An Oncogenic Role of Eif3e&Sol;INT6 in Human Breast Cancer

M Grzmil1, T Rzymski2, M Milani2, AL Harris2, RG Capper1, NJ Saunders1, A Salhan3, J Ragoussis3 and CJ Norbury1

1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK; 2Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK and 3Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

Altered expression of the eukaryotic translation initiation 2005; Bramham and Wells, 2007; Paquin and factor 3 (eIF3) subunit eIF3e/INT6 has been described Chartrand, 2008). Deregulation of translation initiation in various types of human cancer, but the nature of can lead to oncogenic transformation and can support its involvement in tumorigenesis is not yet clear. Using cancer growth, suggesting that targeting translation may immunohistochemical analysis of 81 primary breast be a valuable therapeutic approach (Thumma and cancers, we found that high tumor grade correlated Kratzke, 2007; Wendel et al., 2007; Mavrakis and Wendel, significantly with elevated cytoplasmic eIF3e level in 2008). Protein synthesis is controlled by multiple transla- epithelial tumor cells. Analysis of protein synthesis after tion initiation factors (eIFs), many of which have been siRNA-mediated knockdown in breast cancer cell lines implicated in tumorigenesis (Watkins and Norbury, 2002; indicated that eIF3e is not required for bulk translation. Dong and Zhang, 2006). Nonetheless, despite numerous Microarray analysis of total and polysomal RNAs none- studies of the expression of eIFs in human cancers, a theless identified distinct sets of mRNAs regulated either detailed picture of how deregulation of eIF function might positively or negatively by eIF3e; functional classification modulate the quality of translation in such a way as to of these revealed a marked enrichment of genes involved contribute to tumor progression is currently lacking. in cell proliferation, invasion and apoptosis. Validated Translation initiation factor eIF3, comprising 13 mRNA targets regulated positively at the translational protein subunits in human cells, coordinates interactions level by eIF3e included urokinase-type plasminogen between mRNA and the 40S ribosomal subunit, governs activator and apoptotic regulator BCL-XL, whereas translation reinitiation and functions as a platform for synthesis of proteins including the mitotic checkpoint interactions with other regulatory eIFs (Hinnebusch, component MAD2L1 was negatively regulated. Finally, 2006). Components of eIF3 have previously been eIF3e-depleted breast carcinoma cells showed reduced reported to support malignant transformation and in vitro invasion and proliferation. Taken together, our tumor growth. Individual overexpression of any of five study data suggest that eIF3e has a positive role in breast subunits of human eIF3 (eIF3a, b, c, h or i) promoted cancer progression. It regulates the translation, and in malignant transformation of immortal fibroblasts some cases abundance, of mRNAs involved in key aspects (Ahlemann et al., 2006; Savinainen et al., 2006; Zhang of cancer cell biology. et al., 2007). By contrast, studies of eIF3e/INT6 have Oncogene (2010) 29, 4080–4089; doi:10.1038/onc.2010.152; suggested various roles for this accessory eIF3 subunit published online 10 May 2010 either as an oncoprotein or a tumor suppressor (Marchetti et al., 2001; Rasmussen et al., 2001; Buttitta Keywords: MAD2L1; BCL-XL; PLAU; urokinase-type et al., 2005; Chen et al., 2007). plasminogen activator; breast cancer Here, we describe elevated expression of eIF3e in high-grade breast cancers and identify an eIF3e- regulated subset of messenger RNAs involved in cancer Introduction development. Our study data suggest that eIF3e has an oncogenic role in the promotion of breast cancer cell The overall rate of protein synthesis is an important proliferation and invasion. determinant of cell and tissue function. In addition, alterations in the activities of the translational machin- ery has key functions in controlling gene-specific Results expression, through the selective translation of specific subsets of messenger RNAs (Holcik and Sonenberg, Elevated eIF3e protein levels in human breast cancer The murine Int-6 gene, encoding eIF3e, was identified as Correspondence: Dr CJ Norbury, Sir William Dunn School of a site of mouse mammary tumor virus (MMTV) Pathology, University of Oxford, South Parks Road, Oxford OX1 integration in MMTV-induced tumors and a precancer- 3RE, UK. E-mail: [email protected] ous lesion (Marchetti et al., 1995; Asano et al., 1997). In Received 3 October 2009; revised 6 March 2010; accepted 5 April 2010; each case the MMTV provirus was integrated within an published online 10 May 2010 Int-6 intron, potentially leading to the production of a eIF3e in breast cancer M Grzmil et al 4081 C-terminally truncated form of eIF3e. We therefore staining of eIF3e in paraffin-embedded MDA-MB-231 used western and northern blotting of lysates from cell cells was effectively eliminated by EIF3E siRNA lines to investigate the possibility that truncated forms transfection (Figure 1d), indicating that our antibody of eIF3e might contribute to human breast cancer is also suitable for the analysis of eIF3e expression in development. A single eIF3e species was present at both paraffin sections from primary human breast cancers. the protein and the RNA levels, and was expressed at a Immunohistochemical staining of 81 such cancers comparable level in all the cell lines tested (Figure 1a), showed epithelium-specific, cytoplasmic staining in with no detectable expression of truncated forms. To many tumors (Figure 1e). Interestingly, there was a confirm specificity of the eIF3e signals, we transfected positive correlation (P ¼ 0.001) between expression of cells with two siRNAs against EIF3E; either alone or in eIF3e and tumor grade assessed on the Scarff–Bloom– combination, their transfection led to substantial eIF3e Richardson scale, which takes into account the degree of knockdown as observed by western blotting (Figure 1b) tubular differentiation, mitotic index and nuclear and northern blotting (Figure 1c) analyses. Immuno- pleiomorphism. Grades I–III indicate progression from

