IGF-1R is a molecular determinant for response to p53 reactivation therapy in conjunctival melanoma.

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 ß-arrestin1, the adaptor protein that brings Mdm2 to the IGF-1R, initiating receptor degradation in a ligand-dependent manner. Controlled expression of ß-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 ß-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 ß-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.

Effect of plaque brachytherapy dose and dose rate on risk for disease-related mortality in 1238 patients with choroidal melanoma.

BACKGROUND: Episcleral brachytherapy is the most common treatment for medium-sized choroidal melanomas. Although controversial, inadequate brachytherapy dose and dose rates have at least a hypothetical implication on patient survival. METHODS: All patients who received ruthenium-106 or iodine-125 brachytherapy for choroidal melanoma at St. Erik Eye Hospital 1996 to 2016 were included (n=1238). Cox regression hazard ratios for melanoma-related mortality across deciles, quartiles and individual integers of apex radiation doses (Gy) and dose rates (Gy/hour) were calculated, adjusted for tumour size and location. RESULTS: The average radiation dose at the tumour apex ranged from 73.0 Gy in the first decile to 108.6 Gy in the tenth. Decreasing apex dose by 1 Gy increments or by decile or quartile group was not associated with melanoma-related mortality (p>0.2) The average radiation dose rate at the tumour apex ranged from 0.5 Gy/hour in the first decile to 2.8 Gy/hour in the tenth. Similarly, decreasing apex dose rate by 1 Gy/hour increments or by decile or quartile groups was not associated with melanoma-related mortality (p>0.5). CONCLUSION: There are no increased hazards for choroidal melanoma-related mortality after brachytherapy with decreasing doses between 108.6 and 73.0 Gy, or with decreasing dose rates between 2.8 and 0.5 Gy/hour.

Ruthenium-106 versus iodine-125 plaque brachytherapy of 571 choroidal melanomas with a thickness of ≥5.5 mm.

BACKGROUND: Episcleral brachytherapy is the most common eye-preserving treatment for medium-sized choroidal melanomas. γ-emitting iodine-125 (125I) and ß-emitting ruthenium-106 (106Ru) are widely used. The latter is however generally reserved for thinner tumours (<6 mm). In this study, we compare ocular and patient survival in thicker tumours treated with the respective radioisotope. METHODS: All patients with ≥5.5 mm thick choroidal melanomas who were treated with plaque brachytherapy at a single institution between 1 November 1979 and 31 December 2015 were included (n=571). Size-controlled Cox regression HRs for postbrachytherapy enucleation, repeated brachytherapy and melanoma-related mortality were calculated, as well as Kaplan-Meier disease-specific survival and relative 10-year survival in matched subgroups. RESULTS: 317 patients were treated with 106Ru and 254 with 125I. The rate of repeated brachytherapy was significantly higher among patients treated with 106Ru (8%) than with 125I (1%, p<0.001). Size-controlled Cox regression HRs for postbrachytherapy enucleation (125I vs 106Ru 0.7, p=0.083) and melanoma-related mortality were not significant (125I vs 106Ru 1.1, p=0.63). Similarly, Kaplan-Meier disease-specific and relative 10-year survival was comparable in matched groups of 5.5-7.4 mm (relative survival 106Ru 59%, 125I 56%) and ≥7.5 mm thick tumours (relative survival 106Ru 46%, 125I 44%). CONCLUSIONS: Rates of repeated brachytherapy were significantly higher among patients treated with 106Ru versus 125I for thick choroidal melanomas. There were, however, no significant differences in rates of enucleation or patient survival.

Below the Surface: IGF-1R Therapeutic Targeting and Its Endocytic Journey.

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.

Nuclear HER3 is associated with favorable overall survival in uveal melanoma.

HER3 is a member of the epidermal growth factor receptor (EGFR) family and is expressed in several types of cancer. Both the cytoplasmic and nuclear appearances of the receptor have been reported. Here, we investigate the expression and subcellular distribution of HER3 in uveal melanoma (UM) cells and tissues and its potential impact on clinical outcome of patients. Paraffin-embedded samples from 128 consecutive UM patients, enucleated without alternative treatment on UM diagnosis, were evaluated for HER3 using immunohistochemistry. Immunoreactivity was scored for frequency, intensity of positive cells, and subcellular distribution. The results were correlated with the established clinicopathological parameters using univariate and multivariate statistical analyses. HER3 expression was shown in 70% of the cases (89/128). This contrasts with the other EGFR family receptors (EGFR, HER2 and HER4) that are infrequently expressed in UM. Surprisingly, HER3 was found to be localized solely in the cell nuclei in 56 cases. The remaining 33 HER3 positive cases showed diffuse distribution (cytoplasmic ± nuclear). Nuclear HER3 was independently correlated with a more favorable overall survival (p = 0.043 and hazard ratio = 0.618) compared to cases with diffuse and/or no HER3. Nuclear localization of HER3 was also confirmed in fresh UM material and in UM cell lines. In conclusion, HER3 is frequently localized solely in the cell nuclei in UM and as such it predicts a more favorable overall survival.

