Original Article Expression of Beta-catenin, COX-2 ...

05702#4 27/11/03

Original Article

Expression of Beta-catenin, COX-2 and iNOS in Colorectal Cancer: Relevance of COX-2 and iNOS Inhibitors for Treatment in Malaysia Seok Kwan Hong, Yunus A. Gul,1 Hairuszah Ithnin, Arni Talib2 and Heng Fong Seow, Departments of Clinical Laboratory Science and 1Surgery, Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor, and 2Department of Pathology, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia.

BACKGROUND: Promising new pharmacological agents and gene therapy targeting cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) could modulate treatment of colorectal cancer in the future. The aim of this study was to elucidate the expression of β-catenin and the presence of COX-2 and iNOS in colorectal cancer specimens in Malaysia. This is a useful prelude to future studies investigating interventions directed towards COX-2 and iNOS. METHODS: A cross-sectional study using retrospective data over a 2-year period (1999–2000) involved 101 archival, formalin-fixed, paraffin-embedded tissue samples of colorectal cancers that were surgically resected in a tertiary referral centre. RESULTS: COX-2 production was detected in adjacent normal tissue in 34 samples (33.7 %) and in tumour tissue in 60 samples (59.4%). More tumours expressed iNOS (82/101, 81.2%) than COX-2. No iNOS expression was detected in adjacent normal tissue. Intense β-catenin immunoreactivity was found in both the cytoplasm and nuclei of tumour cells, with a distinct loss of β-catenin immunoreactivity at the cell-to-cell border. Poorly differentiated tumours had significantly lower total β-catenin (p = 0.009) and COX-2 scores (p = 0.031). No significant relationships were established between pathological stage and β-catenin, COX-2 and iNOS scores. CONCLUSIONS: The accumulation of β-catenin does not seem to be sufficient to activate pathways that lead to increased COX-2 and iNOS expression. A high proportion of colorectal cancers were found to express COX2 and a significant number produced iNOS, suggesting that their inhibitors may be potentially useful as chemotherapeutic agents in the management of colorectal cancer. [Asian J Surg 2004;27(1):10–7]

Introduction The incidence of colorectal cancer in Malaysia is increasing; recent data collected by a local cancer registry in Malaysia demonstrate that colorectal carcinoma is the third most common malignancy, with an incidence of 22.5 per 100,000.1 Measures to reduce the morbidity and mortality associated with this malignancy are being implemented primarily through dispersion of information to the public regarding the

importance of seeking early medical advice and via increasing use of endoscopic evaluation. It is imperative, however, for these measures to be supplemented with newer strategies and interventions, apart from surgery, to produce a dramatic improvement in the outcome or survival of patients with colorectal cancer. Vogelstein’s colorectal cancer model outlines at least four sequential mutational changes, including mutations in the adenomatous polyposis coli (APC) gene, which is the earliest

Address correspondence and reprint requests to Dr. Heng Fong Seow, Department of Clinical Laboratory Science, Faculty of Medicine and Health Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia. E-mail: [email protected] • Date of acceptance: 30th May, 2003 © 2004 Elsevier. All rights reserved.

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ASIAN JOURNAL OF SURGERY VOL 27 • NO 1 • JANUARY 2004

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■ EXPRESSION OF β-CATENIN, COX-2, AND INOS IN CRC ■

genetic mutation to occur,2 K-ras, “deleted in colorectal cancer” (DCC), and p53.3,4 APC is necessary for the formation of complexes between glycogen synthase kinase (GSK)-3β and βcatenin. β-Catenin is the target for degradation by the ubiquitinproteosome pathway. 5 Functional loss of APC by genetic mutations in colorectal cancer causes the accumulation of βcatenin.6 β-Catenin can then translocate into the nucleus where it binds to the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of proteins and activates many genes such as c-myc, cyclin D1, matrix metalloproteinase, cyclooxygenase2 (COX-2), and inducible nitric oxide synthase (iNOS).7 COX-2, an enzyme involved in the conversion of arachidonic acid to prostaglandins, is induced by inflammatory and mitogenic stimuli. Thus, the synthesis of prostaglandins is increased in inflamed and neoplastic tissues.8 There is ample evidence suggesting that COX-2 expression is important in carcinogenesis.9 Selective COX-2 inhibitors can interfere with tumorigenesis in experimental systems including colorectal cancer.10 Accumulating evidence indicates that non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the mortality rate by 40% to 50%,11 decrease cell proliferation to give smaller tumours,12 and reduce the number of intestinal polyps. 13 NSAIDs reduce the number and size of polyps in patients with familial adenomatous polyposis.14 Selective COX-2 inhibitors may play an important role in the management of colorectal cancer in the future. Nitric oxide synthase catalyzes the synthesis of nitric oxide (NO) from L-arginine.15 Both iNOS and NO have been reported in various cancers, including colorectal carcinoma.16 NO is a highly reactive free radical and short-lived molecule that is involved in many tumour-promoting functions,17–19 suggesting that iNOS inhibitors may also have therapeutic value in treating colorectal cancer. We investigated 101 paired colorectal carcinoma tissues for β-catenin, COX-2 and iNOS expression to determine the correlation between expression of these molecules and to determine the frequency of COX-2 and iNOS expression, to assess the clinical relevance of new pharmacological agents and gene therapy targeting COX-2 and iNOS. This is the first study in this country to investigate the presence of COX-2 and iNOS in colorectal carcinoma.

