mir-200 can repress breast cancer metastasis through zeb1-independent but moesin-dependent pathways

ORIGINAL ARTICLE

MiR-200 can repress breast cancer metastasis throughZEB1-independent but moesin-dependent pathwaysX Li1, S Roslan1, CN Johnstone2,3,4,5, JA Wright1,6, CP Bracken1,6, M Anderson1, AG Bert1, LA Selth7, RL Anderson2,3,4, GJ Goodall1,6,8,PA Gregory1,6,9 and Y Khew-Goodall1,8,9

The microRNA-200 (miR-200) family has a critical role in regulating epithelial–mesenchymal transition and cancer cell invasionthrough inhibition of the E-cadherin transcriptional repressors ZEB1 and ZEB2. Recent studies have indicated that the miR-200family may exert their effects at distinct stages in the metastatic process, with an overall effect of enhancing metastasis in asyngeneic mouse breast cancer model. We find in a xenograft orthotopic model of breast cancer metastasis that ectopic expressionof members of the miR-200b/200c/429, but not the miR-141/200a, functional groups limits tumour cell invasion and metastasis.Despite modulation of the ZEB1-E-cadherin axis, restoration of ZEB1 in miR-200b-expressing cells was not able to alter metastaticpotential suggesting that other targets contribute to this process. Instead, we found that miR-200b repressed several actin-associated genes, with the knockdown of the ezrin-radixin-moesin family member moesin alone phenocopying the repressionof cell invasion by miR-200b. Moesin was verified to be directly targeted by miR-200b, and restoration of moesin in miR-200b-expressing cells was sufficient to alleviate metastatic repression. In breast cancer cell lines and patient samples, the expression ofmoesin significantly inversely correlated with miR-200 expression, and high levels of moesin were associated with poor relapse-freesurvival. These findings highlight the context-dependent effects of miR-200 in breast cancer metastasis and demonstrate theexistence of a moesin-dependent pathway, distinct from the ZEB1-E-cadherin axis, through which miR-200 can regulate tumour cellplasticity and metastasis.

Oncogene advance online publication, 16 September 2013; doi:10.1038/onc.2013.370

Keywords: miR-200; epithelial–mesenchymal transition; breast cancer; metastasis; actin cytoskeleton

INTRODUCTIONThe majority of cancer-related deaths from carcinomas result frommetastatic progression. For carcinomas to metastasise, epithelialtumour cells undergo a complex series of events, which includeinvasion in the primary site, dissemination and colonisation at asecondary location.1 This metastatic cascade is not onlyorchestrated by numerous signalling pathways operating withinthe tumour cells but can also be modulated through interactionswith stromal cells.2 To acquire invasive capabilities, epithelialtumour cells can undergo an epithelial–mesenchymal transition(EMT), a process that facilitates the loss of cell–cell adhesions andpromotes cell motility, stem cell-like properties and resistance toapoptosis.3 These features are proposed to drive tumourdedifferentiation and enhance metastatic progression.4 However,the analysis of clinical samples indicates that metastases oftenclosely resemble the primary tumour in morphology and geneexpression profile5–7 suggesting that the redifferentiation of themetastasising cell may occur via a mesenchymal to epithelialtransition (MET).8 This concept has been bolstered by recentevidence using genetic and experimental mouse models ofmetastasis,9–12 which demonstrate that the transition between

epithelial and mesenchymal states are important contributors tometastatic progression.4

The microRNA-200 (miR-200) family have emerged recently asimportant regulators of EMT. Across a diverse range of epithelial-derived cancer cell types, the miR-200 family are able to enforcean epithelial state by inhibiting the E-cadherin transcriptionalrepressors ZEB1 and ZEB2.13–16 Furthermore, the ZEB transcriptionfactors can repress miR-200 expression and through this reciprocalfeedback loop modulate epithelial cell plasticity.16–18 Numerousstudies have indicated a role for the miR-200-ZEB feedback loop intumour cell invasion and metastasis, although the dependency ofthis interaction in controlling these processes has not beenevaluated. The loss of miR-200 and the gain of ZEB1 expressionhave each been separately associated with tumour progressionand the acquisition of characteristics of metastatic cells, includingthe invasive and stem-like properties,19–21 as well as resistance toapoptosis and chemotherapeutics.22–25 In mouse xenograftmodels of metastasis, re-expression of miR-200 or inhibition ofZEB1 can attenuate metastasis by inhibiting EMT.26,27 In contrast,studies using the 4T1 series of mouse mammary cancer cells haveshown that miR-200 can enhance metastatic colonisation.28,29

1Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia; 2Peter MacCallum Cancer Centre, St Andrews Place, EastMelbourne, Victoria, Australia; 3Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia; 4Department of Pathology, The Universityof Melbourne, Parkville, Victoria, Australia; 5Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia; 6Discipline of Medicine, The University ofAdelaide, Adelaide, South Australia, Australia; 7Dame Roma Mitchell Cancer Research Laboratories, Discipline of Medicine, University of Adelaide, Hanson Institute, Adelaide,South Australia, Australia and 8School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, Australia. Correspondence: Dr PA Gregory,Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, South Australia 5000, Australia or Associate Professor Y Khew-Goodall, Divisionof Human Immunology, Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, South Australia 5000, Australia.E-mail [email protected] or [email protected] authors contributed equally to this work.Received 20 January 2013; revised 11 July 2013; accepted 26 July 2013

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http://dx.doi.org/10.1038/onc.2013.370

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These seemingly paradoxical findings can be explained by amodel of tumour cell plasticity, whereby miR-200 loss permits EMTand invasion away from the primary tumour, whereas efficientcolonisation requires re-expression of miR-200 and a subsequentMET.4,29 Accordingly, the examination of breast and colorectalpatient samples reveals that miR-200 levels can be reduced at theinvasive front in primary tumours but are often increased inresulting metastases.29–33 Given these observations, it is of clinicalimportance to understand the mechanisms through which themiR-200-ZEB feedback loop controls metastatic progression.

