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Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.
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Glucose is vital for life, serving as both a source of energy and carbon building block for growth. When glucose is limiting, alternative nutrients must be harnessed. To identify mechanisms by which cells can tolerate complete loss of glucose, we performed nutrient-sensitized genome-wide genetic screens and a PRISM growth assay across 482 cancer cell lines. We report that catabolism of uridine from the medium enables the growth of cells in the complete absence of glucose. While previous studies have shown that uridine can be salvaged to support pyrimidine synthesis in the setting of mitochondrial oxidative phosphorylation deficiency1, our work demonstrates that the ribose moiety of uridine or RNA can be salvaged to fulfil energy requirements via a pathway based on: (1) the phosphorylytic cleavage of uridine by uridine phosphorylase UPP1/UPP2 into uracil and ribose-1-phosphate (R1P), (2) the conversion of uridine-derived R1P into fructose-6-P and glyceraldehyde-3-P by the non-oxidative branch of the pentose phosphate pathway and (3) their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Capacity for glycolysis from uridine-derived ribose appears widespread, and we confirm its activity in cancer lineages, primary macrophages and mice in vivo. An interesting property of this pathway is that R1P enters downstream of the initial, highly regulated steps of glucose transport and upper glycolysis. We anticipate that 'uridine bypass' of upper glycolysis could be important in the context of disease and even exploited for therapeutic purposes.
Assuntos
Ribose , Uridina , Ribose/metabolismo , Uridina/metabolismo , RNA/metabolismo , Glicólise , Humanos , Linhagem Celular Tumoral , Fosforilação Oxidativa , Meios de Cultura , Glucose , Células K562 , Proliferação de Células , Via de Pentose FosfatoRESUMO
Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.
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Melanoma , Microftalmia , Animais , Humanos , Fatores de Transcrição/metabolismo , Microftalmia/genética , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Regulação da Expressão Gênica , Proteínas Oncogênicas/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.
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Fator de Transcrição Associado à Microftalmia/metabolismo , NADP Trans-Hidrogenases/metabolismo , Pigmentação da Pele/efeitos da radiação , Raios Ultravioleta , Animais , Linhagem Celular , Estudos de Coortes , AMP Cíclico/metabolismo , Dano ao DNA , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Predisposição Genética para Doença , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Melanossomas/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , NADP Trans-Hidrogenases/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Polimorfismo de Nucleotídeo Único/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/genética , Ubiquitina/metabolismo , Peixe-ZebraRESUMO
Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Limoninas/farmacologia , Mitocôndrias/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Humanos , Limoninas/química , Limoninas/metabolismo , Limoninas/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis. Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals. Fully melanized melanosomes are delivered to keratinocytes of the skin and hair. The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU). Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis. Diseases involving alterations of EMU show various forms of pigmentation phenotypes. This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade.
