# Up-regulation of MET Expression by α-Melanocyte-stimulating Hormone and MITF Allows Hepatocyte Growth Factor to Protect Melanocytes and Melanoma Cells from Apoptosis

Laurent Beuret, Enrica Flori, Christophe Denoyelle, Karine Bille, Roser Buscà, Mauro Picardo, Corine Bertolotto, Robert Ballotti

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Laurent Beuret, Enrica Flori, Christophe Denoyelle, Karine Bille, Roser Buscà, et al.. Up-regulation of MET Expression by α-Melanocyte-stimulating Hormone and MITF Allows Hepatocyte Growth Factor to Protect Melanocytes and Melanoma Cells from Apoptosis. Journal of Biological Chemistry, 2007, 282 (19), pp.14140-14147. 10.1074/jbc.M611563200. hal-02358861

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# Up-regulation of MET Expression by α-Melanocyte-stimulating Hormone and MITF Allows Hepatocyte Growth Factor to Protect Melanocytes and Melanoma Cells from Apoptosis*

Received for publication, December 18, 2006, and in revised form, March 12, 2007 Published, JBC Papers in Press, March 19, 2007, DOI 10.1074/jbc.M611563200

Laurent Beuret<sup>+§1</sup>, Enrica Flori<sup>¶</sup>, Christophe Denoyelle<sup>+§</sup>, Karine Bille<sup>+§</sup>, Roser Busca<sup>+§</sup>, Mauro Picardo<sup>¶</sup>, Corine Bertolotto<sup>+§</sup>, and Robert Ballotti<sup>+§2</sup>

From the <sup>‡</sup>INSERM, U597, Biologie et Pathologies des Cellules Mélanocytaires: de la Pigmentation Cutanée au Mélanome, Equipe labellisée par la Ligue Nationale contre le Cancer, 06107 Nice Cedex 2, France, the <sup>§</sup>Université de Nice Sophia-Antipolis, UFR Médecine, Nice, France, and the <sup>¶</sup>Laboratorio di Fisiopatologia Cutanea, Istituto Dermatologico San Gallicano, Instituti di Ricovero e Cura a Carattere Scientifico, 00144 Rome, Italy

The *MET* proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. In mouse melanoma cells, *Met* expression is increased by α*MSH* via the activation of the cAMP pathway. However, the mechanism by which cAMP regulates *MET* and the biological consequences of this increase were not known. In the present report, we show that α*MSH* regulates *MET* expression in both human melanocytes and mouse melanoma cells through a transcriptional mechanism that requires MITF. Furthermore, the adenovirus driven expression of MITF is sufficient to increase *MET* in melanoma cells. Functional analysis of the *MET* promoter allows us to identify an E-box motif conserved in both human and mouse promoter that mediates the effect of MITF. Interestingly, up-regulation of *MET* expression by cAMP leads to an exacerbated HGF signaling and allows HGF to protect melanocytes and melanoma cells from apoptosis. Thus, physiological stimuli or pathological events that would induce MITF expression may lead to increased *MET* expression thereby favoring melanoma survival. These observations strengthen the roles of MITF and *MET* in melanoma development.

The *MET* proto-oncogene encodes for the hepatocyte growth factor receptor, a plasma membrane tyrosine kinase involved in the pleiotropic physiological effects and tumorigenic processes of numerous tissues.

Upon HGF binding, the intracellular part of b-subunit undergoes autophosphorylation on tyrosine residues creating a unique docking site for PI3K<sup>3</sup> or GRB2-SOS adaptors that allow the activation of ERK signaling pathways. The C-terminal part of *MET* also interacts with the non-receptor tyrosinase kinase Src, the transcription factor STAT3, and the adapters Shc and

Gab1 (1). All of these partner molecules allow *MET* to activate numerous signaling pathways and induce various physiological effects, including cell motility, cell growth, tubulogenesis, B-cell differentiation, and muscle and nervous system development (2).

The role of *MET* in growth and motility, often called “invasive growth” places *MET* at a center stage of tumor and metastasis development. Indeed, *MET* overexpression is very common in human cancer. Gain-of-function mutations in the tyrosine kinase domain of the receptor have been identified in hereditary papillary renal carcinoma type I and in other sporadic tumors (3).

HGF/MET signaling has is. Indeed, in transgenic mice over expressing HGF, melanosis in central nervous system and skin hyperpigmentation were observed (4). HGF can replace steel factor, the Kit ligand, to promote melanoblast growth and differentiation from neural crest explants (5).

*MET* is expressed on melanocytes and HGF is a growth factor for cultured melanocytes (6). HGF/MET has been shown to influence melanocyte transformation and the acquisition of the metastatic phenotype (7). Furthermore, HGF appears to play a key role in the regulation of melanoma interaction with its microenvironment, since HGF controls E-cadherin (8), CD44v6 (9), fibronectin (10), as well as MMP2 (11) expression in melanoma cells.

It is noteworthy that transgenic mice overexpressing HGF in their skin develop melanoma upon UV radiation of the neopates thereby recapitulating the etiologic role of UV in human melanoma genesis (12).

Recently, overexpression of *MET* in human melanoma samples has been correlated with a poor clinical outcome (13).

