title: Smad2/3 linker phosphorylation is a possible marker of cancer stem cells and correlates with carcinogenesis in a mouse model of colitis-associated colorectal cancer.
abstract: BACKGROUND
  Epithelial cells affected by somatic mutations undergo transition from a tumour-suppressive to a carcinogenic Smad pathway during sporadic colorectal carcinogenesis, and the specific linker threonine phosphorylation of Smad2/3 in colon epithelial cells indicates stem-like cells. This study extends previous observations to a model of colitis-associated colorectal cancer.
  
  
  METHODS
  After Crl:CD-1 mice received an administration of azoxymethane [AOM], the mice were given dextran sodium sulfate [DSS] for 7 days. AOM/DSS-treated mice [AOM/DSS mice] were killed at 10 or 20 weeks. After macroscopic observations, a histopathological analysis was conducted. Immunohistochemical staining was performed using the avidin-biotin immunoperoxidase method [pSmad3C-Ser, pSmad3L-Ser, c-Myc] and immunofluorescent methods [Ki67, β-catenin, CDK4, cyclin D1, Sox9, pSmad2/3L-Thr].
  
  
  RESULTS
  The colons from AOM/DSS mice were shorter than those from control mice. The number of colon tumours at Week 20 was higher than at Week 10. The inflammation scores for AOM/DSS mice were greater than those for control mice. Immunostaining-positive cells (staining by Ki67, β-catenin [nuclear and cytoplasmic], cyclin D1, and Sox9) were diffusely distributed in colon tumours. The percentage of pSmad3L-Ser-positive cells in colon tumours was higher than in sites of pre-neoplastic colitis, and that in sites of pre-neoplastic colitis was higher than in control mice. pSmad2/3L-Thr-positive cells were sparsely detected around crypt bases in non-neoplastic colon epithelia and at the tops of tumours, and immunohistochemical co-localisation of pSmad2/3L-Thr with Ki67 was not observed. Immunohistochemical co-localisation of pSmad2/3L-Thr with β-catenin and CDK4 was observed.
  
  
  CONCLUSIONS
  pSmad3L-Ser signalling is an early event in colitis-associated colorectal cancer, and pSmad2/3L-Thr immunostaining-positive cells might be cancer stem cells.
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan fukuitos@hirakata.kmu.ac.jp.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Fukui
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Kishimoto
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Sachi
authors.initials: S
authors.lastname: Miyamoto
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Mitsuyama
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Yutaku
authors.initials: Y
authors.lastname: Sakaguchi
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Kazushige
authors.initials: K
authors.lastname: Uchida
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nishio
authors.affiliation: Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan.
authors.forename: Kazuichi
authors.initials: K
authors.lastname: Okazaki
mesh_terms: D000818:Animals; D001397:Azoxymethane; D014408:Biomarkers, Tumor / Q000378:metabolism; D063646:Carcinogenesis / Q000378:metabolism* / Q000473:pathology; D003092:Colitis / Q000378:metabolism* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000139:chemically induced / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D019938:Cyclin D1 / Q000032:analysis; D016264:Dextran Sulfate; D004195:Disease Models, Animal; D019394:Ki-67 Antigen / Q000032:analysis; D008297:Male; D051379:Mice; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis; D055758:SOX9 Transcription Factor / Q000032:analysis; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism*; D051900:Smad3 Protein / Q000032:analysis / Q000378:metabolism*; D051176:beta Catenin / Q000032:analysis
chemical_list: D014408:Biomarkers, Tumor; D019394:Ki-67 Antigen; C489428:Myc protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D055758:SOX9 Transcription Factor; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C527933:Sox9 protein, mouse; D051176:beta Catenin; D019938:Cyclin D1; D012694:Serine; D016264:Dextran Sulfate; D001397:Azoxymethane
keywords: Mouse model; Smad; cancer stem cell; carcinogenesis; colitis-associated colorectal cancer; phosphorylation