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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.


Related in: MedlinePlus

Three-dimensional culture resulted in dramatically enhanced survival of luminal-type breast cancer cells in a TG2-dependent manner. a MCF7_Cont and MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml). b MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml) and anti-IL-6 monoclonal antibody (10 μg/ml). c MCF7_Cont and MCF7_TG2 cells (1 × 106 cells/each mouse) were injected into the fat pads of NSG mice. Primary tumor growth was measured. Blocking anti-IL-6 antibody (100 μg/mouse) or blocking anti-IL-1β antibody (100 μg/mouse) was injected intraperitoneally every third day, starting 1 day after tumor inoculation. The TG2 inhibitor cysteamine (CyM, 40 mg/kg/day) was injected intraperitoneally starting 1 day after tumor inoculation. Data are given as mean ± SEM of six mice for each group
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Fig4: Three-dimensional culture resulted in dramatically enhanced survival of luminal-type breast cancer cells in a TG2-dependent manner. a MCF7_Cont and MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml). b MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml) and anti-IL-6 monoclonal antibody (10 μg/ml). c MCF7_Cont and MCF7_TG2 cells (1 × 106 cells/each mouse) were injected into the fat pads of NSG mice. Primary tumor growth was measured. Blocking anti-IL-6 antibody (100 μg/mouse) or blocking anti-IL-1β antibody (100 μg/mouse) was injected intraperitoneally every third day, starting 1 day after tumor inoculation. The TG2 inhibitor cysteamine (CyM, 40 mg/kg/day) was injected intraperitoneally starting 1 day after tumor inoculation. Data are given as mean ± SEM of six mice for each group

Mentions: To evaluate the biological behavior of TG2 overexpression and IL-1β stimulation in breast cancer cells, a two-dimensional (2D) matrigel invasion assay was performed. MCF7_TG2 cells showed increased invasiveness compared to MCF7_Cont cells, and IL-1β treatment further increased the invasiveness of MCF7_TG2 breast cancer cells (Fig. 3a and b). Increased invasion of MCF7_TG2 breast cancer cells by IL-1β was attenuated by blocking IL-6 through anti-IL-6 antibody treatment (Fig. 3c and d). A 3D matrigel on top assay also revealed the synergistic effects of TG2 overexpression and IL-1β treatment on the invasion of MCF7 breast cancer cells. MCF7_TG2 breast cancer cells grew more rapidly and formed a large spheroid in the 3D matrigel compared to MCF7_Cont cells, and IL-1β treatment further increased growth and conferred invasiveness and budding-like phenomena in MCF7_TG2 cells (Fig. 4a). Again, these aggressive phenotypes were ameliorated by anti-IL-6 antibody treatment (Fig. 4b). Moreover, an in vivo tumorigenesis assay in NSG mice revealed that, unlike estrogen-dependent MCF7_Cont cells, MCF7_TG2 breast cancer cells obtained tumorigenic capability in vivo without the addition of exogenous estrogen, which were reduced in the presence of blocking anti-IL-6 or anti-IL-1β antibodies or the TG2 inhibitor cysteamine (CyM) (Fig. 4c).Fig. 3


IL-1 β induces IL-6 production and increases invasiveness and estrogen-independent growth in a TG2-dependent manner in human breast cancer cells
Three-dimensional culture resulted in dramatically enhanced survival of luminal-type breast cancer cells in a TG2-dependent manner. a MCF7_Cont and MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml). b MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml) and anti-IL-6 monoclonal antibody (10 μg/ml). c MCF7_Cont and MCF7_TG2 cells (1 × 106 cells/each mouse) were injected into the fat pads of NSG mice. Primary tumor growth was measured. Blocking anti-IL-6 antibody (100 μg/mouse) or blocking anti-IL-1β antibody (100 μg/mouse) was injected intraperitoneally every third day, starting 1 day after tumor inoculation. The TG2 inhibitor cysteamine (CyM, 40 mg/kg/day) was injected intraperitoneally starting 1 day after tumor inoculation. Data are given as mean ± SEM of six mice for each group
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Fig4: Three-dimensional culture resulted in dramatically enhanced survival of luminal-type breast cancer cells in a TG2-dependent manner. a MCF7_Cont and MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml). b MCF7_TG2 cells were grown in 3D culture conditions in the presence or absence of IL-1β (10 ng/ml) and anti-IL-6 monoclonal antibody (10 μg/ml). c MCF7_Cont and MCF7_TG2 cells (1 × 106 cells/each mouse) were injected into the fat pads of NSG mice. Primary tumor growth was measured. Blocking anti-IL-6 antibody (100 μg/mouse) or blocking anti-IL-1β antibody (100 μg/mouse) was injected intraperitoneally every third day, starting 1 day after tumor inoculation. The TG2 inhibitor cysteamine (CyM, 40 mg/kg/day) was injected intraperitoneally starting 1 day after tumor inoculation. Data are given as mean ± SEM of six mice for each group
Mentions: To evaluate the biological behavior of TG2 overexpression and IL-1β stimulation in breast cancer cells, a two-dimensional (2D) matrigel invasion assay was performed. MCF7_TG2 cells showed increased invasiveness compared to MCF7_Cont cells, and IL-1β treatment further increased the invasiveness of MCF7_TG2 breast cancer cells (Fig. 3a and b). Increased invasion of MCF7_TG2 breast cancer cells by IL-1β was attenuated by blocking IL-6 through anti-IL-6 antibody treatment (Fig. 3c and d). A 3D matrigel on top assay also revealed the synergistic effects of TG2 overexpression and IL-1β treatment on the invasion of MCF7 breast cancer cells. MCF7_TG2 breast cancer cells grew more rapidly and formed a large spheroid in the 3D matrigel compared to MCF7_Cont cells, and IL-1β treatment further increased growth and conferred invasiveness and budding-like phenomena in MCF7_TG2 cells (Fig. 4a). Again, these aggressive phenotypes were ameliorated by anti-IL-6 antibody treatment (Fig. 4b). Moreover, an in vivo tumorigenesis assay in NSG mice revealed that, unlike estrogen-dependent MCF7_Cont cells, MCF7_TG2 breast cancer cells obtained tumorigenic capability in vivo without the addition of exogenous estrogen, which were reduced in the presence of blocking anti-IL-6 or anti-IL-1β antibodies or the TG2 inhibitor cysteamine (CyM) (Fig. 4c).Fig. 3

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