Karin Nylander

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 ΔNp63) and the C‐terminal region of the p63α 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 ΔNp63α to be the most widely expressed isoform, compatible with a role for this protein in promoting neoplastic cell growth in these tissues. ΔNp63 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 ΔNp63 or p63α, 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 ΔNp63 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. Copyright

Characterization of the expression pattern of p63α and δnp63α in benign and malignant oral epithelial lesions

The p53 homologue p63 is essential for ectodermal differentiation, such that p63−/− mice lack all squamous epithelia and teeth. The p63 gene expresses at least 6 different transcripts, but information regarding the expression, regulation and function of the different isoforms has remained sparse, due to the lack of adequate reagents directed specifically against the individual proteins. Here we characterize the expression of p63α/ΔNp63α in benign and malignant lesions of the oral epithelium, using a specific antibody raised against a peptide derived from the C‐terminus of p63α, which does not cross‐react with p53 or the other p53 homologue, p73. By immunohistochemical analysis, we show that these p63 isoforms are expressed in the nucleus of many cells. In normal and benign lesions, p63α/ΔNp63α‐expressing cells are mainly found suprabasally, whereas p53‐expressing cells are restricted to the basal‐cell layer. By RT‐PCR, we show that ΔNp63α is the predominant isoform in cell lines from squamous‐cell carcinomas of the head and neck, confirming our immunochemical observations. Our data are consistent with studies suggesting a role for p63 in the transit‐amplifying population of epidermal cells. Over‐expression of p63α, and in particular the ΔN form, was frequently seen in carcinomas. Taken together with previous analyses of p63 expression, our data suggest distinct roles for different p63 isoforms in the regulation of growth and/or differentiation of epithelial cells. Moreover, our data are compatible with the notion that p63 can act to promote neoplastic growth in the oral epithelium. Int. J. Cancer 87:368–372, 2000.

p53 Expression and cell proliferation in squamous cell carcinomas of the head and neck

Background. In squamous cell carcinoma of the head and neck (SCCHN), overexpression of the p53 protein has been found in 34‐80% of the tumors studied. No data are available regarding p53 expression versus tumor cell proliferation and prognosis for this tumor type.

p53 mutations, protein expression and cell proliferation in squamous cell carcinomas of the head and neck

Thirty-three patients with squamous cell carcinoma of the head and neck region were studied concerning p53 protein expression and mutations in exons 4-9 of the p53 gene using immunohistochemistry, polymerase chain reaction (PCR)-single strand conformation polymorphism analysis and DNA sequencing. Immunoreactivity was found in 64% and p53 gene mutations in 39% of the tumours. Thirty-three per cent of the immunopositive and 50% of the immunonegative tumours were mutated within exons 5-8. In one immunopositive tumour three variants of deletions were observed. Sequencing of the p53 mutated, immunonegative tumours revealed four cases with deletions, one case with a transversion resulting in a stop codon and one case with a splice site mutation which could result in omission of the following exon at splicing. All mutations in the immunonegative tumours resulted in a truncated p53 protein. No association between p53 gene status and expression of proliferating cell nuclear antigen (PCNA) or cell proliferation as judged by in vivo incorporation of the thymidine analogue iododeoxyuridine (IdUrd) was found.

DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck.

The human p63 gene codes for multiple protein isoforms and is commonly over‐expressed in squamous cell carcinoma of head and neck (SCCHN). This expression is predominantly of the ΔN‐ and β‐isoforms, the former lacking the p53‐related transactivation domain. p63 can activate or repress transcription of p53 and p73 target genes, but also has unique transcriptional targets and, unlike other p53 family members, is required for normal development and differentiation of squamous epithelia. We have identified novel targets of p63, using microarray analysis of SCCHN cells that stably over‐express individual ΔNp63 isoforms. All three isoforms induced expression of the cancer stem cell marker, CD44, with the ΔNp63β isoform showing strongest induction. Using chromatin immunoprecipitation, we were unable to show direct binding of p63 to the CD44 promoter, but found that p63 specifically increased expression of CD44 lacking variant exon 2. Each of the ΔNp63 isoforms up‐regulated expression of keratins 6A and 14 and down‐regulated expression of keratins 4 and 19, in keeping with their expression patterns in SCCHN. The data strengthen the idea that p63 has key roles in regulating normal and abnormal differentiation processes through both induction and repression of genes with opposite functions. The identification of up‐regulation and differential splicing of CD44 following p63 over‐expression indicates roles in the regulation of adhesion, metastasis and the cancer stem cell phenotype. Copyright

Immunohistochemical detection of oncoprotein 18 (Op18) in malignant lymphomas

SummaryExpression of oncoprotein 18 (Op18), an intracellular phosphoprotein up-regulated in many malignant cell types, was evaluated in a series of normal lymphoid tissue and malignant lymphomas. In normal tonsils and reactive lymph nodes, the majority of Op18-positive cells were present in the germinal centres, whereas cells in the mantle zone were essentially negative and the interfollicular areas showed occasional positive cells. Double staining for PCNA and Op18 revealed that Op18 expression only to some extent was correlated with cell proliferation, as determined by PCNA expression.Non-Hodgkins lymphomas exhibited a variable Op18 expression, and in Hodgkins disease, Reed-Sternberg and Hodgkin cells frequently expressed Op18 with a strong staining intensity. Using Op18-PCNA double staining in malignant lymphomas, Op18 expression could also be partially dissociated from cell proliferation. By using confocal microscopy, the intracellular localization of Op18 was studied, demonstrating diffuse reactivity in the cytoplasm in interphase cells and during mitosis, whereas nuclei and condensed chromosomes were negative. In conclusion, Op18 was expressed at variable levels in most, perhaps all, proliferating lymphocytes in benign lymphoid tissue as well as in malignant lymphomas. However, the Op18 protein was also detected in a significant fraction of apparently non-cycling normal and neoplastic lymphocytes.