HGF and *MET* play a key protective role against apoptosis (14). Indeed, HGF counters anoikis-induced apoptosis (15) that is particularly relevant when cancer cells enter the bloodstream to form distant metastasis. Interestingly, *MET* also favors cell survival, in a HGF-independent process, by interacting and sequestering FAS (16). In melanocytes or in melanoma cells, the role of HGF/MET in apoptosis has not been studied so far, although we have shown that HGF does not protect human melanocytes from Trail induced apoptosis (17).

Induction of melanocyte differentiation by α*MSH* and other cAMP-elevating agents, such as forskolin, has been extensively

<footnote>* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "*advertisement*" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote n="1">¹ Supported by a fellowship from "La Ligue Nationale Contre le Cancer."</footnote>

<footnote n="2">² To whom correspondence should be addressed. Tel.: 33-4-93-37-77-90; Fax: 33-4-93-81-14-04; E-mail: ballotti@unice.fr.</footnote>

<footnote n="3">³ The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HGF, hepatocyte growth factor; αMSH, α-melanocyte-stimulating hormone; PARP, poly(ADP-ribose) polymerase; RT, reverse transcription; siRNA, small interfering RNA; CAT, chloramphenicol acetyltransferase.</footnote>

<footnote>This is an open access article under the CC BY license.</footnote>
studied. During this process, the activation of the cAMP pathway induces the up-regulation of tyrosinase, Tyrp1, and DCT, the key enzymes in melanin synthesis, through a transcriptional mechanism involving MITF (18), the master gene of melanocyte differentiation. Interestingly, in B16 melanoma cells, αMSH-induced differentiation is accompanied by an increase in Met expression (19). This observation suggests that upon differentiation, melanocytes may acquire exacerbate HGF-induced signaling and biological effects.

In the present report, we studied the effect of αMSH- and cAMP-elevating agents, such as forskolin, on MET expression in both B16 melanoma cells and in normal human melanocytes. We demonstrated that the cAMP pathway regulates MET through MITF that binds and activates the *MET* promoter. Finally, we showed that up-regulation of MET expression by cAMP renders melanocytes responsive to the protective effects of HGF against apoptosis.

## MATERIALS AND METHODS

*Antibodies*—Monoclonal procaspase 3 antibody was obtained from Transduction Laboratories. Monoclonal anti-ERK2 (D-2) antibody was from Santa Cruz Biotechnology. Monoclonal anti-MET (25H2) and polyclonal cleaved PARP antibodies were from Cell Signaling Technology Inc. Monoclonal anti-MITF (C5) antibody was from Abcam and polyclonal anti-tyrosinase (Pep7) was provided by V. Hearing (National Institutes of Health, Bethesda, MD).

The anti-mouse and anti-rabbit peroxidase-conjugated secondary antibodies were obtained from Dakopatts.

*Cell Culture; Cell Viability Test*—Mouse melanoma cells B16/F10 were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 7% fetal bovine serum (Invitrogen) and penicillin/streptomycin (100 IU/50 µg/ml (Invitrogen). Human melanoma cells Mel501 and A375 were grown in RPMI (Invitrogen), with the same supplements.

Primary human melanocytes (normal human melanocytes) were obtained from neonatal foreskins of Caucasian individuals, and grown in Medium 254 (Cascade Biologics).

The cell viability was assessed using the cell proliferation kit II (XTT; Roche Applied Science) according to the manufacturer's protocol.

*Quantitative Real-time One-step RT-PCR*—RNAs were extracted using TRIzol reagent (Invitrogen) and RNeasy protect mini kit (Qiagen). mRNA expression levels were quantified by real-time one-step RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems), each reaction containing 100 ng of total sample RNA. The reactions were run for 40 cycles under the following conditions: Stage 1, 48 °C for 30 min; Stage 2, 95 °C for 10 min; Stage 3, 95 °C for 15 s; Stage 4, 60 °C for 1 min.

The following primers were used: mouse *Met*, 5'-GCATGT-CAGCATCGCTCAA-3' (forward) and 5'-TGCAGGCCCAG-CTGTTTC-3' (reverse). For mouse *MItf*, human *MITF*, mouse *Hif1a*, mouse *Tyr*, primers were described previously (20).

All reactions were run in triplicate on an ABI-PRISM 7000 instrument (Applied Biosystems, Foster City, CA) and were normalized to 36B4 or SB34 expression.

*Adenoviral Infection and siRNA*—Adenovirus encoding wild-type Mitf (Ad-Mitf) was described previously (21). siRNA to mouse or human *MITF* were described previously (22, 23).

*Vector Constructions and Mutagenesis*—PCDNA3 vectors encoding wild-type MITF (Mitf) has previously been described (24). The 1.6-kb mouse promoter fused to Cat reporter gene (pMet) was kindly provided by Seol (25). ΔpMet was constructed by digesting pMet by SphI and KpnI. E-box mutations were performed using the QuikChange site-directed mutagenesis kit (Stratagene) and the following primers: 5'-GACAGGGACGAACAGATCCGTGCTGGG-GCGGGTAC-3' (for pMetmE1), 5'-GTTCGGCCCGGGCG-CGTCGGTGACCCGGACGGCCTC-3' (for pMETmE2), and 5'-GTGACCCTGTGCGGAGCTCGATGCTGGGCG-ACCGC-3' (for pMETmE3). An 800-bp fragment of the human MET promoter (AF046925) was cloned using the following primers: 5'-CGACCATCCACTTCTACCTCCGCCTC-3' (forward). Reverse-5'-CCTCCCCGCCTCCTCTCAGCAAG-3'.

