Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines.
BACKGROUND
Activator protein-2 (AP-2) alpha and AP-2gamma 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-2alpha 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-2alpha and AP-2gamma 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-2alpha 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-2alpha 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-2alpha and AP-2gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2alpha and AP-2gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2alpha and AP-2gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter.


CONCLUSIONS
Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.
Nolens
Grégory
G
Laboratory of Molecular Oncology, GIGA Cancer, University of Liège, B34, avenue de l'hopital, Liege, 4000, Belgium. gnolens@gmail.com
Pignon
Jean-Christophe
JC
Koopmansch
Benjamin
B
Elmoualij
Benaïssa
B
Zorzi
Willy
W
De Pauw
Edwin
E
Winkler
Rosita
R
D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006801:Humans; D000072200:Ku Autoantigen; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D000072200:Ku Autoantigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D050656:Transcription Factor AP-2; C000606509:Xrcc6 protein, human