The Expression Of TrKB, MAGE-A4, AR In Breast Cancer And The Effect Of Proliferation To Breast Cancer Cell Lines

Research Objective:To research the expression of tropomyosin-related receptor kinase B(TrKB),melanoma-associated antigen-A4(MAGE-A4)and androgen receptor(AR)in different molecular subtyping breast cancers and their relationship with clinicopathologic characteristics.furthermore,we study the sensitivity of different breast cancer cell lines to bicalutamide(CDX)and preliminary research the mechanism,the AR inhibitor on different breast cancer cell lines growth.We hope for providing some basic research tests in the treatment of breast cancer specially for TNBC.Part One Expression of TrKB,MAGE-A4 and AR in Human Breast Cancer and Their Clinical Significance Researcher Methods:Detected the expression level of TrKB,MAGE-A4,AR in 200 cases breast cancer tissues,and analyzed the relationship with the clinicopathologic characteristics.Research Result:1.The expression of TrKB in TNBC was 46.49%,which had a significance different with other molecular subtyping breast cancers((49)(27)0.05).The positive rate of TrKB had relationship with axillary lymph node status((49)(27)0.05),but not with menopause,age,tumor size,histological grading and clinical TNM staging and estrogen receptor expression((49)>0.05).2.The expression of MAGE-A4 in TNBC was 35.21%,which had no different with other molecular subtyping breast cancers((49)>0.05).The expression of MAGE-A4 had relationship with clinical TNM staging((49)(27)0.05),but not with menopause,age,tumor size,axillary lymph node status,histological grading and estrogen receptor expression((49)>0.05)3.The expression of AR in TNBC was 45.07%,which was the lowest expression in four molecular subtyping breast cancers,and the difference had significance(0.05).The positive rate of AR had relationship with histological grading,menopause station and estrogen receptor expression((49)(27)0.05),but not with clinical TNM staging,age,tumor size,axillary lymph node status,((49)>0.05).Conclusion:1.The high expression of TrKB in TNBC indicates that could take an important role in the invasiveness of breast tumor cell,which could be used as a new molecular to predict the biological behaviors and may be a potential treatment target in TNBC.The expression had relationship with lymph node,which suggests that it could be a predict factor for metastasis.2.The expression of MAGE-A4 had negative relationship with clinical TNM staging which could be a benefit factor in the progress of breast cancer.3.The high expression rate of presented in all kinds of breast cancer subtyping which suggests that it would be a hopeful treatment target in TNBC.The expression had relationship with menopause station and estrogen receptor expression which reminds us that AR could relative with estrogen level.However,the negative relationship with histological staging suggests that it could be associated with differentiated degree and cancer cells atypia.Part Two.The Sensitivity of AR in Different Breast Cancer Cell Lines and The Effect of Alone and Combination with PI3 K Inhibitors Research Methods:1.Detected the expression of AR in three breast cancer cell lines including MCF-7,MAD-MB-231,MDA-MB-453 by immunofluorescence and western blotting.2.Treated breast cancer cell lines with different concentration AR inhibitor bicalutamude(30?mol/l,10?mol/l,1?mol/l)for 6 days respectively.Detected OD value by MTT and drawn the growth curves to fine the relationship between the inhibition effect of cell proliferation and treatment time and dosage of bicalutamide.3.Treated MDA-MB-231,MCF-7,MDA-MB-453 with 30?mol/l bicalutamide for 6 days.Collected the protein and detected the relative expression changes of AR and some key proteome or phosphylated protenome of PI3K/AKT/mTOR pathway before and after treatment.4.Treated MDA-MB-453 with different concentration inhibitors including pan-PI3 K inhibitor LY294002,pan-AKT inhibitor MK2206,mTOR inhibitor everolimus alone and combination with 30?mol/l bicalutamide,Detected OD values by CCK8 kits and calculated inhibition rates at different situation,then valuated the combination effect by Q value.Research Result:1.The result of WB and immunofluorescence suggested that the relative low expression of AR in MCF-7,MDA-MB-231,and the relative high expression of AR in MDA-MB-453.2.The result of MTT suggested that the viability of AR high level breast cancer cell line MDA-MB-453 was reduced significantly after treatment for 6 days by bicalutamide at 30?mol/l((49)(27)0.05),and the AR expression was decrease comparing before,but there was no significance between the control and experiment group((49)>0.05),experients showed that bicalutamide inhibited MDA-MB-453 in dose-dependent and time-dependent.while prolonged the treatment time of MDA-MB-231 and MCF-7 at each concentration bicalutamide,the viability was not inhibited significantly((49)>0.05),and the AR expression was also not decreased(P>0.05).3.From western blotting,we could find that the relative expression of AKT?mTOR?p-mTOR?p-S6 was decrease significantly in MDA-MB-453 experiment group((49)<0.05),p-AKT also decrease,but the difference had no significant((49)>0.05).while in MDA-MB-231 and MCF-7,the expression of AKT?p-AKT?mTOR?p-mTOR?p-S6 had significant decrease((49)>0.05).4.Inhibition rate of all the combination groups were higher than single drug groups in MDA-MB-453,but LY294002 combination group had not synergistic effect at each concentration groups through Q value,while mk-2206 and everolimus combination groups showed synergistic effect at each concentration groups.Conclusion:1.MDA-MB-453 could be as an available experimental model for AR positive breast cancer.2.AR would be as an available treatment target in breast cancer,AR inhibitor bicalutamide could inhibit proliferation of AR strong expression breast cancer cell.3.Bicalutamide inhibits the proliferation ability of AR strong cell line MDA-MB-453 inhibited PI3K/AKT/mTOR activity through inhibite AR.4.When bicalutamide combinated with AKT inhibitor or mTOR inhibitor,which could enhance the inhibition.