ROS Signaling by NOX4 Drives Fibroblast-to-Myofibroblast Differentiation in the Diseased Prostatic Stroma

Abstract

Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGFβ1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGFβ1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGFβ1 induction of NOX4-derived ROS is required for TGFβ1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa.

Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

“…Similar results were observed in other human fibroblast strains including GM21, LF1, and WI38 cells (Supporting Information Figure S3a). The great majority of cells with prominent α‐SMA expression also displayed discrete DDR foci (over 95%; Figure 3a), suggesting a causal link between DDR induction and myofibroblast transdifferentiation, as reported previously (Dimitrijevic‐Bussod, Balzaretti‐Maggi, & Gadbois, 1999; Mellone et al, 2016; Sampson et al, 2011). However, since not all cells with DDR foci expressed high levels of α‐SMA in stress fibers (Supporting Information Figure S3d), our data also suggested that activation of the DDR alone is not sufficient for myofibroblast transdifferentiation and that other factors, such as timing of DDR induction or long‐term persistence of the DDR, are critical for activating the transdifferentiation program.…”

“…TGF‐β1 stimulates the production of ROS by promoting expression of NADPH oxidase‐4 (NOX‐4) in a SMAD3‐dependent manner (Hecker et al, 2009; Jiang, Liu, Dusting, & Chan, 2014; Sampson et al, 2011). While TGF‐β1‐induced myofibroblast transdifferentiation involves the production of such ROS, the mechanisms how these reactive molecules facilitate transdifferentiation are still obscure.…”

“…The observations that a DDR induced by radiation, drugs, or H2O2 similarly promotes myofibroblast transdifferentiation support this conclusion. (Dimitrijevic‐Bussod et al, 1999; Mellone et al, 2016; Sampson et al, 2011). In fact, myofibroblasts have been reported to be nonproliferating cells that show signs of DNA damage (Petrov et al, 2008).…”

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

“…RNA isolation, cDNA synthesis and qPCR using Taqman ® gene expression assays were performed as described 18, 22. TaqMan ® gene expression assays (Applied Biosystems, Vienna, Austria) are listed in Supporting Information, Table 1.…”

“…ROS play a central role in fibroblast activation 17. In particular, we previously demonstrated a pivotal function of Nox4‐derived ROS during TGFβ1‐induced activation of prostate fibroblasts most likely via redox‐dependent phosphorylation of c‐Jun N‐terminal kinase (JNK) 18. More recently, we and others showed that Nox4 expression is higher in PCa patients that experience biochemical relapse following radical prostatectomy and in patients with decreased PCa‐specific survival 19, 20.…”