P1.02-076 DNA Methylation Profiling Unravels a TGF-β Hyperresponse in Tumor Associated Fibroblasts from Lung Cancer Patients

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Tumor associated fibroblasts (TAFs) are drawing increasing attention as potential therapeutic targets owing to its direct implication in major steps of tumor progression in solid tumors including non-small cell lung cancer. Accordingly, there is growing interest in defining the aberrant molecular differences between normal and TAFs that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients, and reported a widespread hypomethylation concomitantly with a focal gain of DNA methylation indicative of a marked epigenetic reprogramming. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-β pathway, including the hypermethylation of SMAD3, which is an important transcription factor of the TGF-β pathway. However, the functional implications of the aberrant TGF-β pathway in lung TAFs remains undefined. Patient-derived TAFs and paired control fibroblasts from either adenocarcinoma (ADC) or squamous cell carcinoma (SCC) patients were stimulated with TGF-β1 and their responses were examined in terms of activation and contractility. Activation markers included expression of alpha-smooth muscle actin (α-SMA) and collagen-I, which were assessed by western-blotting and qRT-PCR, respectively. The contractility of single fibroblasts was assessed by traction force microscopy. We found a larger expression of activation markers including α-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-β1 elicited a larger contractility in TAFs than in control fibroblasts as assessed by traction force microscopy. Of note, these results were consistent with previous observations reported on skin fibroblasts from Smad3 knock-out mice. Our findings reveal that lung TAFs are hyperresponsive to TGF-β1, even though their expression of SMAD3 is epigenetically down-regulated. This aberrant response to TGF-β1 may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer.