Skip Navigation


Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(4):849-857; doi:10.1093/carcin/bgn004
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
29/4/849    most recent
bgn004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Clemo, N. K.
Right arrow Articles by Williams, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Clemo, N. K.
Right arrow Articles by Williams, A. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-{kappa}B activity

Nadine K. Clemo, Tracey J. Collard, Samantha L. Southern, Kieron D. Edwards1, Moganaden Moorghen2, Graham Packham3, Angela Hague4, Christos Paraskeva and Ann C. Williams*

Cancer Research UK Colorectal Tumour Biology Research Group, Department of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK
1 Institute of Molecular Plant Sciences, University of Edinburgh EH9 3JH, Edinburgh, UK
2 Department of Pathology and Histology, Bristol Royal Infirmary, Bristol BS2 8HW, UK
3 Cancer Sciences Division, Cancer Research UK Clinical Centre, University of Southampton SO16 6YD, UK
4 Department of Oral and Dental Science, University of Bristol, Bristol BS1 2LY, UK

* To whom correspondence should be addressed. Tel: 0117 3312070; Fax: 0117 9287896;Email: Ann.C.Williams{at}bristol.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Although expression of the anti-apoptotic protein Bcl-2-associated athanogene-1 (BAG-1) has been reported as up-regulated in a number of malignancies, we show for the first time that BAG-1 is over-expressed in medium/large-sized colorectal adenomas and carcinomas compared with normal epithelium. To investigate whether expression of BAG-1 is important for colorectal tumour cell survival, microarray analysis was carried out on the HCT116 colorectal carcinoma cell line following transfection with BAG-1 small interfering RNA (siRNA). Analysis identified altered expression of a subset of potential nuclear factor-{kappa}B (NF-{kappa}B)-regulated genes. Furthermore, knock down of BAG-1 was shown to inhibit NF-{kappa}B transcriptional activity. Inhibition of NF-{kappa}B activity using BAG-1 siRNA or the NF-{kappa}B inhibitor BAY-117082 suppressed HCT116 cell yield and induced apoptosis; combined treatment had no additive effect, suggesting that the decrease in cell yield associated with knock down of BAG-1 expression is mediated via inhibition of NF-{kappa}B. Of clinical relevance, BAG-1 siRNA sensitized colorectal carcinoma cells to apoptosis induced by potential therapeutic agent TRAIL as well as tumour necrosis factor-{alpha}, both inducers of NF-{kappa}B activity. In summary, knock down of BAG-1 leads to inhibition of NF-{kappa}B, identifying BAG-1 as a novel regulator of NF-{kappa}B. It is proposed that, by inhibiting NF-{kappa}B, suppression of BAG-1 could represent a novel strategy to impede colorectal cancer cell survival and as an adjuvant increase sensitivity to current therapeutic regimes.

Abbreviations: BAG-1, Bcl-2-associated athanogene-1; FBS, fetal bovine serum; NEG, negative; NF-{kappa}B, nuclear factor-{kappa}B; Q-PCR, quantitative polymerase chain reaction; siRNA, small interfering RNA; TNF{alpha}, tumour necrosis factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Since its discovery, the protein Bcl-2-associated athanogene-1 (BAG-1) has been found to interact with a wide variety of molecular targets and, consequently, has been shown to regulate a number of key processes such as proliferation, cell signalling, transcription, differentiation and apoptosis (reviewed in ref. 1).

BAG-1 belongs to a family of six proteins, which all contain an evolutionary conserved BAG domain (reviewed in ref. 1). In humans, there are three major isoforms of BAG-1, generated through alternate translation initiation of a single messenger RNA (2–4). The different BAG-1 isoforms have distinct subcellular localizations within the cell. The most abundant isoform, BAG-1S (~36 kDa), is preferentially localized in the cytoplasm, whereas BAG-1L (the largest isoform, ~50 kDa) contains a nuclear localization signal and is generally restricted to the nucleus. A 46 kDa isoform, BAG-1M, partitions itself between the nucleus and the cytoplasm (2–4). Functional studies of BAG-1 have mainly focused on its anti-apoptotic role. For example, over-expression in tumour cell lines has revealed that BAG-1 confers resistance to multiple stresses including cytotoxic drugs, hypoxia, heat shock, growth factor withdrawal and UV/{gamma}-radiation (5–10). Surprisingly, given the importance of BAG-1 in a number of tumour types, its actual role in tumorigenesis is yet to be clarified.

Nuclear factor-{kappa}B (NF-{kappa}B) is a widely known transcription factor, inducing expression of a number of genes involved in cell survival. The term NF-{kappa}B describes a number of ubiquitous transcription factor complexes formed by the NF-{kappa}B/Rel gene family, which include RelA (p65), RelB, c-Rel, NF-{kappa}B1 (p105) and NF-{kappa}B2 (p100). The members of the family are distinguished by a Rel homology domain, which is the region responsible for DNA binding and dimerization (reviewed in ref. 11). In resting cells, NF-{kappa}B is normally sequestered in the cytoplasm by its inhibitor I{kappa}B and is only activated upon stimulation with agents such as tumour necrosis factor (TNF{alpha}), interleukin-1 or bacterial lipopolysaccharide. Upon stimulation, I{kappa}B is phosphorylated, ubiquitinated and consequently degraded by the proteasome. The loss of I{kappa}B releases NF-{kappa}B, allowing transactivation of target genes (reviewed in ref. 11).

Although NF-{kappa}B is primarily known for its role in inflammatory and immune responses, current evidence clearly indicates that activation of the NF-{kappa}B-signalling pathway is also important for tumorigenesis. For example, chromosomal amplification of genes coding for NF-{kappa}B/Rel factors and mutations that inactivate I{kappa}B or constitutively activate upstream signalling kinases have been observed in many haematopoietic and solid tumours (reviewed in ref. 12). Indeed, NF-{kappa}B has been shown to be up-regulated in human colon cancer cell lines and tumour tissue (13–15). Interestingly, the inflammatory diseases ulcerative colitis and Crohn’s disease, which increase the risk of colorectal cancer, have also been associated with persistent NF-{kappa}B activation in epithelial cells and tissue macrophages of the colonic mucosa (16–18).

The main role of NF-{kappa}B signalling in tumorigenesis appears to prevent cell death. To determine the mechanism for this observation, research has shown that TNF-{alpha}-induced NF-{kappa}B activity is associated with transcription of anti-apoptotic genes such as Bcl-xL, A1, c-FLIP, c-IAP1, c-IAP2, x-IAP, TRAF1 and TRAF2 (reviewed in ref. 11). Highly relevant to tumour development, NF-{kappa}B activation is also known to induce expression of cyclin D1 and cyclooxygenase 2, both which increase cell growth (19–21).

