Apoptosis 2004; 9: 797–805
C 2004 Kluwer Academic Publishers
Bcl-2 and CCND1/CDK4 expression levels predict
the cellular effects of mTOR inhibitors in human
ovarian carcinoma
D. Aguirre∗ , P. Boya∗ , D. Bellet, S. Faivre, F. Troalen, J. Benard, P. Saulnier, S. Hopkins-Donaldson,
U. Zangemeister-Wittke, G. Kroemer∗ and E. Raymond∗
Department of Clinical Biology, Department of Medicine (D. Aguirre, D. Bellet, S. Faivre, F. Troalen, J. Benard, P.
Saulnier, E. Raymond), and CNRS-UMR 8125 (P. Boya, G. Kroemer), Institute Gustave-Roussy, Villejuif, Tumor
Immunology Laboratory FRE2443 CNRS, University René Descartes Paris 5; France; Division of Medical Oncology,
University Hospital, Zurich, Switzerland (S. Hopkins-Donaldson, U. Zangemeister-Wittke)
Molecular markers enabling the prediction of sensitivity/resistance to rapamycin may facilitate further clinical development of rapamycin and its derivatives as anticancer agents. In this study, several human ovarian
cancer cell lines (IGROV1, OVCAR-3, A2780, SK-OV-3)
were evaluated for susceptibility to rapamycin-mediated
growth inhibition. The differential expression profiles of
genes coding for proteins known to be involved in the
mTOR signaling pathway, cell cycle control and apoptosis were studied before and after drug exposure by
RT-PCR. In cells exposed to rapamycin, we observed a
dose-dependent downregulation of CCND1 (cyclin D1)
and CDK4 gene expression and late G1 cell cycle arrest. Among these cell lines, SK-OV-3 cells resistant to
both rapamycin and RAD001 were the sole to show the
expression of the anti-apoptotic gene Bcl-2. Bcl-2/bclxLspecific antisense oligonucleotides restored the sensitivity of SK-OV-3 cells to apoptosis induction by rapamycin
and RAD001. These results indicate that baseline Bcl2 expression and therapy-induced downexpression of
CCND1 and CDK4 may be regarded as molecular markers
enabling the prediction and follow-up of the cellular effects on cell cycle and apoptosis induction of rapamycin
in ovarian cancer. Furthermore, strategies to down regulate Bcl-2 in ovarian cancer may prove useful in combination with rapamycin or RAD001 for ovarian cancer.
Keywords: apoptosis; cell cycle; gene expression; RAD001;
rapamycin; surrogate marker.
Abbreviations: 4EBP, eukaryotic initiation factor 4E binding protein; CDK, cyclin-dependent kinase; EGFR, epithelial
growth factor receptor; IGF: insulin growth factor; MDR-1, multidrug resistance; PI, propidium iodide; PI3K, phosphoinositol-
∗ They equally contributed to this work and should be considered
as joint first and senior authors, respectively.
Correspondence to: Guido Kroemer, M.D., Ph.D. Tumor
Immunology Laboratory, Institut Gustave Roussy, 39, Rue Camille
Desmoulins, 94805 Villejuif, Cedex, France. Tel.: 33 1 4211 6046;
Fax: 33 1 4211 5244; e-mail: Kroemer@igr.fr
3 kinases, PTEN, phosphatase and tensin homologue; S6K
or p70S6K, ribosomal p70 S6 kinase; TBBP, tata box binding protein; mTOR, mammalian target of rapamycin; VEGF,
vascular endothelial growth factor.
