The HER family is composed of four receptor tyrosine kinases, which are frequently deregulated in several types of cancer. Activated HER receptors initiate intracellular signalling pathways by attracting to the plasma membrane a plethora of adaptor and signalling molecules. Although there are more than a dozen HER-interacting proteins that regulate signal transduction and have been extensively studied, recent proteomic studies have shown the existence of many novel but largely uncharacterized factors that may bind HER receptors. In this report, we describe a cell-based identification of several new HER2-binding proteins, including HAX1, YWHAZ, PELO and ACP1. Analysis of these factors showed that one of them, PELO, binds to active HER2 and epidermal growth factor receptor and thereby attenuates phosphatidylinositol 3-kinase (PI3K)/AKT signalling, likely through regulation of the recruitment of p85-PI3K to activated receptor. Functional characterization of PELO showed that it negatively regulates cell migration and metastasis in vivo. These results reveal that PELO is a novel regulator of HER-signalling and therefore is likely to have a role in inhibiting tumour progression and invasion.
The epidermal growth factor receptor (EGFR, also known as HER1 or ERBB1) is the prototype of a receptor tyrosine kinase family that comprises three additional members: HER2 (neu, ERBB2), HER3 and HER4 (ERBB3 and ERBB4). HER receptors react to the binding of epidermal growth factor (EGF)-like ligands by forming homo- or heterodimers. Despite a strong overall similarity, each of the receptors has its own unique functional properties. Remarkably, HER2 does not bind any known ligands, instead the structure of its extracellular domain is constitutively ready for heterodimerization with a ligand-bound family member.1 The dimerization results in juxtaposition and subsequent activation of the intracellular kinase domains. Autophosphorylation of certain tyrosine residues in the carboxyl-terminal domain of the activated receptors triggers the recruitment of proteins bearing phosphotyrosine-binding domains, that is, SRC-homology-2 or phosphotyrosine-binding domains.2 Some of these proteins, such as SHC1 or GRB2, have been extensively characterized and shown to contribute to the initiation of intracellular signalling pathways, which in turn regulate different gene expression programs.3 Other proteins, such as the negative regulator of HER-signalling ERRFI1 (also known as MIG6 or RALT), interact with HER receptors independently of a phosphotyrosine-binding motifs.4
HER receptors often have a causal role in malignant progression. They have been found overactivated in a variety of solid tumours through different mechanisms, including overexpression or acquisition of activating mutations.5 In fact, HER receptors are the targets of different drugs, including tyrosine kinase inhibitors and monoclonal antibodies, currently used to treat different cancers. Furthermore, signalling pathways initiated by the activated HER receptors, such as the RAS/MEK, and the phosphatidylinositol 3-kinase (PI3K)/AKT pathways are frequently overactivated in human tumours, and numerous new anti-cancer drugs are directed against components of these pathways.6
Different in vitro studies have unveiled the existence of many novel partners of the HER receptors.7, 8, 9 The interaction of most of these proteins with HER receptors in vivo remains to be confirmed and their functional relevance are currently unknown. Prompted by these studies, we conducted a proteomic analysis to identify new HER2-binding proteins using as a bait the HER2 fragment known as 100–115-kDa p95HER2 (also known as 611-CTF). This truncated form of HER2, hereafter referred to simply as p95HER2, lacks most of the extracellular domain but is constitutively active because of its ability to form homodimers maintained by disulphide bonds.10
PELO, one of the novel p95HER2-interacting proteins we identified and validated, is an evolutionary conserved and pleiotropic protein. In Drosophila melanogaster males with mutations in pelota, the gene that encodes for PELO, germ cells undergo normal mitoses. However, they are not able to complete the meiotic division as they remain arrested in late prophase. In contrast, in mutant D. melanogaster females, the mitotic division is affected during oogenesis. In addition to these germline defects, the eyes of pelota mutant flies display impaired development.11 In yeast, the accumulation of free ribosomes and a decrease in the number of polyribosomes in dom34 (the ortholog of pelota) mutants, indicates that PELO participates in the regulation of mRNA translation.12 Mice with pelo knockout fail to develop past day 7.5 of embryogenesis due to defects in cell proliferation.13 Interestingly, heterozygous pelo knockout mice showed an increase in the incidence of benign tumours, however, the link between PELO and cancer progression remained unexplored.13
Here we show that PELO binds to active HER2 and EGFR in different breast cancer cell lines. The binding of PELO to these receptors exerts an inhibitory effect as its knockdown resulted in upregulation of the PI3K/AKT pathway activity and in increased cell migration. Consistently with these results in vitro, PELO knockdown cells are more metastatic in vivo. Our results show that PELO is a novel HER receptor-binding protein that participates in the regulation of cell migration and metastasis.
