STAT6 mediates interleukin-4 growth inhibition in human breast cancer cells

Abstract

In addition to acting as a hematopoietic growth factor, interleukin-4 (IL-4) inhibits growth of some transformed cells in vitro and in vivo. In this study, we show that insulin receptor substrate (IRS)-1, IRS-2, and signal transducer and activator of transcription 6 (STAT6) are phosphorylated following IL-4 treatment in MCF-7 breast cancer cells. STAT6 DNA binding is enhanced by IL-4 treatment. STAT6 activation occurs even after IRS-1 depletion, suggesting the two pathways are independent. To examine the role of STAT6 in IL-4-mediated growth inhibition and apoptosis, a full-length STAT6 cDNA was transfected into MCF-7 cells. Transient overexpression of STAT6 resulted in both cytoplasmic and nuclear expression of the protein, increased DNA binding in response to IL-4, and increased transactivation of an IL-4 responsive promoter. In STAT6-transfected cells, basal proliferation was reduced whereas apoptosis was increased. Finally, stable expression of STAT6 resulted in reduced foci formation compared to vector-transfected cells alone. These results suggest STAT6 is required for IL-4-mediated growth inhibition and induction of apoptosis in human breast cancer cells.

Figure 1

IL-4 treatment results in phosphorylation of IRS-1, IRS-2, and STAT6 in MCF-7 breast cancer cells. (A) MCF-7 cells were untreated (-) or treated with IL-4 (10 ng/ml) (+) for 10 minutes. Total cell lysates (total) or immunoprecipitate lysates (IRS-1, IRS-2, and STAT6) separated by SDS-PAGE and then immunoblotted with anti-phosphotyrosine antibody. Total cell lysates were also immunoblotted with antibodies to IRS-1, IRS-2, and STAT6 to show the amount of representative protein in the lysates (lower panels). Data shown are representative of three independent experiments. (B) To degrade IRS-1, MCF-7 cells were incubated in SFM in the absence (-) or presence (+) of IGF-I for 24 hours. Cells were then treated with IL-4 (10 ng/ml) for 10 minutes and levels of IRS-1 and STAT6 in total cell lysates were determined by immunoblotting. Phosphorylated STAT6 was detected by immunoprecipitation with anti-STAT6 antibody followed by anti-phosphotyrosine immunoblotting. Data shown are representative of three independent experiments.

Figure 2

STAT6 DNA binding activity is induced by IL-4 in breast cancer cell lines. (A) MCF-7 cell nuclear extracts were incubated with labeled probe corresponding to the STAT6 binding site in the Fcγ1 gene promoter. Anti-STAT6 or irrelevant antibodies (anti-ER) were added to the same reactions to produce a supershifted complex. (B) Breast cancer cells were treated with IL-4 (10 ng/ml) for 30 minutes. Nuclear extracts were isolated and 10 µg of protein were incubated with labeled probe. Data shown are representative of three independent experiments.

Figure 3

STAT6 overexpression increases IL-4-mediated DNA binding and promoter activity. (A) STAT6 expression was detected by immunofluorescence with anti-STAT6 antibody followed by fluorescein-conjugated anti-mouse secondary antibody in untransfected MCF-7 cells (a) and cells transfected with pcDNA (b) or mSTAT6 (c). Cells overexpressing mSTAT6 are indicated with arrows. Data shown are representative of three independent experiments. Equal magnification (40x) is shown in each panel. (B) MCF-7 cells transiently transfected with 10 µg pcDNA or mSTAT6 were treated with increasing amounts of IL-4. Nuclear extracts were prepared and then 10 µg of each nuclear extract was incubated with labeled probe. Data shown are representative of two independent experiments. (C) COS-7 cells were transfected in triplicate with pKB350luc, β-gal expression construct, and vector alone (pcDNA), wild-type mSTAT6, or antisense mSTAT6 (asSTAT6). After 12 hours, media was changed and cells were incubated in the absence (-) or presence (+) of IL-4 (50 ng/ml). Luciferase and β-gal activities were determined 24 hours later. Results are expressed as relative luciferase activity, which is defined as luciferase divided by β-gal activity in the same sample. Error bars represent the mean of triplicate samples±SEM. (D) Increasing amounts of mSTAT6 were transfected in triplicate into COS-7 cells treated with IL-4 (50 ng/ml). Luciferase was measured and normalized to β-gal activity in the same samples. Error bars represent the mean of triplicate samples±SEM. Data shown are representative of three independent experiments.

Figure 4

STAT6 mediates growth inhibition and apoptosis in MCF-7 cells. (A) MCF-7 cells were transfected with pcDNA or mSTAT6 along with β-gal expression construct as an indicator of transfection in a 10:1 ratio. Cells were treated with IL-4 for 48 hours, fixed with 3.7% formaldehyde, and then stained with X-gal to identify transfected cells. Colonies of blue cells were counted as a measure of proliferation of transfected cells. Error bars represent the mean of three independent experiments±SEM. **P<0.001 (two-way ANOVA) for mSTAT6 control versus pcDNA control. (B) Cells were transfected with pcDNA, mSTAT6, or ΔSTAT6(645) as described in (A) and the percentage of blue cells that had apoptotic morphology was measured. Error bars represent the mean of three independent experiments±SEM. (C) MCF-7 cells were transfected with pcDNA, mSTAT6, and then cultured in the presence of neomycin for 3 weeks. Resulting foci were stained with 0.1% crystal violet and then counted in triplicate. Error bars represent the mean of triplicate counts±SEM. Data shown are representative of three independent experiments. *P< 0.05 (Student's t-test).