Epigenetic <i>SMAD3</i> Repression in Tumor-Associated Fibroblasts Impairs Fibrosis and Response to the Antifibrotic Drug Nintedanib in Lung Squamous Cell Carcinoma.

Abstract

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) 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 <i>in vitro</i> and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFβ transcription factor <i>SMAD3</i> compared with ADC-TAFs, which elicited a compensatory increase in TGFβ1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFβ1/SMAD3 activation, whereas genetic downregulation of <i>SMAD3</i> in ADC-TAFs and control fibroblasts increased TGFβ1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib <i>in vitro</i> and <i>in vivo</i>. 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 <i>SMAD3</i> epigenetic repression, because cigarette smoke condensate selectively increased <i>SMAD3</i> promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential <i>SMAD3</i> promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFβ1/SMAD3 activation. SIGNIFICANCE: This study implicates the selective epigenetic repression of <i>SMAD3</i> in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFβ1/SMAD3.