M2008 Colorectal Adenomas in Patients with H. pylori Infection: Duodenal Ulcer Is a Risk Factor for...
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to determine the impact of TLR signaling on CAC (n=5). Colonoscopy was used to follow tumor progression and the development of inflammation. Inflammation and tumor grade were evaluated histologically and colonic levels of IL-12p40 and TNFα mRNA were measured by real-time PCR. Nuclear β-catenin levels and NF-kB phosphorylation were determined by immunohistochemistry. Results: AOM-treated (6 weekly injections) IL-10-/- mice developed colitis and displayed highly penetrant tumor formation under SPF conditions, while WT mice showed no evidence of intestinal inflammation and had minimal tumor formation. AOM-treated IL10-/- mice showed a dramatic increase in colon tumor multiplicity and progression compared to WT mice. Tumor multiplicity increased 20-fold while progression from low to high-grade/invasive carcinoma increased by ~70% in IL10-/- compared to WT mice. These tumors showed increased nuclear β-catenin accumulation and the presence of phosphorylated RelA. B. vulgatus mono-associated IL10-/- mice failed to develop significant intestinal inflammation and showed greatly reduced tumor formation. AOM-treated IL10-/- ; MyD88-/- mice showed reduced colonic expression of TNFα and IL12p40 mRNA and were devoid of neoplastic lesions compared to AOM-treated IL10-/- mice. Conclusions: Bacterial-induced intestinal inflammation correlates with the progression of colon cancer in AOM-treated IL-10-/- mice. The TLR/MyD88 signaling pathway is essential for the develop- ment of CAC. M2004 Deletion of Matrix Metalloproteinase-9 (MMP-9) Increases the Susceptibility for Colon Cancer Pallavi Garg, Neal R. Patel, Matam Vijay-Kumar, Anupama Ravi, Didier Merlin, Andrew T. Gewirtz, Shanthi V. Sitaraman Background: Inflammatory bowel disease (IBD) is a chronic inflammatory condition of digestive tract. IBD poses increased risk of developing colon cancer. How IBD increases the risk of developing colon cancer is not well documented. Among matrix metalloproteinases (MMPs), gelatinases (MMP-2 and MMP-9) have been observed to be upregulated in IBD and in colorectal cancers. Aim of the present study was to understand the role of MMP-9 in inflammation-induced colon cancer. Methods: C57/B6 wild type (WT) and MMP-9 knock out (KO) mice were administered a single dose of azoxymethane (AOM, 7.6 mg/kg, i.p.). One week later they were exposed to dextran sodium sulfate (DSS, 3% in drinking water, 2 cycles of one week each and with one week apart). Mice were sacrificed 56 days thereafter. Colons were processed for inflammation and polyps. Ki-67 immunohistochemistry was performed to determine cell proliferation. Western blot analysis was done to determine the levels of COX-2, β-catenin, NF-κB and NICD. Results: Compared to WT mice, KO mice were more sensitive to colon cancer as evidenced by increased tumor multiplicity (KO= 4.5, WT=2.5), tumor burden (KO= 9.8, WT=5.7) and incidence of rectal prolapse. In addition, increased erosion of mucosal layer and infiltration of neutrophils were seen in the polyps among KO mice compared to WT mice. Western analysis indicated increased expression of COX-2 (2.3 fold±0.14), β-catenin (2.16±0.03) and NF-κB (4.3 fold±0.25) in the polyps of KO mice compared to that in WT mice. Proliferation marker Ki67 showed increased immunohistostaining in the colons of KO mice compared to WT mice exposed to AOM. AB-PAS staining indicated more goblet cells in the colons of KO mice compared to WT mice exposed to AOM. Western analysis also showed increased NICD (Notch intracellular domain) expression among WT mice treated with AOM+DSS compared to WT mice treated with water alone. Conclusion: Results of the present study show that MMP-9 plays an inhibitory role in the colon cancer contrary to its effects on inflammation. A possible mechanism could be that MMP-9 regulates transcription factors which are involved in epithelial cell differentiation leading to the susceptibility of colon cancer or it may generate cleavage products inhibitory to cell growth. Thus MMP-9 inhibitors used as anti-tumor drugs might cause a paradoxical increase in tumor growth. M2005 Truncated Neurokinin-1 Receptor Mediates Colon Cancer Development Hon Wai Koon, James M. Bugni, Dezheng Zhao, Aatish Kumar, Hua Xu, Charalabos Pothoulakis Background and Aims: We have recently shown that substance P (SP) is involved in human colon cancer cell growth and that in the dextran sulfate sodium (DSS)-exposed ApcMin mouse colon cancer model administration of the SP, neurokinin-1 receptor (NK-1R) antagon- ist reduces tumor size. Evidence also indicates increased NK-1R expression in human colorectal tumors. However, the mechanisms by which NK-1R mediates colon cancer devel- opment have not being fully investigated and this was the goal of this current