ERK3 is transcriptionally upregulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in non-melanoma skin cancers.

BACKGROUND: p63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α 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. METHODS: Fluorescent immunohistochemistry was performed to evaluate the expression levels of ΔNp63α 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 ΔNp63α and ERK3 expression levels (MFI). The regulation of ERK3 by ΔNp63α was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by ΔNp63α on cell migration was measured by performing trans-well migration assay. RESULTS: The expression level of ∆Np63α 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 ∆Np63α and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that ∆Np63α positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with ∆Np63α in NMSCs. Moreover, similar to the effect of ∆Np63α depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing ∆Np63α counteracted the increase in cell migration induced by the depletion of ∆Np63α. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells. CONCLUSIONS: ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.

TIP60 up-regulates ΔNp63α to promote cellular proliferation.

An estimated 5.4 million cases of nonmelanoma skin cancer are reported in the United States at an associated cost of $4.8 billion. ΔNp63α, a proto-oncogene in the p53 family of transcription factors, is overexpressed in squamous cell carcinoma (SCC) and associated with poor prognosis and survival. ΔNp63α elicits its tumorigenic effects in part by promoting cellular proliferation and cell survival. Despite its importance in SCC, the upstream regulation of ΔNp63α is poorly understood. In this study, we identify TIP60 as a novel upstream regulator of ΔNp63α. Using a combination of overexpression, silencing, stable expression, and pharmacological approaches in multiple cell lines, we showed that TIP60 up-regulates ΔNp63α expression. Utilizing cycloheximide treatment, we showed that TIP60 catalytic activity is required for stabilization of ΔNp63α protein levels. We further showed that TIP60 coexpression inhibits ΔNp63α ubiquitination and proteasomal degradation. Stabilization of ΔNp63α protein was further associated with TIP60-mediated acetylation. Finally, we demonstrated that TIP60-mediated regulation of ΔNp63α increases cellular proliferation by promoting G2/M progression through MTS assays and flow cytometry. Taken together, our findings provide evidence that TIP60 may contribute to SCC progression by increasing ΔNp63α protein levels, thereby promoting cellular proliferation.

ΔNp63α and microRNAs: leveraging the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a cellular reprogramming mechanism that is an underlying cause of cancer metastasis. Recent investigations have uncovered an intricate network of regulation involving the TGFß, Wnt, and Notch signaling pathways and small regulatory RNA species called microRNAs (miRNAs). The activity of a transcription factor vital to the maintenance of epithelial stemness, ΔNp63α, has been shown to modulate the activity of these EMT pathways to either repress or promote EMT. Furthermore, ΔNp63α is a known regulator of miRNA, including those directly involved in EMT. This review discusses the evidence of ΔNp63α as a master regulator of EMT components and miRNA, highlighting the need for a deeper understanding of its role in EMT. This expanded knowledge may provide a basis for new developments in the diagnosis and treatment of metastatic cancer.