Luteolin overcomes vemurafenib resistance in melanoma by upregulating RNF125 expression via inhibiting miR-15b-5p

Nearly half of melanoma patients harbor a mutation of valine 600 to glutamine (V600E) in the BRAF gene. BRAF(V600E) inhibitors (BRAFi) including vemurafenib, have good clinical efficacy in treating unresectable or metastatic melanomas with the BRAF(V600E) mutation. However, most patients develop resistance to vemurafenib within 8 months, which severely limits its clinical application. No therapy has been approved for overcoming vemurafenib resistance in melanoma. Novel agents for managing vemurafenib-resistant melanomas are needed.

Luteolin (3',4',5,7-tetrahydroxyflavone) is a flavonoid occurring in diverse edible and medicinal herbs. Previous studies have shown that luteolin inhibits the activation of AKT, STAT3, or ERK to exert anti-melanoma effects. These luteolin-targeted molecules have been shown to promote vemurafenib resistance in melanoma. However, whether luteolin can overcome vemurafenib resistance in melanoma is not known. This study aimed to evaluate the effects of luteolin in overcoming vemurafenib resistance in melanoma and to investigate the mechanisms of action of the compound.

The results of in vitro experiments showed that luteolin reduced the cell viability and colony formation ability, and inhibited tumor sphere growth of, as well as induced apoptosis in, acquired vemurafenib-resistant A375 (A375-VR) melanoma cells. Animal assay data showed that luteolin dose-dependently restrains tumor growth in A375-VR melanoma-bearing BALB/c-nu/nu mice. RNA-seq analyses revealed that the mRNA level of RNF125 (E3 Ubiquitin-Protein Ligase RNF125) was lower in A375-VR cells than in parental A375 melanoma cells, and luteolin abolished the resistance-associated downregulation. RT-qPCR analyses verified the RNA-seq results. Immunoblotting showed that luteolin upregulated the protein level of RNF125 in A375-VR cultures and mouse tumors. Luteolin also downregulated protein levels of the RNF125 substrate JAK1 and JAK1’s downstream molecules PDGFR-β, EGFR, AXL, neuropilin-1, MITF, cyclin D1, RUNX2, caveolin-1, c-JUN, IL7R, and VEGFC, and upregulated the protein level of p21 in A375-VR cells. RNF125 knockdown attenuated the effect of luteolin in decreasing cell viability in A375-VR cells.

MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression. We next explored whether luteolin upregulates RNF125 expression by regulating certain miRNAs in vemurafenib-resistant melanoma cells. Bioinformatics analysis predicted that miR-15b-5p is a potential miRNA that targets RNF125. Our dual luciferase reporter assays verified that miR-15b-5p directly targets RNF125's 3' untranslated region in A375-VR cells. The mRNA and protein levels of RNF125 were significantly downregulated and upregulated upon transfection with the miR-15b-5p mimics and the miR-15b-5p inhibitor, respectively, in A375-VR cells. Also, miR-15b-5p expression was lowered by luteolin treatment in A375-VR cells.

In summary, our novel findings indicate that luteolin overcomes vemurafenib resistance in melanoma cell and mouse models, which is associated with miR-15b-5p inhibition-mediated upregulation of RNF125. This study provides a pharmacological basis for developing luteolin into a new drug for treating vemurafenib-resistant melanomas. Moreover, this work suggests that targeting miR-15b-5p/RNF125 axis is a novel approach for overcoming BRAF inhibitor resistance in melanoma.