RESUMO
Melanocytes evolved to produce the melanin that gives colour to our hair, eyes and skin. The melanocyte lineage also gives rise to melanoma, the most lethal form of skin cancer. The melanocyte lineage differentiates from neural crest cells during development, and most melanocytes reside in the skin and hair, where they are replenished by melanocyte stem cells. Because the molecular mechanisms necessary for melanocyte specification, migration, proliferation and differentiation are co-opted during melanoma initiation and progression, studying melanocyte development is directly relevant to human disease. Here, through the lens of advances in cellular omic and genomic technologies, we review the latest findings in melanocyte development and differentiation, and how these developmental pathways become dysregulated in disease.
Assuntos
Diferenciação Celular , Linhagem da Célula , Melanócitos , Melanoma , Melanócitos/metabolismo , Melanócitos/citologia , Humanos , Animais , Melanoma/patologia , Melanoma/metabolismo , Melanoma/genética , Crista Neural/metabolismo , Proliferação de Células , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genéticaRESUMO
Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.
Assuntos
Acetaldeído , Melanoma , Peixe-Zebra , Melanoma/metabolismo , Melanoma/genética , Melanoma/patologia , Melanoma/tratamento farmacológico , Acetaldeído/metabolismo , Acetaldeído/farmacologia , Animais , Humanos , Linhagem Celular Tumoral , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/genética , Histonas/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Transcrição Gênica/efeitos dos fármacos , Crista Neural/metabolismo , Crista Neural/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.
Assuntos
Melanoma , Fator de Transcrição Associado à Microftalmia , Humanos , Linhagem Celular Tumoral , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Acetilação , Melanoma/genética , Melanoma/metabolismo , Melanócitos/metabolismoRESUMO
Modelling adult diseases to understand their aetiology and progression, and to develop new therapies, is a major challenge for medical biology. We are excited by new efforts in the zebrafish community to develop models of adult diseases that range from cancer to heart, infectious and age-related diseases, and those that relate to toxicology and complex social behaviours. Here, we discuss some of the advances in the field of zebrafish models of adult disease, and where we see opportunities and challenges ahead.
Assuntos
Neoplasias , Peixe-Zebra , Animais , Humanos , Adulto , Comportamento Social , Coração , Modelos Animais de DoençasRESUMO
Phakomatosis pigmentovascularis is a diagnosis that denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement, including CNS disease, asymmetrical growth, and a predisposition to malignancy. Using a tight phenotypic group and high-depth next-generation sequencing of affected tissues, we discover here clonal mosaic variants in gene PTPN11 encoding SHP2 phosphatase as a cause of phakomatosis pigmentovascularis type III or spilorosea. Within an individual, the same variant is found in distinct pigmentary and vascular birthmarks and is undetectable in blood. We go on to show that the same variants can cause either the pigmentary or vascular phenotypes alone, and drive melanoma development within pigmentary lesions. Protein structure modeling highlights that although variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modeling of the missense variants confirms downstream MAPK pathway overactivation and widespread disruption of human endothelial cell angiogenesis. Importantly, patients with PTPN11 mosaicism theoretically risk passing on the variant to their children as the germline RASopathy Noonan syndrome with lentigines. These findings improve our understanding of the pathogenesis and biology of nevus spilus and capillary malformation syndromes, paving the way for better clinical management.
Assuntos
Lentigo , Melanoma , Síndromes Neurocutâneas , Criança , Humanos , Síndromes Neurocutâneas/genética , Síndromes Neurocutâneas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Mosaicismo , Melanoma/genéticaRESUMO
Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models.
Assuntos
Melanoma , Peixe-Zebra , Animais , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Tamoxifeno/farmacologia , Proteínas de Peixe-ZebraRESUMO
Melanocyte stem cells (McSCs) in zebrafish serve as an on-demand source of melanocytes during growth and regeneration, but metabolic programs associated with their activation and regenerative processes are not well known. Here, using live imaging coupled with scRNA-sequencing, we discovered that, during regeneration, quiescent McSCs activate a dormant embryonic neural crest transcriptional program followed by an aldehyde dehydrogenase (Aldh) 2 metabolic switch to generate progeny. Unexpectedly, although ALDH2 is well known for its aldehyde-clearing mechanisms, we find that, in regenerating McSCs, Aldh2 activity is required to generate formate - the one-carbon (1C) building block for nucleotide biosynthesis - through formaldehyde metabolism. Consequently, we find that disrupting the 1C cycle with low doses of methotrexate causes melanocyte regeneration defects. In the absence of Aldh2, we find that purines are the metabolic end product sufficient for activated McSCs to generate progeny. Together, our work reveals McSCs undergo a two-step cell state transition during regeneration, and that the reaction products of Aldh2 enzymes have tissue-specific stem cell functions that meet metabolic demands in regeneration.
