Browsing by Subject "Melanoma"

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    A Side-by-Side comparison of wildtype and variant melanocortin 1 receptor signaling with emphasis on protection against oxidative damage to DNA.
    (MDPI, 2023-09-21) Sánchez-Beltrán, José; Abrisqueta, Marta; Padilla, Lidia; Herraiz Serrano, Cecilia María; Cerdido Ochoa, Sonia; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Lambertos Escudero, Ana; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
    Common variants of the MC1R gene coding the α-melanocyte stimulating hormone receptor are associated with light skin, poor tanning, blond or red hair, and increased melanoma risk, due to pigment-dependent and -independent effects. This complex phenotype is usually attributed to impaired activation of cAMP signaling. However, several MC1R variants show significant residual coupling to cAMP and efficiently activate mitogenic extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Yet, residual signaling and the key actions of wildtype and variant MC1R have never been assessed under strictly comparable conditions in melanocytic cells of identical genetic background. We devised a strategy based on CRISPR-Cas9 knockout of endogenous MC1R in a human melanoma cell line wildtype for BRAF, NRAS and NF1, followed by reconstitution with epitope-labeled MC1R constructs, and functional analysis of clones expressing comparable levels of wildtype, R151C or D294H MC1R. The proliferation rate, shape, adhesion, motility and sensitivity to oxidative DNA damage were compared. The R151C and D294H RHC variants displayed impaired cAMP signaling, intracellular stability similar to the wildtype, triggered ERK1/2 activation as effectively as the wildtype, and afforded partial protection against oxidative DNA damage, although less efficiently than the wildtype. Therefore, common melanoma-associated MC1R variants display biased signaling and significant genoprotective activity.
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    Acriflavine, a Potent Inhibitor of HIF-1α, Disturbs Glucose Metabolism and Suppresses ATF4-Protective Pathways in Melanoma under Non-Hypoxic Conditions
    (MDPI, 2020-12-31) Martí Díaz, Román; Cabezas Herrera, Juan; Goding, Colin; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
    Hypoxia-inducible factor (HIF)-1α is constitutively expressed in melanoma cells under normoxic conditions and its elevated expression correlates with the aggressiveness of melanoma tumors. Here, we used acriflavine, a potent inhibitor of HIF-1α dimerization, as a tool to investigate whether HIF-1α-regulated pathways contribute to the growth of melanoma cells under normoxia. We observed that acriflavine differentially modulated HIF-1α-regulated targets in melanoma under normoxic conditions, although acriflavine treatment resulted in over-expression of vascular endothelial growth factor (VEGF), its action clearly downregulated the expression of pyruvate dehydrogenase kinase 1 (PDK1), a well-known target of HIF-1α. Consequently, downregulation of PDK1 by acrifavine resulted in reduced glucose availability and suppression of the Warburg effect in melanoma cells. In addition, by inhibiting the AKT and RSK2 phosphorylation, acriflavine also avoided protective pathways necessary for survival under conditions of oxidative stress. Interestingly, we show that acriflavine targets activating transcription factor 4 (ATF4) for proteasomal degradation while suppressing the expression of microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development and a melanoma oncogene. Since acriflavine treatment results in the consistent death of melanoma cells, our results suggest that inhibition of HIF-1α function in melanoma could open new avenues for the treatment of this deadly disease regardless of the hypoxic condition of the tumor.
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    An immunohistochemical study of NFE2L2, KEAP1 and 8-hydroxy-2'-deoxyguanosine and the EMT markers SNAI2, ZEB1 and TWIST1 in metastatic melanoma
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2017) Hintsala, Hanna Riikka; Haapasaari, Kirsi Maria; Soini, Ylermi; Karihtala, Peeter
    Background: Little is known regarding the role of redox balance regulators in metastatic melanomas, but there is some evidence for a link between epithelial-to-mesenchymal transition (EMT) and cellular redox status. Methods: We compared the immunohistochemical expression of nuclear factor erythroid-2-related factor 2 (NFE2L2), Kelch-like ECH-associated protein 1 (KEAP1), 8-hydroxy-2'-deoxyguanosine (8-OHdG), TWIST1, SNAI2 and ZEB1 between primary melanomas and metastases in a cohort of 23 nevi, 66 malignant melanomas and 22 metastases. Results: Nuclear NFE2L2 expression was higher (p=0.003) and cytoplasmic KEAP1 lower (p=0.026) in metastatic lesions than at primary sites. Nuclear NFE2L2 expression was associated with the presence of distant metastases (p=0.040) and with nuclear TWIST1 expression (p=0.002). Patients having both NFE2L2 and TWIST1 expression in nuclei had an extremely poor prognosis (p=0.0003). In multivariate analysis nuclear TWIST1 expression was an independent predictor of a poorer prognosis (HR 2.99, 95% CI 1.17-7.69; p=0.023) and the invasive TWIST1/ZEB1 phenotype showed poorer melanoma-specific survival (HR 7.28, 95% CI 2.23-23.77; p=0.001). Nuclear expression of 8-OHdG (p=0.001) was lower at metastatic sites than in primary lesions. Conclusions: EMT signalling and the KEAP1/NFE2L2- axis are likely to be involved in metastatic spread of malignant melanoma and also appear to have potential interactions.
