Abstract

Meningiomas are generally benign neoplasias that arise from the meninges and comprise 20% ofall intracranial tumors. These tumors frequently express the progesterone receptor (PR) andmeningioma growth is affected by treatment with antiprogestins as shown by in vitro and clinical stud-ies. In addition, the PR is associated with recurrence and survival rate. Progesterone could also be im-portant in the regulation of apoptosis since a negative correlation exists between PR and bcl-2. Bcl-2 isan anti-apoptotic protein that is a prognostic factor in meningiomas. The PR exists in at least twoisoforms, PR-A and PR-B each of which are likely to have different biological functions depending oncell and promoter context.

This study was conducted to investigate the expression of other apoptotic proteins than bcl-2, andtheir relationship with the PR. Thus far, nothing is known about the expression of the anti-apoptoticprotein bcl-xl and the pro-apoptotic protein bak in human meningiomas and this issue was addressed inthe present study by using immunoblot analysis. Bcl-xl expression was found in 87/99 meningiomas andPR negative meningiomas expressed significantly less bcl-xl protein. A positive correlation was foundbetween bcl-xl and PR-B protein expression (rs =0.34, P<0.02). Bak was expressed in 52/60meningiomas and appeared to be unrelated to bak and PR expression levels. Bcl-xl and bak were signifi-cantly associated (rs=0.42, P<0.001).

In conclusion, both bcl-xl and bak are variably expressed in human meningiomas. PR ex-pression might be involved in regulating the apoptotic balance in meningioma, since bcl-xl was associ-ated with PR-B, and this may have some biological and clinical significance.

65

Chapter 5Expression of progesterone receptor, bcl-xl and bak

proteins in human meningiomas

F.M. Verheijen, M. de Man, A.J. Schellekens, M. Sprong, J.H.H. Thijssen,M.A. Blankenstein

Department of Endocrinology, University Medical Center Utrecht, The Netherlands

Submitted for publication

66

Chapter 5

Introduction

Meningiomas arise from arachnoidalcells present in the meninges, the mem-branes covering brain and spinal cord, andcomprise as many as 20% of all intra-cranial tumors 1. They are generally be-nign neoplasms that frequently expressthe progesterone receptor (PR), despitethe fact that estrogen receptor expressionis rarely found by ligand binding assay(LBA) 2. The PR belongs to the super-family of ligand activated transcriptionfactors. Clinical studies have shown thatPR expression might be clinically impor-tant since the PR is a marker of recurrenceand survival rate of meningioma dis-ease 3-5. The exact biological function of PRexpression in meningiomas is unknown.

The PR exists as at least two differentisoforms PR-A and PR-B. Both isoformshave distinct transcriptional capacitiesand may exert different biological func-tions 6,7. Meningiomas express both PRisoforms in variable amounts, however,PR-A expression is higher in tumors withhigh total PR expression 8. Since PR isthought to be involved in the proliferationof meningiomas, antiprogestins have beentested for their anti-tumor activity in sev-eral in vitro and clinical studies 9.

In breast cancer, steroid hormone re-ceptors do not only play a role in prolifera-tion but also in cell death or apoptosis. Theprocess of apoptosis is controlled by exter-nal signals in combination with an autono-mous genetic program. Hormone-de-pendency has been described of membersof the Bcl-2 proto-oncogene family in-volved in apoptosis 10. Expression of one of

these proteins, bcl-2 itself, is one of the ma-jor anti-apoptotic proteins and regulatedby estrogen and progesterone 10. Throughthe anti-apoptotic function of bcl-2, it is in-volved in cancer chemotherapy-resistanceand associated with prognosis, recurrenceand sensitivity for adjuvant therapy of sev-eral types of tumors 11.

In meningiomas, bcl-2 expression hasalso been demonstrated. In contrast tobreast cancer, however, meningiomasshowed a negative association betweenbcl-2 and total PR expression levels. Thepro-apoptotic counterpart of bcl-2, bax, isalso expressed in meningiomas. In gen-eral, the ratio bcl-2 to bax expression lev-els is thought to determine apoptoticsensitivity of cells 12. Several meningiomasshow a high ratio bax to bcl-2, indicatingthat at least other anti apoptotic proteinsare involved in maintaining meningiomagrowth.

