Open Access

Functional analysis of polymorphisms in the COX‑2 gene and risk of lung cancer

  • Authors:
    • Joyce L. Moraes
    • Amanda B. Moraes
    • Veronica Aran
    • Marcelo R. Alves
    • Luciene Schluckbier
    • Mariana Duarte
    • Edson Toscano
    • Mauro Zamboni
    • Cinthya Sternberg
    • Emanuela de Moraes
    • José R. Lapa E Silva
    • Carlos Gil Ferreira
  • View Affiliations

  • Published online on: February 16, 2017     https://doi.org/10.3892/mco.2017.1167
  • Pages: 494-502
  • Copyright: © Moraes et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The enzyme cyclooxygenase 2 (COX‑2) is known to be involved in tumorigenesis and metastasis in certain types of cancer. Nevertheless, the prognostic value of COX‑2 overexpression and its polymorphisms in patients with non‑small cell lung cancer (NSCLC) have yet to be fully elucidated. The aim of the present study was to investigate the association between the three most commonly studied COX‑2 gene polymorphisms (‑1195 G/A, ‑765 G/C and 8473 T/C) with COX‑2 expression and lung cancer risk in a Brazilian cohort. In the present hospital based, case‑control retrospective study, 104 patients with NSCLC and 202 cancer free control subjects were genotyped for ‑1195 G/A, ‑765 G/C and 8473 T/C polymorphisms using allelic discrimination with a reverse transcription quantitative polymerase chain reaction method. COX‑2 mRNA expression was analyzed in surgically resected tumors from 34 patients with NSCLC. The results revealed that COX‑2 expression levels were higher in tumor tissue compared with normal lung tissue. However, this overexpression of COX‑2 was not associated with the patient outcome, and furthermore, none of the analyzed polymorphisms were associated with the risk of developing lung cancer, COX‑2 overexpression, or the overall survival of the patients with NSCLC. Taken together, the findings described in the present study do not support a major role for COX‑2 polymorphisms and COX‑2 overexpression in lung carcinogenesis within the Brazilian population.

Introduction

Lung cancer is the most common cause of cancer-associated mortality throughout the world (1). The incidence rates and deaths associated with this cancer type have risen markedly over the last century, correlating with an increase in cigarette consumption (1,2). Although accumulative evidence suggests that >80% of lung cancers are attributed to tobacco exposure, <20% of smokers develop lung cancer, suggesting that genetic susceptibility exerts an important role in the etiology of lung cancer (3,4). Several different pathways are involved in the pathogenesis of lung cancer, of which inflammatory processes and genes involved in the associated functional pathways have been of emerging research interest (5).

Cigarette smoke triggers bronchial epithelial cells to produce pro-inflammatory cytokines (e.g., interleukin-1β), and to up-regulate several inflammation-associated genes, including cyclooxygenase-2 (COX-2) (68). Cyclooxygenase is an important enzyme required for the conversion of arachidonic acid into prostaglandins (PGs) and thromboxane. Two different COX isoforms have been described, termed COX-1 and COX-2, which possess different properties (9,10). COX-1 is constitutively expressed in the majority of the cells and tissues, whereas COX-2 is inducible, and is expressed in response to cytokines, growth factors and other stimuli (11,12). Different solid tumor types have been demonstrated to overexpress COX-2, including those of the colon (13), prostate (14), breast (15), esophagus (16), lung (17), and pancreas (18). These tumors contained high concentrations of prostaglandin E2 (PGE2), a subproduct of the enzymatic action of COX-2 (19). PGE2 is able to affect cell proliferation, apoptosis and angiogenesis, thus contributing to tumor progression (20).

The mechanisms underpinning the regulation of COX-2 expression have yet to be fully elucidated, and may be influenced by genetic variations. A number of genetic variants that may affect enzyme expression have been described in regions proximal to the regulatory sites of the COX-2 gene (21,22), and could contribute to an increased risk of cancer development. It was also suggested that single nucleotide polymorphisms (SNPs) in the COX-2 gene may alter enzyme function, which could influence an individual's risk of any type of cancer (23).

The polymorphism, −1195 A/G, was revealed to influence transcription levels of COX-2, where the A-allele had an increased rate of transcription compared with the G-allele in in vivo studies of esophageal tissues and in luciferase reporter assays performed in HeLa cells (22). Another SNP, −765 G/C, resulted in lower promoter activity, which subsequently led to a lower expression of COX-2 (24). The C-allele of the polymorphism −765 G/C is associated with markedly reduced expression levels of COX-2 compared with the G-allele, and this effect might be mediated by the loss of Sp1 transcription factor binding to its cognate element (21). Recently, the polymorphism −1195 A/G was reported not to be associated with susceptibility to oral cancer, whereas −765 G/C and +837 T/G presented a clear link (25).

Stability of COX-2 mRNA and the synthesis of COX-2 may be increased by genetic variations in the 3′untranslated region (3′UTR) of the prostaglandin endoperoxide synthase 2 (PTGS2) gene (an alternative name for COX-2). The 3′UTR region of COX-2 has been shown to be an important determinant of the stability of the mRNA, and thus, of the enzyme levels (26). Carriers of the C-allele of COX-2 8473 T/C were revealed to exhibit higher basal and induced levels of PGE2 compared with the T-allele (27). In addition, the frequency of SNPs in the PTGS2 gene may vary between different ethnic groups (28,29). Certain polymorphisms in the COX-2 promoter region may explain the different levels of COX-2 expression that have been identified in lung tumors. The frequency of these polymorphisms was previously determined in a Brazilian population (30); however, its influence on COX-2 expression has yet to be elucidated. In the present study, the impact of three different polymorphisms in the COX-2 gene (−1195 G/A, −765 G/C and 8473 T/C) on COX-2 expression was investigated, as well as its influence on the risk of lung cancer in a Brazilian cohort. Furthermore, the expression of COX-2 was analyzed with respect to the overall survival of patients with NSCLC.

Patients and methods

Study population

The present case-control study included 104 patients with NSCLC and 202 cancer-free control subjects. The eligible cases included patients who were diagnosed with primary NSCLC between June 2005 and February 2008 at the Brazilian National Cancer Institute (INCA), Rio de Janeiro, Brazil. There were no age, gender, or stage restrictions; however, patients with prior cancer history were excluded from this study. The control subjects were cancer-free individuals selected from INCA's Tobacco Treatment Program (http://www2.inca.gov.br/wps/wcm/connect/acoes_programas/site/home/nobrasil/programa-nacional-controle-tabagismo).

Volunteers were personally interviewed by trained personnel using a structured questionnaire to determine demographic characteristics and potential risk factors for lung cancer. Information regarding the clinical history was obtained from medical records. All subjects were informed and provided with a written consent form, in order to participate in the study and to allow their biological samples to be genetically analyzed. The study was approved by the Ethics Committee of INCA (Protocol 79/05). The clinicopathological features of the patients with NSCLC are shown in Table I.

Table I.

The general clinical and pathological features of our patients with NSCLC.

Table I.

The general clinical and pathological features of our patients with NSCLC.

Patient characteristicsCases n (%)
Histological cell type
  Adenocarcinoma54 (51.9)
  Squamous cell41 (39.4)
  Other9 (8.7)
Tumor differentiation
  Poor12 (11.5)
  Moderate32 (30.8)
  Well4 (3.8)
  Undifferentiated3 (2.9)
Stage
  I/II38 (38)
  III/IV62 (62)
Performance status
  0–114 (13.9)
  2–387 (86.1)

[i] NSCLC, non-small cell lung cancer.

COX-2 genotyping

Genomic DNA was extracted from peripheral blood from all volunteers (cases and control subjects) using the QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA), following the manufacturer's protocol. All subjects were genotyped by allelic discrimination using a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. The assay reagents for genotyping the SNPs were obtained from the Assays-by-Design service of Applied Biosystems (Applied Biosystems), and consisted of a 40X mix of unlabeled PCR primers and TaqMan minor groove binder probes [fluorescein (FAM) and VIC™ fluorochrome dye-labeled]. These assays were designed for the genotyping of the specific SNPs as follows: −1195 G/A: Forward primer, CCT GAG CAC TAC CCA TGA TAG ATGT, reverse primer, GGA ACA TAG TTG GGT GAG GGA TTAA; probes: VIC-, AAG ATG AAA TTC CAA CTG TCA, FAM-, ATG AAA TTC CAG CTG TCA; −765 G/C: Forward primer, TGC TTA GGA CCA GTA TTA TGA GGA GAA; reverse primer, CCC CCT CCT TGT TTC TTG GAA; probes: VIC-, CTT TCC CGC CTC TCT, FAM-, CCT TTC CCC CCT CTCT; 8473 T/C: Forward primer, GCA TCT TCC ATG ATG CAT TAG AAG TAAC, reverse primer, GCA CTG ATA CCT GTT TTT GTT TGA TGA; probes: VIC-, CTT TTG GTC ATT TTTC, FAM-, ACT TTT GGT TAT TTT TC. Each assay enabled the scoring of the two genotypes in a single well. The probes were distinguished by labeling them with a different fluorescent reporter dye (i.e., FAM dye or VIC dye). RT-qPCR analysis was performed using an ABI Prism 7500 Fast instrument (Applied Biosystems). A marked increase in either FAM or VIC dye fluorescence indicated homozygosis for the FAM- or the VIC-specific allele, respectively, whereas an increase in the two signals indicated heterozygosis.

RNA isolation and relative quantification via RT-qPCR

A total of 34 tumor tissue samples were obtained from surgically removed specimens of individual patients. Total RNA was isolated from tissues, using TRIzol reagent (Invitrogen Life Technologies). FirstChoice® PCR-Ready Human Lung cDNA (cat. no. AM3327; Ambion®; Applied Biosystems) was used for the analysis. An aliquot of total RNA (2 µg) from each specimen was reverse-transcribed into single-strand complementary DNA (cDNA) using oligo(dT) 15 primer and Superscript II (Invitrogen Life Technologies). Relative gene expression quantification for COX-2, with β-actin as an internal reference gene, was performed using the ABI Prism 7500 Sequence Detection system (Applied Biosystems) based on the TaqMan method available at the company's website. The primer used for COX-2 was Hs00153133_m1, and that for β-actin was 4352935E (as featured in the Taqman® gene expression assay).

Relative quantification was performed using the comparative threshold cycle (Cq) method of RT-qPCR, and data were expressed on the logarithmic scale (31). Subsequently, for each tissue sample (n=34), the expression levels of COX-2 and the endogenous control protein, β-actin, were estimated in duplicate using RT-qPCR for 40 cycles, with the arithmetic average threshold cycle (Cq) used for data analysis. To control variations in the amount of RNA input, reactions were performed with the β-actin probe used as the internal control. Furthermore, for each RNA sample tissue, negative control reactions with: i) Negative controls of cDNA synthesis (i.e., without reverse transcriptase) and ii) no-template controls were performed in duplicate. Subsequently, relative gene expression levels for COX-2 were calculated according to the 2ΔCq method (31), with ΔCq [COX-2] values determined using the formula: ΔCq [COX-2]=Cq [COX-2]-Cq [β-actin]. The degree of significance of the mean difference between tumor tissue and the control cDNA (FirstChoice® PCR-Ready Human Lung; Ambion®; Applied Biosystems) was estimated from the log-transformed, normalized expression levels.

Statistical methods

The Hardy-Weinberg equilibrium was examined to compare genotype COX-2 polymorphism frequencies among case subjects and controls. A linkage disequilibrium analysis among the SNPs was performed using the statistical parameters, D' and r2. Odds ratios and 95% confidence intervals were calculated using logistic regression in order to estimate the risk for lung cancer. SNPs and COX-2 expression association was analyzed using Mann-Whitney's t-test. Regarding the overall survival rate, times were obtained from the date of diagnosis to death. Time-to-death parameters were estimated using the Kaplan-Meier method, and data were compared using the log-rank test. Cox proportional hazards models were used to estimate the risk factors in a multivariate model. P<0.05 was considered to indicate a statistically significant value. Statistical analyses were performed using SPSS 20.0 for Windows software (IBM SPSS, Armonk, NY, USA).

Results

Demographic characteristics of patients and control subjects

The general clinical and pathological features of patients with NSCLC are shown in Table I. The demographic variables and risk factors of lung cancer for the cases of the 104 patients with NSCLC and the 200 controls included in the analysis are shown in Table II. The cases and controls appeared to be adequately matched regarding color as suggested by χ2 test. However, the case group had a higher prevalence of men, people aged >60 years old and non-smokers than did the control group (P<0.05). These differences were controlled in the later multivariate analyses.

Table II.

Demographic variables and risk factors of lung cancer of cases and controls.

Table II.

Demographic variables and risk factors of lung cancer of cases and controls.

VariablesCases n (%) 104 (100%)Controls n (%) 200 (100%) P-valuea
Sex
  Male70 (67.3)71 (35.5)<0.001
  Female34 (32.7)129 (64.5)
Color
  White52 (50)84 (42.0)0.412
  Intermediate32 (30.8)71 (65.5)
  Black20 (19.2)45 (22.5)
Age
  >60 years63 (60.6)26 (13.0)<0.001
  ≤60 years41 (39.4)174 (87.0)
Smoking status
  Non-smokers7 (6.7)5 (2.5)0.036
  Light smokers16 (15.4)55 (27.5)
  Moderate smokers41 (39.5)78 (39.0)
  Heavy smokers40 (38.4)62 (31.0)
Quit smoking
  Non-smokers7 (6.7)5 (2.5)<0.001
  ≤12 months42 (40.4)44 (22.0)
  >12a ≤120 months14 (13.5)9 (4.5)
  >120 months16 (15.4)0 (0)
  Smokers25 (24.0)142 (71.0)
Drinking
  No57 (54.8)62 (31.0)<0.001
  Yes47 (45.2)138 (69.0)

a Compared by χ2 test.

COX-2 polymorphisms and occurrence of lung cancer

A total of 340 participants (200 volunteer donors and 104 patients with NSCLC) were genotyped for the COX-2 polymorphisms, −1195G/A, −765G/C and 8473 T/C, as shown in Table III. For COX-2 −1195 G/A, 72 (18%) patients and 36 (17%) controls carried the G-allele. For COX-2 −765 G/C, 299 (75%) patients and 101 (25%) controls carried the G-allele. For COX-2 8473 G/C, 244 (61%) patients and 164 (79%) controls carried the T-allele. All genotypic distributions were consistently within the limits defined by the Hardy-Weinberg equilibrium (P>0.05).

Table III.

ORs (odds ratios) for lung cancer in relation to the studied polymorphisms.

Table III.

ORs (odds ratios) for lung cancer in relation to the studied polymorphisms.

SNP Genotype/alleleCases n (%)Controls n (%)OR (95% IC)aP-value
G1195AA172 (83)328 (82)Ref
G36 (17)72 (18)1.049 (0.675–1.629)0.212
AA71 (68.3)138 (69)Ref
AG30 (28.8)52 (26)1.186 (0.567–2.479)0.651
GG3 (2.9)10 (5)3.498 (0.555–22.037)0.182
AG+GG33 (31.7)62 (31)1.381 (0.692–2.754)0.360
G765CG164 (79)299 (75)Ref
C44 (21)101 (25)0.7943 (0.531–1.187)0.261
GG66 (63.5)112 (56)Ref
GC32 (30.8)75 (37.5)0.967 (0.404–2.314)0.940
CC6 (5.8)13 (6.5)1.224 (0.220–6.819)0.817
GC+CC38 (36.5)88 (44)1.046 (0.456–2.399)0.916
T8473CT131 (63)244 (61)Ref
C77 (37)156 (39)0.919 (0.651–1.299)0.477
TT44 (42.3)69 (34.5)Ref
TC43 (41.3)106 (53)1.803 (0.778–4.182)0.169
CC17 (16.3)25 (12.5)1.414 (0.359–5.567)0.621
TC+CC60 (57.7)131 (65.5)1.726 (0.751–3.966)0.198

a ORs adjusted by gender, age, smoking status and quit smoking. IC, confidence interval.

Subsequently, the association between the SNPs, −1195G/A, −765G/C and 8473 T/C, and the occurrence of lung cancer was examined. No risk association was identified for the distribution of the genotypes between the overall lung cancer cases and the controls (P>0.05), as shown in Table III. Regarding linkage disequilibrium, the findings of the present study indicated strong pairwise linkage disequilibrium involving the SNPs, −765 and 8473 (D'=0.9807, r2=0.58). By contrast, no significant association was demonstrated between the SNPs −765 and −1195 (D'=0.6064, r2=0.027) or −1195 and 8473 (D'=0.7050, r2=0.069).

Overall survival analysis according to COX-2 polymorphisms and COX-2 expression

The overall survival analysis was calculated and correlated with COX-2 polymorphism and expression. The polymorphisms were grouped according to the variant allele: Homozygous for the variant allele compared with homozygous for the wild-type and heterozygous. The 2-year overall survival rates for patients with the −1195 G/A AA and AG/GG genotypes were 43.1% and 52.8, respectively (P=0.360; Fig. 1A). For the −765 G/C polymorphism, these rates were 39.7% for GG, and 64.1% for GC/CC, patients (P=0.758; Fig. 1B). For the 8473 T/C SNP, the 2-year overall survival rates were 51.8 and 42.6% for the TT and TC/CC alleles, respectively (P=0.684; Fig. 1C). No differences in median overall survival were identified with respect to the three COX-2 polymorphisms studied. Subsequently, whether the expression of COX-2 may be a potential prognostic biomarker for patients with lung cancer was examined. The 34 patients in which COX-2 expression had been previously determined were stratified into high and low expression groups according to the median. Neither the high nor the low expression of COX-2 was identified to be a prognostic indicator for patients with NSCLC (P=0.235; Fig. 1D).

COX-2 expression in lung tumor vs. normal lung tissue and according to the COX-2 polymorphism

To evaluate COX-2 expression in tumor tissue, 34 patients with NSCLC who underwent surgery were included in the present study. The clinical and demographic characteristics of these 34 patients are shown in Table IV. The relative mRNA expression of COX-2 in tumor tissues of cancer patients was determined using RT-qPCR and compared with FirstChoice® PCR-Ready Human Lung cDNA as the reference standard. As shown in Fig. 2A, a difference in COX-2 expression levels between tumor tissue and the commercially available normal lung tissue was observed. The tumor tissue had a higher expression level compared with the normal lung tissue (P=0.001), with an average expression of 2,654 (95% confidence interval =212.08 to −5,096.70).

Table IV.

Clinic and demographic variables of lung cancer patients who underwent surgery.

Table IV.

Clinic and demographic variables of lung cancer patients who underwent surgery.

VariableCases n (%) 34 (100)
Gender
  Male19 (55.9)
  Female15 (44.1)
Color
  White20 (58.8)
  Intermediate9 (26.5)
  Black5 (14.7)
Age
  >60 years16 (47.1)
  ≤60 years18 (52.9)
Smoking status
  Non-smokers2 (5.9)
  Light smokers7 (20.6)
  Moderate smokers13 (38.2)
  Heavy smokers12 (35.3)
Quit smoking
  Non-smokers4 (11.8)
  ≤12 months15 (44.1)
  >12a ≤120 months4 (11.8)
  >120 months8 (23.5)
  Smokers2 (5.9)
Drinking
  No16 (47.1)
  Yes18 (52.9)
Histological cell type
  Adenocarcinoma19 (55.9)
  Squamous cell15 (44.1)
  Others
Tumor differentiation
  Poor5 (14.7)
  Moderate20 (58.8)
  Well4 (11.8)
  Undifferentiated0 (0)
Stage
  I/II22 (64.7)
  III/IV10 (29.4)
Performance status
  0–131 (91.2)
  2–31 (2.9)

The expression of COX-2 was also evaluated according to the genotypes for each COX-2 polymorphism studied. As shown in Fig. 2B-D, no significant differences were identified when compared with COX-2 expression for the −1195 G/A (P=0.446; Fig. 2B), −765 G/C (P=0.843, Fig. 2C) and 8473 T/C (P=0.545; Fig. 2D) COX-2 polymorphisms.

Discussion

COX-2 is overexpressed in premalignant and malignant stages of lung, colon, and breast cancer, suggesting that COX-2 may serve an important functional role from the earliest hyperproliferative stages of the disease to the later stages of invasive carcinoma (32). Therefore, the present study has been, to the best of our knowledge, the first to address the role of COX-2 expression and the −1195G/A, −765G/C and 8473 T/C polymorphisms in lung cancer within the Brazilian context.

In the present retrospective study, the frequency and linkage of COX-2 polymorphisms was analyzed. Unlike previous studies (22,23,3335), no significant associations were identified between the SNPs, −765 G/C and −1195 G/A. On the other hand, corroborating the results of another Brazilian study (30), a genetic linkage between the −765 G/C and 8473 T/C polymorphisms was demonstrated. This linkage between them reveals a non-random distribution of these proteins. Therefore, the contributions of −765 G/C and 8473 T/C towards COX-2 expression and activity may be difficult to delineate.

Secondly, the influence of these COX-2 polymorphisms on the occurrence of lung cancer was analyzed. No association was identified between these polymorphisms and this tumor type in our case-control study. Notably, there are conflicting reports concerning the impact of −1195 G/A, −765 G/C and 8473 T/C polymorphisms on the risk of cancer in the literature (22,3537). Supporting our findings, a meta-analysis study performed by Wang et al (38) with 29,487 cancer patients and 3,921 controls demonstrated that −765 C carriers are at a significantly increased risk of contracting gastric cancer, leukemia, and pancreatic cancer, but not of other cancer types, including lung cancer. Furthermore, Tang et al (39) demonstrated that −1195 G/A is a low penetration risk factor for cancer. In addition, Pan et al (40), also in a meta-analysis study with 4,373 lung cancer patients and 5,468 controls, demonstrated that the 8473 T/C polymorphism is not associated with any risk of lung cancer. These negative results could be explained by the fact that the functional mechanisms of such COX-2 polymorphisms may not be responsible for lung carcinogenesis, or they may be attributable to the source of controls for each study or methodological deficiencies in the analysis.

One possible reason for COX-2 polymorphisms being only associated with certain cancer types (22,28,36,37,41) might be due to different cancers having distinct molecular signatures. For example, the lung carcinogenesis activation pathway occurs through a variety of mechanisms, including activating mutations in the genes for epidermal growth factor receptor (EGFR), KRAS, p53, and also echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusions. The oncogene, KRAS, is mutated in ~30% of cases of lung cancer (42); the mutational status of the EGFR gene has been shown to be correlated with responsiveness to small molecule kinase inhibitors (e.g., gefitinib or erlotinib) (43,44); and tumor protein 53 (TP53) inactivation is one of the most significant genetic abnormalities described in lung cancer, occurring in ~90% of small cell carcinomas and ~65% of cases of NSCLC (45). Taken together, these differences may also influence the impact of the COX-2 polymorphisms on the lung carcinogenesis process.

The COX-2 8473 T/C polymorphism may contribute to NSCLC cancer susceptibility in the Kashmiri population (46). Bi et al (47) demonstrated that the −1195 G/A polymorphism may be able to predict survival in patients with lung cancer. Their functional study revealed that the nucleotide base change of −1195 G to A creates a c-MYB binding site in the COX-2 promoter region and, thus, displays a higher promoter activity. Therefore, the −1195 AA genotype, which results in increased COX-2 expression, was associated with poor overall survival in Chinese patients. To validate this hypothesis, the influence of −1195 G/A was analyzed in the present study in addition to, for the first time to the best of our knowledge, the −765 G/C and 8473 T/C polymorphisms in the outcome of the Brazilian patients with NSCLC. However, differences in overall survival regarding the three COX-2 polymorphisms studied were not observed. One reason for this discrepancy may be the fact that certain genetic markers are ethnicity-specific. Further prospective clinical trials with a larger sample size comprising different ethnic populations are required to solve these controversial results.

In the present retrospective study, COX-2 mRNA expression in the tumor lung tissue of 34 patients with NSCLC in comparison with a commercially obtained, normal lung tissue mRNA was also analyzed using RT-qPCR. It was observed that the tumor tissue had higher expression levels of COX-2 mRNA compared with normal tissue (Fig. 2A). Bhat et al (46) also observed a significant increase in the level of COX-2 in NSCLC tumor tissues when compared with normal lung tissues. Generally speaking, COX-2 is expressed in ~40–80% of neoplastic cells, and the level of expression is higher in cancerous cells compared with non-cancer cells (7,48). Krzystyniak et al (49) demonstrated that COX-2 upregulation affects angiogenesis and the production of specific proteases that are critical to lung cancer growth and metastasis. COX-2 also affects tumor progression by stimulating lymphagiogenesis (50).

In spite of these previous results, the prognostic value of COX-2 overexpression in patients with lung cancer remains controversial. Thus, in the present study, the next goal was to analyze whether COX-2 expression was associated with poor prognosis in Brazilian patients with NSCLC. It was revealed that expression of COX-2 was not a prognostic indicator for NSCLC (P=0.235; Fig. 1D) in our Brazilian cohort. Two systematic reviews and meta-analysis studies have corroborated the present study, also demonstrating that COX-2 overexpression does not appear to have any significant impact on the survival of patients with NSCLC (51). By contrast, in a study by Zhan et al (51), statistical significance was identified in stage I lung cancer, suggesting that COX-2 expression may be useful as a prognostic indicator during the early stages of cancer to distinguish between those with a worse prognosis.

Evidence derived from a functional analysis study revealed that the −1195 G/A polymorphism creates a cMYB binding site, thus increasing the transcriptional activity of COX-2 through HeLa cell lines (22). The present study has analyzed, to the best of our knowledge for the first time, whether the overexpression of COX-2 was associated with the −1195 G/A, −765 G/C and 8473 T/C polymorphisms in 34 Brazilian patients with NSCLC, and the conclusion drawn from this analysis is that no significant differences were observed (Fig. 2B-D).

In conclusion, the present study has been the first to describe how, in a Brazilian cohort of patients with NSCLC, the −1195 G/A, −765 G/C and 8473 T/C COX-2 polymorphisms were not associated with any risk of lung cancer, or with the outcome or with COX-2 expression. Nevertheless, it would be interesting to perform a larger prospective study and to compare tumor lung tissue vs. normal adjacent lung tissue in each sample. Even though the present study has disclosed that COX-2 expression was higher in tumor tissue, this parameter was not a prognostic indicator for our cohort of patients with NSCLC. Further functional studies based on a larger sample size are required to determine the effects of COX-2 polymorphisms on the process of lung carcinogenesis.

Acknowledgements

We would like to thank Fundação Ary Frauzino (Brazilian Cancer Foundation), CNPq/Brazil and the Ministério da Saúde (Brazil) for funding. In addition, we thank all the patients involved in this study.

References

1 

Ridge CA, McErlean AM and Ginsberg MS: Epidemiology of lung cancer. Semin Intervent Radiol. 30:93–98. 2013. View Article : Google Scholar : PubMed/NCBI

2 

Freedman ND, Leitzmann MF, Hollenbeck AR, Schatzkin A and Abnet CC: Cigarette smoking and subsequent risk of lung cancer in men and women: Analysis of a prospective cohort study. Lancet Oncol. 9:649–656. 2008. View Article : Google Scholar : PubMed/NCBI

3 

Hecht SS: Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst. 91:1194–1210. 1999. View Article : Google Scholar : PubMed/NCBI

4 

Peto R, Darby S, Deo H, Silcocks P, Whitley E and Doll R: Smoking, smoking cessation, and lung cancer in the UK since 1950 Combination of national statistics with two case control studies. BMJ. 321:323–329. 2000. View Article : Google Scholar : PubMed/NCBI

5 

Coussens LM and Werb Z: Inflammation and cancer. Nature. 420:860–867. 2002. View Article : Google Scholar : PubMed/NCBI

6 

Matanić D, Beg-Zec Z, Stojanović D, Matakorić N, Flego V and Milevoj-Ribić F: Cytokines in patients with lung cancer. Scand J Immunol. 57:173–178. 2003. View Article : Google Scholar : PubMed/NCBI

7 

Koki AT and Masferrer JL: Celecoxib: A specific COX-2 inhibitor with anticancer properties. Cancer Control. 9 2 Suppl:S28–S35. 2002.

8 

Hida T, Yatabe Y, Achiwa H, Muramatsu H, Kozaki K, Nakamura S, Ogawa M, Mitsudomi T, Sugiura T and Takahashi T: Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res. 58:3761–3764. 1998.PubMed/NCBI

9 

Fu JY, Masferrer JL, Seibert K, Raz A and Needleman P: The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. J Biol Chem. 265:16737–16740. 1990.PubMed/NCBI

10 

Tazawa R, Xu XM, Wu KK and Wang LH: Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun. 203:190–199. 1994. View Article : Google Scholar : PubMed/NCBI

11 

Williams CS, Mann M and DuBois RN: The role of cyclooxygenases in inflammation, cancer, and development. Oncogene. 18:7908–7916. 1999. View Article : Google Scholar : PubMed/NCBI

12 

Tanabe T and Tohnai N: Cyclooxygenase isozymes and their gene structures and expression. Prostaglandins Other Lipid Mediat. 68(69): 95–114. 2002. View Article : Google Scholar : PubMed/NCBI

13 

Tsujii M, Kawano S and DuBois RN: Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA. 94:3336–3340. 1997. View Article : Google Scholar : PubMed/NCBI

14 

Gupta S, Srivastava M, Ahmad N, Bostwick DG and Mukhtar H: Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. The Prostate. 42:73–78. 2000. View Article : Google Scholar : PubMed/NCBI

15 

Nam E, Lee SN, Im SA, Kim DY, Lee KE and Sung SH: Expression of cyclooxygenase-2 in human breast cancer: Relationship with HER-2/neu and other clinicopathological prognostic factors. Cancer Res Treat. 37:165–170. 2005. View Article : Google Scholar : PubMed/NCBI

16 

Zimmermann KC, Sarbia M, Weber AA, Borchard F, Gabbert HE and Schror K: Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res. 59:198–204. 1999.PubMed/NCBI

17 

Wolff H, Saukkonen K, Anttila S, Karjalainen A, Vainio H and Ristimäki A: Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res. 58:4997–5001. 1998.PubMed/NCBI

18 

Juuti A, Louhimo J, Nordling S, Ristimäki A and Haglund C: Cyclooxygenase-2 expression correlates with poor prognosis in pancreatic cancer. J Clin Pathol. 59:382–386. 2006. View Article : Google Scholar : PubMed/NCBI

19 

Wang D and Dubois RN: Eicosanoids and cancer. Nature Rev Cancer. 10:181–193. 2010. View Article : Google Scholar

20 

Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB and De Marzo AM: Cyclooxygenases in cancer: Progress and perspective. Cancer Lett. 215:1–20. 2004. View Article : Google Scholar : PubMed/NCBI

21 

Papafili A, Hill MR, Brull DJ, McAnulty RJ, Marshall RP, Humphries SE and Laurent GJ: Common promoter variant in cyclooxygenase-2 represses gene expression: Evidence of role in acute-phase inflammatory response. Arterioscler Thromb Vasc Biol. 22:1631–1636. 2002. View Article : Google Scholar : PubMed/NCBI

