Search

Unsupervised clustering and deconvolution analysis identifies three intrinsic subtypes of Metastatic Castration-Resistant Prostate Cancer (M-CRPC): AR pathway-positive Prostate Cancer (ARPC), Neuro Endocrine Prostate Cancer (NEPC), and a novel subtype endowed with hybrid epithelial/mesenchymal (E/M) and luminal progenitor-like traits (Mesenchymal and Stem-like PC, MSPC). Analysis of large patient datasets and in vitro studies support the notion that MSPC originates from ARPC as a consequence of therapy-induced lineage plasticity. Intriguingly, AR blockade instigates two separate and complementary processes: 1) transcriptional silencing of TP53 and hence acquisition of hybrid E/M and stem-like traits; and 2) inhibition of the BMP-SMAD pathway, which promotes resistance to the pro-apoptotic and anti-proliferative effects of AR inhibition. MSPC cell lines and prostate adenocarcinoma cells reprogrammed in vitro to MSPC exhibit a marked dependency on HER2/3 signaling. Moreover, combinations of the panHER inhibitor neratinib with enzalutamide or the mTORC1 inhibitor MLN0128 exhibit efficacy in preclinical models of mixed ARPC/MSPC or MSPC, respectively. Collectively, these results identify a novel subtype of M-CRPC, trace its origin to therapy induced lineage plasticity, and reveal its dependency on HER2/3 signaling.

While the antiandrogen enzalutamide (Enz) extends the castration resistant prostate cancer (CRPC) patients’ survival an extra 4.8 months, it might also result in some adverse effects via inducing the neuroendocrine differentiation (NED). Here we found that lncRNA-p21 is highly expressed in the NEPC patients derived xenograft tissues (NEPC-PDX). Results from cell lines and human clinical sample surveys also revealed that lncRNA-p21 expression is up-regulated in NEPC and Enz treatment could increase the lncRNA-p21 to induce the NED. Mechanism dissection revealed that Enz could promote the lncRNA-p21 transcription via altering the androgen receptor (AR) binding to different androgen-response-elements, which switch the EZH2 function from histone-methyltransferase to non-histone methyltransferase, consequently methylating the STAT3 to promote the NED. Preclinical studies using the PDX mouse model proved that EZH2 inhibitor could block the Enz-induced NED. Together, these results suggest targeting the Enz/AR/lncRNA-p21/EZH2/STAT3 signaling may help urologists to develop a treatment for better suppression of the human CRPC progression., The induction of neuroendocrine differentiation occurs in enzalutamide treated castration resistant prostate cancer. Here, the authors show that lncRNA-21 mediates enzalutamide induced neuroendocrine differentiation through EZH2/STAT axis and EZH2 inhibition suppresses this differentiation.

Background Patients with advanced primary and recurrent salivary duct carcinoma (SDC), a rare and lethal malignancy, have limited therapeutic options. Novel small-molecule agents aimed at targeting critical signaling associated with SDC tumorigenesis may lead to new therapeutic options for patients with these tumors. The human epidermal growth factor receptor 2 (HER2)/phosphoinositide 3-kinase (PI3K) axis, an important oncogenic pathway, has been targeted for therapy in several solid tumors. Currently, little is known about the role and clinical implications of alterations of the HER2/PI3K pathway in patients with SDC. Methods The authors investigated the clinicopathologic features, genetic alterations, and expression of key members of the HER2/PI3K pathway in 43 primary tumors and conducted in vitro functional and targeted drug-response analyses on cell lines derived from salivary epithelial carcinomas. Results In primary tumors, loss of phosphatase and tensin homolog (PTEN) expression was identified in 22 of 43 tumors (51%), overexpression of HER2 was observed in 12 of 43 tumors (28%), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations were identified in 12 of 43 tumors (28%). Phosphorylated protein kinase B (p-AKT) was highly expressed in most tumors. Most tumors (70%) displayed mutually exclusive alterations of PI3K members, whereas 8 tumors (19%) had 2 or more concurrent abnormalities. In vitro studies demonstrated a direct association between PTEN loss and PI3K pathway activation and evidence of response to combined PI3Kα and PI3Kβ and/or pan-PI3K inhibitors. Conclusions The current analyses reveal frequent PTEN loss and mutually exclusive alterations of key PI3K pathway members in SDC and demonstrate in vitro evidence of a response to pan-PI3K inhibitors. These results provide a framework for a biomarker-based substratification of patients with SDC in future targeted therapy. Cancer 2018;124:3523-32. © 2018 American Cancer Society.

PURPOSE: Androgen receptor (AR) signaling affects prostate cancer (PCa) growth, metabolism and progression. Often PCa progresses from androgen-sensitive to castration-resistant prostate cancer (CRPC) following androgen-deprivation therapy. Clinicopathologic and genomic characterizations of CRPC tumors lead to subdividing CRPC into two subtypes: 1) AR-dependent CRPC containing dysregulation of AR signaling alterations in AR such as amplification, point mutations and/or generation of splice variants in the AR gene, and 2) an aggressive variant PCa (AVPC) subtype that is phenotypically similar to small cell prostate cancer and is defined by chemotherapy sensitivity, gain of neuroendocrine or pro-neural marker expression, loss of AR expression, and combined alterations of PTEN, TP53, and RB1 tumor suppressors. Previously, we reported patient derived xenograft (PDX) animal models that contain characteristics of these CRPC subtypes. In this study, we have employed the PDX models to test metabolic alterations in the CRPC subtypes. PROCEDURES: Mass spectrometry and nuclear magnetic resonance analysis along with in vivo hyperpolarized 1-[(13)C]pyruvate spectroscopy experiments were performed on prostate PDX animal models. RESULTS: Using hyperpolarized 1-[(13)C]pyruvate conversion to 1-[(13)C]lactate in vivo as well as lactate measurements ex vivo, we have found increased lactate production in AR-dependent CRPC PDX models even under low-hormone levels (castrated mouse) compared to AR negative AVPC PDX models. CONCLUSIONS: Our analysis underscores the potential of hyperpolarized metabolic imaging in determining the underlying biology and in vivo phenotyping of CRPC.

Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.

