The molecular basis of carcinogenesis in gastrointestinal carcinoid tumors is not well understood. To clarify the contribution of the Wnt/β-catenin signaling to this type of carcinogenesis, we investigated 72 cases of gastrointestinal carcinoid tumor both immunohistochemically and by direct sequencing of β-catenin. Accumulation of β-catenin in the cytoplasm and/or nucleus was observed in 57 cases (79.2%). We also detected mutations in exon 3 of β-catenin in 27 cases (37.5%) and one mutation in APC (1.4%). Our results suggest that alterations in the Wnt/β-catenin signaling pathway may be involved in the development of gastrointestinal carcinoid tumors.

β-Catenin plays critical roles in both intercellular adhesion and in the Wnt signaling pathway. When localized in the membrane, β-catenin acts as a cadherin-interacting protein by linking cadherin to α-catenin and, hence, mediating anchorage of the cadherin complex to the cortical actin cytoskeleton (1). In addition, the Wnt signaling can stabilize β-catenin and cause it to accumulate in the cytoplasm and nucleus where it can act as a transcriptional coactivator by associating with the TCF/LEF3 family of transcription factors (2). Recent studies have revealed that alterations in the Wnt signaling pathway, including those resulting from mutations in APC, β-catenin, and Axin, play important roles in the carcinogenesis of various types of malignant tumors. Mutations in any of these genes can result in accumulation of β-catenin and activation of TCF/LEF transcription factors (2). Interestingly, cyclin D1 and c-myc have recently been identified as target genes for TCF/LEF transcriptional activation (3, 4). It has been well established that inactivating mutations in APC and activating mutations in β-catenin are not uncommon and, according to adenoma-carcinoma sequence theory, are early events in colorectal carcinogenesis (5). However, in gastrointestinal carcinoid tumors, the genetic changes that lead to carcinogenesis have not received much attention as yet. Therefore, we sought to assess a possible contribution of the Wnt signaling pathway to carcinoid tumor carcinogenesis by conducting immunohistochemical studies of β-catenin and mutational analyses of exon 3 of β-catenin and APC in 72 gastrointestinal carcinoid tumor cases.

Tumor Samples and DNA Extraction.

We analyzed 72 samples of gastrointestinal carcinoid tumors from patients who had undergone surgical operations or endoscopic resections. Each sample was also assessed histologically by pathologists. Eleven of the 72 samples were obtained from the stomach, 13 from the duodenum, 3 from the appendix, and 45 from the rectum. Genomic DNA of tumor cells used in the direct sequence analysis was microdissected from 10-μm thick sections of formalin-fixed and paraffin-embedded tissue and then extracted as described previously (6).

Immunohistochemical Analysis of β-Catenin, Cyclin D1, and Histone H1.

Each tumor section (4 μm) was deparaffinized and subjected to antigen retrieval by microwaving in 0.1 m of citrate buffer (sodium citrate, pH 6.0) for 15–30 min. The sections were incubated with anti-β-catenin (Transduction Laboratories, Lexington, KY), anticyclin D1 (Oncogene Research Products, Boston, MA), and antihistone H1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) monoclonal antibodies at a dilution of 1:250, 1:200, and 1:100, respectively, for 12–16 h at 4°C and stained by the avidin-biotin complex method using a Histofine kit (Nichirei, Tokyo, Japan). The β-catenin and cyclin D1 immunostaining results for both cytoplasm and nucleus were evaluated by comparing the staining intensities of carcinoid tumor cells and adjacent nontumor epithelial cells. Immunostaining was defined as positive when >10% of tumor cells stained more strongly than nontumor cells.

Direct DNA Sequence Analysis.