Intratumoral forkhead box P3-positive regulatory T cells predict poor survival in cyclooxygenase-2-positive uveal melanoma.

BACKGROUND: Forkhead box P3 (FOXP3)-positive regulatory T cells (Tregs) are key mediators of peripheral tolerance and suppress efficient antitumor responses. Prostaglandin E(2) (PGE(2)) produced by inducible cyclooxygenase-2 (COX-2) can lead to Treg induction. COX-2 expression has been linked to tumorigenesis and growth in various malignancies. The objective of the current study was to investigate whether Tregs infiltrate uveal melanomas (UMs) and whether their prevalence is linked to COX-2 expression and the prediction of overall survival (OS). METHODS: One hundred patients who underwent enucleation after they were diagnosed with UM were included in the study. Immunohistochemical staining with monoclonal anti-FOXP3, anti-CD4, and anti-COX-2 antibodies was performed, and immunoreactivity was assessed. Correlations of COX-2 expression with the presence of Tregs, established clinicopathologic parameters, and OS were evaluated in univariate and multivariate analyses. RESULTS: High expression of COX-2 was predictive of shortened OS. FOXP3-positive Tregs were detectable in 24% of UMs and were restricted to malignant tissue. The extent of COX-2 expression was associated significantly with Treg prevalence (P = .004) and Treg intratumoral localization (P = .005). Intratumoral Tregs (but not the prevalence of Tregs) were independent marker for worse OS with a hazard ratio of 5.36 in patients with COX-2-positive tumors. CONCLUSIONS: The current results demonstrated that high COX-2 expression is associated with OS and Treg prevalence in UM. These findings are in line with the observations that COX-2/PGE(2) induces Tregs and that Tregs may alter antitumor responses, resulting in a negative effect on the clinical disease course. Intratumoral Tregs are an independent prognostic marker for COX-2-positive UM, and these results put COX-2 inhibitors and Treg depletion into the spotlight of potential novel treatment modalities for patients with UM.

SUMOylation mediates the nuclear translocation and signaling of the IGF-1 receptor.

The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in developmental and cancer biology. Most of its biological effects have been ascribed to its tyrosine kinase activity, which propagates signaling through the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Here, we report that IGF-1 promotes the modification of IGF-1R by small ubiquitin-like modifier protein-1 (SUMO-1) and its translocation to the nucleus. Nuclear IGF-1R associated with enhancer-like elements and increased transcription in reporter assays. The SUMOylation sites of IGF-1R were identified as three evolutionarily conserved lysine residues-Lys(1025), Lys(1100), and Lys(1120)-in the beta subunit of the receptor. Mutation of these SUMO-1 sites abolished the ability of IGF-1R to translocate to the nucleus and activate transcription but did not alter its kinase-dependent signaling. Thus, we demonstrate a SUMOylation-mediated mechanism of IGF-1R signaling that has potential implications for gene regulation.

Expression and prognostic significance of iNOS in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Disease metastasis occurs in half of the patients and is uniformly fatal despite systemic therapy. Inducible nitric oxide synthase (iNOS) is associated with disease progression in various malignancies including cutaneous melanoma. In this retrospective cohort, we examined the prognostic value of iNOS in UM by performing immunohistochemistry on paraffin-embedded sections of primary tumors (90 patients) and matched primary and metastatic hepatic tumors (19 patients) with complete histopathological and clinical data. We show that iNOS is expressed in UM (57% of the patients) and high iNOS levels significantly (p = 0.04; hazard ratio (HR) = 2.3) predict disease-specific survival (DSS) as assessed by Kaplan-Meier analysis and univariate Cox's proportional hazards regression model. Furthermore, high iNOS expression in the UM primary tissue was significantly associated with metastatic disease and vice versa. Expression of iNOS in hepatic metastases significantly (p = 0.02) predicted a shortened survival as assessed by Kaplan-Meier analysis. However, iNOS did not appear to be a significant (p = 0.16; HR = 1.9) factor in the multivariate Cox's regression analysis performed together with the clinical parameters tumor diameter, tumor cell type, and tumor location in which only tumor diameter predicted DSS. In conclusion, iNOS predicts DSS in UM and may play a role in disease progression but it is not an independent prognostic factor.