miR-206 inhibits cell migration through direct targeting of the actin-binding protein Coronin 1C in triple-negative breast cancer

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available at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/molonc

miR-206 inhibits cell migration through direct targeting of the actin-binding protein Coronin 1C in triple-negative breast cancer Jun Wanga, Efrosini Tsoukoa, Philip Jonssona, Jonas Berghb, Johan Hartmanb, Eylem Aydogdua, Cecilia Williamsa,* a Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, TX 77204-5506, USA b Department of Oncology and Pathology, Karolinska Institutet and University Hospital, S-171 76 Stockholm, Sweden

A R T I C L E

I N F O

A B S T R A C T

Article history:

Patients with triple-negative breast cancer (TNBC) have an overall poor prognosis, which is

Received 22 May 2014

primarily due to a high metastatic capacity of these tumors. Novel therapeutic approaches

Received in revised form

to target the signaling pathways that promote metastasis are desirable, in order to improve

7 July 2014

the outcome for these patients. A loss of function of a microRNA, miR-206, is related to

Accepted 7 July 2014

increased metastasis potential in breast cancers but the mechanism is not known. In

Available online 12 July 2014

this study, we show that miR-206 was decreased in TNBC clinical tumor samples and cell lines whereas one of its predicted targets, actin-binding protein CORO1C, was

Keywords:

increased. Expression of miR-206 significantly reduced proliferation and migration while

miR-206

repressing CORO1C mRNA and protein levels. We demonstrate that miR-206 interacts

Breast cancer

with the 3’-untranslated region (3’-UTR) of CORO1C and regulates this gene post-

CORO1C

transcriptionally. This post-transcriptional regulation was dependent on two miR-206-

Cell migration

binding sites within the 3’-UTR of CORO1C and was relieved by mutations of corresponding sites. Further, silencing of CORO1C reduced tumor cell migration and affected the actin skeleton and cell morphology, similar to miR-206 expression, but did not reduce proliferation. In accordance with this, overexpression of CORO1C rescued the inhibitory effect of miR-206 on cell migration. Our findings suggest that miR-206 represses tumor cell migration through direct targeting of CORO1C in TNBC cells which modulates the actin filaments. This pathway is a novel mechanism that offers a mechanistic basis through which the metastatic potential of TNBC tumors could be targeted. ª 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Abbreviations: TNBC, triple-negative breast cancer; miR-206, microRNA-206; CORO1C, Coronin 1C; 3’-UTR, 3’-untranslated region; NC, negative control; ORF, open reading frame; RFS, relapse-free survival; PARP, poly (ADP-ribose) polymerase; TMSB4X, thymosin beta 4, X-linked; TPM4, tropomyosin 4; TNS3, tensin 3. * Corresponding author. Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, 3605 Cullen Blvd., Houston, TX 77204-5056, USA. Tel.: þ1 832 8428807. E-mail address: [email protected] (C. Williams). http://dx.doi.org/10.1016/j.molonc.2014.07.006 1574-7891/ª 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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1.

Introduction

Breast cancer is the most common female cancer and the second leading cause of cancer death in women. Breast tumors can be subclassified according to their expression of estrogen receptor alpha (ERa), progesterone receptor (PR), and membrane receptor HER2. Targeted treatments are available for ERa/PR-positive and HER2-positive tumors, but triple-negative breast cancers (TNBCs) by definition do not express these receptors (Gluz et al., 2009) and therefore lack targeted therapies. TNBCs constitute 12e17% of all breast cancers and the risk of distant recurrence within 5 years of diagnosis is higher and the survival after recurrence is significantly shorter than for women with other types of breast cancer (Dent et al., 2007). Thus, it is important to understand why the metastatic potential is high in TNBCs so that we can design therapeutic therapies that target these mechanisms. Breast tumors can also be classified based on their gene expression profile, and studies have shown that most TNBCs belong to the basal-like molecular subtype (Foulkes et al., 2010; Sorlie et al., 2001). Given the expression of cell-surface markers, the basal-like subtype has been proposed to owe their high migratory and metastatic capacity to a high fraction of cancer stem cell population (Ben-Porath et al., 2008). Previously, we showed that the gene expression of nontumorigenic murine mammary stem cell-like HC11 cells cluster with that of basal-like breast tumors (Williams et al., 2009). We also defined a number of microRNAs (miRNAs), including miR-206, that were highly regulated during functional differentiation of the HC11 cells (Aydogdu et al., 2012). miRNAs are important gene regulators that regulate multiple stem cell mechanisms, and their dysregulation is associated with tumorigenesis and metastasis (Shenouda and Alahari, 2009; Zhang et al., 2007). Therefore, we suggested that the miRNAs regulated during the transition of the migratory stem-like HC11 cells to its functionally differentiated counterpart may be imperative for these mechanisms in basal-like breast cancer (Aydogdu et al., 2012). miR-206 was regulated during this transition, and is known to be critical in myoblast differentiation and function, and to be downregulated in human rhabdomyosarcoma (RMS) where its re-expression promotes myogenic differentiation and blocks tumor growth (Dey et al., 2010; Missiaglia et al., 2010; Taulli et al., 2009; Yan et al., 2009). Therefore, miR-206 is considered as a potential candidate for novel rhabdomyosarcoma therapy (Mishra and Merlino, 2009). In breast cancers, miR-206 has been found to be downregulated in ERa-positive tumors (Kondo et al., 2008), and to directly repress ERa expression in MCF-7 breast cancer cells (Adams et al., 2007). Low levels of miR-206 in breast cancer are associated with advanced clinical stage, lymph node metastasis, lower overall survival (Li et al., 2013), as well as increased metastatic potential in breast cancer (Tavazoie et al., 2008; Wang et al., 2010), but the molecular mechanism responsible for these correlations is not known. We hypothesized that miR-206 represses migration in breast cancer, and that we could use the transcriptome regulations during HC11 cell differentiation to predict its underlying mechanism.

