Coordinate Expression of Cdc25b and ER- Is Frequent in Low-Grade

[CANCER RESEARCH 63, 6195–6199, October 1, 2003] 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

ABSTRACT driven model of carcinogenesis whereby estrogen stimulation leads to malignant transformation through a stepwise progression from nor- Overexpression of cdc25B, an important cell cycle regulator, has been mal, to atypical hyperplasia, to cancer (5). In contrast, UPSC and CCC shown to result in mammary gland hyperplasia in transgenic mice and to are not estrogen-driven tumors. UPSCs frequently are found to have increase steroid hormone responsiveness as a direct coactivator of the estrogen receptor (ER). We investigated the potential role of cdc25B in the p53 mutations (6). pathogenesis of endometrial carcinomas in conjunction with ER-␣.We Cdc25B has been found recently to function as a direct coactivator examined the expression of cdc25B and phosphorylated ER-␣ in 4 ar- of the ER in mammary glands (7). Cdc25B belongs to a family of chived human specimens of normal endometrium; 7 endometrial hyper- dual-specificity proteins that activate the cyclin/cyclin-dependent ki- plasia with or without atypia; 32 endometrioid endometrial carcinoma nase complex, resulting in cell cycle progression (8). Cdc25B is

(EEC), including 20 low-grade (grade 1) and 12 high-grade (grade 2 or 3) involved in the regulation of the G2/M-phase transition (9). Overex- tumors; and 18 endometrial cancers with aggressive histological subtypes pression of cdc25B in transgenic mice has been shown to cause (uterine papillary serous carcinoma and clear cell carcinoma, UPSC/ mammary gland hyperplasia and increase steroid hormone respon- CCC) by immunohistochemistry with monoclonal antibodies. Expression siveness (10). The direct interactions of cdc25B with steroid receptors of cdc25B and phosphorylated ER-␣ was increased in endometrial hyper- (e.g., ER-␣) in vivo and in vitro suggest a potential direct contribution plasia and atypical hyperplasia compared with normal secretory endometrium. Ninety percent (18 of 20) of the low-grade EEC expressed cdc25B of cdc25B to steroid receptor-mediated transcription (7). at a high level, whereas only 42% (5 of 12) of the high-grade EEC did so On the basis of these observations, we hypothesize that cdc25B, in 2 ␣ -P < 0.01). Sixty-five percent (13 of 20) of the low-grade EEC cooperation with ER- , may contribute to the development and pro ;8.7 ؍ ␹) expressed phosphorylated ER-␣ at high levels, but only 17% (2 of 12) of gression of endometrial cancer. To test this hypothesis, we examined P < 0.01). Coordinate high-level the expression of cdc25B and phosphorylated ER-␣ in normal and ;7.0 ؍ high-grade EEC did so (␹2 expression of phosphorylated ER-␣ and cdc25B occurred in 65% (13 of hyperplastic endometrium, and in low-grade (grade 1) and high-grade 20) of low-grade EEC but in only 17% (2 of 12) of the high-grade EEC (grade 2–3) EEC. We also examined the expression of cdc25B and 2 P < 0.01). In the UPSC/CCC tumors, only 22% (4 of 18) of the phosphorylated ER-␣ in UPSC/CCC to evaluate their roles in the ;7.0 ؍ ␹) ␣ tumors expressed phosphorylated ER- at high-levels. However, 83% (15 development of this disease. To assess the functional consequence of ;13.5 ؍ of 18) of these carcinomas showed high expression of cdc25B (␹2 coordinate high expression of phosphorylated ER-␣ and cdc25B, the P < 0.01). The majority of the UPSC/CCC (15 of 18) did not show coordinate high expression of