Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and

NF-κΒ and IL-6 in oral precancer and cancer

Journal section: Oral Medicine and Pathology doi:10.4317/medoral.20570

Publication Types: Research http://dx.doi.org/doi:10.4317/medoral.20570

Expression of NF-κB and IL-6 in oral precancerous and cancerous

lesions: An immunohistochemical study

Georgios Kamperos 1, Nikolaos Nikitakis 2, Aikaterini Sfakianou 1, Dimitrios Avgoustidis 3, Alexandra

Sklavounou-Andrikopoulou 4

1
DDS, MSc. Postgraduate student, Department of Oral Medicine and Pathology, School of Dentistry, University of Athens,
Greece
2
MD, DDS, PhD. Associate Professor, Department of Oral Medicine and Pathology, School of Dentistry, University of Athens,
Greece
3
MD, DDS. Resident, Department of Oral and Maxillofacial Surgery, “Evaggelismos” Hospital, University of Athens, Greece
4
DDS, MSc, DrDent. Professor and Head, Department of Oral Medicine and Pathology, School of Dentistry, University of
Athens, Greece

Correspondence:
2-4, Alkmaionidon St,
16121, Athens, Greece,
[email protected] Please cite this article in press as: Kamperos G, Nikitakis N, Sfakianou A, Avgoustidis
D, Sklavounou-Andrikopoulou A. Expression of NF-κB and IL-6 in oral precancerous
and cancerous lesions: An immunohistochemical study. Med Oral Patol Oral Cir Bucal.
(2015), doi:10.4317/medoral.20570
Received: 27/12/2014
Accepted: 07/10/2015

Abstract
Background: The purpose of this study was to evaluate the immunohistochemical expression of NF-κB and
IL-6 in oral premalignant and malignant lesions and to investigate their possible correlation with the presence of
subepithelial inflammation.
Material and Methods: Thirty two oral premalignant lesions, clinically compatible with leukoplakia or
erythroplakia, were investigated. Microscopically, 11 of them showed hyperkeratosis and acanthosis (epithelial
hyperplasia) and 21 showed dysplasia of varying degrees. Nine cases of OSCC and four control cases of normal
oral mucosa were also included in the study. Immunohistochemical staining with NF-κB (p65) and IL-6 was
performed. IL-6 and nuclear NF-κB staining were assessed as positive or negative. For cytoplasmic localization
of NF-κB, a total score combining intensity and percentage of positive epithelial cells was additionally calculated.
The presence of inflammation was also recorded.
Results: Intensity and total scores for NF-κΒ cytoplasmic immunostaining showed a statistically significant
gradual increase from normal mucosa to OSCC (p=0.012 and p=0.026 respectively). Non-statistically significant
increased NF-κΒ nuclear localization was detected in dysplasias and OSCCs. Positive statistical correlation was
detected between the presence of inflammation and IL-6 expression (p=0.015). No correlation between NF-κΒ
and IL-6 was detected.
Conclusions: NF-κΒ is activated in the early stages of oral carcinogenesis. IL-6 may have an NF-κΒ-independent
role, possibly through regulation of the inflammatory response.

Key words: NF-κB, IL-6, immunohistochemistry, oral squamous cell carcinoma, oral precancerous lesion.
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

