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Clinical Cancer Research Vol. 11, 7362-7368, October 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Cyclooxygenase-2 Is an Independent Prognostic Factor in Gastric Cancer and Its Expression Is Regulated by the Messenger RNA Stability Factor HuR

Johanna Mrena1, Jan-Patrik Wiksten1, Alexandra Thiel2,3, Arto Kokkola1, Leena Pohjola2,3, Johan Lundin4, Stig Nordling2, Ari Ristimäki2,3 and Caj Haglund1,2

Authors' Affiliations: Departments of 1 Surgery and 2 Pathology, Helsinki University Central Hospital; 3 Molecular and Cancer Biology Research Program, Biomedicum Helsinki; and 4 Folkhälsan Research Centre, University of Helsinki, Helsinki, Finland

Requests for reprints: Caj Haglund, Department of Surgery, Helsinki University Central Hospital, P.O. Box 340, 00029 HUS Helsinki, Finland. Phone: 358-9-471-72427; Fax: 358-9-471-71403; E-mail: caj.haglund{at}hus.fi.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Cyclooxygenase-2 (COX-2) promotes carcinogenesis and its expression associates with clinicopathologic characteristics in gastric cancer. HuR is an mRNA binding protein that controls the stability of certain transcripts including COX-2. We evaluated the prognostic significance of COX-2 and HuR expressions in gastric cancer and whether there exists a link between HuR and COX-2 expressions.

Experimental Design: The study included 342 consecutive patients with histologically confirmed gastric adenocarcinoma, of whom 321 patients had tissue specimens available for COX-2 and 316 for HuR immunohistochemistry. Specimens were stained by COX-2– and HuR-specific monoclonal antibodies and scored by two independent observers. Correlation to clinical data and survival was assessed. TMK-1 gastric adenocarcinoma cells were treated with small interfering RNA against HuR and expressions of HuR and COX-2 were detected by immunofluorescence and Western blot analysis.

Results: Patients with low COX-2 expression had a cumulative 5-year survival of 53% and those with high COX-2 expression had 16% (P < 0.0001). In multivariate analysis, COX-2 was an independent prognostic factor (P = 0.003). Cytoplasmic HuR expression was associated with high COX-2 expression (P < 0.0001) and with reduced survival (P = 0.004) whereas nuclear positivity for HuR was not. When TMK-1 cells were treated with HuR small interfering RNA, expressions of HuR and COX-2 were reduced.

Conclusions: High COX-2 is an independent prognostic factor in gastric cancer. Cytoplasmic expression of HuR associates with high COX-2 expression and with reduced survival, and tissue culture experiments show that HuR can regulate expression of COX-2 in gastric cancer cells.

The etiologic background of gastric cancer is multifactorial, including diet, Helicobacter pylori infection, previous gastric surgery, and genetic factors (e.g., mismatch repair deficiencies, E-cadherin mutations, and interleukin-1 polymorphisms; refs. 1, 2). Epidemiologic studies have suggested that the use of aspirin reduces mortality from digestive tract malignancies, including gastric cancer (3–5). Cyclooxygenase (COX) is the rate-limiting enzyme in prostaglandin synthesis and the best known target of aspirin and other nonsteroidal anti-inflammatory drugs (6, 7). There are two COX isoenzymes: COX-1, which is ubiquitously expressed and responsible for several physiologic functions, and COX-2, which is an inducible enzyme of which the role has been connected to inflammation and carcinogenesis (6, 7). A direct link between COX-2 and carcinogenesis is supported by studies showing that intestinal polyp formation is suppressed by pharmacologic inhibition and genetic deletion of COX-2 in mice (8, 9). We have previously reported that expression of COX-2 is elevated in human gastric cancer tissues (10) and that a selective COX-2 inhibitor, celecoxib, suppressed tumor formation in a trefoil factor 1–deficient mouse model of gastric carcinogenesis (11). In addition, it was recently shown that transgenic expression of COX-2 and microsomal prostaglandin E synthase induced hyperplastic tumors in mouse stomach (12) and that celecoxib reduced chemically induced gastric carcinogenesis in rat (13). Because COX-2 expression is present already in noninvasive gastric dysplasias (14–16), it seems likely that COX-2 has a role in early gastric carcinogenesis.

