Oncogene (2014) 33, 4077–4088 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE MiR-200 can repress through ZEB1-independent but moesin-dependent pathways

XLi1, 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 invasion through inhibition of the E-cadherin transcriptional repressors ZEB1 and ZEB2. Recent studies have indicated that the miR-200 family may exert their effects at distinct stages in the metastatic process, with an overall effect of enhancing metastasis in a syngeneic mouse breast cancer model. We find in a xenograft orthotopic model of breast cancer metastasis that ectopic expression of 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 metastatic potential 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 repression of 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 of moesin significantly inversely correlated with miR-200 expression, and high levels of moesin were associated with poor relapse-free survival. These findings highlight the context-dependent effects of miR-200 in breast cancer metastasis and demonstrate the existence of a moesin-dependent pathway, distinct from the ZEB1-E-cadherin axis, through which miR-200 can regulate tumour cell plasticity and metastasis.

Oncogene (2014) 33, 4077–4088; doi:10.1038/onc.2013.370; published online 16 September 2013 Keywords: miR-200; epithelial–mesenchymal transition; breast cancer; metastasis; actin cytoskeleton

INTRODUCTION epithelial and mesenchymal states are important contributors to The majority of cancer-related deaths from carcinomas result from metastatic progression.4 metastatic progression. For carcinomas to metastasise, epithelial The microRNA-200 (miR-200) family have emerged recently as tumour cells undergo a complex series of events, which include important regulators of EMT. Across a diverse range of epithelial- invasion in the primary site, dissemination and colonisation at a derived cancer cell types, the miR-200 family are able to enforce secondary location.1 This metastatic cascade is not only an epithelial state by inhibiting the E-cadherin transcriptional orchestrated by numerous signalling pathways operating within repressors ZEB1 and ZEB2.13–16 Furthermore, the ZEB transcription the tumour cells but can also be modulated through interactions factors can repress miR-200 expression and through this reciprocal with stromal cells.2 To acquire invasive capabilities, epithelial feedback loop modulate epithelial cell plasticity.16–18 Numerous tumour cells can undergo an epithelial–mesenchymal transition studies have indicated a role for the miR-200-ZEB feedback loop in (EMT), a process that facilitates the loss of cell–cell adhesions and tumour cell invasion and metastasis, although the dependency of promotes cell motility, stem cell-like properties and resistance to this interaction in controlling these processes has not been apoptosis.3 These features are proposed to drive tumour evaluated. The loss of miR-200 and the gain of ZEB1 expression dedifferentiation and enhance metastatic progression.4 However, have each been separately associated with tumour progression the analysis of clinical samples indicates that metastases often and the acquisition of characteristics of metastatic cells, including closely resemble the primary tumour in morphology and gene the invasive and stem-like properties,19–21 as well as resistance to expression profile5–7 suggesting that the redifferentiation of the apoptosis and chemotherapeutics.22–25 In mouse xenograft metastasising cell may occur via a mesenchymal to epithelial models of metastasis, re-expression of miR-200 or inhibition of transition (MET).8 This concept has been bolstered by recent ZEB1 can attenuate metastasis by inhibiting EMT.26,27 In contrast, evidence using genetic and experimental mouse models of studies using the 4T1 series of mouse mammary cancer cells have metastasis,9–12 which demonstrate that the transition between shown 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, East Melbourne, Victoria, Australia; 3Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia; 4Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia; 5Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia; 6Discipline of Medicine, The University of Adelaide, 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, Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, South Australia 5000, Australia. E-mail [email protected] or [email protected] 9These authors contributed equally to this work. Received 20 January 2013; revised 11 July 2013; accepted 26 July 2013; published online 16 September 2013 miR-200 regulates metastasis by targeting moesin XLiet al 4078 These seemingly paradoxical findings can be explained by a elevated in patients with poor metastasis-free survival. Together, model of tumour cell plasticity, whereby miR-200 loss permits EMT these results demonstrate that miR-200 can regulate tumour cell and invasion away from the primary tumour, whereas efficient metastasis through ZEB1-independent but moesin-dependent colonisation requires re-expression of miR-200 and a subsequent pathways. MET.4,29 Accordingly, the examination of breast and colorectal patient samples reveals that miR-200 levels can be reduced at the invasive front in primary tumours but are often increased in resulting metastases.29–33 Given these observations, it is of clinical RESULTS importance to understand the mechanisms through which the Stable expression of miR-200b can alter breast cancer cell miR-200-ZEB feedback loop controls metastatic progression. migration and invasion in the absence of MET The miR-200 family comprises five members that are The miR-200 family causes a MET in a wide range of epithelial- expressed from two distinct polycistronic transcripts (miR- derived cancer cell types with associated reductions in cell 200bB200aB429 and miR-200cB141) and can be separated migration and invasion. However, the ability of individual miR-200 into two functional groups (miR-200b/200c/429 and miR-141/ family members to influence metastasis has not been studied. To 200a) on the basis of their ‘seed’ sequence.16,17 Although determine the role of individual miR-200 family members in differences in the ability of the miR-200 functional groups to controlling breast cancer metastasis, we initially expressed miR- influence cell invasion have been identified,34,35 their individual 200b in the well-characterised MDA-MB-231 LM2 subline, which effect in metastasis has not been investigated. In this study, we can metastasise spontaneously after implantation in the mam- utilise an orthotopic model of breast cancer metastasis (MDA- mary gland.36 Stable expression of miR-200b to B30-fold above MB-231-LM236) and demonstrate that ectopic expression of miR- basal level reduced tumour cell migration and invasion in vitro 200b or miR-200c, but not miR-141, reduces tumour cell invasion (Figure 1a and b) but, contrary to expectation, did not induce and breast cancer metastasis. Surprisingly, these effects were not morphological changes consistent with MET. Although miR-200 mediated directly by the loss of ZEB1 and MET but rather expression reduced ZEB1 and weakly induced E-cadherin expres- through the repression of the cytoskeletal remodelling protein sion (Figure 1c), the E-cadherin protein was localised predomi- moesin. Moesin has previously been shown to be targeted by nantly in the perinuclear region and not at cell–cell junctions as is miR-200c and to influence the ability of miR-200c to repress cell typical of epithelial cells (Figure 1d). Therefore, miR-200b is able to migration.37 Here, we show that moesin expression is inversely alter cellular properties associated with metastasis independently correlated with miR-200 in patient breast cancer samples and is of its ability to induce a MET.

30 120 120 100 100 20 80 80 *** *** 60 60 10 40 40 expression % invaded cells % migrated cells 20 20 Relative miR-200b 0 0 0

Vector Vector Vector miR-200b miR-200b miR-200b

Phase E-cadherin / DAPI

Vector miR-200b Vector E-cadherin

ZEB1

Tubulin miR-200b

Figure 1. miR-200b can reduce migration and invasion independently of a MET. (a) Real-time PCR measurement of miR-200b in LM2 cells transduced with a control or miR-200b lentiviral vector. (b) Comparison of the relative migration (4 hours) and invasion (24 hours) of the vector with miR-200b stable LM2 cells towards a 10% serum gradient. Data are the mean±s.d. of three independent assays. *** denotes P-value of o0.001 as measured by the unpaired two-tailed Student’s t-test. (c) Western blot of E-cadherin and ZEB1 in extracts from LM2 stable cells lines. Tubulin is shown as a loading control. (d) Morphology of the vector and miR-200b stable LM2 cells as shown by phase-contrast imaging. 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.

Oncogene (2014) 4077 – 4088 & 2014 Macmillan Publishers Limited miR-200 regulates metastasis by targeting moesin XLiet al 4079 miR-200b reduces spontaneous breast cancer metastasis to organs by bioluminescence imaging. After 29 days, primary multiple organs without influencing extravasation or colonisation tumours had grown to a similar size; however, cells expressing To determine whether miR-200b directly influences spontaneous miR-200b metastasised less efficiently to the lung, liver and bone breast cancer metastasis, we implanted control or miR-200b- (Figures 2a and b). We verified the presence of tumour cells in the expressing MDA-MB-231 (LM2) cells into the mammary fat pad of lung and bone by immunohistochemical analyses for green nonobese diabetic/severe combined immunodeficient mice and fluorescent protein, which is co-expressed with the luciferase assessed primary tumour growth and metastasis to multiple reporter in LM2 cells (Figure 2c). To assess whether miR-200b-

Vector miR-200b

Primary tumor Lung 1.25 0.40 )

p=0.58 6 0.35 1.00 p=0.064 Primary Tumor 0.30 0.75 0.25 0.50 0.20 0.25 Tumor weight (g) 0.15 Photons/sec (x10 0.10 0 Vector miR-200b Vector miR-200b

Lung Liver Bone 4.0 9.0 ) ) 6 6 3.0 p=0.050 7.0 p=0.005

5.0 Liver 2.0

3.0 1.0 Photons/sec (x10 Photons/sec (x10 1.0 Bone 0 0 Vector miR-200b Vector miR-200b

H & E GFP E-cadherin

Primary Lung Tumor

Epidermis (adjacent to Bone Tumour)

25 Vector )

6 20 miR-200b

15

10

5 photons/sec (x10 p=0.32 0 0 4 8 12 16 20 24 Time (days) Figure 2. miR-200b expression inhibits spontaneous metastasis from the mammary fat pad. (a) Bioluminescent images of primary tumours in 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 (represented by photons/sec). Ten mice were injected per cell line with each point representing a measurement from an individual mouse with the mean values 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 is shown as a positive control. Scale bars represent 100 mm. (e) Control or miR-200b-expressing LM2 cells were injected via tail vein and bioluminescent intensity in the lung in vivo was measured over a 25-day time course. Five mice were injected per cell line with means plotted±s.e.m. The P-value was calculated using two-way analysis of variance (ANOVA).

& 2014 Macmillan Publishers Limited Oncogene (2014) 4077 – 4088 miR-200 regulates metastasis by targeting moesin XLiet al 4080 expressing cells were less metastatic by virtue of the primary metastasis of primary tumour cells without a requirement for their tumour cells having undergone a MET, we stained primary transition to an epithelial phenotype. tumours for E-cadherin. Examination of the LM2-miR-200b Metastasis is a multistep process involving the early steps of primary tumour core revealed very weak cytoplasmic staining of invasion and intravasation followed later by extravasation and E-cadherin, in contrast to the epidermal cells adjacent to the colonisation by tumour cells to establish a distant secondary primary tumour in which prominent membranous staining was tumour. We tested whether miR-200b alters metastasis by observed (Figure 2d). This indicates that miR-200b can repress the influencing the ability of the cells to extravasate and colonise

a miR-200c miR-141 50 100

40 75 30 50 20 Relative miRNA

Relative miRNA 25 10

0 0 Vector miR-200c Vector miR-141

b 0.55 Primary Tumor 7 Lung c Vector miR-200c 6 0.50 ) 6 p=0.68 5 p<0.001 0.45 E-cadherin 4 0.40 3 ZEB1 0.35 2 Tumor weight (g) 0.30 Photons/sec (x10 1 Tubulin 0.25 0 Vector miR-200c Vector miR-200c

d Primary Tumor Lung e 0.7 2.5 p=0.094 p=0.22 Vector miR-141

0.6 )

6 2.0 E-cadherin 0.5 1.5 0.4 1.0 ZEB1 0.3

Tumor weight (g) 0.5

0.2 Photons/sec (x10 Tubulin 0.1 0 Vector miR-141 Vector miR-141

f Vector miR-200c miR-141

Phase

E-cadherin / DAPI

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 stably expressing miR-200c and miR-141, relative to control vectors. (b, d), Quantitation of primary tumour weight and metastasis to the lung (as measured by bioluminescent intensity) measured 28 (b)or35(d) days after mammary fat pad injection with miR-200c, miR-141 or with the respective 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 of extracts derived from the LM2 stable cell lines for E-cadherin, ZEB1 and Tubulin. (f) Phase-contrast and immunofluorescent images of LM2 cells stably expressing vector, miR-200c and miR-141 showing E-cadherin (green) and nuclear DAPI staining (blue). Scale bars represent 50 mm.

