Tumor suppressor SCUBE2 inhibits breast-cancer cell ...Signal peptide-CUB-EGF domain-containing...

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RESEARCH ARTICLE

Tumor suppressor SCUBE2 inhibits breast-cancer cell migration andinvasion through the reversal of epithelial–mesenchymal transition

Yuh-Charn Lin1,2, Yi-Ching Lee3,4, Ling-Hui Li2, Chien-Jui Cheng5 and Ruey-Bing Yang1,2,6,*

ABSTRACT

Signal peptide-CUB-EGF domain-containing protein 2 (SCUBE2)

belongs to a secreted and membrane-associated multi-domain

SCUBE protein family. We previously demonstrated that SCUBE2 is

a novel breast-tumor suppressor and could be a useful prognostic

marker. However, the role of SCUBE2 in breast-cancer cell

migration and invasion and how it is regulated during the

epithelial–mesenchymal transition (EMT) remain undefined. In this

study, we showed that ectopic SCUBE2 overexpression could

enhance the formation of E-cadherin-containing adherens junctions

by b-catenin–SOX-mediated induction of forkhead box A1 (a

positive regulator of E-cadherin) and upregulation of E-cadherin,

which in turn led to epithelial transition and inhibited migration

and invasion of aggressive MDA-MB-231 breast-carcinoma cells.

SCUBE2 expression was repressed together with that of E-cadherin

in TGF-b-induced EMT; direct expression of SCUBE2 alone

was sufficient to inhibit the TGF-b-induced EMT. Furthermore,

quantitative DNA methylation, methylation-specific PCR, and

chromatin immunoprecipitation analyses revealed that SCUBE2

expression was inactivated by DNA hypermethylation at the CpG

islands by recruiting and binding DNA methyltransferase 1 during

TGF-b-induced EMT. Together, our results suggest that SCUBE2

plays a key role in suppressing breast-carcinoma-cell mobility

and invasiveness by increasing the formation of the epithelial

E-cadherin-containing adherens junctions to promote epithelial

differentiation and drive the reversal of EMT.

KEY WORDS: Breast cancer, Tumor suppressor, Epithelial–

mesenchymal transition, Epigenetic regulation, DNA

hypermethylation, DNA methyltransferase

INTRODUCTIONSignal peptide-CUB (complement protein C1r/C1s, Uegf, and

Bmp1)-EGF (epidermal growth factor) domain-containing

protein 2 (SCUBE2) belongs to a novel secreted and

membrane-associated SCUBE protein family (Tsai et al., 2009;

Yang et al., 2002). SCUBE genes are evolutionarily conserved

from zebrafish to humans (Grimmond et al., 2000; Grimmond

et al., 2001; Hollway et al., 2006; Kawakami et al., 2005;Tsai et al., 2009; Woods and Talbot, 2005; Wu et al., 2004;Yang et al., 2002). Three distinct members have been isolated

and designated SCUBE1–3 by the order of their discovery(Grimmond et al., 2000; Grimmond et al., 2001; Hollway et al.,2006; Kawakami et al., 2005; Tsai et al., 2009; Woods andTalbot, 2005; Wu et al., 2004; Yang et al., 2002). These SCUBE

proteins contain ,1000 amino acids and are organized in amodular fashion with five distinct domain motifs: an NH2-terminal signal peptide sequence, followed by nine copies of

EGF-like repeats, a spacer region, three cysteine-rich motifs, andone CUB domain at the COOH terminus (Hollway et al., 2006;Tsai et al., 2009). When overexpressed, SCUBE2 is a secreted

glycoprotein that can form oligomers and is stably anchored onthe cell surface (Tsai et al., 2009; Yang et al., 2002).

Large-scale microarray gene expression analyses have revealeda signature set of genes that can predict breast-cancer prognosis(Paik et al., 2004; Paik et al., 2006; Perou et al., 2000; van’t Veeret al., 2002; van de Vijver et al., 2002; Wang et al., 2005).

However, few genes overlapped in these assays, and only a few ofthe breast-associated genes have been validated at the proteinlevel. Interestingly, a cross-platform comparison of breast-cancer

gene sets from these profiling studies revealed SCUBE2 as theonly common gene (Fan et al., 2006), so SCUBE2 must haveimportant roles in breast-cancer progression. Consistent with this

finding, quantitative RT-PCR analysis of the expression ofSCUBE2, along with that of 15 other breast cancer-related genes,has been clinically used in the Oncotype DX Breast Cancer Assay

to calculate the ‘recurrence score’ for predicting the likelihood ofbreast-cancer recurrence in early-stage, node-negative, estrogen-receptor (ER)-positive breast cancers (Kim and Paik, 2010;Sparano and Paik, 2008). Despite the clinical utility of SCUBE2

mRNA expression in guiding adjuvant treatment for breast tumorpatients, little is known about the biological functions ofSCUBE2 in breast cancer progression.

Our recent clinical association studies with anti-SCUBE2immunohistochemistry showed that patients with SCUBE2-

positive tumors had better disease-free survival than those withnegative tumors (Cheng et al., 2009). Multivariate analysisconfirmed SCUBE2 protein expression as an independent

prognostic factor of disease-free survival (Cheng et al., 2009).Further domain and functional studies demonstrated that SCUBE2could be a novel breast-tumor suppressor through its N-terminalEGF-like repeats or C-terminal CUB domain (Lin et al., 2011).

Our molecular and biochemical analyses showed that theC-terminal CUB domain could directly bind and antagonize bonemorphogenetic protein activity in an autocrine fashion, whereas

the N-terminal EGF-like repeats could mediate cell–cellhemophilic adhesion in a calcium-dependent manner, associatewith E-cadherin, and modulate b-catenin–TCF (T-cell factor)signaling in non-invasive, epithelial-like MCF-7 breast-carcinoma

1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei,114 Taiwan. 2Institute of Biomedical Sciences, Academia Sinica, Taipei, 115Taiwan. 3Institute of Molecular Medicine, National Tsing Hua University, Hsinchu,300 Taiwan. 4Institute of Cellular and Organismic Biology, Academia Sinica,Taipei, 115 Taiwan. 5Department of Pathology, School of Medicine, College ofMedicine, Taipei Medical University and Department of Pathology, Taipei MedicalUniversity Hospital, Taipei, 110 Taiwan. 6Institute of Pharmacology, School ofMedicine, National Yang-Ming University, Taipei, 112 Taiwan.

*Author for correspondence ([email protected])

Received 10 April 2013; Accepted 25 October 2013

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 85–100 doi:10.1242/jcs.132779

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cells (Lin et al., 2011). However, whether SCUBE2 and E-cadherindirectly interact and the specific domains that participate in their

interactions remain unknown. In addition, whether the SOXtranscription factors (structurally related to TCF) that can utilizeb-catenin as a cofactor or antagonize b-catenin–TCF function(Kormish et al., 2010) are involved in SCUBE2-mediated signaling

is unclear. In light of the crucial role of E-cadherin in the formationof adherens junctions and the maintenance of well-differentiated,minimally invasive epithelial traits, E-cadherin has been

considered a potent invasion and/or tumor suppressor of breastcancer (Berx and Van Roy, 2001; Cowin et al., 2005; Strumaneet al., 2004). Likewise, loss of E-cadherin in the epithelial–

mesenchymal transition (EMT) during epithelial tumor progressionleads to mesenchymal morphological features or a phenotype withincreased cell migration and invasion (Perl et al., 1998; Vleminckx

et al., 1991), as well as metastasis (Mareel et al., 1991; Osada et al.,1996).

However, whether SCUBE2 plays a role in controlling themigratory and invasive behaviors in breast-carcinoma cells

remains unknown. In addition, the regulatory mechanismcontrolling the expression of SCUBE2 during EMT is poorlyunderstood. In this study, we demonstrated that SCUBE2 forms

a complex with E-cadherin and increases the formation ofE-cadherin-containing adherens junctions by inducing theexpression of forkhead box A1 (FOXA1; a positive regulator of

E-cadherin) and subsequent transactivation of E-cadherin, whichdrives breast-carcinoma cells to undergo epithelial transition byreversing EMT. Furthermore, we showed that SCUBE2 was

epigenetically inactivated by recruiting DNA methyltransferase 1onto its CpG islands during EMT. Together, these results indicatethat downregulation of SCUBE2 is part of the EMT program thatplays important roles in modulating breast-cancer cell migration

and invasion. Further exploration of SCUBE2 as a diagnostic ortherapeutic target for human breast tumors is warranted.