Purpose: Morphologically heterogeneous prostate cancers that behave clinically like small-cell prostate cancers (SCPC) share their chemotherapy responsiveness. We asked whether these clinically defined, morphologically diverse, “aggressive variant prostate cancer (AVPC)” also share molecular features with SCPC. Experimental Design: Fifty-nine prostate cancer samples from 40 clinical trial participants meeting AVPC criteria, and 8 patient-tumor derived xenografts (PDX) from 6 of them, were stained for markers aberrantly expressed in SCPC. DNA from 36 and 8 PDX was analyzed by Oncoscan for copy number gains (CNG) and losses (CNL). We used the AVPC PDX to expand observations and referenced publicly available datasets to arrive at a candidate molecular signature for the AVPC. Results: Irrespective of morphology, Ki67 and Tp53 stained ≥10% cells in 80% and 41% of samples, respectively. RB1 stained Conclusions: Clinically defined AVPC share molecular features with SCPC and are characterized by combined alterations in RB1, Tp53, and/or PTEN. Clin Cancer Res; 22(6); 1520–30. ©2015 AACR.

Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties.

Combined loss of tumor suppressors (TSPs) PTEN, TP53, and RB1 is highly associated with small cell carcinoma of prostate phenotype. Recent genomic studies of human tumors as well as analyses in mouse genetic models have revealed a unique role for these TSPs in dictating epithelial lineage plasticity – a phenomenon that plays a critical role in the development of aggressive variant prostate cancer and associated androgen therapy resistance. Here, we summarize recently published key observations on this topic, and hypothesize a possible mechanism by which concurrent loss of TSPs could potentially regulate the prostate cancer disease phenotype.

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2cre/Osxf/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osxf/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P

Recently, many therapeutic agents for prostate cancer have been approved that target the androgen receptor and/or the prostate tumor microenvironment. Each of these therapies has modestly increased patient survival. A better understanding of when in the course of prostate cancer progression specific therapies should be applied, and of what biomarkers would indicate when resistance arises, would almost certainly improve survival due to these therapies. Thus, applying the armamentarium of therapeutic agents in the right sequences in the right combination at the right time is a major goal in prostate cancer treatment. For this to occur, an understanding of prostate cancer evolution during progression is required. In this review, we discuss the current understanding of prostate cancer progression, but challenge the prevailing view by proposing a new model of prostate cancer progression, with the goal of improving biologic classification and treatment strategies. We use this model to discuss how integrating clinical and basic understanding of prostate cancer will lead to better implementation of molecularly targeted therapeutics and improve patient survival. Significance: Rapid development of drugs with efficacy against prostate cancer now makes it possible to consider applying these agents with curative intent in men with currently incurable cancers. However, when to apply these new drugs, as well as those under development, to obtain the best outcomes is a challenge that must be addressed. To meet this challenge, better classification of the disease based on the underlying molecular mechanisms of progression will facilitate the implementation of current and emerging therapies. Cancer Discov; 3(8); 849–61. ©2013 AACR.

The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression.

BACKGROUND The interplay between androgen and Hedgehog (Hh) signaling pathways may be associated with prostate cancer progression and resistance to therapy. METHODS Tissue microarrays from prostatectomy specimens were derived from 53 patients treated preoperatively with androgen ablation (AA) with or without chemotherapy, and from 26 stage- and grade-matched controls. A previously characterized androgen-regulated human prostate cancer xenograft was used to conduct parallel murine studies. Expression of markers of interest was determined on both untreated and castrated tumors. RESULTS Four-month exposure to AA or AA with chemotherapy led to a uniform increase in Hh signaling as compared to controls, paired with an inverse trend of androgen receptor (AR) and CYP17 expression in clinically derived specimens. Changes in the expression profiles of Hh signaling were observed in the epithelium and stroma, in response to genotoxic stress of androgen ablation and chemotherapy. A reduced expression of KI67 and increased bcl2 expression was observed in the malignant epithelial compartment. CONCLUSION To our knowledge, this is the first clinical evidence that Hh signaling is induced by AA or the combination of AA and chemotherapy and, by inference, contributes to castrate-resistant progression of prostate cancer as supported by parallel human and murine studies. These data are in agreement with previous reports that implicate Hh signaling in castrate-resistant progression of prostate cancer. Based on these findings, we are pursuing parallel clinical and murine investigations to determine if Hh signaling inhibition combined with AA will be more effective than AA alone. Prostate 73: 153–161, 2013. © 2012 Wiley Periodicals, Inc.

Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation while MDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA-PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCID mice bearing MDA-PCa-118b tumors with LDN-193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone. Cancer Res; 71(15); 5194–203. ©2011 AACR.

Prostate cancer (PCa) accounts for approximately 26,730 deaths each year in the United States. The tremendous heterogeneity observed among prostate cancers contributes to the enormous clinical challenge. Among these tumors, variant Neuroendocrine Prostate Cancers (NEPC) account for up to a third of prostate cancer-related deaths. These tumors are enriched with cancer cells with stem-cell properties (CSCs). Prostate CSCs lack the expression of androgen-receptor (AR), and are hence resistant to AR inhibition. In addition, CSCs are capable of sustaining tumor relapse and metastasis. We previously demonstrated that aberrant induction of epithelial-mesenchymal transition (EMT) in differentiated prostate epithelial cells could result in the activation of CSC features, including ADT resistance and NEPC attributes. Using multiple prostate cancer cell lines as well as patient-derived xenograft samples, we demonstrated a reciprocal relationship between the EMT/CSC phenotype and expression of AR, and also observed a vital role for the central EMT regulator FOXC2 (Forkhead transcription factor C2) in the generation/maintenance of AR-negative CSCs, which are in turn responsible for the formation of NEPCs. We therefore sought to identify conserved EMT-signaling pathways necessary for EMT and neuroendocrine trans-differentiation in PCa cells, and investigate if targeting EMT-promoting pathways renders CSC-enriched PCa cells sensitive to ADT. We demonstrate that FOXC2 is a direct target of p38MAPK (a pleiotropic kinase), and that the p38-FOXC2 signaling cascade is indispensable for NEPC attributes and associated stem-cell functions. Targeting the p38 pathway using a systemic small-molecule inhibitor in animal models in vivo induced epithelial differentiation in NEPCs and indeed rendered them sensitive to androgen-deprivation therapy using enzalutamide, via loss of FOXC2 function. This study therefore highlights a novel (and targetable) signaling pathway that fosters CSC-associated drug resistance in NEPC, and a pioneering treatment potential for patients with NEPC. Acknowledgments: We thank Dr. Nupam Mahajan (Moffitt Cancer Center, University of South Florida, Tampa), Dr. Ignacio Wistuba, Dr. Jaime Rodriguez-Canales, Dr. Sue-Hwa Lin (Department of Translational Molecular Pathology, UT MDACC, Houston) and Dr. Dean Tang (Department of Molecular Carcinogenesis, MDACC, Smithville) for their contributions. We thank the UTMDACC Prostate Cancer SPORE for financial support (RS, SM). Citation Format: Rama Soundararajan, Anurag Paranjape, Robiya Joseph, Neeraja Bhangre, Ana Aparicio, Sankar Maity, Christopher Logothetis, Jeffrey Chang, Sendurai Mani. Modulating a cancer stem cell-specific signaling pathway to reverse the course of neuroendocrine prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A065.

