Activity In Castration-resistant Prostate Cancer Research Articles

Upregulation of Scavenger Receptor B1 Is Required for Steroidogenic and Nonsteroidogenic Cholesterol Metabolism in Prostate Cancer.

Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer initiation and progression. Transcript expression of the high-density lipoprotein-cholesterol receptor scavenger receptor B1 (SR-B1) is elevated in primary prostate cancer. Hypothesizing that SR-B1 expression may help facilitate malignant transformation, we document increased SR-B1 protein and transcript expression in prostate cancer relative to normal prostate epithelium that persists in lethal castration-resistant prostate cancer (CRPC) metastasis. As intratumoral steroid synthesis from the precursor cholesterol can drive androgen receptor (AR) pathway activity in CRPC, we screened androgenic benign and cancer cell lines for sensitivity to SR-B1 antagonism. Benign cells were insensitive to SR-B1 antagonism, and cancer line sensitivity inversely correlated with expression levels of full-length and splice variant AR. In androgen-responsive CRPC cell model C4-2, SR-B1 antagonism suppressed cholesterol uptake, de novo steroidogenesis, and AR activity. SR-B1 antagonism also suppressed growth and viability and induced endoplasmic reticulum stress and autophagy. The inability of exogenous steroids to reverse these effects indicates that AR pathway activation is insufficient to overcome cytotoxic stress caused by a decrease in the availability of cholesterol. Furthermore, SR-B1 antagonism decreased cholesterol uptake, growth, and viability of the AR-null CRPC cell model PC-3, and the small-molecule SR-B1 antagonist block lipid transport-1 decreased xenograft growth rate despite poor pharmacologic properties. Overall, our findings show that SR-B1 is upregulated in primary and castration-resistant disease and is essential for cholesterol uptake needed to drive both steroidogenic and nonsteroidogenic biogenic pathways, thus implicating SR-B1 as a novel and potentially actionable target in CRPC. SIGNIFICANCE: These findings highlight SR-B1 as a potential target in primary and castration-resistant prostate cancer that is essential for cholesterol uptake needed to drive steroidogenic and nonsteroidogenic biogenic pathways.

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

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

C15-162: Phase IB trial of radium 223 and niraparib in patients with castrate resistant prostate cancer (NiraRad).

TPS385 Background: PARP1 is a key regulator of the transition to lethal castrate resistant prostate cancer (CRPC); niraparib is a potent and selective PARP-1 and PARP-2 inhibitor that has shown single agent clinical activity in CRPC. Radium-223 is an alpha particle emitter that causes DNA double strand breaks and is FDA approved for use in CRPC. The addition of a PARP inhibitor (niraparib) may further enhance the clinical benefit of Radium-223 through inhibition of DNA repair and modifications in the bone microenvironment. We hypothesize that targeting the PARP1/androgen receptor (AR) axis in combination with radiopharmaceutical therapy will be safe and may improve clinical outcomes for men with advanced CRPC. Furthermore, combining clinical and pathologic features with correlative studies on tumor tissue, blood, and circulating tumor cells (CTCs) may help us develop biomarkers for future use in the treatment of CRPC. Methods: This is aphase IB dose finding study of patients with metastatic CRPC using a time-to-event continual reassessment (TITE-CRM) design to identify the maximum tolerated dose (MTD) of niraparib (100, 200, or 300 mg QD) in combination with standard doses of Radium-223 in patients with and without prior chemotherapy. Niraparib dosing will continue after 6 cycles of radium-223 until disease progression or intolerable adverse events. Correlative studies include the assessment of tumor biopsy tissues using DNA and RNA next-generation sequencing to assay for the presence of DNA repair aberrations in genes; targeted gene expression profiling of serial RNA (baseline and after cycles 1 and 3) performed on whole blood using the Nanostring PanCancer panel; and evaluation of pre- and post-therapy bone marrow biopsies in a subcohort to characterize the effects of the combined therapy on the bone microenvironment. Additional correlative studies that will be performed on CTC include: assay for nuclear AR expression; immunostaining for the presence of persistent g-H2AX foci, (markers of unrepaired DNA double strand breaks); NFkB activity assayed by immunostaining for p-p65). Enrollment to Dose Level 1 began in October 2017. Clinical trial information: NCT03076203.