To screen for mutations in exon 3 of β-catenin or in APC, extracted genomic DNA was amplified by PCR. The primers for exon 3 of β-catenin were 5′-GCTGATTTGATGGAGTTGGA-3′ and 5′-GCTACTTGTTCTTGAGTGAA-3′ (7). For APC, the primer sets designed for screening codon 1274 to 1523 of APC were used (8). The PCR reaction was performed in 25 μl of reaction mixture containing 2.5 μl of template DNA, 0.5 μm of each primer, 0.16 mm deoxynucleotide triphosphate, 1.2 mm MgCl2, 0.625 units AmpliTaq Gold DNA Polymerase, and 2.5 μl of 10 × PCR buffer containing 100 mm Tris HCl (pH 8.3) and 500 mm KCl (Applied Biosystems, Forester City, CA). The reaction mixture was preincubated for 10 min at 95°C and incubated for 45 cycles (denaturing at 94°C for 1 min, annealing at 54–58°C for 1 min, and extension at 72°C for 1 min) on a GeneAmp PCR System 9700 (Applied Biosystems). Final extension was allowed to continue at 72°C for 5 min. The PCR products were purified from agarose gels, and sequence analysis was performed using BigDye Terminator Cycle Sequencing FS Reaction kits with an ABI PRISM 310 sequencer (Applied Biosystems). All of the mutations detected in this study were confirmed by direct sequence analysis using both forward and reverse primers.

Statistical Analysis.

For comparisons of the frequencies of clinical or pathological characteristics, the χ2 test or Fisher’s exact test was used. The Student t test was used for comparison of means. All of the statistical analyses were performed using StatView software version 5.0 (Abacus Concepts, Berkeley, CA).

Immunohistochemistry of β-Catenin and Cyclin D1.

In normal epithelial cells of the stomach, duodenum, appendix, and rectum, β-catenin staining was localized to the cell membrane, whereas cytoplasmic and nuclear staining was absent (Fig. 1,A). In the carcinoid tumor cases, the immunostaining patterns of β-catenin could be divided into three groups. In 15 cases (20.8%) there was membranous but not cytoplasmic or nuclear staining, exactly as in normal epithelial cells (Fig. 1,B). In an additional 25 cases (34.7%) we observed nuclear staining (Fig. 1,C), whereas in 32 cases (44.5%) there was evidence of cytoplasmic staining but not strong nuclear staining (where present, staining of the nucleus or nuclear membrane was faint; Fig. 1,D). We concluded that the results obtained for the latter two groups (57 cases, 79.2%) were representative of positive β-catenin staining. Furthermore, immunohistochemistry of cyclin D1 showed positive staining in 61 cases (84.7%; Fig. 1,E and Table 2). We also performed immunohistochemistry of histone H1 as a control staining of nuclear proteins, resulting in positive staining of the nucleus in all of the cases (Fig. 1 F).

Mutational Analysis of β-Catenin and APC.

Mutations in exon 3 of β-catenin were detected in 27 (37.5%) of the 72 gastrointestinal carcinoid tumor cases (Fig. 2 and Table 1). In 26 of these 27 cases, Ser was replaced by Ala at codon 37 (S37A) and Gly was replaced by Asp at codon 48 (G48D) in the other sample. The samples, labeled rectum-19 and rectum-22, contained Gly to Asp substitutions at codons 38 (G38D) and 48 (G48D), respectively, in addition to the S37A mutation. Accumulation of β-catenin in the cytoplasm and/or nucleus was observed in 26 of these 27 cases, and there appeared to be a statistically significant correlation between accumulation of β-catenin and mutations in β-catenin (P < 0.0022; Table 2). We also looked for APC mutations in tissues in which β-catenin accumulation had been demonstrated immunohistochemically and detected a mutation in APC in which the Glu at codon 1408 (GAA) had been replaced by the chain-terminating codon TAA in a sample labeled rectum-28 (Table 1). No other APC mutations were detected. We also analyzed the sequences in nontumor regions adjacent to each sample to confirm that the changes in nucleotides and amino acids in β-catenin and APC reported above are not polymorphisms (data not shown).

Carcinoid tumors belong to a family of neuroendocrine tumors that are derived from neuroendocrine cells and show positive staining of synaptophysin, chromogranin, and neuron-specific enolase (9, 10). Although several genetic alterations in pulmonary carcinoid tumors, including loss of heterozygosity of 11q and mutations in p53, have been reported (9), little is known about the nature of the genetic changes that occur in gastrointestinal carcinoid tumors.