The expression of BAG-1 has been studied in a wide range of cancers and accordingly, BAG-1 has been reported to be over-expressed in a number of different tumour types in vivo including breast (22,23), cervical (24), oral squamous cell (25), laryngeal (26) and endometrium (27), as well as tumour cell lines in vitro including leukaemia, breast, prostate, lung and cervical (3,4). Interestingly, high BAG-1 expression has been reported in colon cancer cell lines in vitro (3) and additionally, nuclear BAG-1 expression in colorectal cancer in vivo has been associated with distant metastasis and a shorter overall survival (28). However, the level of expression of BAG-1 during colorectal tumorigenesis relative to the normal colonic epithelium has not been determined; therefore, it remains to be established whether the expression of BAG-1 protein is deregulated during colorectal tumour progression. Since BAG-1 is involved in the regulation of cell survival and proliferation, its altered expression in human cancers may have huge clinical significance and countering BAG-1 function may be a particularly effective anticancer strategy. However, unlike NF-{kappa}B, the BAG-1 anti-apoptotic signalling mechanism is poorly understood and the role of BAG-1 in tumorigenesis is still to be elucidated.

Here, it is reported for the first time that, although unchanged in small adenomas, BAG-1 expression is up-regulated in medium/large-sized adenomas and carcinomas compared with the normal colonic epithelium. Importantly, the level of expression of BAG-1 in the large adenomas was equivalent to that in the carcinomas, suggesting that expression of the pro-survival protein BAG-1 may be important in colorectal tumorigenesis. Indeed, microarray analysis of the HCT116 colorectal cancer cell line transfected with BAG-1 small interfering RNA (siRNA) revealed that suppression of BAG-1 leads to differential expression of a number of genes involved in cell survival and, interestingly, identified altered expression of a small subset of potential NF-{kappa}B-regulated genes. Data presented here further suggest that suppression of BAG-1 expression in HCT116 cells leads to a decrease in cell survival mediated via inhibition of NF-{kappa}B. Knock down of BAG-1 using siRNA also sensitized colorectal carcinoma cells to apoptosis induced by TRAIL and TNF-{alpha}, both known inducers of NF-{kappa}B. In summary, in this report it is shown for the first time that BAG-1 expression is up-regulated during colorectal tumour progression, and through regulating the key transcription factor NF-{kappa}B, BAG-1 promotes the survival of colorectal tumour cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Immunohistochemistry
Samples of formalin-fixed, paraffin-embedded colonic normal, adenoma and carcinoma tissue were obtained from the archives of the Department of Histopathology, Bristol Royal Infirmary, UK, with local Ethics Committee approval. Four micrometer thick sections were deparaffinized, microwaved in citrate buffer (pH 6) for 20 min (antigen retrieval) and incubated with BAG-1 TB3 rabbit polyclonal antibody (29) at a dilution of 1:1000 for 2 h at room temperature. The reaction products were visualized using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA), treated with 3,3'-Diaminobenzidine (DAKO, Ely, UK) as substrate and fixed with enhancer solution. Negative (NEG) controls without the primary antibody were also prepared. Sections were graded as strongly positive (+++), positive (++), weakly positive (+) or background staining (–/+) [similar to a previously used scoring system (30)]. The slides were scored by three independent observers. To verify the specificity of the antibody used, SW480 colorectal carcinoma cells (which had been shown to express BAG-1 protein by western blotting) were transfected with either NEG or BAG-1 siRNA sequence #2 (refer below) and suppression of BAG-1 protein expression verified by western blotting. These cells (NEG or BAG-1 siRNA) were embedded in 1% agar and processed by the standard method for tissue into wax, incubated with BAG-1 TB3 antibody at a dilution of 1:1000 and stained using the same method as described above.

Cell line and cell culture conditions
The human colon carcinoma cell lines HCT116 and SW480 were obtained from the American Type Culture Collection (Rockville, MD). HCT116 cells were maintained on McCoy’s 5A medium (Invitrogen, Paisley, UK) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 10% fetal bovine serum (FBS). SW480 cells were maintained on Dulbecco’s modified Eagle’s medium (Life Technologies, Autogen Bioclear, Calne, UK) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 10% FBS.

siRNA transfection
BAG-1-duplexed siRNA oligonucleotides were obtained from Ambion (TX). The following sequences were used: sequence #1 GGUUGUUGAAGAGGUCAUAtt and sequence #2 GGGAAAAUCUCUGAAGGAAtt. The NEG control siRNA (Ambion Applied Biosystems, Foster City, CA, USA) had no homology to any sequence in the human or mouse genome. Cells were seeded and grown under standard conditions in either T12.5 (Falcon, BD, BD Bioscience Europe, Oxford, UK) or T25 flasks (Corning, Corning Incorporated, Corning, NY, USA) until ~40% confluent. The cells were incubated in Opti-MEM (Invitrogen), exposed to duplexed siRNA oligonucleotides at a final concentration of 50 nM in the presence of Lipofectamine 2000 (Invitrogen) for 6 h as per the manufacturer’s instructions and then returned to normal growth medium. To determine the optimum conditions for BAG-1 down-regulation using siRNA, cells were initially transfected with 0–100 nM of BAG-1 siRNA sequence #1 and BAG-1 siRNA sequence #2 for 0, 24, 48, 72 and 96 h as recommended by Ambion. Controls used in these experiments included the nil (no transfection reagent and no siRNA), mock (no siRNA), NEG siRNA and positive siRNA (Ambion GAPDH siRNA sequence; positive control for the protocol). The down-regulation of BAG-1 was determined by western blot or quantitative polymerase chain reaction (Q-PCR), respectively. It was found that a significant reduction in BAG-1 expression could be achieved for up to 7 days after transfection. Subsequently, cells were transfected with 50 nM BAG-1 siRNA (either sequence) for 24, 48, 72 or 96 h depending on the assay.

Assessment of cell survival
At the time point indicated, the attached epithelial monolayer of cells was trypsinized and the number of cells counted using a Neubauer counting chamber (Weber VWR International, Poole, Dorset, UK). The induction of apoptosis was measured by determining the proportion of total cells that had detached from the epithelial monolayer and that were shed into the medium. Biochemical confirmation of apoptosis was obtained by the presence of cleaved Poly ADP ribose polymerase (PARP) in the shed cell population by western blotting and characteristic apoptotic morphology as demonstrated by acridine orange staining (described in detail previously in ref. 31).

