ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases, blocks VEGF-C-induced activation of VEGFR-3 and cell proliferation in human colon cancer cell lines

Abstract

13171 Background: Vascular endothelial growth factor receptors 1, 2 and 3 (VEGFR-1, -2 and -3) have a key role in activation and proliferation of endothelial cells, with expression of VEGFR-3 largely restricted to lymphatic endothelial cells. Although high expression of VEGFR-3 and its specific ligands, VEGF-C and VEGF-D, has been associated with an increased incidence of lymph node metastasis and a poor prognosis in different human malignancies, little is known about the role of this signaling pathway in tumor cells. This study investigated VEGFR-3 and its specific ligands in human colon cancer cells, and the antiproliferative activity of ZD6474, an inhibitor of VEGFR, epidermal growth factor receptor (EGFR) and RET tyrosine kinases, and of the EGFR inhibitors gefitinib and cetuximab. Methods: The expression of VEGFRs and EGFR was determined by RT-PCR, immunoblotting, flow cytometry and immunohistochemistry in four human colon cancer cell lines (HCT-116, HT-29, HCT-15 and SW480). Secretion of transforming growth factor-α (TGF-α), VEGF-A and VEGF-C by cancer cells was determined by ELISA. The in vitro antiproliferative effects of ZD6474, gefitinib and cetuximab were determined using an MTT assay. Results: All four human colon cancer cell lines expressed functional EGFR and secreted high levels of TGF-α. All four cell lines expressed VEGFR-1 and VEGFR-3, but not VEGFR-2, and secreted both VEGF-A and VEGF-C. Treatment with ZD6474 resulted in a dose-dependent cell growth inhibition (IC50 3–5 μM), whereas treatment with gefitinib or with cetuximab produced little growth inhibition under the same culture conditions. Addition of VEGF-C induced a 2-fold increase in cell growth in vitro in all four colon cancer cell lines. Treatment with ZD6474 blocked both basal and VEGF-C-induced phosphorylation of VEGFR-3, as well as VEGF-C-induced cell proliferation. Conclusions: Human colon cancer cell lines express VEGFR-1 and VEGFR-3, and secrete VEGF-A and VEGF-C. A potential VEGF-C/VEGFR-3 autocrine loop has been identified in human colon cancer cells, which can be inhibited by ZD6474, suggesting that ZD6474 may have direct antitumor activity through inhibition of VEGFR signaling. [Table: see text]