Involvement Of ER Stress In Mechanism Of Trastuzumab Resistance Occurrence And Transmission Of HER2-positive Gastric Cancer

BackgroundGastric cancer is the fourth most common malignant disease and the second leading cause of cancer-related death worldwide.Trastuzumab,a humanized antibody targeting human epidermal growth factor receptor 2(HER2),inhibits HER2-mediated signaling by binding the extracellular domain of HER2.In terms of HER2-positive GC patients,the safety and efficacy of cotreatment of chemotherapy and trastuzumab are remarkably higher than those of chemotherapy treatment alone.However,trastuzumab resistance had seriously influenced the clinical effects in the therapy of HER2-positive gastric cancer.Therefore,trastuzumab resistance becomes one of the major problem in the treatment of GC.Recent studies confirmed that endoplasrnic reticulum stress(ERS)closely related to drug resistance of tumor.ERS is usually triggered under cell hypoxia,carbohydrate undersupply or calcium depletion,and activates the unfolded protein response(UPR),elevating cancer cellular resistance to injury promoting cells survival and regulating cancer drug resistance.The endoplasmic reticulum stress response as an endogenously adaptive mechanism to be able to intervene in drug resistance.However,little is known about the relationship between ERS and trastuzumab resistance of gastric cancer cells and the mechanism of drug resistance.As a newly endogenous gene regulator,miRNA involved in signal regulatory pathways of the ERS,plays an important role in tumor development?progression and drug resistance.In the tumor microenvironment,exosomes are important carriers of miRNAs to prevent degradation.As a form of intercellular communication,exosomes are involving in tumor growth,progression and drug resistance.No data are available about exosomes functions in GC trastuzumab resistance information transmission.The objective of this study was to explore the role of ERS in the mechanism of trastuzumab resistance occurrence and transmission of HER2-positive gastric cancer GC cells.Methods and Contents1.Establish gastric cancer cell endoplasmic reticulum stress models approach with thapsigargin(TG)or-Glu/FBS separation disposal of human gastric cancer cell line NCI-N87 and MKN45,Western blot techniques to detect each time point of GRP78 expression at the protein level.2.CCK8 assay of GC cells pre-treated with TG or-Glu/FBS followed by trastuzumab treatment at indicated concentrations for 72 hr.3.Total RNA was extracted from ER stress gastric cancer cells and normal gastric cancer cells and assessed by Affymetrix microarray V.4.0 platform.Differentially expressed genes among these two groups were compared using whole genome sequences.4.Cells were transiently transfected with miRNA inhibitor and miRNA negative control(miR-NC)by using Lipofectamine 3000 and OPTI-MEM,and subsequently treated with TG to induce ER stress.CCK8 assay of cells transfected with miRNA inhibitor and miR-NC upon trastuzumab treatment.5.Exosomes extracted by differential ultracentrifugation from cells culture supernatants were assessed by transmission electron microscopy,nanoparticle tracking analysis and protein analysis to identify morphology,protein expression,concentration and size distribution of particles.6.CCK8 assay of cells 24hr after incubation with indicated exosomes upon trastuzumab treatment.7.Real-time PCR was used to detected the cell and exosome microRNA expression level.8.Western blotting was used to detected the cell GPR78?ERK?p-ERK?AKT and p-AKT protein expression level.9.All statistical data were displayed as meansz±standard deviation(SD)and statistical significance between groups was determined by a two-tailed Student's t-test and a one-way ANOVA test.Differences were considered to be significant when P<0.05.Results1.ER stress leads to therapeutic resistance to trastuzumab in HER2-positive gastric cancer cells.2.The miRNA microarray analysis showed that miRNAs were differentially expressed in TG-treated cells compared with untreated gastric cancer cells.The eleven of the most differentially expressed miRNAs were subjected to loss-of-function analysis in ERS gastric cancer cells by miRNA inhibitors.Notably,interference of miR-301a-3p suppressed trastuzumab resistance compared with the remaining ten miRNAs and rescued of AKT and ERK signaling from traztuzumab-mediated inhibition,but miR-301a-3p inhibitor does not alter the expression of GRP78.3.Conditioned medium was derived from gastric cancer cells,herein called ERS conditioned medium(ERS CM),treated with TG.Trastuzumab-sensitive cells were cultured in ERS CM or exosomes from ERS cells,and then these "recipient" cells showed significantly lower trastuzumab sensitivity as compared to the cells treated with normal CM.Besides,ERS CM or exosomes from ERS cells rescued of AKT and ERK signaling from traztuzumab-mediated inhibition and inactivated GRP78.4.Exosomal miR-301a-3p levels were significantly higher in ERS gastric cancer cells than in normal gastric cancer cells.The intracellular levels of miR-301a-3p were increased upon incubation with exosomes from ERS cells.Conclusions1.ERS is a possible way to override the effect of trastuzumab in HER2+gastric cancer cells.2.Induction of miR-301a-3p upon ER Stress regulates trastuzumab resistance in HER2-positive gastric cancer.3.Exosomes from ERS HER2+ gastric cancer cells can transmit the drug resistance message,sensitive cells can acquire drug resistance from the exosomes of drug-resistant cells.4.Exosomes transfer of ERS HER2+ gastric cancer cells-derived miR-301a-3p confers trastuzumab resistance in gastric cancer cells.