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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

TIL-derived supernatants (TIL SNs) increase CRC cell proliferation through activation of STAT3 and NF-kB. (a) TIL SNs, lamina propria mononuclear cell-derived supernatants (LPMC SNs) and RPMI 1640 complete medium (control) (all used at 1:20 final dilution) were added to DLD-1 and HT-29 cell cultures. After 24 h, cell proliferation was assessed by 5-bromodeoxyuridine (BrdU) assay. Data indicate mean±s.e.m. of four independent experiments in which culture supernatants derived from TILs and LPMCs isolated from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of four patients who had undergone resection for sporadic CRC were used. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001; HT-29: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001. (b) DLD-1 and HT-29 cells were cultured in the presence of either TIL SNs or LPMC SNs or RPMI 1640 complete medium (control) (all used at 1:20 final dilution) for 15 min. P-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression was assessed by western blotting. β-Actin was used as loading control. Shown is one of four representative experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (c) Representative immunofluorescence pictures showing activated STAT3 colocalization with activated NF-kB/p65 in DLD-1 cells cultured in the presence of TIL SNs for 15 min. Cells were cultured as indicated in (b), fixed and stained with anti-p-STAT3 Tyr705 antibody and secondary Alexa Fluor 546 antibody (red), anti-p-NF-kB/p65 Ser536 and secondary Alexa Fluor 488 antibody (green) or 4',6-diamidino-2-phenylindole (DAPI) nuclear staining (blue). The scale bars are 10 μm. The figure is representative of three separate experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (d) Effect of STAT3 inhibitor BP-1-102 on TIL SN-mediated STAT3 and NF-kB activation. 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 BP-1-102. β-Actin was used as a loading control. Blots are representative of four independent experiments in which culture supernatants derived from TILs isolated from the tumor area of four patients who had undergone resection for sporadic CRC were used. (e) Inhibition of STAT3 and NF-kB activation by BP-1-102 completely suppresses TIL SN-mediated increase of CRC cell proliferation. Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of BP-1-102 for 24 h. Data indicate mean±s.e.m. of four experiments in which culture supernatants derived from TILs isolated from the same patients described in (d) were used. Differences between groups were compared using one-way ANOVA followed by Bonferroni's post hoc test. DLD-1: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001.
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fig1: TIL-derived supernatants (TIL SNs) increase CRC cell proliferation through activation of STAT3 and NF-kB. (a) TIL SNs, lamina propria mononuclear cell-derived supernatants (LPMC SNs) and RPMI 1640 complete medium (control) (all used at 1:20 final dilution) were added to DLD-1 and HT-29 cell cultures. After 24 h, cell proliferation was assessed by 5-bromodeoxyuridine (BrdU) assay. Data indicate mean±s.e.m. of four independent experiments in which culture supernatants derived from TILs and LPMCs isolated from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of four patients who had undergone resection for sporadic CRC were used. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001; HT-29: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001. (b) DLD-1 and HT-29 cells were cultured in the presence of either TIL SNs or LPMC SNs or RPMI 1640 complete medium (control) (all used at 1:20 final dilution) for 15 min. P-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression was assessed by western blotting. β-Actin was used as loading control. Shown is one of four representative experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (c) Representative immunofluorescence pictures showing activated STAT3 colocalization with activated NF-kB/p65 in DLD-1 cells cultured in the presence of TIL SNs for 15 min. Cells were cultured as indicated in (b), fixed and stained with anti-p-STAT3 Tyr705 antibody and secondary Alexa Fluor 546 antibody (red), anti-p-NF-kB/p65 Ser536 and secondary Alexa Fluor 488 antibody (green) or 4',6-diamidino-2-phenylindole (DAPI) nuclear staining (blue). The scale bars are 10 μm. The figure is representative of three separate experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (d) Effect of STAT3 inhibitor BP-1-102 on TIL SN-mediated STAT3 and NF-kB activation. 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 BP-1-102. β-Actin was used as a loading control. Blots are representative of four independent experiments in which culture supernatants derived from TILs isolated from the tumor area of four patients who had undergone resection for sporadic CRC were used. (e) Inhibition of STAT3 and NF-kB activation by BP-1-102 completely suppresses TIL SN-mediated increase of CRC cell proliferation. Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of BP-1-102 for 24 h. Data indicate mean±s.e.m. of four experiments in which culture supernatants derived from TILs isolated from the same patients described in (d) were used. Differences between groups were compared using one-way ANOVA followed by Bonferroni's post hoc test. DLD-1: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001.