study. Methods: In Vitro studies: Human colon tumor lysates were used to assess NK-1R expression and human colon cancer cell lines (SW403 and HT-29) used for signaling experiments. In Vivo studies: We induced colon cancer in mice with azoxymethane (AOM) alone and AOM + DSS, as well as by subcutaneous xenografting of colon cancer cell lines in nude mice. Results: We found significantly increased mRNA NK-1R expression in human colon tumors (n=45) as well as AOM-DSS-exposed mice (n=8). Since NK-1R has two naturally occurring forms, a full-length receptor (407 amino acids) and a truncated receptor (311 amino acids) with different signaling properties, we examined the level of expression of these NK-1R forms. Compared to controls, we found significantly increased levels of truncated NK-1R and reduced full-length NK-1R (mRNA and protein) in both human and animal colon tumors. Moreover, stably transfected human colon cancer HT-29 cells overexpressing truncated NK- 1R (TACR1S) formed larger tumors as xenografts in nude mice than those expressing full- length NK-1R (TACR1L) or LacZ control. In the absence of SP, TACR1S cells had higher cell proliferation rates and elevated cyclooxygenase-2 (COX-2) gene expression than TACR1L or LacZ cells, although compared to TACR1S, TACR1L cells showed more cell proliferation and increased COX-2 gene expression following SP stimulation. Basal and SP-mediated ERK phosphorylation, Rho kinase activation and COX-2 expression in HT-29 expressing TACR1S and SW403 cells expressing endogenous NK-1R were attenuated by pretreatment with the NK-1R antagonist CJ-12255. Moreover, NK-1R antagonism by CJ-12255 reduced tumor size and the number of aberrant crypt foci that developed in mice in response to AOM + DSS as well as to AOM alone. Conclusions: Colon cancers express predominantly the truncated form of NK-1R that can mediate cancer - associated responses In Vitro and In A-465 AGA Abstracts Vivo. Supported by a Research Fellowship from CCFA to HWK and a NIH grant DK47343 to CP. M2006 Elevated Risk of Colorectal Cancer in Inflammatory Bowel Disease and Modification of Risk By Statin Use: A Population-Based Case-Control Study N. Jewel Samadder, Shu-Chen Huang, Bhramar Mukherjee, Gad Rennert, Stephen B. Gruber Background Inflammatory bowel disease (IBD) has long been regarded as a risk factor for colorectal cancer (CRC),however estimates of this risk have varied in the literature. Statins and non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested to decrease the incidence of sporadic CRC in some but not in all studies. The objective of the current study was to determine the prevalence of IBD in a population based case-control study of colorectal cancer (CRC) in Israel and to quantify the relative risk of IBD as risk factor for CRC. Modification of this elevated risk by medication use,including NSAIDs and statins was also assessed. Methods The Molecular Epidemiology of Colorectal Cancer study is a population based case-control study of patients who received a diagnosis of colorectal cancer in northern Israel between 1998 and 2004 and controls matched according to age, sex, clinic and ethnic group. Patients with a self-reported history of IBD and use of statins and NSAIDs in the two groups were identified. The prevalence of IBD in CRC cases and matched controls was measured by self-report and associations were quantified by logistic regression. Results Among 2116 cases of CRC and 2244 matched controls,64 individuals reported a history of IBD. The overall prevalence of IBD among persons with CRC was 2.1%. In individuals with a history of IBD, the relative risk of CRC was 2.4 (95% CI, 1.38-4.02). Kaplan Meier analysis did not show a significant difference in overall survival of patients with and without IBD. Among persons with IBD, distribution of colon cancers was 47.1% left sided, 32.4% right sided and 20.6% rectum. 18% of IBD patients had tumors showing high microsatellite instability (MSI-H) as compared to 12.8% of sporadic tumors, yielding an odds ratio of 1.77 (95% CI, 0.66-4.73). As previously reported, the use of statins for at least five years (vs. never use of statins) was associated with a significantly reduced risk of CRC overall (OR, 0.49, 95% CI, 0.39-0.62). Importantly, statin use for at least five years was associated with a profoundly reduced risk of IBD-associated CRC (OR, 0.07, 95% CI, 0.01-0.62). The use of NSAIDs in patients with a history of IBD at least once per week for 3 years trended towards a reduced risk of CRC, but did not reach statistical significance (OR, 0.30, 95% CI, 0.08-1.13). Conclusions This population based study shows that risk for CRC is elevated in patients with IBD approximately 2.4 fold and suggest that statins may have a chemoprotec- tive role in the development of CRC in this high risk population. Our findings suggest that statins deserve further investigation as chemopreventive agents in