Introduction
Breast cancer is the most common cancer in women in Europe [
1]. Accumulation of different molecular alterations characterizes this complex disease. Five major breast cancer sub-groups have been distinguished according to gene expression signatures [
2,
3]. One of these subgroups is characterized by
ERBB2/
Her2 gene amplification and overexpression. This alteration is present in about 20% of breast cancers and was found to be predictive of poor prognosis before the development of
ERBB2 targeted drugs [
4‐
6].
The
ERBB2 gene encodes for p185
-erbB2, which is a transmembrane protein with intrinsic tyrosine kinase activity belonging to the EGF receptor (EGFR) family. No growth factor recognizing specifically
ERBB2 with high affinity has been identified. Consequently, p185
-erbB2 is assumed to be activated by hetero-dimerization with another ligand-activated member of the EGFR family [
6].
The high levels of p185
-erbB2 measured in breast cancer cells result from gene amplification and increased transcription rates [
7,
8]. In order to investigate the biology of these specific breast cancers, we chose to study the deregulation of
ERBB2 gene expression. Analyses of the
ERBB2 promoter have led to the identification of several regulatory sequences through which the gene is overexpressed. AP-2, Ets and YB-1 transcription factor families bind to some of these regulatory regions and have been shown to play a role in
ERBB2 overexpression. Ets family transcription factors contribute to
ERBB2 overexpression by binding to the proximal promoter [
9]. YB-1 factors act through binding sites located 815 to 1129 bp upstream the main transcription initiation site [
10], whereas AP-2 binding sequences (AP2BS) have been identified in the proximal [
11‐
13] and distal [
14] regions of the promoter.
The AP-2 transcription factor family contains five members: AP-2α, β, γ, δ and ε. All have a similar 50 kDa apparent molecular mass and are able to form homo- and hetero-dimers. They bind specific DNA sequences, AP2BS, through their conserved helix-span-helix DNA binding domain.
The involvement of AP-2α and AP-2γ factors in
ERBB2 overexpression has been described in several breast cancer cell lines [
11‐
13,
15]. Besides the
ERBB2 gene, AP-2 factors control the expression of several target genes implicated in the control of cell growth, differentiation and carcinogenesis [
16].
AP-2 factors control transcription in association with transcriptional cofactors [
17]. Among them, PC4, PARP [
18], CITED-2, CITED-4 and CBP/p300 [
19], as well as YY1 [
20], have been shown to interact with and to contribute to AP-2 transcriptional activity. In our own research, we have observed a good correlation between p185
-erbB2, AP-2α and YY1 expression levels in primary breast tumor samples [
21]. Besides their role in transcription, cofactors are also important for the protection of AP-2 against proteasomal degradation [
22].
In order to improve the current understanding of AP-2 (α and γ) activity, we sought here to identify further AP-2α interacting factors contributing to ERBB2 gene overexpression in breast cancer cells. We used a proteomic approach to isolate proteins interacting with this transcription factor in a BT-474 breast cancer cell line. Ku70 and Ku80 were identified by mass spectrometry among the AP-2α interacting proteins.
Ku 70 and Ku80 hetero-dimers are mostly known for their role, in association with DNA-PK, in the repair of DNA double strand breaks. However, it has been shown that Ku70 and Ku80 are involved in transcription regulation either by binding directly to DNA or through interaction with transcription factors [
23]. Ku factors might also play a role in cancer [
24].
We show that siRNAs targeting Ku mRNAs downregulate ERBB2 mRNA and protein levels. The use of reporter vectors containing the ERBB2 proximal promoter demonstrated that Ku70 and Ku80 proteins are involved in ERBB2 transcription regulation. Moreover, we show by ChIP assays that Ku70 protein is recruited to the ERBB2 gene promoter and its absence decreases AP-2α and AP-2γ recruitment. Furthermore, Ku70 recruitment is dependent on the expression of AP-2α and AP-2γ. These results contribute to a better understanding of the mechanism by which AP-2 factors upregulate ERBB2 gene expression in breast cancer cells.
