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1.
Mol Cancer ; 16(1): 85, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28454577

RESUMO

BACKGROUND: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood. RESULTS: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation. CONCLUSIONS: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.


Assuntos
Melanoma/genética , Neoplasias/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Processamento Alternativo/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Indóis/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/patologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , RNA Mensageiro/genética , Sulfonamidas/administração & dosagem , Vemurafenib
3.
Am J Transl Res ; 12(4): 1275-1292, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32355541

RESUMO

Despite good responses to first-line treatment with platinum-based combination chemotherapy, most ovarian cancer patients will relapse and eventually develop a platinum-resistant disease with a poor overall prognosis. The molecular events leading to the cisplatin resistance of ovarian cancer cells are not fully understood. Here, we performed a proteomic analysis to identify protein candidates deregulated in a cisplatin-resistant ovarian cancer cell line (A2780CP20) in comparison to their sensitive counterpart (A2780). Forty-eight proteins were differentially abundant in A2780CP20, as compared with A2780, cells. Enolase-1 (ENO1) was significantly decreased in cisplatin-resistant ovarian cancer cells. Western blots and RT-PCR confirmed our findings. Ectopic ENO1 expression increased the sensitivity of ovarian cancer cells to cisplatin treatment. In contrast, small-interfering (siRNA)-based ENO1 silencing in A2780 cells reduced the sensitivity of these cells to cisplatin treatment. Whereas glucose consumption was lower, intracellular levels were higher in cisplatin-resistant ovarian cancer cells as compared with their cisplatin-sensitive counterparts. Senescence-associated ß-galactosidase (ß-Gal) levels were higher in cisplatin-resistant ovarian cancer cells as compared with cisplatin-sensitive ovarian cancer cells. ß-Gal levels were decreased in ENO1 overexpressed clones. Protein levels of the cell cycle regulators and senescence markers p21 and p53 showed opposite expression patterns in cisplatin-resistant compared with cisplatin sensitive cells. Our studies suggest that decreased expression of ENO1 promotes glucose accumulation, induces senescence, and leads to cisplatin resistance of ovarian cancer cells.

4.
Oncogene ; 37(30): 4058-4072, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29695835

RESUMO

Targeting RAS is one of the greatest challenges in cancer therapy. Oncogenic mutations in NRAS are present in over 25% of melanomas and patients whose tumors harbor NRAS mutations have limited therapeutic options and poor prognosis. Thus far, there are no clinical agents available to effectively target NRAS or any other RAS oncogene. An alternative approach is to identify and target critical tumor vulnerabilities or non-oncogene addictions that are essential for tumor survival. We investigated the consequences of NRAS blockade in NRAS-mutant melanoma and show that decreased expression of the telomerase catalytic subunit, TERT, is a major consequence. TERT silencing or treatment of NRAS-mutant melanoma with the telomerase-dependent telomere uncapping agent, 6-thio-2'-deoxyguanosine (6-thio-dG), led to rapid cell death, along with evidence of both telomeric and non-telomeric DNA damage, increased ROS levels, and upregulation of a mitochondrial antioxidant adaptive response. Combining 6-thio-dG with the mitochondrial inhibitor Gamitrinib attenuated this adaptive response and more effectively suppressed NRAS-mutant melanoma. Our study uncovers a robust dependency of NRAS-mutant melanoma on TERT, and provides proof-of-principle for a new combination strategy to combat this class of tumors, which could be expanded to other tumor types.


Assuntos
GTP Fosfo-Hidrolases/genética , Melanoma/genética , Proteínas de Membrana/genética , Mutação/genética , Telomerase/genética , Animais , Antioxidantes/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/genética
5.
EMBO Mol Med ; 10(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29650805

RESUMO

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS-mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS-mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS-mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinase Quinase 1/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Acetanilidas/farmacologia , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Proteômica/métodos , Terapia de Salvação/métodos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Oncotarget ; 7(24): 36321-36337, 2016 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-27166999

RESUMO

MicroRNA-21 is overexpressed in most cancers and has been implicated in tumorigenesis. Accumulating evidence supports a central role for the miR-21 guide strand (miR-21-5p) in ovarian cancer initiation, progression, and chemoresistance. However, there is limited information regarding the biological role of the miR-21 passenger strand (miR-21-3p) in ovarian cancer cells. The aim of this study was to investigate the role of miR-21-3p and its target genes in cisplatin-resistant ovarian cancer cells. Expression profiling of miR-21-5p and miR-21-3p was performed in a panel of cancer cells by qPCR. Colony formation and invasion assays were carried out on ovarian and prostate cancer cells transfected with miR-21-5p and miR-21-3p inhibitors. Dual luciferase reporter assays were used to identify the miR-21-3p target genes in ovarian cancer cells. Our results show that miR-21-5p had higher expression levels compared to miR-21-3p on a panel of cancer cells. Moreover, inhibition of miR-21-5p or miR-21-3p resulted in a significant decrease in ovarian and prostate cancer cell proliferation and invasion. Luciferase reporter assays identify RNA Binding Protein with Multiple Splicing (RBPMS), Regulator of Chromosome Condensation and POZ Domain Containing Protein 1 (RCBTB1), and Zinc Finger protein 608 (ZNF608) as miR-21-3p target genes. SiRNA-induced RBPMS silencing reduced the sensitivity of ovarian cancer cells to cisplatin treatment. Immunohistochemical analyses of serous ovarian cancer patient samples suggest a significant decrease of RBMPS levels when compared to normal ovarian epithelium. Taken together, the data generated in this study suggests a functional role for miR-21-3p in ovarian cancer and other solid tumors.


Assuntos
Proliferação de Células/genética , Cistadenocarcinoma Seroso/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Ovarianas/genética , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
7.
Mol Cancer Ther ; 14(10): 2260-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26227489

RESUMO

The purpose of this study was to investigate the molecular and therapeutic effects of siRNA-mediated c-MYC silencing in cisplatin-resistant ovarian cancer. Statistical analysis of patient's data extracted from The Cancer Genome Atlas (TCGA) portal showed that the disease-free (DFS) and the overall (OS) survival were decreased in ovarian cancer patients with high c-MYC mRNA levels. Furthermore, analysis of a panel of ovarian cancer cell lines showed that c-MYC protein levels were higher in cisplatin-resistant cells when compared with their cisplatin-sensitive counterparts. In vitro cell viability, growth, cell-cycle progression, and apoptosis, as well as in vivo therapeutic effectiveness in murine xenograft models, were also assessed following siRNA-mediated c-MYC silencing in cisplatin-resistant ovarian cancer cells. Significant inhibition of cell growth and viability, cell-cycle arrest, and activation of apoptosis were observed upon siRNA-mediated c-MYC depletion. In addition, single weekly doses of c-MYC-siRNA incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000)-based nanoliposomes resulted in significant reduction in tumor growth. These findings identify c-MYC as a potential therapeutic target for ovarian cancers expressing high levels of this oncoprotein.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , Neoplasias Ovarianas/mortalidade , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
PLoS One ; 9(5): e97094, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24865582

RESUMO

Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the pri-mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of pre-mir-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Neoplasias Ovarianas/genética , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
9.
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