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1.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073232

RESUMO

Melanoma, the deadliest form of skin cancer, is still one of the most difficult cancers to treat despite recent advances in targeted and immune therapies. About 50% of advanced melanoma do not benefit of such therapies, and novel treatments are requested. Curcumin and its analogs have shown good anticancer properties and are being considered for use in combination with or sequence to recent therapies to improve patient outcomes. Our group previously published the synthesis and anticancer activity characterization of a novel curcumin-related compound against melanoma and neuroblastoma cells (D6). Here, two hydroxylated biphenyl compounds-namely, compounds 11 and 12-were selected among a small collection of previously screened C2-symmetric hydroxylated biphenyls structurally related to D6 and curcumin, showing the best antitumor potentiality against melanoma cells (IC50 values of 1.7 ± 0.5 µM for 11 and 2.0 ± 0.7 µM for 12) and no toxicity of normal fibroblasts up to 32 µM. Their antiproliferative activity was deeply characterized on five melanoma cell lines by performing dose-response and clonal growth inhibition assays, which revealed long-lasting and irreversible effects for both compounds. Apoptosis induction was ascertained by the annexin V and TUNEL assays, whereas Western blotting showed caspase activation and PARP cleavage. A cell cycle analysis, following cell treatments with either compound 11 or 12, highlighted an arrest in the G2/M transition. Taking all this evidence together, 11 and 12 were shown to be good candidates as lead compounds to develop new anticancer drugs against malignant melanoma.


Assuntos
Antineoplásicos , Apoptose/efeitos dos fármacos , Compostos de Bifenilo , Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade
2.
Nat Commun ; 12(1): 3707, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140478

RESUMO

While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600E PtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.


Assuntos
Carcinogênese/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , Proteínas de Homeodomínio/metabolismo , Melanoma/metabolismo , Fatores do Domínio POU/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Estudos de Coortes , Variações do Número de Cópias de DNA , Progressão da Doença , Técnicas de Silenciamento de Genes , Haploinsuficiência , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Melanoma/genética , Melanoma/mortalidade , Melanoma/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Fatores do Domínio POU/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , RNA Interferente Pequeno , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/secundário
3.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071193

RESUMO

Cutaneous melanoma represents one of the deadliest types of skin cancer. The prognosis strongly depends on the disease stage, thus early detection is crucial. New therapies, including BRAF and MEK inhibitors and immunotherapies, have significantly improved the survival of patients in the last decade. However, intrinsic and acquired resistance is still a challenge. In this review, we discuss two major aspects that contribute to the aggressiveness of melanoma, namely, the embryonic origin of melanocytes and melanoma cells and cellular plasticity. First, we summarize the physiological function of epidermal melanocytes and their development from precursor cells that originate from the neural crest (NC). Next, we discuss the concepts of intratumoral heterogeneity, cellular plasticity, and phenotype switching that enable melanoma to adapt to changes in the tumor microenvironment and promote disease progression and drug resistance. Finally, we further dissect the connection of these two aspects by focusing on the transcriptional regulators MSX1, MITF, SOX10, PAX3, and FOXD3. These factors play a key role in NC initiation, NC cell migration, and melanocyte formation, and we discuss how they contribute to cellular plasticity and drug resistance in melanoma.


Assuntos
Plasticidade Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Melanoma/metabolismo , Crista Neural/metabolismo , Neoplasias Cutâneas/metabolismo , Acrilonitrila/análogos & derivados , Acrilonitrila/farmacologia , Compostos de Anilina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular , Movimento Celular , Resistencia a Medicamentos Antineoplásicos/genética , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Transcrição MSX1/genética , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição PAX3/genética , Fenótipo , Pirimidinonas/farmacologia , Fatores de Transcrição SOXE/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia
4.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072104

RESUMO

Melanoma, the malignancy originating from pigment-producing melanocytes, is the most aggressive form of skin cancer and has a poor prognosis once the disease starts to metastasize. The process of melanin synthesis generates an immunosuppressive and mutagenic environment, and can increase melanoma cell resistance to different treatment modalities, including chemo-, radio- or photodynamic therapy. Recently, we have shown that the presence of melanin pigment inhibits the melanoma cell response to bioactive components of Coriolus versicolor (CV) Chinese fungus. Herein, using the same human melanoma cell line in which the level of pigmentation can be controlled by the L-tyrosine concentration in culture medium, we tested the effect of suppression of melanogenesis on the melanoma cell response to CV extract and investigated the cell death pathway induced by fungus extract in sensitized melanoma cells. Our data showed that susceptibility to CV-induced melanoma cell death is significantly increased after cell depigmentation. To the best of our knowledge, we are the first to demonstrate that CV extract can induce RIPK1/RIPK3/MLKL-mediated necroptosis in depigmented melanoma cells. Moreover, using the co-culture system, we showed that inhibition of the tyrosinase activity in melanoma cells modulates cytokine expression in co-cultured mononuclear cells, indicating that depigmentation of melanoma cells may activate immune cells and thereby influence a host anticancer response.


Assuntos
Leucócitos Mononucleares/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Extratos Vegetais/farmacologia , Polyporaceae/química , Transdução de Sinais/efeitos dos fármacos , Pigmentação da Pele , Biomarcadores , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Melaninas/biossíntese , Melanócitos/patologia , Melanoma/patologia , Necroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
5.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066283

RESUMO

Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: -17.65 kcal mol-1 (D6), -18.07 kcal mol-1 (D2), -18.13 (D5) kcal mol-1, and -10.31 kcal mol-1 (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (-12.84 kcal mol-1) and L-tyrosine (-9.04 kcal mol-1) in melanogenesis.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Pironas/química , Pironas/farmacologia , Domínio Catalítico , Humanos , Levodopa/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Melanoma/metabolismo , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Neoplasias Cutâneas/metabolismo , Relação Estrutura-Atividade , Tirosina/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067690

RESUMO

The melanin fluorescence emitted by pigment cells of the human skin has been a central research topic for decades, because melanin, on the one hand, protects against (solar) radiation in the near-UV range, whereas on the other hand, melanocytes are the starting point for the malignant transformation into melanoma. Until recently, however, melanin fluorescence was not accessible in the context of conventional spectroscopy, because it is ultraweak and is overshadowed by the more intense so-called autofluorescence of endogenous fluorophores. The advent of a new method of laser spectroscopy has made this melanin fluorescence measurable in vivo. A stepwise two-photon absorption with 800 nm photons is used, which more selectively excites melanin (dermatofluoroscopy). Our review summarizes the experimental results on melanin fluorescence of the four types of cutaneous pigment cells from healthy and malignant tissues. Outstanding is the finding that different types of melanocytes (i.e., melanocytes of common nevi, versus dysplastic nevi or versus melanoma cells) show characteristically different fluorescence spectra. The possibilities of using this melanin fluorescence for melanoma diagnosis are shown. Moreover, the uniform fluorescence spectra emitted by different melanoma subtypes are essential. Conclusions are drawn about the molecular processes in the melanosomes that determine fluorescence. Finally, experimental suggestions for further investigations are given.


Assuntos
Melaninas/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Transformação Celular Neoplásica/patologia , Fluorescência , Humanos , Melaninas/análise , Melanoma/classificação , Melanoma/fisiopatologia , Pele/patologia , Neoplasias Cutâneas/patologia , Análise Espectral/métodos
7.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067929

RESUMO

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.


Assuntos
Fibroblastos/fisiologia , Melanoma/metabolismo , Microambiente Tumoral/fisiologia , Fibroblastos Associados a Câncer/metabolismo , Comunicação Celular , Plasticidade Celular/fisiologia , Matriz Extracelular/metabolismo , Humanos , Melanoma/patologia , Melanoma/fisiopatologia , Transdução de Sinais , Neoplasias Cutâneas/patologia , Células Estromais/metabolismo
8.
Nat Commun ; 12(1): 3530, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112755

RESUMO

Targeting tumor-associated macrophages (TAMs) is a promising strategy to modify the immunosuppressive tumor microenvironment and improve cancer immunotherapy. Monoamine oxidase A (MAO-A) is an enzyme best known for its function in the brain; small molecule MAO inhibitors (MAOIs) are clinically used for treating neurological disorders. Here we observe MAO-A induction in mouse and human TAMs. MAO-A-deficient mice exhibit decreased TAM immunosuppressive functions corresponding with enhanced antitumor immunity. MAOI treatment induces TAM reprogramming and suppresses tumor growth in preclinical mouse syngeneic and human xenograft tumor models. Combining MAOI and anti-PD-1 treatments results in synergistic tumor suppression. Clinical data correlation studies associate high intratumoral MAOA expression with poor patient survival in a broad range of cancers. We further demonstrate that MAO-A promotes TAM immunosuppressive polarization via upregulating oxidative stress. Together, these data identify MAO-A as a critical regulator of TAMs and support repurposing MAOIs for TAM reprogramming to improve cancer immunotherapy.


Assuntos
Imunoterapia/métodos , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Neoplasias/tratamento farmacológico , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Linfoma/genética , Linfoma/metabolismo , Linfoma/mortalidade , Melanoma/genética , Melanoma/metabolismo , Melanoma/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Monoaminoxidase/deficiência , Monoaminoxidase/genética , Inibidores da Monoaminoxidase/uso terapêutico , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Análise de Célula Única , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Res Vet Sci ; 137: 226-234, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34023546

RESUMO

Canine oral malignant melanoma (COMM) is considered a chemo-resistant cancer with a poor long-term prognosis. The melanoma-associated antigen A (MAGE-A) genes, which belong to the cancer-testis antigen family, are expressed in several different canine cancers but not in normal somatic tissue. This study evaluates the expression of MAGE-A proteins and their prognostic role in COMM. The study was conducted in 2 parts. During the first part, biopsies from oral malignant melanomas from 43 dogs were examined and immunohistochemically assessed for expression of MAGE-A proteins. For the second part, the association between MAGE-A expression and outcome was assessed using follow-up data which was available for 20 dogs whose primary tumour had been controlled with surgery +/- radiation therapy. MAGE-A proteins were expressed in 88.4% (38/43) of oral malignant melanomas and had a predominantly cytoplasmic expression pattern. Immunopositivity was observed in more than 50% of the cells in 21 dogs (48.8%). Immunostaining intensity was classified as weak, moderate and intense in 16 (37%), 16 (37%) and 6 (14%) cases, respectively. No staining for MAGE-A was seen in 5 dogs (11%). Dogs whose COMM had weak MAGE-A staining intensity had a median survival time (MST) of 320 days while this was 129 days for dogs with moderate and intense immunostaining (p = 0.161). Dogs whose COMM had >50% of positive staining neoplastic cells had an MST of 141 days and dogs with a staining <50% had an MST of 320 days (p = 0.164). MAGE-A expression did not influence survival in our cohort.


Assuntos
Doenças do Cão/metabolismo , Antígenos Específicos de Melanoma/biossíntese , Melanoma/veterinária , Neoplasias Bucais/veterinária , Animais , Doenças do Cão/diagnóstico , Cães , Feminino , Imuno-Histoquímica/veterinária , Masculino , Melanoma/diagnóstico , Melanoma/metabolismo , Neoplasias Bucais/metabolismo , Prognóstico , Resultado do Tratamento
10.
Anticancer Res ; 41(5): 2403-2410, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33952465

RESUMO

BACKGROUND/AIM: Fascin, an actin-bundling protein, plays an essential role in cancer metastasis. The Hippo pathway is critical for carcinogenesis and cancer stem cell self-renewal. Mammalian STE20-like kinase (MST) is a core component of the Hippo pathway. However, whether fascin and MST2 affect melanoma remain largely unknown. This study aimed to investigate the role of fascin and MST2 in melanoma development. MATERIALS AND METHODS: Surgically excised skin melanomas and the adjacent non-tumorous skin tissue from 30 cases were analyzed using immunohistochemistry for fascin and MST2. The melanoma cell line WM793 was employed for fascin and MST2 knock-down followed by western blotting, and melanoma xenografting in BALB/c mice. RESULTS: Immunohistochemistry revealed increased expression of fascin and decreased expression of MST2 in melanoma. The reverse correlation of fascin and MST2 was statistically significant. Fascin siRNA upregulated MST2 expression; however, MST2 siRNA did not significantly affect fascin expression in the WM793. WM793 xenografting followed by fascin knock-down inhibited tumor growth significantly in the animal study. CONCLUSION: Fascin is a regulator of the Hippo pathway and plays an important role in melanoma development. Therefore, fascin could be a potential therapeutic target for melanoma.


Assuntos
Proteínas de Transporte/metabolismo , Melanoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Melanoma/genética , Melanoma/terapia , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Terapêutica com RNAi/métodos , Transdução de Sinais/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
11.
Methods Mol Biol ; 2262: 411-422, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977492

RESUMO

Hyper-activation of RAS signaling pathways causes cancer, including melanoma, and RAS signaling pathways have been successfully targeted using drugs for patient benefit. The available drugs alone cannot cure cancer, however, and so investigation continues into RAS signaling pathways, with the goal of identifying further actionable targets. The zebrafish can be used to model human malignancies, and genetic modification of zebrafish to incorporate selective disease-associated genetic alterations is practicable. The following article describes the methods we are using to genetically modify zebrafish in order to dissect oncogenic RAS signaling in melanoma development.


Assuntos
Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Melanoma/patologia , Mutação , Transgenes/genética , Proteínas ras/metabolismo , Animais , Animais Geneticamente Modificados , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Peixe-Zebra , Proteínas ras/genética
12.
Nat Commun ; 12(1): 2742, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980846

RESUMO

Ultraviolet radiation (UVR) damages the dermis and fibroblasts; and increases melanoma incidence. Fibroblasts and their matrix contribute to cancer, so we studied how UVR modifies dermal fibroblast function, the extracellular matrix (ECM) and melanoma invasion. We confirmed UVR-damaged fibroblasts persistently upregulate collagen-cleaving matrix metalloprotein-1 (MMP1) expression, reducing local collagen (COL1A1), and COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, inhibiting ECM degradation and MMP1 expression restored melanoma invasion. Primary cutaneous melanomas of aged humans show more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen, and collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. Thus, primary melanomas arising over collagen-degraded skin are less invasive, and reduced invasion improves survival. However, melanoma-associated fibroblasts can restore invasion by increasing collagen synthesis. Finally, high COL1A1 gene expression is a biomarker of poor outcome across a range of primary cancers.


Assuntos
Colágeno/metabolismo , Melanoma/metabolismo , Melanoma/terapia , Raios Ultravioleta , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Lentivirus/genética , Espectrometria de Massas , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Microscopia de Força Atômica
13.
Nat Commun ; 12(1): 2550, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953176

RESUMO

Melanoma is the deadliest skin cancer. Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled. Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development. Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma. Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process. Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth. Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Melanoma/genética , Melanoma/metabolismo , Animais , Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Feminino , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Melanoma/patologia , Camundongos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais , Transcriptoma
14.
Nucleic Acids Res ; 49(10): 5760-5778, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037780

RESUMO

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.


Assuntos
Processamento Alternativo/genética , Carcinogênese/metabolismo , Desenvolvimento Embrionário/genética , Hematopoese/genética , Melanoma/metabolismo , Timócitos/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Apoptose/genética , Carcinogênese/genética , Proliferação de Células/genética , Genômica , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Linfopenia/genética , Linfopenia/metabolismo , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Timo/embriologia , Timo/metabolismo
15.
Int J Mol Sci ; 22(7)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917428

RESUMO

Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF V600 mutations are sensitive to vemurafenib whereas class II and class III mutants, non-V600 BRAF mutants are resistant to vemurafenib. Herein we report six pyrimido[4,5-d]pyrimidin-2-one derivatives possessing highly potent anti-proliferative activities on melanoma cells harboring BRAF class I/II/III mutants. Novel and most potent derivative, SIJ1777, possesses not only two-digit nanomolar potency but also 2 to 14-fold enhanced anti-proliferative activities compared with reference compound, GNF-7 against melanoma cells (SK-MEL-2, SK-MEL-28, A375, WM3670, WM3629). Moreover, SIJ1777 substantially inhibits the activation of MEK, ERK, and AKT and remarkably induces apoptosis and significantly blocks migration, invasion, and anchorage-independent growth of melanoma cells harboring BRAF class I/II/II mutations while both vemurafenib and PLX8394 have little to no effects on melanoma cells expressing BRAF class II/III mutations. Taken together, our six GNF-7 derivatives exhibit highly potent activities against melanoma cells harboring class I/II/III BRAF mutations compared with vemurafenib as well as PLX8394.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma , Mutação , Proteínas Proto-Oncogênicas B-raf , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo
16.
Int J Mol Sci ; 22(7)2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33800547

RESUMO

Despite recent groundbreaking advances in the treatment of cutaneous melanoma, it remains one of the most treatment-resistant malignancies. Due to resistance to conventional chemotherapy, the therapeutic focus has shifted away from aiming at melanoma genome stability in favor of molecularly targeted therapies. Inhibitors of the RAS/RAF/MEK/ERK (MAPK) pathway significantly slow disease progression. However, long-term clinical benefit is rare due to rapid development of drug resistance. In contrast, immune checkpoint inhibitors provide exceptionally durable responses, but only in a limited number of patients. It has been increasingly recognized that melanoma cells rely on efficient DNA repair for survival upon drug treatment, and that genome instability increases the efficacy of both MAPK inhibitors and immunotherapy. In this review, we discuss recent developments in the field of melanoma research which indicate that targeting genome stability of melanoma cells may serve as a powerful strategy to maximize the efficacy of currently available therapeutics.


Assuntos
Instabilidade Genômica , Melanoma/genética , Neoplasias Cutâneas/genética , Animais , Ensaios Clínicos como Assunto , Dano ao DNA , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Genoma Humano , Humanos , Imunoterapia/métodos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Camundongos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Radioterapia , Neoplasias Cutâneas/metabolismo
17.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801689

RESUMO

As widely acknowledged, 40-50% of all melanoma patients harbour an activating BRAF mutation (mostly BRAF V600E). The identification of the RAS-RAF-MEK-ERK (MAP kinase) signalling pathway and its targeting has represented a valuable milestone for the advanced and, more recently, for the completely resected stage III and IV melanoma therapy management. However, despite progress in BRAF-mutant melanoma treatment, the two different approaches approved so far for metastatic disease, immunotherapy and BRAF+MEK inhibitors, allow a 5-year survival of no more than 60%, and most patients relapse during treatment due to acquired mechanisms of resistance. Deep insight into BRAF gene biology is fundamental to describe the acquired resistance mechanisms (primary and secondary) and to understand the molecular pathways that are now being investigated in preclinical and clinical studies with the aim of improving outcomes in BRAF-mutant patients.


Assuntos
Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/fisiologia , Neoplasias Cutâneas/genética , Antineoplásicos/administração & dosagem , Ciclo Celular , Quimioterapia Adjuvante , Ensaios Clínicos como Assunto , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Imunoterapia , Sistema de Sinalização das MAP Quinases , Masculino , Oncologia/tendências , Melanoma/metabolismo , Mutação , Metástase Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Recidiva , Neoplasias Cutâneas/metabolismo
18.
Nucleic Acids Res ; 49(W1): W36-W45, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33885790

