RESUMEN
Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and Cyclin B1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assay, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Estradiol/análogos & derivados , Melanoma/tratamiento farmacológico , Invasividad Neoplásica/prevención & control , Neoplasias Cutáneas/tratamiento farmacológico , Piel/efectos de los fármacos , 2-Metoxiestradiol , Apoptosis/efectos de los fármacos , Ciclo Celular , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Estradiol/farmacología , Humanos , Melanoma/patología , Invasividad Neoplásica/patología , Piel/patología , Neoplasias Cutáneas/patologíaRESUMEN
In recent years, melanoma has become a poster-child for the development of oncogene-directed targeted therapies. This approach, which has been exemplified by the development of small-molecule BRAF inhibitors and the BRAF/MEK inhibitor combination for BRAF-mutant melanoma, has brought new hope to patients. Despite these successes, treatment failure seems near inevitable in the majority of caseseven in individuals treated with the BRAF/MEK inhibitor doublet. In the current review, we discuss the future of combination strategies for patients with BRAF-mutant melanoma as well as the emerging therapeutic options for patients with NRAS-mutant and BRAF/NRAS-wild-type melanoma. We also outline some of the newest developments in the in-depth personalisation of therapy that should allow melanoma treatment to continue shaping the field precision cancer medicine.
Asunto(s)
Proteínas Proto-Oncogénicas B-raf/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto , Resistencia a Antineoplásicos/genética , GTP Fosfohidrolasas/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas de la Membrana/genética , Terapia Molecular Dirigida , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genéticaRESUMEN
BACKGROUND: Resistance to BRAF inhibitors is an emerging problem in the melanoma field. Strategies to prevent and overcome resistance are urgently required. METHODS: The dynamics of cell signalling, BrdU incorporation and cell-cycle entry after BRAF inhibition was measured using flow cytometry and western blot. The ability of combined BRAF/MEK inhibition to prevent the emergence of resistance was demonstrated by apoptosis and colony formation assays and in 3D organotypic cell culture. RESULTS: BRAF inhibition led to a rapid recovery of phospho-ERK (pERK) signalling. Although most of the cells remained growth arrested in the presence of drug, a minor population of cells retained their proliferative potential and escaped from BRAF inhibitor therapy. A function for the rebound pERK signalling in therapy escape was demonstrated by the ability of combined BRAF/MEK inhibition to enhance the levels of apoptosis and abrogate the onset of resistance. CONCLUSION: Combined BRAF/MEK inhibition may be one strategy to prevent the emergence of drug resistance in BRAF-V600E-mutated melanomas.
Asunto(s)
Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Factores de TiempoRESUMEN
The discovery of BRAF mutations in melanoma has not yet translated into clinical success, suggesting that BRAF/MEK inhibitors will need to be combined with other agents. In the current review, we discuss other pathways likely to be important for melanoma progression and suggest possible drug combinations for future clinical testing.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Novel anti-cancer treatments use knowledge about the underlying biology of the tumor to find suitable molecular targets. The recent years have seen great advances in understanding the biology of melanoma. In the current review we discuss the most promising molecular targets for melanoma and suggest possible strategies for overcoming resistance.
Asunto(s)
Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Humanos , Melanoma/enzimología , Melanoma/genética , Mutación , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.
