ACTIVATION OF SIGNALING PATHWAYS DOWNSTREAM OF ROR1 IN MELANOMA.

LORENZO D; FERNANDEZ N; PICCO ME; LOPEZ BERGAMI, P

Egmond aan Zee

Congreso; EMBO Meeting - 30 Years of Wnt Signalling; 2012

EMBO - European Molecular Biology Organization

Melanoma represents an important public health problem due to its poor prognosis and the lack ofeffective therapies for the metastatic stage. A hallmark of melanoma cells is the constitutive andsimultaneous activation of several signaling pathways including MAPK, PI3K/Akt, MITF, PKC, NF-κB and p16INK4a. Activation of both canonical and noncanonical Wnt pathway has been alsodemonstrated. The latter is attributed to Wnt5a overexpression and was implicated in melanomametastasis.To assess changes in gene expression that may contribute to activation of the Wnt pathway wedetermined mRNA levels of Fzd and ROR receptors (Fzd3, Fzd5, Fzd6, Fzd7, ROR1 and ROR2).mRNA levels were determined by using Real-Time PCR and normalized to the expression of RNAPol II. Real Time PCR was performed using cDNA from a human normal melanocytic cell line andseveral melanoma cell lines. We observed a marked increase in the levels of ROR1 and Fzd7 inmelanoma cell lines compared to control melanocytes. Increased ROR1 expression was confirmed atthe protein level. It was recently shown that ROR1 is phosphorylated upon incubation of normal cellswith Wnt5a conditioned media. Interestingly, we found that ROR1 is constitutively phosphorylated inmelanoma consistent with Wnt5a overexpression. Of note, we found Dvl2 is also constitutivelyphosphorylated in several melanoma cell lines. To determine ROR1’s contribution to Dvlphosphorylation we stably expressed shRNA for ROR1 in two melanoma cell lines (Lu1205 andA375) displaying constitutive Dvl phosphorylation. Silencing of ROR1 reduced the levels of P-Dvl2and induced a marked shift on the ratio P-Dvl2/un-phosphorylated Dvl2. Similar results wereobserved for Dvl3.To identify signal mediators further downstream of ROR1 and Dvl we determined activation ofsmall GTPases by pull-down assays. ROR1 silencing in both Lu1205 and A375 melanoma cell linesreduced the levels of GTP-bound RhoA/C, indicating a lower GTPase activity. As a first approach tostudy the involvement of protein kinase C (PKC) in ROR1 signaling we determined the protein levelsof PKC isoforms. We found that silencing of ROR1 induced a increase in both PKC alpha and betaisoforms in A375 cells and in PKC beta and epsilon isoforms in Lu1205 cells.Depending on the cellular context, ROR receptors have been shown to activate either the canonicalor noncanonical Wnt pathway. To address this point we performed TOP/FOP reporter assays inmelanoma cells stimulated with Wnt3a conditioned media. Our data reveal that silencing of ROR1does not affect transcription driven by the TOP promoter in response to Wnt3a. In summary, thiswork led to the identification of known downstream mediators (RhoA/C, PKC) of noncanonicalWnt5a signaling in melanoma that are regulated by ROR1. The activation of these pathways mightregulate processes implicated in melanoma development and progression.