IGF-1 induced vascular endothelial growth factor secretion in head and neck squamous cell carcinoma q,qq Mark G. Slomiany a , Leigh Ann Black a , Megan M. Kibbey a , Terry A. Day b , Steven A. Rosenzweig a, * a Department of Cell and Molecular Pharmacology and Experimental Therapeutics and Hollings Cancer Center, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA b Department of Otolaryngology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA Received 7 February 2006 Available online 20 February 2006 Abstract Elevated vascular endothelial growth factor (VEGF) levels correlate with increased progression and poor prognosis of head and neck squa- mous cell carcinomas (HNSCC). VEGF expression is regulated by hypoxia and cytokines, including insulin-like growth factor-1 (IGF-1). In this report, we examined IGF-1 signaling and VEGF expression in SCC-9 cells. IGF-1 and the chemical hypoxia agent, cobalt chloride, each stimulated VEGF secretion and VEGF promoter activation. Cobalt chloride increased Hif-1a protein levels and HIF-1 dependent activation of the enolase promoter. IGF-1 increased these parameters only in the presence of cobalt chloride. IGF-1 stimulated PI-3K/Akt and Erk/MAPK pathways in SCC-9 cells, each contributing to Hif-1a expression and VEGF secretion. SCC-9 cells express the VEGF receptors Flk-1 and neu- ropilin-1 (Np-1), with VEGF treatment increasing VEGF promoter activity and VEGF secretion that was attenuated by the Flk-1 tyrosine kinase inhibitor, ZM 323881. These results demonstrate the presence of an IGF-1 regulated VEGF autocrine loop in HNSCC. Ó 2006 Elsevier Inc. All rights reserved. Keywords: IGF-1; IGFBP; VEGF; Autocrine action; Squamous cell carcinoma Head and neck squamous cell carcinoma (HNSCC) accounts for greater than 90% of all pharyngeal and oral cavity tumors. With nearly 8000 deaths a year nationally, it constitutes about 4% of all cancers in the United States and is one of the six most frequent cancers world- wide. Although tobacco and alcohol are primary risk factors, it is clear that additional factors contribute to this cancer [1]. Under physiologic conditions, angiogenesis is tightly regulated by the net actions of pro-angiogenic versus anti-angiogenic factors [2]. During early carcinogenesis, angiogenesis is essential for the growth and persistence of solid tumors [3] where there is increased release of angio- genic factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and acid- ic/basic-fibroblast growth factors (a/b-FGF; [4]). VEGF, plays a major role in epithelial carcinogenesis, and has been identified as a biomarker for HNSCC [5–7], where its 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.043 q This work was supported, in part, by Grant CA78887 from the National Institutes of Health and Department of Defense grant to Hollings Cancer Center, (N6311601MD10004) to S.A.R. and (N6311602MD200) to T.A.D., M.G.S. was supported by an Abney Foundation Research Scholarship and M.M.K. was supported by National Research Service Award 5F30DE015249 from the NIDCR and by the Dental Medicine Scientist Training Program, Colleges of Dental Medicine and Graduate Studies, Medical University of South Carolina. qq Abbreviations: Akt, protein kinase B (PKB) a-isoform; BCA, bicinch- oninic acid; BLOTTO, bovine lacto transfer technique optimizer; BSA, bovine serum albumin; CoCl 2 , cobalt chloride; Erk, extracellular-signal- regulated kinase; FBS, fetal bovine serum; Flk-1, fetal liver kinase receptor- 1; HIF-1, hypoxia-inducible factor 1; HRE, hypoxia response element; HRP, horseradish peroxidase; HNSCC, head and neck squamous cell carcinoma; IGF-1, insulin-like growth factor 1; IGF-1R, IGF-1 receptor; IGFBP, IGF binding protein; MAPK, MEK/mitogen-activated protein kinase; NP-1, neuropilin-1; PBS, phosphate-buffered saline; PI 3-K, phosphatidylinositol 3-kinase; rhIGFBP-3, recombinant human IGFBP- 3; rhVEGF, recombinant human vascular endothelial growth factor 165 isoform; SPT, second primary tumors; TBS, Tris-buffered saline. * Corresponding author. Fax: +1 843 792 2475. E-mail address: rosenzsa@musc.edu (S.A. Rosenzweig). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 342 (2006) 851–858 BBRC