Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer

Abstract

Introduction: Signal transducer and activator of transcription 3 (Stat3) is constitutively tyrosine-phosphorylated in approximately 50% of primary breast carcinomas. A number of different mechanisms responsible for Stat3 activation, including abnormal activation of receptor tyrosine kinases, Src, and Janus kinases (Jaks), have been implicated in breast cancer.

Methods: We examined six breast cancer-derived cell lines expressing high or low levels of tyrosine-phosphorylated Stat3 (pStat3) as well as primary breast cancer specimens.

Results: Inhibition of Src or EGFR (epidermal growth factor receptor) tyrosine kinases had no effect on pStat3 levels, whereas pan-Jak inhibitor P6 resulted in complete abrogation of Stat3 phosphorylation and inhibition of growth. Jaks are required for cytokine signaling, and the glycoprotein 130 (gp130) receptor-associated Jaks are known mediators of Stat3 phosphorylation. Blockade of the gp130 receptor or sequestration of the interleukin-6 (IL-6) ligand led to a decrease of pStat3 levels. Conditioned media from those cell lines expressing high levels of pStat3 contained IL-6 and were capable of stimulating Stat3 phosphorylation. We examined IL-6 levels in primary breast tumors and found a positive correlation between pStat3 and IL-6 expression.

Conclusion: In summary, a principal mechanism of Stat3 activation in breast cancer is through the IL-6/gp130/Jak pathway.

Figure 1

Signal transducer and activator of transcription 3 (Stat3) phosphorylation in primary breast cancer. Tissue microarrays of primary breast tumors (85) were analyzed for nuclear tyrosine-phosphorylated Stat3 (pStat3) by immunohistochemical analysis. Ten percent expressed high levels (3+), 36% moderate levels (+2), 23% low levels (+1), and 31% no detectable pStat3 (0).

Figure 2

Differential signal transducer and activator of transcription 3 (Stat3) phosphorylation in breast cancer-derived cell lines. Nuclear extracts (20 μg) isolated from MCF10A, MCF7, BT474, MDA-MB-435, MDA-MB-MD-468, and MDA-MB-MD-231 cell lines were analyzed by Western blotting with tyrosine-phosphorylated Stat3 (pStat3) and Stat3 antibodies.

Figure 3

Signal transducer and activator of transcription 3 (Stat3) phosphorylation is mediated through Janus kinases (Jaks) but not epidermal growth factor receptor (EGFR) or Src kinases. (a) Radioimmunoprecipitation assay (RIPA) extracts (50 μg) isolated from MDA-MB-468 cells treated for 4 hours with dimethyl sulfoxide (DMSO), P6 1 μM, BMS 50 nM, and ZD 2 μM were analyzed for pEGFR, total EGFR, tyrosine-phosphorylated Stat3 (pStat3), Stat3, pSrc, Src, pJak2, and Jak2. (b) Nuclear extracts (20 μg) isolated from MDA-MB-435 cells (upper panels) and RIPA extracts (bottom panels) treated as in (a) and probed with the indicated antibodies. (c) Nuclear extracts (20 μg) isolated from MDA-MB-231 cells (upper panels) and RIPA extracts (bottom panels) treated as in (a) and probed with the indicated antibodies. (d) Ten thousand cells (MCF10A, MCF7, MDA-MB-468, and MDA-MB-435) were plated into a 96-well culture dish and treated with DMSO control or P6 (1 μM) for 48 hours, and proliferation was measured using an MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.

Figure 4

Signal transducer and activator of transcription 3 (Stat3) activation is through the interleukin-6/glycoprotein 130/Janus kinase (IL-6/gp130/Jak) pathway. (a) Nuclear extracts isolated from MDA-MB-468 (468) cells treated with immunoglobulin G (IgG) (10 μg/ml), P6 (1 μM), BR3 (10 μg/ml), and α-IL-6 (1 μg/ml) for 16 hours were analyzed for tyrosine-phosphorylated Stat3 (pStat3) and total Stat3 protein. (b) Nuclear extracts isolated from MDA-MB-231 (231) cells treated with IgG (10 μg/ml), P6 (1 μM), BR3 (10 μg/ml), and α-IL-6 (1 μg/ml) for 16 hours were analyzed for pStat3 and total Stat3. (c) Nuclear extracts isolated from MDA-MB-435 (435) cells treated with IgG (10 μg/ml), P6 (1 μM), and BR3 (10 μg/ml) for 16 hours were analyzed for pStat3 and total Stat3. (d) Nuclear extracts isolated from 4175 and 231 cells treated with IgG (10 μg/ml) and BR3 (10 μg/ml) for 16 hours were analyzed for pStat3 and total Stat3. (e) Nuclear extracts isolated from MCF10A cells treated for 30 minutes with conditioned media (CM) from MDA-MB-468 cells after pretreatment for 4 hours with IgG control antibody, BR3 (5 μg/ml), α-IL-6 antibody (5 μg/ml), α-oncostatin M (OSM) antibody (10 μg/ml), and α-leukemia inhibitory factor (LIF) antibody (10 μg/ml) were analyzed for pStat3 and total Stat3.

Figure 5

Immunohisotchemical analaysis of primary breast cancer specimens for interleukin-6 (IL-6) (upper panels) and tyrosine-phosphorylated signal transducer and activator of transcription 3 (pStat3) (bottom panels). Examples of high pStat3 and IL-6 (left panels) as well as low pStat3 and IL-6 (right panels) are indicated.