Cofactor of BRCA1: a Novel Transcription Factor Regulator in Upper Gastrointestinal Adenocarcinomas

Research Article

Cofactor of BRCA1: A Novel Transcription Factor Regulator in Upper Gastrointestinal Adenocarcinomas

Patricia A. McChesney,1 Sarah E. Aiyar,2 Ok-Jae Lee,1,4,5 Alexander Zaika,1,5 Christopher Moskaluk,3 Rong Li,2 and Wa’el El-Rifai1,5

Departments of 1Internal Medicine, 2Biochemistry and Molecular Genetics, and 3Pathology, University of Virginia, Charlottesville, Virginia; 4Department of Internal Medicine and Institute of Health Science, College of Medicine, Gyeongsang National University, Jinju, Korea; and 5Department of Surgery and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee

Abstract regulatory complex, NELF-B (6). In its role as NELF-B, COBRA1 has a Cofactor of BRCA1 (COBRA1) is a newly characterized been shown to bind directly to the active estrogen receptor (ER) member of the negative elongation factor (NELF) complex. and recruit the other NELF subunits to stall RNA polymerase II at In this work, we show that COBRA1 is overexpressed in the the promoter proximal region, thereby preventing transcription majority of primary upper gastrointestinal adenocarcinomas elongation (7, 8). The polymerase stalling mechanism is used by the (UGC), and its overexpression correlates with down-regulation NELF complex to inhibit estrogen-induced transcription of TFF1 in of TFF1. We have detected overexpression of COBRA1 mRNA breast cancer (7). using quantitative real-time reverse transcription-PCR in 28 TFF1 is one of a family of highly conserved, secreted trefoil (79%) primary UGCs. Immunohistochemical analysis of UGC peptide proteins (9). TFF1 is normally expressed only in the upper gastrointestinal system, in the upper portion of the glandular pits tissue arrays that contained 70 tumor samples showed moderate-strong staining for COBRA1 in 60 (84%) tumors. where it is secreted to become part of the protective mucus layer. Interestingly, the tumor samples showed absent-weak staining Tissue damage leads to up-regulation of TFF1, whereas gastric for TFF1 in 45 (65%) of the tumors. Simultaneous loss of TFF1 tumorigenesis is associated with loss of TFF1 expression (10, 11). expression and overexpression of COBRA1 was observed in 42 TFF1 expression is down-regulated in >70% of UCGs (12). Of those, of 70 (60%) tumors. Using small interfering RNA technology approximately one third shows promoter hypermethylation of with gastric cancer cells, we have shown that COBRA1 TFF1 (13), whereas gene mutation is detected in <5% (10). Other inhibition leads to increased TFF1 promoter activity and gene mechanisms that contribute to TFF1 down-regulation remain expression. Promoter analysis of TFF1 indicated that regula- unclear in UGCs. tion of TFF1 by COBRA1 is estrogen independent in contrast to In contrast to studies of TFF1 in UGCs, other tumors, such as breast cancer. Moreover, COBRA1 regulation of TFF1 in gastric carcinomas of the breast and liver, often show high levels of ah cancer cells was independent of NELF-E. Using several expression of TFF1. In the breast cancer model, both ER and the g truncated mutants and site mutants of the TFF1 promoter, ligand-independent ER have been conclusively shown to stimulate TFF1 expression(14). Recent data indicate that estrogen response we have shown that COBRA1 can negatively regulate the activator protein-1 (AP-1) complex at the TFF1 promoter and element is dominant in stimulating TFF1 expression in breast thus down-regulate TFF1 expression in gastric cancer cell cancer cells (15). Similarly, the importance of estrogen stimulation lines. Electrophoretic mobility shift assay showed that in induction of TFF1 expression was observed in hepatic cancers COBRA1 attenuates AP-1 binding to DNA. Our results suggest (16), although cross-talk between multiple pathways seems to be COBRA1 as a novel oncogene in UGCs that regulate AP-1 involved. binding and the expression of TFF1 in upper gastric epithelia. In this work, we have identified COBRA1 as a novel oncogene in (Cancer Res 2006; 66(3): 1346-53) UGCs and showed high expression levels of COBRA1 mRNA and protein in UGCs. We have further examined the regulatory effects of COBRA1 on TFF1 gene expression in gastric adenocarcinoma Introduction cell lines. Upper gastrointestinal adenocarcinomas (UGC) are among the most prevalent causes of cancer-related deaths in the world, and Materials and Methods the disease frequency is rapidly rising (1, 2). This category of Tissue samples. A total of 70 UGCs and 19 normal stomach paraffin- cancers includes adenocarcinomas of the stomach, gastroesopha- embedded tissue samples were available for the immunohistochemical geal junction (GEJ), and lower esophagus. The incidence of Barrett’s analysis. In addition, 28 gastric, GEJ, and lower esophageal tumors and 22 related GEJ and lower esophageal adenocarcinomas is rising faster normal gastric epithelial samples were dissected for optimal tumor content than ever before (3, 4). (>70%) and used for mRNA extraction, cDNA synthesis, and subsequent The cofactor of BRCA1 (COBRA1) was recently identified as a quantitative real-time reverse transcription-PCR (RT-PCR) assays. All tissue BRCA1-interacting protein (5) and was found to be a member of samples were collected in accordance with institutional review board– the negative elongation factor (NELF) transcription elongation approved protocols. Tissues were stained with H&E, and representative regions were selected for inclusion in a tissue array. Tissue cores with a diameter of 0.6 mm were retrieved from the selected regions of the donor blocks and punched to the recipient block using a manual tissue array Requests for reprints: Wa’el El-Rifai, Vanderbilt University Medical Center, 1255 instrument (Beecher Instruments, Silver Spring, MD); samples were Light Hall, 2215 Garland Avenue, Nashville, TN 37232. Phone: 615-322-7934; Fax: 615- punched in triplicates. Control samples from normal epithelial specimens 343-1355; E-mail: [email protected]. I2006 American Association for Cancer Research. were punched in each sample row. Sections (5 Am) were transferred to doi:10.1158/0008-5472.CAN-05-3593 polylysine-coated slides (SuperFrostPlus, Menzel-Gla¨ser, Braunschweig,