Proteomic identification of novel p95HER2-interacting proteins
To identify novel HER2-interacting proteins we used MCF7 Tet-Off cells stably transfected with p95HER2, a truncated and constitutively active form of HER2, tagged with a C-terminal FLAG epitope. Note that p95HER2 migrates as two bands, which correspond to the immature intracellular precursor and the fully glycosylated form located at the cell surface.10 As expected, expression of p95HER2-FLAG resulted in the acute activation of the MEK/ERK1,2 pathway (Figure 1a).
Following a Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) approach, and to subsequently trace proteins from cells treated with or without doxycycline (that is, control cells or cells expressing p95HER2-FLAG), we introduced in the culture media arginine and lysine containing light or heavy isotopes (Figure 1b). After 24 h with or without p95HER2-FLAG expression cell lysates were subjected to anti-FLAG chromatography. Purified proteins were analysed by mass spectrometry (see Materials and Methods) and for every protein identified the ratio of enrichment resulting from p95HER2-FLAG pull-down was calculated (Figure 1c). The levels of the majority of proteins did not vary upon expression of p95HER2-FLAG, indicating that they bound to the beads or the FLAG antibody and not to the active HER2 fragment. However, the heavy/light (H/L) ratio >1 of a group of proteins indicated that they interacted with p95HER2-FLAG (Figure 1c). Validating the proteomic approach, this group of 134 proteins included several factors whose interaction with HER2 has been extensively characterized, such as SHC1, GRB2 and ERRFI15 (Figure 1c, Supplementary Figure S1 and Supplementary Table S1).
As p95HER2 is constitutively active, we expected that some of the proteins with a H/L ratio >1 would bind specifically to active HER2. To test this and to complement the proteomic approach shown in Figure 1b and c, we performed a similar analysis in cells expressing p95HER2-FLAG treated with or without Lapatinib, a small molecule inhibitor that targets the tyrosine kinase activity of HER2 (Supplementary Figure S1). Confirming our expectation, 91 of the 134 factors identified comparing control cells with cells expressing p95HER2 (Figure 1b and c) were also identified by comparing cells expressing p95HER2 treated with or without lapatinib (Supplementary Figure S1 and Supplementary Table S2).
The combined results of the two screenings resulted in a list of 261 factors (91 were identified in both screenings whereas 43 and 127 were identified only in the −/+doxycycline and −/+lapatinib screenings, respectively). Functional annotation of the factors identified showed that 28 of them could be potentially involved in intracellular signal transduction (Supplementary Figure S1C).
Validation of selected candidate p95HER2-interacting proteins
We found specific antibodies against 13 of the 28 candidate proteins putatively involved in signal transduction. To validate the interaction of these 13 factors to p95HER2, using GRB2 as a positive control, we analysed their levels in p95HER2-FLAG immunoprecipitates by western blot. As shown in Figure 2a, the levels of all proteins analysed were enriched in anti-FLAG immunoprecipitates from p95HER2-FLAG-expressing cells. Furthermore, the interaction of several of them with p95HER2 was clearly impaired by Lapatinib. As a result of this analysis, we selected HAX1, YWHAZ, PELO and ACP1 for further characterization because the enrichment of these proteins in p95HER2-FLAG immunoprecipitates was similar to that of GRB2 and because the treatment with Lapatinib clearly impaired the binding of these factors to p95HER2 (Figure 2b). HSP90 was not further analysed because its interaction with HER2 has been previously characterized.14
HAX1 and PELO interact with active full-length HER2 and EGFR
To determine whether the p95HER2-binding proteins identified also interact with full-length HER2, we used breast cancer cells transduced with HER2, as well as BT474 (a breast cancer cell line that naturally overexpresses the tyrosine kinase receptor). All the factors analysed interacted with HER2 expressed in MDA-MB-231 or MCF7 cells (Figures 3a and b). The analysis with BT474 cells confirmed that the binding of HAX1 and PELO to HER2 was impaired upon inhibition of the tyrosine kinase of the latter. The levels of these factors in HER2 immunoprecipitates were reduced by Lapatinib treatment by 61 and 56%, a reduction similar to that of GRB2 (Figure 3c). In contrast, the treatment with Lapatinib only reduced moderately the levels of YWHAZ (36%) and ACP1 (12%).
