Correlation between Loss of PTEN Expression and AktPhosphorylation in Endometrial Carcinoma

Yasunobu Kanamori,1 Junzo Kigawa,Hiroaki Itamochi, Muneaki Shimada,Masakuni Takahashi, Syunji Kamazawa,Shinya Sato, Ryoji Akeshima, andNaoki TerakawaDepartment of Obstetrics and Gynecology, Tottori University Schoolof Medicine, Yonago 683-8504, Japan

ABSTRACTThe tumor suppressor PTEN acts as a lipid phospha-

tase, regulates the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway, and modulates cell cycle progres-sion and cell survival. Somatic mutations ofPTEN havebeen reported in a variety of cancers, especially in endo-metrial carcinoma. To clarify whether and how PTENand the PI3K/Akt pathway relates to endometrial carci-noma, we examined the expression of those pathway-related proteins in patients with endometrial carcinoma.Of 103 endometrial carcinomas, 37 (36%) showed nega-tive immunohistochemical staining of PTEN. Westernblotting revealed that the expression of PTEN in PTEN-negative cases was significantly lower compared with thatin positive cases. In contrast, phospho-Akt level in nega-tive cases was significantly higher. We found a significantinverse correlation between PTEN and phospho-Akt (r 520.796). The expression of phospho-Bad was greater innegative cases, suggesting that Bad might be a target forAkt. The present study demonstrates the phosphorylationof Akt accompanied by the loss of PTEN in clinical spec-imens of endometrial carcinomas.

INTRODUCTIONEndometrial carcinoma, one of the most common malig-

nancies of the female genital tract, is the fourth most commoncancer of women in the United States (1). Despite its preva-lence, the molecular mechanisms of endometrial carcinogenesishave been poorly understood. Alterations of theK-ras andp53genes were reported in endometrial carcinoma, but the extent ofthese alterations is limited (2).PTENis a tumor suppressor genelocated on 10q23, and alterations of this gene have been iden-

tified in a large fraction of cancers including endometrial car-cinoma (3). The incidence ofPTEN mutations (30–50%) inendometrial carcinoma is one of the highest among analyzedtumors (4, 5).PTEN is the most commonly mutated geneidentified in endometrial carcinoma (4). Additionally, the mu-tations were also seen in about 20% of cases of endometrialhyperplasia, a precursor of endometrial carcinoma (6, 7). Ac-cordingly, inactivation of PTEN is considered to be an earlyevent in endometrial carcinogenesis.

PTEN is a lipid phosphatase dephosphorylating the 3-position of phosphatidylinositol 3, 4, 5-triphosphate, a secondmessenger of PI3K2 (3, 8). PTEN antagonizes PI3K activity andnegatively regulates its downstream-target, the serine/threoninekinase Akt (9). Phosphorylated and activated Akt modulates theactivity of a variety of downstream-proteins that relate to cellsurvival and proliferation (3). It is known that activated Aktphosphorylates and then inactivates the proapoptotic factor Bad,which suppresses apoptosis and promotes cell survival (10, 11).On the other hand, overexpression of PTEN inhibits cell growthand induces a G1 arrest with an increase in the cell cycle kinaseinhibitor p27 (12–14), suggesting that PTEN inactivation mayresult in progression of the cell cycle through down-regulationof p27.

Those previous studies suggest that the loss of PTENfunction with subsequent activation of PI3K/Akt signalingpathway contributes to carcinogenesis. However, those find-ings have been not confirmed in patients with endometrialcarcinoma. To clarify whether and how PTEN and the PI3K/Akt pathway relates to endometrial carcinoma, we examinedthe expression of pathway-related proteins such as PTEN,Akt, Bad, and p27 in clinical specimens of endometrialcarcinomas.

MATERIALS AND METHODSSpecimen Collection. Paraffin-embedded specimens

were collected from 103 patients with endometrial carcinomawho underwent hysterectomy at Tottori University Hospital,Yonago, Japan, between 1990 and 1999. The histological typewas endometrioid in all subjects. The number of histologicalgrades included 51 grade 1, 34 grade 2, and 18 grade 3 speci-mens. Specimens of normal endometrium were obtained fromsix patients with benign tumor (three in the proliferative phaseand three in the secretory phase). All patients gave informedconsent before collection of the specimens, according to ourinstitutional guideline.