Treatments
Cells were seeded in triplicate flasks and transfected with BAG-1 or NEG siRNA as described above. 72 hours after transfection, cells were treated with either 0.10 µg/ml TRAIL or 0.10 µg/ml TNF-{alpha} (Autogen Bioclear, Calne, Wiltshire, UK) in 10% FBS McCoy’s 5A medium for 16 h. The control flasks received 10% FBS McCoy’s 5A medium alone. For the BAY-117082 experiment, 24 h after siRNA transfection, cells were treated with 2 µM of BAY-117082 (32) or dimethyl sulfoxide vehicle control in 10% FBS McCoy’s 5A medium for 96 h. In addition, cells were pre-treated with 2 µM BAY-117082 for 24 h and then treated with either 0.10 µg/ml TRAIL or 0.10 µg/ml TNF-{alpha} for 16 h as above.

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis western blotting
Western blotting was carried out using standard techniques as described previously (33). Antibodies against BAG-1 (G3E2) (29) and PARP (Alexis, Nottingham, UK) were used to detect protein expression. A mouse monoclonal {alpha}-tubulin antibody (Sigma, Dorset, UK) was used to assess gel loading.

Microarray analysis
Cells were transfected with siRNA as described above. After 48 h, total RNA was extracted using Tri Reagent (Sigma), chloroform and isopropanol precipitation. All RNA was DNaseI treated (Ambion) prior to use. Microarray analysis was performed using Amersham CodeLink Bioarrays (Amersham, Buckinghamshire, UK) with 20 000 probes. The bioarrays were scanned by Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA) according to the manufacturer’s instructions. Duplicate arrays were carried out for each siRNA condition. Background correction, normalization and gene expression analysis of the array data were performed using GeneSpring version 7.2 (Agilent, Agilent Technologies, Santa Clara, CA).

Quantitative reverse transcription–PCR
RNA was harvested as for the microarray analysis and, following DNaseI treatment, cDNA samples for Q-PCR applications were reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI). Transcript abundance was assessed by Q-PCR in a Stratagene MX3000P cycler using JumpStart SYBR Green PCR Master Mix (Sigma). Transcript levels were normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers for BAG-1, RIN1, GADD45β, C-FLIP and GAPDH were obtained from QuantiTect Primer Assays (Qiagen, Crawley, West Sussex, UK).

NF-{kappa}B reporter assays
Cells were treated with 2 µM BAY-117082 for 24 h or transfected with siRNA as described above. 48 hours after transfection, flasks were co-transfected with either pTA-luc or pNF-{kappa}B-TA-luc reporter constructs (2.5 µg per flask; Clontech, BD Bioscience Europe, Oxford, UK) in combination with the renilla construct pRL-SV40 (0.25 µg per flask; Promega) in Opti-MEM in the presence of Lipofectamine 2000 as described previously. Twenty-four hours later, the cells were treated with 0.10 µg/ml TNF-{alpha} as described above and lysed in passive lysis buffer (Promega) after 16 h according to the manufacturer’s instructions. Reporter activity was measured using the Dual-Luciferase Reporter Assay system (Promega) and a Jade Luminometer (Labtech, Labtech International Ltd, Ringmer, East Sussex, UK). Sample readings were corrected for background autoluminescence using untransfected cells as a control.

Statistical analysis
A Student’s t-test was performed to analyse all data except for the reporter experiment in Figure 4, where a Tukey’s test was used to calculate the significance of BAG-1 expression or BAY-117082 on cell survival.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Expression of the BAG-1 protein is up-regulated in colorectal medium and large-sized adenomas as well as in carcinomas
To determine whether BAG-1 expression is deregulated during colorectal tumour progression, the expression of BAG-1 protein in small (<5 mm), medium (5–10 mm) and large (>10 mm) colorectal adenomas and carcinomas was compared with expression in the normal colonic epithelium (Figure 1a). The specificity of the BAG-1 TB3 antibody, which detects all three BAG-1 isoforms (29,34), was further confirmed using SW480 colorectal carcinoma cells (known to express BAG-1) transfected with BAG-1 siRNA that had undergone the same processing as the tissue samples (Figure 1c). It should be emphasized that knock down of BAG-1 using siRNA results in lower levels of BAG-1 expression as shown by western blotting (Figure 1b) and immunohistochemistry (Figure 1c), rather than complete elimination of BAG-1 protein expression.


Figure 1
View larger version (100K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Expression of the BAG-1 protein is up-regulated in colorectal medium-and large-sized adenomas as well as in carcinomas. (a) BAG-1 protein expression (brown staining) in formalin-fixed, paraffin-embedded normal colon, small adenoma (0–5 mm), medium adenoma (5–10 mm), large adenoma (>10 mm) and carcinoma tissue; objective x10 negative. NEG control is normal colonic epithelium tissue without primary BAG-1 antibody; objective x20. (b) Western blot showing reduced BAG-1 protein level in SW480 cells transfected with 50 nM of BAG-1 siRNA for 72 h compared with cells transfected with 50 nM of NEG siRNA. (c) BAG-1 protein expression (brown staining) in SW480 cells transfected with 50 nM of NEG or BAG-1 siRNA sequence #2 for 72 h. Cells were fixed in agar, paraffin embedded and incubated with a BAG-1 antibody using a similar method as for tissue specimens; objective x10. (d) Insert showing {alpha}-tubulin staining as control to show no generalized suppression of protein in the transfected cells. (e) Scoring for BAG-1 protein expressed as percentage of the total number of samples for each tissue type. –/+, background; +, weakly positive; ++, positive and +++, strongly positive. The samples were scored by three independent observers. (f) Summary of BAG-1 protein expression in colorectal normal, adenoma and carcinoma tissue. Scoring as above.

 
Each tissue sample was assessed for total expression of BAG-1 using a scoring system outlined in the Materials and Methods. Results are summarized in Figure 1e and f. Interestingly, in normal human colonic tissue, BAG-1 staining changes along the colonic crypt axis (Figure 1a); there is more BAG-1 staining in the cytoplasm from the middle to the top of the crypt, similar to that reported in mouse colon tissue (3). It was also noted that there was a striking increase in BAG-1 expression in the colorectal carcinoma tissue when compared with the normal colonic epithelium (Figure 1a and e). This observation has not previously been reported and is significant as it suggests that increased BAG-1 expression may be important in colorectal carcinogenesis. To determine whether BAG-1 is implicated in the development of the tumour (in the progression from adenoma to carcinoma), we investigated BAG-1 expression in adenomas of increasing size (Figure 1a, e and f). Of note, previously such an approach has been used to implicate RAS mutations and cyclooxygenase 2 expression in the development of colorectal tumours (35,36). In the normal colon and the small adenoma, the intensity of BAG-1 staining was low with ~80% of samples having either background (–/+) or weakly positive (+) staining (Figure 1a and e). In contrast, the staining intensity was much higher in the medium adenoma; 55% of samples being positive (++) and 32% of samples being strongly positive (+++) (Figure 1a and e). The high level of BAG-1 expression is maintained in the large adenomas, with 70% of samples staining either positive (++) or strongly positive (+++) (Figure 1a and e), and is similar to that in the carcinomas where 94% of samples stain either positive (++) or strongly positive (+++) (Figure 1a and e). These findings demonstrate for the first time that there is a dramatic increase of BAG-1 expression in colorectal carcinomas when compared with the normal colonic epithelium and that this increase in expression occurs in the medium and large-sized adenomas as well as in carcinomas. As increased expression is detected in the adenoma to carcinoma sequence, our findings suggest a role for BAG-1 in the development of colorectal cancer.