Asunto(s)
Fibronectinas/metabolismo , Melanoma/patología , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Integrina alfa5beta1/metabolismo , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteómica , Proteínas Proto-Oncogénicas B-raf/deficiencia , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The median survival for metastatic melanoma is in the realm of 8-16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan-HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti-melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti-melanoma agents.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/efectos de los fármacos , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Animales , Línea Celular Tumoral , Fase G1/efectos de los fármacos , Histona Desacetilasa 6 , Humanos , Melanoma Experimental/enzimología , Ratones , Ratones Endogámicos C57BLRESUMEN
The retinoblastoma protein (pRB), the product of the retinoblastoma gene, is a key regulator of the cell cycle, affecting apoptosis, proliferation and differentiation. Dysregulation of pRB is implicated in the pathogenesis of many cancers, including malignant melanoma. Recently we demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH)-induced activation of p38 mitogen-activated protein (MAP) kinase leads to differentiation of B16 murine melanoma cells. The current study assesses the ability of alpha-MSH to activate p38 MAP kinase in COLO 853 human melanoma cells and determines whether this is linked to modulation of pRB activity. Treatment of COLO 853 cells with alpha-MSH induced time- and concentration-dependent increases in the phosphorylation of p38 MAP kinase, which corresponded with its ability to induce melanogenesis and inhibit cell growth. SB 203580, a selective inhibitor of p38 MAP kinase, blocked both the alpha-MSH-induced melanogenic response and inhibition of cell growth. Cell cycle analysis using flow cytometry revealed that treatment of COLO 853 cells with alpha-MSH for 72 h led to an increase in the proportion of cells in the G(1) phase and a marked reduction in the amount of phosphorylated pRB. Both of these effects were reversed by pre-treatment of cells with SB 203580. In summary, we have demonstrated for the first time that the alpha-MSH-induced differentiation of COLO 853 human melanoma cells proceeds via a p38 MAP kinase-mediated pathway and is associated with decreased pRB phosphorylation and accumulation of cells in the G(1) phase.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Melanoma/patología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas de Neoplasias/fisiología , Procesamiento Proteico-Postraduccional , Proteína de Retinoblastoma/metabolismo , alfa-MSH/farmacología , Diferenciación Celular/efectos de los fármacos , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fase G1 , Humanos , Imidazoles/farmacología , Cinética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melaninas/biosíntesis , Melaninas/metabolismo , Melanoma/enzimología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Fosforilación , Piridinas/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
The recent years have seen a significant shift in the expectations for the therapeutic management of disseminated melanoma. The clinical success of BRAF targeted therapy suggests that long-term disease control may one day be a reality for genetically defined subgroups of melanoma patients. Despite this progress, few advances have been made in developing targeted therapeutic strategies for the 50% of patients whose melanomas are BRAF wild-type. The most well-characterized subgroup of BRAF wild-type tumors is the 15-20% of all melanomas that harbor activating NRAS (Neuroblastoma Rat Sarcoma Virus) mutations. Emerging preclinical and clinical evidence suggests that NRAS mutant melanomas have patterns of signal transduction and biological behavior that is distinct from BRAF mutant melanomas. This overview will discuss the unique clinical and prognostic behavior of NRAS mutant melanoma and will summarize the emerging data on how NRAS-driven signaling networks can be translated into novel therapeutic strategies.
Asunto(s)
GTP Fosfohidrolasas/genética , Melanoma/genética , Melanoma/terapia , Proteínas de la Membrana/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/terapia , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Farnesil Difosfato Farnesil Transferasa/metabolismo , Humanos , Melanoma/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pronóstico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismoRESUMEN
The 2010 7th International Melanoma Congress sponsored by the Society for Melanoma Research and held in Sydney, Australia, was held together with the International Melanoma and Skin Cancer Centers group and the International Melanoma Pathology Study Group. As a consequence, there were over 900 registrants that included a wide range of clinicians (surgeons, medical oncologists, dermatologists) specialising in the management of melanoma as well as scientists and students carrying out laboratory-based research in melanoma. There was a general consensus that this grouping of clinicians, pathologists and scientists was mutually advantageous and plans are afoot to continue this grouping in future meetings. The meeting was dominated by the advances being made in treatment of melanoma with selective BRAF inhibitors but interest in epithelial mesenchymal transition and phenotypic changes in melanoma was apparent in many of the talks. The authors have attempted to capture many of the new developments in melanoma research but apologize to those speakers and poster presenters who had equally important findings not captured in these summaries.
Asunto(s)
Congresos como Asunto , Melanoma/patología , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/terapia , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Nueva Gales del Sur , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pez Cebra/genéticaRESUMEN
Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.
Asunto(s)
Apoptosis , Resistencia a Antineoplásicos/genética , Melanoma/enzimología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Apoptosis/genética , Bencenosulfonatos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Melanoma/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-raf/genética , Piridinas/farmacología , ARN Interferente Pequeño/genética , Transducción de Señal , Sorafenib , Valina/genética , Valina/metabolismoAsunto(s)
Melanoma/diagnóstico , Melanoma/terapia , Animales , California , Ensayos Clínicos como Asunto , Epigénesis Genética , Predisposición Genética a la Enfermedad , Humanos , Inmunoterapia/métodos , Oncología Médica/métodos , Melanoma/genética , Melanoma/patología , Ratones , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Sociedades MédicasRESUMEN
The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies.