| Objective:Breast cancer(BC)is one of the most common malignant tumors,99%of which occur in women and 1%in men.Among the female malignant tumors in China,the incidence of breast cancer ranks first and the mortality ranks fifth.Until now,treatment of breast cancer is mainly surgery,supplemented by chemotherapy,radiotherapy,etc.,but there is still a high risk of recurrence after surgery.Therefore,it is equally important to find out the causes of recurrence and drug resistance for prevention and early diagnosis and treatment after operation.Some studies have found that Frizzled2(Fzd2)was highly expressed in many malignant tumors,which was related to the adverse prognosis of tumors,but the role of Fzd2 in breast cancer was not clear.The purpose of this study was to investigate the role of Fzd2 in breast cancer.Through the study of Fzd2 involvement in maintaining mesenchymal stemness of breast cancer cells and the signaling mechanism involved in maintaining this stemness,it was revealed that targeted Fzd2 may destroy this signaling network,thereby inhibiting the recurrence,metastasis and drug resistance of breast cancer.Methods:Immunohistochemistry was used to detect the expression of Fzd2 in 44 cases of breast cancer and 10 cases of paracancerous tissues,and the correlation between Fzd2and stage was analyzed.TCGA database was used to analyze the expression of Fzd2 in various types of breast cancer.Relationship between Fzd2 expression and survival was analyzed by Kaplan Meier website.CCLE andGSE12777 databases were used to analyze the correlation between Fzd2 and other genes in various breast cancer cell lines.Combination of Fzd2,Wnt5a/b and Wnt3 is analyzed by using Co-immunoprecipitation(co-IP).After Fzd2 was knocked down stably in MDA-MB-231 cells and BT-549 cells by transfecting with lentivirus,Western blot,immunofluorescence,wound healing experiment and Transwell experiment were used to detect changes of related signal pathway proteins,Epithelial-mesenchymal transition(EMT)related proteins and cell migration and invasion ability.After Fzd2 was knocked down stably in MDA-MB-231cells and MCF7/ADR cells,changes of cell stenness were detected by flow cytometry and sphere formation experiment,and changes of IC50 value of paclitaxel(PTX)and proliferation ability were detected by MTT method.MCF7 cells were stimulated by Adriamycin(Adr),Western blot was used to detect the expression of related pathway proteins.Data were analyzed by graphpad prism 7.0.Differences between two groups were compared by non paired t-test.The correlation between two genes was analyzed by Pearson correlation coefficient.Result:1.Fzd2 was highly expressed in breast cancer and was associated with poor prognosis.2.Expression of Fzd2 in MDA-MB-231,BT-549 and MCF7/Adr cells was higher than that in epithelioid breast cancer cells;Fzd2 could bind to Wnt5a/b and Wnt3by co-IP method;MDA-MB-231 and BT-549 cells could stably knock down Fzd2.The expression of Wnt5a/b and Wnt3 was lower than that of control group.3.Fzd2maintained interstitial phenotype of breast cancer cell lines.Expression of vimentin,epithelial-mesenchymal transition transcription factors Slug and Zeb1 in Fzd2-sh group was lower than that in control group,while the expression of E-cadherin was higher than that in control group.Expression of interstitial-related proteins Tenascin C(TNC),Col1a1 and Col6a1 were also Fzd2-sh in Fzd2-sh group.In Fzd2-sh group,the morphology of cells changed and the motor ability decreased.4.Fzd2 regulates the dryness of breast cancer cells.The subset of G protein-coupled receptor 5 rich in leucine repeat sequence positive(Lgr5~+)cells and the spherogenic ability(number and size of mammospheres)of breast cancer cells in Fzd2-sh group were lower than those in control group.5.Fzd2 functions by interacting with several carcinogenic pathways.Compared with the control transfection group,the expression levels of key molecules in several carcinogenic pathways,such as IL-6,p-Stat3,TGFβ1,Smad3 and yes-associated protein(Yap1),were all lower in Fzd2-sh group.In silico analysis showed that Fzd2 was positively correlated with these molecules mentioned above.6.Fzd2 endowed breast cancer cells with drug resistance.Expression of IL-6,p-Stat3,TGFβ1,Smad3 and Yap1were up-regulated after adriamycin stimulation at different concentrations.MTT test showed that compared with the control group,MDA-MB-231 cells and MCF7/Adr cells Fzd2 knockdown group had lower IC50 value on paclitaxel and slower growth rate.Conclusion:1.Ηigh expression of Fzd2 in breast cancer was related to the adverse prognosis of breast cancer.2.In vitro cell experiments showed that Fzd2 combined with Wnt5a/B and Wnt3 to activate IL-6/STAT3,Yap1 and TGF-β1/Smad3 signaling pathways.3.Fzd2 can induce cell migration and invasion,maintain cell mesenchymal-like phenotype,and improve drug resistance and stemness of breast cancer cells.4.Fzd2 may play a role through the non-canonical Wnt pathway,and involve many carcinogenic pathways,suggesting that targeting Fzd2 can potentially inhibit the metastasis and recurrence of breast cancer,and improve the chemosensitivity of breast cancer. |
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