Epigenetic SMAD3 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 nonsmall 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 was associated with increased promoter methylation of the profibrotic TGFb transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFb1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFb1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGFb1/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, because 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 patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFb1/SMAD3 activation.Significance: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFb1/ SMAD3.

Focal Adhesion Kinase (FAK) Inhibition Synergizes with KRAS G12C Inhibitors in Treating Cancer through the Regulation of the FAK–YAP Signaling

“…FAK signaling has been reported to regulate fibrogenesis within tumors, [ 17 , 48 ] and excessive fibrosis is correlated with drug resistance, because it can create a barrier that prevents interactions between cancer cells and therapeutic agents. [ 49 ] In the present study, AMG510 treatment of three models (1 NSCLC CDX (NCI‐H2122), 1 CRC PDX (CO‐04‐0070), and 1 NSCLC PDX (LU‐01‐0030)) resulted in excessive fibrogenesis, and this was accompanied in each case by hyperactivated FAK signaling. The combination treatment of AMG510 and IN10018 significantly decreased the extent of fibrosis in these tumors, results both supporting that FAK‐related fibrosis may also limit the anticancer effects of KRAS G12C inhibitors and highlighting an additional mechanism through which the combination therapy we tested can help preclude development of drug resistance.…”

Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices

“…Another possible explanation could be that CAF secrete a high basal level of TGF-β1 [39][40][41], which might act in a paracrine fashion inducing maximum proliferation level. It has been also demonstrated that the TGF-β1 receptor and its downstream signaling via SMAD2/3 might be altered in CAF [42]. In addition, other soluble factors e.g., fibroblast growth factor [43] and the differential expression level of cell cycle related genes [44] could also affect the proliferative behavior of CAF.…”

DNA methylome and transcriptome landscapes of cancer-associated fibroblasts reveal a smoking-associated malignancy index

“…Vizoso M et al observed the correlation of SMAD3 hypermethylation in CAFs of 20 NSCLC patients of current smokers with TGF-b1 and ECM expression (37). Increased SMAD3 methylation in CAFs of SCC was also reported as a possible mechanism of resistance to nintedanib that targets TGF-b1/SMAD3 (39). Smoking-associated DNA methylation changes could contribute to lung cancer development (45) thus acting as predictors in tumor screening (46,47).…”

“…Previous studies have reported how the aberrations of DNA methylation in CAFs are correlated with disease resistance and prognosis (38)(39)(40). In this study, we aimed to characterize the behavior of CAFs in the primary cultures of the lung cancer microenvironment and investigate the clinical impact of TME through the evaluation of genome-wide DNA methylation profiling of CAFs in combination with mRNA expression analysis.…”