Mir-218 Suppresses Nasopharyngeal Cancer Progression Through Downregulation of Survivin and the SLIT2-ROBO1 Pathway

Published OnlineFirst March 8, 2011; DOI: 10.1158/0008-5472.CAN-10-2754

Cancer Tumor and Stem Cell Biology Research miR-218 Suppresses Nasopharyngeal Cancer Progression through Downregulation of Survivin and the SLIT2-ROBO1 Pathway

Nehad M. Alajez1, Michelle Lenarduzzi1,2, Emma Ito1,2, Angela B.Y. Hui1, Wei Shi1, Jeff Bruce1,2, Shijun Yue1, Shao H. Huang3, Wei Xu4, John Waldron3,5, Brian O'Sullivan3,5, and Fei-Fei Liu1,2,3,5

Abstract Nasopharayngeal carcinoma (NPC) is an Epstein–Barr virus–associated malignancy most common in East Asia and Africa. Here we report frequent downregulation of the microRNA miR-218 in primary NPC tissues and cell lines where it plays a critical role in NPC progression. Suppression of miR-218 was associated with epigenetic silencing of SLIT2 and SLIT3, ligands of ROBO receptors that have been previously implicated in tumor angiogenesis. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. We used an integrated trimodality approach to identify targets of miR-218 in NPC, cervical, and breast cell lines. Direct interaction between miR-218 and the 30-untranslated regions (UTR) of mRNAs encoding ROBO1, survivin (BIRC5), and connexin43 (GJA1) was validated in a luciferase-based transcription reporter assay. Mechanistic investigations revealed a negative feedback loop wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Pleotropic effects of miR-218 on NPC survival and migration were rescued by enforced expression of miR-218–resistant, engineered isoforms of survivin and ROBO1, respectively. In clinical specimens of NPC (n ¼ 71), ROBO1 overexpression was significantly associated with worse overall (P ¼ 0.04, HR ¼ 2.4) and nodal relapse-free survival (P ¼ 0.008, HR ¼ 6.0). Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management. Cancer Res; 71(6); 2381–91. 2011 AACR.

Introduction In silico analyses predict that approximately one third of the human genome is regulated by miRs, suggesting that several Gene expression is a complex cellular process that is tightly hundred or even thousands of transcripts could potentially be regulated at several levels to ensure proper gene dosage, hence regulated by a single miR. Tumor cells exhibit abnormal miR maintenance of health in the organism. This process is dysre- expression profiles compared with their normal counterparts gulated in human malignancies, leading to overexpression of (5), suggesting possible role for miRs in tumor formation and tumor-promoting genes, and downregulation of tumor sup- disease progression. Using currently available analytic pressor genes. Some of the altered regulatory mechanisms in approaches, several miRs have been identified to mediate tumor cells arise from genomic gains or losses, or aberrant tumor progression via their respective gene targets (6, 7). There protein translation (reviewed in refs. 1, 2). MicroRNAs (miR) remains, however, a majority of miRs whose biological sig- are increasingly recognized to be key regulators of gene expres- nificance remains to be elucidated. miRs target identification is sion in several biological systems, including cancer (3, 4). a complex undertaking given the inherent limitations of the currently available target prediction algorithms, which rely primarily on complementarities, thermodynamics, and inverse Authors' Affiliations: 1Ontario Cancer Institute, University Health Net- correlation analyses associated with high false discovery rates 2 3 work; Department of Medical Biophysics, University of Toronto; Depart- (8, 9). Using primary nasopharyngeal carcinoma (NPC) tissues ment of Radiation Oncology, Princess Margaret Hospital, University Health Network; 4Division of Biostatistics, University Health Network, and the C666-1 NPC cell line, we report underexpression of and 5Department of Radiation Oncology, University of Toronto, Toronto, miR-218 in NPC and identified its many gene targets including Ontario, Canada BIRC5, ROBO1, and GJA1, which seem to play important roles Note: Supplementary data for this article are available at Cancer Research in NPC biology. Epigenetic silencing of SLIT2 and SLIT3 pro- Online (http://cancerres.aacrjournals.org/). moters was discovered as an underlying mechanism by which Corresponding Author: Fei-Fei Liu, Department of Radiation Oncology, Princess Margaret Hospital/Ontario Cancer Institute, 610 University Ave- miR-218 is suppressed in NPC. Reconstituting miR-218 expres- nue, Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2123; Fax: sion in C666-1 cells led to significant toxicity in vitro and 416-946-4586; E-mail: [email protected] delayed tumor growth in vivo, underscoring the importance doi: 10.1158/0008-5472.CAN-10-2754 of miR-218 in NPC biology. Overexpression of miR-218 led to 2011 American Association for Cancer Research. significant inhibition of NPC cell migration toward SLIT2