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Germany) and incubated at 37jC for 2 hours. The resulting tumor tissue (Roche, Indianapolis, IN) cationic lipid transfection reagent. Cells were array was used for immunohistochemical analysis. All tumors and normal harvested and assayed 48 hours after transfection. For promoter analysis, gastric mucosal epithelial tissues were histologically verified. The adeno- the following plasmids were used in transient transfections: TFF1-Luc carcinomas were collected from stomach, GEJ, and lower esophagus and (pGL3-Luc containing the 1,050-bp TFF1 promoter fragment), TFF1-DERE- ranged from well differentiated to poorly differentiated, stages I to IV, with a Luc (18), pcDNA3.1 FLAG-COBRA1, pSUPER-COBRA1, and pSUPER-EGFP. mix of intestinal and diffuse-type tumors. Luciferase assay. pcDNA3.1 FLAG-COBRA, pSUPER-COBRA, pSUPER- Immunohistochemistry. Immunohistochemical analysis of COBRA1 EGFP, or pcDNA3.1 (2.4 Ag) was cotransfected with 0.4 Ag pS2Luc or and TFF1 protein expression was done on a tumor tissue array that pS2DERE-Luc (19) into gastric cells by Fugene 6 reagent. Transfected cells contained 70 UGC samples (stomach, GEJ, and lower esophageal) and 19 were allowed to incubate 48 hours before assay using Dual Luciferase Assay normal gastric tissues. Adjacent normal gastric and esophageal tissues were kit (Promega, Madison, WI). Results from the luciferase assay (Figs. 2-4) available for comparisons. Dewaxing and rehydration by descending were normalized against total protein content in each assay from each concentrations of ethanol was followed by antigen retrieval [20 minutes transfection. All samples were tested in triplicate in each experiment. Data in a microwave, 450 W, 10 mmol/L EDTA (pH 8.0)]. Blocking was done with represent the average luciferase activity per microgram of protein from all 10% goat serum in PBS for 5 minutes. All sections were incubated with samples tested; error bars represent the average SDs from at least two either anti-TFF1 mouse monoclonal antibody (anti-pS2, NeoMarkers, experiments (SE). Estrogen treatments of transfected cells consisted of Fremont, CA) or anti-COBRA1 rabbit polyclonal antibody (R. Li; ref. 5) 10 nmol/L h-estradiol (Sigma, St. Louis, MO) in growth medium (final followed by washing in PBS and incubation with anti-mouse or anti-rabbit concentration) for 24 hours before luciferase assay. secondary antibody (dilution 1:20; MBL; MoBiTec, Goettingen, Germany) for Western blot assay. AGS cells transiently transfected with the COBRA 1 hour at room temperature and then washed in PBS. For revealing positive reagents were lysed in PBS. Protein concentration was measured using a immunohistochemical reaction, the Vectastain ABC-AP kit (mouse IgG, protein assay (Bio-Rad). Samples were suspended in Laemmli loading buffer Vector, Alexis, Gruenberg, Germany) was used as chromogen substrate, and and subjected to sonication and boiling. Protein (2 Ag) was used and the specimens were counterstained with hematoxylin and mounted with transferred to a polyvinylidene difluoride membrane (Amersham, Piscat- DEPEX%. Specificity of immunostaining was checked by omitting single away, NJ). FLAG-COBRA was detected using anti-COBRA mouse monoclo- steps in the protocol and by replacing the primary antibody with non- nal antibody (7). Actin was detected using a monoclonal antibody (sc-8432, immune serum. SantaCruzBiotechnology,SantaCruz,CA).Secondaryanti-mouse Immunohistochemical results were evaluated for intensity and frequency horseradish peroxidase antibody (Pierce, Rockford, IL) was detected using of staining of nuclear and cytoplasmic components and the whole. The chemiluminescence (Pierce SuperSignal Femto kit). intensity of staining was graded as 0 (negative), 1 (weak), 2 (moderate), and TFF1 promoter mutant generation. In addition, five truncation 3 (strong). The frequency was graded from 0 to 4 by percentage of positive mutants and three site mutants (graphed in Fig. 3A) were prepared as cells as follows: grade 0, <3%; grade 1, 3% to 25%; grade 2, 25% to 50%; described below. Electrophoretic mobility shift assay (EMSA) was done grade 3, 50% to 75%; and grade 4, >75%. The index score was the product of using stably transfected AGS cell lines. Stable cell lines were established by multiplication of the intensity and frequency grades, which was then binned transfection (as described) for 48 hours followed by antibiotic selection in into a four-point scale: index score 0, product of 0; index score 1, products 1 800 ng/mL puromycin (Sigma) for