RESUMO
BACKGROUND: Levels of the pro-tumorigenic prostaglandin PGE(2) are increased in colorectal cancer, previously attributed to increased synthesis through COX-2 upregulation and, more recently, to decreased catabolism. The functionally linked genes 15-prostaglandin dehydrogenase (15-PGDH) and the prostaglandin transporter PGT co-operate in prostaglandin degradation and are downregulated in colorectal cancer. We previously reported repression of 15-PGDH expression by the Wnt/ß-catenin pathway, commonly deregulated during early colorectal neoplasia. Here we asked whether ß-catenin also regulates PGT expression. METHODS: The effect of ß-catenin deletion in vivo was addressed by PGT immunostaining of ß-catenin(-/lox)-villin-cre-ERT2 mouse tissue. The effect of siRNA-mediated ß-catenin knockdown and dnTCF4 induction in vitro was addressed by semi-quantitative and quantitative real-time RT-PCR and immunoblotting. RESULTS: This study shows for the first time that deletion of ß-catenin in murine intestinal epithelium in vivo upregulates PGT protein, especially in the crypt epithelium. Furthermore, ß-catenin knockdown in vitro increases PGT expression in both colorectal adenoma- and carcinoma-derived cell lines, as does dnTCF4 induction in LS174T cells. CONCLUSIONS: These data suggest that ß-catenin employs a two-pronged approach to inhibiting prostaglandin turnover during colorectal neoplasia by repressing PGT expression in addition to 15-PGDH. Furthermore, our data highlight a potential mechanism that may contribute to the non-selective NSAID aspirin's chemopreventive efficacy.
Assuntos
Aspirina/farmacologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/prevenção & controle , Mucosa Intestinal/metabolismo , Transportadores de Ânions Orgânicos/biossíntese , beta Catenina/metabolismo , Animais , Anticarcinógenos/farmacologia , Linhagem Celular Tumoral , Células HCT116 , Células HT29 , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Camundongos , Transportadores de Ânions Orgânicos/genética , Transdução de Sinais , beta Catenina/genéticaRESUMO
Understanding the mechanisms that promote aberrant tumour cell survival is critical for the determination of novel strategies to combat colorectal cancer (CRC). We have recently shown that the anti-apoptotic protein BAG-1, highly expressed in pre-malignant and CRC tissue, can potentiate cell survival through regulating NF-κB transcriptional activity. In this study, we identify a novel complex between BAG-1 and the p50-p50 NF-κB homodimers, implicating BAG-1 as a co-regulator of an atypical NF-κB pathway. Importantly, the BAG-1-p50 complex was detected at gene regulatory sequences including the epidermal growth factor receptor (EGFR) and COX-2 (PTGS2) genes. Suppression of BAG-1 expression using small interfering RNA was shown to increase EGFR and suppress COX-2 expression in CRC cells. Furthermore, mouse embryonic fibroblasts derived from the NF-κB1 (p105/p50) knock-out mouse were used to demonstrate that p50 expression was required for BAG-1 to suppress EGFR expression. This was shown to be functionally relevant as attenuation of BAG-1 expression increased ligand activated phosphorylation of EGFR in CRC cells. In summary, this paper identifies a novel role for BAG-1 in modulating gene expression through interaction with the p50-p50 NF-κB complexes. Data presented led us to propose that BAG-1 can act as a selective regulator of p50-p50 NF-κB responsive genes in colorectal tumour cells, potentially important for the promotion of cell survival in the context of the fluctuating tumour microenvironment. As BAG-1 expression is increased in the developing adenoma through to metastatic lesions, understanding the function of the BAG-1-p50 NF-κB complexes may aid in identifying strategies for both the prevention and treatment of CRC.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Subunidade p50 de NF-kappa B/metabolismo , NF-kappa B/fisiologia , Fatores de Transcrição/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos , Receptores ErbB/genética , Fibroblastos , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/genética , Regiões Promotoras Genéticas , Multimerização Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , TransfecçãoRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal neoplasia, an effect that is associated with their ability to induce apoptosis. Although NSAIDs have been reported to inhibit NF-kappaB, more recent studies show activation of NF-kappaB by NSAIDs. NF-kappaB commonly shows antiapoptotic activity and is implicated in the therapeutic resistance of cancer cells. The effects of highly COX-2-selective NSAIDs such as NS-398 on NF-kappaB in colorectal tumour cells have not been reported. Therefore, we addressed whether NF-kappaB has a role in NS-398-induced apoptosis of colorectal cancer cells. Treatment of HT-29 colorectal carcinoma cells with doses of NS-398 (50-75 microM) known to induce apoptosis had no effect on NF-kappaB for up to 48 h. However after 72 and 96 h NF-kappaB DNA-binding activity was increased by NS-398, in parallel with apoptosis induction. NS-398-treated HT-29 cells showed increased p50 homodimer binding and an induction of p50/p65 heterodimers, as demonstrated by supershift assay. However, although NS-398 increased NF-kappaB DNA binding it did not increase NF-kappaB-dependent reporter activity and inhibition of NF-kappaB DNA binding did not enhance NS-398-induced apoptosis. This indicates that NF-kappaB activated by NS-398 is transcriptionally inactive and is an encouraging result for the use of COX-2-selective NSAIDs not only in chemoprevention but also as novel therapies for colon cancer.