Increased PTEN expression due to transcriptional activation of PPARc by
Lovastatin and Rosiglitazone
Rosemary E. Teresi1, Chung-Wai Shaiu2, Ching-Shih Chen2, V. Krishna Chatterjee3, Kristin A. Waite1and Charis Eng1,4,5*
1Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
2Division of Medicinal Chemistry and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA
3Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 1XZ, United Kingdom
4Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, OH, USA
5Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 1XZ, United Kingdom
Germline mutations in the tumor suppressor gene PTEN (protein
phosphatase and tensin homolog located on chromosome ten) pre-
dispose to heritable breast cancer. The transcription factor
PPARc has also been implicated as a tumor suppressor pertinent
to a range of neoplasias, including breast cancer. A putative
PPARc binding site in the PTEN promoter indicates that PPARc
may regulate PTEN expression. We show here that the PPARc
agonist Rosiglitazone, along with Lovastatin, induce PTEN in a
dose- and time-dependent manner. Lovastatin- or Rosiglitazone-
induced PTEN expression was accompanied by a decrease in phos-
phorylated-AKT and phosphorylated-MAPK and an increase in
G1 arrest. We demonstrate that the mechanism of Lovastatin- and
Rosiglitazone-associated PTEN expression was a result of an
increase in PTEN mRNA, suggesting that this increase was tran-
scriptionally-mediated. Compound-66, an inactive form of Rosigli-
tazone, which is incapable of activating PPARc, was unable to
elicit the same response as Rosiglitazone, signifying that the Rosi-
glitazone response is PPARc-mediated. To support this, we show,
using reporter assays including dominant-negative constructs of
PPARc, that both Lovastatin and Rosiglitazone specifically medi-
ate PPARc activation. Additionally, we demonstrated that cells
lacking PTEN or PPARc were unable to induce PTEN mediated
cellular events in the presence of Lovastatin or Rosiglitazone.
These data are the first to demonstrate that Lovastatin can signal
through PPARc and directly demonstrate that PPARc can upre-
gulate PTEN at the transcriptional level. Since PTEN is constitu-
tively active, our data indicates it may be worthwhile to examine
Rosiglitazone and Lovastatin stimulation as mechanisms to in-
crease PTEN expression for therapeutic and preventative strategies
including cancer, diabetes mellitus and cardiovascular disease.
' 2006 Wiley-Liss, Inc.
Key words: PTEN; PPARg; Breast cancer; transcriptional regulation
Breast cancer is the second leading natural cause of death in
women and is thought to affect over 13% of women in the United
States.1Germline mutations in PTEN (protein phosphatase and
tensin homolog located on chromosome ten), a tumor suppressor
gene, are associated with 85% of patients with the autosomal dom-
inant disorder Cowden syndrome (CS).2–4Patients with CS have a
25–50% risk of developing breast cancer.5,6CS patients have also
been reported to have mucocutaneous lesions, thyroid abnormal-
ities, fibrocystic disease, uterine leiomyoma and macrocephaly.7,8
Germline PTEN mutations are also associated with 65% of patients
with Bannayan-Riley-Ravalcaba Syndrome, which is character-
ized by macrocephaly, lipomatosis, hemangiomatosis and speck-
led penis.4,9Somatic alterations in PTEN, whether by genetic or
epigenetic mechanisms, play some role in the pathogenesis of a
broad range of solid tumors, such as carcinomas of the breast, thy-
roid, endometrium and colon.10
PTEN’s protein product, PTEN, is a dual specificity phosphatase
with both lipid and protein phosphatase activity.11Its lipid phosphatase
activity functions as a negative regulator of AKT phosphorylation.
PTEN dephosphorylates phosphatidylinositol-(3,4,5)-triphosphate (PIP3)
at the D3 position generating phosphatidylinositol-(4,5)-biphosphate
(PIP2), subsequently decreasing PIP3 levels. Since PIP3 is required
for AKT phosphorylation, active PTEN leads to a decrease in
P-AKT levels and, consequently, a decrease in AKT-mediated
proliferation pathways.12The protein phosphatase activity of
PTEN has been shown to inhibit the Shc/Grb2/Sos and mitogen-
activated protein kinase (MAPK) pathways. The dephosphorylation
of Shc by PTEN indirectly decreases the phosphorylated form of
MAPK levels, thus reducing MAPK’s activity.13By reducing the
cellular levels of both P-AKT and P-MAPK, PTEN downregulates
cell division and upregulates apoptosis.
Transcriptional regulation of PTEN is beginning to be elucidated,
however, there is still much to be understood. Several groups have
shown that PTEN transcription may be upregulated by Early growth
response-1 (Egr-1),14p5315and Sp1.16In contrast, NF-jB has re-
cently been shown to inhibit PTEN transcription by a yet undeter-
mined mechanism.17In 2001, 2 putative binding sites for the tran-
scription factor peroxisome proliferator-activated receptor-gamma
(PPARg) were identified about 10 kb upstream of the minimal
promoter region of PTEN.18However, the biological importance
of these sites and what role PPARg plays in PTEN transcription
has not been determined. Therefore it remains to be determined if
PPARg has a role in PTEN transcription, and if so, which regions
of the PTEN promoter are responsible. PPARg is a transcription
factor that regulates gene expression by binding to PPARg
response elements (PPRE) within a promoter of a target gene.
Until recently, the focus of PPARg research has been on its role in
regulating fatty acid metabolism. However, recent evidence indi-
cates that PPARg may also act as a tumor suppressor by mediating
the transcription of genes necessary for antiproliferation and pro-
differentiation, which ultimately can downregulate cellular growth
and upregulate apoptosis.17,18Increasing evidence has also shown
that PPARg activation can be anticarcinogenic, indicating the
treatment potential for PPARg agonists in several diseases.19,20
Furthermore, the addition of PPARg agonists to breast cancer cell
lines have been shown to cause them to differentiate.21Previously,
we have shown that somatic loss-of-function mutations in PPARg
play a role in colorectal carcinogenesis22and that PPARg is
frequently downregulated in follicular thyroid carcinomas.23
Additionally, PPARg has been shown to be underexpressed and
Grant sponsor: American Cancer Society; Grant number: RSG02-151-
01-CCE; Grant sponsor: Susan G. Komen Breast Cancer Research Founda-
tion; Grant number: BCTR-2000 462.
*Correspondence to: Genomic Medicine Institute, Cleveland Clinic
Lerner Research Institute, 9500 Euclid Ave NE-30, Cleveland, OH 44195,
USA. Fax: 1-216-636-0009 or 636-0566. E-mail: firstname.lastname@example.org
Received 28 September 2005; Accepted after revision 24 November
Published online 19 January 2006 in Wiley InterScience (www.
Abbreviations: CS, Cowden syndrome; Cmpd-66, compound-66; EtOH,
ethanol; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic
fibroblast; PIP3, phosphatidylinositol-(3,4,5)-triphosphate; PIP2, phospha-
tidylinositol-(4,5)-biphosphate; PTEN, protein phosphatase and tensin
homolog located on chromosome ten; Rosi, Rosiglitazone; NaBut, sodium
butyrate; TZD, thiazolidinediones.
Int. J. Cancer: 118, 2390–2398 (2006)
' 2006 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
correlates with clinical outcomes in breast cancer patients.24In
nude mouse and rat models, treatment of breast cancer cells with
PPARg agonists inhibits tumor progression within the breast
tissue.25,26There is also an overlap of solid tumor data where both
PPARg and PTEN have been implicated in its pathogenesis.27,28
Thus, in vivo and in vitro evidence is mounting to support the idea
of PPARg as a tumor suppressor.