Differential gene expression in HCT116 colorectal carcinoma cells transfected with BAG-1 siRNA
As BAG-1 has been implicated to have a role in colorectal tumour progression, the function of BAG-1 in colorectal carcinogenesis was investigated. BAG-1 expression was suppressed in colorectal carcinoma cells using BAG-1 siRNA sequence #2 (Figure 2a and b; siRNA controls described in Materials and Methods) and differential gene expression assessed using Amersham CodeLink BioArrays, which contained probes for ~20 000 well-characterized human genes. Data generated from the microarray was analysed using GeneSpring version 7.2 (Agilent; refer to Materials and Methods). Gene expression analysis revealed that 750 genes were differentially regulated at least 1.5-fold by BAG-1 knockdown (data not shown).


Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. BAG-1 expression in HCT116 cells transfected with BAG-1 siRNA. (a) Western blot showing reduced BAG-1 protein level in HCT116 cells transfected with 50 nM of BAG-1 siRNA sequence #2 for 48 h compared with cells transfected with 50 nM of NEG siRNA. (b) Q-PCR analysis shows that BAG-1 messenger RNA abundance is reduced following 48 h of transfection with 50 nM of BAG-1 siRNA sequence #2. BAG-1 messenger RNA abundance is presented as a fold of the NEG siRNA control. All values were normalized to the housekeeping gene GAPDH. All data points show the mean result of three separate experiments (±SD).

 
Although potentially regulated by many different transcription factors, it was noted that a number of genes identified as suppressed by BAG-1 siRNA contain a putative NF-{kappa}B consensus binding sequence (Table I). Q-PCR was used to validate the microarray data for two of these genes that have previously been reported to be regulated by NF-{kappa}B; GADD45β, which is induced in response to cell stress (37), and C-FLIP, an inhibitor of caspase-8 (reviewed in ref. 38). Q-PCR confirmed that GADD45β and C-FLIP expressions were down-regulated in cells transfected with the BAG-1 siRNA (Figure 3a and b). RIN1, which encodes a Ras effector (39), was included as a control for the Q-PCR since it is representative of genes identified as up-regulated on the microarray following transfection with BAG-1 siRNA (Figure 3).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Differential gene expression in HCT116 colorectal carcinoma cells transfected with BAG-1 siRNA. To verify the microarray data, Q-PCR was used to assess the messenger RNA abundance of different genes following 48 h after transfection with 50 nM of NEG or BAG-1 siRNA sequence #2. (a) Microarray fold changes of genes in HCT116 cells transfected with BAG-1 siRNA that were validated by Q-PCR. (b) messenger RNA abundance is presented as a fold of the NEG siRNA control. All values were normalized to the housekeeping gene GAPDH. RIN1 was used as a control gene that was up-regulated by BAG-1. All data points show the mean result of three separate experiments (±SD).

 


View this table:
[in this window]
[in a new window]

  		Table I. Genes from the microarray that contain a putative NF-{kappa}B consensus binding sequence (5'-GGGRNNYYCC-3'; where R indicates A or G, Y indicates C or T and N indicates any nucleotide) within 1500 bp upstream of a translation start site

 
BAG-1 regulates NF-{kappa}B activity in colorectal carcinoma cells
Since knock down of BAG-1 in colorectal cancer cells suppressed expression of two pro-survival genes regulated by NF-{kappa}B, and colorectal tumour progression is associated both with increased BAG-1 (shown in this study) and NF-{kappa}B expression in vivo (13–15), it was hypothesized that BAG-1 may promote tumour cell survival through regulation of the NF-{kappa}B pathway. Therefore, the effect of BAG-1 expression on NF-{kappa}B transcriptional activity was investigated using an NF-{kappa}B luciferase reporter assay. HCT116 cells were transfected with BAG-1 siRNA (Figure 4a) and basal NF-{kappa}B activity compared with that in the NEG siRNA control cells (Figure 4b). Although the basal level of NF-{kappa}B activity in HCT116 cells is low, NF-{kappa}B activity was found to be significantly suppressed in the BAG-1 siRNA-transfected cells.


Figure 4
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Knock down of BAG-1 using siRNA inhibits NF-{kappa}B activity, decreases cell yield and increases apoptosis in colorectal carcinoma cells. (a) Western blot analysis confirms that the level of BAG-1 protein was reduced in HCT116 cells transfected with 50 nM of BAG-1 siRNA sequence #2. (b) BAG-1 siRNA suppresses basal levels of NF-{kappa}B activity in HCT116 colorectal carcinoma cells in a luciferase assay. HCT116 cells transfected with NEG or BAG-1 siRNA sequence #2 were subsequently transiently transfected with an NF-{kappa}B luciferase reporter plasmid. (c) Basal levels of NF-{kappa}B activity in HCT116 cells suppressed by treatment with 2 µM BAY-117082 for 24 h. Luciferase activity is expressed as a ratio of firefly:renilla luciferase (the range of readings for the NF-{kappa}B-driven reporter was 817–974 for the NEG and 512–662 for the BAG-1 siRNA-transfected cells). The pTA-luc control was <0.1 for all readings. All data points show the mean result of three separate experiments (±SD). Statistical analysis was carried out using a Student’s t-test (*P < 0.05). (d–g) Decreased cell survival in the BAG-1 siRNA-transfected cells is equivalent to the reduction in cell survival by inhibiting NF-{kappa}B activity using the BAY-117082 inhibitor. HCT116 cells were transfected with 50 nM BAG-1 siRNA sequence #2 [dimethyl sulfoxide (DMSO) + BAG-1], 50 nM NEG siRNA + 2 µM BAY-117082 (+BAY + NEG) or 50 nM BAG-1 siRNA + 2 µM BAY-117082 (+BAY + BAG-1) and cell yield compared with that of cells transfected with 50 nM NEG siRNA [dimethyl sulfoxide (DMSO) + NEG]. (d) Western blot analysis showing the BAG-1 protein levels in the NEG or BAG-1 siRNA-transfected cells. (e) Following treatment with BAY-117082, at the times indicated, the attached cell yield (%) was assessed. Attached cell yield is represented as a percentage of the NEG siRNA control [dimethyl sulfoxide (DMSO) + NEG]. All data points show the mean result of three separate experiments (±SD). Statistical analysis was carried out using a Tukey’s post hoc test (***P < 0.001). (f) Western blot of PARP cleavage confirmed apoptosis in the NEG and BAG-1 siRNA-transfected shed cells, with or without 2 µM BAY-117082 treatment. (g) Following treatment with BAY-117082, at the times indicated, apoptosis (%) was assessed. All data points show the mean result of three separate experiments (±SD). Statistical analysis was carried out using a Tukey’s post hoc test (*P < 0.05; **P < 0.01).

