Blocking TNF-α in mice reduces colorectal carcinogenesis associated with chronic colitis (original) (raw)
Enhanced TNF expression in the colon during the course of colon carcinogenesis. Consistent with previous reports (8, 9), a single intraperitoneal injection of the carcinogen AOM, followed by 3 rounds of 2% DSS intake induced the development of multiple tumors in the middle to distal colon of WT mice (Figure 1, B and C). The essential involvement of a transcription factor, NF-κB, in this colon carcinogenesis model (9) prompted us to investigate the intracolonic expression of TNF-α and its receptor because TNF-α is a potent activator of NF-κB (13, 14). TNF-α mRNA was faintly expressed in untreated WT mice, and AOM treatment alone did not enhance _TNF-_α mRNA expression, but subsequent DSS intake augmented _TNF-_α mRNA expression (Figure 1D). We also detected TNF-α protein expression by immunohistochemical analysis mainly in mononuclear cells present in lamina propria and submucosal regions (Figure 1E). Similarly, immunoreactive TNF-α protein was detected in the colons of patients with active UC and advanced colorectal cancer, but not in normal mucosa (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI32453DS1). Immunohistochemical analysis detected the major receptor for TNF-α, TNF-Rp55, predominantly in leukocytes infiltrating the lamina propria and submucosal regions of the colon during the course of this colon carcinogenesis model (Figure 1F).
Tumor formation in WT and TNF-Rp55–/– (TNF-Rp55KO) mice after AOM and DSS treatment. (A) Schematic overview of this colon carcinogenesis model. (B) Macroscopical changes in colon. Colons were removed at day 56 from treated WT and TNF-Rp55–/– mice, and representative results from 10 independent animals are shown here. (C) The numbers of tumors. Colons were removed at day 56 to determine the numbers of macroscopic tumors. Each value represents the mean ± SD (n = 10 animals). **P < 0.01 versus WT. (D). _TNF-_α gene expression in the colons of WT mice. The levels of _TNF-_α mRNA were quantified by quantitative RT-PCR as described in Methods, and normalized to the level of GAPDH mRNA. *P < 0.05, **P < 0.01 versus untreated (control) mice. (E and F) Immunohistochemical detection of TNF-α and TNF-Rp55 in colons. Colons were obtained from WT mice at the indicated time intervals; insets are higher magnification of the positively stained cells as indicated by arrows. Representative results from 6 independent experiments are shown here (original magnification, ×400; ×1,000 [insets]).
Reduced tumor incidence in TNF-Rp55–/– mice. In order to clarify the role of TNF-Rp55 in this colon carcinogenesis model, we treated both WT and TNF-Rp55–/– mice with AOM and DSS in the same manner. During the course of AOM and DSS treatment, WT mice exhibited profound body weight loss and bloody diarrhea, whereas TNF-Rp55–/– mice had less body weight loss and did not have bloody diarrhea (data not shown). There were no apparent differences in macroscopical and microscopical appearance of the colon of untreated WT and TNF-Rp55–/– mice (Figure 2A). In treated WT mice, edema and hyperemia of the middle to distal colon became evident after day 7, and multiple tumors developed in the same region after day 28, whereas these morphological changes were rare in AOM and DSS–treated TNF-Rp55–/– mice (data not shown). Histological analysis consistently demonstrated massive infiltration of leukocytes into the mucosa and edema of the submucosa, with loss of entire crypts and surface epithelium by day 7, particularly in the middle to distal colon of WT mice (Figure 2A). At day 14, mucosal inflammatory cell infiltration persisted and was accompanied by dysplastic glands with hyperchromatic nuclei, decreased mucin production, and dystrophic goblet cells. By days 28 to 35, macroscopically visible adenocarcinomatous lesions developed, and their size and numbers increased progressively thereafter. Moreover, β-catenin accumulated in the nuclei of the tumor cells after day 28 (Figure 2B). On the contrary, TNF-Rp55–/– mice displayed much milder inflammation of the colon during the course of DSS intake and developed fewer adenocarcinomatous lesions and less nuclear β-catenin accumulation (Figure 1C and Figure 2, A and B). Moreover, the numbers of apoptotic cells were transiently increased at day 7 in WT but not TNF-Rp55–/– mice, as revealed by TUNEL assay (Supplemental Figure 2). However, the incidence of apoptotic cells detected in TNF-Rp55–/– mice was similar in level to those in WT mice except on day 7 (Supplemental Figure 2). These observations suggest a crucial role in this model for TNF-Rp55–mediated signals in the development of chronic inflammation and colon carcinoma but not in the apoptotic reactions.
