Hepatocellular carcinoma (HCC) is the most common form of
liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote
disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and
diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter
ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced
tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (
Ap5A), a broad inhibitor of adenylate
kinases. Mass spectrometry-based proteomics revealed that DEN-induced
tumor mitochondria had increased expression of
adenylate kinase isoform 4 (AK4), which may account for this response to
Ap5A.
Tumor mitochondria also displayed a reduced ability to retain
calcium and generate membrane potential across a physiological span of
ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing
cancer bioenergetics.