Oncogene (2014) 4077 – 4088 & 2014 Macmillan Publishers Limited miR-200 regulates metastasis by targeting moesin XLiet al 4081 the lung. To circumvent the early steps of the spontaneous LM2-miR-200b Primary Tumor metastatic process, control or miR-200b-expressing LM2 cells were cells 0.6 injected directly into the tail vein of mice and subsequent tumour 50 development in the lung monitored. Two days after tail vein 0.5 40 injection when cells that remained in circulation have been p=0.58 cleared, there was no significant difference in lung biolumines- 0.4 cence between mice injected with control or miR-200b-expressing 30 cells (Figure 2e), suggesting that the cells were equally able to 20 0.3

extravasate and deposit in the lung. Furthermore, over a 25-day Tumor weight(g)

period, the rate of tumour formation in the lung was not miR-200b levels 10 significantly different between control and miR-200b-expressing 0.2 cells (Figure 2e), suggesting that the colonisation of secondary (relative to LM2-vector) 0 miR-200b/ miR-200b/ Vector ZEB1 sites was also not affected by elevated levels of miR-200b. By inference, therefore, miR-200b is likely to be inhibiting the early 3.0 Lung steps of metastasis, which is consistent with its repressive effects ZEB1

) Mean on cell migration and invasion. 6 LM2-Vector E-cadherin 2.0 Stable expression of miR-200c, but not miR-141, represses breast cancer metastasis p=0.68 Because the miR-200 family is composed of five members Tubulin 1.0 possessing two distinct ‘seed’ sequences, the miR-200b/200c/429 and miR-141/200a groups likely target different but sometimes (x10 photons/sec overlapping sets of genes (as in the case of ZEB113). To determine 0 whether both seed groups are able to repress breast cancer miR-200b/ miR-200b/ miR-200b/Vector miR-200b/ZEB1 metastasis, we stably expressed either miR-200c or miR-141 in Vector ZEB1 LM2 cells and measured spontaneous metastasis to the lung. To Figure 4. miR-200b can repress metastasis independently of ZEB1. achieve high expression of the introduced microRNA (miRNA), we 38 (a) Real-time PCR and western Blot of miR-200b, ZEB1, E-cadherin used the LMP retroviral system, which resulted in a B40- and and Tubulin levels in miR-200b-expressing LM2 cells transduced B80-fold increase in miR-200c and miR-141 expression above with a control or ZEB1 coding region vector. (b) The measurement of vector-transduced cells (Figure 3a), to levels similar to that primary tumour weight and lung metastasis in LM2-miR-200b cells expressed by epithelial breast cancer cell lines.13 At the same expressing a control vector or ZEB1. A dashed line marking the mean time, we used this system to stably express miR-200b, achieving bioluminescent intensity in LM2-vector cells is shown for compar- Btwofold higher expression levels than with the original (polIII ison (see Supplementary Figure S1). P-values were calculated using a driven) vector (Supplementary Figure S1). This increased expres- two-tailed Mann–Whitney t-test. sion of miR-200b intensified both the changes in E-cadherin and ZEB1 levels and inhibited the degree of metastasis to the lung the functionality of ZEB1 expressed from the exogenous vector (Supplementary Figure S1), although a transition to an epithelial (Figure 4a). Comparing the cells having high miR-200b/low ZEB1 phenotype was again not observed (data not shown). As we found with those having high miR-200b/high ZEB1 in spontaneous with miR-200b, miR-200c was able to efficiently repress metastasis metastasis assays, we found that both cell lines metastasised to the lung without affecting primary tumour growth (Figure 3b). weakly with no significant difference in lung metastasis or tumour MiR-200c also caused similar changes in E-cadherin and ZEB1 weight (Figure 4b). These results were also confirmed in a second expression (Figure 3c) in the absence of a MET (Figure 3f). These group of mice containing additional controls (Supplementary results imply that different members of the miR-200b/200c/429 Figure S2). These results indicate that high miR-200b expression functional group can repress metastasis by similar mechanisms. In inhibits metastasis of LM2 cells, regardless of the level of ZEB1. Of contrast to miR-200b and miR-200c, stable expression of miR-141 note, the related protein ZEB2 is expressed at very low levels in did not significantly reduce the lung metastatic potential of LM2 LM2 cells and is not likely to have a role in these cells, suggesting cells (Figure 3d). Interestingly, miR-141 only modestly reduced that miR-200b may target other genes to regulate metastasis. ZEB1 levels, and E-cadherin expression was not induced in these cells (Figures 3e and f). This may be indicative of the decreased potency of ZEB1 targeting by the miR-141 seed compared with 13 Moesin, cofilin2 and WASF3 are repressed by miR-200b, but not by the miR-200b seed as described previously. Taken together, miR-141 these results indicate that the miR-200b/200c/429, but not the Cell invasion and metastasis require dynamic remodelling of the miR-141/200a, functional group is able to repress the spontaneous actin cytoskeleton to facilitate movement from the primary metastasis of LM2 cells. tumour. Recent reports have shown that the actin-associated factors moesin (MSN), cofilin2 (CFL2)andWASF3 are direct targets miR-200b represses breast cancer metastasis independently of its of the miR-200 family; however, their contribution to metastasis effect on ZEB1 remains largely unexplored.16,37,39 We measured the mRNA Although several studies have demonstrated a role for miR-200 or levels of moesin, cofilin2 and WASF3 in the miR-200b and miR- ZEB1 in cancer cell invasion and metastasis,26,27 the dependency 141 stably transduced LM2 stable cell lines and found that each of their interaction in metastasis has not been investigated. To of these factors was reduced only in the miR-200b cells relative determine whether miR-200b represses metastasis by decreasing to their empty vector cell line (Figure 5a). As a control, the levels ZEB1 levels, we transduced either a control vector or a ZEB1 of ZEB1 mRNA were reduced in both cell lines. This raises the complementary DNA lacking its 3’ untranslated region (30UTR) into possibility that the differential effect of the miR-200b/200c/429 miR-200b-expressing LM2 cells. Both cell lines expressed equi- and miR-141/200a seeds on metastasis may be mediated valent high levels of miR-200b with ZEB1 levels being elevated in through changes in cytoskeletal dynamics. Staining of the LM2 the pLenti4-ZEB1-transduced cells (Figure 4a). In addition, cells stable cell lines for F-actin revealed changes in cell morphology, with high ZEB1 also had reduced E-cadherin levels confirming which were especially evident in LM2 cells that were transiently

