Oncogene (2013) 32, 3130–3138 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

ORIGINAL ARTICLE MEMO1, a new IRS1-interacting , induces epithelial–mesenchymal transition in mammary epithelial cells

AV Sorokin and J Chen

MEMO1 (mediator of ErbB2-driven cell motility 1) regulates HER2-dependent cell migration. Increased MEMO1 expression is associated with cancer aggressiveness. Here, we found that MEMO1 is also involved in breast carcinogenesis via regulating -like growth factor-I -dependent signaling events. We showed that MEMO1 binds to substrate 1, activates the downstream PI3K/Akt signaling pathway, leads to upregulation of Snail1 and thereby triggers the epithelial- mesenchymal transition (EMT) program. In addition, MEMO1 overexpression is accompanied by growth factor-independent proliferation, anchorage-independent growth in soft agar, and enhanced metastatic potential. Together, these findings suggest that MEMO1 acts as an oncogene and is a potential therapeutic target for cancer treatment.

Oncogene (2013) 32, 3130–3138; doi:10.1038/onc.2012.327; published online 23 July 2012 Keywords: MEMO1; IRS1; EMT; Snail1;

INTRODUCTION signaling adaptor , the deregulation of which leads to MEMO1 (mediator of ErbB2-driven cell motility 1), a 297 amino- tumorigenesis. acid protein recently shown to co-precipitate with ErbB2/HER2, is There are limited reports regarding MEMO1 in tumorigenesis. involved in breast carcinoma cell migration.1 MEMO1 regulates The MEMO1 has been shown to be amplified  11 in highly 22 microtubule dynamics and the formation of adhesion sites by metastatic pancreatic . Another study indicated contributing to the localization of small G protein RhoA and its that the tumor suppressor miR-125b, which represses the effector mDia1 at the plasma membrane, and recruiting actin- expression of MEMO1, is significantly underexpressed in invasive 23 binding proteins such as a-actinin.2 MEMO1 also controls the breast cancers. These results indicate that MEMO1 over- plasma membrane localization of APC and CLASP2, which are expression may lead to cell transformation and migration. In this required for efficient microtubule capture and stabilization.3 study, we report that MEMO1 is a new IRS1-binding protein. The The three-dimensional (3D) structure of MEMO1 has been purpose of this study is to elucidate the molecular mechanism by determined and was shown to be homologous to class III which MEMO1 participates in tumorigenesis and the development nonheme iron-dependent dioxygenases. MEMO1 binds directly of a highly metastatic . to a specific ErbB2/HER2-derived phosphopeptide using its vestigial enzymatic active site.4 On the basis of these findings, it has been proposed that MEMO1 should be considered as a new RESULTS class of phosphotyrosine-binding proteins.4 MEMO1 level correlates with IGF-IRb level in breast cancer cell The insulin receptor substrate (IRS) proteins were originally lines identified as signaling intermediates of the insulin receptor (IR), To investigate the potential role of MEMO1 in tumorigenesis and and their functions in metabolic regulation have been studied metastasis of breast cancers, we analyzed the cellular level of extensively.5 Over the past decade, interest in IRSs has grown various receptors and signaling molecules in different breast because of their additional functions as effectors of insulin-like cancer cell lines. Surprisingly, we found that the protein level of growth factor-I receptor (IGF-IR) and several other receptors.6–8 MEMO1 correlates with IGF-IR, but not that of HER2, which was The ability of IRSs to modulate and coordinate multiple signaling originally identified as a MEMO1-signaling partner (Figure 1a).1 We pathways suggests that they may have a role in tumorigenesis. found that stimulation of cells with IGF-I led to MEMO1 Indeed, recent studies have implicated IRSs in human accumulation in membrane ruffle-like structures and MEMO1 co- cancers.7,9–15 In patients with breast cancer, high IRS1 localization with phospho-Tyr staining (Figure 1b). Moreover, expression levels were found to be associated with poor MEMO1 was able to interact not only with HER2, but also with outcomes and correlate with lymph node metastases.16–18 EGFR and HER4 (Supplementary Figure 1a) and stimulation of Recent studies indicated that overexpression of IRS1 or IRS2, as MCF10A cells with EGF also led MEMO1 localization to the well as of their upstream receptor IGF-IR, induced extensive membrane (Supplementary Figure 1b). These findings indicate proliferation and disrupted normal acinar morphogenesis in 3D that MEMO1 could have additional functions besides its already Matrigel cultures.19–21 Furthermore, transgenic mice that over- known role in HER2-driven motility. expressed IRS1 or IRS2 in the mammary gland showed progressive To gain insight into the potential role of MEMO1 in signaling mammary hyperplasia, tumorigenesis and metastasis.20 All of pathways, we investigated whether MEMO1 suppression would these findings support the growing consensus that IRSs are critical affect signaling through either ERK or Akt, two major signaling

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Correspondence: Professor J Chen, Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail: [email protected] Received 14 March 2012; revised 7 June 2012; accepted 20 June 2012; published online 23 July 2012 MEMO1 promotes EMT AV Sorokin and J Chen 3131

Figure 1. MEMO1 level correlates with IGF-IRb level and migration of breast cancer cell lines. (a) The indicated breast cancer cell lines were examined for MEMO1, HER2 and IGF-IRb protein levels by western blotting (WB). (b) MCF10A cells stably expressing HA-Flag-tagged MEMO1 were grown in low-serum medium (Starv.) or were stimulated by IGF-I after serum starvation and then examined for MEMO1 (Flag) and Tyr- phosphorylated proteins (pY) by IF staining. Scale bars, 20 mm. The last column shows an expanded view (scale bars, 5 mm). (c) MDA-MB-231, MCF10A, SKBR3 and BT474 cells stably transfected with control (Scr.) or MEMO1 shRNA (M.) were grown in monolayer in normal (Ctrl.) or low- serum (Starv.) medium or were stimulated by growth factors after serum starvation and then examined for signaling molecules by WB analysis using antibodies as indicated. pathways activated by EGFR and IGF-IR. We found that the PI3K/ predominantly in neuronal cells and ShcD is expressed only in Akt pathway was suppressed in IGF-IR-positive cell lines (MCF10A melanomas.24–26 When we compared the affinity of MEMO1 for and MDA-MB-231) under basal conditions (Figure 1c, compare IRS1 and Shc, we were surprised to find that MEMO1 interacted lanes 1 and 2) or after stimulation by IGF-I (Figure 1c, compare much more strongly with IRS1 than with ShcA (Figure 2d). Only lanes 5 and 6, 7 and 8). MEMO1 suppression had no noticeable MEMO1’s affinity for ShcD was as strong as its affinity for IRS1 effect on PI3K/Akt or MAPK/ERK signaling pathways in IGF-IR- (Figure 2d). This result indicates that MEMO1 binds to and may negative/HER2-positive cell lines (SKBR3 and BT474) (Figure 1c). regulate at least two different families of adapters (IRS and Shc). All these observations led us to suspect a possible HER2- In addition, the observed specificity in binding even among independent but IGF-IR-dependent role of MEMO1 in the members of one family (strong binding to ShcD and markedly regulation of cellular signaling pathways. As it is known that weaker binding to ShcA) indicates the high binding specificity of MEMO1 can bind to HER2 in a phospho-Tyr-dependent manner, MEMO1 (Figure 2d). This finding was further supported by a an obvious assumption is that MEMO1 binds to IGF-IR, IR or its preference of MEMO1 for IRS1 among IRSs (Figure 2e). Collectively, adaptors (IRSs) because these proteins are Tyr-phosphorylated these results indicate that MEMO1 is an IRS1-interacting protein. and membrane-localized in response to IGF-I treatment. MEMO1 interacts with IRS1 in a phospho-Tyr-dependent manner MEMO1 interacts with IRS1 and prevents IRS1 dephosphorylation Using IRS1 fused with a C-terminal triple-epitope (SFB; S protein, We hypothesized that, by analogy with HER2, MEMO1 also binds Flag and streptavidin-binding peptide (SBP)) tag as a bait, we to IRS1 in a phospho-Tyr-dependent manner and modulates IRS1 found that IRS1 formed a stable complex with MEMO1 (Figure 2a). functions in the cell. Indeed, we found that the stimulation with Endogenous and ectopically expressed MEMO1 binds to IRS1 IGF-I causes MEMO1 to concentrate on the plasma membrane (Figure 2b) and partially localized to the ruffle-like membrane (Figure 2c). In addition, we showed that the IRS1–MEMO1 structures and co-localized with IRS1 in MCF10A and HeLa cells interaction correlates with Tyr-phosphorylation of IRS1: the (Figure 2c). MEMO1–IRS1 interaction was markedly upregulated after treat- Interaction of MEMO1 with HER2 seemed to depend on the ment with growth factors that stimulate Tyr-phosphorylation of presence of the Shc adaptor protein.1 The Shc protein family has IRS1 (Figure 2f, lane 2) and substantially suppressed after four members: ShcA–ShcD. ShcA (p46, p52 and p66 splicing treatment with cycloheximide (Figure 2f, lane 3), which inhibits variants) is ubiquitously expressed, ShcB and ShcC are expressed IRS1 Tyr-phosphorylation.27

