Does upregulating RNF125 expression with luteolin overcome vemurafenib resistance in melanoma?

Advanced melanoma is one of the deadliest forms of cancer. Around half of melanoma patients carry a BRAF mutation of valine 600 to glutamine (V600E). Targeted therapies using selective BRAF(V600E) inhibitors (BRAFi), such as vemurafenib, have shown remarkable efficacy in patients with BRAF(V600E)-mutant melanomas, but most patients develop resistance within 8 months. E3 ubiquitin-protein ligase RNF125 is a component of the ubiquitin proteasome system. Its overexpression has been shown to slow the growth of BRAFi-resistant melanoma cells. TCGA data retrieved from the human protein atlas showed that melanoma patients with higher expression levels of RNF125 survive longer. Therefore, upregulating RNF125 is a potential strategy for treating BRAFi-resistant melanomas. However, no RNF125-upregulating agents have been reported. Chinese medicinal herb-derived and multi-targeted natural flavonoids have advantages in fighting drug resistance. Through screening, we found that luteolin (a natural flavone) reduces viability of, and induces apoptosis in, acquired vemurafenib-resistant A375 (A375-VR) and Colo829 (Colo829-VR) melanoma cells. Importantly, prophylactic dosing of luteolin significantly suppresses A375-VR tumor growth in mice without affecting mouse body weight. Moreover, luteolin prolongs survival time of A375-VR melanoma-bearing mice. To understand the mechanisms responsible for overcoming vemurafenib resistance by luteolin, we performed RNA-seq analyses and found that, the mRNA level of RNF125 is lower in A375-VR cells than in parental A375 melanoma cells, and luteolin abolishes the resistance-associated downregulation. We also found that mRNA levels of several genes downstream of RNF125 are altered in A375-VR cells compared to those in A375 cells, and luteolin reverses the alterations of most of these genes. The RNA-seq results have been verified by RT-qPCR. Furthermore, luteolin was found to be able to upregulate the protein level of RNF125 in A375-VR cultures and in mouse A375-VR xenografts. We, therefore, hypothesize that luteolin overcomes vemurafenib resistance in melanoma by upregulating RNF125 expression. The objectives of this study are as follows.
1) To confirm the effects of luteolin in overcoming vemurafenib resistance, and to monitor toxicities of luteolin, in melanoma-bearing mice;
2) To establish the role of RNF125 in the effects of luteolin in overcoming vemurafenib resistance in melanoma cells; and
3) To determine whether luteolin overcomes resistance to other BRAF(V600E)-targeted therapies in melanoma cells.

This study will for the first time demonstrate the ability of luteolin to overcome vemurafenib resistance via, at least in part, upregulating RNF125 in BRAF(V600E)-mutant melanomas; and for the first time demonstrate that pharmacological upregulation of RNF125 is a strategy for treating BRAFi-resistant melanomas. This contribution will be significant because it is one step in a continuum of research that is expected to lead to the development of luteolin and possibly other RNF125-upregulating agents as novel drugs for treating BRAFi-resistant melanomas.