Peroxisome proliferator-activated receptor-gamma inhibits transformed growth of non-small cell lung cancer cells through selective suppression of Snail.
ABSTRACT Work from our laboratory and others has demonstrated that activation of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits transformed growth of non-small cell lung cancer (NSCLC) cell lines in vitro and in vivo. We have demonstrated that activation of PPARgamma promotes epithelial differentiation of NSCLC by increasing expression of E-cadherin, as well as inhibiting expression of COX-2 and nuclear factor-kappaB. The Snail family of transcription factors, which includes Snail (Snail1), Slug (Snail2), and ZEB1, is an important regulator of epithelial-mesenchymal transition, as well as cell survival. The goal of this study was to determine whether the biological responses to rosiglitazone, a member of the thiazolidinedione family of PPARgamma activators, are mediated through the regulation of Snail family members. Our results indicate that, in two independent NSCLC cell lines, rosiglitazone specifically decreased expression of Snail, with no significant effect on either Slug or ZEB1. Suppression of Snail using short hairpin RNA silencing mimicked the effects of PPARgamma activation, in inhibiting anchorage-independent growth, promoting acinar formation in three-dimensional culture, and inhibiting invasiveness. This was associated with the increased expression of E-cadherin and decreased expression of COX-2 and matrix metaloproteinases. Conversely, overexpression of Snail blocked the biological responses to rosiglitazone, increasing anchorage-independent growth, invasiveness, and promoting epithelial-mesenchymal transition. The suppression of Snail expression by rosiglitazone seemed to be independent of GSK-3 signaling but was rather mediated through suppression of extracellular signal-regulated kinase activity. These findings suggest that selective regulation of Snail may be critical in mediating the antitumorigenic effects of PPARgamma activators.
- SourceAvailable from: Ilsup Kim[Show abstract] [Hide abstract]
ABSTRACT: It remains unclear whether patients with non-small-cell lung cancer (NSCLC) develop brain metastasis during or after standard therapy. We attempted to identify biological markers that predict brain metastasis, and investigated how to modulate expression of such markers. A case-control study of patients who were newly diagnosed with NSCLC and who had developed brain metastasis during follow-up was conducted between 2004 and 2009. These patients were compared with a control group of patients who had NSCLC but no evidence of brain metastasis. Immunohistochemical analysis of expression of Ki-67, p53, Bcl-2, Bax, vascular endothelial growth factor, epidermal growth factor receptor, caspase-3, and E-cadherin was conducted. The methylation status of the genes for O(6)-methylguanine-DNA-methyltransferase, tissue inhibitor of matrix metalloproteinase (TIMP)-2, TIMP-3, and death-associated protein-kinase was also determined, by use of a methylation-specific polymerase chain reaction. A significantly increased risk of developing brain metastasis was associated with the presence of primary tumors with low E-cadherin expression in patients with NSCLC. We also investigated the effects of pioglitazone, a peroxisome proliferator-activated receptor γ-activating drug, in tumor-bearing mouse models. We found that E-cadherin expression was proportional to pioglitazone exposure time. Interestingly, pioglitazone pretreatment before cancer cell inoculation prevented loss of E-cadherin expression and reduced expression of MMP9 and fibronectin, compared with the control group. E-cadherin expression could be a predictor of brain metastasis in patients with NSCLC. Preventive treatment with pioglitazone may be useful for modulating E-cadherin expression.Journal of Neuro-Oncology 05/2012; 109(2):219-27. · 3.12 Impact Factor
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ABSTRACT: Glioblastoma is the most common type of astrocytoma in the brain. Due to its high invasiveness and chemoresistance, patients with advanced stage of glioblastoma have a poor prognosis. SNAI1, an important regulator of epithelial-mesenchymal transition, has been associated with metastasis in various carcinoma cells. However, its roles in glioblastoma cells have been poorly characterized. To examine roles of SNAI1 in glioblastoma cells, we knockdowned SNAI1 expression using siRNA. SNAI1 siRNA increased the expression level of E-cadherin and decreased that of vimentin. In the water-soluble tetrazolium salt (WST-1) assay, SNAI1 siRNA inhibited the proliferation of U87-MG and GBM05 glioblastoma cells. Moreover, in the Boyden chamber assay and Matrigel invasion assay, SNAI1 siRNA inhibited serum-induced migration and invasion of glioblastoma cells. These results suggested that SNAI1 is involved in the proliferation and migration of glioblastoma cells.Cellular and Molecular Neurobiology 01/2011; 31(3):489-96. · 2.29 Impact Factor
