Cyclophilin C-associated protein (CyCAP) knock-out mice spontaneously develop colonic mucosal hyperplasia and exaggerated tumorigenesis after treatment with carcinogen azoxymethane.
BACKGROUND
The discovery of a "serrated neoplasia pathway" has highlighted the role of hyperplastic lesions of the colon as the significant precursor of colorectal adenocarcinoma. In mice, hyperplasia of the colonic mucosa is a regular phenomenon after a challenge with colonic carcinogens indicating that mucosal hyperproliferation and thickening, even without cytological dysplasia, represents an early pre-malignant change. Cyclophilin C-associated protein (CyCAP) has been described to down-modulate endotoxin signaling in colorectal murine mucosa and is a murine orthologue of the tumor-associated antigen 90 K (TAA90K)/mac-2-binding protein.


METHODS
Female Balb/c wild-type (WT) and CyCAP knock-out (KO) mice (6-8 weeks old) were administered 2 or 6 weekly subcutaneous injections of azoxymethane. The animals were evaluated post-injection at six weeks for aberrant crypt foci (ACF) study and at five months for colon tumor measurement. The thickness of the colon crypts was measured in microns and the number of colonocytes per crypt was also determined in well-oriented crypts. Morphometric analyses of the colon mucosa were also performed in untreated 6-8 weeks old KO and WT animals. Formalin-fixed/paraffin-embedded colon sections were also studied by immunohistochemistry to determine the Ki-67 proliferation fraction of the colon mucosa, beta-catenin cellular localization, cyclin D1, c-myc, and lysozyme in Paneth cells.


RESULTS
Cyclophilin C-associated protein (CyCAP)-/- mice, spontaneously developed colonic mucosal hyperplasia early in life compared to wild-type mice (WT) (p < 0.0001, T-test) and crypts of colonic mucosa of the (CyCAP)-/- mice show higher proliferation rate (p = 0.039, Mann-Whitney Test) and larger number of cyclin D1-positive cells (p < 0.0001, Mann-Whitney Test). Proliferation fraction and cyclin D1 expression showed positive linear association (p = 0.019, Linear-by-Linear Association). The hyperplasia was even more pronounced in CyCAP-/- mice than in WT after challenge with azoxymethane (p = 0.005, T-test). The length of the crypts (r = 0.723, p = 0.018, Spearman Correlation) and the number of colonocytes per crypt (r = 0.863, p = 0.001, Spearman Correlation) in non-tumorous areas were positively associated with azoxymethane-induced number of tumors. CyCAP-/- developed larger numbers of tumors than WT animals (p = 0.003, T-Test) as well as overall larger tumor mass (p = 0.016, T-Test). Membranous beta-catenin was focally overexpressed in KO mice including proliferative zone of the crypts.


CONCLUSIONS
CyCAP-/- represent the first described model of spontaneous colonic mucosal hyperplasia. We conclude that CyCAP-deficient mice spontaneously and after challenge with carcinogen develop significantly more colorectal mucosal hyperplasia, an early stage in murine colonic carcinogenesis.
Torlakovic
Emina Emilia
EE
Department of Pathology, Royal University Hospital, College of Medicine, University of Saskatchewan, Saskatoon, Canada. emt323@mail.usask.ca
Keeler
Vicki
V
Wang
Chang
C
Lim
Hyun J
HJ
Lining
Leslie Ann
LA
Laferté
Suzanne
S
D000230:Adenocarcinoma / Q000378:metabolism; D000818:Animals; D001397:Azoxymethane; D002273:Carcinogens; D049109:Cell Proliferation; D003106:Colon / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout
D002273:Carcinogens; D006023:Glycoproteins; C082349:Ppicap protein, mouse; D001397:Azoxymethane