Introduction
Genetic alterations that disrupt the regulation of proliferation, cell death, and senescence are hallmarks of malignant transformation of ovarian epithelial cells.1 Ovarian
cancer cells produce and/or respond to various growth factors such as EGF, TGFβ, IGF, PDGF, and stem cell growth
factor.1 Interactions between growth factors and their specific receptors can trigger downstream signaling pathways
involving PI3K, MAPK, AKT and the mammalian target of rapamycin (mTOR).1,2 In cancer cells, AKT and
mTOR act as “master switch” proteins which simultaneously modulate metabolism, the cell cycle, and apoptosis.3
In endometrioid ovarian cancer cells, AKT-dependent cellular proliferation is repressed by PTEN, a phosphatase
that antagonizes the kinase activity of PI3K.1,2 Activation of the AKT/mTOR pathway in ovarian cancers modulates the response to apoptotic stress induced by cytotoxic agents such as cisplatin and paclitaxel.4,5 Rapamycin
(Sirolimus, RapamuneTM ), a macrolide produced by Streptomyces hygroscopicus, binds FKBP-12 (FK506 binding protein), thereby creating a molecular complex that specifically inhibits the function of mTOR.6 The anticancer
effects of rapamycin have stimulated the development
of derivatives such as RAD001 (SDZ RAD, Everolimus,
CerticanTM ), which are being included in clinical trials
and show early evidence of antitumor activity either as a
single agent or in combination with chemotherapy.6 Inhibition of mTOR by rapamycin or RAD001 leads to
the downregulation of G1 cyclin/Cdk complexes, thereby
blocking the cell cycle in the late G1/S phase.7 Recent studies have revealed that rapamycin inhibits the
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oncogenic transformation of human cells induced by either PI3K or AKT and the loss of PTEN function.8
Furthermore, rapamycin antagonizes tumor growth induced by loss of the PI3K antagonist PTEN. PTEN+/−
mice spontaneously develop neoplasia associated with loss
of the normal PTEN allele and increased activation of
AKT/PKB and the ribosomal p70 S6 kinase (S6K), a
known downstream target of mTOR.8 In vivo treatment of
such mice with the rapamycin analog CCI-779 normalizes S6K activity and reduces neoplastic proliferation.9
Similarly, PTEN-deficient human tumors are more sensitive to CCI-779-mediated growth inhibition than PTENexpressing cells. The mechanisms through which mTOR
inhibits apoptosis remain to be elucidated.10–12 As a possibility, its downstream target S6K, which can bind to
mitochondrial membranes, may phosphorylate the proapoptotic molecule BAD on serine 136, a reaction that
disrupts BAD’s binding to the mitochondrial death inhibitors bcl-xL and bcl-2 and thus inactivates BAD.13,14
Rapamycin and its derivatives may thus suppress cancer cell proliferation due to cell cycle inhibition and/or
due to an increase in apoptosis, depending on the type of
cancer cell. The identification of molecular determinants
of cell cycle and apoptotic effects of rapamycin should ultimately facilitate the clinical development of rapamycin
derivatives. This study was designed to identify molecular markers associated with cell cycle and apoptotic effects by quantifying the expression of genes modulated
by rapamycin. The selection of putative marker genes was
based on previous studies identifying essential genes involved in the mTOR signaling pathway, cell cycle control,
and apoptosis in Saccharomyces cerevisiae as well as in mammalian cells.15,16 In this study, we show that expression
of Cyclin D1/CDK4 can be used to monitor the cell cycle
effects of rapamycin in ovarian cancer. Moreover, we show
that Bcl-2 expression is a marker of resistance to apoptosis
induced by rapamycin.
Materials and methods
Cell culture and cytotoxic assays
The ovarian cancer cell lines OVCAR-3, SK-OV-3
(American Type Culture Collection), IGROV1 (kindly
provided by Jean Benard, Institute Gustave-Roussy,
Villejuif, France), and A2780 (kindly provided by Dr.
Larsen, Institute Gustave Roussy, Villejuif, France) were
maintained in RPMI 1640 medium (GIBCO/BRL) supplemented with 10% FCS (BioWhittaker) and 2 mM
L-glutamine (GIBCO/BRL) at 37◦ C in a humidified atmosphere of 5% CO2 . Cells were determined to be free of
mycoplasma infection, and cell viability was assessed by
means of a tetrazolium salt-based colorimetric assay (WST
from Roche Diagnostics GmbH, Mannheim, Germany).
