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Colorectal Cancers Choosing Sides
that loss of intestinal Hai-1/Spint1 in ApcMin+ mice results in shorter lifespan (p < 0.05, Mann-Whitney's U test). On the other hand, Spint1flox/flox-Villin/Cre without Apc mutation did not show such a short lifespan. In fifteen weeks-old male mice, the total number of tumors formed in the small intestine were significantly increased in Spint1flox/flox-Villin/ Cre-ApcMin+ mice (121.4 ± 22.5, n = 5) compared with control mice (45.5 ± 17.5, n = 8) (p < 0.05, Mann-Whitney's U test) (Figure 1). CONCLUSION: These findings indicate that intestinal HAI-1/SPINT1 has an important role in suppressing intestinal tumorigenesis.
Tu1703 Colorectal Cancers Choosing Sides Cristina Albuquerque, Elvira Bakker, Ernst J. Kuipers, Wendy van Veelen, Ron Smits
Tu1705
Sporadic colorectal cancers predominantly occur in the distal colon while most mismatch repair (MMR) deficient tumors occur in the proximal colon. At present no good hypothesis is available to explain these regional preferences of tumor occurrence. Recently, we have screened colorectal tumors for mutations in Wnt-related genes with a specific focus on colorectal location, and combined these results with a meta-analysis of publications in which tumor location, defects in mismatch repair, and somatic mutations were reported. Using this analysis, we present an explanation for the side-related preferences of colorectal cancers. The initiation of colorectal tumors relates to a defect in the β-catenin signal transduction pathway. Originally all mutations in the β-catenin signaling pathway were considered to be equal in their impact on tumor initiation. However, we and others have shown that a strong selective pressure exists on the level of β-catenin signaling resulting from the specific mutations, determining whether an emerging tumor cell is successful in tumor formation (the just-right signaling hypothesis). The APC gene is mostly mutated by truncation of the protein leaving various numbers of repeats intact involved in β-catenin breakdown regulation. Longer truncated APC proteins retaining more repeats also retain more β-catenin breakdown activity. Depending on the tissue type a certain number of those repeats is more readily selected than others. For example, in desmoid and upper GI tumors, APC mutations are selected retaining 2-3 repeats, whereas in colorectal cancer as a whole, 1-2 repeats are preferred. However, whereas 70% of distal colonic tumors acquire mutations retaining no or more often only one 20 aa repeat, 70% of proximal tumors select for truncated APC proteins retaining 2-3 repeats. These results strongly suggest that for tumor initiation a gradient of β-catenin signaling dosage exists along the colorectal tract with moderate levels being preferred proximally, and higher levels towards the rectum. The APC coding sequence coding for truncated proteins with one repeat, comprises half of the sequence commonly mutated in cancers. Accordingly, more than 50% of tumors with a functional MMR obtain truncated proteins with one repeat, which provides the ideal β-catenin signaling level for the distal colon. In MMR deficient cells repetitive sequences are most prone to mutations. Within APC, these are located in the region coding for truncated proteins with 2-3 repeats. Consequently, 70% of APC mutant MMR deficient tumors acquire truncating proteins with 2-3 repeats providing the ideal selective advantage for the proximal colon. In conclusion, the side preference of colorectal tumors can be explained by their specific mutational mechanism, leading to APC mutations providing the β-catenin signaling level ideal for either side of the colon.
