Erk3 Is Transcriptionally Upregulated By Np63 Alpha And Mediates The Role Of Np63 Alpha In Suppressing Cell Migration In Non-Melanoma Skin Cancers

Backgroundp63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. Np63 alpha is the main isoform of p63 and highly expressed in Non-melanoma skin cancer (NMSC). Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose biochemical features and cellular regulation are distinct from those of conventional MAPKs such as ERK1/2. While ERK3 has been shown to be upregulated in lung cancers and head and neck cancers, in which it promotes cancer cell migration and invasion, little is known about the implication of ERK3 in NMSCs.MethodsFluorescent immunohistochemistry was performed to evaluate the expression levels of Delta Np63 alpha and ERK3 in normal and NMSC specimens. Dunnett's test was performed to compare mean fluorescence intensity (MFI, indicator of expression levels) of p63 or ERK3 between normal cutaneous samples and NMSC samples. A mixed effects (ANOVA) test was used to determine the correlation between Delta Np63 alpha and ERK3 expression levels (MFI). The regulation of ERK3 by Delta Np63 alpha was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by Delta Np63 alpha on cell migration was measured by performing trans-well migration assay.ResultsThe expression level of Np63 alpha is upregulated in NMSCs compared to normal tissue. ERK3 level is significantly upregulated in AK and SCC in comparison to normal tissue and there is a strong positive correlation between Np63 alpha and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that Np63 alpha positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with Np63 alpha in NMSCs. Moreover, similar to the effect of Np63 alpha depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing Np63 alpha counteracted the increase in cell migration induced by the depletion of Np63 alpha. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells.ConclusionsERK3 is positively regulated by Np63 alpha and mediates the role of Np63 alpha in suppressing cell migration in NMSC.