*Nuclear Extracts and Gel Mobility Shift Assay*—Nuclear extracts from control cells or cells incubated with forskolin for 5 h were prepared as described previously (26). The *Met* promoter specific probe spanning the E-box2 (E2), 5'-GTTCGGCCGGCGCAGGTGACCCGGACGGCCTC-3' or the mutant probe (mut E2), 5'-GTTCGGCCGGGCCGTCGGTGACCCGGACGGCCTC-3', were γ-³²P-end-labeled using T4 polynucleotide kinase. 5 µg of nuclear proteins were preincubated in a binding buffer containing 10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4% glycerol, 80 µg/ml salmon sperm DNA, 0.1 µg of poly(dI-dC), 10% fetal calf serum, 2 mM MgCl₂, and 2 mM spermidine for 15 min on ice. Then, 50,000 cpm of ³²P-labeled probe were added to the binding reaction for 10 min at room temperature. DNA-protein complexes were resolved by electrophoresis on a 4% polyacrylamide gel (37.5:1 acrylamide-bisacrylamide) in TBE buffer (22.5 mM Tris borate, 0.5 mM EDTA, pH 8) for 1.5 h at 100 V. For super-shift assays, 0.5 µl of preimmune serum or anti-MITF antibody (26) was preincubated with nuclear extracts in the binding reaction buffer before adding the labeled probe.

*Western Blot Assays*—The cells were grown in 6-well dishes and treated as indicated in the figure legends, then solubilized in buffer A containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10 µM leupeptin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 100 units/ml aprotinin, 10 mM NaF, and 1 mM Na₃VO₄. The samples (30 µg) were resolved by 10% SDS-PAGE, transferred to polyvinylidene difluoride, and then exposed to the appropriate antibodies. The proteins were visualized with the ECL system (Amersham Biosciences).

*Transfection, Chloramphenicol Acetyltransferase (CAT), and Luciferase Assays*—B16 melanoma cells were seeded in 6-well dishes, and 24 h later, the cells were co-transfected with 2 µg of promoter/CAT reporter construct, 0.2 µg of β-galactosidase expression vector, and 1 µg of empty pCDNA3 or pCDNA3-Mitf expression vector. The transfection was performed using 10 µl of Lipofectamine reagent (Invitrogen) in 800 µl of Opti-MEM for 6 h. The transfection medium was then replaced with fresh medium and 48 h later the cells were extracted with reporter lysis buffer (Promega).
Up-regulation of *MET* Expression by α*MSH* and *MITF*

<figure><img src="image_2.png" /></figure>

FIGURE 1. cAMP pathway increases *MET* expression in melanocytes and melanoma cells. A, mouse B16 melanoma cells; B, normal human melanocytes (NHM); C, human melanoma cells, MEL501, were treated with 0.1 µM HGF, 1 µM forskolin (Fsk), or 1 µM αMSH for 8 h. The proteins were analyzed by Western blot using monoclonal antibodies against MET and MITF. ERK2 antibody was used as a loading control.

The CAT assay was performed according manufacturer's instructions. The β-galactosidase plasmid was co-transfected to monitor transfection efficiency and the CAT activity was corrected by the activity of β-galactosidase in each experiment. The luciferase assays were performed as described previously (24).

RESULTS

*α*MSH Increases *MET* Expression in Both Human Melanocytes and Mouse Melanoma Cells—In B16F10 melanoma cells and in human melanocytes, we showed that αMSH and forskolin treatments, for 8 h, increased MET and MITF levels (Fig. 1, A and B). MET expression that reached a maximum after 8 h of forskolin was slightly delayed compared with the Mitf expression profile that peaked at 5 h (data not shown). Conversely, treatment of B16 melanoma cells with 0.1 µM HGF for 8 h led to a decrease in Met and Mitf expression (Fig. 1A). HGF has no significant effect on MITF and MET expression in human melanocytes (Fig. 1B).

In human Mel501 melanoma cells, HGF also decreased MET expression but did not significantly affect MITF level (Fig. 1C). Forskolin or αMSH did not increase MITF or MET expression. The cAMP pathway does not seem to be functional in these cells.

Real-time RT-PCR analysis showed a clear increase in *Met* messengers, 4 h after forskolin addition. The maximum stimulation was reached at 8 h and remained stable, at least until 24 h (Fig. 2A). Furthermore, cAMP-elevating agents, forskolin and αMSH, increased (4–6-fold) *Met* promoter activity in CAT reporter assay (Fig. 2B). Interestingly, HGF, which decreased *Met* expression at the protein level, slightly activated *Met* promoter.

This set of observations indicates that cAMP increases MET expression through a transcriptional regulation. The correlation between MET and MITF up-regulation upon activation of the cAMP pathway suggests that MITF might be involved in this process.