798 Apoptosis · Vol 9 · No 6 · 2004
Cells (200 µl per microtiter well) were treated with increasing concentrations of rapamycin (Sigma, France) or
RAD001 (Novartis Institutes for BioMedical Research,
Basel, Switzerland) 6, 24, 48 or 72 h. The assay was terminated by the addition of the WST reagent (20 µl/well)
and incubation of the cells at 37◦ C for 30′ for SK-OV3 and 1 h for the other cell lines. Percent cell survival
was defined as the relative absorbance of control versus
treated cells. All assays were performed in quadruplicate
and repeated at least twice.
Real-time PCR (TaqMan)
Total RNA was extracted from cell lines by RNA-Plus
according to the practical recommendations of the manufacturer (Q-Biogene, Illkirch, France). Total RNA concentration was determined at 260 nm (Gene-Quant,
Pharmacia) and its quality was assessed by conventional
gel electrophoresis. Standard curves were constructed with
5 serial dilutions of total RNA. We used the universal human reference RNA (20, 4, 0.8, 0.16 and 0.032 ng per
tube) from Stratagene (La Jolla, CA, USA) as an internal standard. Real-time PCR was carried out with the
Applied Biosystems PCR system. 1 µg of RNA was
reverse-transcribed under standard conditions (1× RTPCR buffer, 2 mM dNTP, 5 mM MgCl2 , 20 U RNase
inhibitor, 50 U reverse transcriptase, 5 µM random hexamers). The samples were incubated at 42◦ C for 45 min,
and reverse transcriptase was inactivated by heating at
100◦ C for 5 min and cooling at 4◦ C for 10 min. All PCR
reactions were performed using an ABI Prism 7700 Sequence Detection system (Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA). For each PCR run, we
used final concentrations of the master PCR mix (TaqMan
UMM buffer, 20 pmol of each primer, 10 pmol of probe).
10 µl of each appropriately diluted cDNA were added to
40 µl of the PCR master-mix. The thermal cycling conditions comprised an initial denaturation step at 95◦ C for 10
min, 40 cycles at 95◦ C for 15 s, and 60◦ C for 1 min. Each
PCR run included the five points of the standard curve, a
no-template control, the calibrator cDNA, and three samples of cDNAs from a specific cell line exposed to different
drug concentrations (10− 7 , 10− 6 and 10− 5 M). The target
gene mRNA copy value was obtained in 2 h with this assay
format. For each experimental sample, the amounts of the
targets and endogenous reference were determined from
the standard curve. Then the target amount was divided by
the endogenous reference amount to obtain a normalized
target value. The relative gene target expression level was
also normalized to a control without treatment for each
cell line (calibrator), or 1× sample. Each of the normalized target values was divided by the calibrator normalized target value to generate the final relative expression
levels. Primers and probes for the reference gene TBBP
Biomarkers for mTOR inhibitors in ovarian carcinoma
(TATA-box binding protein, a component of protein
complex TFIID) and target genes were chosen with
the assistance of the computer program Primer Express (Perkin-Elmer Applied Biosystems, Foster City, CA,
USA). Primers and probes were purchased from MWGBiotech AG. The nucleotide sequences of the oligonucleotide hybridization probes and primers are available
under request. Experiments were performed with duplicates for each data point. The following genes were analyzed: AKT2, BAD, BCL2, CASP9, CCNB1, CCND1,
CDK1, CDK4, CDKN1A, CDKN2A, EGFR, ERBB2,
FAS, IGF1, KRAS2, MDR1, mTOR, MYC, PIK3CA,
PTEN, RPLPO, TGFB1, TP53, VEGFA, and TBBP.
