4.Discussion
The pathogenesis of laryngeal cancer (LC) which is an important cause of morbidity and mortality, has not been fully elucidated [10]. Despite the developments observed in current treatment methods, the desired satisfactory results have not been obtained in the treatment of LC (11). Understanding the molecular mechanisms involved in the development of LC will be conceivably an important step in developing effective treatment methods [11]. Metastasis is the most important life-threatening risk factor for cancer patients and accounts for more than 90% of cancer-related deaths [12]. Studies have shown that in carcinomas, mesenchymal features induced by EMT play a role in many steps of the invasion- metastasis cascade [13,14]. EMT, which is characterized by the loss of intercellular junctions is defined as the loss of epithelial phenotype in cells (such as E-cadherin, alpha-catenin, beta-catenin) and the acquisition of mesenchymal phenotype (such as N-cadherin, vimentin, αSMA) [2,5]. It is thought that with the activation of EMT programs in tumor cells, tumor cells acquire many features of stem cells and climbing the steps leading to metastasis of the tumor is facilitated [15].
E‑cadherin is a glycoprotein located in the membrane of normal epithelial cells. Intercellular adhesion is provided with the protein complex formed by binding the cytoplasmic domains of E-cadherin with beta-catenin [2]. Beta-catenins are bridges that mediate the binding of E-cadherin to the actin cytoskeleton. Deletions or mutations in the cytoplasmic tail of the E-cadherin result in the breakdown of cell-cell adhesion complexes at the location of the beta-catenin binding site [16]. The released cytoplasmic beta-catenin then displaces the nucleus and interacts with target genes that play a role in cell proliferation [16]. As stated in many stuides, downregulation / loss of E-cadherin and beta-catenin in the cell membrane and expression of nuclear beta-catenin are frequently detected in most types of cancer which suggests that E-cadherin and beta-catenin may be key molecules in tumor development and progression [2, 10].
In the literature, different results have been reported on the ways of evaluating the expressions of E-cadherin and beta-catenin especially in LSCCs and the importance of staining patterns. In their LSCC series of 82 cases, Greco et al reported that in univariate analysis, cytoplasmic and membranous E-cadherin overexpression was associated with shorter OS in advanced stage patients (T3-T4), and cytoplasmic E-cadherin positivity was associated with poor disease-specific survival (DSS) [7]. The authors also stated that in the multivariate analysis, only cytoplasmic E-cadherin overexpression continued to be a negative prognostic factor for OS [7].
On the other hand, in a LC series of 289 cases, Psyri et al. reported that in univariate analysis, disease –free survival (DFS) was longer in cytoplasmic and membranous E-cadherin expressers compared to cytoplasmic E-cadherin expressers [16]. According to the univariate analysis, it is also said that OS is longer in those who express cytoplasmic E-cadherin and beta-catenin than those expressing only beta-catenin. However, it has been reported that these findings have lost their importance in multivariate analysis [16]. Greco et al. reported that both cytoplasmic and membranous staining with beta-catenin can be demonstrated in univariate analysis, and decreased cytoplasmic and membranous staining is associated with higher histological grade [7]. Cytoplasmic overexpression of beta-catenin has been determined as a positive prognostic factor for DSS. In multivariate analysis, cytoplasmic beta-catenin overexpression has been reported to be associated with prolonged DFS [7].
Zhu et al. evaluated E-cadherin staining in their LSCC series of 76 cases, and determined lack of membranous staining in less than 90% of tumor cells [2]. They reported that membranous staining in tumor tissue decreased and diffuse cytoplasmic staining was observed. For beta-catenin, positive cytoplasmic and nuclear staining, but negative membranous staining was reported. They indicated that increased nuclear and cytoplasmic beta-catenin positivity with decreased membranous E-cadherin staining was associated with the presence of LNM, T4 tumor or poorly differentiated tumors [2]. In univariate analysis, negative expression of E-cadherin and positive expression of beta-catenin were associated with a decrease in OS, whereas in multivariate analysis, only beta-catenin continued to be an important factor in OS [2]. In a LSCC series of 37 cases realized by Rocco et al, only membranous staining of E-cadherin was considered positive, and they evaluated the cases with percentages [17]. In their study, they couldn’t detect a significant relationship between E-cadherin and pT, pN, and tumor grade, however, they associated decreased expression of E-cadherin with disease recurrence and shorter DFS [17].
