The HER4-YAP1 Axis Promotes Trastuzumab Resistance In HER2 Positive Gastric Cancer By Inducing EMT Transition

Background and purpose:Gastric cancer is the fifth most commonly diagnosed malignancy and the third leading cause of cancer death worldwide.Adjuvant chemotherapy following radical surgical resection would specifically benefit patients with advanced gastric cancer[20],but the high heterogeneity of the disease leads to metastasis and recurrence,with a significantly poorer prognosis that is less than one year median survival and a 5-year survival rate less than7%[21,22].Among the various genomic events,abnormal expression of HER2,a prognostic factor for patients,is involved in as many as 7%-34%of gastric cancers[23-25].By interacting independently with extracellular ligands or by heterodimerizing with other members of the ErbB family,downstream signaling pathways,including the MAPK and PI3K/AKT/mTOR pathways,are activated to facilitate uncontrolled cell growth and tumorigenesis[26].Trastuzumab,the only target agent approved by the FDA as a first-line treatment of metastatic gastric adenocarcinoma,substantially improves the outcome for patients with HER2-positive gastric cancer[27,28].However,most patients become refractory to the trastuzumab-based treatlent within one year[29].Although new agents have been explored to delay the emergence of resistance,acquired resistance still limits the duration of the response to trastuzumab[30].Thus,a characterization of the resistance mechanism of trastuzumab is urgently needed to offer an alternative option for patients who would suffer from inevitable resistance.Currently,a large proportion of investigations about the molecular mechanisms of trastuzumab resistance stem from breast cancer,and the generally recognized processes include hyperactivation of the phosphatidylinositol-3-kinase(PI3K)pathway by PI3K alterations or PTEN loss[31-33].Scaltriti and colleagues demonstrated that p95HER2,a mutated form of HER2,did not bind to trastuzumab and retained its tyrosine kinase activity,which partly accounted for trastuzumab resistance[34].Recent studies also uncovered that growth factors act as ligands of receptor tyrosine kinases,as well as up-regulation of other growth factors,such as NRGI and HGF,which also bind to HER2,can confer resistance to anti-HER2 drugs[35,36].Piro,G and colleagues found that expression of fibroblast growth factor receptor 3(FGFR3)could stimulate epithelial-to-mesenchymal transition(EMT)after resistance developed in gastric cancer,with the FGFR3/AKT axis as the presumed escape pathway responsible for trastuzumab resistance[29].However,the exactly mechanisms of trastuzumab resistance in HER2-positive gastric cancer still remained unknown.In HER2-positive gastric cancer,dual inhibition of EGFR and HER2 displayed a satisfactory killing ability toward trastuzumab-resistant cells[38,39]HER4,another member of the ErbB family,also reportedly impacts HER2-positive cancer cell survival after cells become resistant to trastuzumab,and nuclear localization of HER2 indicateed a poor prognosis in breast cancer[40,41].The presence of HER4 sensitizes HER2-positive cells to trastuzumab and was considered a potential target for overcoming trastuzumab resistance[42].But the exactly role and mechanism of HER4 in HER2 positive gastric cancer trastuzumab resistance still remains unknown.In gastric cancer,NRG1 associates with its receptors,HER3 and HER4,might be an independent and unfavorable prognostic factor and a therapeutic target.In previous study,we discovered that a somatic mutation in HER4 was associated with peritoneal metastasis of gastric adenocarcinoma[43].The results of this study determined its role in EMT induced trastuzumab resistance by regulating vital downstream target YAP 1 and activating PI3K signal pathway.Importantly,we successfully established HER2 positive PDX model and deepened our understanding on the important role of HER4-YAP1-EMT axis in inducing trastuzumab resistance.Thus,further investigation was conducted to clarify the exact mechanism by which HER4 regulates the acquisition of trastuzumab resistance.Methods:The HER2+GC TR(Trastuzumab resistant)cells were established and the metastatic ability and the expression of HER4,p-HER4 and EMT were detected bylHC,WB,IF,Transwell and in vivo study.Besides,the function of NRG 1-HER4-YAP1 axis in inducing EMT mediated TR was conducted in vitro and in vivo.Besides,HER2+GC PDX model was established and used to uncover the potential mechanism.Result:Part1.HER2 positive gastric cancer cells promote EMT after becoming resistant to trastuzumabIt is possible that multiple resistance mechanisms may coexist in patients with HER2 positive metastatic gastric cancer(Okines et al.,2010).To identify and target the key nodes of trastuzumab resistance,we generated trastuzumab-resistant