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in vitro – , which was reversed by the expression of a miR-218 Table 1. Patient and tumor characteristics resistant ROBO1 open reading frame (ORF). Finally, ROBO1 overexpression was significantly associated with worse overall A. N ¼ 16 % and nodal relapse-free survival in NPC. Age, y Materials and Methods Median age 46 y Range 29–70 y Ethics statement Gender All tissue studies have received institutional Research Male 11 69% Ethics Board approval. All animal experiments were con- Female 5 31% ducted in accordance with the guidelines of the Animal Care Follow-up, y Committee, University Health Network. Median follow-up time 10.54 Range 1.2–12.84 y Quantification of miR-218 expression in NPC tissue Stage The clinical characteristics of the patients involved in this I 2 13% study are provided in Table 1. TaqMan Low Density Array II 6 37% (TLDA) miR expression profiling and single-well qRT-PCR III 4 25% were conducted in primary NPC tissues as previously IV 4 25% described (10). Treatment Concurrent chemoradiation 4 25% Cell culture and transfection experiments Neoadjuvant chemo þ radiation 1 6% The C666-1, CNE-1, HONE-1, C15, C17, HEK293, UT-SCC-8, Radiation alone 11 69% UT-SCC-42a, and NOE (normal oral epithelial) cells were maintained as previously described (11–13). The C666-1 cell B. N ¼ 71 % line was authenticated at the Centre for Applied Genomics (Hospital for Sick Children, Toronto, Ontario, Canada), using Age, y the AmpF/STR Identifiler PCR Amplification Kit (Applied Median age 45.2 Biosystems Inc.). miR precursors (Applied Biosystems Inc.) Range 16–79 transfection was done as previously described (11). Gender Male 56 79% Measurement of cell viability and apoptosis Female 15 21% Cell viability and cell-cycle analyses were conducted as Follow-up, y previously described (11) Median follow-up time 7.2 Range 0.7–14.2 Cell migration Stage Cell migration was assessed using the 6-well BD Transwell I46% migration system (BD Biosciences). On day 1, a total of 5 II 25 35% 5 10 HEK293 cells were seeded in the lower chamber; 18 hours III 25 35% later, cells were transfected with 4 mg of control or hSLIT2 IV 17 24% plasmid (14), using Lipofectamine 2000 (Invitrogen Inc.). Treatment Five hours later, the medium was changed to RPMI plus Concurrent chemoradiation 10 14% 1% FBS. On day 2, C666-1 cells were resuspended in RPMI Neoadjuvant chemo þ radiation 4 6% 5 plus 1% FBS and 3 10 cells were added to the upper Radiation alone 57 80% chamber (2 mL). In some experiments as indicated, C666-1 cells were cotransfected with pre-miR (40 nmol/L) and plasmid (500 ng/24 wells), and 48 hours later, cells were counted and used in the migration experiments. Forty-eight dehydrogenase; antibody (1:12,500; Abcam) as described hours later, inserts were fixed and stained using SIEMENNS before (11). DIFF-QUICK stain set (Siemens Healthcare Diagnostics) and the number of migrating cells in ten 20 fields was Microarray and target prediction analysis quantified. C666-1 cells were transfected with 40 nmol/L pre-miR-Ctrl or Pre-miR-218, and at 16 and 48 hours posttransfection, Immunoblotting total RNA was extracted, hybridized onto the Affymetrix C666-1cellsweretransfectedwiththeindicatedpre-miR Human Genome U133 Plus 2.0 array, and analyzed as and immunoblotting was conducted using anti-Survivin described before (15, 16). Predicted miR-218 gene target list polyclonal antibody (1:1,000 dilution; Novus Biologicals), was generated by compiling a nonredundant list of predicted anti-ROBO1 polyclonal antibody (ab7279; 1:200 dilution; targets using 6 commonly utilized miR target prediction Abcam, Inc.), and anti-GAPDH (glyceraldehyde-3-phosphate algorithms (TargetScan, miRanda, PicTar, GenmiRþþ,

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Figure 1. miR-218 is downregulated in NPC and overexpression is cytotoxic to C666-1 cells. A and B, qRT-PCR analysis for miR-218 expression was conducted on 16 primary NPC biopsy samples showing downregulation of miR-218 compared with normal NP epithelial tissues (A), as did the C666-1, C15, C17, CNE-1, and HONE-1 NPC models compared with NOE cell line (B). C, C666-1 cells were transfected with pre- miR-Ctrl or pre-miR-218 4GyIR delivered on day 3 posttransfection. Cell viability was measured using the MTS assay on day 6. D, NOE, UT-SCC-42a, or UT-SCC-8 cells were transfected as in C and then the MTS assay was conducted on day 6. n.s, not significant; **, P < 0.005; ***, P < 0.0005.

miRBase, and miRDB). Gene overlap was assessed using the Expression of BIRC5 and ROBO1 was then validated using gene list Venn diagram tool (17). Gene Ontology (GO) and qRT-PCR. pathway analyses on miR-218 targets were conducted using DAVID Bioinformatics Database functional-annotation tools 5-Aza-20-deoxycytidine treatment and quantitative CpG (18, 19). promoter methylation analysis C666-1 cells were seeded overnight in a 12-well plate before Luciferase assay adding 5-aza-20-deoxycytidine (5-aza; Sigma Aldrich) at 1 A fragment (150–200 bp) from the 30-untranslated region mmol/L; fresh medium with 5-aza was added on day 3. Cells (UTR) of BIRC5, ROBO1, and GJA1 containing the predicted were harvested and RNA was extracted for qRT-PCR analysis on binding sites for miR-218 (wild type or mutant) was indivi- day 4. The CpG islands in the SLIT2 and SLIT3 promoter regions dually amplified using the indicated primers in Supplementary were identified using Methprimer(20, 21) and were analyzed for Table S1 and then cloned at the SpeI and HindIII sites down- CpG methylation by using the EpiTYPER (SEQUENOM, Inc.; stream of the firefly luciferase gene in the pMIR-REPORT Supplementary Fig. S1) at the Analytical Genetics Technology vector (Applied Biosystems Inc.). Luciferase assay was subse- Centre (AGTC), University Health Network, Toronto. quently conducted as previously described (11). In vivo tumor growth experiments qRT-PCR and cloning Tumor formation experiments were conducted as The expression levels of BIRC5, ROBO1, GJA1, LEF1, RGS1, described before (11). CETN2,EGFR,HOXA1,andLAMB3inpre-miR-218–transfected cells were measured using the primers listed in Statistical analysis Supplementary Table S1 by qRT-PCR. Cloning of BIRC5 Overall survival (OS) and nodal relapse-free survival were ORF and ROBO1 (isoform b) ORF was conducted using calculated using the Kaplan–Meier method. Differences the corresponding primers in Supplementary Table S1, the between survival curves were analyzed by log-rank test. Cox High Fidelity SuperScript III One-Step RT-PCR System, and proportional hazard regression model was applied to estimate pcDNA3.1 Directional TOPO Expression Kit (Invitrogen). the HR and 95% CI, using 2-sided tests. Pearson correlation was

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Figure 2. miR-218 induces apoptosis in vitro and delays tumor growth in vivo. C666-1 cells were transfected with 40 nmol/L pre-miR-Ctrl or pre-miR-218; cell- cycle analysis was done on day 3. A, representative plot with the

percentage of cells in sub-G0,G1, and S/G2M phases of the cell cycle is indicated. B, the average from 3 independent experiments is presented. C, fold change in caspase 3/7 activity in pre-miR- 218–transfected C666-1 cells compared with cells transfected with pre-miR-Ctrl. D, C666-1 cells were transfected with pre-miR- Ctrl or pre-miR-218; 3 days later, cells were exposed to 4 Gy IR and then on day 6, cells were implanted intramuscularly (IM) into the left gastrocnemius of SCID mice. Tumor plus leg diameter was measured twice a week (y-axis). Data are presented as mean SE, n ¼ 5. *, P < 0.05; **, P < 0.005.

