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Clinical Cancer Research Vol. 10, 6938-6945, October 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Concomitant Overexpression of Cyclooxygenase-2 in HER-2–Positive on Smad4-Reduced Human Gastric Carcinomas Is Associated with a Poor Patient Outcome

Hirokazu Okano, Hisashi Shinohara, Akiko Miyamoto, Kyoichi Takaori and Nobuhiko Tanigawa

Department of General and Gastroenterological Surgery, Osaka Medical College, Osaka, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The expression of cyclooxygenase-2 (COX-2) is known to be involved in gastric carcinogenesis and tumor progression, but little is known about the mechanisms responsible for the up-regulation of COX-2. We examined the involvement of two growth factor-signaling systems, HER-2 and transforming growth factor (TGF)-ß, in the induction of COX-2 in human gastric cancer tissue.

Experimental Design: COX-2 expression was detected by immunohistochemistry in surgical specimens obtained from 166 patients with advanced gastric cancer; possible correlations between the expression of COX-2 and the expression of HER-2, TGF-ß1, and Smad4, an intracellular mediator that transmits the TGF-ß signal, were then analyzed.

Results: COX-2 protein was overexpressed in 91 (54.8%) tumors; COX-2 overexpression was correlated with a differentiated histologic type, deep invasion, and positive lymph node metastasis. COX-2 was frequently overexpressed in HER-2–positive tumors (19 of 22, 86.4%) and in Smad4-reduced tumors (67 of 104, 64.4%) but irrelevant to the TGF-ß1 expression status. The expression levels of COX-2 and HER-2 and the reduction in Smad4 were all associated with a poor patient outcome. A multivariate analysis demonstrated a significantly poor outcome for the concomitant overexpression of COX-2 in patients with Smad4-reduced tumors.

Conclusions: These results support the possibility that signal transduction via HER-2 and the TGF-ß/Smad system may be implicated in COX-2 expression and that the reduction of Smad4 may be, in part, of causal significance in the TGF-ß-initiated overexpression of COX-2, which is associated with a poor prognosis for patients with gastric cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastric cancer is one of the most prevalent and lethal malignancies in the world, yet little is known about its molecular process of development and progression (1) . Accumulating evidence suggests that gastric carcinogenesis may be driven by the up-regulation of the inducible form of cyclooxygenase-2 (COX-2), an enzyme responsible for the conversion of arachidonic acid to prostaglandins (PGs) (2, 3, 4) . COX-2 expression occurs in 60–80% of human gastric cancers (5 , 6) and is associated with a poor prognosis (7 , 8) .

The mechanisms that regulate the expression of COX-2 in gastric cancer are not completely understood. COX-2 expression is induced by cytokines, growth factors, and tumor promoters (9) . A link between the signaling of HER-2, a family of receptors for epidermal growth factor (EGF) with tyrosine kinase activity (10) , and COX-2 expression has been established (11 , 12) . Overexpression of HER-2 in the biliary epithelium of transgenic mice led to an increase in COX-2 (11) . Additionally, the activation of the HER-2/HER-3 pathway induced COX-2 in colorectal cancer cells (12) . A recent article by Subbaramaiah et al. (13) demonstrated that HER-2 stimulates COX-2 transcription via the Ras pathway in cultured human mammary epithelial cells. Amplification and/or overexpression of HER-2 occurs in 10–20% of human gastric carcinomas and have been associated with a poor prognosis (14, 15, 16, 17) . However, whether HER-2 signaling is involved in the regulation of COX-2 in gastric cancer remains unknown.

Multiple lines of evidence suggest that the absence of the growth inhibitory effect of the transforming growth factor-ß (TGF-ß) superfamily may be a key step in malignant transformation and tumor progression in intestinal epithelial cells (18 , 19) . Although the exact mechanism by which neoplastic epithelium acquires TGF-ß resistance remains unclear, the inactivation of Smad4, an intracellular signal transducer that acts downstream of the receptors for TGF-ßs (20) , may be involved in the disruption of TGF-ß-induced growth inhibition (21 , 22) . Furthermore, the adverse effects of TGF-ß, one of which is the induction or augmentation of COX-2 expression in intestinal epithelial cells (23 , 24) , may be associated with the malignant conversion. Recent studies have demonstrated that oncogenic Ras activates the transcription of TGF-ßs (25) , which enhances the Ras-induced expression of COX-2 in epithelial cells via mRNA stabilization (26) . These findings suggest that the TGF-ß/Smad system may play an important role in the regulation of COX-2 expression, which, in turn, is involved in the neoplastic transformation of gastric epithelial cells.