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    Bases moleculares de la resistencia del melanoma a los antifolatos: diseño de nuevas estrategias terapéuticas
    (2015-12-10) Fernández Pérez, María Piedad; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Facultad de Biología
    Melanoma is the most aggressive form of skin cancer and it is highly resistant to all current modalities of cancer therapy, including cytotoxic drugs as methotrexate (MTX), an antifolate widely used in clinical oncology against many cancer types. Recently, our lab discovered a new melanoma-specific mechanism of MTX resistance by which folate receptor α (FR-α)–mediated endocytotic transport of MTX facilitates melanosomal drug sequestration and cellular export in melanoma cells, thereby reducing the accumulation of MTX in intracellular compartments. That low MTX intracellular concentration is just able to induce cell growth arrest, but not cell death in melanoma cells. Particularly, we found that melanoma treatment with MTX induces cell cycle arrest without inducing apoptosis, activates melanin synthesis and melanosome biogenesis, and accelerates melanosomal export; but the cellular and molecular processes by which MTX mediates all these effects had not been elucidated. The Main Objective of the present PhD Thesis was the identification of the molecular basis of the cell cycle arrest, the activation of melanogenesis and the acceleration of melanosome transport induced by MTX treatment that, collectively, protect melanoma cells against MTX-induced apoptosis. The Secondary Objective, was the development of new therapeutic strategies combining MTX with drugs directed against molecular targets that are key components of the mechanisms of melanoma resistance to MTX. Regarding the Techniques and Instrumentation, we used several experimental models, including melanoma and non melanoma cell lines, an artificial skin melanoma model, and mice melanoma xenografts. We also employed a battery of cell biology techniques (viability, apoptosis and migration assays; electronic and optical microscopy; flow citometry; immunohistochemistry, immunofluorescence, etc.); biochemistry techniques (western blotting, protein immunoprecipitation, mass spectrometry, enzymatic activity determination by UV-VIS espectroscopy, etc.); molecular biology techniques (nucleic acid extraction, PCR, RT-qPCR, chromatin immunoprecipitation, etc.). The Results of the implementation of these techniques were subjected to the appropriate statistical tests and are summarized bellow. Since the ability of cells to delay cell cycle progression and halt DNA synthesis represents a defensive mechanism that spares potential toxicity, firstly we focused on deciphering the molecular basis of the MTX-induced cell cycle arrest. We identified the transcription factor E2F1 and the checkpoint kinase 1 (Chk1) as key mediators of this mechanism of resistance. The results indicated that MTX stimulated the transcriptional activity of E2F1 on the promoters of dihydrofolate reductase (DHFR) and thymidylate synthase (TS), which lead to an increase in the dTTP levels, instead of dTTP depletion as occurs in MTX-sensible cells. Since dTTP is an allosteric inhibitor of ribonucleotide reductase, which is necessary for the synthesis of all the dNTPs, dTTP excess induced DNA replication fork stress that activates the ATR/Chk1 DNA damage signaling pathway. Under these conditions, melanoma cells were protected from apoptosis by arresting their cell cycle in early S-phase. However, abrogation of this checkpoint by CHEK1 silencing or by UCN-01 (7-hydroxystaurosporine)-mediated Chk1 inhibition, rapidly triggered MTX-induced cell death, suggesting that inhibition of Chk1 in combination with this kind of antimetabolite chemotherapy is a viable therapeutic strategy to overcome melanoma resistance. Secondly, we focused on the study of the activation of melanogenesis observed after MTX treatment. Our results showed that Microphthalmia-associated transcription factor (MITF) that normally acts as a master regulator of melanocyte development, function and survival, is responsible for the activation of melanogenesis in melanoma after MTX treatment. In fact, MTX treatment increases the expression of this transcription factor which in turn induces the expression of melanocytic differentiation genes driving melanin production and melanosome biogenesis as tyrosinase (TYR). The increased TYR expression in response to MTX-mediated MITF activation provided an opportunity to design a two step strategy consisting in the combination of MTX with a prodrug that our group had previously designed to be activated by TYR. This prodrug is a compound called 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG) that inhibits the enzyme DHFR with high affinity. The combination of MTX and TMECG was able to induce depletion of thymidine pools, double-strand DNA breaks, and highly efficient E2F1-mediated apoptosis in vitro and in vivo. Recently, it has been recognized that MITF acts as a melanoma rheostat in determining tumor subpopulation identity. In melanomas, reduced MITF expression leads to G1 arrested, invasive cells with stem-like properties including the ability to initiate tumors with high efficiency. By contrast, elevated MITF leads to activation of differentiation genes. In between, intermediate levels of MITF allow the proliferation of melanoma cells. Consequently, tumors comprise a mix of MITF-positive and negative melanoma cells. Therefore, the increase of MITF expression by MTX treatment would drive heterogeneous populations of tumor cells to a differentiated and less invasive phenotype and, at the same time, would sensitize these differentiated cells to the TYR-processed prodrug TMECG in a cell-specific fashion. In a different approach, we explored the molecular basis of the activation of the transport of melanosomes observed after MTX treatment with the aim of designing a therapeutic strategy driven to block the MTX export within melanosomes. We identified a new MTX-activated molecular pathway involved in the export of melanosomes, in which the motor protein MyosinVa (MyoVa) plays a key role. Our results demonstrated that melanoma treatment with MTX leads to Akt2-dependent MyoVa phosphorylation, which enhances its ability to interact with melanosomes and accelerates their exportation. Due to these findings, we designed a combined therapy driven to block this MyoVa/Akt2 pathway. Because UCN-01 is also a potent inhibitor of PDK1, which activates Akt by phosphorylation, we hypothesized that the inhibition of this Akt2 phosphorylation by UCN-01 may result in the disruption of MTX stimulated melanosome transport. In fact, MTX/UCN-01 combined therapy prevented MTX export by blocking melanosome transport and was able to induce a highly efficient E2F1-mediated apoptosis in culture and in vivo. In summary, we observed that the combination of MTX and UCN-01 may represent a therapeutic option for the treatment of this evasive disease. El melanoma es el tipo de cáncer de piel más agresivo y muestra una alta resistencia frente a todas las terapias anticancerígenas disponibles en la actualidad, incluyendo agentes