==== Front
Int J Mol Sci
Int J Mol Sci
ijms
International Journal of Molecular Sciences
1422-0067 MDPI 

33287464
10.3390/ijms21239246
ijms-21-09246
Review
Role of Cyclooxygenase-2 in Head and Neck Tumorigenesis
Frejborg Ellen 1 Salo Tuula 12345† https://orcid.org/0000-0002-9455-3823Salem Abdelhakim 12*† 1 Department of Oral and Maxillofacial Diseases, Clinicum, University of Helsinki, 00014 Helsinki, Finland; ellen.frejborg@helsinki.fi (E.F.); tuula.salo@helsinki.fi (T.S.)
2 Translational Immunology Research Program (TRIMM), Research Program Unit (RPU), University of Helsinki, 00014 Helsinki, Finland
3 Cancer and Translational Medicine Research Unit, University of Oulu, 90220 Oulu, Finland
4 Medical Research Centre, Oulu University Hospital, 90220 Oulu, Finland
5 Department of Pathology, Helsinki University Hospital (HUS), 00029 Helsinki, Finland
* Correspondence: abdelhakim.salem@helsinki.fi† These authors jointly supervised this work.


03 12 2020 
12 2020 
21 23 924602 11 2020 02 12 2020 © 2020 by the authors.2020Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).The cyclooxygenase-2 (COX-2) is a potent enzyme that converts arachidonic acid to prostaglandins (PG), including PGE2, a key mediator of inflammation and angiogenesis. Importantly, COX-2 is activated in response to inflammatory stimuli, where it is also believed to promote the development and progression of head and neck cancers (HNC). COX-2 can mediate its protumorigenic effect through various mechanisms, such as inducing cell proliferation, inhibition of apoptosis, and suppressing the host’s immune response. Furthermore, COX-2 can induce the production of vascular endothelial growth factors, hence, promoting angiogenesis. Indeed, the ability of COX-2 inhibitors to selectively restrict the proliferation of tumor cells and mediating apoptosis provides promising therapeutic targets for cancer patients. Thus, in this comprehensive review, we summarized the reported differential expression patterns of COX-2 in different stages of head and neck carcinogenesis—from potentially premalignant lesions to invasive carcinomas. Furthermore, we examined the available meta-analysis evidence for COX-2 role in the carcinogenesis of HNC. Finally, further understanding of the biological processes of COX-2 and its role in orchestrating cell proliferation, apoptosis, and angiogenesis may give therapeutically beneficial insight to develop the management plan of HNC patients and improve their clinical outcomes.

cyclooxygenase-2head and neck cancershead and neck squamous cell carcinomaprostaglandinsinflammationcarcinogenesispotentially premalignant lesions
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1. Head and Neck Cancer: An Overview
Head and neck cancers (HNC) represent a heterogeneous group of tumors that arise anywhere in the head and neck region. Approximately 90% of these cancers develop from the squamous cell lining of the oral cavity, oropharynx, hypopharynx, larynx, or nasopharynx [1,2]. In addition, tumors can originate from other tissues, such as the salivary glands, lymphoid tissue, connective tissue, or melanocytes [3]. Epidemiologically, HNC ranks as the sixth most common cancer worldwide, accounting for about 5–10% of all cancers in Europe and North America [4,5].

The risk factors associated with HNC include, e.g., tobacco and alcohol consumption, HPV infection, poor oral hygiene, and improper diet [5]. When consumed together, tobacco and alcohol can produce a synergistic procancerous effect, whereby alcohol increases the body’s exposure to tobacco-derived carcinogens, such as nitrosamines and polycyclic hydrocarbons [4]. Thus, while smoking alone increases the risk of developing oral squamous cell carcinoma (OSCC) by ten times, both smoking and heavy drinking can increase such risk by almost a hundred times [6]. Human papillomavirus (HPV), particularly type 16 and 18, can merge with the host cell DNA and induce a malignant transformation. Interestingly, HPV is mainly associated with oropharyngeal squamous cell carcinomas (OPSCC), which are commonly diagnosed in younger patients with no clear history of smoking or heavy drinking [2,5]. Despite changes in lifestyle, HPV-driven malignancies have been on the rise over the last decade [2]. Luckily, HPV-driven cancers are more responsive to treatments, and thus, patients have a better survival rate compared to other types of HNC [4].

Unfortunately, despite the advances in cancer diagnosis and treatment, the overall survival (OS) for HNC patients has remained low. The survival outcome, however, varies depending on several crucial prognostic factors. For instance, patients with HPV-positive status show a 3-year OS rate of 82% compared with 57% in those with HPV-negative tumors [4]. Other prognostic factors include tumor site and stage at the time of diagnosis, with the most important factor being whether the patient has metastatic involvement in the lymph nodes [3]. Sadly, HNC are commonly diagnosed at later stages when the disease has already progressed and metastasized. At initial presentation, over 40% of patients have regional nodal involvement, and 10% present with distant metastases [2]. Presentation with distant metastases or a recurrent tumor spells an especially grim prognosis with a median survival of only 6–8 months [7]. In this context, recurrence represents another pressing challenge in HNC. Indeed, approximately one-third of OSCC patients relapse with locoregional recurrence. Second primary tumors are also common, with an annual rate of 4–7% [3]. The occurrence of second primary tumors could, in part, be explained by the field cancerization concept. As such, in tobacco- and/or alcohol-driven carcinogenesis, a considerable area of the mucosal tissue has been exposed to the carcinogens, and hence, may harbor mutations. Consequently, the para-cancerous, tumor-free, epithelium may already be in a premalignant change process, and could develop second primary tumors [6].

2. Cyclooxygenase-2An Overview
The cyclooxygenase (COX) enzyme converts arachidonic acid to prostaglandins trough two catalytic steps: First, it adds oxygen to arachidonic acid so that the unstable prostaglandin G2 (PGG2) is formed; second, it reduces PGG2 to the prostaglandin H2 (PGH2), which then can be converted, via specific synthases, to several prostanoids, such as prostaglandin E2 (PGE2), prostaglandin D2, prostacyclin or thromboxane A2 [8]. COX has two isoforms: COX-1 and COX-2. COX-1 is consistently expressed in most cells, where it mediates several physiological functions, such as platelet aggregation and production of protective mucous in the stomach lining [9]. On the other hand, COX-2 is less widely expressed, and it is mainly found in the stomach, kidney, central nervous system, and the female reproductive tract [10]. It can, however, be induced in other cell types by different stimuli, such as growth factors, cytokines, carcinogens and oncogenes, and chronic inflammation [9,11].

An increased expression level of COX-2 has been linked to carcinogenesis [11]. Elevated COX-2 levels have been found in potentially premalignant lesions and malignant tumors, including breast, lung, pancreatic, gastric, esophageal, liver, prostate, and stomach cancers. Supporting these reports, a COX-2 knocked-out mice model of familial adenomatous polyposis reduced the number of polyps, whereas mice that overexpressed COX-2 in mammary glands developed metastatic mammary cancer [11,12]. Furthermore, selectively inhibiting COX-2 in various experimental murine cancer models reduced tumor formation, growth, and metastasis [9].

COX-2 is believed to contribute to carcinogenesis in different ways. For instance, COX-2 overexpression leads to a B-cell lymphoma 2-driven anti-apoptotic effect in epithelial cells. Moreover, elevated COX-2 in these cells leads to increased production of vascular endothelial growth factors (VEGF) and the formation of networks resembling capillaries. COX-2 knocked-out mice models showed less intratumoral vascular density compared with the wild-type group [5,9]. However, COX-2 expression level per se may not directly reflect such carcinogenic potential, which could in part be mediated by its downstream pro-inflammatory products, such as PGE2 [12]. COX-2-derived PGE2 has been found to be one of the most important in carcinogenesis. Noteworthy, PGE2 levels were significantly induced in various cancer types, including OSCC [5]. PGE2 can bind to several receptors (EP1 to EP4) and acts in both autocrine and paracrine fashions, which could enhance protumorigenic processes in OSCC [5,12]. Importantly, PGE2 can suppress the immune system by inhibiting T- and B-cell proliferation and natural killer cell function; suppress the production of tumor necrosis factor-α; induce the production of interleukin-10; and stimulate regulatory T cells [8,9]. Additionally, PGE2 can also mediate chronic inflammation by promoting vasodilation and angiogenesis. Altogether, these activities, when dysregulated, may contribute to carcinogenesis (Figure 1).

3. COX-2 Expression in Head and Neck Cancers
The expression of COX-2 has been examined at both gene and protein levels in different types of HNC. The main findings are summarized in Table 1.

3.1. COX-2 Expression in Head and Neck Tumorigenesis
In general, normal oral mucosa has a very low expression of, or completely lacks, COX-2 [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28]. However, certain tissues of the oral cavity, such as the ductal epithelial cells of salivary glands, normally express COX-2 [29,30,31,32,33]. COX-2 expression in oral mucosa is induced by exposure to tobacco and other carcinogens [25,26,27,28]. Interestingly, normal oral mucosa of smokers exhibits 4-fold more COX-2 mRNA than non-smokers, and oral cancer tissues express 50 times more than para-cancer areas [22,26]. Likewise, COX-2 is typically induced in oral potentially malignant lesions. Hay et al. found that patients with oral lichen planus (OLP) showed significantly higher levels of PGE2 compared with the control group [34].

