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gli2 and m-mitF transcription factors control exclusivegene expression programs and inversely regulate invasionin human melanoma cellsdelphine Javelaud, Vasileia-ismini alexaki,marie-Jeanne Pierrat, Keith s. hoek, sylviane dennler,leon Van Kempen, Corine Bertolotto, robert Ballotti,simon saule, Ve´ronique delmas and alain mauviel
submit your next paper to PCmr online at http://mc.manuscriptcentral.com/pcmr
doi: 10.1111/j.1755-148X.2011.00893.xVolume 24, issue 5, Pages 932-943
GLI2 and M-MITF transcription factors controlexclusive gene expression programs and inverselyregulate invasion in human melanoma cellsDelphine Javelaud1,2,3,4*, Vasileia-Ismini Alexaki1,2,3,4*, Marie-Jeanne Pierrat1,2,3,4*, Keith S. Hoek5,Sylviane Dennler1,2,3,4, Leon Van Kempen6, Corine Bertolotto7, Robert Ballotti7, Simon Saule1,2,3,4,Veronique Delmas1,2,3,4 and Alain Mauviel1,2,3,4
1 Institut Curie, Centre de Recherche, Orsay, France 2 INSERM U1021, Orsay, France 3 CNRS UMR3347, Orsay,France 4 Universit� Paris XI, Orsay, France 5 Department of Dermatology, University Hospital of Zurich, Zurich,Switzerland 6 Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands7 INSERM U895, Nice, France
CORRESPONDENCE Alain Mauviel, e-mail: [email protected]
*These three authors contributed equally to the work.
KEYWORDS GLI2 ⁄ M-MITF ⁄ TGF-b ⁄ Protein
kinase A ⁄ invasion ⁄ gene knock-down
PUBLICATION DATA Received 15 November 2010,revised and accepted for publication 27 July 2011,published online 1 August 2011
doi: 10.1111/j.1755-148X.2011.00893.x
Summary
We recently identified GLI2, the most active of GLI transcription factors, as a direct TGF-b ⁄ SMAD target,
whose expression in melanoma cells is associated with increased invasiveness and metastatic capacity. In this
work, we provide evidence that high GLI2 expression is inversely correlated with that of the melanocyte-
specific transcription factor M-microphthalmia transcription factor (M-MITF) and associated transcriptional
program. GLI2-expressing cell lines were characterized by the loss of M-MITF-dependent melanocytic
differentiation markers and reduced pigmentation. The balance between M-MITF and GLI2 expression did not
correlate with the presence or absence of BRAF-activating mutations, but rather was controlled by two
distinct pathways: the TGF-b pathway, which favors GLI2 expression, and the protein kinase A (PKA) ⁄ cAMP
pathway, which pushes the balance toward high M-MITF expression. Furthermore, overexpression and
knockdown experiments demonstrated that GLI2 and M-MITF reciprocally repress each other’s expression and
control melanoma cell invasion in an opposite manner. These findings thus identify GLI2 as a critical
transcription factor antagonizing M-MITF function to promote melanoma cell phenotypic plasticity and inva-
sive behavior.
Introduction
M-microphthalmia transcription factor (M-MITF), a mem-
ber of the basic Helix-Loop-Helix Leucine Zipper family
of MITF transcription factors, is a critical master-switch
controlling differentiation, morphology, proliferation, and
survival of cells in the melanocyte lineage, from embry-
onic development through malignant transformation and
melanoma progression to metastasis (Levy et al., 2006;
Steingrimsson et al., 2004). M-MITF plays a major role
Significance
The GLI2 transcription factor is a critical mediator of the Hedgehog pathway. We recently identified GLI2
as a TGF-b gene target that controls melanoma cell propensity to metastasize, and whose expression in
melanoma is associated with disease progression. The melanocyte-specific isoform of microphthalmia-
associated transcription factor (M-MITF) is both critical for melanocyte survival and differentiation and for
melanoma progression. In this report, we provide evidence that GLI2 and M-MITF have an exclusive
expression pattern in melanoma cells and demonstrate that GLI2 and M-MITF antagonize each other and
control melanoma cell invasion in an opposite manner.
932 ª 2011 John Wiley & Sons A/S
Pigment Cell Melanoma Res. 24; 932–943 ORIGINAL ARTICLE
in melanoblast and melanocyte migration, as well as in
terminal differentiation by controlling the expression of
tyrosinase (TYR) and other enzymes involved in melano-
genesis. Elevation of intracellular cAMP levels, for
example, as a result of MC1R activation, in melanocytes
results in protein kinase A (PKA) activation, and subse-
quent CREB-dependent M-MITF transcription, leading to
melanogenesis and subsequent skin pigmentation
(Bertolotto et al., 1998). In melanoma, the role of M-MITF
is complex and likely depends on its expression levels
together with concomitantly occurring genetic alterations
such as the frequently occurring activating mutations of
B-RAF and N-RAS (Levy et al., 2006; Steingrimsson
et al., 2004). M-MITF contributes to cell proliferation via
transcriptional regulation of Dia1, CDK2, p21, and p16
(Carreira et al., 2005, 2006; Du et al., 2004). Yet, the role
of M-MITF may be rheostat-like, whereby elevated
expression of M-MITF may also reduce melanoma cell
proliferation and invasiveness (Carreira et al., 2005,
2006; Wellbrock and Marais, 2005), while reduced
M-MITF levels may provide a growth advantage to some
melanoma cells, for example, through redistribution of
energy and oxidative stress normally associated with
pigment biosynthesis (Selzer et al., 2002; Vachtenheim
et al., 2001) and increased invasiveness (Carreira et al.,
2006). In addition to regulating cell growth, M-MITF is
also implicated in melanoma resistance to apoptosis via
transcriptional activation of BCL2 and ML-IAP (Dynek
et al., 2008; McGill et al., 2002). The complexity of
M-MITF’s role is further emphasized by the fact that in
patients with melanoma, M-MITF may either be ampli-
fied or lost (Levy et al., 2006; Steingrimsson et al.,
2004). Finally, studies of melanoma cell plasticity have
suggested a critical role for M-MITF in a process
described as phenotype switching. Specifically, in vitro
and in vivo experiments have linked M-MITF expression
to a so-called proliferative signature, while its absence
correlates with an invasive signature (Hoek et al., 2006).
