AR-regulated Genes Research Articles

Abstract 4313: Abiraterone acetate (AA) treatment of prostate cancer patient-derived xenografts (PDX) demonstrates heterogeneity of responses and identifies potential biomarkers of adaptive resistance

Abstract AA, a prodrug of the CYP17A1 inhibitor abiraterone, is the first second-line hormonal agent shown to improve survival in metastatic castration-resistant prostate cancer (CRPC); however, as with all available treatments, responding tumors eventually develop resistance. This highlights the importance of identification of mechanisms involved in tumor response and resistance to AA. We evaluated the efficacy of AA in LuCaP CRPC PDXs and investigated mechanisms of response and/or resistance, focusing on androgen receptor (AR) and glucocorticoid receptor (GR) signaling. Mice bearing CRPC LuCaP 35CR, 96CR, 77CR, and 136CR PDXs were treated with AA (0.5 mmol/kg/d), and tumor responses and body weights were monitored. Tumors were harvested 7 days after the beginning of the treatment (d7) and at end of study (EOS) (sacrifice when tumor > 1000 mg). Gene expression analyses using Affymetrix HG-U219 gene array and qPCR were performed to evaluate the validity of established biomarkers and identify novel biomarkers related to AA response. Linear regression model analyses showed that AA inhibited tumor progression in 3 of 4 LuCaP PDXs (96CR, 77CR, 136CR), but the initial response was followed by development of resistance. Corresponding analyses of serum prostate-specific antigen (PSA) levels demonstrated a decrease post AA (96CR: p = 0.051; 77CR: p = 0.001; 136CR: undetectable at baseline). AA improved median survival (77CR: 7 vs 9.5 weeks, p = 0.022; and 136CR: 6.8 vs 21.6 weeks, p < 0.0001), and highly variable responses were observed for 96CR (5.75 vs 10 weeks, p = 0.25). 35CR did not exhibit significant growth inhibition, reduction in PSA, or decreased median survival on AA. Evaluation of gene signatures revealed a negative association of proliferation-associated genes with AA at d7 for 35CR and 136CR, while at EOS these signatures were negatively associated with AA for 96CR and 136CR. Signatures of AR-regulated genes were negatively associated with AA at d7 for 35CR and 136CR, while at EOS they were negatively associated with AA for 96CR and 136CR. Targeted sequencing of the AR ligand binding domain coding sequence did not detect any mutations in any PDX at EOS. Trends toward increased expression of full length AR (ARFL) and splice variant ARv7 were associated with AA at d7 for 136CR. At EOS, trends toward increased ARFL and ARv7 expression were associated with AA for 35CR and 96CR. Significantly increased GR expression was associated with AA at EOS for 96CR and 77CR, and a trend was observed for 136CR. Our results demonstrate model-specific heterogeneity of physiological and molecular responses to AA in CRPC. However, our data indicate that increased ARFL, ARv7, and/or GR could be biomarkers of adaptive resistance and potentially play functional roles in conferring resistance. Citation Format: Hung-Ming Lam, Ryan McMullin, Holly M. Nguyen, Michael Gormley, Roman Gulati, Weimin Li, Deborah Ricci, Karin Verstraeten, Shibu Thomas, Elahe A. Mostaghel, Peter S. Nelson, Robert L. Vessella, Eva Corey. Abiraterone acetate (AA) treatment of prostate cancer patient-derived xenografts (PDX) demonstrates heterogeneity of responses and identifies potential biomarkers of adaptive resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2015-4313

A novel prostate cancer therapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variants.

