Oncogenesis rewires signaling networks to confer a fitness advantage to malignant cells.,For instance, the B16F0 melanoma cell model creates a cytokine sink for Interleukin-12 (IL-12) to deprive neighboring cells of this important anti-tumor immune signal.,While a cytokine sink provides an indirect fitness advantage, does IL-12 provide an intrinsic advantage to B16F0 cells?,Acute in vitro viability assays were used to compare the cytotoxic effect of imatinib on a melanoma cell line of spontaneous origin (B16F0) with a normal melanocyte cell line (Melan-A) in the presence of IL-12.,The results were analyzed using a mathematical model coupled with a Markov Chain Monte Carlo approach to obtain a posterior distribution in the parameters that quantified the biological effect of imatinib and IL-12.,Intracellular signaling responses to IL-12 were compared using flow cytometry in 2D6 cells, a cell model for canonical signaling, and B16F0 cells, where potential non-canonical signaling occurs.,Bayes Factors were used to select among competing signaling mechanisms that were formulated as mathematical models.,Analysis of single cell RNAseq data from human melanoma patients was used to explore generalizability.,Functionally, IL-12 enhanced the survival of B16F0 cells but not normal Melan-A melanocytes that were challenged with a cytotoxic agent.,Interestingly, the ratio of IL-12 receptor components (IL12RB2:IL12RB1) was increased in B16F0 cells.,A similar pattern was observed in human melanoma.,To identify a mechanism, we assayed the phosphorylation of proteins involved in canonical IL-12 signaling, STAT4, and cell survival, Akt.,In contrast to T cells that exhibited a canonical response to IL-12 by phosphorylating STAT4, IL-12 stimulation of B16F0 cells predominantly phosphorylated Akt.,Mechanistically, the differential response in B16F0 cells is explained by both ligand-dependent and ligand-independent aspects to initiate PI3K-AKT signaling upon IL12RB2 homodimerization.,Namely, IL-12 promotes IL12RB2 homodimerization with low affinity and IL12RB2 overexpression promotes homodimerization via molecular crowding on the plasma membrane.,The data suggest that B16F0 cells shifted the intracellular response to IL-12 from engaging immune surveillance to favoring cell survival.,Identifying how signaling networks are rewired in model systems of spontaneous origin can inspire therapeutic strategies in humans.,Interleukin-12 is a key cytokine that promotes anti-tumor immunity, as it is secreted by antigen presenting cells to activate Natural Killer cells and T cells present within the tumor microenvironment.,Thinking of cancer as an evolutionary process implies that an immunosuppressive tumor microenvironment could arise during oncogenesis by interfering with endogenous anti-tumor immune signals, like IL-12.,Previously, we found that B16F0 cells, a cell line derived from a spontaneous melanoma, interrupts this secreted heterocellular signal by sequestering IL-12, which provides an indirect fitness advantage.,Normally, IL-12 signals via a receptor comprised of two components, IL12RB1 and IL12RB2, that are expressed in a 1:1 ratio and activates STAT4 as a downstream effector.,Here, we report that B16F0 cells gain an intrinsic advantage by rewiring the canonical response to IL-12 to instead initiate PI3K-AKT signaling, which promotes cell survival.,The data suggest a model where overexpressing one component of the IL-12 receptor, IL12RB2, enables melanoma cells to shift the functional response via both IL-12-mediated and molecular crowding-based IL12RB2 homodimerization.,To explore the generalizability of these results, we also found that the expression of IL12RB2:IL12RB1 is similarly skewed in human melanoma based on transcriptional profiles of melanoma cells and tumor-infiltrating lymphocytes.,Additional file 6:Video abstract.,(MP4 600 kb),Video abstract.,(MP4 600 kb)
To determine underlying correlations in multiplex ligation-dependent probe amplification (MLPA) data and their significance regarding survival following treatment of choroidal melanoma (CM).,MLPA data were available for 31 loci across four chromosomes (1p, 3, 6, and 8) in tumor material obtained from 602 patients with CM treated at the Liverpool Ocular Oncology Center (LOOC) between 1993 and 2012.,Data representing chromosomes 3 and 8q were analyzed in depth since their association with CM patient survival is well-known.,Unsupervised k-means cluster analysis was performed to detect latent structure in the data set.,Principal component analysis (PCA) was also performed to determine the intrinsic dimensionality of the data.,Survival analyses of the identified clusters were performed using Kaplan-Meier (KM) and log-rank statistical tests.,Correlation with largest basal tumor diameter (LTD) was investigated.,Chromosome 3: A two-cluster (bimodal) solution was found in chromosome 3, characterized by centroids at unilaterally normal probe values and unilateral deletion.,There was a large, significant difference in the survival characteristics of the two clusters (log-rank, p<0.001; 5-year survival: 80% versus 40%).,Both clusters had a broad distribution in LTD, although larger tumors were characteristically in the poorer outcome group (Mann-Whitney, p<0.001).,Threshold values of 0.85 for deletion and 1.15 for gain optimized the classification of the clusters.,PCA showed that the first principal component (PC1) contained more than 80% of the data set variance and all of the bimodality, with uniform coefficients (0.28±0.03).,Chromosome 8q: No clusters were found in chromosome 8q.,Using a conventional threshold-based definition of 8q gain, and in conjunction with the chromosome 3 clusters, three prognostic groups were identified: chromosomes 3 and 8q both normal, either chromosome 3 or 8q abnormal, and both chromosomes 3 and 8q abnormal.,KM analysis showed 5-year survival figures of approximately 97%, 80%, and 30% for these prognostic groups, respectively (log-rank, p<0.001).,All MLPA probes within both chromosomes were significantly correlated with each other (Spearman, p<0.001).,Within chromosome 3, the strong correlation between the MLPA variables and the uniform coefficients from the PCA indicates a lack of evidence for a signature gene that might account for the bimodality we observed.,We hypothesize that the two clusters we found correspond to binary underlying states of complete monosomy or disomy 3 and that these states are sampled by the complete ensemble of probes.,Consequently, we would expect a similar pattern to emerge in higher-resolution MLPA data sets.,LTD may be a significant confounding factor.,Considering chromosome 8q, we found that chromosome 3 cluster membership and 8q gain as traditionally defined have an indistinguishable impact on patient outcome.