new signiﬁcanceofdysregulatedmetadherinandmicrorna-375 in … · 2011. 12. 15. · micrornas...

Human Cancer Biology

Significance of Dysregulated Metadherin andMicroRNA-375in Head and Neck Cancer

Angela B.Y. Hui6, Jeff P. Bruce6,7, NehadM. Alajez9, Wei Shi6, Shijun Yue6, Bayardo Perez-Ordonez1, Wei Xu4,Brian O'Sullivan2,8, John Waldron2,8, Bernard Cummings2,8, Patrick Gullane3, Lillian Siu5, and Fei-Fei Liu2,6,7,8

AbstractPurpose: Despite recent improvements in local control of head and neck cancers (HNC), distant

metastasis remains amajor cause of death. Hence, further understanding of HNCbiology, and in particular,

the genes/pathways driving metastasis is essential to improve outcome.

Experimental Design: Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the

expression of miR-375 and metadherin (MTDH) in HNC patient samples. Targets of miR-375 were

confirmed using qRT-PCR, Western blot analysis, and luciferase assays. Phenotypic effects of miR-375

reexpression and MTDH knockdown were assessed using viability (MTS), clonogenic survival, cell

migration/invasion, as well as in vivo tumor formation assays. The prognostic significance of miR-375 or

MTDH in nasopharyngeal carcinoma (NPC) was determined by comparing low versus high expression

groups.

Results: MiR-375 expression was significantly reduced (P ¼ 0.01), and conversely, MTDH was signif-icantly increased (P¼ 0.0001) inNPC samples. qRT-PCR,Western blots, and luciferase assays corroboratedMTDH as a target of miR-375. Reexpression of miR-375 and siRNA knockdown of MTDH both decreased

cell viability and clonogenic survival, cell migration/invasion, as well as in vivo tumor formation. NPC

patients whose tumors expressed high levels of MTDH experienced significantly lower survival and, in

particular, higher distant relapse rates (5-year distant relapse rates: 26% vs. 5%; P ¼ 0.005).Conclusions: Dysregulation of miR-375 and MTDH may represent an important oncogenic pathway

driving humanHNCprogression, particularly distantmetastases, which is now emerging as amajor cause of

death for HNC patients. Hence, targeting this pathway could potentially be a novel therapeutic strategy by

which HNC patient outcome could be improved. Clin Cancer Res; 17(24); 7539–50. �2011 AACR.

Introduction

Head and neck squamous cell carcinomas (HNSCC)constitute the fifth most common malignancy worldwide(1). Patients with locally advanced HNSCC have 5-yearoverall survival (OS) rates hovering around 30% to 45%

(2), underscoring a considerable opportunity for improvingoutcome. Nasopharyngeal carcinoma (NPC) is anothermalignancy of the head and neck region; however, NPCsare distinct fromother head and neck cancers (HNC) due totheir unique etiologic, clinical/biological, and epidemio-logic characteristics. Locally advanced NPC patients have a5-year OS rate of approximately 70% (3), also showing aneed for improvement. Hence, it is imperative to acquire adeeper understanding of HNC biology to guide the devel-opment and evaluation of novel therapies, to improvepatient outcome.

MicroRNAs (miRNA) are a novel class of gene regulatorswhich are involved in many biological systems and recog-nized to play important roles in human cancers (4). Wehave recently completed a miRNA profiling study of locallyadvanced HNSCC and reported downregulation of miR-375 as one of the most frequently detected aberrations (5).Preliminary functional analysis showed a potential tumorsuppressive role for miR-375 in HNSCC (5). In this study,we further investigated the role ofmiR-375 downregulationin HNC and determined that metadherin (MTDH) is atarget of miR-375 in HNC. MTDH, also known as astrocyteelevated gene 1 (AEG-1) was only recently cloned (6);

Authors' Affiliations: Departments of 1Pathology, 2Radiation Oncology,3Surgical Oncology, 4Divisions of Biostatistics and 5Medical Oncology,Princess Margaret Hospital; 6Ontario Cancer Institute, University HealthNetwork; 7Departments of Medical Biophysics and 8Radiation Oncology,University of Toronto, Toronto, Ontario, Canada; and 9Department ofAnatomy, Stem Cell Unit; College of Medicine, King Saud University,Riyadh, Saudi Arabia

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A.B.Y. Hui and J.P. Bruce are equal contributors to this study.

