RESUMO
As colorectal cancer remains the second highest cause of cancer-related deaths in much of the industrialised world, identifying novel strategies to prevent colorectal tumour development remains an important challenge. BAG-1 is a multi-functional protein, the expression of which is up-regulated at relatively early stages in colorectal tumorigenesis. Importantly, BAG-1 is thought to enhance colorectal tumour progression through promoting tumour cell survival. Here, we report for the first time a novel role for BAG-1, establishing it as a suppressor of transforming growth factor ß (TGF-ß1) expression in colorectal tumour cells. Microarray analysis first highlighted the possibility that BAG-1 may regulate TGF-ß1 expression, a key cytokine in normal colonic tissue homoeostasis. Q-RT-PCR and ELISA demonstrated TGFB1 mRNA and protein expression to be significantly increased when BAG1 levels were reduced by small interfering RNA; additionally, induction of BAG-1L caused suppression of TGFB1 mRNA in colorectal tumour cells. Using reporter and chromatin immunoprecipitation assays, a direct association of BAG-1 with the TGFB1 gene regulatory region was identified. Immunohistochemistry and Weiser fraction data indicated that the levels of BAG-1 and TGF-ß1 are inversely correlated in the normal colonic epithelium in vivo, consistent with a role for BAG-1-mediated repression of TGF-ß1 production. In vitro studies showed that the change in TGF-ß1 production following manipulation of BAG-1 is functionally relevant; through induction of anchorage-independent growth in TGF-ß1-dependent normal rat kidney fibroblasts and regulation of SMAD2 phosphorylation in TGF-ß1-sensitive adenoma cells. Taken together, this study identifies the anti-apoptotic protein BAG-1 as a suppressor of the inhibitory growth factor TGF-ß1, suggesting that high expression of BAG-1 can impact on a number of the hallmarks of cancer, of potential importance in promoting the early stages of colorectal tumorigenesis. Establishing BAG-1 as a repressor of TGF-ß1 has important biological implications, and highlights a new role for BAG-1 in colorectal tumorigenesis.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genética , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Fator de Crescimento Transformador beta1/metabolismoRESUMO
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.