Immunohistochemistry. A 4-mm section was cut fromthe paraffin blocks of endometrial carcinoma and normal endome-trium. Each section was mounted on a silane-coated glass slide,

Received 11/27/00; accepted 1/11/01.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 To whom requests for reprints should be addressed, at Department ofObstetrics and Gynecology, Tottori University School of Medicine, 36-1Nishimachi, Yonago 683-8504, Japan. Phone: 81-859-34-8127; Fax:81-859-34-8089; E-mail: kanamori@grape.med.tottori-u.ac.jp. 2 The abbreviation used is: PI3K, phosphatidylinositol 3-kinase.

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deparaffinazed, and soaked for 15 min at room temperature in 0.3%H2O2/methanol to block endogenous peroxidase. A mouse mono-clonal anti-PTEN antibody, PTEN A2B1 (Santa Cruz Biotechnol-ogy, Santa Cruz, CA), was applied for 2 h at 37°C. The primaryantibody was visualized using the Histofine Simple Stain PO(M)kit (Nichirei, Tokyo, Japan) according to the instruction manual.The slide was counterstained with hematoxylin.

Western Blot Analysis. The tumor specimen was ob-tained at the time of surgery, snap-frozen, and stored at280°C.The frozen specimen was homogenized in a lysis buffer con-taining 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% SDS, 1mM DTT, 10 mM NaF, 2 mM Na3VO4, and 13 CompleteProtease Inhibitor Cocktail (Boehringer Mannheim, Ingelheim,Germany). The lysate was centrifuged, and the supernatant was

prepared. Protein concentration of the supernatant was measuredby Bradford’s assay (15).

Seventymg of each protein sample was separated by14% SDS-PAGE, blocked in 2.5% skim milk/TPBS (13PBS; 0.1% Tween-20) for PTEN and p27, or in 5% BSA/TPBS for phospho-Akt and phospho-Bad. Those sampleswere probed with each primary antibody overnight at 4°C.The source of anti-PTEN is described above; anti-p27 wasobtained from Santa Cruz Biotechnology; and anti-phospho-Akt (Ser473) and anti-phospho-Bad (Ser136) were obtainedfrom New England Biolabs (Beverly, MA). After incubationwith horseradish peroxidase-conjugated secondary antibody,protein signals were detected using enhanced chemilumines-cence (Amersham, Buckinghamshire, United Kingdom). The

Fig. 1 PTEN expression in endometrial tissue.A, normal endometrium.B, positive staining for PTEN in endometrial carcinoma (a positivecase).C, heterogeneous staining pattern in endometrial carcinoma (a mixed case).D, negative staining for PTEN in endometrial carcinoma (anegative case).

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immunoblots were quantitated using a public domain NIHimage program (written by Wayne Rasband, NIH3).

Statistical Analysis. We used an unpairedt test to ana-lyze the differences in expression level of proteins. A Pearson’scorrelation test was performed to examine the relationship.

RESULTSIn all normal endometria, PTEN staining was observed in

both epithelial and stromal cells (Fig. 1A; PTEN was stained incytoplasm). In carcinoma specimens, stromal cells were stainedwith PTEN, similarly to normal endometrium, then PTEN-stainingin stromal cells was used as the positive control in each case. Thestatus of staining in endometrial carcinoma was evaluated accord-ing to the following criteria: (a) a positive case was defined as allof the tumor cells showing staining; (b) a mixed case was definedas the presence of both staining and nonstaining cells; and (c) a

3 Available from the Internet by anonymous FTP from zippy.nimh.nih.govor on floppy disk from NTIS, 5285 Port Royal Road, Springfield, VA22161; part no. PB93-504648.

Fig. 2 Western blotting for PTEN,phospho-Akt, phospho-Bad, and p27in PTEN-positive and PTEN-negativecases.