Androgen receptor mutations in patients with castration-resistant prostate cancer treated with apalutamide

Mutations in the androgen receptor (AR) ligand-binding domain (LBD), such as F877L and T878A, have been associated with resistance to next-generation AR-directed therapies. ARN-509-001 was a phase I/II study that evaluated apalutamide activity in castration-resistant prostate cancer (CRPC). Here, we evaluated the type and frequency of 11 relevant AR-LBD mutations in apalutamide-treated CRPC patients. Blood samples from men with nonmetastatic CRPC (nmCRPC) and metastatic CRPC (mCRPC) pre- or post-abiraterone acetate and prednisone (AAP) treatment (≥6 months' exposure) were evaluated at baseline and disease progression in trial ARN-509-001. Mutations were detected in circulating tumor DNA using a digital polymerase chain reaction-based method known as BEAMing (beads, emulsification, amplification and magnetics) (Sysmex Inostics' GmbH). Of the 97 total patients, 51 had nmCRPC, 25 had AAP-naïve mCRPC, and 21 had post-AAP mCRPC. Ninety-three were assessable for the mutation analysis at baseline and 82 of the 93 at progression. The overall frequency of detected AR mutations at baseline was 7/93 (7.5%) and at progression was 6/82 (7.3%). Three of the 82 (3.7%) mCRPC patients (2 AAP-naïve and 1 post-AAP) acquired AR F877L during apalutamide treatment. At baseline, 3 of the 93 (3.2%) post-AAP patients had detectable AR T878A, which was lost after apalutamide treatment in 1 patient who continued apalutamide treatment for 12 months. The overall frequency of detected mutations at baseline (7.5%) and progression (7.3%) using the sensitive BEAMing assay was low, suggesting that, based on this assay, AR-LBD mutations such as F877L and T878A are not common contributors to de novo or acquired resistance to apalutamide. NCT01171898.

Positive feedback loop mediated by protein phosphatase 1α mobilization of P-TEFb and basal CDK1 drives androgen receptor in prostate cancer

P-TEFb (CDK9/cyclin T) plays a central role in androgen receptor (AR)-mediated transactivation by phosphorylating both RNA polymerase 2 complex proteins and AR at S81. CDK9 dephosphorylation mobilizes P-TEFb from an inhibitory 7SK ribonucleoprotein complex, but mechanisms targeting phosphatases to P-TEFb are unclear. We show that AR recruits protein phosphatase 1α (PP1α), resulting in P-TEFb mobilization and CDK9-mediated AR S81 phosphorylation. This increased pS81 enhances p300 recruitment, histone acetylation, BRD4 binding and subsequent further recruitment of P-TEFb, generating a positive feedback loop that sustains transcription. AR S81 is also phosphorylated by CDK1, and blocking basal CDK1-mediated S81 phosphorylation markedly suppresses AR activity and initiation of this positive feedback loop. Finally, androgen-independent AR activity in castration-resistant prostate cancer (CRPC) cells is driven by increased CDK1-mediated S81 phosphorylation. Collectively these findings reveal a mechanism involving PP1α, CDK9 and CDK1 that is used by AR to initiate and sustain P-TEFb activity, which may be exploited to drive AR in CRPC.

Phase II trial of non-pegylated liposomal doxorubicin and low-dose prednisone in second-line chemotherapy for hormone-refractory prostate cancer.

Non-pegylated liposomal doxorubicin (NPLD) (Myocet) has shown marked in vitro activity in castration-resistant prostate cancer (CRPC) and also in docetaxel-resistant cells, higher than that shown by pegylated liposomal doxorubicin. Its activity would seem to be due to a high intracellular drug concentration and induction of Golgi-dependent apoptosis. On the basis of these results, a clinical study was designed to assess the activity of NPLD and low-dose prednisone in second-line therapy. Fifty-four patients were enrolled and evaluated. Eligibility criteria were histologically confirmed CRPC, PSA >20 ng/mL or measurable lesions according to the RECIST criteria, previous docetaxel-based chemotherapy, and adequate cardiac function. Patients were treated with weekly intravenous NPLD 25 mg/m2 and daily prednisone 10 mg until progression. Median patient age was 69 years (range, 52-83) and median baseline PSA concentration was 120 ng/mL (range, 5.35-4350). Sixteen (29.6%) patients had measurable lesions. Objective or PSA responses (>50% reduction) were observed in 8 (14.8%) patients. The median time to progression was 2.8 months and the median overall survival was 11.3 months. Toxicity was generally mild (grade 1-2) and infrequent, with grade 3-4 neutropenia in 12.9% of cases. Grade 3 nonhematological toxicities included nausea in 2 patients (3.7%) and fatigue and stomatitis in 1 case (1.9%). No drug-related serious adverse events were reported. Weekly administration of NPLD is a well tolerated treatment with proven albeit limited activity.

Nucleoporin 62 and Ca2+/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells

Re-activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen-responsive to castrate-resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca(2+) /calmodulin-dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen-responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells. The abundance of CaMKK2 in nuclear fractions of androgen-responsive prostate cancer and CRPC, cells were determined by subcellular fractionation and Western blotting. CaMKK2 association with nuclear pore complexes (NPCs) and nucleoporins (Nups) including Nup62, were imaged by structured illumination and super-resolution fluorescence microscopy and co-immunoprecipitation, respectively. The abundance and subcellular localization of CaMKK2 and Nup62 in human clinical specimens of prostate cancer was visualized by immunohistochemistry. The role of Nups in the growth and viability of CRPC cells was assessed by RNA interference and cell counting. The involvement of CaMKK2 and Nup62 in regulating AR transcriptional activity was addressed by RNA interference, chromatin immunoprecipitation, androgen response element reporter assay, and Western blotting. CaMKK2 was expressed at higher levels in the nuclear fraction of CPRC C4-2 cells, than in that of androgen-responsive LNCaP cells. In C4-2 cells, CaMKK2 associated with NPCs of the nuclear envelope and physically interacted with Nup62. CaMKK2 and Nup62 demonstrated pronounced, and similar increases in both expression and perinuclear/nuclear localization in human clinical specimens of advanced prostate cancer relative to normal prostate. Knockdown of Nup62, but not of Nups, 98 or 88, reduced growth and viability of C4-2 cells. Knockdown of Nup62 produced a greater reduction of the growth and viability of C4-2 cells than of non-neoplastic RWPE-1 prostatic cells. Nup62, CaMKK2, and the AR were recruited to androgen response elements of the AR target genes, prostate specific antigen, and transmembrane protease, serine 2. Knockdown of CaMKK2 and Nup62 reduced prostate specific antigen expression and AR transcriptional activity driven by androgen response elements from the prostate-specific probasin gene promoter. Nup62 and CaMKK2 are required for optimal AR transcriptional activity and a potential mechanism for AR re-activation in CRPC.

Abstract CT134: Androgen receptor (AR) mutations in patients (pts) with castration-resistant prostate cancer (CRPC) with and without prior abiraterone acetate (AA) treatment

