MiR-205/YAP1 Signaling In The Activated Fibroblasts Promotes Breast Tumor Growth And Metastasis Through VEGF-independent Angiogenesis

Objective: Angiogenesis plays a key role in tumor initiation and development.VEGF-VEGFR signal has been recognized to control angiogenesis process.However,it has been challenged by its intrinsic refractoriness and resistance of anti-VEGF and VEGFR TKI in clinic,and it indicates a VEGF-VEGFR independent mechanism in angiogenesis.Our previous data showed that microvesseles have positive relationship with cancer-associated fibroblasts(CAFs)in breast tumor tissues,mi R-205 was significantly downregulated in breast CAFs.To explore miR-205 and its down signaling activated CAFs and stimulate the angiogenic factors,thus influencing breast tumor growth and metastasis via VEGF dependent and(or)independent angiogenesis.Methods: Correlation between activated CAFs biomarker ?-SMA and microvessel density(CD31 a biomarker of microvessels)were accessed in different stage of breast cancer tumor tissues by IHC.A co-cultured system including human umbilical vein endothelial cells(HUVECs)and CAFs was used to assess the effect of CAFs on the proliferation,invation ad tubule formation abilities.Expression of miR-205 in 21 pairs of primary NFs and CAFs were measured by qRT-PCR.Target of miR-205 were determined by bioinformatics,WB and Luciferase reporter assay.The activated characteristics of CAFs were marked with proliferation,migration and invasion abilities.Tubule formation and three-dimensional sprouting assays were used to explore pro-angiogenic effect of CAFs and NFs on HUVECs.CAFs or NFs were engineered to explore fibroblasts' activated condition and its proangiogenic effects.VEGF/VEGFR signaling was explored by measuring VEGF expression and blocking VEGFR1/2/3 by axitinib.1× 106 CAF/Ctrl or engineered CAF cells mixed with 1× 106 MDA-MB-231 cells in 50 ?l PBS: Matrigel at a 1:1 ratio were subcutaneously injected into 4 to 6-week-old female nude mice;Tumor volume and weight were measured,lung metastasis were analyzed by HE and tumor tissue by HE and CD31 staining.Results: We observed abundant vessel-like structures in breast cancer stroma compared with its non-tumor stromal area by HE staining and CD31 staining.A positive correlation between intensity of ?-SMA and MVD was observed in the advanced-stage of breast tumor tissues.Here we found that activation of fibroblasts in breast tumor induced by miR-205,which is generally decreased in stromal CAFs and educated fibroblasts induced by breast cancer cells,acts a critical promoter to angiogenesis in a VEGF-independent manner.Inhibitor of miR-205 reprogramed NFs into CAFs to promote angiogenesis as displayed by tubule formation and sprouting of HUVECs,and rescue of mi R-205 in CAFs induced a functional conversion of CAFs and disrupted angiogenesis.YAP1,the target of miR-205,did not change VEGF expression but specifically regulated IL11 and IL15 expression,thus maintains the tumor angiogenesis even in the presence of Axitinib,an inhibitor of VEGFR.IL11 and IL15 released from CAFs were revealed to activate the STAT3 signaling in HUVECs.Loss of IL11 and IL15 in CAFs using specific siRNA or neutralizing antibodies result an inactivation of STAT3-signaling in HUVEC and repress the CAF-induced angiogenesis.The blunt angiogenesis halts the invasion and metastasis of breast cancer cells in vitro and in vivo.Conclusion: Our study demonstrates that the activated CAFs promote VEGF-independent proangiogenic process in breast tumor microenvironment.Secretion of IL11 and IL15 governed by miR-205/YAP1 in CAFs specifically stimulates STAT3 signaling in vascular endothelial cells to contribute tumor angiogenesis and resistance to anti-VEGF,thus trigger tumor growth and metastasis in breast cancer.