comprehensive microrna profiling for head and...

Published OnlineFirst February 15, 2010; DOI: 10.1158/1078-0432.CCR-09-2166

Human Cancer Biology Clinical
Cancer
Research
Comprehensive MicroRNA Profiling for Head and NeckSquamous Cell Carcinomas
Angela B.Y. Hui1, Michelle Lenarduzzi1,7, Tiffaney Krushel1, Levi Waldron2, Melania Pintilie3,Wei Shi1, Bayardo Perez-Ordonez4, Igor Jurisica2,7,8, Brian O'Sullivan5,9, John Waldron5,9,Pat Gullane6, Bernard Cummings5,9, and Fei-Fei Liu1,5,7,9

Abstract

Authors' A2SignalingBiostatistic6SurgicalNetwork; Dand 9RadiCanada

Note: SuppResearch O

CorresponPrincess Mnue, Toron416-946-65

doi: 10.115

©2010 Am

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Downl

Purpose: The objective of this study is to investigate the significance of microRNAs (miRNA) inpatients with locally advanced head and neck squamous cell carcinoma (HNSCC).Experimental Design: A global miRNA profiling was done on 51 formalin-fixed archival HNSCC

samples using quantitative reverse transcription-PCR approach, correlated with patients' clinical para-meters. Functional characterization of HNSCC-associated miRNAs was conducted on three HNSCC celllines. Cell viability and proliferation were investigated using MTS and clonogenic assays, respectively; cellcycle analyses were assessed using flow cytometry.Results: Thirty-eight of the 117 (33%) consistently detected miRNAs were significantly differentially

expressed between malignant versus normal tissues. Concordant with previous reports, overexpression ofmiR-21, miR-155, let-7i, and miR-142-3p and underexpression of miR-125b and miR-375 were detected.Upregulation of miR-423, miR-106b, miR-20a, and miR-16 as well as downregulation of miR-10a werenewly observed. Exogenous overexpression of miR-375 in HNSCC cell lines reduced proliferation andclonogenicity and increased cells in sub-G1. Similar cellular effects were observed in knockdown studiesof the miR-106b-25 cluster but with accumulation of cells in G1 arrest. No major difference was detectedin miRNA profiles among laryngeal, oropharyngeal, or hypopharyngeal cancers. miR-451 was found to bethe only significantly overexpressed miRNA by 4.7-fold between nonrelapsed and relapsed patients.Conclusion: We have identified a group of aberrantly expressed miRNAs in HNSCC and showed that

underexpression of miR-375 and overexpression of miR-106b-25 cluster might play oncogenic roles inthis disease. Further detailed examinations of miRNAs will provide opportunities to dissect the complexmolecular abnormalities driving HNSCC progression. Clin Cancer Res; 16(4); 1129–39. ©2010 AACR.

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 ∼30%, underscoringsignificant opportunities for improving outcome (2, 3).Hence, there is a need to acquire deeper understandingof HNSCC biology and to develop predictive molecularsignatures, which could improve patient selection for ap-propriate treatment and guide the development and eval-

ffiliations: Divisions of 1Applied Molecular Oncology andBiology, Ontario Cancer Institute and 3Division of

s and Departments of 4Pathology, 5Radiation Oncology, andOncology, Princess Margaret Hospital, University Healthepartments of 7Medical Biophysics, 8Computer Science,

ation Oncology, University of Toronto, Toronto, Ontario,

lementary data for this article are available at Clinical Cancernline (http://clincancerres.aacrjournals.org/).

ding Author: Fei-Fei Liu, Department of Radiation Oncology,argaret Hospital/Ontario Cancer Institute, 610 University Ave-to, Ontario, Canada M5G 2M9. Phone: 416-946-2123; Fax:29; E-mail: [email protected].

8/1078-0432.CCR-09-2166

erican Association for Cancer Research.

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uation of new therapies. MicroRNAs (miRNA) have beenrecently recognized to play important roles in human can-cers and are regarded as key regulators of gene expressionin all biological systems (4). Increasing data support thevalue of miRNA expression profiling to distinguish differ-ent types of human malignancies and to categorize variouscancer subtypes (5–7).To date, several reports have described miRNAs associ-

ated with HNSCC, evaluated in human cancer cell lines orfrozen tissue samples (8–12). With the exception of miR-21, there is no overlap in the identified miRNAs amongany of these studies. In addition, a rate-limiting step inthe broad application of expression profiling to clinicalpractice is the paucity of frozen tissues linked to clinicaloutcome. Archives of formalin-fixed, paraffin-embedded(FFPE) specimens are much more commonly available,providing greater opportunities to acquire biological in-sights. Archival specimens are amenable to miRNA evalua-tions due to their short lengths (∼22 nucleotides in length;ref. 13), and we have recently successfully conducted sucha global miRNA profiling study for breast cancer FFPEsamples (14). In this current study, a comprehensivemiRNAevaluation was conducted on diagnostic biopsies from