Most of the factors known to bind HER2 also interact with other HER receptors.8 Therefore, we tested the interaction of HAX1 and PELO with EGFR. To do so, first we analysed the expression levels of HAX1 and PELO in different breast cancer cell lines. All the cell lines analysed expressed detectable levels of both factors, albeit at different levels. We chose MDA-MB-231 cells because they can be used in a variety of functional assays (see below) and they expresses readily detectable levels of HAX1 and PELO, and high levels of the EGFR (Supplementary Figure S2A). As shown in Figure 3d–f and Supplementary Figure S2B, EGFR efficiently interacted with both HAX1 and PELO in MDA-MB-231 cells. These results demonstrate that HAX1 and PELO bind preferentially to active HER2 in different cell lines and that these factors also interact with the EGFR.
PELO attenuates activation of the PI3K/AKT pathway by inhibiting the interaction between EGFR and p85-PI3K
To functionally characterize the novel HER2- and EGFR-interacting proteins, we analysed the effect of knocking down HAX1 or PELO from MDA-MB-231 cells treated with EGF. Although the knockdown of these factors did not have an effect on activation of the MEK/ERK1,2 pathway (Supplementary Figure S3), reduction in the levels of PELO, but not those of HAX1, resulted in an increase in the activation of the PI3K/AKT pathway as judged by the analysis of the levels of P-AKT levels (Figure 4a–c). As a control, we showed that the knockdown of SHC1 led to a decreased activation of the PI3K/AKT pathway. We confirmed the relevance of the effect PELO knockdown had on PI3K/AKT by analysing the levels of P-PRAS40, a substrate of P-AKT15 (Supplementary Figure S4A and B). In addition, knockdown of PELO also led to an increased activation of the mammalian target of rapamycin pathway, measured as an elevated level of P-S6, after stimulation of EGFR (Supplementary Figure S4C and D).
Although, within HER receptors, HER3 is considered the main activator of the PI3K/AKT pathway (reviewed in Baselga and Swain16), EGFR and HER2 can also activate the PI3K/AKT pathway by recruiting the p85 regulatory subunit of PI3K (PIK3R1), directly or via the adaptor protein Gab1.17, 18 As MDA-MB-231 cells do not express HER3 or HER4,19 we concluded that activation of the PI3K/AKT pathway induced by EGF in these cells (Figures 4a and b) is mediated, at least in part, by the recruitment of p85-PI3K to activated EGFR. Supporting this conclusion, the levels of P-AKT were reduced by Iressa, a specific EGFR tyrosine kinase inhibitor, in MDA-MB-231 cells treated with EGF (Supplementary Figure S4E). Furthermore, the knockdown of PELO induced a clear increase in the levels of p85-PI3K that interacted with EGFR (Figure 4d), indicating that activation of the pathway induced by low levels of PELO is mediated by an increase in the recruitment of p85-PI3K to active EGFR. These results strongly suggest that activation of the PI3K/AKT pathway by EGFR is under the control of PELO, a protein that interacts with the receptor and negatively regulates the recruitment of p85-PI3K.
Knockdown of PELO increases cell migration and invasion
Although the PI3K/AKT pathway is known to positively regulate cell proliferation and survival, the knockdown of PELO led to a slight reduction in the rate of cell proliferation in vitro (Figure 5a). However, it concomitantly resulted in an increased cell migration through uncoated membranes in a Boyden chamber (Figures 5b and c). Twelve hours after seeding cells on top of the filter, the numbers of control and PELO knockdown cells attached at the bottom of the filter were similar (Figure 5b). However, the number of PELO knockdown cells that, after migrating through the filter, detached and accumulated at the bottom of the well was significantly higher than the number found with control cells (Figures 5b and c). A similar reduced adhesion has been observed in cells that shift from mesenchymal-to-amoeboid migration (see, for example, Oppel et al.20). Consistent with its lack of effect on the intracellular signalling pathways initiated by HER receptors, the knockdown of HAX1 did not have an effect on cell migration (Figure 5b and c).
To confirm these results and to generate cells with stable knockdown of PELO, we transduced MDA-MB-231 cells with vectors expressing two different shRNAs targeting PELO. Selected clones of the two shRNAs displayed levels of PELO around 55 and 31% relative to cells expressing a control shRNA (Supplementary Figure S5A–D). The stably reduced levels of PELO did not significantly reduce cell proliferation (Figure 5d), but augmented cell migration through uncoated membranes (Figure 5e, left), confirming the results in Figure 5b and c. Treatment with the PI3K inhibitor GDC-0941 inhibited the migration of PELO knockdown cells to levels similar to those of control cells (Figure 5e, right, and Supplementary Figure S4F), indicating that the activation of the PI3K/AKT pathway induced by the low levels of PELO is responsible for the increase in cell migration. The effect of PELO knockdown on cell migration was also evident when using filters covered with matrigel (Figure 5f), suggesting that downmodulation of PELO enhances cell invasiveness.
Low levels of PELO result in a more metastatic phenotype
Cell migration is a prerequisite for tumour invasion and metastasis. To determine whether PELO exerts an inhibitory effect on metastasis, we transduced cells knocked down for PELO with a vector expressing luciferase, a widely used reporter to monitor metastasis in vivo (see, for example, Kang et al.21), and injected the resulting cells, or corresponding control cells, orthotopically into nude mice.
In agreement with its lack of effect on cell proliferation (Figure 5d), growth of the primary tumours was not affected by the stable knockdown of PELO (Figure 6a; Supplementary Figure S6). When the tumours attained a volume of ∼400 mm3, they were surgically removed. Two months after primary tumour resection we did not detect any sign of metastatic growth in mice injected with cells expressing the control shRNA (Figure 6b, upper lane). In contrast, metastatic growth was evident in three out of eight animals (38%) injected with PELO knockdown cells (Figure 6b, middle lane). When the animals were killed, ex vivo analysis of luminescence confirmed the metastatic growth and showed that the metastasis detected in vivo were located at the axillary lymph nodes and/or the lungs (Figure 6b, lower lane; Supplementary Figure S7).
Collectively, the results presented here show that PELO interacts with HER2 and EGFR, and that it exerts an inhibitory effect on the PI3K/AKT pathway by negatively regulating recruitment of the p85 regulatory subunit of PI3K. The upregulation of the PI3K/AKT pathway induced by lowering the levels of PELO does not significantly affect cell proliferation but increases cell migration and invasion. These effects are important in vivo, as the knockdown of PELO resulted in increased metastasis in mice. This suggests that PELO is likely to be relevant in preventing malignant progression in human cancer.
Recent high-throughput in vitro approaches have analysed the interactome of the intracellular domain of activated HER receptors.7, 8, 9 Despite the limitations of in vitro models, such as aberrant folding of recombinant proteins and peptides or lack of physiological conditions for protein–protein interactions, these reports showed the existence of many uncharacterized HER receptor-interacting proteins. Using a cell-based model we have confirmed the complexity of the interactome of HER receptors; in addition to extensively characterized factors known to bind HER receptors, including ERFFI1, GRB2, SHC1, HSP90 or PIK3R1, we identified several new partners of HER2.
