Expression of survivin, PTEN and p27 in normal, hyperplastic, and carcinomatous endometrium

Int J Gynecol Cancer 2006, 16, 1412–1418

Expression of survivin, PTEN and p27 in normal, hyperplastic, and carcinomatous endometrium S. ERKANLI*, F. KAYASELCUKy, E. KUSCU*, T. BAGIS*, F. BOLATy, A. HABERAL* & B. DEMIRHANy Departments of *Obstetrics and Gynecology and yPathology, Baskent University Faculty of Medicine, Adana, Turkey

Abstract.

Erkanli S, Kayaselcuk F, Kuscu E, Bagis T, Bolat F, Haberal A, Demirhan B. Expression of survivin, PTEN and p27 in normal, hyperplastic, and carcinomatous endometrium. Int J Gynecol Cancer 2006;16:1412–1418. We aimed to investigate if expressions of survivin and p27 proteins are involved in the development of endometrioid carcinoma, along with whether there are any correlations between these proteins and loss of wild-type PTEN that is found in up to 80% of endometrial carcinomas. We also studied their correlations with classical prognostic factors and survival in endometrial carcinoma. To our knowledge, this is the first time survivin expression is investigated in endometrial hyperplasia along with endometrioid adenocarcinoma. For immunohistochemical analysis, 29 endometrioid adenocarcinoma, 38 endometrial hyperplasia, and 10 proliferative endometrium tissue samples were selected in the pathology archives. Staining of cells was scored as 12 if .50%, 11 if ,50%, and negative if none were stained positive. Survivin expression increased from proliferative to hyperplasia to carcinoma cases. PTEN and p27 expressions decreased in hyperplasia and carcinoma cases with respect to proliferative endometrium. All these differences were statistically significant (P , 0.05). PTEN positively correlated to p27 (P , 0.05); however, neither was correlated with survivin. None of these genes were correlated with classical prognostic factors such as grade and myometrial invasion in endometrioid adenocarcinoma. However, mean survival was statistically significantly higher in PTEN-positive cases (46.6 vs 16.4 months) (P , 0.05). Survivin overexpression might be one of the important mechanisms in the development of endometrioid adenocarcinoma along with lost or decreased activity of PTEN and p27. However, survivin seems to exert its role in ways different from those of PTEN or p27 in the development of endometrioid adenocarcinoma. These findings on the role of survivin in endometrioid adenocarcinoma should be confirmed and the pathways through which survivin acts in endometrioid adenocarcinoma studied further with a larger sample size. KEYWORDS:

endometrial carcinoma, endometrial hyperplasia, PTEN, p27, survivin.

Endometrial cancer develops through a series of genetic alterations involving activation of oncogenes and loss of tumor suppressor genes. As the balance between cell proliferation and cell loss or apoptosis is disturbed, the endometrium may go through a spectrum of orderly changes from hyperplasia to carcinoma. Endometrioid adenocarcinoma is the most frequent histologic variant, accounting for up to 80% of the cases. Almost all cases of this histologic type are hormone dependent and associated with obesity, exogenous use of hormones, Address correspondence and reprint requests to: Serkan Erkanli, MD, Department of Obstetrics and Gynecology, Baskent University Faculty of Medicine, Seyhan, Adana 01250, Turkey. Email: serkanli@ tnn.net

and elevated estrogen levels. The tumoral lesion is known to arise from an endometrial hyperplasia(1). Survivin is a recently characterized gene and a member of the inhibitor of apoptosis protein (IAP) family, whose members inhibit apoptosis through a pathway different from that involving the bcl-2 family(2). It is located on chromosome 17q25, and unlike other IAP proteins, survivin is expressed during embryonic and fetal development(2). Survivin suppresses apoptosis induced by Fas, Bax, caspase-7, and anticancer drugs(3). Survivin has been shown to be expressed in 60 cancer cell lines, including breast, colon, brain, leukemia/ lymphoma, lung, melanoma, ovarian, prostate and renal cancers(3). Its overexpression has been found to be associated with poor survival among many cancers(4–6). # 2006, Copyright the Authors Journal compilation # 2006, IGCS and ESGO

Expression of survivin, PTEN, and p27 in endometrial carcinoma

On the basis of these findings, survivin expression may be an important prognostic marker in cancers. In endometrial cancers, the role of survivin is controversial, and in endometrial hyperplasia, its role is unknown. The tumor suppressor gene PTEN is located on chromosome 10q23 and encodes a dual-specificity phosphatase, with both protein and lipid phosphatase activities. It negatively regulates the phosphoinositide 3-kinase signaling pathway, including the downstream effector protein kinase Akt. PTEN, by limiting the level of PIP3, functions as a downregulator of cell proliferation(7,8). Mutations in PTEN gene seem to be a primary cause in the development of endometrial carcinoma. However, little is known about the role of survivin as well as p27 gene in the development of endometrial carcinoma. The entry and progression of cells through the cell cycle are controlled by a family of protein kinases, each of which comprises a regulatory subunit, or cyclin, and a catalytic subunit, termed a cyclin-dependent kinase (Cdk). The levels of specific cyclins peak and then are rapidly degraded as the cell enters the next phase of the cell cycle(9). The currently known members of the kinase inhibitor protein family consist of p21, p27, and p57 that bind and inhibit cyclin E/CDK2 and cyclin A/cdk2 complexes(10). p27 (Kip1) is a member of the Cip1/Kip1 family of Cdk inhibitors and is a potential tumor suppressor gene. The introduction of p27 into mammary cancer cell lines, by means of a recombinant adenovirus that expresses p27 protein, has been shown to induce growth arrest of mammary carcinoma cells(11,12). p27 levels decline as cells are stimulated to enter the cell cycle with specific cytokines and accumulate when cells are arrested by transforming growth factor-b or by contact inhibition(13). As a result, we aimed to investigate further if expressions of survivin and p27 proteins are involved in the development of endometrial hyperplasia and carcinoma, along with whether there are any correlations between these proteins and loss of wild-type PTEN that is found in up to 80% of endometrial carcinomas. To our knowledge, this is the first report in Englishlanguage literature on survivin protein expression in hyperplastic human endometrium. We also investigated if these genes’ expressions are correlated with clinicopathologic prognostic factors of endometrial carcinoma.

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endometrium with proliferative-phase tissue samples were selected in pathology archives. All patients with endometrial carcinoma were surgically treated, histologically diagnosed, and given follow-up care in the Department of Obstetrics and Gynecology at Baskent University Hospital. Surgical staging and grading was determined in accordance with the FIGO(14) criteria; 13 cases were classified as FIGO stage I (IA, IB, IC), 4 cases as stage II, 11 cases as stage III, and one case as stage IV. For statistical analysis, stages I and II and stages III and IV were considered low and high stages, respectively. Clinicopathologic characteristics of adenocarcinoma cases are shown in Table 1. Mean follow-up time was 17.7 (1–52) months. The samples were fixed in 10% neutral-buffered formalin and paraffin embedded. For immunohistochemistry, serial sections of 5-lm thickness were cut from the paraffin blocks. The sections were deparaffinized with xylene and rehydrated with ethanol. Nonenzymatic antigen retrieval was performed on each slide and washed by phosphate-buffered saline. Immunohistochemical staining was performed manually using the standard Table 1. Characteristics of adenocarcinoma patients (n ¼ 29) n (%) Myometrial invasion None 1/2 .1/2 Lymphovascular space invasion None Positive Lymph node metastasis None Positive CA125 Normal High Stage 1 2 3–4 Grade 1 2 3 Menopause Premenopause Postmenopause Status Exitus Alive Unknown Age Mean
SD (minimum to maximum) Follow-up duration (month) Mean
SD (minimum to maximum)

Materials and methods For immunohistochemical analysis, 29 endometrioid adenocarcinoma (6 cases grade 1, 19 cases grade 2, and 4 cases grade 3), 38 endometrial hyperplasia (24 simple and 6 complex hyperplasias without atypia and 8 hyperplasias showing atypia), and 10 normal #

3 (10.3) 17 (58.6) 9 (31.0) 21 (72.4) 8 (27.6) 23 (79.3) 6 (20.7) 22 (75.9) 7 (24.1) 13 (44.8) 4 (13.8) 12 (41.4) 6 (20.7) 19 (65.5) 4 (13.8) 6 (20.7) 23 (79.3) 6 (7.8) 20 (26.0) 3 60.0
59.0 (37–77) 17.7
15.0 (1–52)

2006 IGCS, International Journal of Gynecological Cancer 16, 1412–1418

1414 S. Erkanli et al.

avidin–biotin-peroxidase complex technique with DAKO (LSAB kit; DAKO, Glostrup, Denmark). The primary antibodies for p27 (clone Ab-1, MS-256-R7; Neomarkers, Fremont, CA, USA), PTEN (clone Ab-4, MS-21601-R7; Neomarkers), and survivin (clone Ab-2, MS-1202-P1; Neomarkers) were applied to the slides. Histologic and immunohistochemical evaluation were performed independently by two pathologists. Epithelial glandular cells were stained and scored. Every stained slide was assessed and given a score, in which staining of the cells was 12 when .50%, 11 when ,50%, and negative when none of nuclei were stained positive(15). Statistical analysis Chi-square, Fisher’s exact, and Mantel–Haenszel v2 tests were used for comparison of categorical variables between groups. The Kaplan–Meier method was used to estimate cumulative survival, and log-rank test was applied to compare stratified survival functions. The Cox proportional-hazards model was used to test the statistical independence and significance of PTEN, p27, survivin, and stage. Data were expressed as mean
standard deviation (SD), n (number of cases), and percent (%). A P value 0.05 was considered as significant. SPSS for Windows version 10.0 was used for statistical analyses.

and the difference is statistically significant (P , 0.05) (Table 2; Figs. 1, 3). PTEN was correlated with p27 but not correlated with survivin. Survivin immunohistochemistry Survivin expression increased from proliferative to hyperplasia to carcinoma cases, and the difference was statistically significant (P , 0.05) (Table 2; Figs. 1, 3). Survivin was not correlated with PTEN or p27. p27 immunohistochemistry p27 expression is decreased in hyperplasia and carcinoma cases with respect to proliferative endometrium, and the difference is statistically significant (P , 0.05) (Table 2; Figs. 1, 3). While there was a positive correlation between PTEN and p27 when all groups were analyzed (P ¼ 0.007) (Mantel–Haenszel v2 test), there was no correlation between survivin, PTEN, and p27 within endometrial adenocarcinoma cases. In our study, survivin, PTEN, and p27 protein expressions did not correlate with the classical prognostic factors such as age of the patients, histologic grade, depth of myometrial invasion, or lymphovascular invasion (P . 0.05). Survival

Results PTEN immunohistochemistry PTEN expression is decreased in hyperplasia and carcinoma cases with respect to proliferative endometrium, Table 2.

Of the 29 patients, 3 were lost to follow-up. Overall mean cumulative survival of the 26 patients was 39.6 months (median 48.0). Mean survival according to stage, PTEN, p27, and survivin expression is given in detail in Table 3 and Figure 2. Mean cumulative

Expression patterns of PTEN, p27, and survivin Group

PTEN* Negative ,50% .50% p27** Negative ,50% .50% Survivin*** Negative ,50% .50%

Proliferative, n ¼ 10 (%)

Hyperplasia, n ¼ 38 (%)

Adenocarcinoma, n ¼ 29 (%)

P value (between 3 groups)

— 6 (60.0) 4 (40.0)

14 (36.8) 23 (60.5) 1 (2.6)

12 (41.4) 13 (44.8) 4 (13.8)

0.006

— 7 (70.0) 3 (30.0)

17 (44.7) 17 (44.7) 4 (10.5)

14 (48.3) 11 (37.9) 4 (13.8)

0.076

6 (60.0) 2 (20.0) 2 (20.0)

10 (26.3) 24 (63.2) 4 (10.5)

3 (10.3) 12 (41.4) 14 (48.3)

0.0001

*Proliferative vs hyperplasia, P ¼ 0.001; proliferative vs adenocarcinoma, P ¼ 0.03; hyperplasia vs adenocarcinoma, P ¼ 0.166. **Proliferative vs hyperplasia, P ¼ 0.02; proliferative vs adenocarcinoma, P ¼ 0.02; hyperplasia vs adenocarcinoma, P ¼ 0.830. ***Proliferative vs hyperplasia, P ¼ 0.05; proliferative vs adenocarcinoma, P ¼ 0.006; hyperplasia vs adenocarcinoma, P ¼ 0.002. #