Corresponding Author: Fei-Fei Liu, Department of Radiation Oncology,Princess Margaret Hospital/Ontario Cancer Institute, 610 University Ave-nue, Toronto, Ontario, Canada, M5G 2M9. Phone: 416-946-2123; Fax:1-416-946-4586; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2102

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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on April 6, 2021. © 2011 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 26, 2011; DOI: 10.1158/1078-0432.CCR-11-2102

http://clincancerres.aacrjournals.org/

however, it is rapidly emerging as an important oncogene inmany human cancers (7). Furthermore, MTDH overexpres-sion has been observed to significantly correlate with poorprognosis and distantmetastases in breast (8), lung (9), andgastric (10) cancers, warranting its further examination inHNC.

Materials and Methods

Patient information and tissuesWith approval from the Institutional Research Ethics

Board, 20 diagnostic formalin-fixed paraffin-embedded(FFPE) blocks were collected from HNSCC patients withlocally advanced (Stage III or IV) disease, who wereprevious participants in a phase III randomized study ofhyperfractionated radiotherapy conducted between 1988and 1995 (11). Normal epithelial tissues derived from 3FFPE blocks of individuals who underwent a tonsillecto-my (University Health Network), plus 3 additional FFPEblocks derived from normal laryngeal squamous epithe-lial tissues post-laryngectomy (commercially purchasedfrom Asterand) served as controls. In addition, 94 pri-mary FFPE biopsy samples from NPC patients diagnosedfrom 1993 to 2000 were evaluated. Eight FFPE blocks ofnormal nasopharyngeal epithelial tissues derived frompatients who underwent a diagnostic quadroscopy servedas normal controls. Clinical characteristics of these cancerpatients are provided in Table 1.

RNA purification from FFPE samplesTo ensure that all tissues analyzed contained more than

70% tumor cells, a representative section from each patientwas stained with hematoxylin and eosin stain, thenreviewed by a HNC pathologist (B P-O) to ascertain regionswith malignant epithelial cells for macrodissection. Allblocks were processed randomly, with clinical outcomeunknown, to avoid experimental bias. Total RNA enrichedfor small RNA species was isolated using the RecoverAllTotal Nucleic Acid Isolation Kit for FFPE (Ambion, Inc.),according to the manufacturer’s instructions.

Cell lines and reagentsThe human hypopharyngeal HNSCC cell line, FaDu, was

obtained from American Type Culture Collection and cul-tured according to specifications. The Epstein–Barr virus–positive NPC cell line C666-1 (12) was maintained inRPMI-1640, supplemented with 10% FBS (Wisent, Inc.)and 100mg/L penicillin/streptomycin. The NOE cells (Cel-progen, Inc.) served as normal controls. All cells wereauthenticated at theCentre for AppliedGenomics (Hospitalfor Sick Children, Toronto, Canada) using the AmpF/STRIdentifiler PCR Amplification Kit (Applied Biosystems) andmaintained at 37�C with 5% CO2.

5-aza-20-deoxycytidine treatment and quantitative CpGmethylation analysis

C666-1 and FaDu cells were seeded overnight in 12-wellplates and media containing 3 mmol/L 5-aza-20-deoxycyti-

dine (Sigma-Aldrich) was added to cells, 2 and 4 days afterseeding, respectively. On day 5, cells were harvested andRNAwas extracted for quantitative reverse transcriptasePCR(qRT-PCR) analysis of miR-375 expression levels. The CpGislands in the genomic region of miR-375 were identifiedusing the EMBL-EBI CpG Island Finder and Plotting Tool(Supplementary Fig. S1A), and 3 amplicons spanning theseregions (Supplementary Fig. S1B) were analyzed for CpGmethylation using the EpiTYPER (Sequenom, Inc.) at theAnalytical Genetics Technology Centre (AGTC), UniversityHealth Network, Toronto.

Quantification of miRNA and mRNAmiRNA expression was assessed by qRT-PCR analysis

using TaqMan microRNA Assays (Applied Biosystems) aspreviously described (13). qRT-PCR was also utilized toanalyze expression changes of 4 previously published miR-375 targets: MTPN, USP1, PDK1, ADIPOR2, plus 5 newlyidentified miR-375 targets: MTDH, GMFB, RANBP3,SPAG9, and ZNF462. Total RNA was isolated from cellsusing the Total RNA Purification Kit (Norgen, Inc.). Onemicrogram of total RNA was reverse transcribed usingSuperScript II Reverse Transcriptase (Invitrogen) as speci-fied by the manufacturer. qRT-PCR was done using SYBRGreen PCR Master Mix (Applied Biosystems) and an ABIPRISM 7900 Sequence Detection System (PerkinElmer Bio-systems). Primers for PCR amplifications (SupplementaryTable S1) were designed using Primer 3 Input (version0.4.0). Relative mRNA levels were calculated using the2�DDCt method (14).