gastric cancer cell lines using HER2-positive MKN45 and NCI-N87 cells.Cells were exposed to increasing concentrations of trastuzumab for 8 months,and single-cell clones were isolated from a pool of resistant cells to generate the resultant cell lines.Compared with parental cells,MKN45TR(MKN45 trastuzumab resistant cells)and NCI-N87TR(NCI-N87 trastuzumab resistant cells)exhibited significantly higher resistance to the trastuzumab treatment in vitro(MKN45TR,IC50=99250.70 ?g/ml,RI=31.33,p<0.01,NCI-N87TR,IC50=620570.69?g/ml,RI=1032.95,p<0.01),and the resistance curve was different.Furthermore,trastuzumab cells displayed a long spindle-shaped morphology with thin,long pseudopods,which occasionally resembled finger-like pseudopods that extended from the cell bodies.Wound healing and Transwell assays showed that migration and invasive capacities of resistant cells were higher than parental cells.These findings demonstrated that activation of EMT occurred after parental cells became resistant to trastuzumab.To further dissect this observation,EMT markers were measured using western blot and immunofluorescence.The results revealed that levels of mesenchymal markers Vimentin increased while epithelial markers E-Cadherin decreased.Additionally,Western blot analyses also showed significant expression decrease of other two ErbB family member HER2 and HER3 in trastuzumab resistant lines.Part2.Trastuzumab resistance in MKN45 cells promote metastasis in vivoSince the investigation revealed that migration and invasion ability was elevated upon trastuzumab resistance acquired,the homing capacity of cancer cells in vivo was further investigated.We performed tail vein injections of cancer cells in nude mice to determine the rate of nodule formation in the lungs and livers.Compared with MKN45 group,larger tumor nodules were observed in the lungs and livers in MKN45TR group.Interestingly,we found multiple subcutaneous nodules in both groups.The HE staining analysis confirmed that the subcutaneous nodules in MKN45 group were all inflammatory cell infiltrations while that in MKN45TR group were all metastatic cancer cells.Once resistance acquired,E-Cadherin expression was decreased,and Vimentin was increased in vivo.These observations indicated that during resistance process,the homing capacity became increasingly stronger than parental cells.Part3.HER4 promotes trastuzumab resistance by inducing EMTPrevious research revealed that EMT was involved in trastuzumab resistance in HER2 positive gastric cancer.To gain insight into possible mechanism of the observed phenotypic transition,we examined our previous whole-exome sequencing results,with emphasis on the potential role HER4 played in inducing gastric cancer cell EMT and metastasis.HER4,as a member of the ErbB family,could impact on HER2-positive cancer cell survival after cells become resistant to trastuzumab(Canfield et al.,2015;Mohd et al.,2014).As indicated by Western blot analyses,HER4 and p-HER4 expressions in resistant cells were increased compared to the parental cells.IHC showed that HER4 and p-HER4 were overexpressed in the MKN45TR subcutaneous tumor compared with MKN45 group.To confirm the necessity of HER4 in EMT process of trastuzumab resistance,we selected an efficient HER4-targeted siRNA fragment by Western blot and Q-PCR.Transwell and wound healing assays showed that invasive and migration capacities of the resistant-siHER4 cells were lower than that of resistant-NC cells.After the HER4 knockdown,Vimentin expression decreased while E-Cadherin increased.Meanwhile,the resistance-associated molecular pathway,PI3K pathway,was inactivated after siHER4 silencing.Besides,overexpression of Cleavage-caspase3 and Cleavage-caspase9 indicated that siHER4 could promote resistant cells apoptosis.By using Annexin V staining,flow cytometric analysis showed increased percentage of cells undergoing apoptosis after the siHER4 transfection compared to the NC(p<0.05).The lentivirus-transfected cell lines with stably knocked down expression of HER4 were established and the fluorescence showed transfected efficiency.As shown in Figure 4A and B,growth rate was decreased,and Vimentin expression was reduced while E-Cadherin was increased in tumors of siHER4 group compared with NC group.Besides,HER4 stably knocked down MKN45TR cells were employed to investigate homing capacity of cancer cells in nude mice.Compared to the animal models of NC group,smaller tumor nodules were found in the lungs of the siHER4 group(p=0.0423).These results further confirmed that HER4 played an important role in promoting trasutuzumab resistance by regulating EMT.Part4.The HER4-YAP1 axis,through its induction of EMT,is essential for trastuzumab resistanceTo investigate potential role of HER4 in promoting resistant cells EMT,it is predicted that YAPl might be an important downstream molecule