Wnt5a activates NF-kB and promotes cytokine release in melanoma cells.

BARBERO G; CASTRO MV; VILLANUEVA MB; QUEZADA MJ; LOPEZ BERGAMI P

Mar del Plata

Congreso; Reunión de la Sociedad Argentina de Investigación Clínica; 2018

Sociedad Argentina de Investigación Clínica

Melanoma is the most deadly type of skin cancer and has a poorprognosis when not diagnosed at early stages. Wnt5a is a secretory glycoproteininvolved in the non-canonical Wnt signaling pathway that plays an importantrole in melanoma by increasing motility, invasion, proliferation and resistanceto apoptosis.  Previous results from our lab have demonstrated the abilityof Wnt5a to activate the canonical NF-kB pathway. The aim of this work was tofurther study the molecular mechanism and functional consequences of theWnt5a-NF-kB is crosstalk.Our first goal was to identify proteins from both pathways thatare required for the crosstalk. By using RNA interference and dominant negativemutants we determined that ROR1 and Dvl2 (from the Wnt5a pathway) and TRAF2 andNIK (from the NF-κB pathway) are essential for the phosphorylation of p65dependent of Wnt5a. In contrast, ROR2, Dvl1, Dvl3 and RIP are dispensable.Next, we determined that Akt activity is required for Wnt5a-dependentphosphorylation of p65. Both, the PI3K inhibitor LY-294002 and a shRNA againstRictor prevented the phosphorylation of p65 upon Wnt5a treatment. In contrastpharmacological inhibitors of IKK, PKCδ and GSK3β did not affectWnt5a-dependent p65 phosphorylation.We have previously shown that Wnt5a stimulation of melanomacells activates transcription of genes usually regulated by NF-κB such as RelB, PKCδ, BCL2 and Cyclin D1.To determine whether Wnt5a promotes the production of cytokines, culture mediafrom Mewo melanoma cells stimulated for 32h with Wnt5a were evaluatedusing a Human Inflammatory Antibody Protein Array. This analysis revealedthat Wnt5a increased the production of several cytokines including GM-SCF,IL-6, IL-8, IL-11, MCP-1 and TNF sRI.  We validated these results by ELISAand found a 2-fold increase in IL-8 levels at 4h (p<0.05) and a 14-foldincrease at 32h (p<0.001) upon treatment with Wnt5a. Similar results wereobtained in Skmel-28 and 1205Lu.Taken together these results indicate that Wnt5a might have andimmunomodulatory effect on melanoma promoting pro-inflammatory environment thatenhances tumor survival.