Construction of plasmids and luciferase assaysWild-type and/or mutant fragments (150–200 bp) from

the 30-UTR (untranslated region) of MTDH, MTPN, andUSP1 containing predicted binding sites for miR-375 wereindividually amplified by AmpliTaq gold DNA polymerase(Applied Biosystems) using the primers listed in Supple-mentary Table S2. The PCR products were subsequentlypurified, digested with SPEI and HINDIII, and then cloneddownstream of the firefly luciferase gene in the pMIR-REPORT vector (Ambion, Inc.) to produce the followingplasmids: pMIR-MTDH, pMIR-MTDH-MUT, pMIR-MTPN,and pMIR-USP1. Subsequently, FaDu and C666-1 cellswere seeded onto 24-well plates 1 day and 3 days beforetransfection, respectively. Cells were then transfected with100 nmol/L of pre-miR-NEG or pre-miR-375 in the morn-ing; 6 hours later, cells were cotransfectedwith 100 ng of thereporter plasmid of interest, plus 50 ng of pRL-SV40 (Pro-mega BioSciences) containing the Renilla luciferase gene.Luciferase activity was measured 48 hours posttransfectionusing the Dual-Glo luciferase assay system according tomanufacturer’s instructions (Promega BioSciences). Fireflyluciferase activity was normalized to that of the Renillaluciferase.

Viability and clonogenic assaysViability of pre-miR-375 or siMTDH-transfected FaDu

or C666-1 cells was examined using the CellTiter 96

Hui et al.

Clin Cancer Res; 17(24) December 15, 2011 Clinical Cancer Research7540




Non-Radioactive Cell Proliferation Assay (MTS), accordingto the manufacturer’s protocol (Promega BioSciences). Thecellular effects of these manipulations were further inves-tigated in FaDu cells using clonogenic assays as previouslydescribed (15). Briefly, cells were reseeded at low density in6-well plates in triplicate and incubated at 37�C under 5%CO2 at 3 days posttransfection. After 10 to 12 days, plateswere washed, fixed in 50% methanol, and stained with0.1% crystal violet and then the number of colonies wascounted. The fraction of clonogenically viable cells wascalculatedby comparisonof pre-miR-375or siMTDH-trans-fected cells with pre-miR-NEG or scrambled siRNA (siNEG)transfected cells, respectively.

Cell-cycle analysisCell-cycle analysis of pre-miR-375–transfected C666-1

cells was done as previously described (15). Briefly, cellswere harvested and washed with fluorescence-activated cellsorting (FACS) buffer (PBS/0.5% BSA), then resuspended

and fixed with ice-cold 70% ethanol. After washing, cellswere resuspended in FACS buffer and incubated with pro-pidium iodide in the dark before analysis in the BD FACS-calibur using the FL-2 channel. The flow cytometry datawere analyzed using FlowJo software (Tree Star, Inc.).

Western blot analysisTotal protein extracts were harvested from cell lines and

prepared for immunoblotting as previously described (16).Membranes were probed with anti-Phospho-Akt (Ser473)(D9E) XP rabbit monoclonal antibodies (mAb; Cell Sig-naling Technology), anti-Akt (pan) (C67E7) rabbit mAb(Cell Signaling Technology), anti-MTDH polyclonal (cloneL-19; 1:1,000 dilution; Invitrogen, Inc.), anti–b-actin(8H10D10) mouse mAb (1:20,000 dilution; Cell SignalingTechnology), or anti-GAPDH(glyceraldehyde-3-phosphatedehydrogenase) mAbs (1:15,000 dilution; Abcam, Inc.),followed by secondary antibodies conjugated to horserad-ish peroxidase (1:2,000 dilution; Abcam, Inc.) or IRDye

Table 1. Clinical characteristics of HNC patients studied—clinical descriptors of 94 NPC and 20 HNSCCpatients whose tumor samples were analyzed during this work

