Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells - interplay of ErbB and IGF1 signalling through IGFBP3 regulation
- Jenny Worthington†1,
- Mariana Bertani†2,
- Hong-Lin Chan3,
- Bertran Gerrits2 and
- John F Timms1Email author
© Worthington et al; licensee BioMed Central Ltd. 2010
Received: 23 March 2010
Accepted: 14 September 2010
Published: 14 September 2010
Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression.
Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression.
775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells.
These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.
The expression and activity of the ErbB/HER family of receptor tyrosine kinases is frequently deregulated in human cancers. To date, four members of this family have been described: EGFR, ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4). Signalling through the ErbB family is initiated by ligand-induced receptor homo- or heterodimerzation leading to stimulation of the receptors' intrinsic tyrosine kinase activity and triggering of auto- and cross-phosphorylation of tyrosine residues creating docking sites for adaptor proteins and enzymes that initiate signal transduction events ultimately leading to changes in gene expression and altered cellular phenotype . Numerous tumour, epithelial or stromal-derived growth factors (GFs) bind with different affinities and specificities to the different ErbB family members. These include: EGF, TGFα and amphiregulin (AREG), which bind specifically to EGFR; heparin-binding EGF-like growth factor, betacellulin and epiregulin which bind to both EGFR and ErbB4 ; and the neuregulins/heregulins (HRGs), which are specific for ErbB3 and ErbB4 . Although ErbB2 is an orphan receptor with no ligand described to date, it is the preferred dimerzation partner of the other ErbB family members, acting as a potentiator of signalling and highlighting the importance of heterodimerzation within the ErbB family [3–6].
EGF and HRG can activate many intracellular signalling cascades and appear to exert distinct biological functions that depend on the nature of the receptor complexes induced. Although there is major overlap in the signalling pathways activated by ErbB receptors, specific family members can preferentially modulate distinct pathways. For instance, while all ErbB receptors activate the MAPK pathway via Shc and/or Grb2, ErbB3 is the most potent activator of PI3K signalling due to its multiple binding sites for the p85 regulatory subunit of PI3K [7, 8]. In contrast, Eps15 and Cbl are both EGFR-specific substrates involved in receptor down-regulation [9, 10]. The relative expression of each ErbB receptor influences the cellular response to their ligands. For example, cells expressing high levels of ErbB2 show a greater response to HRG and ErbB3 shows higher affinity for HRG when co-expressed with ErbB2 . This preferential cooperativity extends to oncogenic transformation, with ErbB2-ErbB3 heterodimers reported as the most potent signalling activators [12, 13]. Importantly, the aberrant expression and/or activation of ErbB family members have been reported in a number of different tumour types. In particular, there is an extensive literature on the role of ErbB receptors in breast cancer. ErbB2 is overexpressed in 25-30% of all breast cancers due to gene amplification, and is correlated with disease progression, advanced tumour stage, decreased survival, poor response to therapy and metastasis [14, 15]. Such poor prognosis is a likely reflection of the biological effects of ErbB2 overexpression, including increased cellular proliferation, anti-apoptosis, cell invasiveness and promotion of angiogenesis. The ErbB receptors have consequently become targets for specific anti-cancer therapies [16–20]. Indeed, one of these therapies, herceptin (trastuzumab), a monoclonal antibody against the extracellular domain of ErbB2, has shown significant clinical benefit for patients with ErbB2-positive breast cancers. Indeed, the combined results of several clinical trials have shown that the addition of 1 year of trastuzumab to adjuvant chemotherapy significantly improves disease-free survival by 33%-52% . Despite this, less than 35% of patients respond to trastuzumab as a single agent and those who initially respond well generally acquire resistance within a year (reviewed in ). These data suggest that ErbB2 overexpression alone is not a reliable predictor of therapeutic outcome and that additional factors are involved. Thus, the identification and characterization of genes associated with ErbB2 overexpression would be beneficial, in order to better define the molecular mechanisms involved in ErbB2-dependent transformation and to identify novel drug targets.
Recently, much effort has been put into tumour expression profiling in an attempt to characterize the genes involved in malignant transformation. Microarray analysis has been reported to successfully predict estrogen receptor and lymph-node status of breast cancer [23, 24], to distinguish between cancers associated with BRCA1 or BRCA2 mutations  and to identify subclasses of breast cancer and predict outcome based on gene expression patterns [23, 26–29]. Although these approaches are useful for identifying diagnostic and prognostic markers, few microarray studies have examined ligand-induced signalling events involved in transformation. The aim of this study was to use microarray analysis to investigate ErbB ligand-induced transcriptional responses and diversification of signalling events downstream of ErbB receptors in a human mammary luminal epithelial cell (HMLEC) model. This model comprises an SV40 large T antigen-immortalized HMLEC parental cell line derived from flow-sorted cells from reduction mammoplasty material and a derivative clone stably overexpressing ErbB2 [30, 31]. The cells require serum for proliferation, the removal of which leads to loss of viability. In the absence of serum, treatment with the ErbB-specific ligands HRGβ1 or EGF can support proliferation and survival, with the ErbB2-overexpressing cells displaying increased rates of proliferation, anchorage-independent growth and enhanced mitogenic signalling compared to the parental line [31, 32]. In the present study, cells were serum-starved and treated with EGF or HRGβ1 over a timecourse to establish long-term HRG- and EGF-specific transcriptional responses to examine diversification of signalling through EGFR and ErbB3 receptors and to assess how ErbB2 overexpression alters these responses.
