Correspondence to Richard Marais: richard.marais{at}icr.ac.uk
Abstract
The protein kinase B-RAF is a human oncogene that is mutated in 70% of human melanomas and transforms mouse melanocytes. Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear. In this study, we show that MITF expression is suppressed by oncogenic B-RAF in immortalized mouse and primary human melanocytes. However, low levels of MITF persist in human melanoma cells harboring oncogenic B-RAF, suggesting that additional mechanisms regulate its expression. MITF reexpression in B-RAFtransformed melanocytes inhibits their proliferation. Furthermore, differentiation-inducing factors that elevate MITF expression in melanoma cells inhibit their proliferation, but when MITF up-regulation is prevented by RNA interference, proliferation is not inhibited. These data suggest that MITF is an antiproliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.
Introduction
Melanocytes are pigmented skin cells that protect us from ultraviolet radiation. The processes regulating melanocyte differentiation are intensely studied because melanocytes are thought to be the precursors of melanoma, a skin cancer whose incidence is increasing in Western societies. A master regulator of melanocyte differentiation is the microphthalmia-associated transcription factor (MITF; Widlund and Fisher, 2003). Strikingly, MITF levels are reduced in spontaneously transformed melanocytes (Selzer et al., 2002), and low MITF expression correlates with poor prognosis in melanoma (Salti et al., 2000). MITF regulation is complex. For example, the differentiation factor -melanocytestimulating hormone strongly increases its expression in a cAMP and cAMP response element binding protein (CREB) transcription factordependent manner (Bertolotto et al., 1998). Another signaling module that regulates MITF is the RASRAFMEKERK signaling cascade, which acts downstream of the receptor tyrosine kinase cKIT to stimulate MITF phosphorylation on serine 73 (S73) and enhances its transcriptional activity (Hemesath et al., 1998). However, extracellular regulated protein kinase (ERK)mediated S73 phosphorylation also targets MITF for ubiquitin-dependent degradation through the proteasome pathway (Wu et al., 2000; Xu et al., 2000).
There are three RAS (H-RAS, K-RAS, and N-RAS) and three RAF (A-RAF, B-RAF, and C-RAF) genes in humans. N-RAS is mutated in 520% of melanomas, and B-RAF is mutated in 5070% of melanomas (Davies et al., 2002). The most common mutation in B-RAF (90%) is a glutamic acid for valine substitution at position 600 (formally identified as V599; Wellbrock et al., 2004a), which produces a highly active kinase that stimulates constitutive ERK signaling and stimulates melanoma cell proliferation and survival (Hingorani et al., 2003; Karasarides et al., 2004).
In this study, we show that V600EB-RAF triggers MITF degradation in mouse and human melanocytes and that its reexpression inhibits proliferation. Furthermore, MITF up-regulation suppresses melanoma cell proliferation. These data suggest that high MITF levels are antiproliferative, and, therefore, its expression must be suppressed for transformation by oncogenic B-RAF.
Results and discussion
We previously described the generation of mouse melanocyte lines expressing myc-tagged versions of WTB-RAF (melan-aB-RAF) or V600EB-RAF (melan-aV600E [VE]; Wellbrock et al., 2004b). We demonstrated that melanocytes expressing V600EB-RAF show constitutive ERK signaling and proliferate in a factor-independent manner (Wellbrock et al., 2004b). Importantly, cells expressing high or low levels of WTB-RAF do not have elevated ERK activity or grow in a factor-independent manner, demonstrating that even high levels of WTB-RAF expression are not transforming. Melanocytes expressing V600EB-RAF (clone VE16; Fig. 1 A) display dramatically reduced dendricity and pigmentation, which is similar to the morphology that is observed in melanocytes expressing oncogenic RAS (G12VRAS) or constitutively active MAPK and ERK kinase (MEK; MEKEE; Fig. 1 B). In contrast, clones expressing low or high levels of WTB-RAF (clones B2 and B9) have a parental phenotype (Fig. 1 B). The reduction in pigmentation and dentricity that is induced by oncogenic B-RAF prompted us to examine known regulators of melanocyte differentiation. Importantly, we find that MITF is consistently down-regulated in cell lines expressing V600EB-RAF, G12VRAS, and MEKEE, and this loss correlates with constitutive ERK activation (Fig. 1 C).
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To examine the biological consequences of MITF regulation by B-RAF, we reexpressed MITF in melan-aVE cells. This caused a significant (7384%) reduction in the number of colonies that were formed by these cells (Fig. 3 A). To clarify whether this effect was caused by inhibition of proliferation or induction of apoptosis, we developed melan-aVE cell lines expressing an estrogen receptor (ER) version of MITF (ER-MITF; Carreira et al., 2005) that can be regulated by 4-hydroxy-tamoxifen (4-OHT) and developed a control cell line expressing only the ER fragment. Both proteins are expressed at similar levels (Fig. 3 B). ER-MITF activates the tyrosinase promoter in a 4-OHTdependent manner, whereas the ER fragment does not (Fig. 3 C), demonstrating that ER-MITF is functional. Critically, ER-MITF activation does not induce apoptosis in melan-aVE but significantly impairs its proliferation (38% reduction, P = 0.0116; Fig. 3 D).
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Thus, MITF protein levels are significantly reduced in melanocytes in which B-RAFERK signaling is elevated. However, MITF is present in most melanoma cell lines expressing oncogenic B-RAF or RAS, albeit generally at reduced levels compared with NHM (Fig. 5 A). Our data suggests that MITF is antiproliferative and that one function of oncogenic B-RAF is to suppress its expression to overcome its growth-inhibitory activity. This model is supported by our observation that MITF expression is reduced in NHM expressing V600EB-RAF (Fig. 4, F and G) and the finding that forskolin, which up-regulates MITF (Fig. 5 B), also inhibits DNA synthesis in these cells (Fig. 5 C). Forskolin also up-regulates MITF expression in Colo829 and WM266-4 cells (Fig. 5 D, lanes 1, 2, 5, and 6) and in two melanoma cell lines that express oncogenic B-RAF (Davies et al., 2002), and this is accompanied by reduced proliferation (Fig. 5 E). Importantly, when RNAi is used to prevent MITF up-regulation (Fig. 5 D, lanes 4 and 8), forskolin does not inhibit proliferation of Colo829 and WM266-4 cells (Fig. 5 E), clearly demonstrating that elevated MITF protein levels are growth inhibitory to melanoma cells.
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The antiproliferative effects of high MITF levels in human melanoma is supported by the fact that MITF mRNA expression is frequently low or undetectable in human melanoma cells (Vachtenheim and Novotna, 1999). Importantly, MITF reexpression in transformed MITF-negative human melanocytes and melanoma cells reduces their tumorigenecity in vivo (Selzer et al., 2002), suggesting that elevated MITF is incompatible with melanoma progression. Notably, low MITF levels are linked to reduced survival rates and increased metastases in patients with intermediate thickness melanoma (Salti et al., 2000). Furthermore, MITF target genes such as melan-a/MART-1 or melastatin/TRPM1 are generally down-regulated in more advanced melanomas (Duncan et al., 1998; Hofbauer et al., 1998), which is consistent with MITF not being expressed or being nonfunctional. Our data have clear clinical implications, suggesting that MITF has important prognostic value in melanoma, particularly if used in conjunction with B-RAF mutation status, which is an area that needs urgent investigation.
Previous studies have suggested that MITF protein levels are regulated by ERK-induced degradation (Wu et al., 2000; Xu et al., 2000) and, in agreement with this, we observe that MITF expression is significantly reduced in melanocytes in which ERK is constitutively active as a result of oncogenic B-RAF expression. However, we note that in melanoma cell lines and clinical samples of melanoma, MITF expression is not completely suppressed. We propose that MITF function cannot be completely abolished in melanoma and that low level expression must be maintained to stimulate survival and/or proliferation, possibly by regulating BCL2 (McGill et al., 2002) and CDK2 (Du et al., 2004) expression. Presumably, mechanisms exist to counteract the suppression of MITF expression by oncogenic B-RAF such as maintaining its expression at a level that is compatible with tumor progression but insufficient to suppress cell growth. We are currently developing genetic approaches to test this hypothesis. Our data demonstrate that MITF expression is carefully regulated in melanocytes and melanoma cells and that the regulation of its expression by oncogenic B-RAF warrants further study.
Materials and methods
Cell culture and transfection
Melan-a cells expressing B-RAF, V600EB-RAF, MEKEE, and G12VRAS were described previously (Wellbrock et al., 2004b). Melan-a cells (gift of D. Bennett, St. George's Hospital Medical School, London, UK) and melan-aB-RAF cells were grown in RPMI/10% FCS supplemented with 200 nM TPA and 120 pM cholera toxin. Melan-aVE and melan-aVE-derived cells were cultured in RPMI/10% FCS. Melan-aVE/ER-MITF cells expressed MITF that was fused to the ligand-binding domain of the ER (ER-MITF; gift from C. Goding, Marie Curie Research Institute, Oxted, UK; Carreira et al., 2005), and melan-aVE/ER cells expressed only the ER fragment. They were created by transfecting melan-aVE11 cells with pRK5HA.ER or pRK5HA.ER-MITF and 1:10 of pCDNA3.1/Hygro and were selected in 0.5 mg/ml Hygromycin for 1 wk. pRK5ER and pRK5ER-MITF were generated by cloning HA.ER and HA.ER-MITF as EcoRI fragments from pBABEpuroHA.ER and pBABEpuroHA.ER-MITF (Carreira et al., 2005) into pRK5. Human melanoma cell lines were grown in DME/10% FCS. NHM were cultured in medium 154 with HMGS-2 (Cascade Biologics, Inc.) and transfected with 5 µg DNA using a Nucleofector according to the manufacturer's protocols (Amaxa).
