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

Abstract

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 TGFβ1-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 TGFβ protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF-associated marker expression and migration of TGFβ1-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 TGFβ1-driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGFβ1-activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGFβ1 as conditioned media from PCa cell lines endogenously secreting high TGFβ1 levels induced fibroblast activation in a stromal Nox4- and TGFβ receptor-dependent manner. Importantly, GKT137831 also attenuated PCa cell-driven fibroblast activation. Collectively, these findings suggest the TGFβ-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.

Keywords: cancer-associated fibroblast; in situ hybridization; microenvironment; prostate cancer; reactive oxygen species; transforming growth factor beta.