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.