ΔNp63 isoforms differentially regulate gene expression in squamous cell carcinoma: identification of Cox‐2 as a novel p63 target

The p53 homologue p63 produces six different isoforms that are important in development of epithelial tissues and squamous cell carcinoma of the head and neck (SCCHN). In SCCHN, the expression of p63 isoforms is highly complex, with over‐expression of ΔNp63 and p63β isoforms in many tumours. To date, little is known about the functions of different ΔNp63 isoforms and elucidating the distinctive properties of ΔNp63 isoforms will help to clarify how they influence tumour biology. By gene expression profiling of SCCHN cells over‐expressing the ΔNp63 isoforms we identified different effects of the three isoforms, with ΔNp63β being more effective at gene induction than ΔNp63α and ΔNp63γ, whereas ΔNp63γ was most effective at repressing gene expression. Thus, tumours expressing even low levels of ΔNp63β or ΔNp63γ may have distinct clinicopathological characteristics important for metastasis and therapeutic response. Induction of cyclooxygenase‐2 (Cox‐2) was shown by each isoform and data were confirmed by independent quantitative RT–PCR and western blotting. No direct binding of ΔNp63 to the Cox‐2 promoter could be seen, neither could any evidence for Cox‐2 induction as a consequence of activated NF‐κB pathway responses be found. As Cox‐2 is known to inhibit radiotherapy responses in SCCHN patients, data indicate an additional mechanism through which ΔNp63 acts to promote cell survival and influence therapeutic response of SCCHN. MIAME‐compliant data have been deposited in the MIAME Express database (Accession No. E‐MEXP‐1842). Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

p63 regulates proliferation and differentiation of developmentally mature keratinocytes

p63 is a multi-isoform p53 family member required for epidermal development. Contrasting roles for p63 in either the initial commitment to the stratified epithelial cell fate or in stem cell-based self-renewal have been proposed. To investigate p63 function in a post-developmental context, we used siRNAs directed against p63 to down-regulate p63 expression in regenerating human epidermis. Loss of p63 resulted in severe tissue hypoplasia and inhibited both stratification and differentiation in a cell-autonomous manner. Although p63-deficient cells exhibited hypoproliferation, differentiation defects were not due to tissue hypoplasia. Simultaneous p63 and p53 knockdown rescued the cell proliferation defect of p63 knockdown alone but failed to restore differentiation, suggesting that defects in epidermal proliferation and differentiation are mediated via p53-dependent and -independent mechanisms, respectively. Furthermore, DeltaNp63 isoforms are the main mediators of p63 effects, although TAp63 isoforms may contribute to late differentiation. These data indicate that p63 is required for both the proliferative and differentiation potential of developmentally mature keratinocytes.     

Differential expression of p63 isoforms in normal tissues and neoplastic cells

The p63 gene encodes at least six different proteins with homology to the tumour suppressor protein p53 and the related p53 family member p73. So far, there have been limited data concerning the expression patterns of individual p63 proteins, due to a lack of reagents that distinguish between the different isoforms. Three antibodies have been produced specifically directed against the two N-terminal isoforms (TAp63 and DeltaNp63) and the C-terminal region of the p63alpha proteins. TAp63 proteins are located suprabasally in stratified epithelia compared with the N-terminal truncated forms, which are more abundantly expressed in the basal cell layer, indicating a switch in expression of p63 isoforms during normal cellular differentiation. Analysis of squamous cell carcinomas shows DeltaNp63alpha to be the most widely expressed isoform, compatible with a role for this protein in promoting neoplastic cell growth in these tissues. DeltaNp63 protein expression is also restricted to basal cells in breast and prostate, whilst TAp63 isoforms are more widely expressed in these tissues as well as in tumours at these sites. TAp63, but not DeltaNp63 or p63alpha, is detected in normal colon and in colon carcinoma. TAp63 proteins are also expressed in the nuclei of a sub-population of lymphoid cells and in most malignant lymphomas, whereas DeltaNp63 proteins are not expressed. Taken together, a hitherto unrecognized regulation of p63 isoform expression in vivo has been uncovered, with different p63 proteins expressed during differentiation and in different cell types. The data indicate roles for specific p63 isoforms not only in maintaining epithelial stem cell populations, but also in cellular differentiation and neoplasia.     

