The Foxc2 transcription factor regulates tumor angiogenesis

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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.

Introduction

Tumor initiation and progression involve complex interactions between tumor cells and their microenvironment [1]. One of the critical processes for tumor progression is angiogenesis (neovascularization) in the tumor microenvironment [2], [3], [4]. The tumor vasculature supplies oxygen to the tumor tissue and facilitates cancer cell invasion and metastasis, whereas the formation of tumor blood vessels is regulated by multiple angiogenic factors secreted from tumor cells themselves as wells as various cells in the tumor microenvironment. Tumor blood vessels are usually disorganized and have excessive branching. Perivascular mural cells (vascular smooth muscle cells and pericytes) are loosely associated with the tumor endothelium. Although the roles of angiogenic factors such as vascular endothelial growth factor (VEGF) in promoting tumor angiogenesis have been extensively studied [2], [3], [4], little is known about transcription factors that are involved in tumor neovascularization and growth.

Recent evidence demonstrates that several Forkhead box (Fox) transcription factors, including FoxO and FoxM, are critical for cancer development and progression [5]. For example, FoxM1 in glioma and pancreatic cancer cells promotes angiogenesis [6], [7]. Whereas recent studies show that human FOXC2 is strongly expressed in highly aggressive basal-like breast cancers and is responsible for invasion and metastasis [8], murine Foxc2 is expressed in blood vessels during embryonic development and is required for formation of the aortic arch, remodeling of primitive blood vessels into a vascular network, and arterial-venous specification during embryonic development [9], [10], [11], [12]. Our laboratory has recently shown that Foxc2 is crucial for endothelial cell migration and microvessel formation in vitro[13]. These data indicate that Foxc2 plays important roles in vascular formation [14]; however, its functions in tumor blood vessels need to be revealed.

In this study, we show that the expression of human and mouse FoxC2 is detected in the tumor endothelium. We demonstrate by subcutaneous implantation of B16 melanoma that Foxc2 heterozygous mutant mice exhibit diminished tumor growth, accompanied by reduced formation of tumor blood vessels. Expression of several angiogenic factors, including Vegf, Mmp2, and Pdgfb, is greatly decreased in B16 tumors grown in Foxc2 heterozygous mutant mice, and these tumor blood vessels show impaired smooth muscle coverage and apoptosis of endothelial cells. These findings indicate an essential role of Foxc2 in tumor angiogenesis.

Section snippets

Materials and methods

Cell culture. Murine B16-F10 melanoma cells, provided by Dr. Jin Chen of Vanderbilt University Medical Center, were cultured at 37 °C in a 5% CO2 incubator in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 IU/mL penicillin, and 50 μg/mL streptomycin sulfate.

In vivo tumor development. Foxc2 heterozygous mutant (Foxc2+/−) mice were maintained and genotyped as previously described [10]. Foxc2+/− mice were backcrossed for seven generations onto the C57BL/6J

Human and mouse FoxC2 expression in the tumor endothelium and breast and colonic adenocarcinomas

Although it has recently been shown that FOXC2 is expressed in human invasive breast carcinomas [8], its expression remains less characterized in other types of tumors. We therefore tested FOXC2 expression in various human tumors using T-MTA-6A tissue array (NCI/NIH). FOXC2 immunoreactivity was observed in the majority of breast adenocarcinomas including lobular and ductal adenocarcinoma and about half of colonic adenocarcinoma (Fig. 1A), whereas lung and ovarian tumor tissues did not

Discussion

Since the late Judah Folkman proposed the original hypothesis that the formation of new blood vessels (angiogenesis) is critical for tumor growth [20], significant progress has been made in our understanding of tumor angiogenesis. Indeed, numerous clinical trials are underway to test the efficacy of antiangiogenic drugs that block critical signaling pathways such as VEGF, and a couple of inhibitors of angiogenesis have recently been approved by the U.S. Food and Drug Administration [21]. Yet

Acknowledgments

This work was supported by grants from the NIH (HL74121 to T.K. and CA100562 to M.M.D.), a grant from the March of Dimes Foundation to T.K., and Vanderbilt-Ingram Cancer Center Support Grant to M.M.D.

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    Present address: Feinberg Cardiovascular Research Institute, Northwestern University School of Medicine, IL, USA.

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