VEGF receptor expression and signaling in human bladder tumors Weicheng Wu, Xiaodong Shu, Harut Hovsepyan, Raymond D Mosteller and Daniel Broek Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine at the University of Southern California, CA 90089, USA Overexpression of vascular endothelial growth factor receptors (VEGFRs) has been reported in a variety of tumor types. Here we find that 11 out of the 14 bladder tumor cell lines examined express one or more VEGF receptors. Analysis of the T24 bladder tumor cell line reveals a functional autocrine loop involving VEGF and the Flk-1 receptor. Blocking VEGF expression in T24 cells results in a decrease in DNA synthesis. The Flk-1 receptor in T24 cells is phosphorylated in response to VEGF-121 or VEGF-165, and an Flk-1 inhibitor blocks VEGF to ERK signaling. We report that VEGF stimulation of T24 cells results in activation of H- and N-Rasandthisisdependentoncellularsphingosinekinase 1 (SPK1) activity. Previously, we found VEGF-induced activation of Ras appears to be independent of a Ras- guanine nucleotide exchange factors (GEFs). Here we report that sphingosine can stimulate Ras-GTPase activating protein (GAP) activity in vitro, and sphingo- sine-1-phosphate (SPP) can block the stimulatory effects of sphingosine. We present a model where the balance betweensphingosineandSPPregulatesRas-GAPactivity such that stimulation of SPK1 favors downregulation of Ras-GAP and thereby the activation of Ras proteins. These data highlight a VEGF pathway that may be involvedinthesurvivalandproliferationofbladdertumor cells as well as other tumor cell types. Oncogene (2003) 22, 3361–3370. doi:10.1038/sj.onc.1206285 Keywords: VEGF; sphingosine kinase; bladder tumor Introduction Vascular endothelial growth factor (VEGF) is one of the most important growth factors involved in vasculogen- esis and angiogenesis. Normally, VEGF is produced by cells in close proximity to endothelial cells (such as vascular smooth muscle cells) and the expression is highly regulated by hypoxia as well as some hormones and cytokines (Ferrara, 1999; Carmeliet and Collen, 2000). VEGF receptors (VEGFRs) are mainly expressed in endothelial cells, but have also been reported to be expressed in several nonendothelial cell types including retinal pigment epithelial cells (Guerrin et al., 1995), Schwann cells (Sondell et al., 1999), pancreatic duct cells (Oberg-Welsh et al., 1997), renal mesangial cells (Takahashi et al., 1995), macrophages and monocytes (Sawano et al., 2001) and platelets (Selheim et al., 2002). By binding to VEGFRs, VEGF can protect endothelial cells from apoptosis, as well as stimulate endothelial cell proliferation, migration, and differentiation. VEGF plays a critical role in angiogenesis induced by pathological conditions such as solid tumor growth. VEGF expression is upregulated in most human tumors examined. Many tumor-associated stromal cells also express VEGF (Ferrara, 1999). Tumor cell expression of VEGF is critical for tumor cell-induced angiogenesis. Many tumor cells and tumor-associated vasculature express both VEGFRs and VEGF, and an autocrine/ paracrine function has been proposed (Veikkola et al., 2000). For example, functional VEGFRs are expressed in melanoma (Liu et al., 1995) and mesothelioma (Strizzi et al., 2001) cell lines and exogenous VEGF stimulates cell proliferation. VEGF also stimulates human leuke- mic cell proliferation and migration (Dias et al., 2000). VEGF induces MAPKs activation and cell growth in a human pancreatic cancer cell line (von Marschall et al., 2000). In T-47D breast cancer cells, VEGF stimulates both MAPKs and PI3K (Price et al., 2001). In M21 melanoma cells, VEGF activates integrin avb3, which is involved in cell adhesion and migration (Byzova et al., 2000). Together, these reports highlight the import- ance of VEGF signaling in the biology of a variety of tumor cells in addition to its well-described role in angiogenesis. We recently defined a VEGF-induced signaling path- way in a well-characterized bladder tumor cell line (T24) that affects the growth properties of these tumor cells (Shu et al., 2002). Here we extend those studies by showing that T24 cells express both VEGF and VEGFR-2 (KDR/Flk-1), which act in an autocrine loop to induce DNA synthesis. This mitogenic VEGF pathway involves the activation of PKC, sphingosine kinase (SPK), Ras- and mitogen-activated protein kinases (ERK1/2). VEGF stimulation leads to H-Ras and N-Ras activation (but not K-Ras activation) in a Ras-guanine nucleotide exchange factor (GEF) inde- pendent mechanism. Sphingosine stimulates Ras-GAP activities in vitro and this effect is blocked by excess sphingosine-1-phosphate (SPP). This suggests a novel mechanism of Ras activation that relies on Ras-GTPase activating protein (GAP) modulation rather than regulation of Ras-GEFs. PKC- and SPK-mediated Received 20 June 2002; revised 21 November 2002; accepted 29 November 2002 *Correspondence: D Broek; E-mail: broek@usc.edu Oncogene (2003) 22, 3361–3370 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc