- Email: [email protected]
825 Intestinal Inflammation Targets E. Coli NC101 Transcriptome Response and Promotes Development of Colorectal Cancer (CRC)
AGA Abstracts
Figure 2. (A) A20 interacts with Axin through its N-terminal domain. FLAG-tagged full length (F), N-terminal (N), or C-terminal (C) A20 was co-expressed in RKO cells with MYCtagged Axin and then subject to immunoprecipitation (IP) and immunoblot (IB) for the indicated proteins. (B) A20 supports β-catenin ubiquitylation and degradation. RKO cells were transfected with siRNA to A20 or a scrambled control and then stimulated with recombinant human wnt3a (rhwnt3a) for the indicated times. Lysates were then subject to immunoprecipitation (IP) and immunoblot (IB) for the indicated proteins. (C) Acute deletion of A20 from IECs leads to increased levels of Cyclin D1 and MYC mRNA in vivo. VillinER/Cre A20FL/FL (fl/fl) and control Villin-ER/Cre A20+/+ (+/+) were injected with 1mg of tamoxifen i.p. daily for 5 days. IECs were then isolated and studied for expression of Cyclin D1 (upper panel) and MYC (lower panel) mRNAs by qPCR. Each point represents one mouse. * indicates p , 0.05; ** indicates p , 0.01.
Fig. 1. Patients with the CC genotype of the CCK-B receptor survive significantly longer than those with the AA or AC genotypes. *** p = 0.032. 823 A20 Restricts Canonical Wnt Signaling in Intestinal Epithelial Cells and Suppresses Colon Carcinogenesis Ling Shao, Shigeru Oshima, Bao Duong, Rommel Advincula, Julio Barrera, Barbara Malynn, Averil I. Ma
824 Regulation of Death-Inducing Signaling Complex by Axl Mediates TRAIL Resistance in Esophageal Adenocarcinoma Jun Hong, DunFa Peng, Wael El-Rifai, Abbes Belkhiri
Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Important characteristics of tumors include an ability to proliferate, resist cell death, and benefit from a tumorpromoting inflammatory environment. A well-known regulator of cell death and inflammation is the ubiquitin-editing enzyme, A20 (also known as TNFAIP3). Genome wide associations have linked polymorphisms in A20 to multiple inflammatory and autoimmune diseases including inflammatory bowel disease. Global genetic deficiency of A20 in mice leads to lethal systemic inflammation while intestinal epithelial cell (IEC) specific deletion sensitizes animals to chemical models of colitis. Here, we have studied the role of A20 in intestinal homeostasis and tumorigenesis. An initial search of the NCBI Genome Expression Omnibus revealed that A20 expression is consistently reduced in human colonic adenomas compared to normal surrounding colonic tissues (Fig. 1A). To investigate A20's potential roles in regulating colon carcinogenesis, we generated mice lacking A20 specifically in intestinal epithelial cells (A20FL/FL villin-Cre) and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APCmin). While A20FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20FL/FL villin-Cre APCmin/+ mice spontaneously developed far greater numbers and larger colonic polyps than control APCmin mice (Fig. 1B). Unexpectedly, we found that A20 suppresses colon carcinogenesis, in part, through a direct interaction with the wnt/ β-catenin pathway. Using a human colon cancer cell line with intact wnt signaling, A20 knockdown lead to a seven-fold increase in β-catenindependent luciferase reporter activity compared to a scrambled control siRNA after stimulation with recombinant human wnt3a. β-catenin stability is tightly regulated through sequential phosphorylation, ubiquitylation, and proteasomal degradation by a complex whose core components include APC and the scaffolding protein Axin. We find that A20 binds Axin through its N-terminal domain (Fig. 2A). Furthermore, A20 restricts canonical wnt signaling by promoting ubiquitination and degradation of β-catenin in intestinal epithelial cells (Fig. 2B). To support a role for A20 in regulating β-catenin signaling in vivo, we generated mice expressing a tamoxifen inducible Cre-recombinase under the villin promoter. In this system, acute deletion of A20 from intestinal epithelial cells leads to enhanced epithelial expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer (Fig. 2C). Taken together, these findings demonstrate that A20 is a tumor suppressor in the intestine and uncovers a novel role for A20 in restricting canonical wnt/ β-catenin signaling.
