Inhibition of Nox4-dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal-mediated protumorigenic interactions

Carcinoma-associated fibroblasts (CAFs) play a key onco-supportive role during prostate cancer (PCa) development and progression. We previously reported that the reactive oxygen species (ROS)-producing enzyme NADPH oxidase 4 (Nox4) is essential for TGF beta 1-mediated activation of primary prostate human fibroblasts to a CAF-like phenotype. This study aimed to further investigate the functional relevance of prostatic Nox4 and determine whether pharmacological inhibition of stromal Nox4 abrogates paracrine-mediated PCa-relevant processes. RNA in situ hybridization revealed significantly elevated Nox4 mRNA levels predominantly in the peri-tumoral stroma of clinical PCa with intense stromal Nox4 staining adjacent to tumor foci expressing abundant TGF protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF-associated marker expression and migration of TGF beta 1-activated but not nonactivated primary human prostate fibroblasts. Similar effects were obtained upon shRNA-mediated silencing of Nox4 but not Nox1 indicating that GKT137831 primarily abrogates TGF beta 1-driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGF beta 1-activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGF1 as conditioned media from PCa cell lines endogenously secreting high TGF beta 1 levels induced fibroblast activation in a stromal Nox4- and TGF receptor-dependent manner. Importantly, GKT137831 also attenuated PCa cell-driven fibroblast activation. Collectively, these findings suggest the TGF-Nox4 signaling axis is a key interface to dysregulated reciprocal stromal-epithelial interactions in PCa pathophysiology and provide a strong rationale for further investigating the applicability of Nox4 inhibition as a stromal-targeted approach to complement current PCa treatment modalities. What's new? While the tumor microenvironment plays a critical role in prostate adenocarcinoma pathogenesis, underlying mechanisms remain unclear. This study demonstrates the functional relevance of elevated expression of Nox4which is a major source of cellular reactive oxygen species (ROS)in the tumor microenvironment and downstream consequences on prostate cancer (PCa)-relevant processes. Findings identify stromal Nox4 as a central mediator of reciprocal epithelial-stromal cell crosstalk, fibroblast activation and stromal-driven tumor (cell)-promoting hallmarks. The data thus provide a strong mechanistic rationale for therapeutic inhibition of Nox4 as a stromal-targeted approach to complement current treatment strategies for PCa and restore dysregulated reciprocal stromal-epithelial interactions.