citotóxicos como el metotrexato (MTX), un antifolato ampliamente utilizado en oncología clínica. Recientemente, nuestro laboratorio ha descubierto un nuevo mecanismo de resistencia al MTX específico de las células de melanoma por el cual, la endocitosis de esta droga mediada por el receptor de fólico α (FR-α) conduce al secuestro del MTX en los melanosomas y a su expulsión al exterior celular, reduciendo así la acumulación de esta droga en los compartimentos intracelulares. Esta baja concentración de MTX en el interior celular es capaz únicamente de inducir un arresto del crecimiento, pero no de inducir la muerte de las células de melanoma. Por tanto, se sabía que el tratamiento con MTX es capaz de inducir arresto del ciclo celular sin inducir apoptosis, de activar la síntesis de melanina y la biogénesis de melanosomas, y de acelerar el transporte de melanosomas, pero los mecanismos celulares y moleculares por los cuales el MTX mediaba estos efectos era desconocido. El Objetivo Principal de esta Tesis Doctoral fue la identificación de las bases moleculares del arresto del ciclo celular, de la activación de la melanogénesis y de la aceleración del transporte melanosomal inducidos por el tratamiento con MTX que, en conjunto, protegen a las células de melanoma de la apoptosis inducida por el MTX. El Objetivo Secundario fue el desarrollo de nuevas estrategias terapéuticas combinando el MTX con agentes dirigidos frente a dianas moleculares clave en los mecanismos de resistencia. En cuanto a las Técnicas e Instrumentación, se emplearon como modelos experimentales varias líneas celulares de melanoma y de otros tipos de cáncer, un modelo de melanoma de piel artificial y un modelo de xenoinjerto de melanoma en ratón. Además, empleamos una batería de técnicas de biología celular (ensayos de viabilidad, apoptosis y migración; microscopía óptica y electrónica; inmunohistoquímica; inmunoflorescencia, etc.); técnicas de bioquímica (western blot, inmunoprecipitación de proteínas, espectrometría de masas, determinación de actividad enzimática mediante espectroscopía UV-VIS, etc.); técnicas de biología molecular (extracción de ácidos nucleicos, PCR, RT-qPCR, inmunoprecipitación de cromatina, etc.) Los Resultados de la implementación de las citadas técnicas fueron sometidos a las pruebas estadísticas apropiadas y se resumen a continuación: Dado que se ha descrito que la capacidad de las células de detener la síntesis de ADN constituye un mecanismo de defensa que evita posibles daños, en primer lugar nos centramos en descifrar las bases moleculares del arresto del ciclo celular inducido por el MTX. Así, identificamos al factor de transcripción E2F1 y a la quinasa Chk1 como mediadores de este mecanismo de resistencia. Nuestros resultados demostraron que el tratamiento con MTX estimulaba la actividad transcripcional de E2F1 sobre los promotores de los genes de la dihidrofolato reductasa (DHFR) y la timidilato sintasa (TS). Esto conducía a un incremento en los niveles de dTTP, en lugar de a una depleción de dTTP, como ocurre en células sensibles al MTX. Puesto que el dTTP es un inhibidor alostérico de la ribonucleótido reductasa que es necesaria para la síntesis de todos los dNTPs, este exceso de dTTP inducía un estrés en las horquillas de replicación capaz de activar la vía de señalización del daño al ADN mediada por ATR/Chk1. En estas condiciones, las células de melanoma evitan la apoptosis mediante el arresto de su ciclo celular al inicio de la fase S. Sin embargo, la eliminación de este checkpoint mediante el silenciamiento genético de CHEK1 o mediante la inhibición de esta quinasa con UCN-01 (7-hidroxistaurosporina) tras el tratamiento con MTX desencadena rápidamente la muerte celular, sugiriendo que la inhibición de Chk1 en combinación con este tipo de antimetabolitos es una estrategia terapéutica eficaz para vencer la resistencia del melanoma. En segundo lugar, nos centramos en el estudio de la activación de la melanogénesis por el MTX. Nuestros resultados demostraron que el factor de transcripción MITF (Microphthalmia-associated transcription factor) que, en melanocitos normales actúa como un regulador clave del desarrollo, función y supervivencia, era el responsable de la activación de la melanogénesis tras el tratamiento con MTX. De hecho, el MTX incrementa la expresión de este factor de transcripción el cual, a su vez, induce la expresión de genes implicados en la diferenciación melanocítica que conducen a la producción de melanina, como la enzima tirosinasa (TYR), y a la biogénesis de melanosomas. Este aumento en la expresión de TYR en respuesta a la activación de MITF mediada por MTX nos permitió diseñar una estrategia terapéutica basada en la combinación de MTX con una prodroga activada por TYR que había sido sintetizada previamente en nuestro laboratorio. Esta prodroga es el compuesto denominado 3-O-(3,4,5-trimetoxibenzoil)-(-)-epicatequina (TMECG), capaz de inhibir a la enzima DHFR con una alta afinidad. La combinación MTX/TMECG fue capaz de inducir el agotamiento de las reservas de timidina, roturas de doble cadena en el ADN y la apoptosis mediada por E2F1 con una alta eficacia, tanto in vitro como in vivo. Recientemente se ha descrito que MITF actúa como un reóstato en melanoma, determinando la identidad de las distintas subpoblaciones tumorales. Según este modelo, bajos niveles de MITF dan lugar a células arrestadas en G1, altamente invasivas con propiedades de célula madre, incluyendo una alta capacidad de iniciar tumores. Por el contrario, niveles elevados de MITF conducen a la activación de genes de diferenciación. Entre ambos, niveles intermedios de MITF permiten la proliferación de las células de melanoma. En consecuencia, los tumores suponen una mezcla de células con distintos niveles de MITF. Por tanto, el aumento de la expresión de MITF por el tratamiento con MTX conduciría a las poblaciones heterogéneas de células tumorales hacia un fenotipo común, diferenciado y menos invasivo, y, al mismo tiempo, sensibilizaría a estas células diferenciadas a una prodroga activada por TYR como el TMECG. En una aproximación diferente, estudiamos las bases moleculares de la activación del transporte de melanosomas inducida por el tratamiento con MTX. En este sentido, identificamos una nueva vía molecular implicada en la exportación de melanosomas en la que juega un papel esencial la proteína motora Miosina Va (MyoVa). Nuestros resultados demostraron que el tratamiento del melanoma con MTX conduce a una fosforilación de MyoVa mediada por Akt2 que incrementa la capacidad de esta proteína de interaccionar con los melanosomas y acelera su exportación. En vista de estos hallazgos, diseñamos una terapia combinada encaminada a bloquear la activación del transporte mediada por esta vía. Dado que se ha descrito que el UCN-01 es un potente inhibidor de PDK1, la cual activa a Akt por fosforilación, diseñamos una terapia de MTX combinado con UCN-01 para bloquear el transporte melanosomal. Efectivamente, la terapia combinada MTX/UCN-01 logró evitar la exportación de MTX mediante el bloqueo del transporte melanosomal y fue capaz de inducir la apoptosis mediada por E2F1 con una alta eficiencia tanto in vitro como in vivo.
