Id1 Deficiency Protects against Tumor Formation in Apc(Min/+) Mice but Not in a Mouse Model of Colitis-Associated Colon Cancer

Abstract

Different mechanisms contribute to the development of sporadic, hereditary and colitis-associated colorectal cancer. Inhibitor of DNA binding/differentiation (Id) proteins act as dominant-negative antagonists of basic helix-loop-helix transcription factors. Id1 is a promising target for cancer therapy, but little is known about its role in the development of colon cancer. We used immunohistochemistry to demonstrate that Id1 is overexpressed in human colorectal adenomas and carcinomas, whether sporadic or syndromic. Furthermore, elevated Id1 levels were found in dysplasia and colon cancer arising in patients with inflammatory bowel disease. Because levels of PGE2 are also elevated in both colitis and colorectal neoplasia, we determined whether PGE2 could induce Id1. PGE2 via EP4 stimulated protein kinase A activity resulting in enhanced pCREB-mediated Id1 transcription in human colonocytes. To determine the role of Id1 in carcinogenesis, two mouse models were used. Consistent with the findings in humans, Id1 was overexpressed in tumors arising in both Apc(Min) (/+) mice, a model of familial adenomatous polyposis, and in experimental colitis-associated colorectal neoplasia. Id1 deficiency led to significant decrease in the number of intestinal tumors in Apc(Min) (/+) mice and prolonged survival. In contrast, Id1 deficiency did not affect the number or size of tumors in the model of colitis-associated colorectal neoplasia, likely due to exacerbation of colitis associated with Id1 loss. Collectively, these results suggest that Id1 plays a role in gastrointestinal carcinogenesis. Our findings also highlight the need for different strategies to reduce the risk of colitis-associated colorectal cancer compared with sporadic or hereditary colorectal cancer.

Figure 1. Id1 is expressed in human colorectal neoplasia

A, sporadic colonic adenomas contain straight tubules lined by dysplastic epithelial cells with cigar-shaped nuclei (top) compared to normal entrapped crypts (arrow) with abundant cytoplasm and small nuclei. B, adenomatous epithelium shows strong nuclear Id1 staining (top) compared to non-adenomatous crypts that express much lower levels of Id1 (arrow). Endothelial cells in the lamina propria also stain for Id1 (arrowhead). C, a colon cancer is composed of fused glands that contain malignant epithelial cells. D, Id1 positivity is present in tumor cells and the endothelium of vascular channels within the stroma (arrowhead, see inset). This tumor was from a patient with Familial Adenomatous Polyposis. E, ulcerative colitis-associated low-grade dysplasia (arrowhead) is sharply demarcated from normal mucinous crypts (arrow). F, the dysplastic foci are strongly Id1 positive (arrowhead) compared to normal crypts that are negative (arrow). Id1 positive endothelial cells are present in the lamina propria between dysplastic crypts (inset).

Figure 2. Id1 is overexpressed in mouse models of gastrointestinal neoplasia

A, intestinal adenomas that develop in Apc^Min/+ mice consist of crowded neoplastic crypts with mucin depletion (arrow) compared to the background small bowel mucosa (arrowhead). B, these tumors show strong Id1 staining (arrow), whereas the non-neoplastic epithelium is negative (arrowhead). C, Treatment with azoxymethane followed by several cycles of dextran sodium sulfate produced colonic epithelial neoplasia (arrow) comingled with non-neoplastic epithelium (arrowhead). D, colitis-associated neoplasia show strong, diffuse nuclear staining for Id1 (arrow) compared to entrapped non-neoplastic crypts (arrowhead). E, immunohistochemical stains for myeloperoxidase demonstrate a paucity of granulocytes in intestinal adenomas arising in Apc^Min/+ mice, indicating that these lesions are not associated with inflammation. F, neoplasms associated with experimental colitis contain numerous inflammatory cells (arrow) that show strong staining for myeloperoxidase.

Figure 3. PGE₂ inducesId1transcription

A, cells were treated with 0–500 nmol/L PGE₂ for 24 hours. Cellular protein (100 μg/lane) was subjected to immunoblot analysis. The blots were probed with antibodies to Id1 and β-actin, respectively. B, HCT-15 cells were transfected with 1.8 μg of a human Id1 promoter-luciferase construct -1147/-937 or this promoter construct in which the Egr-1 (mEgr-1), Ebox (mEbox) or cAMP-response element (mCREB) sites were mutagenized. Cells also received 0.2 μg of pSVβgal. Following transfection, cells were treated with vehicle (Control) or 500 nmol/L PGE₂ for 24 hours. Reporter activities were then measured. Id-1 luciferase activity represents data that have been normalized to β-galactosidase activity. C, HCT-15 cells were treated with vehicle or 500 nmol/L PGE₂ for 3 hours. ChIP assays were then performed. Chromatin fragments were immunoprecipitated with antibodies against pCREB and the Id1 promoter was amplified by PCR. DNA sequencing was carried out, and the product was confirmed to be the correct promoter. Mean ± SD are shown, n = 6. *, P < 0.01 compared with vehicle (control) treated cells. D, HCT-15 cells were pre-treated with vehicle or the indicated concentrations of ONO-AE3-208 (top panel), an EP₄ receptor antagonist, or H89 (bottom panel), an inhibitor of PKA, for 2 hours. Subsequently, cells were treated with 500 nmol/L PGE₂ as indicated for 24 hours. Cellular protein (100 μg/lane) was subjected to immunoblot analysis. The blots were probed with antibodies to Id1 and β-actin, respectively.

Figure 4. Id1 deficiency suppresses small intestinal tumor multiplicity and increases survival in Apc^Min/+ mice

A, In comparison to Apc^Min/+ Id1^+/+ mice (n=25), Apc^Min/+ Id1^+/− mice (n=11) and Apc^Min/+ Id1^−/−mice (n=11) developed reduced numbers of small intestinal tumors (P=0.006 and 0.025, respectively). B, among mice that developed tumors, the proportions of tumors in different size categories were not statistically different among the three groups. C, survival of Apc^Min/+ Id1^+/+ (n=17) and Apc^Min/+ Id1^−/− (n=19) mice was assessed. Log-rank test showed statistically significantly lower hazard of death in Apc^Min/+ Id1^−/− compared to Apc^Min/+ Id1^+/+ mice (P=0.04).

Figure 5. Deletion of Id1 does not protect against colitis-associated tumorigenesis

A, study schema of AOM/DSS colitis-associated tumor model. B and C, adenoma number (B) and size (C) were compared between WT (n=20) and Id1^−/− (n=19) mice. Statistical differences were not observed.