Figure 1 Expression of eIF3e in human breast cancer cells. (a) Expression levels of eIF3e protein and EIF3E mRNA were determined by western (upper panels) and northern blotting (lower panels), respectively. Loading controls were provided by staining the western blot with Ponceau S (total protein) and the RNA gel with ethidium bromide (total RNA); the positions of 28S and 18S ribosomal RNAs are indicated. (b–d) Specific downregulation of eIF3e expression in MDA-MB-231 cells transfected with one or both of two pairs of EIF3E-specific siRNA oligonucleotides, as indicated ( þ ); the EIF3E 1 duplex was used in c and d. Reversed sequence oligonucleotides were used as controls. (b) Western blotting of extracts from MDA-MB-231 cells performed 72 h after transfection using an eIF3e-specific antibody. Actin-specific antibodies were used as loading control. Expression levels are shown as the ratio of eIF3e/actin signals; ratios in controls were normalized to 1.0. (c) Northern blotting performed 48 h after transfection, using the human EIF3E ORF as a probe. Methylene blue staining of the same filter served as a loading control (lower panel). (d, e) Immunostaining of eIF3e in sections of formalin-fixed, paraffin-embedded, MDA-MB-231 cells 72 h after siRNA transfection (d) and six examples of the 81 primary human breast cancers screened (e). Sections were stained with an eIF3e-specific antibody (brown) and counterstained with hematoxylin (blue). Bar, 50 mm.

Oncogene eIF3e in breast cancer M Grzmil et al 4082 well differentiated (low grade) to poorly differentiated possible involvement in the regulation of a speciﬁc (high grade); strong cytoplasmic eIF3e staining subset of proteins. was observed in 4 of 9 (44%) grade I, in 38 of 45 To investigate this point further, we transfected (84%) grade II and in 26 of 27 (96%) grade III tumors. MDA-MB-231 cells with siRNA against EIF3E or a These data suggest a potential role for eIF3e in human control duplex for 48 h, at which point total and breast cancer progression. Nuclear staining was gen- polysome-associated (that is, actively translated) RNAs erally weaker and was present in only 25 of 81 (30%) of the tumors, in line with a primary role for eIF3e in translational regulation; there was no signiﬁcant correlation between nuclear eIF3e positivity and tumor grade.

eIF3e regulates a specific subset of mRNAs Previous reports have concluded that, besides binding other components of eIF3, eIF3e can associate separately with subunits of the proteasome (Asano et al., 1997; Yen et al., 2003; Zhou et al., 2005). We therefore further investigated the potential involvement of eIF3e in regulation of global protein levels through translation initiation and/or proteolysis through the ubiquitin– proteasome pathway. Knockdown of eIF3e in MDA- MB-231 or U2OS cells had no significant impact on bulk protein synthesis as measured by 35S-methionine pulse labeling (Figure 2a). Similarly, sucrose density gradient separation revealed no major changes in polysome distribution following eIF3e knockdown in MDA-MB-231 cells (Figure 2b). There was, however, an increase in the relative abundance of free 60S ribosomal subunits in eIF3e-depleted cells. Very similar traces were obtained using lysates from cells transfected with the alternative pair of EIF3E and control siRNA duplexes (data not shown). Protein profiles generated by 2D liquid chromatographic separation of whole cell lysates similarly showed no significant changes on eIF3e knockdown (data not shown) and there was only a minor increase in the level of total protein ubiquitinylation (Figure 2c). Ubiquitination of MCM7 was slightly increased on eIF3e knockdown, whereas total MCM7 level was slightly decreased (Supplementary Figure 1), as reported previously (Buchsbaum et al., 2007). These data indicate that eIF3e does not influence global protein levels in human cells and suggest instead its

