Synergistic And Drug Resistance Of PI3K / AKt / MTOR Signaling Pathway In Gastric

Part I. The combination of Everolimus and MK2206exerts synergistic cytotoxic effects against PTEN deficient Gastric cancer cellsBackground:The prognosis of advanced gastric cancer (AGC) is poor and novel drugs are needed to improve the outcome of the survival. Everolimus (RAD001) is a kind of compound that targeting the mammalian target of rapamycin (mTOR). Preclinical studies showed a compromised anti-tumor effect of this drug because of a negative feed-up loop that triggers Akt, a serine threonine protein kinase in the up-stream of mTOR. As a consequence, we hypothesized the combination of Everolimus and MK2206, an Akt inhibitor, should have a better anti-tumor effect than the single agent.Methods:An Everolimus sensitive human gastric cancer cell line was selected for the following experiment. The in vitro anti-proliferation, induction of cell senescence, apoptosis, autophagy effects were detected in the scenario of single agent Everolimus, MK2206or both of the agents. The effects of Everolimus or MK2206as single agent or their combination on cell signal transduction pathway, cell cycle and autophagy associated proteins were also studied.Results:In the current study, we showed that the combination of Everolimus and MK-2206exerted synergistic cytotoxic effects againstPTEN mutant gastric cancer cells. Everolimus and MK-2206synergistically induced G1/S cell cycle arrest, growth inhibition and cell death in gastric cancer HGC-27cells, without inducing significant cell apoptosis. Everolimus and MK-2206synergistically induced autophagy marker light chain3B (LC3B) and beclin-1expression, two important autophagy indicators. Meanwhile, autophagy inhibitors3-methyladenine (3-MA) and chloroquine inhibited the cytotoxic effects by Everolimus and MK-2206co-administration, suggesting that autophagic, but not apoptotic cell death was important for the cytotoxic effects by the co-administration. For mechanism study, we observed that the combination of Everolimus and MK-2206exerted enhanced effects on Akt/mTOR inhibition, cyclin D1down-regulation and remarkblely, ERK/MAPK activation. Intriguingly, MEK/ERK inhibitors PD98059and U0126suppressed Everolimus plus MK-2206-induced beclin-1expression, autophagy induction and cytotoxicity in HGC-27cells. RNAi knockdown of beclin-1inhibited autophagy of HGC-27without affecting MAPK expression induced by Everolimus plus MK2206.Conclusion:PTEN mutant gastric cancer cell is sensitive to Everolimus, and the synergistic anti-cancer ability by Everolimus and MK-2206involves ERK-dependent autophagic cell death pathway.Part II. Important signal transduction pathway involved in the process of acquiring resistance to EverolimusBackground:The efficacy of Everolimus in advanced gastric cancer patients is far from ideal. Patients are easily to acquire drug resistance and experience disease progression. Considering that mTOR is located in the central of the PI3K/Akt/mTOR pathway and linked to many other pathways in the cell, and it is difficult to find the mechanisms for resistance to Everolimus in the cell. We will first select the tyrosine kinase receptors anchored in the membrane for research. In this study, we will search for the critical pathway mediating the resistance to Everolimus on HGC-27cell line.Methods:By culturing the HGC-27cell in gradually increased concentrations of Everolimus(RAD001), we got HGC-27-RR (HGC-27-resistant to RAD001) cell which can survive and proliferate in MEM containing10μM Everolimus. High throughput protein array was applied to detect the difference in protein expression level between these two cell lines, which have the same genetic background. Then we selected some of the up-regulated tyrosine kinase receptor pathway for further study to verify if the up-regulation of these pathways is involved in the resistance to Everolimus on HGC-27cell line.Results:The result from high throughput protein array showed the phosphorylation of EGFR, PDGFR, FGFR and VEGFR increased in HGC-27-RR cell when compared to HGC-27cell line. Cell immunof luorescence showed a higher expression of pFGFRl in HGC-27-RE cell, and the FGFR1inhibitor TSU68can inhibit the cell viability of HGC-27-RR cell, suggesting FGFR1pathway activation is important in the process of acquired Everolimus resistance on HGC-27cell. HGC-27cell developed resistance to Everolimus after stimulated by bFGF and the selective FGFR inhibitor AZD4547can restore the sensitivity of HGC-27to Everolimus.Conclusion:The activation of FGFR pathway was involved in the acquired resistance to Everolimus on HGC-27cell line, and the detaile^mechanism of how the FGFR pathway mediated Everolimus resistance on HGC-27cell warrant further study.