utilized to assess the correlation between miR-218 and ROBO1 miR (Fig. 1C, left), which was further reduced when cells expression. Statistical analyses were done using version 9.2 of were additionally exposed to ionizing radiation (IR), deliv- the SAS system and User's Guide (SAS Institute), presented ered 3 days posttransfection (Fig. 1C, right). Similar reduc- using the GraphPad Prism software (GraphPad Software). tion in cell viability was observed for the human laryngeal UT-SCC-42a and UT-SCC-8 cells following transfection with Results pre-miR-218, whereas no significant cytotoxicity was observed for the NOE cells (Fig. 1D). To characterize the MiR-218 was downregulated in primary NPC tissues and mode of cytotoxicity inflicted by restoration of miR-218 cell lines, and its overexpression was lethal to tumor expression in C666-1 cells, cell-cycle analyses done at 3 days cells in vitro and in vivo posttransfection have shown significant induction of apop- miR expression profiling of 16 primary NPC samples tosis (sub-G0), reduction in G1, and an increase in S/G2M revealed almost a 10-fold reduction in miR-218 expression phases (Fig. 2A and B). To further confirm induction of observed in all NPC samples compared with normal NP apoptosis in the miR-218–transfected cells, caspase 3/7 tissue (Table 1A and Fig. 1A). Significant downregulation of activation was examined as a function of time in miR- miR-218 was also observed in the C666-1 (EBV positive), C15 218–transfected C666-1 cells, showing a significant increase (EBV positive), C17 (EBV positive), CNE-1 (EBV negative), in caspase 3/7 activity in a time-dependent manner (Fig. 2C). and HONE-1 (EBV negative) NPC models compared with To further understand the biological significance of loss of NOE cells (Fig. 1B). To assess the biological significance of miR-218 on NPC tumorigenesis in vivo, C666-1 cells were miR-218 downregulation, C666-1 cells were transfected with transfected with pre-miR-Ctrl or pre-miR-218 alone or com- pre-miR-Ctrl or pre-miR-218 and then assessed for cell binedwith4GyIRdeliveredonday3posttransfection, viability. Transfection of C666-1 cells with 40 nmol/L pre- implanted into SCID mice 3 days later, and then monitored miR-218 led to significant increase in miR-218 expression for tumor formation. MiR-218 expression in transfected cells (Supplementary Fig. S2). Interestingly, reconstituting miR- was assessed by qRT-PCR prior to implantation (Supple- 218 expression in C666-1 cells led to significant reduction in mentary Fig. S3). Significant reduction in tumor growth was cell viability compared with transfection with control pre- observed in the pre-miR-218 or pre-miR-218 plus IR groups,

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Figure 3. Identification of miR-218 gene targets in NPC. A, Venn diagram showing the intersection between: (i) mRNA transcripts displaying greater than 2-fold increased expression in primary NPC samples [NPC (Up)]; (ii) mRNA transcripts showing greater than 30% reduction in expression level at 16 and 48 hours posttransfection with pre-miR-218 (Exp Det); and (iii) the list of predicted miR-218 targets based on in silico algorithms (in silico). g,the intersection from all 3 "circles"; d, the intersection between the experimentally determined and in silico. B, functional-annotation analyses of the 142 miR-218 targets identified from A and presented as pie-chart. C, qRT- PCR validation of 9 miR-218 gene targets derived from either the g or d lists. D, downregulation of BIRC5, ROBO1, and GJA1 expression at 72 hours posttransfection of SiHa, T47D, and HeLa cells with pre-miR-218. E, Western blotting of BIRC5 and ROBO1 expression in C666-1 cells at 72 hours posttransfection. F, top, schema of the p-MIR- REPORT vector carrying the indicated 30-UTR regions spanning the predicted miR-218 binding sites. Bottom, left, percent relative luciferase activity at 48 hours posttransfection of C666-1 cells with the indicated reporter vector. G, C666-1 cells were cotransfected with pre-miR-218 plus pCDNA3.1 or pBIRC5 and then cell viability was measured on day 6. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

thereby corroborating the in vitro data of miR-218 cytotoxi- cells, we sought to determine the gene targets which might city in a 3-dimensional xenograft model (Fig. 2D). account for these observations. A novel 3-pronged approach was thus developed to address this challenge. First, a non- MiR-218 is a negative regulator of several genes redundant list of predicted miR-218 gene targets was com- involved in NPC pathogenesis piled from 6 miR target prediction databases (TargetScan, Given the profound downregulation of miR-218 in NPC, and miRanda, PicTar, GenmiRþþ, miRBase, and miRDB), result- the dramatic effects of miR-218 overexpression on C666-1 ing in 1,577 potential mRNA targets (Fig. 3A). Second, C666-1