Figure 2 eIF3e knockdown has no major impact on bulk protein synthesis in MDA-MB-231 or U2OS cells. (a) Bulk protein synthesis in MDA-MB-231 or U2OS cells, 48 h after transfection with EIF3E-specific siRNA or control oligonucleotides, as indicated, as measured by 35S-methionine incorporation during a 2 h labeling period followed by SDS–PAGE and autoradiography (upper panel) or Coomassie blue staining (lower panel). Lane M, molecular weight markers; lane U, cells incubated in the absence of radiolabeled methionine. (b) Polysome profiles obtained by sucrose density gradient centrifugation of cell lysates prepared 48 h after transfection with siRNA duplex EIF3E 1 or control 1. The A254 peaks corresponding to ribosomal subunits and polysomes are indicated. (c) Total protein ubiquitinylation was assayed by western blotting using an anti-ubiquitin (Ub) antibody and cell lysates prepared 72 h after transfection with the siRNA duplexes indicated. The same membrane was rehybridized with antibodies against eIF3e and tubulin. A lysate from cells treated for 1 h with the proteasome inhibitor MG132 (10 mM) provided a positive control for the accumulation of ubiquitinylated proteins, which were quantified using the ratio between ubiquitin- and tubulin-specific signals. Ratios in control transfected cells were normalized to 1.0.

Oncogene eIF3e in breast cancer M Grzmil et al 4083 were compared separately using microarray hybridization. We identified 300 differentially expressed mRNAs (Po0.05) using polysomal RNA, and 153 using total RNA (Figure 3a; Supplementary Tables 1 and 2). Of these, 24 targets were common to both polysomal and total RNAs. Notably, the abundance and/or polysome association of some mRNAs was decreased on eIF3e knockdown, whereas others were increased. Four groups of genes, namely those positively or negatively regulated by eIF3e in polysomal or total RNA, were subjected to gene ontology (GO) analysis and functional classification using the DAVID web-based tool (Table 1). This analysis strongly suggested that eIF3e positively regulates a group of mRNAs encoding proteins involved in coagulation, taxis and endocytosis, and negatively regulates genes controlling cell division and adhesion. The altered expression of eight of these targets was validated using reverse transcriptase (RT)– PCR analysis of polysomal and total RNAs (Figures 3b and c). The expression of three eIF3e-regulated targets was analyzed at the protein level (Figures 4a and b). Decreased PLAU and BCLXL, and increased MAD2L1 protein levels were found in eIF3e-depleted MDA-MB- 231 and U2OS cells. In addition, RT–PCR analysis of RNA immunoprecipitated using an eIF3b-specific antibody showed that eIF3-associated BCLXL mRNA was decreased and MAD2L1 mRNA enriched following eIF3e knockdown in MDA-MB-231 cells (Figures 4c and d). The abundance of eIF3e therefore appears to modulate the activity of the whole eIF3 complex in such a way as to determine the association of mRNAs with eIF3 in a selective manner.

Inhibition of eIF3e expression leads to reduced cellular invasion and cell proliferation Our expression profiling data suggested that increased eIF3e levels might promote motility and decrease adhesion, cellular properties relevant to cancer invasion and metastasis. To assess the effect of eIF3e suppression on invasion more directly, we incubated MDA-MB-231 and U2OS cells transfected with control or EIF3E- specific siRNA in Matrigel chambers, which mimic the extracellular matrix and provide the basis for a quantitative assay of invasion (Figure 5a). The invasive capacity of eIF3e-depleted cells 48 h after transfection was reduced to 59% (MDA-MB-231) or 36% (U2OS) that of control transfected cells (Po0.01). At this time Figure 3 Expression profiling of total and polysome-associated point there was no significant difference in the pro- mRNAs from eIF3e-depleted and control transfected MDA-MB- 231 cells. (a) Venn diagram summarizing the numbers of mRNAs liferation of the two cell populations as judged by 3-(4,5- significantly affected by eIF3e knockdown (for further details see dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide- Supplementary Tables 1 and 2). (b) Experimental validation of based assay (Figure 5b). Nonetheless, eIF3e-depleted microarray data by semiquantitative RT–PCR; relative PLAU and MDA-MB-231 and U2OS cells showed significantly ACTB (b-actin control) levels in total and polysomal RNA were measured using 30 or 32 PCR cycles, as indicated. (c) Relative reduced cellular proliferation 72 h after siRNA transfec- expression of further examples of eIF3e targets involved in taxis tion (Po0.05). (SAA1, CMTM7, CXCL1), adhesion (COL5A1), cell-cycle regulation (MAD2L1, CDC34) and apoptosis (BCL2L1) was determined. PCR products were generated using 25 or 30 cycles for total and polysomal RNAs, respectively. Discussion (Figure 1e), plays an oncogenic role by regulating the The data presented here suggest that eIF3e, expression expression of gene products involved in cancer growth of which is elevated in advanced human breast cancer and invasion. Despite identification of the Int-6 gene

Oncogene eIF3e in breast cancer M Grzmil et al 4084 Table 1 GO analysis of microarray data Process (GO term): gene symbols of enriched mRNAs Enrichment

Positively regulated by eIF3e (polysomal RNA) Coagulation (GO:0050817), blood coagulation (GO:0007596): PLAU, SAA1, CD40, LPA 6.7 Taxis (GO:0042330), chemotaxis (GO:0006935): PLAU, SAA1, CMTM7, CXCL1 5.0