& 2014 Macmillan Publishers Limited Oncogene (2014) 4077 – 4088 miR-200 regulates metastasis by targeting moesin XLiet al 4082 Vector miR-200b miR-141 abMSN MSN 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6

0.4 0.4 F-Actin

Relative mRNA 0.2 0.2 0.0 0.0 Vector miR-200b Vector miR-141 Neg Pre-miR 200b Pre-miR 141 Pre-miR MSN

Tubulin

CFL2 CFL2 F-Actin 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4

Relative mRNA 0.2 0.2

0.0 0.0 F-Actin Vector miR-200b Vector miR-141 WASF3 1.2 WASF3 2.4 1.0 2.0 cd 0.8 1.6 1.2 MSN Cell shape 0.6 1.4 6

0.4 0.8 1.2 m) µ * Relative mRNA 0.4 0.2 1.0 4 0.8 0.0 0.0 Vector Vector miR-141 miR-200b 0.6 *** *** 2 0.4 1.2 ZEB1 1.2 ZEB1 Relative mRNA 0.2 Length:width ratio ( 1.0 1.0 0.0 0 0.8 0.8 Pre-miR Neg 200b 141 200c Pre-miR Neg 200b 141 200c 0.6 0.6 MSN 0.4 0.4

Relative mRNA 0.2 0.2 Tubulin 0.0 0.0 Vector miR-200b Vector miR-141 Figure 5. miR-200 family members differentially target actin-associated genes and alter cell morphology. (a) Real-time PCR measuring the expression of several actin-associated miR-200 target genes in LM2 stable cell lines. Protein levels for moesin expression are shown by western blot. (b) Phalloidin staining 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 20 mm. (c) Real-time PCR quantitation of moesin mRNA and western blot analysis of LM2 cells transiently transfected with Pre-miRs. (d) Differences in cell shape 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 are the mean±s.e.m. *Po0.05, ***Po0.001 compared with negative (Neg) using one-way ANOVA, followed by post-hoc Tukey’s multiple comparisons.

transfected with miR-200b (Figures 5b and d). As observed in the Moesin knockdown reduces cancer cell invasion and is inversely stable cell lines (Figure 5a), transient transfection with miR-200b correlated with miR-200b seed in breast cancer or miR-200c specifically decreased moesin mRNA and protein To determine whether moesin, cofilin2 or WASF3 influences levels, whereas transfection with miR-141 did not (Figure 5c). invasion of LM2 cells, we transiently knocked down each of these Cells transfected with miR-200b also displayed a markedly genes and assessed their effect in in vitro invasion assays. Only reduced invasive capacity without forming intercellular junctions moesin knockdown significantly repressed cell invasion (Figure 6a or expressing junctional E-cadherin (Supplementary Figure S3). and Supplementary Figure S4). At the same time, we knocked These effects were less pronounced in miR-141-transfected cells down ZEB1 levels and found that it only marginally altered cell (Figure 5b and Supplementary Figure S3). We therefore further invasion despite increasing E-cadherin levels (Figure 6a and investigated whether repression of actin-related genes contri- Supplementary Figure S4). In contrast, E-cadherin expression was bute to miR-200b-mediated inhibition of cell invasion and not increased after moesin knockdown (Supplementary Figure S4). metastasis. These data suggest that the modulation of moesin rather than the

Oncogene (2014) 4077 – 4088 & 2014 Macmillan Publishers Limited miR-200 regulates metastasis by targeting moesin XLiet al 4083 120 1.4 * MSN full 3'UTR MSN short 3'UTR 100 1.2 * 80 1.0 0.8 60 * 0.6 40 Relative 0.4

% Invaded cells 20 0.2 Luciferase Activity 0 0.0 Neg miR-200b miR-141 trol SN siM siCFL2 ASF3 siZEB1 siCon siW

1.4 HS-578T BT-549 1.2 1.6 1.0 Luminal Basal 1.2 0.8 (High miR-200) (Low miR-200) 0.6 0.8 Relative 0.4 0.4 MSN mRNA 0.2 MSN 0.0 0.0 Pre-miR 141 141 Neg Neg

Tubulin 200b 200b

MSN

MCF-7 T-47D BT-549 ZR-75-1HBL-100HS-578T Tubulin MDA-MB-361MDA-MB-468 MDA-MB-231MDA-MB-435 Pre-miR 141 141 Neg Neg 200b 200b

MCF-7 GSE19783 (n=101) GSE19783 (n=101) 3 14 14

13 13 2 12 12 MSN 1 MSN 11 r= -0.28 11 r= -0.35 p= 0.0053 p= 0.0003 Relative MSN mRNA 0 10 10 Anti-miR 4 6 8 10 12 14 6 8 10 12 14

Neg hsa-miR-200b hsa-miR-200c miR-200 T-47D 779 tumor set UNC337 cohort 3 100 100 Low MSN Low MSN 80 High MSN 80 High MSN 2 60 60

40 40 1 20 20 Relapse free (%)

