Interleukin-12 elicits a non-canonical response in B16 melanoma cells to enhance survival

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?

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).

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

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. Conclusions: 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).