Many solid tumor cells exhibit mitochondrial respiratory impairment. However, the mechanisms of such impairments in cancer development remain unclear. In this study, to investigate an effect of mitochondrial dysfunction on hepatocellular carcinoma, the two projects were studied. The first project was to prove the involvement of mitochondrial DNA repair enzyme in hepatoma cell growth. To address this, it was employed several SNU hepatoma cells, which had been classified as harboring mitochondrial respiratory defects or not in a previous study. Interestingly, SNU cells with declined mitochondrial respiratory activity showed decreased expression of mitochondrial 8-oxoguanine DNA glycosylase/lyase (mtOGG1), a mitochondrial DNA repair enzyme; similar results were obtained with human hepatocellular carcinoma tissues. Among several OGG1-2 variants with a mitochondrial-targeting sequence (OGG1-2a, -2b, -2c, -2d, and -2e), OGG1-2a was the major mitochondrial isoform in all examined hepatoma cells. Interestingly, hepatoma cells with low mtOGG1 levels showed delayed cell growth and increased intracellular reactive oxygen species (ROS) levels. Knockdown of OGG1-2 isoforms in Chang cells, which have active mitochondrial respiration with high mtOGG1 levels, significantly decreased cellular respiration and cell growth, and increased intracellular ROS. Overexpression of OGG1-2a in SNU423 cells, which have low mtOGG1 levels, effectively recovered cellular respiration and cell growth activities, and decreased intracellular ROS. Taken together, these results suggest that mtOGG1 plays an important role in maintaining mitochondrial respiration, thereby contributing to cell growth of hepatoma cells.
The second project was to identify major genes to control hepatoma cell invasiveness in response to mitochondrial respiratory defects. To address this, cDNA microarray was performed using SNU hepatoma cells with and without mitochondrial defects. Interestingly, functional enrichment analysis showed that 1423 commonly up-regulated genes in the hepatoma cells with mitochondrial defects were mainly linked with cell migration and actin organization. To further identify genes that are specifically up-regulated in response to mitochondrial respiratory defects, additional mitochondrial defects models were employed using respiratory inhibitors and mitochondrial DNA depletion (Rho 0). Among 10 genes commonly up-regulated in the three models, three transcription factors, NUPR1, NFIX, and NFE2L1, were further evaluated. All three transcription factors were involved in hepatoma cell invasiveness. Interestingly, NFIX induction was mediated by ROS, whereas NUPR1 and NFE2L1 expressions were mediated by Ca++ increase. Next, granulin (GRN) was identified as a potential downstream target of NUPR1 by employing the cDNA microarray analysis of siRNA-mediated NUPR1-depleted SNU354 cell. Finally, GRN was proved to be a critical molecule to regulate hepatoma cell invasiveness. Taken together, these results suggested that NUPR1 is a key mitochondrial retrograde Ca++ signal-responsive transcription factor to control hepatoma cell invasiveness.
