Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells

Abstract

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and an important prognostic factor in breast cancer. Hypoxia is an important inducer of VEGF expression but less is known of the role of hormones in VEGF regulation. We have studied the regulation of VEGF, VEGF-B, VEGF-C, and VEGF-D mRNAs in human MCF-7 and mouse S115 breast carcinoma cells stimulated by estrogens and androgens, respectively. VEGF, VEGF-B, and VEGF-C were expressed in both cell lines, whereas VEGF-D was expressed only in S115 cells. Addition of estradiol (E₂) caused a biphasic increase of VEGF mRNA in MCF-7 cells and led to accumulation of the VEGF protein in the culture medium. The VEGF-B mRNA was not affected, while a decrease occurred in VEGF-C mRNA. Similarly, testosterone upregulated the expression of VEGF mRNA in the S115 cells. Experiments with actinomycin D and cycloheximide suggested that estrogen induction of VEGF mRNA is dependent on the synthesis of new mRNA and increased mRNA half-life. The antiestrogen ICI 182.780 inhibited E₂ stimulation of VEGF, suggesting that the effect was mediated by the estrogen receptor. In contrast, the antiestrogens tamoxifen and toremifene which inhibit MCF-7 cell growth in vivo and in vitro did not inhibit estrogen effect but induced VEGF mRNA expression when used alone. The antiandrogen cyprosterone acetate inhibited T induction of VEGF mRNA in S115 cells, thus suggesting that activation of androgen receptor must be involved in the increase of VEGF mRNA. Our results suggest that both estrogen and androgen stimulate the expression of VEGF by increasing gene transcription and mRNA stability. In addition, the antiestrogens tamoxifen and toremifene also increased VEGF expression. Estrogen and androgen induction of VEGF expression and promotion of new vessel formation may be an important paracrine mechanism by which these hormones contribute to the early phase of tumor growth of hormonal cancer.

Introduction

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor and vasculotropin, is a potent cytokine that exerts several important actions on the vascular endothelium (for review, see Mustonen and Alitalo, 1995, Ferrara and Davis-Smyth, 1997). VEGF acts on endothelial cells by stimulating cell proliferation, migration, and tubular organization involved in angiogenesis. VEGF is a heparin-binding 46 kDa disulfide-linked dimeric glycoprotein that dissociates upon reduction into two subunits of 23 kDa (Connolly et al., 1989, Ferrara and Henzel, 1989, Gospodarowicz et al., 1989, Leung et al., 1989). Alternative splicing of a single transcript results in the generation of 121-, 165-, 189-, and 206-amino acid encoding molecular forms (Houck et al., 1991, Tischer et al., 1991). Moreover, placental cells and various carcinoma cells of female reproductive tract express an additional 145-aa isoform (Charnock-Jones et al., 1993, Poltorak et al., 1997). The two larger variants, VEGF₁₈₉ and VEGF₂₀₆, remain cell-associated whereas the smaller forms, VEGF₁₂₁, VEGF₁₄₅ and VEGF₁₆₅, are secreted (Houck et al., 1991, Poltorak et al., 1997). Receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2) have been shown to bind VEGF with high affinity and to transduce its cellular signals (reviewed by Mustonen and Alitalo, 1995, Ferrara and Davis-Smyth, 1997).

VEGF-B, VEGF-C and VEGF-D are endothelial cell growth factors identified recently (Joukov et al., 1996, Lee et al., 1996, Olofsson et al., 1996a, Orlandini et al., 1996, Achen et al., 1998). They are 30–43% identical with VEGF, and thus belong to VEGF family of growth factors. VEGF-B mRNA splicing results in transcripts encoding a cell-surface associated VEGF-B₁₆₇ form and a soluble VEGF-B₁₈₆ form (Olofsson et al., 1996b). VEGF-B₁₆₇ can be released into the culture medium with heparin or high salt and it uses only VEGFR-1 for the transduction of its signals (Olofsson et al., 1998). VEGF-C and VEGF-D act via VEGFR-2 and VEGFR-3 (Joukov et al., 1996, Achen et al., 1998). All three growth factors are able to stimulate the endothelial cells, suggesting that they contribute to angiogenesis (Olofsson et al., 1996a, Achen et al., 1998). Recently, VEGF-C has been shown to induce specific lymphatic endothelial proliferation in avian chorioallantoic membrane (CAM) assay and hyperplasia of the lymphatic vasculature in vivo when overexpressed in the skin of transgenic mice (Jeltsch et al., 1997, Oh et al., 1997). The VEGF family also includes placenta growth factor (PlGF), which is 53% homologous with VEGF (Maglione et al., 1991).

The normal growth and differentiation of mammary gland are regulated by several steroid hormones, peptide hormones and growth factors. The hormone regulation of normal growth as well as malignant growth are thought to be largely mediated by autocrine and paracrine factors (Dickson and Lippman, 1995). VEGF mRNA and/or protein expression have been detected at a low level in normal human mammary gland and at much higher levels in primary and metastatic breast cancers (Berse et al., 1992, Brown et al., 1995, Toi et al., 1996, Yoshiji et al., 1996), but little is known of the possible role of VEGF in steroid regulation of mammary gland or breast cancer growth. VEGF expression has been shown to be regulated by estrogen in rat uterus (Cullinan-Bove and Koos, 1993, Shweiki et al., 1993, Hyder et al., 1996) and in human endometrial carcinoma cell lines (Charnock-Jones et al., 1993).

We have here investigated steroid hormone regulation of VEGF mRNA in the estrogen-dependent human MCF-7 (Osborne et al., 1987) and androgen-regulated mouse S115 breast cancer cell lines (Desmond et al., 1976, Darbre and King, 1988). The novel members of the VEGF family were also studied. Our results suggest that the steroid regulation of VEGF expression occurs both at the level of transcriptional and posttranscriptional processing and it is mediated by the specific steroid hormone receptors. Most strikingly, however, the antiestrogens tamoxifen and toremifene tested in this study have agonistic effects on VEGF mRNA although they are antagonistic for tumor cell growth.