Background: The majority of esophageal adenocarcinoma (EAC) is intrinsically resistant to DNA-damaging therapies. Therefore, an alternative therapeutic approach based on activation of death receptors may be warranted. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding to DR4 or DR5 death receptors. TRAILinduced activation of death receptors leads to the formation of death-inducing signaling complex (DISC), which activates caspase-8 and the extrinsic apoptosis pathway. Unfortunately, a significant proportion of cancer cells are refractory to TRAIL-induced cytotoxicity. AXL receptor tyrosine kinase has been reported to regulate cell growth and survival in solid tumors. The aim of this study was to investigate the role of AXL in TRAIL resistance and elucidate the underlying molecular mechanism in EAC. Methods and Results: To investigate the role of AXL in cell survival, we utilized two EAC cell models. The cell viability assay results demonstrated that the reconstitution of AXL expression in OE33 cells significantly enhanced cell survival in response to treatment with increasing concentrations of TRAIL for 5h (p,0.01), and knockdown of endogenous AXL in FLO-1 cells significantly enhanced the sensitivity of cells to TRAIL (p ,0.05). To ascertain the role of AXL in regulating TRAILinduced apoptosis, we selected the TRAIL concentration 40 ng/ml to induce apoptosis. The Annexin V/PI staining and FACS analysis data demonstrated that the reconstitution of AXL expression in OE33 cells suppressed apoptosis events by 53% relative to control in response to TRAIL (p,0.01). In accordance with this finding, Western blot analysis showed considerably higher protein levels of cleaved caspase-8, -9, -3, and cleaved PARP in control cells than AXL-expressing cells in response to TRAIL. On the contrary, knockdown of highly expressed endogenous AXL in FLO-1 cells significantly increased apoptosis by 74% relative to control (p=0.01), and blocked activation of caspase-8, -9 and -3, and cleavage of PARP in response to TRAIL. To determine the mechanism of TRAIL resistance, we evaluated the effect of AXL on the expression of death receptors. Real-time PCR and Western blot data showed that AXL did not down-regulate DR5 and DR4 receptors OE33 and FLO-1 cells. We next examined if AXL regulates death-inducing signaling complex (DISC), thereby blocking TRAIL-induced apoptosis. Indeed, immunoprecipitation and Western blot data indicated that AXL associated with DR5 death receptor and AXL/DR5 association had no effect on FADD interaction, but prevented the association of pro-caspase-8 with FADD in response to TRAIL. Conclusions: Our data uncovered a novel mechanism of TRAIL resistance mediated by AXL through regulation of the DISC, thereby preventing activation of caspase-8, a key molecular event in TRAIL-induced apoptosis. 825 Intestinal Inflammation Targets E. Coli NC101 Transcriptome Response and Promotes Development of Colorectal Cancer (CRC) Marcus Muehlbauer, Raad Gharaibeh, Janelle C. Arthur, Anthony Fodor, Christian Jobin
Figure 1. A20 is a Tumor Suppressor in the Intestine. (A) Human colonic adenomas express less A20 mRNA than normal surrounding colonic mucosa. From Genome Expression Omnibus (GDS2947). (B) Tumor number (left panel) and aggregate tumor size (right panel) in colons from intestinal epithelial cell specific A20 deleted (fl/fl) and wild-type (+/+) mice harboring the APCmin mutation. * indicates p ,0.05, ** indicates p,0.01
AGA Abstracts
Background: We have previously shown that development of CRC depended upon the genotoxic activity of adherent-invasive Escherichia coli (Arthur JC et al., Science 2012). Aim: Assess the interplay between inflammation, E. coli gene expression and CRC development. Methods: Germ-free Il10-/- and Il10-/-; Rag2-/- mice were mono-associated with E. coli NC101. Colon tumors were induced by 6 weekly i.p. injections of azoxymethane (AOM). Changes in E. coli NC101 gene expression were investigated using germ-free Il10-/- mice mono-associated with E. coli NC101 for 2 days (no inflammation), 12 weeks (chronic inflammation) and 20 weeks (inflammation and CRC). Inflammation and dysplasia/tumors were scored histologically. Transcripts of inflammatory mediators in colon tissue were quantified by PCR. Bacterial RNA from the three time points (2 day,12 weeks and 20 weeks) was isolated from stool using RiboPure Bacteria Kit and depleted of ribosomal and transfer RNA using MicrobeExpress. Double stranded cDNA was transcribed and a cDNA library constructed using TruSeq™ RNA Sample Prep Kit v2. Samples were then sequenced using Illumina HiSeq2000. Reads were then aligned to an updated assembly of E. coli NC101 draft genome using Novoalign. Differential gene expression analysis was done using edgeR. Results: In contrast to Il10-/- mice, E. coli NC101 mono-associated Il10-/-; Rag2-/- mice
