The cadherin–catenin complex in laryngeal squamous cell carcinoma

β-Catenin contributes both to cell–cell adhesion and to the Wnt signaling pathway [21, 22]. The present study noted no alteration in β-catenin protein expression between LSCCs and non-tumoral tissues. However, differences were found in the cellular location of β-catenin expression: in 89% of glottic LSCCs, β-catenin was expressed only in the cell membrane, a percentage similar to that found for non-tumoral larynx tissue (73%), whereas in 86% of supraglottic LSCCs, β-catenin was expressed both in membrane and in cytoplasm. Similar findings have recently been reported by Goiliomus et al. [25] in a series of 97 LSCCs comprising 63 glottic and 34 supraglottic carcinomas. The cadherin–catenin complex is a group of membrane proteins that are important in cell–cell adhesion, tumor suppression, cell differentiation and cell migration. Some reports have suggested that downregulated expression of β-catenin might play a role in early and late tumor invasion and metastasis [23, 24]. Downregulation of β-catenin has been found to be closely related to advanced clinical disease and short survival in other head and neck carcinomas, including nasopharyngeral carcinoma [12]. These results suggest that β-catenin may in itself be a major factor for supraglottic LSCC tumor cell invasion and metastasis, and that differences in β-catenin expression between glottic and supraglottic carcinomas may be linked to differences in clinical behavior. Here, β-catenin protein levels were evaluated using inmunohistochemical staining, a semi-quantitative technique enabling assessment of proteins in paraffin-embedded tissues. Further research will evaluate β-catenin expression levels using the Western blot technique, with a view to measure possible alterations in β-catenin protein levels in fresh tissue.

β-Catenin is also involved in the Wnt canonical pathway as a transcriptional activator [21, 22]. The inhibition of glycogen synthase kinase (GSK-3β) and the APC protein ultimately results in the stabilization and nuclear translocation of cytoplastic β-catenin. Finally, nuclear β-catenin interacts with various transcription factors to cause proliferation and differentiation. But further research is required into the link between altered β-catenin levels and the development of supraglottic LSCC. Here, β-catenin was detected in membrane and cytoplasm, but not in the nucleus. Goiliomus et al. [25] in a study of 97 LSCCs, detected nuclear β-catenin in only one sample, perhaps due to differences in tissue processing or to the immunohistochemical staining method used. In the present study, failure to detect nuclear β-catenin suggests that the canonical Wnt pathway may be inactivated in this type of cancer. This cannot be categorically confirmed, since stabilized β-catenin was detected in cytoplasm. Further research is required to determine the possible role of cytoplasmic β-catenin retention in the pathogenesis of LSCC supreglottic and whether the Wnt pathway is activated by these retention, overexpression of its receptors or silencing of its suppressors, and also to investigate the possible activation of the non-canonical Wnt pathway that includes signaling through calcium flux, JNK and heterotrimeric G proteins.

Although none of the LSCC samples expressed Wnt pathway activators such as Wnt-1 and Wnt-5a, strong expression of the inhibitors WIF-1 and Dkk-1 was found in all cases. This would suggest that the Wnt pathway is inactive in this type of tumor. These results enable a preliminary approach to the study of the Wnt pathway in LSCC; future research will analyze the expression of different components of the Wnt pathway using quantitative techniques (Western-blot and real-time PCR), thus overcoming the limitations of immunohistochemical analysis and providing a more accurate view of the activation or inactivation of this signaling pathway.

E-Cadherin is essential for normal cell function, and is downregulated in processes such as wound healing, allowing epithelial cells to move and cover denuded tissue [26]. E-Cadherin, localized to the zonula adherens, complexes with catenins β and γ; β- and γ-catenins in turn bind to γ-catenin which attaches to actin filaments in the cell cytoskeleton [27]. E-Cadherin is thought to act as a tumor suppressor, since it suppresses invasion and metastasis [28]. Low expression of E-cadherin has been shown to predict poor outcome in oral cavity and laryngeal carcinomas [29, 30]. In the present study, strong E-cadherin expression was detected in 82% of non-tumoral tissues and only in 62.5% of glottic and supraglottic LSCCs. In contrast, weak E-cadherin expression was detected in 57% of supraglottic LSCC tissues, while only 22% of glottic LSCCs exhibited weak expression; the remaining 78% displayed strong E-cadherin expression, a percentage similar to that found in non-tumoral tissues. Downregulation of E-cadherin expression in supraglottic LSCC tissues may indicate a greater capacity for invasion and metastasis, and thus account for the distinct clinical behavior of these tumors. Indeed, a positive correlation was observed between E-cadherin expression levels and the presence of lymph node metastasis in supraglottic LSCC tissues [31, 32]. In this respect, several studies have shown that diminished E-cadherin expression is a good predictor of concurrent lymph node metastasis in laryngeal carcinoma and head and neck squamous cell cancer [13, 33‐36]. Kurtz et al. [13] noted a correlation between reduced E-cadherin expression and decreased survival rates and vascular invasion in LSCC patients. These findings may be due to mutations of the E-CADHERIN gene [37] or to loss of heterozygosity of that gene [7]. In some cancer cells, aberrant gene promoter methylation of E-cadherin may be responsible for the decreased expression of E-cadherin [33, 34]; future research will investigate E-cadherin expression using quantitative RT-PCR, in order to obtain a clearer view of potential differences between sites, and thus focus on possible epigenetic changes due to promoter hypermethylation in LSCC tissues.

Finally, no association was observed between E-cadherin protein levels and β-catenin localization, although low E-cadherin levels were more common among LSCCs exhibiting cytoplasmic β-catenin (40 vs. 23.5%). Si et al. [5] observed a significant relationship between abnormal E-cadherin and β-catenin expression and clinical stage and lymph-node metastasis, although they do not distinguish between glottic and supraglottic LSCCs. Other studies of head and neck squamous cell carcinomas report loss of membranous E-cadherin and β-catenin expression and increased cytoplasmic expression, irrespective of the primary carcinoma or nodal carcinoma involved [38]. Impairment of cell–cell adhesion is an essential step in the progression from localized malignancy to stromal and vascular invasion and metastatic disease. This impairment is achieved through a variety of mechanisms involving the cadherin/catenin complex [39].

The results obtained here suggest an enhancement of the potential ability of cancer cells to disperse, a preliminary step in local invasion. A better knowledge of the regulation of the cadherin–catenin system is required for the prevention or treatment of laryngeal carcinoma.

In conclusion, reduced membrane expression of E-cadherin and cytoplasmic retention of β-catenin in supraglottic LSCC seems to be related with more aggressive biological behavior which has been described in clinical studies. Further research is required to clarify the involvement of β-catenin in the mechanism associated with malignant transformation in laryngeal tissues.