The miR-200 family comprises five members that areexpressed from two distinct polycistronic transcripts (miR-200bB200aB429 and miR-200cB141) and can be separatedinto two functional groups (miR-200b/200c/429 and miR-141/200a) on the basis of their ‘seed’ sequence.16,17 Althoughdifferences in the ability of the miR-200 functional groups toinfluence cell invasion have been identified,34,35 their individualeffect in metastasis has not been investigated. In this study, weutilise an orthotopic model of breast cancer metastasis (MDA-MB-231-LM236) and demonstrate that ectopic expression of miR-200b or miR-200c, but not miR-141, reduces tumour cell invasionand breast cancer metastasis. Surprisingly, these effects were notmediated directly by the loss of ZEB1 and MET but ratherthrough the repression of the cytoskeletal remodelling proteinmoesin. Moesin has previously been shown to be targeted bymiR-200c and to influence the ability of miR-200c to repress cellmigration.37 Here, we show that moesin expression is inverselycorrelated with miR-200 in patient breast cancer samples and is

elevated in patients with poor metastasis-free survival. Together,these results demonstrate that miR-200 can regulate tumour cellmetastasis through ZEB1-independent but moesin-dependentpathways.

RESULTSStable expression of miR-200b can alter breast cancer cellmigration and invasion in the absence of METThe miR-200 family causes a MET in a wide range of epithelial-derived cancer cell types with associated reductions in cellmigration and invasion. However, the ability of individual miR-200family members to influence metastasis has not been studied. Todetermine the role of individual miR-200 family members incontrolling breast cancer metastasis, we initially expressed miR-200b in the well-characterised MDA-MB-231 LM2 subline, whichcan metastasise spontaneously after implantation in the mam-mary gland.36 Stable expression of miR-200b to B30-fold abovebasal level reduced tumour cell migration and invasion in vitro(Figure 1a and b) but, contrary to expectation, did not inducemorphological changes consistent with MET. Although miR-200expression reduced ZEB1 and weakly induced E-cadherin expres-sion (Figure 1c), the E-cadherin protein was localised predomi-nantly in the perinuclear region and not at cell–cell junctions as istypical of epithelial cells (Figure 1d). Therefore, miR-200b is able toalter cellular properties associated with metastasis independentlyof its ability to induce a MET.

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Figure 1. miR-200b can reduce migration and invasion independently of a MET. (a) Real-time PCR measurement of miR-200b in LM2 cellstransduced with a control or miR-200b lentiviral vector. (b) Comparison of the relative migration (4 hours) and invasion (24 hours) of thevector with miR-200b stable LM2 cells towards a 10% serum gradient. Data are the mean±s.d. of three independent assays. *** denotesP-value ofo0.001 as measured by the unpaired two-tailed Student’s t-test. (c) Western blot of E-cadherin and ZEB1 in extracts from LM2 stablecells lines. Tubulin is shown as a loading control. (d) Morphology of the vector and miR-200b stable LM2 cells as shown by phase-contrastimaging. Cells were also immunostained for E-cadherin (green) to visualise its intracellular localisation with nuclear 4’,6-diamidino-2-phenylindole (DAPI) counterstaining shown in blue. Scale bars represent 50 mm.

miR-200 regulates metastasis by targeting moesinX Li et al

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Oncogene (2013) 1 – 12 & 2013 Macmillan Publishers Limited

miR-200b reduces spontaneous breast cancer metastasis tomultiple organs without influencing extravasation or colonisationTo determine whether miR-200b directly influences spontaneousbreast cancer metastasis, we implanted control or miR-200b-expressing MDA-MB-231 (LM2) cells into the mammary fat pad ofnonobese diabetic/severe combined immunodeficient mice andassessed primary tumour growth and metastasis to multiple

organs by bioluminescence imaging. After 29 days, primarytumours had grown to a similar size; however, cells expressingmiR-200b metastasised less efficiently to the lung, liver and bone(Figures 2a and b). We verified the presence of tumour cells in thelung and bone by immunohistochemical analyses for greenfluorescent protein, which is co-expressed with the luciferasereporter in LM2 cells (Figure 2c). To assess whether miR-200b-

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Figure 2. miR-200b expression inhibits spontaneous metastasis from the mammary fat pad. (a) Bioluminescent images of primary tumoursin situ and lung, liver and bone (hind limbs) ex vivo generated from the vector and miR-200b stable LM2 cells 28 days post orthotopic injection.(b) Quantitation of primary tumour weight and metastasis to the lung, liver and bone as measured by bioluminescent intensity (representedby photons/sec). Ten mice were injected per cell line with each point representing a measurement from an individual mouse with the meanvalues indicated by a horizontal line. P-values were calculated using a two-tailed Mann–Whitney t-test. (c) Haemotoxylin and Eosin (H&E)and immuno-green fluorescent protein (GFP) staining of a representative lung and bone section derived from LM2-vector cells.(d) Immunostaining of a representative LM2-miR-200b primary tumour section for E-cadherin. Staining of an adjacent region of epidermis isshown as a positive control. Scale bars represent 100 mm. (e) Control or miR-200b-expressing LM2 cells were injected via tail vein andbioluminescent intensity in the lung in vivo was measured over a 25-day time course. Five mice were injected per cell line with meansplotted±s.e.m. The P-value was calculated using two-way analysis of variance (ANOVA).