On the basis of these data, we hypothesize that PPARg, acting
as a tumor suppressor in breast cancer, can upregulate the tran-
scription of PTEN and believe that it is warranted to fully investi-
gate PPARg-mediated transcription of PTEN at the biochemical
and molecular level. To investigate the effect of PPARg agonists
on PTEN expression, we examined the ability of 5 PPARg agonists
that have been or are routinely used in the clinical setting to induce
PTEN expression in the MCF-7 breast cancer cell line. Four of the
agonists, Ciglitazone, Pioglitazone, Rosiglitazone (Rosi) and Trogli-
tazone, are thiazolidinediones (TZD) and are used to treat type II
diabetes mellitus, because of their ability to directly activate PPARg.29,30
Currently, Lovastatin is used clinically for its statin activity of regu-
lating high cholesterol levels. While Lovastatin is generally be-
lieved to be a PPARg agonist, no group has analyzed its ability to
directly activate PPARg. We will show here, substantive data that
indicate that Lovastatin and Rosi induce PTEN protein via a PPARg-
Material and methods
Ciglitazone was obtained from BIOMOL Research Laboratories
Inc (Plymouth Meeting, PA). Rosiglitazone was a generous gift
from Dr. Lisa Yee (The Ohio State University). Both Pioglitazone
and Troglitazone were obtained from Cayman Chemical (Ann Arbor,
MI). Lovastatin and cyclohexamide (CHX) were purchased from
Sigma-Aldrich (St. Louis, MO). Antibodies were obtained from
Cascade Bioscience, Waltham, MA (PTEN 6H2.1), and Cell Sig-
naling (P-p44/42, P-AKT S473, AKT and Actin; Beverly, MA).
Cell culture media was obtained from Gibco-BRL (Rockville,
MD). M-PER Mammalian Protein Extraction Reagent was
obtained from Pierce Biotechnology Inc. (Rockford, IL). All other
reagents were purchased from standard commercial sources.
Cell culture and stimulation
The MCF-7 breast cancer cell line was maintained at 37?C with
5% CO2in DMEM containing 10% FBS and 100 units/ml each pen-
icillin and streptomycin. Both the mouse embryonic fibroblasts
(MEF)-WT and the MEF-PPARg null cell lines were maintained at
37?C with 5% CO2in DMEM containing 14% FBS and 100 units/ml
each penicillin and streptomycin. BT-549 cells were maintained at
37?C with 5% CO2in DMEM containing 10% FBS and insulin.
Cells were plated at 1.0 3 10824 hr prior to treatment. After 24 hr,
stimulates dissolved in EtOH, were added as indicated in figure
legends. The cells were incubated for an additional 48 hr or as
otherwise indicated in the figure legends. In experiments stimulated
with cyclohexamide (CHX), 10 lg/ml CHX was incubated with
MCF-7 cells for either 24 or 48 hr, as indicated. In experiments
stimulated with sodium butyrate (NaBut), 2.5 mM was incubated
with MCF-7 cells for 48 hr.
Synthesis of PPARc analogs
Synthesis of the following analogs were performed as previous
described29: DTG (5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-
2-ylmethoxy)-benzylidene]-2,4-thiazonlidine-dione), DCG (5-[4-
thiazonlidine-2,4-dione) and DPG (5-[4-(2-[5-ethyl-pyridin-2-yl]-
ethoxyl)-benzylidene]-thiazolidine-2,4-dione). The identity and purity
(>99%) of these TZD derivatives were determined by proton mag-
netic resonance, high-resolution mass spectrometry and elemental
MCF-7 cells were plated and stimulated as described above. At
the time of harvest, media was removed and the cells were washed
with PBS. Cells were then harvested into M-PER lysis buffer con-
taining PMSF (0.75 mg/ml), benzamidine hydrochloride (0.5 mg/ml),
leupeptin (2 lg/ml), aprotinin (2 lg/ml), pepstatin (2 lg/ml), b-
glycerophosphate (10 mM), NaOV (0.2 mM) and NaF (25 mM).
Cells were incubated at room temperature with lysis buffer for
1 min before harvesting. Samples were then centrifuged at 16,000g
for 10 min at 4?C to remove cellular debris. The resulting superna-
tant was stored at 280?C. Protein concentration was determined
using the bicotinic method31using BSA as a standard.
Proteins (30 lg) were prepared by the Laemelli method,32then
separated on a 10% SDS-PAGE gel and electrophoretically trans-
ferred onto nitrocellulose.33Equal protein loading between condi-
tions was confirmed by staining with Ponceau S solution. Nonspe-
cific binding was blocked by incubating the nitrocellulose blots
with 5% milk in TBS-T (100 mM Tris, pH, 1 M NaCL, 1% Tween-20)
for 1 hr at room temperature. Blots were then incubated with the
primary antibody (1:1000 in 3% BSA) for 2 hr at room tempera-
ture. Following the primary incubation the blots were washed with
TBS-T for 1 hr with frequent changes of buffer. Blots were then
incubated with the appropriate secondary antibody conjugated to
horseradish peroxidase (Promega; Madison, WI) (1:2500 dilution
in 5% milk) overnight at 4?C, and washed with TBS-T for 1 hr.
Protein bands were visualized using enhanced chemiluminescence
as described by the manufacturer (Amersham Pharmacia Corp;
Piscataway, NJ). The resultant films were then quantified using
NIH-Imager densitometry software.
RT-PCR and real-time PCR
MCF-7 cells were stimulated as described above. After stimula-
tion, cells were released by trypsin treatment and subsequently
washed 3 times with PBS through centrifugation. RNA was ex-
tracted from the cells following the Qiagen RNeasy Mini Protocol
and then converted to cDNA by Superscript II Reverse Transcrip-
tase (SSIIRT). The resultant cDNA was subjected to multiplex
PCR amplification using primers specific to exon 1 and exon 9 of
PTEN (50TCAAGAGGATGGATTCGACTT 30; 50TGAAGT-
ACAGCTTCACCTTAAA 30) and primers to b-actin (Quantum
RNA b-actin; Ambion INC; Austin, TX). Primers were allowed to
anneal at 58?C for 30 sec. The products from the PCR reactions
were run on a 1% agarose gel containing ethidum bromide and
visualized under a UV light. The real-time quantitative PCR and
analysis were carried out using the ABI 7700 Sequence Detector
System (ABI/Perkin Elmer, Foster City, CA) as previously de-
scribed.4PTEN exons 1 and 5 were chosen as targets for the real-
time quantitative PCR assay. The primers and probes were
designed by using PRIMER EXPRESS software (Applied Biosys-
tems). The PTEN exon 3 and exon 5 probes were labeled at the 50
ends with the reporter dye FAM and at the 30end with the
MGBNF quencher. GAPDH was used as an internal control. The
real-time quantitative multiplex PCR assay was optimized accord-
ing to the instructions in User Bulletin No.5. The thermal cycling
conditions were 50oC for 2 min and 95oC for 10 min, followed by
40 cycles of denaturing at 95oC for 15 sec and annealing and
extension at 60oC for 1 min.
Cell cycle assay
MCF-7, MEF-WT, MEF-PPARg null and BT-549 cells were
plated and stimulated as described above, after stimulation cells
were harvested through trypsinization. Cells (1.0 3 106) were then
resuspended in 70% EtOH and stored at 220?C until analysis.