Antisense oligonucleotides and transfection
The chemoresistant line SK-OV-3 was transfected with
the Bcl-2/Bcl-xL antisense oligonucleotide 4625, a
second-generation phosphothioate oligonucleotide with
modifications at the 5′ and 3′ ends consisting of
methoxy-ethoxy residues at the 2′ -alpha-position of the
deoxyribose.17,18 This oligonucleotide has 100% complementarity with Bcl-2 and 3 mismatches with Bcl-xL.
As the scrambled control, we used 4626 (the same nucleotide composition, but in a random order). SK-OV-3
cells were plated at 75,000 cells/ml. On the next day,
oligonucleotides (200 nM) were delivered to cells in the
form of complexes with the transfection reagent Lipofectin
(InVitrogen-Life Technologies) as described previously.
Lipofectin was allowed to complex with oligonucleotides
in serum- and antibiotic-free medium before dilution and
addition to cells, which were exposed to rapamycin or
Rad 001 (10− 5 M or 10− 7 M) 18 h later. To confirm the
downregulation of Bcl-2, cells were harvested after 18 h
transfection evaluated by RT-PCR.
Western blot
Cells were lysed for 15 min in 50 mM HEPES, 150 mM
NaCl, 5 mM EDTA, 0.1% NP-40, supplemented with
protease inhibitor cocktail (Roche), 1 mM dithiothreitol
and 1 mM PMSF, and then centrifuged at 13,000 g for
10 min to remove debris. 40 µg of protein were loaded
onto a 12% SDS-PAGE, transferred on nitrocellulose and
blotted with mouse anti-human Bcl-2 (Ab 124, DAKO,
Denmark) and anti-GAPDH (Chemicon).
V-FITC and propidium iodide for 15 min at room temperature, followed by evaluation with a FACS Vantage
cytofluorometer (Becton Dickinson).
Results
Gene expression profile in ovarian cancer cells
Quantitative comparison of mRNA species revealed that a
panel of four different ovarian cancer cell lines (IGROV1,
SK-OV-3, OVCAR-3, and A2780) all expressed AKT2
and mTOR (Figure 1A), as well as mRNA species coding
for cell cycle regulatory protein required for G1/S transition (cyclin D1 and Cdk4), G2/M transition (CCNB1
and CDK1) and inhibition of cell cycle progression
(CDKN1A and CDKN2A) (Figure 1B). A2780 expressed
the lowest level of several mRNA species (e.g. PI3K,
PTEN, EGFR, CDKN2A), consistent with reports by
the National Cancer Institute using cDNA microarrays (genome-www.edu/nci60/). The expression of mRNA
coding BAD, c-myc and p53 was detected in all ovarian cancer cell lines (Figure 1C). Expression of mRNA
species coding for Bcl-2 and Bcl-xL proteins was found
only in SK-OV-3 cells, a result that was confirmed by immunoblot detection at the protein level (Figure 1C and D).
Antiproliferative effects of mTOR inhibitors
in ovarian cancer cells
Inhibition of cellular proliferation was evaluated using
several concentrations of rapamycin (chemical structures
shown in Figure 2A). In our study, rapamycin more readily inhibited the growth of IGROV1 than that of the
other three ovarian cancer cell lines, with SK-OV-3 cells
being the most resistant (Figure 2B). Attempts to determine the GI50 of RAD001 was not satisfactory due to
the poor solubility of this compound in media. Based on
these data, we focused on IGROV1 and SK-OV-3 and selected concentrations ranging 10− 7 –10− 5 at which subsequent experiments were performed. In our study, steady
state (baseline) expression levels of human homologues
of yeast mRNA species modulated by rapamycin were
found not to be predictive of the antiproliferative effects
of rapamycin in human ovarian cancer cells. Exposure to
rapamycin was shown to induce a downregulation of several genes involved in the the PI3 kinase and AKT pathway. Up regulation of caspase 9 and FAS was observed in
IGROV-1 which was not found in SK-OV-3 (Figure 2C).
