Insulin-like growth factor binding protein-2: Contributions of the C-terminal domain to IGF binding and role in oral and oropharyngeal squamous cell carcinoma

The insulin-like growth factor-1 receptor (IGF-1R) mediates the growth activities of the insulin-like growth factors (IGFs). The IGFs bind with high affinity to the IGF binding proteins (IGFBPs), which effectively inhibit IGF-1R activation due to their greater affinity for the IGFs than that of the IGF-1R. Thus, the IGFBPs are IGF antagonists. Several molecular markers have been associated with oral and oropharyngeal squamous cell carcinoma (OSCC), yet the prognostic and therapeutic relevance of these markers are absent. A component of the IGF system may be a molecular marker of OSCC, and thus may be exploited to develop therapeutic and preventative agents. The objectives of this study were to determine the contributions of the C-terminal domain of IGFBP-2 to IGF binding and to establish a role for IGF-1R signaling in enhancing OSCC tumorigenicity. To ascertain the role of the C-terminus of IGFBP-2 in IGF binding, we selectively cleaved IGFBP-2 with BNPS-skatole, expressed IGFBP-2 truncation mutants (IGFBP-21-248, IGFBP-21-190, and IGFBP-2249-289) and tested their IGF-1 binding activity. Using 6His·IGFBP-2 (EC50 0.76 nM) as the control, recombinant IGFPB-21-190 (EC50 9.2 nM) had similar IGF binding properties to IGFBP-21-248 (EC50 7 nM) with lower efficacy in blocking IGF-binding to the receptor, suggesting that proximal residues (191-248) contribute to blocking IGF-1 from binding to the IGF-1R. The kinetic studies indicated that distal residues (249-289) provide stability to the IGFBP-2/IGF-1 complex accounting for the slower dissociation rates observed for IGFBP-2 versus IGFBP-2 1-248. Furthermore, expression of IGF system components in OSCC cell lines was characterized and functional studies were performed. The level of IGF-1R expression was greater (55,000-89,700 R/cell) than the accepted normal physiologic range (15,000-30,000 R/cell). The cells expressed IGF-1 and -2 and IGFBP-2, -3 and -5 and underwent a 2.5-fold increase in cell number upon addition of 1 nM IGF-1. In conclusion, both the distal and proximal regions of the C-terminal domain of IGFBP-2 provide stability and are necessary for inhibiting IGF-1 binding to the IGF-1R. The OSCC analysis exhibits the prerequisites for demonstrating the dysregulation of the IGF system in contributing to the progression of this cancer.