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    Biopsia selectiva del ganglio centinela (BSGC) en melanoma. Comparación de dos métodos de procedimiento histológico
    (2014-03-18) Martínez Menchón, Teresa; Torre Minguela, Carlos de; Martínez Escribano, Jorge; Sánchez-Pedreño Guillén, Paloma; Departamento de Dermatología, Estomatología, Radiología y Medicina Física
    El melanoma es un tumor de estirpe melanocítica con un elevado potencial metastático y una incidencia en aumento. La técnica de la Biopsia Selectiva del Ganglio Centinela (BSGC) se acepta como la forma más específica, sensible y con menor morbilidad para realizar la estadificación patológica ganglionar. Objetivos:1) Analizar la capacidad de detección de un protocolo extenso, frente a un método simple de procesamiento del ganglio centinela comparando dos cohortes de pacientes; 2) buscar relación entre el resultado de la técnica de BSGC y otras variables con valor pronóstico establecido, tanto clínicas como histológicas; 3) realizar un estudio de supervivencia y de falsos negativos; 4) estudiar subgrupos de pacientes (melanomas ≥1 mm de Breslow, <1 mm de Breslow y melanomas en extremidades);y 5) comparar ambas técnicas desde el punto de vista económico. Metodología: Realizamos un estudio de cohortes con un diseño observacional analítico longitudinal. Se seleccionaron a los pacientes sometidos a la técnica desde el año 1998 hasta el 2010. La muestra a su vez se subdividió en dos series: la serie correspondiente a los años 1998-2004 y, la serie 2007-2010. En la primera de ellas se realizó un procesamiento simple (sección única de hematoxilina-eosina) mientras que en la segunda se realizó un estudio extenso (protocolo transhiliar bivalvo con secciones seriadas cada 250 µm y múltiples tinciones inmunohistoquímicas (HMB 45, Melan A y S100)). Conclusiones: El protocolo histopatológico extenso de análisis ganglionar ha demostrado mejorar la capacidad de detección de la técnica en nuestro medio. No hay diferencias en la tasa de falsos negativos, pero eliminando el factor de confusión correspondiente a los drenajes múltiples, la tasa disminuye a la mitad. El procesamiento por niveles al menos duplica el coste económico de la serie anterior. El porcentaje de pacientes sometidos a la técnica con melanomas delgados (índice de Breslow menor a 1 mm) es cada vez mayor (40%), aunque el rango de positividad es bajo (2%). La aparición de nuevas opciones de tratamiento en el melanoma metastásico obliga a optimizar la técnica con objeto de identificar el subgrupo de pacientes que podrían beneficiarse de estas nuevas terapias. Palabras clave: Melanoma, ganglio centinela. Melanoma is a melanocytic lineage tumor with high metastatic potential and increasing incidence. Sentinel lymph node biopsy (SLNB) is accepted as the most specific, sensitive and with less morbidity technique for pathologic nodal staging. Objectives: 1) To analyze the detection capability of an extensive protocol, versus a simple method of lymph node processing, comparing two cohorts of patients; 2) To seek a relationship between the outcome of the SLNB technique and other variables with established prognostic value, both clinical and histological; 3) To conduct a survival and false negatives study; 4) To study subgroups of patients (melanomas with Breslow thickness of 1 mm or more, <1 mm Breslow melanomas and extremity melanomas) and 5) To compare both lymph node processing methods from the economic point of view. Methods: Cohort study with a longitudinal observational analytic design was conducted. Patients undergoing SLNB in our institution from 1998 to 2010 were selected. The sample was divided into two cohorts, patients treated from 1998 to 2004 and those from 2007 to 2010. In the first subgroup, a simple method of processing was performed (single hematoxylin-eosin slide), while in the second, an extensive protocol (transhiliar bivalving 250µm serial sectioning with multiple immunohistochemical stains (HMB 45, Melan A and S100)) was carried out. Conclusions: Extensive histopathologic protocol for assessing sentinel node has shown an improved detection capability of the technique in our institution. No differences in false negatives rate were detected, but removing multiple drains confusion factor, this rate decreases by half. Extensive histopathologic protocol at least double the economic cost of the previous method. Percentage of patients undergoing SLNB technique with thin melanomas (less than 1 mm of Breslow thickness) is growing (40%), although positivity range is low (2%). New emerging treatment options in metastatic melanoma force to optimize the technique in order to identify the subgroup of patients who might benefit from these new therapies. Keywords: Melanoma, sentinel node.