RESULTSSCUBE2 forms a complex and is co-expressed with E-cadherin inbreast-carcinoma cells during EMTWe recently showed that SCUBE2, through its NH2-terminal EGF-like repeats, acts as a homophilic cell–cell adhesive module that canassociate with E-cadherin and modulate b-catenin-TCF signaling inMCF-7 cells (Lin et al., 2011). E-cadherin is a single-transmembrane

glycoprotein with a large extracellular domain comprising fivecadherin-motif subdomains and a short cytoplasmic domain thatassociates with three catenins (a, b and p120) linking E-cadherin tothe actin cytoskeleton (van Roy and Berx, 2008). We performedfurther analysis of the interaction between E-cadherin and SCUBE2by using a series of E-cadherin cytoplasmic truncation forms (FL,

D1, D2 and D3) and various SCUBE2 deletion mutant constructs(FL, ty97 and D4) including a mutant (D4) that cannot bindE-cadherin as a control (Fig. 1A–C). Our data showed that the

membrane-proximal cytoplasmic region (residues 732–820) iscrucial in its association with the N-terminal EGF-like repeats ofSCUBE2 (Fig. 1C). Using confocal immunofluorescence, SCUBE2was found to be consistently targeted to the plasma membrane

at cell–cell contact sites where it colocalized with E-cadherin orb-catenin, two core proteins of the epithelial adherens junctions(Fig. 1D–G). Further immunoprecipitation analyses identified that it

formed a stable complex with E-cadherin or b-catenin in breastcancer cells (Fig. 1H).

Because this juxtamembrane region is also the binding site for

p120-catenin (van Roy and Berx, 2008), we examined whether

overexpression of p120-catenin could affect the interaction ofSCUBE2 with E-cadherin. As shown by co-immunoprecipitation

assay (supplementary material Fig. S1), p120-catenin overexpressiondid not compete with, but further increased the association ofSCUBE2 with E-cadherin. However, pull-down assays with aGST fusion protein containing the juxtamembrane region of the

E-cadherin cytoplasmic domain (resides 732–820) andrecombinant fragment of SCUBE2 (supplementary material Fig.S2) did not reveal a direct interaction, which suggests that as-yet-

unknown protein(s) may connect the cytoplasmic juxtamembraneregion of E-cadherin with the surface-tethered SCUBE2 into acomplex. Furthermore, SCUBE2 expression is highly correlated

with the expression of E-cadherin in an array of breast-cancer celllines: high in MCF-7 and T-47D breast-cancer cells but absent inMDA-MB-231 and ZR-75-30 cells (supplementary material Fig.

S3). Of note, the former two cell lines were non-invasive and hadan epithelium-like morphology, whereas the latter were highlyinvasive and MDA-MB-231 cells had a spindle-shaped,fibroblastic mesenchymal phenotype (supplementary material

Fig. S3) (Zajchowski et al., 2001).To test whether SCUBE2 is co-regulated with E-cadherin

and involved in EMT, we used a well-documented model of

transforming growth factor b (TGF-b)-induced EMT (Fuxeet al., 2010) to assess expression of SCUBE2 and molecularmarkers associated with EMT by western blot analysis. TGF-b1long-term treatment (14 or 21 days) had no apparent effecton apoptosis as evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay

or caspase-3 activation (supplementary material Fig. S4A–C) butdid induce MCF-7 cells to undergo EMT, as evidenced by theacquisition of fibroblastic mesenchymal morphological features(supplementary material Fig. S4D), loss of the epithelial marker

E-cadherin, and gain of the mesenchymal marker vimentin(supplementary material Fig. S4E,F). Importantly, SCUBE2protein level was suppressed together with decreased

E-cadherin protein level during TGF-b-induced EMT in a dose-and time-dependent manner (supplementary material Fig. S4E,F),which suggests that downregulation of SCUBE2 is part of the

EMT program.

Ectopic SCUBE2 expression drives an epithelial phenotype andinhibits cell migration and invasionTo test whether direct expression of SCUBE2 alone could inhibitthe TGF-b-induced EMT, we used a stable MCF-7 cell line withthe expression of exogenous FLAG-tagged SCUBE2 (FLAG–

SCUBE2) under the control of an inducible Tet-Off promoter asdescribed previously (Cheng et al., 2009). In addition, the MCF-7Tet-Off vector clones containing stable integration of the empty

expression vector were used as a control. These MCF-7 Tet-Offcells were first cultured in the absence of doxycycline for 5 daysto induce the expression of ectopic FLAG–SCUBE2 protein,

followed by treatment of TGF-b1 to induce EMT. Interestingly,in contrast to control cells, ectopic SCUBE2 overexpressionprevented TGF-b-induced cell scattering (Fig. 2A, bottom panel),impeded TGF-b-promoted loss of the epithelial markerE-cadherin at the lateral cell surface (Fig. 2B,C), and inhibitedaccumulation of the mesenchymal marker vimentin (Fig. 2C).The mRNA expression levels of SCUBE2, E-cadherin and

vimentin were generally in agreement with their proteinexpression in these cells (Fig. 2D).

In addition, we investigated whether SCUBE2 could drive

mesenchymal-like breast-cancer cells to undergo an epithelial

RESEARCH ARTICLE Journal of Cell Science (2014) 127, 85–100 doi:10.1242/jcs.132779

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Fig. 1. SCUBE2 colocalizes and forms a stable complex with E-cadherin and b-catenin at adherens junctions. (A) Domain organization of E-cadherinconstructs (Myc epitope-tagged) including the full-length (FL) and the C-terminal deletion mutants (D1–3). SP, signal peptide; PRO, propeptide; EC, extracellularcadherin repeat; TM, transmembrane region; CD, cytoplasmic domain. Amino acid numbers start at the first methionine codon. (B) Domain organization ofthe SCUBE2 expression constructs (FL, ty97 and D4) used in this study. A FLAG epitope was added immediately after the signal peptide sequence at theN-terminus for easy detection. SP, signal peptide; E, EGF-like repeats; Cys-rich, cysteine-rich motifs; CUB, CUB domain. (C) The juxtamembrane region in thecytoplasmic domain of E-cadherin is crucial for its association with SCUBE2. Lysates of HEK-293T cells transiently transfected with the expression plasmidencoding the FLAG-tagged SCUBE2-FL, ty97 or D4 alone or together with the Myc-tagged E-cadherin FL, D1, D2 or D3 constructs were immunoprecipitated(IP) with anti-FLAG antibody, and associated protein levels were determined by western blot analysis (WB) with anti-Myc antibody (top panel) or by a reciprocalco-IP experiment (IP with anti-Myc antibody followed by anti-FLAG immunoblotting; second panel). The expression of FLAG–SCUBE2-FL, ty97 or D4 or all otherMyc-tagged E-cadherin proteins was verified by anti-FLAG or anti-Myc western blot analysis, respectively (third panel and bottom panel). (D–G) SCUBE2colocalizes with E-cadherin and b-catenin at cell–cell contact sites (arrows). Three-color confocal immunofluorescence microscopy of colocalization of SCUBE2with E-cadherin or b-catenin (D, white). SCUBE2 (FLAG-tagged) localization was detected with rabbit anti-FLAG monoclonal antibody and Alexa-Fluor-488-conjugated goat anti-rabbit IgG (E, green). E-cadherin or b-catenin was detected with mouse anti-E-cadherin and Alexa-Fluor-594-conjugated goat anti-mouseIgG (F, red) or anti-b-catenin antibody conjugated with Alexa-Fluor-647 (G, blue). Scale bar: 10 mm. (H) SCUBE2 forms a complex with E-cadherin andb-catenin. Lysates of MDA-MB-231 control or SCUBE2-expressing cells were immunoprecipitated with anti-FLAG antibody to pull down FLAG-tagged SCUBE2protein, and associated E-cadherin or b-catenin protein level was verified by western blot analysis with anti-E-cadherin or anti-b-catenin antibody (upper panel).Protein levels of SCUBE2, E-cadherin, b-catenin and b-actin (a loading control) were confirmed by western blot analysis (lower panel).


