Reconstructing signalling networks regulating intestinal homeostasis and tumour formation

The vast majority of human cancers arise in epithelial tissues. Maintenance of these tissues is based on the virtually infinite capacity of tissue-specific, self-renewing stem cells to generate proliferating and differentiating progenitor cells throughout life. Thereby, the processes of self-renewal, proliferation and differentiation are tightly regulated to ensure correct homeostasis. Several molecular mechanisms have been identified to contribute to these processes and revealed strong parallels in regulatory means between different epithelial tissues. In cancer there is increasing evidence that the maintenance and propagation of tumours depends on stem cell-like cancer cells. Deregulations in self-renewal and proliferation in the stem/progenitor cell compartment contribute to tumour initiation and propagation. Thus, a better understanding of the molecular mechanisms controlling the function of stem/progenitor cell compartments in cancer-prone tissues will essentially increase our knowledge about how tumours arise and may lead to new therapeutic approaches.The best-studied epithelial stem/progenitor cell compartment is contained in the crypt-villus of the gastrointestinal (GI) tract. Numerous genes and molecular pathways have been implicated in GI homeostasis of which many potentiate colorectal cancer (CRC) formation if deregulated. The most important components thereby are the Notch, Wnt and Myb signalling pathways, each of which is essential but not sufficient for the functional integrity of the GI homeostasis. It is proposed here that these three pathways interact to provide the core regulatory force within the GI stem/progenitor cell compartment. Hence, this study aims to reconstruct the interactions within this network to provide insights into the co-ordinate gene regulation central to intestinal homeostasis and colon carcinogenesis. The ultimate goal thereby will be to determine potential targets for novel approaches in cancer therapy.The interaction of Notch, Wnt and Myb pathways will be investigated in models of GI homeostasis and CRC on the levels of gene transcription, stem cell self-renewal and proliferation/differentiation of progenitor cells. This will be done by variably manipulating the activation and inhibition of the three pathways. Different strains of transgenic mice with distinct inducible pathway manipulations will be applied as experimental models, which will be complemented by the use of human cell lines in vitro. Transcriptional interaction will be determined by investigating synergism or antagonism of transcription factors of the Notch, Wnt and Myb pathways on DNA binding and gene activation in transgenic mouse-derived cells, as well as human cells in vitro. Self-renewal, proliferation and differentiation will be investigated by determining tissue regeneration capacity of GI stem/progenitor cells in transgenic mice. This will first be done in specialized 3-D culture model in vitro on a broad range of combinations of pathway manipulation. By focusing on the relevant emerging combinations, investigations will then be expanded to GI regeneration in transgenic mice in vivo.The laboratory of Prof. Ramsay has an extensive expertise in epithelial biology and carcinogenesis with a strong track record in the application of animal models and molecular biology techniques. They revealed the requirement of the Myb pathway in maintaining stem/progenitor cell compartments in different epithelia and provided evidence for its interaction with the Wnt pathway in intestinal cells. Transgenic animals, cell culture models and technical applications necessary to carry out the planned study are all well established in the host laboratory. Significant preliminary results achieved by Prof. Ramsay's group build the basis of and constitute essential cues to the experiments I intend to conduct. The expertise of highly experienced scientists in the host laboratory will further ascertain a successful accomplishment of the foreseen study.