The role of Frizzled-7 in gastrointestinal biology

© 2015 Dr. Dustin FlanaganThe tissues of the gastrointestinal tract; oesophagus, stomach, small and large intestines, are lined by a simple columnar epithelium. An important function of the epithelium is to balance the exchange and absorption of nutrients that are used by cells of the body, whilst simultaneously preventing the entry of potentially harmful pathogens that can induce infection, inflammation and/or cellular transformation. As such, the unrelenting mechanical, chemical and biological stresses stimulate the epithelium of the gastrointestinal tract to undergo constant renewal, as way of eradicating damaged cells. The ability of the epithelium to continually renew is driven by adult stem cells that reside in defined niches throughout the gastrointestinal tract. Despite knowledge of their existence, only over the last decade has the identity and molecular pathways that regulate gastrointestinal stem cells been revealed. Originally discovered from its link to cancer and the genesis of colorectal cancer (CRC), the Wnt signalling pathway, the molecular events of which are detailed in chapter 1, was one of the first signalling pathways shown to play a fundamental role in the regulation of gastrointestinal stem cells and in malignancies of the gastrointestinal epithelium. As such, manipulation of the Wnt pathway is an attractive means to modify the behaviour of gastrointestinal stem cells that can be exploited for regenerative purposes or in the treatment of gastrointestinal cancer. However, prior to the commencement of this thesis, the role of the transmembrane receptors responsible for transducing Wnt signals in gastrointestinal biology, the Frizzled receptors, was poorly defined. The aims of this thesis are to identify the function of Frizzled-7, one of ten receptors of the Frizzled family, in gastric epithelial homeostasis, intestinal regeneration and examine therapeutic benefit of targeting Frizzled-7 for treating gastric cancer. Findings from this thesis provide greater insight into the contribution and function of a specific Frizzled receptor in several gastrointestinal contexts. In chapter 1, I provide an overview of the literature pertaining to gastrointestinal stem cells, the Wnt signalling pathway and the role of Wnt signalling in gastrointestinal homeostasis, regeneration and cancer. In chapter 2, I investigate the role of Frizzled-7 in the homeostasis of the gastric epithelium. I demonstrate that conditional deletion of Frizzled-7 in the gastric epithelium triggers a rapid repopulation of Frizzled-7 deficient cells with wild-type cells, which suggests Frizzled-7 is required for gastric homeostasis. Furthermore, targeted deletion of Frizzled-7 from a known population of gastric stem cells does not trigger epithelial repopulation, demonstrating a requirement for Frizzled-7 in a yet-to-be identified population of gastric epithelial cells. These findings draw close parallels to our recently published observations in the small intestine, demonstrating the critical role for Frizzled-7 in transmitting essential Wnt signals in stem cells of the gastric epithelium. The biological process of intestinal regeneration following injury is contingent on robust Wnt signalling and stem cell activity. In chapter 3, I examine the requirement of Frizzled-7 in intestinal regeneration following irradiation, a well characterised model of intestinal regeneration. Compared to wild-type mice, Frizzled-7 knockout mice have impaired intestinal regeneration, characterised by diminished proliferation, decreased Wnt signalling and an increase in cell cycle inhibition. Interestingly, the integrity of the intestinal epithelium is eventually restored in Frizzled-7 knockout mice, which is considered due to an increase in Paneth cells, which are known to facilitate intestinal regeneration following injury. Similar to the intestine, activating mutations to components of the Wnt pathway are commonly observed in gastric cancer and associated with poor prognosis. In chapter 4, I explore the potential therapeutic benefit of Frizzled-7 inhibition in two independent mouse models of intestinal-type gastric cancer and human-derived gastric cancer cells. Conditional deletion of Frizzled-7 from gastric tumours is sufficient to reduce tumour growth with accompanied decreased angiogenesis. Furthermore, significant reductions in tumourigenicity are observed following targeted inhibition of Frizzled-7 in human gastric cancer cells. Collectively, these data demonstrate that Frizzled-7 is rate-limiting for the growth of gastric tumours, which reveal the therapeutic benefit of targeting Frizzled-7 in the treatment of intestinal-type gastric cancer. Finally, in chapter 5, I bring together the primary conclusions from each study and discuss their relevance with respect to the importance of Wnt signalling in gastrointestinal biology. Collectively, the findings from this thesis demonstrate and highlight a pivotal role for Frizzled-7 in regulating and maintaining gastrointestinal stem cells in dynamic gastrointestinal contexts; homeostasis, regeneration and cancer, which have direct translation to the treatment of gastrointestinal malignancies