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.

Keywords: Warburg effect; invasiveness; mitochondrial DNA copy number; mitochondrial biogenesis; renal cell carcinoma.

Figure 1

The pLKO.1 backbone was found in the NT and TFAM-KD clones, respectively (the 1st row). Western blot showed that the TFAM-KD clone had lower TFAM (the 2nd row), lower COX-2 (the 3rd row) and similar SDHA (the 4th row); higher AKT (the 5th row), MYC (the 6th row), HIF-2α (the 7th row), HK-II (the 9th row), PFK (the 10th row), LDHA (the 11th row), and vimentin (the 12th row) protein expression levels as compared with those of the NT clone. Both the NT and TFAM-KD clones had no detectable HIF-1α (the 8th row). The expression level of β-actin (the 13th row) was used as an internal control.

Figure 2

Trans-well migration assay was performed through a piece of membrane with 8-μm pores (Merck Millipore, Billerica, MA, USA). The more cells invading across the pores denotes a higher invasive activity. Under a light microscope (40×, left side; and 100×, right side), the TFAM-KD clone exhibited a higher trans-well migration activity than did the NT clone.