Studies performed have shown that there is no widely accepted evaluation criterion for E-cadherin and beta-catenin in LSCCs. Studies have demonstrated that the staining patterns of markers may be significant in terms of patient survival, but in multivariate analyzes conducted especially for E-cadherin, markers have lost their significance.
In our study, expressions of tumoral E-cadherin and beta-catenin were evaluated by IRS scoring. There was no significant relationship between downregulation of E-cadherin, beta-catenin and tumor stage, LNM, and OS. Membranous, cytoplasmic and membranous, and only cytoplasmic staining with E-cadherin was seen in 82%, 16%, and 2% of the cases, respectively. OS time was longer in cases with membranous staining, and the lowest OS times were observed in cases with only cytoplasmic staining. However, the findings were not statistically significant which was thought to be due to the limited number of cases with only cytoplasmic staining.
Membranous staining with beta- catenin was observed in 29, cytoplasmic, and membranous staining in 66 and only cytoplasmic staining 5 cases. Besides, the survival was longer in cases with cytoplasmic and membranous staining, and the shortest survival was observed in those with only cytoplasmic staining. However, the results were not statistically significant.
N-cadherin, another member of the cadherin family, is expressed mainly in mesenchymal cells and nerve tissue. N-cadherin stimulates cell motility and migration by interacting with epidermal growth factor receptor-1 and members of the fibroblast growth factor receptor family [18]. N-cadherin is also associated with the MAPK / ERK signaling pathway, which plays a role in tumorigenesis [18]. Unlike E-cadherin, upregulation of N-cadherin increases the migration and invasion capacity of tumor cells [18]. N-cadherin, which is an indicator of the mesenchymal phenotype, is not normally found in epithelial cells. Studies have reported that abnormal N-cadherin expression in epithelial cells is associated with malignancy and tumor progression [18]. It is also stated that increased expression of N-cadherin in the cell membrane or cytoplasm is associated with the progression and metastasis of solid tumors [9]. Zhu et al. evaluated the expression of N-cadherin in LSCCs, and reported that the cytoplasmic N-cadherin in tumor cells was associated with the tumor T stage [2]. However, any significant relationship was not observed with LNM. An association between N-cadherin expression and lower survival rates was reported in univariate analysis which lost its significance in multivariate analysis [2]. In the LSCC series of Greco et al., cytoplasmic and membranous N-cadherin expression in the tumor was taken into consideration, and N-cadherin expression was associated with the histological tumor grade but not with DSS and OS [7]. On the other hand, Rocco et al. considered only membranous staining in their LSCC series of 37 cases, and reported that in most of their cases either N-cadherin staining was not observed or only low levels of immunoreactivity was seen [17].They also reported lack of any relationship between N-cadherin expression and pT, pN, and tumor grade, however, they noted a significant relationship with N-cadherin expression and disease recurrence [17]. In an oral SCC series consisting of 94 cases, Domenico et al. demonstrated cytoplasmic staining with N-cadherin in dysplastic cells and revealed different staining patterns in carcinomatous cells according to the invasion pattern [8]. In their study, they had also observed cytoplasmic and nuclear staining in the droplet invasion pattern, and membranous and cytoplasmic staining in single cell invasions [8]. In our series, similar to the study of Domenico et al, N-cadherin expression was observed in 32 cases, and also nuclear expression in tumor cells was noted. Nine (52.9%) of 17 patients with nuclear N-cadherin expression and 5 (36.7%) of 14 patients with cytoplasmic staining lost their lives. One case with cytoplasmic and membranous staining survived. Considering all cases, there was no significant relationship neither between N-cadherin expression and OS, nor between disease stage and LNM.
