The Role Of Autophagy In Modulation Of Resistance To Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors In Non-small Cell Lung Cancer

Part 1:Involvement of autophagy in EGFR-TKI resistance in NSCLCObjective:Induction of a TKI-resistant cell line and exploration of the correlation between autophagy and TKI-resistanceMethods:First, the EGFR mutation types of 4 NSCLC cell lines HCC827, A549, H460 and H1975 cells were examined, and the corresponding sensitivity to erlotinib was measured with CCK8 tests. HCC827, a TKI-sensitive cell line, was cultured in escalating concentrations of erlotinib for nearly 6 months to induce a TKI-resistant cell line, which was named as HCC827-R and checked for sensitivity to erlotinib with CCK8 tests. Second, the basal autophagy level of HCC827, A549, H460, H1975 and HCC827-R cells were measured with Western blotting. GFP-LC3 and CQ (chloroquine) were further used to check the basal autophagy flux of HCC827 and HCC827-R cells. Last, autophagy inhibitors CQ or 3-MA, or siRNAs targeting the core autophagy initiator Beclin 1, were used to block autophagy to see the dependence of cell proliferation on basal autophagy level.Results:The results of EGFR mutation measurement were:HCC827 (E746-A750 deletion), A549 (wild type), H460 (wild type) and H1975 (L858R and T790M). HCC827 cells were sensitive to erlotinib,while A549,H460,H1975 and HCC827-R cells were resistant to erlotinib, of which the IC50 value of HCC827-R cells against erlotinib were more than 100?M. Among these 5 cell lines, the resistance to erlotinib was positively related to LC3-? expression, indicating the correlation between TKI-resistance and basal autophagy level. Inhibition of basal autophagy with 3-MA or siRNAs targeting Beclin 1 showed no significant influence on the proliferation of HCC827 or HCC827-R cells.Conclusions:When compared with TKI-sensitive cells, TKI-resistant cells showed higher basal autophagy level, indicating the positive correlation between TKI-resistance and basal autophagy level. The basal autophagy was not necessary for proliferation of HCC827 or HCC827-R cells.Part 2:Modulation of EGFR-TKI resistance in NSCLC with autophagyObjective:Measurement of the influence of autophagy inhibition on EGFR-TKI resistance and the underlying mechanisms.Methods:First, to measure the influence of erlotinib on autophagy, HCC827, A549, H1975 and HCC827-R cells were treated with erlotinib with distinct concentrations and time. Autophagy inhibitors, CQ or 3-MA, were used to inhibit the erlotinib-induced autophagy in these 4 cell lines, and CCK8 tests were performed for the measurement of cell proliferation under co-treatment of autophagy inhibition and erlotinib. Second, H1975 and HCC827-R cells were further exposed to co-administration of CQ and erlotinib for measurement of apoptosis with Annexin V and PI double staining. Western blotting tests were used for detection of PARP1 and caspase 3 activation. Last, JC-1 staining and Western blotting were used to check whether mitochondria was involved in the apoptosis induced by CQ and erlotinib co-treatment.Results:Erlotinib showed a time-dependent induction of autophagy in HCC827, A549, H1975 and HCC827-R cells. Autophagy inhibition with CQ or 3-MA inhibited the erlotinib-induced autophagy and overcome resistance to erlotinib. Of note,3-MA showed no significant influence on the cell growth of HCC827, A549, H1975 and HCC827-R cells, but CQ did inhibit the cell proliferation of these 4 cell lines, indicating the distinct mechanisms of autophagy inhibition may have different influence on cell growth of NSCLC cells. Combination of CQ and erlotinib induced apoptosis through PARP1 and caspase 3 activation, which was reversed by a caspase inhibitor, z-VAD fink. Further, Co-treatment of CQ and erlotinib induced mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release from mitochondria into cytosol.Conclusions:Autophagy inhibition with 3-MA or CQ inhibited the erlotinib-induced autophagy and reversed resistance to erlotinib in NSCLC cells. Combination of CQ and erlotinib induced a caspase-dependent apoptosis, which was involved in MOMP and cytochrome c release into cytosol.Part 3:Combination of CQ and erlotinib induces apoptosis and endoplasmic reticulum stressObjective:evaluation of the involvement of mitochondria and endoplasmic reticulum in the synergistical effect of CQ and erlotinib.Methods:To check whether mitophagy was involve in the synergistical effect of CQ and erlotinib, GFP-LC3 and mitotracker were used to locate autophagosomes and mitochondria respectively. Under treatment of CQ and erlotinib, Western blotting was used to detect the activation of EIF2?-CHOP pathway, which is a marker of endoplasmic reticulum stress. SiRNAs targeting CHOP were further added to evaluate the role of CHOP in CQ and erlotinib combination-induced apoptosis and cell growth inhibition. In addition, JC-1 staining and Western blotting were performed to check the influence of CHOP knockdown in CQ and erlotinib combination-induced MOMP and cytochrome c release. Last, the synergistical effect of CQ and erlotinib was measured in vivo.Results:Under co-treatment of CQ and erlotinib, GFP-LC3 failed to co-locate with mitotracker, suggesting co-treatment of CQ and erlotinib did not induce mitophagy. Endoplasmic reticulum stress was involved in the co-treatment of CQ and erlotinib as demonstrated by the activation of EIF2?-CHOP pathway. Knockdown of CHOP with siRNAs reversed not only CQ and erlotinib combination-induced apoptosis and cell growth inhibition, but also the MOMP and cytochrome c release. Last, co-administration of CQ and erlotinib significantly inhibited the growth of H1975 cells in vivo.Conclusions:Combination of CQ and erlotinib induced apoptosis through endoplasmic reticulum stress induced MOMP and cytochrome c release, in which CHOP functioned as a apoptosis signal transducer between endoplasmic reticulum and mitochondria.