Regulation of TGFB2 expression during EMT in lung epithelial cells

Abstract

The epithelial to mesenchymal transition (EMT) process is the driving mechanism behind epithelial-derived tumor metastasis but also plays an important role in developmental and wound healing programs. The EMT programs in these different cellular contexts have very different outcomes but rely on TGF-B signalling pathways to induce the EMT process. In recent years, a number of genome wide association studies (GWAS) of lung function have identified the TGFB2 locus as an important determinant of lung function. Using publicly available GWAS and ChIP-sequencing data from the UCSC genome browser, a cluster of SNPs were found to localize to transcription regulatory epigenetic signatures in A549 lung epithelial cancer cells. In this study we aimed to determine if a regulatory element corresponds with the location of GWAS genetic variants, and if such a regulatory element is responsive to EMT signals. Using ChIP-QPCR to assess the occupancy of H3K4me1 relative to H3K4me3 we identified a novel TGFB2 enhancer. Functional analysis of the enhancer region using a luciferase reporter assay revealed that the enhancer is activated upon TGF-B1-induced EMT in the cancerous A549 cell line but not in normal lung epithelial BEAS-2B cells. Using the online 3DSNP database, which annotates the regulatory function of SNPs by exploring their 3D interactions with genes mediated by chromatin looping, the TGFB2 enhancer region was identified to interact with the promoter of TGFB2. QPCR analysis of A549 and BEAS-2B cells revealed that the mRNA expression of TGFB2 increased in response to TGF-B1 in A549 cells but not BEAS-2B cells. We observed increased invasiveness of A549 and BEAS-2B cells in response to TGF-B2 and using immunohistochemistry we visualized TGF-B2 aggregation in A549 cells during TGF-B1-induced EMT. These results indicate that the aberrant activation of the TGFB2 enhancer increases TGFB2 expression, leading to increased invasiveness of lung epithelial cells. The TGFB2 enhancer could serve as a potential target for therapeutic agents for lung cancer.

Date of Award	2017
Original language	English