RIPK4 Downregulation Reduces ABCG2 Expression, Increasing BRAF-Mutated Melanoma Cell Susceptibility to Cisplatin- and Doxorubicin-Induced Apoptosis.

Melanoma cells remain resistant to chemotherapy with cisplatin (CisPt) and doxorubicin (DOX). The abnormal expression of Receptor-Interacting Protein Kinase 4 (RIPK4) in certain melanomas contributes to tumour growth through the NFκB and Wnt/β-catenin signalling pathways, which are known to regulate chemoresistance and recurrence. Despite this, the role of RIPK4 in response to chemotherapeutics in melanoma has not been reported. In this study, we examined how the downregulation and overexpression of RIPK4 affect the sensitivity of BRAF-mutated melanoma cells (A375 and WM266.4) to CisPt and DOX along with determining the underlying mechanism. Using two RIPK4 silencing methods (siRNA and CRISPR/Cas9) and overexpression (dCas9-VPR), we assessed CisPt and DOX-induced apoptosis using caspase 3/7 activity, annexin V/7AAD staining, and FASC analysis. In addition, qRT-PCR and Western blotting were used to detect apoptosis-related genes and proteins such as cleaved PARP, p53, and cyclin D1. We demonstrated that the overexpression of RIPK4 inhibits, while its downregulation enhances, CisPt- or DOX-induced apoptosis in melanoma cells. The effects of downregulation are similar to those observed with pre-incubation with cyclosporin A, an ABCG2 inhibitor. Additionally, our findings provide preliminary evidence of crosstalk between RIPK4, BIRC3, and ABCG2. The results of these studies suggest the involvement of RIPK4 in the observed resistance to CisPt or DOX.