The Foxc2 transcription factor regulates tumor angiogenesis

Abstract

The Forkhead/Fox transcription factor Foxc2 is a critical regulator of vascular development. However, the role of Foxc2 in pathological angiogenesis in cancer remains unknown. Here we show that FoxC2 is highly expressed in human breast and colonic tumors and in the tumor endothelium in human and mouse melanomas. Using the B16 melanoma tumor model, we investigated the function of Foxc2 in tumor angiogenesis. After subcutaneous injection of B16 melanoma cells, primary tumor growth as well as neovascularization was markedly reduced in mice lacking one copy of the Foxc2 gene (Foxc2+/-). Consistently, expression levels of several angiogenic factors, including vascular endothelial growth factor (Vegf), matrix metallopeptidase 2 (Mmp2), and platelet-derived growth factor-B (Pdgfb), were significantly decreased in B16 tumors grown in Foxc2+/- mice, and tumor blood vessels formed in Foxc2+/- mice showed reduced coverage of mural cells and endothelial cell apoptosis. In addition, the tumor tissue in Foxc2+/- mice had an accumulation of necrotic cells. Taken together, these findings demonstrate that haplodeficiency of Foxc2 results in impaired formation of tumor blood vessels as well as reduced tumor growth and thereby provide evidence that Foxc2 is critical for tumor development and angiogenesis.

Fig. 1

Expression of human and mouse FoxC2. (A) FOXC2 expression in the human breast and colon tumors. FOXC2 expression in lobular adenocarcinoma of breast and colonic adenocarcinoma at 20× (upper panels) and 40× (lower panels) magnifications; the areas presented at higher magnification are indicated by square. Note that FOXC2 immunostaining is primarily limited to cancer cells in tumor tissue. Scale bars, 500 μm and 100 μm for upper and lower, respectively. (B) Expression of FOXC2 is detected in tumor endothelial cells of human malignant melanoma. Frozen sections of malignant melanoma in the human skin were co-immunostained with anti-FOXC2 and anti-PECAM-1 antibodies. FOXC2 protein (green) was detected in tumor endothelial cells positive for PECAM-1 (red). Arrows indicate FOXC2-positive human endothelial cells. Scale bar, 250 μm. (C) Expression of Foxc2 is detected in endothelial cells of mouse B16 melanoma. Co-immunostaining with anti-FOXC2 and anti-PECAM-1 antibodies in mouse B16 tumors grown in C57BL/6 mice for 11 days after subcutaneous injection. Arrows indicate overlapping expression of Foxc2 (red) and PECAM-1 (green) in tumor endothelial cells. Scale bar, 250 μm.

Fig. 2

Tumor development and angiogenesis is impaired in Foxc2+/- mice. (A) Subcutaneous growth of B16 melanoma cells inoculated in WT and Foxc2+/- mice at day 11 after injection. Yellow arrow indicates reduced tumor growth in Foxc2+/- mouse. (B) The difference in tumor weight between WT and Foxc2+/- mice was examined 11 days after injection of B16 melanoma cells. The data reflect five independent experiments. Data are presented as mean ± SD (n = 5). Statistical significance was determined by Student's t-tests. *, P < 0.05 versus WT. (C-E) Tumor angiogenesis is reduced in Foxc2+/- mice. (C) Cryosections from WT and Foxc2+/- mice 11 days after injection of B16 melanoma cells were immunostained with anti-PECAM-1 antibody. Scale bar, 250 μm. (D) The total number of capillaries was calculated by counting PECAM-1 positive endothelial cells in B16 tumors. Results are presented as mean ± SD from four or five independent experiments in which nine microscopic fields (total magnification ×200) were analyzed by the ImageJ program. Statistical significance was determined by Student's t-tests (*p<0.05 versus WT). (E) Total vessel density was calculated by counting PECAM-1 positive vessel area. Results are presented as mean ± SD from four or five independent experiments in which nine fields of each specimen (total magnification ×200) were analyzed by the ImageJ program. The Y axis represents the number of pixels. Statistical significance was determined by Student's t-tests. *, P < 0.05 versus WT.

Fig. 3

Differential expression of (lymph)angiogenesis-related genes in B16 tumors grown subcutaneously in Foxc2+/- mice for 11 days. Fold change in mRNA levels of Foxc genes as well as of genes related to angiogenesis and lymphangiogenesis was measured by real-time RT-PCR. Note a significant reduction in expression of Vegf₁₂₀, Vegf₁₆₄, Dll4, Mmp2, and Pdgfb in B16 tumors grown in Foxc2+/- mice. Results are presented as mean ± SD (n = 4 or 5). Statistical significance was determined by Student's t-tests. *, P < 0.05; **, P < 0.01 versus WT.

Fig. 4

Tumor blood vessels in Foxc2+/- mice show reduced coverage of mural cells and endothelial cell apoptosis. (A) Tumor tissues isolated from WT and Foxc2+/- mice 11 days after implantation of B16 melanoma cells were co-immunostained with PECAM-1 (red) and smooth muscle alpha-actin (αSMA) antibodies. The merged images show the coverage of mural cells in tumor blood vessels. While the tumor endothelium was surrounded by the smooth muscle layer (white arrows) in WT mouse, endothelial cells in tumors of Foxc2+/- mouse exhibited almost no coverage of smooth muscle cells (arrowheads). Tumor-associated fibroblasts expressing αSMA (yellow arrows) were barely detected in Foxc2+/- mouse. Scale bar, 250 μm. (B and C) Analysis of apoptotic cells in B16 tumors by TUNEL staining. (B) Cell counts of positive cells from TUNEL assay on B16 tumor tissue taken from WT and Foxc2+/- mice. Statistical significance was determined by Student's t-tests. *, P < 0.05 versus WT. (C) In contrast with WT mouse, tumor endothelial cells in Foxc2+/- mouse underwent apoptosis detected by TUNEL staining (green) and immunostaining with anti-PECAM-1 antibody (red). Arrowheads indicate TUNEL-positive endothelial cells (yellow). Scale bar, 250 μm. The right panel for Foxc2+/- mouse represents a high magnification (scale bar, 50 μm) of the boxed area in the left panel.