Abstract 2301: The generation of colorectal cancer mouse model based on microsatellite instability and the identification of transforming growth factor-beta signal target
Authors: Miguchi Masashi , Hinoi Takao , Shimomura Manabu , Adachi Tomohiro , Saito Yasufumi , Niitsu Hiroaki , Kochi Masatoshi , Sotomaru Yusuke , Ijichi Hideaki , Ikenoue Tsuneo , Shigeyasu Kunitoshi , Tanakaya Kohji , Sentani Kazuhiro , Oue Naohide , Yasui Wataru , Ohdan Hideki 
Published: August 2015

Abstract
Background & Aims:
The transforming growth factor-beta (TGF-beta) signal is a tumor-suppressor pathway that is commonly inactivated in about 90% of microsatellite instability (MSI) colorectal cancer (CRC). However, there was little evidence what gene is regulated by TGF-beta signal in the multistep progression sequence of CRC. The first aim of the present study was to generate a mouse model that is null for Tgfbr2 and Apc in the colon epithelium and forms tumors in the colon. The second aim was to analyze the tumors that arose in the mice model for the purpose to identify the gene regulated by TGF-beta signal.
Method & Result:
Previously we have described the generation of the ‘CDX2P-G19Cre;Apcflox/flox mice’ (called Apc KO mice) which is randomly null for Apc in the colonic epithelium. By mating Tgfbr2flox/flox mice with Apcflox/flox and CDX2P9.5-G19Cre mice, we have finally generated a mouse model ‘CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice’ (called Apc+Tgfbr2 KO mice) which is null for Apc and Tgfbr2. In these model, the tumors with well differentiated adenocarcinoma arose mainly in proximal colon and most of mice died at 4 weeks age due to tumor bleeding. Therefore the mice were harvested at 3 weeks age to evaluate the development of colon tumors. Total RNAs of only cancerous tissue areas were extracted from frozen samples by the laser capture microdissection method. We compared gene expression profiles of these mice's tumors (n = 3, respectively) with Mouse Exon 1.0 ST Array (Affymetrix). Gene X expression of Apc+Tgfbr2 KO mice tumors was most highly upregurated by 9.25-fold compared with Apc KO mice (p = 0.045). The array data was validated by quantitative PCR. For human CRC samples, mutations of repetitive mononucleotide tracts in the coding regions of TGFBR2 were identified by direct sequencing. By immunohistochemical analysis, the expression of X was classified according to the percentage of stained cancer cells. The expression was considered to be ‘positive’ if ≥30% of cancer cells were stained. An analysis demonstrated that 11 (100%) of 11 mutated TGFBR2 cases were positive for X, whereas 10 (66.7%) of 15 wild type TGFBR2 cases were positive (P = 0.033), indicating that high expression of X was correlated with TGFBR2 mutation in human CRCs samples. Additionally, the cell proliferation assay revealed that silencing of X led to a significant reduction in CRC cell proliferation. Conversely, forced expression of X enhanced CRC cell proliferation in vitro.
Conclusion:
We have generated an in vivo model system that Apc and Tgfbr2 were inactivated only in the colonic epithelium and tumors with well differentiated adenocarcinoma arose mainly in proximal colon. The analysis of this model revealed that Gene X is regulated by a TGF-beta signal and likely promotes cell proliferation in CRC.
Citation Format: Masashi Miguchi, Takao Hinoi, Manabu Shimomura, Tomohiro Adachi, Yasufumi Saito, Hiroaki Niitsu, Masatoshi Kochi, Yusuke Sotomaru, Hideaki Ijichi, Tsuneo Ikenoue, Kunitoshi Shigeyasu, Kohji Tanakaya, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Hideki Ohdan. The generation of colorectal cancer mouse model based on microsatellite instability and the identification of transforming growth factor-beta signal target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2015-2301