Single cell migration in oral squamous cell carcinoma - possible evidence of epithelial-mesenchymal transition in vivo.

BACKGROUND: The invasion of cancer cells into the surrounding normal tissue is one of the defining features of cancer. While the phenomena of tumour budding, epithelial-mesenchymal transition and the presence of myofibroblasts have independently been shown to be related to a poor prognosis of oral carcinomas, their relationship has not been examined in detail. METHODS: Paraffin-embedded tissues from 28 patients with oral squamous cell carcinomas were stained with antibodies to cytokeratin, α-SMA, vimentin, E-cadherin, N-cadherin and Twist and evaluated for their expression in relation to invasive cancer cells and the surrounding tumour stroma. RESULTS AND CONCLUSIONS: A direct, histological relationship between invading, budding tumour cells and myofibroblasts was occasionally seen but was not a general feature. Most of the budding tumour cells at the invasive front had a decreased expression of E-cadherin, but we did not find that this was associated with a consistent or clear increase in either N-cadherin or vimentin. We therefore suggest that the budding of tumour cells is not dependent upon either myofibroblasts or a complete epithelial-mesenchymal transition and that these phenomena most likely represent separate processes in tumour progression.

A reverse Warburg metabolism in oral squamous cell carcinoma is not dependent upon myofibroblasts.

BACKGROUND: The reverse Warburg effect describes the phenomenon that epithelial cancer cells take advantage of the metabolic machinery from nearby cancer-associated fibroblast, inducing them to produce lactate and ketones to fuel the high metabolic demands of the epithelial tumour tissues. This is in breast cancer observed as a lack of stromal caveolin-1 (CAV-1) and an increased expression of monocarboxylate transporter 4 (MCT-4) in the tumour stroma, with a concomitant increase in the expression of monocarboxylate transporter 1 (MCT-1) in the epithelial, tumour compartment. The lack of CAV-1 and increased expression of MCT-4 have been shown to have prognostic importance, primarily in patients with breast cancer. However, this phenomenon has only scarcely been described in oral squamous cell carcinoma (OSCC). Given the prognostic importance of myofibroblasts in OSCC, we also examined a potential relationship between the expression of MCT-4 and the presence of myofibroblasts. METHODS: Paraffin-embedded tissues from 30 patients with OSCC were immunostained with antibodies towards MCT-1, MCT-4, Cav-1, GLUT-1, α-SMA, TOMM20 and KI-67, and evaluated for their specific epithelial and stromal expression. RESULTS AND CONCLUSIONS: In patients with OSCC, we find an increased expression of MCT-1 and MCT-4 in both the epithelial and stromal compartment, with almost no overlap in their spatial expression. We found a large spatial overlap between α-SMA and MCT-1 in the stroma compartment, but no relationship between MCT-4 and myofibroblasts. Interestingly, we did not observe any relationship between the absence of CAV-1 and the presence of MCT-4 as has been shown in breast carcinomas.

MiR-21 expression in the tumor stroma of oral squamous cell carcinoma: an independent biomarker of disease free survival.

Oral squamous cell carcinoma (OSCC) patients have a high mortality rate; thus, new clinical biomarkers and therapeutic options are needed. MicroRNAs (miRNAs) are short noncoding RNAs that regulate posttranscriptional gene expression and are commonly deregulated in OSCC and other cancers. MicroRNA-21 (miR-21) is the most consistently overexpressed miRNA in several types of cancer, and it might be a useful clinical biomarker and therapeutic target. To better understand the role of miR-21 in OSCC, paraffin-embedded tumor tissue samples from 86 patients with primary OSCC were analyzed by in situ hybridization. We found that miR-21 was primarily expressed in the tumor stroma and in some tumor-associated blood vessels with no expression in the adjacent normal epithelia or stroma. Using image analysis, we quantitatively estimated miR-21 expression levels specifically in the stroma of a cohort of OSCC samples. These miR-21 levels significantly correlated with disease free survival with the highest levels being located in the stroma. Stromal miR-21 expression was independently associated with a poorer prognosis, even after adjusting for clinical parameters (perineural invasion and N-stage) in a multivariate analysis. In summary, we have shown that miR-21 is located in the carcinoma cells, stroma and blood vessels of tumors, and its expression specifically in the stromal compartment has a negative prognostic value in OSCC.

Significance of myofibroblasts in oral squamous cell carcinoma.

It is now recognized that the tumor microenvironment makes significant contribution to tumor progression. Activated fibroblast endothelial cells, inflammatory cells, and various extra cellular matrix components are parts of this microenvironment. Most of the activated fibroblasts are α-smooth muscle actin-positive myofibroblast that often represent the majority of tumor stromal cells. Their production of growth factors chemokines and extracellular matrix facilitates tumor growth. Myofibroblast have been demonstrated in close to 50% of oral squamous cell carcinomas. In this review, we highlight the histological distribution of myofibroblast in oral squamous cell and the myofibroblast relation to tumor growth on prognosis.