Introduction Currently, no studies have explored the immunohisto-

The study of potentially malignant disorders offers chemical correlation between NF-κB and IL-6 in oral
insight into the pathogenesis of oral cancer. Furthermore, precancerous lesions. The aim of this study was to eva-
continuing investigation of the molecular basis of oral luate the immunohistochemical expression of NF-κB
carcinogenesis may help to distinguish lesions with and IL-6 in oral precancerous lesions (leukoplakias and
increased malignant potential and to discover new erythroleukoplakias) and to investigate their possible
targets for molecular chemotherapy. It is widely known correlation with the presence of subepithelial inflam-
that carcinogenesis may occur through evolution of matory infiltrate.
precancerous lesions or conditions (1). A precancerous
lesion is a morphologically altered tissue in which oral Material and Methods
cancer is more likely to occur than in its apparently normal * Material
counterpart (2). The most common precancerous lesions Forty five biopsies were retrieved from the files of the
in oral mucosa are leukoplakias and erythroplakias (1). Department of Oral Medicine and Pathology, Dental
The role of inflammation is implicated in the pathogenesis School, National and Kapodistrian University of
of cancer. It is estimated that 15-20% of all cancers are Athens. Thirty two oral premalignant lesions, clinically
etiologically associated with inflammation, especially compatible with leukoplakia or erythroplakia, were
of chronic type (3). Tumor cells are known to produce investigated. Microscopically, 11 of them showed
inflammatory agents in order to alter the stroma and hyperkeratosis and acanthosis (epithelial hyperplasia)
facilitate invasion (4). Increased inflammatory cytokine and 21 showed dysplasia of varying degrees. Nine cases
levels are found in many cancer cell lines including of OSCC and four control cases of normal oral mucosa
oral squamous cell carcinoma (OSCC) (5). Laboratory were also included in the study. The histological
trials showed that even acute inflammation may lead to features were reviewed for confirmation of the diagnosis
increased invasion and metastases (3). Inflammation is according to the WΗΟ 2005 guidelines. The research
also related to the expression of several oncogenes, such protocol was IRB approved.
as RAS and MYC (6). The available epidemiological and clinical characteristics
The transcription factor Nuclear Factor-kappa B (NF- were collected. All precancerous lesions presented
κB) has been extensively studied in carcinogenesis (7). clinically as leukoplakia or erythroleukoplakia without
Mammalian NF-κB protein family is composed of five a history of previous interventions. The normal mucosa
members, RelA (p65), RelB, cRel (Rel), NF-κB1 (p50 cases were harvested from sites adjacent to reactive oral
and its precursor p105) and NF-κB2 (p52 and its precur- mucosal lesions in patients with no medical history of
sor p100) (8). These molecules form homodimeric and oral premalignancy or malignancy and no social history
heterodimeric complexes, the activity of which is regu- of smoking or excessive alcohol drinking.
lated by different pathways (8). The classical pathway of * Methods
NF-κB is the most commonly studied due to its major - Immunohistochemistry
role in the control of innate immunity and inflamma- Paraffin-embedded tissue sections of 5μm thickness
tion (8). It is modulated by the p65:p50 dimer, which is were deparaffinized (5 min in 60oC), immersed in
constantly inactive in the cytoplasm bound to inhibitory xylene and ethanol solutions, and heated for antigen
molecules (Inhibitors of κΒ–ΙκΒs) (8). Various condi- retrieval in 0.01 M citrate buffer for 15 min in a pressure
tions initiate the degradation of the IκΒs, so that the cooker inside a microwave oven. After incubation in
ΝF-κΒ complex is freed to enter the nucleus and acti- 3% hydrogen peroxide (LSAB+ kit Dako Corporation,
vate target genes (8). Specifically, NF-κΒ regulates the Carpinteria USA) to neutralize endogenous peroxidase
expression of proteins that have prominent roles in cell activity and treatment with Protein Free Serum
proliferation, survival, immune response and inflam- (LSAB+ kit Dako Corporation, Carpinteria USA) to
mation, such as Interleukin-6 (IL-6) (7,9). reduce nonspecific binding of primary antibody and
IL-6 is a pro-inflammatory cytokine with autocrine polymer, the sections were incubated with primary
and paracrine functions (10). It binds to a membrane antibodies overnight. The applied antibodies were
receptor, which is a heterodimer-composed of IL-6 monoclonal anti-p65 mouse antibody (sc-8008, Santa
special receptor protein (IL-6Ra/gp80 or CD126) and Cruz Biotechnology, Santa Cruz, California, USA)
gp130 (CD130) (10). IL-6 receptor (IL-6R) activates diluted at 1:200 and monoclonal anti-IL-6 mouse
the Janus kinase family (JAK1, JAK2, TYK2) via antibody (clone 6708.11, Sigma, Saint Louis, Missouri,
gp130 (10). In turn, these kinases activate the molecular USA) diluted at 1:25. Standard streptavidin–biotin–
pathways of Signal transducer and activator of peroxidase complex method was employed to bind to
transcription 3 (STAT3), Phosphoinositide 3-kinase the primary antibody along with multilink concentrated
(PI3K) and Mitogen-activated protein kinases (MAPKs) biotinylated anti-IgG as secondary antibody (LSAB+ kit
with various oncogenic consequences (4,10). Dako). Reaction products were visualized by staining
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

