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995 Proteomic Analysis Separates Barrett's Esophagus and Esophageal Adenocarcinoma from Healthy Controls By Distinct T Cell Populations
colorectal cancer in this group was compared to that among those individuals in the provincial population, who had never used statins, by Poisson regression analysis, with adjustment for age and gender. Stratified analysis was performed to determine the risk after five years of regular statin use. The additional potential confounding factors were identified by linkage to the provincial hospitalization and physicians claims administrative databases and included history of diabetes, inflammatory bowel disease, coronary heart disease, lower gastrointestinal endoscopy, resective colorectal surgery, use of NSAIDs, hormone replacement therapy (among women) and median household income. Results: There were 35,739 individuals (18,500 males; 17, 239 females; mean age 64.8 yrs), who were dispensed statins regularly. The 10,287 (5,069 males; 5,218 females; mean age 63.6 yrs) regular long-term users with history of regular statin dispensation for more than five years, were followed-up for up to five additional years. The ratio of incidence rate of colorectal cancer among those dispensed statins regularly to those who were never dispensed statins was 1.22 (95% CI: 1.11-1.35). The incidence rate ratio of colorectal cancer among the regular long-term users, after five years of use, to those who were never dispensed statins was 0.95 (95% CI: 0.75-1.19). The additional potential confounding factors did not significantly alter the risk ratios. Conclusions: These findings suggest that long-term use of statins for the current clinical indications does not protect against colorectal cancer risk. If there is any benefit to the use of statins in relation to colorectal cancer development it is not in persons who have been prescribed statins for hypercholesterolemia. There may be some added risk inherent to such persons that counters any benefit of statin use, as suggested in experimental studies.
Unraveling Novel Mechanisms By Which the CD24 Oncogene Serves As a Target for Colorectal and Pancreatic Cancer Treatment, Via Down-Regulation By Monoclonal Antibodies or SIRNA Eyal Sagiv, Shiran Shapira, Diana Kazanov, Inna Naumov, Kraus Sarah, Nadir Arber Background: We have previously shown that CD24 is a potential oncogene in the gut, being highly expressed in 90% of malignant and pre-malignant lesions from the entire GI tract, and only in 17% of normal epithelium (N=389). A year ago we confirmed that CD24 is indeed an important oncogene, based on the fact that stably expressed CD24 siRNA reduced the malignant phenotype of colorectal (CRC) and pancreatic (PC) cancer cell lines, and we were able to show the potential of anti-CD24 monoclonal antibodies (MAb) in CRC and PC treatment In Vitro. Aim: To further explore the therapeutic potential of CD24 MAb and siRNA silencing In Vivo and to unravel possible downstream cellular mechanisms. Methods: 1. HT29 (CRC) and Colo357 (PC) cell lines, expressing high levels of CD24, were used as human xenograft models in nude mice bearing: A. Subcutaneous tumors (5x106 cells) N= 20. B. Splenic tumors (1x106 cells) N=20. In each experiment, the mice were randomly divided into two groups; treated IV twice weekly with either MAb or saline. Animals were killed when tumor volume reached 15 mm, or after 18 days in the splenic model. Primary and metastatic tumors were dissected, fixed and stained for H&E. 2. In order to unravel the degradation mechanism of CD24, cells were treated with the MAb ± pre-treatment with chloroquine (lysosome inhibitor) or MG132 (proteosome inhibitor). Lysates were collected and analyzed for CD24 expression by Western blotting. 3. Gene expression array analysis was performed using Genechip®(HG-U133A) for HT29 and Colo357 cells before and after 72hr of exposure to anti-CD24 MAb and for CD24-siRNA expressing HT29 clones. Statistical and cluster analyses were performed using GeneSpring® software; functional analysis was carried out based on Gene Ontology (GO) groups. Results: 1. MAb treatment significantly inhibited subcutaneous tumor growth and formation in the spleen, and metastases shedding. 2. MAb treatment induced lysosomal-mediated degradation of CD24 protein 3. Altered gene expression (>3) following MAb therapy (n=581) and stable expression of CD24 siRNA (N= 1572) was found. siRNA expression in cells mainly affected the Ras and MAPK pathways. The expression of 139 genes was similarly altered by both treatment modalities, including genes of the BCl-2 super-family and p21 WAF1/CIP1. Pathway analysis revealed that genes associated with cell cycle regulation were mostly involved. Conclusions: 1. CD24 is a potential useful target for early intervention and treatment of CRC and PC. 2. Anti-CD24 MAb therapy involves degradation of CD24 through the lysosomal pathway. 3. CD24 initiated a signal transduction pathway that affects cell cycle progression.
995 Proteomic Analysis Separates Barrett's Esophagus and Esophageal Adenocarcinoma from Healthy Controls By Distinct T Cell Populations Uta Berndt, Sebastian Bartsch, Lars Philipsen, Bertram Wiedenmann, Marcus Haemmerle, Heiko Pohl, Andreas Sturm BACKGROUND: Barrett's esophagus (BE) is caused by chronic gastroesophageal reflux with consecutive mucosal inflammation, predisposing the development of esophageal adenocarcinoma (EAC). Mucosal inflammation is triggered by T cells. Thus, we investigated changes in T cell related mucosal combinatorial molecular protein patterns (CMP) using the novel Multi-Epitope-Ligand-Cartography (MELC), a unique robotic whole-cell imaging technology. METHODS: Biopsies were taken during endoscopy from BE, EAC, and normal control tissue (n=9 each group). After fixation and embedding, MELC microscopy with 32 different FITCconjugated antibodies was performed. Thereby this technique allows integrating cell biology and biomathematical tools to simultaneously visualize dozens of proteins in a structurally intact tissue to correlate cellular localization of proteins with function. Identification of molecular networks is possible not only by protein co-localization motifs, even in absence of proteins (anti-co-localization code). RESULTS: When the significance level was set to p<0.0005 (t-test) and the search depth to at most five antibody combinations, controls and BE can be differentiated by 63, controls and EAC by 3227, and BE from EAC by 1521 distinct protein combinations. For example the number of activated apoptotic naïve T cells CD45RA+CD8-caspase3+caspase8+NfkB+, activated apoptotic memory T cells, activated apoptotic regulatory T cells as well as activated apoptotic T lymphocytes was significantly increased only in BE, whereas the number of activated apoptotic helper T cells was significantly elevated in BE and EAC, uncovering a shared role of apoptotic T helper cells in both diseases. In contrast, the number of activated apoptotic cytotoxic T cells was comparably low in each group. Confirming different pathways in BE and EAC, the number of nonactivated T lymphocytes with expression of the tumor-suppressor p53 and downregulation of bcl2 expression (CD3+p53+Bcl2-NfkB-) was significantly increased in EAC compared to BE and controls. Interestingly, the number of thymocytes (CD7+) was significantly elevated only in EAC. These cells lack expression of Bax and caspase-8, hinting an impaired apoptosis in early stages of T cell differentiation. CONCLUSION: Proteomic analysis showed for the first time that CMPs which are critically involved in the mucosal immune system of the esophagus, are distinct in BE and EAC whereas others are comparably changed in both diseases, suggesting that many pathogenic events might be shared by both diseases. Thus, topological proteomic analysis may help to understand the different pathogenic events in the underlying disease pathways.
993 Tissue Transglutaminase Downregulation Potentiates Gemcitabine Efficacy and Blocks Pancreatic Cancer Growth In Vivo Sushovan Guha, Amit Verma, Kapil Mehta Background: Pancreatic ductal adenocarcinoma (PaCa) is virtually incurable. The molecular mechanisms that contribute to intrinsic resistance of PaCa to multiple anticancer therapies are not well understood. We observed that several drug-resistant (including gemcitabine) and metastatic tumors express elevated levels of tissue transglutaminase (TG2). TG2 is an ubiquitous enzyme that leads to activation PLC/MAPK pathways. TG2 also promotes RhoA, FAK, and β-integrin-mediated cell adhesion and cell migration. We showed that elevated expression of TG2 in PaCa cells was associated with increased gemcitabine resistance and invasive potential. Conversely, down-regulation of TG2 by siRNA attenuated gemcitabine resistance and invasive functions In Vitro. Aim: Determine whether liposomal TG2-specific siRNA can block tumor growth and metastasis either alone or in combination with gemcitabine in an orthotopic model of PaCa. Methods and Results: Using Panc-28 cells (TG2hi) stably transfected with luciferase as our model system, we examined the effects of TG2specific siRNA (liposomal formulation) in our orthotopic PaCa model in nude mice. The mice were randomized into 4 groups (n=6/group) after 3 weeks based on luciferase bioluminescence imaging (BLI; Xenogen IVIS200). The 4 groups consisting of scrambled siRNA, gemcitabine (25 mg/kg/twice weekly; IP), TG2-siRNA (150 µg/kg/thrice weekly; IV), and gemcitabine + TG2-siRNA were treated for 4 weeks and tumor volumes were measured weekly using BLI. Animals were sacrificed at 7th week after tumor implantation and tumor volumes were measured. Gemcitabine + TG2-siRNA significantly inhibited growth (p=0.007 vs scrambled and p=0.042 vs gemcitabine) and also blocked metastasis (by 80%; p<0.001). Interestingly, TG2-siRNA alone blocked metastasis similar to combination group. TG2siRNA either alone or in combination significantly decreased Ki-67+ expression (p<0.001 vs scrambled and p<0.05 vs gemcitabine) and the combination group significantly reduced CD31+ microvessel density (p<0.001 vs scrambled). Mechanistically, TG2-siRNA either alone or in combination reduced AKT phosphorylation (S473), and blocked NF-κB activity and expression of its downstream gene product, VEGF. Conclusion: Our results show for the first time that TG2-siRNA (liposomal formulation) given intravenously enhanced anti-tumor effects of gemcitabine by significantly reducing orthotopic PaCa tumor growth and blocking local invasion/metastasis in nude mice. Thus, targeting TG2 in combination with chemotherapeutic agents is a novel therapeutic strategy in PaCa.
996 Implication of Notch Signaling and CDX-2 Expression in Barrett's Carcinogenesis Peter Konturek, Gregor Burnat, Tilman Rau, Eckhart G. Hahn Background/Aims: Barrett esophagus (BE) represents an important precancerous change in the esophageal mucosa. The mechanisms of carcinogenesis in BE remains still poorly understood. The aim of the present study was to investigate the implication of Notch signaling and CDX-2 homebox transcription factor in the Barrett's carcinogenesis. Methods: 10 patients with histologically proved BE were included in this study. The expression of Notch-2 receptor and CDX-2 was analyzed at protein level by Western blot in biopsies obtained during NBIendoscopy from BE and normal squamous esophageal epithelium. In addition, in separate In Vitro studies, Barrett adenocarcinoma cell line (OE-19) was incubated with increasing concentrations of inhibitor of Notch signaling (γ secretase inhibitor, DAPT). Effect of DAPT on cell proliferation and apoptosis was analyzed by MTT assay and FACS analysis, respectively. Moreover, the effect of DAPT on the protein expression of proliferating cell nuclear antigen (PCNA - proliferation marker), p21 (inhibitor of cell cycle) and Notch-2 was assessed by means of Western blot. Finally, in separate experiments, expression of CDX-2 and Notch2 was analyzed in OE-19 cells incubated in neutral and shortly acidified media (pulse acidification) by qRT-PCR and Western blot, respectively. Results: Expression of Notch-2 and CDX-2 was significantly increased in BE as compared to normal squamous esophageal epithelium. Inhibition of Notch-2 signaling by DAPT (6.25-50 µM) was associated with significant decrease in proliferation rate and this effect was accompanied by an increase in p21 expression and decrease in PCNA expression. Interestingly, treatment with DAPT had no effect on cell apoptosis as shown by FACS analysis. Pulse acidification of media was associated with significant increase CDX-2 expression in OE-19 cells. Conclusions: 1) Both Notch signaling and CDX-2 are involved in Barrett's carcinogenesis; 2) Decrease in pH