Cells were stained with 1 lg/ml PI in PBS containing 0.1% Triton
X-100. Flow cytometry was performed on a Beckman-Coulter
elite flow cytometer using a 610 long pass filter for data collection.
INDUCTION OF PPAR?-MEDIATED TRANSCRIPTION OF PTEN BY LOVASTATIN AND ROSIGLITAZONE
Data were filtered, and cell cycle phases were quantified using the
Modfit program (Verity Software, Bowdoin, ME).34
Plasmids were cotransfected into 6-well cultures of MCF-7 cells
in serum-free media with DMRIE-C (Invitrogen). Each well was
cotransfected with 500 ng PPRETKLUC, 50 ng Renilla luciferase
control plasmid and 100 ng of receptor expression vector
(pcDNA3, WT PPARg1 or L468A/E471A PPARg1). Each plas-
mid has previously been described.35After 12 hr, the cultures
were washed and new media containing EtOH, 30 lM Rosi, 3 lM
Cmpd-66 or 3 lM Lovastatin was added for 48 hr. Cells were then
harvested using luciferase lysis buffer as described by the manu-
facturer (Promega). Samples were analyzed on a luminometer
using Renilla luciferase as an internal transfection control.
Lovastatin and Rosiglitazone (Rosi) induce PTEN protein
expression in a dose- and time-dependent manner
To determine PPARg’s role in regulating PTEN expression, we
examined 5 PPARg agonists’ ability to induce PTEN expression
in MCF-7 breast cancer cells. We observed that stimulation with
Ciglitazone, Pioglitazone and Troglitazone did not alter PTEN ex-
pression, despite a wide range of concentrations tested (0–100 lM),
but they did inhibit cyclin D1 in a dose-dependent manner as previ-
ously described (data not shown).36Prior studies, which tested
Rosi’s ability to induce PTEN in MCF-7 cells, studied its effect at
only 1 dose, 1 lM.18Studying Rosi’s effects on PTEN at 1 dose
limits one’s ability to detect a potentially more significant dose effect.
We, therefore, determined the optimal concentration for increased
PTEN expression, by studying our agonists in a dose curve. Cells ex-
pressed a basal level of PTEN (0 lM), which was unchanged when
exposed to the EtOH vehicle control (data not shown). When MCF-7
cells were stimulated with Rosi, we observed a dose-dependent in-
crease in PTEN expression with a maximal induction occurring at
30 lM (Fig. 1a; Rosi). When quantitated to actin levels, we found
that Rosi stimulation resulted in a maximum of ~1.5-fold increase in
PTEN levels (Fig. 1a; Rosi). Similarly, stimulation with Lovastatin
resulted in an increase in PTEN expression (Fig. 1b). Induction of
PTEN protein levels began at 1 lM and was maximal at 3 lM, with
~1.8-fold increase in PTEN (Fig 1b: Lov). This increase in PTEN
due to either Rosi or Lovastatin stimulation was also time-depend-
ent. Stimulation with either Rosi or Lovastatin resulted in an increase
in PTEN expression at 24 hr poststimulation, and maximal effects
were observed at 48 hr poststimulation (data not shown). At 72 hr,
PTEN levels were near basal. Ciglitazone, Pioglitazone and Troglita-
zone did not induce a change in PTEN expression at any time point
tested (0–72 hr; data not shown).
Rosiglitazone induction of PTEN protein expression is
The above data suggest that the specific activation of PPARg by
Rosi, and perhaps Lovastatin, results in an increase in PTEN
expression. To determine if the effect due to Rosi stimulation was
indeed PPARg-dependent, we utilized the Rosi analog, Com-
pound-66 (Cmpd-66).29Analogs to Ciglitazone, Pioglitazone and
Troglitazone were also synthesized, but no analog to Lovastatin
was created because of its complexity. During synthesis of these
analogs, a double bond was added to its respective agonist, render-
ing the compound unable to activate PPARg because of steric hin-
drance. Analogs to Ciglitazone (DCG), Pioglitazone (DPG) and
Troglitazone (DTG) did not induce alterations in PTEN expression
in MCF-7 cells, even over a wide range of concentrations and times
(data not shown). In contrast to Rosi’s ability to induce PTEN
expression, Cmpd-66 was unable to induce PTEN expression to the
same extent as Rosi. Cells stimulated with 3 lM of Cmpd-66 had
an insignificant ~1.2-fold induction of PTEN (Fig. 2; p > 0.2).
Cmpd-66 is not able to activate PPARg; therefore, these results sug-
gest that Rosi induces PTEN expression in a PPARg-dependent
Induction of PTEN protein expression by Lovastatin
and Rosiglitazone subsequently inhibits P-AKT and
P-p44/42 MAPK levels
The above data indicate that stimulation of MCF-7 cells with
Rosi or Lovastatin induces PTEN, but one cannot infer that it is an
active protein. Active PTEN is known to inhibit the phosphorylation
of AKT, via its lipid phosphatase activity, and phosphorylation of
p44/42-MAPK, via its protein phosphatase activity.37Therefore,
PTEN’s activity is commonly studied by examining the levels of
both P-AKT and P-p44/p42. MCF-7 cells have a basal level of
P-AKT or P-p44/42 because of normal cellular proliferation (Fig. 3a
lane 1: control; lane 2: vehicle stimulated). In contrast, stimulation
with 30 lM Rosi (Fig. 3a; lane 3) or 3 lM Lovastatin (Fig. 3a;
lane 5) resulted in a decrease in P-AKT, concomitant with PTEN
expression, indicating an increase in PTEN lipid phosphatase activ-
ity. Furthermore, an inhibition of P-p44/42 MAPK was also
observed (Fig. 3a; lane 3,5), indicating an increase in PTEN protein
phosphatase activity. As expected, stimulation with 3 lM Cmpd-66
(Fig. 3a; lane 4) did not alter P-AKT or P-p44/42 MAPK levels.
These results are additionally represented in a graphical format in
Figure 3b. To verify that the effect of Lovastatin and Rosi on
P-AKT and P-p44/42 MAPK levels was PTEN-dependent, we in-
vestigated the ability of Lovastatin and Rosi to induce these changes
FIGURE 1 – Lovastatin and Rosiglitazone
induce PTEN protein expression in a dose-de-
pendent manner. Cells were stimulated with
Rosiglitazone (a, Rosi) or Lovastatin (b, Lov)
as described in Methods and harvested after
48 hr stimulation as indicated. Levels of
PTEN and actin were detected by western
blot as described in the Methods. Representa-
tive blots of 5 individual experiments for
Rosi stimulation and 3 individual experi-
ments with Lovastatin are displayed. Levels
of PTEN were quantitated by densitometry
and normalized to actin (bottom panels).
Results are depicted as fold-change compared
to nonstimulated cells and are shown in
graphical format below the western blots. (a)
*p < 0.001; (b) *p < 0.001.
TERESI ET AL.
in BT-549 cells, which are PTEN null. As expected, P-AKT and P-
p44/42 MAPK levels did not change in BT-549 cells stimulated
with either Lovastatin or Rosiglitazone (data not shown). These data
suggest that Rosi or Lovastatin stimulation induces functional
Stimulation of MCF-7 cells with Lovastatin or Rosiglitazone
cause G1 cell cycle arrest
Active PTEN has also been shown to result in G1 cell cycle
arrest.34Therefore, to confirm PTEN’s activity in the presence of
our drugs, we analyzed the cell cycle state of MCF-7 cells after
stimulation with either Rosi or Lovastatin. It has previously been
shown that sodium butyrate (NaBut) can induce G1 arrest in
MCF-7 cells by inhibiting histone deacetylases; therefore, NaBut
was used as a positive control.38To replicate the environment
used in the previous experiments, the cells were not synchronized.
