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Stat6 cooperates with Sp1 in controlling breast cancer cell proliferation by modulating the expression of p21Cip1/WAF1 and p27Kip1

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Abstract

Background

The signal transducer and activator of transcription 6 (Stat6), a member of the family of DNA-binding proteins, has been identified as a critical cell differentiation modulator in breast cancer cells. As of yet, the mechanisms underlying this function have remained largely unknown. To further elucidate the role of Stat6 in breast cancer development, we investigated the consequences of exogenous Stat6 expression.

Methods

Proliferation assays and flow cytometry assays were conducted to evaluate the putative role of Stat6 on cell proliferation. To this end, we produced synchronized cells after a double thymidine block, as confirmed by FACS analysis. mRNA levels of Stat6 were measured by RNase protection analysis. To confirm the interaction among proteins, we employed GST pull-down assays and immunoprecipitation assays. Luciferase assays and ChIP assays were used to assess the transcriptional activity.

Results

Compared to control breast cancer cells, we found that exogenous Stat6 expression plays a critical role in controlling cell proliferation. Also in different breast tumor cell lines, endogenous Stat6 expression was found to be positively related to a lower proliferation rate. Interestingly, in human breast cancer cells Stat6 functions in G1/S cell cycle progression, and the growth-inhibitory effect of Stat6 was shown to be mediated by induction of the G1 cyclin-dependent kinase inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Simultaneously, G1-related cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. Stat6 knockdown resulted in enhanced cell proliferation and a decrease in p21 and p27 mRNA levels in the steroid-responsive and non-responsive T-47D and MDA-MB-231 cell lines, respectively. In addition, the stimulatory effect of Stat6 on p21 and p27 gene transcription was found to be associated with interaction of Stat6 with the transcription factor Sp1 at the proximal Sp1-binding sites in their respective promoters.

Conclusions

Together, these results identify Stat6 as an important cell differentiation regulatory protein functioning, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters in breast cancer cells.

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Acknowledgements

This study was supported in part by grant funding of NSFC (Natural Science Foundation of China, 81101847), Doctoral Fund of Ministry of Education of China (20110073120089), project supported by the Shanghai Committee of Science and Technology, China (124119a4801) and NUST Research Funding (NO.2010ZYTS068).

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Authors and Affiliations

  1. Breast Department, International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai, 200030, People’s Republic of China

    Min Wei

  2. Key Laboratory of Shanghai Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200025, People’s Republic of China

    Bingya Liu, Qinlong Gu, Liping Su, Yingyan Yu & Zhenggang Zhu

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  1. Min Wei
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Correspondence to Min Wei.

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SupplementaryFigure 1

The qualifications of the intensity of pRB in Fig. 3b (JPEG 2 kb)

High resolution image (TIFF 206 kb)

SupplementaryFigure 2

The qualifications of P16 of Fig. 4a (JPEG 20 kb)

High resolution image (TIFF 401 kb)

SupplementaryFigure 3

The qualifying of the molecular involved in ChIP assays in Fig. 5d (JPEG 10 kb)

High resolution image (TIFF 496 kb)

SupplementaryFigure 4

Similar results of ChIP assays have been derived from human breast cancer cell line, MCF-7. (JPEG 8 kb)

High resolution image (TIFF 750 kb)

SupplementaryFigure 5

Studies on the putative Stat6 binding site (TTC(N)3GAA) by using luciferase plasmids carrying mutants for this site showed that none obvious reduction of luciferase activity were detected in transfectants of Stat6 mutants compared with control. Stat6-w, wild type of Stat6. Stat6-M, Mutant of Stat6. (JPEG 117 kb)

High resolution image (TIFF 1283 kb)

SupplementaryFigure 6

A. Full-length Sp1 and Sp1 fragments fused to GST. Schematic diagram of Sp1 and Sp1 deletion mutants used in the GST pull-down assay is shown in the top panel. The letters A to D indicate different domains of the Sp1 protein. Three zinc finger DNA binding motifs of Sp1 presented in the C-terminal region are shown. The numbers before the names of each fragment indicate the amino acids of Sp1 linked to the GST protein. The pulldown assay in the center panel and the Coomassie-stained gel of the GST fusion protein inputs in the bottom panel. B. The structural domains of Stat6 and the Stat6 mutants used in this work are diagrammed in the top panel. Domains indicated include the transcriptional activation domain (TAD), DNA binding domain (DBD), Src homology 2 (SH2). The numbers before the names of each fragment indicate the amino acids of Stat6 linked to the GST protein. The pulldown assay in the center panel and the Coomassie-stained gel of the GST fusion protein inputs in the bottom panel. (JPEG 984 kb)

High resolution image (TIFF 13050 kb)

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Wei, M., Liu, B., Gu, Q. et al. Stat6 cooperates with Sp1 in controlling breast cancer cell proliferation by modulating the expression of p21Cip1/WAF1 and p27Kip1 . Cell Oncol. 36, 79–93 (2013). https://doi.org/10.1007/s13402-012-0115-3

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