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5个线粒体OXPHOS配合物（配合物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风险。研究表明，mtdna编码的ND3基因（NADH脱氢酶3）复合物I亚基中的10398 G到A （G10398A）碱基置换增加了非裔美国女性患浸润性乳腺癌的风险（center et al . 2005）。G到A的替换导致氨基酸从丙氨酸（由G等位基因编码）转变为苏氨酸（由A等位基因编码）。在5%的非裔美国人中发现G10398A替代（Canter et al 2005）。为了了解ND3 G10398A基因替代在非裔美国女性浸润性乳腺癌中的作用，我们制造了一个完全缺乏mtDNA的细胞系（mtDNA敲除或rhoo或？0）。我们利用这种rhoo细胞系建立了一种杂交细胞培养模型，将G10398A mtDNA变体引入等基因核背景（跨线粒体杂交）。我们证明，G10398A变体赋予复合物I活性增加，抵抗细胞死亡，并增加致瘤性。基于这些观察结果，我们假设ND3 G10398A影响复合体I的nDNA编码亚基的表达，这有助于提高复合体I的活性，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g10398a诱导的亚基基因表达和复合体I组成的变化。线粒体复合体I代表最大和最不了解的多聚体OXPHOS复合体。它由45个亚基组成。其中，38个亚基由nDNA编码，7个（包括ND3）由mtDNA编码。为了研究与G10398A相关的复合物I活性增加（Kulawiec et al. 2009A）是否由于亚基表达增加，我们将使用实验室开发的聚焦OXPHOS阵列来测量所有45个亚基的基因表达变化。为了研究复合物I基因表达的改变是否会影响复合物I的组成，我们将使用蓝色天然PAGE，它允许在其天然状态下测量呼吸复合物的数量。目的2：确定NDUFA13/GRIM19（一种重要的复合体I调节亚基）在抵抗细胞死亡和肿瘤发生中的作用。在构成复合体I的45个亚基中，NDUFA13/GRIM19（与类视黄素诱导死亡相关的基因）作为一种死亡调节蛋白（Zhang et al . 2003, Lufei et al . 2003）并参与肿瘤发生（Maximo et al . 2008, Nallar et al . 2008）。NDUFA13/GRIM19也是复合物I的重要组成部分，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，G10398A可以抵抗细胞死亡并诱导肿瘤发生（Kulawiec et al . 2009）。因此，我们将确定g10398a相关的抗细胞死亡和肿瘤发生的抗性是否由于基因表达的变化和/或GRIM19/NDUFA13的转录后修饰。我们的资助提案解决了项目公告PAR-09-160的两个关键目标，该公告要求在癌症健康差异的基础癌症研究中进行探索性/开发性资助项目（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)探索由于mtDNA的“遗传差异”，非裔美国妇女对乳腺癌的易感性。我们实验室开发的G10398A线粒体杂交系统有潜力加强非裔美国人的乳腺癌研究。

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. .