Wnt signaling: its transcriptional output in the intestinal crypt and in colon cancer

The transition of an intestinal epithelial cell into a fully transformed, metastatic cancer cell requires mutations in multiple proto-oncogenes and key tumor suppressor genes, including those of the Wnt pathway. We describe a large scale analysis of the downstream genetic program activated by wnt signaling through β-catenin/TCF4 in colorectal cancer cells. Inhibition of wnt signaling in these cells results in a growth arrest with downregulation of genes expressed in the proliferating region of the crypt while forcing these cells into a differentiation program. These results were confirmed by immunohistochemistry on adenomas and early neoplastic lesions in the intestine. This study validated the disruption of the β-catenin/TCF complex as a therapeutic strategy to revert the transformed phenotype in colorectal cancer. Silencing genes that contribute to the survival and progression of tumor cells is a major research objective. Recently RNAi technology for gene knockdown in mammalian cells has been proven to be effective. We describe the generation of a doxycycline-inducible version of a Polymerase III H1-RNA gene promoter driven shRNA vector (pTER). The knockdown of β-catenin/TCF activity by a loss-of-function approach reproduced the phenotypic changes that occur in CRC cells described earlier. This inducible pTER vector is particularly useful for the analysis of genes which functions are difficult to evaluate due to effects on growth or differentiation of cells, like cell-cycle regulators, oncogenes, tumor suppressor genes and genes that control apoptosis. DNA array techniques are rapidly evolving and genome-wide oligonucleotide array platforms are now common use, containing up to 55000 probes. We analyzed the expression profile of LS174 CRC cells stably transfected with inducible dnTCF1 and dnTCF4. The 300-400 selected Wnt responsive genes were subsequently compared to the gene profile of adenomas and adenocarcinomas versus normal colonic epithelium. These genes could be divided into 3 groups based on differential expression in distinct parts of the crypts. Our data demonstrate that in one organ system, the gut, different transcriptional outputs of Wnt signalling correspond with the proposed Wnt functions of maintaining the crypt progenitor phenotype, driving the terminal differentiation of Paneth cells and stem cell renewal, respectively. Mash-2 one of the identified target genes was studied more extensively. In the normal intestine, Mash-2 is expressed at the position of the presumptive crypt stem cell. No Mash-2 expression was found in the TCF4 KO mice and in adenomas of APCmin mice Mash-2 is highly expressed. This confirmed the observation that Mash-2 is indeed a target of the Wnt pathway. To further define the contribution of Mash-2 to the Wnt dependent phenotype in the gut, we generated a Villin-Mash-2-transgenic mouse. Overexpression of Mash-2 in the intestine correlated with overexpression of few wnt target genes implicated in proliferation and in stem cell maintenance, making Mash-2 a possible stem cell regulator. The results described in this thesis has further increased our knowledge of the role of the Wnt pathway. This may be a basis for the ongoing efforts for the development of drugs targeting the inhibition of the Wnt pathway