Epigenetic SMAD3 repression in tumor-associated fibroblasts impairs fibrosis and response to the antifibrotic drug nintedanib in lung squamous cell carcinoma.

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAFs) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non-small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs were associated with increased promoter methylation of the pro-fibrotic TGF-β transcription factor SMAD3 compared to ADC-TAFs, which elicited a compensatory increase in TGF-β1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGF-β1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGF-β1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, since cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs, and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that ADC patients may be more responsive to antifibrotic drugs targeting their stromal TGF-β1/SMAD3 activation.