Integrative Genome-Wide Expression Analysis Bears Evidence of Estrogen Receptor-Independent Transcription in Heregulin-Stimulated MCF-7 Cells

Takeshi Nagashima,Harukazu Suzuki,Noriko Yumoto,Kaoru Takahashi,Toshio Kojima,Kaori Ide,Akihiko Konagaya,Yoko Kuroki,Mariko Hatakeyama,Takahiro Suzuki,Aki Hasegawa,Yoshihide Hayashizaki,Tetsuro Toyoda,Shinji Kondo,Yoshiyuki Sakaki,Dorraya El-Ashry

doi:10.1371/journal.pone.0001803

Abstract

Heregulin ß-1 (HRG) is an extracellular ligand that activates mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathways through ErbB receptors. MAPK and Akt have been shown to phosphorylate the estrogen receptor (ER) at Ser-118 and Ser-167, respectively, thereby mimicking the effects of estrogenic activity such as estrogen responsive element (ERE)-dependent transcription. In the current study, integrative analysis was performed using two tiling array platforms, comprising histone H3 lysine 9 (H3K9) acetylation and RNA mapping, together with array comparative genomic hybridization (CGH) analysis in an effort to identify HRG-regulated genes in ER-positive MCF-7 breast cancer cells. Through application of various threshold settings, 333 (326 up-regulated and 7 down-regulated) HRG-regulated genes were detected. Prediction of upstream transcription factors (TFs) and pathway analysis indicated that 21% of HRG-induced gene regulation may be controlled by the MAPK cascade, while only 0.6% of the gene expression is controlled by ERE. A comparison with previously reported estrogen (E2)-regulated gene expression data revealed that only 12 common genes were identified between the 333 HRG-regulated (3.6%) and 239 E2-regulated (5.0%) gene groups. However, with respect to enriched upstream TFs, 4 common TFs were identified in the 14 HRG-regulated (28.6%) and 13 E2-regulated (30.8%) gene groups. These results indicated that while E2 and HRG may induce common TFs, the regulatory mechanisms that govern HRG- and E2-induced gene expression differ.

Highlights

Heregulin ß-1 (HRG) is an extracellular ligand that binds to and activates ErbB3 and ErbB4 receptors in a process associated with the control of cell proliferation and differentiation [1,2,3].Our previous study using MCF-7 breast cancer cells showed that HRG rapidly up-regulated hundreds of transcripts in a ligand dose-dependent manner, as determined by employing expression arrays using the Affymetrix U133A 2.0 chip that mounts proteincoding and non-protein coding regions of ca. 18,400 transcripts [4]
Since MCF-7 is a cancer cell line, there was a concern that high genome copy numbers in the cells may result in strong gene expression in the absence of HRG that may potentially obscure any HRG-related effects
Integrative analysis of HRG-regulated gene expression in MCF-7 cells was performed using two types of tiling array data, comprising histone H3K9 acetylation and RNA mapping, and compared the results with those based on expression array and array comparative genomic hybridization (CGH)

Summary

Introduction

Heregulin ß-1 (HRG) is an extracellular ligand that binds to and activates ErbB3 and ErbB4 receptors in a process associated with the control of cell proliferation and differentiation [1,2,3].Our previous study using MCF-7 breast cancer cells showed that HRG rapidly up-regulated hundreds of transcripts in a ligand dose-dependent manner, as determined by employing expression arrays using the Affymetrix U133A 2.0 chip that mounts proteincoding and non-protein coding regions of ca. 18,400 transcripts [4]. Heregulin ß-1 (HRG) is an extracellular ligand that binds to and activates ErbB3 and ErbB4 receptors in a process associated with the control of cell proliferation and differentiation [1,2,3]. Our previous study using MCF-7 breast cancer cells showed that HRG rapidly up-regulated hundreds of transcripts in a ligand dose-dependent manner, as determined by employing expression arrays using the Affymetrix U133A 2.0 chip that mounts proteincoding and non-protein coding regions of ca. HRG-stimulated ErbB receptor is known to activate mitogenactivated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathways [5]. The transcriptional regulation induced by HRG was thought to be controlled by activation of the aforementioned signaling pathways and associated transcription factors (TFs). Given that MCF-7 is an ER-positive breast cancer cell line, HRG-induced transcription may involve both ERE- and nonERE-dependent regulation

Methods

Results

Conclusion