Analysis of molecular mechanisms in intestinal stem cells and colorectal cancer progression

Colorectal cancer (CRC) is one of the leading causes of cancer related mortality in Western countries. Activation of the Wnt signaling pathway is the initiating event in the majority of human CRCs. Further mutations in tumor suppressors and oncogenes, such as KRAS and the TGF signaling pathways, are required for carcinoma development. In the intestine, the orphan G-protein coupled receptor LGR5 marks crypt base columnar cells that can differentiate into all cell lineages of the intestinal epithelium. These LGR5 expressing intestinal stem cells provoke rapid adenoma formation upon Wnt pathway activation and are associated with poor clinical outcome. Although several reports have established the importance of cancer stem cells in CRC, factors regulating the maintenance of these cells remain largely unknown. In this study, we employed intestinal stem cell-based 3D cultures, genetic mouse models of intestinal cancer and patient-derived organoid cultures to investigate regulation of the cancer stem cells and CRC progression. We first analyzed mechanisms that modulate resistance to the growth inhibitory function of TGFβ in CRC progression. We demonstrated that normal intestinal cells are resistant to TGFβ-mediated apoptosis, whereas TGFβ induced apoptosis in Apc-mutant cells via upregulation of the BH3-only pro-apoptotic protein BIM. The KRAS oncogene increased resistance to TGFβ via the MAPK/ERK pathway, leading to suppression of BIM. Next, we analyzed the function of the homeobox transcription factor PROX1 in intestinal stem cells and progression of CRC. We showed that activation of Wnt pathway rapidly induces PROX1 expression in the intestine, including in the LGR5 expressing stem cells. By using lineage tracing, we established that some PROX1 expressing cells exhibit stem cell activity in adenomas but not in the normal intestinal epithelium. PROX1 deletion or silencing reduced the number of CRC stem cells via Annexin A1 upregulation and Filamin A downregulation, which was associated with decreased cell proliferation and tumor growth. To further investigate the regulation of CRC stem cells, we analyzed the interaction of the PROX1 and Notch pathway. We showed that Notch inhibition early after Apc deletion decreases the overall number of LGR5-expressing stem cells, whereas the PROX1- expressing LGR5 positive cells exhibited resistance to Notch inhibition. Conversely, NOTCH1 overexpression suppressed PROX1 and consequently decreased the stem cell activity of CRC cells. Furthermore, we demonstrated that PROX1 recruits the Nucleosome remodeling and deacetylase complex to the NOTCH1 promoter to suppress the Notch pathway. Thus, PROX1-induced suppression of the Notch pathway mediates its stem cell function in CRC. This thesis provides important insight into signaling pathways regulating CRC stem cells and progression. We show that BH3-mimetics can override TGFβ insensitivity in late stage CRC, and we identified a cancer specific signaling pathway of PROX1, observations of clinical importance. Expanding the knowledge of molecular mechanisms that regulate cancer stem cells and progression can lead to better treatment of CRC patients.