In the present study, we investigated the possible involvement of two growth factor signaling systems, HER-2 and TGF-ß/Smad, in the expression of COX-2 in human gastric cancer. Surgical specimens obtained from 166 patients with advanced gastric cancer who had undergone a gastrectomy with standard lymph node dissection were examined by immunohistochemistry for the expression of these proteins. We found that both the frequency and the magnitude of COX-2 expression were markedly enhanced in HER-2–positive or Smad4-reduced tumors and that the concomitant overexpression of COX-2 was associated with a poor outcome in patients with advanced gastric cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples.
Formalin-fixed and paraffin-embedded specimens were obtained from 166 consecutive patients with advanced gastric cancer who had undergone a gastrectomy with lymph node dissection at Osaka Medical College Hospital between 1990 and 1995. Patients who received chemotherapy before surgery and those who received surgery for other types of carcinomas were excluded. The patients, 122 males and 44 females, ranged in age from 36 to 91 years old, with a median age of 60 years. The clinicopathological features of these patients were assessed according to the General Rules for the Gastric Cancer Study in Japan (27) . Curative surgery was performed in 136 of 166 patients (82%). The median follow-up period was 48.8 months, ranging from 1.8 to 136.2. All tumor samples were received as coded specimens; for patient confidentiality, the specimens were assigned a number according to the order in which they were received by the laboratory.

Immunohistochemistry.
Serial sections (4-µm thick) were mounted on glass slides. Immunohistochemical staining was performed using the standard avidin-biotin-peroxidase complex technique and the L.V. Dako LSAB kit (DAKO, Carpinteria, CA), as described previously (28) . All cell counts were performed using an Olympus BX-50F photomicroscope at a magnification of x200 (x20 objective and x10 eyepiece). The area counted in each section was randomly selected from a representative tumor field. For each section, 10 areas were assessed; the counts were expressed as the mean percentage of positive tumor cells out of the total number of cells/high-powered field. In each case, serial H&E sections were examined to determine the tumor orientation and confirm the histologic diagnosis. Each case was blindly scored with respect to patient history, presentation, and previous scoring.

COX-2 Expression.
Sections were dewaxed in xylene, rehydrated in ethanol and then heated in a microwave oven (700 W) for 8 min to retrieve the antigens. Endogenous peroxidase was blocked by the incubation of samples with 3% hydrogen peroxide in PBS for 5 min at room temperature. Nonspecific binding was blocked with bovine serum albumin for 10 min at room temperature. The sections were then incubated overnight at 4°C with mouse anti-COX-2 monoclonal antibody (Cayman Chemical Co., Ann Arbor, MI) diluted at 1:200 in a humidified chamber. After washing, biotinylated antimouse and antirabbit IgG (DAKO) were applied for 30 min at room temperature, followed by incubation with streptavidin-conjugated horseradish peroxidase. Finally, 3,3'-diaminobenzidine was used for color development, and hematoxylin was used for the counterstain. Positive COX-2 expression was determined by counting the number of tumor cells with cytoplasmic staining, and semiquantitatively scoring the specimens according to the percentage of positively stained tumor cells: level 0, <10%; level 1, 10–30%; level 2, 30–60%; and level 3, >60% of the cells. Staining in stromal tissues and inflammatory cells was not counted. COX-2 overexpression was defined as a level 2 or 3 score (29) .

HER-2 Expression.
Immunostaining for HER-2 was performed as described above, with the exception of the antigen retrieval step. A mouse anti-HER-2 monoclonal antibody (PharMingen, San Diego, CA) diluted at 1:100 was used. The positive expression of HER-2 was determined by counting the number of tumor cells with membranous staining. The immunoreactivity was scored according to previously published guidelines (30) as follows: score 0, no staining or staining in <10%; score 1, incomplete staining in >10%; score 2, weak to moderate complete staining in >10%; and score 3, strong complete staining in >10% of the cells (30) . A breast cancer specimen previously elevated as score 3 was used as a positive control. Samples with a score of ≥1 were considered to be HER-2–positive (30) .

