As the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome siRNA screen in melanoma cells to expand our understanding of kinases that regulate this pathway, and to illuminate potential therapeutic directions. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAFV600E mutation, negatively regulates Wnt/β-catenin signaling in human melanoma cells. As inhibitors of BRAFV600E show promise in ongoing clinical trials we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAFV600E inhibitor, PLX4720. Surprisingly, endogenous β-catenin is required for PLX4720 to induce apoptosis in melanoma cells, while activation of Wnt/β-catenin signaling strongly synergizes with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlates with a reduction in the abundance of a β-catenin antagonist, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, melanoma cell lines that are resistant to apoptosis after treatment with a BRAFV600E inhibitor become susceptible, and undergo apoptosis, when AXIN1 is reduced by siRNA. These findings point to a role for Wnt/β-catenin signaling and AXIN1 in regulating the efficacy of inhibitors of BRAFV600E, and may stimulate consideration of potential combination therapies and biomarkers for use in conjunction with targeted BRAF therapy.
SUMMARY To identify new protein and pharmacological regulators of Wnt/β-catenin signaling we used a cell-based reporter assay to screen a collection of 1857 human-experienced compounds for their ability to enhance activation of the β-catenin reporter by a low concentration of WNT3A. This identified 44 unique compounds, including the FDA-approved drug riluzole, which is presently in clinical trials for treating melanoma. We found that treating melanoma cells with riluzole in vitro enhances the ability of WNT3A to regulate gene expression, to promote pigmentation, and to decrease cell proliferation. Furthermore riluzole, like WNT3A, decreases metastases in a mouse melanoma model. Interestingly, siRNAs targeting the metabotropic glutamate receptor, GRM1, a reported indirect target of riluzole, enhance β-catenin signaling. The unexpected regulation of β-catenin signaling by both riluzole and GRM1 has implications for the future uses of this drug.
Wnt signaling, which encompasses multiple biochemical pathways that regulate neural development downstream of extracellular Wnt glycoprotein ligands, has been suggested to contribute to major psychiatric disorders including autism spectrum disorders (ASD). We used next-generation sequencing and Sequenom genotyping technologies to resequence 10 Wnt signaling pathway genes in 198 ASD patients and 240 matched controls. Results for single-nucleotide polymorphisms (SNPs) of interest were confirmed in a second set of 91 ASD and 144 control samples. We found a significantly increased burden of extremely rare missense variants predicted to be deleterious by PolyPhen-2, distributed across seven genes in the ASD sample (3.5% in ASD vs 0.8% in controls; Fisher's exact test, odds ratio (OR)=4.37, P=0.04). We also found a missense variant in WNT1 (S88R) that was overrepresented in the ASD sample (8 A/T in 267 ASD (minor allele frequency (MAF)=1.69%) vs 1 A/T in 377 controls (MAF=0.13%), OR=13.0, Fisher's exact test, P=0.0048; OR=8.2 and P=0.053 after correction for population stratification). Functional analysis revealed that WNT1-S88R is more active than wild-type WNT1 in assays for the Wnt/β-catenin signaling pathway. Our findings of a higher burden in ASD of rare missense variants distributed across 7 of 10 Wnt signaling pathway genes tested, and of a functional variant at the WNT1 locus associated with ASD, support that dysfunction of this pathway contributes to ASD susceptibility. Given recent findings of common molecular mechanisms in ASD, schizophrenia and affective disorders, these loci merit scrutiny in other psychiatric conditions as well.
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