The Role And Mechanism Of Lactic Acidosis In Cancer Cell Adaptation To Glucose Deprivation

Background:Solid tumors are dependent on glucose to survive. When glucose is deprived, tumor cells will inevitably initiate several signal transduction pathways, eventually leading to cell death. Previous studies revealed that solid tumors were generally glucose-deprived due to poor vascularization. Nevertheless, cancer cells can generally survive glucose deprivation better than their normal counterparts, although the mechanism is largely unknown.Lactic acidosis is prominent tumor microenvironment factor, generated by glycolysis and other metabolic pathways such as glutaminolysis. It has been recognized as a side product of tumor metabolism, or "metabolic waste", for a long time. Its role in tumor survival under glucose deprivation has not been appreciated yet. We propose that lactic acidosis may allow cancer cells to develop resistance to glucose deprivation-induced death.MethodsIn order to investigate the effect of lactic acidosis on cancer cells adaptation to glucose deprivation, we mimic the tumor lactic acidosis by adding pure lactic acid to culture medium to the final lactate concentration of20mM with corresponding pH6.7. The controls we set include lactosis (by adding lactate to culture medium to20mM and adjusted to pH7.4), acidosis (by adjusting medium pH to6.7using HCl). Tumor cell survival, glucose utilization and lactate generation under the above conditions were determined. Gene chip analysis was used to profile the global influence of lactic acidosis on tumor cell under glucose deprivation. Cell cycle distribution, autophagy induction and apoptosis of the surviving cancer cells were analyzed. Intratumoral lactic acidosis in the4T1mice xenograft model was partially disrupted by sodium bicarbonate buffer, and the xenograft's growth rate and metastasis were determined.Results1. Lactic acidosis renders tumor cells more resistant to glucose deprivation-induced cell death. Under glucose deprivation,4T1tumor cells cultured without lactic acidosis almost died out in a single day, while with lactic acidosis90%of cells died in a period of10days, with viable cells identified even65days after glucose depletion. Other tumor cell lines showed the same tendency, proving that lactic acidosis could assist tumor cells to resist glucose deprivation-induced death. The lactosis did not confer cancer cells with ability to resist glucose deprivation. Acidosis, to some extent, could alleviate glucose deprivation-induced cell death, but not as potent as lactic acidosis.2. The mechanism of lactic acidosis assisting tumor cell to resist to glucose deprivation-induced death. Gene chip analysis showed lactic acidosis regulated several cell mechanisms when glucose was deprived, including cell cycle, autophagy, apoptosis and energy balance, etc. We further proved that:(1) lactic acidosis could arrest cell at GO phase (quiescence or dormancy status) to minimize energy requirement through dowregulating DNA replication and G1/S transition machinery. G2/M transition factors were upregulated on transcription level, presumably to allow tumor cell enriching in GO phase further;(2) lactic acidosis activated autophagy, by which cells could recycle cellular components for survival. Electron microscopy showed typical autophagosomes under glucose deprivation and lactic acidosis. The autophagy marker, LC3-II, increased significantly, and LC3punctates were observed in the presence of lactic acidosis;(3) lactic acidosis also inhibited apoptosis through maintaining cellular energy balance and upregulating anti-apoptosis machinery. NADPH, a critical survival factor which declined sharply in apoptosis, was maintained at a potential threshold level.3. Disrupting lactic acidosis impaired tumor growth and metastasis. Tumor-bearing mice were fed with water containing0.2M NaHCO3, and5%NaHCO3was injected subcutaneously around tumor to neutralize lactic acidosis to lactosis. NaHCO3treated tumor showed significant slower growth rate, fewer lung metastases, and larger intratumoral necrotic region compared with control group.ConclusionWe propose that lactic acidosis could render tumor cells more resistant to glucose deprivation-induced death. This function is executed by arresting tumor cell to quiescence or dormancy status to minimize energy requirement, activating autophagy to recycle cellular content, inhibiting apoptosis to prevent immediate death. These mechanisms work together to rescue tumor cells from glucose deprivation-induced death. We also propose that neutralizing lactic acidosis is logically a potent therapeutic strategy to treat tumor.