with 3,3V-diaminobenzidine reagent (LSAB+ kit acterized as positive. The absence of subepithelial in-
Dako). Sections were counterstained with hematoxylin. flammation resulted in the characterization of the spe-
As positive controls, the following were used: OSCC cific case as negative.
for p65 and papillary thyroid carcinoma for IL-6 with - Statistical analysis
corresponding known positivity. As a negative control, The baseline characteristics of patients were summarized
sections were treated with PBS, with the omission of the as mean and standard deviation (SD) for continuous
primary antibody. or ordinal data and as absolute (n) and relative (%)
- Immunohistochemical scoring frequency for categorical variables. The two tailed
Immunostains were reviewed by three independent Fisher’s exact test was performed in order to evaluate
evaluators (N.N., G.K. and A.S.). Immunohistochemical possible differences in the frequency distribution
reactivity for NF-κB (p65) was assessed separately of clinical and pathologic features of patients or the
in the cytoplasm and in the nucleus of the epithelial parameters of p65 immunohistochemical expression,
cells since it has been suggested that this molecule’s according to the histological classification of lesions.
localization may represent a transition between inactive The same test was also performed to evaluate possible
and active forms (8). NF-κB cytoplasmic staining associations between various parameters of IL-6
was graded according to the percentage of positive immunohistochemical expression and the presence of
epithelial cells (0, 0%; 1, < 20%; 2, 20–50%; 3, > 50%) inflammation. The Mantel-Hæszel method was then
(Fig 1A-C) and intensity of staining (0, no staining; 1, applied for the calculation of odds ratios (OR) and
weak; 2, moderate; 3, strong) (Fig 1D-F) compared to their respective 95% confidence intervals (95% CI) in

Fig. 1. Cytoplasmic NF-κΒ (p65) staining grades according to the percentage of positive epithelial cells [1, < 20% (A); 2, 20–50% (B); 3, > 50%
(C)] and the intensity of staining [1, weak (D); 2, moderate (E); 3, strong (F)]. Note the prominent positive nuclear staining in C. (A-F: 100X).

negative control tissues; a combined score (0-6) was 2 x 2 contingency tables, wherever possible. Pearson
also calculated. NF-κB nuclear staining in the epithelial Correlation Coefficients (r) were calculated between all
cells was characterized as positive or negative (due to dichotomous variables and inflammation presence, as
primarily focal localization, no further assessment well.
was performed). Immunohistochemical cytoplasmic Comparisons concerning the age of patients were based
reactivity for IL-6 was characterized as positive or on one-way ANOVA, while ordinal data were compared
negative. with the use of the Kruskal-Wallis one way analysis of
- Assessment of inflammation variance by ranks. Post – hoc pairwise comparisons
Inflammation was microscopically assessed on hema- after a significant Kruskal-Wallis test were carried out
toxylin and eosin stains. If subepithelial inflammatory with the application of Dunn’s test.
infiltrate (mild to severe) was noted, the case was char- Statistical analyses were performed using the SPSS
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

