RESUMO
BACKGROUND: Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014. METHODS: A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPjκ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC explant tumours was performed by gene array and immunoblotting. RESULTS: We identified a subset of CRC tumours that exhibited elevations of the Notch and Wnt pathways sensitive to PF-03084014. Treatment with the GSI resulted in a significant reduction in cleaved Notch, Axin2 (Wnt-dependent gene) and active ß-catenin. In addition, knockdown of the RBPjκ gene showed that PF-03084014 has specificity for the Notch pathway in an HCT116 cell line xenograft model. Finally, an increase in apoptosis was observed in CRC001- and CRC021-sensitive tumours. CONCLUSION: This study provides evidence that inhibition of γ-secretase may be beneficial in a subset of patients with elevated levels of the Wnt and Notch pathways.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Receptores Notch/metabolismo , Tetra-Hidronaftalenos/farmacologia , Valina/análogos & derivados , Via de Sinalização Wnt/efeitos dos fármacos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Distribuição Aleatória , Valina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase IIα (TopoIIα) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoIIα forms protein-protein interactions with ß-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoIIα and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoIIα N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoIIα and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoIIα-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoIIα, etoposide and merbarone, were ineffective at preventing TopoIIα-dependent TCF transcription. Thus, we propose that TopoIIα participation in TCF transcription may convey a mechanism of MDR to conventional TopoIIα inhibitors. However, our results indicate that TopoIIα N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias do Colo/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , beta Catenina/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Neoplasias do Colo/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica/genética , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Mapas de Interação de Proteínas/genética , beta Catenina/metabolismoRESUMO
Identification of clonal chromosomal abnormalities involving 14q32 and its association with specific histological subtypes of non-Hodgkin lymphoma (NHL) has provided substantial insight to the genetic events leading to the disease. However, in some cases with inferior morphology of tumor cell chromosomes, the additional segment on chromosome 14 remains unidentified by cytogenetic banding techniques alone. To elucidate the origin of the additional chromosomal segment and to correlate the newly determined alterations with histology, metaphases from 15 NHL patients with add(14)(q32) were examined using fluorescence in situ hybridization (FISH) techniques after cytogenetic analysis had been performed. We found the duplication of 14q involving the q32 region in 6 cases with a dup(14) (q32) in 4 cases and a dup(14)(q24q32) in 2 cases. In 8 cases, FISH unveiled known NHL associated translocations; a t(14;18)(q32;q21) in 4 cases, a t(11;14)(q13;q32) in 2 cases, a t(8;14)(q24;q32) and a t(9;14)(p13;q32) in 1 case each. We also noted a t(14;17)(q32;q21) in 1 case. The use of FISH was a valuable asset in determining the origin of the additional material on chromosome 14q32, and helped resolve a group of B-cell NHLs with involvement of a duplicated 14q32 region.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 14 , Duplicação Gênica , Linfoma de Células B/genética , Humanos , Hibridização in Situ Fluorescente , Estudos RetrospectivosRESUMO
Acute lung injury is frequently associated with endotoxemia and is characterized by the accumulation in the lungs of large numbers of neutrophils activated to produce proinflammatory mediators. In the setting of acute lung injury, the percentage of apoptotic cells among lung neutrophils is decreased. The transcriptional regulatory factor NF-kappaB is activated in neutrophils and other pulmonary cell populations after endotoxemia and appears to play a central role in the development of the acute inflammatory process that leads to lung injury. Because NF-kappaB can modulate apoptosis through increasing expression of anti-apoptotic proteins, activation of NF-kappaB may contribute to the alterations in lung neutrophil apoptosis associated with acute lung injury. In the present experiments, endotoxemia resulted in decreased apoptosis and increased expression of anti-apoptotic mediators among lung neutrophils. Amounts of A1, A20, and Bcl-x(L), anti-apoptotic proteins whose transcription is dependent on NF-kappaB, were increased in lung neutrophils after endotoxemia. Inhibition of nuclear translocation of NF-kappaB increased the percentage of apoptotic lung neutrophils after endotoxemia, but not back to the levels found in unmanipulated animals. Although inhibition of nuclear translocation of NF-kappaB prevented endotoxemia-induced increases in Bcl-x(L), A1, and A20 in lung neutrophils, this intervention did not prevent endotoxemia-associated elevation of Mcl-1, an anti-apoptotic protein primarily under the transcriptional regulation of CREB. These results demonstrate that mechanisms independent of NF-kappaB activation play an important role in modulating lung neutrophil apoptosis after endotoxemia.