Negatively regulated by eIF3e (polysomal RNA) M phase (GO:0000279): MAD2L1, TOP3A, DYNC1H1, SHC1, RAD54L, LIG3, POLS, CCNF, SMC2L1 6.8 Cell division (GO:0051301): MAD2L1, LIG1, CCND1, LIG3, POLS, CDCA8, CCNF, SMC2L1 6.7 Mitotic cell cycle (GO:0000278): MAD2L1, CDC34, DYNC1H1, SHC1, CCND1, POLS, CCNF, SMC2L1 5.7 Homophilic cell adhesion (GO:0007156): FREM3, PCDHGB1, PCDHGC3, DSG1, FAT 5.4 Cell adhesion (GO:0007155): COL5A1, COL18A1, FARP1, THBS1, TNC, FREM3, PCDHGB1, PCDHGC3, DSG1, FAT 2.3

Positively regulated by eIF3e (total RNA) Enodocytosis (GO:0006897): SCARF1, RIN2, APLP1, CORO1C 5.6

Enrichment is scored relative to the number of genes corresponding to each GO term that would be expected if the genes were drawn at random from the total gene list. Only those GO terms with a signiﬁcant enrichment (Po0.05, Fisher’s exact test) are listed.

encoding eIF3e as a site of MMTV integration esis in different cell types. The applicability to other (Marchetti et al., 1995), the biological role of eIF3e in tumors of our findings in primary breast cancer there- tumorigenesis has proved difficult to determine. The fore warrants further study. expression of truncated EIF3E mRNA in mammary Human eIF3e was first identified as the p48 subunit epithelial cells induced a transformed phenotype of eIF3 (Asano et al., 1997). In addition, eIF3e has (Mayeur and Hershey, 2002) and in a mouse model been reported to interact with subunits of the protea- truncated eIF3e expression led to persistent hyperplasia some and the COP9 signalosome, suggesting its possible and tumorigenesis in mammary alveolar epithelium involvement in the regulation of both protein synthesis (Mack et al., 2007). These findings could be consistent and degradation (von Arnim and Chamovitz, 2003). either with a gain of function for the truncated alleles, or Inactivation of fission yeast eIF3e compromised protea- with a dominant negative effect. In line with the latter some function and caused the accumulation of interpretation, genetic screens detected loss of EIF3E polyubiquitinylated proteins (Yen et al., 2003). In heterozygosity in human breast carcinomas (Miyazaki Drosophila eIF3e was shown to be a positive regulator et al., 1997); analyses of EIF3E mRNA expression in of cullin neddylation, thus regulating degradation primary human breast and non-small-cell lung carcino- through the ubiquitin–proteasome pathway of sub- mas similarly suggested a potential role of eIF3e as a strates of cullin-containing ubiquitin ligases (Rencus- tumor suppressor (Marchetti et al., 2001). Reduced Lazar et al., 2008). Other studies reported specific expression of eIF3e was observed in 37% of breast interaction of eIF3e with HIF-2a (Chen et al., 2007) cancers and 31% of non-small-cell lung carcinomas, and or with MCM7 (Buchsbaum et al., 2007), leading to appeared to predict poor prognosis in early-stage non- proteasomal degradation or stabilization of the eIF3e small-cell lung carcinoma (Buttitta et al., 2005). How- partner protein, respectively. In our experiments, eIF3e ever, in the latter study 73% of all tumors had EIF3E knockdown induced a slight accumulation of polyubi- RNA levels greater than those observed in matched quitinylated proteins (Figure 2c), including MCM7 normal lung samples, whereas only 27% had reduced (Supplementary Figure 1), leaving open the possibility levels of EIF3E RNA. that some of the biological consequences observed might Our description of a positive correlation between reflect an involvement of eIF3e in ubiquitin-dependent elevated eIF3e protein level and high tumor grade proteolysis. provides evidence of an oncogenic role for eIF3e in The most striking effect of eIF3e downregulation seen breast cancer. In line with this view, other studies of here was, however, at the level of altered polysome eIF3e expression in breast, colon, lung and ovarian association of specific mRNAs (Figures 3 and 4), tumors have suggested a role for eIF3e together with supporting the view that regulation of translational TID1 and Patched proteins in cell growth and tumor- initiation is a major function of eIF3e. This regulatory igenesis (Traicoff et al., 2007). In a zebrafish model we function is clearly selective, as eIF3e knockdown had no found previously that eIF3e was a tissue-specific positive major effect on global translation (Figure 2a), and modulator of the MEK–ERK pathway, a key signaling inhibited polysome association of some mRNAs while pathway in the development and progression of human stimulating that of others, but the basis of this selectivity cancers (Grzmil et al., 2007). The apparently discrepant is currently unclear. eIF3 binds specifically to certain findings of studies of eIF3e function in human cancers viral internal ribosome entry sequences (Buratti et al., might in part reflect the use of different methodologies 1998; Lopez de Quinto et al., 2001; Siridechadilok et al., (for example, measuring mRNA level or loss of 2005) and, with eIF2, can promote mRNA binding to heterozygosity rather than protein expression), or could preinitiation complexes (Jivotovskaya et al., 2006), but indicate that eIF3e contributes differently to tumorigen- eIF3e itself does not possess a known RNA-binding

