FOSL2 Promotes VEGF-Independent Angiogenesis By Regulating Wnt5a In Breast Cancer-Associated Fibroblasts

Objective: Tumor angiogenesis is a crucial process for the progression of solid tumors.Cancer-associated fibroblasts(CAFs),a predominant component in tumor microenvironment,can contribute to aggressive angiogenesis progression not only by VEGF-VEGFR signaling but also the VEGF-independent manner.FOSL2(Fos-like 2)is a member of the Fos family of AP-1 transcription factors which is often up-regulated in mammary carcinomas.Nonetheless,the role of FOSL2 in breast CAFs keep in unveil.To explore the mechanism of transcription factor FOSL2 in the activated stromal fibroblasts on breast tumor growth via VEGF dependent and(or)independent angiogenesis.Methods: FOSL2 expression was assessed by qRT-PCR,western blotting in primary and immortalized CAFs.Immunohistochemistry was used to determine the level and prognostic values of stromal FOSL2 in two breast carcinoma cohorts and correlation between microvessel density(CD31 a biomarker of microvessels)and stromal FOSL2 expression.CAFs or NFs were engineered to explore their proangiogenic effects.A co-cultured system including human umbilical vein endothelial cells(HUVECs)and CAFs was used to assess the effect of CAFs on theinvasion,tubule formation and three-dimensioned sprouting abilities.VEGF/VEGFR signaling was explored by measuring VEGF expression and blocking VEGF-VEGFR by anti-VEGF antibody and axitinib.Activation of FOSL2 upstream signaling pathway confirmed by activators and inhibitors.Bioinformatics,WB,CHIP and LUC assay were analyzed to verify the target secreted factor of FOSL2.Furthermore,the inhibitor of target secreted factor was added to the CM of FOSL2 overexpressed cells,and the human recombinant protein of the target secreted factor was added to the CM of FOSL2 knockdown cells.WB was used to detect the downstream signaling pathway in HUVEC mediated by FOSL2-target secretion factor signal axis.Orthotopic Xenografts was conducted to measure tumor volume and weight and analyze CD31 staining of tumor tissue in vivo.The target secretory factors in the serum of the patients were measured to consider their relationship with angiogenesis.Results: Here we revealed that FOSL2,a transcription factor in breast CAFs,played a critical role for stromal fibroblasts in VEGF-independent angiogenesis.FOSL2 was found to be abundantly expressed in breast CAFs and enhanced FOSL2 was significantly associated with angiogenesis and clinical progress of patients.The supernatant from FOSL2 high expressed CAFs had a strong ability to promote tube formation and sprouting of Human Umbilical VeinEndothelial Cells(HUVECs)under exhaustion of VEGF by neutralizing VEGF antibody or in the presence of Axitinib.Mechanistic analysis showed that expression of stromal FOSL2 in CAFs was regulated by estrogen/cAMP/PKA signaling,blockage of aromatase-induced estrogen/cAMP/PKA pathway diminished FOSL2 expression.Wnt5 a,a direct target of FOSL2,which evidently was approved by luciferase and chromatin immunoprecipitation(CHIP)assay,could specifically activate FZD5/NF-?B/ERK signaling in HUVECs to promote VEGF-independent angiogenesis.In addition,high level of Wnt5 a was commonly detected in the serum of breast cancer patients,and had a close correlation with microvessel density in breast tumor tissues.Conclusions: This work reveals that targeting FOSL2/Wnt5 a signaling axis in CAFs may offer a potential option for anti-angiogenesis therapy in breast cancer.