AKT loss in human epithelial cells treated with severe hypoxia

Abstract

Cancer cells which can survive and or proliferate in hypoxia may be resistant to anti-cancer treatment. In our previous work, we showed that we could group cell lines treated with severe hypoxia into either hypoxia-induced cell cycle arrest-sensitive or resistant phenotypes, and hypoxia-induced cell death (HCD)-sensitive or resistant phenotypes. We showed that the resistant phenotypes were associated with high levels of active-AKT in late hypoxia and sensitive cells were associated with decreased or undetectable levels of AKT in late hypoxia. We have now extended our findings to numerous other cell lines. We show that HCD and loss of AKT is cell density dependent, and both AKT1 and AKT2 isoforms are lost in late hypoxia. Loss of AKT is most likely due to regulated degradation, as transcription of AKT isoforms is unchanged in hypoxia, and AKT is not significantly translocated to the nucleus to account for its disappearance from cytoplasmic lysates. Interestingly, inhibitors of proteosome, calpain or caspase-mediated proteolysis did not significantly block AKT loss. Inhibition of autophagy using diverse lysosome-targeted autophagy inhibitors also did not block AKT loss, however autophagy inhibitors which block general PI3K activity, such as 3-methyladenine or LY294002, were effective inhibitors of AKT loss in late hypoxia. Interestingly, those inhibitors also blocked HCD in an HCD-sensitive cancer cell line. Inhibitors of proteolytic pathways which did not block AKT loss also did not block HCD in HeLa. Our investigations support a model by which AKT is a major switch involved in regulating hypoxia-induced cell death.