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& 2013 Macmillan Publishers Limited Oncogene (2013) 1 – 12

expressing cells were less metastatic by virtue of the primarytumour cells having undergone a MET, we stained primarytumours for E-cadherin. Examination of the LM2-miR-200bprimary tumour core revealed very weak cytoplasmic staining ofE-cadherin, in contrast to the epidermal cells adjacent to theprimary tumour in which prominent membranous staining wasobserved (Figure 2d). This indicates that miR-200b can repress the

metastasis of primary tumour cells without a requirement for theirtransition to an epithelial phenotype.

Metastasis is a multistep process involving the early steps ofinvasion and intravasation followed later by extravasation andcolonisation by tumour cells to establish a distant secondarytumour. We tested whether miR-200b alters metastasis byinfluencing the ability of the cells to extravasate and colonise

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Figure 3. miR-200c, but not miR-141, represses spontaneous metastasis. (a) Real-time PCR measurement of miR-200c or miR-141 in LM2 cells stablyexpressing miR-200c and miR-141, relative to control vectors. (b, d), Quantitation of primary tumour weight and metastasis to the lung (asmeasured by bioluminescent intensity) measured 28 (b) or 35 (d) days after mammary fat pad injection with miR-200c, miR-141 or with therespective control vector-expressing LM2 cells. For the vector/miR-200c- and vector/miR-141-paired experiments ten and nine mice were analysed,respectively, with mean values indicated (horizontal line). P-values were calculated using a two-tailed Mann–Whitney t-test. (c, e) western blot ofextracts derived from the LM2 stable cell lines for E-cadherin, ZEB1 and Tubulin. (f ) Phase-contrast and immunofluorescent images of LM2 cellsstably expressing vector, miR-200c and miR-141 showing E-cadherin (green) and nuclear DAPI staining (blue). Scale bars represent 50mm.


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the lung. To circumvent the early steps of the spontaneousmetastatic process, control or miR-200b-expressing LM2 cells wereinjected directly into the tail vein of mice and subsequent tumourdevelopment in the lung monitored. Two days after tail veininjection when cells that remained in circulation have beencleared, there was no significant difference in lung biolumines-cence between mice injected with control or miR-200b-expressingcells (Figure 2e), suggesting that the cells were equally able toextravasate and deposit in the lung. Furthermore, over a 25-dayperiod, the rate of tumour formation in the lung was notsignificantly different between control and miR-200b-expressingcells (Figure 2e), suggesting that the colonisation of secondarysites was also not affected by elevated levels of miR-200b. Byinference, therefore, miR-200b is likely to be inhibiting the earlysteps of metastasis, which is consistent with its repressive effectson cell migration and invasion.

Stable expression of miR-200c, but not miR-141, represses breastcancer metastasisBecause the miR-200 family is composed of five memberspossessing two distinct ‘seed’ sequences, the miR-200b/200c/429and miR-141/200a groups likely target different but sometimesoverlapping sets of genes (as in the case of ZEB113). To determinewhether both seed groups are able to repress breast cancermetastasis, we stably expressed either miR-200c or miR-141 inLM2 cells and measured spontaneous metastasis to the lung. Toachieve high expression of the introduced microRNA (miRNA), weused the LMP retroviral system,38 which resulted in a B40- andB80-fold increase in miR-200c and miR-141 expression abovevector-transduced cells (Figure 3a), to levels similar to thatexpressed by epithelial breast cancer cell lines.13 At the sametime, we used this system to stably express miR-200b, achievingBtwofold higher expression levels than with the original (polIIIdriven) vector (Supplementary Figure S1). This increased expres-sion of miR-200b intensified both the changes in E-cadherin andZEB1 levels and inhibited the degree of metastasis to the lung(Supplementary Figure S1), although a transition to an epithelialphenotype was again not observed (data not shown). As we foundwith miR-200b, miR-200c was able to efficiently repress metastasisto the lung without affecting primary tumour growth (Figure 3b).MiR-200c also caused similar changes in E-cadherin and ZEB1expression (Figure 3c) in the absence of a MET (Figure 3f). Theseresults imply that different members of the miR-200b/200c/429functional group can repress metastasis by similar mechanisms. Incontrast to miR-200b and miR-200c, stable expression of miR-141did not significantly reduce the lung metastatic potential of LM2cells (Figure 3d). Interestingly, miR-141 only modestly reducedZEB1 levels, and E-cadherin expression was not induced in thesecells (Figures 3e and f). This may be indicative of the decreasedpotency of ZEB1 targeting by the miR-141 seed compared withthe miR-200b seed as described previously.13 Taken together,these results indicate that the miR-200b/200c/429, but not themiR-141/200a, functional group is able to repress the spontaneousmetastasis of LM2 cells.