Clinical descriptors for the 94 NPC patientsAgeMedian 46Range 16–79

Stage Frequency PercentI 8 8II 32 34III 28 30IV 26 28

Relapse Frequency PercentNo relapse 64 68Local relapse only 7 7Local þ Distant relapse 13 14Distant relapse only 10 11

Treatment Frequency PercentRadiation only 69 73Radiation þ chemo 25 27

Clinical descriptors for the 20 HNSCC patientsAgeMedian 59Range 35–75

Stage Frequency PercentIII 8 40IV 12 60

Relapse Frequency PercentNo relapse 4 20Local relapse 6 30Regional relapse 4 20Local þ regional relapse 6 30

Treatment Frequency PercentRadiation #1 (D) 9 45Radiation #2 (T) 11 55

(T ¼ twice daily) and (D ¼ standard).

MTDH As a Target of Mir-375

www.aacrjournals.org Clin Cancer Res; 17(24) December 15, 2011 7541




fluorescent Secondary Antibodies (1:20,000 dilution; LI-COR Biosciences). GAPDH or b-actin protein levels wereused as loading controls.Westernblotswere quantifiedwiththeAdobePhotoshopPixelQuantificationPlug-In (RichardRosenman Advertising & Design), or the Odyssey Applica-tion Software 2.1 (Li-cor Biosciences).

In vitro migration and invasion assaysInvasion and migration of HNC cells were assayed using

the BD BioCoat Matrigel Invasion Chambers and ControlInserts (BD Bioscience), respectively. Each well of a 24-wellplate contained an insert with an 8-mm pore size PET(polyethylene terephthalate) membrane. Inserts coatedwith a thin layer of Matrigel basement membrane matrixwere used to measure the ability of the cells to invadethrough the reconstituted basement membrane. For bothmigration and invasion assays, 1 � 105 cells were seededinside the insertwithmediumcontaining 0.5%serum.Highserum (20%) medium was then added to the bottomchamber of 24-well plates to serve as a chemoattractant.After 48 hours, the membranes were washed, stained, thenseparatedwith a sterile scalpel andmounted on a glass slide.Thenumber ofmigratingor invading cellswas then countedunder a light microscope.

Tumor formation assayAll animal experiments utilized 6- to 8-week-old severe

combined immunodeficient (SCID) BALB/c femalemice inaccordance with the guidelines of the Animal Care Com-mittee, Ontario Cancer Institute, University Health Net-work (Toronto, Canada). Cells were first transfected withsiNEG, siMTDH, pre-miR-NEG, or pre-miR-375 72 hoursbefore they were harvested, and their viability was assessedby the trypan blue exclusion method. Subsequently, 2.5 �105 viable cells were suspended in 100 mL of growth medi-umand injected intramuscularly into the left gastrocnemiusmuscle of female SCIDmice. Tumor growth wasmonitoredbymeasuring tumorplus leg diameter (TLD)3 times aweek.Mice were euthanized by CO2 once TLDs reached 14 mm.

Immunohistochemical detection of MTDH expressionProtein expression of MTDH was evaluated in 94 NPC

patient samples using immunohistochemistry (IHC),which was done on 5-mm FFPE sections of tumor of eachpatient using microwave antigen retrieval, in combinationwith the Level-2 Ultra Streptavidin system (Signet Labora-tories). MTDH expression was detected with the rabbitpolyclonal anti-MTDHantibody (1:50 dilution; Invitrogen,Inc.). Expression levels of MTDH were graded according tointensity of immunoexpression.

Statistical analysesAll experiments were conducted at least 3 independent

times, with the data presented as the mean � SEM. Thestatistical differences between treatment groups were deter-mined using a Student t test when comparing 2 treatmentgroups, or a 1-way ANOVA followed by Tukey’s methodwhen comparing more than 2 treatment groups. Statistical

analyses and graphing were done using Microsoft excel andGraphPad Prism software (GraphPad Software, Inc.).

To determine the potential prognostic significance ofmiR-375 or MTDH expression, NPC patients were dichot-omized into low (

both cell lines with the global demethylating agent 5-aza-20-deoxycytidine (5-aza) significantly reduced methylation atall CpG sites (Fig. 1D), which was associated with thesubsequent reexpression of miR-375 (Fig. 1E), clearly indi-cating that one mechanism for miR-375 suppression inHNC cells is hypermethylation of the promoter or codingregions of miR-375.

