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1.
Int J Cancer ; 144(12): 3070-3085, 2019 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-30556600

RESUMEN

Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.


Asunto(s)
Exosomas/metabolismo , Melanoma/metabolismo , Melanoma/patología , Proteínas rab27 de Unión a GTP/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Medios de Cultivo Condicionados , Exosomas/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanosomas/genética , Melanosomas/metabolismo , Ratones , Invasividad Neoplásica , Nevo/genética , Nevo/metabolismo , Proteómica , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Esferoides Celulares , Proteínas rab27 de Unión a GTP/biosíntesis , Proteínas rab27 de Unión a GTP/genética
2.
Int J Cancer ; 142(9): 1926-1937, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29210065

RESUMEN

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Melanoma/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Sinergismo Farmacológico , Xenoinjertos , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Melanoma/enzimología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos
3.
J Biol Chem ; 288(44): 31761-71, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-24062304

RESUMEN

Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH4Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Proteínas de Neoplasias/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/genética , Lisosomas/genética , Proteínas de Neoplasias/genética
4.
Int J Cancer ; 135(5): 1060-71, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24531984

RESUMEN

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.


Asunto(s)
Sistema de Transporte de Aminoácidos ASC/biosíntesis , Glutamina/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/biosíntesis , Melanoma/patología , Complejos Multiproteicos/antagonistas & inhibidores , Neoplasias Cutáneas/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/genética , Aminoácidos Cíclicos/farmacología , Compuestos de Bencilo/farmacología , Transporte Biológico , Proteína Quinasa CDC2/biosíntesis , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Humanos , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Melanoma/metabolismo , Antígenos de Histocompatibilidad Menor , Complejos Multiproteicos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Serina/análogos & derivados , Serina/farmacología , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Esferoides Celulares , Serina-Treonina Quinasas TOR/genética , Células Tumorales Cultivadas , Enzimas Ubiquitina-Conjugadoras/biosíntesis
5.
Mol Pharmacol ; 83(1): 179-90, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23074173

RESUMEN

Deferasirox is an orally effective iron (Fe) chelator currently used for the treatment of iron-overload disease and has been implemented as an alternative to the gold standard chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO exhibits anticancer activity due to its ability to deplete cancer cells of iron. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines compared with DFO. Furthermore, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular (59)Fe and inhibiting iron uptake from human transferrin depending on the cell type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the antitumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control, and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein N-myc downstream-regulated gene 1 and upregulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1. Collectively, we demonstrate that deferasirox is an orally effective antitumor agent against solid tumors.


Asunto(s)
Antineoplásicos/farmacología , Benzoatos/farmacología , Quelantes del Hierro/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Triazoles/farmacología , Administración Oral , Animales , Antígenos CD/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Benzoatos/uso terapéutico , Ciclo Celular/fisiología , Línea Celular Tumoral , Cobre/metabolismo , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Deferasirox , Femenino , Humanos , Hierro/metabolismo , Quelantes del Hierro/uso terapéutico , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Tumores Neuroectodérmicos Periféricos Primitivos , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Transferrina/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Trasplante Heterólogo , Triazoles/uso terapéutico , Zinc/metabolismo
6.
Pigment Cell Melanoma Res ; 33(6): 889-894, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32511882

RESUMEN

The role of the small GTPase RAB27A as an essential melanosome trafficking regulator in melanocytes is well-accepted. A decade ago, RAB27A was identified as a tumor dependency gene that promotes melanoma cell proliferation. RAB27A has since been linked to another propeller of cancer progression: exosome secretion. We have recently demonstrated that RAB27A is overexpressed in a subset of melanomas. High RAB27A gene and protein expression correlate with poor prognosis in melanoma patients. Mechanistic investigations revealed that the generation of pro-invasive exosomes was RAB27A-dependent and, therefore, silencing RAB27A reduced melanoma cell invasion in vitro and in vivo. However, previous studies have implicated RAB27A to be involved in both proliferation and invasion of melanoma cells. Employing four human cell lines, stratified by RAB27A expression, and one RAB27A-high mouse cell line, we demonstrate in this study that the effects of abrogating RAB27A expression on proliferation are only temporary, in contrast to our previously reported persistent effects on tumor invasion and metastasis. Therefore, we assist in the dissection of the short-term effects of RAB27A knockdown on melanoma cell proliferation versus long-term effects on melanoma invasion and metastasis. We believe that our findings provide novel insights into the effects of RAB27A blockade.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Melanoma/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Proteínas rab27 de Unión a GTP/genética , Muerte Celular , Línea Celular Tumoral , Proliferación Celular/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas rab27 de Unión a GTP/metabolismo
7.
J Invest Dermatol ; 136(7): 1479-1489, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26970356

RESUMEN

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic tumor zones contain clusters of cancer cells that arrest in G1 phase. It is conceivable that neoplastic cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. In this study, we used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of cell cycle phases on clinically used drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1 arrest, are resistant to apoptosis induced by the proteasome inhibitor bortezomib or the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to mitogen-activated protein kinase pathway inhibitor-induced cell death. Of clinical relevance, pretreatment of melanoma cells with a mitogen-activated protein kinase pathway inhibitor, which induced G1 arrest, resulted in resistance to temozolomide or bortezomib. On the other hand, pretreatment with temozolomide, which induced G2 arrest, did not result in resistance to mitogen-activated protein kinase pathway inhibitors. In summary, we established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies.


Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Alquilantes/química , Apoptosis , Bortezomib/química , División Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/química , Fase G1 , Fase G2 , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Temozolomida
8.
J Med Chem ; 55(16): 7230-44, 2012 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-22861499

RESUMEN

We developed a series of second-generation di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands to improve the efficacy and safety profile of these potential antitumor agents. Two novel DpT analogues, Dp4e4mT and DpC, exhibited pronounced and selective activity against human lung cancer xenografts in vivo via the intravenous and oral routes. Importantly, these analogues did not induce the cardiotoxicity observed at high nonoptimal doses of the first-generation DpT analogue, Dp44mT. The Cu(II) complexes of these ligands exhibited potent antiproliferative activity having redox potentials in a range accessible to biological reductants. The activity of the copper complexes of Dp4e4mT and DpC against lung cancer cells was synergistic in combination with gemcitabine or cisplatin. It was demonstrated by EPR spectroscopy that dimeric copper compounds of the type [CuLCl](2), identified crystallographically, dissociate in solution to give monomeric 1:1 Cu:ligand complexes. These monomers represent the biologically active form of the complex.


Asunto(s)
Antineoplásicos/síntesis química , Complejos de Coordinación/síntesis química , Cobre , Cetonas/síntesis química , Neoplasias Pulmonares/tratamiento farmacológico , Piridinas/síntesis química , Tiosemicarbazonas/síntesis química , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Cristalografía por Rayos X , Dimerización , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Inyecciones Intravenosas , Cetonas/química , Cetonas/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Oxidación-Reducción , Piridinas/química , Piridinas/farmacología , Relación Estructura-Actividad , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacología , Transferrina/metabolismo , Trasplante Heterólogo
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