& 2013 Macmillan Publishers Limited Oncogene (2013) 3130 – 3138 MEMO1 promotes EMT AV Sorokin and J Chen 3132

Figure 2. MEMO1 interacts with IRS1 in a phospho-Tyr-dependent manner and regulates IRS1 Tyr-phosphorylation. (a) IRS1-associated proteins in a HEK293T stable cell line identified by mass spectrometric analysis. (b) 293T cells were transfected with empty vector constructs encoding SFB-tagged IRS1 or MEMO1. IP was performed using SBP beads for 2 h at 4 1C, and precipitated materials were then subjected to western blotting (WB) analyses using antibodies as indicated. (c) The localization of endogenous MEMO1 and IRS1 in MCF10A cells (left column), ectopically expressed HA-Flag-tagged MEMO1 and endogenous IRS1 in MCF10A-MEMO1 stable cells (center column), and transiently co-transfected HA-Flag-tagged MEMO1 and -tagged IRS1 in HeLa cells (right column). Scale bars, 20 mm. MEMO1 interacts with IRS1. (d) 293T cells were transfected with constructs encoding SFB-tagged IRS1, ShcA(p66), ShcA(p52) or ShcD or empty vector together with plasmid encoding Myc-tagged MEMO1. IP was performed using SBP beads for 2 h at 4 1C, and precipitated materials were then subjected to WB analyses using antibodies as indicated. (e) 293T cells were transfected with constructs encoding HA-Flag-tagged IRS1, IRS2 or IRS4 together with constructs encoding SFB-tagged MEMO1 (Flag-MEMO1). (f) 293T cells transiently transfected with constructs encoding SFB- tagged IRS1 together with that encoding Myc-tagged MEMO1 were grown in monolayer under normal growth conditions (Ctrl.), treated with cycloheximide (CHX, 10 mg/ml) for 1 h, or stimulated by fetal bovine serum (FBS) (20%, 10 min) after starvation (for 2 h). Cells were collected, and total cell extracts were subjected to IP using SBP beads. WB analysis was performed using antibodies as indicated. (g) MDA-MB-231 cells stably expressing MEMO1 shRNAs were examined for MEMO1, IRS1 and Tyr-phosphorylated IRS1 using antibodies as indicated. (h) IRS1 proteins were immunoprecipitated from MCF10A cells stably expressing HA-Flag-tagged MEMO1 or empty vector and examined for IRS1 Tyr-phosphorylation using antibodies as indicated.

Human IRS1, a 1243 amino-acid protein, contains a pleckstrin Grb2 and SHP27,30,31 (Supplementary Figure 2). Although the homology domain and a phospho binding domain in its binding of PI3K-p85 or Grb2 to IRS1 activates Akt or MEK/ERK N terminus, both of which are highly conserved within the IRS signaling, respectively, binding of SHP2 to IRS1 suppresses the family and are essential for binding of IRSs to IR and IGF-IR28,29 functions of IRS1 via its ability to dephosphorylate the PI3K-p85 (Figure 3a). Upon binding to upstream receptors, IRS1 is and Grb2 binding sites on IRS1.32 We found that downregulation phosphorylated on many tyrosine residues and most of of the MEMO1 level correlates with hypo-Tyr-phosphorylation of them are located at its C terminus (Supplementary Figure 2). IRS1 (Figure 2g), whereas MEMO1 overexpression leads to hyper- Tyr-phosphorylation of IRS1 modulates signal transduction from Tyr-phosphorylation of IRS1 (Figure 2h). These observations led us IGF-IR and IR to downstream effectors via the association of IRS1 to suspect MEMO1 competes with SHP2 for binding with IRS1 and with SH2-domain-containing docking molecules, such as PI3K-p85, thus prevents IRS1 dephosphorylation.

Oncogene (2013) 3130 – 3138 & 2013 Macmillan Publishers Limited MEMO1 promotes EMT AV Sorokin and J Chen 3133

Figure 3. MEMO1 Competes with SHP2 for binding to IRS1. (a) Schematic diagram of wild-type and deletion/point mutants of IRS1 used in this study. (b–e) 293T cells were transiently transfected with plasmids encoding SFB-tagged IRS1 and Myc-tagged MEMO1. IP was performed using SBP beads, and precipitated materials were then subjected to western blotting (WB) analyses using antibodies as indicated. (f) 293T cells were transiently transfected with plasmids encoding SFB-tagged IRS1, Myc-tagged Grb2, SHP2, PI3K-p85 and HA-Flag-tagged MEMO1. IP and WB analyses were performed as above.

Using a set of truncated IRS1 proteins (Figure 3a), we found that As MEMO1 predominantly binds to the two C-terminal deletion of the pleckstrin homology and the phospho tyrosine of IRS1 (Tyr-1179 and Tyr-1229), which are also major SHP2 binding domains, which are essential for binding to upstream binding sites,33 we decided to check whether MEMO1 would receptors and, thereby, critical for IRS1 Tyr-phosphorylation, compete with SHP2 for binding to IRS1. We found that dramatically reduced the interaction between IRS1 and MEMO1 co-expression of MEMO1 reduced the interaction between IRS1 (Figure 3b, N2 deletion mutant). On the other hand, a pleckstrin and SHP2, but not the interaction between IRS1 and Grb2 or PI3K- homology and a phospho tyrosine binding domain only mutant of p85 (Figure 3f). Collectively, these data support our hypothesis IRS1 did not interact with MEMO1 (Figure 3c, C4 deletion mutant). that MEMO1 is a new binding partner of IRS1, which appears to These findings support our hypothesis that MEMO1 interacts with positively regulate its functions. IRS1 in phospho-Tyr-dependent manner. Further analysis revealed that deletion of very C-terminal part of IRS1 (residues 1100–1243) dramatically reduced the interaction between IRS1 and MEMO1 MEMO1 overexpression induces proliferative and migration (Figure 3d). Only two tyrosine residues within this region of IRS1 advantages could be phosphorylated (Tyr-1179 and Tyr-1229 in human IRS1) To examine the effect of MEMO1 overexpression in mammalian (Supplementary Figure 2). Substitution of either of them reduced cells, we introduced this gene by retroviral infection into non- the interaction between IRS1 and MEMO1 (Figure 3e), indicating tumorigenic human mammary epithelial cell line MCF10A. that these two C-terminal Tyr residues are involved in IRS1/ Interestingly, in growth factor-free and serum-free medium, MEMO1 interaction. MCF10A cells that were transfected with control vector underwent

& 2013 Macmillan Publishers Limited Oncogene (2013) 3130 – 3138 MEMO1 promotes EMT AV Sorokin and J Chen 3134 growth arrest, whereas MCF10A-MEMO1 and MCF10A-IRS1 cells that in MCF10A cells, MEMO1 could mediate these migration continued to proliferate (Figure 4a). The proliferative advantages advantages through upregulation of IGF-IR/IRS1 signaling. Indeed, of MEMO1- or IRS1-overexpressing cells were accompanied by the IRS1 suppression reduced IGF-1-stimulated migration, especially in ability of these cells to produce large anchorage-independent MCF10A cells with MEMO1 overexpression (Figure 4e). colonies in soft agar (Figure 4b), a property that frequently correlates with tumorigenicity, indicating that MEMO1 may induce cell transformation. MEMO1 promotes EMT Recent reports about the correlation between MEMO1 over- MCF10A cells have been used extensively to examine the effects expression and metastatic aggressiveness of cancer cells22,23 were of various oncogenes on morphology changes in monolayer and also supported by our observations: MCF10A-MEMO1 cells acinus formation in 3D Matrigel cultures.34 Control vector- displayed a remarkable capacity for migration in response to transfected MCF10A cells grow in epithelial-type islands in growth factors in Transwell assays (Figure 4c) and a higher ability monolayer cultures (Figure 4f) and form well-organized acinar to invade through Matrigel (Figure 4d). Although the role of structures with apical-basal polarity in 3D Matrigel (Figure 4g). We MEMO1 in the regulation of HER2-dependent migration of breast confirmed previous reports that MCF10A-IRS1 cells display cancer cells has been previously studied.1–3 However, MCF10A scattering and loss of cell–cell contacts in monolayer (Figure 4f) cells have a very low level of HER2 (Figure 1a), thus we suspected but form larger disrupted irregular colonies in 3D Matrigel

