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

Rafael Ikemori,Paloma Bragado,Gemma Fuster,Frank Hilberg,Miguel Vizoso,Marselina Arshakyan,Laura Soucek,Cristina Fillat,Luis M Montuenga,Marta Gabasa,Manuel Castro,Paula Duch,Iuliana-Cristiana Luis,Manel Esteller,Jordi Alcaraz,Toni Jauset,Eduard Monsó,A Marín ,Sebastián Morán ,Sabrina Gea‐Sorlí ,Noemı́ Reguart ,Pere Gascón ,Víctor I Peinado

doi:10.1158/0008-5472.can-19-0637

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 in vitro 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 SMAD3 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 SMAD3 in ADC-TAFs and control fibroblasts increased TGFβ1/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 TGFβ1/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 TGFβ1/SMAD3.

Highlights

Lung cancer is the most common cause of cancer-related deaths worldwide, with a 5-year survival rate of approximately 18% in developed countries, and smoking and pollution are its major risk factors [1, 2]
B and C, Densitometry analysis expressed as fold (TAF/control fibroblasts (CF)) of aSMA/b-actin (B) and prolyl-4-hydroxylase a subunit 2 (P4HA2)/b-actin (C; 6 ADC, 5 squamous cell carcinoma (SCC); tumor-associated fibroblasts (TAF) and CFs)
In agreement with our in vitro data, nintedanib elicited significant antitumor growth effects (Fig. 6B) and delayed tumor engraftment (Fig. 6C) in H1437 cancer cells coinjected with control, but not shSMAD3 fibroblasts. Nintedanib reduced both the expression of fibrillar collagens and the percentage of proliferating (Ki-67þ) cancer cells to a much greater extent in tumor-bearing control compared with shSMAD3 fibroblasts (Fig. 6D–F). These results reveal that knocking down SMAD3 in fibroblasts is sufficient to scale down tumor growth and fibrosis as well as to impair the antitumor growth and antifibrotic effects of nintedanib in primary tumor xenografts, thereby mimicking the negative therapeutic responses reported in patients with SCC [13]

Summary

Introduction

Lung cancer is the most common cause of cancer-related deaths worldwide, with a 5-year survival rate of approximately 18% in developed countries, and smoking and pollution are its major risk factors [1, 2]. Most patients with lung cancer are diagnosed as non–small cell lung cancer (NSCLC, $85%), which is further classified into adenocarcinoma (ADC, $50%), squamous cell carcinoma (SCC; $40%), and other less frequent subtypes [3]. The latter subtypes are epithelial in origin, it is increasingly acknowledged that tumor-associated fibroblasts (TAF) are essential. Medicine and Health Sciences, Universitat de Barcelona, Barcelona, Spain.

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Conclusion