<figure><img src="image_3.png" /></figure>

FIGURE 2. cAMP pathway increases *MET* expression at the transcriptional level. A, the relative amount of *Met* mRNA in B16 melanoma cells treated with 1 µM forskolin over the indicated time was determined by quantitative real-time RT-PCR. All results were normalized to 36B4 values. The results are the mean ± S.D. from three independent experiments. B, B16 cells were transfected with pMet reporter plasmid and then treated with HGF (50 ng/ml) or forskolin (1 µM). Met promoter activity was determined using a classical CAT assay. The results are the mean ± S.D. from three independent experiments.

<figure><img src="image_4.png" /></figure>

FIGURE 3. MITF is required for MET expression in melanocytes and melanoma cells. A, real-time RT-PCR assays of *Mitf*, *Tyrosinase*, and *Met* mRNA levels in untreated or forskolin-treated B16 melanoma cells transfected with either siRNA to Mitf (*si*-Mitf) or scrambled siRNA (*si*-SC). In each case, all transcript levels were normalized to 36B4 values. The results are the mean ± S.D. from three independent experiments. B, Western blot analysis of Met, tyrosinase (Tyr), Mitf, and total Erk2 in B16 treated as described for A. C, Western blot analysis of MET, MITF, and ERK2 in Mel501 transfected with either siRNA to Mitf (*si*-Mitf) or scrambled siRNA (*si*-SC).

MITF Mediates the Effect of cAMP on MET and Is Sufficient to Regulate MET Expression—To verify this hypothesis we used specific siRNA to silence Mitf (20). Real-time RT-PCR analysis showed that *Mitf* messengers were dramatically decreased, in both basal and forskolin-stimulated conditions, after transfection with siMitf compared with scramble siRNA (siSc) (Fig. 3A). The messengers of *tyrosinase*, one of the best characterized Mitf target genes, were also decreased in both conditions by siMitf. *Met* mRNA were markedly increased by forskolin treatment in B16 cells transfected by siSc. However, upon transfection with siMitf the levels of *Met* mRNA were decreased, and the effect of forskolin was almost completely lost. At the protein level, Western blot analysis confirmed that siMitf blocked Mitf
Up-regulation of *MET* Expression by αMSH and MITF

C), indicating that MITF is sufficient to up-regulate MET expression.

MITF Stimulates the *MET* Promoter Activity through an *E-box* Motif Conserved in Both Mouse and in Human Sequences—Next, we performed a functional analysis of the *Met* promoter to identify the Mitf binding site. We used the 1.6-kb mouse *Met* promoter fused to CAT reporter gene (pMet) described by Seol et al. (25). This sequence shows a strong homology with the human *MET* promoter. The regions in black in Fig. 5. A display over 70% identity, and the proximal region contains 3 E-boxes that could bind Mitf (Fig. 5A). Transfection of a Mitf expression vector led to an 8-fold stimulation of the promoter activity (Fig. 5B). First, we deleted 1.2 kb from the 5' end to remove E-box1. The new construct ΔpMet contains 400 bp of the *Met* promoter including 2 E-boxes. The response of this construct to Mitf was identical to that of the full-length promoter, indicating that Mitf responses were mediated by cis-acting elements found in this proximal fragment. We analyzed the effect of the individual mutation of the three E-boxes (–13/–19, –83/–89, and –234/–240) contained in this fragment. As expected from the results obtained with the deletion construct, mutation of E-box1 (–234/–240) did not affect Mitf effects. Similarly, mutation of E-box3 (–13/–19) did not impair Mitf effects. However, mutation of the E-box2 (–83/–89) dramatically reduced the activation of the *Met* promoter by Mitf. The same experiments using the human *MET* promoter upstream the luciferase reporter gene (Fig. 5C) confirmed that the *MET* promoter is stimulated by Mitf through the E-box2 that is perfectly conserved in both human and mouse promoters.

Gel shift assay using a probe encompassing the E-box 2 showed an increased binding of nuclear proteins in extract from forskolin treated cells (Fig. 5D). This binding was displaced by an excess of cold E-box2 but not by the mutated E-box2. Furthermore, Mitf was identified as the nuclear protein bound to E-box2, since the E-box2 complex was shifted by antibody to Mitf.

HGF Signaling and Anti-apoptotic Effects Are Exacerbated in Melanocytes and Melanoma Cells Exposed to Forskolin—Analysis of the HGF signaling pathway in B16 melanoma cells (Fig. 6A) showed that in basal conditions, the addition of HGF led to a slight, yet significant activation of Akt and Erk, monitored by Western blot analysis with phospho-Akt and phospho-Erk antibodies. After 8 h of exposure to forskolin, Met and Mitf expression markedly increased. In these conditions, the addition of HGF induced a stronger phosphorylation, and thus activation of Akt compared with basal conditions. Interestingly, after forskolin treatment, HGF led to a huge Erk phosphorylation. This high Erk activity is reflected by the Mitf shift from a doublet to a single upper band, corresponding to Erk phosphorylated Mitf on serine 73 (28). Western blots with anti-Erk2 antibody were performed to ensure the even loading of each lane.

Thus cAMP elevating agents increase Met expression and exacerbate Met signaling.