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cells were transfected with pre-miR-218 or pre-miR-Ctrl, sing miR-218–resistant BIRC5 (which lack the 30-UTR) could followed by microarray analysis. These experimental data rescue NPC cells from cell death upon miR-218 transfection. identified 360 transcripts which were reduced by greater than Data presented in Figure 3G clearly show significant rescue of 30% at both 16 and 48 hours posttransfection with pre-miR- C666-1 cell upon cotransfection with pre-miR-218 and pBIRC5 218. Notably, 40% (142 transcripts) of the experimentally compared with cell transfected with control vector plus pre- determined miR-218 targets were predicted by at least 1 miR-218 whereas transfection of C666-1 with pBIRC5 along algorithm (Supplementary Fig. S4). Functional annotation with pre-miR-Ctrl had no significant effect on cell viability and clustering analyses conducted on these 142 transcripts (data not shown). revealed significant enrichment for genes involved in several important cellular processes such as development, cell divi- Downregulation of miR-218 through epigenetic sion, or gap junctions, and many others described in human silencing of SLIT2 and SLIT3 promoters in NPC cancers (Fig. 3B and Supplementary Table S2). Third, to The mechanism of miR-218 downregulation in NPC determine which miR-218 targets might be relevant to NPC, remained unclear, although several different mechanisms a final list of mRNA transcripts showing greater than 2-fold have been described to regulate miR expression in various overexpression in primary NPC was compiled from 2 publicly biological systems (24). Hence, the Sanger miR database was available data sets (GSE13597 and GSE12452; ref. 22). The interrogated for the chromosomal location of miR-218, which intersection between all 3 gene lists identified 6 overlapping revealed 2 miR-218 primary transcripts (hsa-miR-218-1 and transcripts (ARNTL2, BIRC5, C12orf48, GJA1, ROBO1, and hsa-miR-218-2), which were embedded in the intronic regions STMN1; Fig. 3A). qRT-PCR analyses on selected group of 9 of SLIT2 (4p15.31) and SLIT3 (5q35.1), respectively (Fig. 4A). miR-218 targets validated their downregulation 48 hours Our previous microarray expression profiling indeed docu- posttransfection with pre-miR-218 (Fig. 3C). BIRC5, ROBO1, mented significant downregulation of SLIT3 expression in and GJA1 were the most prominent targets given their bio- primary NPC samples (25). Hence, SLIT2 and SLIT3 expression logical or prognostic significance in NPC (23). To determine was examined in C666-1 cells, showing significant underex- whether miR-218 regulate these targets in other human cancer pression of both transcripts, concordant with miR-218 down- models; SiHa (cervical), HeLa (cervical), and T47D (breast) cell regulation (Fig. 4B). Downregulation of SLIT2 and SLIT3 lines were transfected with pre-miR-218 and 72 hours later, through promoter hypermethylation has been reported in the expression of these 3 targets was measured using qRT-PCR other human cancers (26, 27) but has not been described in compared with cells transfected with pre-miR-Ctrl. The data NPC. Interestingly, 5-aza treatment, a potent inhibitor of DNA presented in Fig. 3D clearly show significant reduction in all 3 methylation, induced reexpression of SLIT2 and SLIT3 tran- targets post-miR-218 transfection; hence, extending the reg- scripts accompanied by significant induction of miR-218 ulation of these targets by miR-218 to additional human reexpression (Fig. 4C, right). Subsequent quantitative analysis cancer models. Nonetheless, overexpression of miR-218 led of SLIT2 and SLIT3 promoter methylation revealed approxi- to significant reduction in BIRC5 and ROBO1 expression at the mately 70% and 90% methylation in C666-1 cells compared protein level as well (Fig. 3E). with approximately 15% and 45% in NOE cells, respectively (Fig. 4D). Significant SLIT2 promoter hypermethylation was MiR-218 directly targets ROBO1, BIRC5, and GJA1 in also observed in the C15 and C17 NPC models (Fig. 4D), NPC thereby confirming that epigenetic silencing of SLIT2 and Direct interaction between miR-218 and the 30-UTR of SLIT3 promoters accounted for their suppressed expression ROBO1, BIRC5, and GJA1 was examined using the luciferase in NPC. assay. Indeed, cotransfection experiments have shown direct regulation of these three 30-UTR regions by miR-218 (Fig. 3F, ROBO1 expression was associated with lower overall bottom), which was significantly abrogated when mutating and nodal relapse-free survival and mediated NPC cell the predicted miR-218 seed regions in the pMIR-REPORT UTR migration in response to SLIT2 in vitro constructs, thereby validating the direct interaction between Given the recent description of ROBO1 as a mediator of miR-218 and the corresponding 30-UTRs. Concordantly, over- cell migration and invasion in the neural system (28, 29), and expressing BIRC5 and GJA1 30-UTR (which includes miR-218- the fact that intracranial invasion is frequent clinical chal- binding region) led to significant reduction in miR-218 expres- lenge in NPC, an independent cohort of 71 primary NPC sion in NOE cells (Supplementary Fig. S5). BIRC5 was the most samples were analyzed using qRT-PCR for miR-218 and notable miR-218 target given its documented prosurvival role ROBO1 expression, showing significant inverse relationship and the significant induction of cell death post–miR-218 between miR-218 and ROBO1 expression (Fig. 5A), corro- transfection; hence, we sought to determine whether expres- borating ROBO1 being relevant biological target for miR-218