Hepatocyte Growth Factor Inhibits Colon Cancer Development in a Mouse Model of Ulcerative Colitis Naohisa Yamaji, Akio Ido, Hitoshi Setoyama, Masatsugu Numata, Akihiro Moriuchi, Hiroshi Fujita, Toshio Sakiyama, Hirohito Tsubouchi Background and aims: Patients with ulcerative colitis often develop colon cancer, so called colitic cancer. Recently, we reported that hepatocyte growth factor (HGF) stimulates the proliferation of colonic epithelial cells, which ameliorates experimental colitis in rats (J Pharmacol Exp Ther 2003;307:146-151; Inflamm Bowel Dis 2005;31:79-87). However, HGF can stimulate the development of colon tumors. The aim of this study was to clarify the effects of HGF on colon tumor development in a mouse model of experimental colitis. Methods: (1)To assess the effects of HGF on colon tumor development, A/J mice were injected intraperitoneally with azoxymethane (AOM) (5 mg/kg) once a week for six weeks. Intraperitoneal injections of recombinant human HGF (0.1, 0.5, or 1.0 mg/kg/day) or the vehicle control (phosphate buffered saline) were initiated with the AOM injection, and continued every second day for 14 weeks. The number, incidence, and multiplicity of colon tumors were determined after the 14-week HGF regimen was complete. (2) To induce colitis-associated cancer, CBA/J mice were injected with a single dose of AOM (12.5 mg/ kg) followed by 3 cycles of administration of 2.5% dextran sulfate sodium (DSS), in which each cycle consists of 5 days of DSS treatment followed by 16 days of regular water. Intraperitoneal injections of HGF or the vehicle control were initiated with the DSS treatment, and continued for 2 weeks (5 days a week) in each cycle. Two weeks after the final administration of DSS, the following parameters were assessed: (i) the number, incidence, and multiplicity of colon tumors, (ii) the proliferation of tumor cells, and (iii) the degree of colitis, including the clinical and histological scores. Results: (1)HGF administration significantly reduced the number, incidence, and multiplicity of colon tumors (p=0.02). (2) (i) HGF treatment suppressed the development of colon tumors in a dose-dependent manner; repeated 1.0 mg/kg/day doses of HGF significantly reduced the number of colon tumors (p=0.011). HGF also decreased the incidence and multiplicity of colon tumors, but this decrease was not statistically significant. (ii) Tumor cell proliferation was not affected by repeated HGF doses. (iii) HGF administration significantly ameliorated both the clinical and histological scores (p=0.024 and p=0.03, respectively). Conclusions: These results suggest that although HGF is a potent mitogen for colonic epithelial cells, it accelerates the healing of mucosal injuries, leading to the inhibition of colon tumor development.
Tu1704 Tu1706
Defect of Hepatocyte Growth Factor Activator Inhibitor Type1, a Cell Surface Serine Protease Inhibitor, Enhances Intestinal Tumorigenesis in ApcMin/+ Mice Shinri Hoshiko, Makiko Kawaguchi, Kenji Yorita, Tsuyoshi Fukushima, Hiroaki Kataoka
Colorectal Cancer After Ionization External Beam Radiation for Prostate Cancers: Are Other Etiologic Factors Involved? Martin Tobi, Yu-Xiao Yang, Rebecca M. Rodriguez, Keith Davies, Bradley Irwin, Steven Marcus
BACKGROUND & AIMS: Hepatocyte growth factor activator inhibitor type 1 (HAI-1) encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membranebound serine protease inhibitor expressed on the basolateral surface of polarized epithelial cells. Evidence is emerging to indicate that HAI-1/SPINT1 modulates cancer cell biology, and in mice, the balance between Hai-1/Spint1 and its target proteinase, matriptase, is critical in the development of skin cancer. Although HAI-1/SPINT1 is abundantly expressed in the intestine, its functional role in the intestinal epithelium is not known. The aim of this study was to determine the role of HAI-1/SPINT1 in intestinal tumorigenesis. METHODS: Mice with intestinal epithelial cell-specific deletion of Spint1 gene were generated by interbreeding of mice carrying the Spint1LoxP homozygous alleles (Spint1flox/flox) with Cre transgenic mice under the control of the intestine-specific villin promoter (Villin/Cre). Successful deletion of Spint1 gene in the intestinal epithelium was confirmed by RT-PCR and immunohistochemical analyses. Then the mice were interbreeded with ApcMin/+ mice generating Spint1flox/flox-Villin/Cre-ApcMin+ and compared intestinal tumor formation and survival with control Spint1flox/flox-APCMin+ mice. RESULTS: The lifespans of Spint1flox/floxVillin/Cre-ApcMin+ and control Spint1flox/flox-APCMin+ mice were 117.0 ± 20.4 (mean ± SD) days (n = 11, male) and 192.9 ± 54.4 days (n = 7, male), respectively, indicating
Case control studies suggest an association between radiation for prostate cancer and the development of CRC after > 5 years. These retrospective findings are controversial but suggestions have been advanced to institute early screening within 5 years after delivery of radiation. Recently, Gillessen et al identified anti-androgen therapy as a cause (JNCI 2010;102:1). To elucidate, we undertook a population survey of the VHA National Database. Methods: We interrogated the VHA National Database using relevant ICD-9 and CPT codes to ascertain the total number of disease and treatment cases for the period of FY 1999 to FY2006. We estimated CRC incidence for the period A. Before diagnosis of prostate cancer up to the day of diagnosis of the same; b. From after the diagnosis of prostate cancer up to 5 years of follow-up; c after 5 years of follow-up. We compared the findings in the test group who received radiation for prostate cancer and a control group who received no irradiation. We analyzed the data by non-parametric chi-square. Results: The number of CRC cases in the entire cohort was 4.9/1,000 patient years, (8.3/1,000py- test group; 4.7/ 1,000py for controls). The proportion of CRC in the test group (1,635/24,706) was greater (15,846/442,238) than controls (RR1.76; CI1.68-1.85; p<0.0001). When considering the