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transformation when introduced into SCUBE2-negative MDA-

MB-231 cells. MDA-MB-231 cells were engineered with thefull-length SCUBE2 expression vector or empty vector (control)by a leniviral transduction system. Control cells were spindly,elongated, and dispersed, whereas overexpression of SCUBE2

transformed the fibroblastic, spindle-shaped MDA-MB-231 cellsinto epithelial clusters with a cobblestone growth pattern inmonolayer culture much like MCF-7 cells (Fig. 3A), and formed

multicellular aggregates in suspension (supplementary materialFig. S5). Consistent with the morphological phenotypes(Fig. 3A), MDA-MB-231 cells expressing ectopic SCUBE2

protein showed increased expression of E-cadherin (anepithelial marker) but decreased expression of the mesenchymalmarkers vimentin and N-cadherin at both the mRNA and protein

levels, similar to epithelial-like MCF-7 control cells (Fig. 3B,C).Interestingly, overexpression of a SCUBE2 mutant (D4) unable tobind E-cadherin produced no effect on epithelial transformationand upregulation of FOXA1 (a positive transcriptional activator

of E-cadherin, see below), suggesting that SCUBE2–E-cadherin

interaction is important for mediating SCUBE2-drived epithelialtransition (supplementary material Fig. S6).

To determine the functional changes in cell behavior thatoccurred following SCUBE2 overexpression, we performed

gap-closure migration (wound-healing) and Matrigel-coatedTranswell invasion assays to characterize the control andSCUBE2-expressing MDA-MB-231 cells. MDA-MB-231

control cells were highly motile and invasive, but SCUBE2overexpression significantly decreased both motility andinvasiveness (Fig. 3D,E). Importantly, cell proliferation did not

differ between the control and SCUBE2-expressing cells (data notshown), which excluded general cytostatic and cytotoxic effectscaused by SCUBE2 expression in MDA-MB-231 cells. Thus,

expression of SCUBE2 induces breast-cancer cells to undergoepithelial transition or a reversal of the EMT and suppressesthe motility and invasive ability of invasive, mesenchyme-likeMDA-MB-231 cells.

Fig. 2. Ectopic SCUBE2 overexpressioninhibited transforming growth factor b(TGF-b)-induced EMT. (A) TGF-b1-induced EMT resulted in morphologicalchanges. MCF-7 Tet-Off vector and FLAG–SCUBE2-expressing cells were treatedwith TGF-b1 (10 ng/ml) for 7, 14 and 21days. Phase-contrast images show themorphological alterations associated withEMT. Scale bar: 50 mm. (B) Lateral cell-surface distribution of E-cadherin in MCF-7vector- and SCUBE2-expressing cellsbefore and after TGF-b1-induced EMT.Immunofluorescent staining of E-cadherinmainly localized to the lateral cell surface inconfluent MCF-7 vector- and SCUBE2-expressing cells in the basal status. Incontrast to control cells showing markedloss of the cell-surface expression ofE-cadherin, ectopic SCUBE2 expressionprevented TGF-b1-promoted decrease ofE-cadherin after TGF-b1 treatment. Scalebar: 10 mm. (C,D) Western blot analysisof protein levels (C) or RT-PCR analysis(D) for mRNA levels of SCUBE2,E-cadherin, vimentin, or b-actin.Exogenous FLAG–SCUBE2 was detectedby anti-FLAG antibody, whereas the totalamount of SCUBE2 (a combination ofendogenous and exogenous SCUBE2)was detected with anti-SCUBE2 antibody.RT-PCR analysis to determine the mRNAlevel of SCUBE2 (total or ectopicexpression level), E-cadherin, vimentin, orGAPDH, which served as an internalcontrol with first-strand cDNA from TGF-b1-treated MCF-7 vector- or SCUBE2-expressing cells at the indicated times.


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Knockdown of endogenous SCUBE2 disrupted the adherensjunctions and concomitantly increased the migratory and invasiveability of MCF-7 breast-cancer cellsTo further confirm the role of SCUBE2 in increasing the

epithelial E-cadherin-containing adherens junctions andmodulating cancer cell motility and invasiveness, we used two

independent SCUBE2-targeting short hairpin RNA (shRNA)lentiviruses (SCUBE2-shRNA #1 and #2) to inhibit endogenousSCUBE2 expression in MCF-7 cells, which exhibit non-invasiveand epithelium-like features (Zajchowski et al., 2001). As a

negative control, cells were infected with an shRNA lentivirustargeting bacterial b-galactosidase (control shRNA). The

Fig. 3. Reversal of epithelial–mesenchymal transition (EMT) in MDA-MB-231 cells ectopically expressing SCUBE2. (A) Phase-contrast images showingthe morphological features of MCF-7, MDA-MD-231 and MDA-MB-231 cells overexpressing SCUBE2. Scale bar: 50 mm. (B) Western blot and RT-PCRanalyses. Cell lysates from MCF-7 and MDA-MB-231 control or SCUBE2-expressing cells were immunoblotted with antibodies for anti-SCUBE2, E-cadherin,vimentin, N-cadherin or b-actin. MCF-7 cells were an epithelial cell control. The first-strand cDNAs derived from MCF-7, MDA-MB-231 control or SCUBE2-expressing cells were used to perform RT-PCR analysis to determine the mRNA level of SCUBE2, E-cadherin, vimentin, N-cadherin, or GAPDH, which servedas an internal control. (C) Immunocytochemical staining of cells with anti-E-cadherin (green) or anti-vimentin (red) antibody. Scale bars: 10 mm. Nuclei werevisualized by DAPI staining (blue). (D,E) SCUBE2 overexpression suppressed MDA-MB-231 cell migration and invasion. (D) SCUBE2 inhibition of MDA-MB-231cell migration was determined by the gap-closure migration assay. Spontaneous motility of MDA-MB-231 control or SCUBE2-expressing cells examined over24 hours in confluent monolayers separated with cell culture insert. After removal of the insert the gap was photographed at 0, 12 and 24 hours (upper panel),and the mean proportion of the gap closure was calculated (lower panel). Scale bar: 500 mm. (E) MDA-MB-231 control or SCUBE2-expressing cells were seededon Transwell plates coated with 30 mg Matrigel. After incubation for 18 hours, the invaded cells were fixed, stained and photographed (upper panel).Quantification of invasion experiment involved counting the number of invaded cells (n53, lower panel). All assays were performed in triplicate. Quantitative dataare means 6 s.d., *P,0.05.


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efficiency of shRNA-mediated SCUBE2 knockdown (,90%)was confirmed by RT-PCR and western blot analyses (Fig. 4A).

Control-shRNA-treated cells retained an epithelial-clusterappearance that was similar to the parental MCF-7 cells(Fig. 4B; Fig. 3A), but the two independent SCUBE2-shRNAknockdown cell clones were dispersed and scattered with an

increase in cell spreading and reduced cell–cell contacts

(Fig. 4B). In agreement with the morphological changes,knockdown of SCUBE2 induced EMT-like events by

downregulating the epithelial E-cadherin and upregulatingmesenchymal markers vimentin and N-cadherin at both themRNA and protein levels (Fig. 4A). Consistently, the intensity ofE-cadherin immunofluorescence was markedly reduced at cell

junction sites, which indicates the disruption and loss of the

Fig. 4. SCUBE2 knockdown leads to the loss of the E-cadherin-containing adherens junctions, induces EMT-like events, and increases the migrationand/or invasion of MCF-7 cells. (A) Effect of endogenous SCUBE2 knockdown on the expression of EMT markers. Endogenous SCUBE2 expression wasinhibited by two independent SCUBE2-targeting short hairpin RNA (shRNA) lentiviruses (SCUBE2-shRNA #1 or #2) in MCF-7 cells. A luciferase shRNAlentivirus was used as a negative control (control-shRNA). Efficiency of SCUBE2 knockdown and its effect on the expression of EMT markers (E-cadherin,vimentin and N-cadherin) were assessed by RT-PCR or western blot analyses using GAPDH or b-actin expression as an internal or loading control. Omission oftemplate cDNA served as a negative PCR reaction control (-). (B) Effect of SCUBE2 knockdown on cellular morphology. Cells cultured under subconfluentconditions were photographed using phase-contrast microscopy. SCUBE2-knockdown cells were dispersed and exhibited migratory features includingprotruding filopodia at the cell edge, whereas control cells retained an epithelial-like appearance that was similar to parental MCF-7 cells (Fig. 3A). Scale bar:50 mm. (C) Effect of SCUBE2 knockdown on the formation of the E-cadherin-containing adherens junctions at the cell–cell contact sites. Confocalimmunofluorescence microscopy with merged images of E-cadherin (green) and b-catenin (red) staining marking the adherens junctions seen in control-shRNAcells but not in two SCUBE2 knockdown cells (SCUBE2 shRNA #1 and #2). Scale bar: 10 mm. (D,E) Knockdown of endogenous SCUBE2 expression increasedthe migratory and invasive capabilities of MCF-7 breast-cancer cells. Migration and invasion assays were performed as described in Fig. 3, except the invadedMCF-7 cells were stained with Crystal Violet and the OD570 was measured to quantify the amount of DMSO-solubilized dye. All assays were performed intriplicate. Quantitative data are means 6 s.d.; *P,0.05, **P,0.01.