Why is p53 protein stabilized in neoplasia? Some answers but many more questions!

The p53 pathway provides a physiological system for integrating signals from diverse insults and eliciting adaptive cellular responses that include (but importantly are not restricted to) growth arrest and apoptosis. Defects in the pathway are prevalent in cancer, most notably being associated with mis‐sense mutations in p53 itself. This leads to the inability of p53 to act as a transcription factor and thus to the non‐occurrence of downstream events. Recent data indicate that the stability (and hence level) of p53 protein in cells is regulated by its interaction with mdm2: this results in enhanced p53 degradation by ubiquitin‐mediated events. Since mdm2 is itself regulated by p53, loss of function of p53 leads to lack of mdm2 and thus to p53 protein accumulation. This provides a mechanistic explanation for the observation that p53 accumulation is associated with neoplasia. It may be that accumulation of p53 in the absence of p53 mutation can occur as a consequence of mdm2 defects, as well as being a physiological response in many situations. Another recent development is the recognition of p53 homologues (p73 alpha, p73 beta, and KET) which have many sequence and probable structural features in common with p53. It seems likely that this will reveal another layer of complexity in the control and regulation of p53 and its role in physiology and pathology.

p63 contributes to cell invasion and migration in squamous cell carcinoma of the head and neck

The transcription factor p63 is commonly over-expressed in squamous cell carcinomas of the head and neck (SCCHN). By microarray analysis of p63-siRNA-treated SCCHN cells we identified 127 genes whose expression relies on over-expression of p63. More than 20% of these genes are involved in cell motility. Chromatin immunoprecipitation and reporter assay revealed PAI-1 and AQP3 as direct p63 transcriptional targets. In addition to PAI-1, most of the key cell motility-related molecules are up-regulated by p63, such as MMP14 and LGALS1. Our findings indicate a contribution by p63 in cell invasion and migration, supporting an oncogenic role for p63 in SCCHN.

Subsite-based alterations in miR-21, miR-125b, and miR-203 in squamous cell carcinoma of the oral cavity and correlation to important target proteins.

Background: MicroRNAs (miRNAs) are small noncoding RNA molecules with an essential role in regulation of gene expression. miRNA expression profiles differ between tumor and normal control tissue in many types of cancers and miRNA profiling is seen as a promising field for finding new diagnostic and prognostic tools. Materials and Methods: In this study, we have analyzed expression of three miRNAs, miR-21, miR-125b, and miR-203, and their potential target proteins p53 and p63, known to be deregulated in squamous cell carcinoma of the head and neck (SCCHN), in two distinct and one mixed subsite in squamous cell carcinoma in the oral cavity. Results: We demonstrate that levels of miRNA differ between tumors of different subsites with tongue tumors showing significant deregulation of all three miRNAs, whereas gingival tumors only showed significant downregulation of miR-125b and the mixed group of tumors in tongue/floor of the mouth showed significant deregulation of miR-21 and miR-125b. In the whole group of oral squamous cell carcinoma (SCC), a significant negative correlation was seen between miR-125b and p53 as well as a significant correlation between TP53 mutation status and miR-125b. Conclusion: The present data once again emphasize the need to take subsite into consideration when analyzing oral SCC and clearly show that data from in vitro studies cannot be transferred directly to the in vivo situation.

Effect of narrow-band ultraviolet B phototherapy on p63 and microRNA (miR-21 and miR-125b) expression in psoriatic epidermis.

Psoriasis is an inflammatory skin disease in which dysregulation of p63, a member of the p53 family that is crucial for skin development and maintenance, has been demonstrated. Involvement of miR-203, miR-21 and miR-125b, small non-coding RNAs implicated in the regulation of p63 or p53, has been suggested in the patho-genesis of psoriasis. To elucidate the roles of p63 and p63-related microRNAs in psoriasis and to increase our understanding of the mechanisms of narrow-band ultraviolet B (NB-UVB) phototherapy, we studied the effects of NB-UVB treatment on the expression of these molecules. Skin biopsies from 12 psoriasis patients were collected before, during and after NB-UVB therapy. Real-time PCR and immunohistochemistry showed that p63 expression was not significantly affected, whereas NB-UVB phototherapy significantly decreased expression of miR-21 (p = 0.003) and increased miR-125b levels (p = 0.003). The results indicate that the unresolved p63 abnormality in treated epidermis may play a role in maintenance of this disease.