Materials and methods Tissue specimens This was a cross-sectional study using retrospective data from 101 archival, formalin-fixed, paraffin-embedded tissue samples


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of colorectal carcinoma that were surgically resected at the Kuala Lumpur Hospital, Malaysia, between 1999 and 2000. None of the patients had received neoadjuvant therapy prior to surgery and none had a known family history of colorectal cancer. Only primary colorectal carcinomas were included. A number of paraffin blocks were made for each surgically resected tumour. Archival haematoxylin and eosin-stained slides were retrieved to find the section that contained the bulk of the tumour, which was representative of the tumour and the paired adjacent normal tissue. The respective tissue blocks were then sectioned at 4 µm and mounted on glass slides coated with 3-aminopropyltrimethoxysilane (Aldrich Chemical Company, Milwaukee, WI, USA).

Primary antibodies Three primary antibodies were used. The anti-β-catenin monoclonal antibody was raised against a peptide corresponding to amino acids 571–781 at the carboxyl terminus of β-catenin (clone 14; Transduction Laboratories, Lexington, KY, USA) and used at a 1:200 dilution. Anti-COX-2 monoclonal antibody was raised against a peptide corresponding to amino acids 368–604 of COX-2 (clone 33; Transduction Laboratories) and used at a 1:50 dilution. The anti-iNOS monoclonal antibody was reactive against a peptide corresponding to amino acids 961–1144 of iNOS (clone 6; Transduction Laboratories) and it was used at a dilution of 1:100.

Immunohistochemistry Prior to immunohistochemical staining, sections were deparaffinized and rehydrated using standard procedures. Antigen retrieval was performed using heat treatment; endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide (Ajax Chemicals, Auburn, NSW, Australia) for 10 minutes. To block non-specific antigen sites, tissue sections were incubated for 1 hour in 1.5% bovine serum albumin (Fluka BioChemika, Deisenhofen, Germany) at room temperature. Incubation with the primary antibodies was performed at room temperature for 30 minutes with anti-βcatenin, and 60 minutes with the anti-COX-2 and anti-iNOS antibodies. After the primary antibody incubation step, a secondary antibody from a streptavidin biotin complex peroxidase kit (LSAB® + kit, Dako, Copenhagen, Denmark) was used according to the manufacturer’s instructions with minor modifications. For sections incubated with anti-βcatenin, both the linking antibody and streptavidin peroxidase complex from the LSAB® + kit were incubated consecutively for 15 minutes. For the remaining antibodies, the incubation

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■ HONG AND OTHERS ■

period was 30 minutes. Peroxidase activity was developed with the substrate 3,3’-diaminobenzidine tetrahydrochloride (DAB; Dako) by incubating the sections in DAB for 10 minutes. Sections were then rinsed gently with distilled water and counterstained with haematoxylin. Negative controls were prepared simultaneously for all 101 samples by replacing the primary antibody with distilled water. Thus, each tumour slide had its own negative control.

Evaluation of immunohistochemical staining At least three fields were randomly selected for each section. A modified semi-quantitative scoring system was used to evaluate staining (Table 1).15,20 The percentage of positive cells and the strength of staining intensity were evaluated for the samples and negative controls. The score for each area was the sum of the percentage of positive cells and strength of staining intensity. A final total score was generated by subtracting the score in the sample from the score in the negative control. The minimum and maximum score for a sample were 0 and 7, respectively. The evaluation was performed by two independent researchers, one of whom was a medically qualified histopathologist, to ensure precision and accuracy.

