The Role And Mechanism Of FOXF2 In Breast Cancer Bone Metastasis

BackgroudPatients with advanced breast cancer prone to metastasize to bone,causing a variety of bone complications,which seriously affects the patient’s quality of life.Clarifying the possible molecular mechanism of breast cancer bone metastasis could contribute to find novel strategies for predicting bone metastasis risk and treatment of bone metastases.Forkhead box F2(FOXF2)is a key transcriptional regulator during the process of embryonic development and tissue differentiation specificly expressed in mesenchymal cells,which promotes the differentiation of mesenchymal cells and inhibits the transformation of adjacent epithelial cells to maintain tissue homeostasis.The previous study of our group found that FOXF2 expression was positively correlated with a set of bone and bone matrix remodeling genes in primary breast cancer tissue,and breast cancer patients with higher FOXF2 expression have higher incidence of bone metastasis,suggesting that FOXF2 may promote breast cancer bone metastasis.To further study the regulation mechanism of FOXF2 in the regulation of bone metastasis of breast cancer,bone morphogenetic protein 4(BMP4)was selected as a potential downstream target gene.BMPs signaling pathway is the key signal pathway in the process of bone matrix remodeling.Therefore,we hypothesized that FOXF2 might promote the bone metastasis of breast cancer by up regulating the BMPs signaling pathway.PurposesThe purpose of this study was to elucidate the role of FOXF2 in bone metastasis of breast cancer and its molecular mechanism to look for novel molecular diagnostic methods and treatment strategies for breast cancer patients with bone metastases.Methods1.Based on the gene expression profile data of primary breast cancer tissue in our dataset and published database,as well as m RNA detected by RT-QPCR,the correlation coefficient between expression of FOXF2 m RNA and a set of bone and bone matrix remodeling genes were analyzed by Pearson correlation coefficient analysis.Kplan Meier survival curves and log rank analysis were used to analyze the correlation of FOXF2 m RNA expression levels and bone metastasis free survival(BMFS).The K-mean clustering analysis was used to analyze the average expression of FOXF2 and bone and bone matrix remodeling related gene level,score level,with the risk of bone-only metastasis in primary breast cancer tissues and in in different organ metastases.2.To overexpress exogenous FOXF2 or knockdown of endogenous FOXF2 expression,the breast cancer cells transfected with FOXF2 instantaneous recombinant eukaryotic expression plasmid of pc DNA3.1-HA-FOXF2 full-length c DNA(FOXF2-HA)and the control plasmid(Vector)or transfected with specific small interfering RNA(si RNA)targeting FOXF2(si FOXF2)and control si RNA(si Control),respectively;Collecting the supernatant from cultured osteoblast-like osteosarcoma cells MG-63 or normal lung epithelial cells BEAS-AB to coculture with breast cancer cells to mimic bone and lung tissue microenvironment in vitro,respectively.To identify the effect of FOXF2 on bone metastasis biological behavior of breast cancer cells in vitro,cell chemotaxis,cell adhesion,anchorage-independent colony formation assay in a mimic bone/lung microenvironment as well as osteoclastogenesis coculture assay were carried out.Western blot was used to detect expression of breast cancer bone and bone matrix remolding related proteins,and key cytokines during osteoblast and osteoclast differentiation.3.According to literature reports,we selected bone metastasis related signaling factors of bone morphogenetic protein 4(BMP4)as a potential target of FOXF2 from genes encoding osteogenic differentiation and bone remodeling.RT-QPCR and western blot were used to analyze the expression changes of BMP4 m RNA and protein levels.Western blot and IF were used to detect p-Smad1 expression level and nuclear localization state,the BMP4 signaling pathway downstream protein.4.To validate FOXF2 promoted breast cancer bone metastasis by the induction of bone and bone matrix remolding related genes in a BMP4 dependent manner,BMP4 cytokine was used to rescue si FOXF2 cells,or cells with FOXF2 over-expressing was rescued by BMP4 signaling pathway inhibitor noggin(NOG),and then the cancer cells were subjected to in vitro cell chemotaxis,adhesion,colony formation assays in mimic bone/lung microenvironment,as well as osteoclastogenesis coculture assay.Bone and bone matrix remolding related genes and osteoclastogenesis related genes in cancer cells,and osteoblastogenesis related genes in cocultured osteoblasts were detected by RT-QPCR and Western blot analysis.5.To validate correlation between FOXF2 and BMP4,as well as significance of BMP4 in predicting bone metastasis(BMFS)in clinical,pearson’ correlation,Kplan Meier survival curve and Log-rank analysis were performed in published database in patients