Finally, we investigated the biological consequences of the increased Met expression and signaling. We studied the anti-apoptotic effect of HGF in normal human melanocytes and in B16 melanoma cells exposed or not to forskolin. An valuation of

<figure><img src="image_5.png" /></figure>

FIGURE 4. **MITF is sufficient for MET expression in melanocytes and melanoma cells.** A, real-time RT-PCR analysis was used to determine the levels of the human *MITF*, mouse *MItf*, *TYR*, *MET*, and *HIF1α* transcripts in A375 human melanoma cells infected with an adenovirus encoding wild-type mouse Mitf (ad-Mitf). All results were normalized to samples infected with control adenovirus and SB34 values. The results are the mean ± S.D. of three independent experiments. B and C, A375 melanoma cells (B) or human normal melanocytes (C) infected with an adenovirus encoding mouse Mitf or an empty adenovirus were analyzed by Western blot using monoclonal antibodies against MET and MITF. ERK2 antibody was used as a loading control.

expression and, consequently, the effect of cAMP on tyrosinase and Met expression (Fig. 3B). Interestingly, the basal levels of Met in B16 melanoma cells as well as in human melanoma, Mel501 (Fig. 3C) were dramatically reduced by MITF silencing, indicating that basal and cAMP-stimulated Met expression, in melanoma cells, required the presence of MITF. However, we still had to determine whether MITF was sufficient to up-regulate MET expression. For this purpose, human A375 melanoma cells, expressing a very low level of MITF, were infected with mouse Mitf encoding adenovirus. The levels of mouse and human *MITF* messengers, as well as the amounts of mRNA of several MITF target genes, were evaluated by Real-time PCR (Fig. 4A). Infection with Ad-Mitf increased the amount of mouse *MItf* mRNA (4-fold), with a minor effect on human *MITF* mRNA (1.5-fold). *BCL2* and *HIF1α* that are MITF target genes (27) were stimulated by Ad-Mitf infection. Although *TYR* is a well known MITF target gene, *TYR* mRNA was not induced by Ad-Mitf infection. In agreement with a previous report (21), this observation indicates that Mitf is required but not sufficient to regulate *TYR* expression. It is noteworthy that Mitf over expression markedly increased *MET* messengers. The same observation has been made at the protein level, in both human melanocytes and in A375 melanoma cells (Fig. 4, B and
Up-regulation of *MET* Expression by α*MSH* and *MITF*

A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

D

<figure><img src="image_9.png" /></figure>

FIGURE 5. **MITF binds to the MET promoter and stimulates its activity.** A, schematic diagram of the human and mouse *MET* promoter sequences. The black boxes show the regions with high homologies. * indicates the transcription initiation. B, mouse pMet reporter constructs were co transfected with the Mitf encoding plasmid or pCDNA3 empty plasmid in B16 cells. CAT activity was measured 48 h after transfection. The results are means ± S.D. of three experiments performed in triplicate. C, human pMET reporter constructs were co-transfected with the Mitf encoding plasmid or pCDNA3 empty plasmid in B16 cells. The luciferase activity (arbitrary units) was measured 48 h after transfection. The results are means ± S.D. of three experiments performed in triplicate. D, electromobility shift assay was performed using B16 nuclear extracts from control or cells treated for 5 h with forskolin (Fsk) with the ³²P-labeled E2 box or ³²P-labeled E2mut box. Binding reactions were performed in the presence of 0.5 µl of preimmune serum (PI) or with 0.5 µl of a specific anti-MITF serum. 25-Fold excess of unlabeled E2 or mE2 were added when indicated (E2° and mE2°).

melanocyte viability showed that TRAIL induced death in nor-
mal human melanocytes (Fig. 6B). HGF was not able to protect
normal human melanocytes from TRAIL-induced death.
When the cells were first exposed to forskolin for 15 h, before
treatment with TRAIL, we observed a slight increase in cell
viability. In these cells, HGF completely blocked cell death
induced by TRAIL.

We also analyzed two classical apoptosis parameters, caspase 3 and PARP cleavage. Consistent with the survival data, in control B16 melanoma cells, staurosporine treatment led to a decrease in caspase 3 expression that reflected its cleavage and activation (Fig. 6C). As a consequence, we observed a cleavage of PARP monitored by an antibody specific for the cleaved form of PARP. In these cells HGF neither prevented caspase 3 nor PARP cleavage. Conversely, in cells pretreated with forskolin, to up-regulate Met, HGF markedly decreased the effect of staurosporine on caspase 3 activation and PARP cleavage.

Taken together, these observations indicate that the up-reg-
ulation of *MET* by cAMP and MITF allows HGF to protect
melanocytes and melanoma cells from apoptosis.