Statistical analysis The average total scores are reported as the mean rank and median score. Since the total score was not normally distributed, the association between immunohistochemical score and clinicopathological features was analysed using the MannWhitney U test or Kruskal-Wallis one-way analysis of variance by ranks. The Spearman rank correlation test was used to analyse the correlation of β-catenin, COX-2 and iNOS Table 1. Scoring system for immunohistochemical staining, adapted from previous reports15,20 Evaluation of % positive cells

Evaluation of stain intensity

0 1+ 2+ 3+ 4+

0 1+ 2+ 3+

no staining < 25% positive 25–50% positive 50–75% positive > 75% positive

no staining mild staining moderate staining intense staining

Score for a tumour area (A) = score for % positive cells + score for stain intensity Score for negative control of similar tumour tissue area (B) = score for % positive cells + score for stain intensity Total score = A – B

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expression with each other. A p value of less than 0.05 was considered significant. Statistical analysis was performed using SPSS for Windows version 10.0 (SPSS Inc, Chicago, IL, USA).

Results Table 2 shows the clinicopathological data of the cases in this study. The study included samples from 59 male and 42 female patients with ages ranging from 23 to 87 years. Fifty-five patients were of Chinese origin and 46 were non-Chinese. Of the 101 cancers, 80 (79.2%) were moderately differentiated, 13 (12.9%) were well differentiated, and eight (7.9%) were poorly differentiated.

Analysis of β-catenin immunoreactivity Positive immunoreactivity for β-catenin was detected in all 101 samples. In normal adjacent tissue, it was mainly localized

Table 2. Clinicopathological data of patients (N = 101) Patient characteristic

n

Gender Male Female

59 42

Age Range, 23–87 yr Median, 63 yr < 40 yr ≥ 40 yr

4 97

Race Chinese Non-Chinese

55 46

Pathological stage (Astler-Coller classification) A B1 B2 C1 C2

4 6 45 4 42

Histological grade Well differentiated Moderately differentiated Poorly differentiated

13 80 8

Tumour site Left colon Right colon Rectum

57 24 20


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in the plasma membrane of the cell-to-cell border and in the cytoplasm of both the colonic epithelium and goblet cells (Figure 1A). No nuclear β-catenin was seen in the normal colonic mucosa. However, in colorectal carcinoma tissue, βcatenin immunoreactivity was found to be markedly intense in both the cytoplasm and nuclei of tumour cells from all 101 cases (Figure 1B). Both loss of and reduced β-catenin immunoreactivity in the cell-to-cell border were observed.

COX-2 and iNOS immunoreactivity COX-2 immunoreactivity was detected in adjacent normal tissue in only 33.7% (34) of samples (Figure 2A) compared to

A

59.4% (60) in tumour tissue (Figure 2B). In all positive cases, COX-2 immunoreactivity was located in the cytoplasm, although some endothelial and stromal cells found at the tumour site were also weakly reactive. iNOS immunoreactivity was not detected in adjacent normal tissue in any samples (Figure 2C), in contrast to positive immunoreactivity in tumour tissue in 81.2% of samples (Figure 2D). The immunoreactivity was located in the cytoplasm of tumour cells, especially at the apical surfaces of cells forming a glandular pattern. Very weak immunoreactivity was detected in endothelial and smooth muscle cells found at or near the tumour site.

B

Figure 1. Immunohistochemical staining for β-catenin is detected at: A) the cell-to-cell border of the plasma membrane and in the cytoplasm of adjacent normal tissue; B) in both the cytoplasm and nuclei of tumour tissue.

A

B

C

D

Figure 2. Representative slides showing immunohistochemical staining for cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). A) COX-2 immunoreactivity is detected in the cytoplasm of normal adjacent colonic tissue (x 100) and B) colorectal carcinoma tissue (x 200). C) There is no iNOS staining in normal adjacent colonic tissue (x 100) but D) iNOS is present in the tumour (x 100).


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Table 3. Correlation among total β-catenin, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) scores β-catenin score β-catenin score r p COX-2 score r p iNOS score r p

COX-2 score

iNOS score

— —

0.323 0.001

0.371 < 0.05

0.323 0.001

— —

0.393 < 0.05

0.371 < 0.05

0.393 < 0.05

— —

Spearman rank correlation test. A 2-sided p < 0.05 indicates statistical significance.