Some of the factors identified (HAX1, YWHAZ, PELO and ACP1) preferentially bound to active HER2. However, none of them contain SRC-homology-2 or phosphotyrosine-binding domains, indicating that if they interact directly with HER2, they do not bind to phosphotyrosine residues. Alternatively, these factors may contain unknown phosphotyrosine-binding domains. The second possibility is feasible as novel phosphotyrosine-binding domains are still being discovered and characterized.22 Another possibility is that binding to phosphotyrosines is indirect, through a protein containing a SRC-homology-2 or phosphotyrosine-binding domain. Future characterization of the binding of these factors to HER2 and EGFR, using recombinant purified proteins, will help to distinguish between these possibilities.
Although we validated the binding of the four factors mentioned to ectopically expressed HER2 in different cell lines, only HAX1 and PELO bound preferentially to active endogenous HER2 and EGFR. Curiously, it has been shown recently that PELO interacts with HAX1.23 However, the interaction of PELO with HER2 or EGFR is not likely to occur via HAX1 as the knockdown of the latter has no effect on PI3K/AKT signalling whereas the knockdown of PELO activates this pathway. However, we cannot rule out that the interaction of HAX1 with the receptor is established via PELO.
Receptor tyrosine kinases activate the PI3K/AKT pathway directly, via the recruitment of the p85 regulatory subunit unit of PI3K, or indirectly, via RAS; the activated small GTPase directly binds and activates PI3K (for a recent review see Engelman6). As HER3 and, to a minor extent, HER4 could mask the activation of the PI3K/AKT by EGFR or HER2, we chose as an experimental system the MDA-MB-231 cell line, which lack HER3 and HER4.19 In these cells, we showed that the PI3K/AKT pathway is activated by the recruitment of the p85 regulatory subunit and that the contribution of activated RAS is limited, if any. This conclusion is based on the fact that although the knockdown of PELO activated the PI3K/AKT pathway, it had no effect on the levels of phospho-ERK1,2 (Supplementary Figure S3), a downstream target of the RAS/MEK pathway. Furthermore, although the knockdown of PELO did not affect the RAS/MEK pathway, it did have a clear effect on the recruitment of p85-PI3K and activation of the PI3K/AKT pathway (Figure 4).
Our demonstration of a novel receptor tyrosine kinase/PI3K regulatory function of PELO does not exclude that the previously described functions in regulation of translation12 and/or cell division11, 13 could be equally important in breast cancer cells. Indeed, very efficient knockdown of PELO with siRNAs (Figure 4c) did lead to a reduction in cell proliferation (Figure 5a), which we did not expect based on the increased activity of the PI3K/AKT/mammalian target of rapamycin pathway in those cells (Figure 4a and Supplementary Figure 4A–D).
Two lines of evidence linking low levels of PELO to cancer progressions have been published. Heterozygous pelo knockout mice show an increase in the incidence of benign tumours.13 Presumably, these mice expressed levels of PELO similar to those in our knockdown cell lines (Supplementary Figure S5 and S7); therefore, the results with knockout mice, as well as ours, point to a tumour-suppressor effect of PELO. In support of this conclusion, a heterozygous non-sense mutation, which introduces a stop codon after amino acid 26 and generates a protein product most likely inactive, has been described recently in an ovary serous carcinoma.24 Interestingly, an inspection of the PELO mRNA levels in two independent publically available genomic data sets revealed that it is particularly underexpressed in the aggressive basal-like breast cancer (Supplementary Figure S8A and B), a subtype that frequently exhibits overactivation of the EGFR.25 Although a correlation analysis including all breast cancer subtypes displayed an inverse correlation between PELO levels and PI3K/AKT activity (Supplementary Figure S8C), only samples of the basal-like subtype had frequent deletion of the pelo gene (Supplementary Figure S8D).
In conclusion, the results presented here unveil the existence of a novel partner of activated HER2 and EGFR with the ability to regulate the PI3K/AKT pathway and as a result of that cell migration and metastasis.
Materials and methods
The cell lines MCF10A, MDA453, MDA-MB-231, BT474, MCF7, T47D and SkBr3 were purchased from ATCC (Manassas, VA, USA). Unless otherwise stated MDA-MB-231, BT474, MCF7, T47D and SkBr3 were grown at 37 °C in presence of 5% CO2 and in DMEM/F-12 (Invitrogen, Paissley, UK) supplemented with 8% fetal bovine serum (FBS) and 4 mM L-glutamine. MDA453 was propagated without CO2 in Leibovitz’s L-15 (Invitrogen) supplemented with 8% FBS and 4 mM L-glutamine. The MCF10A cells were maintained with 5% CO2 in DMEM/F-12 supplemented with 8% FBS, 4 mM L-glutamine, 9 μg/ml insulin (Sigma, Steinheim, Germany), 0.5 μg/ml hydrocortisone and 20 ng/ml EGF. The stable clones MCF7 Tet-Off/p95-HER2-FLAG and MCF7 Dox-Off/HER2-FLAG were selected as describe for 611-CTF and HER2, respectively, in Pedersen et al.,10 except a sequence encoding the FLAG epitope was inserted before the stop codon at the 3′ end of the coding regions.
To follow the proteins from cells with and without expression of p95-HER2-FLAG, we used SILAC with components from Invitrogen and according to the principles described in Blagoev et al.26 MCF7 Tet-Off/p95-HER2-FLAG cells were grown with 1 μg/ml doxycycline in SILAC DMEM medium supplemented with 8% dialyzed serum, 4 mM L-glutamine and the appropriate normal amino acids, in addition to isotopically distinct arginine and lysine (12C14N ‘light’ arginine and 12C ‘light’ lysine or 13C15N ‘heavy’ arginine and 13C ‘heavy’ lysine) at 45 μg/ml and 90 μg/ml, respectively, for 6 cell-doublings. Cells labelled with light or heavy isotopes were detached with trypsin/EDTA and washed three times in a medium with and without doxycycline, respectively, and seeded at 40% confluence. After 12 h the medium was changed and dishes were incubated for an addition 12 h with or without doxycycline. Light and heavy total protein lysates were prepared, 22 ml each of 2 mg/ml, by lysing the cells in immunoprecipitation (IP) lysis buffer: 1% NP-40, 150 mM NaCl, 50 mM Tris pH 7.4, 5 mM β-glycerophosphate, 5 mM NaF, 1 mM Na3VO4 and Complete protease inhibitor cocktail (Roche, Mannheim, Germany). The lysates were incubated with 0.25 ml anti-FLAG M2 agarose beads with gentle tilting at 4 °C for 3 h. After a brief centrifugation, 90% of the supernatants were aspirated and the beads were transferred to Ultrafree-CL Centrifugal Filter Units with 0.22 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and washed with 3 × 2 ml lysis buffer. Bound proteins were eluted with 1 ml 100 μg/ml 3 × FLAG peptide by incubation at room temperature for 1 h. Next the light and heavy lysates were combined and concentrated 10 times with 3 kDa Amicon Ultra-0.5-ml Centrifugal filters (Millipore). Half of the concentrated sample was fractionated in one lane of a precast NuPAGE 4–12% Bis-Tris gel 1.5 mm 10 well (Invitrogen). The one-dimensional gel lane was cut into 20 horizontal slices, and each slice was subjected to in-gel tryptic digestion and liquid chromatography–mass spectrometry analysis as described in Garcia-Castillo et al.27 For protein quantitation, H/L ratios were calculated averaging the measured H/L ratios for the observed peptides, after discarding outliers. For selected proteins of interest, quantitation of data obtained from the automated WARP-LC analysis was manually reviewed.
When mentioned, cells were washed and kept in medium over-night (ON) without FBS, followed by stimulation with 2 nM EGF. Where indicated, EGFR, HER2 or PI3K was inactivated by ON treatment with 1 μM Iressa (AstraZeneca, London, UK), 2 μM Lapatinib (GlaxoSmithKline, London, UK) or 0.5 μM GDC-0941 (Selleckchem, Munich, Germany). Total protein cell lysates were prepared with IP lysis buffer including 0.25% deoxycholate. Fractionation of 15 μg total protein per sample was done in NuPAGE 4–12% Bis-Tris gels. For blotting we used PVDF membranes (Millipore, Carrigtwohill, Ireland). Antibodies against the following proteins were used for probing: AKT, CTNNA1, EEF1A1, EGFR, HSP90, p85-PI3K, P-AKT, P-ERK1/2, P-HER2, P-PRAS40, P-S6 and YWHAZ (Cell Signaling, Leiden, the Netherlands); ACP1, DSTN, NME1, PELO, PFN1, RAB13, TBL2 (Sigma, St Louis, MO, USA); GRB2, SHC1 (BD Bioscience, San Jose, CA, USA); GAPDH (Trevigen, Gaithersburg, MD, USA); GNAS (AbCam, Cambridge, UK); HAX1 (R&D systems, Minneapolis, MN, USA); HER2 (BioGenex, San Ramon, CA, USA).
