Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Reorganization of the osteocyte lacuno-canalicular network characteristics in tumor sites of an immunocompetent murine model of osteotropic cancers.

Mechanosensitive osteocytes are central regulators of bone resorption and formation. However, during the formation of bone metastases, which arise as consequences of breast and prostate cancer and skew homeostatic bone remodeling to favor osteolytic, osteosclerotic or mixed lesions, only a paucity of data exists on tumor-associated osteocyte interaction. Herein, we used a suite of high-resolution imaging and histological techniques to evaluate the effect of osteotropic cancer on cortical bone microarchitecture. Confocal imaging highlighted a direct contact between tumor cells residing in the bone marrow and osteocytes. High-resolution microcomputed tomography revealed a 10-12% larger osteocyte lacuna volume in the presence of tumor cells at day 21 after intratibial injection of EO771-Luc breast and RM1-Luc prostate cancer cells. The 3D representative of the spatial distribution of cortical bone microporosity showed i) a regional accumulation of vascular canals and large lacunae with low connectivity in osteosclerotic regions of interest and ii) an absence of vascular canals and large lacunae in osteolytic regions. These findings pinpoint the relationship between the presence of tumor cells in the bone marrow microenvironment and osteocyte lacunar characteristics and cortical bone blood vessel structure.

High stroma-derived WNT5A is an indicator for low-risk prostate cancer.

Prostate cancer (PCa) is a major cause of cancer-related death in men. Tumor-derived protein derived from Wnt5A gene (WNT5A) plays an important role in primary and metastatic PCa. Surrounding stroma cells also produce WNT5A, which may modulate the biology of PCa. Here, we assessed the role of stroma-derived WNT5A (stWNT5A) in primary PCa. A tissue microarray of samples obtained from 400 patients who underwent radical prostatectomy and control samples from 41 patients with benign prostate hyperplasia (BPH) was immunohistochemically assessed for expression of stWNT5A. The cores were scored for staining intensity: 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong) and the stained stromal surface area: 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), or 4 (76-100%). Gleason Score (GS) and TNM-stage were assessed by stratifying the cohort into high-risk (≥ pT3, pN1, GS ≥ 8) and non-high-risk patients. Ki67 and TUNEL assays were performed to assess proliferation and apoptosis. Expression of stWNT5A in BPH and tumor-free control samples was 1.2-fold higher compared to tumor samples (P < 0.001). Non-high-risk patients had a higher stWNT5A score than high-risk patients (P < 0.05). stWNT5A expression was not correlated with overall and cancer-specific survival. Proliferation (r2  = 0.038, P < 0.001) and apoptosis (r2  = 0.277, P < 0.001) negatively correlated with stWNT5A expression. In summary, we show that expression of stWNT5A is higher in benign tissue and non-high-risk PCa. Stroma-derived Wnt signaling and tumor-derived Wnt may differentially impact on tumor behavior. Future studies are warranted to dissect the Wnt profile in tumor vs. surrounding stroma tissues.

Neuropilin-2 is an independent prognostic factor for shorter cancer-specific survival in patients with acinar adenocarcinoma of the prostate.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa.