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. .

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-OBT-Z (55))
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Salnikow, Konstantin
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University of Alabama Birmingham
Schools of Medicine
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Koochekpour, Shahriar; Marlowe, Timothy; Singh, Keshav K et al. (2013) Reduced mitochondrial DNA content associates with poor prognosis of prostate cancer in African American men. PLoS One 8:e74688