The Mechanism Of FOXF2 In Regulating The Activation Of Breast Cancer Stromal Fibroblasts

Backgroud The breast cancer stromal fibroblasts can be activated into carcinoma-associated fibroblasts(CAF)during the progression of cancer,and they have dual function in tumor promotion and suppression.Taking the tumor stromal cells as target and conditioning the tumor microenvironment can raise the therapeutic effect.Forkhead box F2(FOXF2)is a member of FOX transcriptional factors family,and is specifically expressed in mesenchymal cells which adjacent to the epithelial cells.Our previous research demonstrated that the transcription of ACTA2 was negatively regulated by FOXF2 and then induced the activation of fibroblast.We further found that FOXF2 could bind to the promoter of FGF1 and PDGFD gene and inhibit its transcription.In addition,FOXF2 deficiency could upregulate the expression of PDGFR,and provide the target to Dasatinib,which is a inhibitor of tyrosine kinase.Therefore,we suppose that FOXF2 deficiency can activate the breast cancer stromal fibroblasts by negatively regulating the transcription of FGF1 and PDGFD,and reducing the activity of PDGFR by Dasatinib can inhibit the activation of fibroblasts induced by FOXF2 deficiency.Objective The objective of this sdudy is to clarify the role and mechanism of FOXF2 in activation of breast cancer stromal fibroblast and to provide a strategy of targeted therapy of breast cancer.Methods 1.The CAF and NF were isolated from the primary breast cancer tissues and the paired adjacent normal breast tissue,respectively.The morphology of CAF and NF was observed by microscope.The markers of epithelial and mesenchymal cells were detected by western blot.The localization of α-SMA in NF and CAF was detected using immunofluorescence staining.And to determine the relationship between FOXF2 and activation of fibroblast in tumor microenvironment of breast cancer tissues,and the mRNA and protein expression of FOXF2 were detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR)and western blot,respectively.And to analyze the correlationship of the expression of mRNA and histological grade and TNM stage,FOXF2 mRNA and ACTA2 mRNA in NF and CAF were detected by RT-qPCR.2.To determine the effect of FOXF2 on the fibroblasts activation,silence expression by lipofection transient transfection with FOXF2 RNAi in NF and MRC-5 and Foxf2 RNAi in NIH3T3 and overexpression by lipofection transient transfection with pc DNA3.1-HA-FOXF2 plasmids in CAF.The expression levels of CAF markers were detected by RT-qPCR and western blot.F-actin staining and collagen gel contraction assay were used to detect the abilities of motility and contraction of NF and CAF.To determine the role of FOXF2 in the activation of fibroblasts.3.Chromatin immunoprecitation(Ch IP)were used to validate the transcriptional regulation of FOXF2 on genes associated with activation of fibroblast.Dual luciferase reporter assay the influence of FOXF2 on transcription ability of FGF1 and PDGFD.RT-qPCR was used to detect the mRNA expression of FGF1,FGF2,PDGFA,PDGFB,PDGFC,PDGFD and CXCL12 in FOXF2-silenced and FOXF2-overexpressed fibroblast.To determine the transcriptional role of FOXF2 to FGF1 and PDGFD.4.NIH3T3-si Foxf2 were treated with Dasatinib(10 n M).F-actin staining and collagen gel contraction assay were used to detect the abilities of motility and contraction of NIH3T3-si Foxf2 after treated with Dasatinib.Immunofluorescence staining was used to detect the localization of α-SMA in NIH3T3-si Foxf2 after treated with Dasatinib.To explore the role of Dasatinib in the fibroblasts activation which is transcriptionally regulated by FOXF2.Results 1.The isolation and primary culture of CAF and NF with digestion method were performed successfully,and the CAF and NF expressed the markers of stromal cell.The FOXF2 mRNA level of CAF was downregulated than the level of the paired NF(n = 8,P < 0.05).The FOXF2 protein level was lower in CAF than in the paired NF.The depletion of FOXF2 was associated with the activation of fibroblasts.2.The mRNA levels of ACTA2,FAP and TNC in NF-si FOXF2,MRC-5-si FOXF2 and NIH3T3-si Foxf2 were upregulated than in the control cells,respectively.And the protein levels of α-SMA,PDGFRα and PDGFRβ in NF-si FOXF2 and NIH3T3-si Foxf2 were severally higher than in the control cells,however,the levels of above proteins were downregulated in CAF of which the FOXF2 was overexpressed.The surface area of collagen gel was smaller and F-actin was arrange into fibrous bundles after FOXF2 or Foxf2 gene expression silencing.And we found that FOXF2 deficiency could induce the activation of fibroblast,including upregulating the expression of the markers of fibroblast activation and enhancing the abilities of contraction and motility of fibroblast.3.Ch IP assay showed that FOXF2 could bind to the promoter region of FGF1,FGF2,PDGFC and PDGFD.Dual luciferase reporter system verify that FOXF2 could inhibit transcription ability of FGF1 and PDGFD.And the mRNA expression of FGF1,PDGFC,PDGFD and CXCL12 were negatively related to FOXF2 expression in NF-FOXF2-depleted or CAF-FOXF2-overexpressed.4.The abilities of contraction and motility were lower in NIH3T3-si Foxf2 after treatment of Dasatinib than in control cells.Conclusions FOXF2 can induce the activation of breast cancer stromal fibroblast by the transcriptional negative regulation,and FOXF2 deficiency can upregulate the levels of mRNA expression of FGF1,PDGFC,PDGFD and CXCL12.Dasatinib can inhibit the activity of PDGFR and inhibit the activation of fibroblast arised from FOXF2 deficiency.