with breast cancer.BMP4 m RNA expression level between primary breast cancer tissues patients with bone-only metastasis and non-bone metastasis,as well as between bone metastasis tissue non-bone metastasis tissue were analyzed.Results1.FOXF2 m RNA was positively correlated with a set of bone and bone matrix remodeling genes(39 genes,R > = 0.3)in our microarray dataset and GOBO database,31 of which were the same genes.In organ metastasized breast cancer patients,those who with high FOXF2 m RNA expression level in primary breast cancer tissue had shorter BMFS compared with patients with low FOXF2 m RNA expression level detected by RT-QPCR as shown in total(n = 33,P = 0.05),luminal(n = 21,P = 0.06)and triple negative(n = 12,P = 0.02)subtypes;FOXF2 m RNA was positively related with ITGBL1 and POSTN m RNA expression.It was further confirmed by the positive correlation between FOXF2 and ITGBL1,POSTN,and RUNX2 m RNA expression in public available access database.BMFS analysis revealed that high FOXF2 m RNA level was an independent prognostic predictor for breast cancer bone metastasis.Bone-only metastasis is more likely to occur in patients with high m RNA level of FOXF2 plus bone and bone matrix remolding-related genes evidenced by K mean clustering.FOXF2 plus bone and bone matrix remolding-related genes m RNA level were higher in bone metastases than other metastases.2.FOXF2 over-expression augmented chemotaxis,adhesion,and anchorage independent colony formation,and osteoblastogenesis and osteoclastogenesis abilities abilities in MDA-MB-231 and MCF-7 cells cocultured in the bone mimic microenvironment,whereas MDA-MB-231-FOXF2 cells in mimic lung microenvironment showed decreased chemotaxis and colony formation ability,but had no effect on MCF-7-FOXF2 cells compared with control;Instead,FOXF2-depleted MDA-MB-231 cells displayed reduced malignant phenotype in mimic bone microenvironment,while the chemotaxis and colony formation abilities were enhanced.FOXF2 over-expressed breast cancer cells upregulated expression levels of various bone and bone matrix remodeling genes,and osteoblast and osteoclast differentiation related molecular,wheras silencing of endogenous FOXF2 expression down-regulated expression of these genes in breast cancer cells.3.BMP4 expression is positively related to FOXF2 expression in FOXF2-depleted or overexpressed MDA-MB-231 and MCF-7 cells.Furthermore,silencing FOXF2 expression decreased Smad1 nuclear translocation detected by IF.4.BMP4 partly rescued FOXF2-depleted reduced chemotaxis,adhesion,anchorage independent colony formation,and osteoclastogenesis abilities in cells co-cultured within the bone mimic microenvironment,upregulated FOXF2-depleted repressed expression of bone and bone matrix remodeling genes,while did not affect chemotaxis and anchorage independent colony formation of cells in lung mimic microenvironment;Instead,NOG rescued FOXF2 over-expression induced malignant biological behavior of cells in bone mimic microenvironment and the expression of bone and bone matrix remodeling genes,but had little influence on chemotaxis and anchorage independent colony formation of cells cultured in lung mimic microenvironment.5.In public database,the FOXF2 m RNA level was positively correlated with BMP4(n = 327,p = 0.01);BMP4 m RNA expression level is higher in primary cancer of breast cancer patients with bone-only metastasis than that of non-bone metastasis(n = 368,p = 0.05);Besides,BMP4 m RNA expression level in bone metastasis foci is higher than in other organ metastases(n = 65,p = 0.05).Breast cancer patients with high BMP4 m RNA expression levels in primary tissues have shorter BMFS(n = 368,p = 0.045)than those with low BMP4 m RNA level.Patients with FOXF2 plus BMP4 m RNA high expression level have the shortest BMFS than other groups(n = 368,p = 0.022).Conclusions1.FOXF2 was ectopically co-expressed with a set of bone and bone matrix remolding-related genes,which confer cells with “osteomimicry” phenotype through upregulating expression of these genes.2.The ectopic high expression of FOXF2 promote bone metastasis of breast cancer cells confirmed by study of clinical cases,and in vitro cytological experiments.3.BMP4 is a target gene of FOXF2;BMP4,activated by FOXF2,promotes high expression of bone and bone matrix remodeling genes in breast cancer cells in an autocrine manner to confer cancer cells with osteomimicry phenotype,and elevated chemotactic migration,colonization,clonal formation capacities in the bone microenvironment and enhanced osteoclastogenesis capacity.Thus,FOXF2 may promote the bone metastatic potential of breast cancer through upregulation of BMP4.4.FOXF2 and BMP4 m RNA are highly expressed in primary breast cancer tissue with bone-only metastasis,which could predict risk of bone metastasis;The high expression of FOXF2 combined with BMP4 could better predict risk of bone metastasis with breast cancer patients than each alone.