Knowledge of expression levels ofother members of the bcl-2 proto-onco-gene family in meningiomas is limited. An-other major anti-apoptotic protein besidesbcl-2 is bcl-xl. Bcl-xl prevents bax inducedapoptosis and is described in other hor-mone dependent malignancies, likeglioma and neuroblastoma 13-15. Expres-sion of bcl-xl seems to be hormonally regu-lated by sex steroids, as was revealed bybreast cancer studies 16-19. The expressionof bcl-xl has been reported to be involvedin tumor progression. In addition, bcl-xl

might serve as therapeutic target inantisense anticancer therapy. Therefore,knowledge of the expression level of this

67

Bcl-xl and bak expression in meningiomas

protein in meningiomas is of clinical im-portance.

Another protein from the bcl-2 familyis the pro-apoptotic protein bak. Bak ex-pression has been reported in normal andmalignant breast tissue and other malig-

nancies 20,21, however, thus far, nothing isknown about the expression in menin-giomas. The hormone dependency of bakexpression in breast cancer seems less

than was found for bcl-2 and bcl-xl. Al-

though one study reported that bak ex-

pression was decreased in ER-α negativebreast cancers 21.

Total PR expression in meningiomasis associated with recurrence and survivalrate, but the exact function of this receptorin these tumors is unknown. PR might beinvolved in the regulation of the apoptoticprocess, illustrated by the negative associ-ation between total PR and bcl-2. Thisstudy was conducted to determine the ex-pression of other apoptotic proteins, bcl-xl

and bak, and their relation with PR in hu-man meningiomas.

Patients and methods

Tissues

Human breast cancer or meningiomatissue was placed on ice immediately afterremoval from the patient. Representativespecimens were frozen at -80°C until theywere used for cytosol preparation.

Cytosol preparation

The tissue was chilled in liquid nitro-gen, pulverized with a micro dismem-brator (Braun, Melsungen, FRG) andextracted with 10 mM phosphate buffercontaining 1.5 mM EDTA, 3 mM sodiumazide, 10 mM 1-monothioglycerol and 10%(v/v) glycerol, at pH 7.5. The resulting ho-mogenate was centrifuged at 0-4°C for 30min at 100.000 × g to yield a clear cytosol.The protein content of cytosols was esti-mated with the method of Bradford usingreagents from BioRad (Richmond, CA,USA) and human serum albumin (Kabi

Diagnostica, Stockholm, Sweden) as a

standard 22. The PR was measured imme-

diately after cytosol preparation. Aliquots

for assessment of bcl-xl and bak werestored at -80°C.

Receptor assay

Meningiomas. PR levels were mea-

sured by a ligand-binding assay (LBA) andscatchard plot analysis, according to the

guidelines of the European Organizationfor Research and Treatment of Cancer(EORTC), Breast Cancer Cooperative

Group as described previously 23,24. Thelower cut-off level for PR positivity was set

to be 10 fmol/mg cytosol protein. The be-

tween-assay variability for thess assays

was: PR (n=31): CV=11.3% at 333 fmol/mgprotein, and protein (n=31): 5.8% at3.4 mg/ml.

68

Chapter 5

Breast cancer. For breast cancercytosol samples, total PR was assayed induplicate by enzyme immunoassay ac-cording to the instructions of the manufac-turer (Abbott Laboratories, Chicago, IL,USA). Day-to-day performance of the kitswas monitored by assaying control sam-ples provided with the kits and amyometrium control cytosol prepared atour laboratory. For the kit controls, thefound/target ratio for PR was 0.99 � 0.13(n=8; CV=12.7%). The myometriumin-house control specimen read 717 � 127(n=8; CV=17.7%) fmol/ml in the PR assay.Samples were considered to be receptorpositive when the assay result exceeded 20fmol/mg protein.

Protein Electrophoresis andImmunoblot analysis

Tumor cytosol proteins (50 µg/lane)were separated by electrophoresisthrough 12.5% polyacrylamide (30%Acrylamide/Bis Solution, 37.5:1) resolvinggel and a 3.9% stacking gel both contain-ing 0.10% SDS, using a Mini Protean II ap-paratus (Bio-Rad, Richmond, CA, USA).Proteins were transferred to Immobilon-Pmembrane (Millipore, GB, 125V, 1hr, 4°C)in 25 mM tris, 192 mM glycine, 20% (v/v)methanol as described by Towbin et al. 25.After transfer, unbound sites remaining inthe membrane were blocked with 10%Protifar (Protifar; N.V.-Nutricia, TheNetherlands) in PBS buffer, (Phosphatebuffered saline, pH 7.4) and incubatedwith specific antibodies (AB) against bcl-xl

proteins and bak. Bcl-xl was detected us-ing specific anti-bcl-xl mouse AB at a finaldilution of 1:750 (Bcl-xl (Ab-2),Calbiochem) and bak with anti-bak rabbit

