Gynecologic Tumor Cells Survive In Response To Metabolic Stress Mediated By Site-specific AKT Activation

BackgroundGynecologic malignancies consist of uterus, ovaries, cervix, fallopian tubes, vagina, and vulva cancer. It has been reported there’s already estimated incidence of80,720cases per year and estimated mortality rate of28,120women per year. Cell signaling pathway has emerged as one of the most fascinating new fields of research and drug development. One such pathway that has been proven to be a pivotal player in numerous solid tumors is that of the phosphatidylinositide3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) cell signaling pathway. Several drugs that target the PI3K/AKT/mTOR pathway are being evaluated in gynecologic malignancies. This pathway has been noted to be a key regulator of protein synthesis, apoptosis as well as glucose metabolism.Of importance are two metabolic processes for cell energy:respiration which functions through mitochondria with saturation of oxygen and anaerobic fermentation which produce lactate under hypoxia or anoxia. Compared with differentiated tissues, cancer prefers to adopt unique metabolism process called aerobic glycolysis, which utilizes glucose to generate lactate even in presence of sufficient oxygen. Aberrant metabolism is considered to be one of the hallmarks for cancer. Not only for energy, but for biosynthesis, become cancer cells addicted to glucose due to high flux of glycolysis. As a result, when glucose resource is scarce or the ability to consume up glucose is disturbed, carcinomas are sensitized to death in contrast to normal cells that could be fueled by breakdown of amino acid or lipid. Glucose deprivation withdrawing glucose from culture medium promotes GLUT expression, downregluates glycolysis and induces apoptosis. Moreover, deficiency of glucose could occur when solid tumor grows larger or is treated with anti-angiogenesis agents. Glucose deprivation triggers series of events including oxidative stress, cytotoxicity and inflammation etc mediating cell death through necrosis or through mitochondrial or caspase-8-dependent apoptosis. Cancer cells intends to evade mortality by activating survival signaling pathway network:AMPK (adenosine monophosphate-activated protein kinase), AKT/mTOR, Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular signal-regulated kinase), tyrosine kinase signaling.The serine/threonine kinase AKT has been implicated in the upregulation of glucose transport and aerobic glycolysis in cancer cells. Activated AKT renders cancer cells dependence on glucose. AKT phosphorylates and inhibits TSC1/TSC2leading to activation of mTORCl pathway which promotes protein translation and cell growth. In other aspect, AKT phosphorylation was increased showing the AKT pathway is hyper-activated under chronic glucose deprivation conditions. AKT could stimulate survival pathway to prevent tumor cells from apoptosis.AKT plays a pivotal role in regulating cell growth, proliferation, survival, migration and metabolism. Cancer cells gain survival advantages and manifest immortal by perturbation on death. Compelling evidences have demonstrated that aberrant apoptosis could result in diseases like cancer. AKT pathway has been confirmed to be the principal mediator for cell survival. Activated AKT upregulates pro-survival and inhibit apoptosis to promote cell survival. The fact that cell metabolism and survival converge on AKT proposes a hypothesis that AKT could function as a node to couple these two important processes.PI3K-AKT pathway has been implicated in several malignancies in which aberrant activation or loss of negative control leads to carcinogenesis and progression: PIK3CA gene mutation, loss or mutation of PTEN, and mTOR complexes deregulation. With extensive pharmacological studies, a plethora of proteins including PI3K, AKT, mTOR, and PDK-1in this pathway could be valuable targets for cancer treatment. Herein, the target-based small-molecule inhibitors targeting this pathway undergo investigation and some have progressed into clinical trials.While anti-metabolic agents enhance cancer cells response to apoptosis, subsequently activated survival pathways potentiate opposing effects to protect cells from death. Under energetic stress elicited by2-DG, one of valid and refined glycolytic inhibitor, inhibition of AKT has been proved to sensitize cells to apoptosis. A phase Ⅰ/Ⅱ study has revealed that AKT activation is closely related with resistance to imatinib reinduction therapy in AML patients, coincident with a report that PI3K-AKT pathway plays a crucial role in imatinib-resistant gastrointestinal tumor.As a newly identified hallmark of cancer, deregulated metabolism is emerging as a major target in cancer therapy. We explored the pro-apoptotic AKT activation under long-term glucose deprivation.. Our data suggest that a combination of AKT