Microscopical analysis of colon tissues. (A) Colons were removed at the indicated time intervals, fixed, and stained with hematoxylin and eosin. Representative results from 5 mice are shown here. Original magnification, ×200. (B) Immunohistochemical staining for β-catenin. Colons were removed at the indicated time intervals from WT and TNF-Rp55–/– (TNF-Rp55KO) mice and immunostained with anti–β-catenin antibody as described in Methods. Boxed areas in the left panels are shown at higher magnification in the middle panels. Representative results from 3 independent animals are shown here. Original magnification, ×400 (top and bottom rows), ×1,000 (middle row). (C–G) The numbers of myeloperoxidase- (C), F4/80- (D), CD4- (E), CD8- (F), and DEC205-positive cells (G) were counted as described in Methods and are shown here. All values represent the mean ± SD (n = 10 animals). *P < 0.05, **P < 0.01 versus untreated (control) WT mice.
Inflammatory cell infiltration. We next proceeded to identify the types of cells that were decreased in the absence of TNF-Rp55 by immunohistochemical analysis. In treated WT mice, neutrophils and macrophages infiltrated into lamina propria and submucosa after day 7 and persisted until day 56 (Supplemental Figure 3). In addition, after day 7 aggregates of CD4-positive lymphocytes and DEC205-positive dendritic cells infiltrated into the lamina propria and submucosa (Supplemental Figure 3). In AOM and DSS–treated TNF-Rp55–/– mice, infiltration by both neutrophils and macrophages was markedly decreased, whereas lymphocyte and dendritic cell infiltration was minimally affected (Figure 2, C–G). TNF-α can augment the expression of the chemokines keratinocyte chemoattractant/CXCL1 (KC/CXCL1) and monocyte chemoattractant protein–1/CCL2 (MCP-1/CCL2), which are chemotactic for neutrophils and macrophages, respectively (23, 24). Indeed, after day 7, gene expression of both chemokines was enhanced in WT mice, but their expression was consistently depressed in TNF-Rp55–/– mice (Figure 3). These observations suggest that TNF-Rp55–mediated signals were responsible for the trafficking of neutrophils and macrophages, at least in part by enhancing the expression of chemokines. Moreover, it is plausible that bone marrow–derived cells, neutrophils and macrophages, can be crucially involved in this colon carcinogenesis model. In order to address the contribution of bone marrow–derived cells, we treated various bone marrow chimeric mice with the same combination of AOM and DSS. TNF-Rp55–/– mice transplanted with WT-derived bone marrow cells developed tumors at a similar level as WT mice transplanted with WT-derived bone marrow cells, but at a higher level than either WT or TNF-Rp55–/– mice transplanted with TNF-Rp55–/– mouse–derived bone marrow cells (Figure 4). These observations suggest that TNF-Rp55–mediated signals act mainly on bone marrow, but not non–bone marrow–derived cells in this carcinogenesis model.
Chemokine gene expression in the colons. Quantitative RT-PCR was performed on total RNAs extracted from the colons at the indicated time intervals as described in Methods. The levels of KC/CXCL1 (A) and MCP-1/CCL2 (B) mRNA were normalized to GAPDH mRNA levels. Representative results from 5 independent experiments are shown in here. *P < 0.05, **P < 0.01 versus untreated (control) mice.
Colon tumor formation in bone marrow chimeric mice. Bone marrow chimeric mice were generated and subjected to AOM+DSS treatment as described in Methods. Colons were removed at day 56, and the tumor numbers were determined macroscopically. The bars represent the median of each group; each symbol represents the tumor numbers of each animal. **P < 0.01.