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miRNAs function by post-transcriptionally regulating the stability and/or translation of its target mRNAs. Their target mRNAs can be predicted based on the complementarity between the miRNA, especially its 6e8 nucleotide-long seed sequence, and the target mRNA’s 3’-UTR sequence, using various algorithms. Using target-prediction software in combination with anti-correlation between the mRNA transcriptome (Williams et al., 2009) and corresponding miR-206 regulation in the HC11 cells, we found that Coronin, actinbinding protein, 1C (CORO1C), is a strong candidate for mediating the miR-206 migratory mechanism. CORO1C belongs to the coronin family of actin-binding proteins that are important for the control and remodeling of the actin filaments network (Gandhi and Goode, 2008). As cell motility relies on actin dynamics (Uetrecht and Bear, 2006) and CORO1C has been reported to be upregulated in multiple types of clinically aggressive cancers and its knockdown to reduce cell invasion and metastasis (Ren et al., 2012; Roadcap et al., 2008), we hypothesized that miR-206 post-transcriptionally represses CORO1C expression, and that the loss of miR-206 thereby contributes to higher migratory potential in TNBC. In this study, we explore the relation between miR-206 and CORO1C and their respective function in TNBC to test this hypothesis.

2.

Materials and methods

2.1.

Bioinformatics analysis

2.1.1.

miRNA target gene prediction

Anti-correlation between HC11 miRNA and gene expression data (Williams et al., 2009) of predicted targets using both TargetScan and miRanda algorithms were performed to find potential miR-206 target genes. The full-length mRNA sequences of human CORO1C and mouse Coro1c (ENSG00000110880 and ENSMUSG00000004530) were obtained from the Ensembl Database. The miR-206 mature sequences of human and mouse (MI0000490 and MI0000249) were obtained from the miRBase database.

2.1.2.

Analysis of publicly available breast cancer data sets

Expression levels of CORO1C in human breast cancer were collected from the following four data sets, Yau et al. (Yau et al., 2010), Wang et al. (Wang et al., 2005), The Cancer Genome Atlas (Goldman et al., 2013), and METABRIC (Curtis et al., 2012). Relative CORO1C expression data was classified into the Luminal A, Luminal B, Normal-like, Basal-like, and HER2-positive subtypes of breast cancer. One-way ANOVA was used to test the significance of differences between the tumor groups and differences were considered significant if P < 0.05.

2.1.3.

Survival analysis

CORO1C mRNA levels (AffyID: 221676_s_at) were extracted from publically available microarray data of 3455 breast cancer patients and related to survival (Gyorffy et al., 2010) using the online analysis tool http://kmplot.com. This data set

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includes data from The Cancer Genome Atlas, along with multiple other studies. Relapse-free survival (RFS) in all breast cancer and different subtype patients was observed towards the end point. Hazard ratio and logrank test were calculated for the significance testing. We also extracted CORO1C mRNA levels and patients overall survival from METABRIC date set of 1906 breast cancer patients followed by the same analysis.

2.2.

Clinical samples

Fresh human breast tumors were obtained from patients with tumors larger than 5 mm in diameter, diagnosed at the Karolinska Hospital, Sweden, between January 1 and March 31 2011. In this study, only primary tumors from patients not receiving neo-adjuvant treatment were used. 3  3 mm of fresh tumor pieces were snap-frozen for later RNA processing and analysis. Clinicopathological variables (tumor grade, ER, PR, Her2 and Ki67 status) were measured at diagnosis using formalin-fixed sections of the tumors. Normal human breast tissues were obtained from healthy women under the age of € rans 30, undergoing reduction mammaplasty at Capio St Go Hospital, Stockholm, Sweden. Approximately 5  5 mm of normal tissues were immediately frozen for later RNA isolation. The samples were de-identified and the study was approved by the local ethics board in Stockholm (EPN), Sweden.

2.3.

Cell culture

Mouse HC11 cells were maintained in RPMI1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 5 mg/ml insulin, 10 ng/ml EGF, and 50 mg/ ml gentamicin (all from Sigma, Saint Louis, MO, USA). Human TNBC cell lines MDA-MB-231 and SUM159 cells were cultured in 1:1 DMEM: F12 (Gibco) containing 10% FBS and 1% penicillin streptomycin (Gibco). HEK293 cells were maintained in DMEM supplemented with 10% FBS.

2.4. clone

Transfection with miRNA mimic, siRNA, and ORF

The miRIDIAN miR-206 mimic (Dharmacon, Pittsburgh, PA, USA) and NC-mimic (Dharmacon or Ambion, Grand Island, NY, USA) were transfected into cells at a final concentration of 25 nM using DharmaFECT1 (Dharmacon) as described previously (Aydogdu et al., 2012). Human and mouse have identical mature miR-206 sequences, and the same miR-206 mimic was used for both species. miR-206 inhibitor and corresponding control (both from Dharmacon) were used at a final concentration of 50 nM. Predesigned ON-TARGET plus siRNA pool, targeting CORO1C (target sequence 1: GCACAAGACUGGUC GAAUU; target sequence 2: CAAUCAAGAUGAGCGUAUU; target sequence 3: GAGCAAGUUUCGGCAUGUA; target sequence 4: AAACAGGGAUCUCAAGGUG), or ON-TARGET plus negative control (NC) siRNAs (both from Dharmacon) were transfected into MDA-MB-231 or SUM159 cells in 12well or 24-well plates, at a final concentration of 50 nM using DharmaFECT1. 0.8 mg or 4 mg of human CORO1C ORF cDNA clone (Genecopoeia, Rockville, MD, USA) were transfected

into TNBC cells in 24-well or 6-well plates, using Lipofectamine 2000. When stated, a second transfection was performed after 48 h.

2.5.

RNA extraction, cDNA synthesis and qPCR

Total RNA, including the miRNA population, was extracted using TRIzol (Invitrogen, Grand Island, NY, USA) and miRNeasy kits (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Quantitative and qualitative RNA analyses were performed using NanoDrop 1000 spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA) and the Agilent 2100 BioAnalyzer (Agilent technologies, Palo Alto, CA, USA), respectively. Samples with RNA integrity >9.5 were used. For quantification of protein-coding transcripts, 1 mg of total RNA was subjected to cDNA synthesis using SuperScript III FirstStrand Synthesis reagents and random hexamers, as previously described (Aydogdu et al., 2012). qPCR was performed in triplicates using 10 ng of cDNA and Fast SYBR Green SuperMix (Life Technologies, Grand Island, NY, USA) on an ABI PRISM 7500 series real-time PCR system (Life Technologies). Gene expression levels were normalized to 18S and relative fold-change levels calculated using the DDCT method. For miRNA quantification, the TaqMan small RNA assay kit (Life Technologies) was used according to the manufacturer’s protocol. Briefly, 60 ng total RNA was subjected to cDNA synthesis using miR-206 specific looped reverse primer, and qPCR carried out with primers and probe. U6 spliceosomal RNA was used as an internal control to normalize RNA input to, and relative miRNA levels were calculated using the DDCT method. Student’s unpaired two-tailed t-test was used for significance testing and differences were considered significant if P < 0.05.

2.6.

Western blot analysis

Cells were washed with cold PBS and lysed in RIPA buffer and protein quantified by Pierce 660 nm protein assay (Thermo Scientific). 30 mg of total protein were resolved on a 10% SDSPAGE gel, and transferred to nitrocellulose membranes according to standard procedures. Membranes were washed with TBST, containing 0.1% Tween 20, blocked in 5% milk in TBST and then incubated with primary antibodies against CORO1C (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:400 dilution), PARP (Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), and b-actin (SigmaeAldrich, 1:6000 dilution) overnight. Membranes were then incubated with either anti-rabbit or anti-mouse IgG horseradish peroxidase-linked secondary antibody at room temperature, and visualized using Pierce ECL western blotting substrate (Thermo Scientific) according to the manufacturer’s protocol. Scanned images were quantified using ImageJ, with normalization to the loading control, b-actin.

2.7.

3’-UTR luciferase reporter assay

Wild-type or mutant human CORO1C 3’-UTR and wild-type mouse Coro1c 3’-UTR segments (See Suppl. Table 1 for sequences) were cloned into the luciferase reporter vectors (pEZX-MT01, purchased from Genecopoeia), respectively.

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HEK293 cells were co-transfected with 800 ng CORO1C 3’-UTR luciferase construct and miR-206 mimic or NC-mimic (Ambion, 30 nM) in 24-well plates using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. For mouse Coro1c 3’-UTR, cells were co-transfected with the luciferase construct and miR-206 mimic, miR-148 mimic, or NC-mimic (Ambion, 30 nM each). Cells were collected 24 h after transfection and the luciferase activity was assessed using Dual-Luciferase reporter assay system (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. Experiments were performed in triplicate, and repeated in two independent experiments. Student’s unpaired two-tailed t-test was used for significance testing and differences were considered significant if P < 0.05.

2.8.

Proliferation assay

2.8.1.

Cell counting

Cells were transfected twice with miR-206 mimic, siRCORO1C or corresponding controls (both from Dharmacon) in 24-well plates, as described above. Cells were trypsinized and stained with trypan blue 48 h after the second transfection. The numbers of viable cells were determined using Countess automatic cell counter (Invitrogen). All experiments were repeated in three independent assays, each performed in triplicate. Student’s unpaired two-tailed t-test was used for significance testing and differences were considered significant if P < 0.05.

2.8.2.

Cell cycle analysis

Cells were transfected with miR-206 mimic (30 nM) or NC (Dharmacon) in 6-well plates, and then synchronized in 0.5% BSA medium. After 48 h, full-serum medium was added and cells allowed to grow for 36 h before fixation (70% ethanol) and staining (propidium iodide, 50 mg/ml, SigmaeAldrich). The cell-cycle distribution in G1, S, and G2/M phase was determined using BD FACSAria III (BD Biosciences, San Jose, CA, USA).

2.9.

In vitro wound healing assay

Cells seeded in 24-well plates were transfected twice with miR-206 mimic, siRNA, ORF clone or corresponding controls (NC-mimic from Dharmacon), as described above. A scratch through the monolayer was created 24 h after the second transfection, and cells were washed three times using prewarmed PBS to remove cellular debris. The medium was replaced with fresh serum-reduced (2%) medium and cells were allowed to migrate for 12 h (or 9 h). Cell migration images were photographed at 0 h and 12 h (or 9 h) using inverted microscope with Olympus XM10 camera (Olympus, Center Valley, PA, USA). The relative distance between the leading edge was assessed using Olympus CellSens Dimension software (Olympus) and migratory distance calculated. Experiments were repeated in three independent assays, each performed in triplicate. Student’s unpaired two-tailed t-test was used for significance testing and differences were considered significant if P < 0.05.

2.10.

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Immunofluorescence staining

Cells were cultured and transfected in permanox two-well chamber slides (Nunc, Pittsburgh, PA, USA) and fixed with 3.7% paraformaldehyde for 10 min, followed by three washes with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and washed three times with PBS prior to blocking with 3% BSA-PBS for 30 min. For detection of CORO1C, specimens were incubated overnight at 4  C with CORO1C antibody (Santa Cruz Biotechnology, 1:250 dilution). Specimens were then washed three times with PBS and incubated with Alexa Fluor 488 (Cell Signaling Technology, 1:1000 dilution), Alexa Phalloidin 594 (Cell Signaling Technology, 1:40 dilution), and DAPI in room temperature. Cover slips were mounted with ProLong Gold antifade reagent (Invitrogen). Olympus FV1000 confocal (Olympus) microscopy was used for imaging, followed by ImageJ analysis.

3.

Results

3.1. cells

miR-206 is reduced and CORO1C increased in TNBC

To explore whether miR-206 expression is related to CORO1C expression in human breast cancer, we analyzed the levels of CORO1C in four large-scale breast cancer data sets, including The Cancer Genome Atlas and METABRIC data sets (Curtis et al., 2012; Goldman et al., 2013; Wang et al., 2005; Yau et al., 2010). The five molecular subtypes (luminal A, luminal B, normal-like, basal-like and HER2-positive) of breast cancer were defined by their gene expression profiles. In all data sets, CORO1C levels were significantly increased in basal-like breast cancer compared to ERa-positive and normal-like subtypes (Figure 1A). Furthermore, we compared the patient survival probability between patient cohorts exhibiting high CORO1C expression to those with low expression, using available gene expression and survival data for 3455 patients (Gyorffy et al., 2010). We found that high CORO1C expression was associated with lower survival for the basallike subtype patients (P ¼ 0.015, Figure 1B) as well as for Luminal A and Luminal B breast cancer patients (Suppl. Figure 1A), but not for HER2-positive tumors (Suppl. Figure 1A). However, in another data set METABRIC (Curtis et al., 2012) including 1906 patients, this survival effect was not significant (data not shown). Because neither of these large data sets had high-quality data for miR-206 available, possibly because the low levels of miR-206 in tumors are difficult to reliably detect and measure using high-throughput techniques, we analyzed the levels of miR-206 using quantitative PCR (qPCR) in 24 primary tumors and 13 normal breast (NB) tissue samples. We found that miR-206 expression was significantly suppressed in TNBC tumors (Figure 1C). Using qPCR we also confirmed a significant increase of CORO1C mRNA only in TNBC tumors (Figure 1D). Thus, by analyzing both clinical samples and large-scale data sets, we consistently found high expression of CORO1C in the TNBC subtype. Next, we attempted to explore the relation between miR206 and CORO1C levels by plotting the individual levels for

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Figure 1 e miR-206 is reduced and CORO1C increased in TNBC. A) CORO1C levels are increased in basal-like breast tumors in four human breast cancer data sets collected from Yau et al. (Yau et al., 2010), Wang et al. (Wang et al., 2005), The Cancer Genome Atlas (Gyorffy et al., 2010), and METABRIC (Curtis et al., 2012). One-way ANOVA was used to test the significance of differences between the Luminal A, Luminal B, Normal-like, Basal-like and HER2-positive breast cancer subtypes. B) CORO1C expression in basal-like tumors is correlated with low survival. KaplaneMeier plot, based on data from (Gyorffy et al., 2010), illustrates the survival probability for patients with low or high CORO1C expression in their basal-like breast cancers. CeE) miR-206 and CORO1C levels were determined in clinical breast cancer specimens using qPCR. Box-andwhisker plots illustrate the relative miR-206 and CORO1C levels in 13 normal breast tissues, 3 TNBC, and 21 ERaD/PRD breast tumor samples. One-way ANOVA was used to test the significance of differences. The correlation between miR-206 and CORO1C levels for each individual TNBC sample is illustrated in E). F) miR-206 was significantly downregulated in two TNBC cell lines compared to non-tumor breast epithelial MCF-10A. GeH) CORO1C mRNA and protein levels were high in human TNBC cell lines compared both to MCF-10A and ERa-positive breast cancer cell MCF-7 and T47D. Unpaired two-tailed student’s t-test was used for statistical testing. Significance is presented as *P £ 0.05, **P £ 0.01, ***P £ 0.001.

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each normal breast and TNBC clinical tumor sample. An inverse correlation was indicated for both normal samples and TNBCs, but not for ERa-positive tumors (Figure 1E and Suppl. Figure 1BeC). We further investigated whether the expression pattern of low miR-206 levels and high CORO1C levels was retained in human TNBC cell lines. Compared to levels in non-cancerous mammary epithelial cell line (MCF-10A), miR-206 levels were decreased in both ERa-positive breast cancer cell lines (MCF-7 and T47D) and exhibited the most pronounced downregulation (P < 0.01) in triple-negative MDAMB-231 and SUM159 cells (Figure 1F). Further, in accordance with data from clinical tumors, CORO1C expression was significantly upregulated in TNBC cell lines (Figure 1G), which was also corroborated at the protein level (Figure 1H). This supported a potential direct relationship between miR-206 and CORO1C in TNBCs. Collectively, these results support that miR-206 is repressed in TNBC, and suggests an inverse correlation with CORO1C expression that may be correlated to lower survival.

3.2.

miR-206 directly targets the 3’-UTR of CORO1C

Using target-prediction softwares (TargetScan and miRanda) we scanned the 3’-UTR of both the mouse Coro1c and human CORO1C gene for predicted miR-206 target sites. The eight nucleotides that comprise the seed sequence of miR-206 showed complete complementarity to two target sites in both human and mouse 3’-UTR (Figure 2A and Suppl. Figure 2A). Further, these target sequences were highly conserved among distant species of vertebrates (Figure 2B) indicating an important regulatory role of this sequence. To test whether miR-206 can repress CORO1C mRNA or protein levels in either species, we transfected murine non-tumorigenic SC-like cells (HC11) and human TNBC cells (MDA-MB-231 and SUM159) with miR-206 mimics. Efficient transfection was demonstrated 48 h after transfection using qPCR analysis (Suppl. Figure 2B). CORO1C mRNA and protein levels were significantly reduced 48 h after miR-206 transfection in both mouse and human cells (Figure 2CeD). These results show that miR206 negatively affects the expression of CORO1C. To determine whether miR-206 regulates CORO1C directly, through the interaction with its predicted 3’-UTR binding sites, we used 3’-UTR luciferase reporter assay. Human wild-type or mutant CORO1C 3’-UTR sequences were inserted downstream of the firefly luciferase-coding region in the pEZX-MT01 vector (Figure 2E) and co-transfected with the miR-206 mimic or NC-mimic (Ambion). Three mutated constructs were designed to interrupt the binding of predicted site 1 (Mut01), 2 (Mut02) or both (Mut03). The luciferase activity of wild-type h-CORO1C vector was significantly reduced by miR-206 and rescued by mutation of site 1, Mut01 and double mutation Mut03 (Figure 2F). This demonstrates that binding site 1 is critical for the miR-206 directed CORO1C silencing, and that miR-206 repress CORO1C expression through direct interaction with this site. We note that also Mut02 attenuated the, as evidenced by lack of miR-206 repression in Mut01 (which carries a wild-type site 2); site 2 alone is not sufficient for the repression. Direct binding of miR-206 to mouse Coro1c 3’-UTR was also established (Suppl. Figure 2C). These results demonstrate that miR-206

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regulates CORO1C directly by targeting and interacting with its 3’-UTR in a conserved manner.

3.3. miR-206 represses tumor cell migration but not proliferation through CORO1C Loss of miR-206 has been associated with increased proliferation and metastatic potential in breast cancer (Tavazoie et al., 2008; Wang et al., 2010), and CORO1C overexpression has been associated with invasive properties in other cancers including lung, gastric, glioma and hepatocellular carcinoma (Ren et al., 2012; Thal et al., 2008; Wang et al., 2010; Wu et al., 2010). We hypothesized that the mechanistic basis for the association between loss of miR-206 and metastasis in breast cancer may be attributed to increased levels of its target CORO1C enhancing migratory potential. We first established whether miR-206 or CORO1C affected cell proliferation in TNBC cell lines MDA-MB-231 and SUM159. By transfecting cells with miR-206 mimic or NCmimic (Dharmacon) and investigating cell viability using trypan blue staining and cell counting, we found that miR-206 inhibited cell growth in both cell lines (Figure 3A). Using propidium iodide staining and flow cytometry we observed that miR-206 expression arrested the cells in G1-phase (Figure 3B). Because miR-206 has been reported to induce apoptosis in HeLa cells (Song et al., 2009), we also examined the PARP protein cleavage, which is a marker for apoptotic process. No difference in PARP cleavage was observed after transfection of miR-206 or NC-mimic in MDA-MB-231 or SUM159 cells (Figure 3C). Thus, we conclude that miR-206 represses proliferation by inducing G1-arrest but not apoptosis in TNBC cells. To explore whether it affects these properties through its regulation of CORO1C, we used RNA interference to silence CORO1C (Figure 3D). Silencing of CORO1C, however, did not reduce cell proliferation in either cell line (Figure 3A) demonstrating that miR-206 represses proliferation independently of its regulation of CORO1C. Next, we investigated whether miR-206 modulates cell migration through its regulation of CORO1C. We used reduced-serum medium and short time-points (12 h) in the migration assay to avoid the proliferative effect from skewing the migratory results. By using in vitro wound assays we found that expression of miR-206 significantly decreased cell motility in all three cell lines MDA-MD-231, SUM159 and HC11 cells (Figure 4A and B). Since HC11 cells, but not the TNBC cells, express some level of miR-206, we explored the effect of inhibiting miR-206 using specific inhibitor RNA. Inhibition of miR-206 increased migration significantly (Figure 4B). Further, knockdown of the CORO1C in both TNBC cell lines resulted in a similar reduction of cell migration (Figure 4A) without affecting the proliferation (Figure 3A). To further demonstrate that miR-206 represses tumor cell migration through CORO1C, we added CORO1C lacking the 3’-UTR (Figure 4C, right top) back and found that this rescued the cells form the anti-migratory effect of miR-206 in both MDA-MB-231 and SUM159 cell (Figure 4C). Also, CORO1C overexpression alone significantly promoted cell mobility in TNBC cells (Figure 4C). Collectively, repression of CORO1C by miR-206 reduces cell migration and can be rescued through CORO1C re-expression, demonstrating that

