Characterisation of the functional consequences of PTEN gene mutation on colon cancer
- Publication Type:
- Thesis
- Issue Date:
- 2009
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olon cancer constitutes the second most common cause of cancer death in many Western countries. The PTEN tumour suppressor gene, located on chromosome 10q23.3, is now recognised as the most highly mutated tumour suppressor gene. PTEN, a lipid and protein phosphatase, regulates the phosphatidylinositol 3-kinase (PI3K)/ Akt signalling pathway and modulates cell cycle progression and cell survival. Previous work at University of Technology of Sydney laboratory has shown that a significant proportion of sporadic colorectal tumours harbour PTEN mutations that alter gene function and may therefore contribute to the pathways of colorectal carcinogenesis. A total of 10 novel somatic mutations have been described. In order to determine the functional consequences of these colon cancer-associated PTEN mutations, the wild type (WT) PTEN gene was cloned into a mammalian expression vector system and each of the mutants were generated from this. The WT, and each of the mutant K62R, Y65C, K125E, K125X, E150Q, D153N, D153Y, V217A 319X and N323K PTEN constructs were then transiently transfected into an U87MG glioblastoma PTEN null cell line and HCT116 colon cancer PTEN expressing cell lines that were then assayed for cell cycle phase distribution, Akt phosphorylation levels and cell proliferation. The analyses of endogenous suppression of Phospho Akt assay indicates 50% of PTEN mutants (Y65C, K125E, K125X, D153N, and 319 X) shows deficiency in the U87MG cell line and 70% of the mutants in the EICT116 cell line (Y65C, K125E, K125X, D153N, D153\ V217A and 319X) had deficiency in suppressing endogenous
phosphorylated Akt. The results obtained show 50% (Y65C, K125X, K125E, D153N and 319X) of the PTEN mutants had functional deficiency in cell cycle inhibitory capacity in the S phase in the U87MG cells; in contrast 80% (Y65C, K125X, K125E, E150Q, D153N, D153Y, V217A and 319X) of the PTEN mutants had functional deficiency in cell cycle inhibitory capacity in the S phase in the HCT116 cells. The results obtained show 60% of the PTEN mutants (K62R, Y65C, K125E, K125X, D153N and 319X) had alteration in cell proliferation rate in U87MG cells. In contrast in the HCT116 cell lines, 80% of the PTEN mutants (Y65C, K125E, K125X, E150Q, D153N, D153Y, V217A and 319X) had alteration in cell proliferation rates. These three functional assays of the mutations tested show an alteration of PTEN function. This was observed as a marked reduction in the ability of these PTEN mutants to bring about a level of cycle arrest, reduction of Akt phosphorylation levels and cell proliferation, compared to that observed with the WT PTEN gene product. These studies reveal that PTEN gene somatic mutations do alter PTEN function and are therefore likely to contribute to the process of colorectal carcinogenesis and may mediate a PTEN- associated carcinogenic pathway in these tumours.
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