An autocrine Wnt5a loop is a main mechanism of constitutive NF-κB activation in melanoma.

BARBERO G; CASTRO MV; VILLANUEVA MB; MUÑOZ CONTRERAS I; LOPEZ BERGAMI P.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

Sociedades de BioCiencias

Melanoma is the most deadly type of skin cancer and has a poorprognosis when not diagnosed at early stages. Wnt5a is a secretoryglycoprotein involved in the non-canonical Wnt signaling pathwaythat plays an important role in melanoma by increasing motility, invasion,proliferation and resistance to apoptosis. Wnt5a expressionpositively correlates with melanoma progression, tumor grade andpatient outcome. Constitutive activation of NF-κB is a hallmark ofseveral types of tumors including melanoma. The aim of this workwas to study a possible role of Wnt5a in regulating inflammatoryresponses in melanoma by activating the NF-κB pathway. To thisend, we stimulated 1205Lu melanoma cells with both Wnt5a conditionedmedia and purified recombinant Wnt5a. A kinetic analysisshowed that phosphorylation of the critical S536 residue in p65 isinduced by 5 min and reaches a maximal 8-fold increase at 30 minafter Wnt5a treatment (P<0.005) concomitant with a 2-fold increase results were confirmed in five additional melanoma cell lines. Interestingly,cell lines expressing high levels of Wnt5a also displayedvery high basal levels of P-p65 suggesting an active functioningWnt5a/ NF-κB autocrine loop. This possibility was confirmed sinceinhibition of Wnt5a pathway by IWP-2 (a Wnt5a inhibitor) or a Wnt5ashRNA reduced endogenous P-p65 by 40% and 60% respectively(P<0.005).Wnt5a stimulation induced P-p65 translocation to the nucleus asshown by immunofluorescence and Western blot of subcellular fractions.Wnt5a fully stimulated the transcriptional capacity of NF-κB asdemonstrated by a 7-fold increase in the Luciferase activity of a NF-κB reporter (P<0.005). Moreover, Wnt5a induced a time-dependentincrease on protein levels of known NF-κB targets including RelB,PKCδ, BCL2 and Fas. In summary, these results demonstrate thatautocrine Wnt5a stimulation is partly responsible for NF-κB activityin melanoma.