Quantitation of apoptosis
For the assessment of phosphatidyl serine exposure and
viability, the Annexin V-FITC kit was used (Bender
Medsystems). After the indicated treatments, cells were
trypsinized and incubated in binding buffer with Annexin
Effects of mTOR inhibitors on cell cycle mRNA
species and cellular death
Recent studies have shown that the Cyclin D1 is essential
for mediating the proliferation signaled via the mTOR
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D. Aguirre et al.
Figure 1. Expression of mRNA species coding for key signaling molecules in ovarian cancer cells. RT-PCR analysis of genes coding for
the mTOR pathway (A), cell cycle modulators (B) and apoptosis (C) were analyzed in a panel of 4 human cancer cell lines. Quantification
was normalized using the housekeeping gene TBPP as control. Western blot using anti-Bcl-2 antibody showed that SK-OV-3 cells
express higher Bcl-2 levels than IGROV1 cells (D): 50 µg pf protein from IGROV1 (1), SK-OV-3 (2), and HeLa cells stably transfected
with a plasmid coding for human Bcl-2 (3) were analyzed using anti-GAPDH as loading control.
pathway, and that rapamycin can modulate cellular proliferation by inhibiting cyclin D1 expression. In our study,
IGROV1 and SK-OV-3 cells were incubated for 60 h in
the presence of 10− 6 and 10− 7 M rapamycin, fixed and
DNA content was analyzed by flow cytometry after DAPI
staining.
In ovarian cancer cells sensitive to rapamycin such as
IGROV1, rapamycin and its derivatives blocked cell cycle progression in G1/S (not shown). Furthermore, exposure to rapamycin induced a decrease in mRNA encoding Cyclin D1, CDK4, CDKN1A, CDKN2A, and
CCNB1 (Figure 2D), all genes involved in cell cycle control. Most notably, exposure of IGROV1 to
rapamycin (and RAD001 data not shown) was associated with concentration-dependent inhibition of the
expression of Cyclin D1 and CDK4 mRNA and cyclin D1 protein (data not shown). In contrast, levels
of CDK1 mRNA increased after rapamycin treatment
(Figure 2C). Cyclin D1 and CDK4 mRNA downregulation in IGROV1 was associated with increased expression of caspase 3 (data not shown) and increased cell
death, as indicated by apoptotic shrinkage (Figure 3A
and B).
In rapamycin-resistant SK-OV-3 cells, exposure to
mTOR inhibitors was also associated with a decrease
800 Apoptosis · Vol 9 · No 6 · 2004
in CCND1 and CDK4 expression, cell cycle blockage
in G1/S (Figure 3B and not shown), but no apoptosis
induction (Figure 4B). In contrast to that observed in
IGROV1, exposure to rapamycin was associated with a
decreased expression of CDK1 in SK-OV-3, OVCAR3 and A2780 preventing cells to undergo mitosis
(Figure 2C). To identify factors that mediated resistance to
rapamycin-induced apoptosis in SK-OV-3 cells, we investigated the expression of mRNA species coding for apoptosis regulators and compared it to that of rapamycinsensitive IGROV1 cells. At baseline, we observed that
SK-OV-3 cells expressed higher levels of mRNA coding
for BAD, CD95 and TGFβ than did IGROV1 cells. Conversely, expression of mRNA coding for caspase 9 was
lower in SK-OV-3 than in IGROV1 (Figure 1C and data
not shown). In our panel, SK-OV-3 was the sole ovarian cancer cell line in our panel that expressed mRNA
coding for Bcl-2 (Figure 1C and D). However, since
the level of Bcl-2 mRNA species was low in SK-OV3, we confirmed by western blot that, unlike IGROV1,
SK-OV-3 expressed the bcl-2 protein. Expression of
bcl-xL, another bcl-2 family member, was similar in
SK-OV-3 and IGROV1. Based on these data, we speculated that Bcl-2 could prevent apoptosis induced by rapamycin.