phosphorylated ER-␣ and cdc25B. Our expression level of PR, an ER-regulated protein, was also evaluated. findings show that in endometrial hyperplasia and low-grade EEC, coordinate increase in cdc25B and phosphorylated ER-␣ occurs. However, in MATERIALS AND METHODS UPSC/CCC, cdc25B is highly expressed without coordinate increase in phosphorylated ER-␣. Cdc25B may play important roles in the develop- Endometrial Tissue Samples. Archived formalin-fixed, paraffin-embed- ment and progression of EEC and UPSC/CCC by different mechanisms. ded 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 INTRODUCTION 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 Endometrial carcinoma ranks first in incidence and second in specimens had been collected between 1994 and 2002. The mean age of mortality among female genital tract tumors. In 2003, it is estimated patients was 52 years. The study protocol was approved by the Committee for that there will be 40,100 new cases and 6,800 related deaths among the Protection of Human Subjects at The University of Texas M. D. Anderson women in the United States (1). There are two predominant histolog- Cancer Center. ical subtypes of endometrial carcinoma. EEC2 accounts for Ͼ80% of Immunohistochemical Assessment. Immunostaining for cdc25B and endometrial carcinomas. Nonendometrioid high-risk histological sub- ER-␣ phosphorylated at Ser118 was performed on 5–6 ␮m sections of for- types, including UPSC and CCC, are less common than EEC; how- malin-fixed, paraffin-embedded endometrial specimens using DAKO LSAB ever, they have a more aggressive course (2, 3). A dualistic model of kits (DakoCytomation, Carpinteria, CA). Expression of PRs was also examined by immunohistochemical analysis. Tissue sections were incubated at 60°C carcinogenesis has been proposed on the basis of the distinctive for 1 h, deparaffinized in xylene, and hydrated in a graded series of ethanol clinical and histological features of endometrioid and papillary serous solutions. After a rinse in PBS (137 mM NaCl, 2.7 mM KCl, 1 mM KH PO , adenocarcinoma (4). EEC is associated with a progressive, estrogen- 2 4 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. Received 4/15/03; revised 7/3/03; accepted 7/21/03. Then, after a 10-min rehydration in PBS, the sections were heated in a The costs of publication of this article were defrayed in part by the payment of page microwave oven for 3 min in 10 mM citrate buffer for antigen retrieval. After charges. This article must therefore be hereby marked advertisement in accordance with incubation with blocking serum (4% normal horse serum) for 30 min at room 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at the Department of Gyneco- temperature, the sections were incubated overnight at 4°C with monoclonal logic Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe antibodies against human cdc25B (Cell Signal Technology, Beverly, MA), Boulevard, Houston, TX 77030. Phone: (713) 745-8902; Fax: (713) 792-7586; E-mail: phosphorylated ER-␣ (Cell Signal Technology), and PR (Lab Vision Corpo- [email protected]. ration, Fremont, CA). Each antibody was tested in serial dilutions to achieve 2 The abbreviations used are: EEC, endometrioid endometrial carcinoma; UPSC, uterine papillary serous carcinoma; CCC, clear cell carcinoma; ER, estrogen receptor; PR, maximum sensitivity and specificity. Human breast cancer sections were used progesterone receptor. as positive controls for both antibodies. Negative controls were analyzed on 6195