the pSUPER vectors or 600 Ag/mL G418 and 2; index score 2, products 3 and 4; and index score 3, products 6 to 12. (Sigma) for the pcDNA3.1 vectors. Selection was monitored by incubating Index score 2 or 3 was determined as the overexpression of proteins. untransfected cells in the antibiotic medium and was considered complete Real-time RT-PCR. The mRNA was isolated using RNeasy kit (Qiagen, when all untransfected cells were dead. Stable cells were then plated at low GmbH, Hilden, Germany). Single-stranded cDNA was synthesized using density for cloning, and colonies were screened by Western blot analysis for Advantage RT-for-PCR kit (Clontech, Palo Alto, CA). Quantitative PCR was total COBRA1 expression. done using iCycler (Bio-Rad, Hercules, CA) and threshold cycle number was For generation of promoter fragments and mutants, PCR was used to determined using iCycler software version 3.0 (Bio-Rad) with SYBR Green generate the TFF1 promoter fragments shown in Fig. 3A. The fragments technology (17). Specific primers for COBRA1, TFF1, and HPRT1 were were purified (Qiagen, Valencia, CA), subjected to restriction digest with designed. The primers used for real-time RT-PCR were obtained from XhoI and MluI (Roche), and ligated into pGL3-Luc vector (Promega). GeneLink (Hawthorne, NY), and their sequences are available on request. Following sequence verification, the plasmid constructs were used for Reactions were done in triplicate, and threshold cycle numbers were averaged. transient transfection of cultured cells as described. Site-directed muta- A single melt curve peak was observed for each sample used in data analysis, genesis was done using the QuickChange Site-Directed Mutagenesis kit thus confirming the purity and specificity of all amplified products. The (Stratagene, La Jolla, CA) on the full-length TFF1 promoter. The activator results for COBRA1 and TFF1 were normalized to HPRT1, which had minimal protein-1 (AP-1) consensus binding sites (1V and 2V) were mutated to PstI variation in all normal and neoplastic gastric samples tested. Fold expression and EcoRI restriction sites, respectively. Following sequence verification, the was calculated according to the formula: 2(Rt À Et) /2(Rn À En), where Rt is the 1V mutant was further mutated to a double mutant construct. threshold cycle number for the reference gene observed in the tumor, Et is the Electrophoretic mobility shift assay. Chemiluminescent EMSA was threshold cycle number for the experimental gene observed in the tumor, Rn done on an oligonucleotide containing the AP-1 consensus binding site is the threshold cycle number for the reference gene observed in the normal (Panomics, Redwood City, CA) using the Lightshift EMSA kit (Pierce). sample, and En is the threshold cycle number for the experimental gene Briefly, biotinylated target DNA was incubated with nuclear extract (from observed in the normal sample. Rn and En values were taken from the 22 COBRA1-transfected or siRNA COBRA1-transfected AGS cells; NE-PER kit, normal mucosa samples that were analyzed. The average fold expression is Pierce) and appropriate buffer. The reactions were run on 4% acrylamide shown for all samples in Fig. 1A. gel and transferred to Hybond N+ membrane (Amersham) using semidry Cell culture and vectors. AGS, MKN45, and KATO III gastric electrophoretic transfer (Bio-Rad). Following cross-linking, the membrane adenocarcinoma cells were obtained from American Type Culture was treated according to kit instructions and exposed to Kodak X-ray film Collection (Manassas, VA) and cultured in DMEM with 10% fetal bovine (Fisher Scientific, Pittsburgh, PA). serum (FBS). In addition, T47D breast cancer cells from R. Li (7) were j cultured in RPMI with 10% FBS at 37 C, 5% CO2. RT-PCR revealed that all Results gastric adenocarcinoma cells expressed high levels of COBRA1 and low undetectable levels of TFF1 (data not shown). Cells were passaged regularly Overexpression of COBRA1 and loss of expression of TFF1 and seeded at 50% confluency in 24-well culture plates for transfection with in UGCs. The majority (79%) of primary UCG samples showed pcDNA3.1 FLAG-COBRA1 (5) for COBRA1 overexpression, pSUPER-COBRA1 elevated mRNA expression of COBRA1 and decreased mRNA (7) for expression of small interfering RNA (siRNA) COBRA1, or vector expression of TFF1 (93%) relative to normal tissue samples. The controls. Transient transfections were done using 2 Ag DNA and Fugene 6 real-time RT-PCR of mRNA showed that elevated expression of www.aacrjournals.org 1347 Cancer Res 2006; 66: (3). February 1, 2006