Furthermore, patients with an erosive type of OLP had significantly higher PGE2 than the atrophic type group [34]. In agreement, Prado et al. found that the COX-2 mRNA levels were induced in oral leukoplakia compared to a normal-appearing mucosa from the same patient, as well as to healthy controls [27]. Other studies have found that COX-2 expression is gradually increased along with the transition from normal oral mucosa to cancer, where it is highest in severe dysplasia/carcinoma in situ samples [28,35].

In HNC, a large body of evidence has demonstrated the upregulation of COX-2 in malignant tumors when compared to normal oral mucosa [36,37,38,39,40,41,42,43,44,45]. For instance, one study found that COX-2 mRNA was 11-fold higher in head and neck squamous cell carcinoma (HNSCC) compared to paired normal tissue from the same patient [36]. Chan et al. found that when comparing the levels of COX-2 mRNA in HNSCC tissue, it was around 50 times higher than in the adjacent normal epithelium from the same patients and around 150 times higher when compared to normal oral mucosa from healthy controls [22]. However, some studies did not find a statistically significant difference in COX-2 levels between normal oral mucosa and tumors [38,39]. Additionally, similar amounts of COX-2 have been found in both normal oral mucosa and leukoplakia compared to OSCC samples [43]. Wenghoefer et al. found that irritation fibromas expressed less COX-2 in comparison to the healthy gingiva samples, the leukoplakia, and the OSCC samples [44]. Altogether, these reports highlight the potential involvement of COX-2 in oral carcinogenesis.

3.2. COX-2 Expression in Other Head and Neck Tumors
Several studies have assessed the expression of COX-2 in benign and malignant salivary gland tumors. Interestingly, Sakurai et al. found that the expression of COX-2 was group-dependent and increased from the normal salivary glands, to the salivary gland adenomas, with the highest expression detected in the salivary gland carcinoma group [31]. Furthermore, two studies found that the level of COX-2 in mucoepidermoid carcinoma (MEC) was strongly increased, whereas most of the pleomorphic adenomas and adenoid cystic carcinomas (AdCC) were COX-2-negative [46,47]. In melanomas, Nascimento et al. found that oral melanomas were consistently COX-2-positive compared with the benign oral nevi, which were completely COX-2-negative [48]. However, in another study on odontogenic tumors, both the benign and the malignant tumors expressed COX-2, however, the malignant amelocarcinoma specimens exhibited higher levels of COX-2 compared with the benign ameloblastoma samples. On the contrary, the benign ameloblastic fibromas showed higher COX-2 than the malignant ameloblastic fibrosarcomas [49]. Nevertheless, fibrous hyperplasia was found to express a very low level of COX-2 compared with other premalignant and malignant lesions of the oral cavity [50].

3.3. COX-2 Expression in Tumor Microenvironment
Indeed, the tumor microenvironment (TME) plays a crucial role in tumor development and metastasis [5,6]. The expression of COX-2 seems to be particularly strong at the tumor invasive-front area of the HNSCC [17,18,51]. For instance, Gallo et al. has shown that the median PGE2 protein level was 2.36 μg/mg in the tumoral core compared with 3.85 μg/mg in the invasive-front area of HNSCC [52]. In this study, the cohort included 52 surgical specimens from laryngeal, oral cavity, and oropharyngeal SCC. Positive expression of COX-2 has also been found in the tumoral surrounding stroma of HNSCC, most notably in the inflammatory cells, fibroblasts, and endothelial cells [17,19,41,49,51,53,54,55,56]. Höing et al. compared the expression of various markers, including COX-2, between stroma and tumor nests in 110 laryngeal squamous cell carcinoma (LSCC) patients [57]. Interestingly, and in contrast to the other markers, COX-2 was expressed more in the tumor nest (53%) than in the stroma (39%) of the LSCC patients. Furthermore, this study revealed that tumoral, but not stromal, COX-2 expression correlated with lymph node metastasis and reduced patients’ survival. Hence, since COX-2 can influence immune cell recruitment, the authors proposed that COX-2 could play an important role in establishing tumor-stromal cell crosstalk [57].

4. COX-2 Expression and Cancer Staging
The TNM Classification is a system used for classifying solid tumors and can be employed to assist in prognostic cancer staging [58]. The T stands for tumor size; N stands for nodes, and it describes the regional lymph node involvement of the tumor; and M stands for metastasis, and it informs whether the tumor has metastasized to distant tissues. Cancer stages are usually divided into stages (0 to IV), with stage 0 having the score Tis (i.e., carcinoma in situ), with the numbers increasing gradually (T1-T4, N1-N3, and M1) with the most advanced stage being IV [58].

Importantly, many prognostic studies indicated a significant relationship between the level of COX-2 and the T-stage in patients with HNC. Among these, three studies concluded that induced immunoexpression of COX-2 was significantly associated with the T-stage in OSCC patients [41,59,60]. Similarly, Loong et al. found that advanced T-stage tumors of patients with nasopharyngeal carcinomas (NPC) showed stronger COX-2 expression compared with the lower T-stage tumors [61]. In LSCC patients, COX-2 expression was, likewise, more prevalent in the T3 and T4 tumors than in the lower T1 and T2 tumors [62,63]. Furthermore, Yang et al. reported a similar observation that COX-2 expression was significantly correlated with advanced T-stage in hypopharyngeal SCC (HPSCC) patients [64]. Xu et al. [65] found a significant relationship between COX-2 expression and T stage when looking at NPC samples. However, a correlation between COX-2 immunoexpression and T-stage was not found to be statistically significant in some studies about OSCC [25,51,55,66,67] LSCC [16,68,69], HPSCC [24], MEC [70], NPC [71,72,73], HNSCC [74,75], and tongue squamous cell carcinoma (TSCC) [76]. Nonetheless, some of these studies found a significant correlation with at least one other prognostic parameter, such as the N-stage [24,51,70,71].

Specifically, the N-stage was significantly correlated with COX-2 immunoexpression in OSCC [45,51,59,77,78], LSCC [62], HPSCC [24,64], MEC [70], NPC [65,71,79], TSCC [37], OPSCC [80], and HNSCC [52,81]. Like with T-stage studies, some studies revealed a statistically non-significant relationship between the N-stage and COX-2 expression. These studies included samples from patients with OSCC [20,41,55,60,66,67,82,83], LSCC [16,68], NPC [61,72,73,84], HNSCC [28,74,75,85], and TSCC [76,86]. Almost all the included studies have not assessed the M-stage separately, instead, it was included in the cancer stage. In this regard, a possible link between tumor stage (I-IV) and COX-2 expression has been evaluated in different HNC. On one hand, COX-2 immunoexpression was significantly correlated with the cancer stage in OSCC [20,23,59,60,87], LSCC [62,63], MEC [70], HNSCC [52,81], and TSCC [40,88]. On the other hand, such a link between cancer stage and COX-2 immunoexpression was not statistically significant in some studies that examined patient samples from OSCC [39,41,51,55,66,67,78], TSCC [37,76], LSCC [69], HNSCC [74,75], NPC [52,70,71,82], and glottic cancer [89].

In a meta-analysis study conducted by Yang et al., COX-2 immunoexpression levels were significantly associated with N-stage and cancer stage, but not with T-stage. However, the subgroup analysis revealed that such a significant correlation between N-stage and COX-2 was only seen in patients with OSCC, but not in other HNSCC [90]. For the cancer stage, the correlation was significant in OSCC patients, as well as in no site-specific HNC patients, but not in patients with LSCC or NPC [90].

5. COX-2 Expression and Cancer Grading
Cancer grading is a delineation of the microscopic features of the tumoral cells and tissue. Low-grade, well-differentiated tumors exhibit histological structures that relatively well mimic the normal tissue. On the contrary, higher-grade tumors (i.e., poorly differentiated or undifferentiated tumors) have more abnormal appearing structures, and they tend to be more aggressive and have a worse prognosis [58]. Unlike TNM-staging, most studies did not find any significant correlations between cancer grade and immunoexpression of COX-2, including studies on OSCC [25,39,51,54,55,60,67,78,91], HPSCC [24,64], LSCC [16], MEC [70], TSCC [37,88], HNSCC [52,74,81,85], NPC [72,73], and glottic cancer [89]. Interestingly, the significant correlation was only seen in a few studies, including OSCC [23,41,45] and LSCC [62,63].

6. COX-2 Expression and Survival Outcomes
6.1. COX-2 Expression and Overall Survival
Itoh et al. found that OSCC patients with COX-2 overexpression had worse OS in the univariate analysis, however, COX-2 was not an independent prognostic factor in the multivariate analysis [51]. In a univariate analysis of an LSCC cohort, patients with elevated cytoplasmic expression of COX-2 had shorter OS [63]. Pan et al. showed that Cox-2 was overexpressed in 75.7% of NPCs, and this was associated with the worse OS on both univariate and multivariate analyses [92]. In the same manner, several other studies found a significant association between higher COX-2 level and reduced OS both in univariate and multivariate analyses, including studies on OPSCC [93], OSCC [94], LSCC [62], HNSCC [28,52], NPC [65,84], HPSCC [64], and glottic cancer [89]. Interestingly, Kyzas et al. found that co-expression of COX-2 and VEGF-C meant a significantly shorter OS, which was also an independent prognostic factor in the multivariate analysis [81]. Furthermore, Gallo et al. showed that HNSCC patients with higher PGE2 tumor levels had significantly shorter OS estimates in Kaplan–Meier analysis [52].

