Chk1 Activation Regulates Sensitivity Of Lapatinib In HER2-positive Gastric Cancer Cells

Objective: The ATM and Rad3-related(ATR)/checkpoint kinase 1(Chk1)pathway plays a pivotal role in DNA damage sensor and modulating homologous recombination.Recently,emerging evidence demonstrated that Chk1 phosphorylation was associated with chemotherapy and radiotherapy resistance.However,the relationship between Chk1 phosphorylation and the sensitivity of small-molecule tyrosine kinase inhibitors remains unknown.Lapatinib,a dual tyrosine kinase inhibitor targeting both epidermal growth factor receptor(EGFR)and human epidermal growth factor receptor 2(HER2),has been successfully used in the clinical treatment of advanced HER2 positive breast cancer.Several pre-clinical studies have shown the anti-tumor activity of lapatinib in HER2-positive gastric cancer(GC)cells,indicating that lapatinib might also be effective for treating GC.Unfortunately,present phase II and phase III clinical trials showed that lapatinib combined with chemotherapy did not significantly improve survivals of HER2-positive GC patients.The efficacy of lapatinib was limited by resistance mechanism.Therefore,there is an urgent need to investigate the mechanisms regulating lapatinib sensitivity in HER2-positive GC.In this study,we investigated the regulatory mechanism of ATR / Chk1 pathway on lapatinib sensitivity in HER2-positive GC cells and provided a new therapeutic strategy for improving efficacy of targeted therapies in HER2-positive GC.Methods: 1.Cell viability was measured by MTT assay;2.Long term effects of drugs on cell proliferation was detected by colony formation assay;3.Protein expression was analyzed by Western blotting;4.Cell cycle was evaluated by flow cytometry with PI(Propidium Iodide)staining;5.Expression of the target protein and its intracellular distribution were detected by immunofluorescence assay;6.Lipofectamin 2000 and si RNA transfection were used to silence target genes;7.Lipofectamin 2000 and c DNA plasmids transfection were performed to overexpress target genes;8.Formation of protein complex was demonstrated by immunoprecipitation assay;9.Gene Expression Omnibus(GEO)online database was applied to analyze differentially expressed m RNA between drug-sensitive GC tussues and drug-resistant GC tissues;10.RT-q PCR assay was used to examine m RNA expression level of target gene;11.Statistical analysis: The experimental results were reported as mean ± standard deviation and analyzed by SPSS 16.0 software.Bilateral t test was applied to evaluate the statistical significance.P-values < 0.05 was considered as significantly different.Results: 1.Two HER2-positive GC cell lines possess different sensitivities to lapatinib.Western blotting showed that HER2 protein was expressed in both NCI-N87 and MKN7 cell lines.However,MTT assay demonstrated that MKN7 cells exhibited much less sensitivity to lapatinib.Similar results were found in colony formation assay.These data supported a higher sensitivity of lapatinib in NCI-N87 cells than MKN7 cells.Western blotting demonstrated that in both NCI-N87 and MKN7 cells,the exposure to lapatinib resulted in reduction of phosphorylated HER2 and EGFR as well as the expression level of phosphorylated AKT and ERK,which indicated that phosphorylation of HER2,EGFR and downstream survival signaling was irrelevant with lapatinib sensitivity.2.EGFR / HER2 complex formation is not associated with lapatinib sensitivity.The results of immunoprecipitation assay showed that there existed heterodimers of EGFR and HER2 physically in two cell lines.When treated with lapatinib,the formation of heterodimers was inhibited in both NCI-N87 and MKN7 cells,which suggested that lapatinib sensitivity was probably irrelevant to complex formation of HER2/EGFR.3.Lapatinib induces G1 arrest in sensitive cells.Remarkable G1 arrest induced by lapatinib was observed in NCIN87 cell lines by flow cytometry assay.While this change was not available in MKN7 cell lines,suggesting that lapatinib exerted anti-tumor effect on sensitive GC cells by mediating cell cycle arrest.4.Laptinib induces inhibition of phosphorylated Chk1 in sensitive GC cells,while activaton of Chk1 in resistant GC cells.The results of Western blotting showed that after treatment with lapatinib,Chk1 phosphorylation was inhibited in NCIN87 cells along with downregulation of DNA topoisomerase 2-binding protein 1(Top BP1).On the contrary,the expression level of Chk1 phosphorylation was mildly upregulated in MKN7 cells with Top BP1 upregulated.These results above suggested that the anti-tumor effect of lapatinib by inducing G1 arrest in NCI-N87 cells relied on inhibiting phosphorylated Chk1 along with downregulation of Top BP1.5.Inhibition of Chk1 expression via si RNA enhances lapatinib sensitivity in MKN7 cell line.We transfected MKN7 cells with Chk1 si RNA.MTT assay showed that though lapatinib hardly affected cell growth in MKN7,it significantly suppressed cell viability in Chk1-knockdown cells.Meanwhile,after transfected with Chk1 si RNA,MKN7 cells exhibited G1 arrest after lapatinib treatment.Furthermore,the phosphorylation of AKT and ERK in Chk1-knockdown