Cell culture, growth factor stimulation and RNA isolation
The parental HMLEC line HB4a and an ErbB2-overexpressing derivative C3.6 have been previously described [31, 32]. Cells were cultured in RPMI-1640 media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml hydrocortisone and 5 μg/ml insulin (both Sigma) at 37°C in a 10% CO2 humidified incubator. Before stimulation, HB4a and C3.6 cells were starved of GFs for 48 h in RPMI-1640 media with 0.1% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin and 5 μg/ml hydrocortisone. Cells were then treated with 1 nM EGF or 1 nM HRGβ1 (HRG hereafter) (both R&D Systems) for 4 h, 18 h and 24 h prior to RNA isolation. Two plates were prepared for control (serum-starved) cells and for each time point. Total RNA was isolated using TRIZOL™ reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. Each plate generated two samples of total RNA for reciprocal labelling, resulting in a total of four replicates for microarray experiments. Serum starved cells were also treated with 25 ng/mL IGF1 for the indicated times. SKBR3 cells were maintained in tissue culture flasks containing DMEM/F-12 medium supplemented with 10% (v/v) FCS, 100 μg/mL streptomycin and 100 IU/mL penicillin (Gibco-Invitrogen Corp) in a humidified incubator at 37˚C with 5% CO2.
Microarray experimental design
Hver 1.3.1 arrays used in this study were obtained from the Wellcome Trust Sanger Institute. Each microarray contains a redundant set of 9932 PCR-derived, sequence verified cDNA clones representing around 6,000 genes. 25 μg of total RNA was used to produce labeled cDNA by anchored oligo(dT)-primed reverse transcription with Superscript II Reverse Transcriptase (Invitrogen Life Technologies) in the presence of Cy3- or Cy5-dUTP (Amersham Pharmacia). Unincorporated Cy dye was removed using Autosequ-50 Columns (Amersham Pharmacia) and repetitive DNA sequences were blocked by co-precipitation of labeled cDNA with 8 μg Cot1 (Boehringer Mannheim) and 8 μg poly(dA) DNA (Sigma). The labeled cDNA pellet was re-suspended in hybridization buffer (4 × SSC, 5× Denhardt's solution, 50 mM Tris-HCl pH 7.6, 0.1% sarkosyl, 49% formamide) and hybridized onto arrays at 47°C overnight. Slides were washed twice in 2 × SSC, four times in 0.1 × SSC plus 0.1% SDS, twice in 0.1 × SSC and then dried by centrifugation before scanning. All samples were co-hybridized to a common standard reference comprised of total RNA pooled from cell line BT474 and two grade III invasive ductal breast carcinomas (gift of Dr Alan Mackay, ICR, London), allowing cross-comparison of multiple experimental conditions. A "dye-flip" approach was used to minimize dye-specific bias, where two biological replicates were labeled with Cy3 and hybridized with Cy5-labelled reference and vice versa (four replicates). For the 14 experimental conditions (2 cell lines, 2 GFs and 3 time points plus untreated), a total of 56 hybridizations were thus performed.
Data normalization, filtering and analysis
Slides were scanned using ScanArray 4000XL and spots quantified using QuantArray v3.0 (both Packard BioChip Technology). Cy-dye emission signals were scaled in QuantArray using the median intensity of each channel, and any visible hybridization artifacts were flagged and recorded as absent during data filtering and analysis. Data was background subtracted and exported to GeneSpring v6.1 (Silicon Genetics) for normalization. The fluorescence intensity ratio between each sample and the co-hybridized reference (sample/ref) was calculated and represents the expression level for a given probe on each individual replicate. The dataset was then normalized using the intensity-dependent LOWESS regression technique . Normalized raw data and experimental details were processed to conform with Minimum Information About a Microarray Experiment (MIAME) guidelines and are deposited in the Array Express (http://www.ebi.ac.uk/arrayexpress) repository (accession number E-TABM-106). Genes were then filtered using the excel add-in SAM (Significant Analysis of Microarray)  to identify genes showing significant changes in expression by assigning a score on the basis of change in gene expression relative to the standard deviation of repeated measurements. A false discovery rate threshold of 3% was used as the cut-off to report differentially regulated genes. Averaged values for each of the 14 experimental conditions were then compared to identify genes that were up- or down-regulated generating a list of 775 genes that changed significantly between two or more experimental conditions. TIGR MeV software v2.2 (The Institute for Genomic Research) was used for clustering analysis of the 775 genes using two sets of average ratios: i) the HB4a/C3.6 ratio was taken at each time point for ErbB2-dependent changes in gene expression, and ii) the T*/T0 ratio was taken in each cell line for GF-dependent changes in gene expression. Values were log2 transformed, loaded into TIGR MeV for average-linkage hierarchical clustering using Euclidian Distance and for k-means clustering using 4 (k) groups. Genes in each group were then subjected to hierarchical clustering.