software application (version 21.0: SPSS, Chicago, IL, NF-κB (p65) cytoplasmic expression was detected in the
U.S.A.) with p<0.05 as the threshold of significance. epithelial cells of all cases; as expected, p65 expression
was also seen in the subjacent inflammatory cells. The
Results average percentage, intensity and total scores for NF-
- Cohort characteristics κB cytoplasmic expression in the epithelial cells are
Distribution of the demographic characteristics (sex, presented in table 2. No statistical significant difference
age and location) according to the histologic type is pre- was detected in the percentage score for p65 positive
sented in table 1. No statistically significant difference cells. On the other hand, average intensity scores showed
was observed in the distribution of these parameters be- a statistically significant gradual increase from normal
tween different histologic types. mucosa to OSCC (p=0.012) (Table 2) (Fig. 2). Post-
- Presence of inflammation hoc analysis revealed that the greatest difference was
Subepithelial inflammatory infiltrate (primarily of observed in the transition from epithelial hyperplasia
chronic nature) was observed in 0/4 cases of normal to dysplasia (p=0.011). Moreover, total scores for NF-
epithelium (0%), 9/11 cases of epithelial hyperplasia κB cytoplasmic expression also showed a similar (to
(81.8%), 17/21 cases of dysplasia (81%) and 9/9 cases intensity) statistically significant difference between
of OSCC (100%) (p=0.002) (Table 1). Compared with the histological categories (p=0.026) (Table 2).
normal mucosa, the presence of inflammation was higher NF-κΒ nuclear localization was detected in clusters
in epithelial hyperplasias (p=0.011, 95% confidence of cells in 0/4 (0%) of the normal mucosa cases, 2/11
interval [CI] lower border 2.95), dysplasias (p=0.006, (18.2%) of the epithelial hyperplasia cases, 8/21 (38.1%)
95% CI lower border 3.41) and OSCCs (p=0.001, 95% of the dysplasia cases and 3/9 (33.3%) of the OSCC
CI lower border 9.93). cases (Table 2) (Fig. 1C). Even though there seems to
- NF-κΒ immunohistochemical expression be an increased number of cases exhibiting nuclear

Table 1. Patients’ clinical characteristics and presence of inflammation per histologic type of lesions.
Histologic classification of lesion
Epithelial Epithelial
Normal OSCC Cohort
Characteristics Hyperplasia Dysplasia P*
No of cases 4 (8.9%) 11 (24.4%) 21 (46.7%) 9 (20.0%) 45 (100.0%)
Sex
Men 0 (0.0%) 5 (25.0%) 10 (50.0%) 5 (25.0%) 20 (44.4%)
0.325
Women 4 (16.0%) 6 (24.0%) 11 (44.0%) 4 (16.0%) 25 (55.6%)
Age (years)
Mean ± SD 51.0 ± 9.3 48.2 ± 9.1 55.6 ± 12.8 62.2 ± 10.9 0.059** 54.7 ± 12.0
Range 38 – 60 29 – 57 30 – 69 50 – 82 29 – 82
”55 3 (14.3%) 8 (61.5%) 7 (53.8%) 3 (23.1%) 21 (46.7%)
0.097
>55 1 (4.2%) 3 (14.3%) 14 (66.7%) 6 (28.6%) 24 (53.3%)
Location

Buccal mucosa 4 (30.8%) 3 (23.1%) 4 (30.8%) 2 (15.4%) reference 13 (28.9%)

Tongue &
floor of mouth 0 (0.0%) 2 (11.8%) 11 (64,7%) 4 (23.5%) 0.056 17 (37.8%)

Maxillary /
0 (0.0%) 3 (25.0%) 6 (50.0%) 3 (25.0%) 0.221 12 (26.7%)
mandibular
mucosa

Palate 0 (0.0%) 3 (100%) 0 (0.0%) 0 (0.0%) 0.118 3 (6.7%)

Inflammation
Yes 0 (0.0%) 9 (25.7%) 17 (48.6%) 9 (25.7%) 35 (77.8%)
0.002
No 4 (40%) 2 (20.0%) 4 (40.0%) 0 (0.0%) 10 (22.2%)
Abbreviation: SD, Standard deviation.
*Two-tailed Fisher’s exact test.
**One way analysis of variance.
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