Metastasis free (%) p= 0.0073 p= 0.0313 Relative MSN mRNA 0 0 0 Anti-miR 0 50 100 150 200 250 0 50 100 150

Neg Months Months miR-200 Figure 6. Moesin reduces cell invasion and is inversely associated with miR-200 in breast cancer. (a) Invasion assays of LM2 cells were carried out for 24 hours following small interfering RNA (siRNA)-mediated knockdown of indicated genes (after 72 hours). Data are collated from two to 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 of MDA-MB-231 cells with control (Neg), miR-200b or miR-141 mimic (Pre-miR) for 72 hours. Data are representative of three experiments performed in triplicate. * denotes P-value of o0.05 as calculated using a two-tailed Student’s t-test. (c) Western blot of moesin expression in breast cancer cell lines possessing a luminal or basal expression pattern and previously shown to have either a high or low miR-200 family expression.13 Tubulin is shown as a loading control. (d) Real-time PCR quantitation of moesin mRNA expression and western blot analysis of breast cancer cells transiently transfected with Pre-miRs for 3 days. (e) Real-time PCR quantitation of moesin mRNA expression in breast cancer cell 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 a panel of 101 breast cancer samples (GSE19783). Pearson correlation coefficients and P-values were calculated as indicated. (g) Kaplan–Meier curves showing metastasis-free survival (779 tumour set) or relapse-free survival (UNC311) in patients with high (above median) or low (below median) expression of MSN. Log-rank tests were used to compare the survival distributions (P-value as shown).

ZEB1-E-cadherin axis may be important in miR-200b-mediated plasma membrane.40 A recent study has shown that moesin is a control of metastasis in LM2 cells. direct target of miR-200c and a key contributor to miR-200c- Moesin is a member of the ezrin-radixin-moesin family of mediated repression of cell migration.37 Using reporter assays, we proteins that regulate actin localisation and cross-linking to the showed that the moesin 30UTR is repressed by miR-200b, and not

& 2014 Macmillan Publishers Limited Oncogene (2014) 4077 – 4088 miR-200 regulates metastasis by targeting moesin XLiet al 4084 by miR-141, consistent with the differential regulation of moesin tumours derived from LM2-miR-200b cells with enforced moesin in the LM2 stable cell lines (Figure 6b). To assess whether there is expression (Figure 7d). These data demonstrate that down- an association between the levels of moesin and miR-200 in breast regulation of moesin is required for miR-200b to repress cancer, we analysed their levels in breast cancer cell lines and metastasis of LM2 cells. Collectively, these findings indicate that patient samples. Moesin was strongly enriched in basal subtype miR-200b/200c/429 and miR-141/200a functional groups have cell lines, in which the miR-200 family has been shown previously different roles in the metastatic process, and that miR-200b/ to be lowly expressed,13 in contrast to luminal cell lines in which 200c/429 can operate through moesin-dependent pathways that there is a high miR-200 expression (Figure 6c).13 Transfection of are distinct from the canonical ZEB1-E-cadherin pathway and EMT. two basal subtype cell lines with miR-200b reduced moesin mRNA and protein, whereas transfection with miR-141 did not (Figure 6d). Conversely, inhibition of the miR-200 family in luminal DISCUSSION breast cancer cell lines increased moesin mRNA levels (Figure 6e). The miR-200 family has well-established roles in controlling EMT We next interrogated a published data set of 101 breast cancer and cancer cell invasion, but their functions in metastasis have patient samples where both mRNA and miRNA expression had only begun to be explored. Here, we show that, of the miR-200 been profiled (GSE1978341). Using linear regression analysis, family members, only the miR-200b/200c/429 functional (‘seed’) moesin expression showed a significant negative correlation group inhibited spontaneous metastasis of MDA-MB-231 LM2 with the expression of both miR-200b and miR-200c, consistent human breast cancer cells. Although both miR-200 functional with it being regulated by these miRNAs in breast cancer groups could repress ZEB1, its suppression was not required for (Figure 6f). Furthermore, a strong negative correlation between the inhibition of metastasis. Rather, we uncovered a moesin- moesin and miR-200 expression was also observed in the NCI-60 dependent pathway, distinct from the ZEB1-E-cadherin axis, panel of cancer cell lines (Supplementary Figure S5). In contrast, through which miR-200 regulates tumour cell plasticity and only weak inverse correlations between cofilin2 or WASF3 and the metastasis. miR-200 family members were observed in breast cancers (data Our finding that enforced miR-200 expression reduces metas- not shown). Examining two separate breast cancer cohorts in tasis of a xenografted breast cancer cell line is consistent with which outcome data were available, we also found that patients reports that miR-200 reduces metastasis of xenografted lung whose tumours expressed high moesin levels had reduced adenocarcinoma cells,27 and head and neck squamous cell metastasis- or relapse-free survival (Figure 6g). In contrast, CFL2 carcinoma cells,44 but contrasts with the finding that miR-200 and ZEB1 were generally less predictive of outcome promotes metastasis of 4T07 mouse breast cancer cells.28,29 (Supplementary Figure S6), but, interestingly, WASF3 was pre- Enforced expression of miR-200 in 4T07 cells was shown to alter dictive of outcome in one cohort but not the other. Within these the cancer cell secretome by targeting the anterograde transport cohorts, moesin expression was significantly higher in the basal protein Sec23A.29 Although the invasive capacity of the 4T07 cells and claudin-low classified samples (Supplementary Figure S7), is reduced by miR-200, the net influence of its cell intrinsic and subtypes which are known to be associated with poor prognosis extrinsic effects promotes lung colonisation.29 Thus, the effect of and low miR-200 levels.13,16 Together, these data indicate that the miR-200 on metastasis can depend on the cellular context, and loss of miR-200b leading to the upregulation of moesin expression the effect of loss or gain of miR-200 in human breast cancers may may contribute towards metastasis and that this pathway may be differ with tumour subtype. In LM2 cells, which correspond to the important for breast cancer progression. basal subtype, miR-200b does not induce a full MET in vitro or in vivo but, instead, causes alteration in the cell morphology indicative of cytoskeletal rearrangement. These cells displayed a Restoration of moesin expression prevents miR-200b-mediated reduced invasive capacity, but lung colonisation, as assessed by repression of metastasis the experimental metastasis assay, remained unaffected. Thus, in To determine whether the reduction in moesin expression caused this context, miR-200b most likely acts to reduce metastasis by by miR-200b is necessary for repression of breast cancer repressing pathways that promote primary tumour cell invasion. metastasis, we re-expressed the moesin complementary DNA Control of the ZEB1-E-cadherin axis by the miR-200 family has without its 30UTR in miR-200b-expressing LM2 cells (LM2-miR- been proposed to be an important pathway influencing primary 200b) and performed spontaneous metastasis assays. As shown in tumour cell invasion and metastasis.4,45 Comparison of the three Figure 7a, transduction of the moesin complementary DNA into miR-200 family members (miR-200b, miR-200c and miR-141) LM2-miR-200b cells increased moesin expression to levels similar revealed that each of these miRNAs was able to repress ZEB1 to to LM2-vector cells. Importantly, the miR-200b levels were similar varying extents; however, only the miR-200b/200c/429 functional in control vector and moesin-transduced LM2-miR-200b cells, group inhibited spontaneous metastasis from the mammary both being elevated by B30-fold over LM2-vector cells. Moesin gland, indicating that the repression of ZEB1 may not be expression in LM2-miR-200b cells caused changes from a flattened required to suppress metastasis. We tested this by restoring to a more elongated morphology, which is consistent with recent ZEB1 levels in miR-200b-expressing cells, and, although E-cadherin findings showing that moesin regulates cell polarity and spreading levels were reduced, these cells still retained an impaired (Figure 7b).42,43 In spontaneous metastasis assays, restoration of metastatic ability. These data indicate that miR-200 is able to moesin expression in LM2-miR-200b cells caused them to repress metastasis independent of the ZEB1-E-cadherin axis. ZEB1 metastasise more efficiently than control cells, although not is a key target for miR-200 in several developmental and reaching statistical significance, with a mean level similar to that of pathological scenarios,46,47 with specific knockdown of ZEB1 LM2-vector cells (Figure 7c). However, we noted that the moesin- phenocopying the effects of increased miR-200 expression.19,20 expressing tumours were smaller than the control tumours, and ZEB1 can also reciprocally regulate miR-200 levels, and this the increase in metastasis burden was statistically significant after feedback loop can influence epithelial cell plasticity.16–18 Despite normalising for primary tumour size (Figure 7c). The ability of these studies, the mutual dependence of the miR-200 and ZEB1 moesin, and not ZEB1, to restore metastatic capability to LM2-miR- interaction has not been well investigated. Although the miR-200- 200b cells was confirmed in an additional experiment in which all ZEB1 feedback loop can regulate E-cadherin expression in LM2 groups of cell lines were compared concurrently (Supplementary cells, E-cadherin protein is not efficiently localised to the cell Figure S2). Furthermore, reduced moesin expression was pre- membrane, indicating that these cells do not transition to become served in primary tumours formed from miR-200b-transfected ‘fully epithelial’. Therefore, it is possible that the miR-200-ZEB1-E- LM2 cells and restored moesin expression was maintained in cadherin pathway has a more dominant role in controlling