On the contrary, Ranelletti et al. reported that LSCC patients with COX-2 positive tumors had a longer OS compared to patients with COX-2 negative tumors. In this study, the 5-year OS rate for patients with COX-2-positive tumors was 100%, whereas it was 34% for those with COX-2-negative tumors [69]. In the multivariate analysis, COX-2 retained its significance as an independent prognostic marker. The authors concluded that COX-2 is overexpressed in less aggressive, low-grade laryngeal SCCs, whereas its expression is lost as the tumors progress to a more malignant phenotype [69]. Other studies found no relationship between COX-2 expression and OS, including studies on NPC [71,72,73], TSCC [67,76], OSCC [39,60,66,82,95,96], HNSCC [85], OPSCC [80], and AdCC [32].

6.2. COX-2 Expression and Disease-Specific Survival
In two OSCC studies, patients with higher COX-2 expression had a significantly shorter 5-year disease-specific survival (DSS) [59,60]. In contrast, Loong et al. found that DSS was shorter in patients with low COX-2 expression compared to patients with moderate or strong expression scores [61]. However, this study had a small sample size, and hence, a multivariate analysis could not be performed. Four other studies found no correlation between COX-2 expression and DSS, including patients with LSCC [68], TSCC [97], tonsils, and base of tongue SCC [98].

6.3. COX-2 Expression and Disease-Free Survival
COX-2 expression was found to correlate with disease-free survival (DFS) in HNSCC patients. For instance, Chen et al. found a higher recurrence rate in LSCC patients expressing high COX-2 levels compared with those with low COX-2 expression [62]. In a univariate analysis, Pan et al. found that NPC patients exhibited a significant correlation between COX-2 expression and DFS [92]. In the multivariate analysis, multiple variables, including COX-2, were combined into a principal component (Z), which was an independent prognostic factor in NPC. However, COX-2 expression was not assessed separately [92]. In HNSCC patients, the 5-year relapse-free survival rate in the univariate analysis was worse in patients who had elevated expression of COX-2, however, this was not statistically significant in the multivariate analysis [75,80]. Pannone et al. examined a cohort of OSCC patients and found that COX-2 overexpression was correlated with reduced DFS in the univariate analysis, however, multivariate analysis was not performed [39]. Similarly, Kourelis et al. reported a lower recurrence rate in LSCC patients with higher levels of COX-2 immunostaining, although the multivariate analysis was not performed [68]. Interestingly, higher COX-2 levels were associated with a poor outcome in chemotherapy-naïve OSCC patients compared to those who had received chemotherapy [99]. In the multivariate analysis, Itoh et al. reported that COX-2 overexpression was an independent prognostic factor for shorter DFS in OSCC patients [51]. In agreement with this study, Gallo et al. delineated that HNSCC patients with low or absent COX-2 expression had better DFS than patients with overexpressed COX-2 status, which was also true in the multivariate analysis [52]. However, and despite the aforementioned evidence, several studies found no correlation between COX-2 and DFS, including studies on NPC [71,72,73,84], OSCC [41,45], HNSCC [81,85], TSCC [67,88], glottic cancer [89], and LSCC [100].

6.4. Meta-Analyses of COX-2 Expression and Survival
Two meta-analysis studies examined the prognostic value of COX-2 expression. Wang et al. performed a meta-analysis of 12 studies encompassing 979 OSCC patients. They found that patients with positive COX-2 status had a poor OS rate (hazard ratio (HR) = 2.23) compared with the COX-2-negative group [101]. An analysis conducted by Yang et al. included 29 studies with a total of 2430 patients with HNSCC [91]. They found that positive COX-2 expression was associated with poor outcomes in OS, relapse-free survival, and DFS (HR = 1.93; 2.02; 5.14, respectively). When the authors conducted a subgroup meta-analysis, COX-2 expression predicted reduced, statistically non-significant, survival time [91].

7. COX-2 Polymorphisms and Risk of Cancers
There are some genetic polymorphisms of COX-2 that have been implicated in the risk of developing HNC (Figure 2). The main polymorphisms are:

7.1. COX-2-765G>C
The COX-2-765G>C is a functional polymorphism that disrupts the binding site of stimulatory protein 1 (Sp1), but creates a binding site for E2 promoter binding factor 1 (E2F1), leading to stimulated transcription activity, which could enhance the cancer risk [102,103]. Lin et al. found that the GC and CC genotype was protective against OSCC when compared to the GG genotype in a study that included 297 OSCC patients and 280 healthy controls [102]. In another study on OSCC, Mittal et al. analyzed a single locus and found no significant difference between 193 OSCC patients and 137 controls in the 765 G>C allele frequency [104]. However, in the multivariate logistic regression analysis, the −765 G>C genotype appeared to be protective with an odds ratio of 0.71. Thus, they concluded that the −765 G>C and CC variant might be protective against OSCC compared to the GG variant [104]. However, this was in contrast to another study, in which the GG genotype was more frequent in controls than in OSCC patients (94.66% vs. 73.3%), and thus, could be protective against OSCC. Moreover, the study found that both the GC and the CC genotypes were associated with a significantly increased risk of OSCC [105]. Nonetheless, another two studies found no evidence for the role of −765G>C polymorphisms in the risk of developing OSCC [106,107].

7.2. COX-2-1195G>A
The COX-2-1195G>A polymorphism has also been suggested to influence the risk of oral cancer. The −1195A allele displays an increased transcriptional activity of the COX-2 gene compared to the −1195G allele [108]. Mittal et al. found that −1195GA genotype was relatively higher in OSCC patients compared to the controls, which seemed to confer an increased risk of tobacco-related oral carcinogenesis [104]. Chiang et al. found that the AA genotype was significantly associated with OSCC when compared to the GG genotype and had a 1.55-fold increased risk of OSCC [107]. However, two studies found no association between different COX-2-1195G>A polymorphisms and head and neck cancer risk [106,109].

7.3. COX-2 8473C>T
The 8473 C>T polymorphism is located in the 3′ UTR region of the COX-2 gene, and the T to C change may affect the stability and the secondary structure of the mRNA of COX-2 [110]. COX-2 8473 C>T polymorphisms have also been assessed in patients with HNSCC. Although there was no significant difference between healthy controls and OSCC patients in the single-locus analysis, the CT genotype was less frequent in patients than controls [104]. Campa et al. investigated the SNPs, including the 8473 C>T polymorphism in 811 patients with upper aerodigestive tract cancers, including OSCC, LSCC, and OPSCC [110]. The authors indicated a possible association between esophageal cancer and the 8473C>T polymorphism.

7.4. Meta-Analyses of COX-2 Gene Polymorphisms and Risk of Cancer
Three meta-analyses assessed the potential association between COX-2 gene polymorphisms and the risk of HNC. Deng et al. reported a significantly increased risk of HNSCC in three genetic models of COX-2 polymorphisms. However, the odd ratios were small, and not all models showed an association with HNSCC, which could result from sample size that was too small [111]. Li et al. investigated the polymorphisms: +837T>C, −765G>C, and −1195A>G among seven clinical studies, including a total of 2296 oral cancer patients. Interestingly, the authors found that the +837T>C and the −765G>C polymorphisms are related to the susceptibility of oral cancer and that the gene frequencies in the case group compared to the control group were significantly different both in the allele model and the dominant model [112]. On the other hand, a meta-analysis by Leng et al. included eight case control studies and found no association with either the 8473T>C or the −765G>C polymorphism in the risk of HNSCC. However, they found an association between the −1195G>A polymorphism and HNSCC risk in the pooled result from the crude data in certain models (AA vs. GG, AA vs. GA, and AA vs. GG + GA) [113].

8. COX-2 and Cancer Biomarkers
Several studies investigated the potential correlation between COX-2 and other biomarkers in HNC. However, no significant correlation was found between COX-2 and p53 [19,67,91], Ki67 [19,54], CD68 [54], epidermal growth factor receptor [72,73,84], E-cadherin [37], C-erbB2 [84], p-ERK1/2 [25] or mast cell density [95]. Nevertheless, a positive correlation has been found in a limited number of studies between COX-2 and HGF [33], EP300 [62], matrix metalloproteinase 2 [63], prostate-specific membrane antigen [94], DNA topoisomerase II α [18,77], NF-κB [41], H-Ras [23], cytoplasmic, but not nuclear HuR expression [16,47,60], CD4 + CD25 + Foxp3+ regulatory T cells [75], tumor-associated tissue eosinophilia [87] or platelet-lymphocyte ratio [66].

Importantly, a significant correlation between COX-2 and VEGF was reported in HNSCC [52,59,81,95]. The VEGF family plays a crucial role in tumor-associated angiogenesis and lymphangiogenesis. Cancer cells can secrete VEGF-C and VEGF-D to induce intratumoral and peritumoral lymphangiogenesis, as well as tumor neovascularization [52,59]. COX-2 is believed to stimulate VEGF expression (e.g., VEGF-A and C), and hence, both are associated with lymph node metastasis and tumor angiogenesis [52,59,81]. Co-expression of both factors may also negatively impact the survival of HNSCC patients [76,81].

9. Conclusions
To summarize, there is enormously growing evidence supporting the involvement of COX-2 in tumor-initiating and tumor-promoting events for several solid tumors, including HNC. Furthermore, elevated COX-2 levels were also documented in potentially premalignant lesions of the oral cavity. It is also acknowledged that COX-2 plays vital role in regulating tumorigenesis-related processes, such as apoptosis, angiogenesis, and immunomodulation [5,8,9]. Therefore, there is considerable potential for COX2-based therapeutics, such as COX-2 inhibitors, to serve as either adjuvant therapeutics increasing the overall response rate, or as targeted therapeutics for HNC patients. Recently, Janakiraman et al. determined that inhibition of COX-2 via celecoxib promoted apoptosis in paclitaxel-resistant oral cancer cells, both in vitro and in vivo. Thus, the authors recommended the use of the COX-2 inhibitor celecoxib, in combination with paclitaxel, for the management of paclitaxel-resistant oral cancer cells [114]. However, as aforementioned in this review, some studies showed no significant association between COX-2 and HNC, which, therefore, necessitates more studies with larger sample sizes across different populations. In conclusion, further in vitro and in vivo model studies of COX-2 role in cancer, paralleled with clinical trials, could provide promising therapeutic targets in HNC, and improve the patients’ clinical outcome.

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Author Contributions
Conceptualization, E.F., T.S. and A.S.; methodology, E.F., T.S. and A.S.; software, E.F., T.S. and A.S.; formal analysis, E.F., T.S. and A.S.; resources, T.S.; data curation, E.F., T.S. and A.S.; writing—original draft preparation, E.F.; writing—review and editing, T.S. and A.S.; visualization, A.S.; supervision, T.S. and A.S.; project administration, T.S. and A.S.; funding acquisition, T.S. and A.S. All authors have read and agreed to the published version of the manuscript.

Funding
This research was funded by the Emil Aaltonen Foundation; Minerva Foundation Institute for Medical Research; Cancer Society of Finland; Sigrid Jusélius Foundation; Jane and Aatos Erkko Foundation; Helsinki University Central Hospital Research Funds.

Conflicts of Interest
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations
COX	cyclooxygenase	
HNC	head and neck cancers	
OSCC	oral squamous cell carcinoma	
HPV	human papillomavirus	
OPSCC	oropharyngeal squamous cell carcinoma	
PG	prostaglandin	
NPC	nasopharyngeal carcinoma	
HR	hazard ratio	
EP	prostanoid receptors	
PGE2	prostaglandin E2 	
VEGF	vascular endothelial growth factors	
HNSCC	head and neck squamous cell carcinoma	
AdCC	adenoid cystic carcinomas	
MEC	mucoepidermoid carcinoma 	
TSCC	tongue squamous cell carcinoma	
LSCC	laryngeal squamous cell carcinoma	
OTSCC	oral tongue squamous cell carcinoma	
HPSCC	hypopharyngeal squamous cell carcinoma	
Figure 1 Role of Cyclooxygenase-2 (COX-2) in head and neck carcinogenesis. (a) Inflammatory stimuli, oncogenes or other factors can induce COX-2 expression in epithelial cells; (b) this results in the production of prostaglandins which can influence various protumorigenic processes, such as (c) enhancing anti-apoptotic response, (d) suppression of immune cell response, or (e) inducing angiogenesis in the host tissue.

Figure 2 The main Cyclooxygenase-2 (COX-2) polymorphisms are implicated in the risk of head and neck cancer (HNC).

ijms-21-09246-t001_Table 1Table 1 Summary of COX-2 expression in different types of head and neck cancers.

Cancer Type	Expression	Main Findings	References	
HNSCC	Gene expression	COX-2 mRNA was 11-fold higher than normal control	[36]	
HNSCC	Gene expression,
Immunoexpression	COX-2 mRNA was 50 times higher than para-cancer tissue; and 150 times higher than in healthy controls	[22]	
HNSCC	Gene expression,
Immunoexpression	No statistically significant difference in COX-2 levels between cancer and control	[38,39]	
OSCC	Immunoexpression	Similar levels of COX-2 were found in both normal oral mucosa and leukoplakia	[43]	
OSCC	Gene expression	Irritation fibroma had less COX-2 than cancer tissues	[44]	
Salivary gland carcinomas	Immunoexpression	COX-2 had the highest expression in the salivary gland cancers, including MEC, AdCC, and pleomorphic adenomas	[31,46,47]	
Oral melanoma	Immunoexpression	Tumors were COX-2-positive compared with the benign oral nevi, which were completely COX-2-negative.	[48]	
Odontogenic tumors	Immunoexpression	Amelocarcinoma patients had higher levels of COX-2 compared with the benign ameloblastoma group. Malignant ameloblastic fibrosarcomas had less COX-2 than benign ameloblastic fibromas	[49]	
OSCC	Immunoexpression	COX-2 was increased in the OSCC group compared with the hyperplastic group	[50]	
HNSCC	Immunoexpression	The PGE2 protein level was induced in the invasive-front area more than in the intratumoral core	[52]	
HNSCC	Immunoexpression	Positive expression of COX-2 was found in the para-cancer stroma, mostly in the inflammatory and endothelial cells	[17,19,41,49,51,53,54,55,56]	
LSCC	Immunoexpression	COX-2 was more induced in the tumor nest (53%) than in the stroma (39%). Furthermore, tumoral COX-2 expression correlated with shorter survival outcome	[57]	
AdCC, adenoid cystic carcinomas; COX-2, Cyclooxygenase-2; HNSCC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; LSCC, laryngeal squamous cell carcinoma; MEC, mucoepidermoid carcinoma; PGE2, prostaglandin E2.
==== Refs
References
1. Sanderson R.J.  Ironside J.A.   Squamous Cell Carcinomas of the Head and Neck BMJ 2002 325 822 827 10.1136/bmj.325.7368.822 12376446 
2. Marur S.  Forastiere A.A.   Head and Neck Squamous Cell Carcinoma: Update on Epidemiology, Diagnosis, and Treatment Mayo Clin. Proc. 2016 91 386 396 10.1016/j.mayocp.2015.12.017 26944243 
3. Montero P.H.  Patel S.G.   Cancer of the Oral Cavity Surg. Oncol. Clin. N. Am. 2015 24 491 508 10.1016/j.soc.2015.03.006 25979396 
4. Alfouzan A.F.   Head and Neck Cancer Pathology: Old World versus New World Disease Niger J. Clin. Pract. 2019 22 1 8 10.4103/njcp.njcp_310_18 30666013 
5. Nasry W.H.  Rodriguez-Lecompte J.C.  Martin C.K.   Role of COX-2/PGE2 Mediated Inflammation in Oral Squamous Cell Carcinoma Cancers 2018 10 348 10.3390/cancers10100348 
6. Lippman S.M.  Sudbø J.  Hong W.K.   Oral Cancer Prevention and the Evolution of Molecular-Targeted Drug Development J. Clin. Oncol. 2005 23 346 356 10.1200/JCO.2005.09.128 15637397 
7. Rhee J.C.  Khuri F.R.  Shin D.M.   Advances in Chemoprevention of Head and Neck Cancer Oncologist 2004 9 302 311 10.1634/theoncologist.9-3-302 15169985 
8. Mendes R.A.  Carvalho J.C.  van der Waal I.   An Overview on the Expression of Cyclooxygenase-2 in Tumors of the Head and Neck Oral Oncol. 2009 45 124 128 10.1016/j.oraloncology.2009.03.016 
9. Lin D.T.  Subbaramaiah K.  Shah J.P.  Dannenberg A.J.  Boyle J.O.   Cyclooxygenase-2: A Novel Molecular Target for the Prevention and Treatment of Head and Neck Cancer Head Neck 2002 24 792 799 10.1002/hed.10108 12203806 
10. Goradel N.M.  Najafi M.  Salehi E.  Farhood B.  Mortezaee K.   Cyclooxygenase-2 in Cancer: A Review J. Cell Physiol. 2019 234 5683 5699 10.1002/jcp.27411 30341914 
11. Hamakawa H.  Nakashiro K.  Sumida T.  Shintani S.  Myers J.N.  Takes R.P.  Rinaldo A.  Ferlito A.   Basic Evidence of Molecular Targeted Therapy for Oral Cancer and Salivary Gland Cancer Head Neck 2008 30 800 809 10.1002/hed.20830 18429007 
12. Yang C.  Chang K.   Eicosanoids and HB-EGF/EGFR in Cancer Cancer Metastasis Rev. 2018 37 385 395 10.1007/s10555-018-9746-9 29936588 
13. Seyedmajidi M.  Shafaee S.  Siadati S.  Khorasani M.  Bijani A.  Ghasemi N.   Cyclo-oxygenase-2 Expression in Oral Squamous Cell Carcinoma J. Can. Res. Ther. 2014 10 1024 1029 10.4103/0973-1482.138205 25579548 
14. Thomas N.  Krishnapillai R.  Bindhu P.  Thomas P.   Immunohistochemical Expression of Cyclooxygenase-2 in Oral Squamous Cell Carcinoma Indian J. Dent. Res. 2019 30 102 106 10.4103/ijdr.IJDR_362_17 30900666 
15. Santhi W.S.  Sebastian P.  Varghese B.T.  Prakash O.  Pillai M.R.   NF-κB and COX-2 During Oral Tumorigenesis and in Assessment of Minimal Residual Disease in Surgical Margins Exp. Mol. Pathol. 2006 81 123 130 10.1016/j.yexmp.2006.05.001 16822500 
16. Cho N.  Han H.  Soh Y.  Lee K.  Son H.   Cytoplasmic HuR Over-expression is Associated with Increased Cyclooxygenase-2 Expression in Laryngeal Squamous Cell Carcinomas Pathology 2007 39 545 550 10.1080/00313020701684391 18027256 
17. Nagatsuka H.  Siar C.H.  Tsujigiwa H.  Naomoto Y.  Han P.P.  Gunduz M.  Sugahara T.  Sasaki A.  Nakajima M.   Heparanase and Cyclooxygenase-2 Gene and Protein Expressions During Progression of Oral Epithelial Dysplasia to Carcinoma Ann. Diagn. Pathol. 2012 16 354 361 10.1016/j.anndiagpath.2012.02.004 22575501 
18. Segawa E.  Sakurai K.  Kishimoto H.  Takaoka K.  Noguchi K.  Hashitani S.  Hirota S.  Urade M.   Expression of Cyclooxygenase-2 and DNA Topoisomerase II α in Precancerous and Cancerous Lesions of the Oral Mucosa Oral Oncol. 2008 44 664 671 10.1016/j.oraloncology.2007.08.014 17996482 
19. Shibata M.  Kodani I.  Osaki M.  Araki K.  Adachi H.  Ryoke K.  Ito H.   Cyclo-oxygenase-1 and -2 Expression in Human Oral Mucosa, Dysplasias and Squamous Cell Carcinomas and Their Pathological Significance Oral Oncol. 2005 41 304 312 10.1016/j.oraloncology.2004.09.009 15743693 
20. Li T.  Cui J.   COX-2, MMP-7 Expression in Oral Lichen Planus and Oral Squamous Cell Carcinoma Asian Pac. J. Trop. Med. 2013 6 640 643 10.1016/S1995-7645(13)60110-8 23790336 
21. Mauro A.  Lipari L.  Leone A.  Tortorici S.  Burruano F.  Provenzano S.  Gerbino A.  Buscemi M.   Expression of Cyclooxygenase-1 and Cyclooxygenase-2 in Normal and Pathological Human Oral Mucosa Folia Histochem. Cytobiol. 2010 48 555 563 10.2478/v10042-010-0066-3 21478098 
22. Chan G.  Boyle J.O.  Yang E.K.  Zhang F.  Sacks P.G.  Shah J.P.  Edelstein D.  Soslow R.A.  Koki A.T.  Woerner B.M.    Cyclooxygenase-2 Expression Is Up-Regulated in Squamous Cell Carcinoma of the Head and Neck Cancer Res. 1999 59 991 994 10070952 
23. Moazeni-Roodi A.  Allameh A.  Harirchi I.  Motiee-Langroudi M.  Garajei A.   Studies on the Contribution of Cox-2 Expression in the Progression of Oral Squamous Cell Carcinoma and H-Ras Activation Pathol. Oncol. Res. 2017 23 355 360 10.1007/s12253-016-0114-1 27628320 
24. Peng J.  Su C.  Chang H.  Chai C.  Hung W.   Overexpression of Cyclo-oxygenase 2 in Squamous Cell Carcinoma of the Hypopharynx Hum. Pathol. 2002 33 100 104 10.1053/hupa.2002.30187 11823979 
25. Søland T.M.  Husvik C.  Koppang H.S.  Boysen M.  Sandvik L.  Clausen O.P.  Christoffersen T.  Bryne M.   A Study of Phosphorylated ERK1/2 and COX-2 in Early Stage (T1–T2) Oral Squamous Cell Carcinomas J. Oral Pathol. Med. 2008 37 535 542 10.1111/j.1600-0714.2008.00656.x 18284542 
26. Moraitis D.  Du B.  de Lorenzo M.S.  Boyle J.O.  Weksler B.B.  Cohen E.G.  Carew J.F.  Altorki N.K.  Kopelovich L.  Subbaramaiah K.    Levels of Cyclooxygenase-2 Are Increased in the Oral Mucosa of Smokers: Evidence for the Role of Epidermal Growth Factor Receptor and Its Ligands Cancer Res. 2005 65 664 670 15695412 
27. Prado S.M.  Cedrún J.L.  Rey R.L.  Villaamil V.M.  García A.Á.  Ayerbes M.V.  Aparicio L.A.   Evaluation of COX-2, EGFR, and p53 as Biomarkers of Non-Dysplastic Oral Leukoplakias Exp. Mol. Pathol. 2010 89 197 203 10.1016/j.yexmp.2010.06.004 20599939 
