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Induction of ?Np63 by the newly identified keratinocyte-specific transforming growth factor ? Signaling Pathway with Smad2 and I?B Kinase ? in squamous cell carcinoma.

ABSTRACT: The expression of p63 (TP63/p51) occurs in the basal cells of stratified epithelia and is strongly enhanced at the early stages of squamous cell carcinomas (SCCs) of the head and neck, skin, cervix, and others. We analyzed a promoter/enhancer region (2k?N) that drives the predominant expression of ?Np63 for sensitivity to Smad signaling pathways. Reporter assays in HepG2 cells showed a moderate activation of 2k?N by Smad2 and I?B kinase ? (IKK?), partners of the newly identified keratinocyte-specific transforming growth factor ? (TGF-?) signaling, but not by other Smad molecules. In A431 cells, 2k?N was activated by Smad2 and IKK?, for which a Smad binding element (SMD2) at -204 was essential. Binding of Smad2 to the chromosomal SMD2 site was detectable. The association of Smad2 with IKK? was evident in the nucleus of A431, accounting for the enhancement of ?Np63 expression by TGF-?. Moreover, both ?Np63 and IKK? were necessary to maintain the noninvasive phenotype of this cell line. FaDu, an invasive, Smad4-deficient SCC, also allowed 2k?N transactivation by transfected Smad2 in the presence of endogenous IKK?. Reflecting the lack of chromosomal SMD2-Smad2 association and the absence of nuclear IKK?, however, endogenous ?Np63 was not controlled by TGF-? or IKK? in FaDu. SCC tissue arrays showed nuclear accumulation of IKK? and p63 intensification in well-differentiated noninvasive lesions. This study indicates that p63 is a target gene of the proposed keratinocyte-specific TGF-? signal pathway for suppression of the malignant conversion of SCC.

SUBMITTER: Fukunishi N

PROVIDER: S-EPMC3003132 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Induction of ΔNp63 by the newly identified keratinocyte-specific transforming growth factor β Signaling Pathway with Smad2 and IκB Kinase α in squamous cell carcinoma.

Fukunishi Nahoko N Katoh Iyoko I Tomimori Yoshiya Y Tsukinoki Keiichi K Hata Ryu-Ichiro R Nakao Atsuhito A Ikawa Yoji Y Kurata Shun-Ichi S

Neoplasia (New York, N.Y.) 20101201 12

The expression of p63 (TP63/p51) occurs in the basal cells of stratified epithelia and is strongly enhanced at the early stages of squamous cell carcinomas (SCCs) of the head and neck, skin, cervix, and others. We analyzed a promoter/enhancer region (2kΔN) that drives the predominant expression of ΔNp63 for sensitivity to Smad signaling pathways. Reporter assays in HepG2 cells showed a moderate activation of 2kΔN by Smad2 and IκB kinase α (IKKα), partners of the newly identified keratinocyte-spe ...[more]

PMID: 21170261

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Project description:The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2'-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a "double whammy" for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an "either or" mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.

Project description:BACKGROUND:The simplicity of Transforming Growth Factor ß (TGF?) signaling pathway, linear and non-amplified, hardly sustains its variety of responses. This is often justified by the complex regulation showed by Smad proteins, TGF? signaling intracellular transducers, object of post-translational modifications that modulate TGF?-dependent transcription. Protein acetylation is emerging as a compelling mechanism affecting the activities of significant transcription factors, including p53, FOXO or NF-kB. Smad proteins might be controlled by this mechanism, implying that accessory factors capable of altering Smads-transcriptional complexes acetylation status and hence regulate TGF? responses remain to be identified. Understanding this interaction may help in the assessment of TGF? signaling outcomes, extending from healthy physiology to pathological conditions and cancer. METHODS:A two-hybrid chimera interacting system allowed to identify Sirt1, a NAD+ dependent type III histone deacetylase, as a novel Smad2 interactor. Several well stablished cellular models were applied to characterize this interaction by means of co-immunoprecipitation of tagged proteins and immuno-fluorescence staining. The occurrence of the interaction at Smad2 driven transcriptomic complexes was studied by means of DNA-pull-down and chromatin immunoprecipitation (ChIP), while its effects were assessed by protein over-expression and siRNA applied into a TGF?-dependent reporter gene assay. RESULTS:The interaction was confirmed and observed to be enhanced upon Smad2 acetylation, a known feature of active and nuclear Smad2. However, Sirt1 did not play a major role in Smad2 deacetylation. Anti-Sirt1 ChIP showed increased recovery of promoter regions corresponding to Smad2-driven genes after TGF?-stimulation, while its occurrence at Smad2-dependent transcriptomic complexes on DNA was found to effectively modulate gene expression. CONCLUSIONS:Sirt1 presence on Smad2-driven TGF?-dependent regulatory elements was detected and found to increase after TGF? treatment. Moreover, Sirt1 overexpression resulted in a decrease of the activity of a Smad2-driven TGF?-dependent reporter gene, while Sirt1 interference increased its activity. This would confirm the relevance of the discovered Sirt1-Smad2 interaction for the regulation of TGF?-dependent gene transcription.

Dataset Information

Induction of ?Np63 by the newly identified keratinocyte-specific transforming growth factor ? Signaling Pathway with Smad2 and I?B Kinase ? in squamous cell carcinoma.

Publications

Induction of ΔNp63 by the newly identified keratinocyte-specific transforming growth factor β Signaling Pathway with Smad2 and IκB Kinase α in squamous cell carcinoma.

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