Exploring the role of DNp73 in the intestinal stem cell niche and colorectal cancer

Colorectal cancer (CRC) is one of the common forms of somatic malignancy, and the second most common cause of death (from cancer) in Australia with significant resources committed to the management of the disease. The projected number of new cases of CRC diagnosed in 2015 is estimated at 17,070 and the high mortality rate claims the lives of around 4000 individuals each year. The process by which CRCs arise involves the acquisition of a series of genetic changes that progress via distinct pathological stages such as epithelial hyperplasia, benign adenomatous polyp formation that leads to cancer formation and finally metastatic carcinoma. Accumulating evidence has shown that early diagnosis of CRC or the identification of pre cancerous polyps can notably improve the chances of survival. The aim of this thesis was to investigate the role of the dominant negative form of the p73 protein (DNp73) in regulating intestinal epithelial cell differentiation and tumourigenesis. p73 shares remarkable sequence homology and protein organisation with the p53 tumour suppressor gene and generates two major protein isoforms via the use of alternative promoters. Recently, several reports have provided evidence that the longer TAp73 protein has tumour suppressor capabilities, whereas the N-terminally truncated DNp73 protein can act as an oncogene. The studies in this thesis have shown that p73 is expressed in intestinal epithelial cells within crypts suggesting that p73 expression is associated with undifferentiated stem cells and their immediate progenitors. Previous studies as well as studies in this thesis have indicated that the overall level of p73 is commonly overexpressed in human CRC, with an increase in the DNp73/TAp73 ratio. Investigation of the functional consequences of disrupting the DNp73/TAp73 ratio in the murine intestine using a DNp73α transgenic mouse model that allows inducible conditional expression of DNp73 in the intestinal epithelium identified an intriguing phenotype with an alteration in the crypt-villus architecture that revealed an expansion of the progenitor pool, while differentiated cell types were diminished. This thesis also investigated the relative expression levels of TAp73 and DNp73 isoforms using CRC cell lines and demonstrated that several p73 isoforms that are not found in normal colonic epithelium are expressed in most CRC cell lines. DNp73 was the most common isoform found in the tumour cell lines. Clinical specimens of normal and adjacent tumour tissues from 74 surgical resections (Cabrini Hospital), investigated if either of the two major isoforms of p73 correlated with the expression of intestinal stem cell markers. Two different statistical tests revealed that TAp73 and DNp73 isoforms are upregulated together in tumours, and correlate with tumours that have the EPHB2 signature. A novel investigation to determine whether elevated levels of DNp73 could accelerate polyposis in the Apcmin/+ mouse model revealed an overall enhancement of polyp formation in the AhCre, DNp73, Apcmin/+ mouse. Implications of this work on the role of DNp73 in regulating intestinal epithelial stem cell differentiation and promoting tumourigenesis may assist in the identification of new prognostic biomarkers that have the potential to improve diagnosis or treatment and hence likely to have a significant benefit to the community.