Odontogenic keratocysts (OKCs) are developmental
cysts that have been reclassified according World Health Organization
(WHO), to keratocystic odontogenic tumours (KCOTs), a term that better
reflects their neoplastic nature. The aim of present study is to
evaluate the induction of stress of the endoplasmic reticulum and
execution of the resulting unfolded protein response in keratinocystic
odontogenic tumours.
Material and Methods
We analyzed by
immunohistochemistry the expression of the chaperones BiP/GRP78 and calnexin in
24 cases of KCOTs. As controls, we have used 9 cases of periapical or radicular
cysts (PACs) and 5 cases of Fibromas (FBs). The PACs and the FBs were included
in the analysis, as PACs are the most common type of inflammatory odontogenic
cysts of and FBs, as lesions of the connective tissue with unaffected
epithelium.
Results
Analysis revealed a strong association between
both BiP/GRP78 and calnexin expression and KCOTs: 18 out of 24 (75%) KCOTs
expressed BiP/GRP78 as opposed to 1 out of 9 (13%) PACs, and none of 5 FBs
evaluated (P < 0.001, x2-test). Calnexin was expressed in 11 out of
24 KCOTs (46%) but only one out of 9 (13%) PACs, and none of the 5 FBs analyzed
(P < 0.001, x2-test).
Conclusions
Study results imply
that induction of endoplasmic reticulum stress maybe of diagnostic value in
keratocystic odontogenic tumours characterization. In addition to recent
findings suggesting that endoplasmic reticulum stress plays a causative role in
keratinization of epithelia, pharmacological interference with the execution of
the unfolded protein response should be considered for the management of
keratocystic odontogenic tumours.
The formation of cysts in the jaws possesses
unique features regarding their pathology and implicates the odontogenic
tooth-forming apparatus [1]. In most classification schemes odontogenic
cysts are divided in two major categories, namely the developmental
cysts and the cysts that are initiated in areas of acute or chronic
inflammation, with odontogenic keratocysts (OKC) and periapical or
radicular cysts (PACs) as the most common types, respectively [1,2]. The
World Health Organization (WHO) reclassified OKC in its 2005 edition of
histological classification of odontogenic tumours and according to this
reclassification, the OKC is now considered as a KCOT. It is defined as
“a benign uni- or multicystic, intraosseous tumour of odontogenic
origin, with a characteristic lining of parakeratinized stratified
squamous epithelium that bears the potential for aggressive,
infiltrative behaviour”, a term that better reflects its neoplastic
nature [3]. Several factors form the basis of this decision: a. the
clinical behaviour of the lesion, since KCOT is locally destructive and
highly recurrent, b. the histopathologic characteristics, considering
that the basal layer of the KCOT budding into connective tissue, in
addition to the mitotic figures that are frequently found in the
suprabasal layers [1,3-5], and finally c. genetic alterations, are the
most important parameters [6]. KCOTs comprise approximately 11% of all
cysts of the jaws [7]. They occur most commonly in the mandible,
especially in the posterior body and ramus regions [1,2,7]. It may be
solitary or multiple and the latter is usually characterizing the
inherited nevoid basal cell carcinoma syndrome “NBCCS”. KCOTs have a
high recurrence rate, ranging between 25% and 60% while when associated
with nevoid basal cell carcinoma syndrome “NBCCS” or Gorlin-Goltz
syndrome, the recurrence rate is about 82% [8,9]. So far, there is only
limited evidence regarding the pathogenesis of the developmental cystic neoplasms
of the jaws and for this reason they are considering of unknown
aetiology.
The endoplasmic reticulum (ER) is an
organelle with a major role in the synthesis of lipid and proteins and
leads many cellular processes such as organogenesis, transcriptional
activity, stress responses, and apoptosis [10-14]. ER is responsible for
the proper folding of the newly synthesized proteins that is facilitated
with the assistance of various ER chaperones [10,11]. Unfolded or
malfolded proteins are disposed by mechanisms implicating ER-associated
protein degradation (ERAD). When the amount of unfolded protein exceeds
the folding capacity of the ER, human cells activate a homeostatic
defence mechanism designated as the UPR that follows ER stress [11-16].
Among the various consequences of UPR is
also the upregulation of BiP/GRP78 and of other chaperones that is
considered diagnostic for the induction of ER stress in a given tissue
[16-18]. BiP/GRP78 binds to the hydrophobic region of unfolded proteins
via a substrate-binding domain and facilitates folding through
conformational change evoked by the hydrolysis of ATP by the ATPase
domain [19,20].