Persistent androgen receptor (AR) signaling is the key driving force behind progression and development of castration-resistant prostate cancer (CRPC). In many patients, AR COOH-terminal truncated splice variants (ARvs) play a critical role in contributing to the resistance against androgen depletion therapy. Unfortunately, clinically used antiandrogens like bicalutamide (BIC) and enzalutamide (MDV), which target the ligand binding domain, have failed to suppress these AR variants. Here, we report for the first time that a natural prenylflavonoid, icaritin (ICT), can co-target both persistent AR and ARvs. ICT was found to inhibit transcription of key AR-regulated genes, such as KLK3 [prostate-specific antigen (PSA)] and ARvs-regulated genes, such as UBE2C and induce apoptosis in AR-positive prostate cancer (PC) cells. Mechanistically, ICT promoted the degradation of both AR and ARvs by binding to arylhydrocarbon-receptor (AhR) to mediate ubiquitin-proteasomal degradation. Therefore, ICT impaired AR transactivation in PC cells. Knockdown of AhR gene restored AR stability and partially prevented ICT-induced growth suppression. In clinically relevant murine models orthotopically implanted with androgen-sensitive and CRPC cells, ICT was able to target AR and ARvs, to inhibit AR signaling and tumor growth with no apparent toxicity. Our results provide a mechanistic framework for the development of ICT, as a novel lead compound for AR-positive PC therapeutics, especially for those bearing AR splice variants.

Genome-wide analysis of androgen receptor binding sites in prostate cancer cells.

The transformation of prostate cancer from an androgen-dependent state to an androgen-independent state is a lethal progression. Alterations in transcriptional programs are the basis of prostate cancer deterioration. The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, mediates prostate cancer progression by functioning primarily through the ligand-activated transcription of target genes. Therefore, a detailed map of AR-regulated genes and AR genomic binding sites is required for hormone-naive and castration-resistant prostate cancer. Through the use of chromatin immunoprecipitation in combination with direct sequencing, 4,143 AR binding sites were defined in the LNCaP androgen-sensitive prostate cancer cell line. Using the same method, 2,380 AR binding regions were identified in the LNCaP-AI long-term androgen-deprived cell line. Approximately 8.5% (354/4,143) of the binding regions were mapped to within 2 kb of the transcription start site (TSS) in the LNCaP cells, while ∼12.6% (299/2,380) were mapped to within 2 kb of the TSS in the LNCaP-AI cells. In total, the study mapped 2,796 genes in LNCaP cells and 1,854 genes in LNCaP-AI cells. The cell lines shared 789 mutual genes. In addition, gene ontology (GO) analysis of the genes revealed that there was a notable overlap between the GO terms in the LNCaP cells and LNCaP-AI cells. However, GO terms within the biological process domain that were only observed in the LNCaP-AI cells included the reproduction process, death, immune system process, multi-organism process, pigmentation and viral reproduction. The major genes in the different GO terms were TNFAIP8, RTN4, APP and SYNE1. Through analyzing the AR binding sites in the two cell types, the present study aimed to map potential AR-regulated genes, identify their associated transcription factors and provide a new perspective on the biological processes in the development of prostate cancer. The results provided a valuable data set that furthered the understanding of the genome-wide analysis of AR binding sites in prostate cancer cells, which may be exploited for the development of novel prostate cancer therapeutic strategies.

Generation 2.5 antisense oligonucleotides targeting the androgen receptor and its splice variants suppress enzalutamide-resistant prostate cancer cell growth.

Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (ARFL) or variants (AR-Vs) in disease progression. To define functional roles of ARFL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown ARFL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts. ENZ-R-LNCaP cells express high levels of both ARFL and AR-V7 compared with CRPC-LNCaP; in particular, ARFL levels were approximately 12-fold higher than AR-V7. Both ARFL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of ARFL alone, or ARFL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both ARFL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of ARFL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts. These data identify the AR as an important driver of ENZ resistance, and while the contributions of ARFL and AR-Vs can vary across cell systems, ARFL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both ARFL and AR-Vs is a rational approach for AR-dependent CRPC.

Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer.

Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

Inhibition of Stat5a/b Enhances Proteasomal Degradation of Androgen Receptor Liganded by Antiandrogens in Prostate Cancer.

Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b.

A phase Ib study of BKM120 combined with abiraterone acetate for castrate-resistant, metastatic prostate cancer.

274 Background: There is cross-talk between PI3-kinase (PI3K) pathway and androgen receptor (AR) signaling pathways, respectively, which are both critical for cell survival in castrate-resistant prostate cancer (CRPC). The primary study objective is to determine the safety profile and MTD of BKM120 (B, pan-PI3K inhibitor) in combination with abiraterone/prednisone (A/P) in CRPC patients. The secondary objectives are to assess the impact of PTEN status on duration of response/time to progression in the expansion cohort, and to evaluate the impact of B on a PI3-kinase activation fingerprint in metastatic bone or lymph node tissue samples. An exploratory objective is to assess the effect of B on transcription of a set of AR-regulated genes in metastatic bone biopsy samples. Methods: The trial design involves a 14 day lead-in phase with B alone, to assess single-agent toxicity and perform correlative studies. A/P is combined with B at the end of 14 days using the standard 3+3 dose-escalation design with 3 dose levels of B, and participants are assessed for safety and MTD on the combination therapy. To determine PD impact of single agent B on the PI3K activation signature at a metastatic site, a mandatory CT-guided bone or lymph node biopsy is performed prior to B initiation and at the end of 2 weeks on B single-agent therapy. Immunohistochemical (IHC) stains for three markers (p-AKT, p-S6 and PTEN) are used to obtain a semi-quantitative PI3K activation score, based on the quartile levels of continuous staining scores of each marker. Results: Patient 1 had symptomatic bone pain improvement, marked decline in narcotic pain requirements and a biochemical decline in PSA from 156.5 to a PSA nadir of 9.2 within 4 weeks of combination therapy. Patient 3 had a symptomatic and >90% biochemical improvement and has currently completed 15 cycles on treatment, and remains on study to date. RT-PCR analysis showed that the feedback circuitry between PI3K and AR signaling is heterogeneous in the metastatic prostate cancer-bone microenvironment, and dependent on tumor PTEN status. Conclusions: Preliminary data shows promising anti-tumor activity in CRPC patients from dual targeting of PI3K and AR pathways with B and A/P, respectively. Clinical trial information: NCT01741753.

Anticancer activity of a novel selective CYP17A1 inhibitor in preclinical models of castrate-resistant prostate cancer.

VT-464 is a novel, nonsteroidal, small-molecule CYP17A1 inhibitor with 17,20-lyase selectivity. This study evaluates the anticancer activity of VT-464 compared with abiraterone (ABI) in castrate-resistant prostate cancer cell lines and xenograft models that are enzalutamide (ENZ)-responsive (C4-2) or ENZ-resistant (MR49C, MR49F). In vitro, androgen receptor (AR) transactivation was assessed by probasin luciferase reporter, whereas AR and AR-regulated genes and steroidogenic pathway enzymes were assessed by Western blot and/or qRT-PCR. The MR49F xenograft model was used to compare effects of oral VT-464 treatment to vehicle and abiraterone acetate (AA). Steroid concentrations were measured using LC-MS chromatography. VT-464 demonstrated a greater decrease in AR transactivation compared with ABI in C4-2 and both ENZ-resistant cell lines. At the gene and protein level, VT-464 suppressed the AR axis to a greater extent compared with ABI. Gene transcripts StAR, CYP17A1, HSD17B3, and SRD5A1 increased following treatment with ABI and to a greater extent with VT-464. In vivo, intratumoral androgen levels were significantly lower after VT-464 or AA treatment compared with vehicle, with the greatest decrease seen with VT-464. Similarly, tumor growth inhibition and PSA decrease trends were greater with VT-464 than with AA. Finally, an AR-antagonist effect of VT-464 independent of CYP17A1 inhibition was observed using luciferase reporter assays, and a direct interaction was confirmed using an AR ligand binding domain biolayer interferometry. These preclinical results suggest greater suppression of the AR axis with VT-464 than ABI that is likely due to both superior selective suppression of androgen synthesis and AR antagonism.

SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin.

Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth.

