Loss of mitochondrial DNA enhances angiogenic and invasive potential of hepatoma cells

Mitochondrial DNA (mtDNA) mutations are frequently found in a variety of tumors. However, the role of mtDNA mutations in tumor behavior is poorly understood. We explored the effects of mtDNA mutations on tumor phenotype employing mtDNA-depleted SK-Hep1 rho0 hepatoma cells. Expression of hypoxia inducible factor (HIF)-2alpha mRNA was markedly increased in rho0 cells compared to control cells. Protein level of HIF-2alpha was increased in SK-Hep1 rho0 cells compared to control cells in hypoxic but not in normoxic conditions, suggesting that mitochondrial dysfunction increases angiogenic potential of tumor cells. Expression of HIF-2alpha was increased at the RNA level after treatment of SK-Hep1 hepatoma cells with ethidium bromide (EtBr) or inhibitors of mitochondrial complexes. HIF reporter activity and the expression of vascular endothelial growth factor (VEGF), an angiogenic key molecule induced by HIF, were increased in SK-Hep1 rho0 cells compared to their normal counterparts. Tube formation assay and chick chorioallantoic membrane (CAM) assay showed that conditioned medium (CM) from mtDNA-depleted SK-Hep1 rho0 cells increased formation of tube-like structures and new blood vessels relative to that from control cells. In SK-Hep1 rho0 cells, expression of genes related to invasion such as urokinase-type plasminogen activator (uPA) or matrix metalloproteases (MMPs) was also upregulated compared to control cells, suggesting that mitochondrial dysfunction could also increase invasive potential of tumor cells. These results strongly suggest that HIF-2alpha mRNA expression is increased in tumor cells with mtDNA mutations or deletions, which contributes to the angiogenic and invasive potential of tumor cells.