Expression of Microphthalmia-associated Transcription Factor (MITF), Which Is Critical for Melanoma Progression, Is Inhibited by Both Transcription Factor GLI2 and Transforming Growth Factor-

Institut Curie, Centre de Recherche, INSERM U1021, CNRS UMR3347, and Université Paris XI, 91400 Orsay, France.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2012; 287(22):17996-8004. DOI: 10.1074/jbc.M112.358341
Source: PubMed


The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-β/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-β and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-β. We determined that TGF-β rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.

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Available from: Alain Mauviel, Jul 24, 2015
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    • "The activation of TGF-β signaling induced by HMGA2 was shown to occur preferentially at the invasive front of colorectal tumors and in secondary metastatic lesions [35], and in lymph node metastasis of pancreatic adenocarcinoma [36]. Although the role of HMGA2 in melanoma invasiveness is unknown, TGF-β/GLI2 and MITF inversely regulate invasion and pigmentation in melanoma cells [37], [38], which is consistent with the present results. The lower expression of MITF and related transcriptional networks correlated with increased invasive potential in a panel of cell lines developed from New Zealand patients with metastatic melanoma [34]. "
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    ABSTRACT: The diversity of functional phenotypes observed within a tumor does not exclusively result from intratumoral genetic heterogeneity but also from the response of cancer cells to the microenvironment. We have previously demonstrated that the morphological and functional phenotypes of melanoma can be dynamically altered upon external stimuli. In the present study, transcriptome profiles were generated to explore the molecules governing phenotypes of melanospheres grown in the bFGF(+)EGF(+) serum-free cultures and monolayers maintained in the serum-containing medium. Higher expression levels of MITF-dependent genes that are responsible for differentiation, e.g., TYR and MLANA, and stemness-related genes, e.g., ALDH1A1, were detected in melanospheres. These results were supported by the observation that the melanospheres contained more pigmented cells and cells exerting the self-renewal capacity than the monolayers. In addition, the expression of the anti-apoptotic, MITF-dependent genes e.g., BCL2A1 was also higher in the melanospheres. The enhanced activity of MITF in melanospheres, as illustrated by the increased expression of 74 MITF-dependent genes, identified MITF as a central transcriptional regulator in melanospheres. Importantly, several genes including MITF-dependent ones were expressed in melanospheres and original tumors at similar levels. The reduced MITF level in monolayers might be partially explained by suppression of the Wnt/β-catenin pathway, and DKK1, a secreted inhibitor of this pathway, was highly up-regulated in monolayers in comparison to melanospheres and original tumors. Furthermore, the silencing of DKK1 in monolayers increased the percentage of cells with self-renewing capacity. Our study indicates that melanospheres can be used to unravel the molecular pathways that sustain intratumoral phenotypic heterogeneity. Melanospheres directly derived from tumor specimens more accurately mirrored the morphology and gene expression profiles of the original tumors compared to monolayers. Therefore, melanospheres represent a relevant preclinical tool to study new anticancer treatment strategies.
    PLoS ONE 04/2014; 9(4):e95157. DOI:10.1371/journal.pone.0095157 · 3.23 Impact Factor
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    • "In addition, TGF-β is a potent transcriptional regulator of Gli2, which may activate Gli1 independent of Shh signaling [28]. Moreover, it has been recently discovered that TGF-β inhibits PKA activity while inducing Gli2 and Gli1 expression [43]. PKA blockade may contribute to an increase in the pool of full-length activator Gli proteins, thus inducing an Shh response. "