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ABSTRACT: Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that plays an important role in the control of gene expression linked to a variety of physiological processes, including cancer. Ligands for PPARγ include naturally occurring fatty acids and the thiazolidinedione class of antidiabetic drugs. Activation of PPARγ in a variety of cancer cells leads to inhibition of growth, decreased invasiveness, reduced production of proinflammatory cytokines, and promotion of a more differentiated phenotype. However, systemic activation of PPARγ has been reported to be protumorigenic in some in vitro systems and in vivo models. Here, we review the available data that implicate PPARγ in lung carcinogenesis and highlight the challenges of targeting PPARγ in lung cancer treatments.PPAR Research 01/2012; 2012:362085. · 2.69 Impact Factor
Activated Receptor-γ Inhibits
Transformed Growth of
Non–Small Cell Lung Cancer
Cells through Selective
Suppression of Snail1,2
Rashmi Choudhary*, Howard Li†, Robert A. Winn†,
Amber L. Sorenson*, Mary C.M. Weiser-Evans*
and Raphael A. Nemenoff*
*Division of Renal Diseases and Hypertension, Department
of Medicine, University of Colorado Denver, Aurora, CO,
USA;†Division of Pulmonary Sciences and Critical Care
Medicine, Department of Medicine, University of Colorado
Denver, Aurora, CO, USA
Work from our laboratory and others has demonstrated that activation of the nuclear receptor peroxisome proliferator–
activated receptor-γ (PPARγ) inhibits transformed growth of non–small cell lung cancer (NSCLC) cell lines in vitro and
in vivo. We have demonstrated that activation of PPARγ promotes epithelial differentiation of NSCLC by increasing ex-
pression ofE-cadherin, as well as inhibiting expression of COX-2 and nuclear factor-κB. The Snail family of transcription
factors, which includes Snail (Snail1), Slug (Snail2), and ZEB1, is an important regulator of epithelial-mesenchymal transi-
tion, as well as cell survival. The goal of this study was to determine whether the biological responses to rosiglitazone, a
Our results indicate that, in two independent NSCLC cell lines, rosiglitazone specifically decreased expression of Snail,
with no significant effect on either Slug or ZEB1. Suppression of Snail using short hairpin RNA silencing mimicked the
culture, and inhibiting invasiveness. This was associated with the increased expression of E-cadherin and decreased
expressionofCOX-2andmatrixmetaloproteinases.Conversely, overexpressionofSnailblocked thebiologicalresponses
to rosiglitazone, increasing anchorage-independent growth, invasiveness, and promoting epithelial-mesenchymal transi-
tion. The suppression of Snail expression by rosiglitazone seemed to be independent of GSK-3 signaling but was rather
mediated through suppression of extracellular signal–regulated kinase activity. These findings suggest that selective
regulation of Snail may be critical in mediating the antitumorigenic effects of PPARγ activators.
Neoplasia (2010) 12, 224–234
A considerable amount of current literature suggests that loss of epithe-
lial features and gain of mesenchymal properties play a critical role in
the progression and metastasis of epithelial tumors . These events
are similar to the epithelial-mesenchymal transition (EMT) that has
been well characterized in embryonic development. EMT involves
complex cellular changes including loss of polarity and disruption of
cell-cell contacts, synthesis of extracellular matrix molecules, as well as
in matrix degradation that contribute to cell motility and invasiveness
. Loss of E-cadherin has been shown to be associated with increased
tumor invasiveness, metastasis, and poor prognosis [3–5]. Transcrip-
tional repression has emerged as a mechanism of silencing E-cadherin
during tumor progression. This suppression is mediated by members
of the Snail, ZEB, and basic-helix-loop-helix families of transcription
factors. Snail1 (Snail) and Snail2 (Slug) belong to the Snail superfamily
of zinc finger transcriptional repressors that participate in the devel-
opmental EMT . Snail is required for mesoderm and neural crest
Address all correspondence to: Raphael A. Nemenoff, Division of Renal Diseases and
Hypertension, Department of Medicine, University of Colorado, Denver, C-281,
12700 E 19th Ave, Aurora, CO 80045. E-mail: Raphael.Nemenoff@ucdenver.edu
1This work was supported by grants from the National Institutes of Health (CA103618,
to Dr. Weiser-Evans.