Assuntos
Melanócitos , Peixe-Zebra , Animais , Diferenciação Celular , Crista Neural , Células-TroncoRESUMO
Zebrafish embryos are widely used for drug discovery, however, administering drugs to adult zebrafish is limited by current protocols that can cause stress. Here, we developed a drug formulation and administration method for adult zebrafish by producing food-based drug pellets that are consumed voluntarily. We applied this to zebrafish with BRAF-mutant melanoma, a model that has significantly advanced our understanding of melanoma progression, but not of drug resistance due to the limitations of current treatment methods. Zebrafish with melanomas responded to short-term, precise and daily dosing with drug pellets made with the BRAFV600E inhibitor, vemurafenib. On-target drug efficacy was determined by phospho-Erk staining. Continued drug treatment led to the emergence, for the first time in zebrafish, of acquired drug resistance and melanoma relapse, modelling the responses seen in melanoma patients. This method presents a controlled, non-invasive approach that permits long-term drug studies and can be widely applied to adult zebrafish models.
Assuntos
Melanoma , Peixe-Zebra , Animais , Linhagem Celular Tumoral , Implantes de Medicamento/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Indóis/farmacologia , Melanoma/tratamento farmacológico , Mutação/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Preparações Farmacêuticas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
Melanocytes, the pigment-producing cells, are replenished from multiple stem cell niches in adult tissue. Although pigmentation traits are known risk factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs and lack key lineage markers with which to identify McSCs and study their function. Here we find that Tfap2b and a select set of target genes specify an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and is essential for McSC-dependent melanocyte regeneration. Fate mapping data reveal that tfap2b+ McSCs have multifate potential, and are the cells of origin for large patches of adult melanocytes, two other pigment cell types (iridophores and xanthophores), and nerve-associated cells. Hence, Tfap2b confers McSC identity in early development, distinguishing McSCs from other neural crest and pigment cell lineages, and retains multifate potential in the adult zebrafish.
Assuntos
Melanócitos/metabolismo , Células-Tronco/classificação , Fator de Transcrição AP-2/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Melanócitos/fisiologia , Pigmentação/genética , Pele/metabolismo , Pigmentação da Pele/genética , Células-Tronco/metabolismo , Fator de Transcrição AP-2/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Inibidores de MTOR/administração & dosagem , Melanoma/tratamento farmacológico , Neurofibromina 1/genética , PTEN Fosfo-Hidrolase/genética , Pirimidinas/administração & dosagem , Sulfonamidas/administração & dosagem , Tiofenos/administração & dosagem , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Everolimo/administração & dosagem , Everolimo/farmacologia , Humanos , Mutação com Perda de Função , Inibidores de MTOR/farmacologia , Melanoma/genética , Melanoma/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Numerous drug treatments that have recently entered the clinic or clinical trials have their genesis in zebrafish. Zebrafish are well established for their contribution to developmental biology and have now emerged as a powerful preclinical model for human disease, as their disease characteristics, aetiology and progression, and molecular mechanisms are clinically relevant and highly conserved. Zebrafish respond to small molecules and drug treatments at physiologically relevant dose ranges and, when combined with cell-specific or tissue-specific reporters and gene editing technologies, drug activity can be studied at single-cell resolution within the complexity of a whole animal, across tissues and over an extended timescale. These features enable high-throughput and high-content phenotypic drug screening, repurposing of available drugs for personalized and compassionate use, and even the development of new drug classes. Often, drugs and drug leads explored in zebrafish have an inter-organ mechanism of action and would otherwise not be identified through targeted screening approaches. Here, we discuss how zebrafish is an important model for drug discovery, the process of how these discoveries emerge and future opportunities for maximizing zebrafish potential in medical discoveries.
Assuntos
Ensaios Clínicos como Assunto/estatística & dados numéricos , Modelos Animais de Doenças , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Ensaios de Triagem em Larga Escala , Humanos , Peixe-ZebraRESUMO
There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.
Assuntos
Modelos Animais de Doenças , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Microambiente Tumoral/imunologia , Animais , Humanos , Imunidade/imunologia , Imunoterapia/métodos , Melanoma/patologia , Neoplasias Cutâneas/patologiaRESUMO
The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.