Ab at a final dilution of 1:1000 (Bak, SantaCruz Biotechnology). Blots were incu-bated with a horseradish peroxidase-con-jugated goat anti-mouse (Dako A/S) ordonkey anti-rabbit (Amersham) second-ary antibody at a final dilution of 1:2500 or1:10000, respectively. Visualization usingECL reagents (Amersham) was accordinginstructions of the manufacturer. Blotswere exposed to an autoradiograph. Mo-lecular weight was determined withbiotinylated SDS-PAGE standards broadmolecular weight marker and avidin HRP(Bio-Rad, USA). Three controls ofmyometrium tissue, positive for bcl-xl andbak, were included on each gel containing5 samples.

Band intensities were measureddensitometrically (Sharp JX330, Japan).The linear range of detection of bcl-xl onimmunoblot was established by a standardcurve made using increasing concentra-tions of control cytosol and analyzed bydensitometry. A linear relationship be-tween bcl-xl and bak concentrations anddensitometry could be established. Multi-ple exposures were always made fromeach immunoblot of cytosol samples, andresults were used only from those that fellwithin the linear range. Densities of speci-mens were referenced to those of the con-trols. The between-assay variability ofbcl-xl and bak protein levels in cytosols ofhuman myometrium were 11.6 % (n=31)and 14.2% (n=73), respectively.

Statistical evaluation:

Meningioma and breast cancercytosol samples were ordered by increas-ing levels of PR and divided in four groups,

69

Bcl-xl and bak expression in meningiomas

resulting in quartiles, with total PR nega-tive, low, moderate, and high total PR ex-pression, respectively. Mann-Whitneynon-parametric tests were performed tocompare protein expression between thesubgroups. Spearman rank sum tests wereused to compare PR isoforms, bcl-2, bax,

bcl-xl and bak expression with each other,and Spearman’s correlation coefficient (rs)was used to evaluate the relationship be-tween the expression levels of the variousproteins. P values of <0.05 were consid-ered statistically significant.

Results

Progesterone receptor (PR)expression in meningiomas

Expression levels of total PR inmeningiomas were determined with aligand-binding assay (LBA). In the totalPR positive tumors the PR isoforms, PR-Aand PR-B, were determined by immuno-blotting (example is shown in Figure 5-1).In 67% (66/99) of the meningiomas PR wasexpressed with an average expressionlevel of 174±23 fmol/mg protein (median:94 fmol/mg protein). In 66% of the total PRpositives overexpression of PR-A to PR-Bwas found, median ratio of PR-A to PR-Bwas 1,7. No differences in total PR and PRisoform expression levels in meningiomascould be found between genders.

Expression of apoptotic proteins inmeningioma

Up to now, nothing was known aboutthe expression levels of bcl-xl and bak pro-teins in human meningiomas, both pro-teins belong to the bcl-2 proto-oncogenefamily. Therefore, immunoblot analysiswas performed to analyze expression ofthese proteins in meningiomas. Represen-tative immunoblots are shown in Figure

5-2. The bcl-xl immunoblot revealed a dou-blet band at 32.5kDa and overall analysisshowed variable expression of bcl-xl in 87out of 99 meningiomas. Bak immunoblotanalysis showed also variable expressionof bak in 52 out of 60 meningiomas. Somecytosol samples showed an additional bakband that turned out to be a degradationproduct of bak full-length protein (data

70

Chapter 5

M1 M2

PR-A

PR-B

Figure 5-1. Example of immunoblot analysis ofthe progesterone receptor isoform expressionin meningioma cytosol. PR-B (116-120 kDa)appeared as multiple bands depending onphosphorylation. PR-A shows a single band(81 kDa) and just ahead of PR-A a 78 kDa PR likeprotein migrated as described by Grahamet al.8,35 Both isoforms were expressed in me-ningiomas at different levels.

not shown), therefore those samples werenot used for analysis. Bak expression wassignificantly lower in meningiomas fromfemale than from male patients, as shownin Figure 5-3 (P<0.01; 260% and 695%, re-spectively).

Previous studies have shown thatbcl-2 expression was negatively associatedwith total PR protein expression level,therefore progesterone could play a role inthe regulation of apoptosis in menin-gioma. In this study, therefore, the associ-ation of total PR with other apoptoticproteins was investigated. Figure 5-4shows the bcl-xl and bak expression levelsin groups with different expression levelsof total PR; PR negative, low, moder-ate and high. In total PR negative

71

Bcl-xl and bak expression in meningiomas

0

500

1000

Male Female

Bak expression (% of control)

*

Figure 5-3. Bak expression in meningiomacytosol samples from male (n=19) and female(n=37) patients. Results are expressed as means± sem. *P<0.01.

Meningiomas

Bcl-xl

Bak

C C C

C C C

M1 M2 M3 M4 M5

M6 M7 M8 M9 M10

Figure 5-2. Example of immunoblot analysis of bcl-xl and bak expression in meningioma cytosols. Bothbcl-xl and bak were variably expressed in meningioma cytosols. Meningioma samples were depicted asM1 to M10. Together with 5 samples, 3 control cytosols (C) were measured, as an internal standard.

meningiomas, bcl-xl expression levelswere significantly lower (P<0.01). In allother PR subgroups, bcl-xl and bak ex-pression were not significantly different.

Spearman rank correlation analysis

revealed no significant association be-

tween total PR and both bcl-xl and bak.This, however, could be masked by the dif-

ferential PR isoform expression. There-

fore, correlation analysis was performedwith PR-B and bcl-xl and bak expressionlevels. Figure 5-5 shows that PR-B and

bcl-xl were positively associated, rs=0.34

and P<0.02. Bak expression was not sig-

nificantly associated with PR-A or PR-B.

Inter apoptotic protein analysis

For a large number of meningiomas

used in this study, bcl-2 and bax expres-

sion levels were determined in a previous

study, therefore, multiple correlation

analysis could be performed between the

different apoptotic proteins. A significant

72

Chapter 5

0

100

200

300

400

500

600

800

Neg Low Mod High

PR concentration (fmol/mg protein)

Bak expression (% of control)

700

0

500

1500

1000

2000

Neg Low Mod HighPR concentration (fmol/mg protein)

Bcl-x expression (% of control)l

*

Figure 5-4. Means of bcl-xl and bak expression in meningioma cytosols expressed as a percentage ofcontrol, and divided in three subgroups with low, moderate and high PR expression. Bars and errorbars represent mean ± s.e.m., respectively. The left panel shows that PR negative meningiomas expresssignificantly less bcl-xl (P<0.01). Abbreviations: PR, progesterone receptor; *P<0.01.

association was found between bcl-xl andbak expression levels (rs=0.42; P<0.002).

Bcl-2 was not associated with bcl-xl or withbak expression levels.

Breast cancer cytosols

In breast cancer, bcl-xl is known tobe positively associated with the expres-sion of total PR. Since under our condi-

tions such a positive association couldnot be found in meningioma cytosol,breast cancer cytosols were analyzed as areference for our experimental conditions.Indeed western immunoblot and Spear-

man rank correlation analysis revealed apositive association between total PR andbcl-xl of rs=0.54 and P<0.01.

Discussion

The aim of this study was to elucidate

the involvement of the PR in the regula-

tion of proteins from the apoptotic cascade

in human meningiomas. Expression levels

of the apoptotic proteins bcl-xl and bak

were determined in meningioma cytosolssamples by immunoblotting. These pro-

tein values were then compared with ex-

pression levels of total PR and PRisoforms. This study showed that both

bcl-xl and bak were variably expressed in

meningioma cytosol samples. In addition,

bcl-xl was associated with PR-B and signif-

icantly lower in PR negative menin-

giomas. Bak expression seemed not to be

regulated by progesterone or to be associ-

ated with the PR.

The bcl-x gene is a bcl-2-related gene.