2This article refers to supplementary materials, which are designated by Figures W1
and W2 and are available online at www.neoplasia.com.
Received 22 September 2009; Revised 5 January 2010; Accepted 6 January 2010
Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1522-8002/10/$25.00
Volume 12 Number 3March 2010 pp. 224–234
formation during embryonic development and has been implicated
in the EMT associated with tumor progression. Slug also represses
E-cadherin and induces a complete EMT. However, Slug binds with
lower affinity than Snail to the E-cadherin promoter .
Expression of Snail and/or Slug has been reported in breast, ovarian,
colon, skin, and squamous cell carcinomas and is associated with poor
prognosis [6,8]. Although the function of Snail and Slug can be inter-
changeable in different species , a distinct role for each factor is
supported from analysis of knockout mice. Whereas Snail null mice
present early embryonic lethality , Slug null mice are viable, under-
going a normal program of development . The specific contribu-
tion of Snail and Slug to tumor progression is still poorly defined. Snail
is activated at the invasive front of tumors induced in mouse skin 
and has been associated with breast and hepatocarcinoma invasion
[13,14]. Snail induces a full EMT when overexpressed in epithelial
Madin-Darby canine kidney cells leading to acquisition of a motile/
invasive phenotype [12,15].Recently,thesetranscription repressorshave
also been found to be expressed in lung adenocarcinomas. Knockdown
of Snail, through RNA interference, increases the sensitivity of non–
small cell lung cancer (NSCLC) cells to chemotherapeutic agents .
. Slug expression is a predictor of outcome in lung adenocarcinoma
patients . Overexpression of ZEB-1 has been implicated in mediat-
ing EMT in NSCLC cells .
Peroxisome proliferator–activated receptor-γ (PPARγ) is a mem-
ber of the nuclear receptor superfamily of ligand-activated transcription
factors. In addition to its known role in adipocyte differentiation,
PPARγ has been implicated in regulating carcinogenesis . PPARγ
activators of the thiazolidinedione class (TZDs), such as rosiglitazone
and troglitazone, slow growth of colon and thyroid tumors [21,22].
In NSCLC, activation of PPARγ can inhibit growth of NSCLC cells
in vitro and in xenograft models [23–25]. We have shown that mice
with targeted overexpression of PPARγ in the distal epithelia of the
genesis model .
Mechanistically, several studies have demonstrated that PPARγ acti-
vation can inhibit nuclear factor-κB and COX-2 expression in NSCLC
[26,27]. PPARγ has been shown to increase E-cadherin expression,
suggesting that it may target transcriptional repressors; however, the
effects of PPARγ on Snail family members have not been well studied.
Here, we report that PPARγ-mediated inhibition of Snail expression
represents a critical pathway in mediating the antitumorigenic effects
of TZDs on NSCLC.
Materials and Methods
Antibodies against Snail, Slug, extracellular signal–regulated kinases
(ERKs), phospho (p)-ERKs, GSK-3β, and p-GSK-3β were from Cell
Signaling Technology (Beverly, MA); antibody against E-cadherin was
from BD Biosciences (Franklin Lakes, NJ); and COX-2 antibody was
from Cayman Chemical (Ann Arbor, MI). β-Actin antibody was from
Sigma-Aldrich (St Louis, MO). Rosiglitazone was from Cayman Chemi-
cal (Ann Arbor, MI). PD98059 and GSK-3β inhibitors were from
Cell Culture and Stable Transfections
Human lung adenocarcinoma cell lines, H2009 and H2122, were
obtained from the University of Colorado Health Science Center Tis-
stably transfect NSCLC by retroviral-mediated gene transfer as previ-
ously described . Pools of stable transfectants were screened for
the expression of Snail by immunoblot analysis. Snail and control short
hairpin RNA (shRNA) plasmids obtained from Open Biosystems
(Rockford, IL) were used to transfect NSCLC using ExGen500 trans-
fection reagent (Fermentas, Glen Burnie, MD).
