RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/β-catenin signaling in melanoma cells and tumor growth in vivo.

PURPOSE:The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/β-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. METHODS:We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active β-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. RESULTS:Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active β-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and β-catenin, as manifested by a decrease in the transcriptional activity of β-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on β-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. CONCLUSION:RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.