22 

Zhang X, Miao X, Tan W, Ning B, Liu Z, Hong Y, Song W, Guo Y, Zhang X, Shen Y, et al: Identification of functional genetic variants in cyclooxygenase-2 and their association with risk of esophageal cancer. Gastroenterology. 129:565–576. 2005. View Article : Google Scholar : PubMed/NCBI

23 

Tan W, Wu J, Zhang X, Guo Y, Liu J, Sun T, Zhang B, Zhao D, Yang M, Yu D and Lin D: Associations of functional polymorphisms in cyclooxygenase-2 and platelet 12-lipoxygenase with risk of occurrence and advanced disease status of colorectal cancer. Carcinogenesis. 28:1197–1201. 2007. View Article : Google Scholar : PubMed/NCBI

24 

Kristinsson JO, van Westerveld P, te Morsche RH, Roelofs HM, Wobbes T, Witteman BJ, Tan AC, van Oijen MG, Jansen JB and Peters WH: Cyclooxygenase-2 polymorphisms and the risk of esophageal adeno- or squamous cell carcinoma. World J Gastroenterol. 15:3493–3497. 2009. View Article : Google Scholar : PubMed/NCBI

25 

Li D, Hao SH, Sun Y, Hu CM, Ma ZH, Wang ZM, Liu J, Liu HB, Ye M, Zhang YF, et al: Functional Polymorphisms in COX-2 gene are correlated with the risk of oral cancer. Biomed Res Int. 2015:5806522015.PubMed/NCBI

26 

Gou Q, Liu CH, Ben-Av P and Hla T: Dissociation of basal turnover and cytokine-induced transcript stabilization of the human cyclooxygenase-2 mRNA by mutagenesis of the 3′-untranslated region. Biochem Biophys Res Commun. 242:508–512. 1998. View Article : Google Scholar : PubMed/NCBI

27 

Sanak M, Szczeklik W and Szczeklik A: Association of COX-2 gene haplotypes with prostaglandins production in bronchial asthma. J Allergy Clin Immunol. 116:221–223. 2005. View Article : Google Scholar : PubMed/NCBI

28 

Panguluri RC, Long LO, Chen W, Wang S, Coulibaly A, Ukoli F, Jackson A, Weinrich S, Ahaghotu C, Isaacs W and Kittles RA: COX-2 gene promoter haplotypes and prostate cancer risk. Carcinogenesis. 25:961–966. 2004. View Article : Google Scholar : PubMed/NCBI

29 

Cox DG, Pontes C, Guino E, Navarro M, Osorio A, Canzian F and Moreno V; Bellvitge Colorectal Cancer Study Group, : Polymorphisms in prostaglandin synthase 2/cyclooxygenase 2 (PTGS2/COX2) and risk of colorectal cancer. Br J Cancer. 91:339–343. 2004.PubMed/NCBI

30 

Piranda DN, Festa-Vasconcellos JS, Amaral LM, Bergmann A and Vianna-Jorge R: Polymorphisms in regulatory regions of cyclooxygenase-2 gene and breast cancer risk in Brazilians: A case-control study. BMC Cancer. 10:6132010. View Article : Google Scholar : PubMed/NCBI

31 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

32 

Soslow RA, Dannenberg AJ, Rush D, Woerner BM, Khan KN, Masferrer J and Koki AT: COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer. 89:2637–2645. 2000. View Article : Google Scholar : PubMed/NCBI

33 

Guo Y, Zhang X, Tan W, Miao X, Sun T, Zhao D and Lin D: Platelet 12-lipoxygenase Arg261Gln polymorphism: Functional characterization and association with risk of esophageal squamous cell carcinoma in combination with COX-2 polymorphisms. Pharmacogenet Genomics. 17:197–205. 2007. View Article : Google Scholar : PubMed/NCBI

34 

Lurje G, Nagashima F, Zhang W, Yang D, Chang HM, Gordon MA, El-Khoueiry A, Husain H, Wilson PM, Ladner RD, et al: Polymorphisms in cyclooxygenase-2 and epidermal growth factor receptor are associated with progression-free survival independent of K-ras in metastatic colorectal cancer patients treated with single-agent cetuximab. Clin Cancer Res. 14:7884–7895. 2008. View Article : Google Scholar : PubMed/NCBI

35 

Coskunpinar E, Eraltan IY, Turna A and Agachan B: Cyclooxygenase-2 gene and lung carcinoma risk. Med Oncol. 28:1436–1440. 2011. View Article : Google Scholar : PubMed/NCBI

36 

Ueda N, Maehara Y, Tajima O, Tabata S, Wakabayashi K and Kono S: Genetic polymorphisms of cyclooxygenase-2 and colorectal adenoma risk: The Self Defense Forces Health Study. Cancer Sci. 99:576–581. 2008. View Article : Google Scholar : PubMed/NCBI

37 

Gao J, Kang HF, Ma XB, Tang W, Liu D, Zhao Y, Zhang SQ, Guan HT, Lin S, Ren HT, et al: Functional promoter −765 G > C variant in COX-2 gene is associated with the susceptibility of breast cancer in Chinese Han women. Cancer cell Int. 14:382014. View Article : Google Scholar : PubMed/NCBI

38 

Wang XF, Huang MZ, Zhang XW, Hua RX and Guo WJ: COX-2-765G>C polymorphism increases the risk of cancer: A meta-analysis. PLoS One. 8:e732132013. View Article : Google Scholar : PubMed/NCBI

