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BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.
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Melanoma , Proteínas Quinasas S6 Ribosómicas 90-kDa , Humanos , Animales , Ratones , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas Proto-Oncogénicas B-raf , Evasión Inmune , Línea Celular Tumoral , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ciclo Celular , Melanoma Cutáneo MalignoRESUMEN
Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.
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Melaninas , Trastornos de la Pigmentación , Recién Nacido , Humanos , Ratones , Animales , Melaninas/metabolismo , Melanocitos/metabolismo , Diferenciación Celular , Epigénesis Genética , Trastornos de la Pigmentación/metabolismo , Pigmentación , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Malignant melanoma represents a highly aggressive form of skin cancer. The metastatic process itself is mostly governed by the so-called epithelial mesenchymal transition (EMT), which confers cancer cells migrative, invasive and resistance abilities. Since EMT represents a conserved developmental process, it is worthwhile further examining the nature of early developmental steps fundamental for melanocyte differentiation. This can be done either in vivo by analyzing the physiologic embryo development in different species or by in vitro studies of melanocytic differentiation originating from embryonic human stem cells. Most importantly, external cues drive progenitor cell differentiation, which can be divided in stages favoring neural crest specification or melanocytic differentiation and proliferation. In this review, we describe ectopic factors which drive human pluripotent stem cell differentiation to melanocytes in 2D, as well as in organoid models. Furthermore, we compare developmental mechanisms with processes described to occur during melanoma development. Finally, we suggest differentiation factors as potential co-treatment options for metastatic melanoma patients.
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Transcription factors, extensively described for their role in epithelial-mesenchymal transition (EMT-TFs) in epithelial cells, also display essential functions in the melanocyte lineage. Recent evidence has shown specific expression patterns and functions of these EMT-TFs in neural crest-derived melanoma compared to carcinoma. Herein, we present an update of the specific roles of EMT-TFs in melanocyte differentiation and melanoma progression. As major regulators of phenotype switching between differentiated/proliferative and neural crest stem cell-like/invasive states, these factors appear as major drivers of intra-tumor heterogeneity and resistance to treatment in melanoma, which opens new avenues in terms of therapeutic targeting.
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BACKGROUND: Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF. RESULTS: Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. CONCLUSION: These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.
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Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Melanocitos/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Melaninas/biosíntesis , Melaninas/genética , Melanocitos/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Melanomas are known to exhibit phenotypic plasticity. However, the role cellular plasticity plays in melanoma tumor progression and drug resistance is not fully understood. Here, we used reprogramming of melanocytes and melanoma cells to induced pluripotent stem cell (iPSCs) to investigate the relationship between cellular plasticity and melanoma progression and mitogen-activated protein kinase (MAPK) inhibitor resistance. We found that melanocyte reprogramming is prevented by the expression of oncogenic BRAF, and in melanoma cells harboring oncogenic BRAF and sensitive to MAPK inhibitors, reprogramming can be restored by inhibition of the activated oncogenic pathway. Our data also suggest that melanoma tumor progression acts as a barrier to reprogramming. Under conditions that promote melanocytic differentiation of fibroblast- and melanocyte-derived iPSCs, melanoma-derived iPSCs exhibited neural cell-like dysplasia and increased MAPK inhibitor resistance. These data suggest that iPSC-like reprogramming and drug resistance of differentiated cells can serve as a model to understand melanoma cell plasticity-dependent mechanisms in recurrence of aggressive drug-resistant melanoma.
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Células Madre Pluripotentes Inducidas/metabolismo , Melanoma/metabolismo , Melanoma/patología , Células Madre Neoplásicas/metabolismo , Fenotipo , Biomarcadores de Tumor , Diferenciación Celular , Línea Celular Tumoral , Plasticidad de la Célula , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Melanocitos/metabolismo , Melanoma/genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mutación , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
BACKGROUND: Long-term survival of stage IV melanoma patients has improved significantly with the development of immune checkpoint inhibitors (CIs). Reliable biomarkers to predict response and clinical outcome are needed. METHODS: We investigated the role of melanoma-associated antibodies as predictive markers for CI therapy in two independent cohorts. In cohort 1, a prospective study, we measured specific antibodies before treatment, after one week and after six to nine weeks of treatment. Cohort 2 consisted of serum samples prior to CI therapy initiation. ELISA assays were performed to quantify specific IgG directed against melanocyte differentiation antigens tyrosinase-related proteins 1 and 2 (TRP1/TYRP1 and TRP2/TYRP2), glycoprotein 100 (gp100), MelanA/MART1, and the cancer-testis antigen NY-ESO-1. Response was defined as either complete or partial remission on CT scan according to RECIST 1.1. RESULTS: In cohort 1, baseline levels of these antibodies were higher in the responder group, although statistical significance was only reached for NY-ESO-1 (p = 0.007). In cohort 2, significantly higher antibody baseline levels for MelanA/MART1 (p = 0.003) and gp100 (p = 0.029) were found. After pooling the results from both cohorts, higher levels of MelanA/MART1 (p = 0.013), TRP1/TYRP1 (p = 0.048), TRP2/TYRP2 (p = 0.047) and NY-ESO-1 (p = 0.005) specific antibodies at baseline were independently associated with response. CONCLUSIONS: Melanoma-associated antibodies may be candidate biomarkers for response and survival in metastatic melanoma patients being treated with CIs. These markers may be used to complement patient assessment, in combination with PD-L1 status, tumor-infiltrating lymphocytes and tumor mutational burden, with the aim to predict outcome of CI treatment in patients with metastatic melanoma. TRIAL REGISTRATION: Ethikkommission Ostschweiz, EKOS 16/079 https://ongoingprojects.swissethics.ch/runningProjects_list.php?q=%28BASECID~contains~2016-00998%29&orderby=dBASECID .
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Antineoplásicos/sangre , Antineoplásicos Inmunológicos/uso terapéutico , Ipilimumab/uso terapéutico , Melanoma/sangre , Nivolumab/uso terapéutico , Anciano , Anciano de 80 o más Años , Biomarcadores , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Persona de Mediana EdadRESUMEN
SWI/SNF chromatin remodeling enzymes are multisubunit complexes that contain one of two catalytic subunits, BRG1 or BRM and 9-11 additional subunits called BRG1 or BRM-associated factors (BAFs). BRG1 interacts with the microphthalmia-associated transcription factor (MITF) and is required for melanocyte development in vitro and in vivo. The subunits of SWI/SNF that mediate interactions between BRG1 and MITF have not been elucidated. Three mutually exclusive isoforms of a 60-kDa subunit (BAF60A, B, or C) often facilitate interactions with transcription factors during lineage specification. We tested the hypothesis that a BAF60 subunit promotes interactions between MITF and the BRG1-containing SWI/SNF complex. We found that MITF can physically interact with BAF60A, BAF60B, and BAF60C. The interaction between MITF and BAF60A required the basic helix-loop-helix domain of MITF. Recombinant BAF60A pulled down recombinant MITF, suggesting that the interaction can occur in the absence of other SWI/SNF subunits and other transcriptional regulators of the melanocyte lineage. Depletion of BAF60A in differentiating melanoblasts inhibited melanin synthesis and expression of MITF target genes. MITF promoted BAF60A recruitment to melanocyte-specific promoters, and BAF60A was required to promote BRG1 recruitment and chromatin remodeling. Thus, BAF60A promotes interactions between MITF and the SWI/SNF complex and is required for melanocyte differentiation.