p63 is the molecular switch for initiation of an epithelial stratification program

Development of stratified epithelia, such as the epidermis, requires p63 expression. The p63 gene encodes isoforms that contain (TA) or lack (DeltaN) a transactivation domain. We demonstrate that TAp63 isoforms are the first to be expressed during embryogenesis and are required for initiation of epithelial stratification. In addition, TAp63 isoforms inhibit terminal differentiation, suggesting that TAp63 isoforms must be counterbalanced by DeltaNp63 isoforms to allow cells to respond to signals required for maturation of embryonic epidermis. Our data demonstrate that p63 plays a dual role: initiating epithelial stratification during development and maintaining proliferative potential of basal keratinocytes in mature epidermis.     

TP53 family members and human cancers

Based on gene sequence homologies, a p53 (TP53) gene family become apparent with the addition of the most recently identified p63 (TP73L; formerly TP63) and p73 (TP73) genes to the already known p53. The p53 gene encodes for a unique protein eliciting well-known tumor suppressor gene (TSG) properties that mediate cellular response to DNA damage, e.g., cell cycle arrest or apoptosis. In contrast, both homologues specify an array of isoforms different in their N- and C-terminal domains. Transactivating isoforms, such as TAp63/p73, show TSG properties similar to p53, while isoforms lacking N-terminal transactivating domain such as DeltaNp63/p73, induce a functional block against p53 as well as TAp63/p73 activities. Both p63/p73 types of isoforms are involved in development: p63 is critical for epithelial stem cell renewal and epithelial homeostasis, and p73 is involved in neurogenesis and natural immune response. These facts support interdependent functions for the p53 family members, which appear linked together in a complex and tight regulation network to fulfill cellular functions related to DNA damage and tissue homeostasis maintenance. The lack of p63/p73 mutations in human cancers rule out a typical TSG role for either of the p53 homologues. Nonetheless, p63 and p73 genes seem strongly involved in malignancy acquisition and maintenance process because of: 1) their tissue identities, and 2) their close interplay activities within the p53 family members, and primarily through the negative regulatory role played by DeltaNp63/p73 isoforms for cell death control and differentiation.     

Characterization of specific p63 and p63-N-terminal isoform antibodies and their application for immunohistochemistry

The TP63 gene gives rise to protein isoforms with different properties and functions due to the presence (TAp63) or absence (ΔNp63) of an N-terminal p53-like transactivation domain. Immunohistochemistry for p63 has clinical value for certain tumour types, but investigations have been hampered by a lack of well characterized antibodies and the inability to discriminate between these N-terminal isoforms with opposite functional properties. We have extensively characterized a series of monoclonal antibodies to recombinant human TAp63 and two commercial p63 monoclonals by Western blot, immunostaining and phage display epitope mapping. Twenty-eight of 29 (96.6 %) novel monoclonals that recognized all p63 isoforms showed substantial cross-reactivity with p73, as did the commercial antibody, 4A4. One novel clone, PANp63-6.1, showed slight cross-reaction with p73 by Western blotting but not immunohistochemistry and the SFI-6 monoclonal did not cross-react with p73 or p53. Phage display revealed that the PANp63-6.1 epitope has one amino acid difference between p63 and p73, the 4A4 epitope is identical in both, whereas the SFI-6 epitope is unique to p63, accounting for these findings. We also produced and characterized a TAp63-specific clone that does not recognize p53 or p73, and we prepared polyclonal sera specific for ΔNp63 isoforms. Immunohistochemistry demonstrated that TAp63 is expressed in a variety of epithelial and other cell types during development, often in a converse pattern to ΔNp63, but has a very limited expression in normal adult tissues and is independent of ΔNp63. TAp63 was expressed in 17.6 % of squamous cancers of cervix that expressed p63, unlike normal cervix where TAp63 was not expressed. TAp63 did not associate with proliferative index, but cervical carcinomas with TAp63 expression showed improved survival. These data highlight the need for rigorous antibody characterization and indicate that p63-isoform identification may improve the clinical value of p63 expression analyses.     