As shown in Figure 4, unsynchronized cells stimulated with
30 lM Rosi for 48 hr showed a 10% increase in G1 content (p 5
0.010). This is comparable to the 15% induction of cells in G1 by
NaBut exposure, suggesting that Rosi stimulation results in GI
arrest because of increased PTEN expression. In contrast, Cmpd-
66 stimulation resulted in a minimal increase of cells in G1. We
also observed that Lovastatin stimulation also resulted in an
increase of 13% of cells in G1 (p 5 0.009). As expected, PTEN
null cells (BT-549) did not induce cell cycle arrest in response to
Lovastatin or Rosi (data not shown).
Stimulation with Lovastatin or Rosiglitazone induces
Our results suggest that stimulation of MCF-7 cells with Rosi
increases PTEN in a PPARg-dependent manner and implies that
Lovastatin is also a PPARg agonist. However, it is not clear whether
this increase in PTEN is due to increased PTEN transcription or
decreased protein degradation. We therefore utilized CHX, which
FIGURE 2 – Rosiglitazone’s analog, Com-
pound-66, has a minimal effect on PTEN pro-
tein expression. Cells were stimulated with
Cmpd-66 as described in Methods and har-
vested after 48 hr stimulation as indicated.
Levels of PTEN and actin were detected by
western blot as described. (a) Representative
western blots of PTEN and actin (3 individual
experiments). (b) Protein levels were quanti-
tated by densitometry and normalized against
actin. Solid bars represent mean PTEN levels
(6SEM) after stimulation by Rosi (see Fig. 1
also); Stripped bars represent mean PTEN levels
(6SEM) after exposure to Cmpd-66 (*p<0.02).
FIGURE 3 – Lovastatin and Rosiglitazone
inhibit P-AKT and P-MAPK protein expres-
sion. (a) Representative blot (of 3 separate
experiments) of P-Akt (top panel), P-p44/p42
(middle panel) and total Akt (bottom panel)
from 3 individual experiments. MCF-7 cells
were either untreated (lane 1) or treated with
EtOH (lane 2), Rosi (lane 3), Cmpd-66 (lane
44) or Lovastatin (lane 5) for 48 hr. Cells
were harvested and 30 lg of protein extract
was subjected to western blot analysis with
anti-P-AKT, anti-P-p44/42 or anti-AKT. Both
Ponceau S and anti-AKT antibody analysis
confirmed equal protein loading. (b) Protein
levels were quantitated by densitometry and
normalized against total AKT. Solid bars rep-
resent mean P-AKT levels after stimulation;
Stripped bars represent mean P-p44/42 levels
INDUCTION OF PPAR?-MEDIATED TRANSCRIPTION OF PTEN BY LOVASTATIN AND ROSIGLITAZONE
inhibits protein synthesis, to determine if either Rosi or Lovastatin
stimulation inhibits PTEN degradation. If Rosi or Lovastatin stimu-
lation inhibits PTEN degradation, we would expect to observe no
change in PTEN expression when MCF-7 cells, stimulated with
Rosi, Cmpd-66 or Lovastatin, are treated with CHX, when com-
pared to Rosi, Cmpd-66 or Lovastatin alone. Costimulation with
CHX, for 24 or 48 hr, with either Rosi or Lovastatin, inhibited the
production of PTEN, when compared to agonist alone (Figure 5a).
Cells costimulated with CHX and Cmpd-66, for either 24 or 48 hr,
showed little to no change in PTEN production because of Cmpd-
66 only minimally inducing PTEN (Fig. 5a). We further demon-
strated in Figure 5a that P-AKT levels decreases due to Rosi stimu-
lation; however, its levels do not alter when Rosi and CHX are used
in concert. This suggests that stimulation with Lovastatin and Rosi
does not inhibit PTEN protein degradation, but rather it increases its
To verify that Lovastatin and Rosi stimulation induces PTEN
transcription, we examined the levels of PTEN mRNA, by RT-
PCR, after Rosi or Lovastatin stimulation. Basal levels of PTEN
transcript were observed in unstimulated MCF-7 or cells treated
with EtOH vehicle. In contrast, cells stimulated with 30 lM Rosi
for 48 hr had ~1.8-fold increase in PTEN transcript. No induction
was detected after stimulation with the inactive Rosi analog, Cmpd-
66, when compared to vehicle control. Furthermore, ~1.6-fold in-
duction of PTEN transcript was observed after 48 hr stimulation
with 3 lM Lovastatin (data not shown). Additionally, we investi-
gated the effect of Lovastatin and Rosi on PTEN message levels by
real-time RT-PCR. We found that both Rosi and Lovastatin induced
PTEN mRNA over both EtOH vehicle and Cmpd-66 (Fig. 5b). Both
Rosi (p 5 0.004) and Lovastatin (p 5 0.020) induced ~0.75-fold
induction of PTEN, while Cmpd-66 actually produced a slight inhibi-
tory affect on PTEN transcript. These data suggest that Lovastatin
and Rosi stimulation results in increased transcription of PTEN.
Lovastatin stimulation induces PPARc-specific transcription
Our results indicate that Lovastatin and Rosi induce PTEN tran-
scription. Additionally, our data with Cmpd-66 (Fig. 2) suggest
that this effect is mediated by a PPARg-dependent mechanism. To
date, Rosi has been shown to activate PPARg-mediated transcrip-
tion in several models; however, Lovastatin has never been shown
to be involved in regulating PPARg. Therefore, to definitively
demonstrate that Lovastatin can mediate PPARg-dependent tran-
scription, we utilized several reporter assays.35Rosi was used as a
positive control for the following experiments. Luciferase-tagged
PPRE was cotransfected into MCF-7 cells with empty vector, wild-
type PPARg or dominant-negative PPARg. These cells were subse-
quently either unstimulated or stimulated with Rosi, Cmpd-66 or
Lovastatin. As expected, Rosi stimulation resulted in ~900-fold
induction of luciferase activity in cells transfected with wild-type
PPARg, compared to empty vector or nonstimulated cells (Fig. 6).
In contrast, there was little activity in stimulated cells transfected
with a dominant-negative PPARg (Fig. 6, DN). Furthermore, stimu-
lation with Cmpd-66 resulted in minimal luciferase activity with all
3 vectors, further verifying that this compound is unable to activate
PPARg. In addition, we found that cells stimulated with Lovastatin
had ~600-fold induction of luciferase activity when cotransfected
with wild-type PPARg, as compared to empty vector. Lovastatin
stimulation in cells cotransfected with dominant-negative PPARg
resulted in basal luciferase activity. These results, taken together,
confirm that Lovastatin is signaling through a PPARg-dependent
FIGURE 5 – Lovastatin and Rosiglitazone
induce PTEN mRNA expression. Cells were
stimulated and harvested as described in the
Methods. (a) CHX treatment. MCF-7 cells
were treated with CHX alone or in concert
with either Rosi or Lovastatin as indicated in
the Materials in Methods section. Western
Blots analyzed both PTEN and P-AKT levels.
Ponceau S analysis confirmed equal protein
loading. (b) RT-Real time PCR. mRNA levels
were quantitated by densitometry and normal-
ized against GAPDH. Unstimulated (MCF-7);
3 lM Lovastatin. Bars represent mean 6 SEM
of PTEN levels (from 3 individual experi-
ments). *p 5 0.004; **p 5 0.020.