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Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Transtornos da Pigmentação/metabolismo , Diferenciação Celular , Humanos , Melaninas/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Transtornos da Pigmentação/tratamento farmacológicoRESUMO
MITF and MYC are well-known oncoproteins and members of the basic helix-loop-helix leucine zipper (bHLH-Zip) family of transcription factors (TFs) recognizing hexamer E-box motifs. MITF and MYC not only share the core binding motif, but are also the two most highly expressed bHLH-Zip transcription factors in melanocytes, raising the possibility that they may compete for the same binding sites in select oncogenic targets. Mechanisms determining the distinct and potentially overlapping binding modes of these critical oncoproteins remain uncharacterized. We introduce computational predictive models using local sequence features, including a boosted convolutional decision tree framework, to distinguish MITF versus MYC-MAX binding sites with up to 80% accuracy genomewide. Select E-box locations that can be bound by both MITF and MYC-MAX form a separate class of MITF binding sites characterized by differential sequence content in the flanking region, diminished interaction with SOX10, higher evolutionary conservation, and less tissue-specific chromatin organization.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Genoma Humano , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Motivos de Aminoácidos , Área Sob a Curva , Composição de Bases/genética , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Sítios de Ligação , Epigênese Genética , Humanos , Fator de Transcrição Associado à Microftalmia/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Curva ROC , Fatores de Transcrição SOXE/metabolismoRESUMO
RMEL3 is a recently identified lncRNA associated with BRAFV600E mutation and melanoma cell survival. Here, we demonstrate strong and moderate RMEL3 upregulation in BRAF and NRAS mutant melanoma cells, respectively, compared to melanocytes. High expression is also more frequent in cutaneous than in acral/mucosal melanomas, and analysis of an ICGC melanoma dataset showed that mutations in RMEL3 locus are preponderantly C > T substitutions at dipyrimidine sites including CC > TT, typical of UV signature. RMEL3 mutation does not correlate with RMEL3 levels, but does with poor patient survival, in TCGA melanoma dataset. Accordingly, RMEL3 lncRNA levels were significantly reduced in BRAFV600E melanoma cells upon treatment with BRAF or MEK inhibitors, supporting the notion that BRAF-MEK-ERK pathway plays a role to activate RMEL3 gene transcription. RMEL3 overexpression, in immortalized fibroblasts and melanoma cells, increased proliferation and survival under serum starvation, clonogenic ability, and xenografted melanoma tumor growth. Although future studies will be needed to elucidate the mechanistic activities of RMEL3, our data demonstrate that its overexpression bypasses the need of mitogen activation to sustain proliferation/survival of non-transformed cells and suggest an oncogenic role for RMEL3.
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Citoproteção , Melanoma/genética , Melanoma/patologia , RNA Longo não Codificante/metabolismo , Soro/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Clonais , Citoproteção/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células NIH 3T3 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , RNA Longo não Codificante/genéticaRESUMO
Certain transcription factors have vital roles in lineage development, including specification of cell types and control of differentiation. Microphthalmia-associated transcription factor (MITF) is a key transcription factor for melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes to promote melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis, including genes encoding proteins involved in apoptosis (eg, BCL2) and the cell cycle (eg, CDK2). Loss-of-function mutations of MITF cause Waardenburg syndrome type IIA, whose phenotypes include depigmentation due to melanocyte loss, whereas amplification or specific mutation of MITF can be an oncogenic event that is seen in a subset of familial or sporadic melanomas. In this article, we review basic features of MITF biological function and highlight key unresolved questions regarding this remarkable transcription factor.
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Melanócitos , Melanoma , Fator de Transcrição Associado à Microftalmia , Transdução de Sinais/fisiologia , Animais , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanócitos/fisiologia , Melanoma/metabolismo , Melanoma/fisiopatologia , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Transcrição Associado à Microftalmia/fisiologiaRESUMO
Bioinformatic analysis of genome-wide gene expression allows us to characterize cells, including melanomas. Gene expression profiles have been generated in various stages of melanomas and analyzed by researchers in unique ways. Lauss et al. compared their melanoma subtypes with those of The Cancer Genome Atlas Network and found consistency between the two studies.
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Biologia Computacional/métodos , Melanoma/genética , Melanoma/terapia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Prognóstico , Medição de Risco , Resultado do TratamentoRESUMO
Microphthalmia-associated transcription factor (MITF) acts via pigment epithelium-derived factor (PEDF), an antiangiogenic protein, to regulate retinal pigment epithelium migration. PEDF expression and/or regulation during melanoma development have not been investigated previously. Using immunohistochemistry, we determined expression of PEDF in common and dysplastic melanocytic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma (n = 102). PEDF expression was consistently decreased in invasive and metastatic melanoma, compared with nevi and melanoma in situ (P < 0.0001). PEDF was lost in thicker melanomas (P = 0.003), and correlated with depth of invasion (P = 0.003) and distant metastasis (P = 0.0331), but only marginally with mitotic index, AJCC stage, nodal metastasis, or blood vascular density (0.05 < P < 0.10). Quantitative real-time PCR and microarray analyses confirmed PEDF down-regulation at the mRNA level in several melanoma lines, compared with melanocytes. MITF positively correlated with PEDF expression in invasive melanomas (P = 0.0003). Searching for PEDF regulatory mechanisms revealed two occupied conserved E-boxes (DNA recognition elements) in the first intron of the human and mouse PEDF promoter regions, confirmed by binding assays. Dominant-negative and siRNA approaches in vivo demonstrated direct transcriptional influence of MITF on PEDF, establishing the PEDF gene (SERPINF1) as a MITF target in melanocytes and melanoma cells. These findings suggest that loss of PEDF expression promotes early invasive melanoma growth.