Figure 4. Underexpression of miR-218 in NPC is due to SLIT2 and SLIT3 promoter hypermethylation. A, schema showing the genomic locations of miR-218-1 and miR-218-2 in the introns of SLIT2 and SLIT3 genes, respectively. B, qRT-PCR analysis of basal levels of SLIT2, SLIT3, and miR-218 expression in C666-1 compared with NOE cells. C, representative qRT-PCR analysis of fold change in SLIT2 and SLIT3 (left) or miR-218 (right) expression in C666-1 cells on day 4 posttreatment with 5-aza. D, quantitative methylation analysis showing significantly greater extent of promoter methylation for both SLIT2 (C666-1, C15, and C17) and SLIT3 (C666-1) cells compared with NOE cells.

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Figure 5. Expression of miR-218 and ROBO1 in primary NPC specimens and their roles in regulating cell migration. A, inverse relationship between miR- 218 and ROBO1 expression in second cohort of NPC patients samples [71 FFPE NPC (cohort B, Table 1) and 6 normals]. ROBO1 expression was measured using qRT-PCR and the corresponding primers in Supplementary Table S1, whereas miR-218 expression in the same specimens was measured using single-well TaqMan PCR Assay. Data are

presented as log2 fold change in miR-218 (x-axis) and ROBO1 (y- axis) expression. B and C, actuarial plot of OS (B) or nodal relapse-free survival (C) according to high versus low ROBO1 expression defined by the median expression value. D, left, the Transwell migration assay showing the ability of C666-1 cells to migrate toward SLIT2, which was significantly reduced posttransfection with pre-miR- 218 (middle) and was significantly reversed by cotransfection with miR-218–resistant BIRC5 expression plasmid (right). *, P < 0.05.

in NPC. The potential prognostic role for ROBO1 was Discussion assessed in this same cohort, showing that patients with lower than median ROBO1 expression experienced signifi- Deregulated gene expression is one of the major hallmarks cantly higher 7-year- OS rate of 81% versus 63% for high- of cancer cells, associated with a myriad of mechanisms expressing ROBO1 patients (Fig. 5A; P ¼ 0.04, HR ¼ 2.4). leading to overexpression of tumor-promoting genes, or Surprisingly, nodal relapse-free survival was also signifi- downregulation of tumor-suppressing genes, collectively driv- cantly more favorable for low ROBO1-expressing NPC ing tumor progression. We had previously documented that patients of 95%, versus 67% for high ROBO1 expressors overexpression of the antiapoptotic protein BIRC5 was asso- (Fig. 5B; P ¼ 0.008, HR ¼ 6.0). To acquire greater insight ciated with poor clinical outcome in NPC (23), which has been into the mechanism by which ROBO1 overexpression similarly reported by other groups for different cancers (30). mediated NPC relapse, we evaluated the response of the The mechanisms leading to overexpression of BIRC5 in NPC, C666-1 cells (which express high levels of ROBO1; data not however, remained unknown. Herein, we report for the first shown) to SLIT2 (the ligand for ROBO1) in vitro,usinga time that downregulation of miR-218 is a consequence of Transwell migration assay. The data presented in Figure 5D epigenetic silencing of SLIT2 and SLIT3 promoters, which, in (left) clearly show significant increase in migration of C666-1 turn, resulted in BIRC5 overexpression in NPC. Targeted cells toward SLIT2, thereby supporting the biological rele- depletion of BIRC5 significantly reduced viability and vance of the ROBO1-SLIT2 pathway in mediating NPC cell increased apoptosis (25) and delayed tumor growth in vivo migration. Conversely, overexpression of miR-218 led to (Supplementary Fig. S6), similar to the phenotypic changes significant inhibition of C666-1 migration toward SLIT2 observed in current study (Figs. 1C and 2A, B, and D). In fact, (Fig. 5D, middle), which in turn was reversed by cotransfec- cotransfection with a miR-218-resistant BIRC5 expression tion with a miR-218-resistant ROBO1 expression plasmid plasmid led to significant rescue of the C666-1 cells post- (Fig. 5D, right). transfection with pre-miR-218 (Fig. 3G). Taken together, our

