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The human p63 gene discovered in 1997 encodes a series of protein isoforms that differ in their N- and/or C-terminal sequences. These isoforms have widely differing properties in promoting or repressing p53-related functions such as growth arrest and apoptosis. In addition, p63 appears to play important roles in the maintenance and differentiation of epithelial cell populations. Many studies have shown that p63, particularly Np63, is expressed in normal epithelium and also highly expressed in squamous cell carcinomas of surface epithelia. Methods. We have refined the analysis of the expression patterns of p63 isoforms by the use of a quantitative RT-PCR technique applied to micro-dissected normal oral mucosal samples. We have also studied p63 expression in squamous cell carcinoma of the head and neck (SCCHN) compared to normal oral mucosa taken from the same patient. Furthermore, tobacco-exposed buccal mucosa was compared to age and gender matched non exposed controls. RT-PCR for telomerase and immunohistochemical analysis for detection of p53 and Ki-67 proteins was further performed. We also explored the function of p63 in SCCHN cells by using small interfering RNA (siRNA) to silence the expression of different p63 isoforms in cell lines originating from SCCHN. The effect of p63 knockdown was studied using a Fluorescein Diacetate based cytotoxicity assay and immunohistochemistry looking at expression of differentiation markers. The response of the siRNA treated cells to radiation and cytostatic drug was also investigated. We have further studied normal oral wound healing using immunohistochemistry and quantitative PCR. By the use of a macro array comparing siRNA treated cells with non-treated cells a possible connection between the BRCA1 gene and p63 expression was shown and further studied with the use of cHIP and luciferase reporter assays. Results. The p63 isoforms are expressed in normal epithelium, with the highest levels consistently found in basal and parabasal layers. Extensive use of tobacco had no effect on p63. The quantitative PCR showed statistically increased levels of the ΔNp63 and p63isoforms. No correlation was found between p63-isoform expression patterns and proliferation. The exploration of the function of p63 in SCCHN cells by the use of small interfering RNA (siRNA) showed a statistically significant decreased survival for tumour cells when all p63 isoforms, the N-terminal truncated or the  isoforms were inhibited. No effect on cell proliferation or expression of epithelial differentiation markers was observed. We also demonstrated that inhibition of p63 expression sensitizes cells to the effects of ionizing radiation and cisplatin. The study of normal oral wound healing using immunohistochemistry and quantitative PCR showed significant changes in p63 isoform expression. The Np63 isoform was mainly expressed in the basal layer in the non proliferating and migrating cells while TAp63 was almost absent. The BRCA1 study showed p63 to bind to the BRCA1 promoter and activate the expression of BRCA1 protein. Summary. The p63 proteins have different functions and the balance between the isoforms and their localisation within the epithelium seems to be important for normal wound healing as well as cancer cell survival.