miR-200b represses breast cancer metastasis independently of itseffect on ZEB1Although several studies have demonstrated a role for miR-200 orZEB1 in cancer cell invasion and metastasis,26,27 the dependencyof their interaction in metastasis has not been investigated. Todetermine whether miR-200b represses metastasis by decreasingZEB1 levels, we transduced either a control vector or a ZEB1complementary DNA lacking its 3’ untranslated region (30UTR) intomiR-200b-expressing LM2 cells. Both cell lines expressed equi-valent high levels of miR-200b with ZEB1 levels being elevated inthe pLenti4-ZEB1-transduced cells (Figure 4a). In addition, cellswith high ZEB1 also had reduced E-cadherin levels confirming

the functionality of ZEB1 expressed from the exogenous vector(Figure 4a). Comparing the cells having high miR-200b/low ZEB1with those having high miR-200b/high ZEB1 in spontaneousmetastasis assays, we found that both cell lines metastasisedweakly with no significant difference in lung metastasis or tumourweight (Figure 4b). These results were also confirmed in a secondgroup of mice containing additional controls (SupplementaryFigure S2). These results indicate that high miR-200b expressioninhibits metastasis of LM2 cells, regardless of the level of ZEB1. Ofnote, the related protein ZEB2 is expressed at very low levels inLM2 cells and is not likely to have a role in these cells, suggestingthat miR-200b may target other genes to regulate metastasis.

Moesin, cofilin2 and WASF3 are repressed by miR-200b, but not bymiR-141Cell invasion and metastasis require dynamic remodelling of theactin cytoskeleton to facilitate movement from the primarytumour. Recent reports have shown that the actin-associatedfactors moesin (MSN), cofilin2 (CFL2) and WASF3 are direct targetsof the miR-200 family; however, their contribution to metastasisremains largely unexplored.16,37,39 We measured the mRNAlevels of moesin, cofilin2 and WASF3 in the miR-200b and miR-141 stably transduced LM2 stable cell lines and found that eachof these factors was reduced only in the miR-200b cells relativeto their empty vector cell line (Figure 5a). As a control, the levelsof ZEB1 mRNA were reduced in both cell lines. This raises thepossibility that the differential effect of the miR-200b/200c/429and miR-141/200a seeds on metastasis may be mediatedthrough changes in cytoskeletal dynamics. Staining of the LM2stable cell lines for F-actin revealed changes in cell morphology,which were especially evident in LM2 cells that were transiently

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Figure 4. miR-200b can repress metastasis independently of ZEB1.(a) Real-time PCR and western Blot of miR-200b, ZEB1, E-cadherinand Tubulin levels in miR-200b-expressing LM2 cells transducedwith a control or ZEB1 coding region vector. (b) The measurement ofprimary tumour weight and lung metastasis in LM2-miR-200b cellsexpressing a control vector or ZEB1. A dashed line marking the meanbioluminescent intensity in LM2-vector cells is shown for compar-ison (see Supplementary Figure S1). P-values were calculated using atwo-tailed Mann–Whitney t-test.


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transfected with miR-200b (Figures 5b and d). As observed in thestable cell lines (Figure 5a), transient transfection with miR-200bor miR-200c specifically decreased moesin mRNA and proteinlevels, whereas transfection with miR-141 did not (Figure 5c).Cells transfected with miR-200b also displayed a markedlyreduced invasive capacity without forming intercellular junctionsor expressing junctional E-cadherin (Supplementary Figure S3).These effects were less pronounced in miR-141-transfected cells(Figure 5b and Supplementary Figure S3). We therefore furtherinvestigated whether repression of actin-related genes contri-bute to miR-200b-mediated inhibition of cell invasion andmetastasis.

Moesin knockdown reduces cancer cell invasion and is inverselycorrelated with miR-200b seed in breast cancerTo determine whether moesin, cofilin2 or WASF3 influencesinvasion of LM2 cells, we transiently knocked down each of thesegenes and assessed their effect in in vitro invasion assays. Onlymoesin knockdown significantly repressed cell invasion (Figure 6aand Supplementary Figure S4). At the same time, we knockeddown ZEB1 levels and found that it only marginally altered cellinvasion despite increasing E-cadherin levels (Figure 6a andSupplementary Figure S4). In contrast, E-cadherin expression wasnot increased after moesin knockdown (Supplementary Figure S4).These data suggest that the modulation of moesin rather than the

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Figure 5. miR-200 family members differentially target actin-associated genes and alter cell morphology. (a) Real-time PCR measuring the expressionof several actin-associated miR-200 target genes in LM2 stable cell lines. Protein levels for moesin expression are shown by western blot. (b) Phalloidinstaining of F-actin in LM2 stable cell lines and in LM2 cells transiently transfected with a negative control (Neg), miR-200b or miR-141 mimic (Pre-miR)for 48 hours. For Pre-miR-transfected cells, the lower panel represents a magnified view compared with the upper panel. Scale bars indicate 20mm.(c) Real-time PCR quantitation of moesin mRNA and western blot analysis of LM2 cells transiently transfected with Pre-miRs. (d) Differences in cellshape in Pre-miR-transfected LM2 cells were calculated by measuring the length to width ratio of each cell for 110 cells per treatment. Data shown arethe mean±s.e.m. *Po0.05, ***Po0.001 compared with negative (Neg) using one-way ANOVA, followed by post-hoc Tukey’s multiple comparisons.


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ZEB1-E-cadherin axis may be important in miR-200b-mediatedcontrol of metastasis in LM2 cells.

