The Mechanism Of FOXF2 In Regulating Beast Cancer Bone Metastasis

Background Bone is a common distant metastasis organ for breast cancer.Breast cancer bone metastasis leads to osteolytic lesions,and seriously affects the quality of life and survival of the patients,usually.To elucidate the molecular mechanism of breast cancer osteolytic bone metastasis is helpful to develop early diagnosis and treatment of bone metastasis.Our study group confirmed that the transcription factor Forkhead F2 box(FOXF2)promoted breast cancer bone metastasis.In order to investigate the molecular mechanism of FOXF2 regulating breast cancer bone metastasis,the correlation analysis of gene expression profiles of breast cancer tissues was carried out and the results showed that FOXF2 was related to a set of genes related to bone remodeling,including cathepsin K(CTSK)and urokinase type plasminogen activator(u PA or PLAU).Furthermore,we analyed FOXF2 binding sequence and found multiple FOXF2 binding domains in CTSK and PLAU promoter regions.These results suggested that CTSK and PLAU are potential target genes for FOXF2 transcriptional regulation.Based on the reporters that CTSK and PLAU can promote osteolytic breast cancer bone metastasis,We speculated that FOXF2 may promote the osteolytic bone metastasis of breast cancer by transcriptional regulation of CTSK and PLAU.Purposes The aim of this study was to determine the role of FOXF2 in the regulation of CTSK and PLAU,and the mediating role of CTSK and PLAU in the regulation of osteolytic bone metastasis in breast cancer by FOXF2.Methods 1.The correlation between the level of FOXF2 m RNA and CTSK m RNA/PLAU m RNA expression was analysed in different subtypes of breast cancer,through the public database(GEO: www.nvbi.nlm.nih.gov/geo/info,Accession Number: and GSE11121 GSE20685).2.The binding sites of FOXF2 in CTSK and PLAU promoter was determined by Chromatin immunoprecipitation(Ch IP)assay.The regulatory effect of FOXF2 on the promoter of CTSK and PLAU was confirmed by double luciferase reporter system.To demonstrate the regulation of FOXF2 on CTSK and PLAU in breast cancer cells,we increased or reduced FOXF2 expression in MCF-7,T47-D and MDA-MB-231,and the change of CTSK and PLAU protein levels was mensurated by Western blot.3.We increased the expression of FOXF2 and decreased the expression of CTSK or PLAU,or decreased the expression of FOXF2 and increased the expression of CTSK or PLAU in MDA-MB-231.In vitro those breast cancer cells were used to simulate chemotaxis assay in bone microenvironment induced by mature MC3T3-E1,adhesion assay,Bone matrix protein degradation assay,and the regulation of differentiation and maturation ability of osteoclast precursor cells.4.We want to analyze the difference of the expressions of CTSK and PLAU m RNA expression in bone metastases and non bone metastases and whether the m RNA levels of FOXF2 and CTSK or PLAU can predict the risk of bone metastasis in breast cancer patients,by a public database(GEO:www.nvbi.nlm.nih.gov/geo/info,Accession,Number: GSE14020).Results 1.The expression of FOXF2 m RNA is positively correlated with the expression of CTSK and PLAU m RNA in different subtypes breast cancer.2.We found that FOXF2 has two binding sites(?586/?579 and ?732/–725)in CTSK promoter,and three binding sites(?1082/?1075,?1109/?1102 and ?1784/?1777)in PLAU promoter.The activity of CTSK and PLAU promoter was decreased in MDA-MB-231 with lower expression level of FOXF2 and the activity of CTSK and PLAU promoter was increased in MCF-7 with higher expression level of FOXF2.3.The protein expression levels of secreted and intracellular CTSK and PLAU were reduced in MDA-MB-231 with lower expression of FOXF2,and was enhanced in MDA-MB-231 with higher expression of FOXF2,but,it was not change at all in MCF-7 or T47 D with higher expression of FOXF2.4.The chemotactic migration ability of MDA-MB-231 cells to bone mimic microenvironment created by mature MC3T3-E1 cells was enhanced when the expression of FOXF2 was increased and it was reduced when the expression of CTSK or PLAU was depressed in MDA-MB-231 with lower expression of FOXF2.What's more,the chemotactic migration ability of MDA-MB-231 cells to lung mimic microenvironment created by BEAS-2B cells was weakened after the expression of FOXF2 was increased,but it was not changed when the expression of CTSK or PLAU was depressed in MDA-MB-231 with higher expression of FOXF2.5.The adhesion ability of MDA-MB-231 cells with the lower expression of FOXF2 to osteoblasts and bone matrix proteins was decreased.It was not changed when the expression of CTSK was increased but enhanced when the expression of PLAU was increased in MDA-MB-231 with lower expression of FOXF2.What's more,.The adhesion ability of MDA-MB-231 cells with the higher expression of FOXF2 was enhanced.It was not changed when the expression of CTSK was decreased but depressed when the expression of PLAU was decreased in MDA-MB-231 with higher expression of FOXF2.6.The ability of bone matrix degradation of MDA-MB-231 was weakened when the expression of FOXF2 was reduced and it was enhanced after the expression of CTSK or PLAU was increased in MDA-MB-231 with lower expression of FOXF2.However,the ability of bone matrix degradation of MDA-MB-231 was enhanced when the expression of FOXF2 was increased,and it was weakened after the expression of CTSK or PLAU was depressed in MDA-MB-231 with higher expression of FOXF2.7.The ability that the osteoclast was induced maturation by conditioned medium of MDA-MB-231 was reduced when the expression of FOXF2 was reduced,and it was increased after the expression of CTSK or PLAU was increased in MDA-MB-231 with lower expression of FOXF2.But,the ability was enhanced when the expression of FOXF2 was increased,and it was weakened after the expression of CTSK or PLAU was depressed in MDA-MB-231 with higher expression of FOXF2.8.The expression levels of CTSK and PLAU in breast cancer bone metastases(n = 18)were higher than those in non-bone metastases(n = 94,P < 0.0001)and breast cancer patients with FOXF2 plus CTSK(n = 200,P = 0.0025)or PLAU(n = 125,P = 0.0054)m RNA high expression levels have the shortest bone metastasis free survival than other groups.Conclusions 1.CTSK and PLAU are target genes of FOXF2,and can be transcriptional activated by FOXF2 in BLBC cells,but,there is no transcriptional activation in luminal subtype breast cancer cells.2.FOXF2 promotes the colonization of breast cancer cells in bone microenvironment,degradation of bone matrix,and induction of osteoclast differentiation and maturation,by transcriptional regulating the expression of CTSK and PLAU.3.The m RNA levels of FOXF2 and CTSK or PLAU can predict the risk of bone metastasis in breast cancer patients.