persons with IBD. M2007 Progressive Increase of MGMT (O6-Methylguanine DNA Methyltransferease) CpG Methylation Levels from Colitis to Low-Grade Dysplasia in Ulcerative Colitis Dara L. Aisner, Yuan Yao, Wen Yan, Antonia R. Sepulveda Background: Colorectal carcinoma arising in the setting of ulcerative colitis is thought to progress through a molecular pathway influenced by the longstanding inflammatory and regenerative mucosal changes, including increased quantity or sensitivity to oxidative DNA damage. MGMT plays a key role in the repair of oxidative damage induced-genomic lesions. Downregulation of MGMT through CpG methylation has been reported in human neoplasia. Aim: To examine the potential role of promoter methylation of MGMT in the stepwise progression of ulcerative colitis and associated low-grade dysplasia (LGD). Design: CpG methylation levels of MGMT were determined in tissue samples from 23 colectomy specimens of 23 patients with ulcerative colitis including 21 with low-grade dysplasia. Patients included 13 men and 10 women, 24 to 81 years of age. Non-neoplastic colonic mucosa samples with various grades of colitis, evaluated with a scoring system for severity of colitis, were tested in all patients, for a total of 41 colitis samples. Eighteen patients with LGD had matching colitis distal to the dysplastic lesion in the same colectomy specimen. DNA was extracted from selected paraffin sections and microdissected from selected areas of mucosa with various grades of colitis or low-grade dysplasia. Bisulfite modified DNA was used for Quantitative SYBR green methylation specific PCR (QMSP). The levels of the amplified MGMT CpG locus were measured relative to the internal reference gene (ACTB) and reported as a % of the reference gene. Results: Overall colitis scores ranged from 0-11, mean 4.3. MGMT methylation levels were significantly higher in low-grade dysplasia (mean 11.2%, SE 3.9) compared to colitic mucosa (mean 4.1%, SE 1.42) (P=0.03). Among the 18 pairs of matched LGD and distal colitis, there was significantly increased methylation in LGD as compared to distal colitis, median 3.85 and 0.7, and mean 12.9, and 4.7 respectively (P=0.008), and there was a significant correlation between the methylation levels in colitis and LGD (R=0.62, P= 0.005). No significant difference in MGMT methylation related to the age of patients, colitis grade, location of colitis or location of dysplasia was seen. Conclusions: Quantitative analyses of CpG methylation of MGMT demonstrate a progressive increase of CpG methylation levels from colitis to low-grade dysplasia, suggesting a role for MGMT in the early phase of UC- associated neoplastic development. M2008 Colorectal Adenomas in Patients with H. pylori Infection: Duodenal Ulcer Is a Risk Factor for Colorectal Adenoma Yutaka Yamaji, Takao Kawabe, Toru Mitsushima, Masao Omata Background: H. pylori infection causes chronic inflammation for a lifetime in the stomach. It presents a variety of diseases, which may reflect host systems regulating inflammation. The chronic inflammation and host inflammatory response may also affect lower gastrointesti- nal tract. Thus, we evaluated the colorectal endoscopic findings in H. pylori infected subjects undergoing medical check-up. Methods: We reviewed the initial findings of the subjects of AGA Abstracts
Transcript of M2008 Colorectal Adenomas in Patients with H. pylori Infection: Duodenal Ulcer Is a Risk Factor for...
to determine the impact of TLR signaling on CAC (n=5). Colonoscopy was used to followtumor progression and the development of inflammation. Inflammation and tumor gradewere evaluated histologically and colonic levels of IL-12p40 and TNFαmRNAwere measuredby real-time PCR. Nuclear β-catenin levels and NF-kB phosphorylation were determined byimmunohistochemistry. Results: AOM-treated (6 weekly injections) IL-10-/- mice developedcolitis and displayed highly penetrant tumor formation under SPF conditions, while WTmice showed no evidence of intestinal inflammation and had minimal tumor formation.AOM-treated IL10-/- mice showed a dramatic increase in colon tumor multiplicity andprogression compared to WT mice. Tumor multiplicity increased 20-fold while progressionfrom low to high-grade/invasive carcinoma increased by ~70% in IL10-/- compared to WTmice. These tumors showed increased nuclear β-catenin accumulation and the presence ofphosphorylated RelA. B. vulgatus mono-associated IL10-/- mice failed to develop significantintestinal inflammation and showed greatly reduced tumor formation. AOM-treated IL10-/-; MyD88-/- mice showed reduced colonic expression of TNFα and IL12p40 mRNA andwere devoid of neoplastic lesions compared to AOM-treated IL10-/- mice. Conclusions:Bacterial-induced intestinal inflammation correlates with the progression of colon cancer inAOM-treated IL-10-/- mice. The TLR/MyD88 signaling pathway is essential for the develop-ment of CAC.