Moesin is a member of the ezrin-radixin-moesin family ofproteins that regulate actin localisation and cross-linking to the

plasma membrane.40 A recent study has shown that moesin is adirect target of miR-200c and a key contributor to miR-200c-mediated repression of cell migration.37 Using reporter assays, weshowed that the moesin 30UTR is repressed by miR-200b, and not

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Figure 6. Moesin reduces cell invasion and is inversely associated with miR-200 in breast cancer. (a) Invasion assays of LM2 cells were carriedout for 24 hours following small interfering RNA (siRNA)-mediated knockdown of indicated genes (after 72 hours). Data are collated from twoto three independent experiments performed in triplicate. * denotes P-value of o0.05 as calculated using a two-tailed Student’s t-test.(b) Luciferase reporter assays of a full-length or 740 bp segment of the moesin 30UTR fused to a renilla reporter gene after co-transfection ofMDA-MB-231 cells with control (Neg), miR-200b or miR-141 mimic (Pre-miR) for 72 hours. Data are representative of three experimentsperformed in triplicate. * denotes P-value of o0.05 as calculated using a two-tailed Student’s t-test. (c) Western blot of moesin expression inbreast cancer cell lines possessing a luminal or basal expression pattern and previously shown to have either a high or low miR-200 familyexpression.13 Tubulin is shown as a loading control. (d) Real-time PCR quantitation of moesin mRNA expression and western blot analysis ofbreast cancer cells transiently transfected with Pre-miRs for 3 days. (e) Real-time PCR quantitation of moesin mRNA expression in breast cancercell lines transiently transfected with a miR-200 family Anti-miR for 6 days. (f ) Correlation of moesin and miR-200b or miR-200c expression in apanel of 101 breast cancer samples (GSE19783). Pearson correlation coefficients and P-values were calculated as indicated. (g) Kaplan–Meiercurves showing metastasis-free survival (779 tumour set) or relapse-free survival (UNC311) in patients with high (above median) or low (belowmedian) expression of MSN. Log-rank tests were used to compare the survival distributions (P-value as shown).


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by miR-141, consistent with the differential regulation of moesinin the LM2 stable cell lines (Figure 6b). To assess whether there isan association between the levels of moesin and miR-200 in breastcancer, we analysed their levels in breast cancer cell lines andpatient samples. Moesin was strongly enriched in basal subtypecell lines, in which the miR-200 family has been shown previouslyto be lowly expressed,13 in contrast to luminal cell lines in whichthere is a high miR-200 expression (Figure 6c).13 Transfection oftwo basal subtype cell lines with miR-200b reduced moesin mRNAand protein, whereas transfection with miR-141 did not(Figure 6d). Conversely, inhibition of the miR-200 family in luminalbreast cancer cell lines increased moesin mRNA levels (Figure 6e).We next interrogated a published data set of 101 breast cancerpatient samples where both mRNA and miRNA expression hadbeen profiled (GSE1978341). Using linear regression analysis,moesin expression showed a significant negative correlationwith the expression of both miR-200b and miR-200c, consistentwith it being regulated by these miRNAs in breast cancer(Figure 6f). Furthermore, a strong negative correlation betweenmoesin and miR-200 expression was also observed in the NCI-60panel of cancer cell lines (Supplementary Figure S5). In contrast,only weak inverse correlations between cofilin2 or WASF3 and themiR-200 family members were observed in breast cancers (datanot shown). Examining two separate breast cancer cohorts inwhich outcome data were available, we also found that patientswhose tumours expressed high moesin levels had reducedmetastasis- or relapse-free survival (Figure 6g). In contrast, CFL2and ZEB1 were generally less predictive of outcome(Supplementary Figure S6), but, interestingly, WASF3 was pre-dictive of outcome in one cohort but not the other. Within thesecohorts, moesin expression was significantly higher in the basaland claudin-low classified samples (Supplementary Figure S7),subtypes which are known to be associated with poor prognosisand low miR-200 levels.13,16 Together, these data indicate that theloss of miR-200b leading to the upregulation of moesin expressionmay contribute towards metastasis and that this pathway may beimportant for breast cancer progression.

Restoration of moesin expression prevents miR-200b-mediatedrepression of metastasisTo determine whether the reduction in moesin expression causedby miR-200b is necessary for repression of breast cancermetastasis, we re-expressed the moesin complementary DNAwithout its 30UTR in miR-200b-expressing LM2 cells (LM2-miR-200b) and performed spontaneous metastasis assays. As shown inFigure 7a, transduction of the moesin complementary DNA intoLM2-miR-200b cells increased moesin expression to levels similarto LM2-vector cells. Importantly, the miR-200b levels were similarin control vector and moesin-transduced LM2-miR-200b cells,both being elevated by B30-fold over LM2-vector cells. Moesinexpression in LM2-miR-200b cells caused changes from a flattenedto a more elongated morphology, which is consistent with recentfindings showing that moesin regulates cell polarity and spreading(Figure 7b).42,43 In spontaneous metastasis assays, restoration ofmoesin expression in LM2-miR-200b cells caused them tometastasise more efficiently than control cells, although notreaching statistical significance, with a mean level similar to that ofLM2-vector cells (Figure 7c). However, we noted that the moesin-expressing tumours were smaller than the control tumours, andthe increase in metastasis burden was statistically significant afternormalising for primary tumour size (Figure 7c). The ability ofmoesin, and not ZEB1, to restore metastatic capability to LM2-miR-200b cells was confirmed in an additional experiment in which allgroups of cell lines were compared concurrently (SupplementaryFigure S2). Furthermore, reduced moesin expression was pre-served in primary tumours formed from miR-200b-transfectedLM2 cells and restored moesin expression was maintained in

tumours derived from LM2-miR-200b cells with enforced moesinexpression (Figure 7d). These data demonstrate that down-regulation of moesin is required for miR-200b to repressmetastasis of LM2 cells. Collectively, these findings indicate thatmiR-200b/200c/429 and miR-141/200a functional groups havedifferent roles in the metastatic process, and that miR-200b/200c/429 can operate through moesin-dependent pathways thatare distinct from the canonical ZEB1-E-cadherin pathway and EMT.

