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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

Reduced STAT3/NF-kB activation and reduced expression of IL-17A, IL-21, IL-22, TNF-α and IL-6 are seen in the colonic tumors of BP-1-102-treated Apcmin/+ mice. (a) Representative images showing p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive cells in colonic sections taken from the tumor areas Apcmin/+ mice treated with either dimethyl sulfoxide (DMSO) (CTR) or BP-1-102 and killed on day 56. The scale bars are 20 μm. One of six representative experiments in which similar results were obtained is shown. Right insets. Quantification of p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive infiltrating and epithelial cells in colonic sections taken from the tumor areas of Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Data are presented as mean values of positive cells per high power field (h.p.f.)±s.e.m. of three independent experiments in which two sections per group were analyzed. Differences were calculated using the two-tailed Student's t-test. NT, non-tumor area; T, tumor area. (b) IFN-γ, IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 expression was assessed by real-time PCR in colonic tissues taken from Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Values are mean±s.e.m. of two independent experiments containing at least three mice per group. Differences were calculated using the two-tailed Student's t-test. ND, not detectable; NT, non-tumor area; T, tumor area.
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fig6: Reduced STAT3/NF-kB activation and reduced expression of IL-17A, IL-21, IL-22, TNF-α and IL-6 are seen in the colonic tumors of BP-1-102-treated Apcmin/+ mice. (a) Representative images showing p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive cells in colonic sections taken from the tumor areas Apcmin/+ mice treated with either dimethyl sulfoxide (DMSO) (CTR) or BP-1-102 and killed on day 56. The scale bars are 20 μm. One of six representative experiments in which similar results were obtained is shown. Right insets. Quantification of p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive infiltrating and epithelial cells in colonic sections taken from the tumor areas of Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Data are presented as mean values of positive cells per high power field (h.p.f.)±s.e.m. of three independent experiments in which two sections per group were analyzed. Differences were calculated using the two-tailed Student's t-test. NT, non-tumor area; T, tumor area. (b) IFN-γ, IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 expression was assessed by real-time PCR in colonic tissues taken from Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Values are mean±s.e.m. of two independent experiments containing at least three mice per group. Differences were calculated using the two-tailed Student's t-test. ND, not detectable; NT, non-tumor area; T, tumor area.

Mentions: Endoscopy on day 54 showed that control mice developed multiple large tumors, whereas the number and size of tumors were reduced in the colon of BP-1-102-treated mice (Figure 5b). These results were confirmed by direct assessment of tumors in mice killed on day 56 (unpublished data). Proliferating cell nuclear antigen staining confirmed the antiproliferative effect of BP-1-102 (Figure 5c). By contrast, there was no significant change in proliferating cell nuclear antigen staining in the normal colonic mucosa of mice treated with BP-1-102 (Figure 5c). To determine whether the reduced tumorigenesis observed in BP-1-102-treated mice was associated with a reduced activation of STAT3 and/or NF-kB, we compared p-STAT3 Y705 and p-NF-kB/p65 Ser536 expression in non-tumor and tumor tissue derived from colonic extracts of control and BP-1-102-treated mice killed on day 56. Robust activation of both STAT3 and NF-kB was seen in the neoplastic areas of control mice and inhibited by BP-1-102 treatment, whereas p-STAT3 Y705 and p-NF-kB/p65 Ser536 expression was barely detectable in non-tumor areas of both control and BP-1-102-treated mice (Supplementary Figure 7). Immunohistochemistry confirmed reduced activation of both STAT3 and NF-kB in colonic tumor sections of mice treated with BP-1-102 (Figure 6a). IL-17A, IL-21, IL-22, TNF-α and IL-6 transcripts were reduced in the tumor areas of BP-1-102-treated mice, whereas IFN-γ and IL-17F RNA expression remained unchanged (Figure 6b). Moreover, treatment of mice with BP-1-102 did not change IL-11 transcripts in the tumors as well as the fractions of immune cells in TILs (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)

Reduced STAT3/NF-kB activation and reduced expression of IL-17A, IL-21, IL-22, TNF-α and IL-6 are seen in the colonic tumors of BP-1-102-treated Apcmin/+ mice. (a) Representative images showing p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive cells in colonic sections taken from the tumor areas Apcmin/+ mice treated with either dimethyl sulfoxide (DMSO) (CTR) or BP-1-102 and killed on day 56. The scale bars are 20 μm. One of six representative experiments in which similar results were obtained is shown. Right insets. Quantification of p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive infiltrating and epithelial cells in colonic sections taken from the tumor areas of Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Data are presented as mean values of positive cells per high power field (h.p.f.)±s.e.m. of three independent experiments in which two sections per group were analyzed. Differences were calculated using the two-tailed Student's t-test. NT, non-tumor area; T, tumor area. (b) IFN-γ, IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 expression was assessed by real-time PCR in colonic tissues taken from Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Values are mean±s.e.m. of two independent experiments containing at least three mice per group. Differences were calculated using the two-tailed Student's t-test. ND, not detectable; NT, non-tumor area; T, tumor area.
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fig6: Reduced STAT3/NF-kB activation and reduced expression of IL-17A, IL-21, IL-22, TNF-α and IL-6 are seen in the colonic tumors of BP-1-102-treated Apcmin/+ mice. (a) Representative images showing p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive cells in colonic sections taken from the tumor areas Apcmin/+ mice treated with either dimethyl sulfoxide (DMSO) (CTR) or BP-1-102 and killed on day 56. The scale bars are 20 μm. One of six representative experiments in which similar results were obtained is shown. Right insets. Quantification of p-STAT3 Tyr705- or p-NF-kB/p65 Ser536-positive infiltrating and epithelial cells in colonic sections taken from the tumor areas of Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Data are presented as mean values of positive cells per high power field (h.p.f.)±s.e.m. of three independent experiments in which two sections per group were analyzed. Differences were calculated using the two-tailed Student's t-test. NT, non-tumor area; T, tumor area. (b) IFN-γ, IL-17A, IL-17F, IL-21, IL-22, TNF-α- and IL-6 expression was assessed by real-time PCR in colonic tissues taken from Apcmin/+ mice treated with either DMSO (CTR) or BP-1-102 and killed on day 56. Values are mean±s.e.m. of two independent experiments containing at least three mice per group. Differences were calculated using the two-tailed Student's t-test. ND, not detectable; NT, non-tumor area; T, tumor area.
Mentions: Endoscopy on day 54 showed that control mice developed multiple large tumors, whereas the number and size of tumors were reduced in the colon of BP-1-102-treated mice (Figure 5b). These results were confirmed by direct assessment of tumors in mice killed on day 56 (unpublished data). Proliferating cell nuclear antigen staining confirmed the antiproliferative effect of BP-1-102 (Figure 5c). By contrast, there was no significant change in proliferating cell nuclear antigen staining in the normal colonic mucosa of mice treated with BP-1-102 (Figure 5c). To determine whether the reduced tumorigenesis observed in BP-1-102-treated mice was associated with a reduced activation of STAT3 and/or NF-kB, we compared p-STAT3 Y705 and p-NF-kB/p65 Ser536 expression in non-tumor and tumor tissue derived from colonic extracts of control and BP-1-102-treated mice killed on day 56. Robust activation of both STAT3 and NF-kB was seen in the neoplastic areas of control mice and inhibited by BP-1-102 treatment, whereas p-STAT3 Y705 and p-NF-kB/p65 Ser536 expression was barely detectable in non-tumor areas of both control and BP-1-102-treated mice (Supplementary Figure 7). Immunohistochemistry confirmed reduced activation of both STAT3 and NF-kB in colonic tumor sections of mice treated with BP-1-102 (Figure 6a). IL-17A, IL-21, IL-22, TNF-α and IL-6 transcripts were reduced in the tumor areas of BP-1-102-treated mice, whereas IFN-γ and IL-17F RNA expression remained unchanged (Figure 6b). Moreover, treatment of mice with BP-1-102 did not change IL-11 transcripts in the tumors as well as the fractions of immune cells in TILs (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