Fig. 3 A, comparison of ex-pression of PTEN betweenPTEN-positive and PTEN-negative cases. Means andstandard deviations were indi-cated. B, comparison of ex-pression of phospho-Akt be-tween PTEN-positive andPTEN-negative cases.C, cor-relation between PTEN andphospho-Akt expression. (r520.796;n 5 20; P , 0.001).D, comparison of expressionof phospho-Bad betweenPTEN-positive and PTEN-negative cases.

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negative case was defined as no staining of any tumor cells. As aresult, there were 50 (49%) PTEN-positive cases (Fig. 1B), 16 (15%)mixed cases (Fig. 1C), and 37 (36%) negative cases (Fig. 1D).

According to the immunohistochemical analysis, we se-lected PTEN-positive or PTEN-negative endometrial carci-nomas. Twenty of the protein samples of tumors (10 positiveand 10 negative) were examined for the expression of PTEN,phospho-Akt, phospho-Bad, and p27 by Western blotting.Fig. 2 shows the expression of those proteins according to thedifference of PTEN-staining status. The level of PTEN ex-pression in negative cases was significantly lower than in thepositive cases (Fig. 3A). In contrast, the phospho-Akt level innegative cases was significantly higher (Fig. 3B). A signifi-cant inverse correlation between PTEN and phospho-Aktexpression was observed (Fig. 3C). The phospho-Bad level innegative cases was significantly higher (Fig. 3D). The ex-pression level of p27 did not differ between positive andnegative cases.

DISCUSSIONImmunohistochemical analysis showed that PTEN was ex-

pressed in normal endometrium and tumor stromal cells. Incontrast, PTEN-negative staining was seen in 37% of endome-trial carcinomas, and mixed pattern was seen in 16% of cases.As a result, loss or decrease of PTEN expression was seen inover 50% of endometrial carcinomas. In a previous study, themutations ofPTENwere observed in 50% of endometrial car-cinomas (4). These results support the involvement of PTEN inthe development and/or progression of endometrial carcinomain over 50% of cases.

The loss of PTEN expression is considered to reflect theloss of PTEN function induced by a variety of mechanismssuch as homozygous deletion, nonsense mutation with LOH,and promoter methylation (16). However, there are no reportsconcerning PTEN function in patients with endometrial car-cinoma. In the present study, we first examined the expres-sion of the molecules relating to the Akt pathway accordingto the status of PTEN function in clinical samples of endo-metrial carcinoma. The present study showed that Akt wassignificantly phosphorylated in tumor tissue with a loss ofPTEN expression, and that phospho-Akt expression was neg-atively correlated with PTEN expression. This finding sup-ports the basic evidence that Akt activation accompanied byPTEN inactivation is a key step in the development and/orprogression of carcinomas (3, 9).

Interestingly, Bad, a proapoptotic factor, was more phospho-rylated in PTEN-negative tumors, but not extremely so, suggestingthat Bad may be a target for Akt phosphorylation. Bad inactivationappears to be involved in part in endometrial carcinogenesis.

In a recent study (17), increased growth activity accompaniedby the down-regulation of p27 was observed in PTEN null ES cells.This effect could be dependent on phosphorylation and activationof Akt. The present study failed to find a relationship betweenPTEN and p27. The Akt pathway leading to p27 is not known indetail, although posttranscriptional regulation was shown to beinvolved in p27 expression (18). Additional studies are necessary toaddress the potential roles of p27 in endometrial carcinogenesis.

Mechanisms other than the loss of PTEN function may alsocontribute to the development and/or progression of endometrial

carcinoma, because the expression of PTEN was observed inone-half of endometrial carcinomas. The results of our investi-gation suggest that, in at least one-half of endometrial carcino-mas, the loss of PTEN function relates to carcinogenesis. Acti-vation of Akt caused by the loss of PTEN may be involved inthe mechanism of carcinogenesis by preventing apoptosis byBad inactivation in patients with endometrial carcinoma.

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2001;7:892-895. Clin Cancer Res   Yasunobu Kanamori, Junzo Kigawa, Hiroaki Itamochi, et al.   Phosphorylation in Endometrial CarcinomaCorrelation between Loss of PTEN Expression and Akt

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