DISCUSSIONThe miR-200 family has well-established roles in controlling EMTand cancer cell invasion, but their functions in metastasis haveonly begun to be explored. Here, we show that, of the miR-200family members, only the miR-200b/200c/429 functional (‘seed’)group inhibited spontaneous metastasis of MDA-MB-231 LM2human breast cancer cells. Although both miR-200 functionalgroups could repress ZEB1, its suppression was not required forthe inhibition of metastasis. Rather, we uncovered a moesin-dependent pathway, distinct from the ZEB1-E-cadherin axis,through which miR-200 regulates tumour cell plasticity andmetastasis.

Our finding that enforced miR-200 expression reduces metas-tasis of a xenografted breast cancer cell line is consistent withreports that miR-200 reduces metastasis of xenografted lungadenocarcinoma cells,27 and head and neck squamous cellcarcinoma cells,44 but contrasts with the finding that miR-200promotes metastasis of 4T07 mouse breast cancer cells.28,29

Enforced expression of miR-200 in 4T07 cells was shown to alterthe cancer cell secretome by targeting the anterograde transportprotein Sec23A.29 Although the invasive capacity of the 4T07 cellsis reduced by miR-200, the net influence of its cell intrinsic andextrinsic effects promotes lung colonisation.29 Thus, the effect ofmiR-200 on metastasis can depend on the cellular context, andthe effect of loss or gain of miR-200 in human breast cancers maydiffer with tumour subtype. In LM2 cells, which correspond to thebasal subtype, miR-200b does not induce a full MET in vitro orin vivo but, instead, causes alteration in the cell morphologyindicative of cytoskeletal rearrangement. These cells displayed areduced invasive capacity, but lung colonisation, as assessed bythe experimental metastasis assay, remained unaffected. Thus, inthis context, miR-200b most likely acts to reduce metastasis byrepressing pathways that promote primary tumour cell invasion.

Control of the ZEB1-E-cadherin axis by the miR-200 family hasbeen proposed to be an important pathway influencing primarytumour cell invasion and metastasis.4,45 Comparison of the threemiR-200 family members (miR-200b, miR-200c and miR-141)revealed that each of these miRNAs was able to repress ZEB1 tovarying extents; however, only the miR-200b/200c/429 functionalgroup inhibited spontaneous metastasis from the mammarygland, indicating that the repression of ZEB1 may not berequired to suppress metastasis. We tested this by restoringZEB1 levels in miR-200b-expressing cells, and, although E-cadherinlevels were reduced, these cells still retained an impairedmetastatic ability. These data indicate that miR-200 is able torepress metastasis independent of the ZEB1-E-cadherin axis. ZEB1is a key target for miR-200 in several developmental andpathological scenarios,46,47 with specific knockdown of ZEB1phenocopying the effects of increased miR-200 expression.19,20

ZEB1 can also reciprocally regulate miR-200 levels, and thisfeedback loop can influence epithelial cell plasticity.16–18 Despitethese studies, the mutual dependence of the miR-200 and ZEB1interaction has not been well investigated. Although the miR-200-ZEB1 feedback loop can regulate E-cadherin expression in LM2cells, E-cadherin protein is not efficiently localised to the cellmembrane, indicating that these cells do not transition to become‘fully epithelial’. Therefore, it is possible that the miR-200-ZEB1-E-cadherin pathway has a more dominant role in controlling


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metastasis in scenarios where a stronger MET occurs. However,given that miR-200 targets a number of genes involved in thecytoskeletal organisation16,34,37,39,48 it is perhaps not surprisingthat miR-200 can regulate other pathways that contribute tometastasis.

Examination of miR-200 target genes in the LM2 stable cell linesrevealed that several actin cytoskeleton-associated genes weredownregulated by miR-200b, but not by miR-141, consistent withthe stronger changes in cell morphology and protrusions inducedby miR-200b expression. However, only knockdown of moesinreduced in vitro invasion with restoration of its expression in miR-200b-expressing cells being sufficient to alter cell morphology andalleviate repression of metastasis, implicating it as a key target ofmiR-200b in this process. Moesin is a member of the ezrin-radixin-moesin family that controls cytoskeletal dynamics and linksfilamentous actin to the plasma membrane.40 Although initiallythought to be redundant in function, recent studies havehighlighted divergent roles for the ezrin-radixin-moesin proteins.For example, moesin, but not ezrin, is induced during EMT and

is required for cytoskeletal remodelling of mammary epithelialcells.43 Moesin, but not ezrin, also promotes invasion andlung colonisation of melanoma tumour cells.42 Our findings areconsistent with these studies and indicate that moesin may have aspecific role in enhancing breast cancer progression. Interestingly,knockdown of moesin alone was not able to alter the metastaticpotential of LM2 cells (Supplementary Figure S8) suggesting that itsreduction is necessary, but not sufficient, for metastasis. Therefore,in addition to targeting moesin, miR-200b likely regulates othergenes that contribute to metastatic progression in this context.