Migration/Invasion, Soft Agar Colony Formation, and
Migration and invasion were determined as previously described
. Colony formation in soft agar was performed as described pre-
viously . Colonies formed in 3- to 4-week period were stained with
nitroblue tetrazolium chloride (1 mg/ml), visualized under a micro-
scope and counted. For three-dimensional cultures, cells were grown
in Matrigel using a modification of the procedure of Debnath et al.
, as previously described . Briefly, cells were plated at 5000
per well in 10% Matrigel in full media and were fed with 4% every
other day for a term of 8 to 10 days of culture.
Confocal Microscopy and Image Analysis
Cells grown in two-dimensional or three-dimensional cultures were
fixed in 2% paraformaldehyde in phosphate-buffered saline for 30 min-
utes at room temperature. Cells were permeabilized with 0.5% Triton
X-100 for 15 minutes if needed. All samples were blocked with 1%
BSA in phosphate-buffered saline for 1 hour at room temperature. Cells
were incubated with E-cadherin antibody (1:100; BD Biosciences) for
three-dimensional cultures or anti-Snail antibodies (Santa Cruz Bio-
technology, Santa Cruz, CA) for two-dimensional cultures, overnight
at4°C,followed by 1hour of incubation with the appropriate secondary
antibodies (Alexa Fluor 568 rabbit antigoat immunoglobulin G, 1:250;
Invitrogen, Carlsbad, CA). Slides were mounted and viewed with
TE2000-S IF microscope (Nikon, Tokyo, Japan) or a 510 Meta/FCS
laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Con-
focal images were processed using LSM Image Examiner.
SYBR Green Real-time Reverse Transcription–Polymerase
Inc, Valencia, CA). cDNA was synthesized using iSCRIPT cDNA syn-
thesis kit from BioRad (BioRad, Hercules, CA). For real-time PCR,
relative gene expression was determined by SYBR Green JumpStart
Taq ReadyMix (Sigma) on a Bio-Rad iCycler (BioRad). Primer pairs
and PCR conditions are as follows: SnailF, 5′-CGCGCTCTTTCCT-
CGTCAG-3′; SnailR 5′-TCCCAGATGAGCATTGGCAG-3′; SlugF,
5′-GCCTCCAAAAAGCCAAACTA-3′; SlugR, 5′-CACAGT-
GATGGGGCTGATG-3′; ZEB1F, 5′-AGGAGTGAAAGAGAAGG-
GAATGC-3′; ZEB1R, 5′-GGTCCTCTTCAGGTGCCTCAG3′;
β-ActinF, 5′-AGGGTGTGATGGTGGGTATGG-3′; β-ActinR, 5′-
AATGCCGTGTTCAATGGGG-3′, 55 cycles at 95°C for 15 seconds,
60°C for 1 minute, and 72°C for 30 seconds; MMP-9F, 5′-CCACTT-
CCCCTTCATCTTC-3′; MMP-9R, 5′-CGTCCTGGGTGTA-
GAGTC-3′; 55 cycles at 95°C for 20 seconds, 60°C for 30 seconds,
and 72°C for 30 seconds; MMP-2F, 5′-TCTTGACCAGAATAC-
CATCG-3′; MMP-2R,5′-CACATCGCTCCAGACTTG-3′; 55cycles
at 95°C for 20 seconds, 58°C for 30 seconds, and 72°C for 30 seconds.
The relative gene expression, normalized to β-actin and based on three
separate experiments, was calculated.
Neoplasia Vol. 12, No. 3, 2010PPARγ Activation Suppresses Snail Expression in NSCLCChoudhary et al.
Semiconfluent cultures of cells (∼80% confluent) were placed in
serum-freemedium, treated with or without rosiglitazone,and cultured
for an additional 36 hours. Conditioned medium was concentrated
(Microcon YM-10 centrifugal filter; Millipore,Billerica, MA) and sepa-
rated on 7% SDS–polyacrylamide gel containing 0.1% (wt./vol.) gelatin
under nonreducing conditions. Zymography for MMP-9 and MMP-2
was performed according to Bernhard and Muschel .
