SIRT6 Regulates Glycolysis To Mediate Erotinib Resistance In Non-small Cell Lung Cancer

Background:Lung cancer is the most common malignant tumor with the highest morbidity and mortality in China.Non-small cell lung cancer(NSCLC)accounts for about 80% of lung cancer.Epidermal growth factor receptor(EGFR)abnormal mutations are related to the occurrence and progression of NSCLC,so it is considered an important target for the treatment of cancer.A first-generation tyrosine kinase inhibitor(tyrosine kinase inhibitor,TKI)erlotinib can significantly improve the survival of patients with EGFR-sensitive mutations(19 deletions or L858R)in NSCLC.However,drug resistance has inevitably developed in almost all patients after erlotinib treatment.TKI resistance mechanisms have been discovered including EGFR secondary mutation T790 M,alternative activation,and phenotypic transformation.However,the mechanism of drug resistance in a considerable number of drug-resistant patients has not yet been elucidated.Therefore,we further explore suitable and effective strategies to reverse TKI resistance and improve the survival o f this group of patients.More and more evidence shows that cell metabolism plays an important role in the occurrence and development of tumors.Among them,glycolysis not only provides energy for tumor cell proliferation,but also the intermediate products produced by it are also important raw materials required for tumor cell synthesis.Inhibition of glycolysis can enhance the sensitivity of NSCLC to EGFR-TKI and prevent the development of drug resistance.SIRT6 is a protein deacetylase belonging to the Sirtuins family,which is closely related to tumorigenesis and development;it affects a variety of biological processes such as cell metabolism,oxidative stress,transcription regulation,and DNA repair.It can directly or indirectly interact with genes and enzymes related to glucose metabolism,or regulate upstream factors or pathways of glycolysis to regulate the level of glucose metabolism in cells.Earlier,our research found that the expression of SIRT6 in tumor tissues of NSCLC patients was higher than that in normal tissues,and the overall survival(OS)of the lower expression group was shorter in the lower expression group.It is suggested that the high expression of SIRT6 may be a poor prognostic factor in patients with NSCLC.Our team has successfully induced stable erlotinib-resistant lung adenocarcinoma cell lines(PC9/ER,HCC827/ER),and found that SIRT6 is highly expressed in drug-resistant strains,and the cells of drug-resistant strains have more High glycolytic levels,so we speculate that SIRT6 plays an important role in erlotinib resistance in NSCLC,and its specific mechanism of action remains to be further explored.Methods:1.Expression of SIRT6 in NSCLC tissues and its correlation with prognosisBioinformatics analysis was performed using UALCAN to study the expression of SIRT6 in lung cancer tissues and normal lung tissues;Kaplan-Meier Plotter web was used to obtain survival data,and then analyzed the relationship between the prognosis of lung cancer patients and the expression of SIRT6.2.Expression of SIRT6 in NSCLC cell linesCCK-8 method was used to detect the sensitivity of NSCLC cells to erlotinib.After determining the sensitivity differences of susceptible cells and drug-resistant cells to erlotinib,the expression level of SIRT6 in sensitive and drug-resistant cells was analyzed by western blot.3.SIRT6 changes the sensitivity of NSCLC cells to erlotinibLentiviral transfection technology was used to change the expression of SIRT6 in the cell lines,CCK-8 method was used to detect the sensitivity of stably transfected cells to erlotinib.And Annexin V / 7-AAD was used to detect the apoptosis of stably transfected cells under the intervention of erlotinib.4.SIRT6 regulates glycolysis and promotes cell proliferation in NSCLC cells through the HIF-1a/HK2 signal axisThe lentivirus was used to construct SIRT6 over-expressing cell lines PC9/SIRT6,HCC827/SIRT6,and SIRT6 knockdown cell lines PC9/ER/shSIRT6,HCC827/ER/shSIRT6.Then,the ability of each cell line to produce acid an d ATP was measured.Ed U to detect the effect of SIRT6 on cell proliferation,western blot to analyse HIF-1?,HK2 protein expression changes.Then change the expression of SIRT6 downstream molecules to detect whether the cell glycolysis has changed.5.In vivo experiments verify that SIRT6 induces erlotinib resistance in NSCLC cellsSIRT6 stably overexpressed and knocked down NSCLC cell lines were administered subcutaneously in