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    BRN2 is a non-canonical melanoma tumor-suppressor
    (Springer Nature, 2021-06-17) Hamm, Michael; Sohier, Pierre; Petit, Valérie; Raymond, Jérémy H.; Delmas, Véronique; Le Coz, Madeleine; Gesbert, Franck; Kenny, Colin; Aktary, Zackie; Pouteaux, Marie; Rambow, Florian; Sarasin, Alain; Charoenchon, Nisamanee; Bellacosa, Alfonso; Mosteo, Laura; Lauss, Martin; Meijer, Dies; Steingrimsson, Eirikur; Jönsson, Göran B.; Cornell, Robert; Davidson, Irwin; Goding, Colin R.; Larue, Lionel; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
    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
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    Cancer stem cell as therapeutic target for melanoma treatment
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Alamodi, Abdulhadi A.; Eshaq, Abdulaziz M.; Hassan, Sofie Yasmin; Hmada, Youssef Al; El Jamal, Siraj M.; Fothan, Ahmed M.; Arain, Omair M.; Hassan, Sarah-Lilly; Haikel, Youssef; Megahed, Mosaad; Hassan, Mohamed
    Human malignant melanoma is a highly aggressive skin tumor that is characterized by its extraordinary heterogeneity, propensity for dissemination to distant organs and resistance to cytotoxic agents. Although chemo- and immune-based therapies have been evaluated in clinical trials, most of these therapeutics do not show significant benefit for patients with advanced disease. Treatment failure in melanoma patients is attributed mainly to the development of tumor heterogeneity resulting from the formation of genetically divergent subpopulations. These subpopulations are composed of cancer stem-like cells (CSCs) as a small fraction and non-cancer stem cells that form the majority of the tumor mass. In recent years, CSCs gained more attention and suggested as valuable experimental model system for tumor study. In melanoma, intratumoral heterogeneity, progression and drug resistance result from the unique characteristics of melanoma stem cells (MSCs). These MSCs are characterized by their distinct protein signature and tumor growth-driving pathways, whose activation is mediated by driver mutation-dependent signal. The molecular features of MSCs are either in a causal or consequential relationship to melanoma progression, drug resistance and relapse. Here, we review the current scientific evidence that supports CSC hypothesis and the validity of MSCs-dependent pathways and their key molecules as potential therapeutic target for melanoma treatment.
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    Cathepsin K expression in melanoma is associated with metastases
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2017) Petricevic, Slavica Juric; Pavlovic, Antonia; Capkun, Vesna; Becic, Kristijan; Durdov, Merica Glavina
    Introduction. Melanoma of the skin shows a tendency to metastasize via lymph or blood secreting matrix metalloproteinases and cathepsins, which enable penetration through the dermis. Cathepsin K acts in cytoplasm of atypical melanocytes and completely cleaves internalized collagen. Materials and methods. Expression of cathepsin K was analyzed immunohistochemically in 45 melanomas and correlated to morphological and clinical parameters. Results. During six years follow up, 13 patients developed lymph node metastases and three of them distant metastases. Positive expression of cathepsin K was found in 19 cases. In univariate regression analysis histological type, pagetoid spread, mitotic activity and cathepsin K expression were significantly connected to metastases. Cathepsin K was significantly associated to histologic type, ulceration, pagetoid spread and mitotic rate. In multiple logistic regression adjusted to these variables, cathepsin K was an independent predictor in occurrence of metastases (P=0.015). Median to the occurrence of metastases was 40 months in patients with cathepsin K positive expression and 71 months in patients with cathepsin K negative expression (P<0.001). Conclusions. In this preliminary study positive expression of cathepsin K in melanoma of the skin is associated with other unfavorable prognostic factors. We consider cathepsin K expression in primary tumor would significantly precipitate occurrence of metastases.
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    CircRTTN upregulates EPHA2 to aggravate the malignant process of melanoma via sponging miR-890
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2024) Wang, Yaqin; Gong, Junzuo; Ding, Xiaojie; Luo, Shu
    Background. Malignant melanoma is a kind of tumor derived from melanocytes, which has the characteristics of drug resistance and distant metastasis. Accumulating evidence has demonstrated that circular RNAs (circRNAs) are involved in the pathogenesis of melanoma. Our current study aimed to investigate the role and mechanism of circRTTN in melanoma progression. Methods. The levels of circRTTN, microRNA-890 (miR-890) and EPH receptor A2 (EPHA2) were examined via quantitative real-time PCR (qRT-PCR) and Western blot. Cell Counting Kit-8 (CCK-8), colony formation, 5-Ethynyl-2’-deoxyuridine (EdU) staining, flow cytometry, transwell and tube formation assays were conducted to estimate the effects of circRTTN on growth, apoptosis, migration, invasion and angiogenesis of melanoma cells. Western blot was used to measure related marker protein levels. The interaction between miR-890 and circRTTN or EPHA2 was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. Xenograft assay was used to assess the effect of circRTTN in vivo. Results. CircRTTN and EPHA2 levels were upregulated, while miR-890 was down-regulated in melanoma tissues and cells. CircRTTN knockdown restrained cell proliferation, migration, invasion and angiogenesis, but promoted cell apoptosis in vitro. CircRTTN was an effective molecular sponge for miR890, and negatively regulated miR-890 expression. The suppressive role of circRTTN knockdown on cell growth, metastasis and angiogenesis in vitro was abated by blocking miR-890. MiR-890 directly targeted EPHA2. MiR-890 overexpression elicited a similar antitumor role in melanoma cells, which was abrogated by overexpression of EPHA2. In addition circRTTN knowdown markedly attenuated xenograft tumor growth in vivo. Conclusion. Our findings demonstrated that circRTTN mediated melanoma progression via regulating the miR-890/ EPHA2 axis.
