Clinical Research
Nuclear Accumulation of ß-Catenin in Human Endocrine Tumors: Association with Ki-67 (MIB-1) Proliferative Activity
Shuho Semba, MD, Ryoko Kusumi, MT, Takuya Moriya, MD, and Hironobu Sasano, MD
Abstract
B-Catenin is closely associated with carcinoma invasion/metastasis and poor survival. Recent studies have demonstrated that abnormal expression of ß-catenin, especially its nuclear accumulation, also plays an important role in wingless/Wnt signaling pathway. In this study, we evaluated immunohistochemically the nuclear localization of ß-catenin in a total of 93 human-endocrine-related tumors including 1 medullary carcinoma (thyroid gland), 12 parathyroid tumors, 22 carcinoid tumors (digestive tract and liver), 7 islet cell tumors, 26 adrenocortical tumors, 13 neuroblastoma (adrenal gland), and 12 pheochro- mocytoma (adrenal gland), and also studied genetic alterations of the ß-catenin gene. Nuclear accumulation of ß-catenin was frequently detected in 8 of 22 (36%) carcinoid tumors and 2 of 7 (29%) islet cell tumors. No genetic alteration in exon 3 of the ß-catenin gene encoding serine/threonine rich domain, which was phosphorylated by GSK-3B, was detected in any groups of the endocrine tumors. However, nuclear accumula- tion of ß-catenin in carcinoid tumors was significantly correlated with the proliferative marker Ki-67 (MIB-1) labeling index (p < 0.001). Our findings suggest that nuclear trans- fer and accumulation of the B-catenin may contribute in the tumorigenesis of carcinoid tumor as an oncoprotein.
Key Words: ß-Cantenin; nuclear accumulation; endocrine tumor; carcinoid tumor; immunohistochemistry; Ki-67 (MIB-1).
Address correspondence to: Shuho Semba, Department of Pathology, Tohoku University Hospital, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 E-mail: semba@mail.cc. tohoku.ac.jp
Endocrine Pathology, vol. 11, no. 3, 243-250, Fall 2000 @ Copyright 2000 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/00/11:243-250/$12.00
Introduction
B-Catenin is one of the key proteins of cadherin-catenin mediatintg cell-cell adhesion and forms complex with E-cadherin [1]. Previous reports demonstrated that a decrease of E-cadherin or catenins expres- sion level is closely associated with carcinoma invasion/metastasis and histopathologic fea- tures [1,2]. In addition, recent reports dem- onstrated that ß-catenin plays an important role in the wingless/Wnt signaling path- way, which is considered essential during embryonic development and tissue orga-
nization [3-5]. It has also been reported that nuclear transfer and accumulation of ß-catenin activates cell proliferation, and genetic alterations of the B-catenin gene may prevent its degradation through the expression of phosphorylation protein GSK-3B [5,6]. B-catenin mutation is there- fore thought to stabilize ß-catenin pro- tein itself and may result in its nuclear accumulation. In addition, activation of Tcf-Lef family, which functions as a tran- scription factor, may be further caused by mutant ß-catenin protein, and this results
in providing transfer cell proliferation sig- nals to the nucleus [7,8]. This phenom- enon has been reported in many human malignancies such as colorectal cancer, esophageal cancer, breast cancer, and en- dometrial cancer [8-13].
In this study, we first evaluated nuclear accumulation and decreased expression of membranous ß-catenin protein in various human endocrine tumors, especially those arising from neuroendocrine cells. We then investigated the correlation with cell prolif- erative status examined by Ki-67 (MIB-1) labeling index and histological types of the lesions to study the possible biological sig- nificance of oncogenic mutant ß-catenin protein in these tumors. We also performed polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis to confirm whether somatic mutation of the ß-catenin gene was present in the tumors in which nuclear accumu- lation of the ß-catenin was detected in order to further elucidate its biological significance.