Background: The p63 gene is a member of the p53 transcription factor family and can produce six different proteins using two promoters and differential splicing. Expression of p63 is required for proper formation of epithelial tissues. Studies on the transcriptional control of specific genes involved in cell survival, proliferation, differentiation and adhesion have revealed the contributions of p63 to the continuously renewing stratified epithelium. In this thesis, the aim was to improve our understanding of the roles of p63 in epithelial homeostasis by investigating expression of p63 in normal and benign hyper-proliferative epithelia and exploring the influence of p63 deregulation on cancer progression. Materials and methods: Using quantitative real time RT-PCR and immunohistochemistry, we first examined the expression of different p63 isoforms in patients diagnosed with psoriasis - a benign hyper-proliferative and inflammatory skin disease. Afterwards, we investigated responses of p63 in psoriatic epidermis upon Narrowband-UVB (NB-UVB) phototherapy. At the same time, we studied the potential impact of p63 in carcinogenesis by searching for p63 transcriptional targets in a cell line derived from squamous cell carcinoma of the head and neck (SCCHN) - the sixth most common cancer worldwide with over-expression of the ∆Np63α protein as a common feature. p63 gene silencing and microarray were used to identify p63 regulated genes. Real time RT-PCR, western blot, immunohistochemistry, chromatin immunoprecipitation, transient transfection and reporter assays were performed to confirm specific genes as direct p63 targets. Results: Significant down-regulation of p63 mRNA levels was found in psoriatic lesions compared to patients’ own clinically normal skin. Moreover, a trend of decreased TAp63 mRNA levels was seen in patients’ normal skin compared to age- and sex-matched healthy controls. Following NB-UVB phototherapy, an effective first line therapy for psoriasis, expression of p63 was not significantly affected. However, significant changes in p53, FABP5, miR-21 and miR-125b were found. Surprisingly, location and expression levels of p63 proteins detected by immunohistochemistry were similar under all skin conditions. A direct transcriptional regulation of TRAF4 by p63 was seen in the SCCHN cell line and we further found that the localization of the TRAF4 protein was associated with histological differentiation of SCCHN cells. However, unlike its over-expression in SCCHN, similar TRAF4 mRNA expression levels were seen in psoriatic lesions as compared to healthy controls. Besides TRAF4, a total of 127 genes were identified as potentially p63 regulated in the SCCHN cell line and strikingly, about 20% of these genes are involved in cell adhesion or migration. Conclusions: Dysregulation of p63 isoforms in psoriatic epidermis, especially decreased TAp63 expression, and their resistance to NB-UVB phototherapy implicated a contribution of p63 to the psoriasis phenotype. Transcriptional regulation of genes involved in multiple biological pathways indicated that over-expression of p63 in SCCHN might account for altered cell differentiation, adhesion and migration, thus contributing to SCCHN. In conclusion, our studies have found additional mechanisms through which p63 guarded homeostasis of the established epithelium. Deregulation of p63 might play a role in distinct pathological conditions by participating in diverse cellular pathways under different microenvironments.

Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.

The Wilms’ tumor gene 1 (WT1) was first reported as a tumor suppressor gene in Wilms’ tumor. However, later studies have shown the oncogenic properties of WT1 in a variety of tumors. It was recently proposed that WT1 was a chameleon gene, due to its dual functions in tumorigenesis. We aimed to investigate the clinical significance of WT1 as biomarker in acute myeloid leukemia (AML) and clear cell renal cell carcinoma (ccRCC) and to elucidate the function of WT1 as an oncogene in squamous cell carcinoma of head and neck (SCCHN). In AML, it was suggested that WT1 expression was an applicable marker of minimal residual disease (MRD). In adult patients with AML, we found a good correlation between WT1 expression levels normalized to two control genes, β-actin and ABL. Outcome could be predicted by a reduction in WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis, when β-actin was used as control. Also, irrespective of the control gene used, outcome could be predicted by a reduction in WT1 expression in peripheral blood (≥ 2-log) detected between 1 and 6 months after treatment initiation. Previous studies in RCC demonstrated that WT1 acted as a tumor suppressor. Thus, we tested whether single nucleotide polymorphisms (SNPs) or mutations in WT1 might be associated with WT1 expression and clinical outcome in patients with ccRCC. We performed sequencing analysis on 10 exons of the WT1 gene in a total of 182 patient samples, and we identified six different SNPs in the WT1 gene. We found that at least one or two copies of the minor allele were present in 61% of ccRCC tumor samples. However, no correlation was observed between WT1 SNP genotypes and RNA expression levels. Moreover, none of the previously reported WT1 mutations were found in ccRCC. Nevertheless, we found that a favorable outcome was associated the homozygous minor allele for WT1 SNP. We then further investigated whether WT1 methylation was related to WT1 expression and its clinical significance. Methylation array and pyrosequencing analyses showed that the WT1 promoter region CpG site, cg22975913, was the most frequently hypermethylated CpG site. We found a trend that showed nearly significant correlation between WT1 mRNA levels and hypermethylation in the 5’-untranslated region. Hypermethylation in the WT1 CpG site, cg22975913, was found to be associated with patient age and a worse prognosis. One previous study reported that WT1 was overexpressed in SCCHN. That finding suggested that WT1 might play a role in oncogenesis. We found that both WT1 and p63 could promote cell proliferation. A positive correlation between WT1 and p63 expression was observed, and we identified p63 as a WT1 target gene. Furthermore, several known WT1 and p63 target genes were affected by knocking down WT1. Also, co-immunoprecipitation analyses demonstrated a protein interaction between WT1 and p53. In summary, WT1 gene expression can provide useful information for MRD detection during treatment of patients with AML. In RCC, our results suggested that the prognostic impact of WT1 SNPs was limited to the subgroup of patients that were homozygous for the minor allele, and that WT1 promoter hypermethylation could be used as a prognostic biomarker. In SCCHN, WT1 and p63 acted as oncogenes by affecting multiple genes involved in cancer cell growth.