FIGURE 4 – Lovastatin and Rosiglitazone, but not Compound-66,
induce G1 arrest. MCF-7 cells were either unstimulated (‘‘MCF-7’’
bar) or stimulated with NaBut, Rosi, Cmpd-66 or Lovastatin. After a
48-hr treatment, cells were harvested and incubated with propidium
iodine as described in the Methods. Cells were then analyzed by flow
cytometry. Each bar represents mean percentage of cells in G1 6
SEM (from 3 individual experiments). *p 5 0.009, **p 5 0.010, NS
5 not significant.
TERESI ET AL.
Lovastatin and Rosiglitazone induce PTEN production
in a PPARc-dependent manner
We further studied the effects of dominant-negative PPARg on
PTEN expression. Our above data indicate that PPARg activation
due to stimulation with Rosi or Lovastatin induces PTEN in a
PPARg-dependent manner. To concretely verify this, we deter-
mined if over-expression of dominant-negative PPARg would
ablate the response observed by Lovastatin and Rosi stimulation
in MCF-7 cells. Figure 7a shows that cells transfected with domi-
nant-negative PPARg and stimulated with Rosi did not induce
PTEN, while cells transfected with empty vector or wild-type
PPARg did (~1.5-fold). Additionally, cells transfected with domi-
nant-negative PPARg did not support Lovastatin-induced PTEN
expression. Cells transfected with wild-type PPARg or empty vec-
tor did, however, induce PTEN expression (~1.8-fold), indicating
that PPARg is required for Lovastatin-mediated expression of PTEN.
As would be expected, stimulation with Cmpd-66 did not induce
PTEN expression, regardless of transfection.
Our results indicate that both Lovastatin and Rosi induce PTEN
expression in a PPARg-dependent manner. To further confirm
these results, we stimulated MEF with Rosi, Cmpd-66 or Lova-
statin. Two different MEF cell lines were utilized: a wild-type
MEF (MEF-WT) cell line and a PPARg knock out MEF cell line
(MEF-PPARg null).39Figure 7b shows that both MEF-WT and
MEF-PPARg null cells, which were unstimulated (MEF) or stimu-
lated with vehicle control (EtOH), showed basal PTEN expres-
sion. We demonstrated that MEF-WT cells stimulated with Rosi
or Lovastatin induced PTEN expression, as similarly observed in
the MCF-7 cell line (p 5 0.03). However, neither Rosi nor Lova-
statin stimulation induced PTEN expression in the MEF-PPARg
null cell line. In contrast, Cmpd-66 had no effect on PTEN expres-
sion in both MEF-WT and MEF-PPARg null cells (Fig. 7b). Like
PTEN induced by Rosi and Lovastatin stimulation in MCF-7 cells,
the PTEN induced in MEF-WT cells was active as AKT and
MAPK phosphorylation decreased concomitant with PTEN ex-
pression (Fig. 3c). Additionally, cell cycle arrest was observed in
MEF-WT cells treated with Rosi and Lovastatin compared to un-
stimulated cells (20.4%, 19.6% respectively; data not shown).
Moreover, the MEF-PPARg null cell line did not undergo cell
cycle arrest or have changes in AKT and MAPK phosphorylation
when treated with Rosi or Lovastatin (data not shown). These data
concretely demonstrate that PTEN induction, due to Rosi or Lova-
statin, is PPARg-dependent and not limited to MCF-7 cells.
Our current observations demonstrate that stimulation of MCF-
7 cells with 30 lM Rosi or 3 lM Lovastatin induces PPARg-
mediated transcription of PTEN, subsequently increasing PTEN
protein levels. Furthermore, we show that stimulation with either
Rosi or Lovastatin induces a PTEN protein that is both protein-
and lipid-phosphatase active. We also demonstrate that the induc-
tion of PTEN due to Rosi or Lovastatin is a PPARg-dependent
mechanism through the use of Cmpd-66, dominant-negative
PPARg and PPARg-null MEF’s. Perhaps more importantly, we
have demonstrated for the first time that Lovastatin increases
PTEN via PPARg-mediated transcription, thus suggesting that Lo-
vastatin may also function as an antiproliferative agent.40Taken
together, these data provide further evidence that PPARg is a tu-
mor suppressor and that one of its tumor suppressing mechanisms
is upregulating the transcription of PTEN.
In 2001, Patel et al.’s report suggested that Rosi induces PTEN
expression in MCF-7 cells; however, it remained correlative, as
the mechanism of induction was not investigated fully. We dem-
FIGURE 6 – Lovastatin and Rosiglitazone induce PPRE regulated
transcription. MCF-7 cells were cotransfected with PPRE-luc and ei-
ther empty vector (-; solid bars), vector containing wild-type PPARg
(WT; striped bars) or vector containing dominant-negative PPARg
(DN; spotted bars). Cells were then stimulated with 30 lM Rosi, 3 lM
Cmpd-66 or 3 lM Lovastatin for 48 hr. The cells were then analyzed
for luciferase activity as described. Each bar represents a mean 6 SEM
of 3 individual experiments. *p 5 0.004; **p 5 0.013.
FIGURE 7 – Upregulation of PTEN expression due to Lovastatin
and Rosiglitazone is PPARg-dependent and not cell line specific. (a)
MCF-7 cells were transfected with empty vector (-; solid bars), vector
containing wild-type PPARg (WT; striped bars) or vector containing
dominant-negative PPARg (DN; spotted bars). *p < 0.001; **p 5
0.001. (b) MEF-WT (solid bars) and MEF-PPARg null (striped bars)
cell lines were also analyzed. The MEF-WT and MEF-PPARg null
cell lines were either not stimulated (1) or stimulated with EtOH, (2)
30 lM Rosi, (3) 3 lM Cmpd-66 (4) or 3 lM Lovastatin (5) for 48 hr.
Protein content was analyzed for PTEN expression by western blot
(expressed as mean 6 SEM from 3 individual experiments) as
described in the Methods section. Results are depicted in a quantitated
graphical format. *p 5 0.03. (c) The MEF-WT cell line was either not
stimulated (1) or stimulated with EtOH (2), 30 lM Rosi (3), 3 lM
Cmpd-66 (4) or 3 lM Lovastatin (5) for 48 hr. Protein content was an-
alyzed for both P-AKT and P-p44/42 expression by western blot
(expressed as mean 6 SEM from 3 individual experiments).
INDUCTION OF PPAR?-MEDIATED TRANSCRIPTION OF PTEN BY LOVASTATIN AND ROSIGLITAZONE
onstrate here, using biochemical and molecular techniques, that
stimulation of MCF-7 and MEF-WT cells with either Lovastatin
or Rosi induces PPARg-mediated transcription of PTEN. Previous
work has been in macrophages, leaving the question open if what
is observed in one cell line would equally occur within another. It
is now well known that TZD’s have cell line-dependent functions,
and indeed, we demonstrate that phenomenon here with the other
TZD’s tested. We have previously shown that increases in PTEN
protein does not necessarily correlate with increases in down-
stream PTEN-mediated cellular events;41,42thus, it is imperative
to determine if the increased PTEN protein is active. Patel et al.
did not analyze the normal readouts of PTEN’s activity such as
the phosphorylation state of AKT and MAPK, or G1 arrest. Addi-
tionally, they did not determine if other TZD’s have a similar
affect in MCF-7 cells. Lastly, and of most significance, it has not
been demonstrated, to date, that PTEN induction by Rosi is de-
pendent upon the presence of PPARg.