Oncogene eIF3e in breast cancer M Grzmil et al 4085 domain. One clear possibility is that binding of eIF3e Dissociation of eIF3 from preinitiation complexes is induces a conformational change in the eIF3 complex to associated with 60S ribosomal subunit joining (Benne alter its affinity for regulatory elements on cellular and Hershey, 1978), though it is unclear whether eIF3 mRNAs. This change in affinity could be positive or actively inhibits 60S joining. The increased abundance negative, according to the mRNA in question. of free 60S subunits seen in eIF3e-depleted cells (Figure 2) suggests that the subunit composition eIF3 can indeed influence 60S joining to some extent.

Figure 5 Depletion of eIF3e reduces cancer cell invasion and proliferation. MDA-MB-231 and U2OS cells were transfected either with EIF3E-speciﬁc siRNA or with reversed sequence (control) siRNA. (a) Cells invading through Matrigel were quantiﬁed 48 h after transfection as a percentage of those seen in the control transfected population (mean±s.d. of three independent experiments). (b) Proliferation of control and EIF3E siRNA- transfected cells was assayed using an MTT-based method at the indicated times after transfection. Data shown are the mean±s.d. values of at least three separate experiments.

Figure 4 Decreased PLAU (uPA) and BCL-XL, and increased MAD2L1 protein levels in eIF3e-depleted cells. MDA-MB-231 and U2OS cells were transfected either with EIF3E-specific siRNA duplexes (EIF3E 1 or EIF3E 2) or with reversed sequence duplexes (control 1 or control 2). (a) At 48 h after transfection, growth medium was collected and PLAU (uPA) levels were determined by ELISA. Results are expressed as means±s.d. of three independent measurements. PLAU levels in control transfected cells were normalized to 1.0. (b) At 72 h after transfection, whole-cell lysates were subjected to western blot analysis using antibodies against MAD2L1, BCL-XL or eIF3e; a-tubulin was used as a loading control. (c, d) Whole-cell lysates from untreated MDA-MB-231 (U) and siRNA-transfected cells were used for immunoprecipitation with an eIF3b-specific antibody. (c) Immunoprecipitates and whole-cell lysates were subjected to western blot analysis using antibodies against eIF3e or eIF3b; as a negative control, one sample from untreated cells was processed as for immunoprecipitation but in the absence of primary antibody (no 11). (d) Immuno- precipitates were subjected in parallel to RNA extraction followed by RT–PCR using primers specific for BCL2L1 (which encodes BCL-XL), MAD2L1, EIF3E or ACTB (b-actin control).