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    Comparison of a Pair of Synthetic Tea-Catechin-Derived Epimers: Synthesis, Antifolate Activity, and Tyrosinase-Mediated Activation in Melanoma
    (WILEY, 2011-03-07) Sáez Ayala, Magalí; Chazarra Parres, Soledad; Tárraga Tomás, Alberto; Cabezas Herrera, Juan; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A
    Despite bioavailability issues, tea catechins have emerged as promising chemopreventive agents because of their efficacy in various animal models. We synthesized two catechin-derived compounds, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin (TMCG) and 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), in an attempt to improve the stability and cellular absorption of tea polyphenols. The antiproliferative and pro-apoptotic activities of both compounds were analyzed with various cancer cell systems, and TMCG, which was easily synthesized in excellent yield, was more active than TMECG in both melanoma and non-melanoma cell lines. TMCG was also a better inhibitor of dihydrofolate reductase and was more efficiently oxidized by tyrosinase, potentially explaining the difference in activity between these epimers.
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    Directed Phenotype Switching as an Effective Antimelanoma Strategy
    (Cell Press, 2013-06-20) Sáez Ayala, Magalí; Fernández Pérez, María Piedad; Chazarra Parres, Soledad; Freter, Rasmus; Middleton, Mark; Piñero Madrona, Antonio; Cabezas Herrera, Juan; Goding, Colin R.; Montenegro Arce, María Fernanda; Rodríguez López, José Neptuno; Sánchez del Campo Ferrer, Luis; Bioquímica y Biología Molecular A; Cirugía, Pediatría y Obstetricia y Ginecología
    Therapeutic resistance in melanoma and other cancers arises via irreversible genetic, and dynamic phenotypic, heterogeneity. Here, we use directed phenotype switching in melanoma to sensitize melanoma cells to lineage-specific therapy. We show that methotrexate (MTX) induces microphthalmia-associated transcription factor (MITF) expression to inhibit invasiveness and promote differentiation-associated expression of the melanocyte-specific Tyrosinase gene. Consequently, MTX sensitizes melanomas to a tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(−)-epicatechin (TMECG), that inhibits the essential enzyme DHFR with high affinity. The combination of MTX and TMECG leads to depletion of thymidine pools, double-strand DNA breaks, and highly efficient E2F1-mediated apoptosis in culture and in vivo. Importantly, this drug combination delivers an effective and tissue-restricted antimelanoma therapy in vitro and in vivo irrespective of BRAF, MEK, or p53 status.
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    Ecology of melanoma cell
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Lacina, Lukáš; Kodet, Ondřej; Dvořánková, Barbora; Szabo, Pavol; Smetana Jr, Karel
    Melanoma represents a cancer with increasing incidence worldwide and limited curability of advanced stages of the disease. Similarly to other types of tumors, the microenvironment is an important factor that participates in the control of melanoma biological properties. This review summarizes data regarding the role of the microenvironment, namely fibroblasts, keratinocytes and infiltrating immune cells, on melanoma growth and spreading. The role of embryonic microenvironment on melanoma cell biological properties is also discussed. The potential of therapeutic targeting of the melanoma microenvironment is demonstrated.
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    Efecto paradójico radiosensibilizante del ácido carnósico en células de melanoma metastásico B16F10: una nueva estrategia terapéutica
    (Universidad de Murcia, 2023-12-19) Andreu Gálvez, Marina; Alcaraz Baños, Miguel; Olivares Rueda, Amparo; Escuela Internacional de Doctorado
    El ácido carnósico (AC) es un diterpeno fenólico derivado de la planta Rosmarinus officinalis caracterizado por su elevada actividad antioxidante. Por sus características presenta múltiples aplicaciones industriales, cosméticas y nutricionales. Este estudio pretende evaluar la capacidad radioprotectora del AC en células expuestas directamente a rayos X y en células no irradiadas que reciben las señales de células irradiadas con rayos X, conocidas como células receptoras del efecto bystander inducido por radiación ionizante (RIBE). La capacidad genoprotectora se evalúa mediante ensayos de genotoxicidad aplicando los ensayos de micronúcleos in vivo e in vitro. La capacidad radioprotectora mediante ensayos de supervivencia celular con MTT, clonogénico in vitro, apoptosis y de determinación de glutatión intracelular, comparando células radiosensibles (epitelio prostático humano normal, PNT2) con células radiorresistentes (melanoma metastásico murino, B16F10). El AC presenta una capacidad genoprotectora en todas las células expuestas a la radiación ionizante (p < 0,001) y en las células RIBE (p < 0,01). En las células PNT2, el AC consigue un 97% de supervivencia celular tras la exposición a 20 Gy de rayos X, eliminando el 67% de la muerte celular inducida por la radiación (p < 0,001), disminuyendo la apoptosis (p < 0,001) y aumentando la relación GSH/GSSG (p < 0,01). Sin embargo, la administración de AC a las células B16F10 disminuye la supervivencia celular en un 32%, aumentando la muerte celular en un 200% (p < 0,001), y aumentando la muerte celular en un 100% (p<0,001) en las células bystander (p < 0,01). Además, incrementa la apoptosis celular (p < 0,001) y disminuye la relación GSH/GSSG (p < 0,01), expresando un efecto paradójico radiosensibilizador en estas células. El conocimiento de los posibles mecanismos de acción de sustancias como el AC en estos ámbitos podría ayudar a crear nuevas vías de tratamiento que permitan la protección de las células sanas y el daño exclusivo de las neoplásicas, presentando así una nueva estrategia deseable para los pacientes con cáncer que necesitan radioterapia.