Figure 1. COBRA1 is overexpressed in UGCs. A, real-time RT-PCR of 28 primary UGCs compared with 22 normal samples to measure expression of COBRA1 and TFF1. The threshold cycle number (Ct) from each sample, run in triplicate, was compared with the average Ct of 22 different normal gastric samples to calculate the fold increase or decrease of gene expression. Bars, SE. Y axis, fold expression relative to normal; shaded area, a range of mRNA expression that is considered normal (0.3-3.0 fold expression). B, immunohistochemical staining of UGCs (left) and normal upper gastrointestinal tissues (right) for COBRA1 and TFF1. Brown, protein expression, nuclear localization of COBRA1, and upper gastric pit and epithelial secretion of TFF1. Results also show loss or reduction of TFF1 coincidental to elevated expression of COBRA1.

COBRA1 is associated with attenuated expression of TFF1 in most association with a particular tumor site (stomach, GEJ, and lower of the cases (Fig. 1A). Protein levels of COBRA1 and TFF1 paralleled esophageal), histopathologic subtype, or clinical variables was not the mRNA levels as confirmed by immunohistochemical staining of possible to ascertain. UCG tissue arrays (Fig. 1B). The immunohistochemical staining COBRA1 regulates TFF1 Expression in gastric cancer cells. showed that COBRA1 location is primarily nuclear, whereas TFF1 is Activation of the TFF1 promoter was significantly repressed by cytoplasmic or secreted. There was an inverse relationship between overexpression of COBRA1 (Fig. 2A), whereas reduction of COBRA1 the two proteins in adenocarcinoma tissues, with TFF1 immuno- by siRNA led to a substantial increase in TFF1 promoter activity. histochemical staining intensity being weak-absent in 45 of 70 AGS, MKN45, and KATO III gastric adenocarcinoma cells and (65%) and COBRA1 being moderate-strong in 60 of 70 (84%). T47D breast cancer cells were transiently transfected with lucif- Interestingly, tumors with simultaneously elevated COBRA1 and erase reporter plasmids as described and treated with 10 nmol/L weak-absent TFF1 were 42 of 70 (60%). Our UGC tissue arrays were h-estradiol for 24 hours. In contrast to breast cancer, COBRA1 composed of 7 (10%) female samples and 63 (90%) samples from regulation of TFF1 was not estrogen dependent in gastric males, which represent the expected distribution of these tumors in adenocarcinoma (Fig. 2A). Addition of estrogen to the cell culture our population. Due to the relatively high frequency of TFF1 or mutation of the ER-binding site (ERE versus DERE, where the and COBRA1 alteration and the small number of cases analyzed, binding site sequence is mutated to EcoRI and EcoRV restriction