It is thought that by switching back-and-forth between
these phenotypes through modulation of M-MITF
expression, melanoma is driven to progress (Hoek and
Goding, 2010).
GLI transcription factors were initially identified as
effectors of the Hedgehog (HH) family of growth factors
(Kasper et al., 2006). GLIs are often overexpressed in
cancers and are implicated in the progression of a vari-
ety of neoplasms via regulation of cell cycle progression
and apoptosis (McMahon et al., 2003; Ruiz i Altaba
et al., 2002). We recently identified GLI2, the most
transcriptionally active member of GLIs, as a direct
target of the TGF-b pathway (Dennler et al., 2007,
2009). The latter plays an important role in melanoma
development (Javelaud et al., 2008), and both systemic
and tumor cell-specific inhibition of TGF-b signaling
efficiently prevent melanoma progression in various
experimental models (Divito et al., 2010; Javelaud et al.,
2005, 2007; Mohammad et al., 2011). Recently, we
found that high GLI2 expression in melanoma is associ-
ated with increased aggressiveness and metastatic
potential via acquisition of a mesenchymal phenotype
characterized by the loss of E-cadherin expression and
enhanced MMP2 and MMP9 production (Alexaki et al.,
2010). Similar specific changes in gene expression were
found in human melanoma tumors, suggesting that
GLI2 may represent a marker of poor prognosis for
patients with melanoma (Alexaki et al., 2010). GLI2 may
also contribute to tumor progression and metastasis in
other cancer types [reviewed in (Javelaud et al., 2011)].
In this report, we provide evidence that high GLI2
expression in melanoma cells is associated with
reduced M-MITF levels and subsequent reduction in the
expression of melanocytic differentiation markers and
other M-MITF target genes. We identify the TGF-b and
cAMP ⁄ protein kinase A (PKA) pathways as antagonistic
modulators of GLI2 and M-MITF levels in melanoma
cells, and demonstrate that GLI2 and M-MITF exert
opposite regulatory activity against each other to control
melanoma cell invasiveness.
Results
GLI2 expression in melanoma is associated with
reduced M-MITF and M-MITF-dependent gene
expression
We used quantitative real-time RT-PCR to determine
GLI2 and M-MITF expression levels in a panel of human
melanoma cell lines in culture. As shown in Figure 1A,
B, cell lines expressing high levels of M-MITF (SK28,
888mel, 501mel, WM983A, and WM983B) had low
GLI2 expression. Inversely, cell lines with high GLI2
transcript levels (Dauv1, WM852, 1205Lu, and WM793
cells lines) expressed about 100-fold less M-MITF
mRNA than 888mel, 501mel, or SK28 cell lines. Normal
melanocytes expressed both M-MITF and GLI2. Low
GLI2 mRNA levels were detected in SK28, WM983A,
and WM983B cell lines, together with high M-MITF
expression.
Western analysis demonstrated that GLI2 protein
was abundant in WM852, 1205Lu, and WM793 cells
(Figure 1C, lanes 1–3), while undetectable in 888mel, SK28,
WM983A, WM983B, and 501mel cells (lanes 4–8).
M-MITF protein levels were opposite to those of GLI2
(Figure 1C, center panel). Thus, the GLI2 and M-MITF
expression pattern in all melanoma cell lines showed
parallel variations at both mRNA and protein steady-
state levels. Notably, melanoma cell lines that strongly
expressed GLI2 synthesized little, if any, melanin
(Figure 1D), consistent with their low M-MITF levels.
Data mining from Affymetrix cDNA microarray whole
genome expression profiling of 125 distinct human mel-
anoma cell lines (Hoek et al., 2006) confirmed these
results: gene expression data sorted according to GLI2
transcript levels identified an inverse correlation
between GLI2 expression and that of M-MITF (Figure 2,
Antagonistic roles of GLI2 and M-MITF in melanoma cells
ª 2011 John Wiley & Sons A/S 933
upper left panel) and the pigmentation-related genes
TYR, MLANA, SILV, TRP1, and DCT (Figure 2). Notably,
cell lines with a normalized intensity of 2 and above for
GLI2 expression had almost undetectable levels of
M-MITF, TYR, MLANA, SILV, TRP-1, and DCT transcripts
(Figure 2, orange frame).
250300350400450
050
100150200
M-M
ITF/
GA
PD
H m
RN
A
SK
28
Dau
v1
888m
el
501m
el
FO-1
WM
852
1205
Lu
WM
793
NH
EM
WM
983B
WM
983A
A
100150200250300350400450500
050
SK
28
Dau
v1
888m
el
501m
el
FO-1
WM
983A
1205
Lu
WM
793
NH
EM
GLI
2/G
AP
DH
mR
NA
WM
852
WM
983B
B
1 2 3 4 5 6 7 8
GAPDH
GLI2
M-MITFA-MITF
WM
983A
WM
983B
1205
Lu
WM
793
WM
852
888m
elS
K28
501m
el
C
0.100.120.140.16
00.020.040.060.08
Mel
anin
(arb
itary
uni
ts)
888m
el
WM
983A
501m
el
SK
28
WM
983B
WM
852
1205
Lu
WM
793
D
Figure 1. GLI2 and M-MITF expression are largely exclusive in melanoma cells. Quantitative real-time RT-PCR analysis of M-MITF (A) and
GLI2 (B) expression in a panel of ten different melanoma cell lines as compared to normal human embryonic melanocytes (NHEM). GLI2 and
M-MITF expression was normalized against that of GAPDH. (C) Western analysis for GLI2 and M-microphthalmia transcription factor (M-MITF)
in eight melanoma cell lines (from the 10 shown in panels A and B). GAPDH levels were used as control. (D) Melanin concentration was
measured in several melanoma cell lines. Data were normalized against total cell protein content. All experiments were performed three
times, a representative experiment is shown. Cell lines expressing high levels of GLI2 are marked in red, and cell lines expressing high levels
of M-MITF are in blue. It should be noted that SK28, WM983A, and WM983B cell lines express low but detectable GLI2 levels despite high
M-MITF expression and that cell lines expressing high levels of GLI2 transcripts (1205Lu, WM793, and WM852) are not pigmented.