M2004
Deletion of Matrix Metalloproteinase-9 (MMP-9) Increases the Susceptibilityfor Colon CancerPallavi Garg, Neal R. Patel, Matam Vijay-Kumar, Anupama Ravi, Didier Merlin, Andrew T.Gewirtz, Shanthi V. Sitaraman
Background: Inflammatory bowel disease (IBD) is a chronic inflammatory condition ofdigestive tract. IBD poses increased risk of developing colon cancer. How IBD increases therisk of developing colon cancer is not well documented. Among matrix metalloproteinases(MMPs), gelatinases (MMP-2 and MMP-9) have been observed to be upregulated in IBDand in colorectal cancers. Aim of the present study was to understand the role of MMP-9in inflammation-induced colon cancer. Methods: C57/B6 wild type (WT) and MMP-9 knockout (KO) mice were administered a single dose of azoxymethane (AOM, 7.6 mg/kg, i.p.).One week later they were exposed to dextran sodium sulfate (DSS, 3% in drinking water,2 cycles of one week each and with one week apart). Mice were sacrificed 56 days thereafter.Colons were processed for inflammation and polyps. Ki-67 immunohistochemistry wasperformed to determine cell proliferation. Western blot analysis was done to determine thelevels of COX-2, β-catenin, NF-κB and NICD. Results: Compared to WT mice, KO micewere more sensitive to colon cancer as evidenced by increased tumor multiplicity (KO= 4.5,WT=2.5), tumor burden (KO= 9.8, WT=5.7) and incidence of rectal prolapse. In addition,increased erosion of mucosal layer and infiltration of neutrophils were seen in the polypsamong KO mice compared to WT mice. Western analysis indicated increased expression ofCOX-2 (2.3 fold±0.14), β-catenin (2.16±0.03) and NF-κB (4.3 fold±0.25) in the polypsof KO mice compared to that in WT mice. Proliferation marker Ki67 showed increasedimmunohistostaining in the colons of KO mice compared to WT mice exposed to AOM.AB-PAS staining indicated more goblet cells in the colons of KO mice compared to WTmice exposed to AOM. Western analysis also showed increased NICD (Notch intracellulardomain) expression among WT mice treated with AOM+DSS compared to WT mice treatedwith water alone. Conclusion: Results of the present study show that MMP-9 plays aninhibitory role in the colon cancer contrary to its effects on inflammation. A possiblemechanism could be that MMP-9 regulates transcription factors which are involved inepithelial cell differentiation leading to the susceptibility of colon cancer or it may generatecleavage products inhibitory to cell growth. Thus MMP-9 inhibitors used as anti-tumordrugs might cause a paradoxical increase in tumor growth.
M2005
Truncated Neurokinin-1 Receptor Mediates Colon Cancer DevelopmentHon Wai Koon, James M. Bugni, Dezheng Zhao, Aatish Kumar, Hua Xu, CharalabosPothoulakis
Background and Aims: We have recently shown that substance P (SP) is involved in humancolon cancer cell growth and that in the dextran sulfate sodium (DSS)-exposed ApcMinmouse colon cancer model administration of the SP, neurokinin-1 receptor (NK-1R) antagon-ist reduces tumor size. Evidence also indicates increased NK-1R expression in humancolorectal tumors. However, the mechanisms by which NK-1R mediates colon cancer devel-opment have not being fully investigated and this was the goal of this current study. Methods:In Vitro studies: Human colon tumor lysates were used to assess NK-1R expression andhuman colon cancer cell lines (SW403 and HT-29) used for signaling experiments. In Vivostudies: We induced colon cancer in mice with azoxymethane (AOM) alone and AOM +DSS, as well as by subcutaneous xenografting of colon cancer cell lines in nude mice. Results:We found significantly increased mRNA NK-1R expression in human colon tumors (n=45)as well as AOM-DSS-exposed mice (n=8). Since NK-1R has two naturally occurring forms,a full-length receptor (407 amino acids) and a truncated receptor (311 amino acids) withdifferent signaling properties, we examined the level of expression of these NK-1R forms.Compared to controls, we found significantly increased levels of truncated NK-1R andreduced full-length NK-1R (mRNA and protein) in both human and animal colon tumors.Moreover, stably transfected human colon cancer HT-29 cells overexpressing truncated NK-1R (TACR1S) formed larger tumors as xenografts in nude mice than those expressing full-length NK-1R (TACR1L) or LacZ control. In the absence of SP, TACR1S cells had highercell proliferation rates and elevated cyclooxygenase-2 (COX-2) gene expression than TACR1Lor LacZ cells, although compared to TACR1S, TACR1L cells showed more cell proliferationand increased COX-2 gene expression following SP stimulation. Basal and SP-mediated ERKphosphorylation, Rho kinase activation and COX-2 expression in HT-29 expressing TACR1Sand SW403 cells expressing endogenous NK-1R were attenuated by pretreatment with theNK-1R antagonist CJ-12255. Moreover, NK-1R antagonism by CJ-12255 reduced tumorsize and the number of aberrant crypt foci that developed in mice in response to AOM +DSS as well as to AOM alone. Conclusions: Colon cancers express predominantly thetruncated form of NK-1R that can mediate cancer - associated responses In Vitro and In
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Vivo. Supported by a Research Fellowship from CCFA to HWK and a NIH grant DK47343to CP.
M2006
Elevated Risk of Colorectal Cancer in Inflammatory Bowel Disease andModification of Risk By Statin Use: A Population-Based Case-Control StudyN. Jewel Samadder, Shu-Chen Huang, Bhramar Mukherjee, Gad Rennert, Stephen B.Gruber
Background Inflammatory bowel disease (IBD) has long been regarded as a risk factor forcolorectal cancer (CRC),however estimates of this risk have varied in the literature. Statinsand non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested to decrease theincidence of sporadic CRC in some but not in all studies. The objective of the current studywas to determine the prevalence of IBD in a population based case-control study of colorectalcancer (CRC) in Israel and to quantify the relative risk of IBD as risk factor for CRC.Modification of this elevated risk by medication use,including NSAIDs and statins was alsoassessed. Methods The Molecular Epidemiology of Colorectal Cancer study is a populationbased case-control study of patients who received a diagnosis of colorectal cancer in northernIsrael between 1998 and 2004 and controls matched according to age, sex, clinic and ethnicgroup. Patients with a self-reported history of IBD and use of statins and NSAIDs in thetwo groups were identified. The prevalence of IBD in CRC cases and matched controls wasmeasured by self-report and associations were quantified by logistic regression. ResultsAmong 2116 cases of CRC and 2244 matched controls,64 individuals reported a history ofIBD. The overall prevalence of IBD among persons with CRC was 2.1%. In individuals witha history of IBD, the relative risk of CRC was 2.4 (95% CI, 1.38-4.02). Kaplan Meier analysisdid not show a significant difference in overall survival of patients with and without IBD.Among persons with IBD, distribution of colon cancers was 47.1% left sided, 32.4% rightsided and 20.6% rectum. 18% of IBD patients had tumors showing high microsatelliteinstability (MSI-H) as compared to 12.8% of sporadic tumors, yielding an odds ratio of1.77 (95% CI, 0.66-4.73). As previously reported, the use of statins for at least five years(vs. never use of statins) was associated with a significantly reduced risk of CRC overall(OR, 0.49, 95% CI, 0.39-0.62). Importantly, statin use for at least five years was associatedwith a profoundly reduced risk of IBD-associated CRC (OR, 0.07, 95% CI, 0.01-0.62). Theuse of NSAIDs in patients with a history of IBD at least once per week for 3 years trendedtowards a reduced risk of CRC, but did not reach statistical significance (OR, 0.30, 95%CI, 0.08-1.13). Conclusions This population based study shows that risk for CRC is elevatedin patients with IBD approximately 2.4 fold and suggest that statins may have a chemoprotec-tive role in the development of CRC in this high risk population. Our findings suggest thatstatins deserve further investigation as chemopreventive agents in persons with IBD.
M2007
Progressive Increase of MGMT (O6-Methylguanine DNA Methyltransferease)CpG Methylation Levels from Colitis to Low-Grade Dysplasia in UlcerativeColitisDara L. Aisner, Yuan Yao, Wen Yan, Antonia R. Sepulveda
Background: Colorectal carcinoma arising in the setting of ulcerative colitis is thought toprogress through a molecular pathway influenced by the longstanding inflammatory andregenerative mucosal changes, including increased quantity or sensitivity to oxidative DNAdamage. MGMT plays a key role in the repair of oxidative damage induced-genomic lesions.Downregulation of MGMT through CpG methylation has been reported in human neoplasia.Aim: To examine the potential role of promoter methylation of MGMT in the stepwiseprogression of ulcerative colitis and associated low-grade dysplasia (LGD). Design: CpGmethylation levels of MGMTwere determined in tissue samples from 23 colectomy specimensof 23 patients with ulcerative colitis including 21 with low-grade dysplasia. Patients included13 men and 10 women, 24 to 81 years of age. Non-neoplastic colonic mucosa samples withvarious grades of colitis, evaluated with a scoring system for severity of colitis, were testedin all patients, for a total of 41 colitis samples. Eighteen patients with LGD had matchingcolitis distal to the dysplastic lesion in the same colectomy specimen. DNA was extractedfrom selected paraffin sections and microdissected from selected areas of mucosa with variousgrades of colitis or low-grade dysplasia. Bisulfite modified DNA was used for QuantitativeSYBR green methylation specific PCR (QMSP). The levels of the amplified MGMT CpG locuswere measured relative to the internal reference gene (ACTB) and reported as a % of thereference gene. Results: Overall colitis scores ranged from 0-11,mean 4.3. MGMTmethylationlevels were significantly higher in low-grade dysplasia (mean 11.2%, SE 3.9) compared tocolitic mucosa (mean 4.1%, SE 1.42) (P=0.03). Among the 18 pairs of matched LGD anddistal colitis, there was significantly increased methylation in LGD as compared to distalcolitis, median 3.85 and 0.7, and mean 12.9, and 4.7 respectively (P=0.008), and there wasa significant correlation between the methylation levels in colitis and LGD (R=0.62, P=0.005). No significant difference in MGMT methylation related to the age of patients, colitisgrade, location of colitis or location of dysplasia was seen. Conclusions: Quantitative analysesof CpG methylation of MGMT demonstrate a progressive increase of CpG methylation levelsfrom colitis to low-grade dysplasia, suggesting a role for MGMT in the early phase of UC-associated neoplastic development.
M2008
Colorectal Adenomas in Patients with H. pylori Infection: Duodenal Ulcer Is aRisk Factor for Colorectal AdenomaYutaka Yamaji, Takao Kawabe, Toru Mitsushima, Masao Omata
Background: H. pylori infection causes chronic inflammation for a lifetime in the stomach.It presents a variety of diseases, which may reflect host systems regulating inflammation.The chronic inflammation and host inflammatory response may also affect lower gastrointesti-nal tract. Thus, we evaluated the colorectal endoscopic findings in H. pylori infected subjectsundergoing medical check-up. Methods: We reviewed the initial findings of the subjects of
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sour prospective endoscopic cohort study investigating gastric cancer development (Gut2005;54:764). Among them, the subjects who simultaneously underwent colonoscopyscreening were evaluated. A total of 2,724 subjects (male/female = 1,918/806, mean age =47.6) were entered into analysis. Serum pepsinogen levels (ng/ml) and anti-H. pylori antibodywere measured as indicators of chronic gastritis and H. pylori infection. The risk of colorectaladenomas in subjects with duodenal ulcer and gastric ulcer including scar lesions, and H.pylori positive gastritis without ulcers were compared with the other control subjects. Results:Among 2,724 subjects, duodenal ulcer was found in 183 subjects (6.7%), gastric ulcer in224 subjects (8.2%), including 45 subjects with both ulcers. H. pylori associated gastritiswas found in 1204 subjects (44.2%), and the remaining 1158 subjects served as the control.Among the total subjects, colorectal adenomas were found in 452 subjects (16.6%). Pep-sinogen I and II levels were 58.3 and 16.0 in the patients with colorectal adenoma, whichwere slightly higher than those of 56.9 and 15.0 in the subjects without colorectal adenoma(p=0.28 and p=0.045). The prevalence of colorectal adenomas was 14.7% (170/1158) inthe control subjects, whereas the prevalence was 16.7% (201/1204, p=0.18 vs the control)in H. pylori associated gastritis, 19.6% (35/179, p=0.09) in gastric ulcer alone, 23.9% (33/138, p=0.005) in duodenal ulcer alone, and 28.9% (13/45, p=0.01) in patients with bothulcers. In a multivariate analysis controlling sex and age, duodenal ulcer was shown to bea significant risk factor for colorectal adenoma (adjusted Odds Ratio 1.72, 95%CI 1.2-2.5,p=0.005). Conclusion: Duodenal ulcer was a risk factor for colorectal adenoma. The patientswith duodenal ulcer and those with colorectal adenoma might have a common backgroundfactors for inflammatory response.
M2009
PI3K Inhibition Suppresses Growth of Inflammation-Mediated Colon Cancerin MiceTakaaki Tsushimi, Hiroshi Saito, B. M. Evers
Numerous studies have documented the link between chronic intestinal inflammation andcolorectal cancer (CRC). Phosphatidylinositol 3-kinase (PI3K) stimulates the growth andinvasion of a number of cancers including CRC. We have shown that wortmannin, a PI3Kinhibitor, reduces the growth of human CRC xenografts In Vivo; the effect of PI3K inhibitionon CRCs induced by inflammation is not known. The purpose of this study was to determinewhether PI3K signaling plays a role in the development of CRC using a murine model ofcolonic inflammation and tumor development. METHODS. For all studies, male C57BL/6mice (6-8 wks old) were used. To induce inflammation and tumor formation, mice weretreated by a single intraperitoneal (ip) injection of the tumor promoter azoxymethane (AOM;10 mg/kg) on day 0, followed by 2% dextran sodium sulfate (DSS) in drinking water for7 days. i) In the first experiment, AOM/DSS-treated and control mice were sacrificed onweek 12 and colonic mucosa harvested; activation of PI3K was assessed by Western blotfor expression of PI3K downstream effector proteins (phosphorylated Akt, mTOR, p70S6Kand 4E-BP1). ii) The experiment was repeated; disease activity index (DAI) was calculatedand correlated with tumor formation. iii) Next, the experiment was repeated and micesubdivided to receive wortmannin (0.75 mg/kg, ip) or vehicle (5% EtOH) every 8 h onweeks 4, 6, 8 and 10 for 7 days; mice were sacrificed on week 12.RESULTS. Phosphorylationof Akt, mTOR, p70S6K and 4E-BP1 was significantly increased in the colonic tumors ascompared to normal mucosa. Tumor size was significantly correlated with DAI (R = 0.89;p < 0.01). Wortmannin treatment significantly reduced the average size of colonic tumorscompared to the vehicle-treated group (14.9 mm2 vs 30.3 mm2, respectively). CONCLU-SIONS. PI3K activation is markedly increased in the colonic tumors arising from colonicinflammation. Inhibition of PI3K suppresses the growth of colonic tumors induced by AOM/DSS suggesting that the PI3K/Akt pathway plays a critical role in inflammation-mediateddevelopment of colonic tumors. Inhibition of PI3K may represent a potential preventivetherapy for CRCs induced by chronic inflammation.