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. COBRA Regulates AP-1 in UGC sites; ref. 19) in the TFF1 promoter was ineffective in altering stimulate TFF1 expression (7). To test the function of NELF-E COBRA1 regulatory activity (Fig. 2A). Similar results were obtained on the TFF1 promoter in UGC, the stimulatory activity of siRNA with MKN45 and KATO III gastric adenocarcinoma cells (data not NELF-E was tested in AGS cells (Fig. 2C). For this experiment, we shown). To verify our technique, this procedure was also done in transiently transfected cells with the pSUPER-NELF-E vector, which breast cancer cells (T47D) and resulted in strong TFF1 promoter expresses siRNA NELF-E (ref. 7; Fig. 2C). In contrast to knockdown stimulation as shown previously (Fig. 2B; ref. 7). of COBRA1, knockdown of NELF-E had no stimulatory effect on the In breast cancer, COBRA1 regulation of TFF1 is a function of its TFF1 promoter activity. Thus, COBRA1 can act independently from role as NELF-B; knockdown of NELF-E has been shown to similarly the NELF-E in gastric cancer cells.

Figure 2. COBRA1 regulates TFF1 expression in gastric adenocarcinoma cell lines. A, luciferase reporter assays were used to evaluate TFF1 promoter activity in AGS gastric cancer cells. Cells were transfected with the indicated plasmids (TFF1-Luc, TFF1DERE-Luc, pcDNA3.1 FLAG-COBRA1, pcDNA3.1, or pSUPER-COBRA1) for 24 hours before estrogen treatment (10 mmol/L, 24 hours). Columns, average of three trials; bars, SE. B, luciferase reporter assays show that TFF1 promoter activity in T47D breast carcinoma cells is responsive to estrogen treatment as well as siRNA for COBRA1. Columns, average of two trials; bars, SE. C, luciferase reporter assays show that TFF1 promoter activity is unaffected by siRNA for NELF-E (pSUPER-NELF-E), whereas siRNA for COBRA1 (NELF-B; pSUPER-COBRA1) strongly stimulates promoter activity. Columns, average of three trials; bars, SE. D, real-time RT-PCR of endogenous TFF1 mRNA expression confirms that ectopic expression of FLAG-COBRA1 leads to down-regulation of TFF1, whereas reduction of COBRA1 expression via siRNA leads to elevated levels of TFF1. Columns, fold induction or inhibition relative to vector transfected control cells. Dark columns, reduction of endogenous TFF1 mRNA when COBRA1 is ectopically elevated; light gray columns, elevated levels of TFF1 mRNA relative to controls when COBRA1 is knocked down by transfected siRNA. E, Western blot analysis confirms overexpression of COBRA1 on transient transfection of AGS with pcDNA3.1 FLAG-COBRA1. www.aacrjournals.org 1349 Cancer Res 2006; 66: (3). February 1, 2006