DISCUSSION

In the present report, we demonstrated that cAMP elevating
agents, αMSH and forskolin, increase MET expression in mel-
anocyte and melanoma cells through a transcriptional mecha-
nism that requires MITF. Furthermore, MITF overexpression
is sufficient to increase HIF1A, BCL2, and MET levels but not
tyrosinase expression. The tyrosinase gene, as well as the genes
encoding other melanogenic proteins, appears to have a non-
permissive chromatin conformation. Indeed, MITF needs to
recruit the SWI/SNF enzymes that allow chromatin remodel-
ling and melanogenic gene transcription (29). HIF1A, BCL2,
and MET, which do not require SWI/SNF enzymes, appear to
be in an active genomic region. This suggests that overexpres-
sion of MITF in pathological conditions such as genomic ampli-
fication (30) would be sufficient to induce MET over expres-
sion. However, one should not conclude that MET expression is
controlled exclusively by MITF. Indeed, analysis of several mel-
anoma cells lines did not show a tight correlation between MET
and MITF expression. However, in melanoma cell lines
<figure><img src="image_10.png" /><figcaption>FIGURE 6. HGF protects melanocytes and melanoma cells from apoptosis. A, B16 melanoma cells were incubated or not for 8 h with 1 µM forskolin (Fsk), then treated or not with 0.1 µM HGF for 10 min. Western blot analysis was performed using antibodies against MET, MITF, phospho-AKT (Ser-473), and phospho-ERK2/1 (Thr-202/Tyr-204). ERK2 antibody was used as loading control. B, after stimulation with 1 µM forskolin, MHN cells were stimulated or not with HGF (50 ng/ml) for 30 min and then treated with TRAIL (40 ng/ml) for 4 h. Cell viability was determined using an XTT assay. C, after stimulation with 1 µM forskolin, B16 cells were stimulated or not with HGF (50 ng/ml) for 30 min and then treated with 2 µM staurosporine for 6 h. Then the activation of caspase 3 and the cleavage of PARP were analyzed by Western blot.</figcaption></figure>

expressing both MITF and MET, MITF silencing dramatically reduced MET expression. In melanoma cells, regulation of MET expression probably depends on ubiquitous transcription factors such as SP1 (31) or AP1 (32) that control MET expression in other cell systems. More recently, hypoxia, through HIF1A, has been shown to regulate MET expression in osteosarcoma cells (33). Since we have shown that cAMP, through MITF, regulates HIF1A expression in melanoma, it was tempting to propose that HIF1A mediates the effect of MITF on *MET*. However, in non-hypoxic conditions, HIF1A silencing did not affect MET expression in melanoma cells (data not shown) suggesting that MITF acts directly on *MET*. This hypothesis is strengthened by studies on the *MET* promoter. Analysis of 5' non-coding sequence of the mouse *Met* promoter showed a strong homology with human *MET* gene. In this region 3 E-boxes are conserved in both human and mouse sequences. Functional analysis of the mouse promoter showed a strong stimulation of its activity by Mitf that is mainly mediated by the E-box in position -83/-89 that binds Mitf. This E-box is conserved in the human *MET* gene and also mediates MITF responsiveness of the human *MET* promoter.

Our data, identifying MITF as the transcription factor that mediates the effects of cAMP on MET expression in melanocytes and melanoma cells, agree with the recent paper by McGill *et al.* (34) reporting that MITF mediated the up-regulation of MET by its own ligand, HGF. However, important differences should be highlighted between our data and that of the report mentioned above. In our hands, it has never been possible to show an increase in MET protein expression

induced by HGF. On the contrary, we consistently observe an inhibition of MET at the protein level, with a slight increase in the *MET* messengers. Our observations are consistent with numerous reports demonstrating that HGF increases *MET* transcription through activation of the AP1 transcription factor (32). However, at the same time HGF decreases MET protein level through a classical desensitization mechanism shared by membrane tyrosine kinase receptors (35). Therefore, the results obtained by McGill (34) do not seem to be consistent with the literature and might be explained by the use of an anti-Met antibody that recognizes RON, a closely related tyrosine kinase receptor, that is indeed up-regulated by HGF (36).

On the basis of a gel shift assay, McGill *et al.* (34) have identified E-box at position -300 in the human *MET* promoter as the sequence that binds MITF. According to a previous report from Comoglio's group (33), this E-box is located at +19/

+25 at the 5' end of exon 1 and was recently shown to be the target of the Notch effector, HES-1 (37). In the present report, the functional analysis of the mouse and human promoters shows that this E-box is not involved in the regulation of the *MET* promoter by MITF.

Finally, it should be mentioned that we consistently observe that HGF does not stimulate the *tyrosinase* promoter or a MITF reporter gene composed of 3 M-box upstream the thymidine kinase promoter (data not shown). These observations clearly indicate that HGF does not activate MITF transcriptional activity.

It should be noted that up-regulation of *MET* expression by cAMP has biological consequences in melanocytes and melanoma cells. First, we observe exacerbated HGF signaling in melanoma cells exposed to forskolin. In these cells, HGF is much more efficient in the activation of the PI3K/AKT and ERK pathways. Although HGF/MET signaling plays a key role in the control of cell migration, it also plays a crucial role in protection against apoptosis. Since apoptosis resistance is the hallmark of many tumors including melanomas, we have studied the effect of HGF on apoptosis in cells exposed or not to cAMP.

As reported previously (17), HGF is not able to protect human melanocytes from TRAIL induced apoptosis. However, in melanocytes and melanoma cells exposed to forskolin, the increase in MET expression allows HGF (i) to exert a pro-survival effect and (ii) to activate more strongly PI3 kinase and ERK pathways compared with cells not exposed to forskolin.