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OR, 0.65; 95% CI, 0.45-0.94). In women with low PGE-M (quartile 1), anti-inflammatory drugs were not associated with a lower risk of adenoma (multivariate OR 1.31; 95% CI, 0.62-2.76). Conclusions: Urinary PGE-M is associated with an increased risk of colorectal adenoma, especially advanced, large and multiple tumors. Anti-inflammatory drugs appear to reduce adenoma risk among women with high, but not low, PGE-M levels. Urinary PGEM may serve as a biomarker to define subsets of the population who may obtain differential chemopreventive benefit from anti-inflammatory drugs. 828 Adherence to Annual Fecal Immunochemical Testing in a Large, CommunityBased Population Theodore R. Levin, Christopher D. Jensen, Wei Zhao, Amy R. Marks, Jennifer L. Schneider, Douglas A. Corley, Jeffrey K. Lee, Chyke A. Doubeni, Marjolein van Ballegooijen, Ann G. Zauber, Wendy S. Atkin, Virginia P. Quinn, Joanne E. Schottinger, Nirupa R. Ghai, Richard Contreras, Pauline A. Mysliwiec Background: The advantages of fecal immunochemical testing (FIT) as part of multimodality colorectal cancer (CRC) screening include that it is non-invasive, requires no bowel preparation or dietary/medication restrictions, and is well-suited for outreach programs. Thus, efforts to optimize use of FIT may contribute to reductions in CRC mortality. However, effectiveness of FIT depends on adherence to regular screening (e.g., annually), for which there are minimal data. We evaluated adherence to annual FIT over 4-5 years among two separate fixed cohorts of patients receiving FIT as part of a mailed outreach screening program that was implemented over 2 years, 2007 and 2008. Methods: Patients were Kaiser Permanente Northern California health plan members who received (at home) and completed their first mailed FIT in either 2007 (2007 cohort) or 2008 (2008 cohort); were 50-75 years on either January 1, 2007 or 2008; were health plan members for at least 1 year; and had no sigmoidoscopy in the prior 5 years, colonoscopy in the prior 10 years, or prior history of CRC, colectomy, or inflammatory bowel disease. Patients were followed to the first of the following events: December 31, 2011; diagnosis of CRC; crossover to endoscopy; termination of membership; or death. The primary outcome was proportional adherence to annual FIT over 4-5 calendar years of follow-up. Results: Among the 2007 and 2008 cohorts (Table), 36.3% and 34.8% of the baseline populations crossed over to endoscopy during follow-up, including 20% to colonoscopy and 15%-16% to sigmoidoscopy, respectively. Of those who crossed over to colonoscopy, about 44% (2007 cohort) and 43% (2008 cohort) had a positve FIT beforehand. A total of 48.2% (2007 cohort) and 54.6% (2008 cohort) remained FIT eligible throughout follow-up. During follow-up, annual FIT positivity rates ranged from 4.7%-3.7% and 5.0%-3.6% for the two cohorts, with the highest rates in the baseline year. CRCs were diagnosed in 315 (0.5%) and 328 (0.3%) patients in the respective cohorts; 73% and 80% of cancers were FIT detected (a positive FIT within the prior year). In the 2007 cohort, 87.2% of patients with full follow-up completed three or more FIT over 5 years, including 45.1% who completed FIT annually. In the 2008 cohort, 95.6% of patients with full follow-up completed two or more FIT over 4 years, including 57.8% who completed FIT annually. Conclusions: Our findings from two large and mutually exclusive cohorts in a community-based setting with up to 5 years of follow-up indicate that a mailed FIT outreach program with in-person, mail, secure email, and telephone reminders is effective at achieving a high rate of compliance with repeat testing, although adherence to annual FIT is less than 100%. Patient follow-up and proportional adherence to annual FIT
826 Post-Colonoscopy Colorectal Cancers Are More Likely to Develop Through the Serrated Pathway of Colorectal Neoplasia Reiko Nishihara, Paul Lochhead, Kana Wu, Edward Giovannucci, Charles Fuchs, Shuji Ogino, Andrew T. Chan Background: Post-colonoscopy cancer (PCCRC), defined here as colorectal cancer (CRC) diagnosed within 5 years of a colonoscopy where colorectal neoplasia was not detected, may be due to suboptimal colonoscopy or the unique biology of PCCRC-associated tumors. We examined whether PCCRC is associated with specific tumoral molecular features, including oncogenic mutations (BRAF, KRAS, and PIK3CA), genomic instability (microsatellite instability [MSI]), markers of epigenomic instability (CpG island methylator phenotype [CIMP]), and hypomethylation in long interspersed nucleotide element-1 [LINE-1]), a global marker of DNA methylation. Methods: We utilized biennially-updated information about health and lifestyle (including data on colonoscopy) for participants in two prospective cohort studies, the Nurses' Health Study (NHS, 61,957 women followed since 1984) and the Health Professionals Follow-up Study (HPFS, 32,059 men followed since 1988). Through 2008, we confirmed incident CRC cases by medical record review and obtained formalin-fixed paraffin-embedded tumor tissue from treating hospitals, wherever possible. We conducted a case-case analysis, using a logistic regression model to calculate odds ratio (OR) for PCCRC, adjusted for age at diagnosis, sex (cohort), body mass index, smoking status, family history of colorectal cancer, regular aspirin use, and physical activity level. Results: Over 26 years in the NHS and 22 years in