The role of Frizzled-7 in the normal intestinal epithelium and colorectal cancer

© 2016 Dr. Renate Hilda Marianne SchwabThe Wnt signalling pathway is pivotal for many biological processes, it controls cell fate during embryonic development, is a key regulator of homeostasis in adult self-renewing tissues, is necessary for regeneration and deregulation of the pathway leads to malignant transformation. The intestinal epithelium, which is composed of a single layer of intestinal epithelial cells that line the gastrointestinal tract, is the best-understood example for the roles of Wnt signalling during homeostasis and tumourigenesis. The Wnt signalling pathway was originally linked to the genesis of colorectal cancer (CRC) through linking the genes that are mutated in human CRC to the regulation of Wnt pathway mediated transcription. Through a great deal of attention directed at understanding the link between mutated intracellular components of the pathway and the initiation of CRC, it was shown to have a fundamental role in the regulation of intestinal stem cells and in malignancies of the intestinal epithelium as well as play an important role in intestinal homeostasis. Although our knowledge about the Wnt signalling pathway has increased immensely since its discovery, there are still numerous questions regarding Wnt signalling to explore. Thus, this thesis attempts to expand our knowledge of its role in the intestine and CRC, more specifically the role of the transmembrane receptors responsible for transducing Wnt signals, the Frizzled receptors, particularly Frizzled-7, one of ten seven-transmembrane receptors of the Frizzled family. One of the aims was to determine which Wnt(s) interact with Frizzled-7 (Fzd7), as a role for Fzd7 in CRC morphogenesis was previously shown. Thus in Chapter 2 I demonstrate that Wnt secretion is necessary for the phenotype transitions that are associated with the dissemination of the cells from the primary tumour site and that Wnt2b and Wnt3a can cooperate with Fzd7 in this process. Furthermore, I confirm the interaction between Fzd7 and Wnt3 and demonstrate an interaction between Fzd7 and Wnt2b via co-immunoprecipitation. In chapter 3, the aim was to investigate the role of Fzd7 in the homeostasis of the intestinal epithelium. I demonstrate that conditional deletion of Fzd7 in vitro in 3D “mini-gut” organoid cultures in LGR5+ CBC stem cells is detrimental to the organoids, which verified our previous in vivo findings and suggests that Fzd7 is required for intestinal homeostasis and has a critical role in transmitting essential Wnt signals in stem cells of the intestinal epithelium. In CRC over-expression of Fzds, specifically Fzd7, has been well documented as well as its involvement in the survival, invasion and metastatic capabilities of CRC cell lines. So, in Chapter 4 I explore the potential therapeutic benefit of Fzd7 inhibition in 3d adenoma organoid cultures, by employing antibodies that target Fzd7. Blocking the Fzd7 receptors is sufficient to reduce tumour growth and limit proliferation. Furthermore, conditional deletion of Fzd7 in vitro in the cancer stem cells reveals a strong drive to restore Fzd7+ cells. Collectively, the findings from this thesis demonstrate a role for Frizzled-7 in maintaining intestinal stem cells in homeostasis and potentially in cancer. Moreover a role for the upstream components of the Wnt signalling pathway, i.e. Wnts and Fzd7, in cancer was shown