Cancer associated fibroblasts predict for poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma

Interactions between cancer cells and cancer-associated fibroblasts (CAF) play an important role in tumour development and progression. In this study we investigated the functional role of CAF in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of EAC patients (183 patients) for CAF markers related to disease mortality. We characterized CAF and normal oesophageal fibroblasts (NOF) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3-D organotypic culture and xenograft models were used to examine effects on EAC cell function, and dissect molecular mechanisms regulating invasion. Most EAC (93%) contained CAF with a myofibroblastic (?-SMA-positive) phenotype, which correlated significantly with poor survival (p?=?0.016; HR 7. 1 (1.7-29.4). Primary CAF, isolated from EAC, have a contractile, myofibroblastic phenotype, and promote EAC cell invasion in vitro (Transwell assays, p?=?<0.05; organotypic culture, p?<?0.001) and in vivo (p?=?<0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAF, and acts as a ligand for EAC cell integrins ?v?3 and ?v?5, promoting activation of the PI3kinase/Akt pathway. In patient samples, periostin expression at the tumour cell/stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase/Akt activation and increased tumour cell invasion. Most EAC contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patient

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 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