For Immunoprecipitation of receptors with FLAG-tag, we used the same procedure as described for the SILAC experiment, except the eluted material was not combined but concentrated and fractionated individually. In the case of Myc-tag HAX1, we used 80 μl anti-Myc-coupled beads (Cell Signaling) for 4 mg total protein per sample and enriched proteins were eluted with 1 × SDS loading buffer. To pull-down endogenous HER2 from BT474 cells with or without 24-h pretreatment with 0.25 μM Lapatinib, we incubated 80 mg total protein with 3.2 mg Herceptin (Roche) at 4 °C for 2 h, added 3.2 ml Protein A beads (GE Healthcare, Uppsala, Sweden) and incubated the sample an additional 1 h at 4 °C. Due to the amount of beads used they were washed by spin precipitation, but the 1 × SDS loading buffer elutions were passed through a 0.22-μm PVDF Centrifugal Filter Unit. Likewise, the endogenous EGFR from MDA-MB-231 cells was immunoprecipitated with Cetuximab (Bristol-Myers Squibb, New York, NY, USA) using IgG1 (antibodies-online.com, Aachen, Germany) as a negative control. In samples where a kinase inhibitor had been used in the culture medium it was also included in the IP lysis buffer.
Expression of FLUC, HER2 or HER2-FLAG in MDA-MB-231 cells was accomplished by cloning the corresponding sequences in the retro-viral vector pQCXIH (Clontech, Mountain View, CA, USA), whereas EGFR, EGFR-FLAG, PELO, PELO-FLAG, HAX1 and HAX1-Myc were expressed from the lenti-viral vector pLEX (Open Biosystems, Thermo Scientific ABgene, Epsom, UK). Viruses from pQCXIH were prepared with the GP2-293 packaging cell line, the envelope encoding plasmid pVSV-G and Lipofectamine 2000 (Invitrogen). Lenti viruses were prepared with HEK293T and Trans-Lenti Packaging kit from Open Biosystems. Virus containing supernatants were filtered with 0.45-μm PVDF filters (Millipore), and medium from 1 cm2 packaging cells was used to infect 2 cm2 MDA-MB-231 cells at ∼35% confluence. Approximately 90% infection efficiency was verified by a parallel control expressing GFP around 3 days after transduction.
MDA-MB-231 cells expressing ∼55 and 31% of the levels of PELO found in the parental cells were selected with 0.8 μg/ml puromycin starting 3 days after transduction with the lenti-viral plasmids (Open Biosystems) pGIPZ-shRNAmir-PELO clone V3LHS_328599 and V3LHS_328597. Cells transduced with the pGIPZ-Non-Silencing-Control expressed the same levels of PELO as parental cells.
The levels of HAX1, PELO or SHC1 were transiently knocked down by Lipofectamine RNAiMAX reverse transfection of the corresponding ON-TARGETplus siRNAs (Dharmacon, Chicago, IL, USA). The sequences were the following: PELO (5′-IndexTermGAAAUAAUCUCCACGUACU-3′, 5′-IndexTermUUAAAUGAUUGCCGUACAA-3′, 5′-IndexTermGUGUGGUACUGGAGCGCAU-3′ and 5′-IndexTermGCGUGGAGGCCAUCGACUU)-3′; HAX1 (5′-IndexTermGGAUACGUUUCCACGAUAA-3′, 5′-IndexTermUACAGUAACCCGACACGAA-3′, 5′-IndexTermGGACAGACACUUCGGGACU-3′ and 5′-IndexTermAAUAGCAUCUUCAGCGAUA-3′); SHC1 (5′-IndexTermGACAAUCACUUGCCCAUCA-3′, 5′-IndexTermGAGUUGCGCUUCAAACAAU-3′, 5′-IndexTermCACGGGAGCUUUGUCAAUA-3′ and 5′-IndexTermGACUAAGGAUCACCGCUUU-3′). As a negative control cells were transfected with the ON-TARGETplus Non-Targeting Pool siRNAs (ref 77D–001810–10–20). Cells were lysed or seeded for functional analyses 4 days after transfection.