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    Estudio del polimorfismo de los genes KIR y NKG2D y de sus ligandos HLA clase I y MICA en pacientes con melanoma
    (Universidad de Murcia, 2018-11-05) Martínez Banaclocha, Helios; Campillo Marquina, José Antonio; Esteban Abad, María de los Ángeles; Escuela Internacional de Doctorado
    Los resultados y conclusiones del presente trabajo son los siguientes: 1. El genotipo KIR2DL3+/C1+ se asocia a protección frente al desarrollo de melanoma y de metástasis en ganglio centinela en pacientes diagnosticados de MES y MN. 2. La ausencia de KIR2DL3 (KIR2DL2 en homocigosis) en pacientes portadores de ligandos C1 podría representar un factor de riesgo para el desarrollo de melanoma nodular y para la progresión a la ulceración de la lesión tumoral. 3. El genotipo KIR2DL1+2DS1-C2C2 podría ser considerado como un factor de riesgo para el desarrollo de melanoma y de metástasis en ganglio centinela en individuos diagnosticados de MES. 4. El aumento de clones de células NK KIR2DS1+ observado en pacientes de melanoma sugiere un papel importante de dicha población celular en la respuesta inmunitaria frente al melanoma cutáneo. 5. Los SNPs de la región NKC estudiados individualmente no parece presentar asociación con el desarrollo y/o pronóstico del melanoma cutáneo. 6. El haplotipo NK-3 del bloque hb-2 de la región génica NKC parece estar asociado con un mayor riesgo de desarrollo de melanoma cutáneo. 7. La expresión aumentada del receptor NKG2D en células NK y linfocitos T CD8+ observada en los pacientes con melanoma cutáneo parece indicar un estado de activación de las mismas. 8. El alelo MICA*009 parece estar asociado con un mayor riesgo de desarrollo de melanoma cutáneo. No obstante, se requieren estudios con series más amplias para confirmar esta asociación. 9. El dimorfismo en posición 80 del gen MICA no parece estar asociado con el desarrollo del melanoma cutáneo. 10. La variante MICA-129Met está asociada con un mayor nivel de MICA soluble en plasma y una peor supervivencia de los pacientes de melanoma cutáneo. OBJECTIVES 1. To study the gene polymorphism of KIR receptors and their HLA class I ligands in patients with melanoma and in a control population. 2. To analyze the subtypes of NK cells and CD8 + T lymphocytes that express KIR receptors in patients with melanoma and in a control population. 3. To study the gene polymorphism of the NKG2D receptor and its MICA ligands in patients with melanoma and in a control population. 4. To analyze the expression of the NKG2D receptor in NK cells and CD8 + T lymphocytes of patients with melanoma and a control population. 5. To study the level of soluble MICA in plasma samples from melanoma patients at diagnosis and from a control population. METHODOLOGY The KIR, HLA-A, B, C, and MICA genes typing was performed by SSO-PCR, reverse-SSO-PCR and SSP-PCR techniques, and the NKC gene region typing by an allelic discrimination assay with Taqman probes and by Sanger sequencing. These trials were carried out in 233 patients diagnosed with cutaneous melanoma and 200 healthy controls. The flow cytometry study to evaluate the expression of KIR receptors in NK cells and peripheral blood CD8 + T lymphocytes was performed in 35 patients diagnosed with melanoma and 24 healthy individuals, while the expression of NKG2D was evaluated in 48 patients with melanoma. and 37 healthy individuals. The determination of soluble MICA protein was carried out using a "sandwich" ELISA technique in the plasma of 30 patients diagnosed with cutaneous melanoma and 26 healthy individuals. RESULTS AND CONCLUSIONS The results and conclusions of the present work were the following: 1. The KIR2DL3+/C1+ genotype is associated with protection against the development of melanoma and sentinel lymph node metastasis in patients diagnosed with SSM and NM. 2. The absence of KIR2DL3 (KIR2DL2 in homozygosis) in patients carrying C1 ligands could represent a risk factor for the development of nodular melanoma and for the progression to the ulceration of the tumor lesion. 3. The KIR2DL1+2DS1-C2C2 genotype could be considered as a risk factor for the development of melanoma and sentinel lymph node metastasis in individuals diagnosed with SSM. 4. The increase of KIR2DS1+ NK cell clones observed in melanoma patients suggests an important role of this cell population in the immune response against the cutaneous melanoma. 5. The NKC region SNPs studied individually does not seem to be associated with the development and/or prognosis of cutaneous melanoma. 6. The NK-3 haplotype of the hb-2 block of the NKC gene region seems to be associated with an increased risk of cutaneous melanoma development. 7. The increased expression of NKG2D receptor observed in NK cells and CD8+ T lymphocytes from patients with cutaneous melanoma seems to indicate an activation state of them. 8. The MICA*009 allele seems to be associated with an increased risk of cutaneous melanoma development. However, studies with larger series are needed to confirm this association. 9. The position 80 MICA gene dimorphism does not seem to be associated with the development of cutaneous melanoma. 10. The MICA-129Met variant is associated with a higher level of soluble MICA in plasma and a worse survival of cutaneous melanoma patients.
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    Expression of Skp2 and p27KIP1 in naevi and malignant melanoma of the skin and its relation to clinical outcome
    (Murcia : F. Hernández, 2005) Woenckhaus, C.; Maile, S.; Uffmann, S.; Bansemir, M.; Dittberner, T.; Poetsch, M.; Giebel, J.
