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IL-1 β induces IL-6 production and increases invasiveness and estrogen-independent growth in a TG2-dependent manner in human breast cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: We previously reported that IL-6 and transglutaminase 2 (TG2) were expressed in more aggressive basal-like breast cancer cells, and TG2 and IL-6 expression gave these cells stem-cell-like phenotypes, increased invasive ability, and increased metastatic potential. In the present study, the underlying mechanism by which IL-6 production is induced in luminal-type breast cancer cells was evaluated, and TG2 overexpression, IL-1β stimulation, and IL-6 expression were found to give cancerous cells a hormone-independent phenotype.

Methods: Luminal-type breast cancer cells (MCF7 cells) were stably transfected with TG2. To evaluate the requirement for IL-6 neogenesis, MCF7 cells were stimulated with various cytokines. To evaluate tumorigenesis, cancer cells were grown in a three-dimensional culture system and grafted into the mammary fat pads of NOD/scid/IL-2Rγ−/− mice.

Results: IL-1β induced IL-6 production in TG2-expressing MCF7 cells through an NF-kB-, PI3K-, and JNK-dependent mechanism. IL-1β increased stem-cell-like phenotypes, invasiveness, survival in a three-dimensional culture model, and estrogen-independent tumor growth of TG2-expressing MCF7 cells, which was attenuated by either anti-IL-6 or anti-IL-1β antibody treatment.

Conclusion: Within the inflammatory tumor microenvironment, IL-1β increases luminal-type breast cancer cell aggressiveness by stimulating IL-6 production through a TG2-dependent mechanism.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-016-2746-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


IL-1β induced IL-6 production in MCF7_TG2 cells through the IRAK1, NF-kB, JNK, and PI3K signaling pathway. a MCF7_TG2 cells were treated with IL-1β (10 ng/ml) in the presence of IRAK1/4 inhibitor (20 μM), a NF-kB inhibitor (Bay11-7082, 10 μM), a JNK inhibitor (SP600125, 10 μM), an ERK inhibitor (PD98059, 10 μM), a p38 MAPK inhibitor (SB209580, 10 μM), or a PI3K inhibitor (LY294002, 10 μM) for 48 h. IL-6 levels in culture supernatants were measured by ELISA. b MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. Phospho-p65, p65, Ik-Bα, phospho-JNK, and JNK were detected by Western blot. c MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. IRAK1, IRAK2, and TRAF6 were detected by Western blot. d MCF7_Cont and MCF7_TG2 cells were co-transfected with p3kB-Luc and pRL-TK reporter constructs for 24 h then treated with IL-1β (10 ng/nl) for 18 h. All data shown are representative of three independent experiments
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Fig5: IL-1β induced IL-6 production in MCF7_TG2 cells through the IRAK1, NF-kB, JNK, and PI3K signaling pathway. a MCF7_TG2 cells were treated with IL-1β (10 ng/ml) in the presence of IRAK1/4 inhibitor (20 μM), a NF-kB inhibitor (Bay11-7082, 10 μM), a JNK inhibitor (SP600125, 10 μM), an ERK inhibitor (PD98059, 10 μM), a p38 MAPK inhibitor (SB209580, 10 μM), or a PI3K inhibitor (LY294002, 10 μM) for 48 h. IL-6 levels in culture supernatants were measured by ELISA. b MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. Phospho-p65, p65, Ik-Bα, phospho-JNK, and JNK were detected by Western blot. c MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. IRAK1, IRAK2, and TRAF6 were detected by Western blot. d MCF7_Cont and MCF7_TG2 cells were co-transfected with p3kB-Luc and pRL-TK reporter constructs for 24 h then treated with IL-1β (10 ng/nl) for 18 h. All data shown are representative of three independent experiments

Mentions: To evaluate the underlying molecular mechanisms by which TG2- and IL-1β-dependent IL-6 production results in increased stem-cell characteristics, invasiveness, and hormone-independent in vivo tumorigenesis, we evaluated the key signaling pathways downstream of TG2 and IL-1β by utilizing several signaling pathway inhibitors. IL-6 expression from IL-1β stimulated TG2-overexpressing MCF7_TG2 breast cancer cells was inhibited by an IRAK1/4 inhibitor, BAY11-7082 (a NF-kB inhibitor), SP600125 (a JNK inhibitor), and LY294002 (a PI3K inhibitor), but not by PD98059 (an ERK inhibitor) or SB209580 (a p38 mitogen-activated protein kinase inhibitor) (Fig. 5a). Western blot analysis revealed that activated JNK1 kinase, phospho-JNK1 (P-JNK1), levels were increased by IL-1β stimulation in MCF7_TG2 cells compared to MCF7_Cont cells (Fig. 5b). Basal levels of NF-kB signaling molecules, including p65, phospho-p65, and IkBα, were not changed by TG2 overexpression in MCF7 breast cancer cells (Fig. 5b). IL-1β treatment decreased IkB levels and increased phospho-p65 levels within 10 min of IL-1β stimulation. IkBα levels normalized starting at 60 min post treatment, and phospho-p65 levels began to decrease after 120 min in both MCF7_Cont and MCF7_TG2 breast cancer cells (Fig. 5b). The IL-1β treatment-induced IkBα decrease and the phospho-p65 increase were slightly greater in MCF7_TG2 cells compared to MCF7_Cont cells (Fig. 5b). NF-kB activity was measured using a luciferase reporter assay and found to be increased by IL-1β treatment in MCF7_TG2 and MCF7_Cont cells; however, the degree of increase was greater in MCF7_TG2 cells (Fig. 5d). These results suggest that increased activation of NF-kB signaling by IL-1β in the presence of TG2 is necessary to induce IL-6 expression in MCF7 breast cancer cells.Fig. 5