Abnormal re-epithelialization and lung remodeling in idiopathic pulmonary fibrosis: the role of deltaN-p63

Products of the p63 gene, a recently described member of the p53 family, are constitutively expressed in the basal cells of human bronchi and bronchioli. The truncated isoforms of the p63 gene (deltaN-p63 proteins) counteract the apoptotic and cell cycle inhibitory functions of p53 after DNA damage, and this property is likely to be central in the cell renewal strategy of stratified epithelial tissues. To investigate the dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we immunohistochemically analyzed the expression of the transactivating and dominant-negative isoforms of the p63 gene on 16 tissue samples obtained from patients suffering from this disorder. In most IPF cases herein investigated, epithelial cells expressing deltaN-p63 were observed at sites of abnormal proliferation at the bronchiolo-alveolar junctions, characterized by epithelial hyperplasia, squamous metaplasia, bronchiolization, and abnormal p53 nuclear accumulation. Similar features were not observed in normal lung and in samples taken from other pulmonary diseases used as controls, including acute interstitial pneumonia, idiopathic bronchiolitis obliterans organizing pneumonia, nonspecific interstitial pneumonia, and desquamative interstitial pneumonia. On the basis of these findings, we can hypothesize a new model for UIP pathogenesis, involving a deregulated development of mesenchymal-epithelial interactions and abnormal proliferation of epithelial cells at the bronchiolo-alveolar junction after cell injury. In our view, the progressive loss of alveolar tissue and lung remodeling after injury in IPF/UIP is concomitantly produced by pneumocyte loss and alveolar collapse on one hand and by progressive bronchiolar proliferation and architectural distortion on the other.     

Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells

This study investigated whether cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Primary cultured human corneal epithelial cells were sorted by flow cytometry based on forward scatter profile in comparison with the profile of beads of known size. Four fractions (A, B, C, and D) of cells ranging in size from 10 to 16, 17 to 23, 24 to 30, and >or=31 microm in diameter, respectively, were collected to evaluate their 5-bromo-2-deoxyuridine (BrdU) label retention properties, cell phenotype, and clonal growth capacity on a 3T3 fibroblast feeder layer. Among these four populations, cell size was shown to positively correlate with the expression of the differentiation markers keratin (K) 3, K12, and involucrin and inversely with the levels of stem cell-associated markers DeltaNp63 and ABCG2 and with colony-forming efficiency (CFE) and growth capacity. Population A with the smallest size, accounting for 11.0%+/-4.5% of the entire population, contained the greatest number of BrdU label-retaining slow-cycling cells, displayed the highest percentage of cells immunopositive to p63 and ABCG2 and negative to K3 and involucrin, expressed the highest levels of DeltaNp63 and ABCG2 mRNA and the lowest levels of K3, K12, and involucrin, and possessed the highest CFE and growth capacity. These findings suggest that cell size correlates with cell differentiation phenotypes and proliferative capacity in human corneal epithelial cells. The smallest cells in population A seem to be enriched for putative stem cells, and small cell size may represent one of the important properties of adult corneal epithelial stem cells.     

Expression of p63 in thymomas and normal thymus

The p63 gene, a member of the p53 family, is an epithelial marker expressed in embryonic ectoderm, breast myoepithelium, prostate, oral epithelium, epidermis, and urothelium. The DeltaN-p63 isoforms of p63, which are believed to behave as oncogenes, are expressed in squamous cell carcinoma, basal cell carcinoma, and transitional cell carcinoma. Only a few authors have looked for p63 expression in thymomas and normal thymus. We, therefore, thought of undergoing such a search by taking advantage of our archival material. We studied 66 cases of thymoma (1 type A, 8 type AB, 12 type B1, 19 type B2, 12 type B3, and 14 type C/thymic carcinoma) and 10 specimens of normal human thymus arranged in tissue microarrays. All thymomas (including thymic carcinomas) were positive for p63 regardless of type. Most of the epithelial cells of the normal thymus were also positive for this marker.     

