SUMMARY Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

Communication between cancer cells and the tumor microenvironment results in the modulation of complex signaling networks that facilitate tumor progression. Here, we describe a new mechanism of intercellular communication originating from large oncosomes (LO), which are cancer cell-derived, atypically large (1-10 mm) extracellular vesicles (EV). We demonstrate that, in the context of prostate cancer, LO harbor sustained AKT1 kinase activity, nominating them as active signaling platforms. Active AKT1 was detected in circulating EV from the plasma of metastatic prostate cancer patients and was LO specific. LO internalization induced reprogramming of human normal prostate fibroblasts as reflected by high levels of a-SMA, IL6, and MMP9. In turn, LO-reprogrammed normal prostate fibroblasts stimulated endothelial tube formation in vitro and promoted tumor growth in mice. Activation of stromal MYC was critical for this reprogramming and for the sustained cellular responses elicited by LO, both in vitro and in vivo in an AKT1-dependent manner. Inhibition of LO internalization prevented activation of MYC and impaired the tumor-supporting properties of fibroblasts. Overall, our data show that prostate cancer-derived LO powerfully promote establishment of a tumor-supportive environment by inducing a novel reprogramming of the stroma. This mechanism offers potential alternative options for patient treatment. Cancer Res; 77(9); 2306-17.Ó2017 AACR.

Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelium and its microenvironment. Here, we found that epigenetic changes in prostatic cancer-associated fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified a Ras inhibitor, RASAL3, as epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In an orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castration-resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared with those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of Ras activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT.

Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression and resistance to androgen signaling deprivation therapy (ADT). CAF subjected to extended passaging, compared to low passage CAF, were found to lose tumor expansion potential and heterogeneity. Cell surface endoglin (CD105), known to be expressed on proliferative endothelia and mesenchymal stem cells, was diminished in high passage CAF. RNA-sequencing revealed SFRP1 to be distinctly expressed by tumor-inductive CAF, which was further demonstrated to occur in a CD105-dependent manner. Moreover, ADT resulted in further expansion of the CD105 fibroblastic population and downstream SFRP1 in 3-dimensional cultures and patient-derived xenograft tissues. In patients, CD105 fibroblasts were found to circumscribe epithelia with neuroendocrine differentiation. CAF-derived SFRP1, driven by CD105 signaling, was necessary and sufficient to induce prostate cancer neuroendocrine differentiation in a paracrine manner. A partially humanized CD105 neutralizing antibody, TRC105, inhibited fibroblastic SFRP1 expression and epithelial neuroendocrine differentiation. In a novel synthetic lethality paradigm, we found that simultaneously targeting the epithelia and its microenvironment with ADT and TRC105, respectively, reduced castrate-resistant tumor progression, in a model where either ADT or TRC105 alone had little effect.

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer (PCa). TBX2, a T-box family transcription factor that negatively regulates cell cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here we report that TBX2 is overexpressed in human PCa specimens and bone metastases from xenograft mouse models of human PCa. Blocking endogenous TBX2 expression in PC3 and ARCaPM PCa cell models using a dominant negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro. Blocking endogenous TBX2 in human PCa mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in PCa cells dramatically reduced bone colonizing capability through reduced tumor cell growth and bone remodeling in an intra-tibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in PCa cells with endogenously blocked TBX2 partially restored the TBX2-induced PCa metastatic capability in mice. Conversely, WNT3A neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2 induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in PCa patients.