Table 2. Comparison of p65 immunohistochemical parameters between different histologic types of lesions.
Histologic classification of lesion
Epithelial Epithelial
p65 IHC expression parameters Normal Hyperplasia Dysplasia OSCC
(n=4) (n=11) (n=21) (n=9) P*
Cellular sites of p65
Cytoplasm only 4 (12.5%) 9 (28.1%) 13 (40.6%) 6 (18.8%)
Nucleus only 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
0.448
Cytoplasm & Nucleus 0 (0.0%) 2 (15.4%) 8 (61.5%) 3 (23.1%)
Absent 0 (0.0%) 0 0.0% 0 (0.0%) 0 (0.0%)
Percentage of cells score of
cytoplasmic p65
Mean Score 2.8 2.7 2.9 2.8 0.752**
Range 2–3 1–3 2–3 2–3
Score ”2 1 (14.3%) 2 (28.6%) 2 (28.6%) 2 (28.6%)
0.580
Score 3 3 (7.9%) 9 (23.7%) 19 (50%) 7 (18.4%)
Intensity score of
cytoplasmic p65
Mean Score 1.5 1.7 2.4 2.4 0.012**
Range 1–2 1–3 1–3 1–3
Score ”2 4 (14.3%) 10 (35.7%) 10 (35.7%) 4 (14.3%)
0.022
Score 3 0 (0%) 1 (5.9%) 11 (64.7%) 5 (29.4%)
Total score of
cytoplasmic p65
Mean Score 4.3 4.5 5.3 5.2 0.026**
Range 3–5 2–6 3–6 4–6
Score <=3 1 (20%) 2 (40%) 2 (40%) 0 (0%) 0.387
Score >3 3 (7.5%) 9 (22.5%) 19 (47.5%) 9 (22.5%)
Abbreviations: IHC, Immunohistochemistry.
*Two-tailed Fisher’s exact test.
**The Kruskal–Wallis one way analysis of variance by ranks.

Fig. 2. NF-κΒ (p65) immunohistochemical staining in normal tissue (A), epithelial hyperplasia (B),
epithelial dysplasia (C) and OSCC (D), showing a gradual increase in cytoplasmic intensity from
normal to cancer. (A-D: 100X).
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

localization among dysplasias and OSCCs, no statistical correlation between the presence of inflammation and
significant difference was observed. the expression of IL-6 was detected (p=0.015, 95% con-
There was no statistical correlation between the fidence interval [CI] lower border 1.99) (Table 3).
expression of NF-κΒ (p65) and the presence of the
subepithelial inflammatory infiltrate (data not shown). Discussion
- IL-6 immunohistochemical expression The role of chronic inflammation in oral carcinogenesis
IL-6 had a generally mild cytoplasmic staining in a few is not fully understood. Transcription factor NF-κΒ
subepithelial inflammatory cells and in the adjacent is known to have prominent role in the inflammatory
epithelial cells of the basal or parabasal layer of selective response (3,9). Moreover, proinflammatory cytokines,
cases (Fig. 3). such as IL-6, are often involved in the interaction
Specifically, IL-6 expression was detected in 3/11 between epithelial and stromal cells (6,9). The current
(27.3%) of the epithelial hyperplasia cases, 10/18 (55.6%) study’s objective was to evaluate the expression of
of the dysplasia cases and 2/7 (28.6%) of the OSCC these key molecules in oral precancerous and cancerous
cases, whereas no positive case was detected in normal lesions and correlate it with each other, with the
oral mucosa. No statistical difference was observed histologic subtype of the lesions and with the presence
between the above groups. Overall, epithelial dysplasias of subepithelial inflammatory infiltrate.
showed non-statistically significant increased positivity NF-κΒ pathway is implicated in oral carcinogenesis.
for IL-6 compared to hyperplasias and OSCCs. Among its family members, p65 is the most frequently
There was no statistical correlation between the expres- studied. It is part of the p65(RelA):p50 dimer which
sion of IL-6 and the nuclear or cytoplasmic expression mediates the classical pathway. In the present study,
of NF-κΒ (p65). On the other hand, positive statistical p65 was expressed in the cytoplasm of the epithelial

Fig. 3. IL-6 immunohistochemical staining in subepithelial inflammatory cells (black arrows) and in the adjacent epithelial cells of the basal or
parabasal layer (white arrows) of selective cases. (A-B: 400X).