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone- forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF- β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT- PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.

Purpose: To investigate the molecular events associated with the activation of androgen receptor (AR) as a potential therapeutic target in patients with salivary duct carcinoma (SDC). Experimental Design: Comprehensive molecular and expression analysis of the AR gene in 35 tumor specimens (20 males and 15 females) and cell lines derived from SDC using Western blotting and RT-PCR, FISH analysis, and DNA sequencing was conducted. In vitro and in vivo animal studies were also performed. Results: AR expression was detected in 70% of the tumors and was mainly nuclear and homogenous in both male and female SDCs, although variable cytoplasmic and/or nuclear localization was also found. We report the identification of ligand-independent AR splice variants, mutations, and extra AR gene copy in primary untreated SDC tumors. In contrast to prostate cancer, no AR gene amplification was observed. In vitro knockdown of AR in a female derived SDC cell line revealed marked growth inhibition in culture and in vivo androgen-independent tumor growth. Conclusions: Our study provides new detailed information on the molecular and structural alterations associated with AR gene activation in SDC and sheds more light on the putative functional role of AR in SDC cells. On the basis of these data, we propose that patients with SDC (male and female) can be stratified for hormone-based therapy in future clinical trials. Clin Cancer Res; 20(24); 6570–81. ©2014 AACR.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that most commonly evolves from preexisting prostate adenocarcinoma (PCA). Using Next Generation RNA-sequencing and oligonucleotide arrays, we profiled 7 NEPC, 30 PCA, and 5 benign prostate tissue (BEN), and validated findings on tumors from a large cohort of patients (37 NEPC, 169 PCA, 22 BEN) using IHC and FISH. We discovered significant overexpression and gene amplification of AURKA and MYCN in 40% of NEPC and 5% of PCA, respectively, and evidence that that they cooperate to induce a neuroendocrine phenotype in prostate cells. There was dramatic and enhanced sensitivity of NEPC (and MYCN overexpressing PCA) to Aurora kinase inhibitor therapy both in vitro and in vivo, with complete suppression of neuroendocrine marker expression following treatment. We propose that alterations in Aurora kinase A and N-myc are involved in the development of NEPC, and future clinical trials will help determine from the efficacy of Aurora kinase inhibitor therapy.We report on the largest in-depth molecular analysis of NEPC and provide new insight into molecular events involved in the progression of prostate cancer.

Nuclear transcription factor-Y (NF-Y), a key regulator of cell-cycle progression, often loses its activity during differentiation into nonproliferative cells. In contrast, NF-Y is still active in mature, differentiated neurons, although its neuronal significance remains obscure. Here we show that conditional deletion of the subunit NF-YA in postmitotic mouse neurons induces progressive neurodegeneration with distinctive ubiquitin/p62 pathology; these proteins are not incorporated into filamentous inclusion but co-accumulated with insoluble membrane proteins broadly on endoplasmic reticulum (ER). The degeneration also accompanies drastic ER disorganization, that is, an aberrant increase in ribosome-free ER in the perinuclear region, without inducing ER stress response. We further perform chromatin immunoprecipitation and identify several NF-Y physiological targets including Grp94 potentially involved in ER disorganization. We propose that NF-Y is involved in a unique regulation mechanism of ER organization in mature neurons and its disruption causes previously undescribed novel neuropathology accompanying abnormal ubiquitin/p62 accumulation.

Purpose: To study Wnt/β-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the β-catenin-androgen receptor (AR) interaction. Experimental Design: We carried out β-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of β-catenin-mediated transcription), and sequenced the β-catenin gene inMDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down β-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed β-catenin and AR in 27 bone metastases of human CRPCs. Results: β-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated β-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated β-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant β-catenin. Finally, we found nuclear localization of β-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P=0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/β-catenin signaling. Conclusion: We identified a previously unknown downstream target of β-catenin, HAS2, in prostate cancer, and found that high β-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients. Fil: Wan, Xinhai. University of Texas; Estados Unidos Fil: Liu, Jie. University of Texas; Estados Unidos Fil: Lu, Jing-Fang. University of Texas; Estados Unidos Fil: Tzelepi, Vassiliki. University of Texas; Estados Unidos Fil: Yang, Jun. University of Texas; Estados Unidos Fil: Starbuck, Michael W.. University of Texas; Estados Unidos Fil: Diao, Lixia. University of Texas; Estados Unidos Fil: Wang, Jing. University of Texas; Estados Unidos Fil: Efstathiou, Eleni. University of Texas; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Troncoso, Silvana Patricia. University of Texas; Estados Unidos Fil: Maity, Sankar N.. University of Texas; Estados Unidos Fil: Navone, Nora. University of Texas; Estados Unidos

Purpose: Small-cell prostate carcinoma (SCPC) morphology predicts for a distinct clinical behavior, resistance to androgen ablation, and frequent but short responses to chemotherapy. We sought to develop model systems that reflect human SCPC and can improve our understanding of its biology. Experimental Design: We developed a set of castration-resistant prostate carcinomas xenografts and examined their fidelity to their human tumors of origin. We compared the expression and genomic profiles of SCPC and large-cell neuroendocrine carcinoma (LCNEC) xenografts to those of typical prostate adenocarcinoma xenografts. Results were validated immunohistochemically in a panel of 60 human tumors. Results: The reported SCPC and LCNEC xenografts retain high fidelity to their human tumors of origin and are characterized by a marked upregulation of UBE2C and other mitotic genes in the absence of androgen receptor (AR), retinoblastoma (RB1), and cyclin D1 (CCND1) expression. We confirmed these findings in a panel of samples of CRPC patients. In addition, array comparative genomic hybridization of the xenografts showed that the SCPC/LCNEC tumors display more copy number variations than the adenocarcinoma counterparts. Amplification of the UBE2C locus and microdeletions of RB1 were present in a subset, but none displayed AR nor CCND1 deletions. The AR, RB1, and CCND1 promoters showed no CpG methylation in the SCPC xenografts. Conclusion: Modeling human prostate carcinoma with xenografts allows in-depth and detailed studies of its underlying biology. The detailed clinical annotation of the donor tumors enables associations of anticipated relevance to be made. Future studies in the xenografts will address the functional significance of the findings. Clin Cancer Res; 18(3); 666–77. ©2011 AACR.