Biomarkers for mTOR inhibitors in ovarian carcinoma
Figure 3. Death of ovarian cancer cells after exposure to mTOR inhibitors and/or Bcl-2/Bcl-xL antisense oligonucleotides. (A) Flow
cytometry analysis of IGROV1 cells treated with 10− 5 M rapamycin and RAD001 for 15 h showed increased cell death compared to
control (Co). Conversely, no significantly increased cell death was shown in SK-OV-3 cells exposed to mTOR inhibitors. Thus, SK-OV-3
cells were pretreated with the oligonucleotides antisense oligo4625 and oligo4626 for 16 h, incubated with rapamycin for 16 h and
analyzed by flow cytometry. (B) Quantification of cell death showed that pretreatment of SK-OV-3 with anti bcl-2 oligo4625 (but not
scramble oligo4626) increased cellular death induced by rapamycin and RAD001.
Downregulation of Bcl-2 restores sensitivity
to mTOR inhibitors
To examine the role of bcl-2 in cellular death induced by rapamycin and RAD001, we exposed SK-OV-3
cells to the Bcl-2 anti-sense phosphorothioate olignonucleotide oligo4625 or a scrambled anti-sense oligonucleotide control (oligo4626). Down modulation of bcl2 with oligo4625 consistently increased the apoptotic
demise of SK-OV-3 cells in response to rapamycin and
RAD001, indicating a synergistic death-inducing effect
between mTOR inhibition and Bcl-2 down-regulation. In
contrast, treatment with mTOR inhibitors or oligo4625
alone had no apoptotic effects on SK-OV-3 cells at the
concentrations used. These results were obtained using
two different readouts, namely by measuring the apoptotic shrinkage of cells by cytofluorometry (Figure 3B)
or by determining apoptotic phosphatidylserine exposure
(Annexin V staining) on the plasma membrane surface
(Figure 4A and B), detectable either by cytofluorometry (Figure 4A) or fluorescence microscopy (Figure 4B).
Cell death triggered by the combination of oligo4625
with mTOR inhibitors was clearly apoptotic, as indicated by pronounced chromatin condensation (Figure 4B).
The Bcl-2/Bcl-xL anti-sense oligonucleotide synergized
with rapamycin and RAD001 in terms of induction of
apoptosis (Annexin V+ /PI− ) and secondary necrosis
(PI+ ) (Figure 4C).
Discussion
In the nineteen-eighties, screening performed on the 60human-tumor-cell-line panel of the NCI revealed that raApoptosis · Vol 9 · No 6 · 2004
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D. Aguirre et al.
Figure 2. Effects of rapamycin on cellular proliferation and gene expression ovarian cancer cells. Chemical formula of rapamycin
(A). Antiproliferative effects corresponding to 50% growth inhibition (GI50 ) of rapamycin in our panel of ovarian cancer cell lines were
determined using the MTT assay (B). Semi-quantitative analysis of gene expression in ovarian cancer cells after 48 h exposure to
10− 6 M rapamycin (C). Quantitative RT-PCR analysis of cell cycle genes in IGROV-1 and SK-OV-3 showed concentration-dependent
downexpression of several genes involved in cell cycle progression, such as CCND1 and CDK4 (D).
Figure 4. Apoptosis induction in SK-OV-3 cells exposed to bcl-2 antisense oligonucleotides and mTOR inhibitors. (A). SK-OV-3 cells
were transfected with the oligonucleotides 4625 and 4626 for 16 h and treated with RAPA 10− 7 M for a further 16 h, stained with Annexin
V-FITC conjugate and PI and analyzed by flow cytometry. (B) Cells treated as in A were observed under a fluorescence microscope. (C)
Cells were treated with the antisense oligonucleotides as in A and incubated for 16 h with 10− 7 and 10− 5 with RAPA and stained with
Annexin PI; apoptotic cells (Annexinv+ /PI− ) and dead cells (PI+ ) were determined by flow cytometry.