2006 IGCS, International Journal of Gynecological Cancer 16, 1412–1418

Expression of survivin, PTEN, and p27 in endometrial carcinoma

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mens of normal and cancerous endometrium and concluded that survivin is not a specific marker of endometrial cancer. However, Takai et al.(18) found that survivin expression is increased in endometrial carcinoma cases and suggested that presence of survivin protein could be used as a defining diagnostic marker and also a prognostic marker to discriminate aggressive behavior in endometrial cancers. Furthermore, Saitoh et al.(19) found survivin messenger RNA detectable in 17 endometrial cancer tissues and 6 endometrial cancer cell lines. These authors concluded that these data suggested that survivin might play important roles in the pathway of carcinogenesis of human uterine cancer. Survivin expression in hyperplastic endometrium is not known. We found only one small report in Polish covering six patients(20). In our study, we studied its expression in 38 hyperplastic endometrium cases and found survivin protein expression to be increased in a spectrum from proliferative endometrium to hyperplasic to carcinomatous endometrium, supporting in part the findings of Takai et al.; however, survivin had no correlation with clinicopathologic prognostic factors and survival. Our findings of increased survivin expression in endometrial hyperplasia along with carcinoma suggest that survivin overexpression is one of the important determinants in the developmental pathway of endometrioid carcinoma. The p27 gene, unlike others, has not been found to be mutated in most human tumors. Inverse correlation of p27 expression and degree of malignancy in breast, colorectal, ovarian, and cervical carcinomas are well established, but correlation is not well established in endometrial carcinoma(21,22). The role of p27 in endometrial cancer remains controversial(23,24). In a recent study, Masciullo et al.(23) in their large series of

Figure 1. Ratio of positive expression (,50% 1 .50%) of survivin, PTEN, and p27 between groups.

survival was 46.6 months in the group with positive PTEN; however, survival dropped to 16.4 months in the negative group (P ¼ 0.02) (Table 3). The Cox proportional-hazards model results showed that PTEN and stage are independent and significant factors related with survival rate in adenocarcinoma patients (Table 3). We did not find any statistically significant difference between p27 or survivin and survival.

Discussion Overexpression of survivin has oncogenic potential because it may overcome the G2/M phase checkpoint of the cell cycle to enforce progression of cells through mitosis. Survivin promotes cell proliferation by interacting with cdk-4 and releasing p21 from cdk-4, and then interacts with mitochondrial procaspase-3 to suppress Fas-mediated cell death(16). However, in endometrial cancers, the role of survivin is controversial, where there are only three studies(17–19). Lehner et al.(17) detected survivin messenger RNA in all speciTable 3. Results of survival analysis Kaplan–Meier

Stage Low High PTEN Negative Positive p27 Negative Positive Survivin Negative Positive

Cox regression

Number of events/total

Mean survival (month)

P value (log-rank test)

2/14 4/12

29.2 36.4

0.5

17.5 (1.2–257.1)

0.03

4/12 2/14

16.4 46.6

0.02

0.008 (0.0001–0.52)

0.02

3/13 3/13

39.2 33.0

0.6

0.39 (0.05–2.27)

0.2

1/3 5/23

30.6 40.1

0.7

5.56 (0.29–103.4)

0.3

#

Odds Ratio (Confidence Interval)

P value

2006 IGCS, International Journal of Gynecological Cancer 16, 1412–1418

1416 S. Erkanli et al.

Figure 2. Cumulative survival in patients according to PTEN expression.

endometrial carcinoma cases found, by immunohistochemical analysis, decreased p27 expression from normal to hyperplastic to carcinomatous cases and concluded that loss of this tumor suppressor might represent a novel and distinct molecular alteration involved in estrogen-related endometrial adenocarcinomas (type I). In contrast, Watanabe et al.(24) found p27 to be paradoxically increased in endometrial carcinomas. In our study, all normal proliferative endometrial cells expressed p27, whereas its expression was lost in 44.7% of endometrial hyperplasia and 48.3% of endometrial carcinoma cases. The finding of decreased p27 expression in endometrial hyperplasia and carcinoma compared to normal proliferative endometrium was statistically significant (P , 0.05). These findings suggest that p27 expression is essential for the control of normal endometrial proliferation and confirm that loss of p27 expression may be an important step, although not the only factor in endometrial carcinogenesis. Loss of p27 expression in endometrial carcinoma did not correlate with classical prognostic factors such as myometrial invasion, stage or grade, and survival, supporting similar results from other studies(25,26,23). PTEN tumor suppressor gene is the most commonly mutated gene identified in endometrial carcinoma, where it is mutated in 34–80% of cases(27–29) and in up to 50% of endometrial precancers(29–32). Accordingly, inactivation of PTEN is considered to be an early event in endometrial carcinogenesis. In this study, in keeping with the findings in the literature, we found that PTEN expression is decreased in endometrial #