Oncogene eIF3e in breast cancer M Grzmil et al 4086 This could reflect a direct interaction between the two the mitotic spindle attachment checkpoint; its dysfunc- complexes, or alternatively an influence of eIF3 on other tion, through either reduced or increased expression, can events required for 60S joining, such as GTP hydrolysis cause aberrant chromosome segregation, and hence by eIF2 (Merrick, 1979). In agreement with our results, contribute to malignancy, in mammalian cells (Michel fission yeast strains lacking eIF3e are viable, and et al., 2001; Sotillo et al., 2007). Inhibition of eIF3e polysome profile analysis showed that they have no expression was shown previously to delay mitotic major defects in translation initiation (Bandyopadhyay progression in human cells (Morris and Jalinot, 2005); et al., 2000; Crane et al., 2000), though a decreased level our findings suggest that elevated MAD2L1 expression of de novo protein synthesis was detected by pulse may contribute to this phenotype. labeling in a more recent study (Udagawa et al., 2008). Taken together, the data presented here suggest a Fission yeast eIF3e and eIF3m define two distinct eIF3 model in which high levels of eIF3e support breast complexes that may promote the translation of different cancer progression by regulating translation of mRNAs sets of mRNAs; the eIF3m complex associated with involved in cancer growth, invasion and apoptosis. Our bulk cellular mRNAs, whereas the eIF3e-containing RNA immunoprecipitation data (Figures 4c and d) complex associated with a far more restricted set (Zhou suggest that these effects may be mediated principally et al., 2005). In line with this view, fission yeast eIF3e through the role of eIF3e as a component of the eIF3 was found to be required for maintaining the basal level translation initiation factor, rather than through some of the Atf1 transcription factor (Udagawa et al., 2008). alternative role of eIF3e. Our immunohistochemistry Our expression profiling of breast cancer cells data indicate that measurement of eIF3e levels could in following eIF3 knockdown identified 300 mRNAs future provide valuable prognostic information in breast regulated positively or negatively at the translational cancer. In the longer term, it may even be possible to level (Figure 3a; Table 1), of which 24 were also altered design therapeutic agents to reverse eIF3e-mediated in abundance in total RNA preparations, possibly changes in the pattern of protein synthesis. reflecting their stabilization by increased translation. Intriguingly, GO analysis revealed that numerous genes involved in cell-cycle-related processes were negatively Materials and methods regulated by eIF3e at the level of translation (Table 1). Although this might initially seem at odds with the Chemicals and antibodies positive correlation between eIF3e protein levels and Mouse monoclonal anti-actin antibody (AC-40), horseradish tumor grade and proliferation in vitro (Figure 5), the peroxidase secondary antibodies and proteasome inhibitor relationship between cell proliferation index and malig- MG132 were from Sigma-Aldrich (Gillingham, UK). The nancy is not straightforward; other aspects of tumor cell rabbit polyclonal anti-eIF3e antibody CN24 (Watkins and biology are likely more important determinants of Norbury, 2004) was used in all experiments unless otherwise tumor grade. Several of the targets identified, including indicated. Keith Gull (University of Oxford, UK) generously PLAU, MAD2L1 and BCL2L1, are clearly implicated in provided the mouse monoclonal anti-a-tubulin antibody such aspects of tumorigenesis. Urokinase-type plasmi- (TAT-1). Goat polyclonal antibodies against eIF3b (N-20), PLAU (C-20), MAD2L1(C-19) and rabbit polyclonal anti- nogen activator (PLAU/uPA) is positively regulated by ubiquitin (FL-76) were from Santa Cruz Biotechnology (Santa eIF3e (Figures 3 and 4), and is known to degrade the Cruz, CA, USA), the rabbit monoclonal antibody against extracellular matrix, promoting invasion and metastasis BCL-XL was from Cell Signaling (Danvers, MA, USA). of malignant tumors including breast cancer, in which elevated PLAU expression is correlated with poor Patients and tissue samples outcome (Han et al., 2005). The reduced invasion Formalin-fixed paraffin-embedded tissue blocks and corre- activity of eIF3e-depleted cells (Figure 5) is likely to sponding pathology reports were obtained for sequential result, in part at least, from decreased PLAU levels. patients with breast adenocarcinomas (surgery was performed BCL2L1 mRNA (encoding BCL-XL) was similarly at the John Radcliffe Hospital, Oxford, UK). Tissue micro- positively regulated by eIF3e at the level of polysome arrays were assembled as described previously (Bubendorf association, and our RNA-IP experiments confirmed et al., 2001) with three replicate cores for each tumor. its eIF3e-dependent recruitment to eIF3 complexes Approval was obtained for the use of all human tissue from (Figures 4c and d). Cancer cells overexpressing BCL- the local research ethics committee (reference C02.216). XL are generally resistant to a wide range of anticancer drugs (Minn et al., 1995), suggesting that breast cancer Cell culture and transfection cells with high eIF3e expression might be inherently Breast cancer MDA-MB-231, MDA-MB-435, T47D, MCF-7 resistant to therapy as a result of increased BCL-XL and osteosarcoma U2OS cells were grown in Dulbecco’s translation. Interestingly, eIF3e was previously isolated modified Eagle’s medium medium (Invitrogen, Paisley, UK) through its ability to induce multidrug resistance when containing 10% fetal bovine serum. MCF-10A breast epithe- overexpressed in fission yeast (Crane et al., 2000). lial cells were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium (50:50 ratio) with 10% fetal Although this model organism lacks BCL-2 family bovine serum, 5 mg/ml hydrocortisone, 10 ng/ml epidermal proteins, eIF3e was proposed to target selectively growth factor and 10 mg/ml insulin. All cells were cultured at mRNAs required for protective cellular stress responses 37 1C in a humidified incubator with 5% CO2 and grown to (Zhou et al., 2005). MAD2L1, which was negatively 50–60% confluence for transfection, which was accomplished regulated by eIF3e (Figures 3 and 4), is a component of using Lipofectamine (Invitrogen) according to the supplier’s