Figure 4. MEMO1 enhances growth factor-independent proliferation, anchorage-independent growth and migration and disrupts mammary acinar architecture and promotes EMT in MCF10A cells. (a) Growth curves of MCF10A cells stably transfected with empty vector or expressing HA-Flag-tagged MEMO1 or IRS1 cultivated in serum-free medium. (b) The indicated MCF10A-derivative cell lines were plated in soft agar and allowed to grow for 20 days. Data are the average numbers of colonies counted in five fields of view (±s.d.). Representative fields are shown. Scale bars, 100 mm. (c, d) Migration (c) or invasion (d) of indicated MCF10A-derivative cell lines toward fetal bovine serum (FBS) or IGF-I were determined by Transwell migration or invasion assay. Migration/invasion toward 0.5% FBS was used as a negative control. Data are the means of two independent experiments counted in triplicate (±s.d.). (e) The indicated MCF10A-derivative cell lines were stably infected with viruses overexpressing scrambled (Scr.) or IRS1 shRNAs. Cells were collected and examined by western blotting (WB) analysis (upper panel) using antibodies as indicated or subjected to Transwell migration assay (lower panel) as described above. (f, g) The indicated MCF10A-derivative cell lines were grown in monolayer (f) or Matrigel (g) and examined by phase-contrast microscopy (f) and IF staining (g). Scale bars, 100 mm. (h, i) MCF10A cells stably transfected with empty vector or expressing HA-Flag-tagged MEMO1 were grown in monolayer and examined by WB analysis (h) and IF staining (i) for the expression of epithelial/mesenchymal markers, as indicated. Scale bars, 20 mm.

Oncogene (2013) 3130 – 3138 & 2013 Macmillan Publishers Limited MEMO1 promotes EMT AV Sorokin and J Chen 3135 (Figure 4g).20,35 We found that MCF10A-MEMO1 cells also display medium (Supplementary Figure 3). These results confirmed that cell scattering and loss of cell–cell contacts (Figure 4f), which can EMT was induced by MEMO1 expression in these cells. explain the migration advantages of these cells. MEMO1 expres- sion also disrupted the normal morphogenesis of MCF10A cells in 3D Matrigel: MCF10A-MEMO1 cells failed to form spherical acinar- MEMO1 triggers EMT in an IRS1/Snail1-dependent manner like structures similar to the control vector-transfected cells. Various stimuli within the tumor microenvironment promote Staining of basement membranes of acinar structures with laminin V, EMT of cancer cells.36 To gain insight into the mechanism by a marker of epithelial cell polarity, showed that control vector- which MEMO1 triggers EMT, we investigated whether MEMO1 transfected MCF10A cells formed well-organized acinar structures expression could activate signaling through either ERK or Akt, with apical-basal polarity, whereas the multi-acinar structures of two major signaling pathways that can contribute to EMT in MCF10A-IRS1 and MCF10A-MEMO1 cells had disrupted polarity MCF10A cells. We found that the PI3K/Akt pathway, but not (Figure 4g). the MEK/ERK pathway, was strongly activated even under basal The highly organized cobblestone-like morphology of MCF10A conditions without stimulation by growth factors (Figure 5a). cells was replaced by spindle-like fibroblast morphology in Moreover, we observed that the PI3K/Akt pathway was MCF10A-MEMO1 cells, suggesting that cells overexpressing mainly activated after stimulation of cells by IGF-I, supporting MEMO1 had undergone EMT (Figures 4f and i). Indeed, the the dependence of MEMO1-induced cell signaling activation on mesenchymal markers N-cadherin and vimentin were upregulated IGF-IR/IRSs (Figure 5a). We also observed a mild effect on and the epithelial markers E-cadherin, occludin and b-catenin PI3K/Akt activation following epidermal growth factor (EGF) were all downregulated in MCF10A-MEMO1 cells, as demonstrated treatment. As MEMO1 can also interact with EGFR by immunoblotting and immunofluorescence (IF) analyses (Supplementary Figure 1a), it is possible that the fairly small (Figures 4h and i). effect on PI3K/Akt pathway following EGF stimulation might be Collectively, these morphological changes in monolayer and 3D due to such interaction. Matrigel cultures and the increased motility and invasion in A major driving force of EMT is the repression of the epithelial Matrigel indicated that MEMO1 overexpression triggers EMT in cell–cell junction protein E-cadherin. About a dozen transcriptional MCF10A cells. To examine whether MEMO1-induced EMT can be repressors (for example, Snail, Slug, Twist, ZEB1/2 and TCF reversed, we generated a stable cell line in which the MEMO1 E12/E47) can suppress E-cadherin expression in the cell.37,38 gene was placed under the control of a doxycycline-inducible Reverse transcriptase PCR and immunoblotting analyses showed . We found that doxycycline-induced MEMO1 over- that MEMO1 overexpression resulted in overexpression of Snail expression was accompanied by EMT, but this phenotype was and ZEB1/2 in MCF10A cells (Figures 5b and c), suggesting a reversed when doxycycline was removed from the growth possible mechanism for the effect of MEMO1 on EMT.

