A novel mouse model of inflammatory bowel disease links mammalian target of rapamycin-dependent hyperproliferation of colonic epithelium to inflammation-associated tumorigenesis

Abstract

Inflammatory bowel disease (IBD) is a high-risk condition for human colorectal cancer. However, our mechanistic understanding of the link between inflammation and tumorigenesis in the colon is limited. Here we established a novel mouse model of colitis-associated cancer by genetically inactivating signal transducer and activator of transcription 3 (Stat3) in macrophages, with partial deletion in other myeloid and lymphoid cells. Inflammation developed in the colon of mutant mice spontaneously, and tumor lesions, including invasive carcinoma, arose in the inflamed region of the intestine with a frequency similar to that observed in human IBD patients. The development of both inflammation and tumors in the mutant mice required the presence of microflora. Indeed, inflammation was associated with disruption of colonic homeostasis, fulminant epithelial/tumor cell proliferation, and activation of the mammalian target of rapamycin (mTOR)-Stat3 pathway in epithelial and tumor cells. The activation of this pathway was essential for both the excess proliferation of epithelial/tumor cells and the disruption of colonic homeostasis in the mutant mice. Notably, a similar abnormal up-regulation of mTOR-Stat3 signaling was consistently observed in the colonic epithelial cells of human IBD patients with active disease. These studies demonstrate a novel mouse model of IBD-colorectal cancer progression in which disrupted immune regulation, mTOR-Stat3 signaling, and epithelial hyperproliferation are integrated and simultaneously linked to the development of malignancy.

Figure 1

Inactivation of Stat3-induced inflammation in colon. A and B: Mouse size. Stat3-IKO (IKO) and Stat3^f/f mice (CON) at 8 (A) and 18 (B) weeks of age as indicated. C and D: Colon histology. H&E-stained colon sections from a Stat3^f/f control (C) or a Stat3-IKO (D) mouse. Scale bar = 100 μm. E: Body weight. Stat3-IKO mice were collected between 24 and 29 weeks of age and compared with age-matched Stat3^f/f littermates (CON [n = 6] versus IKO [n = 5]; unpaired t-test, **P = 0.0013). F: Leukocytic infiltration score. The score in the colon of Stat3-IKO and Stat3^f/f littermates (unpaired t-test, **P = 0.0021, n = 3). G or H: Quantitative analysis of F4/80 or CD3 immunohistochemical analysis, respectively (unpaired t-test, *P = 0.0012 for G and P = 0.035 for H, n = 3). I–R: Immunohistochemical analysis for infiltrated leukocytes in colon. I–L: Immunochemical analysis for macrophage (Mϕ) (F4/80⁺) for Stat3^f/f (I and K) or Stat3-IKO colons (J and L). M–P: Immunohistochemical analysis for T lymphocytes (CD3⁺) for Stat3^f/f (M and O) or Stat3-IKO colons (N and P). The inset in I, J, M, or N is shown in K, L, O, or P in higher magnification, respectively. Q and R: Immunohistochemical analysis for granulocyte (Ly6G⁺) for Stat3^f/f (Q) or Stat3-IKO colons (R). Arrows point to some positive cells. Scale bars: 100μm (I, J, M, and N); 30 μm (K, L or O–R).

Figure 2

Tumors developed in the inflamed colon and cecum in Stat3-IKO mice. A–C: Visible nodules. Gross images of colon (A) from a Stat3-IKO (IKO, bottom) or a Stat3^f/f (CON, top) mouse at 19 weeks of age. The proximal end of the colon from the same Stat3^f/f (B) or Stat3-IKO mouse (C). Arrows in C point to the tumor nodules. D and E: Histology of carcinoma. H&E staining of a nodule found in the inflamed colon of a Stat3-IKO mouse. D: The colon section with tumor invasion to muscularis externa as indicated. The inset in D is shown in E in higher magnification. The long arrow in D points to the invasive site and short arrows point to the histology of the colon. Arrows in E indicate cell loss in the invading component of the tumor. Scale bars: 200 μm (D); 30 μm (E). F–K: Colon histology. Representative H&E images of colon from a Stat3^f/f (F and G) and Stat3-IKO mice (H–K) with hyperplasia (H and I) and dysplasia (J and K). The area indicated by the arrow in F, H, or J is shown in G, I, or K, respectively, in higher magnification. Scale bars: 100 μm (F, H, or J); 10 μm (G, I, or K). L: Correlation between inflammation and tumor development. The severity of the dysplastic lesions and the degree of inflammation in the colon of Stat3-IKO mice from 9 to 39 weeks of age were plotted as individual points. Nonparametric Spearman correlation was used for statistical analysis: r = 0.8313, P < 0.0001.

Figure 3

Inflammation and tumor development in Stat3-IKO colon is dependent on intestinal microflora. A–C: Bacterial density in stools of an untreated Stat3^f/f (A), untreated Stat3-IKO (B), or a VNMA-treated Stat3-IKO (C) mouse. D–F: The correlated histology of the colon. Scale bar = 100 μm. Immunohistochemical analysis for macrophages (F4/80) (G and H) and T lymphocytes (CD3) (I and J). Scale bar (in J for all of the images) = 100 μm. K: Comparison of leukocytic infiltration. *Unpaired t-test: IKO versus IKO-T, P = 0.005; CON versus IKO-T, P = 0.174. L: Epithelial injury score. *Unpaired t-test: IKO versus IKO-T, P = 0.019; CON versus IKO-T, P = 0.0013. M: Quantitative immunohistochemical analysis for F4/80. *Unpaired t-test: IKO versus IKO-T, P < 0.001; CON versus IKO-T, P = 0.047. N: Quantitative immunohistochemical analysis for CD3. *Unpaired t-test: IKO versus IKO-T, P = 0.023; CON versus IKO-T, P = 0.29. CON, untreated Stat3^f/f; IKO, untreated Stat3-IKO and IKO-T, VNMA-treated Stat3-IKO mice.

Figure 4

The impact of inflammation on epithelial biology. A: Mucosal thickness. The thickness of the colonic mucosa was measured on the images of H&E stained slides as described in Materials and Methods. CON, Stat3^f/f (n = 3); IKO, Stat3-IKO (n = 5) mice (unpaired t-test, *P = 0.0009). B and C: Epithelium proliferation. Immunochemical analysis for Ki-67 for a Stat3^f/f (B) and Stat3-IKO colon (C). Arrows in B indicate Ki-67⁺ cells and in C indicate Ki-67⁺ cells at the tip of the gland. Scale bar = 50 μm. D–G: Alcian blue/neutral red-stained colon sections from a Stat3^f/f (D and F) and a Stat3-IKO mouse (E and G). The insets in D or F are shown in higher magnification in F and G. Goblet cells are stained in blue, as indicated by arrows. H and I: Immunochemical analysis for endothelial marker CD31 for colon sections from a Stat3^f/f (H) or a Stat3-IKO mouse (I). Arrows point to some of the CD31-positive capillaries. Scale bars: 100 μm (D and E); 30 μm (F–I). J: Quantitative analysis of goblet cell density in colon. The normalized density of goblet cells in the colon of Stat3^f/f (CON) and Stat3-IKO (IKO) mice were plotted (*unpaired t-test, P = 0.02, n = 3). K: Epithelial injury score. The injury scores in the colon of Stat3-IKO (IKO) (n = 5) and Stat3^f/f mice (CON) (n = 6) (*unpaired t-test, P = 0.011) were measured as described in Materials and Methods.

Figure 5

Activation of Stat3 and mTOR pathways in epithelium of inflamed colon. A–H: Activation of Stat3 pathway. Immunochemical analysis for phosphorylated Stat3 (Tyr⁷⁰⁵) (A and B). Red arrows indicate phosphorylated Stat3 (pStat3) (Tyr⁷⁰⁵) positively stained stromal cells and black arrows indicate positively stained intestinal epithelial or tumor cells. Immunochemical analysis for cyclin D1 (C–F). The inset in C or D is shown in E or F in higher magnification. Arrows in E point to cyclin D1-positive cells in the crypt area of the normal colon. Immunochemical analysis for Survivin (G and H). Arrows point to Survivin-positive cells. Samples from Stat3^f/f (CON) and age-matched Stat3-IKO mice (IKO) are indicated. Scale bars: 10 μm (A and B); 100 μm (C and D); 30 μm (E–H). I: Western blotting analysis of Bcl-xL expression in colonic mucosa from Stat3^f/f (Con, lanes 1–4) and age-matched Stat3-IKO mice (IKO, lanes 5–8). The same sample was blotted by anti-β-actin antibody. J–O: Activation of mTOR in epithelium of inflamed colon. J–M: Immunochemical analysis for pS6 in the colon of a Stat3^f/f mouse (J and L) and an age-matched Stat3-IKO mouse (K and M). The inset in J or K is shown in L or M in higher magnification. Arrow in L points to limited pS6-positive epithelial cells in normal colon. Black arrows in K point to the inflamed region of colon that contains a high density of pS6-positive epithelial cells and red arrows point to the adjacent less inflamed area that contains fewer pS6-positive cells. Arrow in M indicates the high density of leukocytic infiltration in the inflamed region of colon. N and O: Immunochemical analysis for pS6 for colon sections from a Stat3-IKO mouse treated with Everolimus (N) and an untreated Stat3-IKO littermate (O). Arrows point to infiltrated leukocytes. P and Q: Immunochemical analysis for Ki-67 in colon sections from a Stat3-IKO mouse treated with Everolimus (P) and an untreated Stat3-IKO littermate (Q). Arrows indicate the high density of leukocytic infiltration adjacent to the colonic glands. Scale bars: 100 μm/L (J and K); 30 μm (L–Q).

Figure 6

mTOR-Stat3 cross-talk in human IBD and Stat3-IKO mouse. A–D: mTOR-Stat3 cross-talk in epithelial cells of Stat3-IKO colon. A and B: Immunochemical analysis for phosphorylated Stat3 (pStat3) (Tyr⁷⁰⁵) for colon sections from a Stat3-IKO mouse treated with Everolimus (A) and a untreated Stat3-IKO littermate (B). C and D: Immunochemical analysis for pStat3 (Ser-727) in colon of a Stat3-IKO mouse treated with Everolimus (C) and a untreated Stat3-IKO littermate (D). E–G: Colon sections from human IBD patients. Immunochemical analysis for pS6 (E), pStat3 (Tyr⁷⁰⁵) (F), and pStat3 (Ser-727) (G). Arrows pointed to leukocytic infiltration in the colon sections. Scale bar = 30 μm.