Our data indicate that while the TZD PPARg agonist Rosi has
the ability to induce PTEN in MCF-7 cells, the other TZD’s Cigli-
tazone, Pioglitazone and Troglitazone do not. Until recently, it has
been thought that all the TZD’s function in a similar manner.
However, it is now becoming more apparent that each agonist
may have its own cellular functions, some of which may be
PPARg-independent.43Our current data with Rosi support this
idea. Additionally, pharmacological affinity studies have previ-
ously shown that of the 4 TZD’s, Rosi has the highest affinity for
PPARg, followed by Pioglitazone > Troglitazone > Ciglitazone.29,30
Previously, PPARg agonists, in particular Rosi, have been
shown to correlate with PTEN expression in MCF-7 breast cancer
cells45and AsPC-1 pancreatic cancer cells.46We show here that
stimulation of MCF-7 cells with 30 lM Rosi results in a ~1.5-fold
increase in PTEN. This level of induction is in agreement with the
ability of other transcription factors to induce PTEN expression.
Egr-1 activation has been shown to induce PTEN at ~1.6-fold
level.47In addition, the level of induction that we observe with
Rosi is comparable to PTEN induction by Rosi in monocytes,
where Rosi stimulation results in a ~1.4-fold increase in PTEN.
Moreover, a modest increase in PTEN expression was also shown
in both the bronchoalveolar lavage and AsPC-1 cell models. Inter-
estingly, Rosi stimulation in macrophages results in a ~10-fold
increase in PTEN.18This may suggest that macrophages respond
to Rosi stimulation in a PPARg-independent manner, which does
not occur in other lines, or that PPARg signaling is higher in mac-
rophages compared to other cell lines. Nevertheless, our data
strongly demonstrate that Rosi stimulates PTEN expression in
MCF-7 cells in a PPARg-dependent manner. Furthermore, our
data also indicate that Rosi’s ability to induce PTEN expression is
not cell line dependent. To confirm these MCF-7 data, we tested
Rosi’s ability to induce PTEN in 3 different cell lines: MEF-WT,
MEF-PPARg null and BT-549. Our results demonstrate that both
Lovastatin and Rosi can induce PTEN expression in MCF-7 cells
and the MEF-WT cell line. However, while PTEN was induced in
MCF-7 and MEF-WT’s, there was no induction of PTEN protein
in response to Lovastatin or Rosi in the MEF-PPARg null cell
line. This indicates that PPARg is necessary for the induction of
PTEN due to Rosi or Lovastatin stimulation and that this induction
of PTEN is not cell line specific.
We have expanded upon the previously published MCF-7
work18and demonstrate here that 30 lM Rosi produced the maxi-
mal induction of PTEN expression in MCF-7 cells. These results
are in concordance with the ability of Rosi to stimulate BRCA1
expression in MCF-7 cells.48Both PTEN (shown here) and
BRCA1 expression is maximal at 30 lM Rosi. Like Patel et al.,18
we see an increase in PTEN expression at 1 lM Rosi; however,
we found that 30 lM Rosi provides the maximal effect. Further-
more, in the BT-549 PTEN null breast cancer cell line, we did not
observe an increase in PTEN expression or an inhibition of P-
AKT and P-MAPK after Rosi or Lovastatin stimulation. This indi-
cates that the response we observed in MCF-7 cells is due to the
production of PTEN and not a nonspecific response to Rosi or Lo-
vastatin. In contrast, we were able to observe the inhibition of
cyclin D1 in the BT-549 cell line with Rosi or Lovastatin stimula-
tion (data not shown), suggesting that the cyclin D1 response to
Lovastatin and Rosi is not dependent upon an increase in PTEN
The TZD’s are used clinically because of their high affinity for
PPARg, but recent data suggest they may play secondary PPARg-
independent roles.43However, to date, it has been assumed that
Rosi is signaling in a PPARg-dependent manner to induce PTEN;
therefore, we utilized Cmpd-66 to determine PPARg-specificity.
Previously, Cmpd-66 has been shown to only minimally compete
with Rosi and have little affinity for PPARg, demonstrating that it
functions in a PPARg-independent manner.29Because of Cmpd-
66’s ability to minimally compete with Rosi, a small induction of
PTEN with Cmpd-66 stimulation should not be unexpected. Our
analog data was then further confirmed through the use of PPARg
plasmids, demonstrating that both Lovastatin and Rosi can induce
PTEN in a PPARg-dependent manner.
It is well known that PTEN can be maintained at an inactive
state within the cell49; therefore, it is important to determine if
Rosi-induced PTEN is active. We were able to demonstrate an in-
crease in PTEN phosphatase activity after Rosi stimulation. This
decrease in P-AKT and P-MAPK levels within the cell thereby
stops cellular proliferation, induces apoptosis and causes G1 arrest.
Our data therefore indicate that the inhibition of cellular prolifera-
tion in MCF-7 cells after stimulation with Rosi, as seen in previ-
ously published data,18is due to an increase in active PTEN.
Lovastatin-simulated MCF-7 cells resulted in decreased P-AKT
and P-MAPK levels, and increased the percentage of cells in G1.
These data resemble previously published data, where Lovastatin
was shown to increase the percentage of cells in G1 using the
MCF-7 model.50These data suggest that the increased G1 arrest af-
ter Lovastatin stimulation could also be due to the increase in active
Until recently, the focus on Lovastatin has been on its ability to
regulate cholesterol levels. However, in leukemia and breast can-
cer cells, Lovastatin was implicated as a regulator of the MAPK
pathway and the cell cycle,51,52respectively, suggesting that it
could have multiple cellular effects. Additionally, several statins,
including atrovastatin and fluvastatin, have proapoptotic functions
within MCF-7 cells, suggesting that the statins, as a group, possess
properties that may be beneficial in the treatment of breast cancer
patients.53Interestingly, women taking statins have a 68% reduc-
tion in the risk of breast cancer.40Recently, work in Sprague–
Dawley rats has demonstrated that atorvastatin treatment increases
PTEN protein expression.54This suggests that the statins as a
group may induce PTEN expression. We now gather the first evi-
dence that a potential mechanism for cellular regulation by Lova-
statin is through PPARg. Our data indicates that Lovastatin stimu-
lation results in the activation of PPARg-mediated transcription.
Currently, it is unclear, in the Sprague–Dawley rat model, whether
the role of atrovastatin is through PPARg-mediated transcription.
However, in light of our data, it is a distinct possibility that all or
most statins induce PPARg, and subsequently PTEN, opening up
an exciting avenue of investigation for both cardiac and cancer
The mechanism behind PTEN induction, due to PPARg agonists,
has yet to be fully understood. Two molecular connections between
PTEN and PPARg have been previously hypothesized: PPARg reg-
ulates the transcription of PTEN18and/or PPARg regulates a sec-
ondary factor, which regulates PTEN.55Furthermore, because
PTEN levels can be regulated by both an increase in transcription or
a decrease in degradation,41,49it remained a possibility that PPARg
agonists induced PTEN expression by decreasing protein degrada-
tion. Our data indicate that both Lovastatin and Rosi stimulation do
not inhibit PTEN degradation, but rather induce PTEN transcription.
