Osterix promotes the migration and angiogenesis of breast cancer by upregulation of S100A4 expression

Abstract

As a key transcription factor required for bone formation, osterix (OSX) has been reported to be overexpressed in various cancers, however, its roles in breast cancer progression remain poorly understood. In this study, we demonstrated that OSX was highly expressed in metastatic breast cancer cells. Moreover, it could upregulate the expression of S100 calcium binding protein A4 (S100A4) and potentiate breast cancer cell migration and tumor angiogenesis in vitro and in vivo. Importantly, inhibition of S100A4 impaired OSX-induced cell migration and capillary-like tube formation. Restored S100A4 expression rescued OSX-short hairpin RNA-suppressed cell migration and capillary-like tube formation. Moreover, the expression levels of OSX and S100A4 correlated significantly in human breast tumors. Our study suggested that OSX acts as an oncogenic driver in cell migration and tumor angiogenesis, and may serve as a potential therapeutic target for human breast cancer treatment.

Keywords: S100A4; angiogenesis; breast cancer; migration; osterix.

Figure 1

Effects of osterix (OSX) on breast cancer cell migration and endothelial cell tube formation. (A) Differential protein expression of OSX in invasive breast cancer cells T‐47D, MDA‐MB‐231 and MDA‐MB‐468, compared with weakly metastatic breast cancer cells MCF7, and non‐cancerous breast epithelium cells MCF 10A. (B) Stable OSX KD and overexpressing MDA‐MB‐231 cells were generated by lentivirus infection. The expression level of OSX was examined by immunoblotting. (C) Cell migration was assessed using a transwell assay. A representative morphological image is shown (left) and the percentage of migrated cells was determined (right). (D) Tube formation by HUVEC and EA.hy926 cells was measured and the results were expressed as the tubule length. Representative morphological images (left) and statistical results (right) are shown. (E) The effects of OSX on the expression levels of CD44, β‐catenin and VEGF were determined by western blotting analysis. The representative figures (left) and the densitometric analysis of the immunoreactive protein bands (right) are presented. Data represent the means ± SD of three independent experiments. CM, conditioned medium. **P < 0.01. ****P < 0.0001

Figure 2

Effects of osterix (OSX) on tumor angiogenesis in vivo. (A) Cells were mixed with matrigel and subsequently implanted onto chicken chorioallantoic membranes (CAMs), as described in Materials and methods. Representative images of angiogenesis on the CAMs are shown. Bar = 1 mm (left). The number of blood vessels was normalized to that of the respective control group, and the results are expressed as the means ± SD (n = 10, right). (B) Cells were mixed with matrigel and injected into right flank of nude mice. Seven days after implantation, gel plugs were collected and photographed (left, bar = 1 mm). Blood vessel formation was quantified by measuring the hemoglobin content using a Drabkin reagent kit 525 (right). (C) Hematoxylin and eosin staining analysis of histological features in plug tissues of nude mice. Arrows point to neovascularization. The right panel shows the quantification of the microvessel density. (D) Immunohistochemical staining analysis of the levels of CD31, CD34 and vascular endothelial growth factor (VEGF) in plug tissues of nude mice. The right panel shows the quantification of the CD31, CD34, and VEGF positive vessels. **P < 0.01. ****P < 0.0001

Figure 3

S100A4 is the target gene in osterix (OSX)‐induced cell migration and angiogenesis. (A) Mass spectrometric analysis of differentially expressed proteins in shOsx‐2, OE‐Osx‐6, and their respective control cells. A total of nine cancer‐related genes are shown. (B) The mRNA and protein expression levels of S100A4 in shOsx‐2, OE‐Osx‐6, and their respective control cells were determined by qRT‐PCR (left) and western blotting (right). (C) The expression levels of OSX and S100A4 were determined by western blotting analysis in shOsx cells transfected with a construct expressing S100A4 and in OE‐Osx cells treated with an S100A4‐siRNA. (D) Cell migration was assessed using a transwell assay. A representative morphological image is shown (left) and the percentage of migrated cells was determined (right). (E) Tube formation by HUVEC and EA.hy926 cells was measured and the results were expressed as the tubule length. Representative morphological images (left) and statistical results (right) are shown. (F) The expression levels of CD44 and vascular endothelial growth factor (VEGF) were determined by western blotting analysis. The representative figures (left) and the densitometric analysis of the bands (right) are presented. Data represent the means ± SD of three independent experiments. CM, conditioned medium. **P < 0.01. ****P < 0.0001

Figure 4

Effects of osterix (OSX) on the expression of migration and angiogenesis‐related genes in vivo. (A) Cells were mixed with matrigel and engrafted into the fourth inguinal mammary fat‐pads of nude mice. Six weeks later, the tumor samples were harvested. Immunohistochemical analysis was used to detect the expression levels of OSX and S100A4 in nude mice tumors. (B) Relative mRNA and proteins expression levels of OSX and S100A4 were determined by qRT‐PCR and western blotting analysis, respectively, in nude mice tumors. (C) Immunohistochemical staining analysis was used to detect the expression levels of CD44, β‐catenin, CD31, CD34 and vascular endothelial growth factor (VEGF) in nude mice tumors. (D) The protein expression levels of CD44, β‐catenin, CD31, CD34 and VEGF were analyzed by western blotting analysis in protein samples from nude mice tumors. *P < 0.05. **P < 0.01. ****P < 0.0001

Figure 5

Correlations between osterix (OSX) and S100A4 expression levels in breast cancer tissues. (A) Representative immunohistochemical peroxidase staining for OSX and S100A4 in breast cancer tissues (upper) and statistic data of OSX and S100A4 expression levels (lower). (B) Schematic diagram representing the role of OSX in cell migration and tumor angiogenesis in breast cancer