Error bars are defined as s.d. To further verify the role of the BMP/SMAD signaling pathway in mediating the promotion of breast cancer bone-specific metastasis by FOXF2, we established stable MDA-MB-231 cells with FOXF2 overexpression (231-FOXF2) or vector control (231-Vector) via lentiviral contamination for mouse xenograft experiments by ventricle injection. of bone differentiation. The epithelial-to-osteomimicry transition regulated by FOXF2 confers a tendency on cancer cells to metastasize to bone which leads to osteolytic bone lesions. The BMP antagonist Noggin significantly inhibits FOXF2-driven osteolytic bone metastasis of breast cancer cells. Thus, targeting the FOXF2-BMP/SMAD axis might be a promising therapeutic strategy to manage bone metastasis. The role of FOXF2 in transactivating bone-related genes implies a biological function of FOXF2 in regulating bone development and remodeling. expression is usually correlated with bone-specific metastasis To investigate the role of AZD4017 FOXF2 in breast cancer bone metastasis, we first analyzed the expression pattern in the luminal and triple-negative/basal-like subtypes of breast cancer based on the GSE12777_GSE15026_GSE65194 data set of human breast cancer cell lines and the E-MTAB-365 and “type”:”entrez-geo”,”attrs”:”text”:”GSE3494″,”term_id”:”3494″GSE3494 data sets of primary breast cancer tissues. The results confirmed our previously published result that mRNA levels were significantly higher in triple-negative/basal-like subtype than in luminal subtype in both cell lines (Fig.?1a) and tissues (Fig.?1b). Then, we analyzed the relationship between expression and organ specificity of metastasis in the luminal and triple-negative subtypes of breast cancer. mRNA levels in primary breast cancer tissues Rabbit Polyclonal to POLE1 that developed distant metastasis were detected by reverse transcriptionCquantitative polymerase chain reaction (RT-qPCR). The patients were divided into high mRNA level (mRNA level (mRNA expression for distinguishing bone metastasis-free survival (BMFS) statuses in overall cases and cases stratified by subtypes. KaplanCMeier survival analysis showed that bone metastasis was a?more frequent occurrence in patients in expression and distant metastasis-free survival (DMFS) or non-bone/other organ metastasis-free survival (NBMFS) in overall cases and in different subtype cases based on our RT-qPCR data of primary breast cancer tissues. The results showed that mRNA level was positively correlated with DMFS in TNBC subtype and with NBMFS in both luminal and TNBC subtypes (Supplementary Fig.?1). These data indicate that breast cancer with high FOXF2 expression has a propensity to metastasize to bone, which is not affected by hormone receptor status. Open in a separate window Fig. 1 Breast cancers with high expression have a propensity to metastasize to bone. aCb expression levels in the luminal and basal-like/triple-negative subtypes of human breast cancer were compared by chi-square assessments. mRNA levels were mined from the GSE12777_GSE15026_GSE65194 data set of breast cancer cell lines (mRNA levels in primary breast cancer tissues that developed distant metastasis (mRNA levels in primary breast cancer tissues (test. Error bars are defined as s.d. FOXF2 enhances bone-specific metastatic potential To investigate the role of FOXF2 in regulating various processes underlying breast cancer bone metastasis, we forced the ectopic expression of FOXF2 in MCF-7 cells and overexpressed or knocked down FOXF2 in MDA-MB-231 cells. The cancer cells with altered FOXF2 expression were evaluated in vitro for chemotactic migration, heterogeneous cellCcell adhesion, and soft agar colony formation in the MC3T3E1 cell-mimic bone microenvironment and BEAS-2B cell-mimic lung microenvironment. The results revealed that this chemotactic migration of MCF-7 and MDA-MB-231 cells toward MC3T3E1 cells (Fig.?3a), heterogeneity adhesion to MC3T3E1 cells (Fig.?3b), and anchorage-independent growth in soft agar with conditioned medium (CM) from MC3T3E1 (Fig.?3c) AZD4017 were significantly increased by forced expression of FOXF2 and decreased by knockdown of FOXF2. In contrast, these properties of TNBC/BLBC MDA-MB-231 cells were suppressed by FOXF2 overexpression and increased by FOXF2 knockdown in the AZD4017 BEAS-2B cell-mimic lung microenvironment. However, forced ectopic expression of FOXF2 did not affect these capabilities of luminal breast cancer MCF-7 cells in the BEAS-2B cell-mimic lung microenvironment (Fig.?3aCc). Since pulmonary fibroblasts and hepatic stellate cells are the most abundant stromal cell types in the lung and liver, primary human pulmonary fibroblasts (HPFs) and human hepatic stellate cells (HHSCs) were also used to mimic the lung and liver microenvironment to evaluate the lung and liver metastatic potential of the above cancer cells. We observed that FOXF2 negatively regulated the chemotactic migration, heterogeneous cellCcell adhesion, and soft agar colony formation of MDA-MB-231 cells in the HPF-mimic lung microenvironment and HHSC-mimic liver microenvironment. Surprisingly, forced ectopic expression of FOXF2 enhanced these capabilities of MCF-7 cells in the HPF-mimic lung microenvironment and HHSC-mimic liver microenvironment (Supplementary Fig.?3aCc). Open in a separate window Fig. 3 FOXF2 enhances the bone-specific metastasis potential of breast cancer cells. MCF-7 and MDA-MB-231 cells were treated as indicated. a Chemotactic migration of cancer cells toward MC3T3E1 or BEAS-2B cells was assessed by transwell assays. b Adhesion of cancer cells to MC3T3E1 or BEAS-2B cells was assessed by.