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    ABSTRACT: Immunosuppressant cyclosporine-A induces gingival hyperplasia, which is characterized by increased fibroblast proliferation and overproduction of extracellular matrix components and regulated by transforming growth factor-beta (TGF-β). The TGF-β and Sonic hedgehog (Shh) pathways both mediate cell proliferation. Crosstalk between these pathways in cancer has recently been proposed, but the hierarchical pattern of this crosstalk remains unclear. In normal fibroblasts, a TGF-β-stimulating Shh pattern was observed in induced fibrosis. However, Shh pathway involvement in cyclosporine-enhanced gingival proliferation and the existence of crosstalk with the TGF-β pathway remain unclear. Cyclosporine enhanced mRNA and protein levels of TGF-β and Shh in human gingival fibroblasts (RT-PCR and western blotting). A TGF-β pathway inhibitor mitigated cyclosporine-enhanced cell proliferation and an Shh pathway inhibitor attenuated cyclosporine-enhanced proliferation in fibroblasts (MTS assay and/or RT-PCR of PCNA). Exogenous TGF-β increased Shh expression; however, exogenous Shh did not alter TGF-β expression. The TGF-β pathway inhibitor mitigated cyclosporine-upregulated Shh expression, but the Shh pathway inhibitor did not alter cyclosporine-upregulated TGF-β expression. The TGF-β and Shh pathways mediate cyclosporine-enhanced gingival fibroblast proliferation. Exogenous TGF-β increased Shh expression, and inhibition of TGF-β signaling abrogated the cyclosporine-induced upregulation of Shh expression; however, TGF-β expression appeared unchanged by enhanced or inhibited Shh signaling. This is the first study demonstrating the role of Shh in cyclosporine-enhanced gingival cell proliferation; moreover, it defines a hierarchical crosstalk pattern in which TGF-β regulates Shh in gingival fibroblasts. Understanding the regulation of cyclosporine-related Shh and TGF-β signaling and crosstalk in gingival overgrowth will clarify the mechanism of cyclosporine-induced gingival enlargement and help develop targeted therapeutics for blocking these pathways, which can be applied in pre-clinical and clinical settings.
    PLoS ONE 07/2013; 8(7):e70128. DOI:10.1371/journal.pone.0070128 · 3.23 Impact Factor
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    • "Although most of the effects caused by BRG1 knockdown on proliferation could be mediated by repression of MITF and hence its targets, several prosurvival factors (OPN, IGF1, survivin and TGFß2), the transcription of which was not reported to be MITF-responsive, were downregulated by BRG1 depletion, as revealed by microarray analysis. All of these proteins were previously implicated as pro-oncogenic factors in melanoma, promoting proliferation, antiapoptosis, invasiveness, or metastasis [30], [34], [37], [40]. To validate the microarray results, we estimated the protein expression and found that protein levels of OPN, survivin, TGFß2, and also livin, a MITF transcriptional target added as a control for MITF-driven gene, were substantially decreased in BRG1-depleted cells (Figure 4A). "
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    ABSTRACT: Metastasized malignant melanoma has a poor prognosis because of its intrinsic resistance to chemotherapy and radiotherapy. The central role in the melanoma transcriptional network has the transcription factor MITF (microphthalmia-associated transcription factor). It has been shown recently that the expression of MITF and some of its target genes require the SWI/SNF chromatin remodeling complex. Here we demonstrate that survival of melanoma cells requires functional SWI/SNF complex not only by supporting expression of MITF and its targets and but also by activating expression of prosurvival proteins not directly regulated by MITF. Microarray analysis revealed that besides the MITF-driven genes, expression of proteins like osteopontin, IGF1, TGFß2 and survivin, the factors known to be generally associated with progression of tumors and the antiapoptotic properties, were reduced in acute BRG1-depleted 501mel cells. Western blots and RT-PCR confirmed the microarray findings. These proteins have been verified to be expressed independently of MITF, because MITF depletion did not impair their expression. Because these genes are not regulated by MITF, the data suggests that loss of BRG1-based SWI/SNF complexes negatively affects survival pathways beyond the MITF cascade. Immunohistochemistry showed high expression of both BRM and BRG1 in primary melanomas. Exogenous CDK2, osteopontin, or IGF1 each alone partly relieved the block of proliferation imposed by BRG1 depletion, implicating that more factors, besides the MITF target genes, are involved in melanoma cell survival. Together these results demonstrate an essential role of SWI/SNF for the expression of MITF-dependent and MITF-independent prosurvival factors in melanoma cells and suggest that SWI/SNF may be a potential and effective target in melanoma therapy.
    PLoS ONE 01/2013; 8(1):e54110. DOI:10.1371/journal.pone.0054110 · 3.23 Impact Factor
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