Semi-quantitative Real Time-PCR
Samples were generated by reverse transcription of 2.5 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen Life Technologies) and random hexamer primers (Applied Biosystems). cDNAs were then treated with RNase H (Invitrogen Life Technologies) to eliminate RNA contamination. Real Time-PCR (qRT-PCR) was performed using the Assay-on-Demand system (Applied Biosystems) according to the manufacturer's protocol. Assay IDs were: Hs99999905_m1 (GAPDH); Hs99999901_s1 (18S); Hs00155832_m1 (AREG); Hs00426287_m1 (IGFBP3); Hs00192713_m1 (G1P2); Hs00175188_m1 (CTSC); Hs00196051_m1 (ISGF3G); Hs00242943_m1 (OAS1); Hs00195584_m1 (S100P); Hs00602835_s1 (SFN); Hs00173626_m1 (VEGF); Hs00185574_m1 (VIL2); Hs00185584_m1(VIM); Hs00170299_m1 (ZYX). Briefly, primer and probe mix was added to PCR Master Mix with 1 μL of cDNA per 50 μL reaction. Sample fluorescence emission was recorded for each cycle on an ABI7700 Sequence Detection System. Cycling conditions were as follows: initial enzyme activation step at 95°C for 10 min and then 39 repeating cycles of 95°C for 10 sec and 60°C for 1 min. Serial dilution experiments of each primer against the endogenous control were prepared to test for amplification efficiency. For primers whose amplification efficiencies were similar to that of the endogenous control (slope of the graph ΔCt vs. log dilutions ≤0.1) the ΔCt method was used, where the equation 2-ΔΔCt determines the amount of a target relative to a calibrator sample. If amplification efficiencies were not similar, the standard curve method was used for relative quantitation of target gene expression. Further information on both of these methods can be found on User Bulletin3 on the ABI website.
Freshly treated cells were lysed in NP40 lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA) with protease and phosphatase inhibitors: pepstatin A (1 μg/mL), leupeptin (1 μg/mL), AEBSF (100 μg/mL), aprotinin (17 μg/mL), sodium orthovanadate (2 mM), okadaic acid (1 μM), fenvalerate (5 μM) and bpV (5 μM). Protein concentration was determined using a Bradford assay and equal amounts of protein used for SDS-PAGE and immunoblotting with commercially-available antibodies (Additional file 1). Antibodies were detected with appropriate HRP-conjugated secondary antibodies and detected by enhanced chemiluminescence (Perkin Elmer). All membranes were reprobed for beta-actin and densitometry performed on all bands using a GS-800 Calibrated Densitometer and QuantityOne software (both BioRad) with local background subtraction. Intensities for each band were then normalized to the actin band in that lane. Normalized values were averaged from 3-5 independent blots and plotted using the standard deviation as the error.
Small interfering RNA (siRNA) reverse transfection
HB4a, C3.6, or SKBR3 cells were withdrawn from antibiotics for a minimum of 2 hrs and subsequently transfected with siRNA pools targeting ErbB2, IGFBP3 or non-targeting scrambled control siRNA (Dharmacon RNA Technologies); the ON-TARGET plus non-targeting control siRNA pool was used in invasion, proliferation and stimulation assays, whilst the #2 ON-TARGET plus non-targeting control siRNA was used in anchorage-independence growth assays. Reverse transfection was performed in 6-well plates according to the manufacturer's instructions using Lipofectamine™ RNAi Max (Invitrogen) and diluting the siRNA with Opti-Mem® reduced serum medium (Invitrogen). A final concentration of 50 nM of siRNA was typically used to transfect 2.5 × 105 cells per well (or 1.5 × 105 for siIGFBP3 knockdown in SKBR3) which were then maintained in their normal growth medium. Cells were typically harvested 96 hrs post-transfection in 200 μL of NP40 lysis buffer and expression knockdown confirmed by western blotting as described.
Transfected SKBR3 cells were subjected to a matrigel-based invasion assay utilizing a 24-well BD Biocoat™ Tumour Invasion Assay System (BD Biosciences) according to the manufacturer's instructions. At 96 hours post-transfection, cells were harvested, counted and plated at 1 × 105 cells/chamber in DMEM/F-12 medium supplemented with 0.1% (v/v) FCS, 100 μg/mL streptomycin and 100 IU/mL penicillin. The lower chamber contained a chemo-attractant of DMEM/F-12 medium supplemented with 10% (v/v) FCS. Invaded cells on the underside of the membrane were fixed and stained after 72 hours in a 99% methanol, 1% crystal violet solution and counted under a bright field microscope with the Image J software. Experiments were performed in triplicate for each siRNA and cells in 5 fields per membrane were counted.
Transfected cells were harvested after 96 hrs, counted and plated at 3 × 103 (SKBR3) and 5 × 103 cells/well (HMLEC) into 96-well plates in DMEM/F-12 medium supplemented with 10% (v/v) FCS, 100 μg/mL streptomycin and 100 IU/mL penicillin with 5 replicates per condition. The number of viable cells was ascertained utilizing a MTT (3-(4, 5-dimehylthiazol-2-yl)-2-5-dyphennyltetrazolium bromide) assay after 48 hrs. For this, cells were incubated with 50 μL/well of 1 mg/mL MTT which is converted to purple formazan crystals by viable cells. After 5 hr crystals were solubilised in 100 μL of DMSO, shaken at room temperature for 10 min and the absorbance measured at 540 nm using a microtitre plate spectrophotometer.
Anchorage-independence growth assays
Transfected SKBR3 cells were harvested after 96 hrs, counted and re-suspended in DMEM/F-12 medium supplemented with 10% (v/v) FCS, 100 μg/mL streptomycin, 100 IU/mL penicillin and 10% (v/v) of a 10 mg/mL bacto-peptone solution which contained 3.3% (v/v) noble agar (both Sigma). Cells were plated at 2 × 104 cells/well into 6-well plates containing DMEM/F-12 medium supplemented with 10% (v/v) FCS, 100 μg/mL streptomycin, 100 IU/mL penicillin and 10% (v/v) of a 10 mg/mL bacto-peptone solution with 6% (v/v) noble agar. Colonies were fixed and stained after 14 days with 1 mg/mL p-iodotertazolium violet (Sigma) prepared in absolute methanol and counted using the Image J software. Experiments were performed in triplicate and 5 fields were counted per plate.
ErbB2 and growth factor cDNA microarray analysis
Our aim was to identify time-dependent and growth factor-specific changes in gene expression associated with long-term EGF and HRGβ1 (hereafter HRG) stimulation of a model HMLEC system. The concentrations of growth factor used (1 nM) were selected based on minimum concentrations required for maximal activation of the ERK1/2 and Akt pathways as measured by western blotting with phospho-specific antibodies (data not shown). We also wanted to assess how such gene expression changes are affected by ErbB2 overexpression in order to understand how ErbB2 contributes to signalling events associated with breast epithelial cell transformation. Microarray experiments were thus carried out using a previously described ErbB2-overexpressing HMLEC system [31, 32]. Serum-starved HB4a parental cells and ErbB2-overexpressing C3.6 cells were stimulated with EGF or HRG for 4 h, 18 h and 24 h or left unstimulated (0 h) prior to microarray analysis of mRNA levels of 9,932 probes, representing ~6,000 genes. These timepoints were chosen to assess the medium-term effects on gene expression that occur within the doubling time of the cells.
Growth factor-induced genes augmented by ErbB2 overexpression
Genes transiently induced by both GFs and whose expressions were induced more strongly in the ErbB2-overexpressing cells are of particular interest in ErbB2-dependent cell transformation. These genes included transcription factors MYC (Fig. 3C), ZFP36L1, ZFP36L2, FOSL1 and ATF4/CREB2, growth factors VEGF and PDGFB and signalling kinases LYN, MAP2K1/MEK1 and MAP2K3/MEK3 (Fig. 3D). Also in this group, were the MYC-induced glycoprotein EMP1, which showed a similar pattern of expression to MYC (Fig. 3E), and BCAR3 (Fig. 3F), a novel SH2 and GEF domain-containing gene. A number of genes associated with cytoskeletal organization and adhesion were also induced more robustly in the ErbB2 overexpressing cells, particularly in response to HRG. These genes included zyxin (ZYX) (Fig. 3G), transgelin (TAGLN), thrombospondin 1 (THBS1), vinculin (VCL), calponin 3 (CNN3), villin 2/ezrin (VIL2), myosin 1E (MYO1E), stathmin 3 (STMN3), Crk-associated substrate-related protein (NEDD9/CASL), ladinin 1 (LAD1) and integrin α2 and α3 (ITGA2 and ITGA3). Members of the plasminogen activator system; tissue-type plasminogen activator (PLAT) (Fig 3H), urokinase-type plasminogen activator receptor (PLAUR), plasminogen activator inhibitor 1 (PAI1/SERPINE1) and the plasminogen and PLAT co-receptor annexin A2 (ANXA2), were also present in this group. The anti-apoptotic genes IER3 and TNFAIP3 (Fig. 3I & 3J) were also more highly induced in the ErbB2-overexpressing cells, as were the poorly characterized genes S100P, CSRP1, HPCAL1 and SMAP, identifying them as potential effectors of ErbB signalling. Finally, and perhaps surprisingly, the genotoxic stress-induced growth arrest gene GADD45A (Fig. 3K) and the MAPK phosphatases DUSP1/MKP1 and DUSP5 were both induced by growth factor treatment.
Growth factor-induced gene expression changes
ErbB2-dependent gene expression
Genes displaying differential expression between HB4a and C3.6 cell lines.