IL-1 β induces IL-6 production and increases invasiveness and estrogen-independent growth in a TG2-dependent manner in human breast cancer cells
IL-1β induced IL-6 production in MCF7_TG2 cells through the IRAK1, NF-kB, JNK, and PI3K signaling pathway. a MCF7_TG2 cells were treated with IL-1β (10 ng/ml) in the presence of IRAK1/4 inhibitor (20 μM), a NF-kB inhibitor (Bay11-7082, 10 μM), a JNK inhibitor (SP600125, 10 μM), an ERK inhibitor (PD98059, 10 μM), a p38 MAPK inhibitor (SB209580, 10 μM), or a PI3K inhibitor (LY294002, 10 μM) for 48 h. IL-6 levels in culture supernatants were measured by ELISA. b MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. Phospho-p65, p65, Ik-Bα, phospho-JNK, and JNK were detected by Western blot. c MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. IRAK1, IRAK2, and TRAF6 were detected by Western blot. d MCF7_Cont and MCF7_TG2 cells were co-transfected with p3kB-Luc and pRL-TK reporter constructs for 24 h then treated with IL-1β (10 ng/nl) for 18 h. All data shown are representative of three independent experiments
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Fig5: IL-1β induced IL-6 production in MCF7_TG2 cells through the IRAK1, NF-kB, JNK, and PI3K signaling pathway. a MCF7_TG2 cells were treated with IL-1β (10 ng/ml) in the presence of IRAK1/4 inhibitor (20 μM), a NF-kB inhibitor (Bay11-7082, 10 μM), a JNK inhibitor (SP600125, 10 μM), an ERK inhibitor (PD98059, 10 μM), a p38 MAPK inhibitor (SB209580, 10 μM), or a PI3K inhibitor (LY294002, 10 μM) for 48 h. IL-6 levels in culture supernatants were measured by ELISA. b MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. Phospho-p65, p65, Ik-Bα, phospho-JNK, and JNK were detected by Western blot. c MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. IRAK1, IRAK2, and TRAF6 were detected by Western blot. d MCF7_Cont and MCF7_TG2 cells were co-transfected with p3kB-Luc and pRL-TK reporter constructs for 24 h then treated with IL-1β (10 ng/nl) for 18 h. All data shown are representative of three independent experiments
Mentions: To evaluate the underlying molecular mechanisms by which TG2- and IL-1β-dependent IL-6 production results in increased stem-cell characteristics, invasiveness, and hormone-independent in vivo tumorigenesis, we evaluated the key signaling pathways downstream of TG2 and IL-1β by utilizing several signaling pathway inhibitors. IL-6 expression from IL-1β stimulated TG2-overexpressing MCF7_TG2 breast cancer cells was inhibited by an IRAK1/4 inhibitor, BAY11-7082 (a NF-kB inhibitor), SP600125 (a JNK inhibitor), and LY294002 (a PI3K inhibitor), but not by PD98059 (an ERK inhibitor) or SB209580 (a p38 mitogen-activated protein kinase inhibitor) (Fig. 5a). Western blot analysis revealed that activated JNK1 kinase, phospho-JNK1 (P-JNK1), levels were increased by IL-1β stimulation in MCF7_TG2 cells compared to MCF7_Cont cells (Fig. 5b). Basal levels of NF-kB signaling molecules, including p65, phospho-p65, and IkBα, were not changed by TG2 overexpression in MCF7 breast cancer cells (Fig. 5b). IL-1β treatment decreased IkB levels and increased phospho-p65 levels within 10 min of IL-1β stimulation. IkBα levels normalized starting at 60 min post treatment, and phospho-p65 levels began to decrease after 120 min in both MCF7_Cont and MCF7_TG2 breast cancer cells (Fig. 5b). The IL-1β treatment-induced IkBα decrease and the phospho-p65 increase were slightly greater in MCF7_TG2 cells compared to MCF7_Cont cells (Fig. 5b). NF-kB activity was measured using a luciferase reporter assay and found to be increased by IL-1β treatment in MCF7_TG2 and MCF7_Cont cells; however, the degree of increase was greater in MCF7_TG2 cells (Fig. 5d). These results suggest that increased activation of NF-kB signaling by IL-1β in the presence of TG2 is necessary to induce IL-6 expression in MCF7 breast cancer cells.Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: We previously reported that IL-6 and transglutaminase 2 (TG2) were expressed in more aggressive basal-like breast cancer cells, and TG2 and IL-6 expression gave these cells stem-cell-like phenotypes, increased invasive ability, and increased metastatic potential. In the present study, the underlying mechanism by which IL-6 production is induced in luminal-type breast cancer cells was evaluated, and TG2 overexpression, IL-1β stimulation, and IL-6 expression were found to give cancerous cells a hormone-independent phenotype.

Methods: Luminal-type breast cancer cells (MCF7 cells) were stably transfected with TG2. To evaluate the requirement for IL-6 neogenesis, MCF7 cells were stimulated with various cytokines. To evaluate tumorigenesis, cancer cells were grown in a three-dimensional culture system and grafted into the mammary fat pads of NOD/scid/IL-2Rγ−/− mice.

Results: IL-1β induced IL-6 production in TG2-expressing MCF7 cells through an NF-kB-, PI3K-, and JNK-dependent mechanism. IL-1β increased stem-cell-like phenotypes, invasiveness, survival in a three-dimensional culture model, and estrogen-independent tumor growth of TG2-expressing MCF7 cells, which was attenuated by either anti-IL-6 or anti-IL-1β antibody treatment.

Conclusion: Within the inflammatory tumor microenvironment, IL-1β increases luminal-type breast cancer cell aggressiveness by stimulating IL-6 production through a TG2-dependent mechanism.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-016-2746-7) contains supplementary material, which is available to authorized users.

No MeSH data available.