MKN7 cells was decreased more significantly with lapatinib treatment,suggesting that Chk1 knockdown by si RNA enhanced lapatinib sensitivity in MKN7 cell line.6.ATR inhibitor VE-821 sensitizes MKN7 cell line to lapatinib.The results of MTT assay and colony formation assay showed that co-treatment with lapatinib and VE-821 further suppressed cell viability and resulted in barely any colony formation in MKN7 cells.VE-821 combined with lapatinib significantly increased G1 percentage of MKN7 cell line detected by flow cytometry.Results of Western blotting demonstrated that the phosphorylation of AKT,ERK and Chk1 were all downregulated in co-treatment group in MKN7 cells compared with monotherapy groups.Results above suggested that application of ATR inhibitor VE-821 possessed the ability of potentiating lapatinib sensitivity in MKN7 cells.7.ATR inhibitor VE-821 potentiates DNA damage induced by lapatinib in MKN7 cells.Western blotting showed upregulation of ?-H2 AX expression,a sensitive marker for DNA damage,in NCI-N87 cells with single lapatinib application whereas no such significant change in MKN7 cells.We combined lapatinib with VE-821 in MKN7 cells and observed increased expression level of ?-H2 AX by Western blotting.Furthermore,the results of immunofluorescence imaging demonstrated that more ?-H2 AX foci were observed upon combination treatment of lapatinib and VE-821 compared with lapatinib or VE-821 alone in MKN7 cells.These observations suggested that ATR inhibitor VE-821 aggravated DNA damage induced by lapatinib in MKN7 cells.8.Overexpression of Chk1 and ATR attenuates lapatinib sensitivity in NCI-N87 cells.We used plasmids to overexpress ATR and Chk1,which led to increased level of total and phosphorylated Chk1.MTT assays showed overexpression of ATR/Chk1 attenuated anti-proliferative effect of lapatinib in NCI-N87 cells.Flow cytometry showed that G1 arrest induced by lapatinib was restored by ATR/Chk1 c DNA transfection in NCI-N87 cells.Overexpression of ATR and Chk1 also reversed the downregulation of phosphorylated AKT and ERK in NCI-N87 cells caused by lapatinib.The above results suggested that overexpression of ATR or Chk1 led to Chk1 activation,and thus attenuated the sensitivity to lapatinib in NCI-N87 cell line.9.ATR inhibitor VE-821 enhances lapatinib sensitivity in NCI-N87 cell line.MTT assays showed that the anti-proliferative effect of combination treatment of VE-821 and lapatinib was more significant than single application of lapatinib in NCI-N87 cells.Furthermore,Western blotting indicated that combination treatment further inhibited the phosphorylation level of Chk1,AKT and ERK compared with lapatinib monotherapy in NCI-N87 cells.Results above suggested that inhibiting phosphorylated Chk1 further enhanced lapatinib sensitivity in NCI-N87 cell line.10.Cisplatin attenuates the sensitivity of lapatinib by activating Chk1 in NCI-N87 cells.MTT assay showed that cisplatin inhibited proliferation of NCI-N87 cells in a dose-dependent manner.Western blotting and immunofluorescence experiments confirmed that cisplatin induced DNA damage and phosphorylation of Chk1 in NCI-N87 cells.Furthermore,MTT assays and Western blotting results suggested that cisplatin phosphorylates Chk1 in NCI-N87 cells,thereby impairing the proliferation inhibitory effect of lapatinib and reversing its inhibitory effects on downstream survival pathways.11.KRT8 positively correlates with trastuzumab resistance in gastric cancer cells.Analysis of GEO database showed that KRT8 m RNA was significantly higher in trastuzumab-resistant gastric cancer cells,suggesting that it is positively correlated with trastuzumab resistance in gastric cancer cells.12.Down-regulation of KRT8 protein expression enhances the sensitivity of MKN7 cells to lapatinib.MTT assay showed that down-regulation of KRT8 protein by si RNA enhanced the anti-proliferative effect of lapatinib on MKN7 cells.Western blotting assay showed that down-regulation of KRT8 expression further enhanced the inhibitory effect of lapatinib on AKT and ERK phosphorylation in MKN7 cells.13.Overexpression of KRT8 reduces the sensitivity of NCI-N87 cells to lapatinib.MTT assay showed that overexpression of KRT8 reversed the anti-proliferative effect of lapatinib on NCI-N87 cells.Western blotting assay suggested that overexpression of KRT8 abrogated inhibition of AKT and ERK phosphorylation by lapatinib in NCI-N87 cells.14.Chk1 regulates the expression of KRT8.Down-regulation of Chk1 protein in MKN7 and NCI-N87 by si RNA led to significant down-regulation of KRT8 m RNA and its protein expression,whereas overexpressing Chk1 led to significant up-regulation of KRT8.Conclusion: 1.Inhibition of Chk1 phosphorylation enhanced lapatinib sensitivity in lapatinib-resistant GC cells by suppressing phosphorylation of AKT and ERK,promoting G1 cell cycle transition and aggravating DNA damage.2.Chk1 activation attenuated sensitivity of lapatinib in lapatinib-sensitive GC cells.On the other hand,ATR inhibitor VE-821 further potentiated lapatinib sensitivity in lapatinib-sensitive GC cells.3.KRT8 was responsible for regulation of lapatinib sensitivity.4.KRT8 was regulated by Chk1.