Targeting the Androgen Receptor Confers In Vivo Cross-resistance Between Enzalutamide and Docetaxel, But Not Cabazitaxel, in Castration-resistant Prostate Cancer

Treatment options for metastatic castration-resistant prostate cancer (CRPC) have evolved with the established benefit of the novel androgen receptor (AR)-targeted agents abiraterone and enzalutamide in the prechemotherapy setting. However, concerns regarding cross-resistance between the taxanes docetaxel and cabazitaxel and these AR-targeted agents have arisen, and the optimal drug treatment sequence is unknown. We investigated the in vivo efficacy of docetaxel and cabazitaxel in enzalutamide-resistant CRPC, and mechanisms of cross-resistance between these agents. Castrated mice harboring enzalutamide-resistant tumors and enzalutamide-naïve tumors were treated with docetaxel and cabazitaxel. Tumor growth kinetics, AR nuclear localization, AR-regulated gene expression, Ki67 expression, and serum levels of prostate-specific antigen, docetaxel, and cabazitaxel were analyzed. Docetaxel inhibited tumor growth, AR nuclear localization, and AR-regulated gene expression in enzalutamide-naïve tumors, but did not in enzalutamide-resistant tumors, demonstrating in vivo cross-resistance. By contrast, cabazitaxel remained highly effective in enzalutamide-resistant tumors and demonstrated superior antitumor activity compared to docetaxel, independent of the AR pathway. These findings demonstrate that the AR pathway is able to confer in vivo cross-resistance between enzalutamide and docetaxel, but not cabazitaxel, in CRPC. Patient summaryWe found reduced efficacy of docetaxel, but not cabazitaxel, in enzalutamide-resistant prostate cancer.

BET bromodomain inhibitors—A novel epigenetic approach in castration-resistant prostate cancer

The androgen receptor (AR) is central to the initiation and progression of prostate cancer, even after castration. There has been some success in therapies targeting AR signaling which have been shown to extend survival in men with castration-resistant prostate cancer (CRPC). However, durable responses to these therapies have been limited and there is a need to identify additional therapeutic targets within the AR-signaling network. Recently a group at University of Michigan Medical School outlined the potential for BET bromodomain protein inhibitors as a novel epigenetic approach to treatment of CRPC. In prostate cancer cell lines, BET bromodomain inhibitor, JQ1, was shown to induce apoptosis and down-regulate AR-regulated gene transcription. Bromodomain and the extra-terminal (BET) subfamily of human bromodomain proteins, with a focus on BRD4, were shown to play a major role in AR signaling and interact with AR via bromodomain (BD) 1/2. JQ1 inhibits this BRD4-AR bond, resulting in removal of RNA polymerase II from AR target genes, causing reduced AR gene transcription and subsequent diminished AR signaling. JQ1 lead to a significant reduction in tumor volume and weight in VCaP xenograft mice.

Androprostamines A and B, the new anti-prostate cancer agents produced by Streptomyces sp. MK932-CF8.

Androgen receptor (AR) is a validated target in all clinical states of prostate cancer. Androprostamines A and B, the new inhibitors of androgen receptor, were isolated from Streptomyces sp. MK932-CF8. Their structures were determined by the spectroscopic analysis, degradation studies and synthesis. Androprostamines showed potent inhibitory effect against androgen-dependent growth of human prostate cancer cells without cytotoxicity and repressed the androgen-induced expression of AR-regulated genes.

