RIPK4 is associated with altered bioenergetics and invasive in melanoma three-dimensional models.

Melanoma is an aggressive cancer characterized by metabolic reprogramming that supports invasion and metastasis. Receptor-interacting protein kinase 4 (RIPK4) has been linked to tumor progression, but its role in melanoma metabolism remains unclear. This study examined whether RIPK4 is associated with melanoma aggressiveness through modulation of cellular bioenergetics. Previously generated RIPK4-knockout A375 and WM266.4 melanoma cell lines (CRISPR/Cas9) were used, and RIPK4 expression was restored by plasmid transfection. Cellular metabolism was assessed using the Seahorse XF Mito Stress Test in 2D monolayer cultures. In 3D spheroid models, ATP assays, qRT-PCR, and Western blotting were performed, together with functional analyses using 3D matrix invasion and CCID assays. In vivo relevance was evaluated by immunohistochemical analysis of RIPK4 and GLUT1 in lung metastases from NOD/SCID mouse xenografts. RIPK4 knockout induced a metabolically compromised state, characterized by reduced mitochondrial respiration and glycolysis, decreased HK2 and GLUT1 expression, and increased SDHB levels also impaired 3D invasive behavior, including reduced formation of invasive protrusions and decreased intravascular invasion. GLUT1 expression was detected in lung metastases but was reduced in RIPK4-deficient tumors. RIPK4 re-expression restored AKT phosphorylation and partially rescued HK2 and GLUT1 levels; however, metabolic flux (OCR/ECAR) was not recovered. These findings suggest that RIPK4 is associated with melanoma metabolic regulation and invasion, potentially involving AKT-linked signaling, but also indicate that additional mechanisms beyond AKT-GLUT1 contribute to the observed metabolic phenotype.