Materials and methods
Cell lines
All the human cell lines (BT-474, ZR-75.1, MDA-MB-231, MCF-7 and SK-BR3, HepG2) were purchased from the American Tissue Culture Collection (Manassas, VA, USA). HCT116 and the derived 70/32 (Ku80+/-) cell lines were gifts from Dr. EA Hendrickson [
25]. All the cells were cultured in the recommended media supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine and 100 μg/ml penicillin/streptomycin (Lonza, Basle, Switzerland).
Antibodies
Mouse anti-AP-2α (3B5) [
20], rabbit anti-AP-2α (C-18) [
20], mouse AP-2γ (6E4/4) [
21], goat anti-Ku70 (C-19) [
26], goat anti-Ku80 (C-20) [
26], mouse anti-Ku80 (B-1) [
27], and control mouse, goat and rabbit IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA. Anti RNA Polymerase II (clone CTD4H8) and anti-β-actin (mAbcam 8226) antibodies were obtained from Abcam (Cambridge, UK).
Plasmids and constructs
The pGEX2T-GST-AP2 and -GST vectors were gifts from Dr. Kannan [
28]. The p86-AP2BS-Luc and p86-AP2BS mut-Luc plasmid reporter vectors (pGL3 basic reporter vector, Promega, Madison, WI, USA) have been previously described by Vernimmen et al [
13]. The SV40-Luc control vector (pGL3 control vector) was purchased from Promega. The AP-2α and the corresponding control expression vectors [
29] have been described previously [
20]. Ku80 expression vector was a gift from Dr. Chen [
30].
GST-pull-down
GST-fusion proteins were expressed and purified according to the procedures provided by Amersham Bioscience (Buckinghamshire, UK). Their purification and the pull-down assay were carried out by using the MagneGST™ Pull-Down System (Promega, Madison, WI, USA). The manipulation (incubation and washing) of the beads was automated by the KingFisher robot (Thermo Fisher Scientific, Waltham, MA, USA). Washing buffer was phosphate buffer saline (PBS)/0,01% Tween. Procedure: 15 μl of MagneGST beads were washed twice with the washing buffer. Beads were incubated with 50 μl of sonicated E. coli (Bl21) transformed with plasmids encoding GST or GST-AP2alpha fusion protein in 250 μl of PBS for 30 min at room temperature (RT). After washing twice with 400 μl of washing buffer, beads bound with GST or GST-AP2 were incubated for 50 minutes with 100 μg (35 μl) of nuclear proteins extracted from BT474 at RT in 180 μl of PBS. After washing the beads three times with 400 μl, the interacting proteins were finally eluted by 8 M urea for 2D gel electrophoresis, or with Laemmli Buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.125 M Tris HCl pH 6.8) for western blotting.
DNase I treatment - GST-AP2 beads incubated with the nuclear protein extracts were washed twice with PBS and suspended in DNase I buffer (40 mM Tris HCl, 10 mM NaCl, 6 mM MgCl2, 1mMCaCl2, pH 7.9). The suspension was incubated with increasing concentrations of DNase I (Roche, Basel, Switzerland) for one hour at 37°C. The suspension was washed three times with PBS, resuspended in Laemmli buffer and the bound proteins were eluted by vortexing during 10 min. Ku70, Ku80 and AP-2 were revealed by western blotting. DNA quantity was estimated by the picoGreen assay (Invitrogen, Carlsbad, CA, USA).
Two-dimensional gel electrophoresis and mass spectrometry
These techniques were performed as described previously [
31], except that the proteins were directly loaded onto IPG strips (non linear IPG strip pH 4-10; Amersham, GE Europe (Buckinghamshire, UK). Liquid chromatography was carried out in an UltiMate™ pump/detection module, FAMOS™ micro autosampler, Switchos™ micro switching module (LC Packings, Dionex, Sunnyvale, CA, USA). The mass analysis was carried out in an ion trap Esquire HCT (Bruker Daltonics, Bremen, Germany) mass spectrometer. The database search was performed using a Mascot local server (Matrix Science, London, UK).