Abstract 4517: Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer

Abstract Prostate cancer is one of the most common malignancy diagnosed in men. Although androgen deprivation therapy is effective at the beginning of treatment, the majority of patients eventually progress to hormone refractory prostate cancer (HRPC), which leads to a poor outcome. HRPC mouse models have been developed to study transition from hormone dependent (HD) to hormone refractory (HR) disease; however, the exact mechanism behind this phenotypic conversion remains unclear. By using serial in vivo transplantation of LNCap xenograft tumors in castrated male SCID mice, we developed, validated and characterized a mouse model of HRPC, HR-LNCap. In vivo passaged tumor fragments form solid tumors in castrated male SCID mice. By comparison, HR-LNCap tumors grow more aggressively than parental LNCap tumors in SCID mice. A tumor cell line established from HR-LNCap xenograft tumors was able to grow in charcoal stripped serum containing medium, indicating it is androgen independent. HR-LNCap tumors lost sensitivity to androgen receptor antagonist bicalutamide, but remained sensitive to MDV-3100, a second generation AR antagonist approved for treating HRPC. Characterization of this model revealed that HR-LNCap tumors had unique AR signaling pathway profiles, including elevated AR expression and decreased expression of the AR-regulated genes, prostate specific antigen (PSA) and FKBP5, when compared with parental LNCap tumors. RNA sequencing was performed on HR-LNCap tumors to determine expression signature of the model. Interestingly, additional pathway analysis showed that HR-LNCap tumors have significantly higher levels of pAKT in comparison with parental LNCap tumors, indicating activation of AKT/mTOR pathway. Treatment of HR-LNCap tumor bearing mice with a small molecule TOR kinase inhibitor CC214-2 showed significant tumor growth inhibition. Further analysis demonstrated that treatment with CC214-2 inhibited TORC1 and TORC2 pathway markers, pAKT, pS6 and p4EBP1, in HR-LNCap tumors. Taken together, this study describes a mouse model of HRPC with a unique molecular profile, which can be used to evaluate novel therapeutics against HRPC. Citation Format: Wenqing Yang, Jing Jiu, Rama Krishna Narla, Heather K. Raymon. Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2014-4517

Abstract CT418: A Phase Ib study of BKM120 combined with abiraterone acetate for castrate-resistant, metastatic prostate cancer

Abstract Introduction: There is significant cross-talk between PI3-kinase (PI3K) and androgen receptor (AR) signaling pathways, respectively, which are both critical for cell survival in castrate-resistant prostate cancer (CRPC). The primary study objective (NCT01741753) is to determine the safety profile and maximum tolerated dose (MTD) of BKM120 (pan-PI3K inhibitor) in combination with abiraterone/prednisone (A/P) in CRPC patients. The secondary study objectives are to assess the impact of PTEN status on duration of response/time to progression in the expansion cohort, and to evaluate the impact of BKM120 on a PI3-kinase activation fingerprint in metastatic bone or lymph node tissue samples. An exploratory objective of the study is to assess the effect of BKM120 on transcription of a set of AR-regulated genes in metastatic bone biopsy samples. Methods: The trial design involves a 14 day lead-in phase with BKM120 alone, to assess single-agent toxicity and perform correlative studies. A/P is combined with BKM120 at the end of 14 days using the standard 3+3 dose-escalation design with 3 dose levels of BKM120 (80 mg, 100 mg, 120 mg, respectively), and participants are assessed for safety and MTD on the combination therapy. To determine pharmacodynamic impact of single agent BKM120 on the PI3K activation signature at a metastatic site, a mandatory CT-guided bone or lymph node biopsy is performed prior to BKM120 initiation and at the end of 2 weeks on BKM120 single-agent therapy. Immunohistochemical (IHC) stains for three markers (p-AKT, p-S6 and PTEN) are used to obtain a semi-quantitative PI3K activation score, based on the quartile levels of continuous staining scores of each marker. Results: The clinical, PSA biochemical responses, immunohistochemical and molecular RT-PCR data for three enrolled patients are summarized here. The first patient had symptomatic bone pain with limited functional mobility at baseline, and was treated with the first BKM120 dose level of 80 mg daily. He had a decline in PSA from 156.5 to 129.6 during the lead-in phase with single-agent BKM120 treatment with partial symptomatic pain relief. Interestingly, comparison of pre- and post-treatment metastatic bone tumor IHC staining on single-agent BKM120 showed inhibition of phospho-S6 staining. Upon adding A/P to BKM120 treatment, he had a striking decline in his PSA to 23.3 within 1 week, which reached a nadir of 9.2 within 4 weeks of combination therapy treatment. This striking biochemical improvement corresponded to a marked decline in narcotic pain requirements, increased mobility and quality-of-life. However, he developed clinical, biochemical and radiographic disease progression after 4 cycles of combination therapy treatment, so had to withdraw from study. The second and third patient had a >90% biochemical improvement within 4 weeks of combination therapy treatment and remain on study to date. To evaluate cross-talk between AR and PI3K signaling in the bone microenvironment, RT-PCR analyses on pre- and BKM120-treated bone biopsy samples were performed. The results showed that the feedback relationship between PI3K and AR signaling are heterogeneous, and dependent on the PTEN status of the tumor. Conclusion: The pan-PI3K inhibitor/abiraterone combination is well-tolerated, and demonstrates promising anti-tumor activity in CRPC patients. Citation Format: Akash Patnaik, Massimo Loda, Justin Kung, Jim Wu, MaryEllen Taplin, Philip Kantoff, Lewis Cantley, Steven Balk, Glenn Bubley. A Phase Ib study of BKM120 combined with abiraterone acetate for castrate-resistant, metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT418. doi:10.1158/1538-7445.AM2014-CT418