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E-cadherin-containing adherens junctions (Fig. 4C). As aconsequence, SCUBE2 knockdown, in contrast to its

overexpression, promoted the motility and invasiveness ofMCF-7 breast-cancer cells (Fig. 4D,E). Again, the cellproliferation rate was comparable between the control andSCUBE2-silenced cells (data not shown). Together, these data

indicate that SCUBE2 expression is essential to maintain theepithelial phenotype, with its long-term loss sufficient to inducesubsequent mesenchymal transition.

SCUBE2 expression upregulates an E-cadherin transcriptionalactivator forkhead box A1 (FOXA1) to maintain the epithelialphenotype in breast-cancer cellsBecause SCUBE2 was colocalized and associated withE-cadherin-positive adherens junctions (Fig. 1), and because

several structural proteins associated with adherens junctions canmediate cellular signaling (Cowin et al., 2005; van Roy and Berx,2008), SCUBE2 expression might induce a transcriptionalactivator to reinstate the expression of E-cadherin in aggressive

mesenchyme-like breast-carcinoma cells. Because E-cadherinexpression during EMT or reversal of EMT could betranscriptionally repressed by members of TWIST, SNAIL and

ZEB protein families (van Roy and Berx, 2008) or positivelyupregulated by FOXA1 (Liu et al., 2005), we sought to profile theexpression of these E-cadherin transcriptional regulators. Western

blot and RT-PCR analyses showed that SCUBE2 overexpressionor knockdown did not substantially alter these E-cadherinrepressors at both the mRNA and protein levels, except for

TWIST, which was upregulated but unable to bind the E-cadherinpromoter with SCUBE2 overexpression in MDA-MB-231(Fig. 5A–E). Because SCUBE2 overexpression inducedE-cadherin expression and drove an epithelial phenotype in

MDA-MB-231 cells (Fig. 3), such TWIST upregulation conflictswith its classical role as an EMT inducer, and its precise functionin this context required further studies. Of note, a chromatin

immunoprecipitation (ChIP) assay showed that SCUBE2overexpression decreased the binding of SNAIL and ZEB1 tothe E-cadherin promoter in MDA-MB-231 cells (Fig. 5E).

However, further studies are required to elucidate theunderlying mechanism and the biological relevance of thesefindings related to the SCUBE2-mediated reversal of EMT.

In contrast, the nuclear protein level of FOXA1, a positive

regulator of E-cadherin, was upregulated and associated withSCUBE2-induced E-cadherin mRNA and protein expression(Fig. 6A,E). Furthermore, FOXA1 transactivates the E-cadherin

promoter containing FOXA1 binding sites on a luciferase reporterassay (Fig. 6B,C). We further examined the direct effect ofFOXA1 on the E-cadherin promoter in MDA-MB-231 control

and MDA-MB-231 SCUBE2-expressing cells by transienttransfection with plasmids encoding full-length FOXA1 or twodifferent FOXA1-targeting shRNAs (#1 and #2), respectively.

With FOXA1 overexpression, activity of the E-cadherin promotercontaining FOXA1-binding sites [E-cad (WT)-Luc] but notmutated FOXA1-binding sites [E-cad (Mut)-Luc] was greatlyincreased in MDA-MB-231 control cells (Fig. 6C, middle panel).

In contrast, knockdown of FOXA1 significantly suppressed theSCUBE2-mediated increase in the E-cadherin promoter activity(Fig. 6C, bottom panel). In addition, a ChIP assay confirmed

that endogenous FOXA1 protein indeed interacted with theDNA region that harbors a consensus FOXA-binding site[59-TGTTTG(T/C)-39] within the E-cadherin promoter (Fig. 6D).Furthermore, we examined the effects of FOXA1 overexpression

in MDA-MB-231 control and FOXA1 silencing in MDA-MB-231SCUBE2-expressing cells on E-cadherin mRNA and protein

expression as described above. FOXA1 overexpression couldtransactivate and upregulate E-cadherin in MDA-MB-231 controlcells, but knockdown of FOXA1 to ,10% of control levels inMDA-MB-231 SCUBE2-expressing cells concomitantly

diminished E-cadherin expression at both the mRNA and proteinlevels by ,90% as compared with control cells (Fig. 6E). Giventhat FOXA1 can activate the E-cadherin promoter in SNAIL- or

ZEB1-expressing MDA-MB-231 cells, we investigated whetherFOXA1 is a dominant activating factor capable of overcomingE-cadherin-suppressing signals. Using MDA-MB-231 SCUBE2

cells, we showed that ectopic overexpression of SNAIL or ZEB1alone was able to repress E-cadherin promoter reporter activitycompared with the control cells (Fig. 5F). Interestingly, co-

expression of FOXA1 can overcome the suppression of theE-cadherin promoter mediated by SNAIL or ZEB1 (Fig. 5F),indicating that FOXA1 has a dominant regulatory role on theE-cadherin promoter in breast cancer cells. Together, these data

suggest that FOXA1 is important in mediating the SCUBE2-induced upregulation of E-cadherin and promotes an epithelialcharacter in invasive MDA-MB-231 breast-cancer cells.

b-catenin is involved in SCUBE2-mediated induction of FOXA1Because SCUBE2 overexpression resulted in increased

expression of b-catenin protein (Fig. 1H) and because b-catenindirectly interacts with Sox17 to transcribe FoxA1 in Xenopusembryos (Sinner et al., 2004), we then investigated how SCUBE2

overexpression regulates the expression of b-catenin and whetherb-catenin participates in upregulation of FOXA1 and E-cadherinin breast cancer cells. RT-PCR and western blot analyseswith anti-b-catenin antibody or anti-b-catenin phosphorylation-specific antibodies showed that SCUBE2 expression is positivelyassociated with b-catenin expression at the mRNA transcriptionallevel (Fig. 7A) but not by post-translational regulation of its

stability and localization such as phosphorylation at Ser33, Ser37,Thr41 or Tyr654 (Fig. 7B). In addition, we found that both SOX4and SOX7, which can interact with b-catenin and modulateb-catenin-mediated transcription (Sinner et al., 2007; Takashet al., 2001), are closely associated with b-catenin and SCUBE2expression in breast cancer cells (Fig. 7C). We then examinedwhether b-catenin is involved in SCUBE2-induced FOXA1upregulation by shRNA silencing experiments. Knockdown ofb-catenin concomitantly suppressed FOXA1 and E-cadherinexpression at both mRNA and protein levels in MDA-MB-231

SCUBE2-expressing cells (Fig. 7D,E), which suggests thatb-catenin, possibly in cooperating with SOX protein, is anessential upstream transcriptional regulator of its target gene

FOXA1 when SCUBE2 is overexpression.

Methylation of CpG sites near the first exon of SCUBE2 gene is crucialfor repression of its expressionBecause SCUBE2 is co-regulated with E-cadherin and loss ofE-cadherin expression is regulated by transcriptional repressorssuch as SNAIL, SLUG and ZEB (van Roy and Berx, 2008) or by

epigenetic regulation, including DNA methylation and histonedeacetylation, in several cancer cell lines (Koizume et al., 2002),we investigated the role of classical repressors of E-cadherin on

SCUBE2 expression and whether these epigenetic modificationsare involved in suppression of SCUBE2 mRNA and proteinexpression in breast-cancer cells. To determine whether these

