Growth Factor Cells via Modulation of Heparin-Binding Epidermal-Like Gastrin Enhances the Angiogenic Potential of Endothelial

This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor–like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycineextended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy. (Cancer Res 2006; 66(7): 3504-12) Introduction Gastrin peptides have multiple carcinogenic activities in gastrointestinal adenocarcinomas through both autocrine/paracrine and endocrine mechanisms (1). Mature amidated gastrin peptides act through the G-protein–coupled cholecystokinin-2/ gastrin receptor (CCK-2R), which mediates the normal endocrine activities of gastrin, such as acid secretion. The classic CCK-2R, together with a number of splice variants, have been shown to play a role in malignancy (2–4). Gastrin gene transcription is normally tightly regulated but may be elevated in the cancer situation, as it is a transcriptional target of h-catenin (5, 6). Post-translational processing of preprogastrin is also disordered in malignant epithelial cells, resulting in constitutive release of poorly processed gastrin forms, such as progastrin, and glycine-extended gastrin species (7). Nonamidated gastrin peptides, such as glycine-extended gastrin-17 (GlyG17), possess biological activity that includes pro-proliferative effects on epithelial cells and enhancement of carcinogen-induced tumorigenicity in the colon (8, 9). Gastrin peptides increase the transcription of a number of key target genes associated with tumorigenesis, including cyclooxygenase-2 (COX-2), plasminogen activator inhibitor-2 (PIA-2), the regenerating protein (Reg ; refs. 10–12), and matrix metalloproteinase (MMP) enzymes (13–15). Importantly, much emphasis has been placed on the ability of gastrin to both up-regulate ligands of the epidermal growth factor (EGF) receptor, in particular amphiregulin and heparin-binding EGF-like growth factor (HBEGF; refs. 16–18). The effect of gastrin on both the shedding of EGF receptor growth factors and related downstream signaling pathways has received particular attention. In terms of a proangiogenic role for gastrin, a recent study showed such an in vivo in a glioma model involving E-selectin activation (19). Our study has explored the role of gastrin peptides on the angiogenic potential of human endothelial cell lines through mediation of HB-EGF expression and shedding. Two gastrin peptides were compared; gastrin-17 (G17), the form involved in endocrine pathways and GlyG17, a form secreted by tumor cells themselves and ascribed a role as an autocrine factor (20). It was confirmed that both G17 and GlyG17 increased tubule formation of human endothelial cells grown on an extracellular matrix and enhanced HB-EGF expression and shedding. MMP-2, MMP-3, and MMP-9 expression also increased, with these factors potentially playing a role in regulating shedding of the HB-EGF. In vivo models confirmed the proangiogenic effects of G17, and these translated to the clinical situation based on the observation that there is increased vessel density and elevated HB-EGF protein expression in the normal mucosa surrounding colorectal adenocarcinomas in patients with elevated serum-amidated gastrin levels. Note: P.A. Clarke and J.H. Dickson are joint first authors. Requests for reprints: Susan A. Watson, Academic Unit of Cancer Studies, D Floor, West Block, Queen’s Medical Centre, University Hospital, Nottingham, NG7 2UH, United Kingdom. Phone: 44-115-9709248; Fax: 44-115-9709902; E-mail: sue.watson@ nottingham.ac.uk. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0280 Cancer Res 2006; 66: (7). April 1, 2006 3504 www.aacrjournals.org Research Article American Association for Cancer Research Copyright © 2006 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-05-0280