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1.
Cell Death Dis ; 8(8): e3029, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29048432

RESUMO

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.


Assuntos
Antígenos CD13/genética , Efrina-A2/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Neoplasias Cutâneas/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Dasatinibe/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Efrina-A2/metabolismo , Humanos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor EphA2 , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Sulfonamidas/uso terapêutico , Transativadores , Vemurafenib
2.
Int J Cancer ; 136(12): 2844-53, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25400033

RESUMO

To investigate the predictive and prognostic value of O(6) -methylguanine DNA methyltransferase (MGMT) inactivation by analyses of promoter methylation in pretreatment tumor biopsies from patients with cutaneous melanoma treated with dacarbazine (DTIC) or temozolomide (TMZ) were performed. The patient cohorts consisted of Belgian and Swedish disseminated melanoma patients. Patients were subdivided into those receiving single-agent treatment with DTIC/TMZ (cohort S, n = 74) and those treated with combination chemotherapy including DTIC/TMZ (cohort C, n = 79). Median follow-up was 248 and 336 days for cohort S and cohort C, respectively. MGMT promoter methylation was assessed by three methods. The methylation-related transcriptional silencing of MGMT mRNA expression was assessed by real-time RT-PCR. Response to chemotherapy and progression-free survival (PFS) and overall survival were correlated to MGMT promoter methylation status. MGMT promoter methylation was detected in tumor biopsies from 21.5 % of the patients. MGMT mRNA was found to be significantly lower in tumors positive for MGMT promoter methylation compared to tumors without methylation in both treatment cohorts (p < 0.005). DTIC/TMZ therapy response rate was found to be significantly associated with MGMT promoter methylation in cohort S (p = 0.0005), but did not reach significance in cohort C (p = 0.16). Significantly longer PFS was observed among patients with MGMT promoter-methylated tumors (p = 0.002). Multivariate Cox regression analysis identified presence of MGMT promoter methylation as an independent variable associated with longer PFS. Together, this implies that MGMT promoter methylation is associated with response to single-agent DTIC/TMZ and longer PFS in disseminated cutaneous melanoma.


Assuntos
Metilação de DNA , Dacarbazina/análogos & derivados , Melanoma/tratamento farmacológico , O(6)-Metilguanina-DNA Metiltransferase/genética , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Coortes , Dacarbazina/administração & dosagem , Dacarbazina/uso terapêutico , Intervalo Livre de Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas , Temozolomida , Melanoma Maligno Cutâneo
3.
Pharmacol Ther ; 142(2): 176-82, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24325952

RESUMO

Activating BRAF mutations, leading to constitutive activation of the MAPK signaling pathway, are common in a variety of human cancers. Several small molecule BRAF inhibitors have been developed during the last years and shown promising results in clinical trials, especially for metastatic melanoma, while they have been less effective in colon cancer. Two inhibitors, vemurafenib and dabrafenib, have been approved for treatment of melanoma. Unfortunately, in most patients who initially respond the tumors eventually develop acquired resistance to the BRAF inhibitors. So far, a number of resistance mechanisms have been identified, including secondary NRAS mutations and BRAF alternative splicing, leading to reactivation of the MAPK pathway. Other alterations, both upstream and downstream of BRAF can have the same effect, and activation of alternative pathways can also play a role in resistance to BRAF inhibitors. In addition, intra-tumor heterogeneity with the presence of clones of tumor cells lacking BRAF mutations needs to be considered, since wildtype BRAF can be activated by inhibitors designed to target mutated BRAF. Combination of the BRAF inhibitor dabrafenib with the MEK inhibitor trametinib has significantly prolonged progression free survival compared to dabrafenib alone in metastatic melanoma. Combination treatments of BRAF inhibitors with other agents may not only circumvent or delay resistance, but may also lead to fewer side effects, such as development of secondary squamous tumors. Several clinical trials are underway for many different BRAF mutation positive cancers with BRAF inhibitors alone or in combination with other small molecule inhibitors, immunotherapies or conventional chemotherapy.


Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Melanoma Res ; 23(5): 360-5, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23921446

RESUMO

Previous studies in cell lines have suggested a role for melanosomes and related protein trafficking pathways in melanoma drug response. We have investigated the expression of six proteins related to melanosomes and melanogenesis (MITF, GPR143, gp100/PMEL, MLANA, TYRP1, and RAB27A) in pretreatment metastases from melanoma patients (n = 52) with different response to dacarbazine/temozolomide. Microphthalmia-associated transcription factor (MITF) and G-protein coupled receptor 143 (GPR143) showed significantly higher expression in nonresponders compared with responders. The premelanosome protein (gp100/PMEL) has been indicated previously in resistance to cisplatin in melanoma cells, but the expression levels of gp100/PMEL showed no association with response to dacarbazine/temozolomide in our clinical material. We also investigated the effects on chemosensitivity of siRNA inhibition of gp100/PMEL in the MNT-1 melanoma cell line. As expected from the study of the tumor material, no effect was detected with respect to response to temozolomide. However, knockdown of gp100/PMEL sensitized the cells to both paclitaxel and cisplatin. Overall, our results suggest that MITF, and several MITF-regulated factors, are associated with resistance to chemotherapy in melanoma and that different MITF targets can be of importance for different drugs.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Melanoma/metabolismo , Melanossomas/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Masculino , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Melanossomas/genética , Melanossomas/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Pessoa de Meia-Idade , Interferência de RNA , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcrição Gênica , Transfecção , Resultado do Tratamento , Antígeno gp100 de Melanoma/genética , Antígeno gp100 de Melanoma/metabolismo
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