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Proteínas do Olho/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Imuno-Histoquímica , Masculino , Melanócitos , Camundongos , Microscopia de Fluorescência , Pessoa de Meia-Idade , Dados de Sequência Molecular , Invasividade Neoplásica , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/metabolismo , Homologia de Sequência do Ácido Nucleico , Adulto JovemRESUMO
The microphthalmia-associated transcription factor (MITF) is essential for melanocyte development. Mutation-induced MAPK pathway activation is common in melanoma and induces MITF phosphorylation, ubiquitination, and proteolysis. Little is known about the enzymes involved in MITF ubiquitination/deubiquitination. Here we report the identification of a deubiquitinating enzyme, named ubiquitin-specific protease 13 (USP13) that appears to be responsible for MITF deubiquitination, utilizing a short hairpin RNA library against known deubiquitinating enzymes. Through deubiquitination, USP13 stabilizes and upregulates MITF protein levels. Conversely, suppression of USP13 (through knockdown) leads to dramatic loss of MITF protein, but not messenger RNA. Through its effects on MITF deubiquitination, USP13 was observed to modulate expression of MITF downstream target genes and, thereby, to be essential for melanoma growth in soft agar and in nude mice. These observations suggest that as a potentially drugable protease, USP13 might be a viable therapeutic target for melanoma.
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Endopeptidases/metabolismo , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/química , Linhagem Celular Tumoral , Endopeptidases/genética , Humanos , Melanoma/enzimologia , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteases Específicas de Ubiquitina , Ubiquitinação , Regulação para CimaRESUMO
How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.
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Melanócitos/metabolismo , Oxirredutases/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Células Cultivadas , Complexo de Golgi/metabolismo , Melaninas/biossíntese , Melanossomas/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Transporte Proteico , Transfecção , Antígeno gp100 de Melanoma/metabolismo , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/imunologia , Doença de Bowen/imunologia , Doença de Bowen/patologia , Linfócitos T CD4-Positivos/imunologia , Contagem de Linfócito CD4 , Humanos , Masculino , Pessoa de Meia-Idade , Remissão EspontâneaRESUMO
Melanosome biogenesis consists of multistep processes that involve synthesis of melanosomal protein, which is followed by vesicle transport/fusion and post-translational modifications such as glycosylation, proteolysis, and oligomerization. Because of its complexity, the details of the molecular mechanism of melanosome biogenesis are not yet fully understood. Here, we report that, in MMAc melanoma cells, wild-type (WT) Rab7 and its dominant-active mutant (Rab7-Q67L), but not its dominant-negative mutant (Rab7-T22N), were colocalized in the perinuclear region with granules containing Stage I melanosomes, where the full-length, immature gp100/Pmel17/Silv was present. It was also found that overexpression of Rab7-Q67L and, to a lesser extent, Rab7-WT increased the amount of proteolytically processed, mature gp100. However, Rab7-T22N did not show such an effect. Moreover, siRNA-mediated Rab7 knockdown considerably inhibited gp100 maturation. These results collectively suggest that the GTP-bound form of Rab7 promotes melanogenesis through the regulation of gp100 maturation in melanoma cells.