BACKGROUND Hedgehog signaling is a stromal-mesenchymal pathway central to the development and homeostasis of both the prostate and the bone. Aberrant Hedgehog signaling activation has been associated with prostate cancer aggressiveness. We hypothesize that Hedgehog pathway is a candidate therapeutic target in advanced prostate cancer. We confirm increased Hedgehog signaling in advanced and bone metastatic castrate resistant prostate cancer and examine the pharmacodynamic effect of Smoothened inhibition by the novel reagent GDC-0449 in an experimental prostate cancer model. METHODS Hedgehog signaling component expression was assessed in tissue microarrays of high grade locally advanced and bone metastatic disease. Male SCID mice subcutaneously injected with the bone forming xenograft MDA PCa 118b were treated with GDC-0449. Hedgehog signaling in the tumor microenvironment was assessed by proteomic and species specific RNA expression and compared between GDC-0449 treated and untreated animals. RESULTS We observe Hedgehog signaling in high grade locally advanced and bone marrow infiltrating disease. Evidence of paracrine activation of Hedgehog signaling in the tumor xenograft, was provided by increased Sonic Hedgehog expression in human tumor epithelial cells, coupled with increased Gli1 and Patched1 expression in the murine stromal compartment, while normal murine stroma did not exhibit Hh signaling expression. GDC-0449 treatment attenuated Hh signaling as evidenced by reduced expression of Gli1 and Ptch1. Reduction in proliferation (Ki67) was observed with no change in tumor volume. CONCLUSIONS GDC-0449 treatment is pharmacodynamically effective as evidenced by paracrine Hedgehog signaling inhibition and results in tumor cell proliferation reduction. Understanding these observations will inform the clinical development of therapy based on Hedgehog signaling inhibition. Prostate 72:1638–1647, 2012. © 2012 Wiley Periodicals, Inc.

The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type–specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention.

BACKGROUND Small-cell carcinoma (SCC) of the prostate is an AR-negative variant of prostate cancer found at progression in 10–20% of castrate-resistant disease. Its finding predicts a distinct clinical course and a poor prognosis. Large-cell neuroendocrine carcinoma (LCNEC) is a much rarer variant that behaves similarly to SCC. The biological mechanisms that drive these disease variants are poorly understood. METHODS Eight tumor fragments from the salvage pelvic exenteration specimen of a patient with castrate-resistant prostate carcinoma were subcutaneously implanted into 6- to 8-week-old male CB17 SCID mice. Serial tissue sections and tissue microarrays of the resulting MDA PCa 144 xenograft lines were used for histopathologic and immunohistochemical characterization of the xenografts and their tissue of origin. RNA from two representative xenograft sublines was used for gene-expression profiling. RESULTS All eight fragments formed tumors: four of the MDA PCa 144 xenograft sublines had morphologic characteristics of SCC and four, of LCNEC. All retained high fidelity to their parent tumor tissue, which remained stable through serial passages. Morphological transitions in the specimen of origin suggested LCNEC represents an intermediate step between adenocarcinoma and SCC. Over 2,500 genes were differentially expressed between the SCC (MDA PCa 144-13) and the LCNEC (MDA PCa 144-4) sublines and enriched in “Nervous System Development” Gene Ontology subtree. CONCLUSION The eight xenograft models described represent the spectrum of neuroendocrine carcinomas in prostate cancer and will be valuable preclinical tools to study the pathogenesis of and therapy targets for this increasingly recognized subset of lethal prostate cancer. Prostate 71:846–856, 2011. © 2010 Wiley-Liss, Inc.

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells. Fil: Zhi, Gang Li. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Mathew, Paul. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Yang, Jun. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Starbuck, Michael W.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Zurita, Amado J.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Liu, Jie. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Sikes, Charles. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Multani, Asha S.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Efstathiou, Eleni. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Lopez, Adriana. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Wang, Jing. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Fanning, Tina V.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Prieto, Victor G.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Kundra, Vikas. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Troncoso, Patricia. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Raymond, Austin K.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Logothetis, Christopher J.. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Lin, Sue-Hwa. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Maity, Sankar. University Of Texas Md Anderson Cancer Center; Estados Unidos Fil: Navone, Nora M.. University Of Texas Md Anderson Cancer Center; Estados Unidos

5055 Background: AVPCa shares clinical features with small cell carcinoma (SCC) such as resistance to androgen signaling inhibition and frequent visceral metastases. A prospective clinical trial of...

149 Background: Unusually aggressive PC behavior is linked to small cell carcinoma (SCC) morphology. We observed SCC clinical features in association with morphologically heterogeneous PC and, in a prospective clinical trial (NCT00514540), also with chemotherapy responsiveness. Our previous studies in patient derived xenografts (PDX) revealed a distinct molecular profile for SCC. Here we sought to support the hypothesis that clinically defined aggressive variant PCs also share relevant biology with SCC. Methods: 59 PC samples, and 8 PDX, from 42 men registered to NCT00514540 were stained for RB, p53, AR, NKX3-1, ASCL1, AURKA, UBE2C and Ki67. Labeling indices (LI) were calculated as the ratio of positive epithelial cells to total of epithelial cells, at 200x. We determined copy number alterations (CNA) by Onco Scan® in 36 of 59 samples and 8 PDX lines. We used Western Blot and qRT-PCR to expand pathway analyses and their associations in PDX models. Results: Donor patients were similar to non-donor patients except for higher ECOG PS and LDH values. Strong positive correlations between nuclear AR (AR-N) and NKX3.1, AR-N and RB, AR-N and nuclear AURKA (AURKA-N), NKX3.1 and RB, p53 and nuclear UBE2C (UBE2C-N), Ki67 and UBE2C-N, Ki67 and cytoplasmic UBE2C (UBE2C-C) as well as a strong negative correlation between NKX3.1 and Ki 67 were observed. Frequent copy number losses were found for PTEN (largely gene-specific) and RB1 (often associated with regional-CNA). Gene-specific AURKA amplifications were not observed. Only RB1 CNA showed a positive correlation with its LI. Samples were segregated by quantity of CNA. PDX models shared these features and showed that only AR-negative tumors expressed pro-neural transcription factors, albeit heterogeneously. Conclusions: Our results support the hypothesis that clinically defined aggressive variant PC (including SCC and non-SCC morphologies) are characterized by frequent Rb, p53 and PTEN alterations, aberrant expression of mitotic, neural development and AR-signaling pathways, and high rates of CNA. Ongoing studies will confirm whether these molecular features distinguish this variant from the more common bone-homing, bone-forming, AR-driven adenocarcinomas of the prostate.

