8626

TP63

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the DeltaNp63alpha and beta isoforms, leaving only DeltaNp63gamma, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the DeltaNp63alpha isoform. We also show that DeltaNp63gamma expressed in a background where all the other DeltaNp63 are knocked down causes EMT with an increase in TGFbeta-1, -2, and -3 and downstream effectors Smads2/3/4. In addition, a p63 binding site in intron 1 of TGFbeta was identified. Inhibition of the TGFbeta response with a specific inhibitor results in reversion of EMT in DeltaNp63alpha- and beta-depleted cells. In summary, we show that p63 is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers.

Proper differentiation of placental epithelial cells, called trophoblast, is required for implantation. Early during placentation, trophoblast cell columns help anchor the developing embryo in the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB) distally, these cells differentiate into invasive extravillous trophoblast. We previously reported that p63, a p53 family member, is highly expressed in proliferative villous CTB and required for induction of the trophoblast lineage in human pluripotent stem cells. We now further explore its function in human trophoblast by using both primary CTB from the early placenta and established trophoblast cell lines. We show that p63 is expressed in epidermal growth factor receptor-positive CTB and that its expression decreases with differentiation into HLA-G(+) extravillous trophoblast. In trophoblast cell lines, p63 is expressed in JEG3 cells but absent from HTR8 cells. Overexpression of p63 in both cell lines enhances cell proliferation and significantly reduces cell migration; conversely, down-regulation of p63 in JEG3 cells reduces cell proliferation and restores cell migration. Analysis of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways shows that p63 blocks epithelial-to-mesenchymal transition, promotes a CTB-specific cell adhesion profile, and inhibits expression of matrix metalloproteinases. Taken together, these data show that p63 maintains the proliferative CTB state, at least partially through regulation of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways.CI - Copyright (c) 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Cell identity signals influence the invasive capability of tumor cells, as demonstrated by the selection for programs of epithelial-to-mesenchymal transition (EMT) during malignant progression. Breast cancer cells retain canonical epithelial traits and invade collectively as cohesive groups of cells, but the signaling pathways critical to their invasive capabilities are still incompletely understood. Here we report that the transcription factor DeltaNp63alpha drives the migration of basal-like breast cancer (BLBC) cells by inducing a hybrid mesenchymal/epithelial state. Through a combination of expression analysis and functional testing across multiple BLBC cell populations, we determined that DeltaNp63alpha induces migration by elevating the expression of the EMT program components Slug and Axl. Interestingly, DeltaNp63alpha also increased the expression of miR-205, which can silence ZEB1/2 to prevent the loss of epithelial character caused by EMT induction. In clinical specimens, co-expression of various elements of the DeltaNp63alpha pathway confirmed its implication in motility signaling in BLBC. We observed that activation of the DeltaNp63alpha pathway occurred during the transition from noninvasive ductal carcinoma in situ to invasive breast cancer. Notably, in an orthotopic tumor model, Slug expression was sufficient to induce collective invasion of E-cadherin-expressing BLBC cells. Together, our results illustrate how DeltaNp63alpha can drive breast cancer cell invasion by selectively engaging promigratory components of the EMT program while, in parallel, still promoting the retention of epithelial character.CI - (c)2015 American Association for cancer Research.

Cervical cancer is one of the most common female cancers worldwide. Although the therapeutic outcomes of patients with early-stage cervical cancer have been significantly improved in the past decades, tumor metastasis and recurrence remain the major causes of cervical cancer-related deaths. In cervical squamous cell carcinoma (SCC), the aberrant activation of epithelial-mesenchymal transition (EMT), a crucial process in invasion and metastasis of epithelial cancer, could promote lymph nodal metastasis and recurrence, and predicts poor prognosis. In this study, we show that the expression levels of EMT markers, beta-catenin and Vimentin, are associated with the p63 isoform DeltaNp63alpha in SCC by using immunohistochemistry staining and analysis. Compared to the control SiHa cells (SiHa-NC), the expression of E-cadherin and beta-catenin are upregulated, while Vimentin and ZEB1 are downregulated in the constructed SiHa cell line with stable DeltaNp63alpha overexpression (SiHa-DeltaNp63alpha). Besides, the migration and invasion abilities are also suppressed in SiHa-DeltaNp63alpha cells with a typical epithelial morphology with cobblestone-like shape, suggesting that DeltaNp63alpha is a vital EMT repressor in SCC cells. In addition, the involvement of miR-205/ZEB1 axis in the inhibition effect of DeltaNp63alpha on EMT program is revealed by a miRNA array and confirmed by the subsequent transfection of the miR-205 mimic and antagomir. Moreover, SCC patients with low DeltaNp63alpha expression and high EMT level show more frequent metastasis and recurrence as well as reduced overall survival. Therefore, EMT program and its vital repressor DeltaNp63alpha could be used as biomarkers for tumor metastasis and recurrence in cervical cancer.