Figure 3. COBRA1 regulates TFF1 expression via AP-1. A, truncation mutants of the TFF1 promoter were generated and inserted into the luciferase reporter vector to precisely locate the site of COBRA1 regulation. Assays indicate that COBRA1 is regulating AP-1 stimulation of the TFF1 promoter. Columns, average of three trials; bars, SE. B, site-directed mutagenesis of the AP-1-binding sites abrogates COBRA1 regulation of the TFF1 promoter. Luciferase reporter assays were done on mutated, full-length, TFF1 promoter/ luciferase constructs. Columns, average of three trials; bars, SE. C, activation of the TFF1 promoter by knockdown of COBRA1 is synergistic with activation by ectopic expression of AP-1 complex components as seen by luciferase reporter assays. Columns, average of three trials; bars, SE.

The luciferase reporter activity data were verified by examination overexpression of c-Jun (Fig. 3B, data set 4, P5 and P6). These data of the expression of endogenous TFF1 under conditions of COBRA1 implied that COBRA1 might regulate the AP-1 complex (Fig. 3B). overexpression and knockdown. Expression of endogenous TFF1 Confirmation of COBRA1/AP-1 interactions was achieved in was measured by real-time RT-PCR in the presence of over- several steps. First, full-length, wild-type TFF1 promoter luciferase expression and attenuated expression of COBRA1 (Fig. 2D) and reporter was cotransfected with siRNA COBRA1 and c-Jun or c-Fos, Western blot analysis (Fig. 2E). Measurement of COBRA1 representative of AP-1 complex members. These data show a expression in these samples verifies transfection efficacy. synergistic activation of the TFF1 promoter when AP-1 members COBRA1 regulation of TFF1 occurs at the AP-1-binding sites. were overexpressed and COBRA1 was knocked down by siRNA Data showed that COBRA1 was not regulating TFF1 at the ER site; (Fig. 3C, lanes 2-4 versus lanes 5 and 6; Fig. 4, data set 4). Second, therefore, localization of COBRA1 influence was done. Truncation the AP-1 consensus binding sites on the full-length TFF1 promoter mutants of the COBRA1 promoter were generated by PCR and at À354 and À710 bp from the 5V untranslated region were each ligated into the pGL3-Basic luciferase reporter vector (Fig. 3A). The mutated by site-directed mutagenesis. Following sequence verifi- mutants serially eliminated consensus-binding sites of transcrip- cation, luciferase reporter assays confirmed the significance of AP-1 tion factors that were known to alter TFF1 expression (7, 16, influence on TFF1 expression and COBRA1 regulation (Fig. 4). 20, 21). Transient transfection of AGS gastric adenocarcinoma cells Cells were cotransfected with the mutant promoter/reporter was followed by luciferase assays. Figure 3B (data set 3) shows that vector and combinations of siRNA for COBRA1 (lanes 5-8) and siRNA COBRA1 stimulated each of the truncated TFF1 promoters c-Fos (lanes 9-12). Both siRNA for COBRA1 and c-Fos activated the until the AP-1 site was lost (P5 versus P6-P8). Loss of the AP-1 site wild-type promoter, and the combination had a synergistic effect was confirmed by stimulation of the truncation mutants with as seen previously (lane 13). The mutation of the first or primary

