Investigating the role of the Pik3ca(H1047R) mutation in colorectal cancer using a novel mouse model

2017-02-15T05:02:53Z (GMT) by Hare, Lauren May
Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases. The PI3K/Akt signalling cascade has crucial roles in many cellular processes associated with malignant behaviour including cell growth, proliferation, migration and survival. Aberrations to this pathway have been detected in many human cancers, including colorectal cancer (CRC). Mutations in the PIK3CA gene, which encodes the p110α catalytic subunit of PI3K, have been identified in up to 30% of sporadic CRCs. However, it is not known at which stage during CRC pathogenesis mutations in PIK3CA are arising. To investigate the effect of this mutation during CRC development in vivo, a novel mouse model was created with a conditional Cre recombinase (Cre) inducible Pik3caH1047R mutation. Initially, expression of the Pik3caH1047R mutation was achieved ubiquitously by crossing the Pik3caH1047R mouse with the Cre-deleter mouse strain. This revealed the Pik3caH1047R mutation to be embryonic lethal when expressed from the zygote stage. Analysis of the cause of this lethality revealed dysfunctional embryonic ‘turning’ and failed vascular system development. Endothelial cell-specific expression of the Pik3caH1047R mutation during embryogenesis confirmed that the failure of the vascular system to develop resulted in embryonic lethality. To combat the lethality, the Pik3caH1047R mutation was targeted specifically to the gastrointestinal tract by crossing it with the A33CrePR2 mouse which has an RU486 inducible Cre under the control of the gastrointestinal specific gpa33 locus. The result was the induction of Pik3caH1047R in A33-expressing cells of the gastrointestinal tract. This study demonstrated that alone, the Pik3caH1047R mutation was insufficient to initiate CRC in vivo. However, when combined with the loss of the tumour suppressor gene Adenomatous polyposis coli (Apc), known to induce intestinal adenomas, the Pik3caH1047R mutation successfully caused progression of these adenomas to invasive adenocarcinomas. This confirmed the hypothesis that mutations in Pik3ca require a ‘second hit’ to initiate tumourigenesis in order for it to exert is effect on CRC progression. This study also investigated the role of the Pik3caH1047R mutation on CRC metastasis. Intestinal carcinomas were induced by a chemical carcinogen in combination with expression of the Pik3caH1047R mutation. Although tumours were observed in the livers of some animals, results were inconclusive as to whether these were the result of intestinal metastasis, as the histology of these tumours was heterogeneous. However, small areas of intestinal cells were observed in some liver tumours suggesting a potential role for constitutively active PI3K/Akt signalling on the migratory ability of intestinal cancer cells. The effect of the Pik3caH1047R mutation on chemosensitivity was also assessed in vitro. While there was no significant differences in the sensitivity of wild type and Pik3caH1047R mutant fibroblasts to several common chemotherapies and PI3K inhibitors, there appeared to be a trend for mutant immortalised mouse mammary epithelial cells requiring higher concentrations of PI3K inhibitors to achieve 50% growth inhibition. This thesis provides evidence that the PI3K/Akt signalling cascade plays a significant role in the correct development of the vasculature of mice during embryogenesis. It has also presents compelling data confirming the role of the Pik3caH1047R mutation in the progression of CRC in vivo.