Mentions: We isolated TILs and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who had undergone resection for sporadic CRC and assessed whether TIL- and LPMC-derived supernatants modulate CRC cell proliferation. TIL-derived supernatants induced a robust proliferation of both DLD-1 and HT-29 cells after 24 h as compared with LPMC-derived supernatants (Figure 1a). No changes in the rate of DLD-1 or HT-29 cell death were seen (not shown). Next, we investigated the mechanism/s underlying this effect, and focused our attention on signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB), two transcription factors whose activation modulates cell proliferation and survival in transformed cells.26 TIL-derived supernatants induced a more pronounced activation of both STAT3 and NF-kB in DLD-1 and HT-29 cells compared with LPMC-derived supernatants (Figure 1b). Immunofluorescence confirmed STAT3 and NF-kB activation in DLD-1 cells and showed a nuclear colocalization of the two activated transcription factors in the presence of TIL-derived supernatants (Figure 1c). Similar results were obtained in HT-29 cells (not shown). Parallel experiments were performed in the presence of BP-1-102, a STAT3 inhibitor that also represses nuclear NF-kB retention both in vitro and in vivo, thereby attenuating NF-kB activation.27 Treatment of DLD-1 and HT-29 cells with BP-1-102 completely suppressed STAT3 and NF-kB activation, as well as the proproliferative effect of TIL-derived supernatants on CRC cells (Figures 1d and e).


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)

TIL-derived supernatants (TIL SNs) increase CRC cell proliferation through activation of STAT3 and NF-kB. (a) TIL SNs, lamina propria mononuclear cell-derived supernatants (LPMC SNs) and RPMI 1640 complete medium (control) (all used at 1:20 final dilution) were added to DLD-1 and HT-29 cell cultures. After 24 h, cell proliferation was assessed by 5-bromodeoxyuridine (BrdU) assay. Data indicate mean±s.e.m. of four independent experiments in which culture supernatants derived from TILs and LPMCs isolated from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of four patients who had undergone resection for sporadic CRC were used. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001; HT-29: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001. (b) DLD-1 and HT-29 cells were cultured in the presence of either TIL SNs or LPMC SNs or RPMI 1640 complete medium (control) (all used at 1:20 final dilution) for 15 min. P-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression was assessed by western blotting. β-Actin was used as loading control. Shown is one of four representative experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (c) Representative immunofluorescence pictures showing activated STAT3 colocalization with activated NF-kB/p65 in DLD-1 cells cultured in the presence of TIL SNs for 15 min. Cells were cultured as indicated in (b), fixed and stained with anti-p-STAT3 Tyr705 antibody and secondary Alexa Fluor 546 antibody (red), anti-p-NF-kB/p65 Ser536 and secondary Alexa Fluor 488 antibody (green) or 4',6-diamidino-2-phenylindole (DAPI) nuclear staining (blue). The scale bars are 10 μm. The figure is representative of three separate experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (d) Effect of STAT3 inhibitor BP-1-102 on TIL SN-mediated STAT3 and NF-kB activation. 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 BP-1-102. β-Actin was used as a loading control. Blots are representative of four independent experiments in which culture supernatants derived from TILs isolated from the tumor area of four patients who had undergone resection for sporadic CRC were used. (e) Inhibition of STAT3 and NF-kB activation by BP-1-102 completely suppresses TIL SN-mediated increase of CRC cell proliferation. Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of BP-1-102 for 24 h. Data indicate mean±s.e.m. of four experiments in which culture supernatants derived from TILs isolated from the same patients described in (d) were used. Differences between groups were compared using one-way ANOVA followed by Bonferroni's post hoc test. DLD-1: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001.
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fig1: TIL-derived supernatants (TIL SNs) increase CRC cell proliferation through activation of STAT3 and NF-kB. (a) TIL SNs, lamina propria mononuclear cell-derived supernatants (LPMC SNs) and RPMI 1640 complete medium (control) (all used at 1:20 final dilution) were added to DLD-1 and HT-29 cell cultures. After 24 h, cell proliferation was assessed by 5-bromodeoxyuridine (BrdU) assay. Data indicate mean±s.e.m. of four independent experiments in which culture supernatants derived from TILs and LPMCs isolated from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of four patients who had undergone resection for sporadic CRC were used. Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. DLD-1: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001; HT-29: TIL SN-stimulated cells vs either LPMC SN-stimulated cells or control, ***P<0.001. (b) DLD-1 and HT-29 cells were cultured in the presence of either TIL SNs or LPMC SNs or RPMI 1640 complete medium (control) (all used at 1:20 final dilution) for 15 min. P-STAT3 Tyr705, STAT3, p-NF-kB/p65 Ser536 and NF-kB/p65 expression was assessed by western blotting. β-Actin was used as loading control. Shown is one of four representative experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (c) Representative immunofluorescence pictures showing activated STAT3 colocalization with activated NF-kB/p65 in DLD-1 cells cultured in the presence of TIL SNs for 15 min. Cells were cultured as indicated in (b), fixed and stained with anti-p-STAT3 Tyr705 antibody and secondary Alexa Fluor 546 antibody (red), anti-p-NF-kB/p65 Ser536 and secondary Alexa Fluor 488 antibody (green) or 4',6-diamidino-2-phenylindole (DAPI) nuclear staining (blue). The scale bars are 10 μm. The figure is representative of three separate experiments in which culture supernatants derived from TILs and LPMCs isolated from the same patients described in (a) were used. (d) Effect of STAT3 inhibitor BP-1-102 on TIL SN-mediated STAT3 and NF-kB activation. 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 BP-1-102. β-Actin was used as a loading control. Blots are representative of four independent experiments in which culture supernatants derived from TILs isolated from the tumor area of four patients who had undergone resection for sporadic CRC were used. (e) Inhibition of STAT3 and NF-kB activation by BP-1-102 completely suppresses TIL SN-mediated increase of CRC cell proliferation. Representative histograms showing cell proliferation of DLD-1 and HT-29 cells stimulated or not with TIL SNs in the presence or absence of BP-1-102 for 24 h. Data indicate mean±s.e.m. of four experiments in which culture supernatants derived from TILs isolated from the same patients described in (d) were used. Differences between groups were compared using one-way ANOVA followed by Bonferroni's post hoc test. DLD-1: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001; HT-29: TIL SN+BP-1-102-treated cells vs TIL SN-treated cells, ***P<0.001.
Mentions: We isolated TILs and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who had undergone resection for sporadic CRC and assessed whether TIL- and LPMC-derived supernatants modulate CRC cell proliferation. TIL-derived supernatants induced a robust proliferation of both DLD-1 and HT-29 cells after 24 h as compared with LPMC-derived supernatants (Figure 1a). No changes in the rate of DLD-1 or HT-29 cell death were seen (not shown). Next, we investigated the mechanism/s underlying this effect, and focused our attention on signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB), two transcription factors whose activation modulates cell proliferation and survival in transformed cells.26 TIL-derived supernatants induced a more pronounced activation of both STAT3 and NF-kB in DLD-1 and HT-29 cells compared with LPMC-derived supernatants (Figure 1b). Immunofluorescence confirmed STAT3 and NF-kB activation in DLD-1 cells and showed a nuclear colocalization of the two activated transcription factors in the presence of TIL-derived supernatants (Figure 1c). Similar results were obtained in HT-29 cells (not shown). Parallel experiments were performed in the presence of BP-1-102, a STAT3 inhibitor that also represses nuclear NF-kB retention both in vitro and in vivo, thereby attenuating NF-kB activation.27 Treatment of DLD-1 and HT-29 cells with BP-1-102 completely suppressed STAT3 and NF-kB activation, as well as the proproliferative effect of TIL-derived supernatants on CRC cells (Figures 1d and e).

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