Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4

Koichi Fujisawa, Shuji Terai, Taro Takami, Naoki Yamamoto, Takahiro Yamasaki, Toshihiko Matsumoto, Kazuhito Yamaguchi, Yuji Owada, Hiroshi Nishina, Takafumi Noma, Isao Sakaida

Adenylate kinase is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. An isoform of this enzyme, adenylate kinase 4 (AK4), is localized in the mitochondrial matrix and is believed to be involved in stress, drug resistance, malignant transformation in cancer, and ATP regulation. However, the molecular basis for the AK4 functions remained to be determined. HeLa cells were transiently transfected with an AK4 small interfering RNA (siRNA), an AK4 short hairpin RNA (shRNA) plasmid, a control shRNA plasmid, an AK4 expression vector, and a control expression vector to examine the effect of the AK4 expression on cell proliferation, sensitivity to anti-cancer drug, metabolome, gene expression, and mitochondrial activity. AK4 knockdown cells treated with short hairpin RNA increased ATP production and showed greater sensitivity to hypoxia and anti-cancer drug, cis-diamminedichloro-platinum (II) (CDDP). Subcutaneous grafting AK4 knockdown cells into nude mice revealed that the grafted cells exhibited both slower proliferation and reduced the tumor sizes in response to CDDP. AK4 knockdown cell showed a increased oxygen consumption rate with FCCP treatment, while AK4 overexpression lowered it. Metabolome analysis showed the increased levels of the tricarboxylic acid cycle intermediates, fumarate and malate in AK4 knockdown cells, while AK4 overexpression lowered them. Electron microscopy detected the increased mitochondrial numbers in AK4 knockdown cells. Microarray analysis detected the increased gene expression of two key enzymes in TCA cycle, succinate dehydrogenase A (SDHA) and oxoglutarate dehydrogenease L (OGDHL), which are components of SDH complex and OGDH complex, supporting the metabolomic results. We found that AK4 was involved in hypoxia tolerance, resistance to anti-tumor drug, and the regulation of mitochondrial activity. These findings provide a new potential target for efficient anticancer therapies by controlling AK4 expression.