EMT is tightly regulated directly or indirectly by transcription factors (TFs) such as Zeb1, Zeb2, Snail, KLF8 and Twist (5,6,19). In addition, there are complex signal networks that regulate TF, such as transforming growth factor- beta [5]. Differences between the potency of these factors have been shown. Besides, as indicated in some studies, the same TF may induce different cellular responses in different carcinoma types [20-23]. ZEB1 is one of the critical members of the zinc finger E-box binding transcription family. Abnormal expression of Zeb 1 has been demonstrated in various tumors including pancreas, lung, liver, and breast carcinomas [19]. Zeb1 is the key factor that regulates EMT in invasive tumor cells and enables tumor cells to acquire a proinvasive and stem cell-like phenotypic characteristics [6]. It is also reported that ZEB1 also facilitates epigenetic silencing of E-cadherin [6].
Wan et al. published a meta-analysis of the results of EMT- inducing transcription factors (EMT-TF) in 2257 cases with HNSCC compiled from 22 articles [24]. Cases with oral SCC, tonsil SCC, LSCC and nasopharyngeal SCC were included in the analysis. As a result of the meta-analysis, it was found that EMT-TF (Zeb1, SNAI1, SNAI2, twist1) overexpression was associated with poor OS. They reported that similar results were observed in LSCCs when tumor subgroups were evaluated individually. In addition, EMT-TF was seen to be associated with DFS, T stage, LNM, distant metastasis, tumor differentiation, and disease recurrence [23]. In the LSCC series of Zhu et al. Zeb2 expression was reported to be associated with LNM, tumor T stage and differentiation [2]. Zeb2 expression was found to be an independent risk factor for OS in univariate and multivariate analysis [2]. Contrarily, in the LSCC series of Rocco et al, any significant relationship between Zeb1 expression in the tumor and pT, pN, tumor grade, disease recurrence and DSS could not be detected [17]. Data on the prognostic significance of Zeb1 expression in the tumor microenvironment, especially in breast carcinomas have been indicated in the literature. It has been shown that Zeb1 in basal-like breast cancers regulates the levels of various inflammatory cytokines such as IL6 / 8 and contributes to the formation of the tumor microenvironment. Increased stromal Zeb1 expression has been associated with extracellular matrix remodeling, immune cell infiltration and angiogenesis [19]. In our series, a statistically significant relationship was observed between tumoral Zeb1 expression and LNM, advanced stage disease and poor OS. In addition, as a remarkable finding, Zeb1 expression observed in the surrounding stroma had a significant relationship with OS. Alpha- SMA is the major component of contractile microflaments used to detect mesenchymal cells, especially myofibroblasts. During the process of EMT, TGF- β stimulates αSMA expression in transitioning epithelial cells, which has been reported to be associated with increased tumor invasion and decreased survival [24]. Benzour et al. evaluated alpha- and gamma- SMA expressions in hepatocellular carcinomas, and reported gamma- SMA was expressed only in tumor cells. They also observed αSMA positivity in only stromal component [25]. Based on this observation, they stated that only gamma-SMA may be expressed in hepatic progenitor cells [25].
In our LSCC series, αSMA expression was not detected in tumor cells. However, a positive reaction with αSMA was detected in the stroma surrounding the tumor. As a remarkable finding, these results were parallel to the data reported by Benzour et al. about αSMA which made us think that, similar to the theories of the researchers, LSCC progenitor cells may also express different SMA subtypes instead of αSMA.
It is known that factors secreted from the tumor, such as TGF- β, activate fibroblasts and these factors have different genetic characteristics from normal fibroblasts [26]. It has been stated that these cancer-related fibroblasts can be found in the ”network pattern” in the whole tumor stroma or in the ”spindle pattern” around the tumor islands [27]. In our series, in the group with lower density (<10%) of αSMA -positive fibroblasts consistent with the spindle pattern around the tumor islands, OS times were prolonged than the other two groups, without any statistically significant intergroup difference.