the HPFS (a total of 2,099,558 person-years of follow-up), we successfully retrieved 669 incident CRCs suitable for molecular analysis, of which 53 were defined as PCCRC. Compared with all other CRCs, patients with PCCRC were more likely to have cancers that exhibited CIMP-high (multivariate OR=2.19; 95% CI, 1.11-4.35 vs. CIMP-low), MSI-high (multivariate OR=2.20; 95% CI, 1.11-4.36 vs. MSS), higher level of LINE-1 methylation (multivariate OR=2.65; 95% CI, 1.01-6.96 per 30% increment of methylation) and BRAF mutation (multivariate OR=2.03; 95% CI, 1.00-4.15 vs. BRAF wildtype), and were less likely to harbor KRAS mutation (multivariate OR=0.38; 95% CI, 0.180.81 vs. KRAS wild-type). PCCRCs did not appear to be significantly associated with PIK3CA mutation (P=0.10). Conclusions: Patients who develop CRC within 5 years of a negative colonoscopy are more likely to harbor molecular features associated with the serrated pathway, particularly CIMP-high. This suggests that current approaches to colonoscopic screening and surveillance do not adequately prevent cancers that arise through the serrated precursors of colorectal neoplasia. 827 Urinary Prostaglandin Metabolites (PGE-M) Are Associated With Risk of Colorectal Adenomas and Chemopreventive Response to Anti-Inflammatory Drugs Navya Bezawada, Kana Wu, Raaj S. Mehta, Mingyang Song, Ginger L. Milne, Shuji Ogino, Charles Fuchs, Edward Giovannucci, Andrew T. Chan Background: Aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colorectal neoplasia at least in part through inhibition of the PTGS2 (COX-2) enzyme. PTGS2 is the rate-limiting step in the production of prostaglandin E2 (PGE2), which promotes carcinogenesis. Overall systemic PGE2 production can be estimated by measuring its major metabolite, PGE-M, in the urine. We examined the potential role of PGE-M as a biomarker for colorectal adenoma risk and chemopreventive response to anti-inflammatory drugs. Methods: We conducted a prospective case-control study nested within the Nurses' Health Study. In 2000, 12338 women provided a urine specimen and data on use of anti-inflammatory drugs and other lifestyle factors and subsequently underwent a lower endoscopy through 2008. We identified 420 cases diagnosed with a colorectal adenoma and matched them to 420 endoscopy-negative controls according to age, race, reason for endoscopy and date of urine collection. We measured urinary PGE-M using a liquid chromatographic/mass spectrometric assay with a coefficient of variation of 14%. We analyzed associations using logistic regression models adjusting for matching and other lifestyle risk factors. Results: The median age of both cases and controls was 67 years. Median urinary PGE-M was 5.73 ng/mg Cr. in cases compared to 5.57 ng/mg Cr. in controls (p=0.06 for difference). Compared with women in the lowest quartile of urinary PGE-M, women in the highest quartile had a multivariate odds ratio (OR) of 1.58 (95% CI, 1.02-2.46) for any adenoma; 1.88 (95% CI, 1.14-3.09) for advanced ( ≥1 cm and/or tubulovillous, villous, or high grade dysplasia) adenoma; 1.93 (95% CI, 1.14-3.29) for large ( ≥1 cm) adenoma; and 2.56 (95% CI, 1.414.65) for multiple (.1) adenoma. PGE-M was not significantly associated with early ( ,1cm and tubular), small (,1cm) or solitary adenoma. The effect of regular use of anti-inflammatory drugs (≥ 2 standard tablets of aspirin or NSAIDs per week) appeared to differ according to baseline PGE-M levels. Among women with high PGE-M (quartiles 2-4), regular use of antiinflammatory drugs was associated with a significant reduction in adenoma risk (multivariate
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AGA Abstracts
AGA Abstracts
failed to develop colitis and CRC at week 20, as measured by histological inflammation score (11.25+0.75 vs. 0.11+0.11 respectively; p,0.001) and dysplasia/tumor score (11.25+1.7 vs. 0.55+0.18 respectively; p,0.0001). Colonic expression of Il12b, Ifng and Il17a mRNA in E. coli NC101 mono-associated Il10-/- mice was increased by 13, 65 and 40-fold respectively compared to mono-associated Il10-/-; Rag2-/- mice. Multidimensional Scale Analysis showed that E.coli NC101 gene expression profile clustered differently at 2 days,12 weeks and 20 weeks in Il10-/- mice. Inflammation predominantly increased E.coli gene expression (2302 genes) compared to day 2 (FDR ,5%). E. coli gene expression in CRC mice (20 weeks) showed 2225 genes up-regulated compared to only 273 upregulated genes during chronic inflammation without CRC(12 weeks), suggesting that the presence of CRC impacts bacterial transcriptome responses. E. coli genes up-regulated during CRC belong to pathways affecting translation and ribosomal structure, energy metabolism and amino acid transport. Importantly, nine genes present in the pks island were significantly induced (FDR ,5%) during development of inflammation and CRC, including the Colibactin-modifying enzyme ClbP gene. Conclusions: Our results indicate that the cancer-inducing activity of E.coli NC101 requires intestinal inflammation. The impact of inflammation on E.coli NC101 transcriptome response suggests a novel mean by which microbes influence development of CRC.