8
10
12 GLI2MITF
TYR
4567
0
2
4
6
Melanoma cell lines01
23
Nor
mal
ized
sig
nal i
nten
sity
Melanoma cell lines
MLANA SILVMLANA
234567
SILV
2
3
4
5
6
01
Nor
mal
ized
sig
nal i
nten
sity
Nor
mal
ized
sig
nal i
nten
sity
Nor
mal
ized
sig
nal i
nten
sity
Melanoma cell lines
TRP1
100120140
01
Nor
mal
ized
sig
nal i
nten
sity
Melanoma cell lines
DCT
67
020406080
Melanoma cell lines012345
Nor
mal
ized
sig
nal i
nten
sity
Melanoma cell lines
Figure 2. GLI2 inversely correlates with
M-MITF and pigmentation-related genes
expression in a cohort of human
melanoma cell lines. Whole genome gene
expression profiling was performed in a
panel of 125 human melanoma cell lines,
using Affymetrix pan-genome cDNA
microarray hybridization. Cell lines were
sorted according to increasing normalized
signal intensity for GLI2. Results for GLI2
(in red), M-MITF (in blue), TYR, MLANA,
SILV, TRP1, and DCT are shown. It should
be noted that the point of inflexion toward
dramatically higher GLI2 expression
(normalized signal intensity >2) coincides
with the loss of expression of M-MITF,
TYR, MLANA, SILV, TRP1, and DCT.
Javelaud et al.
934 ª 2011 John Wiley & Sons A/S
Expression levels of GLI2 and M-MITF were indepen-
dent from the presence of activating mutations of either
BRAF or NRAS, as (i), all melanoma cell lines used for
PCR analysis of gene expression (Figure 1) carry the
V600E-activating mutation of BRAF, except for the
WM852 cell line that carries the Q61R NRAS mutation
(Alexaki et al., 2010; Dumaz et al., 2006), and (ii),
GLI2- and M-MITF-dependent gene signatures in DNA
microarrays are independent from BRAF mutations, as
determined previously (Hoek et al., 2006).
To find genes whose expression patterns show
strong and reproducible correlation or anticorrelation
with GLI2 expression, we compared six distinct data
sets obtained from GEO and generated by several
research groups all using Affymetrix HG-U133 series
platforms to examine in vitro gene expression in mela-
noma cell cultures, as previously described in the
context of the identification of novel M-MITF gene tar-
gets (Hoek et al., 2008). We assessed correlation and
anticorrelation by performing a Pearson correlation and
anticorrelation analyses on each probe set in compari-
son with GLI2 across all samples of each data set. One
hundred and eight different probe sets (79 genes) were
detected that shared a correlation coefficient of at least
0.5 with 207034_s_at (GLI2) in at least three of six data
sets (Table S3). Among the genes exhibiting a positive
correlation with GLI2 were CDH2 and SNAI1, consistent
with our previous observations (Alexaki et al., 2010). On
the other hand, 86 distinct probe sets (69 genes) had a
correlation coefficient below )0.5 (i.e., anticorrelation
coefficient above 0.5) with GLI2 (Table S4). CDH1 was
one of these genes, consistent with recent published
work from our laboratory (Alexaki et al., 2010). Also,
among these genes, at least 25 genes were previously
characterized as M-MITF targets [RAB27A, CDK2, TYR,
MLANA, SILV, SLC45A2, CAPN3, SORT1, PLXNC1,
ASAH1, and HPS4 (Hoek et al., 2008)], some of them
involved in pigmentation (TYR, MLANA, and SILV), sug-
gesting that transcription programs driven by GLI2 and
M-MITF are somehow exclusive. Semi-quantitative PCR
confirmed this hypothesis: cell lines with high GLI2
mRNA levels exhibited low expression of M-MITF and
M-MITF-dependent genes (Figure 3), while expression
of BMP4 and WNT5A, previously identified as GLI2
transcriptional targets (Eichberger et al., 2006), was aug-
mented in melanoma cell lines expressing high levels of
GLI2 (Figure 3).
Expression levels of GLI2 and M-MITF do not
correlate with cell proliferation in vitro or with
subcutaneous tumor growth in vivo
CDK2 expression was slightly higher in melanoma cell
lines expressing high levels of M-MITF, as compared to
those expressing GLI2 (Figure 4A), consistent with its
identification as a M-MITF target (Du et al., 2004).
CDK2 was also found in the group of genes identified
from DNA microarray data mining as inversely corre-
lated with GLI2 (see Table S4). Expression of other cell
cycle–associated genes p16, p21, p27, and CDK4 did
not follow a pattern associated with that of either GLI2
or M-MITF. As expected from melanoma cells, a num-
ber of the cell lines exhibited a loss of p16 expression,
not associated with either GLI2 or M-MITF expression.
Unlike CDK2, expression of CCND1 somewhat
paralleled that of GLI2 (Figure 4A), consistent with its
previous identification as a GLI target (Eichberger et al.,
2006). Yet, its expression was also detected in cells
lines with little GLI2 transcripts, SK28, 888mel, and
501mel.
We next determined the growth rate of each of the
melanoma cell lines by MTS assay. Results shown in
Figure 4B indicate that cell lines expressing high levels
of either GLI2 or M-MITF could not be differentiated
based on their proliferative capacity in vitro (Figure 4B).