as an EMT inducer by using String.com network.Previous IHC and western blot,Q-PCR,immunofluorescence results revealed that YAP1 expression decreased when HER4 was knocked down in vitro and in vivo.Additionally,immunoprecipitation analysis of resistant cells showed mutual interaction between HER4 and YAP1.Then,confocal microscopy analysis revealed that HER4 and YAP1 co-localized to the cytosol and cytoplasmic membrane in MKN45TR cells.Those data indicated that YAP1,interacted with HER4,might play an important role in downstream of HER4.As Immunofluorescence shown,the level of Vimentin decreased while E-Cadherin increased when YAP1 was knocked down.Trans well and wound healing assays also revealed lower invasive and migration capacity of si YAP 1 cells compared to NC group.Western blot analysis confirmed reduction of Vimentin in response to YAP1 silencing in resistant cells.Meanwhile,suppressed YAP1 lead to inhibition of PI3K signaling pathway.Then,resistant cells were simultaneously coinfected into siHER4 and YAP1 cDNA to explore the role of YAP1 in HER4-mediated EMT and trastuzumab resistance.The effects of siHER4 on inhibiting resistant cells EMT and PI3K signal pathway were significantly improved.It was hence proposed that YAP1 might be a demonstrably downstream effector of HER4 and through which HER4 exerted its EMT effects on trastuzumab resistant cells.As shown,the MKN45TR morphology became epithelial in shape and that MKN45TR cells exhibited attenuated levels of p-HER4,HER4,Vimentin and PI3K along with increased levels of epithelial proteins after trastuzumab was withdrawn.These observations indicated that maintenance of EMT required continuous stimulation of trastuzumab.Taken together,those data revealed that HER4-YAP1 axis,through its induction of EMT,might be crucial in process of trastuzumab resistance.Part5.NRG1,through its activation of the HER4-YAP1 axis,is essential for EMT-induced trastuzumab resistanceTo determine whether stimulation of full-length ErbB4 by its ligand,NRG1 was sufficient to induce YAP1 expression and EMT,resistant cells were stimulated with 100 ng/ml NRGl at different time points and optimized the stimulation time to 20 min in MKN45TR cells and 30 min in NCI-N87TR cells,respectively.The results of Transwell assays indicated that NRG1 promoted invasion of resistant cells and that YAP1 silencing reduced activity of NRG 1.The immunofluorescence assay revealed that NRG1 up-regulated Vimentin and down-regulated E-Cadherin,whereas YAP1 silencing reversed the activity.Additionally,NRG1 stimulation reduced the apoptosis rate of resistant cells,and YAP1 silencing offset suppression of apoptosis(p<0.05,).Western blot showed that NRG1 stimulation could reduce expressions of apoptotic protein Cleavage-caspase3 and Cleavage-caspase9 while YAP1 silencing could rescue its expression.Those results indicated that NRG1 was essential for EMT-induced trastuzumab resistance through the activation of the HER4-YAP1 axis.Based on the above,these data revealed that HER4-YAP1 axis might regulate acquired resistance to trastuzumab in HER2-positive gastric cancer cells by promoting EMT.The axis conferred an increased proliferation capacity of HER2-positive gastric cancer cells exposed to trastuzumab.The abnormal expressions of HER4 and YAP1 increased activation of PI3K to promote resistance to the trastuzumab treatment.Part6.The HER4-YAP1 axis is associated with trastuzumab resistance in a HER2-positive patient-derived xenograft modelTo maximize success rate for development of HER2 targeted therapeutics,PDX model reflecting HER2 positive gastric cancer patients were established.In the general gastric cancer model,we observed that all third-generation tumors began to grow in bursts when post-implantation time reached 28 days HE staining and HER2 IHC staining analyses of the PDX model of HER2-positive gastric cancer showed that histological and pathological features were well-maintained from FO to F3 generation.The F3 generation mice were divided into 3 groups randomly,Control group,20mg/kg group and 40mg/kg group.As shown,3 groups grew quickly and reached to potential clinical resistant endpoint.Trastuzumab resistance in the model was confirmed with expression analysis of related proteins.Five candidate trastuzumab-resistant mice were identified in 20 mg/kg dose groups,and 3 were identified in 40 mg/kg dose groups.The western blot analysis revealed decreased expressions of HER2 and E-Cadherin with elevated expressions of p-HER4,YAP1,Vimentin,and PI3K in trastuzumab-resistant mice compare to control group.Consistently,IHC analysis showed the same tendency and confirmed trastuzumab resistance in our PDX model.These results indicated that HER4-YAP1 axis acted as a vital pathway that promoted trastuzumab resistance in HER2-positive gastric cancer by inducing the occurrence of EMT.