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The p53-regulated growth arrest and DNA damage-inducible gene product Gadd45a has been recently identified as a key factor protecting the epidermis against ultraviolet radiation (UVR)-induced skin tumors by activating p53 via the stress mitogen-activated protein kinase (MAPK) signaling pathway. Herein we identify Gadd45a as an important negative regulator of two oncogenes commonly over-expressed in epithelial tumors: the p53 homologue ?Np63a and ß-catenin. ?Np63a is one of the several p63 isoforms and is the predominant species expressed in basal epidermal keratinocytes. ?Np63a lacks the N-terminal transactivation domain and behaves as a dominant-negative factor blocking expression of several p53-effector genes. ?Np63a also associates with and blocks activation of the adenomatous polyposis coli (APC) destruction complex that targets free cytoplasmic ß-catenin for degradation. While most ß-catenin protein is localized to the cell membrane and is involved in cell-cell adhesion, accumulation of free cytoplasmic ß-catenin will translocate into the nucleus where it functions in a bipartite transcription factor complex, whose targets include invasion and metastasis promoting endopeptidases, matrix metalloproteinases (MMP). We show that Gadd45a not only directly associates with two components of the APC complex, namely protein phosphatase 2A (PP2A) and glycogen synthase kinase 3ß (GSK3ß) but also promotes GSK3ß dephosphorylation at Ser9, which is essential for GSK3ß activation, and resultant activation of the APC destruction complex. We demonstrate that lack of Gadd45a not only prevents ?Np63a suppression and GSK3ß dephosphorylation but also prevents free cytoplasmic ß-catenin degradation after UV irradiation. The inability of Gadd45a-null keratinocytes to suppress ß-catenin may contribute to the resulting observation of increased MMP expression and activity along with significantly faster keratinocyte migration in Matrigel in vitro and accelerated wound closure in vivo. Furthermore, epidermal keratinocytes treated with p38 MAPK inhibitors, both in vivo and in vitro, behave very similarly to Gadd45a-null keratinocytes after UVR. Similarly, Trp53-null mice are unable to attenuate ?Np63a expression in epidermal keratinocytes after such stress. These findings demonstrate a dependence on Gadd45a-mediated p38 MAPK and p53 activation for proper modulation of ?Np63a, GSK3ß, and ß-catenin after irradiation. Taken together, our results indicate that Gadd45a is able to repress ?Np63a, ß-catenin, and consequently MMP expression by two means: by maintaining UVR-induced p38 MAPK and p53 activation and also by associating with the APC complex. This implicates Gadd45a in the negative regulation of cell migration, and invasion.Oncogene (2004) 23, 1829-1837. doi:10.1038/sj.onc.1207301 Published online 1 December 2003 [ABSTRACT FROM AUTHOR]

Pan-histone deacetylase (HDAC) inhibitors can induce the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein. However, the underlying mechanism by which this occurs remains unclear. In this study, we show that pan-HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate, were able to induce PTEN mRNA and protein expression via the acetylation of the transcription factor ΔNp63α by inhibiting HDAC1 and HDAC3. ΔNp63α enhanced PTEN promoter activity by binding two newly identified recognition sites on it. Unfortunately, the inhibition of HDAC1 or HDAC3 failed to activate PTEN, as knockdown of HDAC1 inhibited both membrane-bound and nuclear PTEN, and knockdown of HDAC3 only induced cytoplasmic PTEN. Furthermore, the overexpression of ΔNp63α downregulated membrane-bound PTEN but enhanced the nuclear translocation of PTEN, leading to the cisplatin resistance of oral cancer cells. PTEN accumulated in the nuclei of cancerous cells and normal cells when ΔNp63α was highly expressed in specimens from patients with squamous cell carcinoma of the tongue. However, inhibiting either HDAC1 or HDAC6 prevented the nuclear translocation of PTEN and attenuated cisplatin resistance. These results suggest that chemotherapeutic inhibitors of HDAC1 or HDAC6, together with cisplatin, might improve outcomes for patients with squamous cell carcinoma of the tongue., Competing Interests: None., (AJTR Copyright © 2020.)

Targeting metabolic reprogramming is an emerging strategy in cancer therapy. However, clinical attempts to target metabolic reprogramming have been proved to be challenging, with metabolic heterogeneity of cancer being one of many reasons that causes treatment failure. Here, we stratified non-small cell lung cancer (NSCLC) cells, mainly lung adenocarcinoma, based on their metabolic phenotypes and demonstrated that the aerobic glycolysis-preference NSCLC cell subtype was resistant to the OXPHOS-targeting inhibitors. We identified that monocarboxylate transporter 4 (MCT4), a lactate transporter, was highly expressed in the aerobic glycolysis-preference subtype with function supporting the proliferation of these cells. Glucose could induce the expression of MCT4 in these cells through a ΔNp63α and Sp1-dependent pathway. Next, we showed that knockdown of MCT4 increased intracellular lactate concentration and induced a reactive oxygen species (ROS)-dependent cellular apoptosis in the aerobic glycolysis-preference NSCLC cell subtype. By scanning a panel of monoclonal antibodies with MCT4 neutralizing activity, we further identified a MCT4 immunoglobulin M (IgM) monoclonal antibody showing capable anti-proliferation efficacy on the aerobic glycolysis-preference NSCLC cell subtype. Our findings indicate that the metabolic heterogeneity is a critical factor for NSCLC therapy and manipulating the expression or function of MCT4 can be an effective strategy in targeting the aerobic glycolysis-preference NSCLC cell subtype., (© 2020 The Author(s).)