Regulation of COX-2 expression in gastric cancer is poorly known. Eukaryotic gene expression is regulated at both transcriptional and posttranscriptional levels (17) and expression of COX-2 mRNA is modulated by transcriptional control and mRNA stability (18). HuR is an mRNA-binding factor that belongs to the embryonic lethal abnormal vision/Hu protein family (19). It binds to labile transcripts containing AU-rich elements, such as mRNAs for proto-oncogenes, cytokines, and cytokine-response genes. HuR is predominantly present in the nucleus and it can bind to target mRNAs in the nucleus and while in the cytoplasm, it stabilizes these messages (20). Human colon adenocarcinoma cells overexpressing HuR formed larger tumors than the control cells in a mouse model (21). In human colon cancer cells, overexpression of HuR increased expression of COX-2, and in breast and ovarian cancer cells, inhibition of HuR expression resulted in suppression of COX-2 expression (22–24). These data suggest that HuR plays a role in carcinogenesis and part of its effect may be facilitated by induction of COX-2 expression.

In this study, we have investigated the prognostic role of COX-2 and HuR expressions in gastric cancer patients. We have also studied the association between COX-2 and HuR in this material and in tissue culture conditions by evaluating the effect of HuR small interfering RNA on COX-2 expression.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. The study included 342 consecutive patients who underwent surgery for histologically verified gastric adenocarcinoma at the Department of Surgery, Meilahti Hospital, Helsinki University Central Hospital between 1983 and 1999. The approval of the study was obtained from the local ethics committee. Diagnosis and staging were done from specimens according to the Union Internationale Contra Cancrum classification (tumor-node-metastasis 1992). Four patients were excluded from the study due to incomplete information: missing clinical data (one patient), unclear cause of death (one patient), and missing follow-up (two patients). We were able to obtain demonstrative sections containing tumor tissue from 321 cases for COX-2 immunohistochemistry and from 316 specimens for HuR immunohistochemistry. Finally, a total of 328 patients were included in our study. There were 135 (41%) low-stage (I-II) and 193 (59%) high-stage (III-IV) patients. Lymph node metastases were found in 182 (55%) and distant metastases in 92 (28%) patients. Median age was 66 years (range, 30-87 years) and there were 158 females and 170 males. Surgery for cure (total or partial gastrectomy with lymph node dissection) was done in 170 (52%) of the patients and 141 (43%) underwent a palliative procedure (partial gastrectomy, bypass, or laparotomy only). None of the patients received neoadjuvant therapy. Thirty-two patients received postoperative adjuvant therapy, of whom 28 received chemotherapy, 2 radiotherapy, and 2 both chemotherapy and radiotherapy. Twenty-eight of these patients represented stages III to IV. Survival data were obtained until February 2004 from patient records, the Finnish Cancer Registry, and the Population Register Centre of Finland. The clinicopathologic characteristics are described in Table 1. Median follow-up time was 12.7 years (range, 4.7-20.8 years). During follow-up, 210 (64%) patients died of gastric cancer.