Figure 2. (A) A20 interacts with Axin through its N-terminal domain. FLAG-tagged full length (F), N-terminal (N), or C-terminal (C) A20 was co-expressed in RKO cells with MYCtagged Axin and then subject to immunoprecipitation (IP) and immunoblot (IB) for the indicated proteins. (B) A20 supports β-catenin ubiquitylation and degradation. RKO cells were transfected with siRNA to A20 or a scrambled control and then stimulated with recombinant human wnt3a (rhwnt3a) for the indicated times. Lysates were then subject to immunoprecipitation (IP) and immunoblot (IB) for the indicated proteins. (C) Acute deletion of A20 from IECs leads to increased levels of Cyclin D1 and MYC mRNA in vivo. VillinER/Cre A20FL/FL (fl/fl) and control Villin-ER/Cre A20+/+ (+/+) were injected with 1mg of tamoxifen i.p. daily for 5 days. IECs were then isolated and studied for expression of Cyclin D1 (upper panel) and MYC (lower panel) mRNAs by qPCR. Each point represents one mouse. * indicates p , 0.05; ** indicates p , 0.01.
Fig. 1. Patients with the CC genotype of the CCK-B receptor survive significantly longer than those with the AA or AC genotypes. *** p = 0.032. 823 A20 Restricts Canonical Wnt Signaling in Intestinal Epithelial Cells and Suppresses Colon Carcinogenesis Ling Shao, Shigeru Oshima, Bao Duong, Rommel Advincula, Julio Barrera, Barbara Malynn, Averil I. Ma
824 Regulation of Death-Inducing Signaling Complex by Axl Mediates TRAIL Resistance in Esophageal Adenocarcinoma Jun Hong, DunFa Peng, Wael El-Rifai, Abbes Belkhiri
Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Important characteristics of tumors include an ability to proliferate, resist cell death, and benefit from a tumorpromoting inflammatory environment. A well-known regulator of cell death and inflammation is the ubiquitin-editing enzyme, A20 (also known as TNFAIP3). Genome wide associations have linked polymorphisms in A20 to multiple inflammatory and autoimmune diseases including inflammatory bowel disease. Global genetic deficiency of A20 in mice leads to lethal systemic inflammation while intestinal epithelial cell (IEC) specific deletion sensitizes animals to chemical models of colitis. Here, we have studied the role of A20 in intestinal homeostasis and tumorigenesis. An initial search of the NCBI Genome Expression Omnibus revealed that A20 expression is consistently reduced in human colonic adenomas compared to normal surrounding colonic tissues (Fig. 1A). To investigate A20's potential roles in regulating colon carcinogenesis, we generated mice lacking A20 specifically in intestinal epithelial cells (A20FL/FL villin-Cre) and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APCmin). While A20FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20FL/FL villin-Cre APCmin/+ mice spontaneously developed far greater numbers and larger colonic polyps than control APCmin mice (Fig. 1B). Unexpectedly, we found that A20 suppresses colon carcinogenesis, in part, through a direct interaction with the wnt/ β-catenin pathway. Using a human colon cancer cell line with intact wnt signaling, A20 knockdown lead to a seven-fold increase in β-catenindependent luciferase reporter activity compared to a scrambled control siRNA after stimulation with recombinant human wnt3a. β-catenin stability is tightly regulated through sequential phosphorylation, ubiquitylation, and proteasomal degradation by a complex whose core components include APC and the scaffolding protein Axin. We find that A20 binds Axin through its N-terminal domain (Fig. 2A). Furthermore, A20 restricts canonical wnt signaling by promoting ubiquitination and degradation of β-catenin in intestinal epithelial cells (Fig. 2B). To support a role for A20 in regulating β-catenin signaling in vivo, we generated mice expressing a tamoxifen inducible Cre-recombinase under the villin promoter. In this system, acute deletion of A20 from intestinal epithelial cells leads to enhanced epithelial expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer (Fig. 2C). Taken together, these findings demonstrate that A20 is a tumor suppressor in the intestine and uncovers a novel role for A20 in restricting canonical wnt/ β-catenin signaling.