E-cadherin repressors directly regulated SCUBE2 at the


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Fig. 5. Effect of SCUBE2 on the expression of EMT inducers, on E-cadherin promoter binding of classical E-cadherin repressors and on competitionwith E-cadherin activator FOXA1 in breast-cancer cells. (A–D) RT-PCR or western blot analysis of the mRNA or protein expression of the classical EMTinducers TWIST, SNAIL, SLUG, ZEB1 and ZEB2 in SCUBE2-overexpressing MDA-MB-231 cells (A,B) or SCUBE2-silencing MCF-7 cells (C,D). RT-PCRanalysis using first-strand cDNAs derived from MDA-MB-231 (A) or MCF-7 (C) breast-carcinoma cell lines. GAPDH expression was amplified as an internalcontrol and lack of cDNA was a negative control (2). Western blot analysis of protein expression of the EMT inducers in the indicated MDA-MB-231 (B) or MCF-7(D) cells with the specific antibody for each EMT-inducing E-cadherin repressor. Nuclear protein lamin A/C expression was used as a loading control. Theexpression of the EMT inducers remained basically unaltered at both the mRNA and protein levels, except TWIST was induced by SCUBE2 overexpression inMDA-MB-231 cells. Because SCUBE2 overexpression induces E-cadherin expression and drives an epithelial phenotype in MDA-MB-231, such upregulation ofTWIST is contrary to its accepted function as an EMT inducer and its exact function in this context requires further studies. (E) Effect of SCUBE2 overexpressionon the binding of the classical EMT inducers to the E-cadherin promoter. ChIP assay with antibodies specific to each E-cadherin repressor (TWIST, SNAIL,SLUG, ZEB1 or ZEB2) to determine the binding of these repressors to the E-cadherin promoter in MDA-MB-231 control and SCUBE2-expressing cells analyzedby PCR with specific primers for E-cadherin amplifying a 92-bp fragment (see Fig. 6B). n.s., non-specific PCR product. (F) FOXA1 overcomes suppression of theE-cadherin promoter mediated by SNAIL and ZEB1. In MDA-MB-231 SCUBE2 cells, the expression plasmid encoding FOXA1, SNAIL or ZEB1 was transfectedalone or in combination as indicated. Two days after transfection, promoter activity was determined by dual-luciferase assays. Luciferase activity was correctedfor Renilla luciferase activity (pRL-TK) to control for transfection efficiency. Data are means 6 s.d. of three experiments; *P,0.01.


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Fig. 6. FOXA1-mediated SCUBE2-induced upregulation of E-cadherin. (A) Induction of FOXA1 is associated with SCUBE2 expression. Western blot analysisof nuclear FOX1 levels in MCF-7, MDA-MB-231 control and SCUBE2-expressing cells. Nuclear lamin A/C level was used as a loading control. (B) Scheme of theE-cadherin promoter reporter constructs containing the native (WT) or mutated (Mut) FOXA1-binding sites. The location of the promoter (2995,+1) of theE-cadherin gene is indicated by a lateral line. The FOXA1-binding sites are marked by diamonds, whereas the mutant sites are shown as dashed lines. The primersand the amplicon region used in the ChIP assay are labeled (upper panel). (C) FOXA1 transactivates the E-cadherin promoter-driven luciferase activity.Quantification of activity of E-cadherin WT or Mut promoter constructs transiently transfected into MDA-MB-231 control or SCUBE2-expressing cells. Luciferaseactivity was corrected for Renilla luciferase activity (pRL-TK) to control for transfection efficiency. Data are means 6 s.d. of three experiments; *P,0.05 comparedwith the vector control. Note that transactivating activity of the E-cadherin promoter was higher when FOXA1 was overexpressed, whereas the FOXA1-medaitedtransactivation was completely abolished when the FOXA1-binding sites were mutated. (D) FOXA1 directly binds to the E-cadherin promoter. ChIP assay of in vivobinding of FOXA1 protein to the promoter of E-cadherin in MDA-MB-231 control and SCUBE2-expressing cells analyzed by PCR with specific primers forE-cadherin amplifying a 92-bp fragment (see Fig. 6B). Lack of cell lysates (2) or control IgG were used as negative controls. (E) Effects of FOXA1 overexpressionor knockdown on the expression of E-cadherin. FOXA1 was overexpressed in MDA-MB-231 control cells (left panels), but its expression was suppressed by twoindependent FOXA1-targeting short hairpin RNAs (shRNAs; FOXA1-shRNA #1 or #2) in MDA-MB-231 SCUBE2 cells (right panels). An shRNA targeting bacterialb-galactosidase was used as a negative control (Control-shRNA). RT-PCR is shown in the upper panel, western blot (WB) analysis for FOXA1 overexpression orknockdown in nuclear fractions is shown in the middle panel, and its effect on the expression of E-cadherin in cell lysates is shown in the lower panel. Anti-lamin A/Cblotting served as a nuclear marker, whereas anti-GAPDH blotting was a cytosolic marker and loading control.


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Fig. 7. b-catenin is involved in SCUBE2-induced FOXA1 upregulation in breast-cancer cells. (A,B) SCUBE2 expression is positively associated withb-catenin expression at the mRNA transcription level but not after post-translational phosphorylation regulation. RT-PCR or western blot (WB) analysis with theindicated antibodies or phosphorylation-specific antibodies, of the expression of b-catenin (A) or phosphorylation status of b-catenin at Ser33, Ser37, Thr41or Tyr654 (B) with subcellular (cytosolic and nuclear) fractions, total cell lysates or first-strand cDNA from the indicated cell lines. (C) Expression of SOX proteinsis associated with SCUBE2 and b-catenin expression. RT-PCR analysis of the mRNA levels of SOX members (SOX4, SOX7 and SOX17) in the indicatedbreast cancer cell lines. (D,E) Effect of b-catenin knockdown on FOXA1 and E-cadherin expression. b-catenin expression was suppressed by two differentb-catenin-targeting shRNAs (#1 and #2) in MDA-MB-231 SCUBE2 cells. An shRNA targeting bacterial b-galactosidase was used as a negative control(Control-shRNA). Effect of b-catenin knockdown on nuclear expression of b-catenin and FOXA1 or total E-cadherin was verified by RT-PCR (D) or western blot(E) analyses. Anti-lamin A/C blotting served as a nuclear marker and loading control.


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transcriptional level, we first performed a promoter assay inSCUBE2-expressing MCF-7 cells by transfecting the expression

plasmids encoding these repressors individually. SCUBE2promoter reporter activity remained relatively unaltered, whereasE-cadherin promoter activity was significantly suppressed by theseEMT inducers (supplementary material Fig. S7A). Consistently,

overexpression of these E-cadherin repressors has no effect onSCUBE2 expression at both mRNA and protein levels(supplementary material Fig. S7B–D). Our results suggest that

these classical repressors of E-cadherin may not be involved in thedownregulation of SCUBE2 in breast-carcinoma cells. We thentreated SCUBE2-negative MDA-MB-231 cells with 5-aza-29-deoxycytidine (AZA), a DNA demethylation agent; trichostatinA (TSA), a histone deacetylase inhibitor; or both (supplementarymaterial Fig. S8). Treatment with AZA or AZA plus TSA had

greater effects than TSA alone on upregulation of SCUBE2 andE-cadherin at both mRNA and protein levels (supplementarymaterial Fig. S8A,C). Thus, DNA methylation but not histoneacetylation may play a major role in regulating SCUBE2 gene

expression in breast-carcinoma cells.Genomic analysis revealed CpG islands at the 59 flanking

promoter and downstream of the first exon region of SCUBE2

(supplementary material Fig. S9A). To delineate which clustersof CpG sites are involved in regulating SCUBE2 expression, wegenerated three independent luciferase reporter gene constructs

containing: (1) an upstream ,1.5-kb promoter (Prom-Luc), (2) agenomic fragment containing exon 1 and a 59 part of intron 1(Ex1+In1-Luc) or (3) a 39 part of intron 1 and exon 2 (In1+Ex2-Luc) (supplementary material Fig. S9A). We prepared methylatedor unmethylated plasmid DNAs of these three constructs andtransiently transfected them into MCF-7 cells to determinewhether the transcriptional activity of these proximal SCUBE2

promoter regions was sensitive to DNA methylation(supplementary material Fig. S9B). Luciferase activity assayrevealed that in vitro methylation of the Ex1+In1-Luc or

In1+Ex2-Luc but not the Prom-Luc plasmid significantlyinhibited transcriptional activity in MCF-7 cells (supplementarymaterial Fig. S9C). Thus, SCUBE2 expression is indeed repressed

by DNA methylation in breast-cancer cells, which may bespecifically attributed to the CpG sites downstream of exon 1.

Methylation status and recruitment of DNA methyltransferase 1 to theSCUBE2 CpG sites during TGF-b1-induced EMTBecause the Ex1+In1-Luc reporter activity was much reduced byDNA methylation and contained more CpG sites, we focused on

this regulatory region to quantify its DNA methylation levels.Mass spectrometry of base-specific cleaved amplificationproducts (Sequenom EpiTYPER assay) of the four breast-

cancer cell lines revealed the CpG island downstream of exon 1in a single amplicon (position +159 to +592 nt; Fig. 8A). Thelevel of DNA methylation was inversely associated with SCUBE2

mRNA expression (Fig. 8B): MDA-MB-231 and ZR-75-30 cellswith no or low SCUBE2 expression showed a high degreeof methylation (.70% of detectable CpG sites) compared withSCUBE2-positive breast-cancer cells MCF-7 or T47-D.

