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Supplemental Figure 1 (fig. S1). Representative whole body NaF-18 PET scan images after 44 days of (A) vehicle-treated and (B) cabozantinib-treated animals. Supplemental Figure 2 (fig. S2). Effect of cabozantinib on PDX tumor growth, animal weight, and survival. Supplemental Figure 3 (fig. S3). Representative images of viable islets of tumor cells in (A) Patients; (B) MDA PCa-118b grown intrafemurally; and (C) MDA PCa- 118b grown subcutaneously. Supplemental Figure 4 (fig. S4). Time-dependent effect of cabozantinib on expression and phosphorylation of primary and MET downstream targets. Supplemental Figure 5 (fig. S5). Relative expression of c-met mRNA in NT and knockdown cells as determined by qRT-PCR. Supplemental Figure 6 (fig. S6). MET expression and activity in NT and knockdown tumors. Supplemental Figure 7 (fig. S7): Quantitation of CD31 staining on bone tumors. Supplemental Figure 8 (fig. S8). Effects of cabozantinib on bone remodeling markers in the phase-2 clinical trial and growth and differentiation of primary osteoblasts. Supplemental Figure S9 (fig S9). Effect of cabozantinib on bone turnover in the Phase 2 trial and PDX grown intrafemurally. Supplemental Figure 10 (fig. S10). Quantitation of phospho-histone H3 staining.

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PDF file - 150K, A: Mitotic figures in the xenografts; left panel: graphical presentation of the number of mitotic figures in SCPC/LCNEC xenografts (mean 11 mitoses per high power field) and adenocarcinoma xenografts (mean 2 per high power field); right panel: pictures from H&E stained slides of a SCPC and an adenocarcinoma (AdCa) xenograft. Arrows point to the mitotic figures. B: Higher expression levels of ki67 are noted in the SCPC/LCNEC xenografts and clinical samples compared to adenocarcinomas C:Flow cytometry of SCPC xenograft 146-10 and adenocarcinoma xenograft 180-30. (SCPC, small-cell prostate carcinoma; LCNEC, large-cell neuroendocrine carcinoma).

Supplementary Figure S1. Distribution of RET981 cell line. Supplementary Figure S2. Detection of AR splice variants in SDCs. Supplementary Figure S3. Cleaved PARP increased by AR siRNAs.

PDF file - 270K, mRNA levels of A, CDC20 and B, FZR1/CDH1 in SCPC/LCNEC (MDA PCa 144-4, 144-13, 146-10, and 155-2) and adenocarcinoma (MDA PCa 170-4 and 180-30) xenografts, normalized to GAPDH. SCPC, small-cell prostate carcinoma; LCNEC, large-cell neuroendocrine carcinoma.

Purpose: We performed parallel investigations in cabozantinib-treated patients in a phase II trial and simultaneously in patient-derived xenograft (PDX) models to better understand the roles of MET and VEGFR2 as targets for prostate cancer therapy.Experimental Design: In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR2, bone turnover, angiogenesis, and resistance were examined.Results: In responsive patients and PDX, islets of viable pMET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice nor did it affect cabozantinib-induced growth inhibition but did lead to induction of FGFR1. Inhibition of VEGFR2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR2-negative vessel. Reduction of bone turnover was observed in both cohorts.Conclusions: Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic castrate-resistant prostate cancer (CRPC), but inhibition of VEGFR2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents. Clin Cancer Res; 22(1); 107–21. ©2015 AACR.

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.

SUPPLEMENTARY TABLE 1. AVPC ('ANAPLASTIC') ELIGIBILITY CRITERIA FOR NCT00514540, A PHASE II STUDY OF CARBOPLATIN AND DOCETAXEL IN PATIENTS WITH ANAPLASTIC PROSTATE CARCINOMA. SUPPLEMENTARY TABLE 2. CLINICOPATHOLOGICAL FEATURES OF THE PDX DONOR PATIENTS. SUPPLEMENTARY TABLE 3. PDX GROWTH RATE AND PSA PRODUCTION. SUPPLEMENTARY TABLE 4. ANTIBODIES USED FOR IMMUNOHISTOCHEMISTRY AND WESTERN BLOT. SUPPLEMENTARY TABLE 5. qRT-PCR PRIMER SEQUENCES. SUPPLEMENTARY TABLE 6. DONOR PATIENT CHARACTERISTICS. SUPPLEMENTARY TABLE 7. SERUM MARKER LEVELS AT TIME OF REGISTRATION TO NCT00514540. SUPPLEMENTARY TABLE 8. PRINCIPAL COMPONENT VARIATE WEIGHTINGS TCGA/UNSELECTED CRPC/AVPC SAMPLES. SUPPLEMENTARY TABLE 9. LINEAR DISCRIMINANT ANALYSIS WEIGHTS. SUPPLEMENTARY TABLE 10. Tp53 MUTATIONS DETECTED IN TCGA, UNSELECTED CRPC (21) AND AVPC SAMPLES.

PDF file - 180K, A, Growth rate of MDA PCA 144-13, 146-10, 155-2, 170-4 and 180-30 xenografts. Note that the MDA PCA 144-13 xenograft line displays a large amount of necrosis. B. Diagram illustrates unsupervised hierarchal clustering using complete linkage and Pearson's correlation coefficient analysis of the raw expression profiles of androgen receptor-positive adenocarcinoma (MDA PCA 130, 117-9, 79) and androgen receptor-negative SCPC/LCNEC (MDA PCA 146-10, 155-2, 155-12, 144-13, 144-4) xenografts obtained with Affymetrix HGU133Plus2 array. In red are samples assessed on 1 day and in blue, those assessed on a different day. B, diagram illustrates Gene Ontology analysis results showing enrichment in biologic-process subtrees related to mitosis among the differently expressed genes. The biological processes that reached statistical significance are highlighted in red. SCPC, small-cell prostate carcinoma; LCNEC, large-cell neuroendocrine carcinoma.

Supplementary Table S1. PCR primer sequences in this study. Supplementary Table S2. AR IHC and FISH result Supplementary Table S3. AR mutation detail result in this study.

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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.

PDF file - 1.1MB, A, Chromosomal regions commonly amplified (red) and deleted (blue) in the SCPC/LCNEC (left panel) and adenocarcinoma (AdCa, right panel) xenografts. B, AR, cyclin D1, and RB1 promoter methylation of the AR-positive LNCaP prostate cancer cell line, the AR-negative PC-3 and DU145 prostate cancer cell lines, and the SCPC/LCNEC MDA PCa 144-4, 144-13, 146-10, and 155-2 xenografts. SCPC, small-cell prostate carcinoma; LCNEC, large-cell neuroendocrine carcinoma; AR, androgen receptor; RB, retinoblastoma.

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 in MDA 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. Clin Cancer Res; 18(3); 726–36. ©2011 AACR.

supplementary materials and methods, supplementary tables and supplementary figure legends Supplemental Table 1: Patient Characteristics. Supplemental Table 2: Targeting sequences for c-met. Supplemental Table 3: Antibodies and Sources. Supplemental Table 4: Oligonucleotides for PCR Amplification of Mouse Alkaline Phosphatase and Osteonectin.

Supplementary Figure 1. Supervised hierarchical clustering of differentially expressed genes between typical CRPC PDX ("ADENO") and CRPC PDX with small cell carcinoma morphology ("SCPC"). Supplementary Figure 2. Morphologic spectrum of tumors of patients with the aggressive variant phenotype in the primary (A-D) and metastatic tumor sites (E-H) Supplementary Figure 3. Immunohistochemical analysis of all 59 available NCT00514540 samples and 8 PDX lines. Supplementary Figure 4. Principal component analysis of a16-serum-and-IHC-tumor-marker set in "Baseline" samples from 21 patients. Supplementary Figure 5. Total number of copy number alterations per sample. Supplementary Figure 6. mRNA levels of genes of interest. Supplementary Figure 7. Principal component analysis of a 10-CNA-marker set in TCGA, unsupervised CRPC and AVPC samples.

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 MYC (surrogate for 8q) CNG and RB1 CNL showed in 54% of 44 samples each and PTEN CNL in 48%. All but 1 of 8 PDX bore Tp53 missense mutations. RB1 CNL was the strongest discriminator between unselected castration-resistant prostate cancer (CRPC) and the AVPC. Combined alterations in RB1, Tp53, and/or PTEN were more frequent in the AVPC than in unselected CRPC and in The Cancer Genome Atlas samples.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.

IHC, qRT-PCR and Western blot methods.

Supplementary Figure Legends 1-4 from BMP4 Promotes Prostate Tumor Growth in Bone through Osteogenesis

Supplementary Figures 1-4 from BMP4 Promotes Prostate Tumor Growth in Bone through Osteogenesis

Supplementary Table 1 from BMP4 Promotes Prostate Tumor Growth in Bone through Osteogenesis

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.

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.

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.

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.

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

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.