These data were independently validated by L. Larue’s
laboratory (Institut Curie), as BrdU incorporation experi-
ments in four GLI2low (FO-1, 888mel, SK28, and SK29)
versus four GLI2high cell lines (1205Lu, WM852,
Dauv-1, and Quar) showed no differences between the
GAPDH
M-MITF
GLI2
1 2 3 4 5 6 7 8 9 10 11
TYR
DCT
GAPDH
GAPDH
GAPDH
TRP-1GAPDH
SK
28
Dau
v1
WM
852
888m
el
501m
el
FO-1
1205
Lu
WM
793
NH
EM
WM
983A
WM
983B
Figure 3. Exclusive expression pattern for GLI2 and M-MITF
target genes in human melanoma cells. GLI2, M-MITF, TYR, DCT,
and TRP1 mRNA expression was estimated by semi-quantitative
multiplex PCR together with that of GAPDH used as a control in 10
distinct human melanoma cell lines (lanes 1–10) and in normal
human epidermal melanocytes (NHEM, lane 11). For convenience,
lanes corresponding to cell lines expressing high levels of GLI2 are
framed in red. It should be noted that the absence of signal for
M-MITF and M-MITF-dependent gene targets in cell lines
expressing high levels of GLI2.
Antagonistic roles of GLI2 and M-MITF in melanoma cells
ª 2011 John Wiley & Sons A/S 935
two groups (not shown, Larue and coll., personal com-
munication).
We next modulated GLI2 activity and M-MITF expres-
sion levels in 888mel, SK28, and WM983A cells (all
of which express detectable levels of both GLI2 and
M-MITF) to determine whether this would influence
their proliferation capacity. Firstly, we found that stable
overexpression of the constitutively active GLI2DN2 in
888mel cells did not significantly affect their growth in
vitro (doubling time for mock-transfected cells:
47.7 ± 0.24 versus 45.9 ± 1.29 for GLI2DN2-transfected
cells). Likewise, stable knockdown of either M-MITF or
GLI2 in both SK28 and WM983A cells did not modify
cell proliferation in vitro: doubling time was 39.3 ±
0.4 hours for control shRNA-transduced SK28 cells
versus 38.2 ± 2.2, and 39.1 ± 0.1 after M-MITF and
GLI2 knockdown, respectively; mock shRNA-WM983A
doubling time was 71.2 + 2.6 versus 77.1 + 4.5 and
82.45 + 1 for shMITF- and shGLI2-transduced cells,
respectively.
Subsequently, we measured subcutaneous tumor
growth in nude mice of transplanted melanoma cells
expressing various levels of expression of either GLI2 or
M-MITF. As shown in Figure 4C, mice injected with
Daju, SK28, and SK29 cells developed tumors with
tumor take comprised between 75 and 100%, and a
latency comprised between 25 and 35 days, while mice
injected with 888mel and 501mel cells did not, although
these cell lines have similar low levels of GLI2 and high
levels of M-MITF. Similar heterogeneity was found with
cell lines expressing high levels of GLI2. Specifically,
Gerlach and 1205Lu cells exhibited 100% tumor take
with latencies of 60 and 20 days respectively, while only
50% of mice injected with Dauv1 cells and none of
GLI2
1 2 3 4 5 6 7 8 9 10 11GAPDH
9
CDK2GAPDH
p16
GAPDHCCND1
45678
SK28Dauv1888mel
WM852WM983BWM983A1205LuWM793
p21GAPDH
p27GAPDH
CDK4GAPDH
0123
0 2 4 6 8Time (days)
Rel
ativ
e M
TS a
ctiv
ity
501melFO-1
Tumor take Latency(%) (days)
SK
28D
auv1
WM
852
888m
el50
1mel
FO-1
1205
LuW
M79
3N
HE
M
WM
983A
WM
983B
(%) (days)Daju 100 25+/–5Gerlach 100 60 +/–151205lu 95 20+/–5SK28SK29
50 100+/Dauv1 –20888mel501melQuar
90 35+/–575 30 +/–5
0 nd0 nd0 nd
GAPDH
GLI2
Daj
u
Gue
rlach
1205
lu
SK
28
SK
29
Dau
v1
888m
el
501m
el
Qua
r
A B
C
Figure 4. GLI2 and ⁄ or M-MITF
expression levels are not predictive of
either melanoma cell proliferation rate in
vitro or subcutaneous tumor growth in
vivo. (A) GLI2, CCND1, CDK2, p16, p27,
p21, and CDK4 mRNA expression was
estimated by semi-quantitative multiplex
PCR with GAPDH in 10 distinct human
melanoma cell lines (lanes 1–10) and in
normal human epidermal melanocytes
(NHEM, lane 11). (B) Melanoma cell lines
were seeded at the same initial
concentration in full serum conditions, and
cell growth was measured over an 8-day
period using an MTS assay. These
experiments were performed twice or
three times with each cell line, using
triplicate dishes. Standard error was below
or around 5%. (C) Melanoma cells
(1 · 106) were injected subcutaneously
into nude mice, and tumor growth was
followed over a 100-day period. Values for
tumor take and latency are the mean ± SE
of n ‡ 10 for each group. Relative GLI2
levels in each cell line were measured by
semi-quantitative RT-PCR.
Javelaud et al.
936 ª 2011 John Wiley & Sons A/S
those injected with the Quar cell line developed tumors.
Thus, similar to growth rate in vitro, there was no cor-
relation between the GLI2 ⁄ M-MITF expression status in
melanoma cells and their capacity to form subcutaneous
tumors in nude mice.
The TGF-b ⁄ SMAD and PKA ⁄ cAMP pathways
inversely control the GLI2 ⁄ M-MITF expression
balance in melanoma cells
Both the cAMP and TGF-b ⁄ SMAD signaling cascades
are important for melanocyte development, differentia-
tion, and transformation, and have the potential to regu-
late GLI2 and M-MITF expression and ⁄ or activity. We
thus tested the hypothesis that both the cAMP and the
TGF-b ⁄ SMAD pathways may contribute in an opposite
manner to the GLI2 ⁄ M-MITF expression balance identi-
fied above in malignant melanocytes.
