GLI2 and M-MITF transcription factors control exclusive gene expression programs and inversely...

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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: alain.mauviel@curie.fr

*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

100150200

100150200250300350400450500

1 2 3 4 5 6 7 8

M-MITFA-MITF

0.100.120.140.16

00.020.040.060.08

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.

12 GLI2MITF

Melanoma cell lines01

Melanoma cell lines

MLANA SILVMLANA

234567

Melanoma cell lines

100120140

Melanoma cell lines

020406080

Melanoma cell lines012345

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.

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

M-MITF

1 2 3 4 5 6 7 8 9 10 11

TRP-1GAPDH

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.

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

1 2 3 4 5 6 7 8 9 10 11GAPDH

CDK2GAPDH

GAPDHCCND1

SK28Dauv1888mel

WM852WM983BWM983A1205LuWM793

p21GAPDH

p27GAPDH

CDK4GAPDH

0 2 4 6 8Time (days)

501melFO-1

Tumor take Latency(%) (days)

(%) (days)Daju 100 25+/–5Gerlach 100 60 +/–151205lu 95 20+/–5SK28SK29

50 100+/Dauv1 –20888mel501melQuar

90 35+/–575 30 +/–5

0 nd0 nd0 nd

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.

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

SB4315424 GLI2 GLI2

MITF MITF

1205Lu 501mel4

ForskolinH89

1205Lu 501mel

GLI2MITF

1205Lu 501mel 1205Lu 501mel

100120140160180

TGF-βForskolin

––

WM983A

TGF-βForskolin

+WM983A

20406080

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.

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

200400600800

10001200

rter a

5101520253035

A × ×

406080

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.

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

Ad-GFP Ad-MI-DN

shc shMITF

TGF-β0

– + – +

TGF-β0.0

– + – +

TGF-β0.0

– + +

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.

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.

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.

GLI2 and M-MITF transcription factors control exclusive gene expression programs and inversely...

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