Abstract Background: ARN-509 is a second-generation antiandrogen with antitumor activity in CRPC. Activating mutations in the AR ligand-binding domain (LBD) have been associated with resistance to first- (T877A) and second- (F876L) generation antiandrogens. We evaluated the type and frequency of relevant AR LBD mutations in ARN-509-treated CRPC pts at baseline (BL) and disease progression (PD). Methods: ARN-509-001 was a phase I/II study that evaluated ARN-509 activity in nonmetastatic (M0), chemotherapy-naïve, and post-AA CRPC. Of the 97 pts enrolled in phase II at a dose of 240 mg/d, 92 were evaluable for the mutation analysis at BL and 82 at PD. Relevant mutations in circulating tumor DNA were detected using a digital PCR method called BEAMing (Beads, Emulsification, Amplification, and Magnetics) (Richardson AL. Clin Cancer Res. 2012). Results: Median duration of therapy was ∼16 months. One pt in the M0 cohort and one in the chemotherapy-naïve cohort had the F876L mutation at BL. Two pts in the chemotherapy-naïve cohort and one in the post-AA cohort acquired the AR F876L mutation during treatment. Pts with M0 CRPC did not acquire a mutation (Table 1). Three pts in the post-AA cohort had the T877A mutation at BL; the T877A mutation was not detected in any other cohort at BL. In the post-AA cohort, one pt acquired the T877A mutation during treatment while another lost the mutation (Table). The two pts with detectable F876L at BL developed prostate-specific antigen (PSA) progression at 4 and 6 months, respectively, compared with a median time to PSA progression of 16.4 months in the remainder of pts. Conclusions: Pts with metastatic CRPC who were treated with ARN-509 had a low rate of acquisition of the AR F876L (3/82 = 4%) and AR T877A (1/82 = 1%) mutations. These results suggest that ARN-509 may be continued in the setting of a rising PSA. Larger studies are needed to confirm the prevalence of F876L, T877A, and the conversion rate. AR F876LAR T877AEvaluableBLPDAcquiredBLPDAcquiredpatientsn/Nn/Nn/Nn/NTotal922/925/823/823/923/821/82M0491/491/470/470/500/470/47Chemotherapy-naïve241/243/202/200/200/200/20Post-AA190/191/151/153/193/151/15 Citation Format: Dana E. Rathkopf, Matthew R. Smith, Emmanuel S. Antonarakis, Charles J. Ryan, William R. Berry, Neal D. Shore, Glenn Liu, Celestia Higano, Joshi J. Alumkal, Ralph Hauke, Ronald Tutrone, Mansoor Saleh, Edna Chow Maneval, Shibu Thomas, Deborah Ricci, Margaret K. Yu, Carla J. de Boer, Angela Trinh, Thian Kheoh, Rajesh Bandekar, Howard I. Scher. Androgen receptor (AR) mutations in patients (pts) with castration-resistant prostate cancer (CRPC) with and without prior abiraterone acetate (AA) treatment. [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 CT134. doi:10.1158/1538-7445.AM2015-CT134

Abstract 3452: Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer

Abstract Castration resistant prostate cancer (CRPC) still relies on androgens for progression. Abiraterone, an FDA approved CYP17A inhibitor, blocks androgen synthesis and prolongs survival. Abiraterone metabolites are generally thought to be inactive. We have identified a novel abiraterone metabolite, D4A, which inhibits three steroidogenic enzymes and androgen receptor (AR) itself. After abiraterone acetate administration, plasma from mice and patients were analyzed with liquid chromatography-mass spectrometry (LC-MS). The LC-MS results indicate that abiraterone is converted to D4A at detectable concentrations in both mice and patients. To test the function of D4A, the androgen synthesis pathway was analyzed in cell lines and xenografts. After incubation with [3H] labeled androgens, metabolites from cell lines and xenografts were analyzed by HPLC. We found that D4A inhibits activity of the steroidogenic enzymes CYP17A, 3β-hydroxysteroid dehydrogenase (3[[Unsupported Character - Symbol Font ]]HSD) and 5α-reductase. Furthermore D4A competes with the AR agonist R1881 and binds to AR directly. D4A inhibits DHT-induced AR occupancy on the PSA enhancer as detected by chromatin immunoprecipitation and reduces PSA expression. Our findings suggest that D4A is an abiraterone metabolite and a potent inhibitor of the androgen pathway. Citation Format: Zhenfei Li, Nima Sharifi. Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer. [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 3452. doi:10.1158/1538-7445.AM2015-3452

Abstract 4212: Inhibition of mouse PTEN-deficient prostate cancer with next generation antisense oligonucleotide targeting the androgen receptor