To assess the implication of autocrine TGF-b signaling
on GLI2 ⁄ M-MITF expression, 1205Lu (which express
high levels of GLI2 and low levels of M-MITF) and
501mel (which express low levels of GLI2 and high
levels of M-MITF) cells were incubated for 48 h with
the TbRI kinase inhibitor SB431542. A 3- to 4-fold reduc-
tion in GLI2 mRNA steady-state levels was measured
by quantitative RT-PCR, concomitant with a 2-fold eleva-
tion of M-MITF expression (Figure 5A). Inversely, incu-
bation of 1205Lu and 501mel cells with exogenous
TGF-b leads to a 7- and 30-fold elevation of GLI2 mRNA
steady-state levels, respectively, accompanied with a
reduction in M-MITF expression (Figure 5B). Similar
TGF-ββ
4
SB4315424 GLI2 GLI2
1
2
3
2
34
4
1
2
3
3
2
MITF MITF
1205Lu 501mel4
ForskolinH89
×7
3
4
1205Lu 501mel
Fold
indu
ctio
n/re
pres
sion
Fold
indu
ctio
n/re
pres
sion
Fold
indu
ctio
n/re
pres
sion
Fold
indu
ctio
n/re
pres
sion
4
2
GLI2MITF
GLI2MITF
1
2
3
2
4
5
1
2
1205Lu 501mel 1205Lu 501mel
6
8
10
100120140160180
TGF-βForskolin
––
+–
+
+
–+
WM983A
0
2
4
GLI
2/G
AP
DH
mR
NA
TGF-βForskolin
–
–
+
–
+
+
–
+WM983A
20406080
0
M-M
ITF/
GA
PD
Hm
RN
A
A B
C D
E F
×30
Figure 5. The GLI2 ⁄ M-MITF expression
balance is controlled by the TGF-b ⁄ SMAD
and PKA ⁄ cAMP pathways. 1205Lu and ⁄ or
501mel cells were incubated with 5 lM
SB-431542 (A) or 10 lM H-89 (C) for 48 h,
and with 10 ng ⁄ ml TGF-b and 20 lM
forskolin alone (B, D) or in combination in
WM983A cells (E, F) for 8 h in low(1%)-
serum medium. GLI2 and M-MITF
expression was measured by quantitative
RT-PCR and normalized to that of GAPDH.
Experiments were performed at least
twice in triplicates. Representative results
(mean ± SE) are shown. GLI2 expression
is shown in red, M-MITF in blue.
Antagonistic roles of GLI2 and M-MITF in melanoma cells
ª 2011 John Wiley & Sons A/S 937
results were obtained with another GLI2-positive mela-
noma cell line, WM852, and with the highly pigmented
cell line 888mel (not shown).
Inhibition of the cAMP pathway with the protein
kinase A inhibitor H89 reduced M-MITF expression and
concomitantly upregulated GLI2 expression in both
1205Lu and 501mel cell lines (Figure 5C). On the other
hand, forskolin, an activator of adenylate cyclase that
promotes cAMP accumulation, induced M-MITF expres-
sion in both 1205Lu and 501mel melanoma cell lines,
while reducing GLI2 mRNA levels (Figure 5D). Similar
results were obtained with the WM852 and 888mel cell
lines (not shown). In experiments whereby TGF-b and
forskolin were used concomitantly to stimulate
WM983B cells that express detectable basal levels of
both GLI2 and M-MITF, forskolin inhibited the induction
of GLI2 expression elicited by TGF-b in Figure 5E. Inver-
sely, TGF-b antagonized forskolin-induced M-MITF
expression (Figure 5F). Noteworthy, in 1205Lu, 501mel,
and 888mel melanoma cell lines, a 24-h incubation with
TGF-b reduced both basal and forskolin-induced PKA
activity and CREB phosphorylation by approximately
40–50%, accompanied by similarly reduced CRE-driven
transcription in transient cell transfection experiments
(not shown). On the other hand, di-butyryl cAMP and
forskolin inhibited SMAD-driven transcription in mela-
noma cells (not shown), consistent with our previous
demonstration that cAMP-elevating agents antagonize
SMAD-driven gene expression in fibroblasts and keratino-
cytes (Schiller et al., 2003, 2010). Thus, the TGF-b and
PKA pathways reciprocally antagonize each other to
modulate M-MITF and GLI2 levels in melanoma cells.
GLI2 and M-MITF reciprocally inhibit their
expression and inversely control melanoma cell
invasion
The opposite expression pattern of GLI2- and M-MITF-
dependent gene signatures in melanoma cell lines
cannot solely be explained by an imbalance of environ-
mental factors affecting the TGF-b and cAMP pathways
which modulate GLI2 and M-MITF gene expression in
an antagonistic fashion. We tested the hypothesis that
GLI2 and M-MITF may also functionally antagonize each
other directly, thereby providing an amplification mecha-
nism for upstream signals. Using transient cell transfec-
tion assays, we first altered GLI2 function by
overexpressing GLI2DN2, a constitutively active form of
GLI2, in SK28 cells, to determine its effect on M-MITF
transcription. As shown in Figure 6A, GLI2DN2 effi-
ciently increased GLI-dependent transcription (left panel)
while inhibiting M-MITF promoter activity by approxi-
mately 65% (center panel). Stable transfection of
888Mel cells with GLI2DN2 led to similar results (not
shown) and to a dramatic increase in their capacity to
invade Matrigel (right panel).
To determine whether M-MITF is a modulator of GLI2
expression, 1205Lu cells, that express very little
M-MITF (see Figure 1), were infected with recombinant
adenoviruses expressing either GFP as a control, or
M-MITF coupled with GFP. In each case, adenoviral
1400 12040
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A
B
C
Figure 6. Antagonistic activities of GLI2 and M-MITF. (A) SK28 cells
were transiently transfected with either empty (pc) or constitutively
active GLI2 mutant (GLI2DN2) expression vector. GLI-specific
reporter activity (left panel) or M-MITF promoter activity (center
panel) was measured by luciferase assay. Matrigel invasion was
determined in parallel dishes (right panel). (B) 1205Lu cells were
infected with adenoviruses expressing either GFP or M-MITF. M-
MITF (left) and GLI2 (center) mRNA levels were measured by
quantitative RT-PCR and normalized to GAPDH expression. Matrigel
invasion was determined in parallel dishes (right). (C) 501mel cells
were transduced with either a non-targeting shRNA lentiviral vector
(shc), or with an shRNA vector targeting M-MITF (shMITF). M-MITF
(left) and GLI2 (center) mRNA levels were measured by quantitative
RT-PCR and normalized to GAPDH transcript levels. Matrigel invasion
was determined in parallel dishes (right). Experiments were carried
out at least twice in triplicate dishes. Representative experiments are
shown.