Abstract 713: Utilizing a potent and selective nonreplicating adenoviral mutant (Ad5-TV-CU), with gene expression controlled by androgen receptor-dependent activation domains in the TMPRSS2 gene, as a novel prodrug-converting enzyme therapy for prostate cancer

Abstract Prostate cancer (PCa) progression is dependent on transcriptional activation of the androgen receptor (AR) in the majority of cases. Therefore, therapies include anti-androgens, which decrease levels of circulating androgens and inhibit AR activity. However, resistance inevitably develops, resulting in more aggressive, incurable late-stage disease with intact or constitutively active AR signaling. There is currently no cure for castration-resistant metastatic PCa and novel therapies are needed. A major AR-regulated gene is TMPRSS2, which is commonly expressed at higher levels in cancer than normal prostate. In 50% of PCa cases the TMPRSS2 promoter is fused to the oncogenic transcriptional regulator ERG. We generated TMPRSS2 promoter and enhancer constructs to drive the prodrug-converting chimeric enzyme cytosine deaminase uracil phosphoribosyl transferase (CD/UPRT) from an adenoviral vector to investigate the therapeutic efficacy in various PCa models. The selectivity and transcriptional efficiency of various promoter and enhancer regions containing Androgen response elements (ARE's) were investigated after cloning TMPRSS2 constructs driving Luciferase (Luc) expression into the versatile expression vector ‘VISA’ (VP16-GAL4-WPRE integrated systemic amplifier) (Professor Hung, M.D Anderson, TX,USA) to enhance promoter activity. Comparison with the traditional PSA promoter and the chimeric PSA promoter/enhancer in the VISA vector demonstrated the TMPRSS2 construct to be superior by inducing 3.5 fold and 2.3 fold levels of luciferase expression, respectively. When the Luc-gene was replaced with CD/UPRT, specific and dose-dependent cell killing was observed in 22RV1 (AR+) cells when the non-toxic prodrug 5-flourocytosine (5-FC) was administered. No cell death was observed in primary prostate epithelial cells (PrEC), or immortalized prostate cell lines PNT1A or PNT2 (AR-). To increase transfection efficiency of the CD/UPRT gene, the TMPRSS2-VISA-CD/UPRT expression cassette was inserted into a non-replicating adenovirus (Ad5-TV-C/U) replacing the E1-genes. Ad5-TV-C/U was characterized and protein expression of the CD/UPRT gene was detected at high levels post infection in 22RV1, LNCaP, and VCaP cells (AR+). Dose-dependent decreases in EC50-values were observed upon infection with Ad5-TV-C/U in combination with low doses of 5-FC in a number of cell lines, for example 22RV1 cells, where EC50-values decreased from 571ppc ±55 to 174ppc ±30. These results demonstrate that Ad5-TV-C/U is both selective and efficacious in killing AR-positive PCa cells in combination with non-toxic 5-FC. Verification of these findings in preclinical in vivo models is in progress. Citation Format: Emma J. Mercer, Ahmet Imrali, Kevin Sharpe, Gunnel Hallden, Yong-Jie Lu. Utilizing a potent and selective nonreplicating adenoviral mutant (Ad5-TV-CU), with gene expression controlled by androgen receptor-dependent activation domains in the TMPRSS2 gene, as a novel prodrug-converting enzyme therapy for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 713. doi:10.1158/1538-7445.AM2014-713

TMPRSS2:ERG blocks neuroendocrine and luminal cell differentiation to maintain prostate cancer proliferation.