M2010
Tumor Necrosis Factor Receptor Signaling in Intestinal Epithelial Cells MayBe Directly Involved in Colitis-Associated CarcinogenesisMichio Onizawa, Takashi Nagaishi, Shigeru Oshima, Ryuichi Okamoto, Teruji Totsuka,Kiichiro Tsuchiya, Takanori Kanai, Hideo Yagita, Mamoru Watanabe
Background & Aim: Treatment with anti-tumor necrosis factor (TNF) mAb has been acceptedas a successful therapy for patients with inflammatory bowel diseases (IBD). It has beenrecently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoi-etic cells may prevents the development of colitis-associated cancer (CAC). However, itremains unclear whether the TNF signaling in the epithelial cells is also involved in thedevelopment of CAC. To investigate this, we studied the effects of anti-TNF mAb in animalmodels of colitis and CAC. Methods & Results: Wild type C57BL/6 (WT B6) mice weretreated with 3.0% DSS-contained drinking water for 5 days as acute phase followed by 5days of recovering phase with regular water and subsequently sacrificed. Western blotting(WB) and immunohistochemistry (IHC) showed that the NF-κB pathway is activated in thecolonic epithelia from DSS-treated mice in association with increased expression of TNF ininflamed mucosa. It was also observed that TNFR-2, but not TNFR1, was significantlyupregulated in the epithelia from DSS-treated mice by quantitative RT-PCR and WB. Giventhese results, a mouse colonic epithelial cell line, CT26, was examined whether TNFR2signaling is associated with NF-κB activation in these cells. Stimulation with recombinant(r) TNF led to NF-κB activation in CT26 cells in a dose dependent fashion, and blockadeof TNF with a specific mAb, MP6-XT22, diminished such NF-κB activity. The expressionof myosin light chain kinase (MLCK), which has been reported to be a responsible moleculeto the permeability of epithelial barrier in colitis, was downregulated in CT26 cells whenTNFR2 was silenced by specific siRNA transfection even in the presence of rTNF. We nextinduced the CAC model by pretreatment with azoxymethane (AOM) in DSS-receiving mice.Mice administered with AOM/DSS revealed multiple tumors in the middle to distal colonand the NF-κB pathway was further activated in association with more upregulated TNFR2in the tumor compared to non-tumor area. Despite inability to reduce the severity of colitis,sequential MP6-XT22 treatment reduced the numbers and size of tumors in association withthe NF-κB inactivation. IHC with anti-ZO-1 pAb showed that the disrupted epithelial tight
A-466AGA Abstracts
junctions in AOM/DSS-receiving mice, however, MP6-XT22 treatment restored the tightjunctions. Conclusions: Our studies suggest that the TNFR2 signaling in intestinal epithelialcells may be directly involved in the development of CAC with persistent colitis, and implythat the maintenance therapy with anti-TNF mAb may prevent the development of CAC inpatients with IBD.
M2011
Epigenetic Suppression of the Mismatch Repair Protein MLH1 By Hypoxia inMicrosatellite Unstable Colon Cancer in Gia2-/- MiceMavee S. Witherspoon, Kehui Wang, Steven M. Lipkin, Robert A. Edwards
Colorectal cancer (CRC) in patients with either IBD or DNA mismatch repair (MMR) defectsdiffers from sporadic colon cancer. IBD-CRC and MMR-defective CRCs are more likely tobe right sided, multifocal, mucinous, arise from areas of flat dysplasia, and have microsatelliteinstability. The Gia2-/- mouse model of IBD-CRC develops cancers that have all of thesefeatures. We found that the MMR protein MLH1 is downregulated in inflamed mucosa andcancers, and these tumors contain microsatellite instability and have impaired DNA damageresponses. We have therefore investigated mechanisms of MLH1 silencing in colonic mucosa.MLH1 epigenetic silencing via promoter DNA methylation is a well established mechanismof carcinogenesis. We did not observe altered MLH1 promoter methylation in in inflamedGiα2-/- mucosa. Recently, hypoxia has been shown to silence MLH1 expression In Vitro bya mechanism involving histone deacetylation of the proximal promoter. We verified thathypoxic murine colonic epithelial cells (YAMC cells) downregulate MLH1 and have decreasedacetylated histone H3 at the proximal MLH1 promoter by chromatin immunoprecipitation.HDAC inhibitor treatment upregulates acetyl-H3 levels and MLH1 expression In Vitro. Using2-nitroimidazoles as In Vivomarkers of hypoxia, we have identified tissue hypoxia in inflamedGiα2-/- mucosa and cancers that have decreased MLH1 (and its obligate binding partnerPMS2) by qRT-PCR, immunohistochemistry, and western blot. In Vivo, treating Giα2-/-mice with the HDAC inhibitor SAHA increases acetylated histone H3 levels and enhancesMLH1 expression in colonic crypts. These data suggest that chronic hypoxic inflammationcontributes toMMR- defective colon cancer via epigenetic histone modifications and silencingof the MLH1 locus.