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Figure 6. Proposed model for the role of mir-218 in regulating NPC cell survival and migration. Schema showing downregulation of miR-218 in NPC resulting from SLIT2 and SLIT3 promoter hypermethylation, conferring growth advantage and biological aggressiveness of NPC cells through several mechanisms. First, loss of miR-218 leads to overexpression of the antiapoptotic protein BIRC5. Second, the chemotactic interaction between ROBO1 on NPC cells, with SLIT2 secreted by normal neuronal cells, could promote neural invasion by the NPC cells.

data validated BIRC5 as a bona fide target for miR-218 in NPC databases, underscoring the value of combining multiple data- and showed that the cytotoxicity caused by reexpressed miR- bases to discover novel mRNA targets. 218 was mediated by the downregulation of BIRC5. Search of the Sanger database identified the chromosomal These molecular links were discovered through an integration locations of miR-218 transcripts within the intronic regions of of in silico and experimental approaches that was highly efficient SLIT2 and SLIT3, reported to be underexpressed in several in identifying several biologically and clinically relevant targets human malignancies and purported to function as tumor for miR-218. Although miRs are generally believed to regulate suppressors (26, 27, 33). In this study, we observed downregula- gene expression via translational repression, there is mounting tion of the SLIT2 and SLIT3 transcripts in C666-1 cells, con- evidence that miR–mRNA interactionsoftentimes lead to mRNA sistent with our own data documenting downregulation of destabilization and degradation, providing the scientific basis SLIT3 in primary NPC (25). Using 5-aza treatment and quanti- for our combinatorial strategy (31, 32). Using this method, tative promoter methylation analyses, SLIT2 and SLIT3 pro- approximately 40% of the experimentally determined miR-218 moter hypermethylation was documented to be the mechanism targets were indeed predicted by at least 1 miR target prediction leading to their underexpression in NPC (Fig. 4C). Thus, our algorithm (Fig. 3A and Supplementary Fig. S4). Thus, the advan- data provided novel insight into the importance of SLIT2 and tage of this approach is the ability to exclude the vast majority SLIT3 in NPC, wherein their loss via promoter methylation (91%) of false-positive target predictions, defined as mRNA drove tumorigenesis through downregulation of miR-218, targets that could not be experimentally validated. Interestingly, whose target genes increased tumor proliferation, resisted several of the identified targets were predicted by only 1 or 2 apoptosis, and enhanced invasion. While treating NPC cells