肺癌中P63与P53、E-cadherin、Ki-67表达的比较

目的　比较 p6 3及 p5 3、E cadherin(E cad)、Ki 6 7在肺癌中的表达 ,以了解在不同组织类型肺癌发生发展过程中 ,p6 3与抑癌基因 (p5 3)突变、上皮分化标志基因 (E cad)失活及细胞增殖标志基因 (Ki 6 7)激活有无相关性。方法　采用免疫组化S P法分别检测 6 1例原发性肺癌中 p6 3、p5 3、E cad和Ki 6 7的表达情况。 结果　p6 3在肺鳞癌中阳性率为 10 0 0 % ,而在其他组织类型肺癌中 p6 3基本不表达 ,差异有显著性 (P <0 0 5 ) ;在不同分化程度的肺鳞癌中 p6 3、p5 3的表达差异有显著性 (P<0 0 5 ) ,E cad、Ki 6 7的表达差异无显著性 (P >0 0 5 ) ;E cad的表达在小细胞肺癌与肺鳞癌和肺腺癌之间差异有显著性 (P <0 0 5 ) ;Ki 6 7的表达在各种组织类型肺癌之间差异有显著性 (P <0 0 5 ) ;在不同分化程度鳞癌中 p6 3与E cad的表达呈负相关(P <0 0 5 )。结论　p6 3可作为鳞状上皮源性肿瘤标记物 ,是判断鳞状细胞癌的增殖和分化有意义的指标 ,并可作为鉴别分化差的鳞癌和腺癌、小细胞癌的指标。     

Absence of B7.1-CD28/CTLA-4-mediated co-stimulation in human NK cells

Recent studies have suggested that B7-CD28 interactions provide co-stimulatory signals for activation of NK cells. Transduction of the B7.1 (CD80) gene into tumor cells has been shown to trigger proliferation and cytotoxicity of murine NK cells and a human NK cell line, YT2C2. Therefore, transduction of the B7.1 gene into CD80-negative human squamous cell carcinomas of the head and neck (SCCHN) and its stable expression was expected to up-regulate proliferation and cytotoxic activities of human NK cells. However, expression of the B7.1 receptors, CD28 and CTLA-4, could not be demonstrated on the surface or in the cytoplasm of normal human NK cells, irrespective of the state of their activation. In proliferation experiments or various cytotoxicity assays, utilizing highly purified human NK cells as responder or effector cells, no enhancement of NK cell generation or activity, respectively, by B7.1⁺ SCCHN was observed relative to non-transduced or LacZ gene-transduced SCCHN. In contrast, co-incubation of B7.1⁺ SCCHN targets with human NK cells induced significant inhibition of NK cell growth. Thus, the B7.1-CD28/CTLA-4 pathway is not involved in triggering of human adult NK cells.     

Understanding metastatic SCCHN cells from unique genotypes to phenotypes with the aid of an animal model and DNA microarray analysis

Metastasis of squamous cell carcinoma of the head and neck (SCCHN) is a significant health-care problem worldwide. The 5-year survival rate is less than 50% for patients with lymph node metastases. Understanding the molecular basis of SCCHN metastasis would facilitate the development of new therapeutic approaches to the disease. To identify proteins that mediate SCCHN metastasis, we established a SCCHN xenograft mouse model and performed in vivo selection from a SCCHN cell line using the model. In the fourth round of in vivo selection, significant incidences of metastases in lymph nodes (7/10) and lungs (6/10) were achieved from a derived SCCHN cell line as compared with its parental cells, 1/5 in lymph nodes and 0/5 in lungs. Metastatic cell lines from lymph node metastases and parental cell lines from non-metastatic xenograft tumors were subjected to DNA microarray analysis using an Affymetrix gene chip HG-U133A, followed by data mining studies. The identified metastasis-related genes were further evaluated for their encoding protein products and the metastatic cells were examined by biological analyses. DNA microarray analysis highlighted molecular features of the metastatic SCCHN cells, including alteration of expression of cell–cell adhesion proteins, epithelial cell markers, apoptosis and cell cycle regulatory molecules. Further biological analyses of phenotypic alterations revealed that the metastatic cells gained epithelial-mesenchymal transition (EMT) features and were more resistant to anoikis, which are two of the important phenotypes for metastatic SCCHN.     