Several immunohistochemical studies of breast cancers havedemonstrated that moesin is highly expressed in triple (ER/PR/HER2)-negative and basal subtype cancers and in some cases is aprognostic marker of poor outcome.49–51 Moesin has also beendirectly linked with an EMT expression profile in breast cancersamples.52 We showed that moesin is directly targeted by miR-200b, and, using breast cancer cell lines and patient tumours, wefound that moesin mRNA is highly expressed in basal and claudin-low subtypes. Furthermore, its expression shows a significant

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Figure 7. Re-expression of moesin in miR-200b-expressing cells restores metastatic ability. (a) Quantitation of miR-200b and moesinexpression in LM2 stable cell lines as measured by real-time PCR or western blot. The levels of miR-200b and moesin in LM2-miR-200b cellstransduced with a control or moesin coding region vector are shown in relation to parental LM2-miR-200b stable cells and LM2-Vector controlcells. (b) Phalloidin staining of F-actin in LM2-miR-200b cells stably expressing the moesin coding region in comparison with control. Thelower panel represents a magnified view compared with the upper panel. Scale bars indicate 20 mm. (c) Measurement of primary tumourweight and lung metastasis in LM2-miR-200b cells expressing a control vector or moesin. Lung metastatic burden was calculated by dividingthe bioluminescent intensity of lung metastases for each individual mouse by the primary tumour weight from that same mouse. A dashedline marking the mean bioluminescent intensity in LM2-vector cells is shown for comparison (see Supplementary Figure S1). P-values werecalculated using a two-tailed Mann–Whitney t-test. (d) Immunohistochemical staining for moesin of representative primary tumour sectionsfrom vector, miR-200b and miR-200bþMSN xenografts. Scale bars represent 100 mm.


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inverse correlation with miR-200b and miR-200c in these samplesas well as in the NCI-60 panel of cancer cell lines indicating thatthis relationship exists across a wide range of other cancer types.We also demonstrated that high mRNA levels of moesin areassociated with poorer relapse-free survival in two independentpatient cohorts. Given that moesin has been shown to facilitateactin cortical polarisation and induction of tumour cell invasion inthree-dimensional matrices,42 it will be interesting to furtherexamine the interplay between miR-200 and moesin in three-dimensional cultures and within primary tumours.

In summary, we have identified a moesin-dependent pathwaythrough which miR-200 family members act to repress primarytumour cell invasion and metastasis. These findings demonstrate arole for miR-200 in controlling cell plasticity and function apartfrom their regulation of the ZEB1-E-cadherin axis and MET. As themiR-200 family can also have a dual role in enhancing metastaticcolonisation after dissemination,28,29 therapeutic utility of the miR-200 family may require consideration of the stage and degree oftumour progression.

MATERIALS AND METHODSCell cultureA highly metastatic variant of MDA-MB-231, LM2 (4175 TGL), was obtainedfrom Dr Joan Massague.36 Cells were cultured in Dulbecco’s Modified EagleMedium (Gibco, Invitrogen, Carlsbad, CA, USA) þ 10% foetal calf serum(FCS, Bovogen, Bovogen, VIC, Australia). All other human breast cancer celllines were cultured as described previously.13

Lentiviral and retroviral vectors and transductionGenomic regions containing Pri-miRNAs for miR-200b, miR-200c and miR-141 wereamplified from human genomic DNA and cloned into lentiviral and/or retroviralvectors. Cloning procedures are detailed in Supplementary Methods with primersequences shown in Supplementary Table 1. Transductions were performed aspreviously described13 and are detailed in Supplementary Methods.

Transfection with miRNA and small interfering RNATransient transfections of LM2 or breast cancer cell lines were performed insix-well plates (3� 105/well) using synthetic miRNA precursors (Pre-miRs,Ambion, Invitrogen, Carlsbad, CA, USA) or ON-Target Plus SMARTpool smallinterfering RNA (Thermo Fisher, Pittsburgh, PA, USA) at a final concentra-tion of 20 nM using Lipofectamine RNAiMAX (Invitrogen). After 72 h, cellswere processed for downstream applications. Inhibition of the miR-200family in breast cancer lines was performed for 6 days after transfectionwith 100 nM Anti-MiR as described previously.18

Real-time PCRRNA was isolated from adherent cultures and made into complementaryDNA using methods previously described.13 PCR for miRNAs wasperformed using TaqMan microRNA assays (Applied Biosystems,Invitrogen, Carlsbad, CA, USA). For mRNA, PCRs were performed usingQuantitect SyBr green reagents (Qiagen, Hilden, Germany) using thespecific primers listed in Supplementary Table 1. Real-time PCR data formRNA and miRNA are expressed relative to GAPDH or U6, respectively.

Western blotWestern blots were performed as described previously.13 The followingantibodies were used: ZEB1 (1:200 vol:vol, Santa Cruz, Santa Cruz, CA, USA),E-cadherin (1:1000 vol:vol, BD Biosciences; San Jose, CA, USA), moesin (1:1000vol:vol, Cell Signaling Technologies, Danvers, MA, USA) and tubulin (1:5000vol:vol, Abcam, Cambridge, UK). Membranes were developed using enhancedchemiluminescence (ECL Prime, GE Healthcare, Buckinghamshire, UK) andimaged using the LAS4000 luminescent image analyser (Fujifilm, Tokyo, Japan).

Migration and invasion assaysFor migration assays, 1� 105 cells were seeded in Transwells (6.5 mm, 8.0mm poresize) (Corning, Tewksbury, MA, USA) in serum-free medium with 0.01% bovineserum albumin. For invasion assays, 6� 104 cells were seeded into 8mm BiocoatMatrigel chambers (BD Biosciences). After 4 h for migration or 24 h for invasion

towards 10% foetal calf serum, the membranes were fixed with 10% bufferedformalin and cells stained with 4’,6-diamidino-2-phenylindole. Six fields of view foreach membrane were counted using ImageJ software (http://rsbweb.nih.gov/ij/).

