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. 2009 Feb 3;15(2):103-13.
doi: 10.1016/j.ccr.2009.01.001.

IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer

Sergei Grivennikov  1 , Eliad Karin, Janos Terzic, Daniel Mucida, Guann-Yi Yu, Sivakumar Vallabhapurapu, Jürgen Scheller, Stefan Rose-John, Hilde Cheroutre, Lars Eckmann, Michael Karin
Affiliations

IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer

Sergei Grivennikov et al. Cancer Cell. .

Erratum in

  • Cancer Cell. 2009 Mar 3;15(3):241

Abstract

Colitis-associated cancer (CAC) is the most serious complication of inflammatory bowel disease. Proinflammatory cytokines have been suggested to regulate preneoplastic growth during CAC tumorigenesis. Interleukin 6 (IL-6) is a multifunctional NF-kappaB-regulated cytokine that acts on epithelial and immune cells. Using genetic tools, we now demonstrate that IL-6 is a critical tumor promoter during early CAC tumorigenesis. In addition to enhancing proliferation of tumor-initiating cells, IL-6 produced by lamina propria myeloid cells protects normal and premalignant intestinal epithelial cells (IECs) from apoptosis. The proliferative and survival effects of IL-6 are largely mediated by the transcription factor Stat3, whose IEC-specific ablation has profound impact on CAC tumorigenesis. Thus, the NF-kappaB-IL-6-Stat3 cascade is an important regulator of the proliferation and survival of tumor-initiating IECs.

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Conflict of interest statement

All other authors declare no competing financial interests.