or GRP78inhibition with metabolic stress may synergistically kill cancer cells that are resistant to either treatment alone. Small molecule inhibitors of both PDK1and GRP78are in clinical development providing a ready approach for translation to the clinic. Our data reveal a critical AKT-mediated survival mechanism under prolonged metabolic stress, which is of importance to development and implementation of drugs targeting cell metabolism and AKT signaling. Objective:To detect the AKT and its substrate phosphorylation and activation under prolonged glucose deprivation. To Analyze key kinases in AMPK and RTK/PI3K/AKT pathways which could result in selective AKT activation.Methods:Western blotting was used to examined AKT and its substrate phosphorylation under prolonged glucose deprivation in cervical cancer, ovarian cancers, breast cancer etc. And key kinases in AMPK and PI3K-AKT pathway was analyzed by Western blotting, which could account for specific AKT activation.Results:1. Selective AKT T308phosphorylation was recapitulated in tested cell lines under prolonged glucose deprivation, while time course of AKT Ser473was not parallel with that of AKT T308. This observation was different from AKT activation induced by growth factor:phosphorylation of T308and Ser473were coordinated.2. AKT PH domain, not kinase domain, was necessary for selective AKT T308activation.3. PDK1and PI3K, not EGFR and IGFR were indispensable for specific AKT T308phosphorylation under long-term glucose deprivation.4. AKT, PDK1and GRP78complex formation probably result in selective AKT T308phosphorylation.Conclusion:This study demonstrated that transient and prolonged glucose deprivation induce AKT phosphorylation and activation via distinct mechanisms. Transient glucose deprivation modestly induces AKT phosphorylation at both Thr308and Ser473likely via a feedback loop from p70S6K. In contrast, prolonged glucose deprivation induces selective AKT phosphorylation at Thr308through the formation of a complex including GRP78and PDK1. Prolonged glucose deprivation-induced AKT phosphorylation at Thr308is dependent on both PDK1and PI3K. Intriguingly, EGFR, although activated by prolonged glucose deprivation, and IGF1R kinase activity are dispensable for glucose-deprivation induced AKT Thr308phosphorylation. Prolonged glucose deprivation increases GRP78levels and its association with AKT, which may facilitate AKT association with PDK1contributing to the selective AKT Thr308phosphorylation. Glucose deprivation stabilizes the AKT-PDK1complex, likely due to GRP78and potentially other scaffold proteins that bind to the complex, contributing to the selective AKT phosphorylation at Thr308but not Ser473. An intact PH domain of AKT is required for selective AKT phosphorylation at Thr308induced by prolonged glucose deprivation. PART II:Selective AKT phosphorylation promotes tumor cells survival under metabolic stressObjective:To detect intracellular ATP change under prolonged glucose deprivation, study survival mechanisms of tumor cells mediated by selective AKT activation, analyze the effect of combinational treatment of glucose deprivation and AKT inhibitor/GRP78inhibitor on cell viability.Methods:Colorimetric assay was used to detect intracellular ATP level under prolonged glucose deprivation. Gene knockout cells were used to test autophagy’s role in AKT activation under glucose deprivation. RPPA was employed to analyze signaling pathways under glucose deprivation. Cell apoptosis was analyzed by western blotting.Results:1. Intracellular ATP level was declined dramatically under prolonged glucose deprivation. Addition of pyruvate analog, methyl pyruvate, could restore ATP level and inhibit selective AKT phosphorylation.2. Autophagy didn’t play a role in specific AKT T308phosphorylation induced by prolonged AKT activation.3. Specific AKT T308phosphorylation under long-term glucose deprivation activated a group of substrates and pro-apoptotic signaling, while down-regulated signaling that was not closely related with cell survival.4. Apoptosis increased when glucose deprivation prolonged. However, it decreased after certain time point of glucose deprivation.5. Combinational treatment of glucose deprivation and GRP78could decrease colony formation.Conclusion:Selective AKT phosphorylation at Thr308targets AKT to a specific group of substrates contributing to cell survival. GRP78are required for the phosphorylation of AKT and for cell survival under prolonged glucose deprivation. Combination of AKT or GRP78inhibition with metabolic stress may synergistically kill cancer cells that are resistant to either treatment alone. Both AKT and MAPK family members are highly activated during prolonged glucose deprivation, indicating that the signaling network in cells under severe metabolic stress can be rewired and rebalanced to induce effective activation of the cell survival machinery.