Therapeutic targeting of stromal NOX4-derived ROS signaling in prostatic disease

Stromal remodeling via fibroblast-to-myofibroblast transdifferentiation is a critical process in the development of benign prostatic hyperplasia (BPH) and prostate cancer (PCa), two of the most common diseases affecting elderly males. Elevated secretion of transforming growth factor beta 1 (TGFß1) by the diseased epithelium is considered the inducing stromal remodeling stimulus. We recently demonstrated that TGFß1-induced transdifferentiation is mediated by elevated NADPH oxidase 4 (NOX4)-derived reactive oxygen species (ROS) and supported by the concomitant downregulation of selenium-containing ROS scavenging proteins, including Selenoprotein P plasma 1 (SEPP1). Significantly, exogenous selenium inhibits and reverses transdifferentiation by restoring selenoenzyme ROS scavenging activity. The proposed project aims to determine the consequence of dysregulated stromal redox homeostasis on the development/progression of BPH and PCa. Stromal NOX4 and SEPP1 levels will be quantified in BPH/PCa tissue microarrays to identify potential correlations with disease status, progression and outcome. In addition, the subcellular localization of NOX4 in primary human prostatic stromal cells (PrSCs) will be determined. These findings will be applied to identify those direct NOX4-oxidative targets that lead to c-Jun N-terminal kinase activation and downstream transdifferentiation, which as mediators of transdifferentiation, will be functionally characterized with respect to their potential targeting in therapeutic applications. Using PrSC conditioned media and co-culture of PrSCs with prostate epithelial cells, the effects of dysregulated stromal NOX4 on BPH and PCa epithelial cell behavior will be investigated. These findings will be validated in vivo using combined stromal- epithelial xenografts to determine the effect of dysregulated stromal NOX4 on graft development and disease progression. Further, NOX inhibitors and selenium compounds will be applied in vitro and in vivo to evaluate their potential therapeutic applicability in BPH and PCa. In summary, the proposed project is expected to provide a greater understanding of dysregulated stromal-epithelial interactions involved in the pathophysiology of age-related prostatic disease and indicate the potential of targeting NOX4 as a novel strategy for the treatment/prevention of BPH and PCa.

Background: Prostate cancer is a major cause of male cancer death in Western societies. Activated fibroblasts (termed cancer-associated fibroblasts, CAFs) within the stromal tumor microenvironment play a critical role in tumor development, progression and response to current therapies. The project leader previously showed that genetic inhibition of reactive oxygen species (ROS) signaling by the enzyme NADPH oxidase 4 (Nox4) in prostate stromal fibroblasts impairs their activation by transforming growth factor beta 1 (TGF?1), a factor produced in abundant quantities by prostate cancer cells. This project aimed to further investigate the role of Nox4 in fibroblast activation and its consequences with respect to key cancer hallmarks, namely tumor cell proliferation and migration. In addition, studies were to be undertaken to evaluate Nox4 levels in clinical prostate cancer. Methods: Benign prostate fibroblasts isolated from tissue specimens of consenting prostate cancer patients undergoing surgical removal of the prostate were activated using TGF?1. In addition, to more closely mimic human PCa, human prostate-derived CAFs or patient-matched benign adjacent fibroblasts were isolated. Results: Using state-of-the-art downstream analytical methods, the project leader could show that Nox4 is abundantly expressed in the stromal tissue that lies adjacent to or within tumor areas of organ-confined as well as metastatic prostate cancer. Moreover, Nox4 levels were significantly associated with features linked to aggressive prostate cancer. Using a small molecule Nox4 inhibitor the project leader confirmed the essential role of Nox4 with respect to fibroblast activation and maintenance of the activated state in CAFs. Furthermore, the project leader demonstrated that inhibiting Nox4 activity in fibroblasts reduces their tumor-supportive effects on prostate cancer cells. Collectively, these data indicate a pivotal role of Nox4 in mediating tumor-supportive interactions between the tumor and neighboring stromal cells and suggest that the Nox4 inhibitor employed herein should be further investigated with respect to its potential suitability as an adjuvant treatment for current prostate cancer therapeutic strategies.