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  		Table 1. Association of COX-2 with clinicopathologic variables in 321 patients with gastric adenocarcinoma

 
Immunohistochemistry. Formalin-fixed tumor samples were embedded in paraffin. Archive tissue blocks were freshly cut into 4-µm-thick sections, mounted onto slides, and dried for fixation for 12 to 24 hours at 37°C. Tissue sections were then deparaffinized in xylene and rehydrated through graded alcohol and deionized water. For the antigen retrieval, the sections were heated in a microwave oven in 0.3% citrate buffer (pH 6.0) for 4 x 5 minutes and washed with PBS for 2 x 5 minutes. Endogenous peroxidases were inactivated by 30-minute incubation in methanol containing 1.6% hydrogen peroxidase followed by PBS wash for 3 x 5 minutes. To block nonspecific binding sites, the sections were treated with normal horse serum for 15 minutes. The sections were incubated overnight with an anti-human COX-2 mouse monoclonal antibody (160112, Cayman Chemical, Ann Arbor, MI) in a dilution of 1:200. After the overnight incubation with the primary antibody, the sections were first reacted for 30 minutes with biotinylated antimouse immunoglobulin G (Vector Laboratories, Inc., CA), diluted 1:200 in PBS containing 1% normal horse serum, and then for 30 minutes in avidin-biotinylated peroxidase complex (Vectastain ABC Kits, Vector Laboratories). Between each step in the staining procedure, sections were washed with PBS for 3 x 5 minutes. Staining was visualized using 3-amino-9-ethyl-carbazole (A5754, Sigma Chemical Co., St. Louis, MO), 0.2 g/mL in 0.5 mol/L acetate buffer containing 0.03% perhydrol (pH 5.0) for 15 minutes. Subsequently, the sections were washed thoroughly in tap water for 10 minutes, counterstained with Meyer's hematoxylin for 15 to 60 seconds, washed, and mounted in aqueous mounting media (Aquamount, BDH, Poole, United Kingdom). All steps of the procedure were carried out at room temperature. The principle of HuR immunostaining protocol was similar to that of COX-2 immunohistochemistry and has been previously described (24). HuR monoclonal antibody (1:10,000 dilution, 19F12) was a kind gift from Clonegene, LLC (Hartford, CT).

Scoring. Cytoplasmic COX-2 immunoreactivity was assessed by the intensity of staining and percentage of positivity area. The intensity of staining was graded from 0 to 3 (absent, weak diffuse, moderate granular, strong granular) and the area of positivity was estimated as percentage of total area of the tumor (<10%, 10-89%, ≥90%). These two variables consisted the actual score: score 0 (negative staining or intensity 1 under 10% area); score 1 (intensity 1 between 10% and 100% or intensity 2 to 3 under 10% area); score 2 (intensity 2 over 10% or intensity 3 under 90% area); score 3 (intensity 3 over 90% area). Colorectal carcinoma specimens were used as positive controls as previously described (25). Immunoreactivity was scored by two independent interpreters (J.M. and A.R.) without preliminary knowledge of clinical data. The case-by-case final consensus score was discussed and determined in a common session. In statistical analysis, COX-2 scores were handled in all four subgroups (0-3) or in two groups (low: 0-1; high: 2-3).

HuR staining was also observed in a blinded manner as described above. The patients were initially scored to five groups according to nuclear and cytoplasmic intensity of immunoreactivity: nucleus only (1), cytoplasm only (2), nucleus over cytoplasm (3), cytoplasm over nucleus (4), and totally negative (0). The groups were combined and analyses were done between different combinations: cytoplasm negative (0 and 1) versus cytoplasm positive (2, 3, and 4), and nucleus negative (0 and 2) versus nucleus positive (1, 3, and 4).

Statistical analysis. Associations between factors, including clinicopathologic variables and HuR and COX-2 scores, were assessed by {chi}2 test or Fisher's exact test in case of low expected frequencies. Survival rates were calculated using the Kaplan-Meier method and statistical significance between the groups was analyzed by log-rank test or log-rank test for trend in case of three or more ordered groups. Gastric cancer–specific survival was calculated from the date of diagnosis to death of gastric cancer or last day of follow-up. Deaths due to intercurrent causes were censored. Multivariate survival analysis was done with the Cox proportional hazards model, entering the following covariates: Laurén classification, tumor location, gender, tumor size, age, HuR immunoreactivity, COX-2 immunoreactivity, tumor-node-metastasis stage, and intent of surgery, coded as presented in univariate analysis. P < 0.05 was adopted as a limit for inclusion of a covariate. All P values are two sided.