Therefore, CpG methylation status downstream of exon 1 hasan important role in SCUBE2 expression in breast-carcinomacells. To further validate the CpG methylation status, we designed

methylation-specific primers to target CpG sites downstream ofexon 1 of SCUBE2 on the basis of quantitative DNA methylationresults. We detected an unmethylated allele only in SCUBE2-

positive MCF-7 and T-47D cells, but methylated bands were

markedly amplified in SCUBE2-negative MDA-MB-231 andZR-75-30 cells (Fig. 8C). Thus, the downstream region of exon 1

has an important role in methylation-related gene silencing ofSCUBE2 in breast-carcinoma cells.

We further investigated whether epigenetic alterations wereinvolved in repressing SCUBE2 expression in these cells with

TGF-b-induced EMT. Epigenetic modifications in the regulatoryregion of the SCUBE2 gene were assessed by methylation-specific PCR (MSP). DNA methylation of the SCUBE2 locus

started at day 7 and peaked at day 21 with TGF-b1 treatment inMCF-7 cells (Fig. 8D). Therefore, downregulation of SCUBE2,along with decreased E-cadherin level, is a dynamic event

followed by a sequential and progressive increase in DNAhypermethylation of the regulatory CpG sites in the SCUBE2gene during TGF-b-induced EMT.

Because DNA methyltransferase 1 (DNMT1), the major DNMTin adult cells, catalyzes DNA methylation and contributes to theCpG-island hypermethylation of tumor-suppressor genes,including E-cadherin (Suzuki et al., 2004; Wang et al., 2008),

we determined whether DNMT1 is upregulated and binds to theCpG sites near exon 1 of SCUBE2. Western blot analysis and aChIP assay revealed higher DNMT1 protein expression with

predominant binding to the CpG island near exon 1 in MDA-MB-231 cells that did not express SCUBE2 than in SCUBE2-expressing MCF-7 cells (supplementary material Fig. S10). In

line with these findings, treatment with TGF-b1 for 21 days toinduce EMT increased the expression of DNMT1 (Fig. 8E) andbinding of DNMT1 to the regulatory CpG sites at the SCUBE2

locus in MCF-7 cells (Fig. 8F). Furthermore, we performed gain-and loss-of-function experiments to confirm the effect of DNMT1on DNA methylation and expression of SCUBE2. DNMT1overexpression increased DNA methylation and suppressed

SCUBE2 expression in MCF-7 cells, whereas DNMT1knockdown decreased DNA methylation and upregulatedSCUBE2 expression in MDA-MB-231 cells (Fig. 8G). Therefore,

DNMT1 is upregulated and actively recruited to methylate theSCUBE2 CpG sites, which subsequently inactivates the expressionof tumor-suppressor SCUBE2 during TGF-b-promoted EMT.

DISCUSSIONSCUBE2 was initially identified (van’t Veer et al., 2002) andthereafter repeatedly reported as a breast-tumor-associated gene

by several large-scale microarray gene expression profilinganalyses (Paik et al., 2004; Paik et al., 2006; van de Vijveret al., 2002). Interestingly, SCUBE2 is the only gene commonly

described in reports of these studies (Fan et al., 2006), soSCUBE2 must play a crucial role in breast-cancer biology. Insupport of this notion, the expression of SCUBE2 and 15 other

breast-cancer-related genes have been clinically used to calculatea predictive score in guiding adjuvant treatment for breast-cancerpatients (Kim and Paik, 2010; Sparano and Paik, 2008). In

addition, our previous clinical-association study suggested thatSCUBE2 is a prognostic marker for a favorable clinical outcome(Cheng et al., 2009) and is a novel breast-tumor suppressor (Linet al., 2011). However, the functions of SCUBE2 in breast-cancer

cell motility and invasion and the gene regulatory mechanism forthis tumor suppressor during breast-cancer progression are largelyunknown.

In this study, we first demonstrated that SCUBE2 wasco-expressed with E-cadherin in a number of breast-cancercell lines (supplementary material Fig. S3). Overexpressed

SCUBE2 could interact with and increase the formation of the


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Fig. 8. CpGmethylation status and recruitment of DNMT1 to CpG sites of the SCUBE2 gene during TGF-b-activated EMT. (A) Sequence of CpG sites (+159to +592) of human SCUBE2. The location of exons 1 and 2 of SCUBE2 is shown as a boxed number. Vertical lines represent a single CpG site. Methylation statusof CpG sites 1–43, which span positions +159 to +592, was quantified by MassARRAYanalysis. The number of CpG sites (+159 to +592) are indicated. The primersequences are boxed. (B) Quantitative methylation status of CpG sites located in SCUBE2 in four breast-cancer cell lines. Different colors show relative methylationchanges in 10% increments (red50%, yellow5100% methylated). (C) Methylation-specific PCR (MSP) analysis of the SCUBE2 regulatory region in human breast-cancer cell lines. The PCR products labeled ‘M’ (methylated) were generated by methylation-specific primers and those labeled ‘UM’ (unmethylated) weregenerated by primers specific for unmethylated genomic DNA. (D) MSP analysis of the SCUBE2 regulatory region. (E) Western blot analysis of the protein level ofDNMT1. Lamin A/C was a loading control. (F) Chromatin immunoprecipitation of in vivo binding of DNMT1 protein to the exon 1 region of SCUBE2. Primersamplified 246-bp fragments for SCUBE2 (+346–+592). (G) Effect of DNMT1 overexpression and knockdown on DNA methylation and expression of SCUBE2. Theexpression plasmid encoding DNMT1 was transiently transfected into MCF-7 cells. As well, DNMT1 expression was inhibited by transfection of plasmid encodingDNMT1-targeting shRNA (#1 and #2) into MDA-MB-231 cells. An shRNA targeting bacterial b-galactosidase was used as a negative control (Control-shRNA).Three days after transfection, RT-PCR, methylation-specific PCR (MSP) and western blot (WB) analyses were performed to determine the effects of DNMT1 gain-and loss-of-function on DNA methylation and SCUBE2 expression. The MSP products labeled M (methylated) were generated by methylation-specific primers andthose labeled UM (unmethylated) were generated by primers specific for unmethylated genomic DNA.


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E-cadherin–b-catenin complex in invasive MDA-MB-231 breast-cancer cells (Fig. 1D–H). Consistent with these findings,

introduction of SCUBE2 to these cells elevated the expressionof epithelial E-cadherin and downregulated the mesenchymalmarkers vimentin and N-cadherin (Fig. 3), thus promoting anepithelial transition by reversing EMT. Consequently, SCUBE2

overexpression induced the formation of adherens junction-likestrands on the membrane at cell–cell contacts (Fig. 1D–G) andtransformed cells from a fibroblastic phenotype to an epithelia-

like one that formed multicellular aggregates (Fig. 3A andsupplementary material Fig. S5). These SCUBE2-expressingcells also showed markedly reduced cell motility and invasion

activity (Fig. 3D,E). Because cell dissociation, motility andinvasion are key early events in the metastatic process,SCUBE2 may be a breast-cancer invasion and metastasis

suppressor by inhibiting breast-cancer progression at the initialstep of metastasis. Overall, these findings are in agreement withthe prognostic value of SCUBE2 in breast tumors, in that patientswith SCUBE2-positive tumors had better disease-free survival

than those with low or negative SCUBE2-protein-expressingtumors (Cheng et al., 2009). However, further studies are neededto elucidate the signaling pathway underlying the anti-migratory

and anti-invasive functions of SCUBE2 in breast-carcinoma cells.It is of interest to note that the E-cadherin-binding element (i.e.

the EGF-like repeats) in SCUBE2 is extracellular, whereas

SCUBE2 interacts with the cytoplasmic domain of E-cadherin(Fig. 1). In addition, our GST fusion protein pull-down assays didnot reveal a direct interaction between SCUBE2 and E-cadherin,

suggesting that as-yet-unknown protein(s) may connect thecytoplasmic juxtamembrane region of E-cadherin with thesurface-tethered SCUBE2 into adherens junction complexes toelicit b-catenin–SOX signaling and FOXA1 upregulation. Similarto our previous study showing that SCUBE2 can modulateb-catenin signaling (Lin et al., 2011), here we found thatSCUBE2 expression is positively associated with b-cateninexpression at the mRNA transcriptional level but not by post-translational regulation of its stability and localization (Fig. 7).Unlike its conventional role with a reduced b-catenin and TCFtranscriptional activity in epithelial cells, the SCUBE2-inducedexpression of SOX proteins may regulate b-catenin–TCF activityin the context of mesenchymal cells as seen in this study. Insupport of this notion, it has been reported that b-catenin andSOX17 can interact to transcribe endodermal gene expression ofFoxA1 in Xenopus embryos (Sinner et al., 2004) and both SOX4and SOX7 that are closely associated with b-catenin andSCUBE2 expression in breast cancer cells (Fig. 7) can interactwith b-catenin and modulate b-catenin-mediated transcription(Sinner et al., 2007; Takash et al., 2001).

