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




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Breast Cancer Research

, 11:R83

First Online: 11 November 2009Received: 26 April 2009Revised: 07 October 2009Accepted: 11 November 2009

Abstract

IntroductionActivator 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.

MethodsKu 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.

ResultsNuclear 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.

ConclusionsKu proteins in interaction with AP-2 α and γ contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.

AbbreviationsAP-2Activator Protein 2

AP2BSAP-2 binding site

bpbase pairs

ChIPchromatin immunoprecipitation

EGFREGF Receptor

GCKGlucokinase

GSHGlutathion

GSTGlutathione Serine Transferase

LucLuciferase

MSMass Spectrometry

PAGEPoly-Acrylamide Gel Electrophoresis

PBSPhosphate Buffer Saline

Pol IIPolymerase II

RTRoom Temperature

siRNAsmall interfering RNA.

Electronic supplementary materialThe online version of this article doi:10.1186-bcr2450 contains supplementary material, which is available to authorized users.

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