Exploring The Regulation Of Cyclin D3 By Trastuzumab And The Mechanism Of Trastuzumab Resistance In Breast Cell Line

Background:Currently,breast cancer accounts the highest morbidity among all cancers in female globally,and 20%to 30%patients have HER2 gene amplification.Trastuzumab dramatically improved the overall survival of HER2 positive breast cancer patients.However,trastuzumab resistance emerged as a major problem.Thus,the role of elucidating the mechanism of trastuzumab resistance and reverse trastuzumab resistance has great scientific value in treatment of breast cancer.Objective:In this study,we want to explore the critical mechanism of trastuzumab treatment and discover the possible mechanisms of drug resisitance by established trastuzumab resistant cell line model.Obtained the cell cycle associate proteins which would be changed after trastuzumab treatment,then illustrated the regulation of the protein by trastuzumab,to clarify the role of the protein in the target therapy of trastuzumab and the relationship of the protein and drug resistance.Finally,to explore the difference signaling pathway between sensitive and resistant cell line,then try to find new strategy for trastuzumab resistance.Methods:Firstly,we constructed a trastuzumab resistant cell line in BT474,a HER2 positive breast cancer cell line.Then we verified the drug resistance cell line by MTSapoptosis and cell cycle after trastuzumab treatment.Western blot was used to obtain the cell cycle protein which was changed by trastuzumab treatment.RT-qPCR and protein degradation inhibitors were used to verify the reason of the decrease of the protein.After that we explored the relevance of cyclin D3 expression to the overall survival of breast cancer patients from TCGA.Finally we used western blot to analize the different signal pathway between sensitive cell line and trastuzumab resistant cell line,MTS and colony formation analysis were used to find some new treatment strategies for trastuzumab resistance.Results:Trastuzumab could induce G1 phase arrest remarkably in sensitive cells by decreased cyclin D3 expression,but not in trastuzumab resistant cell.The expression of cyclin D3 was not significantly changed in mRNA level between trastuzumab sensitive cell line and resistant cell line.High expression of cyclin D3 was not related to poor prognosis in HER2 positive breast cancer patients in TCGA database.Trastuzumab can cause down-regulation of cyclin D3 protein,trastuzumab caused accumulation of ubiquitinated forms of cyclin D3 protein,indeed we found the degradation of cyclin D3 was through SCF complex.CUL1 can combine with cyclin D3 protein,and the knock down of CUL1 blocked the effect of trastuzumab on cyclin D3.MEK inhibitor?PKC?inhibitor?GLS inhibitor and CDK4/CDK6 inhibitor are more sensitive in trastuzumab resistant cell line.ERK signaling pathway maybe abnormal express in trastuzumab resistant cell line.MEK inhibitor and CDK4/CDK6 inhibitor could well inhibit the proliferation of trastuzumab resistant cell line.And MEK inhibitor may regulate the degradation of cyclin D3 by mTOR signal pathway.Conclusions:Trastuzumab can induce G1 phase arrest by reduce the expression of cyclin D3 through Ubiquitin proteasome system,but not in resistant cell.MEK inhibitor?PKC? inhibitor?GLS inhibitor and CDK4/CDK6 inhibitor may be new treatment strategies for trastuzumab resistance.Innovative points:1.Trastuzumab can induce G1 phase arrest by reduce the expression of cyclin D3 through Ubiquitin proteasome system,but not in resistant cell.2.Trastuzumab can not decrease the expression of cyclin D3 in trastuzumab resistant cell line.3.Cyclin D3 will be a potential protein marker to predict the effect of trastuzumab treatment.4.MEK inhibitor?PKC? inhibitor?GLS inhibitor and CDK4/CDK6 inhibitor are more sensitive in trastuzumab resistant cell line,and they may be new strategies for trastuzumab resistance.