Wnt Pathway Regulation by DOT1L Mediated Methylation of H3K79 in Non-Small Cell Lung Cancer

Abstract

The Wnt pathway is a developmental pathway that is abnormally upregulated in cancer. Our current lab results suggest that decreased H3K79 methylation levels are associated with decreased DNMT1 activity and binding to the WIF1 5’ region, upregulation of WIF1 protein levels, and suppression of Wnt signaling in lung cancer cell lines. We therefore hypothesize that the silencing of the WIF1 gene seen in many lung cancers involves upregulation of H3K79 methylation. We aim to knock down DOT1L and AF10, H3K79me3 mediators, to diminish Wnt expression in non-small cell lung cancers (NSCLCs). NSCLC lines including the A549 and H460 lines will be transfected with shRNAs for DOT1L and AF10; Western blotting will be used to confirm knockdown of DOT1L, AF10, and WIF1 expression. Wnt signaling will be measured by TOPFlash reporter assay, and by western blotting of nuclear β-catenin from total and fractionated cell lysates. We will then measure lung cancer cell growth, apoptosis and senescence.