Identification of mRNA targets of miR-375To identify the mRNA targets of miR-375, genome wide

mRNA expression analysis was done comparing pre-miR-375–transfected HNC cells with that of pre-miR-NEG–transfected cells (data not shown). Transcripts with morethan 2-fold downregulation in pre-miR-375–transfectedcells were compared with in silico predicted targets from 7publicly available databases (miRanda, miRDB, RNA22,PITA, miR-Walk, RNAHybrid, TargetScan), generating alist of 9 candidate target mRNAs (Supplementary TableS3). As confirmation of the validity of our methodology,2 of these genes (MTPN, USP1) had been previouslyreported as targets of miR-375 (18, 19). The effect of

miR-375 transfection on gene expression was analyzed for6 of the 7 identified genes (optimal primers for AEBP2could not be generated), as well as 2 previously reportedmiR-375 targets (PDK1, ADIPOR2; refs. 19, 20). Trans-fection of FaDu and C666-1 cells with pre-miR-375 led todownregulation of all potential mRNA targets, with theexception of ADIPOR2 in FaDu cells (Supplementary Fig.S4). Of note, MTDH was consistently downregulated tothe lowest level in both HNC cell lines; moreover, MTDHwas the only target predicted by all 7 in silico algorithms(Supplementary Table S3).

Downregulation of MTDH by miR-375 was confirmedat both the transcript and protein level by qRT-PCR andWestern blot analysis, respectively (Fig. 2A). The directinteraction between miR-375 and MTDH was then veri-fied using luciferase assays. Supplementary Fig. S5 depictsthe in silico predicted binding sites between the miR-375seed region with the MTDH transcript. The predictedbinding site located at nucleotides 3,518 to 3,539 inthe 30-UTR of the MTDH transcript was supported byall 7 in silico databases and was, therefore, selected for

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Figure 1. Downregulation of miR-375 in HNC cells. A, viability of C666-1 cells as assessed by theMTS assay 3 and 6 days post-pre-miR-375 or pre-miR-NEGtransfection. B, cell-cycle analysis as done by flow cytometry on C666-1 72 hours post-pre-miR-375 or pre-miR-NEG transfection. C, MiR-375 expression in92 primary NPC biopsy samples, compared with 8 normal nasopharyngeal epithelial control tissues as measured by qRT-PCR. D, bisulfite sequencinganalysis of the CpG sites within an approximately 1,200 bp region around the genomic locus encoding miR-375. Methylation status of 3 amplicons wasanalyzed onC666-1 andFaDu cells, treatedwith either 5-aza-20-deoxycytidine (FaDu-AZA/C666-1-AZA), orDMSO (FaDu-V/C666-1-V). E, qRT-PCRanalysisofmiR-375 expression in FaDu andC666-1 cells after treatment with 5-aza-20-deoxycytidine relative to vehicle control (DMSO) treated cells. Each experimentwas conducted at least 3 independent times, with the data presented as the mean � SEM. �, P < 0.05. DMSO, dimethyl sulfoxide.






plasmid construction. In comparison with control cellstransfected with pMIR-REPORT, FaDu and C666-1 cellstransfected with pMIR-REPORT-MTDH showed a signif-icant reduction in luciferase activity of 18% and 20%,respectively, when cotransfected with pre-miR-375 (Fig.2B). Upon mutating the miR-375–binding site, thisinhibitory effect was completely abrogated, corroboratingthat the observed decrease in luciferase activity wasindeed dependent upon an intact miR-375–binding site.Two previously reported mRNA targets, MTPN and USP1,were also shown to be potential targets of miR-375 inboth HNC cell lines (Supplementary Fig. S6).

Effects of MTDH knockdown on HNC cellsThe potential oncogenic role of MTDHwas investigated

by evaluating the phenotypic consequences of transfect-ing HNC cells with siRNA directed against MTDH(siMTDH). MTDH knockdown by this siRNA sequencewas confirmed at both the transcript and protein level(Fig. 2C). Similar to the effects of pre-miR-375 transfec-tion, siMTDH transfection significantly reduced cell via-bility in both C666-1 (>50%) and FaDu (�20%) cells(Fig. 2D). To ensure that the observed cytotoxicity was

not due to off-target effects, the MTS experiments wererepeated using a second distinct siRNA sequence targetingMTDH, showing a similar effect (Supplementary Fig.S7A). As yet another level of corroboration, a rescueplasmid expressing a MTDH transcript refractory to siRNAwas cotransfected with siMTDH, which completely abro-gated any siMTDH-mediated cytotoxicity, confirming thatthis was indeed an MTDH knockdown-specific effect(Supplementary Fig. S7B).