Ensembl Number and Description
GO Biological Process
Fold Change (at T0)
ENSG00000184254:ALDEHYDE DEHYDROGENASE 6
ENSG00000171346:KERATIN, TYPE I CYTOSKELETAL 15
ENSG00000106541:ANTERIOR GRADIENT 2
ENSG00000061676:NCK-ASSOCIATED PROTEIN 1 (NAP 1)
ENSG00000164919:CYTOCHROME C OXIDASE POLYPEPTIDE VIC
ENSG00000167653:PROSTATE STEM CELL ANTIGEN PRECURSOR
ENSG00000171401:KERATIN, TYPE I CYTOSKELETAL 13
ENSG00000164924:14-3-3 PROTEIN ZETA/DELTA
ENSG00000067225:PYRUVATE KINASE, MUSCLE
ENSG00000167283:ATP SYNTHASE G CHAIN, MITOCHONDRIAL
GO:0015992:proton transport; GO:0006754:ATP biosynthesis
ENSG00000155465:Y+L AMINO ACID TRANSPORTER 1
GO:0006832:small molecule transport
ENSG00000106028:SINGLE-STRANDED DNA-BINDING PROTEIN
ENSG00000140264:SMALL EDRK-RICH FACTOR 2
ENSG00000111859:ENHANCER OF FILAMENTATION 1 (HEF1)
GO:0007155:cell adhesion; GO:0000074:regulation of cell cycle
ENSG00000140497:SECRETORY CARRIER-ASSOCIATED MEMBRANE PROTEIN 2
GO:0006886:intracellular protein transport
ENSG00000134531:EPITHELIAL MEMBRANE PROTEIN-1
ENSG00000008394:MICROSOMAL GLUTATHIONE S-TRANSFERASE 1
GO:0032496: response to lipopolysaccharide
ENSG00000128951:DEOXYURIDINE 5'-TRIPHOSPHATE NUCLEOTIDOHYDROLASE
ENSG00000108654:PROBABLE RNA-DEPENDENT HELICASE P68
ENSG00000140319:SIGNAL RECOGNITION PARTICLE 14 KDA PROTEIN
ENSG00000183059:ANNEXIN II (LIPOCORTIN II)
ENSG00000154518:ATP SYNTHASE LIPID-BINDING PROTEIN
GO:0006091:energy pathways; GO:0015992:proton transport
ENSG00000075142:SORCIN (22 KDA PROTEIN)
ENSG00000141618:60S RIBOSOMAL PROTEIN L17
ENSG00000141736:V-ERBB2 ERYTHROBLASTIC LEUKEMIA VIRAL ONCOGENE HOMOLOG 2
GO:0007169:transmembrane receptor protein tyrosine kinase signaling
ENSG00000128524:VACUOLAR ATP SYNTHASE SUBUNIT F
GO:0015992:proton transport; GO:0006754:ATP biosynthesis
ENSG00000159199:ATP SYNTHASE LIPID-BINDING PROTEIN
ENSG00000065361:V-ERBB2 ERYTHROBLASTIC LEUKEMIA VIRAL ONCOGENE HOMOLOG 3
GO:0007169:transmembrane receptor protein tyrosine kinase signaling
ENSG00000167996:FERRITIN HEAVY CHAIN
GO:0008283:cell proliferation; GO:0006826:iron transport
ENSG00000149418:SUPPRESSOR OF TUMORIGENICITY 14
GO:0006508:proteolysis and peptidolysis
GO:0008151:cell growth and/or maintenance
ENSG00000141367:CLATHRIN HEAVY CHAIN 1
GO:0006886:intracellular protein transport
ENSG00000084234:AMYLOID-LIKE PROTEIN 2 PRECURSOR
GO:0007186:G-protein coupled receptor protein signaling pathway
ENSG00000166530:HEAT SHOCK FACTOR BINDING PROTEIN 1
GO:0000122:negative regulation of transcription from Pol II promoter
ENSG00000239672:NON-METASTATIC CELLS 1
GO:0045786:negative regulation of cell cycle; GO:0009142: NTP biosynthesis
ENSG00000213928:INTEREFERON REGULATORY FACTOR 9
GO:0006355:regulation of transcription; GO:0006955: immune response
ENSG00000101557:UBIQUITIN SPECIFIC PEPTIDASE 14
GO:0006511:ubiquitin-dependent protein catabolism
ENSG00000180879:SIGNAL SEQUENCE RECEPTOR DELTA
GO:0006886:intracellular protein transport
ENSG00000075785:RAS-RELATED PROTEIN RAB-7
GO:0007264:small GTPase mediated signal transduction; GO:0006897:endocytosis
GO:0006520:amino acid metabolism
ENSG00000166681:NERVE GROWTH FACTOR RECEPTOR ASSOCIATED PROTEIN 1
ENSG00000128708:HISTONE ACETYLTRANSFERASE 1
GO:0006323:DNA packaging; GO:0006475: internal protein amino acid acetylation
ENSG00000172531:SERINE/THREONINE PROTEIN PHOSPHATASE PP1-ALPHA 1 CATALYTIC SUBUNIT
GO:0006470:protein amino acid dephosphorylation
ENSG00000116030:UBIQUITIN-LIKE PROTEIN SMT3C PRECURSOR
ENSG00000119655:EPIDIDYMAL SECRETORY PROTEIN E1 PRECURSOR
GO:0006164:purine nucleotide biosynthesis
ENSG00000156587:UBIQUITIN-CONJUGATING ENZYME E2L 6
NSG00000115524:SPLICING FACTOR 3B SUBUNIT 1
GO:0006928:cell motility;GO:0006629:lipid metabolism
ENSG00000114416:FRAGILE X MENTAL RETARDATION SYNDROME RELATED PROTEIN 1
ENSG00000105223:SIMILAR TO VACCINIA VIRUS HINDIII K4L ORF
ENSG00000084207:GLUTATHIONE S-TRANSFERASE P
GO:0007417:central nervous system development
ENSG00000115677:VIGILIN (HIGH DENSITY LIPOPROTEIN-BINDING PROTEIN)
GO:0006869:lipid transport; GO:0008203:cholesterol metabolism
GO:0006139:nucleobase, nucleoside, nucleotide and nucleic acid metabolism
ENSG00000101443:MAJOR EPIDIDYMIS-SPECIFIC PROTEIN E4 PRECURSOR
GO:0006508:proteolysis and peptidolysis
ENSG00000089127:2',5'-OLIGOADENYLATE SYNTHETASE 1
ENSG00000149257:COLLAGEN-BINDING PROTEIN 2 PRECURSOR
GO:0006950:response to stress
ENSG00000051523:CYTOCHROME B-245 ALPHA
GO:0006118:electron transport; GO:0006801: superoxide metabolism
ENSG00000185201:INTERFERON-INDUCED TRANSMEMBRANE PROTEIN 2
ENSG00000146674:INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 PRECURSOR
GO:0007165:signal transduction; GO:0001558:regulation of cell growth
ENSG00000165092:ALDEHYDE DEHYDROGENASE 1 FAMILY MEMBER A1
ENSG00000185885:INTERFERON-INDUCED TRANSMEMBRANE PROTEIN 1
GO:0006955:immune response; GO:0008285:negative regulation of cell proliferation
ENSG00000182106:UBIQUITIN CROSS-REACTIVE PRECURSOR
GO:0006955:immune response; GO:0007267:cell-cell signaling
Validation of gene expression changes