Table 3. Correlation of IL-6 immunohistochemical expression with inflammation.

Presence of inflammation
Parameters of IL-6 IHC expression + - P* OR (95% CI)
No of cases 35 (77.8%) 10 (22.2%)

Not available for IL-6 analysis 4 (80.0%) 1 (20.0%)

IL-6 expression
Expressed 15 (100.0%) 0 (0.0%) 0.015 N.C. (1.99 – N.C.)
Not expressed 16 (64.0%) 9 (36.0%) Reference
Abbreviations: N.C., Non calculable. IHC, Immunohistochemistry. OR, odds ratio. CI, confidence intervals
*Two-tailed Fisher’s exact test.
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

cells in every case. This finding may be attributed to carcinoma is formed, needs further investigation.
the sensitivity of the anti-p65 antibody used. The same The expression of IL-6 is commonly regulated by NF-
antibody was used in other studies also, revealing high κB (7). In the present study, no correlation between the
p65 expression rates (7,11). It should be noted that the expression of IL-6 and the nuclear or cytoplasmic ex-
cytoplasmic expression of p65 in OSCC is over 75% pression of NF-κΒ (p65) was noted. This fact indicates
in various studies, while nuclear staining is detected that NF-κΒ may act in an IL-6-independent fashion by
in just 35-45% of the cases (7,11-16). In the present activating different targets in oral carcinogenesis, such
study, nuclear localization was detected in 33.3% of as the anti-apoptotic protein Bcl-2 and various other cy-
OSCC cases. As for the oral precancerous lesions, it tokines and chemokines (30). Moreover, IL-6 might be
is generally believed that p65 expression (percentage activated by molecules other than NF-κΒ, such as In-
and intensity) gradually increases from normal mucosa terleukin-1 (IL-1), Tumor necrosis factor (TNF), pros-
to epithelial dysplasia (13-16). In the present study, taglandin E-2 (PGE-2) and vascular endothelial growth
p65 cytoplasmic intensity and total scores showed a factor (VEGF) (9).
statistically significant gradual increase from normal The results of this study support that the signaling path-
mucosa to OSCC, while non-statistically significant way of NF-κΒ is activated in the early stages of oral car-
increased nuclear localization was detected in epithelial cinogenesis. At the same time, IL-6 might have an in-
dysplasias and OSCCs. These findings suggest that p65 dependent role in the regulation of the inflammatory re-
may be expressed in all stages of oral carcinogenesis sponse. Interestingly, the intensity of NF-κΒ expression
with increased expression especially in the transition is significantly increased in the transition from epithe-
between oral epithelial hyperplasia and dysplasia. lial hyperplasias to dysplasias. The increased presence
Chronic inflammation and IL-6 in particular, are involved of inflammation in epithelial hyperplasias, dysplasias
in colon and prostate carcinogenesis (17-19). In the and OSCCs and its statistical correlation with IL-6 ex-
present study, the non-statistically significant increased pression is consistent with a possible crosstalk between
IL-6 expression in epithelial dysplasias compared to the epithelium and the stroma. On the other hand, the
hyperplasias and normal mucosa suggests a possible absence of correlation between NF-κB expression and
role of this molecular pathway in oral carcinogenesis. IL-6 expression suggests that this cytokine may be the
