The Mechanism Study Of The NRG1/HER3 Activation In The Primary Resistance Of HER2-positive Breast Cancer To Trastuzumab

Objective:To explore the role of NRG1/HER3 pathway activation in trastuzumab primary resistance,and the inhibitory effect of NRG1-dependent HER3 monoclonal antibody in HER2-positive breast cancer.BT474(trastuzumab sensitive)and MDA-MB-453(trastuzumab resistant)HER2-positive breast cancer cells were selected as the study subjects.The HER2/ HER3 activation was blocked by trastuzumab and/ or HER3 antibody,and siRNA was applied to silence HER3 gene.In order to clarify the vital role of NRG1/ HER3 activation in the mechanism of trastuzumab resistance,the changes of downstream signaling transduction molecules,cell apoptosis and cell activity after different treatment were analyzed.Methods:1.BT474(trastuzumab sensitive)and MDA-MB-453 cells(trastuzumab resistant)were treated with NRG1 first and then stimulated with trastuzumab and/ or HER3 antibody.The expression of the p-HER2,p-HER3,p-Akt,p-MAPK were assessed by the western blot method.2.Small interfering RNAs targeting HER3 gene were transfected into MDA-MB-453 cells for gene silencing.The western blot method was performed to detect the changes in expression of p-HER2,p-HER3,p-Akt,p-MAPK.3.BT474(trastuzumab sensitive)and MDA-MB-453 cells(trastuzumab resistant)were treated with NRG1 first and then stimulated with trastuzumab and/ or HER3 antibody.Flow cytometry was performed to detected cell lines apoptosis.4.MTT assay was performed to assess cell viability of BT474(trastuzumab sensitive)and MDA-MB-453 cells(trastuzumab resistant)stimulated with different drugs and concentrations.Results:1.Without NRG1 stimulation,the trastuzumab significantly downregulated the expression of p-HER2,promoted early apoptosis and inhibited cell viability in BT474 cells(P<0.01).2.After NRG1 stimulation,inhibitory effects of trastuzumab weakened in BT474 cells,whereas the HER3 antibody significantly downregulated the expression of p-HER3 and downstream signaling transduction molecules(P<0.01).3.In MDA-MB-453 cells,the HER3 antibody significantly downregulated both pHER2,p-HER3 and downstream signaling p-Akt,p-MAPK after NRG1 stimulation,however,trastuzumab hardly played a role(P<0.01).4.In MDA-MB-453 cells,the expression of p-HER2,p-HER3,p-Akt and p-MAPK was significantly downregulated after HER3 gene silencing compared to the control(P<0.01).5.NRG1-depedent HER3 antibody promoted early apoptosis in BT474 cells(trastuzumab sensitive)and MDA-MB-453 cells(trastuzumab resistant).6.HER3 antibody combined with trastuzumab synergistically inhibited cell viability in BT474 cells(trastuzumab sensitive)and MDA-MB-453 cells(trastuzumab resistant).Conclusion:1.Activation of NRG1-dependent HER3 reduced inhibitory effects of trastuzumab in BT474 cells(trastuzumab sensitive).2.In MDA-MB-453 cells,NRG1-dependent HER3 antibodies might reverse NRG1 initiated primary resistance to trastuzumab by inhibiting the activation of the p-HER2,pHER3,p-Akt,p-MAPK signaling pathway.3.After HER3 gene silencing in MDA-MB-453 cells,changes in the expression of pHER2,p-HER3,p-Akt,and p-MAPK signaling molecules were consistent with that of NRG1-dependent HER3 antibody,which confirmed the inhibitory effect of HER3 antibody.4.NRG1-depedent HER3 antibody promoted early apoptosis and inhibited cell viability in BT474 cells(trastuzumab sensitive)and MDA-MB-453 cells(trastuzumab resistant).5.HER3 monoclonal antibody combined with trastuzumab synergistically inhibited cells viability in HER2 positive breast cancer,which might serve as a treatment choice for primary trastuzumab resistant patients.