TGF-ß1 Expression.
Immunostaining for TGF-ß1 was performed as described above, with the exception of the antigen retrieval step. A rabbit anti-TGF-ß1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:100 was used. The immunoreactivity of TGF-ß1 was defined by counting the number of tumor cells with cytoplasmic staining; in which 10% or more of the cells were positive were regarded as being TGF-ß1 positive (21 , 31) .

Smad4 Expression.
Immunostaining for Smad4 was performed as described previously using a mouse anti-Smad4 monoclonal antibody (Santa Cruz Biotechnology) diluted at 1:300. The expression of Smad4 was determined by counting the number of tumor cells with cytoplasmic or nuclear staining. Samples in which >50% of the tumor cells were strongly stained as normal epithelium were classified as preserved expression, whereas samples in which <50% of the tumor cells were strongly stained were classified as reduced expression (21) . Fig. 1Citation shows examples for each scoring.



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  		Fig. 1. A–D, examples for level 0–3 staining for COX-2 expression in gastric cancer specimens; level 0 (A), level 1 (B), level 2 (C), level 3 (D). E–H, examples for score 0–3 staining for HER-2 expression in gastric cancer specimens; score 0 (A), score 1 (B), score 2 (C); and in a breast cancer specimen (score 3; D). I and J, examples for negative (I) and positive (J) expression of TGF-ß1. K and L, examples for preserved (K) and reduced (L) expression of Smad4. Original magnification, x400.

 
Statistical Analysis.
A {chi}2 test was used to analyze correlations between the immunointensity of the proteins (COX-2, HER-2, TGF-ß1, and Smad4) and the clinicopathological features as well as correlations between COX-2 expression and that of the other proteins. Survival curves were calculated using the Kaplan-Meier method and analyzed using the log-rank test. The prognostic significance of various factors was also determined by univariate and multivariate analyses using the Cox proportional hazard regression model according to a previous study (21) . The statistical significance was defined as P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Correlation with Clinicopathological Features.
The expression of COX-2, HER-2, TGF-ß1, and Smad4 proteins was examined in 166 patients with gastric carcinoma. Table 1Citation shows the correlations between the immunointensity of these proteins and several clinicopathological features, including tumor size, gross type, histology, depth of tumor invasion, peritoneal dissemination, hepatic metastasis, and lymph node metastasis. The overexpression of COX-2 (level 2–3) was detected in 91 (54.8%) tumors and was correlated with a differentiated histologic type, deep invasion, and positive lymph node metastasis. Positive expression of HER-2 and TGF-ß1 was detected in 22 (13.3%) and 51 (30.7%) tumors, respectively, both of which were more frequent in differentiated gastric carcinomas. The expression of Smad4 was preserved in 62 tumors and reduced in 104 tumors (62.7%). A reduction in Smad4 expression was also more frequent in differentiated gastric carcinomas and was significantly correlated with deep invasion and positive lymph node metastasis.


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  		Table 1 Immunointensity of COX-2, HER-2, TGF-ß1, and Smad4 in relation to clinicopathological features

 
Expression Levels of COX-2 in Tumors with or without HER-2.
We examined whether the immunointensity of COX-2 was correlated with the HER-2 status in gastric carcinomas. The results are shown in Fig. 2Citation . Although HER-2 protein was detected in only 22 tumors, 19 of these tumors (86.4%) exhibited COX-2 overexpression. The {chi}2 test revealed that the overexpression of COX-2 was more frequent in HER-2–positive tumors than in HER-2–negative tumors (P = 0.003; Table 2Citation ).



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  		Fig. 2. Expression levels of COX-2 in tumors with presence or absence of HER-2. The intensity of COX-2 expression was scored semiquantitatively according to the percentage of positively stained tumor cells (see text).

