Ligand-independent EphA2 Contributes To Chemoresistance In Small-cell Lung Cancer By Enhancing PRMT1-mediated SOX2 Methylation

Background:Data form WHO shows that,lung cancer is one of the cancers with the highest incidence rate worldwide,and is one of leading causes of death globally.Nowadays,small cell lung cancer(SCLC)occupies approximately 15%of all lung cancers.It is extremely malignant and has a poor prognosis.SCLC is sensitive to chemotherapy in the early stage of treatment,but it is prone to chemoresistance,which leads to treatment failure.Chemoresistance of SCLC needs to be solved urgently in clinical treatment.Therefore,it is urgent to clarify its molecular mechanism to find new and effective therapeutic targets.Purpose:EphA2 is receptor tyrosine kinase,which involves in many biological processes,for example cell morphology,cell adhesion,cell movement,cell proliferation,cell survival,differentiation.While EphA2 has a P817H mutation it is loss of function.Previous study suggested that EphA2 was associated with chemoresistance of SCLC.Through co-immunoprecipitation and tandem mass spectrometry,we found that PRMT1 is not only related to chemoresistance of SCLC,but also EphA2WT,but the relationship between PRMT1 and EphA2 is still unknown,the effect of EphA2P817H on chemoresistance is remain unclear,too.This study will further verify the molecular mechanism of EphA2 and PRMT1,and their role in the regulation of chemoresistance in SCLC,and aim to find out new therapeutic target.Experimental design:Western blot was carried out to verify the correlation between EphA2 and chemoresistance of SCLC in SCLC cell lines and clinical samples.Then EphA2WT,EphA2A785S,EphA2P817H,EphA2Y930D were transfected into SCLC cell lines H69 and H446 respectively.The effects of wild type EphA2 and mutant EphA2 on chemoresistance and stemness in SCLC were detected by CCK8,western blot and sphere formation assay.The relevance between EphA2 and PRMT1 was discovered and proved by co-Immunoprecipitation,tandem mass spectrometry and GST pull down.Meanwhile,we examined the influence of PRMT1 on chemoresistance in SCLC.Western blot,co-immunoprecipitation and tandem mass spectrometry were performed to determine the relationship between PRMT1 and SOX2.Results:We examined the role of cancer stemness on chemoresistance,and the directly interacted with PRMT1,and enhanced cancer stemness to induce chemoresistance in SCLC.While EphA2P817H was loss of function in vitro and in vivo.Moreover,PRMT1 interacted with and methylated SOX2 in SCLC,then augmented chemoresistance of SCLC by enhance cancer stemness.Inhibitor ALW-Ⅱ-41-27 suppressed stemand the growth of SCLC in vitro and in vivo by blocked the activation of EphA2.Conclusions:Collectively,our study demonstrates that wild type EphA2 directly interacts with PRMT1,and PRMT1 combines and methylates SOX2 to induce chemoresistance in SCLC by promoting cancer stemness.