title: Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.
abstract: We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0.007); and drug resistance to doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a drug-resistant phenotype in vitro.
authors.affiliation: Faculty of Medicine, National Yang-Ming University, Taipei 112, Taiwan. doc2765c@ms59.hinet.net.|Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan. doc2765c@ms59.hinet.net.|Division of Thoracic Surgery, Taipei Hospital, Ministry of Health and Welfare, New Taipei City 242, Taiwan. doc2765c@ms59.hinet.net.|Division of Thoracic Surgery, Feng-Yuan Hospital, Ministry of Health and Welfare, Taichung City 420, Taiwan. doc2765c@ms59.hinet.net.
authors.forename: Chen-Sung
authors.initials: CS
authors.lastname: Lin
authors.affiliation: Faculty of Medicine, National Yang-Ming University, Taipei 112, Taiwan. htlee1228@gmail.com.|Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan. htlee1228@gmail.com.|Department of Medicine, Mackay Medical College, New Taipei City 252, Taiwan. htlee1228@gmail.com.|Division of Allergy, Immunology and Rheumatology, Mackay Memorial Hospital, Taipei 104, Taiwan. htlee1228@gmail.com.
authors.forename: Hui-Ting
authors.initials: HT
authors.lastname: Lee
authors.affiliation: Division of Urology, Feng-Yuan Hospital, Ministry of Health and Welfare, Taichung City 420, Taiwan. 470916lee@gmail.com.
authors.forename: Ming-Huei
authors.initials: MH
authors.lastname: Lee
authors.affiliation: Division of Allergy, Immunology and Rheumatology, Mackay Memorial Hospital, Taipei 104, Taiwan. k.elantris@gmail.com.|Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan. k.elantris@gmail.com.
authors.forename: Siao-Cian
authors.initials: SC
authors.lastname: Pan
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan. georgecyk@gmail.com.
authors.forename: Chen-Yeh
authors.initials: CY
authors.lastname: Ke
authors.affiliation: Faculty of Medicine, National Yang-Ming University, Taipei 112, Taiwan. whchiu@ym.edu.tw.|Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan. whchiu@ym.edu.tw.|Division of Urology, Taipei City Hospital, Ren-Ai Branch, Taipei 106, Taiwan. whchiu@ym.edu.tw.
authors.forename: Allen Wen-Hsiang
authors.initials: AW
authors.lastname: Chiu
authors.affiliation: Faculty of Medicine, National Yang-Ming University, Taipei 112, Taiwan. joeman@mmc.edu.tw.|Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan. joeman@mmc.edu.tw.|Department of Medicine, Mackay Medical College, New Taipei City 252, Taiwan. joeman@mmc.edu.tw.|Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan. joeman@mmc.edu.tw.
authors.forename: Yau-Huei
authors.initials: YH
authors.lastname: Wei
mesh_terms: D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000601:surgery*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D056915:DNA Copy Number Variations; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics / Q000601:surgery*; D024101:Mitochondrial Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C499631:TFAM protein, human; D014157:Transcription Factors; D004317:Doxorubicin
keywords: Warburg effect; invasiveness; mitochondrial DNA copy number; mitochondrial biogenesis; renal cell carcinoma