 

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  		Table 2 Correlations of the COX-2 immunointensity with the presence of HER-2, TGF-ß1, and Smad4 in a total of 166 patients

 
Expression Level of COX-2 in Tumors with or without TGF-ß1/Smad4.
We next examined whether the immunointensity of COX-2 could be correlated with the TGF-ß1/Smad4 status in gastric carcinomas. The results are shown in Fig. 3Citation . No significant correlation between COX-2 and TGF-ß1 expression was seen. However, the immunointensity of COX-2 was inversely correlated with the expression of Smad4 (P = 0.002). As shown in Table 2Citation , the overexpression of COX-2 was detected in only 24 (38.7%) of the 62 Smad4-preserved tumors but in 67 (64.4%) of the 104 Smad4-reduced tumors. Furthermore, we examined whether the TGF-ß1/Smad4 system could interact with the HER-2 pathway in the induction of COX-2. The expression of Smad4 was reduced in most of the tumors that expressed HER-2 (17 of 22, 77.2%), and 15 (88.2%) of these 17 tumors overexpressed COX-2. However, no significant interactions between HER-2 and TGF-ß1 or Smad4 expression was seen in the induction of COX-2 (Table 3)Citation .



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  		Fig. 3. Expression levels of COX-2 in tumors with presence or absence of TGF-ß1/Smad4. The intensity of COX-2 expression was scored semiquantitatively according to the percentage of positively stained tumor cells (see text). {circ} Smad 4 preserved, • Smad4 reduced.

 

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  		Table 3 Correlations of the COX-2 immunointensity with the presence of TGF-ß1 and Smad4 in HER-2–positive gastric carcinoma

 
Analysis of Univariate Prognostic Significance.
The postoperative survival of the 136 patients who underwent curative surgery was analyzed according to the expression of COX-2, HER-2, TGF-ß1, and Smad4. The number of patients in each group stratified by the modes of protein expression and the survival results are shown in Table 4Citation and Fig. 4Citation , respectively. The log-rank test revealed that the overexpression of COX-2, the expression of HER-2, and a reduction in Smad4 were all associated with a poor outcome, whereas the presence of TGF-ß1 was not. Factors related to patient prognosis were also evaluated using the Cox proportional hazard univariate regression analysis. The results are shown in Table 5Citation ; these findings indicate that in addition to the established risk factors for gastric cancer (tumor size, depth of invasion, and lymph node metastasis), the expression of COX-2 (P < 0.0001), HER-2 (P = 0.0396), and Smad4 (P < 0.0001) were also related to postoperative survival. Of interest was that these two different statistical methods indicated similar results in terms of survival significance of the biological markers tested.


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  		Table 4 Correlations of the COX-2 immunointensity with the presence of HER-2, TGF-ß1, and Smad4 in 136 patients who underwent curative surgery

 


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  		Fig. 4. Postoperative survival of 136 patients who underwent curative surgery according to the expression of COX-2 (A), HER-2 (B), TGF-ß1 (C), and Smad4 (D). The intensity of COX-2 expression was scored semiquantitatively according to the percentage of positively stained tumor cells (see text). P was determined by the log-rank test. Pre, preserved; Red, reduced.

 

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  		Table 5 Univariate and multivariate analyses of prognostic factors in advanced gastric cancer

 
Analysis of Multivariate Prognostic Significance.
Factors considered to be univariately significant were additionally analyzed in a multivariate analysis. The results, shown in Table 5Citation , demonstrated that in addition to the depth of invasion and lymph node metastasis, the expression of COX-2 and Smad4, but not of HER-2, were also independent prognostic factors in the 136 patients who underwent curative surgery. We additionally analyzed the survival of the 17 patients with tumors that overexpressed HER-2. An increase in the COX-2 levels was not associated with a poorer outcome in patients with HER-2–positive tumors. However, as shown in Fig. 5Citation , in 79 patients with Smad4-reduced tumors, a significant association between COX-2 overexpression and a poor survival rate was detected (P = 0.0201), indicating that these patients have an especially high risk of a poor outcome.



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  		Fig. 5. Postoperative survival of 59 patients with tumors overexpressing COX-2, who underwent curative surgery, according to the HER-2 (A) or Smad4 (B) expression. P was determined by the log-rank test. Pre, preserved; Red, reduced.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although evidence indicating that COX-2 is involved in gastric carcinogenesis is accumulating (2, 3, 4) , the mechanisms responsible for the up-regulation of COX-2 are not completely understood. In the present study, we found that COX-2 is frequently overexpressed in tumors with increased expression levels of HER-2 or with reduced expression levels of Smad4 and that each of these proteins was associated with postoperative survival. Furthermore, a multivariate analysis demonstrated a significantly poor outcome in patients with Smad4-reduced tumors and concomitant COX-2 overexpression.