    Skp2 (S-phase kinase associated protein 2) controls progression from G- to S-phase by promoting the proteolysis of the cyclin dependent kinase inhibitor p27KIP1. Despite the fact that a p27KIP1 decrease has been documented in melanoma progression, the role of Skp2 in these tumours is unknown. We therefore examined by immunohistochemistry the expression of Skp2, p27KIP1 and Ki-67 in 10 naevi (Ns), 15 superficial spreading melanomas (SSMs), 10 nodular melanomas (NMs) and 14 melanoma metastases (Ms). Nuclear Skp2 expression augmented with increasing malignancy (Ns: 1.4%, SSMs: 5.6%, NMs: 17.3%, Ms: 19.1%). In all tumours nuclear Skp2 expression correlated with Ki-67 (p=0.024) and inversely with p27KIP1 (p=0.007). A cytoplasmic reaction for Skp2 was also observed in most tumours and its expression decreased from Ns (12.3%) to SSMs (7.9%) and NMs (4.5%). In contrast, Ms showed an increase of cytoplasmic Skp2 (11.9%) that correlated with its nuclear expression (p=0.016). While nuclear Skp2 expression correlated with the pT-level (p=0.023), Clarklevel (p=0.023) and Breslow index (p=0.019), the cytoplasmic Skp2 expression might be of biological significance only in NMs since it correlated with tumour depth (p=0.02) and pT-level (p=0.025). Our data suggests that Skp2 could contribute to melanoma progression. This is further highlighted by the fact that vertical growth phase (VGP) melanomas show significant higher nuclear Skp2 expressions when compared with the harmless radial growth phase (RGP) (p=0.047). Also nuclear Skp2 expression correlates with a reduced survival time (p=0.025) in melanoma.
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    Fli-1 expression in malignant melanoma
    (Murcia : F. Hernández, 2008) Torlakovic, Emina E.; Slipicevic, Ana; Flørenes, Vivi Ann; Chibbar, Richa; DeCoteau, John F.; Bilalovic, Nurija
    Friend leukemia integration site 1 (Fli-1) has been reported as the first nuclear marker of endothelial differentiation; it is expressed in leukocytes and recently demonstrated in melanomas. Formalin-fixed, paraffinembedded tissue sections from 97 melanomas including 69 cases of primary and 28 metastatic melanomas were evaluated by immunohistochemistry. Five melanoma cell lines were evaluated by Western blot and immunocytochemistry. Fli-1 expression was observed in all cell lines. Fli-1 expression was higher in metastatic than in primary tumors (r=0.208, p=0.041, Spearman correlation), it positively correlated with Ki-67 expression (r=0.233, p=0.022, Spearman correlation), and the presence of an ulcer in the primary tumor (r=0.267, p=0.030, Spearman correlation). Therefore, the expression of Fli-1 in malignant melanoma appears to be associated with biologically more aggressive tumors.
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    Fluorescence in situ hybridization for ambiguous melanocytic tumors
    (F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2012) Gammon, Bryan; Gerami, Pedram
    The large majority of melanocytic lesions can be reliably classified as either benign or malignant based upon morphology alone, but a minority of lesions remains difficult to classify by traditional histologic methods. Recently, a panel of fluorescence in situ hybridization (FISH) probes targeting loci on chromosomes 6 and 11 has emerged as a powerful tool to discriminate melanoma from nevi. This has been validated in numerous difficult diagnostic scenarios. In addition, this same FISH panel has been shown to provide independent prognostic information in traditional melanomas. There is accumulating evidence that FISH targeting these loci as well as several other key chromosomal loci such as 9p21 and 8q24 can provide valuable prognostic information in histologically ambiguous melanocytic tumors. However, since the vast majority of atypical spitz tumors have an indolent course, larger studies including adequate numbers of cases with adverse events is necessary to provide sufficient proof of its role in clinically relevant cases. In this review, we discuss the current literature and studies to date on this topic.
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    Frequent intra-tumoural heterogeneity of promoter hypermethylation in malignant melanoma
    (Murcia : F. Hernández, 2007) Rastetter, M.; Schagdarsurengin, U.; Lahtz, C.; Fiedler, E.; Marsch, V.Ch.; Dammann, R.; Helmbold, P.
    To investigate intra-tumoural coexistence and heterogeneity of aberrant promoter hypermethylation of different tumour suppressor genes in melanoma, we analyzed the intra-tumoural distribution of promoter methylation of RASSF1A, p16, DAPK, MGMT, and Rb in 339 assays of 34 tumours (15 melanoma primaries, 19 metastases) by methylation-specific PCR, correlation to histopathology and RASSF1A expression. We detected promoter hypermethylation of at least one gene in 74% of tumours (30%, 52%, 33%, 20%, and 40% for RASSF1A, p16, DAPK, MGMT and Rb, respectively). 70% of the cases exhibited an inhomogeneous methylation pattern (17%, 45%, 33%, 20%, and 40% for RASSF1A, p16, DAPK, MGMT and Rb, respectively). Samples from the core of the tumours represented the methylation state of the whole tumours more accurately than the periphery. Local intra-tumoural correlation was found between the promoter hypermethylation state of p16 and Rb or p16 and DAPK, or epitheloid tumour cell type and RASSF1A or p16 methylation. Mitosis rate and sex was correlated with methylation of RASSF1A. Histological results confirmed that promoter hypermethylation of RASSF1A led to aberrant expression patterns. We conclude that intra-tumoural inhomogeneity of promoter hypermethylation is frequent in melanoma and this supports the hypothesis of clonal instability during progression of melanomas. In prognosis studies, missing the intra-tumoural sample representativeness may result in a reduction of the sensitivities or specificities.