 
Knock down of BAG-1 using siRNA decreases cell yield and increases apoptosis in colorectal carcinoma cells
The finding that decreased BAG-1 expression inhibited NF-{kappa}B transcriptional activity, and the fact that NF-{kappa}B has previously been reported to promote colorectal tumour cell survival (reviewed in ref. 40), led us to investigate whether knock down of BAG-1 expression inhibited colorectal tumour cell survival and if this was mediated through NF-{kappa}B. HCT116 cells were transfected with either NEG or BAG-1 siRNA alone or additionally treated with the synthetic NF-{kappa}B inhibitor BAY-117082 (32) (2 µM BAY-117082 was confirmed to inhibit NF-{kappa}B in these cells to a similar basal level as the BAG-1 siRNA; Figure 4c) and the attached cell yield measured >96 h. Western blotting confirmed that BAG-1 was successfully knocked down using siRNA for up to 96 h (Figure 4d). Results show that knock down of BAG-1 using siRNA significantly reduced the attached cell yield after 48, 72 and 96 h of treatment when compared with cells transfected with NEG siRNA alone (Figure 4e). Of note, a similar effect on the yield of HCT116 cells was obtained using BAG-1 siRNA sequence #1, and the reduction in attached cell yield was also observed using either siRNA sequence in the SW480 colorectal carcinoma cell line (data not shown). Interestingly, treatment of NEG siRNA-transfected cells with BAY-117082 resulted in a significant decrease of cell yield, which was strikingly similar to the effect of the BAG-1 siRNA (Figure 4e). In addition, when the BAG-1 siRNA-transfected cells were treated with the BAY-117082 inhibitor, there was no additive effect on cell survival; the combined treatment caused a similar decrease in cell yield (Figure 4e). Both treatment with BAG-1 siRNA or NF-{kappa}B inhibitor also resulted in the induction of apoptosis, as confirmed by the presence of cleaved PARP (Figure 4f and g). Importantly, the induction of apoptosis was not further increased by treatment of the BAG-1 siRNA-transfected cells with the NF-{kappa}B inhibitor BAY-117082 (Figure 4g). This finding is consistent with the hypothesis that the decrease in survival observed in cells transfected with BAG-1 siRNA is due to inhibition of NF-{kappa}B. In conclusion, these data raise the possibility that knock down of BAG-1 expression inhibits the survival of colorectal carcinoma cells via inhibition of NF-{kappa}B survival pathway.

BAG-1 siRNA increases the sensitivity of HCT116 cells to TNF-{alpha}- and TRAIL-induced apoptosis
As activation of NF-{kappa}B is associated with resistance to apoptosis (reviewed in ref. 11), we investigated whether suppression of BAG-1 sensitized colorectal tumour cells to death-inducing agents whose efficacy is attenuated by NF-{kappa}B activation. Initially, cells were treated with TNF-{alpha} [a known NF-{kappa}B activator (41)] to enable the effect of reduced BAG-1 expression on activated NF-{kappa}B function to be investigated. Interestingly, reduced BAG-1 expression halved TNF-{alpha}-induced activity of NF-{kappa}B when compared with the NEG siRNA control (Figure 5a and c). This effect was also demonstrated in another colorectal carcinoma cell line (SW480, data not shown). To confirm that reduced expression of BAG-1 inhibited transcriptional activity of NF-{kappa}B, Q-PCR was used to assess the expression of NF-{kappa}B targets, GADD45β and C-FLIP following TNF-{alpha} treatment (Figure 5b). The expression of both GADD45β and C-FLIP was significantly decreased in response to TNF-{alpha} treatment in the BAG-1 siRNA-transfected cells compared with the NEG siRNA cells (Figure 5b). Of note, although BAG-1 siRNA suppressed TNF-{alpha}-induced NF-{kappa}B activity in HCT116 cells by 50%, the messenger RNA level of NF-{kappa}B target genes C-FLIP and GADD45β was only inhibited by 20 and 35%, respectively; consistent with previous reports that the relationship between the activity of a transcription factor and its target genes is not linear (reviewed in ref. 42). In addition, although the colorectal tumour cells already express endogenous BAG-1, we were able to demonstrate that further over-expression of the BAG-1S or BAG-1L isoforms promoted TNF{alpha}-induced NF-{kappa}B activity (Figure 5c).


