DESCRIPTION (provided by applicant): Epithelial tissues are polarized sheets of adherent cells that generate boundaries between different body compartments. Proper epithelial tissue architecture is maintained by a highly polarized network of polarity proteins and proper adhesion between neighboring cells that are aligned along the apical-basal axis. KRAS is an oncogene that is mutated in 30% of all human tumors and in 35-40% of colorectal cancers. Abnormal activation of KRAS up regulates RAS/BRAF/MAPK signaling, which results in hyper proliferation and disruption of apical polarity in Caco-2 colon cancer cells. While several studies indicate that activation of oncogenes such as KRASV12 and BRAFV600E result in loss of cell polarity in most primary tumors, the underlying mechanisms that regulate this crosstalk to govern polarized assembly of epithelial tissue is not known. The goals of my proposed research are to identify and characterize the molecular mechanisms through which KRASV12 and BRAFV600E oncogenes disrupt cell polarity; and to explore genes that cooperate with KRASV12 and BRAFV600E to promote invasion of primary tumors. Specifically, the main aims of this research are to investigate the effects of KRASV12 and BRAFV600E activation on epithelial morphogenesis; to elucidate gene candidates and signaling pathways through which oncogenic KRAS and BRAF regulate apical polarity, and to assess whether RhoGTPases, the cell adhesion machinery and 'putative' genes known to be frequently mutated in colorectal tumors cooperate with KRASV12 to promote invasion of primary tumors. Using a combination of molecular and cell-based approaches, tamoxifen-based inducible cell lines expressing BRAFV600E and KRASV12 will be generated to assess the temporal effects of oncogene activation on polarity maintenance at different stages of cyst development. Using a combination of calcium switch assay, FRAP and photoactivatable (PA) GFP, changes in the dynamic properties of AJ components upon oncogene activation will be determined. Further, the morphological effects of AJ remodeling on regulators of apical polarity proteins will be explored. To identify potential genes that regulate KRAS-dependent polarity defects, based on our DNA microarray analysis, I will use a lenti-viral shRNA-mediated gene knockdown approach to validate ~60 genes that are potentially up regulated upon activation of KRASV12 and BRAFV600E. Further, reporter gene assays will be used to determine oncogene-dependent changes in the activity of signaling pathways. Using confocal microscopy and biochemistry, candidate genes and signaling pathways will be further characterized to determine their role in the regulation of cell polarity. Finally, the role of cell adhesion components, and modulators of RhoGTPases in invasion and cell migration of KRAS-mediated primary tumors will be explored.

PUBLIC HEALTH RELEVANCE: Colorectal cancer (CRC) is the third most common human cancer worldwide and it results from a progressive accumulation of genetic and epigenetic alterations that lead to transformation of normal colonic epithelium to colon adenocarcinoma. Compelling evidence from 3D cell culture systems indicate that activating oncogenes such as K-Ras disrupt establishment of epithelial cell polarity, a characteristic feature of most human cancers, including colon cancer. The objective of this proposal is to use a three-dimensional Caco-2 cell culture system to elucidate the molecular and cellular mechanisms that underlie K-Ras-induced loss of cell polarity, and to explore genes and pathways that cooperate with KRASV12 and BRAFV600E to promote aggressive, more malignant tumors.