FOXP3 inhibits angiogenesis by downregulating VEGF in breast cancer

Abstract

Forkhead box P3 (FOXP3), an X-linked tumor suppressor gene, plays an important role in breast cancer. However, the biological functions of FOXP3 in breast cancer angiogenesis remain unclear. Here we found that the clinical expression of nuclear FOXP3 was inversely correlated with breast cancer angiogenesis. Moreover, the animal study demonstrated that FOXP3 significantly reduced the microvascular density of MDA-MB-231 tumors transplanted in mice. The cytological experiments showed that the supernatant from FOXP3-overexpressing cells exhibited a diminished ability to stimulate tube formation and sprouting in HUVECs in vitro. In addition, expression of vascular endothelial growth factor (VEGF) was downregulated by FOXP3 in breast cancer cell lines. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that FOXP3 can directly interact with the VEGF promoter via specific forkhead-binding motifs to suppress its transcription. Importantly, the inhibitory effects of FOXP3 in the supernatant on tube formation and sprouting in HUVECs could be reversed by adding VEGF in vitro. Nuclear FOXP3 expression was inversely correlated with VEGF expression in clinical breast cancer tissues, and FOXP3 downregulation and VEGF upregulation were both correlated with reduced survival in breast cancer data sets in the Kaplan-Meier plotter. Taken together, our data demonstrate that FOXP3 suppresses breast cancer angiogenesis by downregulating VEGF expression.

Fig. 1. FOXP3 expression is negatively associated with angiogenesis in cancer.

a GO analysis showing several clusters of blood vessel-related genes that may be regulated by FOXP3 in bladder cancer. b Representative nuclear FOXP3 expression and immunohistochemical images of blood vessels in breast cancer samples. Blood vessel density is shown by CD31 staining. Scale bars, 50 μm (×20) and 20 μm (×40). c A significant negative correlation between nuclear FOXP3 and blood vessel density was found in breast cancer specimens. c Student’s t test

Fig. 2. FOXP3 inhibits breast cancer angiogenesis in vivo and in vitro.

a Orthotopic injection of MDA-MB-231 cells was performed to generate xenografts, and adenoviruses carrying FOXP3 or control cDNA were injected into the tumors when their volume reached approximately 50 mm³ (n = 5). Representative immunohistochemical images of FOXP3 and CD31 expression in the primary tumors of mice in the control and FOXP3 groups. Scale bars, 50 μm (×20) and 20 μm (×40). b Quantification of blood vessel density in (a). c Representative images of HUVEC tube formation assays; HUVECs were treated with control MDA-MB-231 cell supernatant or FOXP3-overexpressing MDA-MB-231 cell supernatant. Scale bar, 100 μm. d Quantitation of the cumulative number of tubes in the different groups in (c). e Representative images from the HUVEC spheroid sprouting assays; HUVECs were treated with control cell supernatant or FOXP3-overexpressing MDA-MB-231 cell supernatant. Scale bar, 100 μm. f Quantitation of the cumulative sprout length (CSL) of the different groups in (e). b, d, f Student’s t test

Fig. 3. FOXP3 is a transcriptional suppressor of VEGF.

a FOXP3 suppresses VEGF promoter activity, as evaluated by dual-luciferase reporter assays. b The top panel depicts schematic diagrams of the regions amplified by the ChIP primers. The bottom panel shows the amount of DNA precipitated by either the anti-FOXP3 antibody or control IgG; the results are expressed as a percentage of the input genomic DNA from MDA-MB-231 cells. c FOXP3-mediated suppression of the VEGF promoter requires forkhead-binding motifs, as evaluated by dual-luciferase reporter assays. Relative truncation of the VEGF promoter is illustrated in the left panel. Luciferase activity was detected in cells transfected with the truncated VEGF promoter in the right panel. a, c ANOVA with Tukey’s post hoc test

Fig. 4. FOXP3 downregulates VEGF expression in breast cancer.

Real-time PCR was performed to detect the transcription levels of VEGF in breast cancer cell lines with the gain or loss of FOXP3 expression. Transfection of pcDNA3.1-FOXP3 into (a) MCF-7, (b) T47D or (c) MAD-MB-231 cells. Transfection of FOXP3 shRNA into (d) MCF-7 or (e) T47D cells. Western blotting was performed to detect the expression of VEGF in breast cancer cell lines following the gain or loss of FOXP3 expression. Transfection of pcDNA3.1-FOXP3 into (f) MCF-7, (g) T47D or (h) MAD-MB-231 cells. Transfection of FOXP3 shRNA into (i) MCF-7 or (j) T47D cells. k Evaluation of FOXP3 and VEGF expression in MAD-MB-231 cells by confocal microscopy (control or FOXP3 overexpression). Scale bar, 20 μm. l Orthotopic injection of MDA-MB-231 cells was performed to generate xenografts, and adenoviruses carrying FOXP3 or control cDNA were injected into the tumors when their volume reached ~50 mm³ (n = 5). Serum VEGF levels of mice bearing control or FOXP3-overexpressing tumors were detected by ELISA. a–l Student’s t test

Fig. 5. VEGF is involved in the FOXP3-mediated inhibition of angiogenesis.

a The tube formation activity of HUVECs that were treated with control medium, the culture supernatant of MAD-MB-231 cells (control supernatant), the culture supernatant of FOXP3-overexpressing MAD-MB-231 cells (FOXP3 supernatant), or the culture supernatant of FOXP3-overexpressing MAD-MB-231 cells supplemented with VEGF (FOXP3 supernatant + VEGF). Scale bar, 50 μm. b Quantitation of HUVEC tubulogenesis in the different groups in (a). HUVEC spheroid sprouting activity was determined by 3D culture. c Representative images of spheroid sprouting. Scale bar, 100 μm. d Quantitation of the cumulative sprout length (CSL) in different groups. b, d ANOVA with Tukey’s post hoc test

Fig. 6. Inverse correlation between FOXP3 and VEGF expression in human breast cancer samples.

a Representative immunohistochemical images of nuclear FOXP3 and VEGF expression in breast cancer specimens. Scale bar, 100 μm (×10) and 20 μm (×40). b A significant negative correlation between nuclear FOXP3 and VEGF expression was found in the breast cancer specimens. b Spearman test