Oncogene (2014) 4077 – 4088 & 2014 Macmillan Publishers Limited miR-200 regulates metastasis by targeting moesin XLiet al 4085 LM2-miR-200b 40 40 miR-200b/Vector miR-200b/MSN cells 30 30

20 20 F-Actin 10 10 miR-200b levels 0 0

MSN

Tubulin F-Actin Vector MSN Vector miR-200b miR-200b/ miR-200b/

Primary tumor Lung Lung Metastatic Burden 0.7 8 1.6

p= 0.012 ) p= 0.089 p= 0.019 6 )/g 0.6 7 6 1.2 0.5 4 0.8 0.4 Mean Weight (g) 0.3 2 LM2-Vector 0.4 photons/sec (x10 photons/sec (x10 0.2 0 0 MSN MSN MSN Vector Vector Vector miR-200b/ miR-200b/ miR-200b/ miR-200b/ miR-200b/ miR-200b/

Vector miR-200b/Vector miR-200b/MSN

MSN IHC

Figure 7. Re-expression of moesin in miR-200b-expressing cells restores metastatic ability. (a) Quantitation of miR-200b and moesin expression 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 cells transduced with a control or moesin coding region vector are shown in relation to parental LM2-miR-200b stable cells and LM2-Vector control cells. (b) Phalloidin staining of F-actin in LM2-miR-200b cells stably expressing the moesin coding region in comparison with control. The lower panel represents a magnified view compared with the upper panel. Scale bars indicate 20 mm. (c) Measurement of primary tumour weight and lung metastasis in LM2-miR-200b cells expressing a control vector or moesin. Lung metastatic burden was calculated by dividing the bioluminescent intensity of lung metastases for each individual mouse by the primary tumour weight from that same mouse. A dashed line marking the mean bioluminescent intensity in LM2-vector cells is shown for comparison (see Supplementary Figure S1). P-values were calculated using a two-tailed Mann–Whitney t-test. (d) Immunohistochemical staining for moesin of representative primary tumour sections from vector, miR-200b and miR-200b þ MSN xenografts. Scale bars represent 100 mm. metastasis in scenarios where a stronger MET occurs. However, is required for cytoskeletal remodelling of mammary epithelial given that miR-200 targets a number of genes involved in the cells.43 Moesin, but not ezrin, also promotes invasion and cytoskeletal organisation16,34,37,39,48 it is perhaps not surprising lung colonisation of melanoma tumour cells.42 Our findings are that miR-200 can regulate other pathways that contribute to consistent with these studies and indicate that moesin may have a metastasis. specific role in enhancing breast cancer progression. Interestingly, Examination of miR-200 target genes in the LM2 stable cell lines knockdown of moesin alone was not able to alter the metastatic revealed that several actin cytoskeleton-associated genes were potential of LM2 cells (Supplementary Figure S8) suggesting that its downregulated by miR-200b, but not by miR-141, consistent with reduction is necessary, but not sufficient, for metastasis. Therefore, the stronger changes in cell morphology and protrusions induced in addition to targeting moesin, miR-200b likely regulates other by miR-200b expression. However, only knockdown of moesin genes that contribute to metastatic progression in this context. reduced in vitro invasion with restoration of its expression in miR- Several immunohistochemical studies of breast cancers have 200b-expressing cells being sufficient to alter cell morphology and demonstrated that moesin is highly expressed in triple (ER/PR/ alleviate repression of metastasis, implicating it as a key target of HER2)-negative and basal subtype cancers and in some cases is a miR-200b in this process. Moesin is a member of the ezrin-radixin- prognostic marker of poor outcome.49–51 Moesin has also been moesin family that controls cytoskeletal dynamics and links directly linked with an EMT expression profile in breast cancer filamentous actin to the plasma membrane.40 Although initially samples.52 We showed that moesin is directly targeted by miR- thought to be redundant in function, recent studies have 200b, and, using breast cancer cell lines and patient tumours, we highlighted divergent roles for the ezrin-radixin-moesin proteins. found that moesin mRNA is highly expressed in basal and claudin- For example, moesin, but not ezrin, is induced during EMT and low subtypes. Furthermore, its expression shows a significant

& 2014 Macmillan Publishers Limited Oncogene (2014) 4077 – 4088 miR-200 regulates metastasis by targeting moesin XLiet al 4086 inverse correlation with miR-200b and miR-200c in these samples towards 10% foetal calf serum, the membranes were fixed with 10% buffered as well as in the NCI-60 panel of cancer cell lines indicating that formalin and cells stained with 4’,6-diamidino-2-phenylindole. Six fields of view for this relationship exists across a wide range of other cancer types. each membrane were counted using ImageJ software (http://rsbweb.nih.gov/ij/). We also demonstrated that high mRNA levels of moesin are associated with poorer relapse-free survival in two independent Immunofluoresence patient cohorts. Given that moesin has been shown to facilitate Cells were seeded in eight-well chamber slides (Nunc; Roskilde, Denmark) actin cortical polarisation and induction of tumour cell invasion in pre-coated with 50 ug/ml Fibronectin (Roche, Penzberg, Germany). After three-dimensional matrices,42 it will be interesting to further blocking with 1% bovine serum albumin, cells were stained using anti-E- examine the interplay between miR-200 and moesin in three- cadherin (1:500 vol:vol, BD Biosciences) or Phalloidin (Rhodamine or Alexa dimensional cultures and within primary tumours. Fluor 647-conjugated) to visualise F-actin. Nuclei were visualised by In summary, we have identified a moesin-dependent pathway staining with 4’,6-diamidino-2-phenylindole. Cells were imaged using an through which miR-200 family members act to repress primary Olympus IX81 microscope with a Hamamatsu Orca camera. Images were acquired using the (CellR; Olympus, Munster, Germany) software and tumour cell invasion and metastasis. These findings demonstrate a analysed using AnalySIS LS software (Olympus). Cell size (length:width role for miR-200 in controlling cell plasticity and function apart ratio) was measured using the arbitrary line tool on the AnalySIS