Epithelial to mesenchymal transition (EMT) biomarkers – E-cadherin, beta-catenin, APC and Vimentin – in oral squamous cell carcinogenesis and transformation
Introduction
Oral squamous cell carcinomas (OSCCs) represent the large majority of cancers arising from the oral cavity.1 Despite advances in therapy, the 5-year survival rate remains low at approximately 50%, and this is largely due to the number of patients presenting with advanced stage disease.1, 2, 3, 4 Aggressive treatment modalities for advanced lesions can also be seriously debilitating for the patient.3 Ideally, interception and management of OSCC at a precancerous stage would improve survival and quality of life.
The presence and severity of dysplasia in a potentially malignant oral lesion is currently the standard in predicting the risk of malignant transformation,5 and may be divided into three main categories (mild, moderate and severe) according to the World Health Organisation (WHO) 2005 histopathological classification.6 However, histopathological assessment suffers from intra- and interobserver variation,7, 8 and is non-prognostic in some cases.9, 10, 11 Therefore, a more accurate and reliable system of detecting high risk lesions is required.
Cancer development is a multistep process, where accumulation of genetic defects (reflected by molecular changes), followed by clonal selection and expansion of altered cells, ultimately leads to the development of overt carcinoma.12 Since changes at the molecular level are present before any cellular or clinical changes are evident,2 detection of these molecular changes would ideally allow earlier diagnosis of high risk states, and therefore help improve prognosis.
Malignant transformation in many carcinomas is associated with the loss of epithelial differentiation and gain of a mesenchymal phenotype; a process known as epithelial to mesenchymal transition (EMT).13 EMT is known to play a role in three main processes – embryogenesis, tissue repair, and cancer metastasis.14 Recent studies suggest the presence of EMT may be a predictor of OSCC progression and prognosis.15, 16 The expression of mesenchymal genes with carcinoma progression is often accompanied by an increase in cell motility and the loss of epithelial features, including reduced intercellular adhesion and a loss of epithelial cell polarity.13 These EMT features are seen not only in cases of OSCC progression, but also oral epithelial dysplasia. This suggests EMT changes may be found early in the development of OSCC, and identification of genes and their products that play a role in the transition process may be potential biomarkers of malignant transformation.
Aberrant activation of the Wnt signalling pathway is thought to initiate EMT in OSCCs, promoting tumour invasion and metastasis,17, 18, 19 and may also play an earlier role in carcinogenesis by supporting malignant transformation.20 Binding of Wnt proteins to their transmembrane cell receptors can activate a pathway known more specifically as the canonical Wnt-β-catenin pathway. As the name suggests, the β-catenin protein is a central molecule in this pathway, and is stabilised and translocated into the nucleus where it is able to associate with T-cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF-1) to form a functional transcription factor that mediates the transactivation of target genes involved in tumour progression, invasion and metastasis.21, 22, 23
E-cadherin is a calcium-dependent transmembrane glycoprotein that is expressed in most epithelial cells, and functions in establishing cell polarity and maintaining normal tissue structure.21 The intracellular domain of E-cadherin interacts with catenins, including β-catenin, and provides a link to the actin cytoskeleton.13, 21, 22 Together, the E-cadherin/β-catenin complex functions as intercellular junctions which promote cell adhesion.21, 22 E-cadherin expression is thought to affect, or be affected by Wnt pathway signalling.24 The loss of E-cadherin expression together with the upregulation of Vimentin expression is known to be a marker of EMT changes in epithelial cells.25
Vimentin is a type III intermediate filament protein that is normally found in mesenchymal cells, though it is sometimes expressed in migratory epithelial cells, such as during embryogenesis and wound healing.21 Its expression in oral epithelial cells has been pathologically associated with tumour invasion and metastasis.21, 26, 27 The Vimentin promoter is believed to be a target of the β-catenin-TCF/LEF-1 transcription factor, which suggests its involvement in invasion or migration of epithelial cells.28
The Adenomatous Polyposis Coli (APC) protein prevents aberrant activity of the Wnt-β-catenin signalling system by forming a β-catenin degradation complex with Axin and Gsk-3.21, 22, 23, 29 Aberrant expression or Wnt pathway inhibition of the APC/Axin/Gsk-3 complex allows β-catenin stabilisation and accumulation of the free protein in the cytoplasm of epithelial cells. This also allows for the migration of β-catenin into the nucleus, where it can form a functional transcription factor with TCF/LEF-1 and mediate the activation of certain oncogenes, such as that for Vimentin.21, 22, 23 APC is frequently mutated in colon cancer; however, there have been very few reports of its involvement in OSCC.29
Though Wnt pathway dysregulation is present in OSCC, there is limited knowledge on whether the Wnt pathway contributes to the transformation of potentially malignant oral lesions to malignancy. In this study, we investigated the expression of E-cadherin, β-catenin, APC and Vimentin in normal oral tissue, dysplastic tissue and OSCC, and examined the potential role of EMT in OSCC development in vitro.
