The mutation in BRAF kinase at V600 has been frequently reported in nearly 50% of melanoma patients resulting in the hyper-activation of MAPK pathway. As such, various inhibitors which target mutant BRAF have been developed including vemurafenib and dabrafenib both of which have demonstrated significant clinical outcomes. Nevertheless, both primary and acquired resistance reduce the overall response of patients to the therapy. So, the search of efficient biomarkers to stratify melanoma patients has become an imperative subject of research to enhance the efficacy of RAF inhibitor treatments and to decrease the rate of side effects due to the therapy. The mechanism of ubiquitination in controlling various functions such as stabilization of a protein cannot be neglected. A number of ubiquitin regulatory enzymes have been already shown to act on and regulate various component of MAPK pathway.
In order to investigate the role of deubiquitinating enzymes (DUBs) in MAPK signaling, we performed a RNAi screen using a shRNA library targeting all known human DUBs and phosphorylated ERK level was assessed by western blotting. Biochemical analysis of significant hits and their potential role in response of melanoma patients to RAF inhibitors were investigated.
We identified 9 DUBs which significantly regulate MAPK pathway activity including USP28. Interestingly, USP28 has been reported to be mutated in 10% of melanoma patients. Our analysis demonstrated that the loss of USP28 functionality confers poorer overall survival to melanoma patients. The correlation analysis of expression of USP28 in BRAF600E mutated melanoma patients confirmed the tumor suppressor role of USP28 in melanoma. Our results revealed that cells depleted for USP28 show BRAF stabilization resulting in high MAPK activity. Moreover, tissue microarray analysis of 98 melanoma patients showed the inverse correlation of USP28 and BRAF protein expression. Critically we demonstrated that stabilization of BRAF due to the loss of USP28 results in vemurafenib resistance in melanoma.
Overall, our results demonstrate that the loss of USP28 could be used as a novel and promising biomarker for resistance to RAF inhibitors in melanoma patients.
National University of Singapore
National University of Singapore
All authors have declared no conflicts of interest.