Calnexin and calreticulin are ER chaperones
specifically involved in the folding of glycoproteins. High mannose type
oligosaccharide is attached en
bloc to most proteins
synthesized in the ER, and then trimmed sequentially [21-23]. When two
glucose residues are trimmed by glucosidase I or II and the protein
contains only one glucose residue, calnexin and calreticulin bind and
fold the client protein [21,22]. When the last glucose residue is
trimmed by glucosidase II, the client is released from calnexin and
calreticulin, and binds to UDP-glucose-glycoprotein glucosyltransferase
[10,21]. If the protein is folded properly, it is released from the
enzyme and transported to the Golgi apparatus. If it is not folded
appropriately, UDP-glucose-glycoprotein glucosyltransferase attaches one
glucose residue and returns it to calnexin and calreticulin. This
folding process is called the calnexin cycle. Calnexin and calreticulin
share a similar molecular structure and function, although they are
transmembrane and luminal proteins, respectively [10,11,21,22].
Considering the neoplastic nature of KCOTs,
in combination with their poorly defined aetiology we explored if ER
stress is involved in disease development. Specifically, we evaluated
the expression of the chaperones, BiP/GRP78 and calnexin in a panel of
KCOTs as compared to PACs and FBs. Both of these markers is considered
to accurately reflect the induction of ER stress which has been
associated with neoplastic development [10,11].
MATERIAL AND METHODS
Patients and samples
Paraffin-embedded tissue specimens of KCOTs
(24 cases), PACs (9 cases) and Fibromas (5 cases) were randomly selected
from the archives of the Department of Oral Pathology, of the National
and Kapodistrian University of Athens, Dental School spanning the years
2006 - 2011 and were analyzed by immunohistochemistry. We have analyzed
the expression of chaperones BiP/GRP78 and calnexin in a panel of 24
KCOTs and 9 PACs. The latter represent the most
common type of inflammatory odontogenic cysts. In addition we have also
included in our analysis 5 FBs as controls since they represent lesions
of the connective tissue devoid of pathological findings in the
epithelium.
Details of ethics approval
No ethical issues are related to this study
since only paraffin-embedded archival material has been used and no data
related to the patients’ clinical records have been disclosed. Therefore
the study did not require review by the Institutional Review Board of
the University of Athens.
Immunohistochemistry
Immunohistochemistry was carried out in
formalin fixed, paraffin embedded tissue specimens. The antibodies used
were monoclonal rabbit anti-BiP (C50B12), by Cell Signaling Technology;
1:100 and monoclonal mouse anti-calnexin (sc-46669), by Santa Cruiz
Biotechnology, Santa Cruz, CA, USA; 1:75. Immunostaining was performed
by using the Superpicture Polymer (Dab) Kit (Novocastra), following the
manufacturer’s instructions. Before evaluation, a weak counterstaining
with hematoxylin was performed in all immunostained specimens. Specimens
were evaluated blindly from two authors of the study (I.C., Pathologist
and S.M., MSc in Oral Medicine and Pathology). The positive
immunohistochemical staining, was graded semiquantitatively by using a
5-tier scoring system and classified according to the intensity of the
labelling as: negative (-), marginal (+/-), mild (+), moderate (++) and
intense (+++).
Statistical analysis
Chi-square test was used to statistical
evaluate the results.
RESULTS
In all specimens analyzed and for both
antigens, immunopositivity was relatively homogenous among cells and
varied only in terms of intensity. As shown in
Table 1, BiP/GRP78
immunopositivity was detected in 18 out of 24 (75%) KCOTs. Positivity
was marginal (+/-) in one sample, mild (+) in 10, moderate (++) in 6 and
very intense (+++) in one specimen. With the exception of 3 specimens
exhibiting moderate or very intense immunopositivity and at which
BiP/GRP78 expression was primarily localized in the upper layers of the
epithelium (Figure 1A), in all other cases BiP/GRP78 immunopositivity
spanned full thickness of the epithelium (Figure 1B). As opposed to
KCOTs, PACs exhibited BiP/GRP78 immunopositivity in only 1 out of 9
(13%) cases (Figure 2A) while all five FBs were negative for BiP/GRP78
expression (Figure 2B) suggesting that the overexpression of BiP/GRP78
in KCOTs was statistically significant (P < 0.001, x2-test).