LS from their regulation of the ZEB1-E-cadherin axis and MET. As the software. Cell length was defined by the longest distance between any two miR-200 family can also have a dual role in enhancing metastatic points of the cell, and cell width was measured as the longest line colonisation after dissemination,28,29 therapeutic utility of the miR- perpendicular to the cell-length line. 200 family may require consideration of the stage and degree of tumour progression. Luciferase reporter assay A full-length (B2 kb) or a 740-bp segment of the moesin 30UTR spanning two conserved miR-200b/200c/429 seed sites was amplified from pCMV6- MATERIALS AND METHODS XL5-hMSN (Origene, Rockville, MD, USA) and cloned downstream of Renilla luciferase in the XhoI and NotI sites of the psiCheck2 vector using primers A highly metastatic variant of MDA-MB-231, LM2 (4175 TGL), was obtained shown in Supplementary Table 1. The moesin 30UTR and control vectors from Dr Joan Massague.36 Cells were cultured in Dulbecco’s Modified Eagle (200 ng) were co-transfected with 5 nM of Pre-miR (Ambion) into MDA-MB- Medium (Gibco, Invitrogen, Carlsbad, CA, USA) þ 10% foetal calf serum 231 cells using Lipofectamine 2000 (Invitrogen). Cells were harvested 72 (FCS, Bovogen, Bovogen, VIC, Australia). All other human breast cancer cell hours later for Dual Luciferase assays (Promega, Madison, WI, USA). lines were cultured as described previously.13 Orthotopic and experimental metastasis assay Lentiviral and retroviral vectors and transduction Mice were housed in the SA Pathology Animal Care Facility, and experiments Genomic regions containing Pri-miRNAs for miR-200b, miR-200c and miR-141 were were conducted under the institutional animal ethics guidelines. For amplified from human genomic DNA and cloned into lentiviral and/or retroviral orthotopic and experimental metastasis studies, 5-week- to 6-week-old vectors. Cloning procedures are detailed in Supplementary Methods with primer female mice were used. For orthotopic experiments, mice were anesthetised 6 sequences shown in Supplementary Table 1. Transductions were performed as before injections of 1 Â 10 cells in 50 mlof50%Matrigel(BDBiosciences)into previously described13 and are detailed in Supplementary Methods. the fourth . Approximately 4 weeks after initial tumour implantation, bioluminescent imaging was performed as described below. For experimental metastasis, 2 Â 105 cellsin100ulHBSS(Hank’sBalancedSalt Transfection with miRNA and small interfering RNA Solution (Gibco)) were injected directly into the tail vein of recipient mice. Transient transfections of LM2 or breast cancer cell lines were performed in Bioluminescence images of animals were collected 2 and 4 hours after initial six-well plates (3 Â 105/well) using synthetic miRNA precursors (Pre-miRs, tail vein injection to determine baseline levels of metastasis and weekly Ambion, Invitrogen, Carlsbad, CA, USA) or ON-Target Plus SMARTpool small thereafter to monitor colonisation of tumour cells in the lung. interfering RNA (Thermo Fisher, Pittsburgh, PA, USA) at a final concentra- tion of 20 nM using Lipofectamine RNAiMAX (Invitrogen). After 72 h, cells Bioluminescence imaging were processed for downstream applications. Inhibition of the miR-200 family in breast cancer lines was performed for 6 days after transfection Bioluminescence imaging was performed using the Xenogen IVIS 100 18 with 100 nM Anti-MiR as described previously. imaging system. Mice were injected intraperitoneally with 30 mg/ml of D-Luciferin (in phosphate-buffered saline) 10 min before imaging. Dorsal images of the primary tumour were collected before the animals were culled Real-time PCR and their primary tumour, lung and hind limbs (with muscle tissue removed) RNA was isolated from adherent cultures and made into complementary harvested for ex vivo imaging. Photon emission was quantified using the DNA using methods previously described.13 PCR for miRNAs was Living Image Software (Xenogen) (Perkin Elmer, Hopkinton, MA, USA). performed using TaqMan microRNA assays (Applied Biosystems, Invitrogen, Carlsbad, CA, USA). For mRNA, PCRs were performed using Quantitect SyBr green reagents (Qiagen, Hilden, Germany) using the Histology specific primers listed in Supplementary Table 1. Real-time PCR data for Lung and primary tumours were fixed in 10% buffered formalin for mRNA and miRNA are expressed relative to GAPDH or U6, respectively. 24 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, Western blot Waltham, MA, USA) and human anti-green fluorescent protein (1:1000 Western blots were performed as described previously.13 The following vol:vol, Rockland Immunochemicals, Gilbertsville, PA, USA) antibodies. Bones antibodies were used: ZEB1 (1:200 vol:vol, Santa Cruz, Santa Cruz, CA, USA), were fixed and then decalcified in 0.5 M EDTA þ 0.2% paraformaldehyde for E-cadherin (1:1000 vol:vol, BD Biosciences; San Jose, CA, USA), moesin (1:1000 2 weeks before staining. vol:vol, Cell Signaling Technologies, Danvers, MA, USA) and tubulin (1:5000 vol:vol, Abcam, Cambridge, UK). Membranes were developed using enhanced chemiluminescence (ECL Prime, GE Healthcare, Buckinghamshire, UK) and Bioinformatics and statistical analyses 53 imaged using the LAS4000 luminescent image analyser (Fujifilm, Tokyo, Japan). Expression and clinical data from the previously combined 779 and UNC33754 tumour data sets were obtained from https://genome.unc.edu/ and analysed as detailed in Supplementary Methods. To examine the Migration and invasion assays correlation between miR-200 and moesin expression in breast cancer and For migration assays, 1 Â 105 cells were seeded in Transwells (6.5 mm, 8.0 mmpore the NCI-60 panel, linear regressions were generated using the GSE19783 size) (Corning, Tewksbury, MA, USA) in serum-free medium with 0.01% bovine data set41 or the five-platform gene data from CellMiner (http:// serum albumin. For invasion assays, 6 Â 104 cells were seeded into 8 mmBiocoat discover.nci.nih.gov/cellminer). All statistical analyses were performed Matrigel chambers (BD Biosciences). After 4 h for migration or 24 h for invasion using GraphPad Prism 5 (GraphPad Prism, La Jolla, CA, USA).