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with 5-aza led to substantial reexpression of SLIT2 and SLIT3, target characterized in this study, we then sought to examine miR-218 was only slightly increased in (Fig. 4C). We have 2 the relationship between SLIT2 and SLIT3 (which encodes for plausible explanations for this observation. First, it is possible miR-218) and BIRC5 in several publicly available gene expres- that the cells were unable to fully process the SLIT2 and SLIT3 sion data sets from colorectal (GSE20916), glioma (GDS3627), transcripts to mature miR-218 due to significant toxicity of 5- bladder (GDS1479), and hepatocellular carcinoma (GSE9843). aza treatment. Second, it is possible that miR-218 is subjected to Interestingly, our data revealed strong inverse relationship another level of posttranscriptional miR-processing regulation, between the expression of SLIT2 and SLIT3 versus BIRC5 in similar to regulation of the let-7/miR-98 family by Lin28 (34). these data sets; further strengthening the role of miR-218 in ROBO1 is another mir-218 target that was characterized in regulating BIRC5 expression in several human cancers (Sup- this study. It is a member of the Roundabout family of plementary Fig. S9). receptors involved in several different cellular processes In summary, we report for the first time downregulation of (35). The interaction between ROBO1 and its SLIT receptors miR-218 in NPC as a consequence of epigenetic silencing of plays critical role in mediating axon guidance during neural SLIT2 and SLIT3 genes. The approach we utilized in this study development (28), recruitment of endothelial cells during was highly efficient in characterizing several biologically and angiogenesis (29), and mediating breast cancer cell migration clinically relevant gene targets for miR-218 in NPC. In parti- (36). The observation that ROBO1 is direct target for mir-218, cular, BIRC5 and ROBO1 were among the most prominent which is embedded within the SLIT2 and SLIT3 transcripts, miR-218 targets which are upregulated in NPC, leading to NPC suggests novel role for mir-218 in regulating ROBO-SLIT cell survival and migration. Although several reports docu- signaling in NPC through a negative feedback loop (Fig. 6), mented upregulation of BIRC5 expression in different human which would be consistent with a recent report documenting malignancies, our data are the first to document regulation of similar function for miR-218 in regulating ROBO1 in gastric BIRC5 by a miR (miR-218), hence providing novel regulatory cancer (37). The in vitro demonstration of C666-1 cells migrat- mechanism leading to BIRC5 overexpression. Restoration of ing toward SLIT2 in a ROBO1-SLIT2–dependent manner miR-218 expression (possibly through DNMT inhibitors) (Fig. 5D, left) supports the notion this pathway is biologically might provide potentially useful strategy for the future man- relevant in NPC. Indeed, overexpression of miR-218 led to agement of this disease. significant inhibition of C666-1 migration. A previous study had reported overexpression of ROBO1 in an EBV-positive Disclosure of Potential Conflicts of Interest NPC cell line posttreatment with TPA/SB carcinogen (38); therefore, our data provide additional miR-dependent The authors declare that there are no conflicts of interest. mechanism leading to ROBO1 overexpression in NPC. Very Acknowledgments importantly, ROBO1 overexpression was associated with sig- P ¼ ¼ nificantly lower probability of overall (Fig. 5B; 0.04, HR We thank Dr. Jonathan Raper (University of Pennsylvania) for kindly 2.4) and nodal relapse-free survival (Fig. 5C; P ¼ 0.008, HR ¼ providing the SLIT2 expression vector and Drs. Xiangdong Liu and Cheryl 6.0) in NPC. There was also a trend suggesting that low miR- Crozier (ACTC, University Health Network, Toronto) for their help with the methylation analysis. 218 expression was associated with a slightly higher probability of relapse in NPC (Supplementary Fig. S7). Neural Grant Support tissues are known to express high levels of SLIT2 (39); hence, we sought to examine the proportion of patients with brain This work has been supported by funds from the Canadian Institutes of involvement in a group of 11 patients with local plus nodal Health Research, and the Dr. M. Elia Chair in Head and Neck Cancer Research. relapse (Supplementary Fig. S8A). Interestingly, the 4 patients We acknowledge the philanthropic support from the Wharton Family, Joe's Team, and Mr. Gordon Tozer. N.M. Alajez is a recipient of a scholarship from the who showed skull base and/or brain invasion at the time of Terry Fox Foundation Strategic Training Initiative for Excellence in Radiation relapse had tumors, which tended to express higher levels of Research for the 21st Century (EIRR21) at the Canadian Institutes of Health Research (CIHR). Support is also provided from the Campbell Family Institute ROBO1 (Supplementary Fig. S8B). for Cancer Research and the Ministry of Health and Long-Term Planning. While these novel miR-218 targets were identified using an The costs of publication of this article were defrayed in part by the payment NPC model, BIRC5, ROBO1, and GJA1 were also validated as of page charges. This article must therefore be hereby marked advertisement in miR-218 targets in cervical (SiHa and HeLa) and breast (T47D) accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells, thus potentially extending our observations to other Received July 27, 2010; revised November 17, 2010; accepted January 11, 2011; human cancers. Since BIRC5 was the most notable miR-218 published OnlineFirst March 8, 2011.

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www.aacrjournals.org Cancer Res; 71(6) March 15, 2011 2391

miR-218 Suppresses Nasopharyngeal Cancer Progression through Downregulation of Survivin and the SLIT2-ROBO1 Pathway

Nehad M. Alajez, Michelle Lenarduzzi, Emma Ito, et al.

Cancer Res 2011;71:2381-2391. Published OnlineFirst March 8, 2011.

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