A set of genes of interest from the microarray analysis were chosen for validation using semi-quantitative real-time PCR and/or immunoblotting. In general, there was good agreement between the microarray and RT-PCR datasets for genes examined, although fold-changes were generally higher and data more reproducible for the RT-PCR analyses (Fig. 5). Thus, EGF and/or HRG treatment induced AREG, VEGF, SFN, VIL2, ZYX and CTSC expression, with a more potent induction of VIL2 and ZYX in C3.6 cells, in agreement with the microarray data. Also in agreement, IGFBP3 and three ISGs (ISGF3G, G1P2 and OAS1) were all expressed at lower levels in C3.6 cells with IGFBP3 potently down-regulated by both GFs in the HB4a cells, whilst S100P was overexpressed in the C3.6 cells. One exception was vimentin (VIM), where RT-PCR showed increased expression in C3.6 cells with potent induction by EGF and HRG, rather than the down-regulation suggested from the microarray. The reason for this discrepancy is unclear, although the RT-PCR data is likely to provide a more accurate measure of regulated expression. The protein expression of several targets was also examined to test if the observed mRNA changes were indeed translated at the protein level (Fig. 6 and Additional file 6). When comparing the effect of ErbB2 overexpression alone (i.e. between cell lines), there was reasonable concordance between relative protein and mRNA expression for MYC, CLDN4, ZYX, PHB, MAP2K1, NME1, AGR2, PKM2 and ANXA2, (all up-regulated) and IGFBP3, ISGF3G and G1P2 (all down-regulated) (Fig. 6B). However, GF-induced changes in protein level were only apparent for MYC, CLDN4, S100A6, ZYX and G1P2 (Fig. 6C). In particular, the robust repression of IGFBP3 mRNA by GF treatment was not observed at the protein level or the induction of DUSP1 and SFN.
Altered expression of IGFBP3 may alter IGF1-dependent signalling
This study has identified genes whose differential expression may contribute to ErbB2-dependent transformation and which define common and specific signalling events induced through EGFR and ErbB3 receptor-containing complexes. Although we and others have previously examined ErbB2-dependent gene expression changes in the same cell model, and find overlap in the genes identified [37, 38], to the best of our knowledge, this is the first study to simultaneously investigate long-term ErbB2- and GF-dependent gene expression using ligands that activate specific ErbB receptor complexes in the same cell system. A number of gene expression changes were further validated using qRT-PCR and we report a good correlation between the datasets, indicating the robustness of the microarray protocol employed.
There were significantly more HRG-responsive genes than EGF-responsive genes and in many cases the HRG response was elevated in the ErbB2-overexpressing cells. This is likely to be a consequence of the higher expression of ErbB2 and ErbB3 in these cells  and the preferred heterodimerzation of these receptors [3–6], which would act to augment the response to HRG. We do not think that ErbB4 (also a HRGβ1 receptor) plays a major role in orchestrating signalling events in this cell system, since it appears to be expressed at very low levels, if at all, in these cell lines (data not shown). Although HRG-induced expression was generally of a lower magnitude than for EGF, it was often sustained compared to EGF, consistent with our previous finding that HRG-dependent mitogenic signalling is sustained in these cells . Such temporal differences may be connected with differential rates of receptor or signal down-regulation, but also highlight the fact that the two growth factors initiate diverse responses which are likely to be relevant in vivo. Genes induced robustly by HRG (ZNF236, ZFP36L1, ZFP36L2, MADH4, TRIO, HMGCR, SLC16A1, SLPI, GYS1, SFRS5, CTNND1, LCAT, LYN, STAT1, KRT15, C20orf16 and FN1) are likely candidates for regulation by the PI3K/Akt pathway which is potently activated by HRG through ErbB2-ErbB3 heterodimers [7, 8]. Since HRG expression itself correlates with tumourigenicity and metastasis in breast cancer cells lines [39, 40], it will be interesting to assess whether the induction of these genes is affected by chemical inhibition of the PI3K pathway, or whether such inhibitors would make clinically useful therapeutics for breast cancer treatment.
Notably, a group of EGF-specific genes (e.g. AREG, S100A2 and CTSC) were induced exclusively in the HB4a cells, potentially through EGFR homodimers which predominate in these cells . One of these genes, AREG, is a ligand of EGFR itself, suggesting that EGF could drive autocrine signalling to enhance EGF-specific responses. Members of the MT family were also potently induced by EGF. Whilst induction of MT1 expression by EGF has been shown in rat hepatocytes , this is the first report of MT1 (and MT3) induction by EGF in human epithelial cells. Since the altered expression of MT family members has been implicated in neoplasia and drug resistance [42, 43], it will be interesting to investigate whether MT expression is linked to deregulated GF signalling in cancer.
