A Study On The Effect Of The Conditioned Media From M1 Macrophages On The Epithelial-mesenchymal Transition Of Breast Cancer Cells And Its Signaling Mechanism

Objective: Tumor-associated macrophages(TAMS)play an important role in the development of malignant tumors.TAMS are usually divided into M1(classic activation)and M2(alternative activation)two subtypes.M2 macrophages highly express arginine and MMP,etc,which are shown to promote tumor development.while M1 macrophages are generally considered to promote immune response and anti-tumor effect.However,a recent report shows that the M1 macrophages are linked to epithelialmesenchymal transition in breast cancer.the role of M1 macrophages in the development of breast cancer remains to be further confirmed,and its underlying signal mechanism is not yet clear.This study was to explore the role of M1 macrophages in the process of EMT in breast cancer cells and its related mechanism.Methods: ER(estrogen receptor,ER)-positive breast cancer cell lines(MCF-7 and T47D),and ERnegative breast cancer cell lines(MDA-MB-231)were used as model cells.M1-like macrophages conditioned medium(M1-CM)were used to induce epithelialmesenchymal transition(EMT)in the model cells.The levels of m RNA and proteins were measured using q RT-PCR and Western Blot.Cell proliferation and invasion were observed by wound healing assay and transwell coated with matrigel.Pharmacological intervention was used to investigate the potential mechanisms for M1-CM induced EMT.Results:(1)M1-CM significantly enhanced migration of MCF-7 and T47 D,as well as the invasion ability of MCF-7(p<0.05),But M1-CM has no significant effect on the migration(p>0.05)and invasion(p<0.05)of MDA-MB-231 cells.(2)M1-CM promoted EMT in ER positive breast cancer cells.Compared with the control,M1-CM treatment caused upregulation of the FN(Fibronectin,FN)and Vim(Vimentin,Vim)proteins in MCF-7 cells and T47 D,while E-cad(E-cadherin,E-cad)protein was downregulated(p<0.05);(3)Calpain Inhibator suppressed M1-CM induced ER+ migration and invasion(p<0.05).(4)Calpain Inhibator suppressed the levels of EMT markers in ER+ cells(p<0.05).(5)M1-CM caused increased protein levels of phophorylated ERK,Notch1 and its target Hes1 in MCF-7 cells(p<0.01).(6)Notch pathway inhibitor DAPT attenuated the induction of EMT by M1-CM in MCF-7 cells.the M1-CM induced migration and invasion were significantly suppressed by DAPT(p<0.01).The FN and Vim protein expressions were inhibited while E-cad protein expression was reversed(p<0.05).(7)ERK inhibitor U0126 weakened the induction of EMT by M1-CM in MCF-7 cells.The M1-CM induced migration and invasion were significantly suppressed by U0126(p<0.01).The protein levels of FN and Vim by M1-CM were inhibited by U0126.While the expression of E-cad protein was reversed(p<0.05).(8)The levels of Notch1 and its target protein Hes1 induced by M1-CM were inhibited by the ERK inhibitor U0126.Compared with the control group(M1-CM),both the protein levels of Notch1 and target Hes1 induced by M1-CM were inhibted by U0126(p<0.01),but the DAPT cann't inhibited the levels of p-ERK introduced by M1-CM(p>0.05).(9)The levels of Notch1 and its target Hes1 induced by M1-CM were down-regulated by Calpeptin and Calpain inhibitor III(p<0.05).Conclusion: M1-type macrophages can promote EMT in ER positive breast cancer cells,and its underlying signal mechanism may be related to the ERK/Calpain-Notch1 signaling pathway.