Data are expressed as mean ± SEM of at least three independent ex-
periments. Statistical significance was determined by analyzing the data
using one-way analysis of variance (GraphPad InStat 3 Software). P <
.05 was considered statistically significant.
PPARγ Selectively Inhibits Expression of Snail in NSCLC
To begin to define the molecular mechanisms whereby PPARγ can
control EMT in NSCLC, we examined the effects of the PPARγ acti-
vator rosiglitazone on transcriptional repressors of E-cadherin: Snail,
Slug, and ZEB1 in two lung adenocarcinoma cell lines, H2009 and
H2122. Stimulation with rosiglitazone (10 μM) decreased the expres-
sion of Snail in a time-dependent manner. Expression was significantly
decreased at 6 hours; by 24 hours, the expression was inhibited in both
cell lines by approximately 75% (Figure 1A, left panel). No significant
changes were observed in expression of Slug (Figure 1A, right panel).
Levels of messenger RNA (mRNA) for Snail, Slug, and ZEB1 were as-
sessed by real-time reverse transcription–polymerase chain reaction
(RT-PCR). Rosiglitazone significantly decreased Snail mRNA levels
in both cell lines; no significant effect was observed on expression of
by reporter assays, wherein cells were transiently transfected with
Snail, Slug,and ZEB1promoter constructs driving a luciferase reporter.
Rosiglitazone inhibited Snail promoter activity in both cell lines (Fig-
ure 1C) but had no effect on either Slug or ZEB promoter activity.
To confirm if the effects on Snail expression were PPARγ specific, we
also examined Snail expression in H2122 cells overexpressing PPARγ.
Expression was markedly decreased in these cells compared with con-
trols transfected with empty vector (Figure 1D). No change in Slug ex-
pression was observed (Figure 1D). Finally, a pharmacological inhibitor
of PPARγ, T0070907 (T007) , was able to reverse the decrease in
Snail mRNA induced by rosiglitazone (Figure W1).
Snail Silencing Mimics Effects of PPARγ Activation on
To assess the role that decreased expression of Snail plays in mediat-
ing the effects of PPARγ, we silenced Snail in both adenocarcinoma
celllines.Cellswere transfectedwith lentiviral vectors encodingshRNA
against Snail or with nonsilencing control shRNA (NS). Pools of cells
transfected with two independent Snail shRNA constructs (A12 and
E8) showed decreased expression of Snail protein compared with NS
controls in both cell lines (Figure 2A). A third shRNA construct (B9)
did not significantly decrease Snail expression in either cell line. In
addition, Snail promoter activity was also decreased in the silenced
cells compared with NS controls (data not shown). We have previ-
ously reported that overexpression of PPARγ increases E-cadherin
levels in NSCLC . Here, we examined the effects of rosiglitazone
on E-cadherin expression. As shown in Figure 2B (top panels, left)),
rosiglitazone increased E-cadherin expression in both H2122 and
expression (Figure 2B, top panels, right). In both H2122 and H2009,
rosiglitazone decreased COX-2 expression, and this was also observed
in the cells silenced for Snail expression (Figure 2B, bottom panels).
Silencing Snail Mimics Biological Responses to Rosiglitazone
Consistent with the work of other investigators , exposure to
rosiglitazone inhibited soft agar colony formation, a measure of trans-
formed growth in both NSCLC cell lines (Figure 2C, left panel, top).
Importantly, silencing Snail using two independent shRNA constructs
(A12 and E8), also resulted in a marked decrease in colony formation
(Figure 2C, right panel, top). Rosiglitazone decreased invasion of
H2122 cells and inhibited both migration and invasion of H2009 cells
(Figure 2C). Silencing Snail expression similarly decreased migration
(Figure 2C, right panel, middle) and invasion (Figure 2C, right panel,
bottom) in both cell lines compared with NS cells. Finally, we exam-
ined the growth of NSCLC in three-dimensional Matrigel cultures.