Several observations of the expression of COX-2 in human gastric cancer have been made using immunohistochemistry or Western blotting analyses (5, 6, 7, 8) . These data show a high frequency (58–68%) of COX-2 immunoreactivity, whereas the reported correlations between COX-2 expression and the clinicopathological characteristics and patient outcome were controversial. Lim et al. (32) did not find any significant correlations between COX-2 protein expression and several clinicopathological characteristics such as Tumor-Node-Metastasis staging, tumor histology, and lymphatic invasion and suggested that COX-2 overexpression may be involved in the initial development, but not in the progression, of gastric cancer. In contrast, a recent study by Saukkonen et al. (6) reported that the COX-2 protein was abundantly found in intestinal-type (well-differentiated) gastric cancer specimens; this group concluded that the expression of COX-2 mRNA and its enzymatic activity were consistently higher in well-differentiated gastric cancer cell lines. Furthermore, evidence that the overexpression of COX-2 is involved in tumor growth and metastasis, and thus associated with a poor prognosis, has been obtained in several studies (7 , 8) . Our data indicate that COX-2 is frequently expressed in differentiated-type tumors and that the overexpression of COX-2 was significantly correlated with deep invasion, lymph node metastasis, and a poor postoperative outcome. Several studies have indicated an association between COX-2 overexpression and a mutation in p53, a major tumor suppressor gene involved in apoptosis, in gastric cancer specimens (29 , 33) . Tsujii and DuBois (34) demonstrated that the overexpression of COX-2 in intestinal epithelial cells made the cells resistant to apoptosis via BCL2 induction and was also associated with an increased adhesion to extracellular matrix protein. These alterations could imply the involvement of COX-2 in tumor progression.

Growth factor receptors such as HER-1 and HER-2 play essential roles in the regulation of gastric epithelial cells (15) . Although an increase in the expression of HER-2 occurs in only 10–20% of human gastric cancers (14, 15, 16, 17) , it has been associated with a poor patient outcome (16 , 17) , consistent with the present results. The overexpression of HER-2 is known to activate the Ras pathway and to increase mitogenic signaling (35) , although the exact mechanism is not completely understood. Recently, a link between HER-2 signaling and COX-2 expression has been established (11, 12, 13) . Vadlamudi et al. (12) demonstrated that the HER-2 signal activates the COX-2 promoter, leading to the expression of COX-2 mRNA and protein, and causes PGE2 to accumulate in human colorectal cancer cell lines. Subbaramaiah et al. (13) showed that HER-2 stimulates COX-2 transcription via the Ras pathway in cultured human mammary epithelial cells. In the present study, both the frequency and the magnitude of COX-2 expression were markedly enhanced in HER-2–positive tumors. These findings and results support the hypothesis that the HER-2 status may be one of the determinants of COX-2 expression in human gastric cancer.

Although insensitization of the growth inhibitory effects of TGF-ß may be a key step in the escape of intestinal epithelial tumors from normal growth control (18 , 19) , the exact role of TGF-ß in neoplastic transformation remains unclear. Recent evidence has ascribed the TGF-ß-induced malignant conversion of epithelial cells to the up-regulation of COX-2 (23, 24, 25, 26 , 36) . Shao et al. (23) found that the TGF-ß-immunostaining pattern in metastatic human colon cancers was similar to that observed for COX-2. Several studies have demonstrated that TGF-ß collaborates with oncogenic Ras to induce the expression of COX-2 mRNA and protein in rodent colonocytes (24 , 26 , 36) . Therefore, we compared the immunointensity of the COX-2 protein in TGF-ß1-positive and -negative human gastric carcinomas; however, COX-2 overexpression was rare in TGF-ß1-positive tumors. To elicit their biological effects, TGF-ßs require both TGF-ß type I and type II receptors (37) . After dimerization upon binding to the ligand, the type II receptors phosphorylate the type I receptors, which then transmit the TGF-ß signal to the intracellular mediators known as Smad (20) . The type II receptors and Smad4 are often defective in transformed epithelial cells (38 , 21) , resulting in the loss of growth inhibition in response to TGF-ß. Sheng et al. (39) recently reported that the tumorigenic transformation of intestinal epithelial cells by exposure to TGF-ß1 was associated with the down-regulation of TGF-ß type II receptors and the induction of COX-2. Interestingly, the immunointensity of COX-2 was significantly enhanced in Smad4-reduced tumors in the present study; thus, COX-2 may contribute to the TGF-ß-induced transformation of gastric epithelial cells, and the reduction in Smad4 may be, in part, of causal significance.

Epidemiologic studies showing a decreased risk of gastrointestinal cancer in individuals taking nonsteroidal anti-inflammatory drugs (40) and a number of cancer prevention studies demonstrating that COX-2 is a target of nonsteroidal anti-inflammatory drugs (41 , 42) have prompted the development of selective COX-2 inhibitors. Celecoxib is one such drug that inhibits COX-2-derived PG production, resulting in the prevention of tumor growth in vitro (43) and in vivo (44) . Our results, indicating that the concomitant overexpression of COX-2 in tumors with an increased expression of HER-2 or a reduced expression of Smad4 is associated with a poor outcome, support the idea that selective COX-2 inhibitors may be of therapeutic value for the treatment of gastric cancer. Saha et al. (45) found that TGF-ß1 and EGF synergistically induced the expression of COX-2 and PGE2 production in mink lung epithelial cells. Interestingly, AG1478, a selective EGF receptor kinase inhibitor, completely suppressed the induction of COX-2 by either EGF or TGF-ß1 plus EGF, suggesting a collaborative interaction between TGF-ß1 and EGF signaling in the induction of COX-2 in vitro (45) . Furthermore, a recent study by Pai et al. (46) demonstrated that PGE2 transactivates the EGF receptor in human colon cancer cell lines and that this PGE2 effect is significantly attenuated by AG1478 inhibition. Although our data from clinical samples failed to demonstrate a clear interaction between HER-2 and the TGF-ß/Smad system in the induction of COX-2, these studies imply that the combinatorial use of selective COX-2 inhibitors and other agents targeting HER-2 and TGF-ß signaling may be effective for the treatment of cancer in the gastrointestinal tract. The present study supports the significance of COX-2 in the development of human gastric carcinoma and suggests that COX-2 may be a promising therapeutic target for future pharmaceutical therapies.


   ACKNOWLEDGMENTS
 
We thank Dr. Yasuichiro Nishimura (Associate Professor, Department of Mathematics, Osaka Medical College) for his expert contribution in statistical evaluation of multiple markers.


   FOOTNOTES
 
Grant support: Grant-in-Aid for Young Scientists-B 13770682 (H. Shinohara) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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.

Requests for reprints: Nobuhiko Tanigawa, Department of General and Gastroenterological Surgery, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan. Phone: 81-72-683-1221, ext. 2361; Fax: 81-72-685-2057; E-mail: sur001{at}poh.osaka-med.ac.jp