Figure 5. IRS1 and Snail1 are both required for MEMO1-induced EMT. (a) MCF10A cells stably transfected with empty vector (V.) or expressing HA-Flag-tagged MEMO1 (M.) were grown in monolayer in normal (Ctrl.) or low-serum (Starv.) medium or were stimulated by growth factors after serum starvation and then examined for signaling molecules by western blotting (WB) analysis using antibodies as indicated. (b, c) MCF10A cells, as in (a), were examined for levels of signaling molecules and transcription factors by WB analysis and reverse transcriptase PCR. (d, e) MCF10A cells stably expressing HA-Flag-tagged MEMO1 were transiently infected with viruses overexpressing scrambled (Scr.), IRS1 or Snail shRNAs and were examined by WB analysis (d) and IF staining (e) using antibodies as indicated. Scale bars, 20 mm. (f) The role of MEMO1 in a current model of how the activation of the IGF-IR/IRSs signaling pathway triggers the EMT program.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3130 – 3138 MEMO1 promotes EMT AV Sorokin and J Chen 3136 MEMO1 depletion suppresses aggressive cancer MEMO1-induced EMT was consistent with the proposed As MEMO1 overexpression led to enhanced proliferation and mechanism of IGF-IR/IRSs/Akt/Snail-dependent EMT. Akt was migration in non-tumorigenic MCF10A cells, we asked whether activated in MEMO1-overexpressing cells (Figure 5a), resulting in MEMO1 would be required for the invasive phenotypes in breast the accumulation of Snail and ZEB1 proteins (Figure 5c). Moreover, cancer cells using short hairpin RNAs (shRNA) to knockdown the PTEN level was markedly decreased, whereas the IRS1 level was MEMO1 expression in highly metastatic MDA-MB-231 cells increased (Figure 5c). The decrease in the PTEN level could be (Figure 6a). Decreased proliferation of MEMO1-depleted cells explained by the suppression of its transcription by Snail; the (Figure 6b) was accompanied by the decreased ability of these increased IRS1 level was probably due to protein stabilization cells to form anchorage-independent colonies in soft agar (Figure 5c). More directly, we showed that knockdown of IRS1 or (Figure 6c). Normal mammary epithelial cell forms spherical Snail1 in MCF10A-MEMO1 cells de-repressed E-cadherin synthesis acinar-like structures in 3D Matrigel (Figure 4g), whereas MDA- (Figures 5d and e). Collectively, these findings indicate that MEMO1 MB-231 cells forms large disorganized invasive colonies triggers EMT in MCF10A cells in an IRS1/Snail-dependent manner. (Figure 6d). We found that MEMO1 suppression dramatically Information about the expression and functions of MEMO1 is reversed this invasive phenotype in MDA-MB-231 cells, as colonies very limited. Transcriptome analysis revealed that the level of formed with MEMO1-knockdown cells were smaller and did not MEMO1 expression in mice is significantly higher during have projections into Matrigel (Figure 6d). Indeed, we also embryogenesis than in adult mice (http://www.ebi.ac.uk). A similar observed decreased migration and invasion in MEMO1-knock- result was shown for Drosophila melanogaster: the level of CG8031 down cells (Figure 6e). Together, these data indicate that MEMO1 mRNA (the fly ortholog of MEMO1) was elevated during knockdown in MDA-MB-231 cells suppress aggressive cancer embryogenesis and metamorphosis stages. These findings indi- phenotypes in these cells. cate a possible role of MEMO1 in developmental processes such as gastrulation and organogenesis. All known MEMO1 functions support this possibility, because they all, in one way or another, are linked with the regulation of cell motility, which is essential for DISCUSSION early development.1,3,55 Here, we propose a role of MEMO1 in EMT, which is also essential during early embryogenesis and Here, we identified MEMO1 as a new mediator of IRS1: MEMO1 37 binds to IRS1 and activates the downstream PI3K/Akt signaling required for tumor dissemination. Our conclusion is consistent with reports that high level of MEMO1 correlates with a highly pathway. MEMO1/IRS1-dependent activation of Akt leads to 22,23 upregulation of Snail1, thereby triggering EMT, which is accom- metastatic phenotype in some cancers. panied by enhanced metastatic potential. A current model of how the activation of the IGF-IR/IRSs signaling pathway triggers EMT is shown in Figure 5f. The binding MATERIALS AND METHODS of IGF-I/IGF-II to the IGF-IR induces its autophosphorylation and Antibodies leads to recruitment and Tyr phosphorylation of IRS docking AKT, phospho-Ser-473-AKT, GSK3b, phospho-Ser-9-GSK3b, PTEN, MEK1/2, proteins.39 Concomitant binding and phosphorylation of the p85 phospho-Ser-217/221-MEK1/2, ERK1/2, phospho-Thr-202/Tyr-204-ERK1/2, regulatory subunit of PI3K cause PI3K activation and, via Grb2, IRS1, phospho-Tyr-612-IRS1, phospho-Tyr-1229-IRS1, IGF-IR, Snail, Slug, ZEB1, p85 and p110a antibodies were obtained from Cell Signaling PI(3,4,5)P3, lead to stimulation of PI-dependent kinase and Technology (Danvers, MA, USA). FLAG(M2), HA, b-actin, MEMO1 and activation of Akt.40–42 Activation of Akt leads to the suppression 43 a-tubulin monoclonal antibodies were obtained from Sigma-Aldrich of GSK3b, which is known to phosphorylate and destabilize Snail (St Louis, MO, USA). Occludin, c-Myc(9E10) and Twist antibodies were 44,45 46 protein. NF-kB is activated in an Akt-dependent manner and obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and activates transcription of Snail and ZEB1/2 mRNAs.46–49 In b-catenin, phospho-Tyr(4G10), BrdU-Alexa488, E-cadherin and N-cadherin addition, Snail may also be involved in the activation of ZEB1/2 antibodies were obtained from BD Biosciences (San Jose, CA, USA). .50 An increase in Snail and ZEB1/2 protein expression leads to the suppression of E-cadherin, and thereby Plasmids 51,52 triggers EMT. Moreover, Snail suppresses expression of the All constructs were generated by PCR and subcloned into pDONR201 53 54 PTEN gene, which is an Akt suppressor. vector using Gateway technology (Invitrogen, Carlsbad, CA, USA).

