The Role And Underlying Mechanism Of CA916798 In Acquired EGFR-TKI-Resistance In NSCLC

Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide.Non-small cell lung cancer(NSCLC)is the main histological type of lung cancer,accounting for about 85% of all lung cancers.Most lung cancer patients are in advanced stages at the time of diagnosis,thus have poor prognosis.In recent years,molecular targeted drugs represented by epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI),such as Gefitinib,have replaced traditional cytotoxic drugs for treatment of patients with advanced NSCLC containing EGFR-sensitive mutations(mainly including exon 19 deletion mutation and exon 21 L858 R point mutation)and achieved great success.However,after a period of treatment(median time: 8-12months),an acquired resistance to EGFR-TKIs is developed in most patients,which limits the clinical application and efficacy of EGFR-TKIs.Therefore new strategies for overcoming or delaying resistance to EGFR-TKIs are urgently neededMany studies have demonstrated that EGFR-TKI resistance is related to EGFR secondary mutation,alternative activation,and phenotypic change.In response to the discovered drug resistance mechanisms,EGFR-TKI has been continuously updated,and various regimens combining chemotherapy are also being clinically developed,but the results are still not satisfactory.Therefore,further exploration of the underlying resistance mechanism will help to discover new potential targets for overcoming of EGFR-TKIs resistance.The CA916798 gene is a new gene related to cisplatin resistance in lung cancer that was first discovered,reported,and confirmed by our research group.It has been demonstrated that CA916798 may be involved in cisplatin resistance through the PI3 K / AKT pathway,and the resistance mechanism is related to anti-apoptosis.However,the roles and underlying molecular mechanism of CA916798 gene in EGFR-TKI resistance have not been reported so far.The subgroup analysis of IPASS study revealed that there was a significant difference in the objective response rate(ORR)of chemotherapy between patients with EGFR mutations and those without mutations,suggesting that there may be a connection between chemotherapy resistance and targeted resistance.Therefore,we hypothesized that CA916798,which promotes cisplatin resistance,may also play an important role in the resistance of EGFR-TKI-targeted drugs in NSCLC cells.In this study,this hypothesis is confirmed through a series of in vivo and in vitro experiments and the underlying molecular mechanisms are also explored preliminarily.The main methods,results and conclusions of this study are as follows:1.CA916798 promotes the resistance of NSCLC cells to EGFR-TKI(1)CA916798 is highly expressed in acquired EGFR-TKI-resistant NSCLC cells We induced two acquired EGFR-TKI-resistant NSCLC cell lines PC9 / GR(Gefitinib-resistant strain)and HCC827 / IR(Icotinib-resistant strain)through the intermittent induction method.The results of q RT-PCR and Western Blot analyses showed that the expressions of CA916798 were significantly higher in the two EGFR-TKI-resistant cells(p <0.01)than their parental cells at both m RNA and protein levels.These results suggest that CA916798 may be involved in the acquired resistance of NSCLC cells to EGFR-TKIs.(2)Silencing / overexpressing CA916798 significantly changes the sensitivity of NSCLC cells to EGFR-TKITo investigate the role of CA916798 in NSCLC acquired EGFR-TKI-resistance,we constructed PC9 / GR and HCC827 / IR cells of stably silencing CA916798 by using sh RNA and PC9 and HCC827 cells of stably overexpressing CA916798.The results showed that after knocking down the expression of CA916798,the sensitivity of PC9 / GR and HCC827 / IR cells to EGFR-TKIs was significantly increased(p <0.01)as compared with control cells;on the contrary,stably overexpressing CA916798(over-CA916798)significantly reduced the sensitivity of PC9 and HCC827 to EGFR-TKIs.These results indicate that CA916798 is an important molecule that promotes acquired EGFR-TKI-resistance in NSCLC cells.2.Knocking down CA916798 attenuates the growth of xenograft tumors derived from NSCLC cells resistant to EGFR-TKITo further verify the role of CA916798 in acquired resistance to EGFR-TKIs in NSCLC cells,we performed in vivo experiments in mice.We established a xenograft model in NOD / SCID mice by subcutaneous injection of PC9 / GR-sh CA916798 and its control cells,and followed by treatment with gefitinib / placebo.Then the growth of xenograft tumors was observed.The experimental results showed that the growth of xenografts derived from CA916798-silecing gefitinib-resistant cells was significantly slower than that of control cells(p <0.05).The results also showed that gefitinib treatment showed a better effect in the CA916798-knockdown group,that is,the volume and weight of the xenograft tumors in the CA916798 knockdown combined with gefitinib treatment group were significantly smaller than those in the control group(p <0.001).3.CA916798 attenuates the apoptosis-inducing ability of EGFR-TKI in NSCLC cellsWe investigated the effect of CA916798 on acquired EGFR-TKI-resistance from the aspect of apoptosis.Annexin V-APC / PI double staining was used to measure the apoptosis rate induced by EGFR-TKIs after knockdown or overexpression of CA916798.The experimental results showed that knocking down CA196798 significantly increased the apoptosis rate of drug-resistant cells under the action of EGFR-TKI drugs(p <0.01)as compared to the control cells;on the contrary,overexpression of CA196798 in sensitive cells significantly reduced apoptosis cells under the induction of EGFR-TKI(p <0.05)as compared to the control cells.These experimental results suggest that CA916798 may affect the sensitivity of NSCLC cells to EGFR-TKI by affecting apoptosis.4.HMGA2 may be a key mediator for CA916798 promoting the resistance of NSCLC cells to EGFR-TKIsIn order to explore the mechanism of CA916798 promoting the resistance of NSCLC cells to EGFR-TKIs,we performed transcriptome sequencing(RNA-Seq)to compare the gene expression profiles of PC9 / GR cells with or without CA916798 knockdown.RNA-Seq results showed that after knocking down CA916798,the HMGA2 molecule was significantly down-regulated,which was verified by q RT-PCR.The expression of HMAG2 was positively related to the expression of CA916798,suggesting that HMGA2 may be a key molecule for CA916798 to promote EGFR-TKI resistance.5.CA916798 may regulate the expression of Bcl-2 / Bax to inhibit the apoptosis of NSCLC cellsIn order to further explore the molecular mechanism that CA916798 promotes the anti-apoptosis of NSCLC cells,we screened BCL-2 family-related molecules through Western Blot.As a result,knocked down of C916798 markedly decreased the expression of HMGA2 in drug-resistant cells.Meanwhile the expression of pro-apoptosis protein BAX was increased,and the expression of anti-apoptotic protein BCL-2 was decreased.On the contrary,overexpression of CA916798 in sensitive cell lines increased the expression of HMGA2 and anti-apoptotic protein BCL-2,but decreased the expression of pro-apoptotic BAX.These results indicate that CA916798 may inhibit the apoptosis of NSCLC cells by regulating the expression of Bcl-2 / Bax through HMGA2,thereby promoting the resistance of NSCLC cells to EGFR-TKIs.In conclusions:1.CA916798 is highly expressed in acquired EGFR-TKI-resistant NSCLC cells.2.CA916798 attenuates the apoptosis-inducing ability of EGFR-TKI in NSCLC cells and thereby promotes the resistance of NSCLC cells to EGFR-TKI.3.CA916798 may inhibit the apoptosis of NSCLC cells by regulating the expression of Bcl-2 /Bax through HMGA2,thereby promoting the resistance of NSCLC cells to EGFR-TKI.