Interestingly, no other studies have tested IL-6 downstream target of other NF-κB-unrelated molecu-
immunohistochemical expression in oral precancerous lar pathways. Further studies regarding these processes
lesions. As for OSCC, only 2/7 cases (28.6%) were may be of significance in the quest for better prognosti-
positive for IL-6. Other immunohistochemical studies cation and/or management of potentially malignant dis-
have found that 35.7-100% of OSCC tissues or cell orders of the head and neck.
lines express IL-6 (20-24). This disagreement can be
attributed to the small sample (n=7) of the research. On References
the other hand, these tumors may still express IL-6R in 1. Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral
order to receive IL-6 signaling from the subepithelial epithelial dysplasia classification systems: predictive value, utility,
inflammatory infiltrate, as suggested by other studies weaknesses and scope for improvement. J Oral Pathol Med.
2008;37:127-33.
(24). 2. Warnakulasuriya S. Histological grading of oral epithelial
In the present study, increased presence of inflammation dysplasia: revisited. J Pathol. 2001;194:294-7.
was detected in epithelial hyperplasias, dysplasias 3. Balkwill F, Charles KA, Mantovani A. Smoldering and polarized
and OSCCs, compared with normal mucosa. In inflammation in the initiation and promotion of malignant disease.
Cancer Cell. 2005;7:211-7.
addition, a statistically significant correlation between 4. Ara T, Declerck YA. Interleukin-6 in bone metastasis and cancer
IL-6 expression and the presence of subepithelial progression. Eur J Cancer. 2010;46:1223-31.
inflammatory infiltrate was observed. These findings 5. Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC,
should be interpreted in the context of a possible Smith CW et al. Expression of proinflammatory and proangiogenic
cytokines in patients with head and neck cancer. Clin Cancer Res.
crosstalk between the epithelium and the stroma in oral 1999;5:1369-79.
carcinogenesis. The inflammatory cells (macrophages 6. Porta C, Larghi P, Rimoldi M, Totaro MG, Allevena P, Mantovani
and lymphocytes) typically produce IL-6 that binds A et al. Cellular and molecular pathways linking inflammation and
to the epithelial cells via IL-6R and/or sIL-6R, thus cancer. Immunobiol. 2009;214:761–77.
7. Squarize CH, Castilho RM, Sriuranpong V, Pinto DS Jr, Gutkind
facilitating cellular growth and invasion (4,9,25,26). JS: Molecular cross-talk between the NFkappaB and STAT3
Moreover, inflammatory cells also express IL-6R and signaling pathways in head and neck squamous cell carcinoma.
are able to receive signals from the epithelium (27,28). Neoplasia. 2006;8:733-46.
These molecular signals cause oncogenic differentiation 8. Baud V, Karin M. Is NF-κB a good target for cancer therapy?
Hopes and pitfalls. Nat Rev Drug Discov. 2009;8:33–40.
of the inflammatory infiltrate towards Th2 lymphocytes 9. Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in
and M2 macrophages (29). The exact role of IL-6 in inflammation and cancer. Eur J Cancer. 2005;41:2502-12.
the early stages of oral carcinogenesis, before invasive
Med Oral Patol Oral Cir Bucal-AHEAD OF PRINT - ARTICLE IN PRESS NF-κΒ and IL-6 in oral precancer and cancer

