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The ability of a receptor to preferentially activate only a subset of available downstream signal cascades is termed biased signaling. Although comprehensively recognized for the G protein–coupled receptors (GPCR), this process is scarcely explored downstream of receptor tyrosine kinases (RTK), including the cancer-relevant insulin-like growth factor-1 receptor (IGF1R). Successful IGF1R targeting requires receptor downregulation, yet therapy-mediated removal from the cell surface activates cancer-protective β-arrestin–biased signaling (β-arr-BS). As these overlapping processes are initiated by the β-arr/IGF1R interaction and controlled by GPCR-kinases (GRK), we explored GRKs as potential anticancer therapeutic targets to disconnect IGF1R downregulation and β-arr-BS. Transgenic modulation demonstrated that GRK2 inhibition or GRK6 overexpression enhanced degradation of IGF1R, but both scenarios sustained IGF1–induced β-arr-BS. Pharmacologic inhibition of GRK2 by the clinically approved antidepressant, serotonin reuptake inhibitor paroxetine (PX), recapitulated the effects of GRK2 silencing with dose- and time-dependent IGF1R downregulation without associated β-arr-BS. In vivo, PX treatment caused substantial downregulation of IGF1R, suppressing the growth of Ewing's sarcoma xenografts. Functional studies reveal that PX exploits the antagonism between β-arrestin isoforms; in low ligand conditions, PX favored β-arrestin1/Mdm2-mediated ubiquitination/degradation of IGF1R, a scenario usually exclusive to ligand abundancy, making PX more effective than antibody-mediated IGF1R downregulation. This study provides the rationale, molecular mechanism, and validation of a clinically feasible concept for “system bias” targeting of the IGF1R to uncouple downregulation from signaling. Demonstrating system bias as an effective anticancer approach, our study reveals a novel strategy for the rational design or repurposing of therapeutics to selectively cross-target the IGF1R or other RTK. Significance: This work provides insight into the molecular and biological roles of biased signaling downstream RTK and provides a novel “system bias” strategy to increase the efficacy of anti–IGF1R-targeted therapy in cancer.

As the p53 tumor suppressor is rarely mutated in conjunctival melanoma (CM), we investigated its activation as a potential therapeutic strategy. Preventing p53/Mdm2 interaction by Nutlin-3, the prototypical Mdm2 antagonist, or via direct siRNA Mdm2 depletion, increased p53 and inhibited viability in CM cell lines. The sensitivity to Nutlin-3 p53 reactivation with concomitant Mdm2 stabilization was higher than that achieved by siRNA, indicative of effects on alternative Mdm2 targets, identified as the cancer-protective IGF-1R. Nutlin-3 treatment increased the association between IGF-1R and β-arrestin1, the adaptor protein that brings Mdm2 to the IGF-1R, initiating receptor degradation in a ligand-dependent manner. Controlled expression of β-arrestin1 augmented inhibitory Nutlin-3 effects on CM survival through enhanced IGF-1R degradation. Yet, the effect of IGF-1R downregulation on cell proliferation is balanced by β-arrestin1-induced p53 inhibition. As mitomycin (MMC) is a well-established adjuvant treatment for CM, and it triggers p53 activation through genotoxic stress, we evaluated how these alternative p53-targeting strategies alter the cancer-relevant bioactivities of CM. In 2D and 3D in vitro models, Nutlin-3 or MMC alone, or in combination, reduces the overall cell tumor growth ~30%, with double treatment inhibition rate only marginally higher than single-drug regimens. However, histopathological evaluation of the 3D models revealed that Nutlin-3 was the most effective, causing necrotic areas inside spheroids and complete loss of nuclear staining for the proliferative marker Ki67. These findings were further validated in vivo; zebrafish xenografts demonstrate that Nutlin-3 alone has higher efficacy in restraining CM tumor cell growth and preventing metastasis. Combined, these results reveal that β-arrestin1 directs Mdm2 toward different substrates, thus balancing IGF-1R pro-tumorigenic and p53-tumor suppressive signals. This study defines a potent dual-hit strategy: simultaneous control of a tumor-promoter (IGF-1R) and tumor-suppressor (p53), which ultimately mitigates recurrent and metastatic potential, thus opening up targeted therapy to CM.

Ligand-activated plasma membrane receptors follow pathways of endocytosis through the endosomal sorting apparatus. Receptors cluster in clathrin-coated pits that bud inwards and enter the cell as clathrin-coated vesicles. These vesicles travel through the acidic endosome whereby receptors and ligands are sorted to be either recycled or degraded. The traditional paradigm postulated that the endocytosis role lay in signal termination through the removal of the receptor from the cell surface. It is now becoming clear that the internalization process governs more than receptor signal cessation and instead reigns over the entire spatial and temporal wiring of receptor signaling. Governing the localization, the post-translational modifications, and the scaffolding of receptors and downstream signal components established the endosomal platform as the master regulator of receptor function. Confinement of components within or between distinct organelles means that the endosome instructs the cell on how to interpret and translate the signal emanating from any given receptor complex into biological effects. This review explores this emerging paradigm with respect to the cancer-relevant insulin-like growth factor type 1 receptor (IGF-1R) and discusses how this perspective could inform future targeting strategies.

Background IGF-1R, a member of the tyrosine receptor family, is well documented in various experimental models to be an important factor in cell transformation, tumor progression, protection from apoptosis and metastasis. In the absence of IGF-1R, most oncogenes are unable to induce malignant transformation, suggesting that some signaling pathways activated by IGF-1R are essential for transformation. We have recently demonstrated that β-arrestin1 (β-arr1), a key regulator of G Protein Coupled Receptor (GPCR), is involved in IGF-1R signaling. Upon ligand binding, β-arr1 brings Mdm2, an E3 ubiquitin ligase, to the IGF-1R resulting in receptor ubiquitination. β-arr1 also functions as a scaffold to activate the MAPK pathway like GPCR. This study aims to investigate whether the β-arr1 mediated signal of IGF-1R is necessary for malignant transformation. Methods Mouse embryonic fibroblasts (MEFs) and a bladder cancer cell line, T24 cells were used as target cells. β-arr1 knockdown was performed by siRNA transfection. Activation of MAPK and PI-3K pathways was analysed by western blot. Cell proliferation was evaluated by PrestoBlue assay. Cellular transformation was estimated by colony formation assay in soft agar. Results To investigate whether β-arr1 is important for transformation, we stably transfected WT MEF and β-arr1 deficient MEFs with some oncogenes such as HRasV12, PvMT, and vSrc. In the absence of β-arr1, HRasV12 was not able to induce malignant transformation. Similar sensitivity to absence of β-arrestin1 was not observed with PyMT or vSrc. β-arr1 regulates IGF-1-induced activation of the MAPK and PI-3K pathways, both pathways being less active in the absence of β-arr1. These data suggested that β-arr1 is involved in Ras-mediated malignant transformation. In T24 cells with the same mutation in HRas, β-arr1 knockdown attenuate IGF-1 induced MAPK and PI-3K pathways like MEFs. Consistent with less activation of the downstream, IGF-1 induced cell proliferation and colony formation are decreased in β-arr1 knockdown cells, indicating that β-arr1 is required for malignant phenotypes in T24 cells. Conclusions These data suggest that β-arr1-dependent signaling by the IGF-1R regulates mechanisms involved in malignant transformation and progression of cancer cells. Legal entity responsible for the study Karolinska institutes, Department of Oncology-Pathology, Leonard Girnita’s group. Funding Swedish Research Council, Swedish Cancer Society, The Swedish Childhood Cancer Foundation. Disclosure All authors have declared no conflicts of interest.