802 Apoptosis · Vol 9 · No 6 · 2004
Biomarkers for mTOR inhibitors in ovarian carcinoma
pamycin and its derivatives exerted an original spectrum
of antiproliferative activity, presumably via mechanisms
that differed from those of other anticancer drugs.6 Recently, several rapamycin derivatives including CCI-779
and RAD001 have entered clinical trials, either as single
agents or in combination with additional chemotherapeutic drugs.6 Surprisingly, while rapamycin derivatives were
expected only to slow down tumor progression by interfering with the mTOR-controlled metabolic pathway and
protein synthesis, some evidence, including early tumor
regression, suggested that mTOR-inhibitors were also capable of triggering cancer cell death. The precise molecular mechanisms by which mTOR inhibitors are capable of
inducing cancer cell death are elusive.11,13,14,17–19 Recent
reports have indicated that phosphorylation of mTOR
by AKT can be obtained through the activation of upstream molecules including PI3K (resulting from mutated or deleted PTEN) and/or through the MAPK pathway. Indeed, rapamycin was found to exert more potent
antiproliferative effects in cells characterized by intrinsically phosphorylated (activated) AKT and loss of expression of PTEN (see 6 for review). More recently, mTOR was
also discovered to serve as an important apoptosis modulator in human cells.13,14 However, the downstream targets
connecting mTOR inhibition to cellular demise remain
poorly understood. Two recent studies have suggested that
members of the Bcl-2 family could be downstream mediators of IGF-I-stimulated PI-3 kinase-dependent survival
of cells.14,17,19 Furthermore, several growth factors that
activate the PI-3 kinase and S6K pathways were recently
shown to increase expression of Bcl-2, thereby promoting cell survival in myeloid progenitor cells.19 The role of
Bcl-2 in resistance to anticancer agents has been investigated extensively in in vitro systems, animal models and
clinical studies. In studies with patients suffering from
ovarian carcinoma, high levels and/or aberrant patterns of
Bcl-2 expression have been correlated with resistance to
commonly used anticancer agents.20
In this study, we found, within a panel of 4 ovarian
cancer cell lines, that the IGROV1 cell line derived from a
polymorphous endometrioid tumor was most sensitive to
rapamycin. Attempts to identify molecular markers that
predict sensitivity to rapamycin were based on previous
studies performed in Saccharomyces cereviciae.15,16 We first
evaluated the human homologues of yeast genes were
associated with rapamycin sensitivity in yeast. However,
baseline expressions of mRNA species coding for those
selected signaling molecules involved in the mTOR
pathway and cell cycle control did not determine the rapamycin response in ovarian carcinoma cells. We observed
that under exposure to rapamycin, IGROV1 displayed
downregulation of mRNA coding for Cyclin D1 and
CDK4, an alteration that could lead to cell cycle arrest in
the late G1 phase. Therefore, downregulation of Cyclin
D1 was considered as a potential biomarker to evaluate the
cell cycle effects of rapamycin and rapamycin derivatives
at the cellular level in clinical trials. However, downregulation of Cyclin D1 was observed in both sensitive and resistant cells and was not predictive of rapamycin-induced
antiproliferative effects. Therefore, other mechanisms
downstream from Cyclin D1/CDK4 downregulation
were suspected to play a role in cytotoxicity induced
by rapamycin. CDK1/cyclin B kinase acts as a universal M-phase promoting factor and might also play
a role in cellular apoptosis.13 Interestingly, exposure
to rapamycin was associated to an increased CDK1
expression in IGROV1 contrasting with CDK1 downexpression in other less sensitive ovarian cancer cell
lines. This suggested that ovarian cancer cells enable to
maintain or increase CDK1 expression under exposure
to rapamycin would have reduced cell proliferation.
Intriguingly, CDK1 overexpression was associated with
FAS overexpression in IGROV1. Although specific
molecular interactions linking mTOR, Cyclin B1/cdk1,
and Fas ligand in cancer cell are not fully determined,
authors have recently found that activation-induced Fas
ligand expression appears to be mediated by the Cyclin
B1/cdk1 complex.21 Taken together; our data suggest
that Cyclin D1/CDK4 downregulation together with
CDK1 overexpression may represent a molecular pattern
associated with sensitivity to rapamycin.
