Xiaolian Gu

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

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.

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.

Altered expression of miR-21, miR-125b, and miR-203 indicates a role for these microRNAs in oral lichen planus

BACKGROUND Oral lichen planus (OLP), which is a chronic inflammatory disease of the oral mucosa with unknown etiology, affects about 2% of the population. MicroRNAs are small non-coding RNAs involved in normal processes such as development and differentiation as well as progression of human diseases. The aim of this study was to investigate the expression of miR-21, miR-125b, and miR-203 and to compare RNA levels of their potential targets, the tumor suppressor p53 and its relative p63, both known to be deregulated in OLP. METHODS In biopsies from 20 patients with OLP and 20 age- and sex-matched healthy controls, epithelium was laser dissected and analyzed for the expression of miR-21, miR-125b, miR-203, p53, and p63 using qRT/PCR. RESULTS Increased expression of miR-21 and miR-203, decreased expression of miR-125, and down-regulation of p53 and ΔNp63 RNA were seen in OLP compared to normal oral mucosa. When comparing microRNA expression to levels of p53 and p63 RNA, a significant negative correlation was seen between ΔNp63 and miR-203 and between miR-21 and p53, respectively. CONCLUSION Results indicate a role for the studied microRNAs in changes seen in OLP.

DeltaNp63 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

TRAF4 is potently induced by TAp63 isoforms and localised according to differentiation in SCCHN

p63, a member of the p53 family, is overexpressed in squamous cell carcinoma of the head and neck (SCCHN) and some other tumors of epithelial origin. As a transcription factor, p63 can bind to p53-type response elements and there is some overlap between p53 family transcriptional targets. Tumor necrosis factor receptor associated factor 4 (TRAF4) is a p53 regulated gene which is overexpressed in many human carcinomas. We investigated the involvement of p63 in regulation of TRAF4 and the expression of the TRAF4 protein in SCCHN. Disrupting endogenous p63 expression resulted in downregulation of TRAF4 mRNA and protein in an SCCHN cell line. Endogenous p63 bound to the TRAF4 promoter in vivo and reporter assays showed that p63, p73 and p53 can all transactivate TRAF4, with TAp63 isoforms being the most potent activators. The level of TRAF4 activation by TAp63 was two-fold higher than by p53, and TRAF4 was 10-fold more responsive to TAp63 than another p63-target, IGFBP3. Nuclear expression of TRAF4 was seen in normal oral epithelium and highly/moderately differentiated SCCHN, whereas cytoplasmic expression of TRAF4 was seen in poorly differentiated SCCHN. These results indicate that TRAF4 is a common target of p53 family members and that localization of TRAF4 is associated with differentiation of SCCHN cells.

Correlation between Reversal of DNA Methylation and Clinical Symptoms in Psoriatic Epidermis Following Narrow-Band UVB Phototherapy

Epigenetic modifications by DNA methylation are associated with a wide range of diseases. Previous studies in psoriasis have concentrated on epigenetic changes in immune cells or in total skin biopsies that include stromal-associated changes. In order to improve our understanding of the role of DNA methylation in psoriasis, we sought to obtain a comprehensive DNA methylation signature specific for the epidermal component of psoriasis and to analyze methylation changes during therapy. Genome-wide DNA methylation profiling of epidermal cells from 12 patients undergoing narrow-band UVB phototherapy and 12 corresponding healthy controls revealed a distinct DNA methylation pattern in psoriasis compared with controls. A total of 3,665 methylation variable positions (MVPs) were identified with an overall hypomethylation in psoriasis patient samples. DNA methylation pattern was reversed at the end of phototherapy in patients showing excellent clinical improvement. Only 7% of phototherapy-affected MVPs (150 out of 2,108) correlate with nearby gene expression. Enrichment of MVPs in enhancers indicates tissue-specific modulation of the transcriptional regulatory machinery in psoriasis. Our study identified key epigenetic events associated with psoriasis pathogenesis and helps understand the dynamic DNA methylation landscape in the human genome.

Oxidation Reduction is a Key Process for Successful Treatment of Psoriasis by Narrow-band UVB Phototherapy

Narrow-band UVB (NB-UVB) phototherapy is commonly used for treatment of psoriasis, though the mechanisms underlying its efficacy have not been completely elucidated. We used gene expression profiling to characterise gene expression in lesional epidermis from psoriasis patients in the middle and late stages of NB-UVB photo-therapy. Increased melanogenesis gene expression was the earliest response to phototherapy. At the end of treatment, genes responding to phototherapy and correlated to treatment outcome were involved in oxidation reduction, growth and mitochondria organisation. Particularly, SPATA18, a key regulator of mitochondrial quality, was significantly down-regulated in psoriasis (p < 0.05). Poly(dA:dT) and poly(I:C) stimulation increased SPATA18 level in primary keratinocytes, indicating the importance of mitochondria quality control under innate immune induced oxidative stress. Normalised SPATA18 expression after phototherapy indicates improved mitochondrial quality control and restored cellular redox status. Our data suggest that oxidation reduction is critical for the resolution of psoriatic plaques following NB-UVB phototherapy.

Expression of the long non-coding RNA HOTAIR as a prognostic factor in squamous cell carcinoma of the head and neck: a systematic review and meta-analysis

Introduction Long noncoding RNAs (lncRNAs) are often dysregulated in cancer tissue and seem to play an important role in neoplastic processes. Recent studies have shown that the HOX transcript antisense intergenic RNA (HOTAIR) may play a role as a marker of prognosis in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to perform a meta-analysis of studies focused on the prognostic role of HOTAIR in SCCHN. Results At the end of the selection process, four studies were considered eligible for inclusion in the meta-analysis, comprising a total of 271 patients. Meta-analysis revealed that high expression of HOTAIR was associated with poor overall survival (HR, 1.90; 95% CI: [1.42, 2.53]; p < 0,0001), advanced tumor stage (OR, 3.44; 95% CI: [1.84, 6.43]; p < 0,001) and lymph-node metastasis (OR, 3.31; 95% CI: [1.24, 8.79]; p = 0,02). Materials and Methods The literature search was performed in the following databases: PUBMED, SCOPUS, EMBASE and Web of Science, in order to find studies that met the inclusion criteria. Conclusions Findings from this systematic review and meta-analysis revealed that HOTAIR represents a potential biomarker of prognosis in patients with squamous cell carcinoma of the head and neck.

Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck

BackgroundWilms’ tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu.MethodsSilencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN.ResultsWe found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples.ConclusionsOur findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth.