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1.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33803640

RESUMEN

The LATS1 kinase has been described as a tumor suppressor in various cancers. However, its role in melanoma has not been fully elucidated. There are several processes involved in tumorigenesis, including melanin production. Melanin content positively correlates with the level of reactive oxygen species (ROS) inside the cell. Accordingly, the purpose of the study was to assess the role of LATS1 in melanogenesis and oxidative stress and its influence on tumor growth. We have knocked down LATS1 in primary melanocytes and melanoma cells and found that its expression is crucial for melanin synthesis, ROS production, and oxidative stress response. We showed that LATS1 ablation significantly decreased the melanogenesis markers' expression and melanin synthesis in melanocyte and melanoma cell lines. Moreover, silencing LATS1 resulted in enhanced oxidative stress. Reduced melanin content in LATS1 knocked down tumors was associated with increased tumor growth, pointing to melanin's protective role in this process. The study demonstrated that LATS1 is highly engaged in melanogenesis and oxidative stress control and affects melanoma growth. Our results may find the implications in the diagnosis and treatment of pigmentation disorders, including melanoma.


Asunto(s)
Melaninas/biosíntesis , Melanoma/patología , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Cinética , Melanocitos/metabolismo , Melanoma/genética , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Hipoxia Tumoral/genética
2.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33803618

RESUMEN

A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The conformational preferences of the thiosemicarbazone system were characterized using theoretical calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions. The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes, with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites presence and the expression of oxidative stress and DNA damage response genes were evaluated. The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant enzymes expression.


Asunto(s)
Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Cobre/farmacología , Melanoma/patología , Tiosemicarbazonas/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/química , Cobre/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Iones , Melanoma/genética , Conformación Molecular , Necrosis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Temperatura , Tiosemicarbazonas/química
3.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800878

RESUMEN

Uveal melanoma (UM) is a malignant tumor that arises in the melanocytes of the uveal tract. It is the most frequent eye cancer, and despite new therapeutic approaches, prognosis is still poor, with up to 50% of patients developing metastasis with no efficient treatment options available. In contrast to cutaneous melanoma, UM is considered an "immune-cold" tumor due to the low mutational burden and the unique immunosuppressive microenvironment. To gain insight into the role of the UM microenvironment in regard to prognosis and metastatic progression, we have performed a pool analysis characterizing the UM microenvironment by using a bioinformatic approach. A variety of scores based on gene expression measuring stromal infiltration were calculated and used to assess association with prognosis. As a result, the highest immune and stromal scores were associated with poor prognosis. Specifically, stromal cells (fibroblasts and endothelial cells), T cells CD8+, natural killer (NK) cells, and macrophages M1 and M2 infiltration were associated with poor prognosis. Contrary to other tumors, lymphocytic infiltration is related to poor prognosis. Only B cells were associated with more favorable prognosis. UM samples scoring high in both angiogenesis (Angio) and antigen presentation (AP) pathways showed a poor prognosis suggesting an additive role of both functions. Almost all these tumors exhibited a chromosome 3 monosomy. Finally, an enrichment analysis showed that tumors classified as high Angio-high AP also activated metabolic pathways such as glycolysis or PI3K-AKT-MTOR. In summary, our pool analysis identified a cluster of samples with angiogenic and inflammatory phenotypes exhibiting poor prognosis and metabolic activation. Our analysis showed robust results replicated in a pool analysis merging different datasets from different analytic platforms.


Asunto(s)
Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Neovascularización Patológica/fisiopatología , Neoplasias de la Úvea/patología , Anciano , Animales , Presentación de Antígeno , Análisis por Conglomerados , Conjuntos de Datos como Asunto , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoterapia , Estimación de Kaplan-Meier , Subgrupos Linfocitarios/patología , Macrófagos/patología , Masculino , Melanoma/irrigación sanguínea , Melanoma/genética , Melanoma/inmunología , Redes y Vías Metabólicas , Persona de Mediana Edad , Neovascularización Patológica/genética , Pronóstico , Transducción de Señal , Células del Estroma/patología , Microambiente Tumoral , Neoplasias de la Úvea/irrigación sanguínea , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/inmunología
4.
Nat Commun ; 12(1): 1434, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33664264

RESUMEN

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.


Asunto(s)
Aneuploidia , Variaciones en el Número de Copia de ADN/genética , Genoma Humano/genética , Melanoma/genética , Neoplasias Cutáneas/genética , Progresión de la Enfermedad , Exoma/genética , Humanos , Mutación INDEL/genética , Melanocitos/patología , Mutación Puntual/genética , Polimorfismo de Nucleótido Simple/genética , Secuenciación del Exoma Completo , Secuenciación Completa del Genoma
5.
Methods Mol Biol ; 2265: 277-286, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704722

RESUMEN

Molecular testing of tumor biopsies allows for the identification of the key mutations driving a patient's cancer. However, this is limited to singular nodes and may not accurately reflect cancer heterogeneity. Circulating tumor cell (CTC) analyses offer a noninvasive method of interrogating the genomic profile of patient-derived tumor material. To date, molecular analysis of CTCs has relied on the characterization of bulk or pooled CTC lysates, limiting the detection of minor tumorigenic CTC subclones. Here, we show a workflow enabling BRAFV600E/NRASQ61R mutation detection from single cultured melanoma cells by combining micromanipulation and genomic material amplification methods. This workflow can be directly integrated into circulating tumor cell analysis applications.


Asunto(s)
GTP Fosfohidrolasas/genética , Melanoma/genética , Proteínas de la Membrana/genética , Mutación Missense , Células Neoplásicas Circulantes , Proteínas Proto-Oncogénicas B-raf/genética , Análisis de la Célula Individual , Sustitución de Aminoácidos , Línea Celular Tumoral , Humanos , Melanoma/patología
6.
Methods Mol Biol ; 2265: 377-384, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704728

RESUMEN

We present the assay based on multimarker analysis of mRNA transcripts associated with melanocytic cells detected in lymphatic fluid collected after lymph node dissection. Positive results of reverse transcriptase polymerase chain reaction (RT-PCR) test have a strong relationship with melanoma recurrence and disease-specific survival time in stage III melanoma.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Ganglios Linfáticos/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Biomarcadores de Tumor/genética , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática , Melanocitos/patología , Melanoma/genética , Melanoma/patología , Recurrencia Local de Neoplasia , Estadificación de Neoplasias
7.
Methods Mol Biol ; 2265: 487-512, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704736

RESUMEN

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.


Asunto(s)
Biología Computacional , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Melanoma , MicroARNs , ARN Mensajero , Línea Celular Tumoral , Humanos , Melanoma/genética , Melanoma/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
8.
Methods Mol Biol ; 2265: 529-541, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704738

RESUMEN

We describe here a protocol to measure gene expression, T cell receptor (TCR) sequence, and protein expression by single T cells extracted from melanoma, using 10× Chromium technology. This method includes freezing and thawing of the melanoma infiltrating lymphocytes, staining of cells with fluorescent and barcode-conjugated antibodies, sorting of T cells, and loading the cells on the 10× Chromium Controller. After sequencing, analysis includes quality control, genetic demultiplexing to resolve genetically different samples, and T cell clonality and clustering analysis. Single cell RNA sequencing paints the complete portrait of individual T cells, including their clonality and phenotype, and it reconstructs a complete picture of the T cell infiltrate in a tumor that is represented as cell clustering similar to a pointillism painting.


Asunto(s)
Linfocitos Infiltrantes de Tumor/inmunología , Melanoma , RNA-Seq , Receptores de Antígenos de Linfocitos T , Análisis de la Célula Individual , Humanos , Melanoma/genética , Melanoma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología
9.
Methods Mol Biol ; 2265: 621-634, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704743

RESUMEN

RNA interference (RNAi) is a posttranscriptional regulatory mechanism that employs siRNA. It typically results in the degradation of a target mRNA that encodes a particular protein. Treatment with siRNA therapeutics requires the use of an effective drug delivery system to assist in delivering these therapeutics into the cytoplasm of the transfected cells. Here we describe the transfection of melanoma cancer cells with siRNA using cationic niosome nanoparticles as a delivery system. The method of niosome preparation is first introduced and is followed by complex formation with siRNA and the transfection method.


Asunto(s)
Melanoma , Nanopartículas , ARN Interferente Pequeño , Transfección , Humanos , Liposomas , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Nanopartículas/química , Nanopartículas/uso terapéutico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología
10.
Methods Mol Biol ; 2265: 635-644, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704744

RESUMEN

Gene electrotransfer (GET) is a reliable and effective physical method for in vivo delivery of plasmid DNA (pDNA). Several preclinical and clinical studies have utilized GET to deliver plasmids encoding immune stimulating genes for treatment of melanoma and other tumor types. Intratumor delivery of plasmids encoding cytokines directly to tumors can induce not only a local immune response, but a systemic one as well. To obtain an effective immune response, it is critical to achieve the appropriate expression pattern of the delivered transgene. Expression pattern (levels and kinetics) can be modified by manipulating the electrotransfer parameters. These parameters include the tissue target and the electric pulse parameters of pulse width, electric field, and pulse number. We have found that to induce a robust immune response, we needed only low to moderately elevated expression levels compared to controls. When developing a therapeutic protocol, it is important to establish what expression profile will enable the appropriate response. In this chapter we describe how to determine the appropriate GET protocol to achieve the expression profile that can result in the desired clinical response.


Asunto(s)
Electroporación , Técnicas de Transferencia de Gen , Melanoma , Plásmidos , Transgenes , Línea Celular Tumoral , Humanos , Melanoma/genética , Melanoma/metabolismo , Plásmidos/genética , Plásmidos/farmacología
11.
Methods Mol Biol ; 2265: 591-620, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704742

RESUMEN

Melanoma accounts for 4% of all skin cancer malignancies, with only 14% of diagnosed patients surviving for more than 5 years after diagnosis. Until now, there is no clear understanding of the detailed molecular contributors of melanoma pathogenesis. Accordingly, more research is needed to understand melanoma development and prognosis.All the treatment approaches that are currently applied have several significant limitations that prevent effective use in melanoma. One major limitation in the treatment of cancer is the acquisition of multidrug resistance (MDR). The MDR results in significant treatment failure and poor clinical outcomes in several cancers, including skin cancer. Treatment of melanoma is especially retarded by MDR. Despite the current advances in targeted and immune-mediated therapy, treatment arms of melanoma are severely limited and stand as a significant clinical challenge. Further, the poor pharmacokinetic profile of currently used chemotherapeutic agents is another reason for treatment failure. Therefore, more research is needed to develop novel drugs and carrier tools for more effective and targeted treatment.Nucleic acid therapy is based on nucleic acids or chemical compounds that are closely related, such as antisense oligonucleotides, aptamers, and small-interfering RNAs that are usually used in situations when a specific gene implicated in a disorder is deemed a therapeutically beneficial target for inhibition. However, the proper application for nucleic acid therapies is hampered by the development of an effective delivery system that can maintain their stability in the systemic circulation and enhance their uptake by the target cells. In this chapter, the prognosis of the different types of melanoma along with the currently used medications is highlighted, and the different types of nucleic acids along with the currently available nanoparticle systems for delivering these nucleic acids into melanoma cells are discussed. We also discuss recently conducted research on the use of different types of nanoparticles for nucleic acid delivery into melanoma cells and highlight the most significant outcomes.


Asunto(s)
Antineoplásicos , Sistemas de Liberación de Medicamentos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Melanoma/tratamiento farmacológico , Nanopartículas , Ácidos Nucleicos , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Nanopartículas/química , Nanopartículas/uso terapéutico , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología
12.
Methods Mol Biol ; 2265: 25-46, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704703

RESUMEN

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.


Asunto(s)
Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Melanoma/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Factores de Transcripción Forkhead/genética , Vectores Genéticos , Ratones , Transfección
13.
Methods Mol Biol ; 2265: 1-21, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704702

RESUMEN

Since the first resection of melanoma by Hunter in 1787, efforts to treat patients with this deadly malignancy have been ongoing. Initial work to understand melanoma biology for therapeutics development began with the employment of isolated cancer cells grown in cell cultures. However, these models lack in vivo interactions with the tumor microenvironment. Melanoma cell line transplantation into suitable animals such as mice has been informative and useful for testing therapeutics as a preclinical model. Injection of freshly isolated patient melanomas into immunodeficient animals has shown the capacity to retain the genetic heterogeneity of the tumors, which is lost during the long-term culture of melanoma cells. Upon advancement of technology, genetically engineered animals have been generated to study the spontaneous development of melanomas in light of newly discovered genetic aberrations associated with melanoma formation. Culturing melanoma cells in a matrix generate tumor spheroids, providing an in vitro environment that promotes the heterogeneity commonplace with human melanoma and displaces the need for animal care facilities. Advanced 3D cultures have been created simulating the structure and cellularity of human skin to permit in vitro testing of therapeutics on melanomas expressing the same phenotype as demonstrated in vivo. This review will discuss these models and their relevance to the study of melanomagenesis, growth, metastasis, and therapy.


Asunto(s)
Modelos Animales de Enfermedad , Melanoma/genética , Neoplasias Cutáneas/genética , Animales , Línea Celular Tumoral , Perros , Caballos , Humanos , Melanoma/inducido químicamente , Melanoma/patología , Ratones , Organoides , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Porcinos , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra
14.
Methods Mol Biol ; 2265: 203-212, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704716

RESUMEN

Early detection of cancer has been a goal of cancer research in general and melanoma research in particular (Birnbaum et al., Lancet Glob Health 6:e885-e893, 2018; Alendar et al., Bosnian J Basic Med Sci 9:77-80, 2009). Early detection of metastasis has been targeted as pivotal to increasing survival rates (Menezes et al., Adv Cancer Res 132:1-44, 2016). Melanoma, though curable in its early stages, has a dramatic decrease in survival rates once metastasis has occurred (Sharma et al., Biotechnol Adv 36:1063-1078, 2018). The transition to metastasis is not well understood and is an area of increasing interest. Metastasis is always premeditated by the shedding of circulating tumor cells (CTCs) from the primary tumor. The ability to isolate rare CTCs from the bloodstream has led to a host of new targets and therapies for cancer (Micalizzi et al., Genes Dev 31:1827-1840, 2017). Detection of CTCs also allows for disease progression to be tracked in real time while eliminating the need to wait for additional tumors to grow. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify and quantify these cells in order to track disease progression. Additionally, these CMCs are captured and isolated allowing for future analysis such as RNA-Seq or microarray analysis.


Asunto(s)
Citometría de Flujo/métodos , Melanoma/diagnóstico , Células Neoplásicas Circulantes , Técnicas Fotoacústicas/instrumentación , Técnicas Fotoacústicas/métodos , Reología/instrumentación , Reología/métodos , Neoplasias Cutáneas/diagnóstico , Progresión de la Enfermedad , Detección Precoz del Cáncer/métodos , Citometría de Flujo/instrumentación , Biblioteca de Genes , Humanos , Inmunohistoquímica/métodos , Melanoma/sangre , Melanoma/genética , Melanoma/patología , Metástasis de la Neoplasia/diagnóstico , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Ultrasonografía/métodos
15.
Methods Mol Biol ; 2265: 235-245, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704719

RESUMEN

Circulating tumor cells (CTCs) are cancer cells shed by the primary tumor or its metastases that circulate in the peripheral blood. CTCs are potential seeds for metastases, and their detection may allow early uncovering of metastatic dissemination and disease prognostication. To fully ascertain the biomarker potential of melanoma CTCs, sensitive and reliable methods are required. Melanoma-specific transcript analysis has been widely utilized as a standard approach for measuring the presence of CTCs. Here we describe a method for the analysis of CTCs through the detection of specific transcripts in CTC-enriched fractions.


Asunto(s)
Biomarcadores de Tumor/sangre , Melanoma/sangre , Células Neoplásicas Circulantes/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Biomarcadores de Tumor/genética , ADN/sangre , ADN/aislamiento & purificación , Humanos , Melanoma/diagnóstico , Melanoma/genética , Melanoma/patología , Células Neoplásicas Circulantes/patología , ARN/sangre , ARN/aislamiento & purificación
16.
Methods Mol Biol ; 2265: 265-276, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704721

RESUMEN

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.


Asunto(s)
Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Dispositivos Laboratorio en un Chip , Melanoma/metabolismo , Microfluídica/instrumentación , Microfluídica/métodos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Técnicas Biosensibles/instrumentación , Técnicas de Cultivo de Célula/métodos , Humanos , Inmunoensayo/instrumentación , Biopsia Líquida/métodos , Melanoma/genética , Melanoma/patología , Mutación , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología
17.
Methods Mol Biol ; 2265: 247-263, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704720

RESUMEN

In recent years, circulating tumor DNA (ctDNA) has emerged as a promising prognostic and monitoring biomarker of various cancers, including melanoma. However, sensitive methods are required for its preservation, isolation, and detection. Here we describe a sensitive method for plasma ctDNA isolation using a column-based extraction kit, followed by quantification using a single mutational target with a droplet digital PCR system. This sensitive protocol has been successfully used to quantify diverse mutations present in plasma-derived ctDNA from cancer patients. The full procedure, from blood processing to the analysis of results, takes approximately a day of work.