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AP-1-binding site (À710 bp) had little effect on COBRA1 or c-Fos in UGCs. First, we showed that the COBRA1 transcriptional activation (lanes 6, 10, and 14). However, mutation of the second or cofactor negatively regulates TFF1 expression in vitro and likely secondary AP-1 site (À354 bp) dramatically reduced both COBRA1 in vivo. Second, we showed that, unlike breast cancer, COBRA1 and c-Fos effects (lanes 7, 11, and 15), and mutation of both regulation of TFF1 is estrogen independent and NELF-E indepen- (double DAP-1) resulted in further promoter inhibition (lanes 8, 12, dent. Finally, we have shown that COBRA1 inhibits AP-1 activation and 16), suggesting a small amount of compensation between of the TFF1 gene in UGC. the two sites when one is mutated. As a control for site-directed COBRA1 regulation of TFF1 may occur at a variety of different mutagenesis, we tested the activity of a second transcription sites on the promoter (Fig. 3A; refs. 7, 20–22). As COBRA1 does not factor-binding site on the mutated promoters. TFF1 expression is have a DNA-binding domain, it acts solely as a cofactor. In breast inhibited by nuclear factor-nB (21); therefore, InB superrepressor cancer, COBRA1 binds to the ER to inhibit hormone-dependent was used to successfully stimulate the TFF1 promoter activity (data activation of TFF1 (7). However, in gastric cancer cells, we found not shown), thus showing that the other transcription factor- that TFF1 activation is hormone independent but, nevertheless, binding sites on the TFF1 promoter remained unaltered by inhibited by COBRA1. Therefore, we conducted a search for other mutation of the AP-1 sites. transcription factors that may bind COBRA1 and lead to inhibition COBRA1 regulates AP-1 complex members. COBRA1 nega- of TFF1 expression. Our data led to COBRA1 inhibiting AP-1 tively regulates AP-1 binding to DNA. EMSA data (Fig. 5) showed stimulation of TFF1 expression. Data regarding the interaction of that reduction of COBRA1 by siRNA led to increased DNA shift AP-1 with COBRA1 are supported by another recent work (23), (lane 4), whereas elevation of COBRA1 expression led to attenuated where the authors showed direct binding between COBRA1 and DNA shift (lane 2). The AP-1 shift was verified by antibody c-Fos. Whereas our site-directed mutagenesis of AP-1-binding supershift using anti-phospho-c-Jun (lane 6). We chose to use a locations was effective in showing COBRA1/AP-1 regulatory simple, direct oligonucleotide mobility shift assay to examine interactions, the results did not show that COBRA1 has absolute COBRA1 interactions with the AP-1 transcription complex because control of TFF1 promoter activity. We hypothesize that, because the TFF1 promoter contains numerous transcription factor-binding there are many transcription factor-binding sites in the 1,050-bp sites, and our EMSAs using a short fragment of the TFF1 promoter TFF1 promoter and our experiments only mutated two of resulted in a complex band pattern (data not shown). these, influence on the expression of this gene is likely to be multifactorial. Interactions between AP-1 and the ER have been studied for Discussion several years. In hepatic carcinoma cells, the AP-1 transcription The current work provides the first evidence for overexpression factor complex [stimulated by the mitogen-activated protein kinase of COBRA1 and its physiologic role as inhibitor of TFF1 expression (MAPK) pathway] is known to enhance the estrogen-mediated

Figure 4. Mutation of the AP-1 consensus binding sites results in reduced promoter sensitivity to siRNA for COBRA1. AP-1 sites at À354 and À710 bp of the TFF1 promoter were mutated to EcoRI and PstI endonuclease sites, respectively. Promoter activity was measured by luciferase reporter assay under treatment conditions shown. Columns, average of three trials; bars, SE.

