Inflammation-induced tumorigenesis in the colon is regulated by caspase-1 and NLRC4 - PubMed (original) (raw)

Inflammation-induced tumorigenesis in the colon is regulated by caspase-1 and NLRC4

Bo Hu et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association is currently unknown. The inflammasome, a multiprotein complex formed by NOD-like receptor (NLR) family members, has recently been shown to orchestrate multiple innate and adaptive immune responses, yet its potential role in inflammation-induced cancer has been little studied. Using the azoxymethane and dextran sodium sulfate colitis-associated colorectal cancer model, we show that caspase-1-deficient (Casp1(-/-)) mice have enhanced tumor formation. Surprisingly, the role of caspase-1 in tumorigenesis was not through regulation of colonic inflammation, but rather through regulation of colonic epithelial cell proliferation and apoptosis. Consequently, caspase-1-deficient mice demonstrate increased colonic epithelial cell proliferation in early stages of injury-induced tumor formation and reduced apoptosis in advanced tumors. We suggest a model in which the NLRC4 inflammasome is central to colonic inflammation-induced tumor formation through regulation of epithelial cell response to injury.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Caspase-1 deficiency exacerbates AOM-DSS–induced colon cancer. (A) Schematic overview of the inflammation-induced cancer model. (B) Tumor load and tumor numbers/mouse in WT and caspase-1 KO (Casp1−/−) mice (n = 8). P values < 0.05 were considered statistically significant. The experiments were repeated five times. (C) Representative colonoscopic appearance of WT and Casp1−/− mice colon on day 65 of AOM-DSS–induced color cancer. (D) Representative histopathologic sections of colon adenocarcinomas (I) and foci of tumor invasion (II) from WT and Casp1−/− mice. Invasive tumor foci (arrows) were smaller and less frequent in WT mice than Casp1−/− mice, where tumor foci were surrounded by abundant amounts of pale blue mucin. H&E staining; *muscularis mucosae; m, mascularis externa. (Scale bars, 200 μm.)

Fig. 2.

Fig. 2.

No differences in inflammation between WT and Casp1−/− mice during acute DSS colitis. There were no significant difference in percent-mass change (A), colonoscopy inflammation severity score (B), severity of histopathological morphology (C) (H&E staining), or histopathology parameters for edema, inflammation, ulceration or overall severity of injury (D) between WT and Casp1−/− mice. (Scale bars in C, 200 μm.) (E) Likewise, the severity of chronic DSS colitis was similar between WT and Casp1−/− mice. All experiments were repeated twice.

Fig. 3.

Fig. 3.

Enhanced colon epithelial and tumor cell proliferation in Casp1−/− mice during inflammation-induced colorectal cancer. Representative H&E, Ki67, and BrdU immunohistochemistry at day 15 (A–F) and at day 65 (G–I), and TUNEL-positive cells in colons (day 8) and tumors (day 65) in WT and Casp1−/− mice given AOM-DSS. The majority of crypts at day 15 appeared normal (A); however, there were increased Ki67+ (B) and BrdU+ (C) crypt epithelial cells in Casp1−/− mice compared with WT mice. In foci of crypt hyperplasia/dysplasia (D), there were no significant difference in the number of Ki6+ cells (E) and a moderate increase in the number of BrdU+ cells (F) in Casp1−/−compared with WT mice. At day 65 there were numerous large colon adenocarcinomas (G) with increased numbers of Ki67+ tumor cells (H) and BrdU+ tumor cells (I) in Casp1−/− compared with WT mice. (A, D, G: H&E staining; B, C, E, F, H, and I: DAB, Hematoxalyn) (Scale bars, 200 μm). (J) The number of TUNEL-positive cells in colon is similar in WT compared with Casp1−/− mice. In contrast, there is reduced cell death of tumor tissue in Casp1−/− mice (average cells counted: WT: 34.27 ± 8.964 positive cells/293.6 ± 86.39 total cells per field; Casp1−/−: 16.87 ± 4.533 positive cells/364.2 ± 98.70 total cells per field). DNA fragmentation in WT and Casp-1−/− mice was determined on either whole-colon sections on day 8 of AOM-DSS model or tumor tissues at day 65. Error bars represent ± SEM, P < 0.001. The experiments were repeated two to three times.

Fig. 4.

Fig. 4.

Caspase-1–mediated tumor enhancement is mediated by the NLRC4 inflammasome. (A) Caspase-1, NLRP3, and NLRC4 mRNA expression in colon epithelial cells and CD45+ cells. The levels of the indicated mRNAs were quantitated by real-time PCR and normalized to the level of HPRT mRNA. (B) Inflammation–induced tumor formation is similar in WT and NLRP3−/− mice (n ≥ 10). The experiments were repeated three times. (C) NLRC4−/− mice feature enhanced inflammation-induced colon cancer compared with WT mice (n ≥ 6); P < 0.05 was considered statistically significant. The experiments were repeated twice. Severity of DSS colitis in WT and NLRC4−/− mice, as evident by (D) mass change and (E) inflammation colonoscopy severity score. The experiments were repeated twice. (F) Caspase-1 mRNA expression in normal and colon and adjacent tumors from WT mice. mRNA levels were assessed by real-time PCR and normalized to the level of HPRT (n ≥ 5). The experiment was repeated twice. P < 0.05 was considered statistically significant. (G) Caspase-1 mRNA expression in naive and AOM-DSS treated proximal and distal colonic epithelial cells from WT mice. mRNA levels were assessed by real-time PCR and normalized to the level of HPRT (n ≥ 8). The experiment was repeated twice. P < 0.05 was considered statistically significant.

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