MTDH is frequently reported to promote metastasis inhuman cancers; hence, the effect of miR-375 and MTDHon HNC cell migration and invasion were evaluatedusing in vitro trans-well migration assays. Compared withtheir corresponding negative controls, transfection withpre-miR-375 significantly reduced migration of bothC666-1 and FaDu cells by 57% and 80%, respectively(Fig. 3A). Similar inhibition of migration was observedin cells transfected with siMTDH, with 75% reduction inC666-1 cells and 86% in FaDu cells (Fig. 3A). Moreover,siMTDH and pre-miR-375 transfection also resulted in86% and 92% reduction in invasion of FaDu cells,respectively (Fig. 3B). C666-1 cells are unable to pene-trate the Matrigel coating used in the invasion assay;

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Figure 2. Identification of mRNA targets of miR-375. A, MTDH expression at both the mRNA (left) and protein (right) level after pre-miR-375 or pre-miR-NEGtransfection in C666-1 and FaDu cells. B, relative luciferase activity in C666-1 or FaDu cells after cotransfection with pMIR-REPORT, pMIR-MTDH, or pMIR-MTDH-MUT vectors and pre-miR-375 or pre-miR-NEG. C, MTDH expression at both the mRNA (left) and protein (right) level after siMTDH or siNEGtransfection in C666-1 and FaDu cells. D, Viability of C666-1 and FaDu cells as assessed by the MTS assay 3 days post-siMTDH or siNEG transfection. OD,optical density; �, P < 0.05.

Hui et al.





hence, this property could not be assessed in the NPCmodel.

Effects of miR-375 transfection andMTDH knockdownon PI(3)K-Akt signalingWestern blot analyses were done to investigate the sig-

naling pathways downstreamofmiR-375 overexpression orMTDH knockdown in FaDu and C666-1 cells. The PI(3)K-Akt pathway has been previously reported to be activated byMTDH overexpression; specifically increased phosphoryla-tion of Akt (p-Akt), although the mechanism by which thisinductionoccurs remainspoorly understood (21–23).Con-cordantly, a significant reduction in p-Akt was observed inbothC666-1 and FaDu cells after siMTDHknockdown (Fig.4A and B), phenocopied by miR-375 transfection in FaDucells (Fig. 4B), but not so forC666-1 cells (Fig. 4A). Total Aktexpression remained unchanged under these conditions.

MTDH knockdown and miR-375 transfection delayedtumor formation in vivoTo investigate the effects of miR-375 overexpression

and MTDH knockdown on head and neck tumor formingability, FaDu and C666-1 cells transfected with siMTDH,pre-miR-375, or corresponding negative controls wereinjected intramuscularly into SCID mice. The resultsshowed that both miR-375 overexpression and MTDH

knockdown resulted in a significant delay in tumor for-mation, wherein such transfected tumors attained thehumane endpoint (TLD ¼ 14 mm) approximately 10 to15 days later than tumors transfected with the corre-sponding negative controls (Fig. 4C and D). Tumors fromboth C666-1 and FaDu xenografts were FFPE for immu-nohistochemical analyses for CD31 and Ki-67, which didnot show any significant difference in their expressions(data not shown).

Overexpression of MTDH in primary HNC samplesThe relevance of MTDH in HNC was further investi-

gated in primary human HNC samples in the previouslyevaluated 20 HNSCC (5), as well as the same 92 NPCsamples for which miR-375 expression has already beenexamined (Fig. 1C). Significantly higher MTDH transcriptlevels were detected in both HNSCC (P¼ 0.048) and NPCpatient samples (P < 0.0001) compared with correspond-ing normal controls as measured by qRT-PCR (Fig. 5A andB). Using a 2-fold cut-off, MTDH mRNA overexpressionwas detected in 65% and 93% of HNSCC and NPCsamples, respectively. Overexpression of MTDH at theprotein level was corroborated in 94 NPC patient samples(including the 92 previously investigated cases) usingIHC, illustrating that MTDH expression was predomi-nantly localized to the cell membrane or cytoplasm, with