Immunohistochemical
expression of BiP/GRP78 in keratocystic odontogenic tumours
(x10). (A) Brown colour (DAB) indicates staining in the upper
layers of the epithelium or (B) in the full thickness with
stronger intensity in the upper layers. Weak counterstaining
with haematoxylin was performed in all samples following
immunostaining (original magnification x20).
The
microphotograph shows negative staining for BiP/GRP78 in the
epithelium of periapical cyst (A) and negative staining for
calnexin in the epithelium of a fibroma (B). Weak
counterstaining with haematoxylin was performed in all samples
following immunostaining (original magnification x20).
The same panel of specimens was also
analyzed for the expression of calnexin. Our results that are summarized
in Table 1 show that calnexin expression is significantly higher (P <
0.001, x2-test) in KCOTs since it was expressed in 11 out of
24 KCOTs (46%) but only one out of 9 (13%) PACs and none of the 5 FBs
analyzed. Immunopositivity in KCOTs ranged from marginal (+/-) in 5,
mild (+) in 5 and moderate (++) in one specimen while the single PAC
that was positive for calnexin expression exhibited marginal
immunopositivity.
Expression levels and localization of BiP/GRP78 and calnexin in KCOTs, PACs and FBs
SpecimenNo.
Age
BiP/GRP78 expression
intensity
BiP/GRP78expression
localization
Calnexinexpression
intensity
Calnexinexpression
localization
KCOTs
1
47
-
+
FT
2
74
+
FT
±
FT
3
58
+
FT
±
FT
4
40
-
-
5
65
++
UL
-
6
62
-
-
7
60
-
-
8
43
++
FT/UL
+
UL
9
24
+++
UL
+
UL
10
33
++
FT/UL
+
UL
11
51
++
FT/UL
++
FT/UL
12
70
-
-
13
66
-
-
14
44
+
FT
-
15
40
±
FT
±
FT
16
46
+
FT
-
17
47
+
FT
+
FT
18
8
++
FT/UL
+
FT
19
28
++
FT
++
FT
20
57
+
FT
±
FT
21
48
+
FT
-
22
45
+
UL
-
23
75
+
FT/UL
-
24
32
+
FT
-
PACs
1
57
+
FT
±
FT
2
45
-
-
3
32
-
-
4
30
-
-
5
53
-
-
6
40
-
-
7
52
-
-
8
40
-
-
9
45
-
-
FBs
1
10
-
-
2
56
-
-
3
67
-
-
4
50
-
-
5
34
-
-
KCOTs = keratocystic odontogenic tumors; PACs = periapical or radicular cysts; FBs = fibromas; FT = full thickness of the epithelium; UL = upper layers; FT/UL = full thickness with stronger intensity in the upper layers.
In all but 3 cases that exhibited mild
immunopositivity and was localized in the upper layers of the epithelium
(Figure 3A), calnexin expression was spanned the full thickness of the
epithelium (Figure 3B).
Immunohistochemical
expression of calnexin in keratocystic odontogenic tumours
(x10). (A) Brown colour (DAB) indicates staining in the upper
layers of the epithelium or (B) in the full thickness. Weak
counterstaining with haematoxylin was performed in all samples
following immunostaining (original magnification x20).
Considering that calnexin and BiP/GRP78 are
both indicators of ER stress we asked if their expression is correlated
in the same specimens. Indeed, out of the 38 specimens subjected to our
analyses, 29 were either positive or negative for both antigens while
only 9 exhibited expression for either BiP/GRP78 or calnexin. This
observation confirms that BiP/GRP78 and calnexin are co-expressed (P <
0.001, x2-test).
DISCUSSION
In order to better understand the pathogenic
mechanisms that underline the development of KCOTs we have hypothesized
that ER stress and the resulting UPR may be associated with their onset.
In order to test this hypothesis a bank of KCOTs specimens were analyzed
by immunohistochemistry for the expression of BiP/GRP78 and calnexin,
two widely used chaperones that are considered diagnostic for cells
undergoing ER stress [19-21,23]. The expression of BiP/GRP78 and
calnexin in the KCOTs were compared to that in PACs that are cysts with
distinct pathologic features and pathogenetic mechanism from that of
KCOTs as well as in FBs at which the cell type affected does not involve
the epithelium but rather the stroma. Our results show a strong
overexpression of the ER stress markers BiP/GRP78 and calnexin in the
KCOTs but not in the PACs and the FBs. Furthermore, these two chaperones
were frequently co-expressed in the same specimens, an observation that
implies that the overexpression of BiP/GRP78 and calnexin was not
coincidental but rather indicative for ER stress induction and execution
of the UPR [23].
