STAT3 can be activated through paracrine signaling in breast epithelial cells - PubMed (original) (raw)

STAT3 can be activated through paracrine signaling in breast epithelial cells

Jacqueline C Lieblein et al. BMC Cancer. 2008.

Abstract

Background: Many cancers, including breast cancer, have been identified with increased levels of phosphorylated or the active form of Signal Transducers and Activators of Transcription 3 (STAT3) protein. However, whether the tumor microenvironment plays a role in this activation is still poorly understood.

Methods: Conditioned media, which contains soluble factors from MDA-MB-231 and MDA-MB-468 breast cancer cells and breast cancer associated fibroblasts, was added to MCF-10A breast epithelial and MDA-MB-453 breast cancer cells. The stimulation of phosphorylated STAT3 (p-STAT3) levels by conditioned media was assayed by Western blot in the presence or absence of neutralized IL-6 antibody, or a JAK/STAT3 inhibitor, JSI-124. The stimulation of cell proliferation in MCF-10A cells by conditioned media in the presence or absence of JSI-124 was subjected to MTT analysis. IL-6, IL-10, and VEGF levels were determined by ELISA analysis.

Results: Our results demonstrated that conditioned media from cell lines with constitutively active STAT3 are sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels. This signaling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and is persistent for at least 24 hours. ELISA analysis confirmed a correlation between elevated levels of IL-6 production and p-STAT3. Neutralization of the IL-6 ligand or gp130 was sufficient to block increased levels of p-STAT3 (Y705) in treated cells. Furthermore, soluble factors within the MDA-MB-231 conditioned media were also sufficient to stimulate an increase in IL-6 production from MCF-10A cells.

Conclusion: These results demonstrate STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signaling through soluble factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The induction of STAT3 phosphorylation is through the IL-6/JAK pathway and appears to be associated with cell proliferation. Understanding how IL-6 and other soluble factors may lead to STAT3 activation via the tumor microenvironment will provide new therapeutic regimens for breast carcinomas and other cancers with elevated p-STAT3 levels.

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Figures

Figure 1

Figure 1

Soluble factor(s) secreted by MDA-MB-231, MDA-MB-468, and breast cancer associated fibroblasts are sufficient to amplify p-STAT3 (Y705) levels in MDA-MB-453 and MCF-10A. A. MDA-MB-453 cells were co-cultured with MDA-MB-468 cells for 120 hours. B. MCF-10A cells were treated with MDA-MB-468 conditioned media for 6 and 24 hours. C. MCF-10A cells were treated with MDA-MB-231 conditioned media for 0.5 hours. D. MDA-MB-453 cells were treated with MDA-MB-231 conditioned media for various time points between 0.5 and 24 hours. E. MCF-10A cells were treated with MDA-MB-231 conditioned media for 1, 24, and 48 hours. F. MDA-MB-453 cells were treated with conditioned media from breast cancer associated fibroblasts for 0.5 hours. 453: MDA-MB-453; 468: MDA-MB-468; 231: MDA-MB-231; BCF: breast cancer associated fibroblasts; No Tx: no treatment; CM: conditioned media; hr: hour.

Figure 2

Figure 2

Secretion of VEGF, IL-10, and IL-6 cytokines by various breast cell lines. A. VEGF secretion by breast cell lines after 24 hours. VEGF production does not correlate with p-STAT3 levels. B. IL-10 secretion by breast cell lines after 24 hours. IL-10 production does not correlate with p-STAT3 levels. C. IL-6 secretion by breast cell lines after 48 hours. p-STAT3-positive cell lines secrete high levels of IL-6 and cell lines that do not express constitutive p-STAT3 have low levels of IL-6 secretion. Cell lines that do not express constitutive p-STAT3: MDA-MB-453 and MCF-10A; p-STAT3-positive cells: MDA-MB-468, MDA-MB-231, and breast cancer associated fibroblasts (BCF).

Figure 3

Figure 3

Soluble factor(s) from MDA-MB-231 conditioned media were able to confer increased growth rates in MCF-10A. A. MCF-10A cells were treated with MDA-MB-231 conditioned media and analyzed by MTT assay for cell viability. B. MCF-10A cells were treated with MDA-MB-231 conditioned media and/or 5 μM JSI-124. C. MCF-10A cells were treated with MDA-MB-231 conditioned media for varying amounts of time. Cell viability was analyzed by MTT assay. 231: MDA-MB-231; No Tx: no treatment; CM: conditioned media.

Figure 4

Figure 4

Activation of STAT3 occurs through the JAK/STAT3 signaling pathway. A. MCF-10A cells were treated with MDA-MB-468 conditioned media for 0.5 hours. Treatment with 10 μM JSI-124 one hour prior to treatment with conditioned media reduced STAT3 activation. B. MCF-10A cells were treated with MDA-MB-231 conditioned media for 0.5 hours. Treatment with 10 μM JSI-124 one hour prior to treatment with conditioned media reduced STAT3 activation. C. MDA-MB-453 cells were treated with MDA-MB-231 conditioned media for 0.5 hours. Treatment with 10 μM JSI-124 one hour prior to treatment with conditioned media reduced STAT3 activation. 468: MDA-MB-468; 231: MDA-MB-231; 453: MDA-MB-453; No Tx: no treatment; CM: conditioned media.

Figure 5

Figure 5

Addition of IL-6 is sufficient to increase levels of p-STAT3 in MDA-MB-453 and MCF-10A cell lines. A. MDA-MB-453 cells were treated with 50 ng/ml IL-6 for 30 minutes. B. MCF-10A cells were treated with 25 ng/ml IL-6 for 30 minutes.

Figure 6

Figure 6

Inhibition of IL-6 or gp130 is sufficient to block increased levels of p-STAT3 (Y705) in MDA-MB-453. A. MDA-MB-231 conditioned media was subjected to 4 μg/ml IL-6, gp130, or GAPDH antibody neutralization. This neutralized conditioned media was then used to treat MDA-MB-453 cells for 0.5 hours. B. BCF conditioned media was subjected to 4 μg/ml IL-6, gp130, or GAPDH antibody neutralization. This neutralized conditioned media was then used to treat MDA-MB-453 cells for 0.5 hours. 453: MDA-MB-453; 231: MDA-MB-231; BCF: breast cancer associated fibroblasts; No Tx: no treatment; CM: conditioned media.

Figure 7

Figure 7

Soluble factor(s) from MDA-MB-231 stimulate MCF-10A to increase production of IL-6. A. MCF-10A cells were treated with MDA-MB-231 conditioned media for 24, 48, or 96 hours. After this treatment time, MCF-10A cells were washed and allowed to produce their own cytokines for 24 hours, after which ELISA analysis was performed to assay for IL-6 secretion. For the 0 hour treatment, conditioned media was added, but was washed away immediately and collected for analysis. This was done to show that any soluble factors from the MDA-MB-231 conditioned media were washed away and were not contaminating our results. For untreated MCF-10A cells, the cells were mock-treated with fresh 2% FBS, 1× DMEM instead. B. To confirm that soluble factors from the MDA-MB-231 conditioned media were inducing MCF-10A to produce IL-6, RNA collected from these cells and used to perform non-quantitative RT-PCR. RNA from MDA-MB-231 was used as a positive control. 231: MDA-MB-231; No Tx: no treatment; CM: conditioned media.

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