Thymidine incorporation, long-term growth, and colony formation assay
Cells were incubated with 0.4 µCi/ml 3H-thymidine for 4 h before harvesting and were quantified by liquid scintillation counting. For long-term growth, melan-aVE/ER and melan-aVE/ER-MITF cells were seeded at 0.5 x 106 cells per 10-cm dish in 5% FCS (+/ 4-OHT), counted every 3 d, and replated at 0.5 x 106 cells per dish. For clonogenic survival, cells were transfected with pCDNA3.1/V5-HisMITF (provided by H. Arnheiter, Porter Neuroscience Research Center, Bethesda, MD) or pCDNA3.1/V5-His plus 1:10 of pCDNA3.1/Hygro and were selected in Hygromycin for 5 d. After a further 10 d without Hygromycin, colonies were stained with crystal violet.
Cell lysis, Western blotting, and antibodies
Western blot analysis was performed by standard protocols with the following antibodies: A-RAF (C-20; Santa Cruz Biotechnology, Inc.), B-RAF (F-7; Santa Cruz Biotechnology, Inc.), C-RAF (Transduction Laboratories), phosphorylated ERK (ppERK; MAPK-YT; Sigma-Aldrich), myc tag (9E10), HA tag (12CA5), ERK2 (C-14; Santa Cruz Biotechnology, Inc.), MITF (C5 and D5; Neomarkers and provided by D. Fisher, Dana-Farber Cancer Institute, Boston, MA), rabbit anti-myc (Abcam), and phosphorylated CREB (Cell Signaling).
RNAi
Cells were transfected as described previously (Wellbrock et al., 2004b) using 20120 nM A-RAF, C-RAF, scrambled (Karasarides et al., 2004), B-RAF (5'-AACAGUCUACAAGGGAAAGUG-3'), or melanocyte-specific MITF (5'-AGCAGTACCTTTCTACCAC-3') siRNA oligonucleotides.
Luciferase assays
2.5 x 105 cells were transfected with 0.6 µg pGL2 or pGL2htyr (Hemesath et al., 1998) with 0.4 µg pSVß-galactosidase (Promega) using LipofectAMINE (GIBCO BRL) and were treated with 4-OHT after 24 h for a further 24 h before lysis in reporter lysis buffer (Promega) for luciferase activity analysis. Experiments were performed in triplicate, and luciferase activity was corrected for ß-galactosidase expression.
RT-PCR
RNA was isolated using TRIzol (GIBCO BRL), and first-strand cDNA synthesis was performed with 1 µg of total RNA and random hexanucleotides. Specific genes were amplified under conditions in which amplification was still linear. Primers that were used are listed as follows: gapdh (5'-CGGAGTCAACGGATTTGGTCGTAT-3' and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3') and melanocyte-specific mitf (5'-ATGCTGCAAATGCTAGAATACAGTCACTA-3' and 5'-GTTGCTGTAGAGGTCGATCAAGTTTCC-3').
Immunofluorescence
Cells that were grown on glass coverslips were fixed in methanol/acetone, blocked with 1% BSA/PBS, and incubated with rabbit anti-myc (Abcam) and/or anti-MITF (clones C5 and D5; Neomarkers). Staining was revealed by using secondary antibodies that were conjugated to Cy2 or Cy3 (Dianova), and nuclei were counterstained with DAPI. Cells were mounted in DABCO-glycerol.
Microscopy and image analysis
Fluorescence images were acquired at RT with a 40x/0.55 long working distance objective lens on a microscope (model TS-100; Nikon) with fluorescence optics. Bright field images were acquired with a 20x/0.4 long working distance objective lens on the same microscope. A camera (model DN-100; Nikon) was used for image processing using Nikon acquisition software.
Online supplemental material
Fig. S1 shows Western blot analysis of p16INK-4a in melanoma cell lines SKMel-2, A375, WM266-4, Colo829, and SKMel-28 and in NHM. ERK2 serves as a loading control. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200505059/DC1.
Acknowledgments
We thank Prof. D. Bennett for providing the melan-a cells; Dr. C. Goding for the pCMVHA-MITF, pCMVHA-S73A-MITF, pBABEpuroHA.ER, and pBABEpuroHA.MITF-ER plasmids; Dr. D. Fisher for the pGL2htyr construct and C5 MITF antibody; and Dr. H. Arnheiter for the pCDNAV5MITF plasmid.
This work was funded by Cancer Research UK (grant C107/A3096) and The Institute of Cancer Research. The authors have no conflicting interests involving this work.
Submitted: 10 May 2005
Accepted: 15 July 2005
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