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Figure 2 e miR-206 directly targets CORO1C 3’-UTR. A) Putative target sites for miR-206 are indicated in the 3’-URT of the human CORO1C and mouse Coro1c genes (upper panel). The position refers to distance from the start of the 3’-UTR. Predicted binding between miR-206 and the 3’-UTR of CORO1C was analyzed using TargetScan (lower panel). B) The target sites are conserved in vertebrate species. Highlighted nucleotides indicate the miR-206 seed sequence. C) Expression of miR-206 in murine HC11, and human MDA-MB-231 and SUM159 cells reduced CORO1C mRNA levels. Data illustrate three independent experiments. D) miR-206 also reduced the CORO1C protein levels. Bottom panel illustrates the quantification of CORO1C protein levels based on three experiments. E) The ability of miR-206 to directly bind to the 3’-UTR of CORO1C was tested using the illustrated pEZX-MT01 construct. F) HEK293 cells were co-transfected with wild type h-CORO1C 3’-UTR luciferase construct, or construct containing mutations in predicted miR-206-binding site 1 (Mut01), site 2 (Mut02) or both sites (Mut03), and either miR-206 mimics or NC-mimic (Ambion). Luciferase expression was normalized to Renilla luciferase and depicted as the mean ± S.D. The figure illustrates data from three independent experiments performed in triplicate. Unpaired two-tailed student’s t-test was used in CeF) to test the significance of differences between two parallel groups. Significance is presented as *P £ 0.05, **P £ 0.01, ***P £ 0.001.

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Figure 3 e miR-206 but not siR-CORO1C arrests the cell cycle in G1-phase. A) miR-206 but not siR-CORO1C decreased cell proliferation in MDA-MB-231 and SUM159 cell lines. Trypan blue viability assays were performed 48 h after second transfection with NC-mimic (Dharmacon), miR206-mimic, NC-siRNA or siR-CORO1C. Three independent experiments were performed in triplicate. Data are represented as mean ± S.D. and unpaired two-tailed student’s t-test was used for significant testing (*P £ 0.05, **P £ 0.01, ***P £ 0.001). B) miR-206 induced G1 cell cycle arrest in MDA-MB-231 cells. Cells transfected with NC-mimic or miR206-mimic were subjected to the PI staining and flow cytometry analysis of DNA content 36 h after transfection. This experiment was repeated three times and similar results were observed. One representative data is shown in upper panel and quantification in lower panel. C) miR-206 did not significantly affect apoptosis. MDA-MB-231 and SUM159 cells were transfected with NC-mimic or miR206-mimic twice, and analyzed for PARP cleavage using western blot 48 h after transfection. MCF-7 cells treated with cisplatin were used as a positive control of PARP cleavage. D) Western blot confirmed knockdown of CORO1C protein levels by siCORO1C transfection in MDA-MB-231 and SUM159.

miR-206 represses cell migration through the targeting and silencing of CORO1C.

3.4. miR-206 remodels actin filaments and affects cell shape through the repression of CORO1C We next explored the mechanism whereby miR-206-mediated silencing of CORO1C affects cell mobility in TNBC cells. CORO1C is an actin-binding protein that can influence actin filaments networks (Gandhi and Goode, 2008), and thereby affects the migratory potential. To investigate whether miR-206 and CORO1C expression modified the structure of actin

filaments in TNBC, we used immunofluorescence staining of CORO1C and F-actin along with DAPI staining and confocal microscopy. We examined the localization and expression of CORO1C and F-actin, and the effect on cell shape that miR206, siR-CORO1C, and ORF-CORO1C treatment mediate in MDA-MB-231 cells. As expected, we noted a widespread colocalization of CORO1C and actin filaments throughout the cytoplasm and cell membranes in the controls (Figure 5A, upper panels). Upon treatment with miR-206 mimic or siRCORO1C, CORO1C expression in the membrane and cytoplasm decreased and co-localization was not detectable (Figure 5A, lower panels). Interestingly, the miR-206 and siR-CORO1C

