ROLE OF MITOCHONDRIAL DNA IN BREAST CANCER

根据乳腺癌进展审查小组（BC-PRG）的说法，乳腺癌病因学研究的首要目标是确定疾病因果途径中的风险因素，如果改变这些因素，将改变患乳腺癌的风险。其中一项建议是研究与肿瘤发生有关的基因多态性，以便确定乳腺癌风险增加的亚群。在本提案中，我们计划研究线粒体DNA （mtDNA）变异在乳腺癌危险因素中的作用。线粒体氧化磷酸化在能量代谢、活性氧的产生和细胞凋亡中的重要作用暗示了线粒体在衰老和癌症生长过程中的重要性。线粒体是动物中唯一含有自身基因组且在个体间高度多态性的细胞器。DNA的变异可能影响mtDNA的稳定性、复制、mtRNA的合成和加工、功能酶的活性以及细胞核与线粒体之间的通讯，从而调控细胞的死亡和生长。此外，由于细胞器环境中产生的活性氧（ROS）水平高，加上DNA修复水平低，线粒体基因组特别容易发生突变。我们假设线粒体DNA变异正在改变乳腺癌的危险因素。mtDNA的多态性变异可能引起线粒体氧化磷酸化活性的微妙变化，从而导致ROS水平略高。活性氧的积累和氧化DNA损伤在一生中可能是有害的。我们还假设体细胞mtDNA突变在肿瘤发生中起作用，因为突变的线粒体可能适应复制优势，导致异常细胞生长并最终导致乳腺癌。我们计划使用多重PCR/ASO（等位基因特异性寡核苷酸）点印迹法分析正常对照和乳腺癌患者中已知与DNA损伤、肿瘤发生和线粒体疾病相关的32种线粒体DNA突变/多态性。如果多态线粒体DNA变异在改变乳腺癌风险中发挥作用，则将观察到某些线粒体DNA变异与患者的显著关联。我们还将通过时间温度梯度凝胶电泳（TTGE）方法筛选乳腺肿瘤线粒体基因组中的体细胞突变，然后进行DNA测序。如果线粒体突变在癌症生长中起作用，则会检测到体细胞mtDNA突变。直接鉴定mtDNA突变将为未来的研究提供假设，从而阐明线粒体在肿瘤发生过程中的作用。

According to The Breast Cancer Progress Review Group (BC-PRG), an overarching goal for breast cancer etioloogical research is to identify risk factors in the causal pathway of disease that, if changed, will alter the risk of developing breast cancer. One of the recommendations is the study of polymorphisms of genes involved in tumorigenesis, so that subpopulations at increased risk of breast cancer may be identified. In this proposal we plan to investigate the role of mitochondrial DNA (mtDNA) variations a a risk factor for breast cancer. The essential role of mitochondrial oxidative phosphorylation in energy metabolism, the generation of reactive oxygen species, and the initiation of apoptosis have implicated the importance of mitochondria in the process of aging and cancerous growth. Mitochondria are the only animal cellular organelles containing their own genomes that are highly polymorphic among individuals. The DNA variations may affect the stability of the mtDNA, its replication, mtRNA synthesis and processing, the activity of the functional enzymes, and the communication between the nucleus and mitochondria in regulating cell death and growth. In addition, the mitochondrial genome is particularly susceptible to mutation because of the high level of reactive oxygen species (ROS) generated in the organelle environment, coupled with a low level of DNA repair. We hypothesize that mitochondrial DNA variations are modifying risk factors for breast cancer. The polymorphic variations in mtDNA may cause subtle changes in mitochondrial oxidative phosphorylation activity that lead to slightly higher levels of ROS. The accumulation of ROS and oxidative DNA damage during the course of a lifetime may be deleterious. We also hypothesize that somatic mtDNA mutations play a role in tumorigenesis because mutant mitochondria may adapt a replicative advantage leading to abnormal cell growth and ultimately breast cancer. We plan to use the multiplex PCR/ASO (Allele Specific Oligonucleotide) dot blot method to analyze 32 mitochondrial DNA mutations/polymorphisms, known to be associated with DNA damage, tumorigenesis, and mitochondrial disorders, in normal control and breast cancer patients. If the polymorphic mitochondrial DNA variations play a role in modifying breast cancer risk, significant associations of certain mtDNA variations with patients will be observed. We will also screen breast tumors for somatic mutations in the mitochondrial genome by the Temporal Temperature Gradient Gel Electrophoresis (TTGE) method followed by DNA sequencing. If mutant mitochondria play a role in cancerous growth, somatic mtDNA mutations will be detected. Direct identification of mtDNA mutations will generate hypotheses for future research that will elucidate the role of mitochondria in the process of tumorigenesis.