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Breast Cancer Research
Published in: Breast Cancer Research 6/2009

Open Access 01-12-2009 | Research article

Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2overexpression in breast cancer cell lines

Authors: Grégory Nolens, Jean-Christophe Pignon, Benjamin Koopmansch, Benaïssa Elmoualij, Willy Zorzi, Edwin De Pauw, Rosita Winkler

Published in: Breast Cancer Research | Issue 6/2009

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Abstract

Introduction

Activator protein-2 (AP-2) α and AP-2γ transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity.

Methods

Ku proteins were identified as AP-2α interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression.

Results

Nuclear proteins from BT-474 cells overexpressing AP-2α and AP-2γ were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2α activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2α on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2α and AP-2γ siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2α and AP-2γ or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2α and AP-2γ. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter.

Conclusions

Ku proteins in interaction with AP-2 (α and γ) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.
Appendix
Available only for authorised users
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Metadata
Title
Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2overexpression in breast cancer cell lines
Authors
Grégory Nolens
Jean-Christophe Pignon
Benjamin Koopmansch
Benaïssa Elmoualij
Willy Zorzi
Edwin De Pauw
Rosita Winkler
Publication date
01-12-2009
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 6/2009
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/bcr2450

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