Integrin β6 Regulates Epithelial to Mesenchymal Transition

OBJECTIVES: TGFβ1 which promotes Epithelial to Mesenchymal transition (EMT) is activated by αvβ6 integrin. The object of this study was to determine if EMT can be reversed by deleting the β6 C-terminal sequence responsible for TGFβ1 activation. METHODS: Four SCC lines (SCC9, SCC96, SCC9β6D1, SCC9β6D2) were used. The parental SCC9 cells were originally derived from a tongue lesion and purchased from American Type Culture Collection (ATCC). The SCC9β6,SCC9β6D1 and SCC9β6D2 cell lines were derived in our laboratory by retroviral expression of the full length β6 cDNA, and β6 constructs missing the C-terminal 11 amino acids(AA)(D1)and C-terminal 18 AA (D2), respectively. All β6 cDNA constructs activated TGFβ1 except D2. All cells were routinely cultured in Dulbecco's Modified Essential Medium plus 10% fetal bovine serum. Tumor cells were followed for network formation using a serum-free, Matrigel vascular mimicry assay (Ramos et al 2009. Expression of E-cadherin, N-cadherin, Notch, Snail, pMAPK, and TGFβ1 was determined by Western blotting. Immunohistochemistry was performed using anti-β6 antibodies (4B5; Robert Pytela). RESULTS: We identified β6 positive, tumor cell lined “vascular” channels (mimics) containing red blood cells in oral SCC in vivo. In vitro, β6 positive oral SCC cells formed “vascular” channels within Matrigel and this was disrupted by expressing the D2 construct which does not activate TGFβ1. Activation of the Notch/Snail/MAPK pathway which promotes EMT is dependent upon β6 activation of TGFβ1. Expression of the D2 construct significantly reduced expression of Notch, Snail, N-cadherin and the phosphorylation of pMAPK. CONCLUSION: These results indicate that activation of TGFβ1 is central to regulation of the Notch/Snail/MAPK pathway that promotes EMT and oral cancer invasion. In the absence of active TGFβ1, EMT is reversed and may provide insight on how to limit oral cancer progression. Grant support is from University of California CRCC 2-512508-36647.