Mesenchymal and stem-like prostate cancer linked to therapy-induced lineage plasticity and metastasis.

Bioinformatic analysis of 94 patient-derived xenografts (PDXs), cell lines, and organoids (PCOs) identifies three intrinsic transcriptional subtypes of metastatic castration-resistant prostate cancer: androgen receptor (AR) pathway + prostate cancer (PC) (ARPC), mesenchymal and stem-like PC (MSPC), and neuroendocrine PC (NEPC). A sizable proportion of castration-resistant and metastatic stage PC (M-CRPC) cases are admixtures of ARPC and MSPC. Analysis of clinical datasets and mechanistic studies indicates that MSPC arises from ARPC as a consequence of therapy-induced lineage plasticity. AR blockade with enzalutamide induces (1) transcriptional silencing of TP53 and hence dedifferentiation to a hybrid epithelial and mesenchymal and stem-like state and (2) inhibition of BMP signaling, which promotes resistance to AR inhibition. Enzalutamide-tolerant LNCaP cells re-enter the cell cycle in response to neuregulin and generate metastasis in mice. Combined inhibition of HER2/3 and AR or mTORC1 exhibits efficacy in models of ARPC and MSPC or MSPC, respectively. These results define MSPC, trace its origin to therapy-induced lineage plasticity, and reveal its sensitivity to HER2/3 inhibition.

Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y.

A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 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.

Androgen Receptor Signaling in Castration-Resistant Prostate Cancer Alters Hyperpolarized Pyruvate to Lactate Conversion and Lactate Levels In Vivo.

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-[13C]pyruvate spectroscopy experiments were performed on prostate PDX animal models. RESULTS: Using hyperpolarized 1-[13C]pyruvate conversion to 1-[13C]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.

Frequent PTEN loss and differential HER2/PI3K signaling pathway alterations in salivary duct carcinoma: Implications for targeted therapy.

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 PI3Kß 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.

Function of Tumor Suppressors in Resistance to Antiandrogen Therapy and Luminal Epithelial Plasticity of Aggressive Variant Neuroendocrine Prostate Cancers.

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 (PCa) 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 PCa disease phenotype.

Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer.

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.

Targeting DNA Damage Response in Prostate Cancer by Inhibiting Androgen Receptor-CDC6-ATR-Chk1 Signaling.

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.

NF-Y (CBF) regulation in specific cell types and mouse models.

The CCAAT-binding factor CBF/NF-Y is needed for cell proliferation and early embryonic development. NF-Y can regulate the expression of different cell type-specific genes that are activated by various physiological signaling pathways. Dysregulation of NF-Y was observed in pathogenic conditions in humans such as scleroderma, neurodegenerative disease, and cancer. Conditional inactivation of the NF-YA gene in mice demonstrated that NF-Y activity is essential for normal tissue homeostasis, survival, and metabolic function. Altogether, NF-Y is an essential transcription factor that plays a critical role in mammalian development, from the early stages to adulthood, and in human pathogenesis. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Targeting of CYP17A1 Lyase by VT-464 Inhibits Adrenal and Intratumoral Androgen Biosynthesis and Tumor Growth of Castration Resistant Prostate Cancer.

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 < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status.

Combined Tumor Suppressor Defects Characterize Clinically Defined Aggressive Variant Prostate Cancers.

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 <10% cells in 61% of samples and AR in 36%. 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.

Integrating Murine and Clinical Trials with Cabozantinib to Understand Roles of MET and VEGFR2 as Targets for Growth Inhibition of Prostate Cancer.

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.

Context-specific role of SOX9 in NF-Y mediated gene regulation in colorectal cancer cells.

