Tumorigenic role of mitochondria in African-American women

描述（由申请人提供）：线粒体执行多种细胞功能。这些功能包括通过氧化磷酸化（OXPHOS）产生ATP，控制细胞生长，细胞死亡，发育和各种代谢途径。OXPHOS通过由五种线粒体OXPHOS复合物（复合物I、II、III、IV和V）催化的一系列氧化还原反应偶联。在构成线粒体OXPHOS系统的86个亚基中，线粒体DNA（mtDNA）编码13个亚基。其余的由核DNA（nDNA）编码。有趣的是，过去几年的研究已经描述了与线粒体OXPHOS基因多态性相关的各种癌症的风险。研究表明，复合物I的mtDNA编码的ND 3基因（NADH脱氢酶3）亚基中的10398 G至A（G10398 A）碱基取代增加了非洲裔美国妇女患浸润性乳腺癌的风险（Canter et al 2005）AG至A取代导致氨基酸从丙氨酸（由G等位基因编码）变为苏氨酸（由A等位基因编码）。在5%的非洲裔美国人人群中发现了这种G10398 A置换（Canter et al 2005）。为了了解ND 3 G10398 A基因替换与非裔美国妇女浸润性乳腺癌相关的作用，我们建立了一个完全缺乏mtDNA的细胞系（mtDNA敲除或rhoo或？0）。我们使用该rhoo细胞系开发了一个胞质杂种细胞培养模型，用于将G10398 A mtDNA变体引入等基因核背景（trans-mitochondrial cybrid）。我们证明G10398 A变体赋予增加的复合物I活性、对细胞死亡的抗性和增加的致瘤性。基于这些观察结果，我们假设ND 3 G10398 A影响复合物I的nDNA编码亚基的表达，这有助于增加复合物I活性，抵抗细胞死亡和致瘤性。为了解决上述假设，我们将：目的1：确定G10398 A诱导的亚基基因表达和复合物I组成的变化。线粒体复合物I代表最大和最不了解的多聚体OXPHOS复合物。它由45个亚单位组成。其中，38个亚基由nDNA编码，7个（包括ND 3）由mtDNA编码。为了研究与G10398 A相关的复合物I活性增加（Kulawiec等人，2009 A）是否是由于亚基表达增加，我们将使用我们实验室开发的聚焦OXPHOS阵列来测量所有45个亚基的基因表达的变化。为了研究复合物I基因表达的改变是否影响复合物I的组成，我们将使用蓝色非变性PAGE，其允许测量其天然状态下呼吸复合物的量。目的2：确定NDUFA 13/GRIM 19（一种必需的复合物I调节亚基）在抵抗细胞死亡和肿瘤发生中的作用。在组成复合物I的45个亚基中，NDUFA 13/GRIM 19（与类维生素A干扰素诱导的死亡相关的基因）充当死亡调节蛋白（Zhang et al 2003，Lufei et al 2003）并且参与肿瘤发生（Maximo et al 2008，Nallar et al 2008）。NDUFA 13/GRIM 19也是复合物I的重要组成部分。它在复合物I的组装和酶活性中起着不可或缺的作用（Huang et al 2004）。我们的研究表明，G10398 A赋予对细胞死亡的抗性并诱导肿瘤发生（Kulawiec等人2009）。因此，我们将确定G10398 A相关的抗细胞死亡和肿瘤发生的抗性是否是由于GRIM 19/NDUFA 13的基因表达和/或转录后修饰的变化。我们的赠款提案解决了计划公告PAR-09-160的两个关键目标，要求在癌症健康差异的基础癌症研究中进行探索性/发展赠款计划（R21）。这些目标包括：1）开发“旨在研究癌症差异的新细胞培养模型/系统”，2）探索非裔美国妇女因mtDNA“遗传差异”而对乳腺癌的易感性。在我们实验室开发的G10398 A transmitochondrial cybrid系统有可能加强非洲裔美国人乳腺癌的研究。 公共卫生相关性：在过去的几年中，一些流行病学研究报告了与非裔美国人mtDNA多态性相关的乳腺癌（和其他）癌症风险增加。然而，缺乏对某些mtDNA多态性如何诱导癌症的机制的理解。这项研究将有助于确定G10398 A在非洲裔美国妇女中诱导致瘤性的机制。该项目的创新方面包括开发G10398 A transmitochondrial cybrid细胞系。该细胞系将作为拟议研究的重要工具。它将允许在等基因核背景下对非洲裔美国人的多态性mtDNA进行机制分析。所提出的胞质杂种模型将为分析非裔美国人群体中线粒体基因组内其他mtDNA多态性和个体多态性位点之间的上位相互作用（即协同作用）的相关性铺平道路。.