Figure 5
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Knock down of BAG-1 increases the sensitivity of HCT116 cells to TNF-{alpha}- and TRAIL-induced apoptosis. (a) BAG-1 siRNA inhibits NF-{kappa}B activity induced by TNF-{alpha}. HCT116 cells transfected with either 50 nM NEG or BAG-1 siRNA sequence #2 were subsequently transfected with an NF-{kappa}B reporter plasmid and treated with 0.10 µg/ml TNF-{alpha} for 16 h. Luciferase activity was expressed as a ratio of firefly:renilla luciferase (the range of readings for the NF-{kappa}B driven reporter was 135–274 for the control untreated NEG siRNA, 816–1255 for the TNF-{alpha}-treated NEG siRNA, 138–179 for the control untreated BAG-1 siRNA and 536–655 for the TNF-{alpha}-treated BAG-1 siRNA-transfected cells. The pTA-luc control was <0.1 for all readings. All data points show the mean result of three separate experiments (±SD). Data were analysed by a Student’s t-test (***P<0.001). (b) Q-PCR was used to assess the messenger RNA abundance of GADD45β and C-FLIP in HCT116 cells transfected with 50 nM of either NEG or BAG-1 siRNA sequence #2 and treated with 0.10 µg/ml TNF-{alpha} for 16 h. messenger RNA abundance is presented relative to the NEG siRNA-transfected cells following treatment with TNF-{alpha} (set as 1.0). All values were normalized to the housekeeping gene GAPDH. All data points show the mean result of three separate experiments (±SD). Statistical analysis was carried out using a Student’s t-test (*P< 0.05;**P < 0.01). (c) BAG-1 siRNA inhibits NF-{kappa}B activity induced by TNF-{alpha} whereas over-expression of BAG-1 increases TNF-{alpha}-induced NF-{kappa}B activity. Inhibition of TNF-{alpha}-induced NF-{kappa}B activity in HCT116 cells transfected with 50 nM BAG-1 siRNA sequence #2 is represented as a fold change of TNF{alpha}-treated cells transfected with 50 nM NEG siRNA (NEG). Also shown is TNF-{alpha}-induced NF-{kappa}B activity in HCT116 cells over-expressing BAG-1S and BAG-1L. Results are represented as fold change of the treated vector control cell line (vector). All cells were transfected with an NF-{kappa}B reporter plasmid and treated with 0.10 µg/ml TNF-{alpha} for 16 h. HCT116 cells transfected with 50 nM of either NEG or BAG-1 siRNA sequence #2 for 48 h or pre-treated with solvent control [dimethyl sulfoxide (DMSO)] or 2 µM BAY-117082 for 24 h were then further treated with either (d) 0.10 µg/ml TNF-{alpha} or (e) 0.10 µg/ml TRAIL for 16 h and apoptosis (%) assessed. All data points show the mean result of three separate experiments (±SD). Data were analysed using Student’s t-test (*P < 0.05; **P < 0.01). Protein was extracted from cells treated with either (f) 0.10 µg/ml TNF-{alpha} or (g) 0.10 µg/ml TRAIL and analysed for BAG-1 protein levels using western blotting.

 
To determine whether the inhibition of NF-{kappa}B activity in cells with reduced BAG-1 expression increased the sensitivity of the cells to the induction of apoptosis, the effect of BAG-1 knockdown on death receptor-mediated apoptosis was investigated. As stated previously, NF-{kappa}B activation has been reported to attenuate both TNF-{alpha}- and TRAIL-induced apoptosis (43,44). Since BAG-1 knockdown inhibited NF-{kappa}B activity, it was hypothesized that BAG-1 siRNA would sensitize cells to TNF-{alpha}- and TRAIL-induced apoptosis. As predicted, knock down of BAG-1 significantly sensitized HCT116 cells to both TNF-{alpha}- and TRAIL-induced apoptosis similar to that resulting from treatment with the NF-{kappa}B inhibitor BAY-117082 (2 µM) (Figure 5d and e). These data are consistent with a previous report showing that over-expression of BAG-1 attenuates TNF-{alpha}-induced apoptosis in endothelial cells (45). The expression of BAG-1 did not alter following treatment with either TNF-{alpha} or TRAIL (Figure 5f and g). As activation of NF-{kappa}B is associated with resistance to therapy, these data suggest that inhibition of BAG-1 expression in both medium/large-sized adenomas and carcinomas would suppress NF-{kappa}B activity and hence increase the sensitivity of tumour cells to therapeutic agents.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Despite having been reported as associated with a poor prognosis and distant metastasis in colorectal cancer, surprisingly little is known about the role of BAG-1 in colorectal tumorigenesis. In this report, we have shown that there is significantly higher expression of BAG-1 in colorectal carcinoma tissue than in the normal colonic epithelium, demonstrating that BAG-1 expression is deregulated in colorectal carcinogenesis. Although BAG-1 has previously been reported to be over-expressed in colon cancer cell lines (3), the current study is the first to show that the expression of BAG-1 is up-regulated in colorectal carcinoma tissue in vivo compared with the normal colonic epithelium. Furthermore, we have established that increased expression occurs before acquisition of the invasive phenotype; BAG-1 is highly expressed in medium/large-sized adenoma tissue and retained in the carcinoma. This suggests that, although not required for the formation of the adenoma, increased BAG-1 expression is important for further growth and progression of the tumour. In addition, as increased BAG-1 expression is retained in the carcinoma, continued expression may be necessary for survival of the cancer cells. Indeed, previous reports have shown that over-expression of BAG-1 in different tumour cell types confers growth and/or survival in in vitro tumour cell lines and in vivo tumour xenograft models (reviewed in ref. 1,9,10). Consistent with such data, in this report we have shown that knock down of BAG-1 expression suppressed the attached cell yield and increased apoptosis in colorectal carcinoma-derived cells, establishing a pro-survival function for BAG-1 in colorectal cancer cells. Interestingly, as BAG-1 is over-expressed in medium- and large-sized adenomas, it raises the possibility that expression of BAG-1 could be important for cell survival during tumour progression and that BAG-1 may therefore be a novel target for chemoprevention as well as cancer therapy.

Data presented here shows that expression of BAG-1 regulates the activity of the pro-survival transcription factor NF-{kappa}B in colorectal epithelial cells. NF-{kappa}B is present throughout the colonic crypt and, importantly, has been shown to be constitutively active/over-expressed in colorectal cancer (13–15). Furthermore, persistent activation of NF-{kappa}B has been reported in macrophages and epithelial cells from colonic mucosal inflammatory tissue (colitis) (16,17), a condition associated with a 10-fold greater risk of colorectal cancer than in the general Western population (reviewed in ref. 46). Recently, experimental data has begun to support such correlative evidence for the association between NF-{kappa}B signalling, inflammation and tumorigenesis. For example, it was shown in the mouse model of colitis-associated cancer that selective inhibition of IKKβ within colonic enterocytes resulted in a significant decrease in the number of tumours (47). Therefore, given the evidence that both NF-{kappa}B and BAG-1 are over-expressed in colorectal tumorigenesis, the finding that BAG-1 regulates the activity of NF-{kappa}B suggests a novel mechanism of tumour promotion whereby high levels of BAG-1 expression in colorectal tumours in vivo promote the activity of NF-{kappa}B and hence increase colorectal tumour cell survival.