The correlation between total scores for β-catenin, COX-2 and iNOS is shown in Table 3. Using the Spearman rank correlation test, we found that total β-catenin, COX-2 and iNOS scores were significantly related to each other. There was a positive linear relationship between total β-catenin score and both total COX-2 score and total iNOS score. There was also a significant positive linear relationship between COX-2 and iNOS scores.

total β-catenin, COX-2 and iNOS scores (Table 5). Total βcatenin (p = 0.009, Kruskal-Wallis one-way analysis of variance by ranks) and COX-2 (p = 0.031) scores were statistically significantly associated with histological grade (Table 6). In general, poorly differentiated tumours had significantly lower total β-catenin and COX-2 immunohistochemical scores. There was no significant correlation between tumour site and total COX-2 and iNOS scores (Table 7). Tumours found in the left

Correlations between total immunoreactivity scores and patient characteristics

Table 4. Correlation between gender and total β-catenin, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) scores

There was no significant correlation between gender and total β-catenin, COX-2 and iNOS scores (Table 4). There was no significant correlation between age or race and total β-catenin, COX-2 and iNOS scores (data not shown).

Male (n = 59)

Female (n = 42)

p

52.34 48.83 49.97

49.12 54.05 52.45

0.566 0.359 0.654

Correlations between total immunoreactivity scores and tumour characteristics

Mean β-catenin rank Mean COX-2 rank Mean iNOS rank

No statistically significant correlations were established between pathological stage (Astler-Coller classification) and

Mann-Whitney U-test. p < 0.05 indicates statistical significance.

Table 5. Correlation between pathological stage (Astler-Coller classification) and total β-catenin, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) scores Astler-Coller classification


A (n = 4)

B1 (n = 6)

B2 (n = 45)

C1 (n = 4)

C2 (n = 42)

p

39.50 62.38 54.88

37.33 49.08 56.17

56.29 55.27 53.83

57.75 31.38 56.50

47.74 47.49 46.33

0.333 0.366 0.709

Kruskal-Wallis one-way analysis of variance by ranks. p < 0.05 indicates statistical significance.

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Table 6. Relationship of total β-catenin, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) scores to histological grade Histological grade


Well differentiated (n = 13)

Moderately differentiated (n = 80)

Poorly differentiated (n = 8)

p

52.35 63.00 54.65

53.67 51.19 52.41

22.06 29.63 31.00

0.009 0.031 0.096


colon and rectum had significant total β-catenin scores, whereas tumours in the right colon did not.

Discussion β-Catenin is a critical signal transducer in the Wnt/Wingless pathway that regulates cell growth, morphogenesis, organ development and cancer development.21 The localization of βcatenin immunoreactivity to the plasma membrane and cellto-cell border of the normal colonic mucosa is consistent with the findings of Iwamoto et al.22 This is not surprising; βcatenin binds to the cytoplasmic tail of β-catenin, E-cadherin and, indirectly, to the cytoskeleton, so it is localized to the adherens junction of the cell-to-cell plasma membrane.6 Formation of multiprotein complexes consisting of proteins such as APC, GSK-3β, axin and β-catenin makes βcatenin a target for degradation, so that no cytoplasmic or nuclear β-catenin will be detected in normal tissue. Our data indicate that the amount of β-catenin is increased in both the cytoplasm and nuclei of all colorectal carcinoma tissue, as shown by the increased intensity of immunostaining. This could be due to the inability of GSK-3β to phosphorylate βcatenin, or to mutations in the genes encoding the proteins that form the multiprotein complexes, which will result in the accumulation of cytoplasmic β-catenin and translocation to the nucleus.23 Furthermore, mutations in E-cadherin may also

cause the dissociation of β-catenin from the E-cadherin–βcatenin complex, resulting in the release of unbound β-catenin into the cytoplasm and subsequent translocation to the nucleus. Once in the nucleus, β-catenin can interact with members of the TCF/LEF DNA-binding family and induce the expression of c-myc, cyclin D1 and matrilysin, which are responsible for tumour proliferation and malignant progression. 23–25 Other examples of genes that are transcriptionally activated include COX-2 and iNOS.26 Our results show, for the first time, the presence of accumulated cytoplasmic and nuclear β-catenin in colorectal carcinoma specimens in Malaysia. They concur with those reported previously.21,22 We need to further investigate the reasons for the inhibition of β-catenin degradation in our colorectal carcinoma specimens. Immunoreactivity to COX-2, detected in 59.4% of specimens in our study, was less frequent than in previous studies, which report positivity in as much as 85% to 90% of samples.27,28 Only 33.7% of samples demonstrated increased COX-2 production in adjacent normal tissue. The reason for the increased production of COX-2 by these apparently normal cells is not clear. It is possible that the presence of soluble factors in the tumour microenvironment could have contributed to this increased COX-2 expression. The positive COX-2 immunoreactivity in our study further supports the role of COX-2 in carcinogenesis. COX-2 intermediates are