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Figure 3. Migration and invasion of miR-375 or siMTDH in HNC cells. A, representative images (left) and bar graphs (right) depicting the migratory ability ofC666-1 and FaDu cells after pre-miR-375 or siMTDH transfection compared with corresponding negative controls. B, representative images (left) and bargraphs (right) depicting the invasive ability of FaDucells after pre-miR-375 or siMTDH transfection comparedwith correspondingnegative controls. �,P

little to no immunostaining observed in the surroundingstroma (Fig. 5C). The vast majority of NPC samples hadcytoplasmic MTDH immunoexpression (91 of 94 cases,or 97%), with only 1 sample showing nuclear staining(Fig. 5D). Strikingly, when dichotomized based onMTDH transcript expression level, NPC patients withhigh expression (�median) had significantly worse over-all (HR ¼ 2.34; P ¼ 0.04), disease-free (HR ¼ 2.08; P ¼0.04), and distant relapse-free (HR ¼ 6.38; P ¼ 0.005)survival compared with patients with low MTDH(

samples had relapsed disease), survival analysis was notdone on this cohort.

Discussion

Our recent study profiling miRNA expression in locallyadvanced HNSCC reported a high frequency of miR-375underexpression in these tumors and suggested its potentialtumor suppressor function (5). This article shows that miR-375 is also downregulated in 75% of NPCs and confirms atumor suppressor function for this miRNA in HNC celllines. Furthermore, MTDH has been verified as bona fidetarget of miR-375 in HNC. During the preparation of this

article,MTDHwas also identified independently by anothergroup as a target of miR-375 in HNSCC (24). However, tothe best of our knowledge, this study is the first to support arole for the dysregulation of miR-375 and MTDH in pro-moting metastases in HNCs, particularly in NPC.

MiR-375 was first described in the development andfunction of pancreatic islet cells (25, 26) and,more recently,in human embryonic stem cell development (27). Mount-ing data, however, suggest that miR-375 might also beplaying an equally important role inhuman cancers. Severalgroups have recently shown a tumor suppressor role formiR-375 (20, 28–31), congruent with our own data in thisstudy. Reduced miR-375 expression has, in fact, been

Figure 5. MTDH expression inHNC patient samples. MTDHexpression measured by qRT-PCRin (A) 20 HNSCC and (B) 92 NPCpatient biopsy samples, incomparison with 6 normal laryngealepithelial tissues and 7 normalnasopharyngeal epithelial tissues,respectively. C, representativeimages of immunohistochemicalanalysis of MTDH expression inprimary NPC biopsy samples.Arrows indicate tumor cellsexhibiting (C) cytoplasmicmembrane or (D) nuclearexpression ofMTDH. T, tumor cells;E, endothelial cells. E, Kaplan–Meier plots of OS, disease-freesurvival, and distant relapse-freesurvival for NPC patientsdichotomized on the basis of high(�median) versus low (

reported to be associated with worse outcome for patientswith esophageal carcinoma (32, 33); high miR-221 to lowmiR-375 ratio has also been suggested as a potential diag-nostic marker for HNSCC (30). Conversely, several groupshave also described a potential oncogenic role for miR-375(34–36), underscoring that similar to many other miRNAs,miR-375 can function as both a tumor suppressor and anoncogene, probably dependent upon context and cancertype.

Chromosomal loss andpromoter hypermethylation are 2commonmechanisms for inactivation of tumor suppressorgenes. Deletion of chromosome 2q35 has indeed beenreported in HNSCC (37). In this work, bisulfite sequencingshowed that CpG islands proximal to themiR-375 locus arehighly methylated (Fig. 1D), and exposure to 50-aza-2-deoxycytidine resulted in a significant reexpression ofmiR-375 (Fig. 1E), corroborating that hypermethylation isindeed one important mechanism for miR-375 underex-pression in HNC, similar to the previous report in HCC(17). Hence, chromosomal loss and hypermethylation-mediated silencing are 2 likely mechanisms leading tomiR-375 downregulation in HNC.

In terms of MTDH overexpression in HNC, miR-375downregulation is clearly one documentedmechanism, butthe downregulation of other miRNAs could also play a rolein this process. As an example, miR-26a was recentlyreported to be underexpressed in breast cancer, targetingbothMTDHand enhancer of zeste homolog 2 (EZH2) (38).Our own group has previously reported significantmiR-26adownregulation in primary HNSCC (5), as well as NPC(39); hence, underexpression of miR-26a might indeed beanother mechanism for MTDH overexpression in HNC. Athird potentialmechanism forMTDHoverexpression couldbe chromosomal amplification, given that 8q22 (genomiclocation for MTDH) is also frequently amplified in bothNPC and HNSCC (40, 41). Indeed, this mechanism forMTDH overexpression has already been reported for bothbreast (8) and HCCs (42). Taken together, these conglom-erate reports illustrate thatMTDHoverexpression inHNC islikely mediated by a complex interplay between severalfactors, including chromosomal amplification and target-ing by multiple miRNAs.