Many of the identified genes have been previously implicated in tumour progression, found to be aberrantly expressed in different tumour types and/or to be linked with poor prognosis, hyper-proliferation, cell survival or tumour invasiveness. Our findings suggest that dysregulated ErbB signalling can account for changes in the expression of these genes, and may thus contribute to the establishment and progression of ErbB2-overexpressing breast tumours. For example, of the genes induced by both GFs and augmented by ErbB2, the proto-oncogenic transcription factor MYC has been associated with many forms of cancer often indicating poor prognosis . Importantly, patient survival was significantly reduced in breast cancers where MYC and ErbB2 are co-amplified . The MYC-induced glycoprotein EMP1 was also similarly regulated and whilst its function is unknown, it has reported tumourigenic activity  and was identified as a marker of gefinitib-resistance in xenograft models . Thus, one possible scenario that warrants further investigation is that EMP1 acts in concert with MYC to promote ErbB2-dependent proliferation and drug resistance. A pattern of ErbB2-augmented GF-induction was also observed for other genes known to be involved in proliferation, autocrine signalling and anti-apoptosis (e.g. ATF4, FOSL1, IER3, MAP2K1/MEK1, MAP2K3/MEK3, PDGF, TNFAIP3, VEGF) and it is possible that these changes contribute to the reported hyper-proliferative phenotype of these ErbB2-overexpressing cells [31, 32]. Induction of the pro-angiogenic factor VEGF is particularly relevant to tumour progression and confirms previous data [48, 49]. Notably, VEGF expression was shown to depend upon ATF4 expression under certain conditions  and we hypothesize that such a regulatory circuit exists in these cells, whereby ErbB2-augmented GF signalling would promote VEGF expression through up-regulation of ATF4. The induction of some genes was perhaps surprising given their reported functions. GADD45A, SFN and the dual-specificity phosphatases DUSP1/MKP1 and DUSP5 were induced by GF treatment and are involved in genotoxic stress-induced growth arrest , p53-dependent negative regulation of G2/M progression  and down-regulation of MAPK signalling, respectively . We propose that these may be negative feedback mechanisms adapted to self-regulate proliferative signalling.
Conversely, the down-regulation of genes with anti-proliferative functions identifies mechanisms by which increased ErbB2 signalling may promote proliferation and survival. Examples include the multiple ISGs that were identified and IGFBP3. G1P2/ISG15 was the most down-regulated gene in the dataset. Like ubiquitin, G1P2 is conjugated to proteins in a process called ISGylation which appears to modulate protein activity during the immune response and signalling . The other ISGs were UBE2L6 (the proposed E2 enzyme for ISGylation ), IFIT1, IFITM1, IFITM2, OAS1 and ISGF3G/p48/IRF9. Notably, ISGF3G is a component of a transcription factor complex that with STAT1 and STAT2 controls type I IFN-mediated induction of ISGs containing interferon-stimulated regulatory elements (ISREs) . The lowered expression of ISGF3G could thus account for the down-regulation of the other ISGs in the ErbB2-overexpressing cells, as suggested by our previous work . Whilst the ISGs were induced by IFN treatment in the HMLECs, induction of ISGF3G (particularly with IFNγ) was blocked by GF co-treatment, revealing a possible cross-talk between the IFN and ErbB signalling pathways (data not shown). Although preliminary, our data suggested an inverse correlation between ErbB2 and ISG expression, supporting a role for repressed basal ISG expression in the pathogenesis of ErbB2-dependent breast cancer.
IGFBP3 mRNA and protein expression were both markedly lower in the ErbB2-overexpressing cells, whilst mRNA levels were decreased by GF treatment, particularly in the parental cells. Given IGFBP3's putative role as a negative regulator of IGF1 signalling , its anti-proliferative role  and the negative correlation between serum IGFBP3 levels and cancer risk [58–60], we investigated a possible link between its expression and IGF1 signalling. We found that IGF1-mediated ERK and Akt activation and proliferation were increased in the ErbB2-overexpressing cells and that the signalling effect was reversed by siRNA-mediated knockdown of ErbB2. The mechanism by which this occurs is unclear, although does not involve altered IGF1R expression, and may be mediated through interaction between ErbB receptors and IGF1R as previously reported in other cell models [61–63]. ErbB2 may also down-regulate IGFBP3 expression to promote IGF1 signalling. We propose that ErbB2-dependent suppression of IGFBP3 expression is a long-term adaptive response and would be the reason why IGFBP3 protein levels were not affected by transient ErbB2 knockdown. We speculate that this may be due to IGFBP3 promoter methylation, as previously reported for other cancers [64, 65]. In the C3.6 cells, IGFBP3 expression is suppressed, allowing maximal IGF1 signalling through ErbB2-IGF1R interaction [61–63]. Knocking down ErbB2 in these cells therefore does not affect IGFBP3 levels, but abrogates IFG1 signalling. In HB4a cells, IGF1 signalling is restricted by normal IGFBP3 expression with knockdown of IGFBP3 enhancing basal ERK1/2 and Akt activation, thus supporting its role as a negative regulator of proliferation and survival. Although reduced IGFBP3 expression did not affect acute IGF1 triggering, our data partly support findings in primary and immortalized human esophageal cells, where EGF-mediated down-regulation of IGFBP3 was shown to determine cellular response to IGF1 . However, this effect may be mediated by the as yet unknown IGF1-independent actions of IGFBP3 (reviewed in [67, 68])
The observed increases in invasiveness and anchorage-independent growth of ErbB2-overexpressing SKBR3 cells following knockdown of IGFBP3 supports a role for IGFBP3 as a negative regulator of cellular transformation in breast cancer and we propose that its down-regulation is a mechanism whereby ErbB2 promotes tumour cell growth through increased IGF1-dependent proliferation, survival and invasion. Indeed, a requirement for IGF1 in EGF-mediated cell cycle progression has been shown in primary murine mammary epithelial cells . Whilst an attractive model, other studies report that IGFBP3 can potentiate EGF-stimulated proliferation in MCF10A cells  and that IGFBP3 expression is associated with growth stimulation of T47D human breast cancer cells . These differences may be explained by cell type-specific effects and are possibly dependent upon the extent of interaction with the ErbB receptor system . Future experiments should explore the effects of overexpressing IGFBP3 on IGF1 signalling, proliferation, survival and invasion and to investigate the level of IGFBP3 promoter methylation in this cell system.