Oncogene (2014) 4077 – 4088 & 2014 Macmillan Publishers Limited miR-200 regulates metastasis by targeting moesin XLiet al 4087 CONFLICT OF INTEREST 22 Schickel R, Park SM, Murmann AE, Peter ME. miR-200c regulates The authors declare no conflict of interest. induction of apoptosis through CD95 by targeting FAP-1. Mol Cell 2010; 38: 908–915. 23 Morel AP, Hinkal GW, Thomas C, Fauvet F, Courtois-Cox S, Wierinckx A et al. EMT ACKNOWLEDGEMENTS inducers catalyze malignant transformation of mammary epithelial cells and drive tumorigenesis towards claudin-low tumors in transgenic mice. PLoS Genet 2012; We thank Professor Joan Massague for providing the MDA-MB-231 LM2 cell line 8: e1002723. and Dr Ross Dickins for providing the pLMP-puro-GFP construct. We thank Dr Agatha 24 Cochrane DR, Spoelstra NS, Howe EN, Nordeen SK, Richer JK. MicroRNA-200c Labrinidis and Ms Yuka Harata-Lee for assistance with bioluminescence imaging and mitigates invasiveness and restores sensitivity to microtubule-targeting che- inoculation of tumour cells, respectively, Dr Peter Diamond for help with bone motherapeutic agents. Mol Cancer Ther 2009; 8: 1055–1066. histological analysis and Professors Andreas Evdokiou and Shaun McColl for insightful 25 Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL discussions. This work was supported by fellowships from the National Breast Cancer et al. Epithelial to mesenchymal transition contributes to drug resistance in Foundation of Australia (PAG, CPB and RLA, nos ECF-09-08 and PF-09-03) and grants pancreatic cancer. Cancer Res 2009; 69: 5820–5828. from the National Health and Medical Research Council of Australia (PAG, YK-G, GJG, 26 Spaderna S, Schmalhofer O, Wahlbuhl M, Dimmler A, Bauer K, Sultan A et al. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 4077 – 4088 & 2014 Macmillan Publishers Limited