The humanMTDH gene was first cloned in astrocytes, inwhich it was shown to be upregulated in response totreatment with TNF-a, gp120, or HIV infection, thereforeinitially denoted as "Astrocyte elevated gene 1" (AEG-1;ref. 6). Themouse homologwas cloned shortly thereafter ina murine model for metastatic breast cancer, discoveredbecause of its role in selectively homing metastatic breastcancer cells to the lung (43). Since these seminal observa-tions,MTDHhas rapidly emerged as an importantmediatorin the development and progression for several humancancers (7). In fact, MTDH overexpression has already beenassociated with poor outcome for breast (8), liver (42),esophageal (44), renal cell (45), colorectal (46), and non–small cell lung cancers (9), frequently associated withmetastases and chemoresistance. However, the molecularmechanisms by which MTDH mediates metastases and/or

chemoresistance remain to be clearly elucidated. Some ofthe suggested downstream signaling events have includedactivation of PI3K-Akt (21), NF-kB (47), mitogen-activatedprotein kinase andWnt (42), upregulation of MMP-9 (48),and FOXO3a (22, 44), suppression of FOXO1 (23), as wellas induction of EMT (47).

Herein, we report the overexpression of MTDH in bothHNSCC and NPC. Due to small sample size (n ¼ 20), thesignificance of MTDH overexpression observed in theHNSCC samples was marginal (P ¼ 0.048); therefore, wesought out additional evidence to support these findings.Using a publicly available online (http://www.ebi.ac.uk/gxa/array/U133A) data set mined by Lukk and colleagues(49), we identified that MTDH was also significantly over-expressed in their HNSCC samples by 2.1- to 4.2-fold (P <0.08). Furthermore, another group has also recentlyreported the significant overexpressionofMTDHinHNSCCusing 20 paired HNSCC/normal samples (24); hence, weare confident about MTDH overexpression in primaryHNSCC. We also report, for the first time, a role for MTDHin promoting a metastatic phenotype in HNC, particularlyin NPC. The oncogenic function of MTDHwas shown bothin vitro and in vivo, wherein MTDH affected cell migrationand invasion, potentially mediated in part via phosporyla-tion of Akt. The clinical impact of this observation isextremely important, due to the advent of intensity-mod-ulated radiation therapy, the 5-year local control rates forNPC are excellent, at approximately 90% (3). However, the5-year OS for NPC remains modest at approximately 70%due to distant metastases (3). Hence, therapies that effec-tively reducemetastases would be of extreme importance inimproving survival for NPC patients. As MTDH is primarilyexpressed at the plasma membrane of breast cancer cells(43), a DNA vaccine therapy targeting MTDH has beendeveloped, showing some preliminary success in reducingtumor burden and lung metastases in a mouse model ofbreast cancer (50). However, such approaches might not beapplicable to HNC, given that MTDH expression seems tobe predominantly cytoplasmic in this disease (Fig. 5C).

In conclusion, a novel pathway of miR-375 downre-gulation, leading to MTDH overexpression, has beendocumented in human HNC, which has significant clin-ical implications as a mechanism by which HNC metas-tases could develop. Further unraveling of the MTDHprotein structure and function, as well as the molecularmechanisms by which it activates a metastatic cascade,could lead to the development of small molecule inhi-bitors, which can be examined as a potential therapeuticstrategy by which outcome can be improved for futurepatients with HNC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank theWharton family, Joe’s Team, and Gordon Tozer fortheir philanthropic support.

Hui et al.





Grant Support

This work was supported by funds from the Ontario Institute for CancerResearch, the Canadian Institutes of Health Research, and the Dr. MarianoElia Chair in Head & Neck Cancer Research, and also in part from theCampbell Family Institute for Cancer Research, and the Ministry of Healthand Long-Term Planning.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 15, 2011; revised October 4, 2011; accepted October 12,2011; published OnlineFirst October 26, 2011.

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2011;17:7539-7550. Published OnlineFirst October 26, 2011.Clin Cancer Res Angela B.Y. Hui, Jeff P. Bruce, Nehad M. Alajez, et al. Head and Neck CancerSignificance of Dysregulated Metadherin and MicroRNA-375 in

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