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ROS-Generating Mitochondrial DNA Mutations Can Regulate Tumor Cell Metastasis. Kaori Ishikawa, et al. Science. 2008; 661–664 Adviser : Chin-Tin Chen Speaker: Yi-Chen Tsai D98B47402 1 Cancer The 5-year relative survival rate is 98% for breast cancer patients with localised disease and only 26% for those with distant-stage disease. (Ries et al, 2006) 2 Mitochondria of tumor cells have been reported to differ functionally and morphologically from those of normal cells. Normal gastric cell line 3 Gastric cancer cell line (Kim, 2007) Mitochondrion – “cellular power plants” Membrane-enclosed organelle found in most eukaryotic cells inner membrane Perform the redox reactions of oxidative phosphorylation 4 Generate ATP Electron transport chain couples a chemical reaction between an electron donor (such as NADH) and an electron acceptor (such as O2) to the transfer of H+ ions across a membrane, through a set of mediating biochemical reactions. These H+ ions are used to produce adenosine triphosphate (ATP), the main 5 energy intermediate in living organisms. (Wallace, 1999) Many chemical carcinogens have been shown to bind preferentially to mtDNA rather than to nuclear DNA. (Allen and Coombs, 1980) High frequencies of homoplasmic mutations in mtDNA of tumors rather than in mtDNA of normal tissues of the same patients. (Fliss, 1978) Preferential accumulation of mutated mtDNAs in tumor cells. (Gallardo, 2006) 6 7 8 9 Are mtDNA mutations involved in oncogenic transformation of normal cells to develop tumors? mtDNA was considered to be the major cellular target of chemical carcinogens, and resultant creation of mutations in mtDNA could be responsible for oncogenic transformation of normal cells to develop tumors. (Shay and Werbin, 1987) ? Tumorigenicity 10 mtDNA mutation Nuclear but not mitochondrial genome involvement in 3methylcholanthrene (MCA)-induced expression of tumori(Hayashi et al., 1989) genicity. nucleus Mitochondrion 11 Normal cell MCA-induced tumor cell No Tumorigenicity Still express tumorigenicity Maternal inheritance of mtDNA Mitochondria are normally inherited exclusively from the mother. Only 12 Tumorigenicity Mother Offspring With cancer ? 13 However, there has been no statistical evidence for the presence of maternal inheritance of tumor development. Mitochondrial defect disease In humans, complex I defects caused by mtDNAs with pathogenic mutations and the resultant overproduction of ROS are reported to be associated with the pathogenesis in Leber’s hereditary optic neuropathy (LHON). According to the statistical analysis, the patients with LHON develop cancers that are already metastatic. (Wallace, 1999) 14 Motive Mitochondrion Tumorigenicity ? mtDNA mutation Metastasis 15 Are mtDNA mutations involved in malignant progression of tumor cells to develop metastasis? High metastatic cells showed significant reduction of complex I (NADH dehydrogenase) activity. P29 16 A11 B82 B82M P29 A11 B82 B82M Complex I consists of subunits encoded by both nuclear (Wallace, 1999) DNA and mtDNA. Difficulty of excluding possible involvement of nuclear DNA mutations in these processes mtDNA exchange technology 17 mtDNA exchange technology Nuclear DNA P29 P29 P29 A11 mtDNA A11 A11 P29 A11 The mtDNA of high metastatic cells is therefore likely to harbor a mutation(s) responsible for complex I defects. 18 Does the mtDNA that induce metastasis in tumor cells also induce tumor development in normal cells? Nuclear DNA P29 P29 P29 B82M mtDNA 19 B82 B82 B82 A11 NIH NIH NIH NIH A11 B82M NIH3T3, normal cell Nuclear mtDNA DNA B82 B82 B82 A11 NIH NIH NIH A11 NIH B82M These mutations do not control the development of tumorigenicity, at least in nontransformed NIH3T3 cells. 20 P29 A11 G13997A mutation B82 B82M 13885insC mutation Complex I defects ? Metastasis 21 ND6 gene mutation NADH dehydrogenase subunit 6 How does Complex I defects regulate metastasis, and which nuclear genes (if any) are involved in this process? A11 cells, but not P29 cells, show resistance to hypoxiainduced apoptosis, accompanied by up-regulation of antiapoptotic MCL-1 (myeloid cell leukemia–1). (Takasu, 1999) A11 cells showed higher expression levels of two genes associated with neoangiogenesis, HIF-1α (hypoxia-inducible factor–1α) and VEGF (vascular endothelial growth factor), in comparison with P29 cells. (Koshikawa, 2006) 22 Nuclear DNA P29 P29 mtDNA P29 A11 A11 A11 P29 A11 The mutated mtDNA and the resultant complex I defects induce up-regulation of the MCL-1, HIF-1α, and VEGF genes and are associated with high metastatic potential. 23 Mitochondrion ND6 gene mutation NADH dehydrogenase subunit 6 Complex I defects ROS production (Koshikawa, 2006) 24 ? Nuclear-coded genes related to metastasis ? Nuclear-coded genes ROS production Nuclear DNA mtDNA P29 A11 ROS scavenger 25 ROS scavenger Treatment of the cybrids with ROS scavenger in cell culture reduced the amount of ROS. Nuclear DNA P29 A11 mtDNA ROS scavenger ROS scavenger Cells 26 Treatment of the cybrids with ROS scavenger in cell culture down-regulated MCL-1 reduced theirROS metastatic potential ROS and scavenger scavenger in mouse models. Mitochondrion ND6 gene mutation NADH dehydrogenase subunit 6 Complex I defects ROS production ? Metastasis 27 Nuclear-coded genes related to metastasis Nuclear DNA P29 A11 mtDNA Cells Down-regulation of MCL-1 in P29mtA11 cybrids by small interfering RNA also suppressed their metastatic potential. 28 Mitochondrion ND6 gene mutation NADH dehydrogenase subunit 6 Complex I defects ROS production Metastasis 29 Nuclear-coded genes related to metastasis Can contribution of mtDNA to tumor cell metastasis be extended to human tumors? Human cell lines Cell type Metastatic potential human breast cancer HeLa MDAMB231 human breast cancer Low High Nuclear DNA HeLa MDAMB231 mtDNA 30 Can contribution of mtDNA to tumor cell metastasis be extended to human tumors? HeLa Nuclear DNA HeLa HeLa MDAMB231 mtDNA Cells Transfer of mtDNA from human cancer cells expressing high metastatic potential into low metastatic cells induces complex I defects, increased ROS production, and high metastatic potential. 31 Summary Mitochondrion ND6 gene mutation NADH dehydrogenase subunit 6 Complex I defects ROS production 32 Metastasis Nuclear-coded genes related to metastasis Discussion 33 Mitochondrion Discussion mtDNA mutation Metastasis Resolve the question Why does the patients with LHON develop cancers that are already metastatic? Conventional mitochondrial theories 34 ROS-mediated genetic instability is responsible for aging, age-associated disorders.