Javelaud et al.
938 ª 2011 John Wiley & Sons A/S
infection was in the range of 80–90%, as estimated by
GFP fluorescence (not shown). High M-MITF expres-
sion, verified by quantitative real-time RT-PCR (Fig-
ure 6B, left panel) and Western blotting (not shown),
resulted in a dramatic reduction (approximately 95%) in
GLI2 mRNA steady-state levels, as measured using
quantitative real-time RT-PCR (Figure 6B, center panel),
while expression of the M-MITF target genes TYR and
DCT was increased [·6.7- and ·7-fold respectively,
DCt(TYR): 35.92 ± 0.74 in Ad-GFP-infected 1205Lu cells
versus 32.64 ± 0.01 in AdM-MITF-infected 1205Lu cells;
DCt(DCT): 30.19 ± 0.01 in Ad-GFP-infected cells versus
27.38 ± 0.17 in AdM-MITF-infected 1205Lu cells]. Over-
expression of M-MITF also inhibited 1205Lu cell capac-
ity to invade Matrigel by approximately 50% (Figure 6B,
right panel). Inversely, in 501mel cells, which express lit-
tle GLI2, M-MITF knockdown by specific shRNA expres-
sion (Figure 6C, left panel) resulted in a 10-fold increase
in GLI2 expression (Figure 6C, center panel) associated
with an approx. threefold increase in Matrigel invasion
(right panel). Likewise, expression of a dominant-nega-
tive form of M-MITF via adenoviral infection allowed a
solid increase (approximately 8-fold) in GLI2 mRNA
steady-state levels, associated with reduced TYR and
DCT expression (8.64- and 1.8-fold, respectively,
DCt(TYR): 28.24 ± 0.26 in Ad-GFP-infected 501mel cells
versus 31.34 ± 0.14 in AdDN-M-MITF-infected 501mel
cells; DCt(DCT): 17.61 ± 0.11 in Ad-GFP-infected cells
versus 18.51 ± 0.4 in AdDN-M-MITF-infected 501mel
cells).
In SK28 melanoma cells, which express detectable
levels of both GLI2 and M-MITF, GLI2 knockdown
resulted in increased M-MITF, TYR, DCT, and CDK2
expression, together with reduced WNT5A expression
(2.5-fold). Inversely, M-MITF knockdown resulted in
increased GLI2 and WNT5A expression (·4.5- and ·
3.9-fold, respectively), associated with reduced DCT and
CDK2 expression (2.6- and 4.3-fold, respectively).
M-MITF attenuates TGF-b-induced GLI2 expression
We previously demonstrated that GLI2 is an immediate-
early gene downstream of TGF-beta signaling in a
variety of cell types, including melanoma cells (Dennler
et al., 2007). Yet, in the melanocyte lineage, M-MITF
may interfere and exert a repressory activity on TGF-
b-driven gene expression. To ascertain this hypothesis,
we performed several experiments. Firstly, we overex-
pressed M-MITF in 1205Lu cells by means of adenoviral
vector transduction. Under these conditions, GLI2 induc-
tion by TGF-b was considerably reduced as compared to
that measured in mock-transduced 1205Lu cells (Fig-
ure 7A). Secondly, expression of a dominant-negative
form of M-MITF in 501mel cells resulted in enhanced
activation of GLI2 expression in response to TGF-b (Fig-
ure 7B). Thirdly, TGF-b response was examined in
501mel cells after M-MITF knockdown. Non-targeting
shRNA lentivirus-transduced cells were used as control.
In both instances, GLI2 upregulation was observed in
response to TGF-b, yet the amplitude of TGF-b response
was strongly enhanced in cells with reduced M-MITF
expression (Figure 7C).
Taken together, these experiments demonstrate that
GLI2 and M-MITF antagonize each other and that
M-MITF reduces the capacity of TGF-b to induce GLI2
expression in melanoma cells.
Discussion
In this report, we provide definitive evidence for exclu-
sive expression of GLI2 and M-MITF, and associated
transcriptional programs, in melanoma cells. While we
recently demonstrated a critical role for GLI2 in driving
Ad-GFP Ad-M-MITF
15
20
25
PD
Hm
RN
A
Ad-GFP Ad-MI-DN
1.5
2.0
2.5
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mR
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shc shMITF
1.5
2.0
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mR
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TGF-β0
5
10
– + – +
GLI
2/G
A
TGF-β0.0
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– + – +
GLI
2/G
A P
TGF-β0.0
0.5
1.0
– + +
GLI
2/G
A P
–
A B C
Figure 7. M-microphthalmia transcription factor (M-MITF) antagonizes TGF-b-induced GLI2 expression. (A) 1205Lu cells were infected with
adenoviruses expressing either GFP or M-MITF. After 48 h of infection, cells were treated (+) or not ()) with TGF-b for 4 h, after which GLI2
mRNA levels were measured by quantitative RT-PCR and normalized to GAPDH expression. (B) 501mel cells were infected with adenoviruses
expressing either GFP or a dominant-negative form of M-MITF (MI-DN). After 48 h of infection, cells were treated (+) or not ()) with TGF-bfor 4 h, after which GLI2 mRNA levels were measured by quantitative RT-PCR and normalized to GAPDH expression. (C) SK28 cells were
transduced with either a non-targeting shRNA lentiviral vector (shc), or with an shRNA vector targeting M-MITF (shMITF). GLI2 mRNA levels
were measured by quantitative RT-PCR following a 24-h stimulation with TGF-b and normalized to GAPDH transcript levels.
