108297 and CORT118335(1μM). Quantification of cell numbers in vitro in response to these treatments was determined by trypan blue exclusion/live cell counting and the CellTiter Glo 2.0 ATP-luciferase assay. For in vivo studies, PC cells were injected subcutaneously into male nude mice and grown to a predetermined tumor size, mice were then castrated, and fifteen days later treated with mifepristone (12mg/kg), SGRM (16mg/kg, 20mg/kg), or vehicle by daily intra-peritoneal injection to assess time to CRPC. (C) Results: AR antagonism with concurrent GR activation resulted in increased expression of canonical AR/GR-target genes and SGRMs (1μM) effectively inhibited expression of glucocorticoid-mediated genes at the RNA and protein levels. SGRMs did not affect AR-mediated gene expression. When AR was antagonized, activated GR promoted cell survival, which was inhibited by SGRM treatment to varying degrees. Preliminary studies support the hypothesis that these novel SGRMs delay CRPC tumor xenograft progression in vivo. (D) Conclusion: In vitro, new generation SGRMs inhibit GR-mediated, but not AR-selective gene expression and result in decreased tumor cell number. SGRM administration to nude mice bearing PC xenografts can delay castration-resistant tumor growth. Therefore, new SGRMs may be an effective therapy for CRPC treatment worthy of further exploration. Studies are ongoing to determine how SGRMs modulate GR target gene expression globally and whether they decrease expression of tumorigenic pro-survival genes in vivo. Citation Format: Jacob Kach, Phillip Selman, Diana C. West, Donald J. Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant 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 334.