277 Background: Androgen deprivation, anti-androgen and androgen biosynthesis inhibitor treatment can initially control the metastatic prostate cancer (PCa), but treatment-refractory progression frequently follows, with the loss of tumor suppressors (TSPs) and increased expression of cell cycle proteins. Inhibition of the nuclear export protein, Exportin 1 (XPO1) leads to nuclear accumulation of cargo proteins such as TSPs & cell-cycle regulators implicated in castration-resistant PCa (CRPC) progression. XPO1 and specific cargo genes are overexpressed in metastatic CRPC relative to benign & primary prostate tumors, implicating XPO1 activity as playing a role in disease progression. Selinexor (KPT-330), a novel, oral SINE currently in Phase 1/2 for both hematological and solid tumors, has potent activity against CRPC. We hypothesized this activity is due selinexor induced nuclear expression of TSPs. Methods: To test this hypothesis, we treated selected PCa cell lines and patient-derived xenografts (PDXs, two adenocarcinomas and one small cell carcinonoma) with selinexor to determine the effect on survival and cargo protein localization. Results: Treatment with selinexor markedly inhibited PCa cell proliferation in vitro, activated the tumor suppressor TP53 & inhibited cell-cycle regulators. Also, treatment of the PDXs with selinexor for at least 3 weeks significantly inhibited tumor growth & reduced the prostate-specific antigen level in the adenocarcinomas. Selinexor increased cell death in all three PDX tumors and reduced cell proliferation in the adenocarcinomas, but not in the small-cell tumor. Expression analyses demonstrated that selinexor induced nuclear accumulation of different cargo proteins unique to the PCa model, accounting for PDX-specific regression. Conclusions: These results point to an anti-tumorigenic effect of selinexor treatment across a spectrum of hormone-refractory PCa that may provide insight into the drivers of PCa treatment resistance and heterogeneity.

158 Background: CRPC typically responds to anti-androgen therapy but resistance is common. CYP17 inhibitors that block lyase (L) and not hydroxylase (H), do not require prednisone, and may delay tumor resistance are needed. VT-464 is an oral, non-steroidal inhibitor of CYP17 L in clinical development for CRPC. The present studies characterized: 1) the kinetic mechanism of VT-464 inhibition of h-CYP17 L and H, and 2) VT-464 effects compared to abiraterone acetate (AA) in CRPC models. Methods: CYP17 Enzyme Assays: r-h-CYP17 inhibition studies were conducted using substrates pregnenolone (for H) or 17-a-hydroxypregnenolone (for L). Product rates (17-a-hydroxypregnenolone (H) and DHEA (L)) were assessed by LC/MS/MS. Data were fit to inhibition models using SigmaPlot 11.2. In vitro CRPC studies: VT-464 and AA effects on AR transcription (luciferase) and PSA and NKX3.1 gene expression (QRT-PCR) were compared in C4-2B cells. Mouse xenograft model: Effects of oral VT-464 or AA on tumor growth and tumoral steroids (T, DHT, P) were compared in the MDA-PCa-133 castrate mouse model. Results: VT-464 was a reversible uncompetitive inhibitor of CYP17 L (Ki = 84 nM, K’i=200 nM) and a competitive inhibitor of H (Ki = 620 nM). VT-464 CYP17 L/H selectivity was 7.4 at low [S] but selectivity increased with increasing [S]. VT-464 L/H selectivity was 60-x greater than AA. In C4-2B cells VT-464 and AA inhibited AR transactivation but VT-464 suppressed PSA and NKX3.1 more potently than AA. In the xenograft model, VT-464 decreased tumor volume as effectively as AA. VT-464 more potently decreased T and DHT, while AA increased P. Conclusions: VT-464 demonstrated much greater CYP17 L selectivity than AA and equivalent or superior activity in several CRPC models. Less tumor resistance may arise from treatment with the CYP17 lyase-selective inhibitor VT-464 than with AA, since it more effectively blocked androgen biosynthesis, did not cause an accumulation of progesterone, and should not require co-administration of prednisone.

Molecular markers that identify clinically meaningful, biologically based, subsets of PC are lacking. This results in inefficient clinical trial designs and suboptimal therapy selection off-protocol. The androgen receptor (AR)-negative small cell PC (SCPC) is an often-unrecognized morphological variant of the disease found on repeat biopsies during the castrate-resistant (CR) progression of PC. Its presence predicts for a poor response to hormonal therapies, a distinct clinical behavior and a short survival. Characteristic clinical features of SCPC are frequently present in patients with CRPC, constituting a clinical syndrome termed anaplastic PC. We hypothesized that the anaplastic PC share underlying biology with SCPC, irrespective of morphology. Preclinical studies show that SCPC are characterized by loss of tumor suppressors and AR signaling, as well as overexpression of mitotic genes and proneural transcription factors. To test our hypothesis we used immunohistochemistry to analyze the expression of markers in these pathways, in tumor biopsies from chemotherapy-naïve men with anaplastic PC participating in a prospective phase II clinical trial. To date we have analyzed 6 markers in 25 samples (6 with pure or mixed SCPC morphology) from prostate biopsies (n=10), transurethral resections of prostate tumors (n=4), bone marrow or bone biopsies (n=9), lymph node (n=1) or brain (n=1). Results are shown in Table 1. Table 1. Immunohistochemistry Anaplastic PC % Positive Cells AR RB1 p53 UBE2C Ki67 CYP17A1 0 8 11 11 5 1 0 < or = 10% 3 6 9 9 8 1 > 10% 14 8 5 11 16 24 Of the 17 samples that had ≤10% RB1+ cells, 10 had ≤10% AR+ cells and 9 had >10% UBE2C+ cells. Of the 11 samples that had ≤10% AR+ cells, 10 also had ≤10% RB1+ cells and 6 had >10% UBE2C+ cells. These data support our hypothesis that the anaplastic prostate carcinomas are characterized by loss of tumor suppressors and overexpression of mitotic genes. Updated results in additional samples and of additional markers will be presented. Citation Format: Ana M. Aparicio, Sankar Maity, Xuemei Wang, Anh G. Hoang, Eleni Efstathiou, Patricia Troncoso, Christopher J. Logothetis. A molecular characterization of the anaplastic prostate carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2013-1195