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. cdc25B AND ER-␣ IN ENDOMETRIAL CANCER adjacent sections incubated without antibody. After a PBS rinse, the sections (2 of 12) of the high-grade EEC (␹2 ϭ 7.0; P Ͻ 0.01). Cdc25B was were treated with biotinylated goat antimouse antibody for 15 min. The expressed at high levels in 90% (18 of 20) of low-grade EEC, but in sections were washed with PBS three times and incubated with an avidin- only 42% (5 of 12) of high-grade EEC (␹2 ϭ 8.7; P Ͻ 0.01). biotinylated horseradish peroxidase macromolecular complex for 10 min ac- Furthermore, coordinate high expression of phosphorylated ER-␣ and cording to the manufacturer’s instructions for the LASB kit. Visualization of cdc25B was present in 65% (13 of 20) of low-grade EEC but in only peroxidase was carried out by incubating the sections with diaminobenzidine 17% (2 of 12) of high-grade EEC (␹2 ϭ 8.5; P Ͻ 0.01; Table 2). for 10 min. The sections were counterstained with hematoxylin, dehydrated in a series of ethanol solutions, and cleared with xylene, and coverslips were Among the 18 UPSC/CCC, most showed negative or low-level ␣ placed over Permount mounting medium for evaluation by light microscopy. staining for phosphorylated ER- , and only 4 had moderate or high All of the immunostained tissue sections were evaluated and scored coop- levels of expression. In contrast, 83% (15 of 18) of UPSC/CCC had 2 eratively by two investigators (W. W. and K. H. L.) who were blinded to any high expression of cdc25B (␹ ϭ 13.5; P Ͻ 0.01). The majority of the clinical or pathological information about the sections. For each case a total of UPSC/CCC tumors (15 of 18) did not show coordinate high expres- 1000 cells were assessed in three to four different fields at ϫ400 magnitudes. sion of phosphorylated ER-␣ and cdc25B (␹2 ϭ 20.6; P Ͻ 0.01; Staining was repeated to check for possible technical errors, but similar results compared with low-grade EEC; Table 2). were obtained. Scores for the expression of cdc25B and phosphorylated ER-␣ To assess the functional consequence of coordinate high expression were assigned semiquantitatively according to the percentage of cells stained of phosphorylated ER-␣ and cdc25B, the expression level of PR, an (Ͻ25%, score 1; 25–75%, score 2; and Ͼ75%, score 3) and the intensity of ER regulated protein, was determined by immunohistochemical anal- staining (weak, score 1; moderate, score 2; and strong, score 3). The two scores were then multiplied. When Ͻ25% of the cells were stained or the intensity of ysis. Of the 32 EEC tumors (20 low-grade and 12 high-grade), 15 ␣ the stain was weak, then the product of the two scores was Յ3, and expression showed coordinate high expression of phosphorylated ER- and was categorized as “ low.” When at least 25% of the cells were stained, and the cdc25B. High-level expression of PR was present in 93% (14 of 15) intensity of the stain was moderate or strong, then the product of the two scores of cases with coordinate expression. In contrast, only 41% (7 of 17) of was at least 4, and expression was categorized as “high.” cases without coordinate expression showed high-level PR expression Statistical Analysis. ␹2 test was used to evaluate the association between (␹2 ϭ 9.6; P Ͻ 