28. Saba N.F.  Choi M.  Muller S.  Shin H.J.  Tighiouart M.  Papadimitrakopoulou V.A.  El-Naggar A.K.  Khuri F.R.  Chen Z.  Shin D.M.   Role of COX-2 in Tumor Progression and Survival of Head and Neck Squamous Cell Carcinoma Cancer Prev. Res. 2009 2 823 829 10.1158/1940-6207.CAPR-09-0077 
29. Branco K.F.  Ribeiro A.L.  de Mendonça R.P.  de Jesus Viana Pinheiro J.  da Silva Kataoka M.S.  Arnaud M.V.  de Melo Alves Junior S.   Abnormal Activation of the Akt Signaling Pathway in Adenoid Cystic Carcinoma Eur. Arch. Otorhinolaryngol. 2018 275 3039 3047 10.1007/s00405-018-5182-2 30367261 
30. Lipari L.  Mauro A.  Gallina S.  Tortorici S.  Buscemi M.  Tete S.  Gerbino A.   Expression of Gelatinases (MMP-2, MMP-9) and Cyclooxygenases (COX-1, COX-2) in Some Benign Salivary Gland Tumors Int. J. Immunopathol. Pharmacol. 2012 25 107 115 10.1177/039463201202500113 22507323 
31. Sakurai K.  Urade M.  Noguchi K.  Kishimoto H.  Ishibashi M.  Yasoshima H.  Yamamoto T.  Kubota A.   Increased Expression of Cyclooxygenase-2 in Human Salivary Gland Tumors Pathol. Int. 2001 51 762 769 10.1046/j.1440-1827.2001.01280.x 11881728 
32. Bell D.  Roberts D.  Karpowicz M.  Hanna E.Y.  Weber R.S.  El-Naggar A.K.   Clinical Significance of Myb Protein and Downstream Target Genes in Salivary Adenoid Cystic Carcinoma Cancer Biol. Ther. 2011 12 569 573 10.4161/cbt.12.7.17008 21785271 
33. Aoki T.  Tsukinoki K.  Kurabayashi H.  Sasaki M.  Yasuda M.  Ota Y.  Watanabe Y.  Kaneko A.   Hepatocyte Growth Factor Expression Correlates with Cyclooxygenase-2 Pathway in Human Salivary Gland Tumors Oral Oncol. 2006 42 51 56 10.1016/j.oraloncology.2005.06.012 16139560 
34. Abdel Hay R.M.  Fawzy M.M.  Metwally D.  Kadry D.  Ezzat M.  Rashwan W.  Rashed L.A.   DNA Polymorphisms and Tissue Cyclooxygenase-2 Expression in Oral Lichen Planus: A Case-Control Study J. Eur. Acad. Dermatol. Venereol. 2012 26 1122 1126 10.1111/j.1468-3083.2011.04229.x 21923838 
35. Akita Y.  Kozaki K.  Nakagawa A.  Saito T.  Ito S.  Tamada Y.  Fujiwara S.  Nishikawa N.  Uchida K.  Yoshikawa K.    Cyclooxygenase-2 is a Possible Target of Treatment Approach in Conjunction with Photodynamic Therapy for Various Disorders in Skin and Oral Cavity Br. J. Dermatol. 2004 151 472 480 10.1111/j.1365-2133.2004.06053.x 15327557 
36. Celenk F.  Bayramoglu I.  Yilmaz A.  Menevse A.  Bayazit Y.   Expression of Cyclooxygenase-2, 12-Lipoxygenase, and Inducible Nitric Oxide Synthase in Head and Neck Squamous Cell Carcinoma J. Craniofac. Surg. 2013 24 1114 1117 10.1097/SCS.0b013e31828f2491 23851751 
37. Fujii R.  Imanishi Y.  Shibata K.  Sakai N.  Sakamoto K.  Shigetomi S.  Habu N.  Otsuka K.  Sato Y.  Watanabe Y.    Restoration of E-cadherin Expression by Selective Cox-2 Inhibition and the Clinical Relevance of the Epithelial-to-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma J. Exp. Clin. Cancer Res. 2014 33 40 10.1186/1756-9966-33-40 24887090 
38. Nasry W.H.  Wang H.  Jones K.  Tesch M.  Rodriguez-Lecompte J.C.  Martin C.K.   Cyclooxygenase and CD147 Expression in Oral Squamous Cell Carcinoma Patient Samples and Cell Lines Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2019 128 400 410 10.1016/j.oooo.2019.06.005 31350224 
39. Pannone G.  Sanguedolce F.  de Maria S.  Farina E.  Muzio L.L.  Serpico R.  Emanuelli M.  Rubini C.  de Rosa G.  Staibano S.    Cyclooxygenase Isozymes in Oral Squamous Cell Carcinoma: A Real-Time RT-PCR Study with Clinic Pathological Correlations Int. J. Immunopathol. Pharmacol. 2007 20 317 324 10.1177/039463200702000211 17624243 
40. Renkonen J.  Wolff H.  Paavonen T.   Expression of Cyclo-oxygenase-2 in Human Tongue Carcinoma and its Precursor Lesions Virchows Arch. 2002 440 594 597 10.1007/s00428-002-0616-y 12070598 
41. Sawhney M.  Rohatgi N.  Kaur J.  Shishodia S.  Sethi G.  Gupta S.D.  Suryanaryana V.S.  Shukla N.K.  Aggarwal B.B.  Ralhan R.   Expression of NF-κB Parallels COX-2 Expression in Oral Precancer and Cancer: Association with Smokeless Tobacco Int. J. Cancer 2007 120 2545 2556 10.1002/ijc.22657 17354234 
42. Tan K.  Putti T.C.   Cyclooxygenase 2 Expression in Nasopharyngeal Carcinoma: Immunohistochemical Findings and Potential Implications J. Clin. Pathol. 2005 58 535 538 10.1136/jcp.2004.021923 15858127 
43. Amirchaghmaghi M.  Mohtasham N.  Mozaffari P.M.   Comparison of COX2 Expression between Oral Squamous Cell Carcinoma, Leukoplakia and Normal Mucosa J. Contemp. Dent. Pract. 2012 13 205 209 10.5005/jp-journals-10024-1122 22665749 
44. Wenghoefer M.  Pantelis A.  Dommisch H.  Reich R.  Martini M.  Allam J.P.  Novak N.  Bergé S.  Jepsen S.  Winter J.   Decreased Gene Expression of Human β-defensin-1 in the Development of Squamous Cell Carcinoma of the Oral Cavity Int. J. Oral Maxillofac. Surg. 2008 37 660 663 10.1016/j.ijom.2008.02.003 18346877 
45. Byatnal A.A.  Byatnal A.  Sen S.  Guddattu V.  Solomon M.C.   Cyclooxygenase-2–An Imperative Prognostic Biomarker in Oral Squamous Cell Carcinoma- An Immunohistochemical Study Pathol. Oncol. Res. 2015 21 1123 1131 10.1007/s12253-015-9940-9 25962348 
46. Da Rocha Tenório J.  da Silva L.P.  de Aguiar Xavier M.G.  Santana T.  do Nascimento G.J.  Sobral A.P.   Differential Expression of Cyclooxygenase-2 and Cyclin D1 in Salivary Gland Tumors Eur. Arch. Otorhinolaryngol. 2018 275 2341 2347 10.1007/s00405-018-5058-5 30003391 
47. Cho N.  Han H.  Soh Y.  Son H.   Overexpression of Cyclooxygenase-2 Correlates with Cytoplasmic HuR Expression in Salivary Mucoepidermoid Carcinoma but Not in Pleomorphic Adenoma J. Oral Pathol. Med. 2007 36 297 303 10.1111/j.1600-0714.2007.00526.x 17448140 
48. De Souza do Nascimento J.  Carlos R.  Delgado-Azañero W.  Mosqueda-Taylor A.  de Almeida O.P.  Romañach M.J.  de Andrade B.A.   Immunohistochemical Expression of Cyclooxygenase-2 (COX-2) in Oral Nevi and Melanoma J. Oral Pathol. Med. 2016 45 440 443 10.1111/jop.12385 26608958 
49. Sánchez-Romero C.  Mosqueda-Taylor A.  Delgado-Azañero W.  de Almedia O.P.  Bologna-Molina R.   Comparison of Fatty Acid Synthase and Cyclooxygenase-2 Immunoexpression in Embryonal, Benign, and Malignant Odontogenic Tissues Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2019 127 309 317 10.1016/j.oooo.2018.12.020 30692057 
50. Pontes H.A.  Pontes F.S.  Fonseca F.P.  de Carvalho P.L.  Pereira É.M.  de Abreu M.C.  de Freitas Silva B.S.  dos Santos Pinto D. Jr.   Nuclear Factor κ B and Cyclooxygenase-2 Immunoexpression in Oral Dysplasia and Oral Squamous Cell Carcinoma Ann. Diagn. Pathol. 2013 17 45 50 10.1016/j.anndiagpath.2012.04.008 22818026 
51. Itoh S.  Matsui K.  Furuta I.  Takano Y.   Immunohistochemical Study on Overexpression of Cyclooxygenase-2 in Squamous Cell Carcinoma of the Oral Cavity: Its Importance as a Prognostic Predictor Oral Oncol. 2003 39 829 835 10.1016/S1368-8375(03)00105-2 13679206 
52. Gallo O.  Masini E.  Bianchi B.  Bruschini L.  Paglierani M.  Franchi A.   Prognostic Significance of Cyclooxygenase-2 Pathway and Angiogenesis in Head and Neck Squamous Cell Carcinoma Hum. Pathol. 2002 33 708 714 10.1053/hupa.2002.125376 12196922 
53. Banerjee A.G.  Gopalakrishnan V.K.  Bhattacharya I.  Vishwanatha J.K.   Deregulated Cyclooxygenase-2 Expression in Oral Premalignant Tissues Mol. Cancer Ther. 2002 1 1265 1271 12516959 
54. Bôas D.S.  Takiya C.M.  Coelho-Sampaio T.L.  Monção-Ribeiro L.C.  Ramos E.A.  Cabral M.G.  dos Santos J.N.   Immunohistochemical Detection of Ki-67 is not Associated with Tumor-Infiltrating Macrophages and Cyclooxygenase-2 in Oral Squamous Cell Carcinoma J. Oral Pathol. Med. 2010 39 565 570 10.1111/j.1600-0714.2010.00883.x 20412400 
55. Pannone G.  Bufo P.  Caiaffa M.F.  Serpico R.  Lanza A.  Muzio L.L.  Rubini C.  Staibano S.  Petruzzi M.  de Benedictis M.    Cyclooxygenase-2 Expression in Oral Squamous Cell Carcinoma Int. J. Immunopathol. Pharmacol. 2004 17 273 282 10.1177/039463200401700307 15461861 
56. Peltanova B.  Raudenska M.  Masarik M.   Effect of Tumor Microenvironment on Pathogenesis of the Head and Neck Squamous Cell Carcinoma: A Systematic Review Mol. Cancer 2019 18 63 10.1186/s12943-019-0983-5 30927923 