Cell culture and RNA interference. The cells were cultured in RPMI 1640 supplemented with 10% FCS (PromoCell GmbH, Heidelberg, Germany), 2 mmol/L L-glutamine, and antibiotics (Bio Whittaker Europe, Verviers, Belgium), and maintained at 37°C and 5% CO2. The small interfering RNA duplexes were synthesized by Dharmacon, Inc. (Lafayette, CO) and the sequences were for HuR: sense 5'-AACAUGACCCAGGAUGAGUUA-dTdT-3' and antisense 5'-UAACUCAUCCUGGGUCAUGUU-dTdT-3', and for ß-actin: sense 5'-AAUGAAGAUCAAGAUCACUGC-dTdT-3' and antisense 5'-GCAAUGAUCUUGAUCUUCAUU-dTdT-3'. The day before transfection, TMK-1 cells were trypsinized and diluted 1:20 with optiMEM 1 medium (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 10% FCS without antibiotics and transferred to 12-well plates at 1 mL per well with the final split ratio being 1:4. Transient transfection of small interfering RNAs was carried out using Oligofectamine reagent (Invitrogen Life Technologies, Carlsbad, CA) following the instructions of the manufacturer and as previously described (24). The final small interfering RNA concentrations ranged from 0.5 to 150 nmol/L. The cells were lysed for 78 hours after the transfection at 95°C in 400 µL of 1x lysis buffer [60 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, 0.002% bromophenol blue]. The samples were heated for 4 minutes at 95°C, centrifuged at room temperature at 14,000 x g for 10 minutes, and 50 µL of the sample were taken and separated by 12% SDS-PAGE. In selected experiments, interleukin-1ß (10 ng/mL, R&D Systems, Minneapolis, MN) was added for the last 24 hours of the incubation period.

Immunofluorescence. For immunofluorescence experiments, TMK-1 cells were grown on coverslips in 24-well plates overnight and transfected with 50 nmol/L HuR small interfering RNA as described above. After 72 hours, the cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature and washed once with PBS. Cells were permeabilized with 0.5% NP40 in PBS for 5 minutes at room temperature and washed thrice with PBS. Nonspecific binding of antibodies was blocked with 3% bovine serum albumin/TBS for 10 minutes at room temperature. The samples were then incubated at 1:10,000 dilution with HuR monoclonal antibody in 3% bovine serum albumin/TBS for 1 hour at 37°C and washed thrice with PBS. The secondary antibody tetramethyrhodamine isothiocyanate–conjugated rabbit anti-mouse (1:50 dilution; DAKO A/S, Glostrup, Denmark) was incubated for 1 hour at 37°C. After three washes with PBS, samples were incubated for 2 minutes at room temperature with Hoechst (1:1,000; Sigma). Images were obtained using a Zeiss Axiplan imaging fluorescence microscope (Carl Zeiss, Jena, Germany).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cyclooxygenase-2 and HuR immunoreactivity. No COX-2 immunoreactivity was found in 20% (65 of 321) and it was score 1 in 25% (80 of 321), score 2 in 48% (153 of 321), and score 3 in 7% (23 of 321) of the gastric adenocarcinoma specimens. Combining these groups as COX-2 low (score 0-1) and COX-2 high (score 2-3), the values were 45% (145 of 321) and 54% (176 of 321), respectively. The positive staining was observed in the cytoplasm of the malignant cells and in the perinuclear region whenever strong positivity was observed. Stromal cells were either negative or weakly positive. In respect of HuR staining, there were 60% (189 of 316) cytoplasm-negative and 40% (127 of 316) cytoplasm-positive specimens whereas 12% (37 of 316) were nucleus negative and 88% (279 of 316) were nucleus positive.