0.01). In 18 of the UPSC/CCC, high-level expression two categorical variables. P Ͻ 0.05 was considered to be statistically signif- of PR was shown in all of the tumors (3 of 3) with coordinate ER-␣ icant. and cdc25B expression, but only 20% (3 of 15) of tumors without coordinate ER-␣ and cdc25B expression (␹2 ϭ 7.2; P Ͻ 0.01; RESULTS Table 3). Microscopic evaluation of the immunohistochemical staining of the endometrial specimens resulted in the categorization of their expres- DISCUSSION sion of cdc25B and phosphorylated ER-␣ as “low” or “high” (Table 1). Examples of the immunostaining (Figs. 1 and 2) show that ex- We hypothesized that cdc25B, in cooperation with ER-␣, may pression of cdc25B and phosphorylated ER-␣ was confined to the cell contribute to the stepwise tumorigenesis of EEC. We found that the nuclei. All 3 specimens of normal endometrium from the proliferative immunostaining for cdc25B is weak in normal endometrium. The phase expressed phosphorylated ER-␣ at a moderate level. Expression immunoreactivity is increased in endometrial hyperplasia, with higher of phosphorylated ER-␣ was absent or very low in the only specimen intensity of immunostaining when atypia is present, suggesting that of normal endometrium from the secretory phase. Expression of cdc25B might contribute to oncogenic transformation in endometrial cdc25B was absent or low in 2 of the 4 specimens of normal endo- glandular epithelium. In addition, 57% (4 of 7) of our specimens of metrium, regardless of the menstrual phase. The expression of phos- hyperplasia with or without atypia showed coordinate high expression phorylated ER-␣ was increased in 6 of the 7 specimens of hyperplastic of phosphorylated ER-␣ and cdc25B, implying that cdc25B might endometrium. Five of the 7 specimens of hyperplastic endometrium function as a coactivator of ER-␣ in the tumorigenesis of EEC. We showed increased expression of cdc25B. In the 6 cases of endometrial showed that low-grade EEC is much more likely than high-grade EEC hyperplasia with increased expression of phosphorylated ER-␣,4 to highly express phosphorylated ER-␣ (70% versus 17%; ␹2 ϭ 7.0; showed coordinate high expression of cdc25B. The expression of both P Ͻ 0.01) or cdc25B (90% versus 42%; ␹2 ϭ 8.7; P Ͻ 0.01). phosphorylated ER-␣ and cdc25B was higher in specimens of hyper- Furthermore, coordinate high expression of phosphorylated ER-␣ and plasia with atypia than in the specimens of hyperplasia without atypia, cdc25B was significant in the low-grade EEC compared with the although statistical analysis of ␹2 test was not done because of the high-grade EEC (65% versus 17%; ␹2 ϭ 7.0; P Ͻ 0.01). High-level limited sample size. expression of PR was present in 93% (14 of 15) of the cases with Expression of phosphorylated ER-␣ and cdc25B was high in the coordinate expression of phosphorylated ER-␣ and cdc25B. In con- majority of the low-grade EEC specimens, but not present in the trast, only 41% (7 of 17) of the cases without coordinate expression majority of high-grade EEC. Phosphorylated ER-␣ was expressed at showed high-level PR (␹2 ϭ 6.8; P Ͻ 0.01). These findings support high levels in 65% (13 of 20) of the low-grade EEC, but in only 17% our hypothesis that cdc25B in cooperation with phosphorylated ER-␣