Background: The majority of esophageal adenocarcinoma (EAC) is intrinsically resistant to DNA-damaging therapies. Therefore, an alternative therapeutic approach based on activation of death receptors may be warranted. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding to DR4 or DR5 death receptors. TRAILinduced activation of death receptors leads to the formation of death-inducing signaling complex (DISC), which activates caspase-8 and the extrinsic apoptosis pathway. Unfortunately, a significant proportion of cancer cells are refractory to TRAIL-induced cytotoxicity. AXL receptor tyrosine kinase has been reported to regulate cell growth and survival in solid tumors. The aim of this study was to investigate the role of AXL in TRAIL resistance and elucidate the underlying molecular mechanism in EAC. Methods and Results: To investigate the role of AXL in cell survival, we utilized two EAC cell models. The cell viability assay results demonstrated that the reconstitution of AXL expression in OE33 cells significantly enhanced cell survival in response to treatment with increasing concentrations of TRAIL for 5h (p,0.01), and knockdown of endogenous AXL in FLO-1 cells significantly enhanced the sensitivity of cells to TRAIL (p ,0.05). To ascertain the role of AXL in regulating TRAILinduced apoptosis, we selected the TRAIL concentration 40 ng/ml to induce apoptosis. The Annexin V/PI staining and FACS analysis data demonstrated that the reconstitution of AXL expression in OE33 cells suppressed apoptosis events by 53% relative to control in response to TRAIL (p,0.01). In accordance with this finding, Western blot analysis showed considerably higher protein levels of cleaved caspase-8, -9, -3, and cleaved PARP in control cells than AXL-expressing cells in response to TRAIL. On the contrary, knockdown of highly expressed endogenous AXL in FLO-1 cells significantly increased apoptosis by 74% relative to control (p=0.01), and blocked activation of caspase-8, -9 and -3, and cleavage of PARP in response to TRAIL. To determine the mechanism of TRAIL resistance, we evaluated the effect of AXL on the expression of death receptors. Real-time PCR and Western blot data showed that AXL did not down-regulate DR5 and DR4 receptors OE33 and FLO-1 cells. We next examined if AXL regulates death-inducing signaling complex (DISC), thereby blocking TRAIL-induced apoptosis. Indeed, immunoprecipitation and Western blot data indicated that AXL associated with DR5 death receptor and AXL/DR5 association had no effect on FADD interaction, but prevented the association of pro-caspase-8 with FADD in response to TRAIL. Conclusions: Our data uncovered a novel mechanism of TRAIL resistance mediated by AXL through regulation of the DISC, thereby preventing activation of caspase-8, a key molecular event in TRAIL-induced apoptosis. 825 Intestinal Inflammation Targets E. Coli NC101 Transcriptome Response and Promotes Development of Colorectal Cancer (CRC) Marcus Muehlbauer, Raad Gharaibeh, Janelle C. Arthur, Anthony Fodor, Christian Jobin
Figure 1. A20 is a Tumor Suppressor in the Intestine. (A) Human colonic adenomas express less A20 mRNA than normal surrounding colonic mucosa. From Genome Expression Omnibus (GDS2947). (B) Tumor number (left panel) and aggregate tumor size (right panel) in colons from intestinal epithelial cell specific A20 deleted (fl/fl) and wild-type (+/+) mice harboring the APCmin mutation. * indicates p ,0.05, ** indicates p,0.01
AGA Abstracts
Background: We have previously shown that development of CRC depended upon the genotoxic activity of adherent-invasive Escherichia coli (Arthur JC et al., Science 2012). Aim: Assess the interplay between inflammation, E. coli gene expression and CRC development. Methods: Germ-free Il10-/- and Il10-/-; Rag2-/- mice were mono-associated with E. coli NC101. Colon tumors were induced by 6 weekly i.p. injections of azoxymethane (AOM). Changes in E. coli NC101 gene expression were investigated using germ-free Il10-/- mice mono-associated with E. coli NC101 for 2 days (no inflammation), 12 weeks (chronic inflammation) and 20 weeks (inflammation and CRC). Inflammation and dysplasia/tumors were scored histologically. Transcripts of inflammatory mediators in colon tissue were quantified by PCR. Bacterial RNA from the three time points (2 day,12 weeks and 20 weeks) was isolated from stool using RiboPure Bacteria Kit and depleted of ribosomal and transfer RNA using MicrobeExpress. Double stranded cDNA was transcribed and a cDNA library constructed using TruSeq™ RNA Sample Prep Kit v2. Samples were then sequenced using Illumina HiSeq2000. Reads were then aligned to an updated assembly of E. coli NC101 draft genome using Novoalign. Differential gene expression analysis was done using edgeR. Results: In contrast to Il10-/- mice, E. coli NC101 mono-associated Il10-/-; Rag2-/- mice
