IGF-1R expression and activation levels generally cannot be correlated in cancer cells, suggesting that cellular proteins may modulate IGF-1R activity. Strong candidates for such modulation are found in cell-matrix and cell–cell adhesion signaling complexes. Activated IGF-1R is present at focal adhesions, where it can stabilize β1 integrin and participate in signaling complexes that promote invasiveness associated with epithelial mesenchymal transition (EMT) and resistance to therapy. Whether IGF-1R contributes to EMT or to non-invasive tumor growth may be strongly influenced by the degree of extracellular matrix engagement and the presence or absence of key proteins in IGF-1R-cell adhesion complexes. One such protein is PDLIM2, which promotes both cell polarization and EMT by regulating the stability of transcription factors including NFκB, STATs, and beta catenin. PDLIM2 exhibits tumor suppressor activity, but is also highly expressed in certain invasive cancers. It is likely that distinct adhesion complex proteins modulate IGF-1R signaling during cancer progression or adaptive responses to therapy. Thus, identifying the key modulators will be important for developing effective therapeutic strategies and predictive biomarkers.

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, β1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.

Although insulin-like growth factor 1 (IGF-1) signaling promotes tumor growth and cancer progression, therapies that target the IGF-1 receptor (IGF-1R) have shown poor clinical efficacy. To address IGF-1R activity in cancer cells and how it differs from that of the closely related insulin receptor (IR), we focused on two tyrosines in the IGF-1R C-terminal tail that are not present in the IR and are essential for IGF-1–mediated cancer cell survival, migration, and tumorigenic growth. We found that Tyr1250 and Tyr1251 (Tyr1250/1251) were autophosphorylated in a cell adhesion–dependent manner. To investigate the consequences of this phosphorylation, we generated phosphomimetic Y1250E/Y1251E (EE) and nonphosphorylatable Y1250F/Y1251F (FF) mutant forms of IGF-1R. Although fully competent in kinase activity and signaling, the EE mutant was more rapidly internalized and degraded than either the wild-type or FF receptor. IGF-1 promoted the accumulation of wild-type and EE IGF-1R within the Golgi apparatus, whereas the FF mutant remained at the plasma membrane. Golgi-associated IGF-1R signaling was a feature of migratory cancer cells, and Golgi disruption impaired IGF-1–induced signaling and cell migration. Upon the formation of new cell adhesions, IGF-1R transiently relocalized to the plasma membrane from the Golgi. Thus, phosphorylation at Tyr1250/1251 promoted IGF-1R translocation to and signaling from the Golgi to support an aggressive cancer phenotype. This process distinguishes IGF-1R from IR signaling and could contribute to the poor clinical efficacy of antibodies that target IGF-1R on the cell surface.

The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (

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.