RESUMO
BACKGROUND AND AIMS: The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer. METHODS: The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues. RESULTS: Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts. CONCLUSION: CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.
Assuntos
Apoptose/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Repressoras/fisiologia , Animais , Apoptose/efeitos dos fármacos , Caspases Efetoras/metabolismo , Sobrevivência Celular/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Terapia Genética/métodos , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas de Neoplasias/fisiologia , Transplante de Neoplasias , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Fatores de Transcrição , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Previously, we have identified the transcription factor CUTL1 as an important mediator of tumor invasion and target of tumor growth factor-beta. Using high-throughput approaches, we identified several putative downstream effectors of CUTL1, among them WNT5A, a secreted member of the Wnt multigene family. The aim of this study was to investigate the role of WNT5A as a novel target of CUTL1 in pancreatic cancer. CUTL1 and WNT5A were stably over-expressed as well as transiently and stably knocked down by RNA interference. Effects on proliferation, migration and invasiveness were investigated by thymidine incorporation, Boyden chamber experiments and time-lapse microscopy. Expression of WNT5A in pancreatic cancer tissues was analyzed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. We found that CUTL1 transcriptionally up-regulated WNT5A on RNA, protein and promoter level. WNT5A significantly enhanced migration, proliferation and invasiveness, mediating the pro-invasive effects of CUTL1 to a major extent. WNT5A effects were accompanied by a marked modulation of marker genes associated with epithelial-mesenchymal transition. Using RT-PCR and immunohistochemistry, we found that WNT5A is up-regulated early during pancreatic cancerogenesis in pancreatic intraepithelial neoplasias lesions and in invasive pancreatic adenocarcinomas, as compared with normal pancreas tissues. These data identify WNT5A as important target of CUTL1 and as novel mediator of invasiveness and tumor progression in pancreatic cancer.