Signaling Pathway Differences Between EGFR TKI Sensitive And Resistant NSCLC Cells And Preliminary Study Of MTOR Targeted Treatment

Lung cancer is the most common cause of malignancy-related death worldwide, non-small cell lung cancer (NSCLC) accounts for80-85%of cases of lung cancer. Median survival of advanced NSCLC is about8to10months with the traditional chemotherapy. EGFR signalling pathway plays a central role in the development and progression of lung cancer. EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are effective clinical therapies for patients with advanced NSCLC who have EGFR-activating mutations (such as deletion in exon19and L858R point mutation in exon21). However, despite these dramatic benefits from EGFR TKIs,all of these patients inevitably developed resistance to gefitinib and erlotinib, usually6-12months after the TKI treatment.Several mechanisms, including T790M secondary mutation in EGFR; MET amplification and overexpression of hepatocyte growth factor (HGF), were reported to induce acquired resistance to reversible EGFR-TKI for NSCLC with EGFR-activating mutations. How to overcome TKI resistance was still a big problem to clinical practice. Generally, the strategies for overcome resistance could be from the following two aspects:one is to consider from resistance mechanism itself, for example, the second generation of the irreversible TKI such as BIBW for T790M mutations patients; c-Met inhibitors combined with EGFR TKIs for c-Met amplification; the other strategy is to find another new molecules or mechanisms to overcome the resistance such as mTOR.mTOR is a conserved serine/threonine kinase and exists in two complexes, mTORC1and mTORC2. It integrates signals from growth factors, nutrient supply and energy status to activate cell growth and is upregulated in various cancers. Therefore, studies of targeting mTOR for cancer therapy have become intense focuses in recent years. However, the clinical response to rapamycin and its analogues has been feeble. Many studies have well demonstrated the mechanisms of its poor response in vitro and in vivo. In fact, rapamycin and its analogue only target for mTORC1not for mTORC2. Meanwhile, more recent studies have indicated that mTORC2regulated cell survival and cytoskeletal organization. Therefore, we hypothesised whether mTORC2signals in EGFR TKI-resistant NSCLC cells were hyperactive and whether targeting mTOR not mTORC1could gain better anti-tumor effect after TKI-acquired resistance.In the present study, we first analysed signalling pathways differences between the EGFR TKI sensitive and resistant NSCLC cells in vitro under basic state. Then we assayed the mTORC2and mTORC1Kinase activities within EGFR TKI sensitive and resistant NSCLC cells in vitro. At last we used the second generation ATP-competitive mTOR inhibitor, ku-0063794, to assess the antiproliferative effects and its influence on cell cycle between the four cells. Furthermore, we also analysed the expression and cellular localization of p-STAT3between sensitive and resistant NSCLC cells in vitro and these results were verified in the clinical EGFR TKI sensitive and resistant lung adenocarcinoma tumor specimens.Chapter1Signaling pathway differences between EGFR TKI-sensitive and resistant non-small cell lung cancer cellsMethodsWe selected four types of NSCLC cell lines:PC-9cell (exonl9del, sensitive to TKI), PC9GR cell (gefitinib-acquired resistant cell), H1650cell (exon19del, resistant to TKI), H1975cell (exon21L858R and exon20T790M double mutations, resisrant to TKI) as cell models, signaling pathway differences between PI2K/Akt/mTOR, Erk and STAT3were analysed through western blot, immunoprecipitation and confocal microscope.ResultsWestern blot analysis showed that mTOR, Raptor and Rictor protein had high basic expression levels in all of the four NSCLC cell lines, and Raptor was the core molecule for mTORCl and Rictor for mTORC2, so the expression levels of Raptor and Rictor protein respectively represented expression levels of mTORC1and mTORC2. The further study showed that activation status of EGFR super signaling pathways:PI3K-Akt, Erk and STAT3between EGFR TKI-sensitive and resistant NSCLC cells were different under basic state. The phosphorylation levels of Akt ser473, FOXO1and Erk in EGFR TKI-resistant cells, especially in H1650cell, were higher than that in PC9cell except for p-STAT3.mTORC2and mTORC1kinase activity assay between EGFR TKI sensitive and resistant NSCLC cells in vitro showed that EGFR TKI resistant cells had higher mTORC2kinase activities while sensitive cell had higher mTORC1kinase activity.Cellular localization of p-STAT3in EGFR TKI-sensitive cell was different than that in resistant cells. Our studies showed that p-STAT3expression levels in PC9cell and H1975cell were significantly higher than that in PC9GR cell and H1650cell. p-STAT3was almost distributed in neucleus in PC9cell and H1975cell while in PC9GR cell and H1650cell, p-STAT3were almost expressed in cytoplam.ConclusionWe found that EGFR TKI sensitive and resistant NSCLC cells had different activation status of signaling pathway and different mTORC2and mTORCl kinase activities in vitro under basic state. EGFR TKI resistant cells had higher activation status in the mTORC2associated pathway and Erk pathway and higher mTORC2kinase activities while sensitive cell have higher mTORCl kinase activity than resistant cells. STAT3signaling pathways in PC9cell and H1975cell were more hyperactived. These results indicated that targeting mTOR with specific mTOR inhibitors would be a good strategy for NSCLC treatment after EGFR TKI resistance and activation status differences of STAT3in these cells maybe related with the antiproliferative effect differences of mTOR inhibitor.Chapter2Preliminary study of mTOR targeted treatment between EGFR TKI sensitive and resistant non-small cell lung cancer cellsMethodsCells were exposed to ku-0063794and the effects of inhibition were analyzed with MTT assay. The antiproliferative effects were analysed through western blot. Using FCM (flow cytometry) to detect cell cycle between the EGFR TKI sensitive and resistant NSCLC cells treated with ku-0063794. Western blot analysed the interference effects of targeting mTORC2by Rictor RNAi in H1975cell.Using IHC (Immunohistochemistry) to assay the expression and cellular localization of p-STAT3between human EGFR TKI sensitive and resistant lung adenocarcinoma specimens.ResultsIn our evaluation of the antiproliferative effects of ku-0063794treatment on PC9, PC9GR, H1650and H1975cells, we observed dose-response growth inhibition effects. Comparison of IC50of ku-0063794in EGFR TKI sensitive and resistant NSCLC showed statistically significant differences, F=18.64, P=0.001. Comparison of IC50of ku-0063794in PC9cell and H1975cell showed no significant difference, P=0.218. And comparison of IC50of ku-0063794in PC9with the other two resistant cells PC9GR and H1650cell show statistically significant differences, P value is0.001and0.009respectively. We also detected cell cycle after treated with Ku-0063794at IC50levels between the four cells through flow cytometry, we found that Ku-0063794at IC50levels could effectively lead to G1cell cycle arrest.Antiproliferative effects of Ku-0063794at IC50level were analysed through western blot. Our results showed that Ku-0063794could effectively inhibit phosphorylation status of mTOR, deeply inhibit phosphorylation levels of p70S6K and also partly inhibit phosphorylation levels of Akt ser473and Foxol.The study of targeting mTORC2by siRNA interference of Rictor showed that effectively RNAi of Rictor dramatically decrease the phosphorylation levels of Foxol in H1975cell.Expression and cellular localization of p-STAT3between the EGFR TKI sensitive and resistant lung adenocarcinoma tissues through IHC identified the results of our cell experiments. These results showed that EGFR TKI sensitive lung cancer tissues and EGFR TKI resistant lung cancer tissues harbouring T790M mutation had higher p-STAT3expressions and p-STAT3were nearly distributed in nucleus in these tumor tissues.Both EGFR TKI sensitive and resistant lung adenocarcinoma tissues had high expressions of total and phosphorylated p70S6K and p70S6K maybe a good predictor of response to selective mTOR inhibitors.Conclusionku-0063794as a novel ATP-competitive mTOR inhibitor showed good antiproliferative effects and Gl cell cycle arrest both in EGFR TKI sensitive and resistant NSCLC cells in vitro. The difference of antiproliferative effect maybe associated with the expression and cellular localization of p-STAT3. Expression and cellular localization of p-STAT3in clinical EGFR TKI sensitive and resistant lung adenocarcinoma tumor specimens further confirmed the results of our cell experiments.