Figure 6. MEMO1 depletion suppresses aggressive cancer phenotypes in MDA-MB-231 cells. (a, b) Growth curves of MDA-MB-231 cells stably transfected with control (Scr.) or MEMO1 shRNA (b). (c) The indicated MDA-MB-231-derivative cell lines were plated in soft agar and allowed to grow for 14 days. Data are the average numbers of colonies counted in five fields of view (±s.d.). Representative fields are shown. Scale bars, 200 mm. (d) The indicated MDA-MB-231-derivative cell lines were grown in Matrigel for 5 days and examined by phase-contrast microscopy. Scale bars, 100 mm. (e) Migration and invasion of indicated MDA-MB-231-derivative cell lines toward IGF-I or fetal bovine serum (FBS) were determined by transwell migration or invasion assay. Migration toward 0.5% FBS was used as a negative control. Data are the means of two independent experiments counted in triplicate (±s.d.).

Oncogene (2013) 3130 – 3138 & 2013 Macmillan Publishers Limited MEMO1 promotes EMT AV Sorokin and J Chen 3137 The entry clones with corresponding cDNAs were transferred into medium containing 500 mg/ml G418 and experiments were conducted Gateway-compatible destination vectors with an N-terminal Myc epitope, immediately afterwards. HA-Flag epitope, MBP or GST protein tag or a C-terminal SFB tag, or into a doxycycline-inducible vector with a C-terminal SFB tag. All constructs were Transwell migration and invasion assays verified by sequencing. Cells were starved in medium without fetal bovine serum and growth factors for 12 h, and then 5 Â 104 cells (1 Â 105 cells for the invasion assay) Reverse transcriptase PCR were placed into the top chambers of 24-well Transwell plates (8 mm; Reverse transcriptase PCR analysis was performed as described pre- Trevigen, Gaithersburg, MD, USA). For invasion assays, the chamber viously.56 The sequences of the PCR primer pairs (all listed from 50 to 30) membrane was coated with 100 ml of Matrigel (200 mg/ml). The bottom were as follows: IRS1-F: ATGGCGAGCCCTCCGGAGAGCGATGGC; IRS1-R: chambers were filled with growth medium supplemented with 10% fetal CTGACGGTCCTCTGGCTGCTTCTGGAAAC; PTEN-F: ATGACAGCCATCATCAAA bovine serum, 100 ng/ml EGF or 100 ng/ml IGF-I. Cultures were maintained GAGATCGTTAG; PTEN-R: TCAGACTTTTGTAATTTGTGTATGCTGATC; E-cadherin-F: for 18 h, and then nonmotile cells on the upper surface of the membrane TCCATTTCTTGGTCTACGCC; E-cadherin-R: TCAGGATCTTGGCTGAGGAT; SNAIL-F: were removed, and migrated/invaded cells in the bottom chamber were TTCTTCTGCGCTACTGCTGCG; SNAIL-R: GGGCAGGTATGGAGAGGAAGA; SLUG-F: stained with Crystalline violet solution. Percentages of migrated/invaded ATGCCGCGCTCCTTCCTGGTCAAGA; SLUG-R: GTGTGCTACACAGCAGCCAG cells were calculated from standard curves generated for each cell line. ATTCC; TWIST-F: ATGATGCAGGACGTGTCCAGCTCGC; TWIST-R: GTGGGA CGCGGACATGGACCAGGCC; ZEB1-F: ATGAAAGTTACAAATTATAATACTG 3D matrigel and soft-agar assays TGG; ZEB1-R: GGCTTCATTTGTCTTTTCTTCAGACACT; ZEB2-F: ATGAAGCAG The 3D Matrigel assay was performed as described previously.19 Cells CCGATCATGGCGGATGGCC; ZEB2-R: CATGCCATCTTCCATATTGTCTTCCTCG; 3 TCF E12-F: ACCAGCCCAGACGAGGACGAGGACGACC; TCF E12-R: GGGCTTC (2.5 Â 10 ) were added to 1.5 ml of growth medium with 0.25% agar and TCGCTGTTGAGGTGCAG; TCF E47-F: CAGTACGGACGAGGTGCTGTCCCTGG; layered onto 2 ml of 0.5% agar beds in six-well plates. Cells were fed with TCF E47-R: CGCTTTGTCCGACTTGAGGTGCAT; actin-F: ATGGATGATGATA 0.5 ml of medium every 3 days for 20 days, after which colonies were TCGCCGCGCTCGT; actin-R: CTAGAAGCATTTGCGGTGGACGATGG. examined by phase-contrast microscopy and photographed. Colonies larger than 30 mm in diameter were counted as positive colonies.