S-815
AGA Abstracts
AGA Abstracts
MyD88 are protected from intestinal tumorigenesis. The receptor for advanced glycolysation endproducts (Rage) is a pattern recognition receptor that activates the innate immune system in response to HMGB1, a protein released by necrotic cells. Such activation by necrotic cells may be particularly relevant in the context of tumor development. Moreover it has previously been shown that Rage and its ligands are upregulated in human colon carcinomas. Therefore we investigated the role of Rage signaling in a model of sporadic intestinal adenoma development in the mouse. Methods: Rage knockout (Rage-/-) mice were crossed with Apcmin mice to homozygosity (n=15). In addition, a Green Fluorescent Protein (GFP) reporter gene was used, which is expressed upon Rage deletion. Ragewt/Apcmin mice were used as controls. At 17 weeks of age, mice were sacrificed. The number and size of the polyps in the intestine were analyzed. Rage expression was analyzed by immunostaining for GFP. As well immunohistochemistry for neutrophils, macrophages, T cells and mast cells was performed. These immune cells were counted and analyzed. Further apoptosis and proliferation in polyps was assessed. Results: Rage-/- animals exhibited a marked reduction in the number of polyps compared to control animals (111.3 ± 51.65 vs 46.86 ± 27.58, p<0.001). The size of the polyps did not differ. Immunostaining for GFP showed increased Rage expression in tumor epithelium. Analysis of tumor associated immune cells showed a 14-fold increase in the number of mast cells in polyps of Rage-/- versus control animals (75.31 vs 5.25 cells per vision field p<0.05). In contrast, there was no difference in the number of infiltrating neutrophils, macrophages or T cells. Number of caspase positive cells per polyp was increased in Rage-/- animals (34.3 ± 21.8 vs 11.8 ± 6.0 p<0.05). There was no significant difference in the number of proliferating cells (7.92 ± 4.36 vs 9.98 ± 5.39). Conclusion: Our results suggest that Rage signaling plays an important role in the initiation of spontaneous intestinal tumorigenesis. Rather than leading to diminished infiltration of immune cells, absence of Rage signaling results in a considerable recruitment of mast cells.
Tu1703 Colorectal Cancers Choosing Sides Cristina Albuquerque, Elvira Bakker, Ernst J. Kuipers, Wendy van Veelen, Ron Smits
Tu1705
Sporadic colorectal cancers predominantly occur in the distal colon while most mismatch repair (MMR) deficient tumors occur in the proximal colon. At present no good hypothesis is available to explain these regional preferences of tumor occurrence. Recently, we have screened colorectal tumors for mutations in Wnt-related genes with a specific focus on colorectal location, and combined these results with a meta-analysis of publications in which tumor location, defects in mismatch repair, and somatic mutations were reported. Using this analysis, we present an explanation for the side-related preferences of colorectal cancers. The initiation of colorectal tumors relates to a defect in the β-catenin signal transduction pathway. Originally all mutations in the β-catenin signaling pathway were considered to be equal in their impact on tumor initiation. However, we and others have shown that a strong selective pressure exists on the level of β-catenin signaling resulting from the specific mutations, determining whether an emerging tumor cell is successful in tumor formation (the just-right signaling hypothesis). The APC gene is mostly mutated by truncation of the protein leaving various numbers of repeats intact involved in β-catenin breakdown regulation. Longer truncated APC proteins retaining more repeats also retain more β-catenin breakdown activity. Depending on the tissue type a certain number of those repeats is more readily selected than others. For example, in desmoid and upper GI tumors, APC mutations are selected retaining 2-3 repeats, whereas in colorectal cancer as a whole, 1-2 repeats are preferred. However, whereas 70% of distal colonic tumors acquire mutations retaining no or more often only one 20 aa repeat, 70% of proximal tumors select for truncated APC proteins retaining 2-3 repeats. These results strongly suggest that for tumor initiation a gradient of β-catenin signaling dosage exists along the colorectal tract with moderate levels being preferred proximally, and higher levels towards the rectum. The APC coding sequence coding for truncated proteins with one repeat, comprises half of the sequence commonly mutated in cancers. Accordingly, more than 50% of tumors with a functional MMR obtain truncated proteins with one repeat, which provides the ideal β-catenin signaling level for the distal colon. In MMR deficient cells repetitive sequences are most prone to mutations. Within APC, these are located in the region coding for truncated proteins with 2-3 repeats. Consequently, 70% of APC mutant MMR deficient tumors acquire truncating proteins with 2-3 repeats providing the ideal selective advantage for the proximal colon. In conclusion, the side preference of colorectal tumors can be explained by their specific mutational mechanism, leading to APC mutations providing the β-catenin signaling level ideal for either side of the colon.