Association of cyclooxygenase-2 and HuR with clinicopathologic variables. We observed a significant association (P < 0.0001) between elevated COX-2 expression and high stage, lymph node metastasis, advanced penetration depth, intestinal type of the tumor, and noncurative resection (Table 1). High COX-2 levels associated also with male gender, metastatic disease, proximal location of the tumor, and tumor size (P < 0.05). Cytoplasmic HuR immunoreactivity correlated with high COX-2 expression (P < 0.0001; Table 2). Cytoplasmic HuR was also associated with old age, male gender, high stage, distant metastases, proximal location, intestinal type, and nonresectability (Table 2). Similarly to cytoplasmic HuR, nuclear HuR immunoreactivity correlated to old age (P = 0.006), but in contrast to cytoplasmic staining, nuclear HuR was found more frequently in tumors located distally (P = 0.014) and representing diffuse type (P = 0.048). There was no association between nuclear HuR and other clinicopathologic variables or COX-2 (P > 0.29).


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  		Table 2. Association of cytoplasmic HuR positivity with clinicopathologic variables in 316 patients with gastric adenocarcinoma

 
Prognostic role of cyclooxygenase-2 and HuR. High COX-2 expression and cytoplasmic HuR positivity associated with poor survival. Nuclear positivity of HuR did not have prognostic significance. Cumulative 5-year survival rates according to COX-2 and HuR scores and clinicopathologic variables are shown in Table 3. In addition to elevated COX-2 expression and cytoplasmic positivity of HuR, age over 66 years, high stage, penetration depth, lymph node metastasis, distant metastasis, proximal location of the tumor, tumor size over 5 cm, and nonresectability of the tumor were significant variables for decreased 5-year survival (Table 3).


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  		Table 3. Univariate analysis of the relationship between preoperative characteristics and survival in 328 patients with gastric cancer

 
Multivariate survival analysis. A multivariate survival analysis showed that tumor-node-metastasis stage (P < 0.0001), high COX-2 (P = 0.003), and intent of surgery (P = 0.004) were independent prognostic factors (Table 4). No other variables, including HuR immunoreactivity, were retained in the model or affected the magnitude of the hazard ratios of the variables in the final model. Separate Cox regression models were also fitted for patients with stage I to II as well as stage III to IV disease. In both of these, COX-2 and intent of surgery added significant prognostic information [in stage I-II: COX-2 hazard ratio, 2.54 (95% confidence interval, 1.32-4.86); intent of surgery hazard ratio, 5.15 (95% confidence interval, 1.79-14.8); in stage III-IV: COX-2 hazard ratio, 1.72 (95% confidence interval, 1.22-2.45); intent of surgery hazard ratio, 2.63 (95% confidence interval, 1.78-3.89)].


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  		Table 4. Cox multivariate survival analysis in 304 patients with gastric cancer

 
Effect of HuR small interfering RNA on cyclooxygenase-2 expression in gastric adenocarcinoma cells. To test the hypothesis that HuR regulates COX-2 expression in gastric carcinoma cells, we treated TMK-1 cells with HuR small interfering RNA molecules. This treatment suppressed HuR expression as detected by immunofluorescence and immunoblotting (Fig. 1A). Furthermore, we observed a concentration-dependent suppression of COX-2 protein expression after the HuR small interfering RNA treatment (Fig. 1B). ß-Actin small interfering RNA showed no effect on HuR or COX-2 expression. These results indicate that HuR can regulate COX-2 expression in gastric cancer cells.



