Introduction: Insulin-like growth factor binding protein-3 (IGFBP-3), a major carrier protein for insulin-like growth factors (IGFs) in circulation, exhibits an antiproliferative effect. We have recently observed IGFBP-3 overexpression in esophageal cancer with epidermal growth factor receptor (EGFR) overexpression (Takaoka et al. Cancer Res. 2004 64:7711-23). However, IGFBP-3 is subject to complex regulation by EGF as well as other serum factors, such as growth hormone and IGF-I, and it remains unclear how EGFR regulates IGFBP-3 expression and its cellular functions. Methods: IGFBP-3 was stably transduced with a retroviral vector. Gene expression was determined by real-time RT-PCR, Western blotting or enzyme linked-immunosorbent assay. Pharmacological inhibitors were used to dissect the signaling pathways involved. Cell proliferation was assessed by 3H-thymidine incorporation assay. Results: In all esophageal cancer cell lines, EGF greatly decreased IGFBP-3 mRNA and protein levels within 24 hours in a dose dependent fashion. However, a subset of cell lines demonstrated induction of IGFBP-3 protein in cell lysates and conditioned media between 24 and 48 hours after EGF treatment, which was antagonized by AG1478, an EGFR specific inhibitor. Furthermore, such induction of IGFBP-3 by EGF was enhanced by increased cellular density. Cycloheximide abrogated the induction of IGFBP-3, suggesting a requirement of de novo protein synthesis. Furthermore, LY294002 (PI3K inhibitor) and rapamycin, but not PD 98059 (MEK inhibitor), prevented the induction of IGFBP-3 with reduced phosphorylation levels of p70S6 kinase and 4E-BP1, suggesting the involvement of the phosphatidylinositol-3 kinase (PI3K)-mTOR pathway. When overexpressed in immortalized human esophageal cells, IGFBP-3 greatly reduced IGF-I, but not EGF, mediated AKT activation, resulting in inhibition of DNA synthesis. These results suggested that EGFR may indirectly regulate IGF signaling by regulation of IGFBP-3 expression. Conclusions: EGFR appears to regulate IGFBP-3 in a biphasic manner in a subset of esophageal cancer cell lines. While EGF transcriptionally suppresses IGFBP-3, its induction involved translational regulation through the PI3K-mTOR pathway. EGFR may regulate the IGF signaling pathway by modulating IGFBP-3 levels, a novel mechanistic finding.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]