Cell–cell adhesion plays an important role in regulation of cell proliferation, migration, survival, and drug sensitivity. Metformin, a first line drug for type 2 diabetes, has been shown to possess anti-cancer activities. However, whether cell–cell adhesion affects metformin anti-cancer activity is unknown. In this study, Microscopic and FACS analyses showed that metformin induced cancer cell–cell adhesion exemplified by cell aggregation and anoikis under glucose restriction. Furthermore, western blot and QPCR analyses revealed that metformin dramatically upregulated integrin β1 expression. Silencing of integrin β1 significantly disrupted cell aggregation and reduced anoikis induced by metformin. Moreover, we showed that p53 family member ΔNp63α transcriptionally suppressed integrin β1 expression and is responsible for metformin-mediated upregulation of integrin β1. In summary, this study reveals a novel mechanism for metformin anticancer activity and demonstrates that cell–cell adhesion mediated by integrin β1 plays a critical role in metformin-induced anoikis. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are key regulators in cell proliferation, survival, tumor initiation and development. However, the role of ROS in tumor metastasis is less clear. Here, we show that oxidative stress inhibited tumor metastasis via activation of Hippo kinase MST1/2, which led to the phosphorylation and nuclear accumulation of FoxO3a, resulting in upregulation of ΔNp63α expression and suppression of cell migration independent of YAP. Strikingly, while loss of MST1 led to and disruption of cell-cell junction exemplified by reduced E-cadherin expression, resulting in scattered cell growth, loss of MST2 led to disruption of cell-matrix adhesion as evidenced by reduced integrin β4, resulting in increased cell migration and tumor metastasis. Furthermore, expression of MST1 and MST2 was down-regulated in human breast carcinoma. Furthermore, oxidative stress inhibited HER2-or PI3K-mediated tumor metastasis via the MST2-FoxO3a-ΔNp63α pathway. Together, these results that this noncanonical Hippo MST2-FoxO3a-ΔNp63α pathway may play a critical role in ROS-mediated regulation of cell migration and tumor metastasis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tobacco smoke (TS) critically contributes to the development of lung cancer; however, the underlying molecular mechanisms remain unclear. The induction of cancer stem cells (CSCs) by TS represents an early event in tumor initiation. The lung cancer-related gene ΔNp63α is highly expressed in epithelial tissues and drives tumor formation and cancer stem cell properties. This study investigated the role of ΔNp63α in the long-term acquisition of TS-induced lung CSC-like properties. Methods: The expression levels of ΔNp63α, lung CSC markers, and interleukin (IL)-6 in lung carcinoma specimens were determined by western blotting and enzyme linked immunosorbent assays. Human bronchial epithelial (HBE) cells were chronically exposed to 2 % cigarette smoke extract for 55 passages, following which colony formation capacity, expression of proteins associated with malignant transformation, lung CSC markers, and tumor incidence were investigated. The effects of ΔNp63α on long-term TS exposure-induced lung CSC-like properties and Notch activation were analyzed using tumorsphere formation ability, immunofluorescence assays, luciferase reporter assays, and western blotting. The roles of IL-6 on chronic TS exposure-induced lung CSC-like properties and ΔNp63α expression were also examined. Moreover, the effects of sulforaphane (SFN) on TS-transformed lung CSC-like properties, IL-6 and ΔNp63α expression, and Notch signaling were investigated in vitro and in vivo . Results: Higher levels of ΔNp63α were observed in the lung cancer tissues of smokers than in those of non-smokers, whereas ΔNp63α was positively correlated with CD133 and Oct4 expression in lung cancer tissues. Data from the in vivo and in vitro experiments demonstrated that long-term TS exposure-transformed HBE (THBE) cells acquired lung CSC-like properties. Furthermore, ΔNp63α transcriptionally activated the Notch signaling pathway to promote the acquisition of CSC-like properties by the THBE cells. TS upregulated IL-6, which increased ΔNp63α expression in THBE sphere-forming cells. Finally, SFN inhibited the TS-induced CSC-like properties of THBE cells via the IL-6/ΔNp63α/Notch axis. Conclusion : Our data suggest that the IL-6/ΔNp63α/Notch axis plays an important role in the long-term TS exposure-induced acquisition of lung CSC-like properties and SFN intervention., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objective: To investigate the expressions of ΔNp63 α , DPC4/Smad4 and P21 in cervical squamous cell carcinomas and explore their implications in tumorigenesis, progression and prognosis of the malignancy., Methods: The expressions of ΔNp63 α , DPC4/Smad4 and P21 were examined with immunohistochemistry in 100 specimens of cervical squamous cell carcinoma, 40 specimens of cervical intraepithelial neoplasia (CIN) and 40 specimens of normal cervical tissues to explore their associations with the occurrence, progression and prognosis of cervical squamous cell carcinoma., Results: The expressions of ΔNp63 α and DPC4/Smad4 decreased and P21 expression increased significantly in the order of normal cervical tissue, CIN and cervical squamous cell carcinoma ( P < 0.01), and their expressions were associated with the differentiation, clinical stages and lymph node metastasis of cervical squamous cell carcinoma ( P < 0.01). The expression of ΔNp63 α was positively correlated with the expression of DPC4/Smad4 ( r =0.581, P < 0.05), and they were both negatively correlated with P21 expression ( r =-0.449 and -0.254, respectively; P < 0.05). Kaplan-Meier survival analysis showed that patients with cervical squamous cell carcinoma positive for ΔNp63 α and DPC4/Smad4 had a significantly higher 5-year survival rate than those negative for ΔNp63 α and DPC4/Smad4 ( P < 0.001); the patients positive for P21 had a significantly lower 5-year survival rate than the P21-negative patients ( P < 0.005)., Conclusions: The expressions of ΔNp63 α , DPC4/Smad4 and P21are related with the differentiation, invasion, lymph node metastasis, pTNM stage and prognosis of in cervical squamous cell carcinomas, suggesting their value as potential markers for prognostic evaluation of patients with cervical squamous cell carcinoma.

