The DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. MMR deficient tumours are often resistant to standard chemotherapies, therefore there is a critical need to identify new therapeutic strategies to treat MMR deficient disease. This study demonstrates that MLH1 deficient tumours are synthetically lethal with the mitochondrial-targeted agent Parthenolide which is known to induce reactive oxygen species (ROS) as one of its main mechanisms of action. Upon functional analysis, I show for the first time that loss of MLH1 is associated with deregulated mitochondrial function evidenced by a reduction in complex I expression and activity, reduced basal oxygen consumption rate and reduced spare respiratory capacity. This mitochondrial phenotype in the MLH1-deficient cell lines is accompanied by a reduction in mitochondrial biogenesis as evidenced by down regulation of pgc1β and decreased mitochondrial copy number. Furthermore, MLH1-deficient cancer cells have a decreased antioxidant defence capacity with reduced expression of the antioxidant genes NRF1, NRF2, Catalase, Glutathione peroxidase and SOD1 as well as increased ROS production when treated with Parthenolide. I further demonstrate that both MSH2- and MSH6-deficient cell lines also display deficiencies in complex I compared to their MMR-proficient counterparts. Taken together, the results of this study show a novel role for MLH1 in mitochondrial function and biogenesis. The MMR proteins MSH2 and MSH6 are also likely to have a role in the mitochondria. My results suggest that targeting the mitochondria may be a potential therapeutic strategy for the treatment of MMR and specifically MLH1 deficient disease.

Defects in mismatch repair genes, particularly the hMLH1 and hMSH2 genes, are associated with pathogenesis and prognosis of some cancers. The lack of correlation between replication error phenotype and mutations in hMLH1 in sporadic human cancers suggested that inactivation of the hMLH1 gene may be associated with promoter hypermethylation. This study was to investigate the association of hMLH1 promoter hypermethylation and hMLH1 protein expression in oral cancer. Our results indicated that all 75 cases (100%) were without any methylation of hMLH1 promoter by use of methylation-specific PCR (MSP). Nineteen of 99 cases (19.2%) were partial methylation by HpaII-based PCR. In addition, 24 (26.1%) of 92 cases of OSCC had reduced levels of hMLH1 protein. The concordance analysis showed that the expression level of hMLH1 protein was not correlated with methylation of hMLH1 promoter. Furthermore, the prognosis significance of hMLH1 or hMSH2 proteins on OSCC was also investigated. We analyzed the association of hMLH1 and hMSH2 protein expression with clinicopathological data of 92 cases of OSCC at KSVGH. We found that 24 (26.1%) of 92 cases of OSCC had reduced levels of hMLH1 protein, however only 10 cases (10.9%) had reduced hMSH2 by use of IHC. In addition, the reduced expression of hMLH1 correlated with the tumor differentiation and N classification. However, none of these clinical and pathological characteristics of the OSCC patients were associated with the extent of hMSH2 expression. Finally, previous studies reports that the hMLH1 and Aurora-A are directly involved in the prognosis of several cancers. The expression levels of hMLH1 and Aurora-A protein were investigated in the 138 tumor samples for consecutive patients with pathological confirmed primary buccal carcinoma (BC). Then the association of the protein expression with clinicopathological data and survival were also evaluated. The loss of hMLH1 protein was found in 15 (10.9%) of 138 tumor sections by IHC. In addition, loss of hMLH1 protein expression was not any correlated with clinical features and patients¡¦ prognosis. The up-regulation of Aurora-A protein was found in 118 (85.5%) of 138 tumor sections by IHC. In addition, the up-regulation of Aurora-A protein expression was correlated with the pathological stage and T classification, but Aurora-A protein up-regulation was not correlated with prognosis. In conclusion, promoter methylation of hMLH1 might not play a potent role in the gene expression in oral cancer. Defective expression of hMLH1 but not hMSH2 was associated with the development of OSCC. In addition, the Aurora-A protein expression but not hMLH1 may affect the malignant behavior of BC. However, the hMLH1 and Aurora-A protein expression might be not the prognostic factors for BC patients.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

Thesis (M.S.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 50-55.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 132-147).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 56-75).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20073号 / 医博第4166号 / 新制||医||1018(附属図書館) / 33189 / 京都大学大学院医学研究科医学専攻 / (主査)教授 高田 穣, 教授 武藤 学, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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