Hanley, Christopher J., Mellone, Massimiliano, Ford, Kirsty, Thirdborough, Steve M., Mellows, Toby, Frampton, Steven J., Smith, David M., Harden, Elena, Szyndralewiez, Cedric, Bullock, Marc, Noble, Fergus, Moutasim, Karwan A., King, Emma V., Vijayanand, Pandurangan, Mirnezami, Alex H., Underwood, Timothy J., Ottensmeier, Christian H. and Thomas, Gareth J. (2018) Targeting the myofibroblastic cancer-associated fibroblast phenotype through inhibition of NOX4. JNCI Journal of the National Cancer Institute, 110 (1), 109-120, [djx121]. (doi:10.1093/jnci/djx121).
Abstract
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 and neck cancer (oral, n ¼ 260; oropharyngeal, n ¼ 271), and colorectal cancer (n ¼ 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4’s rolein CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypicculture assays, and in vivo, using xenograft (n ¼ 9–15 per group) and isograft (n ¼ 6 per group) tumor models. All statistical tests were two-sided.
Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specificsurvival rates in each cancer type analyzed (hazard ratios [HRs] ¼ 1.69–7.25, 95% confidence intervals [CIs] ¼ 1.11 to 31.30,log-rank P .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significantupregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic CAFs(r ¼ 0.65–0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic CAF phenotype ex vivo (54.3% decrease in a-smooth muscle actin [a-SMA], 95% CI ¼ 10.6% to 80.9%, P ¼ .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%–79.0% decrease in a-SMA across different models, P .02) and slow tumor growth (30.6%–64.0% decrease across different models, P .04).
Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.
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