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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

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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.


Related in: MedlinePlus

IL-1β induced IL-6 production from breast cancer cells in a TG2-dependent manner. a TG2-overexpressing MCF7 cells (TG2) and control vector-transfected MCF-7 cells (Cont) were treated with various cytokines (10 ng/ml) for 48 h and IL-6 levels in culture supernatants were measured by ELISA. b Cells were treated with IL-1β (10 ng/ml) in the presence of TGFβ (10 ng/ml), EGF (10 ng/ml), or TNFα (10 ng/ml) for 48 h and secreted IL-6 levels in culture supernatants were measured by ELISA. c Cells were treated with IL-1β (10 ng/ml) for the indicated times. d Cells were treated with IL-1β at various concentrations for 48 h. a-d All data shown are representative of three independent experiments. Data are presented as mean ± SD
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Fig2: IL-1β induced IL-6 production from breast cancer cells in a TG2-dependent manner. a TG2-overexpressing MCF7 cells (TG2) and control vector-transfected MCF-7 cells (Cont) were treated with various cytokines (10 ng/ml) for 48 h and IL-6 levels in culture supernatants were measured by ELISA. b Cells were treated with IL-1β (10 ng/ml) in the presence of TGFβ (10 ng/ml), EGF (10 ng/ml), or TNFα (10 ng/ml) for 48 h and secreted IL-6 levels in culture supernatants were measured by ELISA. c Cells were treated with IL-1β (10 ng/ml) for the indicated times. d Cells were treated with IL-1β at various concentrations for 48 h. a-d All data shown are representative of three independent experiments. Data are presented as mean ± SD

Mentions: In our previous report, expression of TG2 and expression of IL-6 were found to correlate with one another, and TG2 was found to promote aggressive phenotypes in breast cancer cells through IL-6. A knockdown (KD) of TG2 in MDA-MB-231 breast cancer cells reduced IL-6 expression, and a knockdown of both TG2 and IL-6 inhibited tumor growth and metastasis [14]. In contrast to our expectations, simple overexpression of TG2 in otherwise TG2- and IL-6-negative luminal-type breast cancer MCF7 cells did not lead to IL-6 expression (Fig. 2a). The behavior and gene expression of cancer cells are affected by the microenvironment surrounding the tumor, and this environment includes cytokines and growth factors released by stromal cells such as leukocytes and fibroblasts. To evaluate the effect of paracrine signals, MCF7 cells were treated with IL-1β, TNF-α, TGF-β, and EGF. The results show that IL-1β induced expression of IL-6 in breast cancer cells, and that TG2 overexpressing cells expressed over twenty times more than control cells after IL-1β treatment. Treating cells with TGF-β or EGF alone did not increase IL-6, but TNF-α treatment slightly increased IL-6 expression (Fig. 2a). Treatment with TGF-β, EGF, and TNF-α after IL-1β further increased IL-6 expression in MCF7_TG2 breast cancer cells (Fig. 2b). Other inflammatory/immune-stimulating reagents, including lipopolysaccharide (LPS), Pam3Cys (Pam), peptidoglycan (PGN), CpG, and bleomycin (BLM), did not induce IL-6 expression in either MCF7_Cont or MCF7_TG2 breast cancer cells (Additional file 1: Figure S2).Fig. 2


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 from breast cancer cells in a TG2-dependent manner. a TG2-overexpressing MCF7 cells (TG2) and control vector-transfected MCF-7 cells (Cont) were treated with various cytokines (10 ng/ml) for 48 h and IL-6 levels in culture supernatants were measured by ELISA. b Cells were treated with IL-1β (10 ng/ml) in the presence of TGFβ (10 ng/ml), EGF (10 ng/ml), or TNFα (10 ng/ml) for 48 h and secreted IL-6 levels in culture supernatants were measured by ELISA. c Cells were treated with IL-1β (10 ng/ml) for the indicated times. d Cells were treated with IL-1β at various concentrations for 48 h. a-d All data shown are representative of three independent experiments. Data are presented as mean ± SD
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Fig2: IL-1β induced IL-6 production from breast cancer cells in a TG2-dependent manner. a TG2-overexpressing MCF7 cells (TG2) and control vector-transfected MCF-7 cells (Cont) were treated with various cytokines (10 ng/ml) for 48 h and IL-6 levels in culture supernatants were measured by ELISA. b Cells were treated with IL-1β (10 ng/ml) in the presence of TGFβ (10 ng/ml), EGF (10 ng/ml), or TNFα (10 ng/ml) for 48 h and secreted IL-6 levels in culture supernatants were measured by ELISA. c Cells were treated with IL-1β (10 ng/ml) for the indicated times. d Cells were treated with IL-1β at various concentrations for 48 h. a-d All data shown are representative of three independent experiments. Data are presented as mean ± SD
Mentions: In our previous report, expression of TG2 and expression of IL-6 were found to correlate with one another, and TG2 was found to promote aggressive phenotypes in breast cancer cells through IL-6. A knockdown (KD) of TG2 in MDA-MB-231 breast cancer cells reduced IL-6 expression, and a knockdown of both TG2 and IL-6 inhibited tumor growth and metastasis [14]. In contrast to our expectations, simple overexpression of TG2 in otherwise TG2- and IL-6-negative luminal-type breast cancer MCF7 cells did not lead to IL-6 expression (Fig. 2a). The behavior and gene expression of cancer cells are affected by the microenvironment surrounding the tumor, and this environment includes cytokines and growth factors released by stromal cells such as leukocytes and fibroblasts. To evaluate the effect of paracrine signals, MCF7 cells were treated with IL-1β, TNF-α, TGF-β, and EGF. The results show that IL-1β induced expression of IL-6 in breast cancer cells, and that TG2 overexpressing cells expressed over twenty times more than control cells after IL-1β treatment. Treating cells with TGF-β or EGF alone did not increase IL-6, but TNF-α treatment slightly increased IL-6 expression (Fig. 2a). Treatment with TGF-β, EGF, and TNF-α after IL-1β further increased IL-6 expression in MCF7_TG2 breast cancer cells (Fig. 2b). Other inflammatory/immune-stimulating reagents, including lipopolysaccharide (LPS), Pam3Cys (Pam), peptidoglycan (PGN), CpG, and bleomycin (BLM), did not induce IL-6 expression in either MCF7_Cont or MCF7_TG2 breast cancer cells (Additional file 1: Figure S2).Fig. 2

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.


Related in: MedlinePlus