RESUMO

Efficient integration and visualization of heterogeneous biomedical information in a single view is a key challenge. In this study, we present Arena3Dweb, the first, fully interactive and dependency-free, web application which allows the visualization of multilayered graphs in 3D space. With Arena3Dweb, users can integrate multiple networks in a single view along with their intra- and inter-layer connections. For clearer and more informative views, users can choose between a plethora of layout algorithms and apply them on a set of selected layers either individually or in combination. Users can align networks and highlight node topological features, whereas each layer as well as the whole scene can be translated, rotated and scaled in 3D space. User-selected edge colors can be used to highlight important paths, while node positioning, coloring and resizing can be adjusted on-the-fly. In its current version, Arena3Dweb supports weighted and unweighted undirected graphs and is written in R, Shiny and JavaScript. We demonstrate the functionality of Arena3Dweb using two different use-case scenarios; one regarding drug repurposing for SARS-CoV-2 and one related to GPCR signaling pathways implicated in melanoma. Arena3Dweb is available at http://bib.fleming.gr:3838/Arena3D or http://bib.fleming.gr/Arena3D.


Assuntos
Algoritmos , Visualização de Dados , Internet , Mapas de Interação de Proteínas , Software , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Cor , Reposicionamento de Medicamentos , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Linguagens de Programação , Receptores de Endotelina/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais
19.
Int J Mol Sci ; 22(7)2021 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-33916572

RESUMO

Cold atmospheric plasma (CAP) is partially ionized gas near room temperature with previously reported antitumor effects. Despite extensive research and growing interest in this technology, active components and molecular mechanisms of CAP are not fully understood to date. We used Raman spectroscopy and colorimetric assays to determine elevated nitrite and nitrate levels after treatment with a MiniFlatPlaster CAP device. Previously, we demonstrated CAP-induced acidification. Cellular effects of nitrite and strong extracellular acidification were assessed using live-cell imaging of intracellular Ca2+ levels, cell viability analysis as well as quantification of p21 and DNA damage. We further characterized these observations by analyzing established molecular effects of CAP treatment. A synergistic effect of nitrite and acidification was found, leading to strong cytotoxicity in melanoma cells. Interestingly, protein nitration and membrane damage were absent after treatment with acidified nitrite, thereby challenging their contribution to CAP-induced cytotoxicity. Further, phosphorylation of ERK1/2 was increased after treatment with both acidified nitrite and indirect CAP. This study characterizes the impact of acidified nitrite on melanoma cells and supports the importance of RNS during CAP treatment. Further, it defines and evaluates important molecular mechanisms that are involved in the cancer cell response to CAP.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Nitritos/farmacologia , Gases em Plasma/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Humanos , Melanoma/metabolismo , Melanoma/patologia
20.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922284

RESUMO

BACKGROUND: Melanoma patients stop responding to targeted therapies mainly due to mitogen activated protein kinase (MAPK) pathway re-activation, phosphoinositide 3 kinase/the mechanistic target of rapamycin (PI3K/mTOR) pathway activation or stromal cell influence. The future of melanoma treatment lies in combinational approaches. To address this, our in vitro study evaluated if lower concentrations of Celecoxib (IC50 in nM range) could still preserve the chemopreventive effect on melanoma cells treated with trametinib. MATERIALS AND METHODS: All experiments were conducted on SK-MEL-28 human melanoma cells and BJ human fibroblasts, used as co-culture. Co-culture cells were subjected to a celecoxib and trametinib drug combination for 72 h. We focused on the evaluation of cell death mechanisms, melanogenesis, angiogenesis, inflammation and resistance pathways. RESULTS: Low-dose celecoxib significantly enhanced the melanoma response to trametinib. The therapeutic combination reduced nuclear transcription factor (NF)-kB (p < 0.0001) and caspase-8/caspase-3 activation (p < 0.0001), inhibited microphthalmia transcription factor (MITF) and tyrosinase (p < 0.05) expression and strongly down-regulated the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway more significantly than the control or trametinib group (p < 0.0001). CONCLUSION: Low concentrations of celecoxib (IC50 in nM range) sufficed to exert antineoplastic capabilities and enhanced the therapeutic response of metastatic melanoma treated with trametinib.


Assuntos
Adjuvantes Farmacêuticos/farmacologia , Celecoxib/farmacologia , Inflamação/prevenção & controle , Melanoma/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Piridonas/farmacologia , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
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