p63 overexpression associates with poor prognosis in head and neck squamous cell carcinoma

p63 belongs to a protein family that includes 2 structurally related proteins, p53 and p73. The aim of this study was to investigate the biologic role of p63 in oral tumorigenesis and its possible role as prognostic marker in oral cancer. Ninety-four cases of oral squamous cell carcinoma and 10 cases of normal mucosa were analyzed for p63 expression by immunohistochemistry. Normal oral mucosa showed a basal and parabasal expression of p63. Five (5.3%) cases of oral cancer showed less than 10% of positive tumor cells; in 33 (35.1%) cases the positive tumor cells comprised between 10% and less than 30%, in 36 (38.3%) cases the positive tumor cells comprised between 30% and less than 50%, and in 20 (21.3%) cases the positive tumor cells were more than 50%. There was also a statistically significant correlation between p63 expression and tumor differentiation: p63 expression was amplified in poorly differentiated tumors (P < .05). When analyzed for prognostic significance, patients with perineural infiltration had poorer survival rates than the group with no perineural infiltration (P < .05) and patients with increased p63 expression had poorer survival rates than the group with reduced p63 expression (P < .05). The statistical analysis showed no significant correlation between p63 expression, sex, age, tumor size, staging, recurrence, and metastasis. Cases with diffuse p63 expression were more aggressive and poorly differentiated and related to a poorer prognosis. These data suggest that p63 expression may be useful to identify cases of oral squamous cell carcinoma with more aggressive and invasive phenotype providing novel diagnostic and prognostic information on individual patient survival with oral cancers.     

Immunohistochemical detection of p53 homolog p63 in solid cell nests, papillary thyroid carcinoma, and hashimoto's thyroiditis: A stem cell hypothesis of papillary carcinoma oncogenesis

Most models suggest that the cell of origin of papillary carcinoma is the mature thyroid follicular epithelial cell. In a recent study, p63 was detected in papillary carcinoma, Hashimoto's thyroiditis, and in squamoid aggregates and solid cell nests (SCNs), embryonic remnants found sporadically in the fully developed thyroid. In the present study, the relationship between solid cell nests and papillary carcinoma was investigated further. Four-micrometer sections from 88 routinely fixed and processed archival thyroidectomy specimens were pretreated with citric acid pH 6.0 for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained with a streptavidin-biotin kit and diaminobenzidine as chromogen and were counterstained with hematoxylin. Squamoid aggregates or SCNs were noted in 21 specimens. Several morphologic variants of SCNs were found, all of which displayed p63 positivity. These included undifferentiated SCNs and those displaying commitment toward squamoid and ciliated glandular differentiation. Small, morphologically inconspicuous aggregates of p63-positive cells were commonly found in Hashimoto's thyroiditis. Commitment of p63-positive undifferentiated cells toward thyroid follicular epithelial differentiation was occasionally noted. One SCN variant, also associated with Hashimoto's thyroiditis, was a floretlike arrangement of p63-positive cells with fusiform nuclei. p63 staining was strong and uniform in some SCNs, but in other SCNs it was compartmentalized and homologous to stem cell-staining patterns in normal squamous or bronchial epithelia. Stem cell-like staining, associated with compartmentalized p63 staining or p63-positive undifferentiated cells, was noted in 7 of 27 papillary carcinomas. p63 immunostaining is a highly sensitive means of detecting SCNs. p63 expression patterns in SCNs and a subset of papillary carcinomas are closely homologous to stem cell-associated p63 staining patterns that have been described elsewhere in squamous and bronchial epithelia. We propose a stem-cell-associated model of papillary carcinoma oncogenesis that suggests that (1) p63-positive embryonal remnants rather than mature follicular cells are the cells of origin of a subset of papillary carcinomas; (2) these p63-positive cells are pluripotent and may stay undifferentiated or undergo benign squamoid or glandular maturation, may undergo thyroid follicular epithelial differentiation, may undergo oncogenic change leading to papillary carcinoma, or may trigger an immune reaction, resulting in lymphoid infiltration and Hashimoto's thyroiditis; and (3) Hashimoto's thyroiditis and papillary carcinoma may therefore be linked etiologically, because both disorders may be initiated by the same population of pluripotent p63-positive embryonal stem cell remnants.