Akt/protein kinase B, an onco-protein with serine/threonine kinase activity that plays a central role in cell signaling downstream of growth factors, mediates mitogenic and anti-apoptotic responses by activating multiple signaling cascades. Aberrant activation of Akt promotes cell growth, survival and proliferation, and is associated with cancer development and progression. Thus, Akt represents a promising therapeutic target against cancer, and inhibitors of Akt are being developed and tested in clinic as anticancer agents. However, these compounds often show limited activity as a single agent. How to enhance the efficacy of Akt inhibitors remains a challenge in cancer treatment. It is known that cytotoxicity of Akt inhibitors results from induction of apoptotic cell death via suppressing survival-associated signaling pathways. Autophagy, an evolutionarily conserved lysosomal degradation process in which a cell degrades long-lived proteins and damaged organelles, has been reported to be activated by, and involved in, the modulation of the cytocidal activity caused by inhibition of Akt. Yet, whether and how autophagy has an impact upon the antitumor activity induced by Akt inhibitors is unclear. We investigated the regulatory mechanisms underlying the crosstalk between apoptosis and autophagy in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt, which has entered clinical development as an anticancer agent. The results of our study demonstrated that activation of autophagy indeed affects the cytotoxicity of Akt inhibitors. We also identified eEF-2 kinase as a crucial player in mediating the autophagic response induced by Akt inhibition.
eEF-2 kinase, a negative regulator of protein synthesis through terminating peptide elongation, is overexpressed in various types of neoplasms such as malignant glioma and breast cancer. Inhibition of eEF-2 kinase results in a decreased viability of tumor cells. Previous studies from our group and others have demonstrated that eEF-2 kinase participates in the induction of autophagy in response to multiple cellular stresses, underscoring the importance of eEF-2 kinase as a regulator of autophagy. Since Akt is considered an effective therapeutic target for cancer treatment, we wanted to know whether eEF-2 kinase was involved in the activation of autophagy caused by Akt inhibition. We found that silencing of eEF-2 kinase blocks the autophagic response triggered by Akt inhibition either by MK-2206 or by RNA interference in human glioma cells. Our demonstration of the role of eEF-2 kinase in modulating the autophagic response induced by Akt inhibition adds a new meddler in the Akt-mediated cell survival/death pathway. Considering that Akt is a positive regulator of the mTOR-S6 kinase signaling pathway that phosphorylates eEF-2 kinase and control its activity, this makes perfect sense. Indeed, we showed that the TSC2-mTOR-S6 kinase pathway mediates the effects of eEF-2K on autophagy triggered by Akt inhibition. Although eEF-2 kinase has been demonstrated to play an essential role in regulating autophagy in cells experiencing various cellular insults such as Akt inhibition, the precise molecular mechanisms by which eEF-2 kinase controls autophagy remain unknown. Currently, we are investigating whether or not eEF-2K activates autophagy by regulating specific proteins of the Atg family.
Consistent with previous reports, we demonstrated that inhibition of Akt could induce both autophagy and apoptosis; furthermore, we elucidated how apoptosis and autophagy affect cellular sensitivity to Akt inhibitors. Although activation of autophagy (also termed type II programmed cell death) can promote autophagic cell death, this form of intracellular degradation also supports cell survival. A better understanding of what exact roles autophagy plays in the response of tumor cells to therapeutic stress, and how to exploit autophagy as an effective strategy for cancer therapy, would be therapeutically significant and beneficial. We demonstrated that blunting autophagy by the small molecule inhibitor 3-MA or by RNA interference enhances the apoptotic cell death induced by MK-2206 and the cytocidal activity of this compound, indicating that autophagy activated by Akt inhibition is cytoprotective and could counter the cytotoxicity of the Akt inhibitor. We also found that inhibition of eEF-2 kinase not only decreases autophagic activity, but also significantly enhances the effectiveness of MK-2206 against human glioma cells.
One type of autophagy, termed mitophagy, contributes to survival by the selective degradation of damaged mitochondria. We found that mitochondria were dysfunctional in the tumor cells treated with MK-2206, as evidenced by the generation of ROS, a critical regulator of both apoptosis and autophagy, and by the permeability of mitochondria that play a central role in mediating the apoptotic pathway and are the source and target of ROS. The ROS scavenger NAC blocks the autophagy and apoptosis activated by this agent, suggesting that ROS serves as a key regulator in these cellular processes. We further showed that MK-2206 activates mitochondrial autophagy, as there was a decrease in COX IV, a marker of mitochondria and an increase in LC3-II in the mitochondrial fractions in cells treated with MK-2206. We demonstrated that inhibition of Akt causes mitochondrial defects, and mitophagy is activated to protect cells from apoptosis by removing damaged mitochondria and thus eliminating the source of apoptogenic signals or reducing ROS levels. It is noteworthy that when autophagy is blunted by inhibition of eEF-2 kinase, degradation of the damaged mitochondria is retarded, and this might further increase the accumulation of ROS, subsequently resulting in more apoptotic cell death.
In summary, inhibition of autophagy by silencing eEF-2 kinase augments apoptotic cell death and enhances the sensitivity of tumor cells to Akt inhibition, even under hypoxic conditions. These findings not only identify a new regulator of Akt-activated autophagy, but also provide a rationale for carefully investigating the clinical use of autophagy inhibitors, including eEF-2 kinase inhibitors (such as NH125) and hydroxychloroquine, in combination with Akt inhibitors. Suppression of autophagy may represent a novel strategy to reinforce the apoptotic effect of Akt inhibitors to achieve a more complete tumor cell killing.