A distinct diagnostic feature of the KCOTs
is the keratinization of the epithelium [1,3,4]. It is conceivable that
this feature of the KCOTs, namely the misexpression of keratin, is
causatively associated with ER stress. For reasons not yet well
understood commitment to this keratinocyte differentiation program, may
induce ER stress and activate the UPR [24,25]. Whether this is due to
the ectopic overexpression of a specific protein such as keratin(s) or
it is related to the perturbation of tissue homeostasis due to aberrant
differentiation remains unclear. However, since neither PACs nor FBs
display evidence of ER stress it is likely that the latter is linked to
KCOTs’ pathogenesis. Consistently with this notion, recent findings
showed that in the skin, at which keratinization represents a normal and
physiological process, ER stress-related chaperones BiP/GRP78 and HRD1
was elevated in cells of normal human epidermis that contain
keratinocytes undergoing differentiation [24]. These findings suggest
that keratinization, either in its physiological context such as in the
skin or in abnormal context such as in the KCOTs, are associated with ER
stress induction. Furthermore, in the same study it has been
demonstrated that pharmacological interference with the execution of the
UPR affected the differentiation of keratinocytes in
vitro, providing further clues regarding the causative link between
ER stress and keratinocyte differentiation [24]. Our results are
consistent with these findings and suggest that ER stress is induced in
the odontogenic epithelium when cells are committed to the keratinocyte
differentiation program, during KCOT development. This is also supported
by the observation that especially BiP/GRP78 and to some extent calnexin
immunopositivity was frequently more intense in the upper layers of the
epithelium at which keratinization is more prominent. Studies comparing
the involvement of ER stress to parakeratinized normal tissues,
hyperplastic and precancerous/dysplastic lesions of the oral epithelium
would bestow insightful understanding if BiP/GRP78 or calnexin may be
involved in parakeratinization. Indeed, a variety of diseases associated
with aberrant keratinization, such as hereditary keratoses, including
Darier’s disease, keratosis linearis with ichthyosis congenita and
keratoderma syndrome, erythrokeratoderma variabilis, and ichthyosis
follicularis with atrichia and photophobia syndrome, have been linked to
UPR [24]. It could be argued that keratinization and thus ER stress
induction is irrelevant to the oncogenic stimulus that induces KCOTs’
development. However, to the extent that keratinization reflects
aberrant differentiation of the cells and considering that neoplastic
development is indeed a disease of aberrant differentiation, ER stress
is causatively linked to the disease development.
The results of the present study may provide
clues regarding the pathogenesis of KCOTs. Which specific branches of
UPR are activated during KCOT development and whether its inhibition is
sufficient for the reversal of keratinization and regression of KCOTs
remains to be explored. Furthermore, it would be of particular interest
to investigate the involvement of epithelial to mesenchymal
transition-associated markers in the pathogenesis of KCOT and their
potential link to ER stress. In view of recent findings linking
mutations in PTCH1 gene to KCOTs’ development, and considering that the
PTCH1 gene’s product is a secreted ligand, an interesting hypothesis
would be that its expression induces ER stress [26,27]. This is also
supported by the ability of PTCH1 to act on post ER SMO and to modulate
its activity without affecting its overall expression levels [28].
These findings may have implications in the
clinical practice at two major directions. The detection of ER stress
and activation of the UPR may have diagnostic value for KCOT
characterization especially in cases at which diagnosis is not clear
especially in inflamed KCOTs [4] at which infiltration of the epithelium
by inflammatory cells masks their typical histopathologic
characteristics, raising issues of differential diagnosis. More
importantly, considering that ER stress influences keratinocyte
differentiation we may postulate that inhibition of UPR may have
adjuvant therapeutic value for the management of KCOTs, particularly
those with aggressive behaviour and often recurrences, at which surgery
is the treatment of choice [29]. Indeed, recently developed chemical
chaperones that can inhibit specific branches of the UPR may be
beneficial for the therapy of KCOTs’ patients.
CONCLUSIONS
This is the first demonstration of the
involvement of endoplasmic reticulum stress in the pathogenesis of
keratocystic odontogenic tumours. Understanding the precise mechanism by
which endoplasmic reticulum stress is involved the development of
keratocystic odontogenic tumours may find application in the diagnosis
and management of the disease.
ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS
This study was supported by a Research Grant
from the Empeirikion Foundation (granted to I.C.).
The authors declare that there are no
conflicts of interest.
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