Received 4/30/03; revised 2/25/04; accepted 3/30/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stadtlander CT, Waterbor JW. Molecular epidemiology, pathogenesis and prevention of gastric cancer. Carcinogenesis (Lond.) 1999;20:2195-2208.[Abstract/Free Full Text]
  2. Van Rees BP, Saukkonen K, Ristimaki A, et al Cyclooxygenase-2 expression during carcinogenesis in the human stomach. J Pathol 2002;196:171-179.[CrossRef][Medline]
  3. Tsuji S, Tsuji M, Kawano S, Hori M. Cyclooxygenase-2 up-regulation as a perigenetic change in carcinogenesis. J Exp Clin Cancer Res 2001;20:117-129.[Medline]
  4. Ristimaki A, Honkanen N, Jankala H, Sipponen P, Harkonen M. Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res 1997;57:1276-1280.[Abstract/Free Full Text]
  5. Uefuji K, Ichikura T, Mochizuki H, Shinomiya N. Expression of cyclooxygenase-2 protein in gastric adenocarcinoma. J Surg Oncol 1998;69:168-172.[CrossRef][Medline]
  6. Saukkonen K, Nieminen O, van Rees B, et al Expression of cyclooxygenase-2 in dysplasia of the stomach and in intestinal-type gastric adenocarcinoma. Clin Cancer Res 2001;7:1923-1931.[Abstract/Free Full Text]
  7. Chen CN, Sung C-T, Lin M-T, Lee P-H, Chang K-J. Clinicopathologic association of cyclooxygenase 2 expression in gastric adenocarcinoma. Ann Surg 2001;233:183-188.[CrossRef][Medline]
  8. Murata H, Kawano S, Tsuji S, et al Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma. Am J Gastroenterol 1999;94:451-455.[CrossRef][Medline]
  9. Williams CS, DuBois RN. Prostaglandin endoperoxide synthase: why two isoforms?. Am. J Physiol 1996;270:G393-G400.
  10. Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990;61:203-212.[CrossRef][Medline]
  11. Kiguchi K, Carbajal S, Chan K, et al Constitutive expression of ErbB-2 in gallbladder epithelium results in development of adenocarcinoma. Cancer Res 2001;61:6971-6976.[Abstract/Free Full Text]
  12. Vadlamudi R, Mandal M, Adam L, Steinbach G, Mendelsohn J, Kumar R. Regulation of cyclooxygenase-2 pathway by HER2 receptor. Oncogene 1999;18:305-314.[CrossRef][Medline]
  13. Subbaramaiah K, Norton L, Gerald W, Dannenberg AJ. Cyclooxygenase-2 is overexpressed in HER-2/neu-positive breast cancer. J Biol Chem 2002;277:18649-18657.[Abstract/Free Full Text]
  14. Yokota J, Yamamoto T, Miyajima N, et al Genetic alterations of the c-erbB-2 oncogene occur frequently in tubular adenoma of the stomach and are often accompanied by amplification of the v-erbA homologue. Oncogene 1988;2:283-287.[Medline]
  15. Lemoine NR, Jain S, Silvestre F, et al Amplification and overexpression of the EGF receptor and c-erbB-2 proto-oncogenes in human stomach cancer. Br J Cancer 1991;64:79-83.[Medline]
  16. Yonemura Y, Ninomiya I, Yamaguchi A, et al Evaluation of immunoreactivity for erbB-2 protein as a marker of poor short-term prognosis in gastric cancer. Cancer Res 1991;51:1034-1038.[Abstract/Free Full Text]
  17. Allgayer H, Babic R, Gruetzner KU, Tarabichi A, Schildberg FW, Heiss MM. c-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumor-associated protease systems. J Clin Oncol 2000;18:2201-2209.[Abstract/Free Full Text]
  18. Roman C, Saha D, Beauchamp R. TGF-ß and colorectal carcinogenesis. Microsc Res Tech 2001;52:450-457.[CrossRef][Medline]
  19. Maehara Y, Kakeji Y, Kabashima A, et al Role of transforming growth factor-ß1 in invasion and metastasis in gastric carcinoma. J Clin Oncol 1999;17:607-614.[Abstract/Free Full Text]
  20. Lagna G, Hata A, Hemmatibrivanlou A, Massague J. Partnership between DPC4 and Smad proteins in TGF-ß signaling pathways. Nature (Lond.) 1996;383:832-836.[CrossRef][Medline]
  21. Xiangming C, Natsugoe S, Takao S, et al Preserved Smad4 expression in the transforming growth factor ß signaling pathway is a favorable prognostic factor in patients with advanced gastric cancer. Clin Cancer Res 2001;7:277-282.[Abstract/Free Full Text]
  22. Powell SM, Harper JC, Hamilton SR, Robinson CR, Cummings OW. Inactivation of Smad4 in gastric carcinomas. Cancer Res 1997;57:4221-4224.[Abstract/Free Full Text]
  23. Sho J, Sheng H, Aramandla R, et al Coordinate regulation of cyclooxygenase-2 and TGF-ß1 in replication error-positive colon cancer and azoxymethane-induced rat colonic tumors. Carcinogenesis (Lond.) 1999;20:185-191.[Abstract/Free Full Text]
  24. Sheng H, Shao J, Hooton EB, Tsujii M, DuBois RN, Beauchamp RD. Cyclooxygenase-2 induction and transforming growth factor ß growth inhibition in rat intestinal epithelial cells. Cell Growth Differ 1997;8:463-470.[Abstract]
  25. Geiser AG, Kim S, Roberts AB, Sporn MB. Characterization of the mouse transforming growth factor-ß1 promoter and activation by the Ha-ras oncogene. Mol Cell Biol 1991;11:84-92.[Abstract/Free Full Text]
  26. Sheng H, Shao J, Dixon DA, et al Transforming growth factor-ß1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA. J Biol Chem 2000;275:6628-6635.[Abstract/Free Full Text]
  27. Japanese Gastric Cancer Association. Japanese classification of gastric carcinoma, 2nd English edition. Response assessment of chemotherapy and radiotherapy for gastric carcinoma: clinical criteria. Gastric Cancer 2001;4:1-8.[CrossRef][Medline]
  28. Kawasaki H, Toyoda M, Shinohara H, et al Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during colorectal tumorigenesis. Cancer (Phila.) 2001;91:2026-2032.
  29. Leung W, To K-F, Ng Y-P, et al Association between cyclo-oxygenase-2 overexpression and missense p53 mutations in gastric cancer. Br J Cancer 2001;84:335-339.[CrossRef][Medline]
  30. Carter P, Presta L, Gorman CM, et al Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992;89:4285-4289.[Abstract/Free Full Text]
  31. Saito H, Tsujitani S, Oka S, et al The expression of transforming growth factor-ß1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric cancer. Cancer (Phila.) 1999;86:1455-1462.
  32. Lim HY, Joo HJ, Choi JH, et al Increased expression of cyclooxygenase-2 protein in human gastric carcinoma. Clin Cancer Res 2000;6:519-525.[Abstract/Free Full Text]
  33. Kawabe A, Shimada Y, Uchida S, et al Expression of cyclooxygenase-2 in primary and remnant gastric carcinoma: comparing it with p53 accumulation, Helicobactor pylori infection, and vascular endothelial growth factor expression. J Surg Oncol 2002;80:79-88.[CrossRef][Medline]
  34. Tsujii M, DuBois RN. Alteration in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxidase synthase 2. Cell 1995;83:493-501.[CrossRef][Medline]
  35. Reese DM, Slamon DJ. HER-2/neu signal transduction in human breast and ovarian cancer. Stem Cells 1997;15:1-8.[Abstract/Free Full Text]
  36. Roman CD, Morrow J, Whitehead R, Beauchamp RD. Induction of cyclooxygenase-2 and invasiveness by transforming growth factor b1 in immortalized mouse colonocytes expressing oncogenic Ras. J Gastrointest Surg 2002;6:304-309.[CrossRef][Medline]
  37. Massague J. TGF-ß signal transduction. Annu Rev Biochem 1998;67:753-791.[CrossRef][Medline]
  38. Chen R-H, Ebner R, Derynck R. Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-ß activities. Science (Wash. DC) 1993;260:1335-1338.[Abstract/Free Full Text]
  39. Sheng H, Shao J, O’Mahony CA, et al Transformation of intestinal epithelial cells by chronic TGF-ß1 treatment results in down-regulation of the type II TGF-ß receptor and induction of cyclooxygenase-2. Oncogene 1999;18:855-867.[CrossRef][Medline]
  40. Peleg II, Maibach HT, Brown SH, Wilcox CM. Aspirin and nonsteroidal anti-inflammatory drug use and the risk of subsequent colorectal cancer. Arch Intern Med 1994;154:394-399.[Abstract/Free Full Text]
  41. Dubois RN, Abramson SB, Crofford L, et al Cyclooxygenase in biology and disease. FASEB J 1998;12:1063-1073.[Abstract/Free Full Text]
  42. Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (Part II). J Natl Cancer Inst (Bethesda) 1998;90:1609-1620.[Abstract/Free Full Text]
  43. Williams CS, Watson AJ, Sheng H, Helou R, Shao J, Dubois RN. Celecoxib prevents tumor growth in vivo without toxicity to normal gut: lack of correlation between in vitro and in vivo models. Cancer Res 2000;60:6045-6051.[Abstract/Free Full Text]
  44. Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki AT, Masferrer JL. Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Res 2002;62:625-631.[Abstract/Free Full Text]
  45. Saha D, Datta PK, Sheng H, et al Synergistic induction of cyclooxygenase-2 by transforming growth factor-ß1 and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia 1999;1:508-517.[CrossRef][Medline]
  46. Pai R, Soreghan B, Szabo IL, Pavelka M, Baatar D, Tarnawski AS. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nat Med 2002;8:289-293.[CrossRef][Medline]



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