Although MET signaling involves Src kinase, PLCγ (38) or NFκB (39), the anti-apoptotic function of MET is mediated by
Up-regulation of MET Expression by αMSH and MITF

<figure><img src="image_11.png" /><figcaption>FIGURE 7. Up-regulation of MET expression by α-melanocyte stimulating hormone and MITF allows HGF to protect melanocytes and melanoma cells from apoptosis.</figcaption></figure>

ERK and PI3K pathways (14). Thus, the robust activation of these two pathways by HGF in melanocytes and melanoma cells exposed to forskolin might account for the anti-apoptotic effect of HGF in these cells (Fig. 7).

Interestingly, the tyrosine phosphatase SHP2 (PTPN11) is required for MET signaling to ERK and PI3K pathways and mutations in SHP2 lead to generalized lentigines found in the LEOPARD syndrome. These observations support the role of MET in melanocyte biology.

The role of HGF/MET in the survival of melanoma cells has not been extensively studied and as far as we know this is the first report demonstrating that HGF/MET can protect melanoma cells from apoptosis. This effect of the HGF/MET cascade might be particularly important for the pro survival effects provided by HGF derived from the stromal environment of the tumor (40). Furthermore, at the beginning of the invasive process, melanoma cells enter the bloodstream to disseminate and form distant metastasis. Up-regulation of MET could favor melanoma survival in anoikis conditions and allow transient growth in an anchorage-independent manner.

These effects of MET are not necessarily dependent on HGF, since MET interacts with other membrane receptors with adhesive functions such as CD44 and integrin (41). MET can also block apoptosis through its direct interaction with Fas (16) and melanoma cells are particularly resistant to Fas ligand-induced apoptosis. Since melanoma cells are more sensitive to TRAIL, it would be interesting to investigate the possible interaction of MET with TRAIL receptors (DR4, DR5).

The up-regulation of MET by the activation of the cAMP pathway is particularly relevant in the case of solar irradiation. Indeed, UV radiations are known to increase the production of αMSH and stimulate the synthesis of pro-inflammatory agents such as prostaglandins. Both αMSH and prostaglandins through G-protein coupled receptor activate the cAMP and could stimulate MET expression as a response to solar exposure or acute inflammation.

Taken together the data presented in this report shows that the up-regulation of MITF expression by cAMP or by other physiological or pathological stimuli would lead to increased

expression of MET thereby favoring melanoma survival. These observations strengthen the roles of MITF and MET in melanoma development.

Acknowledgment—We are grateful to Dr. Seol for pMet-Cat construct.

REFERENCES

1. Bolanos-Garcia, V. M. (2005) *Mol. Cell. Biochem.* **276**, 149–157

2. van der Voort, R., Taher, T. E., Derksen, P. W., Spaargaren, M., van der Neut, R., and Pals, S. T. (2000) *Adv. Cancer Res.* **79**, 39–90

3. Danilkovitch-Miagkova, A., and Zbar, B. (2002) *J. Clin. Invest.* **109**, 863–867

4. Takayama, H., La Rochelle, W. J., Anver, M., Bockman, D. E., and Merlino, G. (1996) *Proc. Natl. Acad. Sci. U. S. A.* **93**, 5866–5871

5. Kos, L., Arzon, A., Takayama, H., Maina, F., Ponzetto, C., Merlino, G., and Pavan, W. (1999) *Pigment Cell Res.* **12**, 13–21

6. Matsumoto, K., Tajima, H., and Nakamura, T. (1991) *Biochem. Biophys. Res. Commun.* **176**, 45–51

7. Otsuka, T., Takayama, H., Sharp, R., Celli, G., LaRochelle, W. J., Bottaro, D. P., Ellmore, N., Vieira, W., Owens, J. W., Anver, M., and Merlino, G. (1998) *Cancer Res.* **58**, 5157–5167

8. Li, G., Schaider, H., Satyamoorthy, K., Hanakawa, Y., Hashimoto, K., and Herlyn, M. (2001) *Oncogene* **20**, 8125–8135

9. Recio, J. A., and Merlino, G. (2003) *Cancer Res.* **63**, 1576–1582

10. Gaggioli, C., Deckert, M., Robert, G., Abbe, P., Batoz, M., Ehrengruber, M. U., Ortonne, J. P., Ballotti, R., and Tartare-Deckert, S. (2005) *Oncogene* **24**, 1423–1433

11. Hamasuna, R., Kataoka, H., Moriyama, T., Itoh, H., Seiki, M., and Koono, M. (1999) *Int. J. Cancer* **82**, 274–281

12. Noonan, F. P., Otsuka, T., Bang, S., Anver, M. R., and Merlino, G. (2000) *Cancer Res.* **60**, 3738–3743

13. Cruz, J., Reis-Filho, J. S., Silva, P., and Lopes, J. M. (2003) *Oncology* **65**, 72–82

14. Xiao, G. H., Jeffers, M., Bellacosa, A., Mitsuuchi, Y., Vande Woude, G. F., and Testa, J. R. (2001) *Proc. Natl. Acad. Sci. U. S. A.* **98**, 247–252

15. Zeng, Q., Chen, S., You, Z., Yang, F., Carey, T. E., Saimis, D., and Wang, C. Y. (2002) *J. Biol. Chem.* **277**, 25203–25208