β-Catenin is a component of the Wnt signaling pathway of which the stability appears to be regulated by the Wnt signaling. In the present study, we analyzed exon 3 of β-catenin, which includes Ser and Thr at codons 29, 33, 37, 41, and 45. These amino acids can be directly phosphorylated by GSK-3β, and amino acid changes at these codons protect β-catenin from phosphorylation and degradation. We found an S37A mutation in 26 gastrointestinal carcinoid tumor cases. It is believed that wild-type β-catenin can be ubiquitinated efficiently but that the S37A mutant β-catenin is not ubiquitinated in SKBR3 cells that have been treated with a proteasomal inhibitor (11). Consistent with this finding, it has been shown by pulse-chase analysis that the half-life of the S37A mutant β-catenin is longer than that of the wild-type β-catenin (12). These findings suggest that the S37A mutant β-catenin accumulates in cells and, hence, may promote transmission of the oncogenic signals that lead to gastrointestinal carcinoid tumor formation. Therefore, it is important to note that cyclin D1 was overexpressed in 84.7% of our cases, and c-myc was reported previously to be overexpressed in gastrointestinal carcinoid tumors (13). Such overexpression of cyclin D1 and c-myc may be a downstream effect of mutations in β-catenin and the consequential activation of TCF/LEF. In addition, we identified two other mutant β-catenins, G38D and G48D. Although the Gly residues at codons 38 and 48 are located in the so-called negative regulatory domain of β-catenin (14) and are highly conserved amino acids in various species, and although mutations at codon 38 have been reported in other tumors (7, 15), we still do not know whether these mutant β-catenins can in fact be phosphorylated by GSK-3β and then ubiquitinated.

The results of the immunohistochemical and direct sequencing analyses in this study enabled us to identify 31 cases in which there was positive β-catenin staining but no mutation in β-catenin. Therefore, we conducted a mutational analysis of APC but detected a mutation in only one sample. Presumably, other molecules (e.g., Axin) are altered in the remaining tumor samples. Axin has been shown to act as a negative regulator of the Wnt signaling pathway by forming a complex with GSK-3β, β-catenin, and APC, and, hence, regulating β-catenin degradation (16, 17). Therefore, loss of function of Axin might lead to the accumulation of β-catenin and activation of the Wnt signaling. Thus, it is of interest to note that mutations in Axin have been detected recently in hepatocellular carcinomas and in various cell lines (18, 19). It is also possible that mutations in other regions or deletions of β-catenin or the APC gene that we did not look for in this study are present in these samples. Moreover, we were unable to detect the accumulation of β-catenin in one of the S37A mutant samples (labeled duodenum-1). Although we cannot explain this contradiction between our immunohistochemical and sequencing results, one possibility is that an epigenetic alteration involving methylation of the promoter region in the β-catenin gene occurred in tandem with the S37A mutation.

The results of various statistical analyses revealed no significant correlation between β-catenin mutation and biological attributes (Table 2). This may indicate that mutation in β-catenin tends to occur at an early stage in the carcinogenesis of gastrointestinal carcinoid tumor, as has been reported in colon cancer and ovarian cancer with microsatellite instability (20). Interestingly, we were able to detect both the accumulation of β-catenin and presence of an S37A mutation in one particular sample, which had been resected by endoscopic biopsy from a very small tumor (0.1 cm in diameter) in the submucosa (labeled rectum-33).

We believe that this is the first evidence that alterations in the Wnt signaling pathway and mutations in β-catenin are frequently encountered in gastrointestinal carcinoid tumors. The accumulation of β-catenin and the presence of mutations in very small and noninvasive tumors indicate that these alterations may well be an early event and perhaps even an initiating event in the development of gastrointestinal carcinoid tumors.

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.

      
1

This work was supported by a Tsuchiya Memorial Foundation Grant (1999).

            
3

The abbreviations used are: TCF, T-cell factor; LEF, lymphoid enhancer-binding factor; APC, adenomatous polyposis coli; GSK-3β, glycogen synthase kinase-3β.

Fig. 1.

