A century of scientific research has established that mitochondria dysfunction is one of the most prevalent and profound phenotypes of human cancer cells. At the genetic level, the differences between normal and tumor cells include both the depletion of mitochondrial DNA (mtDNA) and somatic mtDNA mutations in all human cancers examined to date. The rate of mutation in mtDNA appears to be 20 times higher than that in nuclear DNA. This high rate of mutation is due to the high concentration of reactive oxygen species (ROS) produced by the mitochondria, limited DNA repair and lack of DNA protective histones in the mitochondria. Until now, studies have focused on the identification and characterization of mutations in mtDNA, with little insight into the contribution of these mutations to the etiology of cancer. To understand the contribution of mtDNA in etiology of cancer, we have generated the complete mtDNA- knockout ( rho(o)) in breast epithelial cells. We demonstrate that mtDNA knockout in epithelial cells leads to neoplastic transformation that shows increased invasiveness and anchorage-independent growth. Interestingly, these properties of (rho(o) epithelial cells are restored by reintroduction of mtDNA (transmitochondrial cybrid) suggesting a role for mtDNA in the induction of the cancer phenotype. We also demonstrate that mitochondrial gene knockout leads to i) gross chromosomal rearrangements (GCR) 2) resistance to apoptosis 3) altered expression of ROS generating NADPH oxidase (Nox1) involved in tumorigenesis. Based on these observations we hypothesize that mutant mtDNA contributes to the etiology of cancer. MtDNA knockout cells established in our laboratory will serve as a novel tool for transfer of mutant mtDNA in a uniform genetic background. The mtDNA mutations could contribute to neoplastic transformation of epithelial cells by increasing oxidative stress, modulating cell proliferation, modulating apoptosis, affecting the expression of ROS producing Noxi protein and increasing nuclear genome instability. To test this hypothesis we plan to: i) Construct a mtDNA mutator system and characterize those mutants that are found in primary breast tumors. 2) Generate isogenic mutant mtDNA cybrids, and determine the consequences of mutations on mitochondrial function. 3) Using isogenic transmitochondrial cybrids determine the consequences of mtDNA mutations on cell proliferation, apoptosis and nuclear genome instability. 4) Using isogenic transmitochondrial cybrids determine the importance of superoxide generating Nox1 in tumorigenesis. 5) Using isogenic mutant mtDNA cybrids assess the significance of mtDNA mutations on in vitro and in vivo tumorigenesis and metastasis in severe combined immunodeficient (SCID) mice. The proposed study will help determine the contribution of mutant mtDNA to the development of human cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA121904-12
Application #
8272973
Study Section
Cancer Genetics Study Section (CG)
Program Officer
Okano, Paul
Project Start
1999-02-01
Project End
2014-06-30
Budget Start
2011-07-06
Budget End
2014-06-30
Support Year
12
Fiscal Year
2010
Total Cost
$211,621
Indirect Cost
Name
University of Alabama Birmingham
Department
Genetics
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Zhang, Hengshan; Singh, Keshav K (2014) Global genetic determinants of mitochondrial DNA copy number. PLoS One 9:e105242
Donthamsetty, Shashikiran; Brahmbhatt, Meera; Pannu, Vaishali et al. (2014) Mitochondrial genome regulates mitotic fidelity by maintaining centrosomal homeostasis. Cell Cycle 13:2056-63
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
Minocherhomji, Sheroy; Tollefsbol, Trygve O; Singh, Keshav K (2012) Mitochondrial regulation of epigenetics and its role in human diseases. Epigenetics 7:326-34
Kim, Seog-Young; Rhee, Juong G; Song, Xinxin et al. (2012) Breast cancer stem cell-like cells are more sensitive to ionizing radiation than non-stem cells: role of ATM. PLoS One 7:e50423
Hall, Brandon M; Owens, Kjerstin M; Singh, Keshav K (2011) Distinct functions of evolutionary conserved MSF1 and late embryogenesis abundant (LEA)-like domains in mitochondria. J Biol Chem 286:39141-52
Gonçalves, António Pedro; Máximo, Valdemar; Lima, Jorge et al. (2011) Involvement of p53 in cell death following cell cycle arrest and mitotic catastrophe induced by rotenone. Biochim Biophys Acta 1813:492-9
Chandra, Dhyan; Singh, Keshav K (2011) Genetic insights into OXPHOS defect and its role in cancer. Biochim Biophys Acta 1807:620-5
Owens, Kjerstin M; Kulawiec, Mariola; Desouki, Mohamad Mokhtar et al. (2011) Impaired OXPHOS complex III in breast cancer. PLoS One 6:e23846
Graham, Kelly A; Kulawiec, Mariola; Owens, Kjerstin M et al. (2010) NADPH oxidase 4 is an oncoprotein localized to mitochondria. Cancer Biol Ther 10:223-31

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