[Cancer Research 63, 6195-6199, October 1, 2003]
© 2003 American Association for Cancer Research
Coordinate Expression of Cdc25B and ER-
Is Frequent in Low-Grade Endometrioid Endometrial Carcinoma but Uncommon in High-Grade Endometrioid and Nonendometrioid Carcinomas
Weiguo Wu,
Brian M. Slomovitz,
Joseph Celestino,
Linda Chung,
Angela Thornton and
Karen H. Lu1
Department of Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT
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Overexpression of cdc25B, an important cell cycle regulator, has been shown to result in mammary gland hyperplasia in transgenic mice and to increase steroid hormone responsiveness as a direct coactivator of the estrogen receptor (ER). We investigated the potential role of cdc25B in the pathogenesis of endometrial carcinomas in conjunction with ER-
. We examined the expression of cdc25B and phosphorylated ER- in 4 archived human specimens of normal endometrium; 7 endometrial hyperplasia with or without atypia; 32 endometrioid endometrial carcinoma (EEC), including 20 low-grade (grade 1) and 12 high-grade (grade 2 or 3) tumors; and 18 endometrial cancers with aggressive histological subtypes (uterine papillary serous carcinoma and clear cell carcinoma, UPSC/CCC) by immunohistochemistry with monoclonal antibodies. Expression of cdc25B and phosphorylated ER- was increased in endometrial hyperplasia and atypical hyperplasia compared with normal secretory endometrium. Ninety percent (18 of 20) of the low-grade EEC expressed cdc25B at a high level, whereas only 42% (5 of 12) of the high-grade EEC did so (
2 = 8.7; P < 0.01). Sixty-five percent (13 of 20) of the low-grade EEC expressed phosphorylated ER- at high levels, but only 17% (2 of 12) of high-grade EEC did so (2 = 7.0; P < 0.01). Coordinate high-level expression of phosphorylated ER- and cdc25B occurred in 65% (13 of 20) of low-grade EEC but in only 17% (2 of 12) of the high-grade EEC (2 = 7.0; P < 0.01). In the UPSC/CCC tumors, only 22% (4 of 18) of the tumors expressed phosphorylated ER- at high-levels. However, 83% (15 of 18) of these carcinomas showed high expression of cdc25B (2 = 13.5; P < 0.01). The majority of the UPSC/CCC (15 of 18) did not show coordinate high expression of phosphorylated ER- and cdc25B. Our findings show that in endometrial hyperplasia and low-grade EEC, coordinate increase in cdc25B and phosphorylated ER- occurs. However, in UPSC/CCC, cdc25B is highly expressed without coordinate increase in phosphorylated ER-. Cdc25B may play important roles in the development and progression of EEC and UPSC/CCC by different mechanisms.
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INTRODUCTION
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Endometrial carcinoma ranks first in incidence and second in mortality among female genital tract tumors. In 2003, it is estimated that there will be 40,100 new cases and 6,800 related deaths among women in the United States (1)
. There are two predominant histological subtypes of endometrial carcinoma. EEC2
accounts for >80% of endometrial carcinomas. Nonendometrioid high-risk histological subtypes, including UPSC and CCC, are less common than EEC; however, they have a more aggressive course (2
, 3)
. A dualistic model of carcinogenesis has been proposed on the basis of the distinctive clinical and histological features of endometrioid and papillary serous adenocarcinoma (4)
. EEC is associated with a progressive, estrogen-driven model of carcinogenesis whereby estrogen stimulation leads to malignant transformation through a stepwise progression from normal, to atypical hyperplasia, to cancer (5)
. In contrast, UPSC and CCC are not estrogen-driven tumors. UPSCs frequently are found to have p53 mutations (6)
.
Cdc25B has been found recently to function as a direct coactivator of the ER in mammary glands (7)
. Cdc25B belongs to a family of dual-specificity proteins that activate the cyclin/cyclin-dependent kinase complex, resulting in cell cycle progression (8)
. Cdc25B is involved in the regulation of the G2/M-phase transition (9)
. Overexpression of cdc25B in transgenic mice has been shown to cause mammary gland hyperplasia and increase steroid hormone responsiveness (10)
. The direct interactions of cdc25B with steroid receptors (e.g., ER-) in vivo and in vitro suggest a potential direct contribution of cdc25B to steroid receptor-mediated transcription (7)
.