Immunohistochemical analysis of β-catenin and cyclin D1 in gastrointestinal carcinoid tumors. A, in normal epithelial cells (sample labeled rectum-2), β-catenin staining is localized to the cell membrane. B, membranous staining of β-catenin without cytoplasmic and nuclear staining in a duodenal carcinoid tumor (sample labeled duodenum-1). C, strong nuclear staining of β-catenin in a rectal carcinoid tumor (sample labeled rectum-2). D, cytoplasmic-positive staining of β-catenin without strong nuclear staining is observed in a stomach carcinoid tumor (sample labeled stomach-6). In this sample, the nucleus and nuclear membrane are also faintly immunostained by β-catenin. E, nuclear-positive staining of cyclin D1 (sample labeled rectum-2). F, immunohistochemistry of histone H1 (sample labeled stomach-6). Scale bar (F), 50 μm.

Fig. 1.

Immunohistochemical analysis of β-catenin and cyclin D1 in gastrointestinal carcinoid tumors. A, in normal epithelial cells (sample labeled rectum-2), β-catenin staining is localized to the cell membrane. B, membranous staining of β-catenin without cytoplasmic and nuclear staining in a duodenal carcinoid tumor (sample labeled duodenum-1). C, strong nuclear staining of β-catenin in a rectal carcinoid tumor (sample labeled rectum-2). D, cytoplasmic-positive staining of β-catenin without strong nuclear staining is observed in a stomach carcinoid tumor (sample labeled stomach-6). In this sample, the nucleus and nuclear membrane are also faintly immunostained by β-catenin. E, nuclear-positive staining of cyclin D1 (sample labeled rectum-2). F, immunohistochemistry of histone H1 (sample labeled stomach-6). Scale bar (F), 50 μm.

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Fig. 2.

Direct sequence analysis of exon 3 of β-catenin in gastrointestinal carcinoid tumors. A, S37A mutation in β-catenin (TCT to GCT substitution in nucleotide) in a rectal carcinoid tumor (sample labeled rectum-16). B, G48D mutation in β-catenin (GGT to GAT substitution in nucleotide) in rectal carcinoid tumor (sample labeled rectum-17). C, S37A and G38D mutations in β-catenin (TCT to GCT and GGT to GAT substitutions in nucleotide, respectively) exist simultaneously in a rectal carcinoid tumor (sample labeled rectum-19).

Fig. 2.

Direct sequence analysis of exon 3 of β-catenin in gastrointestinal carcinoid tumors. A, S37A mutation in β-catenin (TCT to GCT substitution in nucleotide) in a rectal carcinoid tumor (sample labeled rectum-16). B, G48D mutation in β-catenin (GGT to GAT substitution in nucleotide) in rectal carcinoid tumor (sample labeled rectum-17). C, S37A and G38D mutations in β-catenin (TCT to GCT and GGT to GAT substitutions in nucleotide, respectively) exist simultaneously in a rectal carcinoid tumor (sample labeled rectum-19).

Close modal
Table 1

Summary of immunohistochemistry and mutations in β-catenin and APC in 72 gastrointestinal carcinoid tumor cases

Sample no.Affected codonMutationAmino acid changeImmunohistochemistry of β-catenin
NucleusCytoplasm
β-catenin      
 stomach      
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
10 37 TCT→GCT Ser→Ala − 
 duodenum      
37 TCT→GCT Ser→Ala − − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
11 37 TCT→GCT Ser→Ala 
12 37 TCT→GCT Ser→Ala − 
13 37 TCT→GCT Ser→Ala − 
 appendix      
37 TCT→GCT Ser→Ala 
 rectum      
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
16 37 TCT→GCT Ser→Ala 
17 48 GGT→GAT Gly→Asp − 
19 37 TCT→GCT Ser→Ala − 
 38 GGT→GAT Gly→Asp   
22 37 TCT→GCT Ser→Ala − 
 48 GGT→GAT Gly→Asp   
26 37 TCT→GCT Ser→Ala − 
27 37 TCT→GCT Ser→Ala 
33 37 TCT→GCT Ser→Ala − 
36 37 TCT→GCT Ser→Ala 
45 37 TCT→GCT Ser→Ala − 
APC      
 rectum      
28 1408 GAA→TAA Glu→Terminal codon − 
Sample no.Affected codonMutationAmino acid changeImmunohistochemistry of β-catenin
NucleusCytoplasm
β-catenin      
 stomach      
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
10 37 TCT→GCT Ser→Ala − 
 duodenum      
37 TCT→GCT Ser→Ala − − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
11 37 TCT→GCT Ser→Ala 
12 37 TCT→GCT Ser→Ala − 
13 37 TCT→GCT Ser→Ala − 
 appendix      
37 TCT→GCT Ser→Ala 
 rectum      
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
37 TCT→GCT Ser→Ala − 
16 37 TCT→GCT Ser→Ala 
17 48 GGT→GAT Gly→Asp − 
19 37 TCT→GCT Ser→Ala − 
 38 GGT→GAT Gly→Asp   
22 37 TCT→GCT Ser→Ala − 
 48 GGT→GAT Gly→Asp   
26 37 TCT→GCT Ser→Ala − 
27 37 TCT→GCT Ser→Ala 
33 37 TCT→GCT Ser→Ala − 
36 37 TCT→GCT Ser→Ala 
45 37 TCT→GCT Ser→Ala − 
APC      
 rectum      
28 1408 GAA→TAA Glu→Terminal codon − 
Table 2

