Abstract 5296: The role of SOCS-1 in stimulating melanoma development through growth factor receptors and MAPK pathways

Abstract

Abstract Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that multiple signaling pathways that affect motility, growth control, invasion, metabolism, cytokine release and the ability to escape the immune response, regulate melanoma development. Silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with shRNAi inhibits in vitro growth by cell cycle regulation with S phase arrest and increased size of cells and nuclei. It also inhibited tumor cell migration and invasion. Down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptors. Based on the results of SOCS-1 silencing, the present work aims at analyzing the expression of proteins relevant to tumor development, searching for signaling pathways related with SOCS-1. A protein microarray analysis of murine melanoma cells silenced for SOCS-1 by shRNAi SOCS-1 lentivirus transduction was undertaken using as controls, cells transduced with the empty vector. Among the 614 differentially expressed genes some cytokine/growth factor receptors were down regulated, e.g. c-kit, met and EphA3. Western blotting of B16 shR-SOCS-1 cell extracts showed a significant decrease in the expression of these receptors, indicating that SOCS-1 positively regulates the MAPK pathways in these cells. We analyzed the main MAPK pathways that were up regulated in melanoma and observed a decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727). These results demonstrate that SOCS-1 can play an important role in tumor development and progression up regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) was up regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments showed that using a prophylactic protocol, the subcutaneous immunization of syngeneic mice with B16 shR-SOCS-1 viable cells, rendered a protective effect against subcutaneously grafted and metastatic murine melanoma, with significant reduction in tumor growth and metastates in the lungs. A flow cytometry assay showed decreased expression of PD-L1 at the surface of B16 shR-SOCS-1 cells. This might explain the protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells that overexpress PD-L1, which negatively regulates T-cell immune response. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma stimulating cell growth and migration, allowing for the invasive phenotype. Citation Format: Rodrigo Berzaghi, Felipe V. Pereira, Vera S.C. Maia, Jorge B. Scutti, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through growth factor receptors and MAPK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2014-5296