Squamous cell carcinoma of the head and neck (SCCHN), the 6th most common cancer worldwide, has a low 5-year survival. Disease as well as treatment often causes patients severe functional and aesthetic problems. In order to improve treatment and diagnosis at earlier stages of tumour development it is important to learn more about the molecular mechanisms behind the disease. p63, an important regulator of epithelial formation, has been suggested to play a role in the development of SCCHN. Six different isoforms of p63 have been found and shown to have various functions. The aim of the studies in this thesis was to learn more about the role of p63 and proteins connected to p63 in SCCHN. Expression of p63, Cox-2, EGFR, beta-catenin, PP2A and p53 isoforms was mapped in tumours and normal tumour adjacent tissue from patients with SCCHN using western blot or RT-PCR. Results showed no significant difference between tumours and normal tumour adjacent tissue concerning expression of EGFR and beta-catenin. Cox-2 and PP2A showed significantly higher expression in tumours while p63 was more expressed in normal tumour adjacent tissue. However, expression of all these proteins in normal tumour adjacent tissue differed from tissue from disease-free non-smoking individuals. Smoking in itself did not affect expression of these proteins. The p53 isoforms p53, p53beta, p53gamma, ∆133p53, ∆133p53beta and ∆133p53gamma were expressed at RNA level in samples both from tumours and normal tumour adjacent tissue, though most of them at fairly low levels. The functional properties of the different p63 isoforms have not been fully mapped. By establishing stable cell lines over-expressing the different p63 isoforms we investigated their specific effect on tumour cells from SCCHN. Only the ∆Np63 isoforms could be stably over-expressed, whereas no clones over-expressing TAp63 could be established. Using microarray technique, cell lines stably expressing the ∆Np63 isoforms were studied and CD44, Keratins 4, 6, 14, 19 and Cox-2 were found to be regulated by p63. In conclusion, the present project adds new data to the field of p63 and SCCHN. For example, we have shown that clinically normal tumour adjacent tissue is altered compared to normal oral mucosa in non tumour patients, and that smoking does not change expression of p63, Cox-2, EGFR, beta-catenin or PP2A in oral mucosa. Novel p53 isoforms are expressed in SCCHN, and even though levels are very low they should not be overlooked. Furthermore, CD44, keratins 4, 6, 14, 19 and Cox-2 were identified as p63 targets in SCCHN.