β-catenin status, cyclin D1 immunohistochemistry, and clinicohistological characteristics

CategoriesTotalMutation in β-cateninP
PositiveNegative
Number of samples 72 27 (37.5%) 45 (62.5%) — 
Immunohistochemistry of β-catenin     
 negative 15 14 0.0022 
 positive 57 26 31  
Gender     
 male 29 10 19 0.663 
 female 43 17 26  
Mean age 55.4 ± 13.7 57.3 ± 13.5 54.4 ± 13.9 0.386 
Tumor location     
 stomach 11 0.077 
 duodenum 13  
 appendix  
 rectum 45 12 33  
Tumor size (cm) 0.82 ± 0.77 0.98 ± 1.19 0.72 ± 0.34 0.180 
Depth of invasion     
 T1 63 21 42 0.375 
 T2  
 T3  
 unknown  
Lymph node metastasis     
 absent 68 25 43 0.959 
 present  
Lymphatic invasion     
 absent 61 21 40 0.662 
 present  
 unknown  
Venous invasion     
 absent 61 21 40 0.662 
 present  
 unknown  
Immunohistochemistry of cyclin D1     
 positive 61 51a 10b 0.0289 
 negative 11 6a 5b  
CategoriesTotalMutation in β-cateninP
PositiveNegative
Number of samples 72 27 (37.5%) 45 (62.5%) — 
Immunohistochemistry of β-catenin     
 negative 15 14 0.0022 
 positive 57 26 31  
Gender     
 male 29 10 19 0.663 
 female 43 17 26  
Mean age 55.4 ± 13.7 57.3 ± 13.5 54.4 ± 13.9 0.386 
Tumor location     
 stomach 11 0.077 
 duodenum 13  
 appendix  
 rectum 45 12 33  
Tumor size (cm) 0.82 ± 0.77 0.98 ± 1.19 0.72 ± 0.34 0.180 
Depth of invasion     
 T1 63 21 42 0.375 
 T2  
 T3  
 unknown  
Lymph node metastasis     
 absent 68 25 43 0.959 
 present  
Lymphatic invasion     
 absent 61 21 40 0.662 
 present  
 unknown  
Venous invasion     
 absent 61 21 40 0.662 
 present  
 unknown  
Immunohistochemistry of cyclin D1     
 positive 61 51a 10b 0.0289 
 negative 11 6a 5b  
a

Immunohistochemistry of β-catenin, positive.

b

Immunohistochemistry of β-catenin, negative.

We thank Dr. Donald G. MacPhee for helpful discussion and critical review of the manuscript and doctors at Hiroshima Prefectural Hospital, Hiroshima General Hospital of West Japan Railway Company, Onomichi General Hospital, Inokuchi Hospital, Mitsubishi Mihara Hospital, Matsuyama Red Cross Hospital, Chugoku Rosai Hospital, Kureshi Ishikai Hospital, Miharashi Ishikai Hospital, National Sanatorium Yanai Hospital, Ohtake National Hospital, Kawaishi Memorial Hospital, and Yoshijima Hospital for their kind advice regarding the experiments and specimen collections. We also thank the staff of the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.

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