The Prognostic Value And Relevant Mechanisms Of Growth-arrest Specific Protein 6(Gas6)in Triple Negative Breast Cancer

Background and Purpose:Triple negative breast cancer(TNBC)is a heterogeneous disease.The prognoses are different among different subtypes.Currently,there is still no consensus clinical prognostic biomarker in patients with TNBC.Growth-arrest specific protein 6(Gas6)exerts mitogenic activity when bound to their receptors.Several studies have indicated that Gas6 plays a role in tumor invasion and metastasis in a number of cancers,and correlated to prognosis in variety of cancers.Vitro studies indicated that Gas6 production related to tumor-associated macrophages(TAMs).However,due to the lacking of relevant clinical study,it is not clear whether Gas6 is related to prognosis of TNBC.The aim of this study is to investigate the prognostic value of growth-arrest specific protein 6(Gas6)in triple negative breast cancer(TNBC),and the relationship between Gas6 expression and tumor-associate macrophages(TAMs)infiltration.Try to determine the relevant mechanisms of Gas6 in the development and progression of TNBC.Methods:Immunohistochemistry(IHC)was used to verify the Gas6 expression and stromal TAMs infiltration in 278 TNBC primary breast cancer tissues samples.Then,the correlations of Gas6 expressions,stromal TAMs infiltrations,clinicopathologic features and patients' survivals were analyzed.Epithelioid TNBC cell line MDA-MB-468 and mesenchyme-like cell line MDA-MB-231 were co-cultured with macrophages using transwell co-culture system.Real-time quantitative PCR(RT-q PCR)and Enzyme linked immunosorbent assay(ELISA)were used to detect the Gas6 expression differences before and after co-culture.We extract the supernatant A(epithelioid TNBC cell line MDA-MB-468 and mactophages co-culture system medium)and supernatant B(mesenchyme-like TNBC cell line MDA-MB-231 and mactophages co-culture system medium).We divided supernatant B into to two parts.Gas6 neutralizing antibody was added into one part.We added the three kind of supernatants into epithelioid TNBC cell line MDA-MB-468 medium,then used Western Blot to detect the epidermal mesenchymal transition(EMT)biomarkers(Vimentin and E-cadherin)expression of MDA-MB-468.Results:High Gas6 expression was significantly associated with lymph node metastasis(P=0.001)and TAMs infiltrations(P<0.001).However,no significant association was observed between Gas6 overexpression and age(P=0.734),tumor size(P=0.186),TNM stage(P=0.185),histologic grade(P=0.110),P53(P=0.100)and epidermal growth factor receptor(EGFR)(P=0.624).High levels of Gas6 expression were associated with worse overall survival(OS,P=0.002).Multivariate analysis indicated that Gas6 expression(Hazard Ratio(HR): 0.654,95% Confidence Interval(CI):0.432-0.990,P=0.045)?TNM stage(HR: 0.523,95%CI: 0.339-0.807,P=0.003)and EGFR(HR: 0.619,95%CI: 0.339-0.960,P=0.032)were independent predictor.The OS of patients in risk score 0-1 group was better than patients in risk score 2 group(P=0.001).RT-q PCR and ELISA indicated that mesenchyme-like TNBC cell line MDA-MB-231 induced macrophages Gas6 expression increased significantly.There is no significant Gas6 expression difference of macrophages before and after co-culture with epithelioid TNBC cell line MDA-MB-468.Western Blot showed vimentin expression of MDA-MB-468 was up-regulated and E-cadherin expression was down-regulated by supernatant B.There is no significant vimentin and E-cadherin expression difference of MDA-MB-468 before and after the addition of supernatan A.Conclusions:Mesenchymal-like TNBC cells induce upregulation of Gas6 expression significantly in TAMs.Gas6 is a poor prognostic factor for TNBC patients.Gas6 promote EMT of epithelial TNBC cells.Combined Gas6 with TAMs was a significant prognostic biomarker for patients with TNBC.