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Cancer Research 67, 9039, October 1, 2007. doi: 10.1158/0008-5472.CAN-06-3690
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

A Novel Variant of Ileal Bile Acid Binding Protein Is Up-regulated through Nuclear Factor-{kappa}B Activation in Colorectal Adenocarcinoma

Changming Fang, Jarrod Dean and Jeffrey W. Smith

Cancer Research Center, Burnham Institute for Medical Research, La Jolla, California

Requests for reprints: Jeffrey W. Smith, Cancer Research Center, Burnham Institute for Medical Research, La Jolla, CA 92037. Phone: 858-646-3121; Fax: 858-795-5221; E-mail: jsmith{at}burnham.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ileal bile acid binding protein (IBABP) is the only cytosolic protein known to bind and transport bile acids. Because IBABP is reportedly up-regulated in colorectal cancer, it has been suggested as a link between bile acids and the risk of colorectal cancer. However, in this study, we show that IBABP is not up-regulated. Rather, a novel transcript of the IBABP gene, which encodes an additional 49 NH2-terminal amino acid residues, is up-regulated in colorectal cancer (P < 0.001). The novel transcript, called IBABP-L, is also distinct from IBABP because its transcription is controlled by nuclear factor-{kappa}B (NF-{kappa}B) rather than by the farnesoid X receptor. Most significantly, IBABP-L is necessary for the survival of HCT116 colon cancer cells in the presence of physiologic levels of the secondary bile acid deoxycholate. Collectively, the studies point toward a unique bile acid response pathway involving NF-{kappa}B and IBABP-L that could be useful for diagnosis and could potentially be targeted for therapeutic benefit. [Cancer Res 2007;67(19):9039–46]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colorectal cancer is the third most common type of cancer and the second leading cause of cancer mortality. It is estimated that ~150,000 cases of colorectal cancer will be diagnosed, and 55,000 people will die of colorectal cancer in the United States in 2006 (1). There are two well-described colorectal cancer syndromes that are inherited: familial adenomatous polyposis, which arises because of germ-line mutations in the APC gene, and hereditary nonpolyposis colorectal cancer, which is caused by germ-line mutations in mismatch repair genes (2). These conditions account for ≤5% of all cases of colorectal cancer; importantly though, the majority of colorectal cancer is of sporadic nature with no unique and clear genetic etiology. Consequently, there is a great need to understand the mechanisms underlying the onset and progression of sporadic colorectal cancer.

One factor that may be involved in sporadic colorectal cancer is bile acid homeostasis. Epidemiologic studies indicate that secondary bile acids, produced by colonic bacteria, are linked to the incidence of colorectal cancer (see ref. 3 for reviews). Experimental studies show that deoxycholic acid (DCA), a major secondary bile acid, strongly enhances the protumorigenic effects of chemical mutagens in animal models of colorectal cancer (4, 5). DCA causes mitochondrial oxidative stress and DNA damage and was proposed as a carcinogen (3). It is well known that DCA can kill intestinal epithelial cells by apoptosis at a concentration usually seen in colon lumen of subjects with high fat diet; however, it also has been suggested that DCA can either induce cell death resistance or select death-resistant cells to promote tumorigenesis (3).

Another pathway receiving increased attention in colorectal cancer is that controlled by the nuclear factor-{kappa}B (NF-{kappa}B) transcription factor. In a resting state, the p50 and p65 subunits of NF-{kappa}B form a heterotrimeric complex with I{kappa}B{alpha} that sequesters them in the cytoplasm. On various stimuli, I{kappa}B{alpha} is degraded and the p50-p65 heterodimer is translocated to the nucleus where it binds specific regulatory elements and drives gene expression (6). NF-{kappa}B is up-regulated in colorectal adenoma and adenocarcinoma (7–10) and controls the expression of many colorectal cancer–linked genes, including cyclooxygenase-2 and Bcl-2 (6). NF-{kappa}B is also believed to be involved in the onset of colorectal cancer (6), in the inflammatory process associated with colorectal cancer (11), and in the development of resistance of colorectal cancer to chemotherapy (12, 13).

