Abstract 111: A specific phosphorylation on the IGF-1 receptor controls internalization and signaling in cancer cells
Authors: Rieger Leonie , O'Shea Sandra , Godsmark Grant , Stanicka Joanna , Kelly Geraldine M. , O'Connor Rosemary 
Published: September 2020

Insulin-like Growth Factor-1 signaling facilitates tumorigenesis and promotes tumor growth by acting through its cell surface receptor (IGF-1R). Despite promising pre-clinical results, efforts to target the IGF1-1R in many different cancers have been largely unsuccessful, and this remains un-explained. The IGF-1R shares significant sequence homology with the Insulin receptor (IR), with most divergence in the C-terminal tail, which contains two tyrosines (Y1250/Y1251) that are not present in the IR. Previous studies have shown that mutation of these tyrosines to phenylalanine (FF) is sufficient to impair IGF-1 receptor activation and signaling for cell survival, migration and tumorigenesis (reviewed in 1, 2). The Y1250/Y1251 site is required for integrating IGF-1R and Integrin signaling (2). However, whether, where and how these tyrosines are phosphorylated, and how this site controls receptor activity have not been established.Here, we show that the Y1250/Y1251 site is auto-phosphorylated by the IGF-1R kinase and this is enhanced by cell adhesion. Using a phospho-specific monoclonal antibody we observe that Y1250/1251 phosphorylation is evident in a range of cancer cell lines, is responsive to IGF-1 stimulation, and is more prominent in migratory cancer cells than non-migratory cells. The phosphorylated Y1250/Y1251 site was further investigated by generating an IGF-1R phosphomimetic where the tyrosines were mutated to glutamates (Y1250E/Y1251E). Upon transient or stable expression of the EE mutant in either receptor-negative MEFs (R- cells) or Crspr-generated IGF-1R knockout cancer cells, we observed that the EE mutant is expressed at lower levels than either the wild type (WT) or FF mutant IGF-1R. The EE mutant can be partially recovered by proteasomal inhibition, indicating that it is less stable than the other receptors. Immunofluorescence analysis established that in serum-starved cells, the EE mutant becomes rapidly internalized within intracellular vesicles, while the WT IGF-1R and FF mutant remain at the cell surface. IGF-1 stimulates rapid of internalization of WT and EE but not the FF mutant does. Thus, phosphorylation on this site is required for internalization and intracellular trafficking of the IGF-1R. Interestingly, enhanced EE mutant intracellular trafficking is accompanied by enhanced signaling output compared to the YY mutant or WT IGF-1R.We conclude that phosphorylation of the IGF-1R on Y1250/Y1251 is required for specific intracellular trafficking of the IGF-1R, which enhances its activity in cancer cells. This phospho-site distinguishes the signaling potential of the IGF-1R and IR, and may also explain the lack of efficacy observed with therapeutic monoclonal antibodies that target cell surface IGF-1R in different cancers. References:1. Cox, O.T et al., (2015) Front. Endocrinology 6,(10.3389/fendo.2015.00106).2. Stanicka, J., et al., (2018) Oncogene 27: 3131-3150.3. Kiely. P.A. et al., (2005) J. Biol. Chem. 280: 7624-7633.

Citation Format: Leonie Rieger, Sandra O'Shea, Grant Godsmark, Joanna Stanicka, Geraldine M. Kelly, Rosemary O'Connor. A specific phosphorylation on the IGF-1 receptor controls internalization and signaling in cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 111.