Affiliation of authors: Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD
Correspondence to: Hynda K. Kleinman, PhD, Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg. 30, Rm. 433, 30 Convent Dr. MSC 4370, Bethesda, MD 20892 (e-mail: hkleinman{at}dir.nidcr.nih.gov)
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) is a predominant inducer of tumor angiogenesis and an important prognostic factor in breast cancer (3). VEGF binds two high-affinity receptors, VEGFR-1 and VEGFR-2, that have ligand-stimulated tyrosine kinase activity (4). Although VEGFR-2 is recognized as the predominant receptor involved in VEGF-stimulated angiogenesis (4,5), the function of VEGFR-1 is less clear. VEGFR-1 has several unique structural and functional characteristics. The VEGFR-1 gene encodes a full-length membrane receptor (200 kd) and a soluble receptor (
110 kd), which are generated by alternative splicing of the VEGFR-1 pre-mRNA and contain the extracellular ligand-binding domains but lack the signaling tyrosine kinase domains (4). VEGFR-1 has more than a 40-fold higher affinity than VEGFR-2 for VEGF (4,6). It is believed that VEGFR-1 functions as an inert decoy by binding endogenous VEGF, thereby negatively regulating the availability of VEGF and the activation of angiogenesis through VEGFR-2. Such a decoy function is attributable mainly to soluble VEGFR-1 (4). Several studies have used soluble VEGFR-1 as a VEGF-blocking reagent (i.e., negative regulator of angiogenesis) and as an inhibitor of tumor growth (69).
Although expression of VEGFR-1 was previously believed to be restricted to the vascular endothelium, VEGFR-1 has been detected in several types of non-endothelial cellsbreast cancer cells in particular (10,11). We used western blot analysis (Fig. 1) and immunofluorescent staining (data not shown) to determine levels of VEGFR-1 protein in normal breast cell lines (Hs578Bst; obtained from American Type Culture Collection [ATCC], Manassas, VA) and in two breast carcinoma cell lines (MCF-7, obtained from ATCC, and MDA-MB-231, obtained from Dr. P. Steeg, National Cancer Institute, Bethesda, MD) and compared the levels to those in human umbilical vein endothelial cells (HUVECs). Whereas vascular endothelial cells expressed a substantial fraction of the total VEGFR-1 protein as the full-length, membrane-spanning form of the receptor, normal and breast cancer cell lines expressed soluble VEGFR-1 almost exclusively (Fig. 1).
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Our results suggest that the inhibition of VEGFR-1 expression could present a novel mechanism by which estrogen exerts pro-angiogenic effects and thus promotes breast cancer development or progression. It is conceivable that normal breast tissue cells constitutively express soluble VEGFR-1 at levels sufficient to absorb endogenous VEGF, thus preventing the induction of inappropriate angiogenesis and cell growth. In agreement with this proposed mechanism, a recent study (16) has shown the presence of endogenous soluble VEGFR-1 at concentrations as high as 440 pg/mL in biologic fluids from normal human subjects. In normal breast tissue, estrogen is unlikely to inhibit VEGFR-1 expression because only a minority of normal breast epithelial cells (7%17%) express detectable ER levels (17,18). However, in primary breast tumors, the majority of proliferating breast cancer cells are ER-positive (18,19), and estrogen, by reducing the levels of VEGFR-1, may increase the levels of VEGF available to activate angiogenesis. Thus, estrogen triggers an angiogenic switch and further promotes tumor progression. This proposed mechanism is supported by the fact that patients whose tumors had soluble VEGFR-1 levels at least 10-fold higher than their tumor VEGF levels had a markedly favorable prognosis compared with patients whose tumors had a much lower VEGFR-1/VEGF ratio (20).
The classical mechanism of estrogen action involves formation of an estrogenER complex that binds to the estrogen response element (ERE) in target promoters and modulates (by increasing or decreasing) gene transcription (21). However, analysis of the VEGFR-1 gene 1.5-kilobase regulatory sequence (22) using MatInspector software (23) revealed a lack of formal EREs in the promoter region, suggesting that the effect of estrogen on soluble VEGFR-1 expression is mediated by interactions between the estrogenER complex and additional transcription modulators. Such an ER-mediated but ERE-independent pathway has been reported for a number of estrogen-regulated genes (21). Some transcription factors regulating VEGFR-1 expression (i.e., Egr-1, Ets) (24,25) have been previously shown to mediate the cellular effects of estrogen (26,27). Because steroid hormones simultaneously control gene transcription and alternative splicing (28), the observed action of estrogen on VEGFR-1 expression may reflect effects on both transcriptional and post-transcriptional (i.e., alternative RNA processing) regulatory events that are involved in the generation of soluble VEGFR-1.
Regulation of soluble VEGFR-1 by estrogen may represent one of the molecular pathways responsible for the angiogenic switch during breast tumorigenesis. Detailed understanding of the role of estrogen and antiestrogens (i.e., tamoxifen) used in clinical settings to control VEGFR-1 expression may help in the design of new strategies for preventing resistance to endocrine therapy and may also help clarify the emerging role of estrogen in controlling vascularization.
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Supported in part by the German-Israeli Foundation for Scientific Research & Development (GIF).
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Manuscript received November 6, 2003; revised March 17, 2004; accepted March 26, 2004.
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