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1.
J Pathol ; 244(2): 242-253, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29160922

RESUMO

Testicular germ cell tumours (TGCTs) are the most frequent malignancy and cause of death from solid tumours in the 20- to 40-year age group. Although most cases show sensitivity to cis-platinum-based chemotherapy, this is associated with long-term toxicities and chemo-resistance. Roles for receptor tyrosine kinases other than KIT are largely unknown in TGCT. We therefore conducted a phosphoproteomic screen and identified the insulin growth factor receptor-1 (IGF1R) as both highly expressed and activated in TGCT cell lines representing the nonseminomatous subtype. IGF1R was also frequently expressed in tumour samples from patients with nonseminomas. Functional analysis of cell line models showed that long-term shRNA-mediated IGF1R silencing leads to apoptosis and complete ablation of nonseminoma cells with active IGF1R signalling. Cell lines with high levels of IGF1R activity also showed reduced AKT signalling in response to decreased IGF1R expression as well as sensitivity to the small-molecule IGF1R inhibitor NVP-AEW541. These results were in contrast to those in the seminoma cell line TCAM2 that lacked IGF1R signalling via AKT and was one of the two cell lines least sensitive to the IGF1R inhibitor. The dependence on IGF1R activity in the majority of nonseminomas parallels the known role of IGF signalling in the proliferation, migration, and survival of primordial germ cells, the putative cell of origin for TGCT. Upregulation of IGF1R expression and signalling was also found to contribute to acquired cisplatin resistance in an in vitro nonseminoma model, providing a rationale for targeting IGF1R in cisplatin-resistant disease. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Testiculares/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia
2.
Oncotarget ; 7(36): 57525-57544, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27438153

RESUMO

MYC oncoproteins deliver a potent oncogenic stimulus in several human cancers, making them major targets for drug development, but efforts to deliver clinically practical therapeutics have not yet been realized. In childhood cancer, aberrant expression of MYC and MYCN genes delineates a group of aggressive tumours responsible for a major proportion of pediatric cancer deaths. We designed a chemical-genetic screen that identifies compounds capable of enhancing proteasomal elimination of MYCN oncoprotein. We isolated several classes of compound that selectively kill MYCN expressing cells and we focus on inhibitors of PI3K/mTOR pathway in this study. We show that PI3K/mTOR inhibitors selectively killed MYCN-expressing neuroblastoma tumor cells, and induced significant apoptosis of transgenic MYCN-driven neuroblastoma tumors concomitant with elimination of MYCN protein in vivo. Mechanistically, the ability of these compounds to degrade MYCN requires complete blockade of mTOR but not PI3 kinase activity and we highlight NVP-BEZ235 as a PI3K/mTOR inhibitor with an ideal activity profile. These data establish that MYCN expression is a marker indicative of likely clinical sensitivity to mTOR inhibition, and provide a rationale for the selection of clinical candidate MYCN-destabilizers likely to be useful for the treatment of MYCN-driven cancers.


Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Imidazóis/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Neuroblastoma/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Quinolinas/química , Transdução de Sinais , Transgenes
3.
Clin Cancer Res ; 19(21): 5940-51, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23918606

RESUMO

PURPOSE: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths. EXPERIMENTAL DESIGN: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA-mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations. RESULTS: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP-ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations. CONCLUSIONS: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Modelos Animais de Doenças , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Rabdomiossarcoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Clin Cancer Res ; 18(3): 796-807, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22065083

RESUMO

PURPOSE: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. EXPERIMENTAL DESIGN: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. RESULTS: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. CONCLUSION: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo.


Assuntos
Terapia Genética/métodos , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Ácidos Nucleicos Peptídicos/farmacologia , Rabdomiossarcoma/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Dosagem de Genes , Genes myc/genética , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma/terapia , Transcrição Gênica , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Cancer Ther ; 3(11): 1467-84, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15542786

RESUMO

Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas WT1/genética , Processamento Alternativo/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Desoxirribonucleases/metabolismo , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Proteínas WT1/metabolismo
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