M2012
Vav1 Acts As An Essential Modulator in Colitis Associated Colon CancerStefanie Zenker, Imke Atreya, Christoph Becker, Markus F. Neurath
Introduction: The important intracellular adapter molecule and guanine nucleotide exchangefactor Vav1 plays key roles in various intracellular signalling cascades. In contrast to thespecific characterization of Vav1 in T lymphocytes, the impact of Vav1 in macrophages hasnot been studied extensively yet. We now analysed the effects of Vav1 deficiency in macro-phages and hence the influence and function of Vav1 in the setting of intestinal inflammationand tumorigenesis. Methods: The susceptibility of Vav1 deficient (Vav1-/-) mice was testedin the dextran sulfate sodium (DSS)/azoxymethane (AOM) model of colitis associated coloncancer. Severity of mucosal inflammation and tumour burden was evaluated by mini-endoscopy. In Vivo and In Vitro behaviour of Vav1-/-macrophages was further specified byimmunhistology, ELISA, real time PCR and Western blotting. Results: Compared to wild-type (WT) Balb/c mice, treatment with DSS/AOM resulted in enhanced susceptibility ofVav1 deficient mice for colonic tumorigenesis. A higher number of colonic tumours and anincreased tumour size could be detected in Vav1-/-mice compared to WT Balb/c animals.The evident infiltration of F4/80+macrophages in the inflamed intestinal mucosa of WTbalb/c mice as well as of Vav1-/-mice exemplified that this cell population seemed to bevery important in our tumour model. Interestingly, the early secretion of pro-inflammatorycytokine IL-6 was significantly enhanced by LPS-stimulated Vav1-/-macrophages In Vitrocompared to WT cells. At the same time, secretion of TNF-alpha by macrophages was notmarkedly affected by the lack of Vav1 in our experimental setting. Furthermore, the lackof Vav1 in macrophages revealed in reduced cDNA- and protein level of SOCS3. Theseresults implicate that active Vav1 is essential for LPS-mediated induction of SOCS3 inmacrophages and thereby plays a central role for the regulation of IL-6 secretion. Conclusion:Vav1 exhibits a protective key role in inflammation associated colonic tumour genesis.Overall our results give evidence to the working hypothesis that the tumour-suppressivepotential of Vav1 is trigged by its capacity to induce the expression of SOCS3 and thus thedown regulation of pro-inflammatory cytokines.
M2013
Effect of Targeting the Neurokinin-1 Receptor On the Transition of ChronicInflammation to DysplasiaBeatriz Pagan, Angel A. Isidro, Domenico Coppola, Jie Wu, Caroline B. Appleyard
Patients with long-standing colitis have a significantly increased risk of developing colorectalcancer. Substance P and its neurokinin-1 receptor (NK-1R) are known to be involved inchronic colonic inflammation but their role in the transition to dysplasia is unclear. Aim:To investigate the effects of targeting the NK-1R in a novel model of colitis-associateddysplasia with a selective NK-1R antagonist. Methods: Chronic colitis was induced in maleSprague Dawley rats by the administration of trinitrobenzene sulfonic acid (TNBS; 30 mgin 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) forthree days. To induce colitis-associated dysplasia the rats then received TNBS (iv) twice aweek for ten weeks. One group received the NK-1R antagonist SR140333 (1 mg/kg ip)twice a week for ten weeks; the rest received vehicle (n=22/group). After sacrifice the colonswere removed and analyzed for total macroscopic damage, microscopic damage and presenceof dysplasia. The expression of the downstream signalling components and cell proliferationwere analyzed by immunohistochemical staining and quantitative real-time RT-PCR. Results:Those animals receiving the NK-1R antagonist gained significantly less weight than thevehicle treated group throughout the study, however, these animals had significantly lessmacroscopic (p<0.01) and microscopic damage (p<0.01) than the vehicle treated-controls.
