Abstract A146: The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression
Authors: Berzaghi Rodrigo , Maia Vera SC , Pereira Felipe V. , Melo Filipe Manegatti De , Travassos Luiz R. , Travassos Luiz R. 
Published: May 2016

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 lentivirus (shR-SOCS-1) 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 614 differentially expressed genes, c-kit, met and EphA3 cytokine/tyrosine-kinase (TK) receptors were downregulated. A significant decrease in the expression of TK receptors as observed by Western blotting of B16 shR-SOCS-1 extracts showed that SOCS-1 positively regulates the MAPK signaling pathways in these cells. A decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727) was observed. These results demonstrate that SOCS-1 plays an important role in tumor development and progression up-regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) and total AP-2 alpha were up-regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments using a syngeneic model and a prophylactic protocol showed that the subcutaneous immunization with shR-SOCS-1-transduced viable tumor cells, rendered protection against either subcutaneously grafted or metastatic murine melanoma cells, with significant reduction in tumor growth and lung metastatic nodules. Such immunization increased the expression of CD11c, CD80, CD86 and MHC-II in splenic dendritic cells, as compared to DCs from a group that received only B16F10-Nex2 cells. Of notice was the decreased expression of PD-L1 in SOCS-1-silenced B16F10-Nex2 cells (B16shR-SOCS-1). Such molecular phenotype may have a role in the immune response and protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells. By overexpressing PD-L1, WT-melanoma cells negatively regulates T-cell proliferation in the tumor environment. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma development and the potential of SOCS-1-silenced tumor cells in raising an effective anti-tumor immune response, inducing protection even against a metastatic melanoma challenge.

Funded by FAPESP 2010/51423-0 and 2012/17473-6.

Citation Format: Rodrigo Berzaghi, Esq., Vera SC Maia, II, Felipe V. Pereira, III, Filipe Manegatti De Melo, Luiz R. Travassos, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A146.