ß-catenin negatively regulates expression of the prostaglandin transporter PGT in the normal intestinal epithelium and colorectal tumour cells: a role in the chemopreventive efficacy of aspirin?

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.

BAG-1 interacts with the p50-p50 homodimeric NF-κB complex: implications for colorectal carcinogenesis.

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.

Increased NF-kappaB DNA binding but not transcriptional activity during apoptosis induced by the COX-2-selective inhibitor NS-398 in colorectal carcinoma cells.

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.