473 DUSP4-DUSP6 axis contributes to sustained ERK activity and high proliferation of the melanoma cells

Abstract

A subset of dual-specificity phosphatases (DUSPs) act as major negative regulators of mitogen-activated protein kinases (MAPKs), and their possible involvement in tumorigenesis has been suggested. Among them, DUSP4 preferentially dephosphorylates ERK1/2 and Jnk over p38. In the present study, we aimed to identify the possible role of DUSP4 in melanomagenesis. Examination of publicly available large-scale data on cancer cell-lines revealed a remarkably high DUSP4 expression and dependency of most melanoma cell lines compared with other tumor-derived cell lines. No such high gene dependency specific to melanoma cells was evident for the other 24 DUSPs encoded in the human genome. Using two melanoma cell lines SK-MEL-28 and A375 as representatives, we confirmed that DUSP4 depletion impaired cell growth without notably inducing apoptosis for up to several days. Immunoblotting and kinase translocation reporter assay showed that the depletion simultaneously induced a decrease, not an increase, in ERK1/2 phosphorylation and an increase in DUSP6 protein level and that it barely affected Jnk phosphorylation. This observation indicates that neither ERK nor Jnk was a direct target of DUSP4 in our experimental setting. Quantitation of DUSP6 mRNA suggested that change in DUSP6 protein level mainly occurred through a post-transcriptional process, although it is not clear whether this took place by an increase in translation rate or inhibition of degradation kinetics. CRISPR/CAS9-mediated knock-out of DUSP6 mostly eliminated a decrease in the ERK phosphorylation and growth retardation following DUSP4 depletion suggesting the existence of the DUSP4-DUSP6-ERK serial regulation pathway. Our data support a model that DUSP4 plays a role in maintaining high ERK1/2 activity by negatively regulating DUSP6, thus contributing to the survival and growth of melanoma cells.