ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration, and invasion. ΔNp63α has been shown to regulate several microRNAs that are involved in development and cancer. We identified miRNA miR-320a as a positively regulated target of ΔNp63α. Previous studies have shown that miR-320a is downregulated in colorectal cancer and targets the small GTPase Rac1, leading to a reduction in noncanonical WNT signaling and EMT, thereby inhibiting tumor metastasis and invasion. We showed that miR-320a is a direct target of ΔNp63α. Knockdown of ΔNp63α in HaCaT and A431 cells downregulates miR-320a levels and leads to a corresponding elevation in PKCγ transcript and protein levels. Rac1 phosphorylation at Ser71 was increased in the absence of ΔNp63α, whereas overexpression of ΔNp63α reversed S71 phosphorylation of Rac1. Moreover, increased PKCγ levels, Rac1 phosphorylation and cell invasion observed upon knockdown of ΔNp63α was reversed by either overexpressing miR-320a mimic or Rac1 silencing. Finally, silencing PKCγ or treatment with the PKC inhibitor Gö6976 reversed increased Rac1 phosphorylation and cell invasion observed upon silencing ΔNp63α. Taken together, our data suggest that ΔNp63α positively regulates miR-320a, thereby inhibiting PKCγ expression, Rac1 phosphorylation, and cancer invasion.

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.

Advances in high-throughput sequencing have enabled profiling of microRNAs (miRNAs), however, a consensus pipeline for sequencing of small RNAs has not been established. We built and optimized an analysis pipeline using Partek Flow, circumventing the need for analyzing data via scripting languages. Our analysis assessed the effect of alignment reference, normalization method, and statistical model choice on biological data. The pipeline was evaluated using sequencing data from HaCaT cells transfected with either a non-silencing control or siRNA against ΔNp63α, a p53 family member protein which is highly expressed in non-melanoma skin cancer and shown to regulate a number of miRNAs. We posit that 1) alignment and quantification to the miRBase reference provides the most robust quantitation of miRNAs, 2) normalizing sample reads via Trimmed Mean of M-values is the most robust method for accurate downstream analyses, and 3) use of the lognormal with shrinkage statistical model effectively identifies differentially expressed miRNAs. Using our pipeline, we identified previously unrecognized regulation of miRs-149-5p, 18a-5p, 19b-1-5p, 20a-5p, 590-5p, 744-5p and 93-5p by ΔNp63α. Regulation of these miRNAs was validated by RT-qPCR, substantiating our small RNA-Seq pipeline. Further analysis of these miRNAs may provide insight into ΔNp63α’s role in cancer progression. By defining the optimal alignment reference, normalization method, and statistical model for analysis of miRNA sequencing data, we have established an analysis pipeline that may be carried out in Partek Flow or at the command line. In this manner, our pipeline circumvents some of the major hurdles encountered during small RNA-Seq analysis.

Abstract 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 DNp63a 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 DNp63a and ERK3 expression levels (MFI). The regulation of ERK3 by DNp63a was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by DNp63a 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.

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 DNp63a 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 (i.e., Actinic Keratosis (AK), a precursor to invasive squamous cell carcinoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC)). A mixed effects (ANOVA) test was used to determine the correlation between DNp63a and ERK3 expression levels (MFI). The regulation of ERK3 by DNp63a was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by DNp63a on cell migration was measured by performing trans-well migration assay. Results: The expression levels of both ∆Np63α and ERK3 proteins are upregulated in NMSCs compared to normal tissues. There is a strong positive correlation between ∆Np63α and ERK3 expression in normal skin and patients with AK, SCC and 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. Conclusions: ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.