10. Hammacher A, Ward LD, Weinstock J. Structure-function 28. Matsumoto S, Hara T, Mitsuyama K, Yamamoto M, Tsuruta
analysis of human IL-6: identification of two distinct regions that are O, Sata M et al. Essential roles of IL-6 trans-signaling in colonic
important for receptor binding. Protein Sci. 1994;3:2280–93. epithelial cells, induced by the IL-6/soluble-IL-6 receptor derived
11. Mishra A, Bharti AC, Varghese P, Saluja D, Das BC. Differential from lamina propria macrophages, on the development of colitis-
expression and activation of NF-kappaB family proteins during oral associated premalignant cancer in a murine model. J Immunol.
carcinogenesis: Role of high risk human papillomavirus infection. 2010;184:1543-51.
Int J Cancer. 2006;119:2840-50. 29. Martinez FO, Sica A, Mantovani A, Locati M. Macrophage
12. Fukuda M, Ehara M, Suzuki S, Ohmori Y, Sakashita H. IL- activation and polarization. Front Biosci. 2008;13:453–61.
23 promotes growth and proliferation in human squamous cell 30. Molinolo AA, Amornphimoltham P, Squarize CH, Castilho RM,
carcinoma of the oral cavity. Int J Oncol. 2010;36:1355-65. Patel V, Gutkind JS. Dysregulated molecular networks in head and
13. Nakayama H, Ikebe T, Beppu M, Shirasuna K. High expression neck carcinogenesis. Oral Oncol. 2009;45:324-34.
levels of nuclear factor kappaB, IkappaB kinase alpha and Akt
kinase in squamous cell carcinoma of the oral cavity. Cancer.
Conflicts of interest
2001;92:3037–44.
None
14. Bindhu OS, Ramadas K, Sebastian P, Pillai MR. High expression
levels of nuclear factor kappa B and gelatinases in the tumorigenesis
of oral squamous cell carcinoma. Head Neck. 2006;28:916-25.
15. Santhi WS, Sebastian P, Varghese BT, Prakash O, Pillai MR.
NF-kappaB and COX-2 during oral tumorigenesis and in assessment
of minimal residual disease in surgical margins. Exp Mol Pathol.
2006;81:123-30.
16. Sawhney M, Rohatgi N, Kaur J, Shishodia S, Sethi G, Gupta
SD et al. Expression of NF-kappaB parallels COX-2 expression in
oral precancer and cancer: association with smokeless tobacco. Int J
Cancer. 2007;120:2545-56.
17. Brozek W, Bises G, Fabjani G, Cross HS, Peterlik M. Clone-specific
expression, transcriptional regulation, and action of interleukin-6 in
human colon carcinoma cells. BMC Cancer. 2008;18:13-20.
18. Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY,
Vallabhapurapu S et al. IL-6 and Stat3 are required for survival
of intestinal epithelial cells and development of colitis-associated
cancer. Cancer Cell. 2009;15:103–13.
19. Inokuchi J, Lau A, Tyson DR, Ornstein DK. Loss of annexin A1
disrupts normal prostate glandular structure by inducing autocrine
IL-6 signaling. Carcinogenesis. 2009;30:1082-8.
20. Wang YF, Chang SY, Tai SK, Li WY, Wang LS. Clinical
significance of interleukin-6 and interleukin-6 receptor expressions
in oral squamous cell carcinoma. Head Neck. 2002;24:850-8.
21. Shibahara T, Nomura T, Cui NH, Noma H. A study of osteoclast-
related cytokines in mandibular invasion by squamous cell carcinoma.
Int J Oral Maxillofac Surg. 2005;34:789-93.
22. Shinriki S, Jono H, Ota K, Ueda M, Kudo M, Ota T et al.
Humanized anti-interleukin-6 receptor antibody suppresses tumor
angiogenesis and in vivo growth of human oral squamous cell
carcinoma. Clin Cancer Res. 2009;15:5426-34.
23. Van Tubergen E, Vander Broek R, Lee J, Wolf G, Carey T,
Bradford C et al. Tristetraprolin regulates interleukin-6, which is
correlated with tumor progression in patients with head and neck
squamous cell carcinoma. Cancer. 2011;117:2677-89.
24. Kayamori K, Sakamoto K, Nakashima T, Takayanagi H, Morita
K, Omura K et al. Roles of interleukin-6 and parathyroid hormone-
related peptide in osteoclast formation associated with oral cancers:
significance of interleukin-6 synthesized by stromal cells in response
to cancer cells. Am J Pathol. 2010;176:968-80.
25. Becker C, Fantini MC, Wirtz S, Nikolaev A, Lehr HA, Galle
PR et al. IL-6 signaling promotes tumor growth in colorectal cancer.
Cell Cycle. 2005;4:217-20.
26. Huang WH, Chang MC, Tsai KS, Hung MC, Chen HL, Hung SC.
Mesenchymal stem cells promote growth and angiogenesis of tumors
in mice. Oncogene. 2013;32:4343-54.
27. Hobisch A, Rogatsch H, Hittmair A, Fuchs D, Bartsch G,
Klocker H. Immunohistochemical localization of interleukin-6 and
its receptor in benign, premalignant and malignant prostate tissue. J
Pathol. 2000;191:239–44.