Asunto(s)
ADN Tumoral Circulante/sangre , ADN de Neoplasias/sangre , Melanoma/sangre , Reacción en Cadena de la Polimerasa/métodos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Humanos , Melanoma/genética , Melanoma/patología , Plasma/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética
18.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-33670365

RESUMEN

MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.


Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , MicroARNs/biosíntesis , Proteínas Mitocondriales/biosíntesis , Proteínas de Neoplasias/biosíntesis , ARN Neoplásico/biosíntesis , Factores de Transcripción/biosíntesis , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Femenino , Genómica , Humanos , Masculino , Melanoma/genética , Melanoma/patología , MicroARNs/genética , Proteínas Mitocondriales/genética , Proteínas de Neoplasias/genética , ARN Neoplásico/genética , Factores de Transcripción/genética
19.
Nat Commun ; 12(1): 1536, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750776

RESUMEN

Hyperactivation of the MAPK signaling pathway motivates the clinical use of MAPK inhibitors for BRAF-mutant melanomas. Heterogeneity in differentiation state due to epigenetic plasticity, however, results in cell-to-cell variability in the state of MAPK dependency, diminishing the efficacy of MAPK inhibitors. To identify key regulators of such variability, we screen 276 epigenetic-modifying compounds, individually or combined with MAPK inhibitors, across genetically diverse and isogenic populations of melanoma cells. Following single-cell analysis and multivariate modeling, we identify three classes of epigenetic inhibitors that target distinct epigenetic states associated with either one of the lysine-specific histone demethylases Kdm1a or Kdm4b, or BET bromodomain proteins. While melanocytes remain insensitive, the anti-tumor efficacy of each inhibitor is predicted based on melanoma cells' differentiation state and MAPK activity. Our systems pharmacology approach highlights a path toward identifying actionable epigenetic factors that extend the BRAF oncogene addiction paradigm on the basis of tumor cell differentiation state.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Epigenómica/métodos , Melanoma/metabolismo , Dependencia del Oncogén , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Femenino , Histona Demetilasas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanocitos/metabolismo , Melanoma/genética , Ratones , Ratones Desnudos , Mutación , Dependencia del Oncogén/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
20.
Medicine (Baltimore) ; 100(8): e24840, 2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33663104

RESUMEN

ABSTRACT: Malignant melanoma is a highly malignant tumor originating from the melanocytes of the neural crest, which is prone to metastasis and has a poor prognosis. Previous research demonstrated that melanoma inhibitory activity (MIA) and lactate dehydrogenase (LDH) could serve as serum markers in malignant melanoma and indicate prognosis in the Caucasian race. Researchers suspected that both MIA and LDH could prompt the prognosis of malignant melanoma in the Chinese population. This study aimed to investigate the value of MIA and LDH in the prognosis of acral malignant melanoma.From January 1, 2014, to December 31, 2017, in Jiangsu Province, 44 acral malignant melanoma patients with complete data were chosen from the clinic. The LDH levels were extracted from their clinical data, and MIA levels were measured by enzyme-linked immunosorbent assay method. 8 paired advancing samples before and after metastasis were examined. 22 health donors were matched to the patient group. Receiver operating characteristic (ROC) curves of MIA and LDH were drawn to determine acral malignant melanoma tumorigenesis and metastasis and finally got the cut-off value. Cumulative survival was illustrated with the Kaplan-Meier plot, and factors were compared using the Log-rank test.Compared with age-matched healthy donors, MIA was significantly high in patients (P < .001). Moreover, serum MIA was significantly higher in III-IV stage patients than I-II stage patients (P < .001). However, there was no such association between LDH and melanoma stage and risk. Further study indicated that the MIA cut-off > 914.7pg/mL predicted disease progression with 86.4% specificity and 95.5% sensitivity. In the Kaplan-Meier analysis, MIA levels were independent risk factors for long-term mortality of acral malignant melanoma patients.It concluded that the quantification of MIA in the serum should be performed as a general standard of care in patients at risk of developing metastatic melanoma.


Asunto(s)
Proteínas de la Matriz Extracelular/sangre , Melanoma/sangre , Melanoma/genética , Proteínas de Neoplasias/sangre , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/genética , Anciano , Grupo de Ascendencia Continental Asiática , Biomarcadores/sangre , Estudios de Casos y Controles , China , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Estimación de Kaplan-Meier , L-Lactato Deshidrogenasa/sangre , Masculino , Melanoma/mortalidad , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Retrospectivos , Neoplasias Cutáneas/mortalidad
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