57. Höing B.  Kanaan O.  Altenhoff P.  Petri R.  Thangavelu K.  Schlüter A.  Lang S.  Bankfalvi A.  Brandau S.   Stromal Versus Tumoral Inflammation Differentially Contribute to Metastasis and Poor Survival in Laryngeal Squamous Cell Carcinoma Oncotarget 2018 9 8415 8426 10.18632/oncotarget.23865 29492204 
58. Rosen R.D.  Sapra A.   TNM Classification Available online: https://www.ncbi.nlm.nih.gov/books/NBK553187/  (accessed on 2 November 2020) 
59. Kono M.  Watanabe M.  Abukawa H.  Hasegawa O.  Satomi T.  Chikazu D.   Cyclo-Oxygenase–2 Expression is Associated with Vascular Endothelial Growth Factor C Expression and Lymph Node Metastasis in Oral Squamous Cell Carcinoma J. Oral Maxillofac. Surg. 2013 71 1694 1702 10.1016/j.joms.2013.04.015 23809761 
60. Cha J.  Li S.  Cha I.   Association between Expression of Embryonic Lethal Abnormal Vision-like Protein HuR and Cyclooxygenase-2 in Oral Squamous Cell Carcinoma Head Neck 2011 33 627 637 10.1002/hed.21507 20960565 
61. Loong S.L.  Hwang J.S.  Li H.H.  Wee J.T.  Yap S.P.  Chua M.L.  Fong K.W.  Tan T.W.   Weak Expression of Cyclooxygenase-2 is Associated with Poorer Outcome in Endemic Nasopharyngeal Carcinoma: Analysis of Data from Randomized Trial between Radiation Alone Versus Concurrent Chemo-radiation (SQNP-01) Radiat. Oncol. 2009 4 23 10.1186/1748-717X-4-23 19591688 
62. Chen Y.  Luo R.  Li Y.  Cui B.  Song M.  Yang A.  Chen W.   High Expression Levels of COX-2 and P300 Are Associated with Unfavorable Survival in Laryngeal Squamous Cell Carcinoma Eur. Arch. Otorhinolaryngol. 2013 270 1009 1017 10.1007/s00405-012-2275-1 23179937 
63. Dong P.  Li X.  Yu Z.  Lu G.   Expression of Cyclooxygenase-2, Vascular Endothelial Growth Factor and Matrix Metalloproteinase-2 in Patients with Primary Laryngeal Carcinoma: A Tissue Microarray Study J. Laryngol. Otol. 2007 121 1177 1183 10.1017/S002221510700031X 17888194 
64. Yang Q.  Liu Y.  Huang Y.  Huang D.  Li Y.  Wu J.  Duan M.   Expression of COX-2, CD44v6 and CD147 and Relationship with Invasion and Lymph Node Metastasis in Hypopharyngeal Squamous Cell Carcinoma PLoS ONE 2013 8 e71048 10.1371/journal.pone.0071048 24019861 
65. Xu L.  Jiang Y.  Zheng J.  Xie G.  Li J.  Shi L.  Fan S.   Aberrant Expression of β -catenin and E-cadherin is Correlated with Poor Prognosis of Nasopharyngeal Cancer Hum. Pathol. 2013 44 1357 1364 10.1016/j.humpath.2012.10.025 23375645 
66. Sano Y.  Kogashiwa Y.  Araki R.  Enoki Y.  Ikeda T.  Yoda T.  Nakahira M.  Sugasawa M.   Correlation of Inflammatory Markers, Survival, and COX2 Expression in Oral Cancer and Implications for Prognosis Otolaryngol. Head Neck Surg. 2018 158 667 676 10.1177/0194599817745284 29359615 
67. Atula T.  Hedström J.  Ristimäki A.  Finne P.  Leivo I.  Markkanen-Leppänen M.  Haglund C.   Cyclooxygenase-2 Expression in Squamous Cell Carcinoma of the Oral Cavity and Pharynx: Association to p53 and Clinical Outcome Oncol. Rep. 2006 16 485 490 10.3892/or.16.3.485 16865247 
68. Kourelis K.  Vandoros G.  Kourelis T.  Papadas T.  Goumas P.  Sotiropoulou-Bonikou G.   Low COX2 in Tumor and Upregulation in Stroma Mark Laryngeal Squamous Cell Carcinoma Progression Laryngoscope 2009 119 1723 1729 10.1002/lary.20569 19554635 
69. Ranelletti F.O.  Almadori G.  Rocca B.  Ferrandina G.  Ciabattoni G.  Habib A.  Galli J.  Maggiano N.  Gessi M.  Lauriola L.   Prognostic Significance of Cyclooxygenase-2 in Laryngeal Squamous Cell Carcinoma Int. J. Cancer 2001 95 343 349 10.1002/1097-0215(20011120)95:63.0.co;2-d 11668514 
70. Zyada M.M.  Grawish M.E.  Elsabaa H.M.   Predictive Value of Cyclooxygenase 2 and Bcl-2 for Cervical Lymph Node Metastasis in Mucoepidermoid Carcinoma Ann. Diagn Pathol. 2009 13 313 321 10.1016/j.anndiagpath.2009.06.003 19751908 
71. Chan C.M.  Ma B.B.  Hui E.P.  Wong S.C.  Mo F.K.  Leung S.F.  Kam M.K.  Chan A.T.   Cyclooxygenase-2 Expression in Advanced Nasopharyngeal Carcinoma—A Prognostic Evaluation and Correlation with Hypoxia Inducible Factor 1α and Vascular Endothelial Growth Factor Oral Oncol. 2007 43 373 378 10.1016/j.oraloncology.2006.04.004 16979927 
72. Fang F.  Li C.  Chien C.  Rau K.  Huang H.   Immunohistochemical Expression of Epidermal Growth Factor Receptor and Cyclooxygenase-2 in Pediatric Nasopharyngeal Carcinomas: No Significant Correlations with Clinicopathological Variables and Treatment Outcomes Int. J. Pediatr. Otorhinolaryngol. 2007 71 447 455 10.1016/j.ijporl.2006.11.019 17208308 
73. Huang T.  Li C.  Huang H.  Fang F.   Correlations between Expression of Epidermal Growth Factor Receptor (EGFR), Phosphorylated EGFR, Cyclooxygenase-2 and Clinicopathological Variables and Treatment Outcomes in Nasopharyngeal Carcinomas Chang. Gung. Med. J. 2010 33 619 627 21199607 
74. Bron L.  Jandus C.  Andrejevic-Blant S.  Speiser D.E.  Monnier P.  Romero P.  Rivals J.   Prognostic Value of Arginase-II Expression and Regulatory T-cell Infiltration in Head and Neck Squamous Cell Carcinoma Int. J. Cancer 2013 132 E85 E93 10.1002/ijc.27728 22815199 
75. Sun D.  Zhao M.  Xia M.  Li L.  Jiang Y.   The Correlation between Tumor-Infiltrating Foxp3+ regulatory T cells and Cyclooxygenase-2 Expression and Their Association with Recurrence in Resected Head and Neck Cancers Med. Oncol. 2012 29 707 713 10.1007/s12032-011-9903-2 21424865 
76. Morita Y.  Morita N.  Hata K.  Nakanishi M.  Kimoto N.  Omata T.  Nakamura Y.  Yoneda T.   Cyclooxygenase-2 Expression is Associated with Vascular Endothelial Growth Factor-C and Lymph Node Metastasis in Human Oral Tongue Cancer Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2014 117 502 510 10.1016/j.oooo.2013.12.410 24560404 
77. Sakurai K.  Urade M.  Noguchi K.  Hashitani S.  Takaoka K.  Segawa E.  Kishimoto H.   Prognostic Significance of Cyclooxygenase-2 and DNA topoisomerase IIα Expression in Oral Carcinoma Head Neck 2007 29 1002 1009 10.1002/hed.20627 17427971 
78. Sappayatosok K.  Maneerat Y.  Swasdison S.  Viriyavejakul P.  Dhanuthai K.  Zwang J.  Chaisri U.   Expression of Pro-Inflammatory Protein, iNOS, VEGF and COX-2 in Oral Squamous Cell Carcinoma (OSCC), Relationship with Angiogenesis and Their Clinico-Pathological Correlation Med. Oral Patol. Oral Cir. Bucal. 2009 14 E319 E324 19300368 
79. Chen W.  McBride W.H.  Chen S.  Lee K.  Hwang T.  Jung S.  Shau H.  Liao S.  Hong J.  Chen M.   Prediction of Poor Survival by Cyclooxygenase-2 in Patients with T4 Nasopharyngeal Cancer Treated by Radiation Therapy: Clinical and in vitro Studies Head Neck 2005 27 503 512 10.1002/hed.20178 15772955 
80. Sekimizu M.  Ozawa H.  Saito S.  Ikari Y.  Nakahara N.  Nakamura S.  Yoshihama K.  Ito F.  Watanabe Y.  Imanishi Y.    Cyclo-oxygenase-2 Expression is Associated with Lymph Node Metastasis in Oropharyngeal Squamous Cell Carcinoma Under the New TNM Classification Anticancer. Res. 2019 39 5623 5630 10.21873/anticanres.13758 31570459 
81. Kyzas P.A.  Stefanou D.  Agnantis N.J.   COX-2 Expression Correlates with VEGF-C and Lymph Node Metastases in Patients with Head and Neck Squamous Cell Carcinoma Mod. Pathol. 2005 18 153 160 10.1038/modpathol.3800244 15272284 
82. Kim K.  Li S.  Cha J.  Zhang X.  Cha I.   Significance of Molecular Markers in Survival Prediction of Oral Squamous Cell Carcinoma Head Neck 2012 34 929 936 10.1002/hed.21856 22128038 
83. Kim K.  Cha I.   A Novel Algorithm for Lymph Node Status Prediction of Oral Cancer before Surgery Oral Oncol. 2011 47 1069 1073 10.1016/j.oraloncology.2011.07.017 21840750 
84. Kim T.  Lee Y.S.  Kang J.  Kim Y.  Kang C.S.   Prognostic Significance of Expression of VEGF and Cox-2 in Nasopharyngeal Carcinoma and its Association with Expression of C-erbB2 and EGFR J. Surg. Oncol. 2011 103 46 52 10.1002/jso.21767 21031415 
85. Erovic B.M.  Pelzmann M.  Turhani D.  Pammer J.  Niederberger V.  Neuchrist C.  Grasl M.C.  Thurner D.   Differential Expression Pattern of Cyclooxygenase-1 and -2 in Head and Neck Squamous Cell Carcinoma Acta OtoLaryngol. 2003 123 950 953 10.1080/00016480310016118 14626203 
