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UBC Theses and Dissertations

Characterization of cellular abnormalities due to loss of TSC2 Moosa, Alym Platinum


Tuberous sclerosis complex (TSC) is a disorder characterized by multiple benign tumours in all major organs that can result in neurological manifestations including mental retardation and autism. TSC results from mutation in TSC1 or TSC2, which together form a complex that serves as a negative regulator of protein kinase mTORC1, a key regulator of cell growth and metabolism. Thus, cells with diminished TSC1:TSC2 function display elevated mTORC1 signaling, leading to the formation of benign tumours with very large cells. Rapamycin is a potent mTORC1 inhibitor, and rapamycin derivative everolimus has been approved for treatment of TSC patients with inoperable subependymal giant cell astrocytomas. However, these drugs can have serious side effects and should not be used for extended periods of time. In a screen of approved human drugs, amiodarone, dronedarone, perhexiline, and niclosamide were found to inhibit the elevated mTORC1 signaling seen in mouse embryo fibroblast (MEF) cells lacking TSC2. The goal of this project was to determine whether the many abnormal cell phenotypes associated with loss of TSC2 are directly related to elevated mTORC1 levels, and whether these drugs can ameliorate these abnormal phenotypes. Unlike wild type MEFs, TSC2-null MEFs showed an epithelial-like morphology with an increase in localization of actin to the cell periphery, focal adhesions, localization of β-catenin to cell-cell junctions, and localization of N-cadherin to cell-cell junctions. Exposure of TSC2-null MEFS to the mTORC1 inhibitors for seven days caused a transition from an epithelial-like to a fibroblast-like morphology in all of the aforementioned phenotypes, resembling that of wild type MEFs. TSC2-null MEFs were shown to express E-cadherin, a cell adhesion protein not normally found in MEFs. Knocking down levels of TSC2 in wild-type MEFs did not induce expression of E-cadherin, but restoring TSC2 expression in TSC2-null MEFs slightly reduced E-cadherin expression. A tentative model was proposed to explain how TSC2 can control E-cadherin expression, which has not yet been described in literature.

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