Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4

Background: Cancer-associated fibroblasts (CAFs) are tumour-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head & neck cancer (oral and oropharyngeal), and colorectal cancer was analysed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by PCR, Western blotting, immunofluorescence and functional assays. RNA-Sequencing/bioinformatics and immunohistochemistry was used to analyse NAD(P)H Oxidase-4 (NOX4) expression in different human tumours. NOX4’s role in CAF-mediated tumour progression was assessed in vitro –using CAFs from multiple tissues in Transwell and organotypic culture assays; and in vivo ¬–using xenograft and isograft tumour models. All statistical tests were two-sided. Results: We found myofibroblastic-CAF correlated significantly with poor survival in multiple solid cancers (HRs=1.7-7.3;p≤0.014). Fibroblast-to-myofibroblast transdifferentiation was shown to be dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. NOX4 expression was significantly upregulated in multiple human cancers (p<0.0001), strongly correlating with myofibroblastic-CAFs (r=0.647-0.913; adj. p<1x10-24). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex-vivo (54% decrease in α-SMA, p<0.01); prevent myofibroblastic-CAF accumulation in vivo (53-79% decrease in α-SMA across different models; p≤0.02) and slow tumour growth (30-64% decrease across different models, p≤0.04).