Roles for SOX9 have been extensively studied in development and particular emphasis has been placed on SOX9 roles in cell lineage determination in a number of discrete tissues. Aberrant expression of SOX9 in many cancers, including colorectal cancer, suggests roles in these diseases as well and recent studies have suggested tissue- and context-specific roles of SOX9. Our genome wide approach by chromatin immunoprecipitation sequencing (ChIP-seq) in human colorectal cancer cells identified a number of physiological targets of SOX9, including ubiquitously expressed cell cycle regulatory genes, such as CCNB1 and CCNB2, CDK1, and TOP2A. These novel high affinity-SOX9 binding peaks precisely overlapped with binding sites for histone-fold NF-Y transcription factor. Furthermore, our data showed that SOX9 is recruited by NF-Y to these promoters of cell cycle regulatory genes and that SOX9 is critical for the full function of NF-Y in activation of the cell cycle genes. Mutagenesis analysis and in vitro binding assays provided additional evidence to show that SOX9 affinity is through NF-Y and that SOX9 DNA binding domain is not necessary for SOX9 affinity to those target genes. Collectively, our results reveal possibly a context-dependent, non-classical regulatory role for SOX9.

Secretome analysis of an osteogenic prostate tumor identifies complex signaling networks mediating cross-talk of cancer and stromal cells within the tumor microenvironment.

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-ß2, GDF15, FGF3, FGF19, CXCL1, galectins, and ß2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFß2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFß2, 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.

Alterations associated with androgen receptor gene activation in salivary duct carcinoma of both sexes: potential therapeutic ramifications.

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.

Targeting poly(ADP-ribose) polymerase and the c-Myb-regulated DNA damage response pathway in castration-resistant prostate cancer.

Androgen deprivation is the standard treatment for advanced prostate cancer (PCa), but most patients ultimately develop resistance and tumor recurrence. We found that MYB is transcriptionally activated by androgen deprivation therapy or genetic silencing of the androgen receptor (AR). MYB silencing inhibited PCa growth in culture and xenografts in mice. Microarray data revealed that c-Myb and AR shared a subset of target genes that encode DNA damage response (DDR) proteins, suggesting that c-Myb may supplant AR as the dominant regulator of their common DDR target genes in AR inhibition-resistant or AR-negative PCa. Gene signatures including AR, MYB, and their common DDR-associated target genes positively correlated with metastasis, castration resistance, tumor recurrence, and decreased survival in PCa patients. In culture and in xenograft-bearing mice, a combination strategy involving the knockdown of MYB, BRCA1, or TOPBP1 or the abrogation of cell cycle checkpoint arrest with AZD7762, an inhibitor of the checkpoint kinase Chk1, increased the cytotoxicity of the poly[adenosine 5'-diphosphate (ADP)-ribose] polymerase (PARP) inhibitor olaparib in PCa cells. Our results reveal new mechanism-based therapeutic approaches for PCa by targeting PARP and the DDR pathway involving c-Myb, TopBP1, ataxia telangiectasia mutated- and Rad3-related (ATR), and Chk1.

NF-Y inactivation causes atypical neurodegeneration characterized by ubiquitin and p62 accumulation and endoplasmic reticulum disorganization.

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.

Molecular classification of prostate cancer progression: foundation for marker-driven treatment of prostate cancer.

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.

Activation of ß-catenin signaling in androgen receptor-negative prostate cancer cells.

PURPOSE: To study Wnt/ß-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the ß-catenin-androgen receptor (AR) interaction. EXPERIMENTAL DESIGN: We carried out ß-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of ß-catenin-mediated transcription), and sequenced the ß-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down ß-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed ß-catenin and AR in 27 bone metastases of human CRPCs. RESULTS: ß-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 ß-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated ß-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 ß-catenin. Finally, we found nuclear localization of ß-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/ß-catenin signaling. CONCLUSION: We identified a previously unknown downstream target of ß-catenin, HAS2, in prostate cancer, and found that high ß-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.

Combined Inhibition of IGF-1R/IR and Src family kinases enhances antitumor effects in prostate cancer by decreasing activated survival pathways.

BACKGROUND: Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways. MATERIALS AND METHODS: Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively). RESULTS: In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers. CONCLUSIONS: Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.