[The oral cavity: what shall we look for--and what can we do?]. / Hvad skal vi se efter i mundhulen--og hvad kan vi gøre?

Patients present with symptoms of the oral cavity at their general practitioner (GP). As a general rule, the GP should refer the patient to a dentist for diagnosis and treatment. However, treatment of acute infections can be started by the GP. In the present paper, the diagnosis and treatment of such cases is described.

Epigenetic alterations of the SERPINE1 gene in oral squamous cell carcinomas and normal oral mucosa.

A high level of plasminogen activator inhibitor-1 (PAI-1 or SERPINE1) in tumor extracts is a marker of a poor prognosis in human cancers, including oral carcinomas. However, the mechanisms responsible for the upregulation of PAI-1 in cancers remain unclear. Investigating specific PAI-1 expressing cells in oral carcinomas by immunohistochemistry, we found that PAI-1 was expressed in 18 of the 20 patients, mainly by cancer cells. Two showed PAI-1 positive stromal cells surrounding the tumor areas and five showed PAI-1 positive cells in tumor-adjacent normal epithelium. By real-time RT-PCR analysis, 17 of 20 patients with oral carcinoma were found to have between 2.5- and 50-fold increased tumor PAI-1 mRNA level, as compared with the matched tumor-adjacent normal tissues. The PAI-1 mRNA level in connective tissues from 15 healthy volunteers was similar to the level in tumor-adjacent normal tissues, but the level in epithelium was 5- to 10-fold lower. Analyzing DNA methylation of 25 CpG sites within 960 bp around the transcription initiation site of the SERPINE1 gene by bisulfite sequencing, we did the surprising observation that both tumors and tumor-adjacent normal tissue had a significant level of methylation, whereas there was very little methylation in tissue from healthy volunteers, suggesting that tumor-adjacent normal tissue already contains transformation-associated epigenetic changes. However, there was no general inverse correlation between PAI-1 mRNA levels and SERPINE1 gene methylation in all tissues, showing that CpG methylation is not the main determinant of the PAI-1 expression level in oral tissue.

Molecular markers in the surgical margin of oral carcinomas.

BACKGROUND: Local or regional lymph node recurrence is the most common pattern of treatment failure in oral squamous cell carcinoma (SCC). The local recurrence rate is 30% even when the surgical resection margin is diagnosed as tumour free. Accumulation of genetic changes in histologically normal epithelium in the surgical resection margin may explain the local recurrence rate. The purpose of this study is to investigate the presence of senescence markers, which may represent early malignant changes in the margin that in routine pathological evaluations are classified as histologically normal. METHODS: Formalin-fixed, paraffin-embedded surgical specimens from 16 consecutive patients with oral SCC and a clear surgical margin were obtained. The margin was analysed by immunohistochemistry for p53, p16, Chk2, Laminin-5 and glycosylated oncofetal fibronectin. RESULTS: Two patterns of p53 expression were found in the histologically normal epithelium in the surgical resection margin. One was characterized by no protein expression in the majority of cells, except for small clusters of basal and parabasal cells with nuclear staining. The other was characterized by p53 expression in the nuclei of most basal cells. The expression of p16 was confined to small groups of cells in the basal cell layer whereas Chk2 was only seen in one case. Upregulation of the stromal proteins, Laminin-5 or glycosylated oncofetal fibronectin, was only seen at regions of invasion. CONCLUSION: Small groups of cells expressing p53 and p16 were found in the surgical resection margin that appeared to be histologically normal and may represent early malignant changes.

Overexpression of protease nexin-1 mRNA and protein in oral squamous cell carcinomas.

Protease nexin-1 (PN-1) belongs to the serpin family of serine protease inhibitors. It is the phylogenetically closest relative of plasminogen activator inhibitor-1 (PAI-1). Whilst there are numerous studies of the occurrence and functions of PAI-1 in cancer, a possible tumour biological role of PN-1 has been almost totally neglected. We have now compared the level of PN-1 mRNA in 20 cases of oral squamous cell carcinomas and in matched samples of the corresponding normal oral tissues. We found that the average PN-1 mRNA level in tumours and normal tissues was significantly different, being increased up to 13 fold in tumour samples compared with the average level in normal tissues. The PN-1 mRNA level was significantly higher in tumours from patients with lymph node metastasis than in tumours from patients without. We could conclude that PN-1 is frequently overexpressed in oral squamous cell carcinomas and that its level may correlate with the occurrence of lymph node metastasis. We hypothesise that PN-1 may have a tumour biological function similar to that of PAI-1.

Molecular basis for the presence of glycosylated onco-foetal fibronectin in oral carcinomas: the production of glycosylated onco-foetal fibronectin by carcinoma cells.

Glycosylated onco-foetal fibronectin (GOF) deposited in the stroma of oral squamous cell carcinomas correlates with survival. One of the two polypeptide GalNAc-transferases, GalNAc-T3 or GalNAc-T6, is required for the biosynthesis of GOF by the initiation of a unique O-glycan in the alternative spliced IIICS region. Using cell culture experiments, immunohistochemical staining of primary tissue, and RT-PCR of tumour cells isolated by laser capture techniques we have examined the molecular basis for the production of GOF in oral carcinomas. Immuno-histochemical investigation confirmed the stromal deposition of GOF in oral carcinomas. However, neither GalNAc-T3 nor GalNAc-T6 could be detected in stromal fibroblasts. In contrast both transferases were present in the oral squamous carcinoma cells, suggesting that GOF is produced by the oral cancer cells and not only the stromal cells. RT-PCR analysis of RNA isolated from carcinoma cells provided further support for this conclusion by demonstrating in-splicing of the alternative spliced IIICS domain in GOF.

Function and importance of p63 in normal oral mucosa and squamous cell carcinoma of the head and neck.

BACKGROUND/AIMS: Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common malignancy worldwide with a 5-year survival that has not improved over the last 20-25 years. Factors of prognostic significance for this tumour type include the presence of regional lymph node metastasis and amplification of chromosome 3q21-29, where the p63 gene is located. This gene encodes 6 proteins and is crucial for formation of the oral mucosa, teeth, salivary glands and skin. Each of the 6 different p63 proteins has different characteristics and functions, where some resemble the tumour suppressor protein p53, whilst others have functions that oppose p53. METHODS: To understand the function and importance of p63 in oral mucosa and tumour development we have studied protein as well as mRNA expression in normal oral mucosa and tumours. RESULTS/CONCLUSION: Expression of p63 proteins differs between the cell layers in normal oral mucosa, and primary HNSCC has a high expression level of p63 isoforms normally expressed in basal cells. Data suggest that p63 expression in HNSCC influences tumour cell differentiation.

Complex p63 mRNA isoform expression patterns in squamous cell carcinoma of the head and neck.

The human p63 gene encodes a series of protein isoforms that differ in their N- and/or C-terminal sequences and possess widely varying activities in promoting or repressing p53-related functions and in regulating the proliferation and differentiation of epithelial cells. To gain further information on the role of p63 expression in human tumours, we used quantitative real-time RT-PCR to study individual p63 isoforms in squamous cell carcinomas of the head and neck (SCCHN). In keeping with previous reports, expression of the deltaN- and p63alpha-isoforms predominated and deltaNp63 mRNA was expressed at significantly higher levels in tumours compared to matched normal tissues. Some tumours also expressed the highly efficient transactivator TA- and p63beta-isoforms, and p63beta was significantly increased in tumours compared to matched normal tissue. We could not identify any correlations between different p63-isoform expression patterns and proliferation, p53 status, or telomerase expression. All p63 isoforms could be identified in normal surface epithelium, and micro-dissection showed that the high levels present in basal layers were similar to those seen in tumour tissues. Thus, high-level expression of deltaNp63 in tumour cells may represent maintained expression by the basal cells from which the tumour arose, rather than representing a true over-expression of p63 during tumourigenesis. Tobacco usage, a genotoxic predisposing factor for SCCHN, had no effect on p63 expression in oral epithelium. Taken together, our data indicate that SCCHN maintain expression of high levels of deltaNp63alpha in combination with varying levels of other p63 isoforms, some of which are highly efficient transcriptional activators. The complexity of these p63 expression patterns seen in primary SCCHN indicates that p63 has multifaceted roles in tumour biology.

Histo-blood group ABO antigen in oral potentially malignant lesions and squamous cell carcinoma--genotypic and phenotypic characterization.

Loss of histo-blood group A/B antigens is frequent in oral cancer. It is unclear whether this alteration is due to loss of the chromosomal region encoding the genes. The aim was to investigate genotypic alterations in the ABO locus in oral potentially malignant lesions and carcinomas. Seventy-three cases which expressed A/B antigen in normal epithelium by immunohistochemical (IHC) staining were investigated. Both tumour and normal cells were collected from paraffin-embedded tissue by laser microdissection. DNA was extracted and analysed by PCR coupled with restricted digestion analysis in order to establish the ABO genotype. Total and patchy loss of A/B antigen expression was found in 24/32 carcinomas, 6/7 leukoplakias with severe dysplasia, 12/17 leukoplakias with mild and moderate dysplasia, and 6/17 leukoplakias without dysplasia. Specific A/B allele loss was found in 8/24 cases with carcinoma and 3/24 cases with mild and moderate dysplasia by genotyping analysis. O allele loss was found in 10 cases involving all four groups. In patients with heterozygous genotypes, A/B allelic loss by genotyping analysis was always followed by loss of A/B antigen expression by IHC staining. Loss of A/B antigen expression in tissues which had intact ABO alleles was, however, found and may be explained by other genetic and epigenetic changes.

