Effect And Mechanism Of LncRNA H19 Downregulation Confers EGFR-TKI Resistance In EGFR-mutant Lung Cancers

Background and Aims: First-generation EGFR tyrosine kinase inhibitors(TKIs)such as erlotinib have significant activity in NSCLC patients with activating EGFR mutations.However,EGFR-TKI resistance inevitably occurs after approximately 12 months of treatment.Acquired mechanisms of resistance,other than secondary mutations in EGFR(T790M)which account for 50-60%,are less well understood.Here,we identified LncRNA H19 as a significantly downregulated LncRNA in in vitro models and clinical specimens with acquired EGFR-TKI resistance,and we aimed to investigate its role in EGFR-TKI resistance in NSCLC.Methods: The first part: Expression of LncRNA H19 in clinical samples and prognostic analysis.1.RNA sequencing and quantitative real-time RT-PCR was used to profile LncRNA expression in human NSCLC and detect relative expression level of H19.2.H19 expression levels in lung cancer tissues assessed by RT-PCR in patients with EGFR-TKI-sensitive NSCLC(before treatment)and in patients who developed acquired resistance to EGFR-TKI.Progression-free survival(PFS)and the objective response rate(ORR)in patients with high and low H19 expression levels before EGFR-TKI treatment was analyzed to investigate the correlation of H19 levels with survival in NSCLC patients harbouring EGFR activating mutations.The second part: Effect of low H19 levels on erlotinib resistance in lung cancer cell lines.1.Drug-resistant cell lines were established as an acquired erlotinib resistant model in vitro.The IC50 value and the expression of H19 were detected by MTT test and q RT-PCR.2.Stable H19 knockdown and overexpression cell lines were established to further validate the role of H19 in the process of erlotinib resistance.3.MTT method was used to compare cell viability in H19-overexpressing, H19-knockdown and control cells.4.colony formation assays were used to compare colony formation abilities between H19-overexpressing,H19-knockdown and control cells in the presence of erlotinib The third part: The regulation mechanism of low H19 levels on EGFR-related signal pathway and drug resistance-related EMT.1.Western blot analysis was used to detect the expression of EGFR-related signaling pathway proteins and EMT-related proteins.2.AKT inhibitors LY294002 and erlotinib were used to treat the cells,and MTT and clone formation assay were used to detect the survival rate and clone formation ability of cells under the action of drugs.The fourth part: The regulation mechanism of H19 regulating AKT activation and affecting drug resistance through interaction with PKM2.1.Chromatin isolation by RNA purification(Ch IRP)and LC-MS/MS was used to explore the H19 interacting proteins in non-small-cell lung cancer cells.2.RNA immunoprecipitation assay was used to validate the interaction between PKM2 and H19.3.Parallel reaction monitoring(PRM)assays were used to separate and detect PKM2 peptides in HCC827 cells 4.LncRNA fluorescence in situ hybridization(FISH)and immunofluorescence staining were used to detect the intracellular localization of H19 and PKM2,respectively.5.CHX assays were used to detect PKM2 degradation rate in H19-overexpressing,H19-knockdown and control cells.6.In vitro ubiquitination assay was used to examined whether H19 downregulated PKM2 through ubiquitin-mediated degradation.The fifth part: In vivo verification of the effect of low H19 levels on erlotinib resistance in CDX model.1.Cell line-derived xenograft(CDX)models was used to determine whether H19 affected the sensitivity of EGFR mutated NSCLC cells to erlotinib in vivo.2.Immunohistochemistry staining was used to detect the levels of Ki-67,PKM2 and cleaved-caspase3.The sixth part: Statistical analysis.SPSS version 22.0 software was used for statistical analyses,and Prism version 7.0(Graph Pad)was used to generate graphics.For the comparisons,Student’s t-test was performed for two groups,and analysis of variance(ANOVA)was performed for multiple groups.Results: The first part: Expression of LncRNA H19 in clinical samples and prognostic analysis.1.Downregulation of H19 was detected in EGFR-TKI resistant samples by LncRNA-seq and q RT-PCR.2.A Kaplan–Meier survival analysis and log-rank test showed that low H19 levels were significantly related to a shorter progression-free survival(PFS).3.The objective response rate(ORR)in the low H19 expression group was significantly lower than in the high expression group.4.Multivariate Cox regression analysis indicated that H19 expression was an independent prognostic factor for PFS.The second part: Effect of low H19 levels on erlotinib resistance in lung cancer cell lines.1.H19 was down-regulated in erlotinib-resistant cells,compared with the corresponding parental cells.2.Compared with the control conditions,H19 knockdown decreased,while H19 overexpression enhanced the sensitivity of both cell lines to erlotinib or gefitinib.The third part: The regulation mechanism of low H19 levels on EGFR-related signal pathway and drug resistance-related EMT.1.H19 silencing weakened,and H19 overexpression enhanced the inhibitory effect of erlotinib on p AKT.PARP and caspase3 cleavage increased in H19-overexpressing cells with increasing concentrations of erlotinib,and decreased in H19-knockdown cells.2.LY294002 restored the sensitivity of H19-knockdown and erlotinib-resistant cells to erlotinib.3.The expression of H19 reversed the EMT phenotype in erlotinib-resistant cells,as evidenced by elevated E-cadherin and reduced vimentin.The fourth part: The regulation mechanism of H19 regulating AKT activation and affecting drug resistance through interaction with PKM2.1.Pyruvate kinase M2(PKM2)was one of the H19-interacting proteins identified by mass spectrometry and validated by parallel reaction monitoring.2.The interaction between PKM2 and H19 was further validated by RNA immunoprecipitation.3.RNA FISH-IF analysis showed that H19 colocalized with PKM2 in the cytoplasm.4.H19 knockdown increased,whereas H19 overexpression decreased PKM2 protein levels in PC9,HCC827 and H3255 cells.PKM2 protein expression was higher in erlotinib-resistant cells than in the parental cells,5.Cycloheximide chase assay and in vitro ubiquitination assay showed that PKM2 was downregulated by H19 induced ubiquitin-mediated degradation.6.inhibition of PKM2 suppressed the increase of p AKT levels caused by downregulation of H19 and restored the sensitivity of cells to erlotinib.7.A negative correlation was observed between H19 and PKM2 expression in 10 pairs of tumor tissues and corresponding non-tumor tissues.The fifth part: In vivo verification of the effect of low H19 levels on erlotinib resistance in CDX model.1.Cell line-derived xenograft(CDX)models and Western blot showed that H19 downregulation confers resistance to erlotinib by decreasing the effect of erlotinib in the EGFR-AKT pathway in vivo.2.IHC analysis showed lower Ki67(proliferation marker)and PKM2 and higher cleaved Cleaved-Caspase-3(apoptosis marker)levels in HCC827 H19 tumors and HCC827 ER H19 tumors.Conclusion: We have found that inhibition of H19 expression is capable of causing EGFR-TKI resistance in lung cancer cells with EGFR activating mutations.H19 regulates EGFR-TKI resistance by interacting with PKM2,which mediates AKT activation and affects drug resistance-associated EMT.Furthermore,H19 low expression has been validated in EGFR-mutant lung adenocarcinoma specimens from patients who developed resistance to first-generation EGFR-TKI.Low levels of H19 were correlated with poor PFS in patients with EGFR-mutant lung adenocarcinomas treated with EGFR-TKI.Our findings thus unveil a novel mechanism of H19 in acquired EGFR-TKI resistance and provides a potential way to overcome EGFR-TKI resistance by the combination of EGFR-TKI and AKT inhibitors in some EGFR-mutant NSCLC patients.