Hepatocyte Growth Factor Inhibits Colon Cancer Development in a Mouse Model of Ulcerative Colitis Naohisa Yamaji, Akio Ido, Hitoshi Setoyama, Masatsugu Numata, Akihiro Moriuchi, Hiroshi Fujita, Toshio Sakiyama, Hirohito Tsubouchi Background and aims: Patients with ulcerative colitis often develop colon cancer, so called colitic cancer. Recently, we reported that hepatocyte growth factor (HGF) stimulates the proliferation of colonic epithelial cells, which ameliorates experimental colitis in rats (J Pharmacol Exp Ther 2003;307:146-151; Inflamm Bowel Dis 2005;31:79-87). However, HGF can stimulate the development of colon tumors. The aim of this study was to clarify the effects of HGF on colon tumor development in a mouse model of experimental colitis. Methods: (1)To assess the effects of HGF on colon tumor development, A/J mice were injected intraperitoneally with azoxymethane (AOM) (5 mg/kg) once a week for six weeks. Intraperitoneal injections of recombinant human HGF (0.1, 0.5, or 1.0 mg/kg/day) or the vehicle control (phosphate buffered saline) were initiated with the AOM injection, and continued every second day for 14 weeks. The number, incidence, and multiplicity of colon tumors were determined after the 14-week HGF regimen was complete. (2) To induce colitis-associated cancer, CBA/J mice were injected with a single dose of AOM (12.5 mg/ kg) followed by 3 cycles of administration of 2.5% dextran sulfate sodium (DSS), in which each cycle consists of 5 days of DSS treatment followed by 16 days of regular water. Intraperitoneal injections of HGF or the vehicle control were initiated with the DSS treatment, and continued for 2 weeks (5 days a week) in each cycle. Two weeks after the final administration of DSS, the following parameters were assessed: (i) the number, incidence, and multiplicity of colon tumors, (ii) the proliferation of tumor cells, and (iii) the degree of colitis, including the clinical and histological scores. Results: (1)HGF administration significantly reduced the number, incidence, and multiplicity of colon tumors (p=0.02). (2) (i) HGF treatment suppressed the development of colon tumors in a dose-dependent manner; repeated 1.0 mg/kg/day doses of HGF significantly reduced the number of colon tumors (p=0.011). HGF also decreased the incidence and multiplicity of colon tumors, but this decrease was not statistically significant. (ii) Tumor cell proliferation was not affected by repeated HGF doses. (iii) HGF administration significantly ameliorated both the clinical and histological scores (p=0.024 and p=0.03, respectively). Conclusions: These results suggest that although HGF is a potent mitogen for colonic epithelial cells, it accelerates the healing of mucosal injuries, leading to the inhibition of colon tumor development.
Tu1704 Tu1706
Defect of Hepatocyte Growth Factor Activator Inhibitor Type1, a Cell Surface Serine Protease Inhibitor, Enhances Intestinal Tumorigenesis in ApcMin/+ Mice Shinri Hoshiko, Makiko Kawaguchi, Kenji Yorita, Tsuyoshi Fukushima, Hiroaki Kataoka
Colorectal Cancer After Ionization External Beam Radiation for Prostate Cancers: Are Other Etiologic Factors Involved? Martin Tobi, Yu-Xiao Yang, Rebecca M. Rodriguez, Keith Davies, Bradley Irwin, Steven Marcus
BACKGROUND & AIMS: Hepatocyte growth factor activator inhibitor type 1 (HAI-1) encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membranebound serine protease inhibitor expressed on the basolateral surface of polarized epithelial cells. Evidence is emerging to indicate that HAI-1/SPINT1 modulates cancer cell biology, and in mice, the balance between Hai-1/Spint1 and its target proteinase, matriptase, is critical in the development of skin cancer. Although HAI-1/SPINT1 is abundantly expressed in the intestine, its functional role in the intestinal epithelium is not known. The aim of this study was to determine the role of HAI-1/SPINT1 in intestinal tumorigenesis. METHODS: Mice with intestinal epithelial cell-specific deletion of Spint1 gene were generated by interbreeding of mice carrying the Spint1LoxP homozygous alleles (Spint1flox/flox) with Cre transgenic mice under the control of the intestine-specific villin promoter (Villin/Cre). Successful deletion of Spint1 gene in the intestinal epithelium was confirmed by RT-PCR and immunohistochemical analyses. Then the mice were interbreeded with ApcMin/+ mice generating Spint1flox/flox-Villin/Cre-ApcMin+ and compared intestinal tumor formation and survival with control Spint1flox/flox-APCMin+ mice. RESULTS: The lifespans of Spint1flox/floxVillin/Cre-ApcMin+ and control Spint1flox/flox-APCMin+ mice were 117.0 ± 20.4 (mean ± SD) days (n = 11, male) and 192.9 ± 54.4 days (n = 7, male), respectively, indicating
Case control studies suggest an association between radiation for prostate cancer and the development of CRC after > 5 years. These retrospective findings are controversial but suggestions have been advanced to institute early screening within 5 years after delivery of radiation. Recently, Gillessen et al identified anti-androgen therapy as a cause (JNCI 2010;102:1). To elucidate, we undertook a population survey of the VHA National Database. Methods: We interrogated the VHA National Database using relevant ICD-9 and CPT codes to ascertain the total number of disease and treatment cases for the period of FY 1999 to FY2006. We estimated CRC incidence for the period A. Before diagnosis of prostate cancer up to the day of diagnosis of the same; b. From after the diagnosis of prostate cancer up to 5 years of follow-up; c after 5 years of follow-up. We compared the findings in the test group who received radiation for prostate cancer and a control group who received no irradiation. We analyzed the data by non-parametric chi-square. Results: The number of CRC cases in the entire cohort was 4.9/1,000 patient years, (8.3/1,000py- test group; 4.7/ 1,000py for controls). The proportion of CRC in the test group (1,635/24,706) was greater (15,846/442,238) than controls (RR1.76; CI1.68-1.85; p<0.0001). When considering the
S-815
AGA Abstracts
AGA Abstracts
MyD88 are protected from intestinal tumorigenesis. The receptor for advanced glycolysation endproducts (Rage) is a pattern recognition receptor that activates the innate immune system in response to HMGB1, a protein released by necrotic cells. Such activation by necrotic cells may be particularly relevant in the context of tumor development. Moreover it has previously been shown that Rage and its ligands are upregulated in human colon carcinomas. Therefore we investigated the role of Rage signaling in a model of sporadic intestinal adenoma development in the mouse. Methods: Rage knockout (Rage-/-) mice were crossed with Apcmin mice to homozygosity (n=15). In addition, a Green Fluorescent Protein (GFP) reporter gene was used, which is expressed upon Rage deletion. Ragewt/Apcmin mice were used as controls. At 17 weeks of age, mice were sacrificed. The number and size of the polyps in the intestine were analyzed. Rage expression was analyzed by immunostaining for GFP. As well immunohistochemistry for neutrophils, macrophages, T cells and mast cells was performed. These immune cells were counted and analyzed. Further apoptosis and proliferation in polyps was assessed. Results: Rage-/- animals exhibited a marked reduction in the number of polyps compared to control animals (111.3 ± 51.65 vs 46.86 ± 27.58, p<0.001). The size of the polyps did not differ. Immunostaining for GFP showed increased Rage expression in tumor epithelium. Analysis of tumor associated immune cells showed a 14-fold increase in the number of mast cells in polyps of Rage-/- versus control animals (75.31 vs 5.25 cells per vision field p<0.05). In contrast, there was no difference in the number of infiltrating neutrophils, macrophages or T cells. Number of caspase positive cells per polyp was increased in Rage-/- animals (34.3 ± 21.8 vs 11.8 ± 6.0 p<0.05). There was no significant difference in the number of proliferating cells (7.92 ± 4.36 vs 9.98 ± 5.39). Conclusion: Our results suggest that Rage signaling plays an important role in the initiation of spontaneous intestinal tumorigenesis. Rather than leading to diminished infiltration of immune cells, absence of Rage signaling results in a considerable recruitment of mast cells.