Cell proliferation was followed by measurement of WST1 (Roche) conversion. Cells were detached with trypsin/EDTA, at time 0 cells (2000/well) were seeded in a 96-well plate and incubated.
Cell migration and invasion were determined with 24-well format Boyden chambers containing FluoroBlok PET membranes with 8-μm pores without or with precoated matrigel (BD biosciences). Wells were filled with medium containing 0.5% FBS and 2 nM EGF. ON FBS-starved cells were detached with 10 mM EDTA, washed and resuspended in medium without FBS. At time 0 cells (20 000/insert) were seeded on top of membranes and an identical volume of cell suspension was seeded in a standard 24-well plate as a control of cell number. The assay was terminated by fixing membranes with cells in 4% formaldehyde for 15 min at room temperature and staining with Green CMFDA CellTracker (Invitrogen). Where shown, the PI3K inhibitor GDC-0941 was included in the well (0.5 μM). For each experiment and condition, migration across the membranes was quantified for eight independent inserts by acquiring an image of the bottom of the membrane and one of the well both at the centre with a × 10 objective and manual counting of cell numbers.
Mouse model of breast cancer metastasis
Mice were maintained and treated in accordance with institutional guidelines of Vall d'Hebron University Hospital Care and Use Committee. 1.5 × 106 cells were injected into the mammary fat pad of gland number 4 of 7-week-old female BALB/c nude mice purchased from Charles Rivers Laboratories (L'Arbresle Cedex, France). Tumour xenografts were measured with calliper every 7 days, and tumour volume was calculated using the formula: (length × width2) × (pi/6). At the end of the experiment, the animals were anaesthetized with a 1.5% isoflurane air mixture and killed by cervical dislocation. Results are presented as mean±s.d. of tumour volume. Metastatic colonization was monitored by in vivo bioluminescence imaging using the IVIS-200 imaging system from Xenogen (PerkinElmer, Waltham, MA, USA) as described in Minn et al.28
Genomic bioinformatics analyses
Gene expression microarray data, DNA copy number data, and reverse-phase protein array data were obtained from The Cancer Genome Atlas (TCGA) project portal (http://cancergenome.nih.gov/) of the following tumour types: breast cancer,29 lung squamous cell carcinoma,30 ovarian cancer,31 glioblastoma multiforme32 and colorectal cancer.33 Subtypes calls were used as provided in each TCGA publication. For gene expression microarray analyses, 244K Agilent-based data (Agilent Technologies, Santa Clara, CA, USA) from all tumour types was filtered by requiring the Lowess normalized intensity values in both sample and control to be >10. The normalized log2 ratios (Cy5 sample/Cy3 control) or log2 intensity of probes mapping to the same gene (Entrez ID as defined by the manufacturer) were averaged to generate independent expression estimates. In each cohort, genes were median centred and standardized to zero mean and unit variance. To confirm the findings in breast cancer, an independent, and publicly available, gene expression microarray data set was evaluated, which is composed of 337 breast samples (UNC337 data set: GSE18229).34 Subtype calls were used as provided. Finally, we estimated the activation of the PI3K pathway using the TCGA RRPA data and the formula provided in the TCGA breast cancer report.
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This work was supported by the Instituto de Salud Carlos III (Intrasalud PI081154 and the network of cooperative cancer research (RTICC-RD06/0020/0022)), the Breast Cancer Research Foundation, the Asociación Española Contra el Cancer (AECC) and AVON Foundation. KP was supported by the postdoctoral program from the AECC.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Pedersen, K., Canals, F., Prat, A. et al. PELO negatively regulates HER receptor signalling and metastasis. Oncogene 33, 1190–1197 (2014). https://doi.org/10.1038/onc.2013.35
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