Our previous studies demonstrated that H2122 cells form loose dis-
organized aggregates in three-dimensional culture (Figure 2D, panel a)
like structures . Treatment of NSCLC with rosiglitazone also led
to the formation of organized structures (Figure 2D, panel b). Cells si-
lenced for Snail expression also formed ordered structures that were
indistinguishable from those observed with rosiglitazone (Figure 2D,
panel d), whereas NS controls formed structures analogous to wild-type
cells (Figure 2D, panel c).
Because MMPs are important in promoting invasion through the
extracellular matrix and are involved in tumor invasion and progression
, we determined whether the decreased migratory and invasive
properties of cells in response to PPARγ activation or silencing of Snail
were associated with changes in the activity or expression of MMPs. As
shown in Figure 3A (top panel), rosiglitazone markedly decreased the
mRNA expression of MMP-9 in H2122 cells and modestly decreased
the expression in H2009 cells. No significant effects were seen on
MMP-2 expression (Figure 3A, bottom panel). By zymography, expo-
sure to rosiglitazone was associated with a marked decrease in the ac-
tivity of both MMP-9 and MMP-2 in both cell lines (Figure 3C, left
panel), indicating that PPARγ activation of MMPs involves additional
posttranscriptional mechanisms. Silencing of Snail had similar effects
in both cell lines. Levels of MMP-9 were decreased in both cell lines
(Figure 3B, top panel), with minimal effects on MMP-2 mRNA (Fig-
ure 3B, bottom panel) compared with NS controls. Zymography indi-
cated that silencing Snail selectively decreased MMP-9 in H2122 cells
while modestly decreasing both MMP-9 and MMP-2 in H2009 cells
(Figure 3C, right panel).
Snail Overexpression Blocks Biological Responses
To complement the studies silencing expression Snail in NSCLC,
we stably overexpressed the protein in the two NSCLC cell lines. Mul-
tiple clones of stably transfected cells were selected by immunoblot
analysis. As shown in Figure 4A, cells transfectants showed an in-
crease inSnail proteinexpression comparedwith empty vector controls,
and expression was largely localized to the nucleus (Figure 4B). As
PPARγ Activation Suppresses Snail Expression in NSCLCChoudhary et al.Neoplasia Vol. 12, No. 3, 2010
expected, Snail-overexpressing cells had lower levels of E-cadherin pro-
tein (Figure 4C) and decreased E-cadherin promoter activity (data
not shown). Rosiglitazone reduced Snail expression in the empty vec-
tor controls but had no effect on Snail expression in the overexpressing
In two independent clones of H2122 cells, Snail-overexpressing
cells showed a marked increase in colony formation in soft agar com-
pared with empty vector control (Figure 5A, top). In H2009, there was
a more modest but still statistically significant increase (Figure 5A, top
right). Importantly, whereas rosiglitazone decreased colony formation
Figure 1. Rosiglitazone selectively decreases expression of Snail in NSCLC. (A) Left panel: H2122 or H2009 cells treated with rosiglitazone
or vehicle (0.1% DMSO) for the indicated times were immunoblotted for Snail expression. Levels of β-actin were used as a loading control.
panel) was determined by real-time PCR. (C) Cells were transiently transfected with constructs encoding either the Snail promoter, the Slug
promoter, or the ZEB1 promoter, ligated to a luciferase reporter, along with a plasmid encoding β-gal under the control of CMV promoter to
normalize for transfection efficiency. Cells were stimulated with rosiglitazone for 24 hours, and luciferase activity normalized to β-gal activ-
ity was determined. Results are presented as percent of control for each promoter. **P < .01 versus controls, ***P < .001 versus controls.
All data represent the means of three independent experiments. (D) H2122 cells overexpressing PPARγ (H2122-PPARγ) or empty vector
(H2122-LNCX) were grown under standard conditions. Nuclear extracts were prepared as described in Materials and Methods and immuno-
blotted for Snail (left panel) or Slug (right panel) expression. Levels of β-actin were used as a loading control. Results are representative of
three independent experiments.
Neoplasia Vol. 12, No. 3, 2010 PPARγ Activation Suppresses Snail Expression in NSCLC Choudhary et al.
PPARγ Activation Suppresses Snail Expression in NSCLCChoudhary et al.Neoplasia Vol. 12, No. 3, 2010