Hintergrund: Die Prognose von Patienten mit einem Plattenepithelkarzinom des Kopf-Hals-Bereichs konnte trotz multimodaler Therapieregime in den letzten Jahren nicht wesentlich verbessert werden. Neue Therapieansätze sind also von hoher Bedeutung. Methoden: In dieser Arbeit wurde ein Patientenkollektiv von 66 Patienten untersucht, welches im Zeitraum von Oktober 2010 bis Oktober 2012 wegen eines Plattenepithelkarzinoms des Kopf-Hals-Bereichs mit einer konkomitanten Radiochemotherapie (RCT) in der Abteilung für Strahlentherapie und Radioonkologie der Universitätsmedizin Göttingen behandelt wurde. Die Plasmen dieser Patienten wurden zu drei unterschiedlichen Zeitpunkten der Behandlung auf das Vorliegen der vier Chemokine CCL2, CCL5, CCL20 und CXCL12 und des Akut-Phase-Zytokins IL-6 und deren Konzentrationsveränderungen während der RCT mittels ELISA (Enzyme-linked-Immunosorbent-Assay) untersucht. Dabei wurde sowohl ein möglicher Zusammenhang zwischen der Konzentration und der Behandlungsmodalität als auch ein Zusammenhang zwischen der Konzentration und dem Lymphknotenstatus analysiert.  Ergebnisse: Unsere Ergebnisse zeigen sowohl, dass CCL2 im Plasma dieses Kollektivs nachweisbar ist, als auch einen signifikanten Anstieg der CCL2-Konzentration während einer RCT. Bei Patienten, die zu Beginn der Therapie noch einen manifesten Tumor hatten, ließ sich ein signifikant niedrigerer CCL2-Spiegel messen als bei Patienten, die im adjuvanten Rahmen eine RCT erhielten. Im Vergleich der CCL2-Konzentrationen bei Patienten mit unterschiedlichem Lymphknotenstatus  konnten wir keine signifikanten Werte messen. Während der RCT maßen wir einen signifikanten Abfall der CCL5-Konzentration im Plasma. Patienten, die aufgrund der Ausdehnung des Tumors inoperabel waren, hatten signifikant höhere CCL5-Spiegel als Patienten, die vor Beginn der RCT operiert wurden. Ein signifikanter Unterschied zwischen einem positiven oder negativen Lymphknotenstatus ließ sich bei CCL5 nicht feststellen. Die CCL20-Konzentration fiel während der RCT signifikant ab. Ein Vergleich des unterschiedlichen Lymphknotenstatus zeigte keine Signifikanz. Unsere Ergebnisse zeigen weiterhin, dass die CXCL12-Konzentration nicht signifikant während einer RCT anstieg. Der Unterschied der CXCL12-Konzentration getrennt nach positivem oder negativem Lymphknotenstatus war ebenfalls nicht signifikant. Während einer RCT analysierten wir einen signifikanten Anstieg der IL-6-Konzentration. Die Konzentration des Akut-Phase-Zytokins war zu zwei Zeitpunkten unserer Messung in primär behandelten Patientenplasmen signifikant höher als in adjuvant behandelten Patientenplasmen. Einen signifikanten Unterschied der IL-6-Konzentration zwischen einem positiven oder negativen Lymphknotenstatus konnten wir nicht messen. Diskussion: Die klinische Relevanz der dargestellten Ergebnisse ist aufgrund der einfach zugänglichen Probengewinnung und Methodik und einer möglichen Verwendung als Biomarker für das Tumoransprechen und die Prognose als hoch einzuschätzen. Wie in dieser Arbeit z. B. bei dem Chemokin CCL5 gezeigt, könnte es in Zukunft von klinischer Bedeutung sein, eine individualisierte Therapie für Patienten mit unterschiedlichem Krankheitsstadium (Inoperabilität oder Operabilität des Tumors) etablieren zu können. In der vorgelegten Arbeit ließ sich eine signifikant höhere CCL5-Konzentration im Plasma bei Patienten mit Vorliegen des Primärtumors messen im Vergleich zu Patientenplasma, welches adjuvant behandelt wurde. Dies könnte ein Hinweis darauf sein, dass CCL5 direkt vom bestehenden Tumor ins Blut sezerniert wird und somit auch als möglicher Biomarker in der Diagnose und Therapie des Kopf-Hals-Malignoms genutzt werden könnte.  Sollten sich durch weiterführende Untersuchungen die hier beschriebenen Effekte bestätigen, ist die Anwendung im klinischen Alltag als eine sinnvolle Ergänzung zur bisherigen Behandlung in der Therapie von Patienten mit einem Malignom des Kopf-Hals-Bereichs denkbar.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

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