Pathology - Theses

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    Targeting DNA-dependent protein kinase promotes accelerated senescence of irradiated human cancer cells
    AZAD, ARUN ( 2012)
    Ionizing radiation is a widely used anti-cancer modality. Unfortunately however, relapse rates are high following radiation treatment indicating an urgent need for novel radiosensitizing strategies. Since radiation potently induces DNA double-strand breaks (DSBs), targeting signaling networks involved in DSB repair is a promising approach for enhancing cellular radiosensitivity. In mammalian cells the primary repair mechanism of radiation-induced DSBs is the non-homologous end-joining (NHEJ) pathway, in which DNA-dependent protein kinase (DNA-PK) plays a critical role. As a result, DNA-PK potentially represents an important molecular target for inhibiting DSB repair and enhancing the cytotoxicity of radiation. Using BEZ235, a novel small molecule inhibitor of DNA-PK and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) currently in clinical trials, the first aim of this thesis was to characterize the effects of inhibiting DNA-PK on tumor radiosensitivity in vitro and in vivo. The second aim of this thesis was to examine the mechanisms through which DNA-PK inhibition improves tumor radiosensitivity as little is known about the mechanisms involved in the radiation-enhancing effects of DNA-PK blockade. BEZ235 was seen to abrogate radiation-induced DSB repair and potently increase the radiosensitivity of H460 and A549 cells, human non-small cell lung cancer (NSCLC) cell lines. BEZ235 also potentiated the anti-tumor activity of ionizing radiation in H460 xenografts. Significantly, radiation enhancement by BEZ235 coincided with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression and senescence-associated cytokine secretion. Subsequent experiments sought to examine the mechanisms involved in the pro-senescence response of irradiated cells to BEZ235, and specifically whether selective inhibition of DNA-PK is sufficient to promote accelerated senescence after radiation. Significantly, it was shown that specific pharmacological inhibition of DNA-PK but not PI3K or mTORC1 delays DSB repair leading to accelerated senescence after radiation. It was additionally demonstrated that PRKDC knock down using small interfering RNA promotes a striking accelerated senescence phenotype in irradiated cells comparable to that of BEZ235. Collectively, these data establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade and underline the emerging link between unrepaired DSBs and enforcement of p53-dependent accelerated senescence. These data highlight the potential benefits of using DNA-PK blockade to modulate repair of therapeutically-induced DSBs and thereby promote radiation-induced accelerated senescence. In turn, these findings provide a rationale for further pre-clinical and clinical evaluation of DNA-PK inhibitors in combination with anti-cancer agents that induce DSBs or inhibit DSB repair.