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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

IL-17A, IL-22, TNF-α and IL-6 contribute to TIL-derived supernatant (TIL SN)-mediated STAT3/NF-kB activation and mitogenic effect in CRC cells. (a) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 (all used at 25 ng/ml) for 15 min. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (b) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-17F, anti-IL-21, anti-IL-22, anti-TNF-α and anti-IL-6 (all used at 10 μg/ml) as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (c) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (b). Data indicate mean±s.e.m. of four experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. (d) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-22, anti-TNF-α and anti-IL-6, used in combination as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (e) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (d). Data indicate mean±s.e.m. of five experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, *P<0.05, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, **P<0.01, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001.
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fig4: IL-17A, IL-22, TNF-α and IL-6 contribute to TIL-derived supernatant (TIL SN)-mediated STAT3/NF-kB activation and mitogenic effect in CRC cells. (a) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 (all used at 25 ng/ml) for 15 min. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (b) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-17F, anti-IL-21, anti-IL-22, anti-TNF-α and anti-IL-6 (all used at 10 μg/ml) as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (c) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (b). Data indicate mean±s.e.m. of four experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. (d) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-22, anti-TNF-α and anti-IL-6, used in combination as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (e) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (d). Data indicate mean±s.e.m. of five experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, *P<0.05, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, **P<0.01, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001.

Mentions: IL-22 and IL-6 activated STAT3 in DLD-1 and HT-29 cells, whereas activation of NF-kB was seen in cells stimulated with IL-17A or TNF-α. In contrast, no activation of STAT3 and NF-kB was seen in cells stimulated with IL-17F or IL-21 (Figure 4a). Both these cytokines were however biologically active, as IL-17F induced IL-6 in stromal cell cultures and IL-21 activated STAT3 in human LPMCs (not shown). To investigate the contribution of Th17-related cytokines, TNF-α and IL-6, in TIL-derived supernatant-driven CRC cell growth, supernatants were preincubated with neutralizing anti-cytokine antibodies before adding to CRC cell lines. Initially, we verified the functional activity of each neutralizing antibody by testing its ability to suppress the function of the corresponding cytokine in cultures of CRC cells (Supplementary Figure 3). Blockade of IL-6 partially reduced p-STAT3 Y705, but not p-NF-kB/p65 Ser536, in DLD-1 and HT-29 cells cultured with TIL-derived supernatants. In contrast, individual neutralization of IL-17A, IL-17F, IL-21, IL-22 or TNF-α did not change TIL-derived supernatant-driven STAT3 and NF-kB activation (Figure 4b). Moreover, no significant change in TIL-derived supernatant-driven CRC cell growth was seen by the blockade of a single cytokine (Figure 4c). Next, we assessed the effect of neutralization of multiple cytokines on STAT3 and NF-kB activation as well as on the promitogenic effect of TIL-derived supernatants. Neutralization of both IL-17A and TNF-α did not alter activation of STAT3 and NF-kB nor TIL-derived supernatant-induced CRC cell proliferation (Figures 4d and e). Both anti-IL-22 and anti-IL-6 reduced TIL-derived supernatant-driven p-STAT3 Y705 but not p-NF-kB/p65 Ser536, and this was associated with a decreased CRC cell growth (Figures 4d and e). Simultaneous neutralization of IL-17A, TNF-α, IL-22 and IL-6 reduced both STAT3 and NF-kB activation and significantly inhibited growth of cells induced by TIL-derived supernatants compared with cells preincubated with anti-IL-22 and anti-IL-6 (Figures 4d and e). No change in cell viability was observed, thus ruling out the possibility that changes in cell proliferation were secondary to induction of cell death (data not shown).


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)

IL-17A, IL-22, TNF-α and IL-6 contribute to TIL-derived supernatant (TIL SN)-mediated STAT3/NF-kB activation and mitogenic effect in CRC cells. (a) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 (all used at 25 ng/ml) for 15 min. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (b) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-17F, anti-IL-21, anti-IL-22, anti-TNF-α and anti-IL-6 (all used at 10 μg/ml) as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (c) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (b). Data indicate mean±s.e.m. of four experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. (d) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-22, anti-TNF-α and anti-IL-6, used in combination as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (e) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (d). Data indicate mean±s.e.m. of five experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, *P<0.05, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, **P<0.01, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001.
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fig4: IL-17A, IL-22, TNF-α and IL-6 contribute to TIL-derived supernatant (TIL SN)-mediated STAT3/NF-kB activation and mitogenic effect in CRC cells. (a) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 (all used at 25 ng/ml) for 15 min. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (b) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-17F, anti-IL-21, anti-IL-22, anti-TNF-α and anti-IL-6 (all used at 10 μg/ml) as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (c) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (b). Data indicate mean±s.e.m. of four experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. (d) Representative western blotting showing p-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression in DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of anti-IL-17A, anti-IL-22, anti-TNF-α and anti-IL-6, used in combination as indicated. β-Actin was used as a loading control. One of three representative experiments in which similar results were obtained is shown. (e) Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated as indicated in (d). Data indicate mean±s.e.m. of five experiments. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, *P<0.05, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, **P<0.01, TIL SN+anti-IL-17A+anti-TNF-α+anti-IL-22+anti-IL-6- vs TIL SN-treated cells, ***P<0.001.
Mentions: IL-22 and IL-6 activated STAT3 in DLD-1 and HT-29 cells, whereas activation of NF-kB was seen in cells stimulated with IL-17A or TNF-α. In contrast, no activation of STAT3 and NF-kB was seen in cells stimulated with IL-17F or IL-21 (Figure 4a). Both these cytokines were however biologically active, as IL-17F induced IL-6 in stromal cell cultures and IL-21 activated STAT3 in human LPMCs (not shown). To investigate the contribution of Th17-related cytokines, TNF-α and IL-6, in TIL-derived supernatant-driven CRC cell growth, supernatants were preincubated with neutralizing anti-cytokine antibodies before adding to CRC cell lines. Initially, we verified the functional activity of each neutralizing antibody by testing its ability to suppress the function of the corresponding cytokine in cultures of CRC cells (Supplementary Figure 3). Blockade of IL-6 partially reduced p-STAT3 Y705, but not p-NF-kB/p65 Ser536, in DLD-1 and HT-29 cells cultured with TIL-derived supernatants. In contrast, individual neutralization of IL-17A, IL-17F, IL-21, IL-22 or TNF-α did not change TIL-derived supernatant-driven STAT3 and NF-kB activation (Figure 4b). Moreover, no significant change in TIL-derived supernatant-driven CRC cell growth was seen by the blockade of a single cytokine (Figure 4c). Next, we assessed the effect of neutralization of multiple cytokines on STAT3 and NF-kB activation as well as on the promitogenic effect of TIL-derived supernatants. Neutralization of both IL-17A and TNF-α did not alter activation of STAT3 and NF-kB nor TIL-derived supernatant-induced CRC cell proliferation (Figures 4d and e). Both anti-IL-22 and anti-IL-6 reduced TIL-derived supernatant-driven p-STAT3 Y705 but not p-NF-kB/p65 Ser536, and this was associated with a decreased CRC cell growth (Figures 4d and e). Simultaneous neutralization of IL-17A, TNF-α, IL-22 and IL-6 reduced both STAT3 and NF-kB activation and significantly inhibited growth of cells induced by TIL-derived supernatants compared with cells preincubated with anti-IL-22 and anti-IL-6 (Figures 4d and e). No change in cell viability was observed, thus ruling out the possibility that changes in cell proliferation were secondary to induction of cell death (data not shown).

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