Materials and Methods
Tissue Samples and DNA Extraction
We examined a total of 93 endocrine tumors removed at Tohoku University Hospital (Sendai, Japan). Tumors examined in this study are as follows: 1 medullary carcinoma of the thyroid gland, 12 parathyroid tumors (2 hyperplasias, 9 adenoma, and 1 carcinoma), 22 carcinoid tumors arising from digestive tract and the liver, 7 islet cell tumors of the pancreas, adenocortical tumors (16 adenomas and 10 carcinomas), 13 neuroblastomas, and 12 pheochromocytomas of the adrenal gland. The locations of carcinoid tumors are sum- marized in Table 2. A total of 93 formalin- fixed-paraffin-embedded tissues was used
for histological diagnosis, immunohis- tochemical analysis, and DNA extraction. DNA was extracted according to methods with proteinase K treatment and phenol- chloroform described previously [14].
Expression of ß-Catenin and Ki-67 Proteins
Monoclonal antibodies to ß-catenin and Ki-67 (MIB-1) proteins were purchased from Transduction Laboratories (Lexing- ton, KY, USA) and Immunotech (Marseille, France), respectively. Modifications of the immunoglobulin enzyme bridge technique (ABC method) were used as described else- where [15]. Briefly, deparaffinized tissue sec- tions were immersed in methanol containing 0.03% hydrogen peroxide for 30 min to block the endogenous peroxide activity. Microwave (B-catenin) and autoclave (Ki-67) pretreatments in citrate buffer were per- formed for 15 min to retrieve the antige- nicity. After incubation with normal horse serum (diluted 1:20) for 30 min to block the nonspecific antibody binding sites, the sections were treated consecutively at room temperature with anti-ß-catenin (diluted 1:200) or anti-Ki-67 (diluted 1:100) monoclonal antibodies for 90 min, biotinylated by antimouse IgG horse se- rum (diluted 1:100) for 30 min, and with avidin DH-biotinylated horseradish per- oxide complex (Histofine kit, Nichirei, Tokyo, Japan) for 30 min. Peroxide stain- ing was performed for 10-15 min using a solution of 3,3’-diaminobenzidine tetrahy- drochloride in 50 mM’Tris-HCI (pH 7.5) containing 0.001% hydrogen peroxide. The sections were counterstained with 0.1% hematoxylin. All the immunostained slides were independently observed by SS and HS. Immunoreactivity of anti-ß- catenin antibody was graded as - to 3+ according to the number of stained cells and the staining intensity in individual cells
as follows: - , almost no positive cells; 1+, 5-25% of tumor cells showed weak to moderate immunoreactivity; 2+, 25-50% of tumor cells showed moderate immu- noreactivity or 10-50% of tumor cells showed intense immunoreactivity; 3+, over 50% of tumor cells showed intense immu- noreactivity. The Ki-67 labeling index was determined by counting the number of positive cells (%o) in a total of 1000 or more tumor cells observed in 10 or more repre- sentative high-power fields (x 400).
Statistical Analysis
The relationships between the results of the immunohistological study and clinico- pathologic parameters were investigated by Mann-Whitney U test and x2-test. A level of p < 0.05 was taken to be statistically sig- nificant.
PCR-SSCP Analysis of the ß-catenin Gene
The cases with nuclear accumulations of ß-catenin were examined for genetic alteration by PCR-SSCP of exon 3 of the gene. Primers used for PCR amplification and sequencing were as follows: forward primer, 5’ TTAGTCACTGGCAG- CAACAG-3’ and reverse primer, 5’ CT CTTCCTCAGGATTGCCTT-3’. Each 15 uL reaction mixture containing 10 ng of DNA, 6.7 mM Tris-HCI (pH 8.8), 16.6 mM (NH)2SO 4 10 mMB-mercaptoe- thanol, 6.7 uM ethylenediaminetetraacetic acid (EDTA), 6.7 mM MgCl2, 1 uM of the primer pair, 1.5 uM of each deoxynu- cleotide, 10% (v/v) dimethylsulfoxide, and 0.75 U of Taq DNA polymerase was amplified for 40 cycles with the following regime: denaturation at 94℃ for 30 s; annealing at 55℃ for 30 s, and extension at 72℃ for 30 s. PCR-SSCP was performed to screen the mutations of the B-catenin gene as described previously [16].
Results
ß-Catenin and Ki-67 Immunohistochemistry
We first examined ß-catenin in endo- crine tumors immunohistochemically. Results are summarized in Table 1. In the normal rectal mucosa, ß-catenin immuno- histochemistry was detected mainly at the cell membrane but weakly in the cytoplasm (Fig. 1A). Normal cells in endocrine organs examined in this study also demonstrated membranous and weak cytoplasmic stain- ing of ß-catenin (data not shown). Nuclear accumulation of ß-catenin protein was detected in 8 (36%) of 22 carcinoid tumors and 2 of 7 (29%) of islet cell tumors (see Table 2 and Fig.1B,C). In other tumors, the tumor cells demonstrated cytoplasmic and/or membranous ß-catenin immunore- activity. In some carcinoid tumors, hetero- geneous expression (membranous and nuclear staining) of ß-catenin was detected (Fig. 1B,C). No statistical relationship was detected in age, sex, size, and location of each tumor. The carcinoid tumor cases in which nuclear accumulation of ß-catenin was detected had significantly higher Ki-67 labeling index than those that did not (Fig. 1D).
PCR-SSCP Analysis
We further examined genetic alterations of the ß-catenin gene at exon 3, which har- bors the gene. PCR-SSCP analyses were performed in all the cases that showed abnormal accumulations of ß-catenin in their nuclei immunohistochemically. In carcinoid and islet cell tumors with abnor- mal accumulation of ß-catenin, no abnor- malities were detected by PCR-SSCP analysis (Fig. 2), which was also confirmed by DNA direct sequencing using ABI Prizm autosequencer (data not shown).
| Tumor type | Location | Nuclear expression of ß-catenin protein |
|---|---|---|
| Pituitary adenomaª | Pituitary gland | 21/37 (57%) |
| Medullary carcinoma | Thyroid gland | 0/1/(0%) |
| Parathyroid tumors | Parathyroid gland | 1/12 (8%) |
| Hyperplasia | 0/2 (0%) | |
| Adenoma | 1/9 (11%) | |
| Carcinoma | 0/1/(0%) | |
| Carcinoid tumor | Digestive tract and liver | 8/22 (36%) |
| Islet cell tumor | Pancreas | 2/7 (29%) |
| Adrenocortical tumor | Adrenal gland | 0/26 (0%) |
| Adenoma | 0/16 (0%) | |
| Carcinoma | 0/37 (0%) | |
| Neuroblastoma | Adrenal gland | 0/13 (0%) |
| Pheochromocytoma | Adrenal gland | 0/12 (0%) |
“The frequency of ß-catenin protein in pituitary adenoma has been published in our previous report (see ref. 17).
A
C
B
D
Đ
| Age (yr) | Sex | Location | Size (mm) | Immunoreactivity of ß-catenin | Ki-67 labeling index | |||
|---|---|---|---|---|---|---|---|---|
| N | C | M | ||||||
| Carcinoid C-1 | 28 | M | Rectum | 8 | 3+ | + | – | 37.3 |
| tumor C-2 | 43 | F | Rectum | 2 | 2+ | + | 2+ | 22.5 |
| C-3 | 64 | F | Rectum | 8 | 2+ | + | 1+ | 46.7 |
| C-4 | 73 | F | Stomach | 10 | 2+ | + | 1+ | 31.1 |
| C-5 | 63 | M | Stomach | 7 | 2+ | + | – | 23.3 |
| C-6 | 48 | F | Rectum | 7 | 2+ | + | – | 50.4 |
| C-7 | 44 | M | Rectum | 10 | 2+ | + | – | 14.3 |
| C-8 | 38 | M | Rectum | 2 | 1+ | + | 1+ | 20.6 |
| C-9 | 57 | F | Liver | 2 | – | 2+ | – | 8.3 |
| C-10 | 49 | F | Rectum | 9 | – | 2+ | 2+ | 8.3 |
| C-11 | 48 | F | Liver | 7 | – | 2+ | 1+ | 28.3 |
| C-12 | 68 | M | Colon | 6 | – | + | 2+ | 3.9 |
| C-13 | 78 | F | Rectum | 5 | – | + | 1+ | 5.7 |
| C-14 | 56 | F | Rectum | 38 | – | + | 1+ | 4.3 |
| C-15 | 22 | M | Rectum | 3 | – | + | 1+ | 16.7 |
| C-16 | 70 | F | Rectum | 7 | – | + | 1+ | 4.1 |
| C-17 | 58 | F | Rectum | 20 | – | + | + | 0.2 |
| C-18 | 72 | F | Duodenum | 2 | – | + | 1+ | 3.8 |
| C-19 | 71 | M | Rectum | 3 | – | + | 1+ | 7.9 |
| C-20 | 48 | M | Rectum | 30 | – | + | 1+ | 0.8 |
| C-21 | 71 | M | Rectum | 12 | – | + | 1+ | 14.2 |
| C-22 | 48 | M | Rectum | 4 | – | + | 1+ | 8.75 |
| Islet cell I-1 | 68 | F | Pancreas | 6 | 3+ | 2+ | 1+ | 7.8 |
| tumor I-2 | 50 | M | Pancreas | 25 | 2+ | 1+ | 1+ | 4.5 |
| I-3 | 69 | F | Pancreas | 26 | – | 2+ | 1+ | 3.3 |
| I-4 | 44 | F | Pancreas | 20 | – | 1+ | – | 5.8 |
| I-5 | 33 | F | Pancreas | 29 | – | 1+ | – | 2.2 |
| I-6 | 27 | F | Pancreas | 14 | – | 1+ | – | 1.8 |
| I-7 | 13 | M | Pancreas | 13 | – | – | – | 8.7 |
Immunoreactivity of B-catenin was graded as - to 3+ according to the number of stained cells (see Materials and Method) and Ki-67 (MINB-1_ labelling index was determined by counting the number of positive cells in a total of 1000 or more tumor cells observed in ten or more representative hihg-power fields (x400).
Discussion
B-Catenin was originally detected as a membranous protein, but nuclear accumulation of ß-catenin has been detected in various human malignancies including carcinomas of the gastrointesti- nal tract, liver, breast, endometrium, and thyroid gland [9-13]. The presence of nuclear accumulation of ß-catenin in some carcinoid and islet cell tumors demon-
strated that abnormal nuclear expression of ß-catenin plays an important role in tumorigenesis and tumor development and progression of these tumors. To the best of our knowledge, this is the first report demonstrating abnormal nuclear accumula- tion of ß-catenin in a series of human neo- plasms arising from neuroendocrine cells.
We previously investigated the frequency of nuclear accumulation of ß-catenin protein
K Z 1 Z NTNTNTNTNTNTNT 2
C-7
C-6
C-5
C-4
C-3
C- 2
C-1
HCT116
Water blank
Genomic control
in human pituitary adenomas. Twenty-one (57%) of 37 tumors showed monotonous nuclear accumulation of ß-catenin, and we detected somatic mutation in exon 3 of the B-catenin gene in four cases (11%) of this tumor [17]. In this report, we performed a further study in various human endocrine tumors. “Carcinoid tumors” is the generic term applied to low-grade malignant neo- plasms originating from the diffuse endo- crine system outside of the pancreas and the thyroid C cell. It is interesting that nuclear accumulation of ß-catenin was frequently detected in both carcinoid and islet cell tumors. These two tumors may have the same molecular events during multistep carcinogenesis. Some of these tumors dem- onstrated different expression patterns of ß-catenin, i.e., heterogeneity in its nuclear and membranous ß-catenin expression. This abnormal nuclear accumulation of B-catenin may be a late event in their
tumorigenesis. Carcinoid and islet cell tumors were both previously regarded as low-grade malignancies associated with a slow growth but also with high invasive and metastasizing properties in some cases. Such biological behavior of these tumors may be derived from decreased membra- nous ß-catenin expression level, but it awaits further investigation for clarification.