Abstract Resistance to androgen deprivation therapies is characterized by the persistent activation of the androgen receptor (AR). Traditional approaches for the development of novel androgen deprivation therapies have focused on inhibition of AR activity by blocking the production or binding of androgens to the AR. These novel treatment strategies have shown improvements, however, patients still develop castration-resistant disease. Continued AR activity in castration-resistant prostate cancer (CRPC) may be the result of ligand dependent or ligand independent mechanisms. Antisense oligonucleotides (ASO) have the ability to block specific genes inhibiting the associated proteins thus providing an alternate treatment strategy for CRPC. Here, we investigated the in vivo antitumor effects of a generation 2.5 ASO targeting the mouse AR in a PTEN-deficient mouse model of prostate cancer. Pharmacodynamic analysis demonstrated strong inhibition of target RNA by the AR-ASO in prostate tumor tissues. The AR-ASO also exhibited strong suppression of AR protein levels in prostate tumors as demonstrated by western blot analysis. Immunohistochemical analysis confirmed reduced levels of AR expression and transcriptional activity in tumor tissue. Furthermore, AR was also inhibited in normal tissues of the accessory sex glands. The treatment efficacy of the AR-ASO was evaluated in pharmacological intervention trials in mice with castration-naïve prostate cancer (CNPC) and CRPC. Treatments effects in CNPC were also compared to androgen deprivation by surgical castration. Compared to saline treated controls, tumor growth was significantly inhibited by the AR-ASO (34%) and was comparable to that of surgical castration (38%). In the CRPC model, significant suppression of tumor growth (10% vs control) was also observed in mice treated with the AR-ASO. Furthermore, strong inhibition of AR expression and activity was observed in AR-ASO-treated mice harboring either CNPC or CRPC, but not in mice treated with surgical castration. Gene expression profiling using the Fluidigm Biomark platform revealed that 40 AR-dependent target genes were significantly altered in CNPC after treatment with the AR-ASO; 34 of these genes were also significantly changed following castration. A decrease of ERK phosphorylation in was observed in AR-ASO-treated mice in both CNPC and CRPC intervention models and was associated with decreased protein levels of AR. These findings demonstrate that treatment with generation 2.5 AR-ASO is effective against preclinical mouse models of prostate cancer and may provide a promising approach to overcoming treatment resistance of CRPC. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Emiko Fukushima, Barry R. Davies, Hayley Campbell, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Inhibition of mouse PTEN-deficient prostate cancer with next generation antisense oligonucleotide targeting the androgen receptor. [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 4212. doi:10.1158/1538-7445.AM2015-4212

Abstract 2065: TMEFF2 is a novel androgen receptor co-activator in prostate cancer cells

Abstract The goal of this study is to delineate the role of a novel androgen receptor (AR) coactivator in advanced prostate cancer (PCa). While great advances have been made in the ability to detect PCa, the disease remains the second leading cause of cancer related deaths in men. The AR is a steroid nuclear receptor that serves a central function in both normal prostate cell homeostasis and in PCa; in fact, the vast majority of PCa tumors require androgens for growth. This dependency has led to the development and use of androgen deprivation therapies (ADTs) for the treatment of PCa. ADT is initially a successful form of treatment for most PCa patients; however, the majority of patients eventually relapse with castration-resistant prostate cancer (CRPC), which is currently incurable. Because sustained AR activity under androgen depleted conditions is a major contributor to the development of CRPC, identifying molecular mechanisms that contribute to AR activity in CRPC is paramount for the development of successful therapies. Our lab has been studying the role of the transmembrane protein with epidermal-like growth factor domain and two follistatin domains 2 (TMEFF2) in PCa. While TMEFF2 gene expression is repressed by hypermethylation in multiple cancers, TMEFF2 is overexpressed in PCa and CRPC, suggesting that TMEFF2 may have a unique function in PCa. Importantly, TMEFF2 expression is activated by the androgen receptor (AR) at the level of transcription and translation, which may contribute to its overexpression in PCa. Our data presented here indicate that TMEFF2 functions in PCa cells as a coactivator of the AR. Using shRNA to stably knockdown TMEFF2 expression in androgen dependent PCa and CRPC cell lines, we demonstrate that TMEFF2 activates the basal and androgen-induced expression of multiple androgen responsive genes, including prostate-specific antigen (PSA), at the mRNA and protein level. Importantly, the knock-down of TMEFF2 does not alter AR protein levels, indicating that TMEFF2 influences AR activity. Additionally, by coimmunoprecipitation analysis, we demonstrate that TMEFF2 interacts with the AR, and western blot analyses with cellular fractionation samples indicate that TMEFF2 has the ability to translocate to the nucleus of PCa cells. Based on these results, we conclude that TMEFF2 is a novel AR coactivator in PCa. Future studies will focus on understanding the nature and biological effects of the interaction between TMEFF2 and the AR, and delineating the molecular mechanism through which TMEFF2 activates AR-mediated gene expression. Because sustained AR activity in androgen depleted conditions is a major contributor to CRPC relapse and PCa mortality, the TMEFF2-mediated AR activation mechanism may be clinically useful as a therapeutic target in CRPC. Citation Format: Joshua Corbin, Thomas Green, Maria J. Ruiz-Echevarria. TMEFF2 is a novel androgen receptor co-activator in prostate cancer cells. [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 2065. doi:10.1158/1538-7445.AM2015-2065

Adaptive responses of androgen receptor signaling in castration-resistant prostate cancer.

Prostate Cancer (PCa) is an important age-related disease being the most common cancer malignancy and the second leading cause of cancer mortality in men in Western countries. Initially, PCa progression is androgen receptor (AR)- and androgen-dependent. Eventually advanced PCa reaches the stage of Castration-Resistant Prostate Cancer (CRPC), but remains dependent on AR, which indicates the importance of AR activity also for CRPC. Here, we discuss various pathways that influence the AR activity in CRPC, which indicates an adaptation of the AR signaling in PCa to overcome the treatment of PCa. The adaptation pathways include interferences of the normal regulation of the AR protein level, the expression of AR variants, the crosstalk of the AR with cytokine tyrosine kinases, the Src-Akt-, the MAPK-signaling pathways and AR corepressors. Furthermore, we summarize the current treatment options with regard to the underlying molecular basis of the common adaptation processes of AR signaling that may arise after the treatment with AR antagonists, androgen deprivation therapy (ADT) as well as for CRPC, and point towards novel therapeutic strategies. The understanding of individualized adaptation processes in PCa will lead to individualized treatment options in the future.

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.

IND 205B: A phase II study of the PI3K inhibitor PX-866 and continued abiraterone/prednisone in patients with recurrent or metastatic castration resistant prostate cancer (CRPC) with PSA progression on abiraterone/prednisone.

279 Background: PX-866 is an irreversible, pan-isoform inhibitor of Class I PI-3K. In part A of this study, PX-866 as a single agent was well tolerated and showed modest activity in CRPC but did not meet a priori benchmarks as a single agent in unselected patients (pts). As androgen receptor inhibition promotes PI3K activity in PTEN-loss PC models, the addition of PX-866 in patients whose PSA is rising on abiraterone acetate plus prednisone (AA+P) may reverse resistance and phase B of the study tested this hypothesis clinically. Methods: In this multicentre, phase II study, CRPC pts with PSA progression on AA+P received PX-866, 8mg daily on a 6-week cycle while continuing AA+P. Primary endpoint was lack of progression at 12 weeks (PCWG2 criteria). Secondary endpoints included PSA and objective response rates. PX-866 would be deemed worthy of further study if at least four of 25 pts were progression free at 12 weeks. Results: 25 pts were accrued and all are evaluable for toxicity and PSA response. Eleven pts had measurable disease of which 11 are evaluable for objective response. Median age was 72, ECOG PS was 0/1 in 10/15 pts. Eighteen pts received prior chemotherapy. Median number of cycles was 2 (range 1–3). 52% of pts received at least 90% of planned dose of PX-866. Most common adverse events (AEs) were fatigue (21 patients), diarrhea, (18), nausea (13), vomiting (11) and anorexia (12); one patient discontinued because of protocol therapy toxicity (grade 3 anemia). Other grade 3 AEs (one patient for each) were diarrhea, vomiting, fatigue, AST and ALT elevation and lymphocyte and platelet changes. Six pts were progression free at 12 weeks. No objective or PSA responses were seen. Of the pts with measurable disease, best objective response was stable disease in six (2.3-3.6m) and best PSA response was non-response in eight pts. Conclusions: The addition of PX-866 to AA+P in unselected pts progressing on AA+P shows no evidence of antitumour effects. Strategies to combine PI3K inhibition with AR targeted therapies should consider initiation earlier in the disease course and/or recruiting a selected population. Clinical trial information: NCT01331083.