Oncogene eIF3e in breast cancer M Grzmil et al 4087 Table 2 Oligonucleotide primers used for RT–PCR analysis shearing through a 25-gauge needle, lysates were clarified by centrifugation (5 min, 13 000 g) and were precleared with Gene Forward primer Reverse primer protein G-Sepharose. Clarified lysates were incubated over- EIF3E 50-atggcggagtacgacttgact 50 -tcagtagaagccagaatcttgagt night at 4 1C with 1 mg of appropriate antibody and 50 mlof SAA1 50-tgacatgagagaagccaattacat 50-tctctggatattctctctggcatc protein G-Sepharose (Cancer Research UK). Immunoprecipi- CMTM7 50-aactacagcgcctacagctacttt 50-gactgggttacacacgagatctta tates were washed three times with NP-40 lysis buffer, half of CXCL1 50-ccaagaacatccaaagtgtgaa 50-agttggatttgtcactgttcagc the immunoprecipitates were used for RNA extraction using COL5A1 50-tgatggaataacaaagacaacagg 50-tttcacagttgttaggatggagaa RNAeasy Mini Kit (Qiagen) and the rest was separated by BCL2L1 50-atgtctcagagcaaccgggagctg 50-tcatttccgactgaagagtgagcc 10% SDS–PAGE and subjected to immunoblotting as MAD2L1 50-ggccatggcgctgcagctctcccg 50-tgtcatcctcagtcattgacagga 0 0 described above. For uPA detection in growth medium human CDC34 5 -atggctcggccgctagtgcccagct 5 -tcaggactcctccgtgccagagtc uPA ELISA Kit (Assay Pro, St Charles, MO, USA) was used ACTB 50-cgtgatggtgggcatgggtca 50-cttaatgtcacgcacgatttcc PLAU 50-tccaacaagtacttctccaacatt 50-gatcacccagcaagggctgatgag according to the manufacturer’s protocol. All assays were performedintriplicate. instructions with either gene-specific siRNA duplexes (EIF3E Immunohistochemistry 1, EIF3E 2) or reversed sequence control RNA oligonucleo- Formalin-fixed, paraffin-embedded tissue or transfected tides (control 1 and control 2) at a final concentration of MDA-MB-231 cell pellet sections were treated with DeWax 100 nM in Optimem (Gibco). EIF3E 1 sense RNA: 50- solution (Biogenex, San Ramon, CA, USA) according to user CAGGGAUGGUAGGAUGCUCdTdT-30; EIF3E 2 sense manual followed by incubation with sodium citrate buffer (pH RNA: 50-GAACCACAGUGGUUGCACAUU-30. Cells and 8) for 2 min and with 0.03% hydrogen peroxide (Dako, Ely, growth medium were collected at 24, 48 and 72 h after UK) for the next 5 min. Horse serum (2.5%) was used for transfection for subsequent analysis. For proliferation assays, blocking. Sections were incubated overnight with diluted 1:50 cells were split into 24-well plates 6 h after transfection. After (in phosphate-buffered saline, PBS) eIF3e-specific antibody 2, 24, 48 and 72 h of incubation, EZ4U substrate (50 ml per (CN24) and washed with PBS. The EnVision system (Dako) well; Biozol, Eching, Germany) was added and absorbance at was used for signal visualization according to the manufac- 450 nm was measured with a plate reader (GE Healthcare, turer’s instructions and we counterstained sections with Amersham, UK). All assays were performed in triplicate. hematoxylin. The levels of cytoplasmic and nuclear eIF3e staining were assessed semiquantitatively to produce an Northern blot analysis and RT–PCR reactions intensity distribution score (IDS) on a 12-point scale as Total RNA was isolated using an RNeasy Mini Kit (Qiagen, described previously (Winters et al., 2001). Average IDS values Crawley, UK) according to the manufacturer’s instructions. were determined by examination of 10 fields. Data were For northern blot analysis, total RNA was separated by analyzed using STATA (STATA Corporation, College Sta- denaturing agarose gel electrophoresis and transferred to a tion, TX, USA) and Pearson’s correlation coefficients were Hybond-C nylon membrane (GE Healthcare). An EIF3E ORF used to determine the association between eIF3e IDS (using a generated by RT–PCR (1338 bp) was cloned into pGEM-T cutoff value for IDS of 2 to distinguish between ‘high’ and Easy plasmid (Promega, Southampton, UK), sequenced and ‘low’ eIF3e staining) and tumor grade. used as a probe. The probe was labeled with [a-32P] dCTP using a Rediprime II labeling kit (GE Healthcare) and Sucrose density gradient centrifugation hybridized to membranes in Rapid-hyb buffer (GE Health- Cells were incubated for 3 min at 37 1C with 0.1 mg/ml care) containing 100 mg/ml denatured salmon sperm DNA at cycloheximide, washed in PBS, harvested in polysome lysis 65 1C for 16 h. Membranes were washed at room temperature buffer (1% Triton X-100, 300 mM NaCl, 15 mM MgCl2,15mM for 15 min in 2 Â SSC, then in 0.5 Â SSC, 0.5% (w/v) SDS for Tris-HCl (pH 7.4), 0.1 mg/ml cycloheximide and 0.33 U/ml 15 min at 65 1C. Hybridization signals were detected with a RNAse inhibitor), incubated for 5 min on ice, mixed by Fuji FLA-5000 imager (Fujifilm, Milton Keynes, UK) and vortexing and centrifuged at 2000 g for 5 min. The super- quantified using Aida software (Raytest Isotopenmessgera¨te, natants were supplemented with heparin to a final concentra- Straubenhardt, Germany). RT–PCR and semiquantitative tion of 200 mg/ml, centrifuged at 10 000 g for 5 min to remove RT–PCR analyses were performed using a one-step RT– cell debris and layered onto 20–50% sucrose gradients for PCR kit (Qiagen) with the primers listed in Table 2. fractionation (90 min at 39 000 r.p.m. in a Beckman SW40.1 rotor and L8 ultracentrifuge at 4 1C). After centrifugation, 30 Immunoblotting, RNA immunoprecipitation and ELISA fractions (0.5 ml) were harvested with continuous monitoring Whole-cell lysates were prepared using lysis buffer (150 mM of A254 using a gradient fractionator (Bio-Rad, Hemel NaCl, 10 mM EDTA, 50 mM Tris–HCl (pH 7.6), 1% Triton Hempstead, UK). The fractions were used for polysomal RNA X-100, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml isolation using TriReagent (Sigma-Aldrich) and the RNA was phenylmethylsulfonyl fluoride). Lysates (50 mg per lane) were further purified using RNeasy Mini columns (Qiagen). separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE). Proteins were electrotransferred to Hybond-C Microarray analysis nitrocellulose membranes (GE Healthcare) before being Before labeling, RNA concentration and integrity were incubated with appropriate antibodies and processed for determined using Nanochips on a 2100 Bioanalyzer (Agilent ECL detection (ECL Plus; GE Healthcare) according to the Technologies, Stockport, UK), according to the manufac- manufacturer’s protocol. Signals were analyzed using Lab- turer’s instructions. Two labeling and hybridization protocols Works software (UV Products, Cambridge, UK). For im- were used in conjunction with two types of arrays. For Human munoprecipitation, cell lysates were prepared at 0 1C in NP-40 OpArrays (Operon, Huntsville, AL, USA), containing lysis buffer (1% NP-40, 150 mM NaCl, 20 mM Tris–HCl (pH oligonucleotide probes representing approximately 39 600 7.5), 2 mM EDTA, 1 mg/ml leupeptin, 1 mg/ml aprotinin and transcripts, a dendrimer-based system was used according to 1 mg/ml phenylmethylsulfonyl fluoride) supplemented with the manufacturer’s instructions, with minor modifications. 0.2 U/ml RNaseOUT inhibitor (Invitrogen). After repeated Briefly, RNA (1 mg of total or polysomal RNA) was labeled