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OR, 0.65; 95% CI, 0.45-0.94). In women with low PGE-M (quartile 1), anti-inflammatory drugs were not associated with a lower risk of adenoma (multivariate OR 1.31; 95% CI, 0.62-2.76). Conclusions: Urinary PGE-M is associated with an increased risk of colorectal adenoma, especially advanced, large and multiple tumors. Anti-inflammatory drugs appear to reduce adenoma risk among women with high, but not low, PGE-M levels. Urinary PGEM may serve as a biomarker to define subsets of the population who may obtain differential chemopreventive benefit from anti-inflammatory drugs. 828 Adherence to Annual Fecal Immunochemical Testing in a Large, CommunityBased Population Theodore R. Levin, Christopher D. Jensen, Wei Zhao, Amy R. Marks, Jennifer L. Schneider, Douglas A. Corley, Jeffrey K. Lee, Chyke A. Doubeni, Marjolein van Ballegooijen, Ann G. Zauber, Wendy S. Atkin, Virginia P. Quinn, Joanne E. Schottinger, Nirupa R. Ghai, Richard Contreras, Pauline A. Mysliwiec Background: The advantages of fecal immunochemical testing (FIT) as part of multimodality colorectal cancer (CRC) screening include that it is non-invasive, requires no bowel preparation or dietary/medication restrictions, and is well-suited for outreach programs. Thus, efforts to optimize use of FIT may contribute to reductions in CRC mortality. However, effectiveness of FIT depends on adherence to regular screening (e.g., annually), for which there are minimal data. We evaluated adherence to annual FIT over 4-5 years among two separate fixed cohorts of patients receiving FIT as part of a mailed outreach screening program that was implemented over 2 years, 2007 and 2008. Methods: Patients were Kaiser Permanente Northern California health plan members who received (at home) and completed their first mailed FIT in either 2007 (2007 cohort) or 2008 (2008 cohort); were 50-75 years on either January 1, 2007 or 2008; were health plan members for at least 1 year; and had no sigmoidoscopy in the prior 5 years, colonoscopy in the prior 10 years, or prior history of CRC, colectomy, or inflammatory bowel disease. Patients were followed to the first of the following events: December 31, 2011; diagnosis of CRC; crossover to endoscopy; termination of membership; or death. The primary outcome was proportional adherence to annual FIT over 4-5 calendar years of follow-up. Results: Among the 2007 and 2008 cohorts (Table), 36.3% and 34.8% of the baseline populations crossed over to endoscopy during follow-up, including 20% to colonoscopy and 15%-16% to sigmoidoscopy, respectively. Of those who crossed over to colonoscopy, about 44% (2007 cohort) and 43% (2008 cohort) had a positve FIT beforehand. A total of 48.2% (2007 cohort) and 54.6% (2008 cohort) remained FIT eligible throughout follow-up. During follow-up, annual FIT positivity rates ranged from 4.7%-3.7% and 5.0%-3.6% for the two cohorts, with the highest rates in the baseline year. CRCs were diagnosed in 315 (0.5%) and 328 (0.3%) patients in the respective cohorts; 73% and 80% of cancers were FIT detected (a positive FIT within the prior year). In the 2007 cohort, 87.2% of patients with full follow-up completed three or more FIT over 5 years, including 45.1% who completed FIT annually. In the 2008 cohort, 95.6% of patients with full follow-up completed two or more FIT over 4 years, including 57.8% who completed FIT annually. Conclusions: Our findings from two large and mutually exclusive cohorts in a community-based setting with up to 5 years of follow-up indicate that a mailed FIT outreach program with in-person, mail, secure email, and telephone reminders is effective at achieving a high rate of compliance with repeat testing, although adherence to annual FIT is less than 100%. Patient follow-up and proportional adherence to annual FIT
826 Post-Colonoscopy Colorectal Cancers Are More Likely to Develop Through the Serrated Pathway of Colorectal Neoplasia Reiko Nishihara, Paul Lochhead, Kana Wu, Edward Giovannucci, Charles Fuchs, Shuji Ogino, Andrew T. Chan Background: Post-colonoscopy cancer (PCCRC), defined here as colorectal cancer (CRC) diagnosed within 5 years of a colonoscopy where colorectal neoplasia was not detected, may be due to suboptimal colonoscopy or the unique biology of PCCRC-associated tumors. We examined whether PCCRC is associated with specific tumoral molecular features, including oncogenic mutations (BRAF, KRAS, and PIK3CA), genomic instability (microsatellite instability [MSI]), markers of epigenomic instability (CpG island methylator phenotype [CIMP]), and hypomethylation in long interspersed nucleotide element-1 [LINE-1]), a global marker of DNA methylation. Methods: We utilized biennially-updated information about health and lifestyle (including data on colonoscopy) for participants in two prospective cohort studies, the Nurses' Health Study (NHS, 61,957 women followed since 1984) and the Health Professionals Follow-up Study (HPFS, 32,059 men followed since 1988). Through 2008, we confirmed incident CRC cases by medical record review and obtained formalin-fixed paraffin-embedded tumor tissue from treating hospitals, wherever possible. We conducted a case-case analysis, using a logistic regression model to calculate odds ratio (OR) for PCCRC, adjusted for age at diagnosis, sex (cohort), body mass index, smoking status, family history of colorectal cancer, regular aspirin use, and physical activity level. Results: Over 26 years in the NHS and 22 