Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs.

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbß3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbß3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

A Novel FGFR3 Splice Variant Preferentially Expressed in African American Prostate Cancer Drives Aggressive Phenotypes and Docetaxel Resistance.

Alternative splicing (AS) has been shown to participate in prostate cancer development and progression; however, a link between AS and prostate cancer health disparities has been largely unexplored. Here we report on the cloning of a novel splice variant of FGFR3 that is preferentially expressed in African American (AA) prostate cancer. This novel variant (FGFR3-S) omits exon 14, comprising 123 nucleotides that encode the activation loop in the intracellular split kinase domain. Ectopic overexpression of FGFR3-S in European American (EA) prostate cancer cell lines (PC-3 and LNCaP) led to enhanced receptor autophosphorylation and increased activation of the downstream signaling effectors AKT, STAT3, and ribosomal S6 compared with FGFR3-L (retains exon 14). The increased oncogenic signaling imparted by FGFR3-S was associated with a substantial gain in proliferative and antiapoptotic activities, as well as a modest but significant gain in cell motility. Moreover, the FGFR3-S-conferred proliferative and motility gains were highly resistant to the pan-FGFR small-molecule inhibitor dovitinib and the antiapoptotic gain was insensitive to the cytotoxic drug docetaxel, which stands in marked contrast with dovitinib- and docetaxel-sensitive FGFR3-L. In an in vivo xenograft model, mice injected with PC-3 cells overexpressing FGFR3-S exhibited significantly increased tumor growth and resistance to dovitinib treatment compared with cells overexpressing FGFR3-L. In agreement with our in vitro and in vivo findings, a high FGFR3-S/FGFR3-L expression ratio in prostate cancer specimens was associated with poor patient prognosis. IMPLICATIONS: This work identifies a novel FGFR3 splice variant and supports the hypothesis that differential AS participates in prostate cancer health disparities.