Genetic and epigenetic alterations of the blood group ABO gene in oral squamous cell carcinoma.

Loss of histo-blood group A and B antigen expression is a frequent event in oral carcinomas and is associated with decreased activity of glycosyltransferases encoded by the ABO gene. We examined 30 oral squamous cell carcinomas for expression of A and B antigens and glycosyltransferases. We also examined DNA from these tumors for loss of heterozygosity (LOH) at markers surrounding the ABO locus at chromosome 9q34, for loss of specific ABO alleles, and for hypermethylation of the ABO promoters. Loss of A or B antigen expression was found in 21 of 25 tumors (84%) and was a consistent feature of tumors lacking expression of A/B glycosyltransferases. LOH at 9q34 was found in 7 of 27 cases (26%), and one case showed microsatellite instability. Among 20 AO/BO cases, 3 showed loss of the A/B allele and 3 showed loss of the O allele. Analysis of the proximal ABO promoter by methylation-specific PCR and melting curve analysis showed hypermethylation in 10 of 30 tumors (33.3%), which was associated with loss of A/B antigen expression. ABO promoter hypermethylation was also found in hyperplastic or dysplastic tissues adjacent to the tumors, suggesting that it is an early event in tumorigenesis. Collectively, we have identified molecular events that may account for loss of A/B antigen expression in 67% of oral squamous cell carcinomas.

Cell-cycle regulatory proteins in human wound healing.

Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas the abundance of most of the CKIs, including p27Kip1, p57Kip2, p15ink4b and p18ink4c, was relatively maintained in the migrating epithelial tongue. These data indicate that downmodulation of several G(1)/S-phase cyclins and a relative excess of CKIs may cooperate to ensure the quiescent state of migrating keratinocytes during wound healing.

Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts.

Migration of fibroblasts from surrounding normal tissue into the wound bed is an important requirement for successful wound healing. This study investigated the motility pattern of buccal, periodontal and skin fibroblasts to determine whether differences in the wound healing efficiency at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer-assisted microscope work-station. For evaluation of cell morphology, cell contours were recognized semiautomatically and used for determination of cell area, cell spreading and number and length of processes. We found that the cellular displacement of the buccal fibroblasts was only approximately 50% of the cellular displacement of periodontal and skin fibroblasts. The decreased cellular displacement of the buccal fibroblasts was found to be due to both lower cellular speed and less persistence in direction. The buccal fibroblasts also displayed smaller areas and longer processes. The differences in cellular morphology and motility pattern amongst the three fibroblast types could not be explained by differences in secretion of extracellular matrix components and are therefore believed to reflect phenotypic differences amongst fibroblast subpopulations.

ABO blood group antigens in oral mucosa. What is new?

Histo-blood group ABH (O) antigens are major alloantigens in humans. These antigens are widely distributed in human tissues and undergo changes in expression during cellular differentiation and malignant development. The ABH antigens have been characterized as terminal disaccharide determinants which represent secondary gene products. They are synthesized in a stepwise fashion from a precursor by the action of different glycosyltransferases. In non-keratinized oral mucosa, a sequential elongation of the carbohydrates is associated with differentiation of epithelial cells, resulting in expression of precursors on basal cells and A/B antigens on spinous cells. Reduction or complete deletion of A/B antigen expression in oral carcinomas has been reported, a phenotypic change that is correlated with invasive and metastatic potential of the tumours and with the mortality rates of the patients. Disappearance of the antigens is ascribed to the absence of A or B transferase gene expression. Several studies have shown that loss of A and B antigen expression is associated with increased cell motility, invasion in matrigel, and tumourigenecity in syngenic animals. In vivo studies of human oral wound healing show similarly decreased expression of A/B antigens on migrating epithelial cells. Some studies suggest that the relationship between expression of blood group antigens and cell motility can be explained by different degrees of glycosylation of integrins. Changes in ABO expression in tumours have, in some cases, been due to the A/B gene promoter, although little is known about the regulation of A, and B expression, in normal tissue.

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes.

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5 days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether with or without stimulation. The results demonstrate that the secretion of KGF and HGF in both unstimulated fibroblasts and in fibroblasts co-cultured with keratinocytes is dependent on the type of fibroblasts. In general, the periodontal fibroblasts had the highest level of cytokine production. This high level of growth factor production may influence the proliferation and the migration of junctional epithelium and thereby influence the development of periodontal disease.