Investigating the functional significance of the upregulation of Cyclin D2 and p21 following Apc loss in vivo

Cole, Alicia M. (2010) Investigating the functional significance of the upregulation of Cyclin D2 and p21 following Apc loss in vivo. PhD thesis, University of Glasgow.

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The Apc gene encodes the Adenomatous polyposis coli tumour suppressor protein, the germ line mutation of which characterizes Familial Adenomatous Polyposis (FAP), an autosomal syndrome characterized by multiple colorectal lesions. Inactivation of the Apc gene is recognized as a key early event in the development of colorectal cancers and leads to the deregulation of the Wnt pathway and the activation of TCF/LEF target genes. This project focuses on the proto-oncogene c-Myc as it is a key Wnt target gene which is activated following loss of Apc in vivo. This upregulation is noteworthy as c-Myc is implicated in stem cell survival, proliferation, apoptosis and tumourigenesis. Previous studies have shown c-Myc dependency for both apoptosis and proliferation following activation of the Wnt pathway, however little is known about the role c-Myc plays in inducing apoptosis following DNA damage in vivo. To study this I have conditionally deleted c-Myc from the intestinal epithelium and examined the response of intestinal enterocytes following DNA damage. Remarkably, following DNA damage, c-Myc deficient enterocytes were unable to upregulate p53 and induce apoptosis, which was mechanistically due to an upregulation of MDM2. Taken together, results from this study showed for the first time in vivo, a key role for c-Myc in inducing apoptosis following DNA damage through control of p53.
Previous studies from this lab have shown that within the intestinal epithelium, c-Myc is absolutely required for the hyper-proliferative phenotype that is observed following loss of Apc. Therefore one of the key aims of this thesis is to look downstream of c-Myc in order to delineate how c-Myc induces and controls this proliferation. Given that one of the key postulated functions of c-Myc is the transcriptional repression of p21, this thesis examines this hypothesis by investigating the significance of the upregulation of p21 following c-Myc deletion in Apc deficient intestinal enterocytes. To do this, I have generated triple knockout (TKO) intestines by intercrossing p21 knockout mice to mice where we can conditionally delete both Apc and c-Myc within the murine intestinal epithelium. Surprisingly, the levels of proliferation were the same between double knockout Apc Myc and TKO intestines, which had markedly less proliferation than Apc deficient intestines. However, unlike double knockout enterocytes, TKO intestinal enterocytes no longer moved up the crypt-villus axis and failed to generate villus. To examine which of these phenomena were key to tumourigenesis (differentiation or proliferation), we investigated whether TKO intestines could form intestinal adenomas and found that even in the absence of p21, c-Myc deficient cells were unable to form tumours. Taken together we have identified a novel role for p21 in driving differentiation following Apc and Myc deletion. This is consistent with the expression of p21 in the normal crypt at the crypt villus junction. Remarkably this function of p21 is independent of its key role as a cell cycle inhibitor.
Moreover, this study also examined the importance of the upregulation of the Cyclin D/CDK4 complexes following Apc loss and their role in c-Myc dependent proliferation. Results from these studies showed that Cyclin D2 is required for efficient proliferation immediately following loss of Apc as well as for tumourigeneis in the Apc Min/+ mouse. Taken together, results from these studies showed that the upregulation of Cyclin D2 and CDK4 are c-Myc dependent and that the upregulation of these complexes are key for Wnt driven proliferation and tumourigenesis.
Lastly, in this study I have examined whether Apc loss within the intestinal epithelium, where it is a bona fide tumour suppressor gene, can provoke senescence, and compared this to the ability of Apc gene deletion to trigger senescence in the renal epithelium, where it is not mutated in human cancer. This study showed that deletion of Apc within the renal epithelium invoked a p21 dependent senescence response, and Apc deficient renal epithelial cells were cleared and very rarely initiated tumourigenesis. However, combined Apc and p21 gene deletion rapidly initiated tumourigenesis, with all mice developing renal carcinoma by 2 months of age. In contrast to Apc deficient intestinal epithelium, this process was unaffected by loss of c-Myc. However within the intestinal epithelium, deletion of Apc did not invoke senescence, but lead to a highly proliferative, p21 independent response. Combined Apc and p21 gene loss had no impact on either the short term phenotypes of Apc loss or upon tumourigenesis.
Taken together these results show for the first time that Apc loss in vivo can invoke a senescence program but in a context dependent fashion. This implies escape from senescence is not a crucial pathway in colorectal cancers that are initiated by Apc loss, and goes to explain why renal carcinoma is not observed in FAP patients who are germline heterozygous for APC. Therefore the aims for this thesis are:

• To investigate the role of c-Myc in inducing apoptosis within the intestinal crypt, and whether this is p21 dependent?

• To investigate the role of p21 in causing senescence of Apc deficient cells, and whether this is c-Myc dependent?

• To determine the functional importance of repression of p21 by c-Myc in Apc deficient cells.

• To determine the significance of Cyclin D2 upregulation within Apc deficient cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: colon cancer, Apc, Cyclin D2, c-Myc, p21, senescence, mouse models of cancer
Subjects: R Medicine > RB Pathology
Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Supervisor's Name: Sansom, Dr. Owen
Date of Award: 2010
Depositing User: Miss Alicia Cole
Unique ID: glathesis:2010-1539
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 15 Mar 2010
Last Modified: 10 Dec 2012 13:42

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