Oncogene eIF3e in breast cancer M Grzmil et al 4088 using the 3DNA Array 900 kit (Genisphere, Hatfield, PA, USA), Invasion assay using Superscript III reverse transcriptase (Invitrogen). The Transfected cells were suspended at 5 Â 104 cells per ml in hybridization and detection steps were performed using a complete medium, and 500 ml of each cell suspension was two-step procedure on a SlideBooster (Advalytix, Munich, placed in a BioCoat Matrigel Invasion Chamber (BD Germany), with a power setting of 25 and a pulse ratio of 3:7 Biosciences, Oxford, UK) and incubated for 20 h at 37 1C. at 55 1C. The first hybridization was for 16 h using hybridization Invasive cells adhered to the chamber membranes, which were buffer EB, and the second was for 4 h using SDS buffer. For fixed in methanol, washed with PBS and mounted on human 22K genome-wide printed cDNA arrays (v1.0.0) from microscope slides in Vectashield mounting medium with DAPI Cancer Research UK, an Amino Allyl MessageAmp aRNA Kit (Vector Laboratories, Peterborough, UK). Images were (Applied Biosystems, Warrington, UK) was used for labeling acquired and cells were counted using a Zeiss Axioskop with Cy3 and Cy5 dyes according to the manufacturer’s microscope (Carl Zeiss Ltd, Welwyn Garden City, UK) and instructions. Hybridization and washing steps were performed MetaMorph software (Molecular Devices, Sunnyvale, CA, using a Lucidea SlidePro platform (GE Healthcare). The USA). All experiments were repeated in triplicate, and the OpArray and 22K Cancer Research UK slides were scanned noninvasive breast cancer cell line MCF-7 was used as a using a ScanArray ExpressHT system (PerkinElmer, Cambridge, negative control. UK) and GenePix 4000B microarray scanner, respectively. Images were obtained using autocalibration with 100% laser power, a variable PMT and a target saturation of 90%. Poor- Conflict of interest quality spots were manually flagged, and intensity values were extracted using BlueFuse for microarrays version 2 (BlueGnome, The authors declare no conflict of interest. Cambridge, UK). Three OpArrays were used to compare total RNAs from three independent transfections and two OpArrays for polysomal RNA from two independent transfections, in each case using two pairs of siRNAs. In addition, six 22K Cancer Acknowledgements Research UK slides were used to analyze total RNA from three independent transfections together with a dye swap for each We thank Ben Thomas and Sasha Akoulitchev for help and experiment. Intensity values, extracted using BlueFuse, were advice with 2D liquid chromatography, Cheng Han for help analyzed using BASE (Saal et al., 2002). Only median fold ratio with the statistical analysis, Dan Scott and other members of values with Po0.05 (Cyber t-test) were used for subsequent the laboratory for their comments on the article. This work analysis. The DAVID web-based tool was used for GO terms was supported by Cancer Research UK, the Association for identification and functional classification of the differentially International Cancer Research and the Wellcome Trust expressed genes (Dennis et al., 2003). (through grant 075491/Z/04 to JR).

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