994 Long-Term Users of Statins Are Not Protected Against Colorectal Cancer Harminder Singh, Donna Turner, Salaheddin M. Mahmud, Lin Xue, Anita L. Kozyrskyj, Alain Demers, Charles N. Bernstein Statins have chemo-preventive effect against colorectal cancer in laboratory studies. However human studies have been conflicting. We conducted a population-based cohort study to determine the risk of colorectal cancer among long-term users of statins. Methods: The cohort of individuals, dispensed statins regularly was identified from the Manitoba's population-based prescription drug data base. Those with prior history of colorectal cancer were excluded. The cohort was followed-up to diagnosis of colorectal cancer in the provincial cancer registry, migration out-of province, death or December 2005. The incidence of
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AGA Abstracts
992
Unraveling Novel Mechanisms By Which the CD24 Oncogene Serves As a Target for Colorectal and Pancreatic Cancer Treatment, Via Down-Regulation By Monoclonal Antibodies or SIRNA Eyal Sagiv, Shiran Shapira, Diana Kazanov, Inna Naumov, Kraus Sarah, Nadir Arber Background: We have previously shown that CD24 is a potential oncogene in the gut, being highly expressed in 90% of malignant and pre-malignant lesions from the entire GI tract, and only in 17% of normal epithelium (N=389). A year ago we confirmed that CD24 is indeed an important oncogene, based on the fact that stably expressed CD24 siRNA reduced the malignant phenotype of colorectal (CRC) and pancreatic (PC) cancer cell lines, and we were able to show the potential of anti-CD24 monoclonal antibodies (MAb) in CRC and PC treatment In Vitro. Aim: To further explore the therapeutic potential of CD24 MAb and siRNA silencing In Vivo and to unravel possible downstream cellular mechanisms. Methods: 1. HT29 (CRC) and Colo357 (PC) cell lines, expressing high levels of CD24, were used as human xenograft models in nude mice bearing: A. Subcutaneous tumors (5x106 cells) N= 20. B. Splenic tumors (1x106 cells) N=20. In each experiment, the mice were randomly divided into two groups; treated IV twice weekly with either MAb or saline. Animals were killed when tumor volume reached 15 mm, or after 18 days in the splenic model. Primary and metastatic tumors were dissected, fixed and stained for H&E. 2. In order to unravel the degradation mechanism of CD24, cells were treated with the MAb ± pre-treatment with chloroquine (lysosome inhibitor) or MG132 (proteosome inhibitor). Lysates were collected and analyzed for CD24 expression by Western blotting. 3. Gene expression array analysis was performed using Genechip®(HG-U133A) for HT29 and Colo357 cells before and after 72hr of exposure to anti-CD24 MAb and for CD24-siRNA expressing HT29 clones. Statistical and cluster analyses were performed using GeneSpring® software; functional analysis was carried out based on Gene Ontology (GO) groups. Results: 1. MAb treatment significantly inhibited subcutaneous tumor growth and formation in the spleen, and metastases shedding. 2. MAb treatment induced lysosomal-mediated degradation of CD24 protein 3. Altered gene expression (>3) following MAb therapy (n=581) and stable expression of CD24 siRNA (N= 1572) was found. siRNA expression in cells mainly affected the Ras and MAPK pathways. The expression of 139 genes was similarly altered by both treatment modalities, including genes of the BCl-2 super-family and p21 WAF1/CIP1. Pathway analysis revealed that genes associated with cell cycle regulation were mostly involved. Conclusions: 1. CD24 is a potential useful target for early intervention and treatment of CRC and PC. 2. Anti-CD24 MAb therapy involves degradation of CD24 through the lysosomal pathway. 3. CD24 initiated a signal transduction pathway that affects cell cycle progression.
995 Proteomic Analysis Separates Barrett's Esophagus and Esophageal Adenocarcinoma from Healthy Controls By Distinct T Cell Populations Uta Berndt, Sebastian Bartsch, Lars Philipsen, Bertram Wiedenmann, Marcus Haemmerle, Heiko Pohl, Andreas Sturm BACKGROUND: Barrett's esophagus (BE) is caused by chronic gastroesophageal reflux with consecutive mucosal inflammation, predisposing the development of esophageal adenocarcinoma (EAC). Mucosal inflammation is triggered by T cells. Thus, we investigated changes in T cell related mucosal combinatorial molecular protein patterns (CMP) using the novel Multi-Epitope-Ligand-Cartography (MELC), a unique robotic whole-cell imaging technology. METHODS: Biopsies were taken during endoscopy from BE, EAC, and normal control tissue (n=9 each group). After fixation and embedding, MELC microscopy with 32 different FITCconjugated antibodies was performed. Thereby this technique allows integrating cell biology and biomathematical tools to simultaneously visualize dozens of proteins in a structurally intact tissue to correlate cellular localization of proteins with function. Identification of molecular networks is possible not only by protein co-localization motifs, even in absence of proteins (anti-co-localization code). RESULTS: When the significance level was set to p<0.0005 (t-test) and the search depth to at most five antibody combinations, controls and BE can be differentiated by 63, controls and EAC by 3227, and BE from EAC by 1521 distinct protein combinations. For example the number of activated apoptotic naïve T cells CD45RA+CD8-caspase3+caspase8+NfkB+, activated apoptotic memory T cells, activated apoptotic regulatory T cells as well as activated apoptotic T lymphocytes was significantly increased only in BE, whereas the number of activated apoptotic helper T cells was significantly elevated in BE and EAC, uncovering a shared role of apoptotic T helper cells in both diseases. In contrast, the number of activated apoptotic cytotoxic T cells was comparably low in each group. Confirming different pathways in BE and EAC, the number of nonactivated T lymphocytes with expression of the tumor-suppressor p53 and downregulation of bcl2 expression (CD3+p53+Bcl2-NfkB-) was significantly increased in EAC compared to BE and controls. Interestingly, the number of thymocytes (CD7+) was significantly elevated only in EAC. These cells lack expression of Bax and caspase-8, hinting an impaired apoptosis in early stages of T cell differentiation. CONCLUSION: Proteomic analysis showed for the first time that CMPs which are critically involved in the mucosal immune system of the esophagus, are distinct in BE and EAC whereas others are comparably changed in both diseases, suggesting that many pathogenic events might be shared by both diseases. Thus, topological proteomic analysis may help to understand the different pathogenic events in the underlying disease pathways.
993 Tissue Transglutaminase Downregulation Potentiates Gemcitabine Efficacy and Blocks Pancreatic Cancer Growth In Vivo Sushovan Guha, Amit Verma, Kapil Mehta Background: Pancreatic ductal adenocarcinoma (PaCa) is virtually incurable. The molecular mechanisms that contribute to intrinsic resistance of PaCa to multiple anticancer therapies are not well understood. We observed that several drug-resistant (including gemcitabine) and metastatic tumors express elevated levels of tissue transglutaminase (TG2). TG2 is an ubiquitous enzyme that leads to activation PLC/MAPK pathways. TG2 also promotes RhoA, FAK, and β-integrin-mediated cell adhesion and cell migration. We showed that elevated expression of TG2 in PaCa cells was associated with increased gemcitabine resistance and invasive potential. Conversely, down-regulation of TG2 by siRNA attenuated gemcitabine resistance and invasive functions In Vitro. Aim: Determine whether liposomal TG2-specific siRNA can block tumor growth and metastasis either alone or in combination with gemcitabine in an orthotopic model of PaCa. Methods and Results: Using Panc-28 cells (TG2hi) stably transfected with luciferase as our model system, we examined the effects of TG2specific siRNA (liposomal formulation) in our orthotopic PaCa model in nude mice. The mice were randomized into 4 groups (n=6/group) after 3 weeks based on luciferase bioluminescence imaging (BLI; Xenogen IVIS200). The 4 groups consisting of scrambled siRNA, gemcitabine (25 mg/kg/twice weekly; IP), TG2-siRNA (150 µg/kg/thrice weekly; IV), and gemcitabine + TG2-siRNA were treated for 4 weeks and tumor volumes were measured weekly using BLI. Animals were sacrificed at 7th week after tumor implantation and tumor volumes were measured. Gemcitabine + TG2-siRNA significantly inhibited growth (p=0.007 vs scrambled and p=0.042 vs gemcitabine) and also blocked metastasis (by 80%; p<0.001). Interestingly, TG2-siRNA alone blocked metastasis similar to combination group. TG2siRNA either alone or in combination significantly decreased Ki-67+ expression (p<0.001 vs scrambled and p<0.05 vs gemcitabine) and the combination group significantly reduced CD31+ microvessel density (p<0.001 vs scrambled). Mechanistically, TG2-siRNA either alone or in combination reduced AKT phosphorylation (S473), and blocked NF-κB activity and expression of its downstream gene product, VEGF. Conclusion: Our results show for the first time that TG2-siRNA (liposomal formulation) given intravenously enhanced anti-tumor effects of gemcitabine by significantly reducing orthotopic PaCa tumor growth and blocking local invasion/metastasis in nude mice. Thus, targeting TG2 in combination with chemotherapeutic agents is a novel therapeutic strategy in PaCa.
996 Implication of Notch Signaling and CDX-2 Expression in Barrett's Carcinogenesis Peter Konturek, Gregor Burnat, Tilman Rau, Eckhart G. Hahn Background/Aims: Barrett esophagus (BE) represents an important precancerous change in the esophageal mucosa. The mechanisms of carcinogenesis in BE remains still poorly understood. The aim of the present study was to investigate the implication of Notch signaling and CDX-2 homebox transcription factor in the Barrett's carcinogenesis. Methods: 10 patients with histologically proved BE were included in this study. The expression of Notch-2 receptor and CDX-2 was analyzed at protein level by Western blot in biopsies obtained during NBIendoscopy from BE and normal squamous esophageal epithelium. In addition, in separate In Vitro studies, Barrett adenocarcinoma cell line (OE-19) was incubated with increasing concentrations of inhibitor of Notch signaling (γ secretase inhibitor, DAPT). Effect of DAPT on cell proliferation and apoptosis was analyzed by MTT assay and FACS analysis, respectively. Moreover, the effect of DAPT on the protein expression of proliferating cell nuclear antigen (PCNA - proliferation marker), p21 (inhibitor of cell cycle) and Notch-2 was assessed by means of Western blot. Finally, in separate experiments, expression of CDX-2 and Notch2 was analyzed in OE-19 cells incubated in neutral and shortly acidified media (pulse acidification) by qRT-PCR and Western blot, respectively. Results: Expression of Notch-2 and CDX-2 was significantly increased in BE as compared to normal squamous esophageal epithelium. Inhibition of Notch-2 signaling by DAPT (6.25-50 µM) was associated with significant decrease in proliferation rate and this effect was accompanied by an increase in p21 expression and decrease in PCNA expression. Interestingly, treatment with DAPT had no effect on cell apoptosis as shown by FACS analysis. Pulse acidification of media was associated with significant increase CDX-2 expression in OE-19 cells. Conclusions: 1) Both Notch signaling and CDX-2 are involved in Barrett's carcinogenesis; 2) Decrease in pH
994 Long-Term Users of Statins Are Not Protected Against Colorectal Cancer Harminder Singh, Donna Turner, Salaheddin M. Mahmud, Lin Xue, Anita L. Kozyrskyj, Alain Demers, Charles N. Bernstein Statins have chemo-preventive effect against colorectal cancer in laboratory studies. However human studies have been conflicting. We conducted a population-based cohort study to determine the risk of colorectal cancer among long-term users of statins. Methods: The cohort of individuals, dispensed statins regularly was identified from the Manitoba's population-based prescription drug data base. Those with prior history of colorectal cancer were excluded. The cohort was followed-up to diagnosis of colorectal cancer in the provincial cancer registry, migration out-of province, death or December 2005. The incidence of
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AGA Abstracts
AGA Abstracts
992