The specific regions in the promoter that is required for PPARg
mediated transcription by Lovastatin and Rosi remain to be eluci-
TERESI ET AL.
dated and are beyond the scope of this paper. While 2 putative
PPRE’s have been suggested,18these sites are well upstream of the
minimal PTEN promoter and it remains unclear what role, if any,
to demonstrate that these are indeed Lovastatin and Rosi responsive
PPARg binding sites in breast cancer cell lines (data not shown).
Nonetheless, PTEN’s minimal promoter has several sites that are
potential candidates for PPARg-mediated transcription mediated by
Lovastatin and Rosi. One must also keep in mind that these data do
not limit PPARg’s ability to regulate PTEN only through inducing
ting the transcription of another transcription factor that binds
In conclusion, we have demonstrated that PPARg directly regu-
lates PTEN at the transcriptional level. Furthermore, since PTEN
is constitutively active, it may be worthwhile to examine the
effects of Lovastatin and Rosi as a mechanism for increasing
PTEN expression for both the treatment and prevention of cancer.
Indeed, the data with the Sprague–Dawley rats may begin to sug-
gest that treatment of patients (e.g. CS) or cancers that have hemi-
zygous deletions or haploinsufficiency of PTEN with Lovastatin
or Rosi may be conceivable. Despite these encouraging results, we
must be aware that these medications may indeed harm patients
with germline intragenic PTEN mutations or those with neoplasias
with somatic intragenic mutations by raising levels of mutant protein.
R.E.T. is a graduate student of the Integrated Biomedical Gradu-
ate Program of The Ohio State University and a Predoctoral Fellow
of the Cleveland Clinic Genomic Medicine Institute. The authors
wish to thank Rick Meister (The Ohio State University Veterinary
Flow Cytometry Core) for flow cytometry support and Dr. Lisa Yee
(The Ohio State University) for the generous gift of Rosiglitazone.
We would also like to thank both Drs. Larry Kirschner (The Ohio
State University) and Bruce Spiegelman (Dana-Farber Cancer Insti-
tute) for contributing the MEF-WT and MEF-PPARg null cell lines,
respectively. We are grateful to Drs. Shipra Agrawal and Yufang
Tang, and Michelle Sinden for critical review of this manuscript
and helpful discussions. This work was partially funded by the
American Cancer Society (RSG02-151-01-CCE to C.E.), and the
Susan G. Komen Breast Cancer Research Foundation (BCTR-2000
462 to C.E.). C.E. is a recipient of the Doris Duke Distinguished
Clinical Scientist Award.
1. Ries LAG, Eisner MP, Kosary CL, Hankey BF, Miller BA, Clegg L,
Mariotto A, Fay MP, Feuer EJ, Edwards BK. SEER cancer statistics
review, 1975–2000. National Cancer Institute, 2003.
Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, Zheng Z, Bose S, Call KM,
Tsou HC, Peacocke M, Eng C, Parsons R. Germline mutations of the
PTEN gene in Cowden disease, an inherited breast and thyroid cancer
syndrome. Nat Genet 1997;16:64–7.
Marsh DJ, Kum JB, Lunetta KL, Bennett MJ, Gorlin RJ, Ahmed SF,
Bodurtha J, Crowe C, Curtis MA, Dasouki M, Dunn T, Feit H, et al.
PTEN mutation spectrum and genotype-phenotype correlations in
Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with
Cowden syndrome. Hum Mol Genet 1999;8:1461–72.
Zhou XP, Waite KA, Pilarski R, Hampel H, Fernandez MJ, Bos C,
Dasouki M, Feldman GL, Greenberg LA, Ivanovich J, Matloff E,
Patterson A, et al. Germline PTEN promoter mutations and deletions
in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant
PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt
pathway. Am J Hum Genet 2003;73:404–11.
Eng C. Cowden syndrome. J Genet Couns 1997;6:181–92.
Eng C, Hampel H, de la Chapelle A. Genetic testing for cancer predis-
position. Annu Rev Med 2001;52:371–400.
Eng C. Will the real Cowden syndrome please stand up: revised diag-
nostic criteria. J Med Genet 2000;37:828–30.
Pilarski R, Eng C. Will the real Cowden syndrome please stand up
(again)? Expanding mutational and clinical spectra of the PTEN hamar-
toma tumour syndrome. J Med Genet 2004;41:323–6.
Gorlin RJ, Cohen MM, Jr, Condon LM, Burke BA. Bannayan-Riley-
Ruvalcaba syndrome. Am J Med Genet 1992;44:307–14.
10. Eng C. PTEN: one gene, many syndromes. Hum Mutat 2003;22:183–98.
11. Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons
R, Tonks NK. P-TEN, the tumor suppressor from human chromosome
10q23, is a dual-specificity phosphatase. Proc Natl Acad Sci USA
12. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1,
dephosphorylates the lipid second messenger, phosphatidylinositol
3,4,5-trisphosphate. J Biol Chem 1998;273:13375–8.
13. Gu J, Tamura M, Yamada KM. Tumor suppressor PTEN inhibits
integrin- and growth factor-mediated mitogen-activated protein
(MAP) kinase signaling pathways. J Cell Biol 1998;143:1375–83.
14. Virolle T, Adamson ED, Baron V, Birle D, Mercola D, Mustelin T, de
Belle I. The Egr-1 transcription factor directly activates PTEN during
irradiation-induced signalling. Nat Cell Biol 2001;3:1124–8.
15. Stambolic V, MacPherson D, Sas D, Lin Y, Snow B, Jang Y, Benchimol
S. Regulation of PTEN transcription by p53. Mol Cell 2001;8:317–25.
16. Han B, Dong Z, Liu Y, Chen Q, Hashimoto K, Zhang JT. Regulation
of constitutive expression of mouse PTEN by the 50-untranslated
region. Oncogene 2003;22:5325–37.
17. Vasudevan KM, Gurumurthy S, Rangnekar VM. Suppression of PTEN
expression by NF-kappa B prevents apoptosis. Mol Cell Biol 2004;24:
18. Patel L, Pass I, Coxon P, Downes CP, Smith SA, Macphee CH. Tumor
suppressor and anti-inflammatory actions of PPARgamma agonists are
mediated via upregulation of PTEN. Curr Biol 2001;11:764–8.
19. Celi FS, Shuldiner AR. The role of peroxisome proliferator-activated
receptor gamma in diabetes and obesity. Curr Diab Rep 2002;2:179–85.
20. Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and dis-
ease. Nature 2000;405:421–4.
21. Mueller E, Sarraf P, Tontonoz P, Evans RM, Martin KJ, Zhang M,
Fletcher C, Singer S, Spiegelman BM. Terminal differentiation of human
breast cancer through PPAR gamma. Mol Cell 1998;1:465–70.
22. Sarraf P, Mueller E, Smith WM, Wright HM, Kum JB, Aaltonen LA,
de la Chapelle A, Spiegelman BM. Loss-of-function mutations in PPAR
gamma associated with human colon cancer. Mol Cell 1999;3:799–804.
23. Aldred MA, Morrison C, Gimm O, Hoang-Vu C, Krause U, Dralle H,
Jhiang S, Eng C. Peroxisome proliferator-activated receptor gamma is
frequently downregulated in a diversity of sporadic nonmedullary thy-
roid carcinomas. Oncogene 2003;22:3412–6.
24. Jiang WG, Douglas-Jones A, Mansel RE. Expression of peroxisome-
proliferator activated receptor-gamma (PPARgamma) and the PPAR-
gamma co-activator, PGC-1, in human breast cancer correlates with
clinical outcomes. Int J Cancer 2003;106:752–7.
25. Clay CE, Namen AM, Atsumi G, Willingham MC, High KP, Kute TE,
Trimboli AJ, Fonteh AN, Dawson PA, Chilton FH. Influence of J series
prostaglandins on apoptosis and tumorigenesis of breast cancer cells.
26. Suh N, Wang Y, Williams CR, Risingsong R, Gilmer T, Willson TM,
Sporn MB. A new ligand for the peroxisome proliferator-activated
receptor-gamma (PPAR-gamma), GW7845, inhibits rat mammary
carcinogenesis. Cancer Res 1999;59:5671–3.
27. Eng C. Role of PTEN, a lipid phosphatase upstream effector of pro-
tein kinase B, in epithelial thyroid carcinogenesis. Ann NY Acad Sci
28. Dwight T, Thoppe SR, Foukakis T, Lui WO, Wallin G, Hoog A, Frisk T,
Larsson C, Zedenius C. Involvement of the PAX8/peroxisome prolifera-
tor-activated receptor gamma rearrangement in follicular thyroid tumors.
J Clin Endocrinol Metab 2003;88:4440–5.
29. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans
RM. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipo-
cyte determination factor PPAR gamma. Cell 1995;83:803–12.
30. Young PW, Buckle DR, Cantello BC, Chapman H, Clapham JC,
Coyle PJ, Haigh D, Hindley RM, Holder JC, Kallernder H, Latter AJ,
Lawrie KW, et al. Identification of high-affinity binding sites for the
insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adi-
pocytes using a radioiodinated ligand for peroxisomal proliferator-
activated receptor gamma. J Pharmacol Exp Ther 1998;284:751–9.
31. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano
MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of
protein using bicinchoninic acid. Anal Biochem 1985;150:76–85.
32. Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 1970;227:680–5.
33. Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis for the separation of proteins in the range
from 1 to 100 kDa. Anal Biochem 1987;166:368–79.
34. Ginn-Pease ME, Eng C. Increased nuclear phosphatase and tensin
homologue deleted on chromosome 10 is associated with G0-G1 in
MCF-7 cells. Cancer Res 2003;63:282–6.
INDUCTION OF PPAR?-MEDIATED TRANSCRIPTION OF PTEN BY LOVASTATIN AND ROSIGLITAZONE
35. Gurnell M, Wentworth JM, Agostini M, Adams M, Collingwood TN, Download full-text
Provenzano C, Browne PO, Rauanayagam O, Burris TP, Schwabe JW,
Lazar MA, Chatterjee VK. A dominant-negative peroxisome prolifera-
tor-activated receptor gamma (PPARgamma) mutant is a constitutive
repressor and inhibits PPARgamma-mediatedadipogenesis. J Biol Chem
36. Qin C, Burghardt R, Smith R, Wormke M, Stewart J, Safe S. Peroxi-
some proliferator-activated receptor gamma agonists induce protea-
some-dependent degradation of cyclin D1 and estrogen receptor alpha
in MCF-7 breast cancer cells. Cancer Res 2003;63:958–64.
37. Waite KA, Eng C. Protean PTEN: form and function. Am J Hum
38. Chopin V, Toillon RA, Jouy N, Le Bourhis X. P21(WAF1/CIP1) is dis-
pensable for G1 arrest, but indispensable for apoptosis induced by so-
dium butyrate in MCF-7 breast cancer cells. Oncogene 2004;23:21–9.
39. Mueller E, Drori S, Aiyer A, Yie J, Sarraf P, Chen H, Hauser S, Rosen
ED, Ge K, Roeder RG, Spiegelman BM. Genetic analysis of adipo-
genesis through peroxisome proliferator-activated receptor gamma
isoforms. J Biol Chem 2002;277:41925–30.
40. Cauley JA, Zmuda JM, Lui LY, Hillier TA, Ness RB, Stone KL,
Cummings SR, Bauer DC. Lipid-lowering drug use and breast cancer in
older women: a prospective study. J Womens Health (Larchmt) 2003;
41. Waite KA, Eng C. BMP2 exposure results in decreased PTEN protein
degradation and increased PTEN levels. Hum Mol Genet 2003;12:
42. Waite KA, Sinden MR, Eng C. Phytoestrogen exposure elevates
PTEN levels. Hum Mol Genet 2005;14:1457–63.
43. Na HK, Surh YJ. Peroxisome proliferator-activated receptor gamma
(PPARgamma) ligands as bifunctional regulators of cell proliferation.
Biochem Pharmacol 2003;66:1381–91.
44. Hong G, Davis B, Khatoon N, Baker SF, Brown J. PPAR gamma-
dependent anti-inflammatory action of rosiglitazone in human mono-
cytes: suppression of TNF alpha secretion is not mediated by PTEN
regulation. Biochem Biophys Res Commun 2003;303:782–7.
45. Lee KS, Park SJ, Hwang PH, Yi HK, Song CH, Chai OH, Kim JS,
Lee MK, Lee YC. PPAR-gamma modulates allergic inflammation
through up-regulation of PTEN. FASEB J 2005;19:1033–5.
46. Farrow B, Evers BM. Activation of PPARgamma increases PTEN
expression in pancreatic cancer cells. Biochem Biophys Res Commun
47. Tell G, Pines A, Arturi F, Cesaratto L, Adamson E, Puppin C, Presta
I, Russo D, Filetti S, Damante G. Control of phosphatase and tensin
homolog (PTEN) gene expression in normal and neoplastic thyroid
cells. Endocrinology 2004;145:4660–6.
48. Pignatelli M, Cocca C, Santos A, Perez-Castillo A. Enhancement of
BRCA1 gene expression by the peroxisome proliferator-activated re-
ceptor gamma in the MCF-7 breast cancer cell line. Oncogene 2003;
49. Vazquez F, Ramaswamy S, Nakamura N, Sellers WR. Phosphoryla-
tion of the PTEN tail regulates protein stability and function. Mol Cell
50. Zhou Y, Mi MT, Zhu JD, Zhang QY. [Effects of lovastatin on prolif-
eration and gap junctional intercellular communication of human
breast cancer cell MCF-7]. Ai Zheng 2003;22:257–61.
51. Wu J, Wong WW, Khosravi F, Minden MD, Penn LZ. Blocking the
Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells
to lovastatin-induced apoptosis. Cancer Res 2004;64:6461–8.
52. Rao S, Lowe M, Herliczek TW, Keyomarsi K. Lovastatin mediated
G1 arrest in normal and tumor breast cells is through inhibition of
CDK2 activity and redistribution of p21 and p27, independent of p53.
53. Muck AO, Seeger H, Wallwiener D. Inhibitory effect of statins on the
proliferation of human breast cancer cells. Int J Clin Pharmacol Ther
54. Mensah K, Mocanu MM, Yellon DM. Failure to protect the myocar-
dium against ischemia/reperfusion injury after chronic atorvastatin
treatment is recaptured by acute atorvastatin treatment: a potential
role for phosphatase and tensin homolog deleted on chromosome ten?
J Am Coll Cardiol 2005;45:1287–91.
55. Aldred MA, Ginn-Pease ME, Morrison CD, Popkie AP, Gimm O,
Hoang-Vu C, Krause U, Dralle H, Jhiang SM, Plass C, Eng C. Caveolin-
1 and caveolin-2, together with three bone morphogenetic protein-related
genes, may encode novel tumor suppressors down-regulated in sporadic
follicular thyroid carcinogenesis. Cancer Res 2003;63:2864–71.
TERESI ET AL.