ERRP

Abstract

Purpose: Activation of EGFR (epidermal growth factor) and its family member(s) stimulates many processes of carcinogenesis, including angiogenesis. Thus interference with EGFR activation represents a promising strategy for the development of novel and selective anticancer therapies. ERRP (EGFR Related Protein) is a negative regulator of EGFR. The present study was undertaken to determine whether ERRP would affect angiogenesis and the regulation this process. Methods: Colon cancer and rat endothelial cells were maintained in enriched medium. Cell growth was assessed by MTT assay. ERRP dependent apoptosis was determined using the Cell Death Detection ELISA kit. ERRP-dependent regulation of genes in colon cancer cells was investigated utilizing Atlas human 1.2 array membranes (Clontech). Secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) following treatments with ERRP was investigated by utilizing immunoassay kits for respective growth factors. Tubule formation by aortic endothelial cells, a measure of angiogenesis, was carried out utilizing the In-vitro Angiogenesis Assay Kit. Migration of cells through the matrigel in the absence or presence of ERRP was studied by utilizing the cell invasion assay kit. The invasive cells were stained, photographed and counted in random fields. Results: ERRP inhibited the growth of rat endothelial cells in a dose-dependent manner resulting in a 65% reduction with the highest dose of ERRP (10 μg/ml) after 48 hrs. In the absence of ERRP (controls), tubule formation was completed by 6 hrs and remained stable for the next 3 hrs. In the presence of ERRP, tubule formation was disrupted at 6 hrs, and after 9 hrs there was a complete disintegration of the tubules. In addition 30–40% of colon cancer cells failed to pass through the matrigel in the presence of ERRP. Treatment also resulted in attenuated levels of porangiogenic factors such as bFGF, VEGF and TGF-α. ERRP attenuated expression of several genes regulating the processes of proliferation, angiogenesis and invasion, while inducing expression of apoptosis promoting molecules. ERRP inhibited expression of proliferation regulatory genes such as Stratifin, FAK and MEK-2. Conclusions: ERRP inhibits pathways of angiogenesis and proliferation by specifically attenuating multiple mediators of EGFR signaling pathway and by causing diminished secretion of proangiogenic growth factors.