Immunoblotting
Proteins were separated on an SDS-PAGE (10%) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Primary antibodies were used at a 1:1000 dilution. Secondary antibodies coupled with peroxydase (DAKO, Glostrup, Denmark) at a 1:4000 dilution were detected using the ECL system (Thermo Fisher Scientific).
Immunoprecipitation was carried out using Dynabeads Protein G (Invitrogen), according to the manufacturer's recommended protocol, using acetate sodium buffer for antibodies binding. Anti-AP-2α (C-18), Ku70 (C-19), Ku86 (C-20) antibodies and control antibody were used.
Transient transfection assays of reporter vectors
HCT116, 70/32 (HCT116 Ku80 +/-), BT-474 and SKBR3 cells were transfected using FuGENE HD reagent (Roche Applied Science). The cells (3 × 105) were plated onto 24 mm tissue culture dishes, treated with FuGENE HD/DNA (ratio of 3:1) and incubated for 40 h in complete medium. Cells were then harvested. Lysis and enzymatic activity measures were carried out using the Luciferase Reporter Gene Assay kit (Roche Applied Science). Enzymatic activity was measured in a Wallac Victor™ luminometer (PerkinElmer, Waltham, MA, USA). The data were normalized to total protein content.
Transient siRNA transfection
siRNAs were transfected at a 30 nM final concentration using the Calcium Phosphate precipitation technique [
32]. Cells were transfected twice at 48 h intervals. As a control, cells were transfected with the negative control siRNA OR-0030-neg05 (Eurogentec, Seraing, Belgium). The AP-2 siRNAs used were as previously described [
14]. Other siRNAs were: Ku70-1, 5-GUGUGUACAUCAGUAAGAU; Ku70-2, 5-CAGGCAUCUUCCUUGACUU; Ku80-1, 5-GAAGAGGCAUAUUGAAAUA; Ku80-2, 5-CUCCAUUCCUGGUAUAGAA.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted after 90 hours of siRNA transfection treatment, using the High Pure RNA Isolation kit (Roche Applied Sciences). RNA quantification was carried out on a Nano Drop 1000 (Thermo Fisher Scientific). Reverse transcription was performed on 1 μg of total extracted RNA. Real time PCR analysis was performed on an ABI Prism 5700 apparatus (Applied Biosystems, Foster City, CA, USA) using the standard protocol. All the results were reported to the β-2-microglobulin mRNA quantity. The primers used were as previously described [
21], except for Ku70: 5-AGAAGCAAACCGCCTGTA and 5-CAAGCCTCCTCCAATAAAGC; and Ku80: 5-TGCAGCAAGAGATGATGAGG and 5-GAAAGGCAGCTGCACATACA.
Chromatin Immunoprecipitation (ChIP)
Chromatin Immunoprecipitation was carried out using Dynabeads Protein G (Invitrogen). The previously described protocol [
33] was adapted for higher chromatin quantities. On average for each immunoprecipitation reaction, 30 μg of chromatin DNA was precipitated with 4 μg of antibody. Rabbit anti-AP-2α (C-18), goat anti-Ku70 (C-19) and mouse anti-Ku80 (C-20) were used. Immunoprecipitation with pre-immune sera from the same animal species (Ig) or mock immunoprecipitation (NoAb) served as negative controls. Recovered DNA was quantified by real time PCR or by end-point PCR followed by agarose gel electrophoresis and the results were compared to known quantities of chromatin. The gene-specific primer sequences were: -6900 primers: 5-GCAGTAGCAAGCATCGAGTT and 5-TGGATCATCACAAAGGTTTTCA (-6981 bp to -6780 bp); -500-bp
ERBB2 primers, 5-GACTGTCTCCTCCCAAATTT and 5-CTTAAACTTTCCTGGGGAGC (fragment -575 to -349 bp); -100-bp
ERBB2 primers, 5-GCGAAGAGAGGGAGAAAGTG and 5-GGGGAATCTCAGCTTCACAA; GCK primers, 5-GGTAGAGCAGATCCTGGCAGAG and 5-TGAGCCTTCTGGGGTGGAGCGCA. Dilutions of known quantities of input DNA were used to quantify the PCR products.
