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

• Published in: Journal of Crohn's and colitis Vol. 9; no. 7; pp. 565 - 574
• Main Authors: Suzuki, Ryo, Fukui, Toshiro, Kishimoto, Masanobu, Miyamoto, Sachi, Takahashi, Yu, Takeo, Masahiro, Mitsuyama, Toshiyuki, Sakaguchi, Yutaku, Uchida, Kazushige, Nishio, Akiyoshi, Okazaki, Kazuichi
• Format: Journal Article
• Language: English
• Published: UK Oxford University Press 01.07.2015
• Subjects: Animals; Azoxymethane; beta Catenin - analysis; Biomarkers, Tumor - metabolism; Carcinogenesis - metabolism; Carcinogenesis - pathology; Colitis - metabolism; Colitis - pathology; Colorectal Neoplasms - chemically induced; Colorectal Neoplasms - chemistry; Colorectal Neoplasms - metabolism; Colorectal Neoplasms - pathology; Cyclin D1 - analysis; Dextran Sulfate; Disease Models, Animal; Ki-67 Antigen - analysis; Male; Mice; Neoplastic Stem Cells - metabolism; Phosphorylation; Proto-Oncogene Proteins c-myc - analysis; Serine - metabolism; Signal Transduction; Smad2 Protein - metabolism; Smad3 Protein - analysis; Smad3 Protein - metabolism; SOX9 Transcription Factor - analysis; Mouse model; Smad; carcinogenesis; cancer stem cell; phosphorylation; colitis-associated colorectal cancer
• ISSN: 1873-9946; 1876-4479
• DOI: 10.1093/ecco-jcc/jjv073

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.