Section snippets
Patient selection and tissue specimens
A total of 100 formalin-fixed paraffin-embedded (FFPE) oral biopsy specimens (18 normal, 27 mild dysplasia, 8 moderate-severe dysplasia, 47 OSCC) from patients diagnosed and followed between 2000 and 2008 were obtained from archival material at the University of Queensland Centre for Clinical Research, University of Queensland, and the Melbourne Dental School, University of Melbourne. The study population included men and women of all ages, and details of clinicopathological parameters for each
Immunohistochemical analysis
E-cadherin, β-catenin, APC and Vimentin expression was analysed by assessing immunoreactivity for each protein, and the immunoreactivity scores are presented in Table 1.
E-cadherin IHC
In all normal, dysplastic and cancerous tissues, E-cadherin was localised to the epithelial cell membranes, and was expressed in the lower, middle and upper thirds of the epithelium. Immunoreactivity for E-cadherin in normal tissue was high (mean IHC score = 9.176) (Fig. 1a). A decrease in E-cadherin expression was observed when
Discussion
This study used immunohistochemistry to quantify and analyse expression patterns of E-cadherin, β-catenin, APC and Vimentin in normal oral mucosa, oral epithelial dysplasia and OSCC in order to investigate the role of these molecules in oral carcinogenesis, and their ability to predict transformation in potentially malignant lesions. The pattern of expression for E-cadherin and Vimentin were further investigated by RT-PCR and with a novel organotypic culture model of neoplastic cell invasion.
A
Conflict of interest statement
None declared.
Acknowledgement
The authors thank Michael McCullough at the Melbourne Dental School for provision of tissue specimens for this study.
References (59)
- et al.
Applying the molecular biology and epigenetics of head and neck cancer in everyday clinical practice
Oral Oncol
(2009) - et al.
Dysregulated molecular networks in head and neck carcinogenesis
Oral Oncol
(2009) - et al.
Pitfalls and procedures in the histopathological diagnosis of oral and oropharyngeal squamous cell carcinoma and a review of the role of pathology in prognosis
Oral Oncol
(2009) Potentially malignant disorders of the oral and oropharyngeal mucosa; terminology, classification and present concepts of management
Oral Oncol
(2009)- et al.
Intraexaminer and interexaminer reliability in the diagnosis of oral epithelial dysplasia
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
(1995) - et al.
Why oral histopathology suffers inter-observer variability on grading oral epithelial dysplasia: an attempt to understand the sources of variation
Oral Oncol
(2007) - et al.
Long-term treatment outcome of oral premalignant lesions
Oral Oncol
(2006) - et al.
Head and neck cancer: molecular carcinogenesis
Ann Oncol
(2005) - et al.
Hypoxia-inducible factor-1 alpha, in association with TWIST2 and SNIP1, is a critical prognostic factor in patients with tongue squamous cell carcinoma
Oral Oncol
(2011) - et al.
Upregulation of vimentin and aberrant expression of E-cadherin/beta-catenin complex in oral squamous cell carcinomas: correlation with the clinicopathological features and patient outcome
Mod Pathol
(2010)
Expression of adenomatous polyposis coli (APC) in tumorigenesis of human oral squamous cell carcinoma
Oral Oncol
Expression of E-cadherin and vimentin correlates with metastasis formation in head and neck squamous cell carcinoma patients
Radiother Oncol
Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays
Lab Invest
Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays
Am J Pathol
Product differentiation by analysis of DNA melting curves during the polymerase chain reaction
Anal Biochem
Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
Anal Biochem
Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model
Br J Plast Surg
Significance of tumor satellite variables in reflecting the epithelial–mesenchymal transition of tongue cancer
Oral Oncol
Aberrant expression of beta- and gamma-catenin is an independent prognostic marker in oral squamous cell carcinoma
Int J Oral Maxillofac Surg
Alterations of adenomatous polyposis Coli (APC) gene in oral squamous cell carcinoma
Int J Oral Maxillofac Surg
Disease mechanism and biomarkers of oral squamous cell carcinoma
Curr Opin Oncol
Advances in the diagnosis of oral premalignant and malignant lesions
J Can Dent Assoc
Longitudinal studies in oral leukoplakias
J Oral Pathol
Malignant transformation and natural history of oral leukoplakia in 57,518 industrial workers of Gujarat, India
Cancer
Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis
Cancer Res
Cancer-associated fibroblasts and epithelial-mesenchymal transition in metastatic oral tongue squamous cell carcinoma
Int J Cancer
Overexpression of SIP1 and downregulation of E-cadherin predict delayed neck metastasis in stage I/II oral tongue squamous cell carcinoma after partial glossectomy
Ann Surg Oncol
Involvement of the Wnt-beta-catenin pathway in invasion and migration of oral squamous carcinoma cells
Int J Oncol
Activation of WNT family expression and signaling in squamous cell carcinomas of the oral cavity
J Dent Res
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