Previous molecular and cell biology studies revealed thatFOXA1, a forkhead family transcription factor, can directly bindand transactivate the promoter of E-cadherin (Liu et al., 2005).

When overexpressed, it induces the formation of E-cadherin-containing adherens junctions and transforms invasive MDA-MB-231 cells from being spindle-shaped to more epithelial like,with a concomitant decrease of motility (Liu et al., 2005).

Furthermore, FOXA1 has been shown to be a ‘pioneer factor’ ofchromatin remodeling that can open compacted chromatinthrough its C-terminal domain to facilitate further transcription

(Carroll et al., 2005; Cirillo et al., 2002). Therefore, highlyexpressed FOXA1 protein probably can activate a silentE-cadherin promoter by its dual functions not only as a potent

transactivator (Fig. 6) but also a chromatin remodeler to decrease

the binding of the transcriptional repressors SNAIL and ZEB1to the E-cadherin promoter (Fig. 5E,F). However, further

investigations are needed to verify this hypothesis. In contrast,silencing of FOXA1 is sufficient to promote EMT (Song et al.,2010). Consistent with these findings, clinical studies showed thatFOXA1 expression is a positive prognostic factor among patients

with ER-positive tumors (Badve et al., 2007; Hisamatsu et al.,2012). In this study, we identified that SCUBE2 functionsupstream, on the plasma membrane at adherens junctions to

trigger induction of FOXA1 possibly by b-catenin–SOXtranscriptional activity and subsequent E-cadherin upregulation(Fig. 7). Because this SCUBE2–b-catenin/SOX–FOXA1–E-cadherin pathway does not depend on the regulation of well-established E-cadherin repressors such as TWIST, SNAIL,SLUG, ZEB1 or ZEB2 (Fig. 5), it adds a novel mechanism

to the regulatory circuits defining the differentiation state ofbreast-cancer cells.

Besides genetic mutations, epigenetic modifications such asDNA methylation of the E-cadherin gene promoter play a role in

malignant tumor transformation (Grady et al., 2000; Graff et al.,1995; Yoshiura et al., 1995). Likewise, the loss or downregulationof SCUBE2 expression is regulated by DNA hypermethylation

at the CpG islands downstream of exon 1, as evidenced bybisulphite sequencing and MSP (Fig. 8). Consistently, treatingSCUBE2-negative MDA-MB-231 breast-cancer cells with AZA (a

DNA methyltransferase inhibitor) reversed the hypermethylationstatus and restored both SCUBE2 and E-cadherin mRNA andprotein expression (supplementary material Fig. S8). Most

importantly, during TGFb-induced EMT, SCUBE2 wasdownregulated by DNA hypermethylation with upregulation andrecruitment of DNMT1 to the regulatory CpG sites at the SCUBE2locus in MCF-7 breast-cancer cells (Fig. 8). DNMT1 may play a

role in regulating SCUBE2, together with E-cadherin, for bothEMT and the reversal of EMT, during breast-cancer progression.

Of note, unlike E-cadherin, which is silenced by 59 promoterCpG-island methylation (Grady et al., 2000; Graff et al., 1995;Yoshiura et al., 1995), with SCUBE2 expression, the intrinsicCpG sites immediately downstream of exon 1 appear to play an

essential role in DNA-methylation-mediated inactivation byactive recruitment of DNMT1 in breast-carcinoma cells.Although CpG islands are generally found at the 59 promoterregion of genes, methylation of CpG islands far downstream of

the transcription start site (Medvedeva et al., 2010) or within bothexons and introns (Kim et al., 2008; Lu et al., 2001) can regulategene expression. However, the precise mechanisms of the binding

of DNMT1 to these SCUBE2 intrinsic CpG sites remain to befurther explored. One possible mechanism, described forE-cadherin repression during EMT (Dong et al., 2012), is

whether G9a (a histone lysine methyltransferase) could interactwith DNMT1 directly for recruitment to the SCUBE2 CpG sitesby associating with SNAIL, thus leading to SCUBE2 DNA

hypermethylation and EMT induction.In summary, our data unveiled previously unknown anti-

migratory and anti-invasive functions of a newly discoveredbreast-tumor-associated gene, SCUBE2. Mechanistically, SCUBE2

acts as a tumor migratory and invasive suppressor by increasingthe formation of E-cadherin–b-catenin adhesive complexes on theplasma membrane and drives epithelial differentiation through the

reversal of EMT. Furthermore, TGF-b-mediated upregulation andrecruitment of DNMT1 to regulatory CpG islands are crucialmolecular events epigenetically inactivating this novel breast-

tumor suppressor during EMT. This study highlights the important


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roles of SCUBE2 in breast-cancer progression and suggests furtherexploration of SCUBE2 as a diagnostic or therapeutic target for

breast tumors.

MATERIALS AND METHODSAntibodiesAnti-SCUBE2, anti-DNMT1 and anti-N-cadherin polyclonal antibodies

were from GeneTex (Irvine, CA, USA). E-cadherin and b-actinantibodies were from BD Biosciences (San Jose, CA, USA) and

NOVUS Biologicals (Littleton, CO, USA), respectively. Vimentin,

SNAIL, SLUG and lamin A/C antibodies were from Cell Signaling

Technology (Danvers, MA, USA). FOXA1 and TWIST antibodies were

from Abcam (Cambridge, MA, USA). ZEB1 and ZEB2 antibodies were

from Santa Cruz Biotechnology (Dallas, Texas, USA).

Expression plasmids and the luciferase reporter vectorsThe expression plasmids encoding SCUBE2 or E-cadherin were

constructed as described previously (Lin et al., 2011). An expression

plasmid encoding FOXA1 (pcDNA3-FOXA1) and the E-cadherin

promoter luciferase reporter plasmid were kind gifts from Ji Hshiung

Chen (Tzu Chi University, Taiwan) as described (Liu et al., 2005).

Cell cultureMCF-7, T-47D, MDA-MB-231 and ZR-75-30 human breast-cancer cell

lines were obtained from the American Type Culture Collection

(Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were cultured in

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%

fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin.T-47D and ZR-75-30 breast cancer cells were maintained in RPMI 1640

medium supplemented with 10% fetal bovine serum, 1 mM sodium

pyruvate, 100 units/ml penicillin, and 100 mg/ml streptomycin.

TGF-b1 treatmentMCF-7 cells (56105 cells) were seeded into 6-cm dishes for 18 hours.Cells were washed with phosphate-buffered saline (PBS) and incubated

in serum-free medium for 24 hours. Cells were treated with 10 ng/ml

TGF-b1 (in 1% fetal bovine serum medium) for 7, 14 and 21 days, withmedium replaced with fresh TGF-b1 every 3 days.

Immunoprecipitation and western blot analysisCancer cells were washed once with PBS and lysed for 15 minutes on ice

in lysis buffer. Lysates were clarified by centrifugation at 10,000 g for

15 minutes at 4 C̊. Samples underwent immunoprecipitation and western

blot analysis as described (Tsai et al., 2009).

RT-PCRTotal RNA was prepared from cultured cells by the TRIzol method (Life

Technologies, Grand Island, NY). First-strand cDNA synthesis with

SuperScript II reverse transcriptase (Life Technologies) used 5 mg RNA.One-tenth of the first-strand cDNA reaction was used for each PCR as a

template. Primers are listed in supplementary material Table S1. The

PCR products were run on a 1% agarose gel.