Reduced COX-2 expression in TNF-Rp55–/– mice. Accumulating evidence indicates the causal involvement of COX-2 in colon carcinogenesis. Hence, we examined COX-2 mRNA expression by real-time RT-PCR. After day 7, intracolonic COX-2 expression was markedly enhanced in treated WT but not TNF-Rp55–/– mice (Figure 5A). COX-2 protein was detected mainly in infiltrating inflammatory cells (Figure 5B), and the numbers of COX-2–positive cells increased from day 7 to day 56 in WT but not TNF-Rp55–/– mice (Figure 5, B and C). Double-color immunofluorescence analysis detected COX-2 protein in F4/80-positive macrophages and to a lesser extent in Ly-6G–positive neutrophils (Figure 5D). These observations suggest that in the absence of TNF-Rp55 the infiltration of macrophages and neutrophils, which are a major source of COX-2, was reduced, leading to decreased COX-2 expression.
COX-2 expression in the colons. (A) Quantitative RT-PCR was performed on total RNAs extracted from the colons at the indicated time intervals as described in Methods. The levels of COX-2 mRNA were normalized to the levels of GAPDH mRNA. **P < 0.01 versus untreated (control) mice. (B–D) Immunohistochemical and immunofluorescence detection of COX-2–expressing cells. Colons were obtained from WT mice at the indicated time intervals and processed for immunohistochemical analysis using anti–COX-2 antibodies, and representative results from 5 independent animals are shown in B. The numbers of COX-2–expressing cells were determined as described in Methods and are shown in C and expressed as mean ± SD. *P < 0.05, **P < 0.01 versus untreated. Double-color immunofluorescence analysis was performed with the combination of anti–COX-2 and anti–F4/80 (D, top row) or that of anti–COX-2 and anti-Ly6G antibodies (D, bottom row). Representative results from 5 independent experiments are shown here. Original magnification, ×400 (B); ×800 (D). Scale bars, 10 μm.
Effect of TNF-α antagonist, etanercept, on tumor formation in WT mice. Because the human TNF antagonist, etanercept, can inhibit the biological activity of murine TNF (22), we explored its effects on tumor progression by administering it to mice from day 56 to day 60, after the AOM and 3 cycles of DSS treatments (Figure 6A). Compared with the vehicle-treated group, etanercept treatment reduced the numbers and size of macroscopical tumors remarkably when administered even over this short and delayed time period (Figure 6, B–D). Concomitantly, etanercept treatment reduced intracolonic infiltration by inflammatory cells, particularly neutrophils and macrophages (Figure 6, E and F, and Supplemental Figure 4), together with a decrease in mRNA levels of the neutrophil-tropic chemokine, KC/CXCL1, and the macrophage-tropic chemokine, MCP-1/CCL2 (Figure 6, G and H). Furthermore, etanercept reduced COX-2 mRNA expression (Figure 7A) and the numbers of COX-2 expressing cells (Figure 7, B and C). Because COX-2–derived PGE2 is an important stimulant of tumor angiogenesis (25), we next examined the effect of etanercept on the intratumoral vascular density by immunostaining with anti-CD31 antibody. At 56 and 67 days after the initiation of DSS intake, WT mice exhibited a marked increase in vascular densities, and this increment was markedly depressed by etanercept (Figure 7, D and E).
The effects of a TNF antagonist, etanercept, on colon carcinogenesis. (A) Schematic overview of etanercept administration. Colons were removed at day 67 after the mice were administered etanercept (Et) or a vehicle control between days 56 and 60. (B) The tumor sizes and numbers were determined macroscopically. The bars represent the median of each group. Each symbol represents the tumor numbers of each animal or the average size of the tumors of each animal. (C) Macroscopic evaluation of the tumors. Colons were removed on day 67 from WT mice, treated with etanercept or with vehicle. Representative results from 10 independent animals are shown here. Original magnification, ×6. (D) Colons were processed for hematoxylin and eosin staining and representative results from 5 independent animals are shown here. Original magnification, ×40. (E and F) Myeloperoxidase- (E) and F4/80-positive cells (F) were enumerated as described in Methods. All values represent the mean ± SD (n = 10 animals). *P < 0.05, **P < 0.01 versus etanercept-untreated WT mice. (G and H) Quantitative RT-PCR analysis for KC/CXCL1 (G) and MCP-1/CCL2 (H) was performed on total RNAs extracted from the colons at the indicated time intervals as described in Methods. KC/CXCL1 and MCP-1/CCL2 mRNA levels were normalized to the levels of GAPDH mRNA. *P < 0.05, **P < 0.01 versus etanercept untreated WT mice.
The effect of etanercept on COX-2 expression and angiogenesis. (A) Quantitative RT-PCR analysis for COX-2 was performed on total RNAs extracted from the colons at the indicated time intervals as described in Methods. The levels of COX-2 mRNA were normalized to the levels of GAPDH mRNA. *P < 0.05 versus untreated (control) mice. (B and C) Immunohistochemical analysis with anti–COX-2 antibody was performed on colons from WT mice as described in Methods. Boxed area in B is shown at higher magnification. Representative results from 5 independent animals are shown in B (original magnification, ×400; ×1,000 [insets]). The numbers of COX-2 expressing cells were determined on 5 independent animals as described in Methods. The mean and SD were calculated on all values and are shown in C. *P < 0.05 versus untreated mice. (D and E) Colon tissues were immunostained with anti-CD31 antibody as described in Methods. Representative results from 5 independent animals are shown in D. Arrows in D indicate capillary vessels. Original magnification, ×400. The vascular densities were determined as described in Methods and are shown in E. All values represent the mean ± SD. *P < 0.05 versus untreated mice.
PGE2 has also been reported to have direct effects on the β-catenin axis (26). This prompted us to evaluate the state of β-catenin in the tumors of mice treated with etanercept. Indeed, etanercept also decreased the nuclear accumulation of β-catenin at the tumor sites (Figure 8, A and B). The amounts of unphosphorylated (active) β-catenin protein were increased in WT mice at days 56 and 67, and etanercept markedly reduced this increase (Figure 8C). Moreover, etanercept markedly decreased the numbers of cytokeratin 20–positive cells (Figure 8, A and D), which represent colon adenocarcinoma cells (27). Takahashi and colleagues observed that in these AOM-induced tumors, the β_-catenin_ gene, particularly at its glycogen synthase kinase–3β (GSK-3β) phosphorylation sites, mutated more frequently than adenomatous polyposis coli (APC) gene (28). Hence, we examined the effects of TNF blockade on the mutations of the GSK-3β phosphorylation sites of the β_-catenin_ gene, located in its exon 3. Mutations of β_-catenin_ gene were detected in all tumors, and etanercept treatment reduced the mutation frequency markedly from 10/10 positive in the untreated group to 3/10 in the etanercept-treated group (Supplemental Table 1). These observations suggest that blocking of TNF signaling can reverse tumorigenesis even when colon carcinoma is already present, probably by reducing the infiltration of inflammatory cells. Such cells are a major source of COX-2, which is presumed to be involved in both tumor neovascularization and β-catenin activation.
The effect of etanercept on β-catenin nuclear translocation. (A) Colons were immunostained with anti–β-catenin (upper panels) or anti–cytokeratin 20 antibody (lower panels) and representative results from 5 independent animals are shown. Insets are higher magnifications of the positively stained cells, indicated by arrows. Original magnification, ×400; ×1,000 (insets). (B) The β-catenin nuclear localization ratio was determined as the ratio of the numbers of tumor nuclei with β-catenin localization to the total number of tumor nuclei per field. At least 5 randomly chosen fields at ×400 magnification were examined. All values represent the mean ± SD. **P < 0.01 versus etanercept untreated WT mice. (C) Immunoblotting analysis with anti–β-catenin antibodies was performed on cell lysates from colon tissues as described in Methods. Representative results from 3 independent experiments are shown here. (D) The numbers of cytokeratin 20–positive cells were determined on 5 randomly chosen visual fields at ×400 magnification. All values represent the mean ± SD. **P < 0.01 versus etanercept untreated WT mice.