Functional Investigation Of Anxa2 In Breast Cancer Epithelial-Mesenchymal Transition And Metastasis

Objectives Metastasis is responsible for most of therapeutic failure and cancer-associated mortality including breast cancer.Epithelial-mesenchymal transition(EMT)has been proved to be the key event in cancer metastasis and epidermal growth factor receptor(EGFR)signaling plays an important role in EMT.Overexpression of annexin A2(Anxa2)has been reported to be associated with increased metastasis and poor prognosis in many types of carcinomas,however,the clinical significance and detailed mechanism of Anxa2 in breast cancer progression remains unclear.Recent studies reported Anxa2 is involved in some EMT processes and in EGFR signaling activation,which suggest the possible role of Anxa2 in EGFR signaling induced EMT.In the present study,we aimed to determine the prognostic effect of Anxa2 in breast cancer,and investigate its role in EGF induced EMT.Methods 1.Immunohistochemistry(IHC)and Western blotting were used to detect expression of Anxa2,EGFR and EMT markers in breast cancer tissues and cell lines.2.Knockdown Anxa2 expression by lentivirus mediated sh RNA to confirm the role of Anxa2 in EGF induced EMT,and rescue experiments using Anxa2 mutants were carried out.3.Wound healing assay and Transwell cell invasion assay were used to explore the migration and invasion ability in vitro,and SCID mice were used as animal models for analysis of metastasis ability in vivo.4.Western blotting and immunofluorescence were used to analyze the effect of Anxa2 on STAT3 phosphorylation and activation.Then STAT3 was knocked down by sh RNA to identify the role of STAT3 in this EMT model.5.Co-immunoprecipitation and Dual-luciferase reporter assay were performed to affirm the interaction between Anxa2 and STAT3,and to identify the mediated effect of Anxa2 on transcription activity of STAT3.Results 1.Elevated expression of Anxa2 is correlated with worse pathological parameters and outcome,and is correlated with EGFR and EMT markers.2.EGF-induced EMT is inhibited by Anxa2 knockdown and depends on 23 tyrosine phosphorylation of Anxa2.Depletion of Anxa2 expression impaired migration and invasion abilities of T47 D cells in vitro,and decreases metastasis ability of T47 D cells in vivo.3.Anxa2 is essential for EGF-induced STAT3 phosphorylated activation and translocation in nucleus,and STAT3 is required for EGF-induced EMT in breast cancer cells.4.Anxa2 has a direct interaction with STAT3,the N-terminal fragment of Anxa2 had stronger interaction with C-terminal domain of STAT3.5.Anxa2 enhanced the transcription activity of STAT3,the N-terminal fragment of Anxa2 containing Tyr 23 phosphorylation site had the strongest enhancive effect.Conclusion This study identified Anxa2 as a predictor of poor outcomes for breast cancer patients.We demonstrated in vitro and in vivo that Anxa2 promoted breast cancer metastasis through its essential function in mediating EGFR signaling-triggered EMT.The modulatory effect of Anxa2 on EMT depends on its phosphorylation at 23 tyrosine.Anxa2 directly interacts with STAT3 and regulates STAT3 phosphorylation and nuclear translocation,finally enhancing the transcription activity of STAT3,which results in the upregulation of EMT driver Slug.The EGFR/Anxa2/STAT3/Slug pathway is a novel demonstration of the EMT process in breast cancer.Anxa2 may be a good therapeutic target for the inhibition of breast cancer metastasis and reversal of anticancer therapies.