In the present study, we uncover an unanticipated link between NF-{kappa}B and bile acids. We have identified a variant of ileal bile acid binding protein (IBABP) that arises from an alternative transcription start site (TSS). Unlike IBABP, which is transcribed by the farnesoid X receptor/bile acid receptor (FXR), the new variant, called IBABP-L, is regulated by a NF-{kappa}B binding site in a distal promoter. IBABP-L contains 49 amino acids at its NH2 terminus that are absent in IBABP. More significantly, the transcript for IBABP-L is up-regulated in all stages of colorectal adenocarcinoma. In fact, we show that the up-regulation of IBABP in colorectal cancer reported in prior studies (14, 15) can be attributed to the up-regulation of IBABP-L, whereas the expression of the previously defined form of IBABP is unchanged in colorectal cancer. Most significantly, IBABP-L is necessary for the survival of colon cancer cells in the presence of secondary bile acids. These observations provide an important mechanistic link between bile acids, NF-{kappa}B, and colorectal cancer that can likely be exploited for therapeutic benefit.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tissue samples. Caco-2, HCT116, and HEK293 cells, obtained from the American Type Culture Collection, were grown in DMEM (Irvine Scientific) containing 1 mmol/L sodium pyruvate, 4.5 g/L D-glucose, and 4 mmol/L L-glutamine and supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Omega Scientific) and with 10% fetal bovine serum (FBS; Irvine Scientific). Cells were maintained in 100-mm standard cell culture dishes (BD Biosciences) and grown at 37°C under 5% CO2.

Matched human colorectal adenoma, adenocarcinoma, and adjacent normal mucosa were purchased from Asterand, Inc. or obtained from the Cooperative Human Tissue Network (service of the National Cancer Institute, Bethesda, MD). Patients have provided written consent for use of tissues for scientific purpose. Tissue array slides containing human colorectal cancer and matching normal tissues were ordered from Imgenex.

Assessing the expression of IBABP variants by PCR. Expression of mRNA encoding IBABP-L (Genbank accession number DQ132786) and IBABP (Genbank accession number NM_001445) along the digestive tract was measured by quantitative reverse transcription-PCR (RT-PCR) with RNA from normal human intestine and liver purchased from Invitrogen and from BioChain Institute, Inc. Expression of acidic ribosomal phosphoprotein P0 (ARPP0) was used as a control. The expression of IBABP-L, IBABP, and total IBABP in human tumor and adjacent normal tissue was measured with tissues purchased from Asterand, Inc. and from the Cooperative Human Tissue Network as described above.

Total RNA was isolated from tissues using Trizol reagent (Invitrogen) in a protocol combined with the RNeasy Mini kit (Qiagen). For each sample, frozen tissue (~0.1 g) was cut and soaked in prechilled RNAlater-ICE stabilizing solution (1.0 mL; Ambion) for 24 h at –20°C. Tissue was minced using a surgical scalpel, immersed in Trizol (1.0 mL), and homogenized using a Tissue-Tearor (BioSpec Products). Chloroform (200 µL) was added to homogenized tissue and sample was mixed by vortexing for 30 s. Samples were centrifuged (12,000 x g, 10 min at 4°C) to separate phases. The aqueous phase was removed, added to an equal volume of 70% ethanol, mixed by pipetting, and loaded into the RNeasy column. Following RNA binding, an on-column DNase digestion protocol using RNase-Free DNase Set (Qiagen) was done according to the manufacturer's instructions.

To determine the relative expression levels of IBABP-L and IBABP, a two-step quantitative RT-PCR procedure was used. In the first step, cDNA was synthesized from total RNA. For each sample, RNA (2.0 µg) was reverse transcribed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) in a 20 µL final reaction volume containing 10 mmol/L deoxynucleotide triphosphate mix (1.0 µL), 0.5 µg/mL oligo(dT)12-18 (1.0 µL), 0.1 mol/L DTT (2.0 µL), 25 mmol/L MgCl2 (4.0 µL), 10x reverse transcriptase buffer (2.0 µL), RNaseOUT Recombinant RNase Inhibitor (1.0 µL), and SuperScript II Reverse Transcriptase (1.0 µL). Reverse transcription was done at 42°C for 50 min and terminated by heating to 70°C for 15 min followed by chilling samples on ice. Template RNA was cleaved by incubating with RNase H (1.0 µL) for 20 min at 37°C. In the second step, quantitative PCR (qPCR) was carried out on a Mx 3000P Real-time PCR System (Stratagene) using a solution containing diluted cDNA (1:20; 2.0 µL), 1x SYBR Green PCR Master Mix (Applied Biosystems), and primers for IBABP-L, IBABP, or ARPP0 (0.25 µmol/L). The primer sets are as follows: IBABP, 5'-CCACCCATTCTCCTCATCCCTCTGCTC-3' (in exon 4a) and 5'-ACCAAGTGAAGTCCTGCCCATCCTG-3' (in exon 5), and IBABP-L, 5'-ACATGGGTGAGCCGGAAAGGAGAC-3' (in exon 3) and 5'-CCGGAGTAGTGCTGGGACCAAGTGAAGT-3' (in exon 5). The primer set that cannot distinguish IBABPL and IBABP is 5'-AGGATGGGCAGGACTTCACTTG-3' (in exon 5) and 5'-GCTCACGCGCTCATAGGTCAC-3' (in exon 7); the primer set of ARPP0 is 5'-CAAGACTGGAGACAAAGTGG-3' and 5'-AATCTGCAGACAGACACTGG-3'. All primers were designed using PrimerSelect (DNAStar) and synthesized by Integrated DNA Technologies, Inc. The following cycling variables were used: denaturation at 95°C for 15 s, annealing at 56°C for 20 s, extension at 72°C for 30 s, and detection at 78°C for 5 s. After 40 cycles, PCR products were subjected to dissociation curve analysis to check the PCR specificity. Each sample was done in duplicate.