The mechanism by which BAG-1 regulates NF-{kappa}B remains to be elucidated. As described previously, the activity of NF-{kappa}B can be regulated by the I{kappa}B inhibitor proteins. Additionally, post-translational modifications such as acetylation and phosphorylation have been shown to enhance the transcriptional response of NF-{kappa}B in the nucleus (reviewed in ref. 48). In the current study, we have found that expression not only of BAG-1S but also the nuclear BAG-1L isoform increases NF-{kappa}B activity, suggesting that BAG-1 may modulate NF-{kappa}B transcriptional activity in the nucleus. Interestingly, during the preparation of this manuscript, it has been reported that another member of the BAG family, BAG-3, can regulate NF-{kappa}B activity; BAG-3 was shown to suppress the interaction of the p65 subunit of NF-{kappa}B with the {kappa}B DNA motif of the HIV-1 long terminal repeat in microglial cells (49). Although BAG-3 has an opposing activity to that identified for BAG-1 in colorectal tumour cells, this report does support a role for the BAG proteins in the regulation of NF-{kappa}B signalling. We suggest that through acting as a scaffolding molecule (whether directly or mediated through a biding protein such as Hsp70), BAG-1 may represent a tissue-specific mechanism for directing NF-{kappa}B transcriptional activity, promoting expression of target genes important for colorectal tumour cell survival.

In this report inhibition of NF-{kappa}B using the chemical compound BAY-117082 resulted in reduced cell yield and induction of apoptosis consistent with a role for NF-{kappa}B in colorectal tumour cell survival. Interestingly, despite other potential off-target effects of the BAY-117082 inhibitor, knock down of BAG-1 using siRNA caused a similar reduction in cell yield and induction of apoptosis. Combined treatment with the BAY-117082 inhibitor and BAG-1 siRNA did not further suppress colorectal carcinoma cell survival suggesting that BAG-1 may regulate the survival of colorectal carcinoma cells via regulation of NF-{kappa}B transcriptional activity. This has clinical implications as inhibition of BAG-1 suppresses NF-{kappa}B activity and may therefore sensitize cells to therapeutic strategies. Indeed, strategies to inhibit NF-{kappa}B are currently of significant clinical interest since constitutive activity of NF-{kappa}B has been reported in a number of different tumour types (12) and NF-{kappa}B has been shown to be activated following radiotherapy and chemotherapy, reducing tumour sensitivity to treatment (50,51). However, the use of broad-spectrum NF-{kappa}B inhibitors in the treatment of cancer has been limited by their effects on a wide range of pathways, including the immune system (reviewed in ref. 52). Targeting BAG-1 may therefore be an attractive strategy to inhibit NF-{kappa}B in those cells with high activity, leading to decreased colorectal tumour cell survival and increasing the sensitivity of colorectal tumours to cancer therapy. Additionally, TRAIL has the potential to be exploited as a cancer therapeutic agent (reviewed in ref. 53). The data presented in this study shows that, through inhibition of NF-{kappa}B, suppression of BAG-1 increases the sensitivity of colorectal carcinoma cells to both TNF-{alpha}- and TRAIL-induced apoptosis. This suggests that, in combination, inhibition of BAG-1 may increase the therapeutic potential of TRAIL.

In summary, we report that BAG-1 is over-expressed in colorectal carcinomas as well as medium/large-sized adenomas when compared with the normal colonic epithelium, implicating a role for BAG-1 in colorectal tumorigenesis. We have established that knock down of BAG-1 leads to inhibition of NF-{kappa}B, identifying BAG-1 as a novel regulator of NF-{kappa}B. Data presented suggest that, through inhibition of NF-{kappa}B, suppression of BAG-1 represents a potential novel strategy to impede colorectal cancer cell survival and as an adjuvant may increase sensitivity to current therapeutic regimes.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Citrina Foundation; John James Foundation; Cancer Research UK.


   Acknowledgments
 
We would like to thank Feng Lin for carrying out the microarray, Catherine Wallam, Victoria Skeen and Jenny Baker for their help with the immunohistochemistry and Alex Greenhough for his help with the figures. We would also like to thank Dr Marion MacFarlane for the kind gift of the TRAIL.