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Figure 4 e miR-206 represses tumor cell migration through CORO1C. A) Both miR-206 (25 nM) and siR-CORO1C (50 nM) reduce migration in MDA-MB-231 and SUM159 cells. The experiments were repeated three times and pictures from one representative experiment are shown. The solid white line highlights the wound edge at 0 h and 12 h. The decrease of the gap area between the migrating cells from the opposite wound edge was quantified using Olympus CellSens Dimension software (Olympus) and quantified at the right panel. B) HC11 cells treated with either miR206 mimic (25 nM), inhibitor (50 nM), or corresponding controls, were analyzed by scratch wound assays 48 h after transfection. C) TNBC cells were transfected with 800 ng ORF-CORO1C alone or in combination with miR-206 mimic and migration assessed 48 h after transfection. The quantification of three assays is shown to the lower right. Upper right panel: Western Blot verifies the overexpression of CORO1C protein in both MDA-MB-231 and SUM159 cells. All data from A) to C) are presented as mean ± S.D. from three independent experiments. Unpaired two-tailed student’s t-test was used for significant testing, and presented as *P £ 0.05, **P £ 0.01, ***P £ 0.001.

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Figure 5 e Silencing of CORO1C remodels the actin filaments and changes the cell morphology. A) Immunofluorescence staining of DNA (blue), CORO1C (green) and F-Actin (red) in MDA-MB-231 cells. Scale bar: 10 mm. Cells were transfected with NC-mimic (Dharmacon, first row), NC-siRNA (second row), miR206-mimic (third row), and siR-CORO1C (fourth row). Merged pictures (right column) show the co-localization of CORO1C and actin filaments in control cells (upper panels). CORO1C expression was decreased and co-localization undetectable after miR-206 mimic or siR-CORO1C treatment (lower panels). Actin dynamic was also suppressed as CORO1C was silenced, by either siRNA or miR-206, resulting in a more compact and rounder cell shape. B) Upper panel: Completely complimentary alignment between miR-206 and TMSB4X and TPM4 3’-UTR was identified using TargetScan. Highlighted nucleotides indicate the predicted miR-206 seed sequence binding. Lower panel: miR-206 downregulated additional actin filament regulators TMSB4X, TPM4, and TNS3 in murine HC11, and human TNBC cells. Unpaired two-tailed student’s t-test was used to test the significance, presented as *P £ 0.05, **P £ 0.01, ***P £ 0.001. C) Illustration of a proposed mechanism for how miR-206 affects tumor cell migration through the regulation of actin filaments dynamics.

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treatment resulted in a more compact and rounder cell shape, indicative of actin network remodeling. The co-localization of CORO1C and F-actin reoccurred when we overexpressed CORO1C, which also moderately rescued actin filaments turnover, especially in the cytoplasm (Suppl. Figure 3). We also propose that this rescue was not complete because miR-206 may target additional actin filament regulators. Using prediction software, we found that several more actin filament regulators were predicted targets of miR-206, including TMSB4X, TPM4, and TNS3 (Figure 5B, upper panels.), and miR-206 expression resulted in reduced levels also of these regulators (Figure 5B, lower panels). Collectively, we demonstrate that miR-206 by directly repressing CORO1C remodels actin filaments and inhibits migration in TNBC cells.

4.

Discussion

Existing data imply that the suggested tumor suppressor miR206 regulates cancer-associated processes such as proliferation, apoptosis, migration, and metastasis in various cancers in a tissue-specific manner (Chen et al., 2010; Song et al., 2009; Tavazoie et al., 2008; Wang et al., 2010; Zhang et al., 2011). In this study, we show that loss of miR-206 is correlated to upregulation of its predicted target CORO1C in human TNBCs (Figure 1) and that CORO1C expression may be associated with lower relapse-free survival among cancer patients (Figure 1B and Suppl. Figure 1A). We show that miR-206 by repressing CORO1C reduces migration in TNBC and propose that this mechanism is involved in breast cancer metastasis and distant recurrence. We confirmed the higher expression of CORO1C in primary TNBC samples and explored miR-206 levels in primary tumors using qRT-PCR. The difference between TNBC and ER-positive samples is statistically significant. However, due to the small cohort of TNBC samples, the power of the statistics is low. We also attempted to confirm the survival effect of CORO1C expression in a separate data set, the METABRIC cohort. However, in this data set only patient overall survival was recorded and data for relapse-free survival were not available. The relapse-free survival phenotype of CORO1C expression, could not be replicated in the METABRIC overall survival data set, although a higher expression of CORO1C in basal-like tumors were significant here as in all data sets. It is also possible that the lack of significance for the METABRIC data set is affected by different methodologies to define high and low levels of CORO1C from microarray or sequencing, as well as varying follow-up time and/or varying patients’ treatment in different data sets. We showed that miR-206 targets the 3’-UTR of CORO1C and reduces its expression (Figure 2). Repression of CORO1C, either by introduction of siRNA or miR-206, remodels the actin filaments and cell shape (Figure 5), and dramatically inhibits tumor cell mobility in TNBC cells (Figure 4A). While miR-206 represses also proliferation in TNBC cells, the targeting of CORO1C only mediates its effect on migration. Our findings are consistent with clinical data associating a loss of miR206 with increased metastatic potential in breast cancers (Tavazoie et al., 2008; Wang et al., 2010), and offer a mechanistic basis for this observation.