Small cell prostate cancer (SCPC) is an androgen receptor (AR) negative variant that often emerges in the castration-resistant progression of typical AR-positive prostate adenocarcinomas. Its presence predicts for an aggressive clinical course with poor response to hormonal therapies and a short median survival. Evidence suggests that the emergence of (SCPC) occurs through cellular transdifferentiation. Therefore, we hypothesized that loss of AR gene transcription may depend on epigenetic events. Given the central role of AR in prostate cancer, we reasoned that identifying the mechanisms involved in its downregulation could improve our understanding of how the lethal SCPC subtype arises. Here we investigated the DNA methylation and histone modifications of AR in a group of six prostate tumor xenografts developed from men with CRPC (two AR-positive [MDA PCa 170.4 and MDA PCa 180.30] and four AR-negative SCPC [MDA PCa 144.13, MDA PCa 144.4, MDA PCa 155 and MDA PCa 146.10]) and three established prostate cancer cell lines (one AR-positive [LNCaP] and two AR-negative [PC3 and DU145]). We first evaluated the methylation status of selected CpG sites in the AR promoter-associated CpG island using bisulfite-sequencing followed by pyrosequencing analysis. Except for the cancer cell lines PC-3 and DU145, all other cell lines and xenografts were unmethylated regardless of the AR expression status. Next, we used chromatin immunoprecipitation (ChIP) to evaluate marking of the AR promoter and a putative proximal enhancer by active (H3K4me3 and H3K9ac) and repressive (H3K9me2 and H3K27me3) histone modifications. Marking of the constitutively expressed genes (ACTB and GAPDH) and a repressed gene (HBB) served as control for these experiments. As expected, the only samples with AR marking by H3K4me3 and H3K9ac were the two AR-positive xenografts and LNCaP. Marking by repressive histone modifications was more variable: H3K27me3 was a universal finding in AR-negative samples except for Du-145, and was accompanied by H3K9me2 in two out of four xenografts. H3K27me3 and H3K9me2 also marked the putative enhancer region when AR was repressed. Altogether, these data indicate that promoter DNA methylation of AR is an infrequent finding in prostate cancer, while polycomb protein-based silencing is nearly universal in AR-negative variants. Also, it shows that H3K9me2 promotes reinforcement of silencing. Thus, we propose that reactivation of AR may by achieved in selected cases by inhibiting H3K27me3 alone, while concomitant inhibition of both polycomb and SET domain proteins may be necessary for double-positive H3K27/K9 methylation cases. Given the purported differentiating and tumor suppressor effects of AR, its reactivation in SCPC may be of therapeutic benefit. Citation Format: Brittany N. Kleb, Marcos RH Estecio, Guanglin Wu, Jing-Fang Lu, Christopher J. Logothetis, Sankar Maity, Ana M. Aparicio. Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2986. doi:10.1158/1538-7445.AM2013-2986

Cytochrome P450 17α-hydroxylase /17,20-lyase (CYP17) is a validated treatment target for metastatic castrate-resistant prostate cancer (mCRPC). Abiraterone acetate (AA; Zytiga) inhibits 17α-hydroxylase and 17,20-lyase reactions catalyzed by CYP17 and thus reduces androgen biosynthesis. However, co-administration of prednisone is required to suppress the mineralocorticoid excess from 17α-hydroxylase inhibitions. VT-464, a non-steroidal small molecule inhibits androgen production without mineralocorticoid excess or cortisol depletion by selective inhibition of CYP17 17,20-lyase. We determined the impact of VT-464 on tumor growth of a mCRPC xenograft, MDA-PCa-133, in vivo, and on androgen signaling in C4-2B prostate cancer cells in vitro. The MDA-PCa-133 xenograft is derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumor expresses PSA, full-length androgen receptor (AR) and AR-V7 isoform. We determined the effect of VT464 and AA on MDA-PCa-133 growing in tumor-bearing castrated male mice: randomization into three groups; oral treatment with vehicle only, VT-464, (100 mg/kg bid), or AA (100 mg/kg bid) for 25 days. Both VT-464 and AA reduced tumor volume (>two fold compared to vehicle; p We determined the effects of VT-464 on AR-dependent luciferase reporter activity and expression of AR signaling genes, PSA and NKX3.1, in cultured C4-2B prostate cancer cells with or without VT-464 or AA treatment. VT-464 or AA highly reduced AR-dependent reporter activity and specific expression of PSA and NKX3.1. AR-dependent signaling in the presence of VT-464 or AA was restored however by 10 nM DHT, consistent with VT-464- or AA-mediated depletion of androgen in C4-2B cells. The AR-dependent reporter activity was also measured following overexpression of full-length AR or AR-V7 isoform in C4-2B cells. Full-length AR-dependent reporter activity was (>80%) inhibited; AR-V7-dependent reporter activity was inhibited by 50% by 1 μM VT-464 or AA. We conclude that VT-464 inhibited AR signaling as effectively as AA in C4-2B prostate cancer cells. The findings suggest that AR signaling in cells, which express high levels of AR isoform (AR-V7) are more resistant to androgen biosynthesis inhibitors than those with wild type AR. Preliminary observations suggest C4-2B cells with AR-V7 are less sensitive to AA than VT- 464. Ongoing clinical and co-clinical studies will determine if the observed differences in steroid profile and relative efficacy of VT-464 compared to AA are clinically and biologically meaningful. Citation Format: Sankar N. Maity, Guanglin Wu, Jing-fang Lu, Joel R. Eisner, Stephen W. Rafferty, Robert J. Schotzinger, William R. Moore, Christopher J. Logothetis, John C. Araujo, Eleni Efstathiou. Efficacy of VT-464, a novel selective inhibitor of cytochrome P450 17,20-lyase, in castrate-resistant prostate cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2013-4772