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  		Fig. 1. Expression of COX-2 is suppressed by HuR small interfering RNA (siRNA) in gastric cancer cells. A, HuR-targeted small interfering RNA (50 nmol/L) inhibited HuR protein expression in TMK-1 gastric adenocarcinoma cells as detected by immunofluorescence. Hoechst was used for staining nuclear DNA. B, HuR small interfering RNA (50-150 nmol/L) inhibited HuR and COX-2 protein expression in interleukin-1ß–treated TMK-1 cells as detected by immunoblotting. ß-Actin small interfering RNA did not have an effect on HuR or COX-2 expression. ß-Actin was used as a loading control.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We found that high COX-2 protein expression is an independent prognostic factor in patients with gastric adenocarcinoma. In addition to survival, COX-2 expression is associated with several clinicopathologic factors, such as advanced tumor penetration depth, lymph node metastases, distant metastases, and noncurative operation (i.e., factors reflecting advanced stage). A number of studies have previously shown COX-2 expression to correlate with clinicopathologic variables in gastric cancer, such as depth of tumor invasion, size, lymph node metastasis, stage, and microvessel density (26–37), but the association of COX-2 with survival has been controversial. Several groups have reported no correlation with survival (30, 32, 38, 39) whereas others have described COX-2 to correlate with survival, but in a nonindependent manner (28, 37) or in patients with an advanced disease (36). However, Shi et al. (35) have reported that COX-2 is an independent prognostic factor in gastric cancer. In their series, there were no tumors invading beyond the serosa and patients with COX-2–positive and COX-2–negative tumors had 5-year cumulative survivals of 67.9% and 91.4%, respectively, whereas in our material the survival rates were 16% for high COX-2 and 53% for low COX-2 expression. To this end, our consecutive material indicates that tumor expression of COX-2 is an independent prognostic factor also in an unselected gastric cancer patient material.

The results of our multivariate analysis show that the only independent prognostic factors were stage, intent of surgery, and COX-2. In separate Cox regression models for stage groups I to II and III to IV, COX-2 and intent of surgery were independent prognostic factors. Hazard ratio (i.e., risk for gastric cancer related death) for patients with high tumoral COX-2 expression in stage group I to II was 2.54 whereas it was 1.72 in stage group III to IV. Although COX-2 was an independent prognostic factor in both of these models, the prognostic role of COX-2 seems to be more pronounced in the nonadvanced disease. Stage and intent of surgery are superior to COX-2 as prognosticators. However, because COX-2 is so tightly related to gastric carcinogenesis and clinicopathologic factors, it reveals the biological properties of the tumor in a whole different manner than the established prognostic factors. Whether this justifies clinicians to reconsider treatment modalities is too early to say. More controlled studies should be done and should also include prospective data.

Mechanisms controlling COX-2 expression in gastric cancer are poorly understood. We show that down-regulation of the mRNA stability factor HuR inhibits COX-2 expression in TMK-1 gastric cancer cells. Previously, it has been reported that expressions of HuR and COX-2 are linked in colon, breast, and ovarian carcinoma cells (22–24), which is in line with our results in gastric cancer cells. Our data also show that cytoplasmic, but not nuclear, HuR immunoreactivity correlated with high COX-2 expression, high stage, and reduced survival in the gastric adenocarcinoma material. Previously, cytoplasmic HuR expression has been shown to associate with high grade and reduced survival in ovarian and breast cancers (24, 40, 41).

In conclusion, our data show that high COX-2 expression is an independent prognostic marker for poor outcome in gastric cancer. Furthermore, we report that cytoplasmic HuR expression correlates to COX-2 expression and poor survival in gastric cancer and that reduction in HuR expression leads to inhibition of COX-2 expression. These results suggest that HuR regulates expression of COX-2 in gastric cancer and that COX-2 may be one factor that facilitates carcinogenic properties of HuR.


   Acknowledgments
 
We thank Elina Laitinen, Päivi Peltokangas, Elina Malkki, and Tuija Hallikainen for technical assistance.


   Footnotes
 
Grant support: Finnish Cancer Society, Finska Läkaresällskapet, Medicinska Understödsföreningen Liv och Hälsa, Helsinki University Central Hospital Research Funds, Helsinki University Research Funds, The Academy of Finland, and the Sigrid Jusélius Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. Ristimäki and C. Haglund have equal last authorship.

Received 4/19/05; revised 6/28/05; accepted 7/21/05.


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 Results
 Discussion
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