Missence mutation of the B-catenin gene has been reported in many carcino- mas, especially in cancers in which nuclear accumulation of ß-catenin was detected [12,13,17-19], although no alteration of exon 3 phosphorylated site of the B-catenin gene was detected by PCR-SSCP analysis in carcinoid and islet cell tumors. In our study, the expression pattern in these tumors demonstrating nuclear transfer of B-catenin was not seen equally in all the cells but was scattered. Ubiquitin-mediated proteasome is required for degradation of B-catenin protein [20-22]. Therefore, this nuclear localization of ß-catenin may not be due to genetic change including DNA methylation of the promoter region of this gene but possibly to the posttranscriptional system. In addition, it is well known that loss or abnormalities of the adenomatous polyposis coli (APC) proteins can also cause mislocation of ß-catenin [23,24]. Tumors without genetic alterations in the B-catenin gene may have functional abnormalities in APC protein, but these proteins are required for clarification. Possible involve- ment of abnormal nuclear accumulation of ß-catenin protein varied among endo- crine tumors. This nuclear accumulation is considered to play especially important roles in the development of pituitary adenomas, carcinoid tumors, and pancre- atic islet cell tumors, but it awaits further investigation for clarification.
Our present findings of close association of nuclear ß-catenin accumulation may be
related to the biological behavior of this tumor. In this study, tumors with nuclear accumulations of ß-catenin were associated with high Ki-67 expression as we reported previously [17]. This may suggest that abnormally expressed ß-catenin plays an important role in this series of tumorigenesis through upregulation of cell proliferation and progression. It was reported that the com- plex of mutant ß-catenin and Tcf protein may induce expression of the c-myc and/or cyclin D genes in colon cancer cell lines [7,8] and further investigations including the analysis of these genes are required for clarification.
Acknowledgments
This work is supported by the Ministry of Education, Science, Sports and Culture of Japan. This work is in part supported by The Grant-in-aid for Cancer Research 7-1 from The Ministry of Health and Wel- fare, Japan, a grant-in-aid for scientific research area on priority area (A-11137301) from The Ministry of Education, Science and Culture, Japan, a grant-in-aid for Sci- entific Research (B-11470047) from Japan Society for the Promotion of Science and a grant from The Naitou Foundation and Suzuken Memorial Foundation.
References
1. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251:1451-1455, 1991.
2. Takayama T, Shiozaki H, Shibamoto S, Oka H, Kimura Y, Tamura S, et al. ß-catenin expression in human cancers. Am J Pathol 148:39-46, 1996.
3. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H. Constitutive transcriptional activation by a B-catenin-Tcf complex in APCZ colon carcinoma. Science 275:1784-1787, 1997.
4. Rubinfeld B, Souza B, Albert I, Muller O, Chamberlain SH, Masiarz FR, et al. Associa- tion of the APC gene product with ß-catenin. Science 262:1731-1734, 1993.
5. Hart MJ, de los Santos R, Albert IN, Rubinfeld B, Polakis P. Down-regulation of B-catenin by human Axin and its association with the APC tumor suppressor, ß-catenin and GSK-3B. Curr Biol 8:573-581, 1998.
6. Sakanaka C, Weiss JB, Williams LT. Bridging of ß-catenin and glycogen synthase kinase-3฿ by axin and inhibition of ß-catenin-mediated transcription. Proc Natl Acad Sci USA 95:3020-3023, 1997.
7. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, et al. Identification of c-MYC as a target of the APC pathway. Sci- ence 281:1509-1512, 1998.
8. Tetsu O, McCormick F. Beta-catenin regu- lates expression of cyclin D1 in colon carci- noma cells. Nature 398:422-426, 1999.
9. Krishnadath KK, Tilanus HW, van Blankenstein M, Hop WC, Kremers ED, Dinjens WN, Bosman FT. Reduced expression of the cadherin-catenin complex in oesophageal adenocarcinoma correlates with poor progno- sis. J Pathol 182:331-338, 1997.
10. Bukholm IK, Nesland JM, Karesen R, Jacobsen U, Borresen-Dale AL. E-cadherin and a-, B-, and y-catenin protein expression in relation to metastasis in human breast car- cinoma. J Pathol 185:262-266, 1998.
11. Cerrato A, Fulciniti F, Avallone A, Benincasa G, Palombini L, Grieco M. ß- and y-catenin expression in thyroid carcinomas. J Pathol 185:267-272, 1998.
12. Mirabelli-Primdahl L, Gryfe R, Kim H, Millar A, Luceri C, Dale D, et al. ß-catenin muta- tions are specific for colorectal carcinomas with microsatellite instability but occur in endometrial carcinomas irrespective of muta- tor pathway. Cancer Res 59:3346-3351, 1999.
13. Kobayashi K, Sagae S, Nishioka Y, Tokino T, Kudo R. Mutations of the ß-catenin gene in endometrial carcinomas. Jpn J Cancer Res 90:55-59, 1999.
14. Ford N, Nolan, C, Ferguson, M. In: Sambrook J, Fritsh EF, Maniatis T, eds. Molecular Clon- ing, vol. 9 2nd Ed. New York: Cold Spring Harbor Laboratory Press; 16-23, 1989.
15. Yasui W, Kuniyasu H, Yokozaki H, Semba S, Shimamoto F, Tahara E. Expression of cyclin
E in colorectal adenomas and adenocarcino- mas: Correlation with expression of Ki-67 antigen and p53 protein. Virchows Arch 429:13-19, 1996.
16. Mayama T, Mori T, Abe K, Sasano H, Nishihira T, Satomi S, Horii A. Analysis of the p53 gene mutations in patients with multiple primary cancers. Eur J Surg Oncol 23:298-303, 1997.
17. Semba S, Han S-Y, Ikeda H, Horii A. Frequent nuclear accumulation of ß-catenin (CTNNB1) in human pituitary adenoma. Cancer (in press).
18. Sagae S, Kobayashi K, Nishioka Y, Sugimura M, Ishioka S, Nagata M, Terasawa K, Tokino T, Kudo R. Mutational analysis of ß-catenin gene in Japanese ovarian carcinomas: frequent mutations in endometrioid carcinomas. Jpn J Cancer Res 90:510-515, 1999.
19. Gumbiner BM. Carcinogenesis: a balance between ß-catenin and APC. Curr Biol 7:R443-446, 1997.
20. Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. ß-catenin is a target for the
ubiquitin-proteasome pathway. EMBO J 16:3797-3804, 1997.
21. Papkoff J, Rubinfeld B, Schryver B, Polakis P. Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mol Cell Biol 16:2128-2134, 1996
22. Funayama N, Fagotto F, McCrea P, Gumbiner BM. Embryonic axis induction by the arma- dillo repeat domain of beta-catenin: evidence for intracellular signaling. J Cell Biol 128:959-968, 1995.
23. Weidner N, Moore DH, Vartanian R. Cor- relation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel “paraffin”- reactive MIB1 antibody. Hum Pathol 25:337-342, 1994.
24. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, et al. Activation of B-catenin-Tcf signaling in colon cancer by mutations in ß-catenin or APC. Science 275: 1787-1790, 1997.