Confocal immunofluorescence microscopyMDA-MB-231 control, SCUBE2-expressing or MCF-7 cells were fixed

in 4% formaldehyde, blocked with 2% fetal bovine serum for 1 hour, and

incubated with mouse anti-E-cadherin antibody and rabbit anti-vimentin

antibody for 1 hour. Slides were washed three times with PBS and stained

with Alexa-Fluor-488-labeled anti-mouse IgG antibody and Alexa-Fluor-

594-labeled anti-rabbit IgG antibody for 1 hour, then washed three times in

PBS and mounted with VECTASHIELD mounting medium and DAPI

(Vector Laboratories, Burlingame, CA, USA). Fluorescence images were

captured at room temperature under a confocal microscope (model LSM

510; Carl Zeiss, Thornwood, NY, USA).

Cell aggregation assayMDA-MB-231 control and SCUBE2-expressing cells were detached by

treatment with trypsin (0.01%)-EDTA (2.5 mM) and suspended in

DMEM containing 5 mM CaCl2 or Ca2+ free DMEM, at 16106 cells/ml

in polystyrene tubes. Then tubes were incubated on a rotating platform

(10 rpm) at 37 C̊ for 9 hours. The cell aggregation was viewed

microscopically and photographed. For quantification, cell clusters of

more than four cells were considered as aggregates. The data are means

6 s.d. of three independent experiments performed in duplicate.

Cell migration assayFor the breast cancer cell migration assay, a culture insert (IBIDI GmbH,

Martinsried, Germany) was placed into one well of a 24-well plate. An

equal number of MDA-MB-231 control, SCUBE2-expressing, MCF-7

control-shRNA, or SCUBE2-shRNA cells (100 ml; 56104 cells/ml) wereadded into the two reservoirs of the same insert and incubated at 37 C̊,

5% CO2. After 16 hours, the insert was gently removed, thus creating a

gap of ,500 mm. The well was filled with complete growth medium andmigration was observed by live-cell imaging with a microscope (model

IX71, Olympus, Center Valley, PA, USA).

Cell invasion assayFor invasion assays, cells were suspended in serum-free medium and

plated in duplicate in the top well of Matrigel invasion chambers (8-mmpore size; BD, Franklin Lakes, NJ, USA). Complete medium was

placed in the lower chamber and cells were allowed to invade for

18 hours at 37 C̊, 5% CO2. Cells in the upper chamber were removed

using a cotton swab, and cells on the lower chamber were fixed in

methanol and stained with Crystal Violet. The number of invasive cells

was counted for three random fields per experiment from three

independent experiments.

Luciferase reporter assayMCF-7, MDA-MB-231 control, and SCUBE2 cells were plated in

24-well plates (16105 cells/well) and incubated overnight at 37 C̊. Thenext day, cells were transfected with 0.5 mg SCUBE2 promoter, exon 1,exon 2 (MCF-7 cells) or E-cadherin promoter (MDA-MB-231 cells)

luciferase constructs and internal control (0.05 mg pRL-TK Renillaluciferase plasmid) with FuGENE HD (Promega, Madison, WI, USA)

according to the manufacturer’s instructions. Cells were cultured for an

additional 2 days, harvested and prepared for reporter assay with the

Dual-Luciferase reporter assay system (Promega).

Pull-down assayRecombinant HA-tagged p120-catenin protein (HA–p120-catenin),

FLAG-tagged SCUBE2-FL, -ty97 or -D4 (FLAG–SCUBE2-FL, -ty97,

-D4) protein was produced by overexpression from HEK-293T cells. The

GST–E-cadherin protein was mixed with FLAG–SCUBE2 protein bound

to anti-FLAG M2-antibody agarose beads or control beads in 0.5 ml

binding buffer [40 mM HEPES (pH 7.5), 100 mM KCl, 0.1% Nonidet

P-40 and 20 mM 2-mercaptoethanol]. After incubation for 4 hours at

4 C̊, the beads were washed extensively, and interacting protein was

visualized by immunoblotting using anti-GST antibody.

Drug treatment5-aza-29-deoxycytidine (AZA) and trichostatin A (TSA) were obtainedfrom CalBiochem (San Diego, CA, USA) and Sigma (St Louis, MO,

USA), respectively; dissolved in DMSO and prepared as a 1000-fold

concentrated stock solution; and added to the culture medium at 1:1000

dilution according to the experimental procedure. MDA-MB-231 cells

were treated with 10 mM AZA for 96 hours, with medium replaced withfresh AZA every 12 hours. TSA (300 nM) was added to cells 24 hours

before the end of experiments.

Preparation of methylated and unmethylated SCUBE2 reporter-gene plasmid DNAsThe reporter plasmid DNAs were methylated in vitro by use of bacterial

CpG methylase M.SssI (New England BioLabs, Ipswich, MA, USA)

and 1 mM S-adenosylmethionine according to the manufacturer’sinstructions. Complete methylation of plasmid DNAs was confirmed

by digestion with methylation-sensitive endonucleases (HpaII).


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Quantitative DNA methylation analysisAnalysis of the regulatory regions of SCUBE2 involved the use of the

EpiTYPER methylation assay (Sequenom, San Diego, CA, USA). We

designed two bisulfite reactions that covered 43 CpG sites for SCUBE2.

The DNA of breast-cancer cells was extracted, and 1 mg DNA wastreated with bisulfite, in vitro transcribed, cleaved by RNase A, and

subjected to matrix assisted laser desorption ionization time of flight

(MALDI-TOF) mass spectroscopy analysis to determine methylation

patterns. Primers used to amplify the regulatory regions for SCUBE2 are

in supplementary material Table S1.

DNA extraction, bisulfite treatment and methylation-specific PCRGenomic DNA was extracted from breast-cancer cells by use of the QIAamp

DNA mini kit (Qiagen GmbH, Hilden, Germany). We used the EZ DNA

Methylation Kit (ZYMO Research, Irvine, CA, USA). Briefly, extracted DNA

samples were denatured and subjected to bisulfite treatment using CT

conversion reagent for 16 hours at 50 C̊. Modified DNA was purified by use of

the Zymo-Spin IC column according to the manufacturer’s instructions.

Modified DNA was then used for MSP to detect the methylation status of the

SCUBE2 exon 1 to exon 2 region. The primer sequences designed to amplify

specifically methylated (M) or unmethylated (UM) forms of SCUBE2 exon 1

are in supplementary material Table S1. The amplification products of M and

UM forms were 217 and 220 bp, respectively. For both PCR reactions, 100 ng

modified DNA was amplified in a 25-ml reaction at 94 C̊ for 5 minutes; then 35cycles at 94 C̊ for 15 seconds, 60 C̊ for 30 seconds and 72 C̊ for 30 seconds;

then 72 C̊ for 5 minutes. The PCR products were run on a 1.5% agarose gel.

Chromatin immunoprecipitationWe used the EZ-Magna ChIP G Kit (Millipore, Billerica, MA, USA).

Briefly, MCF-7 or MDA-MB-231 cells (16107 cells) were cross-linkedusing 1% formaldehyde, lysed in 500 ml lysis buffer and sonicated to,500-bp fragments. ChIP involved antibodies against DNMT1, FOXA1or IgG. The input control DNA or immunoprecipitated DNA was

amplified in a 50-ml reaction volume consisting of 2 ml eluted DNAtemplate with the primers (supplementary material Table S1). Taq

polymerase was used for PCR with 35 cycles of 94 C̊ for 30 seconds,

63.5 C̊ for 30 seconds, 72 C̊ for 50 seconds, then 5 minutes at 72 C̊. A

10-ml aliquot from each PCR reaction was separated on 1.5% agarose gel.

Statistical analysesSPSS v10.0 (SPSS Inc., Chicago, IL, USA) was used for statistical

analyses. P,0.05 was considered statistically significant.

AcknowledgementsWe thank the National Center for Genome Medicine at Academia Sinica, Taiwan,for DNA methylation analysis.

Competing interestsThe authors declare no competing interests.

Author contributionsY.C.L, Y.C.L., L.H.L. designed and performed experiments, and analyzed andinterpreted data. C.J.C and R.B.Y. designed research, analyzed and interpreteddata, and wrote the manuscript.

FundingThis work was supported by the Taiwan National Science Council [grant numbersNSC 101-2325-B-001-030, 102-2325-B-001-028, 102-2320-B-001-015-MY3 toR.B.Y., and NSC 100-2320-B-038-029 to C.J.C.]. The National Center forGenome Medicine at Academia Sinica, Taiwan is supported by grants from theNational Core Facility Program for Biotechnology, National Science Council,Taiwan [grant numbers NSC 101-2319-B-001-001 and 102-2319-B-001-001].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132779/-/DC1

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