Doctor of Philosophy in Biochemistry and Molecular Biology (PhD) 
Small molecules as modulators of mitotic arrest and senescence in cancer
Autophagy is a cellular “self-eating” process that enables cells to degrade and recycle cytoplasmic materials both as a housekeeping mechanism and in response to extracellular stress. Based on preclinical studies, autophagy promotes cell survival in the nutrient-deprived tumour environment and in response to several cancer therapy agents, making it a prospective therapeutic target. Due to a lack of pharmacologically suitable and selective autophagy inhibitors, a phenotypic automated fluorescence microscopy assay was designed and used to screen > 3,500 drugs and pharmacological agents for novel inhibitors of autophagosome accumulation. Verteporfin, a benzoporphyrin derivative used in photodynamic therapy, was the only active compound identified. As a photosensitizer, verteporfin generates large amounts of singlet oxygen upon light irradiation, which elicits a cell-death response. In the absence of light activation, verteporfin is nontoxic and inhibits starvation- and drug-induced autophagy. Biochemical and microscopy assays revealed that verteporfin prevents autophagic sequestration and degradation downstream of LC3 lipidation and membrane association.p62 is a scaffold protein that oligomerizes and links poly-ubiquitinated proteins to the autophagosome membrane by binding LC3, thus delivering both its cargo and itself for lysosomal degradation. Western blot analysis revealed that verteporfin produces SDS-stable high-MW p62, which is highly oxidized, and is likely a product of p62 crosslinking. The mechanism of high-MW p62 generation by verteporfin was discovered to be low-level singlet oxygen production, and the appearance of high-MW p62 correlated with autophagy inhibition. p62 co-immunoprecipitation experiments revealed that its association with EGFP-LC3 was not affected by verteporfin, but binding to poly-ubiquitinated cargo was disrupted. Therefore, non-photoactivated verteporfin generates low-level singlet oxygen that induces p62 oxidation and high-MW products that may interfere with autophagosome formation.Verteporfin was used to evaluate the therapeutic potential of autophagy inhibition using two different tumour xenograft models. Verteporfin did not show anti-tumour activity in a JIMT-1 breast cancer model, but it did enhance the anti-tumour and survival effects conferred by gemcitabine in a BxPC-3 pancreatic cancer model. The characterization of an early autophagy inhibitor among FDA-approved drugs that shows in vivo potential has significant implications for understanding autophagy modulation as a therapeutic strategy.
Manipulation of the cell cycle is an extensively used and promising strategy for cancer therapy. To identify novel cell cycle modulators, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic arrest and senescence. 8-azaguanine, IC 261, erysolin and SKF 96365 were identified as chemicals that stimulate senescence, a state of prolonged growth arrest, in a p53-mutated growth arrest-deficient cell line. Microtubule-targeting cancer therapies such as paclitaxel block cell cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time, or undergo mitotic slippage through degradation of cyclin B1 in the presence of an active mitotic checkpoint and enter interphase without having separated their chromosomes. Regulation of extended mitotic arrest and mitotic slippage and their contribution to subsequent cell death or survival is incompletely understood. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic exit through mitotic slippage. Using these drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. SU6656 and geraldol were identified as chemicals that induce mitotic slippage. Cells arrested at mitosis with paclitaxel or vinblastine and induced by these compounds to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death. Mitotic slippage induced by SU6656 or geraldol involved proteasome-dependent degradation of cyclin B1, but also required proteasome- and caspase-3-dependent inactivation of themitotic checkpoint through degradation of the mitotic checkpoint protein BubR1. Caspase-3 and p53, both apoptotic effectors, did not affect cell death after exposure to paclitaxel, with or without mitotic slippage induction. The requirement for caspase-3 for chemically induced mitotic slippage reveals a new mechanism for mitotic exit and a link between mitosis and apoptosis that has implications for the outcome of cancer chemotherapy.
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.