Subsequently, we focused on two cell lines representative of the highest and lowest resistance to rapamycin cell
lines in our panel. We found that exposure of IGROV1
cells to rapamycin and RAD001 was associated with substantial cellular apoptosis. Conversely, SK-OV-3 cells were
only marginally sensitive to rapamycin and appeared incapable of undergoing apoptosis. In both SK-OV-3 and
IGROV1, exposure to rapamycin and RAD001 was associated with a decrease in the expression of Cyclin D1 and
CDK4, as well as G1 arrest. This confirmed that decreased
expression of Cyclin D1 resulting from pharmacological
mTOR inhibition did not necessarily indicate apoptotic
effects in our cancer cells. Thus, we sought parameters
other than cell cycle gene modulation that might play a
role in sensitivity/resistance to rapamycin. In this study,
we found that mRNA species of genes involved in apoptosis were expressed at different levels in our ovarian cancer
cell lines. However, Bcl-2 was the sole gene to be expressed (although at a relatively low level) in SK-OV-3
cells and not in other cancer cell lines. Conversely, expression of bcl-xL was found in both SK-OV-3 and IGROV1.
To determine whether Bcl-2 could play a role in preventing SK-OV-3 cells from undergoing apoptosis, we used
anti-Bcl-2 oligonucleotides designed to interact with expressed Bcl-2 mRNA species. We found that sensitivity to
rapamycin was restored by prior exposure to oligo-4625,
which targets bcl-2/bcl-xL mRNA. This was associated
with an increase in SK-OV-3 cells undergoing apoptosis.
As expected, the use of anti-Bcl-2 oligonucleotides had
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no effect in IGROV1. Therefore, our data suggest that
bcl-2 plays a pivotal role in preventing apoptosis induction by rapamycin and derivatives in some ovarian cancers.
Recent data also showed that in mice bearing transgenes
encoding both AKT and Bcl-2, prostate intraepithelial
neoplastic cells remain sensitive to RAD001-induced inhibition of proliferation but are resistant to apoptosis.22
In those transgenic mice as well as in our human ovarian cancer cells, apoptosis induction is mediated through
caspase 3 suggesting a mechanism that requires an intact
mitochondrial apoptotic pathway. Taken together, those
data suggest that mTOR inhibition may be less effective
in human cancers characterized by Bcl-2 overexpression,
a situation eventually associated with the loss of PTEN
in cancer cells. In that case, apoptosis induction by rapamycin derivatives could be restored using combinations
with Bcl-2 inhibitors. Those results will be used in future
clinical trials to better characterize tumor types likely to
respond to mTOR inhibitors.23
Conclusion
In summary, our data indicate that Cyclin D1/CDK4 and
Bcl-2 may serve as surrogate markers to monitor sensitivity and to detect resistance to rapamycin and its derivative
RAD001. Attempts will be made to use these surrogate
molecular markers to monitor the biological activity doses
of rapamycin derivatives in clinical trials, for instance by
performing RT-PCRs on cancer cells isolated from blood,
from ascites, or from solid tumors. Moreover, our data provide a rationale for exploring the combination of mTORand Bcl-2-targeted drugs in the treatment of ovarian carcinoma.
Acknowledgments
We thank Isabelle Pouillon, Nelly Motté and Jean Louis
Bobot for their excellent technical assistance with cell
cultures and quality control of mRNA analysis, and Heidi
Lane, Novartis Institutes for BioMedical Research, for her
critical reading of the manuscript. This work has been
supported by a special grant from the Ligue contre le
Cancer (to G.K.), as well as by grants from the European
Commission (QLK3-CT-2002-01956 to G.K. and MCFI2000-00943 to P.B.).
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