86. Lim S.  Zhang S.  Ishii G.  Endoh Y.  Kodama K.  Miyamoto S.  Hayashi R.  Ebihara S.  Cho J.  Ochiai A.   Predictive Markers for Late Cervical Metastasis in Stage I and II Invasive Squamous Cell Carcinoma of the Oral Tongue Clin. Cancer Res. 2004 10 166 172 10.1158/1078-0432.CCR-0533-3 14734465 
87. Rakesh N.  Iyengar A.  Majumdar K.  Vidya G.S.  Kumar S.S.   Quantitative Evaluation of Tumour-Associated Tissue Eosinophilia and Cyclo-oxegenase-2 Gene in Oral Cancer Patients with Assessment of Long Term Outcomes Pathol. Oncol. Res. 2016 22 385 392 10.1007/s12253-015-0016-7 26577687 
88. Ryott M.  Marklund L.  Wangsa D.  Elmberger G.  Munck-Wikland E.   Cyclooxygenase-2 Expression in Oral Tongue Squamous Cell Carcinoma J. Oral Pathol. Med. 2011 40 385 389 10.1111/j.1600-0714.2010.00992.x 21481000 
89. Sackett M.K.  Bairati I.  Meyer F.  Jobin E.  Lussier S.  Fortin A.  Gélinas M.  Nabid A.  Brochet F.  Têtu B.   Prognostic Significance of Cyclooxygenase-2 Overexpression in Glottic Cancer Clin. Cancer Res. 2008 14 67 73 10.1158/1078-0432.CCR-07-2028 18172254 
90. Yang B.  Jia L.  Guo Q.  Ren H.  Hu Y.  Xie T.   Clinicopathological and Prognostic Significance of Cyclooxygenase-2 Expression in Head and Neck Cancer: A Meta-Analysis Oncotarget 2016 7 47265 47277 10.18632/oncotarget.10059 27323811 
91. Filho J.A.  Nonaka C.F.  da Costa Miguel M.C.  de Almeida Freitas R.  Galvão H.C.   Immunoexpression of Cyclooxygenase-2 and p53 in Oral Squamous Cell Carcinoma Am. J. Otolaryngol. 2009 30 89 94 10.1016/j.amjoto.2008.02.012 19239949 
92. Pan J.  Tang T.  Xu L.  Lu J.J.  Lin S.  Qiu S.  Chen G.  Tham I.W.   Prognostic Significance of Expression of Cyclooxygenase-2, Vascular Endothelial Growth Factor, and Epidermal Growth Factor Receptor in Nasopharyngeal Carcinoma Head Neck 2013 35 1238 1247 10.1002/hed.23116 22972415 
93. Chang B.W.  Kim D.H.  Kowalski D.P.  Burleson J.A.  Son Y.H.  Wilson L.D.  Haffty B.G.   Prognostic Significance of Cyclooxygenase-2 in Oropharyngeal Squamous Cell Carcinoma Clin. Cancer Res. 2004 10 1678 1684 10.1158/1078-0432.CCR-03-0354 15014019 
94. Haffner M.C.  Laimer J.  Chaux A.  Schäfer G.  Obrist P.  Brunner A.  Kronberger I.E.  Laimer K.  Gurel B.  Koller J.    High Expression of Prostate-Specific Membrane Antigen in the Tumor-Associated Neo-Vasculature is Associated with Worse Prognosis in Squamous Cell Carcinoma of the Oral Cavity Mod. Pathol. 2012 25 1079 1085 10.1038/modpathol.2012.66 22460809 
95. Baghban A.A.  Taghavi N.  Shahla M.   Combined Analysis of Vascular Endothelial Growth Factor Expression with Cyclooxygenase-2 and Mast Cell Density in Oral Squamous Cell Carcinoma Pathobiology 2017 84 80 86 10.1159/000447778 27577989 
96. Seki S.  Fujiwara M.  Matsuura M.  Fujita S.  Ikeda H.  Asahina I.  Ikeda T.   Prediction of Outcome of Patients with Oral Squamous Cell Carcinoma Using Vascular Invasion and the Strongly Positive Expression of Vascular Endothelial Growth Factors Oral Oncol. 2011 47 588 593 10.1016/j.oraloncology.2011.04.013 21602095 
97. Hwa J.S.  Kwon O.J.  Park J.J.  Woo S.H.  Kim J.P.  Ko G.H.  Seo J.H.  Kim R.B.   The Prognostic Value of Immunohistochemical Markers for Oral Tongue Squamous Cell Carcinoma Eur. Arch. Otorhinolaryngol. 2015 272 2953 2959 10.1007/s00405-014-3254-5 25169079 
98. Lindquist D.  Ahrlund-Richter A.  Tarján M.  Tot T.  Dalianis T.   Intense CD44 Expression is a Negative Prognostic Factor in Tonsillar and Base of Tongue Cancer Anticancer. Res. 2012 32 153 161 22213301 
99. Kekatpure V.D.  Singh M.  Selvam S.  Shetkar G.  Hedne N.C.  Trivedi N.P.  Siddappa G.  Govindan S.V.  Suresh A.  Rangarajan B.    Factors Predicting Outcome After Salvage Treatment for Stage IV Oral Squamous Cell Carcinoma: Evidence of the Potential Importance of the Cyclooxygenase-2–Prostaglandin E2 Pathway Head Neck 2015 37 1142 1149 10.1002/hed.23721 24771596 
100. Wildeman M.A.  Gibcus J.H.  Hauptmann M.  Begg A.C.  van Velthuysen M.L.  Hoebers F.J.  Mastik M.F.  Schuuring E.  van der Wal J.E.  van den Brekel M.W.   Radiotherapy in Laryngeal Carcinoma: Can a Panel of 13 Markers Predict Response? Laryngoscope 2009 119 316 322 10.1002/lary.20069 19160427 
101. Wang Z.  Liu J.  Liu H.  Ye M.  Zhang Y.  Yang D.   Abnormal COX2 Protein Expression May Be Correlated with Poor Prognosis in Oral Cancer: A Meta-Analysis Biomed. Res. Int. 2014 2014 364207 10.1155/2014/364207 25028647 
102. Lin Y.  Huang H.  Wang L.  Tsai C.  Lung O.  Dai C.  Yu M.  Ho C.  Chen C.   Polymorphisms of COX-2 -765G > C and p53 Codon 72 and Risks of Oral Squamous Cell Carcinoma in a Taiwan Population Oral Oncol. 2008 44 798 804 10.1016/j.oraloncology.2007.10.006 18234542 
103. Pu X.  Lippman S.M.  Yang H.  Lee J.J.  Wu X.   Cyclooxygenase-2 Gene Polymorphisms Reduce the Risk of Oral Premalignant Lesions Cancer 2009 115 1498 1506 10.1002/cncr.24157 19197984 
104. Mittal M.  Kapoor V.  Mohanti B.K.  Das S.N.   Functional Variants of COX-2 and Risk of Tobacco-Related Oral Squamous Cell Carcinoma in High-Risk Asian Indians Oral Oncol. 2010 46 622 626 10.1016/j.oraloncology.2010.06.002 20620096 
105. Lakshmi A.  Muralidhar S.  Kumar C.K.  Kumar A.P.  Chakravarthy P.K.  Anjaneyulu V.  Kaiser J.   Cyclooxygenase-2−765G>C Functional Promoter Polymorphism and Its Association with Oral Squamous Cell Carcinoma J. Investig. Clin. Dent. 2012 3 182 188 10.1111/j.2041-1626.2011.00104.x 
106. Peters W.H.  Lacko M.  te Morsche R.H.  Voogd A.C.  Ophuis M.B.  Manni J.J.   COX-2 Polymorphisms and the Risk for Head and Neck cancer in White Patients Head Neck 2009 31 938 943 10.1002/hed.21058 19280666 
107. Chiang S.  Chen P.  Lee C.  Ko A.M.  Lee K.  Lin Y.  Ho P.  Tu H.  Wu D.  Shieh T.    Up-regulation of Inflammatory Signalings by Areca Nut Extract and Role of Cyclooxygenase-2 -1195G>A Polymorphism Reveal Risk of Oral Cancer Cancer Res. 2008 68 8489 8498 10.1158/0008-5472.CAN-08-0823 18922923 
108. Zhu W.  Wei B.  Shan X.  Liu P.   -765G>C and 8473T>C Polymorphisms of COX-2 and Cancer Risk: A Meta-Analysis Based on 33 Case–Control Studies Mol. Biol. Rep. 2010 37 277 288 10.1007/s11033-009-9685-1 19669667 
109. Lee W.T.  Huang C.C.  Chen K.C.  Wong T.Y.  Ou C.Y.  Tsai S.T.  Yen C.J.  Fang S.Y.  Lo H.I.  Wu Y.H.    Genetic Polymorphisms in the Prostaglandin Pathway Genes and Risk of Head and Neck Cancer Oral Dis. 2015 21 207 215 10.1111/odi.12244 24724948 
110. Campa D.  Hashibe M.  Zaridze D.  Szeszenia-Dabrowska N.  Mates I.N.  Janout V.  Holcatova I.  Fabiánová E.  Gaborieau V.  Hung R.J.    Association of Common Polymorphisms in Inflammatory Genes with Risk of Developing Cancers of the Upper Aerodigestive Tract Cancer Causes Control. 2007 18 449 455 10.1007/s10552-007-0129-8 17356794 
111. Deng D.  Xia L.  He B.  Guo J.  Huang C.  Zeng X.   Cyclooxygenase-2-1195G>A Polymorphism and Head and Neck Squamous Cell Carcinoma Susceptibility: A Meta-Analysis of 1564 Cases and 2346 Controls Med. Sci. Monit. 2015 21 3514 3520 10.12659/MSM.894948 26568042 
112. Li D.  Hao S.  Sun Y.  Hu C.  Ma Z.  Wang Z.  Liu J.  Liu H.  Ye M.  Zhang Y.    Functional Polymorphisms in COX-2 Gene Are Correlated with the Risk of Oral Cancer Biomed. Res. Int. 2015 2015 580652 10.1155/2015/580652 25977924 
113. Leng W.  Wen X.  Kwong J.S.  Huang W.  Chen J.  Zeng X.   COX-2 rs689466, rs5275, and rs20417 Polymorphisms and Risk of Head and Neck Squamous Cell Carcinoma: A Meta-Analysis of Adjusted and Unadjusted data BMC Cancer 2016 16 457 10.1186/s12885-016-2535-3 27412115 
114. Janakiraman H.  House R.P.  Talwar S.  Courtney S.M.  Hazard E.S.  Hardiman G.  Mehrotra S.  Howe P.H.  Gangaraju V.  Palanisamy V.   Repression of caspase-3 and RNA-binding protein HuR cleavage by cyclooxygenase-2 promotes drug resistance in oral squamous cell carcinoma Oncogene 2017 36 3137 3148 10.1038/onc.2016.451 27941877