2006 IGCS, International Journal of Gynecological Cancer 16, 1412–1418

hyperplasia and carcinoma compared to normal proliferative endometrium, and this difference was statistically significant (P , 0.05). PTEN expression was not correlated with classical prognostic factors; however, it was positively correlated with mean survival rates (P ¼ 0.02). In the literature, data on the prognostic significance of PTEN are not conclusive. Some authors have found loss of PTEN expression to be a favorable prognostic factor(33), while others have found its negative expression to be associated with aggressive behavior in endometrial carcinoma(34,35). Being a tumor suppressor gene, it is more plausible that PTENpositive status comforts a survival advantage. Our results suggest that PTEN expression might be an independent prognostic factor along with stage. In endometrial cell lines, introduction of PTEN gene induces apoptosis, whereas in glioblastoma cell lines, it causes cell cycle arrest at G1 phase through elevation of Cdk inhibitor, p27(36,37). Although upregulation of p27 induced by PTEN has been reported in different cell lines, suggesting that p27 may be a target of the PTEN cell cycle arrest pathway, there is little and controversial information regarding PTEN and p27 correlation in endometrial carcinoma cases. In endometrial carcinoma, An et al.(26), in their elegant study, have found p27 to be a downstream target of PTENdependent cell cycle arrest, whereas Kanamori et al.(38) showed that PTEN expression is decreased causing activation of the Akt pathway; however, they did not find a relationship between PTEN and p27. Keeping up with the former study, we also found PTEN and p27 to be positively correlated when we analyzed these in proliferative, hyperplasia, and adenocarcinoma cases, which supports the finding that p27 might be the downstream target of PTEN in endometrial carcinoma. We could not detect a relation when we analyzed PTEN and p27 within endometrial adenocarcinoma cases probably due to low number of cancer patients. Zhu et al.(39) in their study showed that PTEN induced a specific reduction of cyclin D3 levels and an associated increase in the amount of p27 complexed with cdk2 in endometrial carcinoma cell lines. The authors stated that p27’s regulation is indirect and its role in PTEN signaling seems complex and dependent upon cell type and the context of the growth-regulatory signal. There are data that PI3K pathway inhibition downregulates survivin expression and enhances apoptosis in neuroblastomas and treatment with inhibitors of PI3K may provide potential novel therapeutic options(40). We do not know if such a relationship exists in endometrial carcinoma. In this study, we also investigated whether there is any correlation between survivin and PTEN,

Expression of survivin, PTEN, and p27 in endometrial carcinoma

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Figure 3. a) Proliferative endometrium and b) endometrial carcinoma showing strongly positive and weak-positive p27 expression, respectively (immunohistochemistry [IHC], 3200, 3200). c) Proliferative and d) carcinomatous endometrium showing strongly positive and negative PTEN expression, respectively (IHC, 3200, 3200). e) Proliferative, f) hyperplastic, and g) carcinomatous endometrium showing negative, weak-positive, and strong-positive survivin expression, respectively (IHC, 3200, 3200, 3200).

noma. Neither survivin nor PTEN or p27 is correlated with classical prognostic factors; however, PTEN-positive status might be related to a better survival rate in endometrial carcinoma. These findings, especially on the role of survivin in endometrioid carcinoma, should be confirmed and the pathways through which survivin acts in endometrioid carcinoma studied further with a larger sample size.

which is known to inhibit PI3K/Akt pathway. Yet, we did not find any relationship between PTEN and survivin, which suggests that these genes act in different ways from each other in the development of endometrial carcinoma. In conclusion, although our sample size in this study is small for drawing firm conclusions, we observed that survivin overexpression might be one of the important mechanisms in the development of type I endometrial carcinoma along with lost or decreased activity of PTEN and p27. However, it seems that survivin exerts its role in ways different from those of PTEN or p27 in the development of endometrial carci-

Acknowledgment We would like to thank Dr Gulsah Saydeoglu for her contributions in statistical analysis. #

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Accepted September 11, 2004