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Diferenciación Celular , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , Diferenciación Celular/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Melaninas/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/química , Modelos Biológicos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Subunidades de Proteína/metabolismoRESUMEN
Melanoma is the most aggressive form of skin cancer. Current challenges to melanoma therapy include the adverse effects from immunobiologics, resistance to drugs targeting the MAPK pathway, intricate interaction of many signal pathways, and cancer heterogeneity. Thus combinational therapy with drugs targeting multiple signaling pathways becomes a new promising therapy. Here, we report a family of stilbene-like compounds called A11 that can inhibit melanoma growth in both melanoma-forming zebrafish embryos and mouse melanoma cells. The growth inhibition by A11 is a result of mitosis reduction but not apoptosis enhancement. Meanwhile, A11 activates both MAPK and Akt signaling pathways. Many A11-treated mouse melanoma cells exhibit morphological changes and resemble normal melanocytes. Furthermore, we found that A11 causes down-regulation of melanocyte differentiation genes, including Pax3 and MITF. Together, our results suggest that A11 could be a new melanoma therapeutic agent by inhibiting melanocyte differentiation and proliferation.
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Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Melanocitos/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Estilbenos/farmacología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/efectos de los fármacos , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
During development, melanocyte progenitors originate from the neural crest, a transient embryonic structure in vertebrates that gives rise to a variety of cell types including neurons and glia of the peripheral nervous system, smooth muscle cells of the cardiovascular system, chondrocytes and osteoblasts of the craniofacial elements, and pigment cells in the skin. In this chapter, we describe a method for the differentiation of multipotent embryonic neural crest stem cells into differentiated pigmented melanocytes by using in vitro explant culture system. This protocol allows the dissection of genetic and cellular mechanisms regulating neural crest stem cell and melanocyte development. Based on this knowledge it is possible to make predictions about processes that might also be implicated in melanoma initiation and progression.
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Immune checkpoint blockade has formally demonstrated the clinical benefit of immunotherapy against melanoma. New immunotherapeutic modalities are currently explored to improve the management of relapsing/refractory patients. Potent antitumor vaccines would have the advantage to promote long-lasting tumor control while limiting autoimmunity. Alphavirus vectors and nonreplicating particles offer versatile platforms to deliver antigen expression and immunize against cancer. They have shown promising preclinical results and initial proof of clinical activity in melanoma. The growing number of clinically available immunomodulatory agents provides a tremendous opportunity to exploit and revisit anticancer vaccines in the setting of powerful immunotherapeutic combinations. Accelerating the evaluation of alphavirus-based vaccines in patients with immune sensitive, but still very deadly malignancies, such as melanoma, is thus extremely important.
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Alphavirus/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/uso terapéutico , Vacunas contra el Cáncer/inmunología , Vectores Genéticos/genética , Inmunoterapia , Melanoma/terapia , Animales , Presentación de Antígeno , Antígenos de Neoplasias/genética , Humanos , Activación de Linfocitos , Melanoma/inmunología , Mejoramiento de la CalidadRESUMEN
BACKGROUND: The formation of dendrites by melanocytes is highly analogous to that process in neural cells. We previously reported that a C2 domain-containing protein, copine-1, is involved in the extension of dendrites by neural cells. However, the effect of C2 domain-containing proteins in dendrite formation by melanocytes has not yet been elucidated. OBJECTIVE: The aim of this study was to screen novel C2 domain-containing proteins related to dendrite outgrowth in melanocytes and to investigate their precise roles in melanocyte dendrite formation during differentiation. METHODS: We transduced mouse melan-a melanocytes with a recombinant adenovirus expressing a C2 domain library. Dendrite elongation, melanin content, tyrosinase activity and Western blot analyses were conducted to elucidate the possible underlying mechanisms of action in melanocytes. RESULTS: Sixteen sets of C2 domain-containing proteins were identified whose over-expression resulted in dendrite lengthening. Among those, we focused on the C2 domain of SYT14L (truncated mutant of SYT14L) in this study. Forced expression of full length SYT14L or the C2 domain of SYT14L induced a significant elongation of dendrite length accompanied by the induction of melanocyte differentiation-related markers, including melanin synthesis, tyrosinase catalytic activity and the expression of tyrosinase (TYR), tyrosinase related protein-1 (TRP-1) and TRP-2. In addition, over-expression of either the C2 domain or the full length form of SYT14L significantly increased the phosphorylation of ERK and CREB. CONCLUSION: These results suggest that SYT14L, especially its C2 domain, may play an important role in regulating melanocyte differentiation through the modulation of ERK and (or) CREB signaling.