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1.
Cell ; 141(4): 583-94, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20478252

RESUMO

Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.


Assuntos
Melanoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Histona Desmetilases com o Domínio Jumonji , Proteínas de Membrana/metabolismo , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Receptor Notch1/metabolismo , Proteínas Repressoras/genética , Proteínas Serrate-Jagged , Transdução de Sinais
2.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35121657

RESUMO

Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically "cold" pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling, and all but one lack functional cytosolic DNA sensing via cGAS-STING. However, double-stranded RNA (dsRNA) sensing via Toll-like receptor 3 (TLR3) was heterogeneous, as was signaling through other dsRNA sensors and TLRs more broadly. Seven cell lines showed robust response to dsRNA, six of which are in the mesenchymal epigenetic state, while all unresponsive cell lines are in the adrenergic state. Genetically switching adrenergic cell lines toward the mesenchymal state fully restored responsiveness. In responsive cells, dsRNA sensing results in the secretion of proinflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T cells in vitro. Using single-cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intratumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.


Assuntos
Epigênese Genética/genética , Inflamação/genética , Neuroblastoma/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/genética , Humanos , Fatores Imunológicos/genética , Imunoterapia/métodos , Masculino , Camundongos , Camundongos SCID , Nucleotidiltransferases/genética , RNA de Cadeia Dupla/genética , Transdução de Sinais/genética , Receptor 3 Toll-Like/genética , Transcriptoma/genética
4.
Nature ; 546(7658): 431-435, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28607484

RESUMO

Therapies that target signalling molecules that are mutated in cancers can often have substantial short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can result from secondary mutations, but in other cases there is no clear genetic cause, raising the possibility of non-genetic rare cell variability. Here we show that human melanoma cells can display profound transcriptional variability at the single-cell level that predicts which cells will ultimately resist drug treatment. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signalling pathways, partially mediated by the activity of the transcription factors JUN and/or AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general program in which expression is displayed in rare subpopulations of cells.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/genética , Melanoma/patologia , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Marcadores Genéticos/efeitos dos fármacos , Marcadores Genéticos/genética , Humanos , Hibridização in Situ Fluorescente , Indóis/farmacologia , Masculino , Proteínas Nucleares/metabolismo , Proteína Oncogênica p65(gag-jun)/metabolismo , Fatores de Transcrição SOXE/deficiência , Fatores de Transcrição SOXE/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Análise de Célula Única , Sulfonamidas/farmacologia , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Vemurafenib , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Cancer Ther ; 5(5): 1136-44, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16731745

RESUMO

Although >66% of melanomas harbor activating mutations in BRAF and exhibit constitutive activity in the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway, it is unclear how effective MEK inhibition will be as a sole therapeutic strategy for melanoma. We investigated the anticancer activity of MEK inhibition in a panel of cell lines derived from radial growth phase (WM35) and vertical growth phase (WM793) of primary melanomas and metastatic melanomas (1205Lu, 451Lu, WM164, and C8161) in a three-dimensional spheroid model and found that the metastatic lines were completely resistant to MEK inhibition (U0126 and PD98059) but the earlier stage cell lines were not. Similarly, these same metastatic melanoma lines were also resistant to inhibitors of the phosphatidylinositol 3-kinase/Akt pathway (LY294002 and wortmannin). Under adherent culture conditions, the MEK inhibitors blocked growth through the induction of cell cycle arrest and up-regulation of p27, but this was readily reversible following inhibitor washout. However, when the phosphatidylinositol 3-kinase and MEK inhibitors were combined, the growth and invasion of the metastatic melanoma three-dimensional spheroids were blocked. Taken together, these results suggest that the most aggressive melanomas are resistant to strategies targeting one signaling pathway and that multiple signaling pathways may need to be targeted for maximal therapeutic efficacy. It is further suggested that BRAF mutational status is not predictive of response to MEK inhibition under three-dimensional culture conditions.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/patologia , Transdução de Sinais , Butadienos/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Cromonas/metabolismo , Cromonas/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/metabolismo , Morfolinas/metabolismo , Morfolinas/farmacologia , Metástase Neoplásica , Nitrilas/metabolismo , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares
6.
Cancer Res ; 77(21): 5873-5885, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28887323

RESUMO

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of ß-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low ß-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased ß-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing ß-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873-85. ©2017 AACR.


Assuntos
Proteína 5 Relacionada à Autofagia/genética , Autofagia/genética , Melanoma/genética , Via de Sinalização Wnt/genética , Aminoquinolinas/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Western Blotting , Linhagem Celular Tumoral , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Poliaminas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
7.
Mol Cancer Res ; 14(9): 767-75, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27297629

RESUMO

UNLABELLED: The majority of patients with melanoma harbor mutations in the BRAF oncogene, thus making it a clinically relevant target. However, response to mutant BRAF inhibitors (BRAFi) is relatively short-lived with progression-free survival of only 6 to 7 months. Previously, we reported high expression of ribonucleotide reductase M2 (RRM2), which is rate-limiting for de novo dNTP synthesis, as a poor prognostic factor in patients with mutant BRAF melanoma. In this study, the notion that targeting de novo dNTP synthesis through knockdown of RRM2 could prolong the response of melanoma cells to BRAFi was investigated. Knockdown of RRM2 in combination with the mutant BRAFi PLX4720 (an analog of the FDA-approved drug vemurafenib) inhibited melanoma cell proliferation to a greater extent than either treatment alone. This occurred in vitro in multiple mutant BRAF cell lines and in a novel patient-derived xenograft (PDX) model system. Mechanistically, the combination increased DNA damage accumulation, which correlated with a global decrease in DNA damage repair (DDR) gene expression and increased apoptotic markers. After discontinuing PLX4720 treatment, cells showed marked recurrence. However, knockdown of RRM2 attenuated this rebound growth both in vitro and in vivo, which correlated with maintenance of the senescence-associated cell-cycle arrest. IMPLICATIONS: Inhibition of RRM2 converts the transient response of melanoma cells to BRAFi to a stable response and may be a novel combinatorial strategy to prolong therapeutic response of patients with melanoma. Mol Cancer Res; 14(9); 767-75. ©2016 AACR.


Assuntos
Melanoma/terapia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/genética , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Masculino , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Distribuição Aleatória , Ribonucleosídeo Difosfato Redutase/deficiência , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Clin Cancer Res ; 22(7): 1592-602, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26673799

RESUMO

PURPOSE: To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models. EXPERIMENTAL DESIGN: We established 12 PDXs from BRAF inhibitor-progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches. RESULTS: We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals. CONCLUSIONS: Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. See related commentary by Hartsough and Aplin, p. 1550.


Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Melanoma/genética , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Análise por Conglomerados , Modelos Animais de Doenças , Progressão da Doença , Amplificação de Genes , Perfilação da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Proteômica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Pigment Cell Melanoma Res ; 23(6): 820-7, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20973932

RESUMO

Targeted intervention of the B-Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next-generation inhibitor with exquisite specificity against the V600E oncogene and striking anti-melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E-positive cells in a variety of in vitro experimental systems; follow-up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.


Assuntos
Substituição de Aminoácidos/genética , Indóis/farmacologia , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise Mutacional de DNA , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Melanoma/patologia , Camundongos , Modelos Biológicos , Indução de Remissão , Transdução de Sinais/efeitos dos fármacos , Vemurafenib
10.
Cancer Res ; 69(13): 5312-20, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19549918

RESUMO

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (N(IC)) was exploited to determine if Notch activation is a "driving" event in melanocytic transformation or instead a "passenger" event associated with melanoma progression. N(IC)-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. N(IC)-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease.


Assuntos
Melanócitos/fisiologia , Receptor Notch1/fisiologia , Antígeno CD146/genética , Antígeno CD146/fisiologia , Divisão Celular , Progressão da Doença , Prepúcio do Pênis/citologia , Humanos , Masculino , Melanócitos/patologia , Melanoma/genética , Melanoma/patologia , Melanoma/prevenção & controle , Fenótipo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controle , Regulação para Cima
11.
Stem Cells ; 24(7): 1668-77, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16574754

RESUMO

Because of their undifferentiated nature, human embryonic stem cells (hESCs) are an ideal model system for studying both normal human development and the processes that underlie disease. In the current study, we describe an efficient method for differentiating hESCs into a melanocyte population within 4-6 weeks using three growth factors: Wnt3a, endothelin-3, and stem cell factor. The hESC-derived melanocytes expressed melanocyte markers (such as microphthalmia-associated transcription factor and tyrosinase), developed melanosomes, and produced melanin. They retained the melanocyte phenotype during long-term cell culture (>90 days) and, when incorporated into human reconstructed skin, homed to the appropriate location along the basement membrane in the same manner as epidermis-derived melanocytes. They maintained a stable phenotype even after grafting of the reconstructs to immunodeficient mice. Over time in culture, the hESC-derived melanocytes lost expression of telomerase and underwent senescence. In summary, we have shown for the first time the differentiation of hESCs into melanocytes. This method provides a novel in vitro system for studying the development biology of human melanocytes.


Assuntos
Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Indução Embrionária , Melanócitos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Substâncias de Crescimento/farmacologia , Humanos , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos , Pele/citologia , Proteínas Wnt/fisiologia
12.
Semin Cancer Biol ; 15(6): 451-9, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16043361

RESUMO

Cells grow within defined environmental niches and are subject to microenvironmental control. Outside of their niche, the environment is hostile, the normal cells lack appropriate survival signals which leads to anoikis. During tumor development and progression, malignant cells must escape the local tissue control and resist anoikis. The inherent genetic instability of tumor cells makes their phenotype very plastic, which changes under continuous environmental selection pressure. In this way the microenvironment drives the somatic evolution of the tumor. In the current review, we assess how this environmental selection pressure fits into the classical scheme of tumor progression.


Assuntos
Evolução Molecular , Instabilidade Genômica , Neoplasias/genética , Animais , Progressão da Doença , Humanos , Células Estromais/patologia
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