ImmunofluoresenceCells were seeded in eight-well chamber slides (Nunc; Roskilde, Denmark)pre-coated with 50 ug/ml Fibronectin (Roche, Penzberg, Germany). Afterblocking with 1% bovine serum albumin, cells were stained using anti-E-cadherin (1:500 vol:vol, BD Biosciences) or Phalloidin (Rhodamine or AlexaFluor 647-conjugated) to visualise F-actin. Nuclei were visualised bystaining with 4’,6-diamidino-2-phenylindole. Cells were imaged using anOlympus IX81 microscope with a Hamamatsu Orca camera. Images wereacquired using the (CellR; Olympus, Munster, Germany) software andanalysed using AnalySIS LS software (Olympus). Cell size (length:widthratio) was measured using the arbitrary line tool on the AnalySIS LSsoftware. Cell length was defined by the longest distance between any twopoints of the cell, and cell width was measured as the longest lineperpendicular to the cell-length line.

Luciferase reporter assayA full-length (B2 kb) or a 740-bp segment of the moesin 30UTR spanningtwo conserved miR-200b/200c/429 seed sites was amplified from pCMV6-XL5-hMSN (Origene, Rockville, MD, USA) and cloned downstream of Renillaluciferase in the XhoI and NotI sites of the psiCheck2 vector using primersshown in Supplementary Table 1. The moesin 30UTR and control vectors(200 ng) were co-transfected with 5 nM of Pre-miR (Ambion) into MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen). Cells were harvested 72hours later for Dual Luciferase assays (Promega, Madison, WI, USA).

Orthotopic and experimental metastasis assayMice were housed in the SA Pathology Animal Care Facility, and experimentswere conducted under the institutional animal ethics guidelines. Fororthotopic and experimental metastasis studies, 5-week- to 6-week-oldfemale mice were used. For orthotopic experiments, mice were anesthetisedbefore injections of 1� 106 cells in 50ml of 50% Matrigel (BD Biosciences) intothe fourth mammary gland. Approximately 4 weeks after initial tumourimplantation, bioluminescent imaging was performed as described below. Forexperimental metastasis, 2� 105 cells in 100 ul HBSS (Hank’s Balanced SaltSolution (Gibco)) were injected directly into the tail vein of recipient mice.Bioluminescence images of animals were collected 2 and 4 hours after initialtail vein injection to determine baseline levels of metastasis and weeklythereafter to monitor colonisation of tumour cells in the lung.

Bioluminescence imagingBioluminescence imaging was performed using the Xenogen IVIS 100imaging system. Mice were injected intraperitoneally with 30 mg/ml ofD-Luciferin (in phosphate-buffered saline) 10 min before imaging. Dorsalimages of the primary tumour were collected before the animals were culledand their primary tumour, lung and hind limbs (with muscle tissue removed)harvested for ex vivo imaging. Photon emission was quantified using theLiving Image Software (Xenogen) (Perkin Elmer, Hopkinton, MA, USA).

HistologyLung and primary tumours were fixed in 10% buffered formalin for24 hours before processing and embedding in paraffin. Sections were (4 um)stained with Haematoxylin and Eosin (H&E), anti-E cadherin (1:1000 vol:vol,BD Biosciences), anti-moesin (1:400 vol:vol, clone 38/87, Thermo Scientific,Waltham, MA, USA) and human anti-green fluorescent protein (1:1000vol:vol, Rockland Immunochemicals, Gilbertsville, PA, USA) antibodies. Boneswere fixed and then decalcified in 0.5 M EDTAþ 0.2% paraformaldehyde for2 weeks before staining.

Bioinformatics and statistical analysesExpression and clinical data from the previously combined 77953 andUNC33754 tumour data sets were obtained from https://genome.unc.edu/and analysed as detailed in Supplementary Methods. To examine thecorrelation between miR-200 and moesin expression in breast cancer andthe NCI-60 panel, linear regressions were generated using the GSE19783data set41 or the five-platform gene data from CellMiner (http://discover.nci.nih.gov/cellminer). All statistical analyses were performedusing GraphPad Prism 5 (GraphPad Prism, La Jolla, CA, USA).


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http://rsbweb.nih.gov/ij/

https://genome.unc.edu/

http://discover.nci.nih.gov/cellminer

http://discover.nci.nih.gov/cellminer

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe thank Professor Joan Massague for providing the MDA-MB-231 LM2 cell lineand Dr Ross Dickins for providing the pLMP-puro-GFP construct. We thank Dr AgathaLabrinidis and Ms Yuka Harata-Lee for assistance with bioluminescence imaging andinoculation of tumour cells, respectively, Dr Peter Diamond for help with bonehistological analysis and Professors Andreas Evdokiou and Shaun McColl for insightfuldiscussions. This work was supported by fellowships from the National Breast CancerFoundation of Australia (PAG, CPB and RLA, nos ECF-09-08 and PF-09-03) and grantsfrom the National Health and Medical Research Council of Australia (PAG, YK-G, GJG,RLA and CNJ, nos 566871 and APP1020280), Cancer Council South Australia (GJG,PAG and YK-G) and Prostate Cancer Foundation of Australia (LAS, no. YI 0810).

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)


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http://www.nature.com/onc

mir-200 can repress breast cancer metastasis through zeb1-independent but moesin-dependent pathways

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