Antagonistic roles of GLI2 and M-MITF in melanoma cells
ª 2011 John Wiley & Sons A/S 939
melanoma invasion and metastasis, by controlling a
mesenchymal transition of melanoma cells, character-
ized by the loss of E-cadherin expression and increased
secretion of metalloproteinases (Alexaki et al., 2010),
the current data further identify GLI2 as a major regula-
tor of melanoma cell behavior, and as a potent inhibitor
of the melanogenic differentiation pathway leading
to melanin biosynthesis. We found that the cAMP
pathway, known to promote pigmentation via M-MITF
upregulation (D’Orazio et al., 2006; Price et al., 1998)
and the TGF-b pathway, which inhibits M-MITF function
(Kim et al., 2004), exert antagonistic activities on GLI2
and M-MITF expression, a phenomenon that is accentu-
ated by a direct repressory activity of GLI2 and M-MITF
against each other. The latter phenomenon is likely to
amplify any imbalance initiated by alterations in the
signals originating from the extracellular milieu and
provides a logical explanation for the remarkably oppo-
site expression of GLI2 and M-MITF found in melanoma
cells. Notably, in all melanoma cell lines tested, both
genes remained inducible by appropriate stimuli, indicat-
ing that low expression levels of either gene did not
reflect epigenetic suppression mechanisms such as
hypermethylation of their regulatory promoter regions,
but rather an absence of proper stimuli capable of
driving their expression (e.g., TGF-b or cAMP-elevating
hormones, either secreted in an autocrine or paracrine
fashion, or present in the microenvironment), which
could subsequently impact on the invasive capacity of
melanoma cells. Our data are consistent with previous
reports of an inhibitory activity of M-MITF on melanoma
cell invasion (Carreira et al., 2006; Goodall et al., 2008)
and with our previous demonstration that GLI2 pro-
motes cell migration, invasion, and metastasis (Alexaki
et al., 2010).
Remarkably, we previously found a 100% penetrance
of bone metastases with melanoma cell lines with high
levels of GLI2 (very low M-MITF levels), while pene-
trance was significantly lower for cell lines with low
GLI2 levels (high M-MITF levels) (Alexaki et al., 2010).
Furthermore, penetrance was drastically reduced when
GLI2 expression was knocked down in the GLI2high
1205Lu cell line, associated with the loss of MMP2
and MMP9 expression. Xenograft growth assays, on
the other hand, could not differentiate GLI2high from
GLI2low cell lines. These data are consistent with
those by Goding and coworkers who previously demon-
strated that M-MITF knockdown in SK28 cells induces
a hyper-invasive phenotype associated with increased
MMP2 expression, while inversely, no tumors were
observed with SK28 cells overexpressing MITF expres-
sion (Carreira et al., 2006). Remarkably, in the latter
study, the authors observed no difference between
MITF- and mock-transfected cell growth in vitro, consis-
tent with our own data indicating that GLI2high and
GLI2low melanoma cell lines (which could also be
described as MITFlow versus MITFhigh) cannot be
segregated upon in vitro growth yet exhibit distinct
behaviors in vivo.
The latter results are in line with those previously
published by Selzer et al. (2002), whereby the authors
used two distinct melanoma cell lines described as
exhibiting very low basal M-MITF expression, and
showed significantly reduced subcutaneous xenograft
tumor growth in SCID mice by MITF-transfected mela-
noma cells as compared to mock-transfected cells.
While M-MITF can act as an oncogene in human
melanoma (Garraway et al., 2005), M-MITF expression
is highly variable across melanoma specimens, and it
has been suggested that M-MITF is commonly downregu-
lated in advanced melanoma, except for those where it
is amplified (Salti et al., 2000; Selzer et al., 2002).
Remarkably, we recently identified GLI2 as a master
controller of a mesenchymal transition in melanoma
cells favoring metastatic dissemination and observed
that GLI2 expression is increased with advancing tumor
stage in melanoma samples (Alexaki et al., 2010). The
current identification of GLI2 as a potent negative regu-
lator of M-MITF expression and function provides a
possible explanation for the loss of M-MITF in a subset
of metastatic melanoma.
Another critical function assigned to M-MITF in mela-
noma is to control cell proliferation, in part via direct
transcriptional activation of CDK2 (Du et al., 2004). We
consistently found that CDK2 expression parallels that
of M-MITF and is inversely correlated with that of GLI2.
However, melanoma cell lines expressing high levels of
GLI2 (and thus low levels of CDK2 and M-MITF) could
not be distinguished according to their proliferative
capacities in vitro or in a subcutaneous xenograft tumor
growth assay in nude mice, suggesting that GLI2 is able
to compensate for diminished M-MITF expression and
maintain the proliferative activity of melanoma cells
while enhancing their invasive capacity. In this context,
we observed elevated levels of CCND1 transcripts in
GLI2-expressing melanoma cell lines. How these two
transcription factors precisely regulate cell cycle pro-
gression in melanoma cells will be investigated in the
future.
In conclusion, we recently identified GLI2 as a master
controller of a mesenchymal transition in melanoma
cells favoring metastatic dissemination. We now provide
clear definitive evidence that GLI2 is a potent negative
regulator of M-MITF function, further emphasizing the
importance of GLI2 in melanoma biology.
Methods
Cell cultures and reagentsHuman melanoma cell lines, described in the study by Alexaki
et al., 2008, 2010; Javelaud et al., 2007; MacDougall et al., 1993;
Moore et al., 2004; Rodeck et al., 1991, 1999; were grown in RPMI
1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS
and antibiotics, at 37�C, 5% CO2 in a humidified atmosphere. The
Javelaud et al.
940 ª 2011 John Wiley & Sons A/S
GLI-specific reporter plasmid (GLI-BS)8-luc and constitutively active
GLI2 mutant, GLI2DN2, and expression vector (Sasaki et al., 1997,
1999) were gifts from H. Sasaki (Osaka University). Adenoviruses
expressing human M-MITF and the human M-MITF promoter con-
struct have been described previously (Bertolotto et al., 1996;
Gaggioli et al., 2003). The pRL-TK vector was from Promega
(Madison, WI, USA). Forskolin, H89, and the ALK5 ⁄ TbRI inhibitor
SB431542 were purchased from Sigma-Aldrich (St Louis, MO,
USA). TGF-b1 was purchased from R&D Systems Inc. (Minneapolis,
MN, USA).