Table 7. Relationship of total β-catenin, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) scores to tumour site Tumour site


Left colon (n = 57)

Right colon (n = 24)

Rectum (n = 20)

p

55.29 54.79 54.63

36.85 49.52 47.90

55.75 41.97 44.38

0.017 0.207 0.291



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highly reactive molecules that can derivatize DNA and oxidize certain compounds such as oestrogens into mutagens.29 Hence, COX-2 and its by-products of arachidonic acid metabolism may contribute to the “genetic stability” and malignant progression of tumour cells. The absence of COX-2 in the nucleus is consistent with the results of some studies,30,31 but not with those of other studies where both cytoplasm and nuclear membranes were positive.32 We have yet to elucidate whether COX-2 is the downstream target of the Wnt signalling pathway in both normal and tumour tissue in our colorectal carcinoma cases. The alternative pathway for induction of COX-2 expression is the nuclear factor (NF)-κβ signalling pathway.33 Overexpression of βcatenin alone has no effect on COX-2 gene expression, which suggests a Wnt signalling pathway that cannot be mimicked by β-catenin expression.34 This may explain our finding that 40.6% of our samples did not express COX-2 in tumour cells, although there was an accumulation of β-catenin in the cytoplasm and nucleus. Our findings of iNOS production are similar to those of previous studies. iNOS has been reported in macrophages, vascular smooth muscle and a human colonic adenocarcinoma cell line.35–37 Takahashi et al reported iNOS expression in rat colonic carcinoma epithelial cells, especially at the luminal surfaces of cancer cells, forming a glandular pattern, with all rat colonic epithelium testing positive.31 This differs from our findings in human cells, where 81.2% of colorectal cancer specimens demonstrated positive iNOS expression. This difference may be due to the fact that chemical-induced carcinogenesis in experimental animal models is not identical to human colorectal carcinoma. iNOS expression in the tumour cells and surrounding stroma may result from interferon γ, tumour necrosis factor and other cytokines produced by tumour cells or inflammatory cells at the tumour site, since the activation of the NF-κβ signalling pathway by certain cytokines can also induce iNOS expression.38 Further studies are required, however, to ascertain the reasons for the increased iNOS production. No significant relationship was found between gender, age or race and total β-catenin, COX-2 and iNOS scores. This agrees with the findings of a previous study that there was no significant correlation between COX-2 expression and gender or age. 32 We failed to establish statistically significant relationships between pathological grade and total β-catenin, COX-2 and iNOS scores, even though the total β-catenin and COX-2 scores were at the higher end for well-differentiated tumours. The latter finding should be interpreted with caution

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as a large number of specimens in this study were moderately differentiated (79.2%) while few were well differentiated or poorly differentiated. The localization of β-catenin was both cytoplasmic and nuclear, irrespective of the state of differentiation. Our results are similar to those of one study which showed that low-grade adenomas have lower nuclear localization scores,39 but contrast with those of another study which reported that the most significant correlation of nuclear β-catenin was not with dysplasia grade but with tumour size.40 We were not able to use these results to determine the prognosis because we were unable to access the data for patients who were subsequently followed. Because the evaluation of immunohistochemical staining intensity can be subjective, two researchers independently evaluated the sections and counter-checked them to obtain accurate results. In addition, negative controls for each section were included in all immunostaining experiments. Our current findings need to be supplemented with Western blotting. Each tumour specimen is heterogeneous and one limitation of the study is that results presented were mostly from one section per specimen. To counteract this, we took care to select blocks that contained the bulk of the tumour with adjacent apparently normal tissue, as determined by a qualified histopathologist using microscopy. A high proportion (59.4%) of colorectal cancers in our population produced COX-2 and an even higher proportion produced iNOS (81.2%). Our results suggest that there is a possibility that COX-2 and iNOS inhibitors may be useful as chemotherapeutic agents in the treatment of colorectal carcinoma in some of our patients. In addition, our study suggests that the overexpression of β-catenin is not sufficient to induce COX-2 and iNOS gene expression since 40.6% and 18.8% of tumour cells did not express COX-2 and iNOS, respectively, even though β-catenin had accumulated. It is likely that additional events need to occur for induction of COX-2 and iNOS expression and further investigation is warranted to better understand the pathogenesis of this disease.

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