Systematic analyses of CTNNB1-, BRAF-, KRAS- and PIK3CA-induced oncogenic activities in the intestinal epithelium

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Colon cancer cells contain sets of recurring mutations that alter the activities of major signalling pathways, among these the Wnt/beta-Catenin, MAPK, and the PI3K pathways that regulate stem cell traits, proliferation, differentiation and apoptosis in the intestinal epithelium. The contribution of a specific oncoprotein to the cancer phenotype is difficult to dissect, since many mutated proteins contribute simultaneously to aberrant signalling. We have therefore in the last years established a series of transgenic mice that allow induction of oncogenic forms of beta-Catenin (gene symbol CTNNB1), KRAS, BRAF or PIK3CA alone or in combinations, using tetracycline-controlled transgene expression. By this approach, oncogenes can be induced in a generalized manner in the intestine, allowing to study early oncoprotein-driven signalling events and effects on cell fate within a few days. Preliminary analyses of all models, and in-depth analyses of the CTNNB1 and BRAF models, showed that following oncogene induction the intestines of mice rapidly develop characteristics of different subtypes of colon tumors. We propose here to systematically analyze cellular signalling and cell composition of the intestines of the transgenic mice. First, we propose to evaluate changes in the intestinal cell hierarchy instigated by each oncogene in a time- and spatially-resolved manner (i.e. numbers and localizations of stem cells, proliferative cells, differentiated cells, apoptotic and senescent cells), using a combination of immunohistochemical (IHC), gene expression and fluorescent-activated cell sorting (FACS) analyses. In parallel, we will assess the localisation, amount and phosphorylation status of the central signal transducers CTNNB1, MEK, ERK and AKT, using IHC and Phospho-Western analyses. The functional roles of important signalling pathways will subsequently be interrogated using interference with small molecule inhibitors in oncogene-inducible organotypic primary cell cultures derived from the mice.Second, we propose to determine comprehensive sets of genes whose expression is de-regulated following oncogene activation in intestinal stem cells and in differentiated cells, using RNA-seq of FACS-sorted cell populations. Importantly, no such analyses have been performed in intestinal stem cells before. These experiments are thus suited to shed light on novel and relevant oncoprotein-induced changes of the cancer cell transcriptome. In summary, this proposal aims to disentangle and isolate the effects of the recurring CTNNB1, KRAS, BRAF and PIK3CA oncogenes in the intestine. The expected results will allow to link specific oncogenic signals to activation levels of target genes, to changes in cell fate, and finally to experimental therapeutic intervention. The proposed research will thus aid our understanding of how the clinically relevant subtypes of colon cancer form and will help to identify novel vulnerabilities of cancer cells.