DESCRIPTION (provided by applicant): Mitochondria perform multiple cellular functions. These functions include ATP production via oxidative phosphorylation (OXPHOS), control of cell growth, cell death, development and various metabolic pathways. OXPHOS is coupled through a series of oxido-reduction reactions catalyzed by five mitochondrial OXPHOS complexes (Complex I, II, III, IV and V). Of the 86 subunits that make up the mitochondrial OXPHOS system, mitochondrial DNA (mtDNA) encodes 13 subunits. The rest are encoded by nuclear DNA (nDNA). Interestingly, studies in the past few years have described the risk of various cancers associated with polymorphism in mitochondrial OXPHOS genes. Studies show the 10398 G to A (G10398A) base substitution in the mtDNA-encoded ND3 gene (NADH dehydrogenase 3) subunit of complex I increases the risk of invasive breast cancer in African-American women (Canter et al 2005).The G to A substitution results in an amino acid change from alanine (encoded by the G allele) to threonine (encoded by the A allele). This G10398A substitution is found in 5% of the African-American population (Canter et al 2005). To understand the contribution of ND3 G10398A gene substitution associated with invasive breast cancer in African-American women, we generated a cell line completely devoid of mtDNA (mtDNA- knockout or rhoo or ?0). We used this rhoo cell line to develop a cybrid cell culture model for introducing the G10398A mtDNA variant into the isogenic nuclear background (trans-mitochondrial cybrid). We demonstrate that the G10398A variant confers increased complex I activity, resistance against cell death, and increased tumorigenicity. Based on these observations, we hypothesize that ND3 G10398A affects expression of nDNA encoded subunits of complex I, which contributes to increased complex I activity, resistance against cell death and tumorigenicity. To address the above hypothesis, we will: Aim 1: Determine G10398A-induced changes in subunit gene expression and composition of complex I. Mitochondrial complex I represents the largest and the least understood multimeric OXPHOS complex. It is composed of 45 subunits. Of these, 38 subunits are encoded by nDNA and 7 (including ND3) are encoded by mtDNA. To investigate whether increased complex I activity associated with G10398A (Kulawiec et al. 2009A) is due to increased expression of subunits, we will use a focused OXPHOS array developed in our laboratory to measure changes in the gene expression of all 45 subunits. To investigate whether alterations in complex I gene expression affect complex I composition, we will use blue native PAGE, which allows for measurement of the amounts of the respiratory complexes in their native state. Aim 2: Determine a role for NDUFA13/GRIM19 (an essential complex I regulatory subunit) in resistance to cell death and tumorigenesis. Of the 45 subunits that make up complex I, NDUFA13/GRIM19 (the gene associated with retinoid interferon induced mortality) acts as a death regulator protein (Zhang et al 2003, Lufei et al 2003) and is involved in tumorigenesis (Maximo et al 2008, Nallar et al 2008). NDUFA13/GRIM19 is also an essential component of complex I. It plays an indispensable role in the assembly and enzymatic activity of complex I (Huang et al 2004). Our study suggests that G10398A confers resistance against cell death and induces tumorigenesis (Kulawiec et al 2009). We will therefore determine whether the G10398A-associated resistance against cell death and tumorigenesis is due to changes in gene expression and/or posttranscriptional modification of GRIM19/NDUFA13. Our grant proposal addresses the two key objectives of the program announcement PAR-09-160 requesting exploratory/development grants program in basic cancer research in cancer health disparities (R21). These objectives include: 1) the development of "new cell culture models/systems designed to investigate cancer disparities" and 2) explores the susceptibility to breast cancer in African-American women due to "genetic differences" in mtDNA. The G10398A transmitochondrial cybrid system developed in our laboratory has the potential to enhance breast cancer research in the African-American population. PUBLIC HEALTH RELEVANCE: In the last few years, several epidemiological studies have reported the increased risk of breast (and other) cancer associated with mtDNA polymorphisms in the African-American population. However, a mechanistic understanding of how certain mtDNA polymorphisms induce cancer is lacking. The proposed study will help determine the mechanism underlying G10398A induced tumorigenicity in African-American women. Innovative aspects of this project include the development of a G10398A transmitochondrial cybrid cell line. This cell line will serve as an important tool for the proposed studies. It will permit the mechanistic analysis of African-American polymorphic mtDNA in an isogenic nuclear background. The proposed cybrid model will pave the way to analyze the relevance of other mtDNA polymorphisms and epistatic interaction (i.e. synergy) between individual polymorphic loci within the mitochondrial genome in the African-American population. .