Discussion
The aim of this study was to identify novel proteins interacting with and contributing to AP-2 transcription factor activity. Using GST-pull-down coupled with two-dimensional gel electrophoresis and mass spectrometry, Ku70 and Ku80 proteins were identified as AP-2α interactors. AP-2/Ku interaction was confirmed by co-immunoprecipitation. We showed that downregulation of Ku proteins by siRNAs induced a strong reduction in ERBB2 mRNA and protein levels in BT-474 and SKBR3 cells. These siRNAs also inhibited the activity of reporter vectors containing the ERBB2 proximal promoter. ChIP experiments revealed that Ku70 proteins were recruited to the ERBB2 promoter. Interestingly, the inhibition of AP-2α and AP-2γ expression by siRNA strongly reduced Ku70 recruitment to the ERBB2 promoter. Ku70 siRNA reduced by half the recruitment of AP-2 factors to the -500 bp region of the ERBB2 promoter containing a high affinity AP2BS. More importantly, Ku70 siRNA downregulated PolII recruitment to the ERBB2 promoter. These results show that the Ku proteins are involved in ERBB2 gene expression regulation by AP-2 in breast cancer cells.
In addition to their role in the repair of DNA double strand breaks, Ku proteins have been shown to control other important cellular processes such as transcription and apoptosis. Ku proteins modulate transcription by several mechanisms and these properties seem to be gene and cell specific. For example, DNA binding of the Ku70/Ku80 heterodimer is responsible for the downregulation of glycoprotein glycophorin B in non erythroid cells [
38]. In contrast, Ku binding to apolipoprotein C-IV promoter stimulates the expression of the gene [
39]. Another study showed that interleukins -13/-4 induced expression of the lipooxygenase-1 gene is mediated by the binding of Ku dimmers to the promoter [
40]. C-jun expression is also stimulated by Ku80 and possibly by Ku70 binding to gene promoter [
41]. Ku proteins can also influence transcription by interaction with transcription factors or cofactors. So, Ku binding inhibits ESE1, an Ets family transcription factor, from binding to DNA and thus its transcriptional activity [
42]. Ku proteins might also be involved in elongation [
37] and transcription reinitialization [
43].
Ku proteins were also shown to modulate gene expression by binding to PARP-1 and to YY1, which were shown to interact with and influence AP-2 transcription factor activity. Indeed, PARP-1 is necessary to preserve the transcriptional activity of overexpressed AP-2 transcription factors [
18]. The Ku - PARP interaction has opposing effects on transcription. Ku proteins inhibit the transcriptional activity of β-catenin-TCF4 complex by interfering with PARP-1 binding [
44]. In contrast, the Ku70/Ku80 dimmer - PARP-1 complex stimulates the expression of the S10019 gene [
45]. Ku - YY1 interaction inhibits α myosin heavy-chain gene expression in the heart [
46], contrary to the consequence of YY1 interaction with AP-2 on ERBB2 gene expression [
20].
Acknowledgements
We thank Dr Kannan, Dr Hendrickson and Dr. Chen for their gifts. We thank Dr. Gabriel Mazzucchelli for his help in Mass Spectrometry analyses. This work was supported by grants from Televie (Belgium), Anticancer Centre attached to the University of Liege (Belgium).
GN and JCP are recipients of Televie grants from the FNRS; BK is research fellow of the FNRS; BE and WZ are researchers financially supported by Région Wallonne, contract BA4 grant 114915, contract EPH331030000092-430001, EPH331030000022-130033, Fonds Social Européen contract W2002134, W1000346, contract 14531, iPCRq; EDP is professor at the University of Liege, director of the Mass Spectrometry Laboratory; RW is research director of the FNRS. Support is via FRSM Grant no. 3.4.542.04 and from the Centre anticancéreux at the University of Liège.
Competing interests
The authors declare that they have no financial competing interests.
Authors' contributions
GN carried out all the studies and drafted the manuscript. JCP and BK helped in the studies and interpretation of the results. BE, WZ and EDP participated in the study design, revised the manuscript and provided important intellectual support. RW conceived of the study, participated in its design, coordination and interpretation of the results and finalized the manuscript. All authors read and approved the final manuscript.