Due to alternative splicing, different

mRNA products are formed. A large bcl-x

form, bcl-xl, shows close similarity with

bcl-2. Bcl-xl has been reported as a potent

inhibitor of apoptotic cell death in various

cell types 26. It exhibits regulated expres-

sion during lymphoid development, and is

expressed in developing B cells as shown

by immunoblot analysis 27. Bcl-xl expres-

sion has also been reported in various

types of cancers; such as gynecological tu-

mors, like ovarian tumors, breast cancer

and in breast cancer cell lines 14 28,29; lym-

phomas 30; and in prostate cancer 31. Bcl-xl

expression levels have been reported to be

clinical important. Transfection of bcl-xl in

73

Bcl-xl and bak expression in meningiomas

PR-B (as percentage of total PR)

Bcl-x (as percentage of control)l

3000

2000

1000

00 20 40 60 80 100

Figure 5-5. Association between expressionlevels of PR-B and bcl-xl in meningiomas. Theexpression levels are positively associated(rs=0.34, P<0.02).

human ovarian carcinoma cells resulted inhighly resistance of the cells to chemo-therapeutic agents 29. In addition, promis-ing results were obtained in studies in

which cell proliferation of breast cancercells was inhibited by bcl-xl-bispecificantisense oligonucleotide treatment ofthese cells 32,33.

The other protein invloved inapoptosis and investigated in this studywas bak. Bak is a pro-apoptotic proteinand expressed in normal breast tissue, as

well as in breast cancer, malignant gliomacells, gastric ad colorectal cancer 20,21. Reg-ulation of bcl-xl and bak by sex steroidshas been described in several hormonally

regulated tissues. Marone et al. estab-

lished a negative correlation between PRand bcl-xl in neoplastic ovarian tissue 14. Inaddition, progestins increase the ratio ofbcl-xl to bcl-xs in a rat endometrial cell

line 17. Less is known about the hormonesensitivity of bak expression. Gompel et al.findings showed that estrogen and proges-terone did not affect bak expression lev-

els 10. In contrast, Leung et al. reported in-

hibition of a time dependent increaseof bak mRNA in MCF-7 cell line 34. In addi-tion, a decrease in bak expression has

been described in ER-α negativebreast cancers 21.

Previous investigations have showeda negative association between Bcl-2 and

total PR expression levels in meningiomacytosols. Treatment of meningioma tissuefragments with the progesterone agonistorg2058 altered the bcl-2 expression levels(data not shown) suggesting a direct effect

of progesterone on bcl-2 expression regu-

lation. This study showed that bak seemsnot associated with PR. In contrast, bcl-xl

seems positively associated with PR-B.

These findings suggest that bcl-2 expres-

sion is most likely regulated differentlythan bcl-xl or bak.

For comparative purposes, the rela-tion between bcl-xl and total PR as ana-lyzed in breast cancer cytosol samples. For

breast cancer, a positive correlation be-

tween PR and bcl-xl based on IHC data hasalready been described in literature.Breast cancer cytosol analysis revealed apositive association between bcl-xl and PR

in PR positive tumors. This result furthervalidates our experimental method andshowed that the hormonal regulation ofbcl-x gene seems tissue specific.

For bcl-2, bax and bcl-xl no differencebetween male and female patients could

be detected. Bak expression, however, wassignificantly lower in meningiomas of fe-

male patients. Involvement of sex steroidsin the origin of this observation is not verylikely. First, this study showed that PR

was not involved in bak expression regula-

tion. Second, the group of meningioma pa-tients in this study mainly consisted out ofman or post-menopausal woman, thus

with low estrogen levels. The biologicalimportance of lower bcl-xl levels in womanis not known.

In summary, estrogen and progestindependency has been reported for bcl-2,bcl-xl and bak in other tissues. Also, corre-lation between ER, PR and these apoptotic

74

Chapter 5

proteins has been found. In meningioma,only PR is abundantly expressed and onlynegatively associated with bcl-2 expres-sion levels. Bcl-xl was positively associ-

ated with PR-B and bak seemed notregulated by progesterone in menin-giomas. The hormone dependent regula-tion of bcl-2 seems distinct from the

regulation of that of Bcl-xl and bak. This isconfirmed by the absence of a correlationbetween bcl-2 with the latter two proteins.In contrast is the association between

bcl-xl and bak, indicating that they mayshare a common progesterone independ-ent regulation pathway in meningioma.

In conclusion, the apoptotic proteinsbcl-xl and bak are expressed in menin-giomas at variable amounts and this mightbe clinically important. Our hypothesis is

that the PR may regulate the balance be-

tween pro- and anti-apoptotic proteins bydifferentially regulating the anti-apoptoticproteins bcl-2 and bcl-xl.

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