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Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth.

De Simone V, Franzè E, Ronchetti G, Colantoni A, Fantini MC, Di Fusco D, Sica GS, Sileri P, MacDonald TT, Pallone F, Monteleone G, Stolfi C - Oncogene (2014)

Bottom Line: Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells.In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells.Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Medicine, University of Rome 'Tor Vergata', Rome, Italy.

ABSTRACT
Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.

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Related in: MedlinePlus

Analysis of cytokine production and T-bet/RORγt expression in LPMC- and TIL-derived CD3+CD8− subsets. (a) Representative histograms showing the fraction of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-expressing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of IFN-γ- and/or IL-21-, IL-17A- and/or IL-17F-, IL-22- and/or IL-6- and TNF-α-producing CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. (b) Representative histograms showing the fraction of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. Staining of LPMCs with APC- and PE-Cy7-conjugated control isotype IgG is also shown. (c) Representative dot plots showing the ability to produce IFN-γ and/or IL-17A by the indicated subsets of TILs. The numbers indicate the percentage of cells in the designated quadrants. (d) Representative histograms showing the percentage of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of one patient undergoing colectomy for sporadic CRC . IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells were gated and analyzed for the indicated markers. The example is representative of 10 independent experiments in which cells isolated from 10 patients undergoing colectomy for sporadic CRC were analyzed.
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fig3: Analysis of cytokine production and T-bet/RORγt expression in LPMC- and TIL-derived CD3+CD8− subsets. (a) Representative histograms showing the fraction of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-expressing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of IFN-γ- and/or IL-21-, IL-17A- and/or IL-17F-, IL-22- and/or IL-6- and TNF-α-producing CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. (b) Representative histograms showing the fraction of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. Staining of LPMCs with APC- and PE-Cy7-conjugated control isotype IgG is also shown. (c) Representative dot plots showing the ability to produce IFN-γ and/or IL-17A by the indicated subsets of TILs. The numbers indicate the percentage of cells in the designated quadrants. (d) Representative histograms showing the percentage of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of one patient undergoing colectomy for sporadic CRC . IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells were gated and analyzed for the indicated markers. The example is representative of 10 independent experiments in which cells isolated from 10 patients undergoing colectomy for sporadic CRC were analyzed.

Mentions: Next, LPMC- and TIL-derived supernatants were analyzed for cytokines known to control cancer cell proliferation. Higher expression of IL-17A, IL-17F, IL-21, IL-22, TNF-α and IL-6 was found in TIL-derived supernatants compared with LPMC-derived supernatants, whereas there was no difference in terms of IFN-γ concentrations (Figure 2a). To ascertain whether these changes in cytokine production reflected accumulation of specific immune cell types in the tumor area, LPMCs and TILs were analyzed by flow cytometry. The percentage of CD3+CD8−, CD3+CD8+, CD3+CD56+, CD3−CD56+, CD19+ and CD68+ cells did not differ between LPMCs and TILs (Figure 2b). However, while TILs and LPMCs contained similar fractions of IFN-γ-producing CD45+ cells, the percentages of IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-expressing CD45+ cells were increased in TILs (Figure 2c). Further analysis showed that, in both LPMCs and TILs, IFN-γ was mainly produced by CD3+CD8− and CD3+CD8+ cells (Supplementary Figure 1). Lower production of IFN-γ by CD3+CD8− cells and higher production of IFN-γ derived from CD3+CD8+ cells was seen in TILs as compared with LPMCs (Supplementary Figure 1). IL-17A, IL-17F, IL-21, IL-22 and IL-6 were almost totally derived from CD3+CD8− cells. TNF-α was also produced mainly by CD3+CD8− cells and, to a lesser extent, by CD3+CD8+ cells in both LPMCs and TILs (Supplementary Figure 1). As the majority of IFN-γ, Th17-related cytokine-, TNF-α- and IL-6-producing CD45+ cells in both TILs and LPMCs were CD3+CD8− and CD3+CD8+ cells, we restricted our further analysis to these cell types. The percentage of IFN-γ-producing CD3+CD8− cells was not different between TILs and LPMCs (Figure 3a). In contrast, the percentage of CD3+CD8− cells expressing IL-17A, IL-17F, IL-21, IL-22, TNF-α and IL-6 was increased in TILs (Figure 3a). Next, we assessed the expression of T-bet and receptor-related orphan receptor gamma t (RORγt) in CD3+CD8− cells in TILs and LPMCs. The majority of CD3+CD8− cells coexpressed T-bet and RORγt both in TILs and LPMCs (Figure 3b). In contrast, TIL samples contained more T-bet-/RORγt+ CD3+CD8− cells and less T-bet+/RORγt− CD3+CD8− cells than LPMC samples (Figure 3b). As expected, T-bet+/RORγt− CD3+CD8− cells produced IFN-γ but not IL-17A, whereas T-bet−/RORγt+ CD3+CD8− cells produced IL-17A but not IFN-γ. Double-positive T-bet/RORγt CD3+CD8− cells coexpressed both IFN-γ and IL-17A, whereas double-negative T-bet/RORγt CD3+CD8− cells produced neither IFN-γ nor IL-17A (Figure 3c). Further analysis of cytokine production revealed that the double-positive T-bet/RORγt CD3+CD8− cells were the major source of IFN-γ, Th17-related cytokines, TNF-α and IL-6 in both TILs and LPMCs (Figure 3d). However, TILs contained higher fraction of T-bet−/RORγt+ CD3+CD8− cells producing Th17 cytokines, TNF-α and IL-6, as compared with LPMCs (Figure 3d). A higher fraction of IFN-γ- and TNF-α-producing CD3+CD8+ cells was also observed in TILs as compared with LPMCs, whereas IL-17A-, IL-17F-, IL-21-, IL-22- and IL-6-producing CD3+CD8+ cells were barely detectable in both TILs and LPMCs (Supplementary Figure 2A). The majority of CD3+CD8+ cells in both TILs and LPMCs coexpressed T-bet and RORγt (Supplementary Figure 2B). In contrast, TIL samples contained more T-bet+/RORγt+ CD3+CD8+ cells and less T-bet+/RORγt− CD3+CD8+ cells than LPMC samples (Supplementary Figure 2B). Analysis of cytokine production revealed that double-positive T-bet/RORγt CD3+CD8+ cells were the major source of IFN-γ and TNF-α in both TILs and LPMCs (Supplementary Figure 2C). Altogether, these findings show that transition from the uninvolved colonic mucosa to the neoplastic area is marked by a shift in the CD3+CD8− and CD3+CD8+ cell phenotype leading to the accumulation of Th17-related cytokines, TNF-α and IL-6.


Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth.

De Simone V, Franzè E, Ronchetti G, Colantoni A, Fantini MC, Di Fusco D, Sica GS, Sileri P, MacDonald TT, Pallone F, Monteleone G, Stolfi C - Oncogene (2014)

Analysis of cytokine production and T-bet/RORγt expression in LPMC- and TIL-derived CD3+CD8− subsets. (a) Representative histograms showing the fraction of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-expressing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of IFN-γ- and/or IL-21-, IL-17A- and/or IL-17F-, IL-22- and/or IL-6- and TNF-α-producing CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. (b) Representative histograms showing the fraction of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. Staining of LPMCs with APC- and PE-Cy7-conjugated control isotype IgG is also shown. (c) Representative dot plots showing the ability to produce IFN-γ and/or IL-17A by the indicated subsets of TILs. The numbers indicate the percentage of cells in the designated quadrants. (d) Representative histograms showing the percentage of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of one patient undergoing colectomy for sporadic CRC . IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells were gated and analyzed for the indicated markers. The example is representative of 10 independent experiments in which cells isolated from 10 patients undergoing colectomy for sporadic CRC were analyzed.
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fig3: Analysis of cytokine production and T-bet/RORγt expression in LPMC- and TIL-derived CD3+CD8− subsets. (a) Representative histograms showing the fraction of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-expressing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of IFN-γ- and/or IL-21-, IL-17A- and/or IL-17F-, IL-22- and/or IL-6- and TNF-α-producing CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. (b) Representative histograms showing the fraction of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of 14 patients undergoing colectomy for sporadic CRC. Data are expressed as mean±s.e.m. and differences were calculated using the two-tailed Student's t-test. Right insets. Representative dot plots showing the percentage of T-bet+ and/or Rorγt+ CD3+CD8− cells in LPMCs and TILs. The numbers indicate the percentage of cells in the designated quadrants. Staining of LPMCs with APC- and PE-Cy7-conjugated control isotype IgG is also shown. (c) Representative dot plots showing the ability to produce IFN-γ and/or IL-17A by the indicated subsets of TILs. The numbers indicate the percentage of cells in the designated quadrants. (d) Representative histograms showing the percentage of IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells in LPMCs and TILs isolated from adjacent tumor and non-tumor areas of one patient undergoing colectomy for sporadic CRC . IFN-γ-, IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-producing CD3+CD8− cells were gated and analyzed for the indicated markers. The example is representative of 10 independent experiments in which cells isolated from 10 patients undergoing colectomy for sporadic CRC were analyzed.
Mentions: Next, LPMC- and TIL-derived supernatants were analyzed for cytokines known to control cancer cell proliferation. Higher expression of IL-17A, IL-17F, IL-21, IL-22, TNF-α and IL-6 was found in TIL-derived supernatants compared with LPMC-derived supernatants, whereas there was no difference in terms of IFN-γ concentrations (Figure 2a). To ascertain whether these changes in cytokine production reflected accumulation of specific immune cell types in the tumor area, LPMCs and TILs were analyzed by flow cytometry. The percentage of CD3+CD8−, CD3+CD8+, CD3+CD56+, CD3−CD56+, CD19+ and CD68+ cells did not differ between LPMCs and TILs (Figure 2b). However, while TILs and LPMCs contained similar fractions of IFN-γ-producing CD45+ cells, the percentages of IL-17A-, IL-17F-, IL-21-, IL-22-, TNF-α- and IL-6-expressing CD45+ cells were increased in TILs (Figure 2c). Further analysis showed that, in both LPMCs and TILs, IFN-γ was mainly produced by CD3+CD8− and CD3+CD8+ cells (Supplementary Figure 1). Lower production of IFN-γ by CD3+CD8− cells and higher production of IFN-γ derived from CD3+CD8+ cells was seen in TILs as compared with LPMCs (Supplementary Figure 1). IL-17A, IL-17F, IL-21, IL-22 and IL-6 were almost totally derived from CD3+CD8− cells. TNF-α was also produced mainly by CD3+CD8− cells and, to a lesser extent, by CD3+CD8+ cells in both LPMCs and TILs (Supplementary Figure 1). As the majority of IFN-γ, Th17-related cytokine-, TNF-α- and IL-6-producing CD45+ cells in both TILs and LPMCs were CD3+CD8− and CD3+CD8+ cells, we restricted our further analysis to these cell types. The percentage of IFN-γ-producing CD3+CD8− cells was not different between TILs and LPMCs (Figure 3a). In contrast, the percentage of CD3+CD8− cells expressing IL-17A, IL-17F, IL-21, IL-22, TNF-α and IL-6 was increased in TILs (Figure 3a). Next, we assessed the expression of T-bet and receptor-related orphan receptor gamma t (RORγt) in CD3+CD8− cells in TILs and LPMCs. The majority of CD3+CD8− cells coexpressed T-bet and RORγt both in TILs and LPMCs (Figure 3b). In contrast, TIL samples contained more T-bet-/RORγt+ CD3+CD8− cells and less T-bet+/RORγt− CD3+CD8− cells than LPMC samples (Figure 3b). As expected, T-bet+/RORγt− CD3+CD8− cells produced IFN-γ but not IL-17A, whereas T-bet−/RORγt+ CD3+CD8− cells produced IL-17A but not IFN-γ. Double-positive T-bet/RORγt CD3+CD8− cells coexpressed both IFN-γ and IL-17A, whereas double-negative T-bet/RORγt CD3+CD8− cells produced neither IFN-γ nor IL-17A (Figure 3c). Further analysis of cytokine production revealed that the double-positive T-bet/RORγt CD3+CD8− cells were the major source of IFN-γ, Th17-related cytokines, TNF-α and IL-6 in both TILs and LPMCs (Figure 3d). However, TILs contained higher fraction of T-bet−/RORγt+ CD3+CD8− cells producing Th17 cytokines, TNF-α and IL-6, as compared with LPMCs (Figure 3d). A higher fraction of IFN-γ- and TNF-α-producing CD3+CD8+ cells was also observed in TILs as compared with LPMCs, whereas IL-17A-, IL-17F-, IL-21-, IL-22- and IL-6-producing CD3+CD8+ cells were barely detectable in both TILs and LPMCs (Supplementary Figure 2A). The majority of CD3+CD8+ cells in both TILs and LPMCs coexpressed T-bet and RORγt (Supplementary Figure 2B). In contrast, TIL samples contained more T-bet+/RORγt+ CD3+CD8+ cells and less T-bet+/RORγt− CD3+CD8+ cells than LPMC samples (Supplementary Figure 2B). Analysis of cytokine production revealed that double-positive T-bet/RORγt CD3+CD8+ cells were the major source of IFN-γ and TNF-α in both TILs and LPMCs (Supplementary Figure 2C). Altogether, these findings show that transition from the uninvolved colonic mucosa to the neoplastic area is marked by a shift in the CD3+CD8− and CD3+CD8+ cell phenotype leading to the accumulation of Th17-related cytokines, TNF-α and IL-6.

Bottom Line: Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells.In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells.Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Medicine, University of Rome 'Tor Vergata', Rome, Italy.

ABSTRACT
Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.

Show MeSH
Related in: MedlinePlus