A major basis for cell motility is the elongation of actin filaments in the cytoskeleton towards the cell membrane that results in a leading edge protrusion, the lamellipodia (Arjonen et al., 2011). The actin branching and remodeling at the lamellipodia is regulated by Arp2/3 complex and cofilin. Coronins have been shown to co-localize with F-actin (Rosentreter et al., 2007) and recruit Arp2/3 complex to ATPF-actin at the fast growing end (plus end) of actin filaments (Cai et al., 2007), which facilitate nucleation branching. Coronins also inhibit cofilin activity at the plus end to prevent filaments disassembling and recruit cofilin to ADP-F-actin increasing filaments disassembly at the slow growing end (minus end) of actin filaments (Cai et al., 2007). Therefore, it is not surprising if CORO1C is upregulated and contributes to cell migration, invasion and even tumor metastasis in aggressive triple-negative breast cancer. We showed that overexpression of miR-206 in TNBC cells leads to a reduction in the co-localization of F-actin and CORO1C and a corresponding change in the actin structure (Figure 5A). The actin filaments in the cytosol are fewer and more spread out, and the F-actin staining is overall weaker (Figure 5A). As overexpression of CORO1C partially rescued the F-actin turnover mediated by miR-206 (Supp. Figure 3) and induced increased cell mobility (Figure 4B), we suggest that the mechanism whereby miR206 reduces migration is through actin monomer turnover and the extension of actin filaments, functions known to be executed by CORO1C in other cells (Chan et al., 2012). Thus, the loss of miR-206 and corresponding high expression of CORO1C found in TNBC would contribute to their aggressive feature and metastasis. We suggest that the repression of CORO1C by miR-206 is an important mechanism that regulates cell migration. The progression of tumor metastasis is a sequential process, that include the acquisition of several traits by which malignant cells can disseminate from the primary tumor and colonize at a secondary site (Gupta and Massague, 2006). Via remodeling of actin networks, coronin proteins contribute to the regulation of cell adhesion, invasiveness, cell mobility and epithelial-to-mesenchymal transition. Therefore, the coronin family could be involved both in the initiation of this aggressive phenotype, and in the intravasation and extravasation during the development process of tumor metastasis. For the first time, we show that a member of the actin-binding coronin family can be regulated by a miRNA. The potential role of miRNAs in regulating coronins adds a new dimension to how miRNAs regulate migration through actin network remodeling and how this can contribute to tumor metastasis. The conserved miR-206 target site is also found in the 3’-UTR of CORO2A, suggesting that the direct repression of actin dynamics by this miRNA could be widespread. In fact, we found indications that miR-206 represses several other actin regulator genes, such as TMSB4X, TPM4, and TNS3. We showed that these are all predicted targets of miR-206 and their expression was repressed by miR-206 in both TNBC and murine SC-like mammary cells (Figure 5B). TMSB4X contributes to F-actin bundles and cell motility (Maelan et al., 2007), TPM4 also binds to actin filaments (Helfman et al., 1999) and TNS3 has been associated to cell migration and tumorigenesis in lung and breast cancer (Qian et al., 2009). Therefore, it is possible that miR-206 targets a

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cluster of actin-binding proteins (including CORO1C) and this combination results in drastic effects which will further affect cell focal adhesion and migration. This would make miR-206 a powerful regulator of migration. Considering the high metastatic potential of TNBCs and the imminent need for better treatments, targeting the coronin family proteins and thereby controlling the actin dynamics, perhaps using miR-206 therapy, should be explored. Our results indicated that the mechanism underlying miR206’s effect on cell proliferation was independent on CORO1C. Although miR-206 has been reported to activate apoptotic processes in HeLa cells (Song et al., 2009), we found no evidence that apoptosis was induced by miR-206 in TNBC cells. These data suggest that the different tumor suppressor roles of miR-206, inducing G1 cell cycle arrest and repressing migration in TNBC cells, are mediated through different direct targets. In conclusion, we show that miR-206 represses both proliferation and migration in TNBC cells. We demonstrate that CORO1C is post-transcriptionally regulated by miR-206 and that this mechanism reduces migration but not proliferation. The targeting of CORO1C results in a remodeling of the actin filaments and thereby a changed morphology and migratory behavior of the cells. Our work detailing the miR-206 e CORO1C e actin filament e migration axis offers a mechanistic basis for the metastatic potential of TNBC and has the potential to be utilized for novel therapeutic or diagnostic approaches.

Conflicts of interest The authors disclose no potential conflicts of interest.

Acknowledgments We are grateful to Melissa Landis (The Houston Methodist Research Institute), Daniel E. Frigo, Seemah Khurana and Preethi Gunaratne (all University of Houston) for valuable advice on this project. This work was supported by faculty start-up funding (CW) from the University of Houston.

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2014.07.006. R E F E R E N C E S

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