years in the HPFS (a total of 2,099,558 person-years of follow-up), we successfully retrieved 669 incident CRCs suitable for molecular analysis, of which 53 were defined as PCCRC. Compared with all other CRCs, patients with PCCRC were more likely to have cancers that exhibited CIMP-high (multivariate OR=2.19; 95% CI, 1.11-4.35 vs. CIMP-low), MSI-high (multivariate OR=2.20; 95% CI, 1.11-4.36 vs. MSS), higher level of LINE-1 methylation (multivariate OR=2.65; 95% CI, 1.01-6.96 per 30% increment of methylation) and BRAF mutation (multivariate OR=2.03; 95% CI, 1.00-4.15 vs. BRAF wildtype), and were less likely to harbor KRAS mutation (multivariate OR=0.38; 95% CI, 0.180.81 vs. KRAS wild-type). PCCRCs did not appear to be significantly associated with PIK3CA mutation (P=0.10). Conclusions: Patients who develop CRC within 5 years of a negative colonoscopy are more likely to harbor molecular features associated with the serrated pathway, particularly CIMP-high. This suggests that current approaches to colonoscopic screening and surveillance do not adequately prevent cancers that arise through the serrated precursors of colorectal neoplasia. 827 Urinary Prostaglandin Metabolites (PGE-M) Are Associated With Risk of Colorectal Adenomas and Chemopreventive Response to Anti-Inflammatory Drugs Navya Bezawada, Kana Wu, Raaj S. Mehta, Mingyang Song, Ginger L. Milne, Shuji Ogino, Charles Fuchs, Edward Giovannucci, Andrew T. Chan Background: Aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colorectal neoplasia at least in part through inhibition of the PTGS2 (COX-2) enzyme. PTGS2 is the rate-limiting step in the production of prostaglandin E2 (PGE2), which promotes carcinogenesis. Overall systemic PGE2 production can be estimated by measuring its major metabolite, PGE-M, in the urine. We examined the potential role of PGE-M as a biomarker for colorectal adenoma risk and chemopreventive response to anti-inflammatory drugs. Methods: We conducted a prospective case-control study nested within the Nurses' Health Study. In 2000, 12338 women provided a urine specimen and data on use of anti-inflammatory drugs and other lifestyle factors and subsequently underwent a lower endoscopy through 2008. We identified 420 cases diagnosed with a colorectal adenoma and matched them to 420 endoscopy-negative controls according to age, race, reason for endoscopy and date of urine collection. We measured urinary PGE-M using a liquid chromatographic/mass spectrometric assay with a coefficient of variation of 14%. We analyzed associations using logistic regression models adjusting for matching and other lifestyle risk factors. Results: The median age of both cases and controls was 67 years. Median urinary PGE-M was 5.73 ng/mg Cr. in cases compared to 5.57 ng/mg Cr. in controls (p=0.06 for difference). Compared with women in the lowest quartile of urinary PGE-M, women in the highest quartile had a multivariate odds ratio (OR) of 1.58 (95% CI, 1.02-2.46) for any adenoma; 1.88 (95% CI, 1.14-3.09) for advanced ( ≥1 cm and/or tubulovillous, villous, or high grade dysplasia) adenoma; 1.93 (95% CI, 1.14-3.29) for large ( ≥1 cm) adenoma; and 2.56 (95% CI, 1.414.65) for multiple (.1) adenoma. PGE-M was not significantly associated with early ( ,1cm and tubular), small (,1cm) or solitary adenoma. The effect of regular use of anti-inflammatory drugs (≥ 2 standard tablets of aspirin or NSAIDs per week) appeared to differ according to baseline PGE-M levels. Among women with high PGE-M (quartiles 2-4), regular use of antiinflammatory drugs was associated with a significant reduction in adenoma risk (multivariate
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AGA Abstracts
failed to develop colitis and CRC at week 20, as measured by histological inflammation score (11.25+0.75 vs. 0.11+0.11 respectively; p,0.001) and dysplasia/tumor score (11.25+1.7 vs. 0.55+0.18 respectively; p,0.0001). Colonic expression of Il12b, Ifng and Il17a mRNA in E. coli NC101 mono-associated Il10-/- mice was increased by 13, 65 and 40-fold respectively compared to mono-associated Il10-/-; Rag2-/- mice. Multidimensional Scale Analysis showed that E.coli NC101 gene expression profile clustered differently at 2 days,12 weeks and 20 weeks in Il10-/- mice. Inflammation predominantly increased E.coli gene expression (2302 genes) compared to day 2 (FDR ,5%). E. coli gene expression in CRC mice (20 weeks) showed 2225 genes up-regulated compared to only 273 upregulated genes during chronic inflammation without CRC(12 weeks), suggesting that the presence of CRC impacts bacterial transcriptome responses. E. coli genes up-regulated during CRC belong to pathways affecting translation and ribosomal structure, energy metabolism and amino acid transport. Importantly, nine genes present in the pks island were significantly induced (FDR ,5%) during development of inflammation and CRC, including the Colibactin-modifying enzyme ClbP gene. Conclusions: Our results indicate that the cancer-inducing activity of E.coli NC101 requires intestinal inflammation. The impact of inflammation on E.coli NC101 transcriptome response suggests a novel mean by which microbes influence development of CRC.