16. Wang, X., DeFrances, M. C., Dai, Y., Pediaditakis, P., Johnson, C., Bell, A., Michalopoulos, G. K., and Zarnegar, R. (2002) *Mol. Cell* **9**, 411–421

17. Larribere, L., Khaled, M., Tartare-Deckert, S., Busca, R., Luciano, F., Bille, K., Valony, G., Eychene, A., Auberger, P., Ortonne, J. P., Ballotti, R., and Bertolotto, C. (2004) *Cell Death Differ.* **11**, 1084–1091

18. Widlund, H. R., and Fisher, D. E. (2003) *Oncogene* **22**, 3035–3041

19. Rusciano, D., Lorenzoni, P., and Burger, M. M. (1999) *J. Cell Sci.* **112**, 623–630

20. Busca, R., Berra, E., Gaggioli, C., Khaled, M., Bille, K., Marchetti, B., Thyss, R., Fitsialos, G., Larribere, L., Bertolotto, C., Virolle, T., Barbry, P., Pouyssegur, J., Ponzio, G., and Ballotti, R. (2005) *J. Cell Biol.* **170**, 49–59

21. Gaggioli, C., Busca, R., Abbe, P., Ortonne, J. P., and Ballotti, R. (2003) *Pigment Cell Res.* **16**, 374–382

22. Carreira, S., Goodall, J., Denat, L., Rodriguez, M., Nuciforo, P., Hoek, K. S., Testori, A., Larue, L., and Goding, C. R. (2006) *Genes Dev.* **20**, 3426–3439

23. Larribere, L., Hilmi, C., Khaled, M., Gaggioli, C., Bille, K., Auberger, P., Ortonne, J. P., Ballotti, R., and Bertolotto, C. (2005) *Genes Dev.* **19**, 1980–1985

24. Bertolotto, C., Bille, K., Ortonne, J. P., and Ballotti, R. (1996) *J. Cell Biol.* **134**, 747–755

25. Seol, D. W., and Zarnegar, R. (1998) *Biochim. Biophys. Acta* **1395**, 252–258

26. Bertolotto, C., Abbe, P., Hemesath, T. J., Bille, K., Fisher, D. E., Ortonne, J. P., and Ballotti, R. (1998) *J. Cell Biol.* **142**, 827–835

27. McGill, G. G., Horstmann, M., Widlund, H. R., Du, J., Motyckova, G., Nishimura, E. K., Lin, Y. L., Ramaswamy, S., Avery, W., Ding, H. F., Jordan, S. A., Jackson, I. J., Korsmeyer, S. J., Golub, T. R., and Fisher, D. E. (2002) *Cell* **109**, 707–718
28. Wu, M., Hemesath, T. J., Takemoto, C. M., Horstmann, M. A., Wells, A. G., Price, E. R., Fisher, D. Z., and Fisher, D. E. (2000) *Genes Dev.* **14**, 301–312

29. de la Serna, I. L., Ohkawa, Y., Higashi, C., Dutta, C., Osias, J., Kommajosyula, N., Tachibana, T., and Imbalzano, A. N. (2006) *J. Biol. Chem.* **281**, 20233–20241

30. Garraway, L. A., Widlund, H. R., Rubin, M. A., Getz, G., Berger, A. J., Ramaswamy, S., Beroukhim, R., Milner, D. A., Granter, S. R., Du, J., Lee, C., Wagner, S. N., Li, C., Golub, T. R., Rimm, D. L., Meyerson, M. L., Fisher, D. E., and Sellers, W. R. (2005) *Nature* **436**, 117–122

31. Zhang, X., Li, Y., Dai, C., Yang, J., Mundel, P., and Liu, Y. (2003) *Am. J. Physiol.* **284**, F82–F94

32. Seol, D. W., Chen, Q., and Zarnegar, R. (2000) *Oncogene* **19**, 1132–1137

33. Pennacchietti, S., Michieli, P., Galluzzo, M., Mazzone, M., Giordano, S., and Comoglio, P. M. (2003) *Cancer Cell* **3**, 347–361

34. McGill, G. G., Haq, R., Nishimura, E. K., and Fisher, D. E. (2006) *J. Biol. Chem.* **281**, 10365–10373

35. Petrelli, A., Gilestro, G. F., Lanzardo, S., Comoglio, P. M., Migone, N., and Giordano, S. (2002) *Nature* **416**, 187–190

36. Chen, Q., Seol, D. W., Carr, B., and Zarnegar, R. (1997) *Hepatology* **26**, 59–66

37. Stella, M. C., Trusolino, L., Pennacchietti, S., and Comoglio, P. M. (2005) *Mol. Cell. Biol.* **25**, 3982–3996

38. Kochhar, K. S., and Iyer, A. P. (1996) *Cancer Lett.* **104**, 163–169

39. Muller, M., Morotti, A., and Ponzetto, C. (2002) *Mol. Cell. Biol.* **22**, 1060–1072

40. Kankuri, E., Cholujova, D., Comajova, M., Vaheri, A., and Bizik, J. (2005) *Cancer Res.* **65**, 9914–9922

41. Corso, S., Comoglio, P. M., and Giordano, S. (2005) *Trends Mol. Med.* **11**, 284–292

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