High Endogenous Expression of Chitinase 3-Like 1 and Excessive Epithelial Proliferation with Colonic Tumor Formation in MOLF/EiJ Mice

Colorectal cancer (CRC) development is mediated by uncontrolled survival and proliferation of tumor progenitor cells. Using animal models to identify and study host-derived factors that underlie this process can aid interventions in preventing tumor expansion and metastasis. In healthy steady states in humans and mice (e.g. C57BL/6 strain), colonic Chitinase 3-like 1 (CHI3L1) gene expression is undetectable. However, this expression can be induced during intestinal inflammation and tumorigenesis where CHI3L1 plays an important role in tissue restitution and cell proliferation. Here, we show that a wild-derived mouse strain MOLF/EiJ expresses high levels of colonic epithelial CHI3L1 at the steady state due to several nucleotide polymorphisms in the proximal promoter regions of the CHI3L1 gene. Interestingly, these mice spontaneously developed polypoid nodules in the colon with signs of immune cell infiltrations at steady state. The CHI3L1 positive colonic epithelial cells were highly proliferative and exhibited malignant transformation and expansion when exposed in vivo to azoxymethane, one of the well-known colonic carcinogens

MOLF mice spontaneously developed polyp-like nodules in colon at steady state.

<p>[<b>A</b>] Six months old MOLF and B6 mice were sacrificed and the colons were opened up longitudinally. [<b>B</b>] Multiplicity and size of polyp-like nodules were determined under a dissection microscope (n = 7 in each group). [<b>C</b>] H&E sections of colons from B6 and MOLF are shown. [<b>D</b>] IHC staining of colonic sections from B6 and MOLF mice was performed using anti-Ki67 primary antibody.</p

MOLF express high levels of CHI3L1 in colonic epithelial cells but not in immune cells.

<p>Immunohistochemical analysis was performed on colonic serial sections (non-polypoid area) from six months old MOLF mice using anti-CHI3L1, -TROMA–1 (cytokeratin 8), -CD4, -CD11b, and -F4/80 antibodies.</p

MOLF express high levels of CHI3L1 in the colon and polypoid area and associates with high immune cellular infiltrates.

<p>Two-color Immunofluorescence analysis was performed on colonic sections from six months old B6 and MOLF mice using anti-CHI3L1 and anti-F4/80 (<b>A</b>) -CD11b (<b>B</b>) or -CD4 (<b>C</b>) primary antibodies respectively.</p

High endogenous colonic CHI3L1 expression state MOLF/EiJ mice colon at steady state.

<p>[<b>A</b>] Colonic CHI3L1 mRNA level in MOLF/Eij (MOLF) and C57BL/6 (B6) mice were measured using real time PCR (n = 5 in each group). [<b>B</b>] Serum CHI3L1 protein level in MOLF (n = 5) and B6 (n = 5) mice were quantified using ELISA. [<b>C</b>] Schema showing the genomic structure of the CHI3L1 locus. Arrows indicating single nucleotide polymorphisms between MOLF and B6 mice identified by in silico comparison using the Jackson mouse phenome database. [<b>D</b>] Proximal promoter from MOLF and B6 mice was PCR amplified, sequenced and aligned using Clustal W program. [<b>E</b>] SW480 cells were transfected with pGL3-basic vector containing MOLF- or B6-derived CHI3L1 proximal promoter and renilla-normalised luciferase assay was performed (n = 5 in each group). **P<0.01, ***P<0.001 between the two groups as indicated.</p

Alignment of Human, MOLF and B6 mice CHI3L1 proximal promoter.

<p>Approximately 600 bp upstream sequence of the CHI3L1 transcription start site from human, MOLF and B6 mice were aligned using Clustal W program.</p

Chitinase 3-like 1 induces survival and proliferation of intestinal epithelial cells during chronic inflammation and colitis-associated cancer by regulating S100A9

Many host-factors are inducibly expressed during the development of inflammatory bowel disease (IBD), each having their unique properties, such as immune activation, bacterial clearance, and tissue repair/remodeling. Dysregulation/imbalance of these factors may have pathogenic effects that can contribute to colitis-associated cancer (CAC). Previous reports showed that IBD patients inducibly express colonic chitinase 3-like 1 (CHI3L1) that is further upregulated during CAC development. However, little is known about the direct pathogenic involvement of CHI3L1 in vivo. Here we demonstrate that CHI3L1 (aka Brp39) knockout (KO) mice treated with azoxymethane (AOM)/dextran sulphate sodium (DSS) developed severe colitis but lesser incidence of CAC as compared to that in wild-type (WT) mice. Highest CHI3L1 expression was found during the chronic phase of colitis, rather than the acute phase, and is essential to promote intestinal epithelial cell (IEC) proliferation in vivo. This CHI3L1-mediated cell proliferation/survival involves partial downregulation of the pro-apoptotic S100A9 protein that is highly expressed during the acute phase of colitis, by binding to the S100A9 receptor, RAGE (Receptor for Advanced Glycation End products). This interaction disrupts the S100A9-associated expression positive feedback loop during early immune activation, creating a CHI3L1hi S100A9low colonic environment, especially in the later phase of colitis, which promotes cell proliferation/survival of both normal IECs and tumor cells