The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of Np63alpha, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of Np63alpha expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of Np63alpha alone is sufficient to induce EMT. Both H-RasV12 expression and Np63alpha depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of Np63alpha.

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 TGFbeta, 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, DeltaNp63alpha, has been shown to modulate the activity of these EMT pathways to either repress or promote EMT. Furthermore, DeltaNp63alpha is a known regulator of miRNA, including those directly involved in EMT. This review discusses the evidence of DeltaNp63alpha 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.

OBJECTIVES: We previously showed that DeltaNp63beta, a splicing variant of DeltaNp63, mediated EMT and affected cell motility. DNA microarray was thus performed to elucidate the mechanism that DeltaNp63beta affects cell motility. As the results, Wnt5a was significantly down-regulated by DeltaNp63beta overexpression in tongue SCC cell line (SQUU-B) with EMT phenotype. MATERIALS AND METHODS: Seven OSCC cell lines were used. expression of DeltaNp63, Wnt5a, its receptor Ror2, and matrix metalloproteinases (MMPs) were analyzed by RT-PCR, real-time PCR, and western blotting, and gelatin zymography. Furthermore, we examined the effects of siRNA for Wnt5a or Ror2 and recombinant human Wnt5a (rhWnt5a) on motility of tongue SCC cells. Biopsy specimens from tongue SCC patients were used for immunohistochemical staining of Wnt5a and Ror2. RESULTS: Wnt5a and Ror2 were expressed only in SQUU-B cells without DeltaNp63 expression, and negatively associated with DeltaNp63 expression in other cells. DeltaNp63beta overexpression in SQUU-B cells decreased Wnt5a and Ror2 expression. By Wnt5a or Ror2 knockdown, cell motility was remarkably inhibited, but EMT markers expression was unaffected. MMP-2 expression and the activities inversely correlated with DeltaNp63 expression, and were inhibited by Wnt5a or Ror2 knockdown. Cell motility and MMP-2 activities were recovered by adding rhWnt5a in the cells with Wnt5a knockdown, but not in those with Ror2 knockdown. Moreover, immunohistochemical analyses in tongue SCC specimens found that high expression of Wnt5a or Ror2 was associated with poorer prognosis. CONCLUSION: Wnt5a-Ror2 signaling enhanced tongue SCC cell aggressiveness and promoted production of MMP-2 following DeltaNp63beta-mediated EMT.CI - Copyright (c) 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

P63, a p53 family member, is expressed as TA and DeltaN isoforms. Interestingly, both TAp63 and DeltaNp63 are transcription factors, and regulate both common and distinct sets of target genes. p63 is required for survival of some epithelial cell lineages, and lack of p63 leads to loss of epidermis and other epithelia in humans and mice. Here, we explored the role of p63 isoforms in cell proliferation, migration and tubulogenesis by using Madin-Darby Canine Kidney (MDCK) tubular epithelial cells in two- or three-dimensional (2-D or 3-D) culture. We found that like downregulation of p53, downregulation of p63 and TAp63 decreases expression of growth-suppressing genes, including p21, PUMA and MIC-1, and consequently promotes cell proliferation and migration in 2-D culture. However, in 3-D culture, downregulation of p63, especially TAp63, but not p53, decapacitates MDCK cells to form a cyst structure through enhanced epithelial-to-mesenchymal transition (EMT). In contrast, downregulation of DeltaNp63 inhibits MDCK cell proliferation and migration in 2-D culture, and delays but does not block MDCK cell cyst formation and tubulogenesis in 3-D culture. Consistent with this, downregulation of DeltaNp63 markedly upregulates growth-suppressing genes, including p21, PUMA and MIC-1. Taken together, these data suggest that TAp63 is the major isoform required for tubulogenesis by maintaining an appropriate level of EMT, whereas DeltaNp63 fine-tunes the rate of cyst formation and tubulogenesis by maintaining an appropriate expression level of genes involved in cell cycle arrest and apoptosis.