The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains poorly understood. To address this, we compared the transcriptional effects of TMPRSS2:ERG expression in a transgenic mouse model with those of ERG knockdown in a TMPRSS2:ERG-positive PC cell line. This reveals that ERG represses the expression of a previously unreported set of androgen receptor (AR)-independent neuronal genes that are indicative of neuroendocrine (NE) cell differentiation-in addition to previously reported AR-regulated luminal genes. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. Inhibition of ERG expression in TMPRSS2:ERG-positive PC cells through blockade of AR signaling is tracked with increased NE gene expression. We also provide evidence that these NE cells are resistant to pharmacological AR inhibition and can revert to the phenotype of parental cells upon restoration of AR/ERG signaling. Our findings highlight an ERG-regulated mechanism capable of repopulating the parent tumor through the transient generation of an anti-androgen therapy-resistant cell population, suggesting that ERG may have a direct role in preventing resistance to anti-androgen therapy.

AB46. Screening and identification for the target genes of androgen receptor in mouse Sertoli cells

Androgen and androgen receptor (AR) play important roles in spermatogenesis, yet detailed androgen/AR signals in Sertoli cells remain unclear. To identify AR target genes in Sertoli cells, we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR) and their littermate wild-type (WT) mice. Digital gene expression analysis identified 2,276 genes downregulated and 2,865 genes upregulated in the S-AR mice testis compared to WT ones. To further nail down the difference within Sertoli cells, we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells. Interestingly, additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing ten times more androgen sensitivity than TM4 cells. In the condition where maximal androgen response was demonstrated, we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone. Among these genes, 603 androgen-/AR-regulated genes, including 164 up-regulated and 439 down-regulated, were found in both S-AR mice testis and TM4/AR cells.Ubiquitin-conjugating enzyme E2B (Ube2b) is one of the regulated genes from the digital gene expression analysis. The expression of UBE2B was decreased in the testes of the S-AR mice analyzed by quantitative RT-PCR (qRT-PCR) and immunofluorescence. The up-regulation of Ube2b gene by testosterone was further demonstrated by Western blot and qRT-PCR in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay validated that the ligand-bound AR activated Ube2b transcription via directly binding to the androgen-responsive element of the Ube2b promoter. In vitro analyses showed that testosterone increased UBE2B expression and activated H2A ubiquitylation, while downregulation of UBE2B blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly down-regulated and 71 were up-regulated by UBE2B in TM4 cells. These results suggest that Ube2b, as a direct AR transcriptional target in Sertoli cells, mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation.Our previous digital gene expression analysis data also showed that heat shock transcription factors 1 (HSF1) was regulated by androgen in mouse Sertoli cells. We found that the expression of Hsf1 was increased in the testes of S-AR mice compared with wild-type mice by quantitative real-time PCR and the expression of HSF1 in the S-ARSertoli cells was significantly increased as examined by immunofluorescence analysis. Besides, in vitro study showed that testosterone repressed the expression of Hsf1 in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to Hsf1 promoter. Our experiment also demonstrated that testosterone downregulated the expression of heat shock protein HSP105 and HSP60 by inhibiting the transcription of Hsf1. Taken together, these results reveal that HSF1 is a novel target of androgen receptor in mouse Sertoli Cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting HSF1 expression.

The pluripotency factor Nanog is directly upregulated by the androgen receptor in prostate cancer cells.