Values obtained from qPCR were normalized to expression of ARPP0. The difference in RNA expression of IBABP-L and IBABP between colon cancer tissue and matched adjacent normal mucosa was represented as fold change (cancer versus normal). The correlation between IBABP-L expression level and clinical variables (gender, age, race, tumor size, tumor locale, differentiation level, and clinical stage) was analyzed by t test and one-way ANOVA.

Regulation of expression of the IBABP variants in Caco-2 cells. Caco-2 cells were seeded in six-well plates (BD Biosciences) at 2 x 105 per well in phenol-free DMEM (Mediatech) supplemented with charcoal-stripped FBS (Hyclone). Medium was replaced every 2 days until cells reached 100% confluence and began spontaneous differentiation. Stock solutions of CDCA (Sigma-Aldrich), as free acids, were prepared in absolute ethanol (100 mmol/L) and stored at –20°C. Confluent Caco-2 cells were incubated at 37°C for 24 h in medium containing 100 µmol/L CDCA or solvent (0.1% ethanol). Cells were harvested, cellular RNA was isolated using the RNeasy Mini kit according to the manufacturer's instruction, and expression of IBABP variants was determined by qPCR as described above. Values obtained from qPCR were normalized to the expression of ARPP0. The difference in RNA expression of IBABP-L and IBABP between CDCA and control Caco-2 cells is represented as fold change.

Determine the TSS. Primer extension was conducted using Human Small Intestine Marathon-Ready cDNA with Advantage 2 PCR kit (Clontech) according to the manufacturer's manual. Briefly, first PCR was done using primers AP1 (included in the kit) and gene-specific primer 1 (GSP1) 5'-ACATTATATTTTCTTGCCAAGTAGAGGA-3' (exon 2) followed by a nested PCR with primer AP2 (included in the kit) and GSP2 5'-ACTTGCCAGCTGCCTTCCT-3' (exon 3). The longest PCR product was cloned into pCR2.1 T/A cloning vector (Invitrogen) and 12 individual clones were sequenced. The TSS was unanimously located 78 nucleotides away from GSP2. To simplify the description, the position of the TSS "C" was set to as +1.

Construct the wild-type and modified IBABP-L promoter-luciferase reporters. A 1.6-kb region (from –1563 to +79) of IBABP-L promoter was amplified from human bacterial artificial chromosome clone RP11-725E20 (Children's Hospital Oakland Research Institute, Oakland, CA) by PCR using Pfu DNA polymerase (Stratagene) with primers 5'-ACATTATATTTTCTTGCCAAGTAGAGGA-3' (–1563/–1534) and primer GSP2 described above (+61/+79). The –1188/+79 and –1154/+79 fragments were amplified using primer 5'-GAAGTAGAGCTTCCTCTTC-3' (–1188/–1168) or primer 5'-CCTGTCTAATTAGGAATAA-3' (–1154/–1137), respectively, together with primer GSP2. PCR product was cloned into pCRII-Blunt-TOPO (Invitrogen) for sequencing, and verified clone was digested by SpeI/XhoI (New England Biolabs) restriction enzymes. The promoter fragment was retrieved and inserted into pGL3-Basic vector (Promega) between NheI and XhoI (New England Biolabs). The single site–mutated pGL3_–1165 G/C IBABP-L reporter was generated from pGL3_–1563/+79 IBABP-L using QuikChange Multisite-Directed Mutagenesis kit (Stratagene) with primers 5'-CTTCCTCTTCAAAGCGACTTTCCTTCCCG-3' and 5'-CGGGAAGGAAAGTCGCTTTGAAGAGGAAG-3' (–1179/–1150, the –1165 G/C substitution is italicized).

Transient transfection and luciferase reporter assay. Individual promoter construct (300 ng) and 100 ng of pRL-CMV vector (Promega) were cotransfected into 2 x 105 HEK293 or HCT116 cells grown in 24-well plate using 3 µL Lipofectamine 2000 (Invitrogen). The cells were then either treated with 25 ng/mL of tumor necrosis factor-{alpha} (TNF-{alpha}) dissolved in PBS (pH 7.4) for 5 h or left alone. For cotransfection with NF-{kappa}B complex in HEK293 cells, 300 ng of individual promoter construct and 100 ng of pRL-CMV were cotransfected with 300 ng each of plasmids encoding p65 and p50 (gift from Dr. Marty W. Mayo, University of Virginia, Charlottesville, VA; ref. 16). For control cotransfection without p65/p50 constructs, pUC19 was used to make the DNA content equal. After 24 h of incubation, cells were harvested in 1x Passive Lysis Buffer (Promega), and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Assay System (Promega) with Veritas Microplate Luminometer. All activity data are the mean ± SD of three independent experiments done in triplicate.

Produce IBABP-L–specific antiserum and immunohistochemical studies. A peptide unique to IBABP-L (CTWVSRKGDLQRMKQTHKGKPPSS) was synthesized, conjugated to keyhole limpet hemocyanin, and used to immunize rabbits. Antisera were tested for reactivity against recombinant IBABP-L and IBABP by Western blot. The antibody is highly specific for IBABP-L, and its binding can be blocked by the synthetic peptide antigen. The antisera (1:2,000) were used to stain paraffin-embedded array slides of human colorectal carcinomas and normal adjacent tissues (Imgenex). Binding of the antibody was detected with diaminobenzidine using a horseradish peroxidase (HRP) system. The slides were counterstained with hematoxylin. The microscopic photographs were taken at 100, 200, and 400 magnification with an Inverted TE300 Nikon wild field microscope equipped with color CCD SPOT RT Camera (Diagnostic Instruments).

IBABP short hairpin RNA transfection and cell death assays. Short hairpin RNA (shRNA)-encoding constructs were purchased from Open Biosystems. The sequence of the shRNA targeting IBABP is 5'-CCCGCAACTTCAAGATCGTC-3'. The sequence of the nonsilencing scrambled shRNA is 5'-ATCTCGCTTGGGCGAGAGTAAG-3'. These constructs are in the pSM2c vector and transcribed by type III RNA polymerase through the U6 promoter. HCT116 cell suspension was mock transfected or transfected with either pSM2c-IBABP, pSM2c-scrambled, or pSM2c at 200 ng DNA/1,000 cells and then seeded into 96-well plate at 1,000 cells per well. Following transfection, cells were incubated at 37°C for 72 h and treated with DCA in different concentration for 24 h. The cell apoptosis was measured using the Cell Death Detection ELISAplus kit (Roche Applied Science), which detects the amount of cleaved DNA/histone complexes using a sandwich enzyme immunoassay–based method. The value was determined using a colorimetric 96-well plate reader (Bio-Rad) and represented as mean ± SD of three independent experiments done in quadruplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a new variant of IBABP. While doing preliminary PCR analysis to determine if IBABP was up-regulated in human colorectal cancer tissue, we noticed significant up-regulation of IBABP in tumor tissue compared with normal tissue when using a primer set that hybridized in the coding sequence of IBABP. Yet, when using a 5' primer hybridizing far upstream of the start site of IBABP, up-regulation was not detected. This observation suggested that there could be an alternative form of IBABP.

To test this possibility, we searched the National Center for Biotechnology Information human expression sequence tag (EST) database1 using the IBABP sequence NM_001445. This search revealed two ESTs (BM974219 and BU683560) that were identical to IBABP except that they encoded a protein with a predicted 49-amino acid extension on the NH2 terminus. To verify the presence of this transcript, the two EST clones were obtained and sequenced and compared with the sequence of human BAC clone RP11-725E20 containing the IBABP gene [fatty acid binding protein 6 (fabp6)]. These studies led to the finding that the mRNA encoding IBABP-L contains seven exons, three of which are unique and are present at the 5' end of the gene. The shorter transcript, IBABP, contains only four exons and its transcription is initiated within the third intron of the fabp6 gene (Fig. 1A ). Thus, the two variants of IBABP share exons 5 to 7 and part of exon 4. Both variants could be detected in mRNA extracted from human intestine by RT-PCR (Fig. 1B). Interestingly, searches of the sequence databases indicate that IBABP-L is also present in primates such as Rhesus monkeys and chimpanzees, but it is absent in mouse, rat, and rabbit (data not shown). The complete nucleotide sequence of IBABP-L transcript was deposited in Genbank with accession number DQ132786.


Figure 1
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  		Figure 1. Identification of IBABP-L, a new transcript of IBABP. A, the gene encoding both forms of IBABP was originally annotated as fabp6. The structure of this gene is shown but is not drawn to scale. Exons are labeled E1 to E7 and promoters are labeled P1 and P2. P1 drives the expression of IBABP-L, whereas P2 controls the transcription of IBABP. IBABP-L shares exons 4b to 7 with IBABP and contains the complete open reading frame for IBABP. B, primers specific for each variant of IBABP were used to assess their expression by RT-PCR in mRNA extracted from intestine. Reactions were done in duplicate. A housekeeping gene, ARPP0, was used as a control to monitor the efficiency of reverse transcription and PCR. The PCR product was separated in 1.6% agarose gel. The PCR product was also confirmed by direct sequencing (data not shown).

 
IBABP and IBABP-L are differentially expressed in the gastrointestinal tract. IBABP is primarily expressed in the intestine (17). Therefore, we compared the expression of transcripts encoding IBABP and IBABP-L in the gastrointestinal tract, particularly in tissues associated with the enterohepatic bile acid cycle (human liver, gallbladder, and intestinal sections). The studies were conducted with tissue RNA extracted from donors who did not have gastrointestinal diseases. Oligonucleotides capable of selective amplification of each variant were used to initiate real-time qPCRs. The copy number of mRNA transcripts was normalized to the expression of the housekeeping gene ARPP0, also known as ribosomal protein large P0, which is often used as an endogenous control in research on prostate and colon cancer (18, 19). The transcript encoding IBABP-L was found at similar weak levels in all tissues tested with the exception of the rectum where it was expressed at lower levels (Fig. 2A ). In contrast, IBABP is highly expressed within a section of the intestine extending from the jejunum through ascending colon with peak expression in the ileum. In these sections, the expression of IBABP was 10- to 1,000-fold higher than the expression of IBABP-L.


Figure 2
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  		Figure 2. Differential tissue expression pattern and regulation of IBABP variants. A, primers specific for each variant of IBABP were used to quantify their expression in mRNA extracted from human liver, gallbladder, and sections of the gastrointestinal tract (duodenum through rectum) by quantitative RT-PCR. The expression of each variant was normalized using the housekeeping gene ARPP0. B, Caco-2 cells, which respond to bile acids by activation of FXR, were incubated with 100 µmol/L CDCA for 24 h. The expression of IBABP-L and IBABP was measured and normalized. Columns, mean of four separate assays with each assay done in duplicate; bars, SD.

 
IBABP and IBABP-L are differentially regulated by chenodeoxycholic acid. Like many other genes associated with bile acid homeostasis, IBABP is regulated by the FXR. In fact, bile acids, especially chenodeoxycholic acid (CDCA), bind directly to FXR and activate its transcriptional function (20–22), which in turn leads to up-regulation of IBABP (23, 24). Studies were conducted to determine if IBABP-L is regulated in a similar manner. The effect of CDCA on the expression of IBABP-L and IBABP was studied in Caco-2 cells, which respond to CDCA by up-regulating IBABP (25). Confluent Caco-2 cells were treated with 100 µmol/L of CDCA or buffer, and the relative expression of transcripts encoding IBABP-L and IBABP was measured by quantitative RT-PCR. As anticipated, CDCA increased expression of IBABP by 14-fold. However, this bile acid was without effect on expression of IBABP-L (Fig. 2B). These results are consistent with the idea that the two variants of IBABP arise from separate TSSs (Fig. 1A).

Transcription of IBABP-L is controlled by NF-{kappa}B. As a first step toward understanding the transcriptional regulation of IBABP-L, the TSS was successfully identified through 5'-rapid amplification of cDNA ends method. This information provided an anchor point for mapping regions of the promoter of IBABP-L. The genomic sequence of IBABP-L extending 1.6 kb upstream of TSS was analyzed for putative transcription factor binding sites using the P-Match program.2 A putative NF-{kappa}B binding motif was identified 1.16 kb upstream of the TSS (Fig. 3A ).


Figure 3
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  		Figure 3. The IBABP-L promoter is regulated by NF-{kappa}B. A, the IBABP-L promoter containing a putative NF-{kappa}B binding motif (–1563/+79) was amplified from the human genomic DNA BAC clone by PCR. Deletion constructs of this promoter were made through PCR. The G->C mutation was made through site-directed mutagenesis. The wild-type and modified promoters were inserted into pGL3 vector containing firefly luciferase (LUC) reporter. B, reporter constructs were introduced into HCT116 cells together with pRL-CMV that encodes Renilla luciferase. The luciferase activity was measured in 24 h and normalized to the Renilla signal, and the fold of change was calculated by comparing with the control vector pGL-Basic. C, same as B except that the transfected HCT116 cells were treated with or without 25 ng/mL TNF-{alpha} for 5 h before measurement. D, wild-type and modified IBABP-L promoter activity reporter constructs were introduced into HEK293 cells with or without cotransfection with constructs encoding NF-{kappa}B complex p65/p50. The luciferase activity was measured in 24 h and normalized. As in B, pRL-CMV was included in every transfection. Columns, mean of three experiments done in triplicate; bars, SD.

 
To verify that NF-{kappa}B binds the response element and drives the expression of IBABP-L, several studies were done with deletions and mutations of the promoter linked to luciferase reporter in the colon cancer cell line HCT116. Reporter vectors were constructed to either delete the entire putative NF-{kappa}B binding motif or mutate the most conserved "G" to "C" within this motif (Fig. 3A, underlined). The wild-type promoter and modified reporters were introduced into HCT116 cells, and luciferase activity was analyzed at 24 h. The wild-type promoter for IBABP-L produced a 7.5-fold increase in luciferase activity compared with the basic reporter (Fig. 3B). Deletion of the sequences upstream of the putative NF-{kappa}B binding motif had no effects on the luciferase activity. However, when the whole binding motif was deleted, the reporter constructs nullified its transcriptional activity. More importantly, a single nucleotide substitution from G to C at the most conserved region of the NF-{kappa}B binding motif also resulted in loss of promoter activity (Fig. 3B). Furthermore, TNF-{alpha}, which is an activator of NF-{kappa}B, dramatically increased the transcription activity of the wild-type promoter but not the mutated promoter (Fig. 3C).

The activity of the IBABP-L promoter was also tested in HEK293 cells, which have low endogenous levels of NF-{kappa}B. In these cells, the IBABP-L promoter had low activity (Fig. 3D), but its activity significantly increased when constructs encoding protein p65 and p50 of NF-{kappa}B were cotransfected into HEK293 cells (Fig. 3D). This activation by p65/p50 was not observed when the IBABP-L promoter lacked a functional NF-{kappa}B binding site (Fig. 3D). Together, these findings provide strong support for the idea that NF-{kappa}B regulates the transcription activity of the IBABP-L promoter.

IBABP-L is up-regulated in colorectal cancer. NF-{kappa}B is implicated in multiple cancers, including colorectal cancer (6). Because IBABP-L is regulated by NF-{kappa}B, we tested its expression in colorectal tumors. The expression of IBABP and IBABP-L was measured in 68 cases of colorectal cancer by quantitative RT-PCR. The clinicopathologic data of all cases are shown in Supplementary Table S1. Experiments were done with primers that selectively amplified each form of IBABP and also with a pair of primers that amplified both forms together (total IBABP). The gene expression level of the transcript in adenocarcinoma was compared with its levels in adjacent normal tissue. The transcript encoding IBABP-L was massively up-regulated in colorectal adenocarcinoma; in some cases, it was expressed at levels >100-fold higher than in normal tissue (Fig. 4A ). IBABP-L was up-regulated in 76% (52 of 68) of the colorectal cancers using a 2-fold cutoff (P < 0.001). In contrast, there was no significant change in the expression of IBABP in cancer tissue. Because of the up-regulation of IBABP-L, total IBABP is also up-regulated.


Figure 4
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  		Figure 4. Up-regulation of IBABP-L mRNA in colorectal cancer. Total RNA was isolated from 68 sets of matched human colorectal and adjacent normal mucosa and used as template in a two-step quantitative RT-PCR procedure as described in Materials and Methods. Specific primer sets were used to quantify mRNA encoding IBABP-L, IBABP, and total IBABP. The qPCR was done in duplicate, and values were normalized to expression of ARPP0 before calculating the fold change. Statistical analysis was done using one-way ANOVA. Points, value of individual patient; long bars, mean; short bars, SE. A, the differential expression of IBABP, IBABP-L, and total IBABP from 68 adenocarcinomas and matched normal mucosa is presented as fold change of IBABP variants between cancer and normal tissues. Note that total IBABP up-regulation in adenocarcinomas is entirely contributed by IBABP-L. B, the fold change of IBABP-L was grouped by clinical stage and statistical analysis was done. The fold change of IBABP-L in stage I and stage II to IV adenocarcinoma is statistically significant (P < 0.05).

 
Further statistical analysis was conducted to determine if the up-regulation of IBABP-L is associated with any clinical variables such as gender, age, race, tumor size, tumor locale, differentiation level, and clinical stage. The expression of IBABP-L was not associated with patient age, gender, differentiation level, and tumor locale (data not shown). However, there is a trend toward increased expression of IBABP-L from stage I to stage II to IV adenocarcinoma.

An IBABP-L–specific antiserum was raised against the peptide CTWVSRKGDLQRMKQTHKGKPPSS, which falls within the 49 NH2-terminal residues that are unique to IBABP-L. The resulting antiserum selectively detected IBABP-L but not IBABP by Western blot analysis. The protein expression of IBABP-L was then investigated using the validated antiserum with tissue sections of human colorectal adenocarcinomas and their adjacent normal mucosa. The microscopic view of two patient samples is shown at several magnifications (Fig. 5 ). IBABP-L is weakly expressed in some normal epithelial cells. In contrast, nearly all of the cancer cells stained positively and strongly for IBABP-L. The IBABP-L staining is confined to a region close to nucleus on the apical side of both normal and neoplastic cells. These data strongly support that IBABP-L protein is increased in colorectal cancer.


Figure 5
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  		Figure 5. IBABP-L protein expression in colorectal cancer. Paraffin-embedded tissue sections of human colorectal adenocarcinoma and its adjacent normal tissue were stained by IBABP-L–specific antiserum followed by a diaminobenzidine-based detection method using HRP system. Two matched colorectal adenocarcinomas and their adjacent normal tissues are shown. Magnifications, x200 (A) and x400 (C). B and D, panels are x20 magnified view of A and C, respectively. Bar, 100 µm. B and D, arrows, staining for IBABP-L.

 
IBABP-L is necessary for the survival of HCT116 colon cancer cells in the presence of DCA. Studies were conducted to determine if IBABP-L has a role in conferring bile acid resistance in colon cancer cells. As a model system, we chose the HCT116 colon cancer cell line because it expresses high levels of IBABP-L but barely detectable levels of IBABP (Fig. 6A ). Therefore, this cell line recapitulates the expression pattern of IBABP-L and IBABP that we observe in human colon cancer tissue. RNA interference was used to knock down the expression of IBABP-L in HCT116 cells. Quantification of mRNA encoding IBABP-L showed that the expression was reduced by 50% and that this level of repression was maintained 4 days. Two days after the knockdown of IBABP-L, the cells were incubated with 100 µmol/L DCA for 24 h, and the number of cells undergoing apoptosis was determined. The level of apoptosis in cells where IBABP-L was knocked down was substantially higher than that in cells transfected with scrambled shRNA or other control groups (Fig. 6B). We found nearly identical results when activation levels of caspase-8 and caspase-9 were analyzed (see Supplementary Fig. S1). This observation is consistent with reports in the literature showing that DCA activates both caspases (26).


Figure 6
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  		Figure 6. IBABP-L knockdown increases sensitivity to DCA-induced cell death. A, HCT116 cells express 13-fold greater levels of IBABP-L mRNA than IBABP mRNA. B, HCT116 cells are resistant to cell death induced by DCA (100 µmol/L); knockdown of the expression levels of IBABP-L causes the cells to become sensitive to DCA-induced cell death.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both bile acids and NF-{kappa}B have been shown to have a role in colorectal cancer. In the present study, we identify a new transcript of the gene encoding IBABP, called IBABP-L, which provides a missing mechanistic link between NF-{kappa}B and bile acids. In colorectal cancer, IBABP-L is up-regulated by NF-{kappa}B and this up-regulation seems to be necessary for resistance of colon cancer cells to bile acid–induced apoptosis. Together, these findings suggest the existence of a bile acid response pathway controlled by NF-{kappa}B and requiring IBABP-L that is essential for the survival of colon cancer cells in the presence of secondary bile acids.

The fact that IBABP-L is up-regulated in colorectal cancer forces a reinterpretation of two prior studies that reported that the shorter transcript of IBABP is up-regulated in colorectal cancer (14, 15). Those studies were done without knowledge of IBABP-L so the PCR-based studies were done with primers that failed to distinguish between the two forms of IBABP. Our work shows that the up-regulation previously attributed to IBABP is actually the up-regulation of IBABP-L. We found the mRNA level for IBABP-L is on average 30-fold higher in colorectal cancer tissue than in matched normal tissue, whereas the levels of IBABP remained negligible; the IBABP-L protein is also up-regulated in colorectal cancer.

There are several important distinctions between IBABP and IBABP-L. First, the proteins have distinct NH2 termini. Because there are six potential methionine start sites in the predicted NH2-terminal peptide of IBABP-L, we have yet to pinpoint the precise NH2 terminus, but the open reading frame encodes 49 residues following the initiator ATG that are absent in IBABP. A polyclonal antibody raised against a synthetic peptide corresponding to residues 25 to 49 was reactive with IBABP-L, so we conclude that some of this region is present in IBABP-L. Additional analysis will be required to define the exact NH2 terminus.

IBABP and IBABP-L are also differentially expressed in normal intestinal tissue. Whereas IBABP is highly expressed in the region of the normal intestine extending from the jejunum to the ascending colon, the expression of IBABP-L in normal intestine is several orders of magnitude lower. Interestingly though, in the limited number of samples we could obtain, the IBABP-L protein is highly expressed in human fetal intestine (data not shown), suggesting that the protein may have a role in development.

Perhaps, the most important distinction between IBABP and IBABP-L is the way in which their expression is regulated. IBABP is part of the FXR transcription pathway (23) that responds to bile acids and regulates their reabsorption across the ileum. In contrast, IBABP-L is not regulated by FXR; it is regulated by NF-{kappa}B. One of the paradoxical findings in the literature on IBABP is the observation that it is up-regulated in colorectal cancer, whereas its key regulator FXR is down-regulated (14). The discovery that the seeming up-regulation of IBABP is actually up-regulation of IBABP-L and the fact that this transcript is regulated by NF-{kappa}B resolve this paradox.

In addition to the differential expression and regulation of IBABP-L, we provide a biological function for IBABP-L in colon tumorigenesis; IBABP-L is necessary for the survival of colon cancer cells in the presence of physiologic levels of DCA, a toxic secondary bile acid (27). The concentration of DCA in the fecal water of normal subjects is ~100 µmol/L (3). We observed that colon cancer cells are resistant to cell death induced by these levels of DCA; however, when the expression of IBABP-L is knocked down, the cells undergo apoptosis. IBABP-L is also necessary for survival of cells in the presence of concentrations of DCA observed in colorectal cancer patients, ~2-fold higher than normal levels (28). These observations show that IBABP-L promotes survival of colorectal cancer cells in the presence of toxic bile acids and is likely to contribute to colon tumorigenesis.

The intriguing link between bile acids, NF-{kappa}B, and IBABP-L may provide a novel target for intervention in colorectal cancer. In conjunction with the findings in the literature, our study indicates that IBABP-L is up-regulated as a result of the constitutive activation of NF-{kappa}B in colorectal cancer (29, 30). This coupling of NF-{kappa}B and IBABP-L enables colon cancer cells to buffer toxic bile acids, protecting the cells from apoptosis. IBABP-L can be exploited as a therapeutic target in two ways. First, inhibitors of IBABP-L may enhance the chemopreventative and therapeutic effects of NF-{kappa}B inhibitors (31). Second, because IBABP-L is necessary for tumor cell survival in the presence of bile acids, its inhibition would be expected to cause tumor cell death in the colon.


   Acknowledgments
 
Grant support: NIH grants R21 CA 116329 and R01 CA 108959.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Cooperative Human Tissue Network for supplying human tissue specimens used in this research, Dr. Marty W. Mayo for providing plasmids encoding p65 and p50, and Christina Niemeyer for editorial assistance with this manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1 http://www.ncbi.nlm.nih.gov/ Back

2 http://www.gene-regulation.com Back

Received 10/ 5/06. Revised 6/15/07. Accepted 7/11/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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