Abstract 675: Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer

Abstract Inhibition of the androgen receptor (AR) results in activation of the PI-3K/mTOR pathway in PTEN deficient prostate cancer cells by reciprocal feedback inhibition (Carver BS, 2011). Enzalutamide or PI-3K/mTor inhibition result in feedback activation and combined inhibition abrogated this effect. We explored AR inhibition in CRPC cell lines with low, intermediate and high AR expression in the context of PI-3K/mTor inhibition to identify biomarkers of response and resistance. We utilized no/low (DU145, PC3), intermediate (LNCaP) and high (22Rv1,VCap) AR expressing prostate cancer (PC) cell lines to evaluate anti-tumor activity with 4 pan-PI3K/mTOR inhibitors (GDC-0980, GDC-0941, LY294002, PF-04691502), enzalutamide and abiraterone as single agents or various in combinations at clinically relevant doses. Cytotoxic activity of enzalutamide was more potent than abiraterone (IC50's was < 1µM and > 10µM) in LNCaP cells, however, the opposite was seen in DU145 and PC-3 cell lines. High AR expressing cells (22Rv1, VCap) were more sensitive to AR inhibition. PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 had IC50's in the range 50-500nM while LY294002 had an IC50 16-24 µM except for DU145. This cell line had increased IC50 values comparatively for PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 (1.5-3.75 µM) and LY29002 (48.5 µM). The combination index (CI) isobologram method indicated significant synergism for PI-3K/mTOR SMIs plus enzalutamide or abiraterone in all PC cell lines irrespective of AR status. Combination treatments were associated with increased apoptosis (Annexin-V staining and PARP-cleavage assay) in LNCaP and VCaP > 22Rv1 > DU145 and PC3. Cell cycle analysis by flow cytometry demonstrated that enzalutamide induces G1 arrest in LNCaP and DU145 cells and G2/M arrest in PC3 cells. PI-3K inhibition induces S arrest in LNCaP and DU145 and G2/M arrest in PC3 cells. The combination of enzalutamide plus PI-3K inhibition enhanced S arrest in LNCaP while in DU145 and PC3, G0/G1 arrest was prominent. PI-3K SMI plus enzalutamide completely inhibited pAkt308/473, pS6 and p4EBP1 in LNCaP cells, pAkt473 in PC3 cells and pS6 and p4EBP1 in 22Rv1 cells. Enzalutamide alone or in combination with PI-3K inhibition also suppressed AR expression in 22Rv1 cells. A 22Rv1 mouse xenograft model is evaluating safety and efficacy of the combination. This is a novel therapeutic strategy for CRPC patients to be evaluated in a clinical trial. Citation Format: Daruka Mahadevan, Carla Morales, Laurence S. Cooke, Bradley Somer, Wenqing Qi. Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant 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 675. doi:10.1158/1538-7445.AM2014-675

Lyn tyrosine kinase regulates androgen receptor expression and activity in castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) progression is a complex process by which prostate cells acquire the ability to survive and proliferate in the absence or under very low levels of androgens. Most CRPC tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes owing to reactivation of AR. Protein tyrosine kinases have been implicated in supporting AR activation under castrate conditions. Here we report that Lyn tyrosine kinase expression is upregulated in CRPC human specimens compared with hormone naive or normal tissue. Lyn overexpression enhanced AR transcriptional activity both in vitro and in vivo and accelerated CRPC. Reciprocally, specific targeting of Lyn resulted in a decrease of AR transcriptional activity in vitro and in vivo and prolonged time to castration. Mechanistically, we found that targeting Lyn kinase induces AR dissociation from the molecular chaperone Hsp90, leading to its ubiquitination and proteasomal degradation. This work indicates a novel mechanism of regulation of AR stability and transcriptional activity by Lyn and justifies further investigation of the Lyn tyrosine kinase as a therapeutic target for the treatment of CRPC.