Circulating markers that inform of the adaptive biology and the clinical virulence of prostate cancer do not exist. Small cell carcinoma of the prostate (SCPC) is an aggressive, androgen receptor (AR)-negative, morphological variant that arises often during the castration-resistant progression of typical adenocarcinoma. Its presence is associated with a poor response to hormonal therapies and predicts for a short survival. Micro-RNAs (miRNAs) regulate a range of processes, including establishment and maintenance of tissue differentiation, and are well-preserved in serum. Based on the observation that SCPC are characterized by a loss of prostate epithelial marker expression (such as AR and HOXB13) and an increase in the expression of proneural transcription factors (such as MYCN and ASCL1) we formulated the hypothesis that the SCPC display a miRNA profile distinct from that of typical AR-driven prostate carcinomas. We profiled the miR extracted from the serum samples of 13 men with biopsy-proven SCPC, and of 9 men with bone-predominant metastatic CRPC. Because SCPC is associated with large tumor burdens, we selected patients with bone-predominant CRPC that had abnormally high alkaline phosphatase levels and PSA > 20ng/mL. 322 miR were detected in at least one of the 22 samples. We identified two miR that were present in 10 and 11 of 13 (77% and 85%) patients with SCPC and 3 and 1 of 9 (33% and 11%) of men with unselected typical bone-predominant CRPC. Both of these miRs have been shown to control multiple genes regulating neuronal differentiation and to induce the conversion of human fibroblasts into neurons, a mechanism which may be shared by prostate adenocarcinoma cells in their transdifferentiation to small cell prostate cancer cells. Moreover, one of them was recently shown to silence AR expression. Therefore we conclude that two circulating miRs may serve to monitor adaptive responses and identify virulent subsets of prostate cancer, thus serving to guide therapy and selection of patients for clinical trials. Validation of these findings is ongoing in a larger cohort of patients. Citation Format: Ana M. Aparicio, Emily Gallichotte, Heather Cheng, Suk-Young Yoo, Sankar Maity, Christopher Logothetis, Muneesh Tewari. Circulating micro-RNAs can detect adaptive response to therapy and aggressive subset of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2013-1205

Lethal prostate cancer is predominantly a castrate-resistant disease. However, castrate-resistant prostate cancer (CRPC) is clinically heterogeneous and a classification that is predictive of outcome and response to therapies is lacking. Previous epigenetic profiling studies in CRPC have been limited to a small number of genes or sequences and therefore we explored the DNA methylome of CRPC and its capacity to classify the disease. We performed methylated CpG island amplification microarrays (MCAMs) using a panel of xenografts derived from 24 patients with CRPC tumors, 8 prostate tumors, 1 local recurrence, 5 liver metastases, 3 pleural fluid samples, 2 adrenal gland metastases and 1 each of the following: bone, subcutaneous tissue, retroperitoneal lymph node, ascitic fluid and brain. The xenograft morphology consisted of 15 adenocarcinomas, 11 small cell carcinomas, 3 large cell neuroendocrine carcinomas, 1 squamous cell carcinoma, 2 mixed morphologies and 2 unknown. The Lowess-normalized M values were used for analysis and the MCAM results were validated by pyrosequencing of 20 sequences associated with 12 genes with a sensitivity of 61.9%, a specificity of 84.3%, a positive predictive value of 73.6% and a negative predictive value of 75.7%. An unsupervised hierarchal clustering analysis using M values for probes located in CpG islands was performed and these results were compared with the clinicopathological features of the tissues. We observed that, different xenograft sublines derived from the same donor tumor clustered together suggesting that the DNA methylome is homogenous throughout a given tumor mass, despite the presence of histological heterogeneity, and that it is maintained through serial passages in mice. Unsupervised hierarchal clustering classified the xenografts into 3 groups, which showed trend towards distinct clinicopathological features including overall survival and androgen receptor (AR) status. In summary, we found that DNA methylation is a frequent event in CRPC and well preserved in xenograft tumors. Furthermore, the methylation profile of CRPC may represent a marker of clinicopathological subgroups with differential survival and AR status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 991. doi:1538-7445.AM2012-991

Small cell and large cell neuroendocrine carcinoma belong to the spectrum of poorly differentiated neuroendocrine carcinomas (NECa) of the prostate, characterized by a distinct clinical course. Often unrecognized, they can emerge either de novo or in the castrate-resistant progression of typical acinar adenocarcinoma (AdCa) and are found in 10-20% of men who die of the disease. Although sensitive to chemotherapy, responses are short lived and prognosis is dismal. An understanding of the biology underlying NECa and identification of novel therapeutic targets for this variant are urgently needed. In this study we used xenograft models and tissue specimens obtained from castrate-resistant prostate carcinomas (CRPC) to gain insight into the pathways implicated in the development of the highly aggressive NECa. Xenograft models with features of typical AdCa (n=3) and NECa (n=4) were subjected to gene-expression profiling using Affymetrix HGU133 Plus 2.0. One-way ANOVA was applied to identify differentially expressed probes (DEPs). The Web-based Gene Ontology (GO) Tree Machine was used to examine GO hierarchies. Selected genes were validated by quantitative real-time PCR (qRT-PCR) and Western blotting in the xenografts and by immunohistochemistry in the xenografts, and the tumor specimens of 62 patients with CRPC. Unsupervised hierarchal clustering of the raw data classified the xenografts correctly according to their morphology. Using stringent FDR 0.05, we identified 140 DEPs (0.3%) between the two groups. GO analysis revealed significant enrichment in the “M phase of mitotic cell cycle” biological process subtree (adjP=7.04e-08). Amongst the genes differentially expressed between the two groups, UBE2C, (E2 ubiquitin-conjugating enzyme UBCH10), a member of the anaphase promoting complex (APC), was significantly upregulated in NECa relative to AdCa by qRT-PCR (P=0.003) and immunohistochemistry (P=0.007) in xenografts, and by immunohistochemistry in the CRPC specimens (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 36. doi:10.1158/1538-7445.AM2011-36

Background Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a “master switch” in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies. Methodology/Principal Findings The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9. Conclusions/Significance Our results provide a broad understanding of the strategies used by a “master” transcription factor of differentiation in control of the genetic program of chondrocytes.

