P2.03-16 TGF-β Induced EMT and Stemness Characteristics are Associated with Epigenetic Regulation in Lung Cancer

Abstract

Transforming growth factor-β (TGF-β) promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). EMT is often associated with acquisition of stem-like characteristics. In this study, we investigated whether EMT and stem-like characteristics induced by TGF-β could associated with epigenetic regulation in lung cancer. Human normal epithelial (BEAS-2B) and cancer (A549, H292, H226 and H460) cell lines were incubated with 10 ng/ml of TGF-β for 3 days. Transcriptome analysis of BEAS-2B and A549 cells treated with TGF-β were performed by using next-generation sequencing (HiSeq 2500 system). Western blotting was performed to analyze the expression of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin, fibronectin, vimentin and α-SMA). Wound healing assay, Matrigel invasion assay, sphere formation assay and in vivo mice tumor model were used to assess functional characteristics of EMT and stemness acquisition. TGF-β induced DNA demethylation was identified by methylation-specific PCR and bisulfite sequencing. Next-generation sequencing revealed significant changes in the expression of stem cell markers, CD44, CD87 and CD90 in both BEAS-2B and A549 cells. Functional analysis revealed increased wound healing, Matrigel invasion, sphere formation and in vivo mice tumor formation after TGF-β treatment. TGF-β induced EMT was associated with acquisition of stem-like characteristics. CD44, CD87 and CD90 were activated by either TGF-β and treatment with AZA. MSP showed decreased CD44, CD87 and CD90 promoter methylation after TGF-β treatment. These results suggest that TGF-β induces stem cell characteristics which are related with CD44, CD87 and CD90 reactivation by promoter demethylation.