39 

Tang Z, Nie ZL, Pan Y, Zhang L, Gao L, Zhang Q, Qu L, He B, Song G, Zhang Y and Shukui Wang: The Cox-2 −1195 G >A polymorphism and cancer risk: A meta-analysis of 25 case-control studies. Mutagenesis. 26:729–734. 2011. View Article : Google Scholar : PubMed/NCBI

40 

Pan F, Tian J, Pan Y and Zhang Y: Lack of association of the cyclooxygenase 8473 T>C polymorphism with lung cancer: Evidence from 9841 subjects. Asian Pac J Cancer Prev. 12:1941–1945. 2011.PubMed/NCBI

41 

Vogel U, Christensen J, Wallin H, Friis S, Nexø BA, Raaschou-Nielsen O, Overvad K and Tjønneland A: Polymorphisms in genes involved in the inflammatory response and interaction with NSAID use or smoking in relation to lung cancer risk in a prospective study. Mutat Res. 639:89–100. 2008. View Article : Google Scholar : PubMed/NCBI

42 

Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, Sougnez C, Greulich H, Muzny DM, Morgan MB, et al: Somatic mutations affect key pathways in lung adenocarcinoma. Nature. 455:1069–1075. 2008. View Article : Google Scholar : PubMed/NCBI

43 

Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350:2129–2139. 2004. View Article : Google Scholar : PubMed/NCBI

44 

Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, et al: EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science. 304:1497–1500. 2004. View Article : Google Scholar : PubMed/NCBI

45 

Wistuba II, Berry J, Behrens C, Maitra A, Shivapurkar N, Milchgrub S, Mackay B, Minna JD and Gazdar AF: Molecular changes in the bronchial epithelium of patients with small cell lung cancer. Clin Cancer Res. 6:2604–2610. 2000.PubMed/NCBI

46 

Bhat IA, Rasool R, Qasim I, Masoodi KZ, Paul SA, Bhat BA, Ganaie FA, Aziz SA and Shah ZA: COX-2 overexpression and 8473 T/C polymorphism in 3′ UTR in non-small cell lung cancer. Tumour Biol. 35:11209–11218. 2014. View Article : Google Scholar : PubMed/NCBI

47 

Bi N, Yang M, Zhang L, Chen X, Ji W, Ou G, Lin D and Wang L: Cyclooxygenase-2 genetic variants are associated with survival in unresectable locally advanced non-small cell lung cancer. Clin Cancer Res. 16:2383–2390. 2010. View Article : Google Scholar : PubMed/NCBI

48 

Koki AT, Khan NK, Woerner BM, Seibert K, Harmon JL, Dannenberg AJ, Soslow RA and Masferrer JL: Characterization of cyclooxygenase-2 (COX-2) during tumorigenesis in human epithelial cancers: Evidence for potential clinical utility of COX-2 inhibitors in epithelial cancers. Prostaglandins Leukot Essent Fatty Acids. 66:13–18. 2002. View Article : Google Scholar : PubMed/NCBI

49 

Krzystyniak KL: Current strategies for anticancer chemoprevention and chemoprotection. Acta Pol Pharm. 59:473–478. 2002.PubMed/NCBI

50 

Guo X, Chen Y, Xu Z, Xu Z, Qian Y and Yu X: Prognostic significance of VEGF-C expression in correlation with COX-2, lymphatic microvessel density, and clinicopathologic characteristics in human non-small cell lung cancer. Acta Biochim Biophys Sin (Shanghai). 41:217–222. 2009. View Article : Google Scholar : PubMed/NCBI

51 

Zhan P, Qian Q and Yu LK: Prognostic value of COX-2 expression in patients with non-small cell lung cancer: A systematic review and meta-analysis. J Thorac Dis. 5:40–47. 2013.PubMed/NCBI

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Spandidos Publications style
Moraes JL, Moraes AB, Aran V, Alves MR, Schluckbier L, Duarte M, Toscano E, Zamboni M, Sternberg C, de Moraes E, de Moraes E, et al: Functional analysis of polymorphisms in the COX‑2 gene and risk of lung cancer. Mol Clin Oncol 6: 494-502, 2017
APA
Moraes, J.L., Moraes, A.B., Aran, V., Alves, M.R., Schluckbier, L., Duarte, M. ... Gil Ferreira, C. (2017). Functional analysis of polymorphisms in the COX‑2 gene and risk of lung cancer. Molecular and Clinical Oncology, 6, 494-502. https://doi.org/10.3892/mco.2017.1167
MLA
Moraes, J. L., Moraes, A. B., Aran, V., Alves, M. R., Schluckbier, L., Duarte, M., Toscano, E., Zamboni, M., Sternberg, C., de Moraes, E., Lapa E Silva, J. R., Gil Ferreira, C."Functional analysis of polymorphisms in the COX‑2 gene and risk of lung cancer". Molecular and Clinical Oncology 6.4 (2017): 494-502.
Chicago
Moraes, J. L., Moraes, A. B., Aran, V., Alves, M. R., Schluckbier, L., Duarte, M., Toscano, E., Zamboni, M., Sternberg, C., de Moraes, E., Lapa E Silva, J. R., Gil Ferreira, C."Functional analysis of polymorphisms in the COX‑2 gene and risk of lung cancer". Molecular and Clinical Oncology 6, no. 4 (2017): 494-502. https://doi.org/10.3892/mco.2017.1167