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, β-catenin and Vimentin, are associated with the p63 isoform ΔNp63α in SCC by using immunohistochemistry staining and analysis. Compared to the control SiHa cells (SiHa-NC), the expression of E-cadherin and β-catenin are upregulated, while Vimentin and ZEB1 are downregulated in the constructed SiHa cell line with stable ΔNp63α overexpression (SiHa-ΔNp63α). Besides, the migration and invasion abilities are also suppressed in SiHa-ΔNp63α cells with a typical epithelial morphology with cobblestone-like shape, suggesting that ΔNp63α is a vital EMT repressor in SCC cells. In addition, the involvement of miR-205/ZEB1 axis in the inhibition effect of ΔNp63α 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 ΔNp63α expression and high EMT level show more frequent metastasis and recurrence as well as reduced overall survival. Therefore, EMT program and its vital repressor ΔNp63α could be used as biomarkers for tumor metastasis and recurrence in cervical cancer.

Corneal epithelial stem cells are thought to reside in the limbus, the transition zoon between cornea and conjunctiva. Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are two paracrine factors that regulate the proliferation, migration and differentiation of the limbal epithelial cells; however, the underlying mechanisms are still poorly understood. In an ex vivo limbal explant culture, we found that KGF is a more potent growth stimulator for the epithelial outgrowth than HGF. Immunofluorescence studies of the epithelial outgrowth from cells treated with HGF or KGF showed similar expression patterns of keratin-3 and keratin-14. Interestingly, p63 was highly expressed in KGF-treated limbal epithelial sheets but not in those treated with HGF. Kinase inhibitor studies showed that induction of ΔNp63α expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous ΔNp63α was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of ΔNp63α. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with ΔNp63α expression as one of the downstream targets. [ABSTRACT FROM AUTHOR]

The p63 transcription factor has a pivotal role in epithelial morphogenesis. Multiple transcripts of the TP63 gene are generated because of alternative promoter usage and splicing. δNp63α is the predominant isoform of p63 observed during epithelial morphogenesis and in human cancers. Loss of δNp63α expression has been shown to promote invasiveness in a subset of human cancer cell lines. Here, we studied whether the regulation of VDR by δNp63α controls the invasiveness of an epidermoid cancer cell line. We demonstrate that VDR expression is induced by all p63 isoforms, including δNp63α. Endogenous δNp63α protein was observed to bind to the VDR promoter, and silencing of endogenous δNp63α resulted in diminished VDR expression. Although silencing of p63 inhibits VDR expression leading to an increase in cell migration, overexpression of p63 or VDR results in reduced cell migration as a result of increased VDR expression. Therefore, it is conceivable that p63 inhibits cell invasion by regulating VDR expression. Finally, we observed that expression of p63 and VDR overlaps in the wild-type mouse skin, but a reduced or complete absence of VDR expression was observed in skin from p63-null mice and in p63-null mouse embryonic fibroblasts. In conclusion, we demonstrate a direct transcriptional regulation of VDR by δNp63α. Our results highlight a crucial role for VDR in p63-mediated biological functions. [ABSTRACT FROM AUTHOR]