Tandem affinity purification of SFB-tagged protein complexes BrdU incorporation and IF staining 293T cells were transfected with plasmids encoding SFB-tagged proteins. BrdU solution (final concentration of 10 mM) was added directly into Cell lines stably expressing the tagged protein were selected by culturing 40–60% confluent MCF10A cells grown on coverslips. Cells were harvested in medium containing 2 mg/ml puromycin and confirmed by IF and after 30 min of incubation, and IF staining was performed. We counted 500 western blotting. For affinity purification, 293T cells were lysed in lysis cells for each of the three independent experiments. IF was analyzed as buffer (20 mM Tris–HCl (pH 7.6), 100 mM NaCl, 10 mM NaP buffer (pH 7.6), described previously.57 For growth factor stimulation, cells were serum- 0.5% NP-40, 1 mM Na VO ,10mM NaF, 2 mM dithiothreitol, 1 mM EDTA 3 4 starved for 2 h and then stimulated with 100 ng/ml EGF or IGF-I for 10 min (pH 8.0), 2 mg/ml leupeptin, 2 mg/ml pepstatin and 1 mM phenylmethane- before fixation. sulfonylfluoride) at 4 1C for 40 min. Crude lysates were centrifuged at 4 1C at 14 000 r.p.m. for 15 min. Supernatants were incubated with streptavidin- conjugated beads (Amersham, Piscataway, NJ, USA) for 2 h at 4 1C. The beads were washed three times with lysis buffer, and bound proteins were CONFLICT OF INTEREST eluted with lysis buffer containing 2 mg/ml biotin (Sigma-Aldrich). The The authors declare no conflict of interest. eluates were incubated with S protein beads (Novagen, Madison, WI, USA). The beads were washed 3 times with lysis buffer and subjected to SDS–polyacrylamide gel electrophoresis. Protein bands were excised and ACKNOWLEDGEMENTS subjected to mass spectrometric analysis. We thank all our colleagues in the Chen laboratory for insightful discussions and technical assistance. We thank Dr Douglas Yee at the University of Minnesota for Cell culture and transfection sharing hIRS2 cDNA and Dr Cosima Baldari at the University of Siena (Italy) for sharing ShcA(p66) cDNA. This work was supported in part by an Era of Hope Research HEK293T and HeLa cells were purchased from American Type Culture award to JC (W81XWH-09-1-0409). JC is also a recipient of an Era of Hope Scholar Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s award from the Department of Defense (W81XWH-05-1-0470). This work was modified Eagle medium supplemented with 10% fetal bovine serum at supported by MD Anderson’s Cancer Center Support Grant (CA016672). 37 1Cin5%CO2 (v/v). 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2013) 3130 – 3138 & 2013 Macmillan Publishers Limited