Currently no therapy effectively controls the progression of advanced human prostate cancer. We previously reported that fibroblast growth factor 9 (FGF9) contributes to the osteoblastic progression of human prostate cancer in bone (J Clin Invest 2008;118:2697). We examined the effect of TKI258 (a receptor tyrosine kinase inhibitor [TKI] with strong activity against FGF receptor 1-3 [FGFR1-3; IC50 < 40 nM]; [Novartis Pharma Corp.]) on primary mouse osteoblasts treated with and without FGF9 and TKI258. TKI258 blocked FGF9's induction of p-FRS2α (a gatekeeper that mediates FGFR downstream signals) an indication that TKI258 specifically blocks FGF signaling in osteoblasts. These observations provide strong evidence implicating FGF signaling in the osteoblastic progression of prostate cancer in bone. We hypothesized that pharmacologic blockade of the FGFR signaling has an antitumor effect in prostate cancer. To test our hypothesis, we studied the antitumor efficacy of TKI258 in the osteoblastic growth of MDA PCa 118b, a prostate cancer tumor graft that depends on FGF9 for growth. We assessed the effect of TKI258 on the growth of MDA PCa 118b prostate cancer cells in the femurs of male SCID mice. Ten mice were treated with TKI258 (20 and 60 mg/kg body weight daily by oral gavage), and another 10 mice were treated with vehicle only. Treatment started the day after we identified tumor on MRI scanning at the site of tumor cell injection and continued for 3 weeks, when we used MRI to assess tumor volumes in the femurs and microCT to assess bone mass. Femurs bearing MDA PCa 118b in mice treated with TKI258 had significantly smaller tumors (P = 0.019) and less prostate cancer-induced cortical bone (P = 0.034) than did control mice. Histopathologic analysis indicated that tumors in the treated mice were smaller than in the controls. Initial analysis of tumor-associated osteoclasts (assessed by their expression of tartrate-resistant acidic phosphatase) shows no difference between the treated and control mice. The femurs bearing MDA PCa 118b tumors were then analyzed by Western blotting to measure signaling; we found that TKI258 specifically inhibited p-ERK1/2 (an FGF signaling target gene) but not p-AKT. Taken together, these results suggest that TKI258 inhibits the growth of prostate cancer cells in bone by targeting both those cells and osteoblasts (but not osteoclasts), possibly by blocking FGF signaling. Subsequent molecular analysis of FGF downstream target genes will help elucidate the mechanism underlying TKI258's inhibition of prostate cancer growth in bone. Results from these studies will also help identify markers of response to TKI258 that will be used to interpret an ongoing clinical study with TKI258 in selected men with castrate-resistant prostate cancer with bone marrow infiltration. The results will serve as the foundation for the development of candidate predictive markers and further therapies based on targeting the FGF pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 344.

Background: The tumor microenvironment is involved in prostate cancer progression. Several stromal-epithelial-interacting pathways associated with prostate cancer (PCa) progression in the bone, are also considered candidates of resistance to current therapeutic strategies applied. Hedgehog (Hh) signaling has been associated with PCa aggressiveness. Therefore Hh pathway could be a novel diagnostic and therapeutic target, also contributing to therapy against prostate carcinoma metastasis in bone. Experimental work on specific chemical inhibitors of Hh signaling suggests that they may represent an entirely new class of therapeutic agents. In this study we tested the efficacy of Hh pathway inhibition with Smoothened inhibitor GDC-0449 in 118b prostate cancer xenograft model. Experimental Design: We employed the xenograft MDA PCa 118b, derived from the bone metastasis of a patient with castrate-resistant prostatic adenocarcinoma. This is characterized by unique bone producing feature when injected subcutaneously in SCID mice. Bone is the most common site for metastasis of prostate carcinoma, therefore a model with the new bone formation distinctive characteristic is really informative. Following MDA PCa 118b sc injection, animals were treated with Smoothened inhibitor GDC-0449 (oral administration, for 21 days). Hh pathway components expression was measured both in the tumor cells and stromal compartment using species specific primers for real-time PCR, western blot analysis and immunohistochemistry. Results: Paracrine Hedgehog signaling was confirmed in the tumor xenograft microenvironment. Baseline Shh expression was present in human tumor cells and significantly higher compared to stromal compartment, while nuclear Gli1 (a hallmark of pathway activation) and Ptch1 expression in the stromal compartment were present and significantly higher compared to human tumor cells. Smoothened inhibition resulted in attenuation of stromal expression of Gli1 and Ptch1, in the treated group compared to the control thus confirming the pharmacodynamic effect of the agent. A trend for slower tumor growth at the treated group was observed, while a decline in proliferation rate was noted when Hh pathway was suppressed in the stromal compartment. Conclusion: Hh signaling pathway demonstrates paracrine activation in the 118b prostate cancer bone producing xenograft model. GDC-0449 is effective in blocking Hh signaling in PCa. Future directions: A combination of Hedgehog signaling inhibition and cytotoxic therapies, such as androgen ablation would likely increase efficacy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C253.

Background The genetic control of prostate cancer development is poorly understood. Large numbers of gene-expression datasets on different aspects of prostate tumorigenesis are available. We used these data to identify and prioritize candidate genes associated with the development of prostate cancer and bone metastases. Our working hypothesis was that combining meta-analyses on different but overlapping steps of prostate tumorigenesis will improve identification of genes associated with prostate cancer development. Methods A Z score-based meta-analysis of gene-expression data was used to identify candidate genes associated with prostate cancer development. To put together different datasets, we conducted a meta-analysis on 3 levels that follow the natural history of prostate cancer development. For experimental verification of candidates, we used in silico validation as well as in-house gene-expression data. Results Genes with experimental evidence of an association with prostate cancer development were overrepresented among our top candidates. The meta-analysis also identified a considerable number of novel candidate genes with no published evidence of a role in prostate cancer development. Functional annotation identified cytoskeleton, cell adhesion, extracellular matrix, and cell motility as the top functions associated with prostate cancer development. We identified 10 genes--CDC2, CCNA2, IGF1, EGR1, SRF, CTGF, CCL2, CAV1, SMAD4, and AURKA--that form hubs of the interaction network and therefore are likely to be primary drivers of prostate cancer development. Conclusions By using this large 3-level meta-analysis of the gene-expression data to identify candidate genes associated with prostate cancer development, we have generated a list of candidate genes that may be a useful resource for researchers studying the molecular mechanisms underlying prostate cancer development.