Looking beyond Androgen Receptor Signaling in the Treatment of Advanced Prostate Cancer

This review will provide a description of recent efforts in our laboratory contributing to a general goal of identifying critical determinants of prostate cancer growth in both androgen-dependent and -independent contexts. Important outcomes to date have indicated that the sustained activation of AR transcriptional activity in castration-resistant prostate cancer (CRPC) cells results in a gene expression profile separate from the androgen-responsive profile of androgen-dependent prostate cancer (ADPC) cells. Contributing to this reprogramming is enhanced FoxA1 recruitment of AR to G2/M phase target gene loci and the enhanced chromatin looping of CRPC-specific gene regulatory elements facilitated by PI3K/Akt-phosphorylated MED1. We have also observed a role for FoxA1 beyond AR signaling in driving G1/S phase cell cycle progression that relies on interactions with novel collaborators MYBL2 and CREB1. Finally, we describe an in-depth mechanism of GATA2-mediated androgen-responsive gene expression in both ADPC and CRPC cells. Altogether these efforts provide evidence to support the development of novel prostate cancer therapeutics that address downstream targets of AR activity as well as AR-independent drivers of disease-relevant transcription programs.

Activity of Cabazitaxel in Castration-resistant Prostate Cancer Progressing After Docetaxel and Next-generation Endocrine Agents

BackgroundCabazitaxel, abiraterone, and enzalutamide are survival-prolonging treatments in men with castration-resistant prostate cancer (CRPC) progressing following docetaxel chemotherapy. The sequential activity of these agents has not been studied and treatment sequencing remains a key dilemma for clinicians. ObjectiveTo describe the antitumour activity of cabazitaxel after docetaxel and next-generation endocrine agents. Design, setting, and participantsWe report on a cohort of 59 men with progressing CRPC treated with cabazitaxel, 37 of whom had received prior abiraterone and 9 of whom had received prior enzalutamide. Outcome measurements and statistical analysisChanges in prostate-specific antigen (PSA) level were used to determine activity on abiraterone, enzalutamide, and cabazitaxel treatment. Radiologic tumour regressions according to Response Evaluation Criteria in Solid Tumors (RECIST) and symptomatic benefit were evaluated for cabazitaxel therapy. Results and limitationsThe post–endocrine-therapy patients received abiraterone (n=32), sequential abiraterone and enzalutamide (n=5) or enzalutamide (n=4). These patients received a median of 7 mo of abiraterone and 11 mo of enzalutamide. A median of six cabazitaxel cycles (range: 1–10 cycles) were delivered, with ≥50% PSA declines in 16 of 41 (39%) patients, soft tissue radiologic responses in 3 of 22 (14%) evaluable patients, and symptomatic benefit in 9 of 37 evaluable patients (24%). Median overall survival and progression-free survival were 15.8 and 4.6 mo, respectively. Antitumor activity on cabazitaxel was less favourable in the abiraterone- and enzalutamide-naïve cohort (n=18), likely reflecting biologic differences in this cohort. These data were obtained from a retrospective analysis. ConclusionsThis is the first report of cabazitaxel activity in CRPC progressing after treatment with docetaxel and abiraterone or enzalutamide. We demonstrate significant cabazitaxel activity in this setting. Patient summaryWe looked at the antitumour activity of the chemotherapy drug cabazitaxel in men previously treated with docetaxel chemotherapy and the hormonal drugs abiraterone and enzalutamide. Cabazitaxel appeared active when given after abiraterone and enzalutamide. We can reassure men that cabazitaxel can be used after these novel endocrine treatments.

Mechanisms of androgen receptor activation in castration-resistant prostate cancer.

Systemic treatment of advanced prostate cancer is initiated with androgen deprivation therapy by gonadal testosterone depletion. Response durations are variable and tumors nearly always become resistant as castration-resistant prostate cancer (CRPC), which is driven, at least in part, by a continued dependence on the androgen receptor (AR). The proposed mechanisms that underlie AR function in this clinical setting are quite varied. These include intratumoral synthesis of androgens from inactive precursors, increased AR expression, AR activation through tyrosine kinase-dependent signaling, alterations in steroid receptor coactivators, and expression of a truncated AR with constitutive activity. Various pharmacologic interventions have clinically validated some of these mechanisms, such as those that require the AR ligand-binding domain. Clinical studies have failed to validate other mechanisms, and additional mechanisms have yet to be tested in patients with CRPC. Here, we review the mechanisms that elicit AR activity in CRPC, with a particular focus on recent developments.

Regulation of the Transcriptional Coactivator FHL2 Licenses Activation of the Androgen Receptor in Castrate-Resistant Prostate Cancer

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.