The Androgen Receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in all stages of prostate cancer progression, including progression to castration-resistance following androgen-deprivation therapy. Thus, identifying and targeting critical AR-regulated genes is one potential method to block castration-resistant cancer proliferation. Of particular importance are transcription factors that regulate stem cell pluripotency; many of these genes are emerging as critical oncogenes in numerous tumor cell types. Of these, Nanog has been previously shown to increase the self-renewal and stem-like properties of prostate cancer cells. Thus, we hypothesized that Nanog is a candidate AR target gene that may impart castration-resistance. We modulated AR signaling in LNCaP prostate cancer cells and assayed for Nanog expression. Direct AR binding to the NANOG promoter was tested using AR Chromatin Immunoprecipation (ChIP) and analyses of publically available AR ChIP-sequencing data-sets. Nanog over-expressing cells were analyzed for cell growth and cytotoxicity in response to the AR antagonist enzalutamide and the microtubule stabilizing agent docetaxel. AR signaling upregulates Nanog mRNA and protein. AR binds directly to the NANOG promoter, and was not identified within 75 kb of the NANOGP8 pseudogene, suggesting the NANOG gene locus was preferentially activated. Nanog overexpression in LNCaP cells increases overall growth, but does not increase resistance to enzalutamide or docetaxel. Nanog is a novel oncogenic AR target gene in prostate cancer cells, and stable expression of Nanog increases proliferation and growth of prostate cancer cells, but not resistance to enzalutamide or docetaxel.

Arrestin2 modulates androgen receptor activation.

Androgen receptor (AR) has a pivotal role in the growth and survival of prostate cancer (PCa). Arrestin2 (Arr2) is a ubiquitous scaffolding/adaptor protein first characterized as a regulator of G protein-coupled receptor signaling. In this study, we report that Arr2 additionally functions as a positive regulator of AR expression and function in PCa cells. Expression level of Arr2 correlates with that of AR, and knockdown of Arr2 inhibits the expression of AR and its effectors prostate-specific antigen, transmembrane protease serine 2, FK506-binding protein 51 and fatty acid synthase. Mechanistically, the knockdown of Arr2 attenuates the binding of AR to androgen response elements and consequently decreases transcription of AR-regulated genes. The inhibition of AR by Arr2 knockdown occurs in both androgen-dependent and castration-resistant PCa (CRPC) cells, although the effect is more prominent in CRPC. Arr2 knockdown inhibits the in vitro CRPC cell proliferation, prostasphere growth and invasion, as well as the in vivo prostate tumor formation, local invasion and distant metastasis. These results illustrate a new role for Arr2 in the expression and activation of AR and its potential relevance as a target for therapeutic intervention and monitoring of disease progression.

Vitamin D, intermediary metabolism and prostate cancer tumor progression.

Epidemiological data have demonstrated an inverse association between serum vitamin D3 levels, cancer incidence and related mortality. However, the effects of vitamin D on prostate cancer biology and its utility for prevention of prostate cancer progression are not as well-defined. The data are often conflicting: some reports suggest that vitamin D3 induces apoptosis in androgen dependent prostate cancer cell lines, while others suggest that vitamin D3 only induces cell cycle arrest. Recent molecular studies have identified an extensive synergistic crosstalk between the vitamin D- and androgen-mediated mRNA and miRNA expression, adding an additional layer of post-transcriptional regulation to the known VDR- and AR-regulated gene activation. The Warburg effect, the inefficient metabolic pathway that converts glucose to lactate for rapid energy generation, is a phenomenon common to many different types of cancer. This process supports cell proliferation and promotes cancer progression via alteration of glucose, glutamine and lipid metabolism. Prostate cancer is a notable exception to this general process since the metabolic switch that occurs early during malignancy is the reverse of the Warburg effect. This “anti-Warburg effect” is due to the unique biology of normal prostate cells that harbor a truncated TCA cycle that is required to produce and secret citrate. In prostate cancer cells, the TCA cycle activity is restored and citrate oxidation is used to produce energy for cancer cell proliferation. 1,25(OH)2D3 and androgen together modulates the TCA cycle via transcriptional regulation of zinc transporters, suggesting that 1,25(OH)2D3 and androgen maintain normal prostate metabolism by blocking citrate oxidation. These data demonstrate the importance of androgens in the anti-proliferative effect of vitamin D in prostate cancer and highlight the importance of understanding the crosstalk between these two signaling pathways.