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independently of NELF-E. Therefore, it is possible that COBRA1 exclusively inhibits AP-1, preventing AP-1 binding to the TFF1 promoter and thus inhibiting gene transcription, and that COBRA1 does not bind to RNA polymerase II as seen in similar gene regulation involving NELF. Recent work characterizing the NELF complex in Drosophila melanogaster has shown that the A and E subunits of NELF do not have conserved sequences when compared with mammalian coun- terparts. However, the C/D and B subunits are uniquely conserved (28), suggesting that COBRA1 is an ancient and important regulator of gene expression. Therefore, COBRA1 may be an important cofactor in regulation of gene expression during tumorigenesis. Whereas TFF1 expression is lost in UGCs, TFF1 is aberrantly expressed in other cancers around the body. Studies show that TFF1 may be important for tumor metastasis in breast and hepatic cancers, and regulation of this gene has been under investigation for some time (14, 15). Data show that whereas the estrogen response element is dominant in stimulating TFF1 expression in breast cancer cells (7), the binding of the AP-1 complex is Figure 5. COBRA1 decreases AP-1 binding specificity at the TFF1 promoter. mandatory and dominant for estrogen stimulation of this gene in EMSA was done using an oligonucleotide target containing an AP-1 consensus binding site. Nuclear extracts from AGS cells treated with COBRA1 reagent hepatic cancer cell lines (16). plasmids were incubated with the target DNA and anti-phospho-c-Jun The decline of TFF1 expression during upper gastrointestinal monoclonal antibody. carcinogenesis is well documented (10, 21, 29). Nearly 50% of all UGCs tested show marked reduction of TFF1 expression (9). activation of the TFF1 promoter (16). Binding between c-Jun The TFF1 knockout mouse develops gastric dysplasia and and ERa was shown by mammalian two-hybrid assay and was gastric cancer similar to human (29, 30), leading to the hypoth- independent of ER binding to DNA (24), and mutation of a esis that TFF1 is a UGC tumor suppressor. Evidence has shown conserved lysine on ERa results in hyperstimulated AP-1 that less than one third of cases where TFF1 expression is lost activation of gene promoters (25), further supporting a direct can be explained by promoter hypermethylation, gene mutation, interaction between ER and AP-1. However, the ER/AP-1 or deletion (10, 31). Therefore, the current data suggest that pathways described thus far are all ligand dependent. AP-1 COBRA1 as a novel oncogene that can inhibit TFF1 expression and ER are also known to be activated by the MAPK pathway and may account for the loss of TFF1 expression in the majority (16, 26), and the possibility remains that ER may bind to AP-1 of tumors where mutations and DNA methylation cannot be in response to MAPK influence via protein-protein interactions. found. COBRA1 regulation of ER in breast cancer was also shown Inconclusion,wehaveshownthatCOBRA1isanovel to be ligand dependent (7). The data presented in this article transcription cofactor in upper gastrointestinal carcinogenesis. show a ligand-independent regulation of TFF1 (Fig. 2A). DNA COBRA1 negatively regulates TFF1 in gastric cancer cells by microarray data on UCG show a basal level of ER expression, inhibiting AP-1 activation of transcription. As AP-1 affects which remains unchanged during carcinogenesis (data not transcription for multiple genes, the oncogenic potential of shown), and data presented in this article do not exclude the COBRA1 could result in widespread effects that require further possibility of ligand-independent interactions between ER and studies. AP-1, nor potential influence of MAPK on either of these proteins. COBRA1 acts as NELF-B in breast cancer along with other Acknowledgments subunits A, C/D, and E. NELF-B (COBRA1) binds to the ER (7), Received 10/5/2005; revised 11/8/2005; accepted 11/15/2005. whereas NELF-E is responsible for interactions with RNA (27) and Grant support: National Cancer Institute grants CA93999 and CA106176 is hypothesized to be critical for the RNA polymerase II ‘‘tethering’’ (W. El-Rifai) and DK064604 (R. Li). The costs of publication of this article were defrayed in part by the payment of page or transcriptional ‘‘pausing’’ caused by the NELF complex. charges. This article must therefore be hereby marked advertisement in accordance However, our studies with gastric cancer show that COBRA1 acts with 18 U.S.C. Section 1734 solely to indicate this fact.

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