We have previously reported a high correlation between mRNA and protein expression for a subset of genes in these cell lines , and a previous proteomic study found reduced expression of GSTP1, PRDX5 and USP14 and increased expression of KRT13, ALDH1A3 and NME1 in the C3.6 cells , in agreement with the mRNA data presented here. In the present study, the mRNA expression of several targets (MYC, CLDN4, S100A6, ZYX, PHB, MAP2K1, NME1, AGR2, PKM2, IGFBP3, ISGF3G, G1P2 and ANXA2) correlated with altered protein expression, signifying that these changes are likely to be functionally relevant. However, correlation between protein and mRNA expression was not apparent for some targets in response to the GF treatments. For example, the repression of IGFBP3 mRNA by GF treatment was not confirmed at the protein level and neither was induction of DUSP1 or SFN. This suggests that the IGFBP3 protein may be relatively stable over the time course of the assay or that the DUSP1 and SFN mRNAs are not translated. Such post-transcriptional regulatory mechanisms are likely to be important, and whilst some mRNA changes appear to be redundant, they may be relevant in other circumstances, for example, during development, differentiation or stress.
A relatively large group of genes involved in regulating the cytoskeleton, cell adhesion and motility were identified. Whilst various patterns of gene expression were apparent, genes up-regulated to a greater degree by either GF in the ErbB2-overexpressing cells (ZYX, VIM, VCL, TAGLN, VIL2, PDLIM1, ITGA2, ITGA3, PLAT, PLAUR, SERPINE1 and ANXA2) are perhaps the most interesting, since they may promote the ErbB2-mediated anchorage-independent growth and reduced cellular adhesion previously observed in this cell model system [31, 38]. Notably, some of these genes are members of the plasminogen activator system and have been implicated in tumour progression and invasiveness through proteolysis of the extracellular matrix. Indeed, increased levels of PLAUR and SERPINE1 have been associated with poor prognosis in breast cancer patients [73, 74]. Our data thus implicates ErbB2-mediated signalling in the regulation of the plasminogen activator system, as well as cell adhesion-related events.
Finally, a number of genes of unknown or poorly-defined function were identified and several were validated. These include BCAR3, CPNE3, CSRP1, HPCAL1, LCP1, MGC10471, NME1, SMAP, ZFP36L1, ZFP36L2 and ZNF236, which were differentially regulated by GF in an ErbB2-dependent manner and AGR2, LOC402057, NPC2, PSCA, S100P and SERF2, which were differentially expressed in an ErbB2-dependent manner. Our data reveals that the expression of these genes can be regulated by ErbB receptor signalling and thus implicates them as possible biomarkers and effectors of ErbB2-dependent tumourigenesis. Indeed, AGR2, LCP1 and S100P overexpression have been previously correlated with breast cancer progression [75–77], and we now link the aberrant expression of these genes with ErbB2 expression.
Fully understanding and characterising the interactions and outcomes of the identified gene expression changes is a huge undertaking and will require additional studies addressing the functional consequences of such changes. However our data provides a valuable resource and a number of testable hypotheses with potentially important implications in GF signalling and ErbB2-dependent tumourigenesis. One assumption of this work is that the measured effects are indeed ErbB2-dependent and not an artefact of clonal selection and variation. With this in mind, future validation work should involve testing of candidate genes in other clones, mammary cell lines or breast tumour samples that overexpress ErbB2 and by RNAi-mediated knockdown of ErbB2 expression to see if the observed effects can be reversed. Indeed, for one candidate, IGFBP3, we demonstrate it to be a negative regulator of transformation using siRNA-dependent knockdown and propose that its down-regulation enhances IGF1-dependent signalling in ErbB2-overexpressing cells.
human mammary luminal epithelial cell
foetal calf serum, qRT-PCR: quantitative real time-PCR
small interfering RNA
insulin-like growth factor 1
Insulin-like growth factor binding protein 3
We thank Dr Alan Mackay for his gift of reference RNA and Prof Michael Waterfield for supervision and useful discussion. This work was funded by the Ludwig Institute for Cancer Research and the Association for International Cancer Research (project grant award 05-426) and was partly undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme.
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