Quantitative Proteomics and Transcriptomics Addressing the Estrogen Receptor Subtype-mediated Effects in T47D Breast Cancer Cells Exposed to the Phytoestrogen Genistein

Maarten D. Sollewijn Gelpke,Jacques Vervoort,Ana M. Sotoca,Anders Ström,Albertinka J. Murk,Ivonne M.C.M. Rietjens,Jan-Åke Gustafsson,Sjef Boeren

doi:10.1074/mcp.m110.002170

Abstract

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERβ cells with inhibited ERβ expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERβ breast cancer cells with low levels of ERα and no expression of ERβ. In addition, data from our study indicate that ERβ-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERβ cells expressing ERβ and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERβ decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERβ cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERβ ratio) in the cells or tissue of interest.

Highlights

From the ‡Toxicology section, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands, ʈDepartment of Molecular Design and Informatics, Schering-Plough, 5340 BH, Oss, The Netherlands, §Biochemistry section, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, ‡‡Department of Biosciences and Nutrition, Karolinska Institutet, Novum, 14186 Huddinge, Sweden, and **Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, Texas 77204
To understand the critical role of estrogens in the regulatory cascade involved in the progression of breast cancers, several studies focused on the evaluation of global transcriptomics and showed the association of ER␣ activation with cell proliferation and the opposing effects for activation of ER␤ (8 –10)
It was observed that mRNA levels are only a partial reflection of the functional state of an organism and that a comprehensive understanding of the genomic information will require means of analyzing quantitative differences in protein expression on a proteome-wide scale (38 – 41)

Summary

Introduction

From the ‡Toxicology section, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands, ʈDepartment of Molecular Design and Informatics, Schering-Plough, 5340 BH, Oss, The Netherlands, §Biochemistry section, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, ‡‡Department of Biosciences and Nutrition, Karolinska Institutet, Novum, 14186 Huddinge, Sweden, and **Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, Texas 77204. High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer [13, 14] These two flavonoids, genistein and quercetin, have been shown to have potent antiproliferative effects on tumor cells in vitro by halting the cell cycle and inducing apoptosis [15,16,17]. The aim of the present study was to provide omics data on the effects of the phytoestrogen genistein in cells with a variable intracellular ratio of the two estrogen receptors ER␣ and ER␤ and to compare these outcomes qualitatively with those reported earlier for estradiol [8]. Using the human T47D breast cancer cell line with tetracycline-dependent ER␤ expression (T47D-ER␤), the effect of a varying intracellular ER␣/ER␤ ratio on estradiol- or genistein-induced cell proliferation was characterized [20]. The results of our previous studies revealed that in this T47D-ER␤ model system the effects of genistein on cell proliferation with varying cellular ER␣/ER␤ ratios were comparable with the effects induced by estradiol itself [20, 22]

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Methods

Conclusion