nude mice to establish a nude mouse tumor formation model,and the volume change of the transplanted tumor was used to reflect the therapeutic effect of erlotinib.Immunohistochemical staining was used to analyze the expression of SIRT6,HIF-1?,HK2,and ki-67 in each tumor tissue.6.Statistical analysisThe experimental data are expressed as mean ± standard deviation.Graphpad Prism 5.0 is used for statistical analysis.The comparison of variable data between the two groups uses paired t test.For pairwise comparison between multiple groups,single factor analysis of variance is used.P <0.05 indicates that the difference is statistically significant.Results:1.SIRT6 overexpression is associated with poor prognosis in NSCLC patientsUsing UALCAN to download genetic data of lung cancer patients in the TCGA database,analysis showed that SIRT6 is highly expressed in lung adenocarcinoma and lung squamous cell carcinoma,suggesting that SIRT6 is a potential marker for NSCLC.Kaplan-Meier Plotter was used to obtain survival data,and the results showed that high expression of SIRT6 was significantly associated with poor prognosis in patients with lung cancer and lung adenocarcinoma;indicating that high expression of SIRT6 may be a poor prognostic factor for lung cancer,especially patients with lung adenocarcinoma.2.Compared with NSCLC Erlotinib-sensitive cell lines,SIRT6 is highly expressed in Erlotinib-resistant cell linesStudies have confirmed that PC9 and HCC827 cells are sensitive to e rlotinib,while PC9/ER and HCC827/ER cells show strong resistance to erlotinib.We then detected by Western blot whether SIRT6 was highly expressed in PC9/ER and HCC827/ER cells with or without erlotinib intervention.3.SIRT6 promotes erlotinib resistance in NSCLC cellsCompared with the corresponding parental cells,the IC50 value of erlotinib in SIRT6 overexpressing cells(PC9/SIRT6,HCC827/SIRT6)was significantly increased,in contrast,the IC50 value of SIRT6 knockdown cells PC9/ER/shSIRT6,HCC827/ER/sh SIRT6 was decreased significantly.And the apoptosis of SIRT6 knockdown cells increased with the intervention of erlotinib.4.SIRT6 regulates glycolysis and promotes cell proliferation of NSCLC cells through HIF-1?/HK2 signal axisCompared with the corresponding control cells,SIRT6 over-expressing cells PC9/SIRT6 and HCC827/SIRT6 produced more lactic acid and ATP,even when erlotinib was used.In contrast,whether or not erlotinib was used,the levels of lactate and ATP produced by PC9/ER/sh SIRT6 and HCC827/ER/sh SIRT6 cells were significantly reduced.EdU test results also showed that the proliferation ability of PC9/SIRT6 and HCC827/SIRT6 cells was greatly improved,while the proliferation ability of PC9/ER/shSIRT6 and HCC827/ER/sh SIRT6 cells was reduced.We further examined some genes related to glycolysis,and found that the expression of HIF-1? and HK2 can increase with the increase of SIRT6.Therefore,we used a HIF-1? specific inhibitor(PX-478 2HCL)to inhibit the expression of HIF-1? in SIRT6 overexpressing cells(PC9/SIRT6,HCC827/SIRT6).When HIF-1? was successfully inhibited,the expression of HK2 also decreased,and the simultaneous detection of lactic acid and ATP levels also decreased,suggesting that HIF-1? inhibitors can successfully reverse the glycolysis promoted by SIRT6.5.SIRT6 can induce erlotinib resistance in vivoCompared with the nude mice inoculated with the PC9/vector cell group,the nude mice inoculated with the PC9/SIRT6 cell group had larger tumor volumes,and their growth could not be inhibited by erlotinib.The results of immunohistochemistry also suggested that SIRT6 was overexpressed,the expression levels of HIF-1? and HK2 in tumor tissues were also higher,and ki-67,which reflected proliferation,was also increased.The nude mice transplanted with SIRT6 knockdown(PC9/ER/shSIRT6 cell group)had a relatively small volume(compared with PC9/ER/shCtrl cell group),and the expression levels of HIF-1?,HK2,and ki-67 also reduced.It has been shown that SIRT6 can induce erlotinib resistance in vivo.Conclusions:1.SIRT6 is highly expressed in NSCLC patients and is associated with poor prognosis.2.SIRT6 overexpression can enhance the glycolytic ability of NSCLC cells,while knocking down SIRT6 decreases the glycolytic ability of NSCLC cells.3.SIRT6 regulates glycolysis through HIF-1?/HK2 signal axis and induces NSCLC erlotinib resistance.4.SIRT6 overexpression can reduce the inhibitory effect of erlotinib on NSCLC cells and xenografts,while knocking down SIRT6 can restore the inhibitory effect of erloti nib on NSCLC cells and xenografts.