Table 1 Expression of cdc25B and phosphorylated ER-␣ in normal, hyperplastic, and malignant endometrial tissuea Expression of cdc25B Expression of phosphorylated ER-␣

Histological feature Total no. Lowb Highc Lowb Highc Normal 4 2 2 1 (secretary) 3 (proliferative) Hyperplasia 7 3 4 1 6 EEC 32 9 (28%) 23 (72%) 17 (53%) 15 (47%) Low grade (1) 20 2 (10%) 18 (90%) 7 (35%) 13 (65%) High grade (2 or 3) 12 7 (58%) 5 (42%) 10 (83%) 2 (17%) UPSC/CCC 18 3 (17%) 15 (83%) 14 (78%) 4 (22%) a Expression of cdc25B and phosphorylated ER-␣ was assessed by immunohistochemical staining with monoclonal antibodies. b Stain weak in intensity, or Ͻ25% of cells stained. c Stain moderate or strong in intensity, and Ն25% of cells stained. 6196

Fig. 1. Immunohistochemical staining for phosphorylated ER-␣ (left) and cdc25B (right) in selected samples of normal and hyperplastic endometrial gland epithelium. A, normal endometrium, proliferative phase. B and C, normal endometrium, secretory phase. D and E, endometrial hyperplasia. F and G, endometrial hyperplasia with atypia. Staining for phosphorylated ER-␣ was moderate in proliferative endometrium (A) and weak in secretory endometrium (B). Staining for cdc25B was weak in normal endometrium (C). Expression of phosphorylated ER-␣ and cdc25B was increased in a majority of samples of hyperplastic endometrium (D–E), with the strongest staining in hyperplasia with atypia (F–G). (Immunostained with peroxidase and diaminobenzidine; counterstained with hematoxylin. Original magnification: ϫ400.) may play a functional role in the development of EEC. However, it is coordinate expression of phosphorylated ER-␣ and cdc25B (17% unclear why expression of cdc25B and phosphorylated ER-␣ was versus 65%, compared with low-grade EEC; ␹2 ϭ 20.6; P Ͻ 0.01). decreased or lost in high-grade EEC. A link has been demonstrated Expression of PR in UPSC/CCC was significantly correlated with between methylation of the ER-␣ gene, and loss of ER-␣ expression coordinate expression of ER-␣ and cdc25B (100% versus 20%, com- in breast carcinomas and in breast cancer cell lines (11). However, the pared with noncoordinate expression tumors, ␹2 ϭ 7.2; P Ͻ 0.01). hypothesis that loss of ER expression in endometrial carcinomas is However, PR expression was not associated with cdc25B. Overex- associated with aberrant de novo methylation of the 5Ј end of the ER pression of cdc25B has been shown in many human tumors (14, 15) gene has been refuted by studies from other groups (12, 13). Thus, the and correlated with c-myc in non-Hodgkin’s lymphoma (16). How- basis for the loss of ER-␣ gene expression in high-grade EEC remains ever, the mechanism of overexpression of cdc25B in UPSC/CCC to be elucidated. tumors is unclear. Although overexpression of cdc25B may play UPSC and CCC have a distinct clinical course and a different important roles in both EEC and UPSC/CCC, its oncogenic action in molecular pathogenesis from EEC tumors. In these tumors, we found tumor development and progression may be different in these two that high expression of cdc25B is much more common than high types. Interestingly, it appears unlikely that cdc25B functions mainly expression of phosphorylated ER-␣ (83% versus 22%; ␹2 ϭ 13.5; as a coactivator of ER-␣ in UPSC/CCC, because coordinate expres- P Ͻ 0.01). Furthermore, the majority of these tumors do not show sion of phosphorylated ER-␣ and cdc25B was uncommon. 6197

Fig. 2. Immunohistochemical staining for phosphorylated ER-␣ (left) and cdc25B (right) in selected samples of endometrial carcinoma. A and B, grade 1 EEC. C and D, grade 3 EEC. E and F, UPSC. Staining for phosphorylated ER-␣ (A) and cdc25B (B) was strong in a majority of samples of low-grade EEC, but absent (C) or weak (D) in a majority of samples of high-grade EEC. In UPSC, most samples had absent or very weak staining for phosphorylated ER-␣ (E), but strong staining for cdc25B (F). (Immunostained with peroxidase and diaminobenzidine; counterstained with hematoxylin. Original magnification: ϫ400.)

In summary, our findings indicate that cdc25B and phosphoryl- Whereas cdc25B may function as a coactivator of ER-␣ in the ated ER-␣ may play a role in the development and progression of transformation of endometrial hyperplasia and the development endometrial carcinomas. However, the oncogenic action of cdc25B of EEC, its oncogenic action in UPSC/CCC may be unrelated may be different in various types of endometrial carcinoma. to ER-␣.

Table 2 Coordinate high expression of cdc25B and phosphorylated ER-␣ in hyperplastic endometrium, low-grade EEC, high-grade EEC, and UPSC/CCC High expression of phosphorylated Coordinate high expression Histological feature Total no. High expression of cdc25B ER-␣ of cdc25B and ER-␣ P Hyperplasia 7 4 6 4 (57%) Nea EEC 32 23 15 15 (47%) Nea Low grade (1) 20 18 14 14 (70%) High grade (2 or 3) 12 5 2 2 (17%) Ͻ0.01b UPSC/CCC 18 15 4 3 (17%) Ͻ0.01b a Ne, P not determined. b P was evaluated in comparison to low-grade EEC.

Table 3 Correlation of PR expression with coordinate expression of cdc25B and phosphorylated ER-␣ in EEC and UPSC/CCC PR expression in tumors with PR expression in tumors without Histology Total no. PR expression coordinate expression coordinate expression P EEC 32 66% (21/32) 93% (14/15) 41% (7/17) Ͻ0.01 UPSC/CCC 18 33% (6/18) 100% (3/3) 20% (3/15) Ͻ0.01 6198

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. Coordinate Expression of Cdc25B and ER-α Is Frequent in Low-Grade Endometrioid Endometrial Carcinoma but Uncommon in High-Grade Endometrioid and Nonendometrioid Carcinomas

Weiguo Wu, Brian M. Slomovitz, Joseph Celestino, et al.

Cancer Res 2003;63:6195-6199.

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