On the basis of these observations, we hypothesize that cdc25B, in cooperation with ER-, may contribute to the development and progression of endometrial cancer. To test this hypothesis, we examined the expression of cdc25B and phosphorylated ER- in normal and hyperplastic endometrium, and in low-grade (grade 1) and high-grade (grade 2–3) EEC. We also examined the expression of cdc25B and phosphorylated ER- in UPSC/CCC to evaluate their roles in the development of this disease. To assess the functional consequence of coordinate high expression of phosphorylated ER- and cdc25B, the expression level of PR, an ER-regulated protein, was also evaluated.
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MATERIALS AND METHODS
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Endometrial Tissue Samples.
Archived formalin-fixed, paraffin-embedded endometrial tissue specimens were obtained from 61 patients surgically treated at The University of Texas M. D. Anderson Cancer Center (Houston, TX), including 4 specimens of normal endometrial tissue, 7 of endometrial hyperplasia, 32 of EEC, and 18 of UPSC/CCC (16 UPSC and 2 CCC). Twenty of EEC was low grade (grade 1), and 12 were high grade (grade 2 or 3). The specimens had been collected between 1994 and 2002. The mean age of patients was 52 years. The study protocol was approved by the Committee for the Protection of Human Subjects at The University of Texas M. D. Anderson Cancer Center.
Immunohistochemical Assessment.
Immunostaining for cdc25B and ER- phosphorylated at Ser118 was performed on 5–6 µm sections of formalin-fixed, paraffin-embedded endometrial specimens using DAKO LSAB kits (DakoCytomation, Carpinteria, CA). Expression of PRs was also examined by immunohistochemical analysis. Tissue sections were incubated at 60°C for 1 h, deparaffinized in xylene, and hydrated in a graded series of ethanol solutions. After a rinse in PBS (137 mM NaCl, 2.7 mM KCl, 1 mM KH2PO4, and 10 mM Na2HPO4; pH 7.4), endogenous peroxidase activity was quenched by incubating the sections for 15 min with 0.3% H2O2 in absolute methanol. Then, after a 10-min rehydration in PBS, the sections were heated in a microwave oven for 3 min in 10 mM citrate buffer for antigen retrieval. After incubation with blocking serum (4% normal horse serum) for 30 min at room temperature, the sections were incubated overnight at 4°C with monoclonal antibodies against human cdc25B (Cell Signal Technology, Beverly, MA), phosphorylated ER- (Cell Signal Technology), and PR (Lab Vision Corporation, Fremont, CA). Each antibody was tested in serial dilutions to achieve maximum sensitivity and specificity. Human breast cancer sections were used as positive controls for both antibodies. Negative controls were analyzed on adjacent sections incubated without antibody. After a PBS rinse, the sections were treated with biotinylated goat antimouse antibody for 15 min. The sections were washed with PBS three times and incubated with an avidin-biotinylated horseradish peroxidase macromolecular complex for 10 min according to the manufacturer’s instructions for the LASB kit. Visualization of peroxidase was carried out by incubating the sections with diaminobenzidine for 10 min. The sections were counterstained with hematoxylin, dehydrated in a series of ethanol solutions, and cleared with xylene, and coverslips were placed over Permount mounting medium for evaluation by light microscopy.
All of the immunostained tissue sections were evaluated and scored cooperatively by two investigators (W. W. and K. H. L.) who were blinded to any clinical or pathological information about the sections. For each case a total of 1000 cells were assessed in three to four different fields at x400 magnitudes. Staining was repeated to check for possible technical errors, but similar results were obtained. Scores for the expression of cdc25B and phosphorylated ER- were assigned semiquantitatively according to the percentage of cells stained (<25%, score 1; 25–75%, score 2; and >75%, score 3) and the intensity of staining (weak, score 1; moderate, score 2; and strong, score 3). The two scores were then multiplied. When <25% of the cells were stained or the intensity of the stain was weak, then the product of the two scores was 
3, and expression was categorized as " low." When at least 25% of the cells were stained, and the intensity of the stain was moderate or strong, then the product of the two scores was at least 4, and expression was categorized as "high."