Figures

Figure 1
Figure 1. IL-6 controls tumor formation and growth in a mouse CAC model
WT and Il6-/- mice were subjected to AOM-based CAC induction protocol using three cycles of 2.5% (A) or 1.5% (B) DSS in drinking water. Tumors were counted at the end of the 12 week CAC induction regimen. Data represent average tumor numbers ± s.d., n>10. p=0.003 for (A); p=0.0001 for (B). (C) Tumor sizes were determined using imaging Spot software (Zeiss) for microscopic tumors or with a caliper for macroscopic tumors. Average tumor size ± s.d. is shown; * p=0.012. (D) Histogram showing size distribution of tumors. (E) Average tumor load was determined by summing all tumor diameters for a given animal. Results are averages ± s.d. (n>7); * p=0.047.
Figure 2
Figure 2. IL-6 is required for maintenance of mucosal integrity
(A) WT and Il6-/- mice exhibit colon shortening after 7 days of 2.5% DSS exposure. (B) Mucosal histology was examined in WT or Il6-/- mice 4 or 10 days after initiation of 2.5% DSS treatment by H&E staining of paraffine embedded sections. Scale bar- 50 μm. (C) Colitis severity score after 3% DSS exposure was determined on day 10. Results are averages ± s.d. (n=5), * p≤0.05. (D) Apoptosis in colons of 3% DSS treated mice was evaluated on day 4 after DSS administration by TUNEL staining. Scale bar- 50 μm. (E) The extent of IEC proliferation in colons of DSS-treated mice was determined by BrdU labeling and immunohistochemistry. Scale bar- 50 μm. (F) The percentage of Ki-67 positive cells among all crypt cells in colons of DSS treated mice was enumerated. Results are averages ± s.d. (n=6), * p= 0.001. (G) Lysates of distal colons prepared on the indicated days after initiation of 3% DSS treatment were analyzed for PCNA expression by immunoblotting.
Figure 3
Figure 3. IL-6 produced by bone marrow derived cells is required for CAC tumorigenesis
(A) Tumor multiplicity in radiation chimeras subjected to induction of CAC using 2.5% DSS. Results are averages ± s.d. (n>8), * p<0.01. (B) Paraffine embedded sections of adenoma-containing colons stained with H&E. Scale bar- 100 μm. (C) Average tumor loads in radiation chimeras. Results are averages ± s.d. (n=5), NS- not significant, * p<0.01. (D) Immunohistochemical analysis of IL-6 expression in DSS-treated colons (normal mucosa) or CAC-bearing colons of WT mice. Scale bar- 50 μm. (E,F) Intracellular IL-6 cytokine staining of PMA+ionomycin restimulated tumor infiltrating cells analyzed by flow cytometry. Results are averages ± s.d. (n=3). (E) Percentages of IL-6 expressing cells in each given population (DC, macrophages, T and B cells) (F) Percentages of each population positive for IL-6 cells among all tumor infiltrating cells. (G) Macrophages (CD45+CD11b+CD11c+), dendritic cells (CD45+CD11b+CD11c+) and T cells (CD45+CD4+ or CD45+CD8α+) isolated by FACS sorting were analyzed for IL-6 mRNA by Q-RT-PCR. Tumor population- tumor infiltrating cells from pooled tumors. LP population- lamina propria cells from colons from which the tumors were excised. (H) Percentage of macrophages (CD11b+), T cells (CD3+) and dendritic cells (CD11c+) in total lamina propria cells isolated from pooled CAC tumors from WT mice.
Figure 4
Figure 4. IL-6 regulates cell proliferation and expression of genes involved in proliferation, survival and inflammation
(A) Colons of adenoma-bearing mice were stained with anti-PCNA antibody. Scale bar- 50 μm. (B) Lysates of distal colons of adenoma-bearing mice were prepared and PCNA mRNA expression was analyzed by Q-RT-PCR. Results are averages ± s.d. (n=3). (C) Sections of adenoma-bearing colons were stained with antibodies to cyclin D. Scale bar- 50 μm. (D) Percentages of Ki-67-positive cells within colonic crypts. Results are averages ± s.d. (n=6), * p=0.005. (E) IEC from mice treated with 2.5% DSS were purified on day 10. Expression of the indicated proteins was analyzed by immunoblotting after gel separation. (F) RegIIIγ and Tff3 mRNA expression was analyzed by Q-RT-PCR. Results are averages ± s.d. (n=3).
Figure 5
Figure 5. STAT3 maintains mucosal integrity during acute colitis
(A). Mice were exposed to 2.5% DSS. Colonic lysates were prepared at the indicated timepoints and analyzed for expression and phosphorylation of the indicated proteins. (B) Deletion of STAT3 in IEC of Stat3ΔIEC mice was examined by immunoblotting of IEC lysates. (C) Deletion of STAT3 in colons of Stat3ΔIEC mice was analyzed by immunohistochemistry with anti-STAT3 antibody. Scale bar- 50 μm (D) Total colon lysates of WT and Stat3ΔIEC mice were analyzed for STAT3 expression and phosphorylation by immunoblot analysis (E) Increased susceptibility of Stat3ΔIEC mice to DSS colitis. Colons of WT and Stat3ΔIEC mice were analyzed by sectioning and H&E staining 10 days after initiation of 2.5% DSS exposure. Scale bar- 100 μm. (F) Bodyweights of the indicated mice were measured 10 days after initiation of 2.5% DSS exposure. Results show % of body weight on day 0 and are averages ± s.d. (n=6), p<0.05. (G;H) Total colon lysates (G) or IEC lysate (H) from mice on day 10 after 2.5% DSS administration were analyzed for expression of the indicated proteins by immunoblotting.
Figure 6
Figure 6. STAT3 is critical for CAC tumorigenesis
(A) Percentage of Ki-67-positive cells in WT and Stat3ΔIEC colonic crypts 10 days after initiation of DSS exposure. Results are averages ± s.d. (n=3). * p=0.03. (B) Tumor multiplicity in WT and Stat3ΔIEC mice subjected to induction of CAC. Results are averages ± s.d. (n=8), *p=0.004. (C) Tumor sizes in WT and Stat3ΔIEC mice. Results are averages ± s.d. (n=8), * p=0.012. (D) Paraffine embedded sections of adenoma-containing colons of WT and Stat3ΔIEC mice were stained with H&E. Scale bar- 100 μm. (E) Apoptosis in colons of DSS treated mice was evaluated on day 4 after DSS administration by TUNEL staining. Amount of TUNEL-positive cells per microscope field was determined. Results are averages ± s.d. (n=3). * p=0.05.
Figure 7
Figure 7. IL-6 signaling stimulates tumor formation and growth
(A) Scheme of treatment with IL-6 agonists during late stage of CAC growth. Mice were i.p. injected with 2 μg Hyper-IL-6 or 5 μg recombinant IL-6 every 3 days after the last DSS cycle. Tumors were analyzed on day 100 after AOM injection. (B) Numbers of tumors larger than > 2mm. Results are averages ± s.d. (n=7), * p<0.05. (C) Tumor multiplicity; Results are averages ± s.d., NS- not significant. (D) Scheme of treatment with IL-6 agonists during CAC induction. Mice were i.p. injected with the same amounts of IL-6 agonists as in (A), on day 1, 5 and 8 of each DSS cycle. Tumors were analyzed 100 days after AOM injection. (E) Average tumor load. Results are averages ± s.d. (n=6). * p<0.05. (F) Tumor multiplicity. Results are averages ± s.d. (n=6). * p<0.05. (G) Immunoblot analysis of colonic lysates from mice treated with hyper-IL-6, rec IL-6 or PBS after exposure to 2.5% DSS for 7 days. Mice were sacrificed on day 10 30 min after the last treatment.
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