Conflict of Interest Statement: None.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Townsend PA, et al. BAG-1: a multifunctional regulator of cell growth and survival. Biochim. Biophys. Acta (2003) 1603:83–98.[Medline]
  2. Packham G, et al. Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation. Biochem. J. (1997) 328:807–813.[Web of Science][Medline]
  3. Takayama S, et al. Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines. Cancer Res. (1998) 58:3116–3131.[Abstract/Free Full Text]
  4. Yang X, et al. Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells. Oncogene (1998) 17:981–989.[CrossRef][Web of Science][Medline]
  5. Naishiro Y, et al. BAG-1 accelerates cell motility of human gastric cancer cells. Oncogene (1999) 18:3244–3251.[CrossRef][Web of Science][Medline]
  6. Zeiner M, et al. The hsp70-associating protein Hap46 binds to DNA and stimulates transcription. Proc. Natl Acad. Sci. USA (1999) 96:10194–10199.[Abstract/Free Full Text]
  7. Yang X, et al. BAG-1 promotes apoptosis induced by N-(4-hydroxyphenyl)retinamide in human cervical carcinoma cells. Exp. Cell Res. (2000) 256:491–499.[CrossRef][Web of Science][Medline]
  8. Chen J, et al. Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-1-transfected C33A human cervical carcinoma cell line. Oncogene (2002) 21:7050–7059.[CrossRef][Web of Science][Medline]
  9. Townsend PA, et al. BAG-1 prevents stress-induced long-term growth inhibition in breast cancer cells via a chaperone-dependent pathway. Cancer Res. (2003) 63:4150–4157.[Abstract/Free Full Text]
  10. Barnes JD, et al. Nuclear BAG-1 expression inhibits apoptosis in colorectal adenoma-derived epithelial cells. Apoptosis (2005) 10:301–311.[CrossRef][Web of Science][Medline]
  11. Karin M, et al. NF-kappaB at the crossroads of life and death. Nat. Immunol. (2002) 3:221–227.[CrossRef][Web of Science][Medline]
  12. Rayet B, et al. Aberrant rel/nfkb genes and activity in human cancer. Oncogene (1999) 18:6938–6947.[CrossRef][Web of Science][Medline]
  13. Lind DS, et al. Nuclear factor-kappa B is upregulated in colorectal cancer. Surgery (2001) 130:363–369.[CrossRef][Web of Science][Medline]
  14. Yu HG, et al. Increased expression of RelA/nuclear factor-kappa B protein correlates with colorectal tumorigenesis. Oncology (2003) 65:37–45.[Web of Science][Medline]
  15. Yu LL, et al. Nuclear factor-kappaB p65 (RelA) transcription factor is constitutively activated in human colorectal carcinoma tissue. World J. Gastroenterol. (2004) 10:3255–3260.[Medline]
  16. Neurath MF, et al. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Nat. Med. (1996) 2:998–1004.[CrossRef][Web of Science][Medline]
  17. Rogler G, et al. Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa. Gastroenterology (1998) 115:357–369.[CrossRef][Web of Science][Medline]
  18. Xavier RJ, et al. Unravelling the pathogenesis of inflammatory bowel disease. Nature (2007) 448:427–434.[CrossRef][Medline]
  19. Yamamoto K, et al. Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells. J. Biol. Chem. (1995) 270:31315–31320.[Abstract/Free Full Text]
  20. Guttridge DC, et al. NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol. Cell. Biol. (1999) 19:5785–5799.[Abstract/Free Full Text]
  21. Hinz M, et al. NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol. Cell. Biol. (1999) 19:2690–2698.[Abstract/Free Full Text]
  22. Zapata JM, et al. Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors. Breast Cancer Res. Treat. (1998) 47:129–140.[CrossRef][Web of Science][Medline]
  23. Turner BC, et al. BAG-1: a novel biomarker predicting long-term survival in early-stage breast cancer. J. Clin. Oncol. (2001) 19:992–1000.[Abstract/Free Full Text]
  24. Yang X, et al. Overexpression of anti-apoptotic gene BAG-1 in human cervical cancer. Exp. Cell Res. (1999) 247:200–207.[CrossRef][Web of Science][Medline]
  25. Shindoh M, et al. BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma. Oral Oncol. (2000) 36:444–449.[CrossRef][Web of Science][Medline]
  26. Yamauchi H, et al. Nuclear BAG-1 localization and the risk of recurrence after radiation therapy in laryngeal carcinomas. Cancer Lett. (2001) 165:103–110.[CrossRef][Web of Science][Medline]
  27. Moriyama T, et al. BAG-1 expression in normal and neoplastic endometrium. Gynecol. Oncol. (2004) 94:289–295.[CrossRef][Web of Science][Medline]
  28. Kikuchi R, et al. Nuclear BAG-1 expression reflects malignant potential in colorectal carcinomas. Br. J. Cancer (2002) 87:1136–1139.[CrossRef][Web of Science][Medline]
  29. Brimmell M, et al. High level expression of differentially localized BAG-1 isoforms in some oestrogen receptor-positive human breast cancers. Br. J. Cancer (1999) 81:1042–1051.[CrossRef][Web of Science][Medline]
  30. Chell SD, et al. Increased EP4 receptor expression in colorectal cancer progression promotes cell growth and anchorage independence. Cancer Res. (2006) 66:3106–3113.[Abstract/Free Full Text]
  31. Williams AC, et al. Insulin-like growth factor binding protein 3 (IGFBP-3) potentiates TRAIL-induced apoptosis of human colorectal carcinoma cells through inhibition of NF-kappaB. Cell Death Differ. (2007) 14:137–145.[CrossRef][Web of Science][Medline]
  32. Pierce JW, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J. Biol. Chem. (1997) 272:21096–21103.[Abstract/Free Full Text]
  33. Williams AC, et al. In vitro models of human colorectal cancer. Cancer Surv. (1993) 16:15–29.[Web of Science][Medline]
  34. Cutress RI, et al. The nuclear BAG-1 isoform, BAG-1L, enhances oestrogen-dependent transcription. Oncogene (2003) 22:4973–4982.[CrossRef][Web of Science][Medline]
  35. Bos JL, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature (1987) 327:293–297.[CrossRef][Medline]
  36. Elder DJ, et al. Human colorectal adenomas demonstrate a size-dependent increase in epithelial cyclooxygenase-2 expression. J. Pathol. (2002) 198:428–434.[CrossRef][Web of Science][Medline]
  37. Abdollahi A, et al. Sequence and expression of a cDNA encoding MyD118: a novel myeloid differentiation primary response gene induced by multiple cytokines. Oncogene (1991) 6:165–167.[Web of Science][Medline]
  38. Kataoka T. The caspase-8 modulator c-FLIP. Crit. Rev. Immunol. (2005) 25:31–58.[CrossRef][Web of Science][Medline]
  39. Bliss JM, et al. The RIN family of Ras effectors. Methods Enzymol. (2005) 407:335–344.[Web of Science]
  40. Amit S, et al. NF-kappaB activation in cancer: a challenge for ubiquitination- and proteasome-based therapeutic approach. Semin. Cancer Biol. (2003) 13:15–28.[CrossRef][Web of Science][Medline]
  41. Israel A, et al. TNF stimulates expression of mouse MHC class I genes by inducing an NF kappa B-like enhancer binding activity which displaces constitutive factors. EMBO J. (1989) 8:3793–3800.[Web of Science][Medline]
  42. Sanguinetti G, et al. Probabilistic inference of transcription factor concentrations and gene-specific regulatory activities. Bioinformatics (2006) 22:2775–2781.[Abstract/Free Full Text]
  43. MacFarlane M, et al. Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J. Biol. Chem. (1997) 272:25417–25420.[Abstract/Free Full Text]
  44. Van Antwerp DJ, et al. Inhibition of TNF-induced apoptosis by NF-kappa B. Trends Cell Biol. (1998) 8:107–111.[CrossRef][Web of Science][Medline]
  45. Nakagami H, et al. Lack of association between the hepatocyte growth factor receptor, c-met, and the anti-apoptotic action of bag-1 in endothelial cells. Hypertens. Res. (2004) 27:359–365.[CrossRef][Web of Science][Medline]
  46. Clevers H. At the crossroads of inflammation and cancer. Cell (2004) 118:671–674.[CrossRef][Web of Science][Medline]
  47. Greten FR, et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell (2004) 118:285–296.[CrossRef][Web of Science][Medline]
  48. Chen LF, et al. Shaping the nuclear action of NF-kappaB. Nat. Rev. Mol. Cell Biol. (2004) 5:392–401.[CrossRef][Web of Science][Medline]
  49. Rosati A, et al. Evidence for BAG3 modulation of HIV-1 gene transcription. J. Cell Physiol. (2007) 210:676–683.[CrossRef][Web of Science][Medline]
  50. Arlt A, et al. Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin. Oncogene (2001) 20:859–868.[CrossRef][Web of Science][Medline]
  51. Yang CR, et al. Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. FASEB J. (2000) 14:379–390.[Abstract/Free Full Text]
  52. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat. Rev. Mol. Cell Biol. (2007) 8:49–62.[CrossRef][Web of Science][Medline]
  53. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat. Rev. Cancer (2002) 2:420–430.[CrossRef][Web of Science][Medline]
Received October 10, 2007; revised December 17, 2007; accepted December 27, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
29/4/849    most recent
bgn004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Clemo, N. K.
Right arrow Articles by Williams, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Clemo, N. K.
Right arrow Articles by Williams, A. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?