Statistical Analysis.
2 test was used to evaluate the association between two categorical variables. P < 0.05 was considered to be statistically significant.
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RESULTS
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Microscopic evaluation of the immunohistochemical staining of the endometrial specimens resulted in the categorization of their expression of cdc25B and phosphorylated ER- as "low" or "high" (Table 1)
. Examples of the immunostaining (Figs. 1
and 2)
show that expression of cdc25B and phosphorylated ER- was confined to the cell nuclei. All 3 specimens of normal endometrium from the proliferative phase expressed phosphorylated ER- at a moderate level. Expression of phosphorylated ER- was absent or very low in the only specimen of normal endometrium from the secretory phase. Expression of cdc25B was absent or low in 2 of the 4 specimens of normal endometrium, regardless of the menstrual phase. The expression of phosphorylated ER- was increased in 6 of the 7 specimens of hyperplastic endometrium. Five of the 7 specimens of hyperplastic endometrium showed increased expression of cdc25B. In the 6 cases of endometrial hyperplasia with increased expression of phosphorylated ER-, 4 showed coordinate high expression of cdc25B. The expression of both phosphorylated ER- and cdc25B was higher in specimens of hyperplasia with atypia than in the specimens of hyperplasia without atypia, although statistical analysis of 2 test was not done because of the limited sample size.
Expression of phosphorylated ER- and cdc25B was high in the majority of the low-grade EEC specimens, but not present in the majority of high-grade EEC. Phosphorylated ER- was expressed at high levels in 65% (13 of 20) of the low-grade EEC, but in only 17% (2 of 12) of the high-grade EEC (2 = 7.0; P < 0.01). Cdc25B was expressed at high levels in 90% (18 of 20) of low-grade EEC, but in only 42% (5 of 12) of high-grade EEC (2 = 8.7; P < 0.01). Furthermore, coordinate high expression of phosphorylated ER- and cdc25B was present in 65% (13 of 20) of low-grade EEC but in only 17% (2 of 12) of high-grade EEC (2 = 8.5; P < 0.01; Table 2
).
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Table 2 Coordinate high expression of cdc25B and phosphorylated ER- in hyperplastic endometrium, low-grade EEC, high-grade EEC, and UPSC/CCC
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Among the 18 UPSC/CCC, most showed negative or low-level staining for phosphorylated ER-, and only 4 had moderate or high levels of expression. In contrast, 83% (15 of 18) of UPSC/CCC had high expression of cdc25B (2 = 13.5; P < 0.01). The majority of the UPSC/CCC tumors (15 of 18) did not show coordinate high expression of phosphorylated ER- and cdc25B (2 = 20.6; P < 0.01; compared with low-grade EEC; Table 2
).
To assess the functional consequence of coordinate high expression of phosphorylated ER- and cdc25B, the expression level of PR, an ER regulated protein, was determined by immunohistochemical analysis. Of the 32 EEC tumors (20 low-grade and 12 high-grade), 15 showed coordinate high expression of phosphorylated ER- and cdc25B. High-level expression of PR was present in 93% (14 of 15) of cases with coordinate expression. In contrast, only 41% (7 of 17) of cases without coordinate expression showed high-level PR expression (2 = 9.6; P < 0.01). In 18 of the UPSC/CCC, high-level expression of PR was shown in all of the tumors (3 of 3) with coordinate ER- and cdc25B expression, but only 20% (3 of 15) of tumors without coordinate ER- and cdc25B expression (2 = 7.2; P < 0.01; Table 3
).
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Table 3 Correlation of PR expression with coordinate expression of cdc25B and phosphorylated ER- in EEC and UPSC/CCC
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DISCUSSION
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We hypothesized that cdc25B, in cooperation with ER-, may contribute to the stepwise tumorigenesis of EEC. We found that the immunostaining for cdc25B is weak in normal endometrium. The immunoreactivity is increased in endometrial hyperplasia, with higher intensity of immunostaining when atypia is present, suggesting that cdc25B might contribute to oncogenic transformation in endometrial glandular epithelium. In addition, 57% (4 of 7) of our specimens of hyperplasia with or without atypia showed coordinate high expression of phosphorylated ER- and cdc25B, implying that cdc25B might function as a coactivator of ER- in the tumorigenesis of EEC. We showed that low-grade EEC is much more likely than high-grade EEC to highly express phosphorylated ER- (70% versus 17%; 2 = 7.0; P < 0.01) or cdc25B (90% versus 42%; 2 = 8.7; P < 0.01). Furthermore, coordinate high expression of phosphorylated ER- and cdc25B was significant in the low-grade EEC compared with the high-grade EEC (65% versus 17%; 2 = 7.0; P < 0.01). High-level expression of PR was present in 93% (14 of 15) of the cases with coordinate expression of phosphorylated ER- and cdc25B. In contrast, only 41% (7 of 17) of the cases without coordinate expression showed high-level PR (2 = 6.8; P < 0.01). These findings support our hypothesis that cdc25B in cooperation with phosphorylated ER- may play a functional role in the development of EEC. However, it is unclear why expression of cdc25B and phosphorylated ER- was decreased or lost in high-grade EEC. A link has been demonstrated between methylation of the ER- gene, and loss of ER- expression in breast carcinomas and in breast cancer cell lines (11)
. However, the hypothesis that loss of ER expression in endometrial carcinomas is associated with aberrant de novo methylation of the 5' end of the ER gene has been refuted by studies from other groups (12
, 13)
. Thus, the basis for the loss of ER- gene expression in high-grade EEC remains to be elucidated.
UPSC and CCC have a distinct clinical course and a different molecular pathogenesis from EEC tumors. In these tumors, we found that high expression of cdc25B is much more common than high expression of phosphorylated ER- (83% versus 22%; 2 = 13.5; P < 0.01). Furthermore, the majority of these tumors do not show coordinate expression of phosphorylated ER- and cdc25B (17% versus 65%, compared with low-grade EEC; 2 = 20.6; P < 0.01). Expression of PR in UPSC/CCC was significantly correlated with coordinate expression of ER- and cdc25B (100% versus 20%, compared with noncoordinate expression tumors, 2 = 7.2; P < 0.01). However, PR expression was not associated with cdc25B. Overexpression of cdc25B has been shown in many human tumors (14
, 15)
and correlated with c-myc in non-Hodgkin’s lymphoma (16)
. However, the mechanism of overexpression of cdc25B in UPSC/CCC tumors is unclear. Although overexpression of cdc25B may play important roles in both EEC and UPSC/CCC, its oncogenic action in tumor development and progression may be different in these two types. Interestingly, it appears unlikely that cdc25B functions mainly as a coactivator of ER- in UPSC/CCC, because coordinate expression of phosphorylated ER- and cdc25B was uncommon.
In summary, our findings indicate that cdc25B and phosphorylated ER- may play a role in the development and progression of endometrial carcinomas. However, the oncogenic action of cdc25B may be different in various types of endometrial carcinoma. Whereas cdc25B may function as a coactivator of ER- in the transformation of endometrial hyperplasia and the development of EEC, its oncogenic action in UPSC/CCC may be unrelated to ER-.
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FOOTNOTES
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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 To whom requests for reprints should be addressed, at the Department of Gynecologic Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 745-8902; Fax: (713) 792-7586; E-mail: khlu{at}mdanderson.org 
2 The abbreviations used are: EEC, endometrioid endometrial carcinoma; UPSC, uterine papillary serous carcinoma; CCC, clear cell carcinoma; ER, estrogen receptor; PR, progesterone receptor.
Received 4/15/03.
Revised 7/ 3/03.
Accepted 7/21/03.
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ABSTRACT
INTRODUCTION