Biochemical methodsProtein extraction and Western blotting were performed as previ-
ously described (Alexaki et al., 2010). Rabbit anti-MITF and
anti-GLI2 antibodies were purchased from Bioscience Innovations
(Interchim SA, Montlucon, France) and Santa Cruz Biotechnology
(Santa Cruz, CA, USA), respectively. Mouse monoclonal anti-GAPDH
was from Abcam (Cambridge, MA, USA). Secondary donkey anti-
rabbit and anti-mouse HRP-conjugated antibodies were from Santa
Cruz Biotechnology.
Cells transfectionsFor reporter assays, melanoma cells were seeded in 24-well plates
and transfected at approximately 70–80% confluency with the poly-
cationic compound Fugene� (Roche Diagnostics, Indianapolis, IN,
USA) in fresh medium containing 1% FCS. Following a 16-h incuba-
tion, luciferase activities were determined with a Dual-Glo� luciferase
assay kit (Promega). For stable expression of GLI2DN2, melanoma
cells were transfected with Fugene at approximately 70–80% con-
fluency with 10 lg of either the empty expression vector (ctrl), or
the same vector carrying constitutively active GLI2DN2 (Sasaki
et al., 1999) per 100 mm diameter culture dish. Three days later,
G418 (Sigma-Aldrich, 0.7 mg ⁄ ml) was added to the culture med-
ium. Selection of stably transfected cell populations occurred within
a 3-week period. Semi-quantitative RT-PCR was used to verify the
expression of the transfected gene constructs.
Gene silencing in human melanoma cellsSubconfluent melanoma cells were infected with lentiviral parti-
cules expressing either control, non-targeting, short hairpin RNAs
(shRNAs) (shCtrl, Sigma-Aldrich SHC002V) or shRNA targeting GLI2
(Sigma-Aldrich SHVRS clone ID TRCN0000033329 and TRCN00000-
33330) or M-MITF (Sigma-Aldrich SHVRS clone ID TRCN0000019-
123) at a multiplicity of eight plaque forming units per cell in presence
of 8 lg ⁄ ml hexadimethrine bromide. Transduced cell populations
were selected with puromycin (2 lg ⁄ ml). Efficient and stable reduc-
tion in gene expression over time was verified by real-time RT-PCR
after each passage and prior to experiments.
RNA extraction and gene expression analysisRNA extraction procedure and reverse transcription polymerase
chain reaction (RT-PCR) methodologies (either semiquantitative or
real-time) have been described previously (Alexaki et al., 2010).
Primer sequences for multiplex semi-quantitative RT-PCR and for
real-time RT-PCR are provided in Tables S1 and S2, respectively.
Gene array data miningWe performed correlation and anticorrelation studies based on the
Pearson product-moment correlation coefficient and assumed both
a normal distribution of data and a strictly linear relationship
between GLI2 gene expression and GLI2 function (i.e., target gene
transcription) in six distinct DNA microarray data sets derived from
experiments using HG-U133 series microarrays (Affymetrix, Santa
Clara, CA, USA): melanoma cell line data from GSE8332 (Wagner
data set, 18 samples), GSE7127 (Johansson data set, 63 samples),
GSE4843 (Mannheim data set, 45 samples), GSE4841 (Philadelphia
data set, 30 samples), and GSE4840 (Zurich data set, 15 samples)
(Hoek et al., 2006; Johansson et al., 2007; Wagner et al., 2007)
extracted from the NCBI Gene Expression Omnibus (http://
www.ncbi.nlm.nih.gov/geo/), and melanoma cell line data published
by Ryu and coworkers (Ryu data set, 10 samples) (Ryu et al., 2007)
extracted from the Public Library of Science (http://www.plo-
sone.org). Extensive details concerning statistical analyses and cri-
teria for significance of the results are published in (Hoek et al.,
2008).
Matrigel invasion assaysMelanoma cell invasion through Matrigel (Biocoat; BD Biosciences,
San Jose, CA, USA) was assayed using Matrigel-coated Transwell
inserts (8-lm pore size; Falcon, Franklin Lakes, NJ, USA) as previ-
ously described (Javelaud et al., 2005).
Melanin content determinationMelanin content in melanoma cells was assayed as described pre-
viously (Tsuboi et al., 1998). Briefly, cell pellets were dissolved in
1 N NaOH at 100�C for 30 min, samples were centrifuged at
16 000 g for 20 min, and optical densities of the supernatants were
measured at 400 nm.
Xenograft growth assaysFemale 8-week-old Swiss nu ⁄ nu (nude) mice were housed at the
animal facilities of the Curie Institute, Orsay, France, in specific
pathogen-free conditions. Their care was in accordance with the
institutional guidelines of the French Ethical Committee (Ministere
de l’Agriculture et de la Foret, Direction de la Sante et de la Protec-
tion Animale, Paris, France) and under supervision of authorized
investigators. 106 of each melanoma cell line (Daju, Gerlach,
1205Lu, SK28, SK29, Dauv1, 888mel, 501mel, and Quar) in loga-
rithmic growth phase was injected subcutaneously into the flank of
at least 10 nude mice. The viability of cells, estimated by Trypan
blue exclusion before injection into mice, was over 95%. Tumor
take was calculated as the % of mice in each group with palpable
tumors 100 days after implantation (or earlier when tumor size
required early euthanasia). Latency represents the number of days
after which tumors are first palpable.
Acknowledgements
This work was supported by institutional funding from INSERM,
CNRS and Institut Curie, grants from INCa (PLBIO08-126), Canc-
eropole Ile-de-France (RS-013), and Ligue Nationale Contre le
Cancer (Equipe Labellisee LIGUE 2011–2015), and by a donation
from Emile and Henriette Goutiere to A.M. V.I.A. was recipient of a
Canceropole Ile-de-France ⁄ Region Ile-de-France post-doctoral
fellowship; M.J.P. received a Canceropole doctoral scholarship.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Sequences of primers used for semi-quan-
titative mutiplex RT-PCR.
Table S2. Sequences of primers used for quantitative
real-time RT-PCR.
Table S3. Genes that may be positively regulated by
the GLI2 transcription factor.
Table S4. Genes that may be negatively regulated by
the GLI2 transcription factor.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
Antagonistic roles of GLI2 and M-MITF in melanoma cells
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