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Translational Relevance

We have conducted a global microRNA (miRNA)profiling of 51 archival formalin-fixed, paraffin-embedded samples of locally advanced stage headand neck squamous cell carcinoma (HNSCC). Ourresults show that approximately one third (38 of117) of consistently detected miRNAs were aber-rantly expressed in this cancer. These included bothpreviously reported HNSCC-associated miRNAs andnovel miRNAs of potential biological significance.Many of the dysregulated miRNAs map to fragile sitesof the chromosome, indicating that genomic gains orlosses account for one mechanism of this aberration.One of the novel miRNAs is miR-106b-25, whichseems to play an oncogenic role in HNSCC develop-ment and progression, as well as miR-375. GlobalmiRNA profiles were similar between SCCs arisingfrom the larynx, oropharynx, or hypopharynx. miR-451was the only miRNA that was significantly downregu-lated in relapsed compared with nonrelapsed patients.These data illustrate the usefulness of miRNA profilingusing archival formalin-fixed, paraffin-embedded sam-ples; further interrogation of these miRNAs will providenovel insights into the complexity of miRNA biologyin HNSCC.

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patients with locally advanced HNSCC, showing miRNAdysregulation and potentially oncogenic roles for miR-106b-25 and miR-375.

Materials and Methods

Patient information. Patient samples were collected froma phase III randomized study (331 participants) of hyper-fractionated radiotherapy conducted in 1988 to 1995(15), with approval from the Institutional Research EthicsBoard. Only fifty-four tissue samples out of the collectedsamples had follow-up data of >4 y and sufficient RNAfor miRNA profiling. All patients presented with locallyadvanced (stage III or IV) HNSCC; the clinical charac-teristics of these 54 patients are shown in SupplementaryTable S1. The predominant subsite was oropharynx (24 of54, 44%); the majority of patients were males (44 of 54,82%). With a minimum follow-up of 4 y, 44 patients hadexperienced disease relapse; only 10 had no relapse. FourFFPE blocks of normal laryngeal squamous epithelial tis-sues derived from laryngectomy specimens served as “nor-mal” controls (commercially purchased from Asterand).RNA purification from FFPE samples. For each sample, a

representative section was stained with H&E and re-viewed by a head and neck cancer pathologist (B.P-O.)to identify regions containing >70% malignant epithelialcells for macrodissection. All blocks were processed ran-

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domly, with clinical outcome unknown, to avoid experi-mental bias. Total RNA enriched for small RNA specieswas isolated using the RecoverAll Total Nucleic Acid Iso-lation kit for FFPE (Ambion) according to the manufac-turer's instructions.Real-time quantification of miRNAs. The quality of the

54 HNSCC and 4 normal laryngeal tissue samples wasassessed by reverse transcription-PCR (RT-PCR) analysisof the endogenous control RNU44 using Applied Bio-systems Taqman MicroRNA Assay. This assay includes aRT step using the Taqman MicroRNA Reverse Transcrip-tion kit (Applied Biosystems), wherein a stem-loop RTprimer specifically hybridizes to a miRNA molecule andis then reverse transcribed with a MultiScribe reverse tran-scriptase (14).Global miRNA profiling of the 54 FFPE samples was

done using an in-house prepared RT and PCR plates.Using Taqman MicroRNA Assays Human Panel (AppliedBiosystems), a panel of 312 human miRNAs plus 10 en-dogenous control miRNAs was simultaneously assayed.With the Biomek FX Laboratory Automation Workstationprovided by the Samuel Lunenfeld Research InstituteHigh-Throughput Screening Robotics Facility (Toronto,Ontario, Canada), 322 Taqman human miRNA stem-loopRT primers were transferred to 384-well plates, sealed, andkept at −20°C for storage. PCR plates were prepared sim-ilarly, except that each Taqman miRNA probe was pipettedin triplicate. Similar to the individual Taqman miRNA as-say, total RNA was first reverse transcribed with the RTplates. For each sample, 1 to 3 μg of total RNA were mixedwith the RT reagent and then added to the 384-well RTplates. This was followed by transfer of correspondingcDNA to the PCR plates and then analyzed using the Ap-plied Biosystems 7900HT Real-Time PCR System.Cell lines and reagents. The human hypopharyngeal

FaDu HNSCC cell line was obtained from the AmericanType Culture Collection and cultured according to specifi-cations. The human laryngeal squamous UTSCC-8 andUTSCC-42a (kind gifts from R. Grénman, Turku UniversityHospital, Turku, Finland) were maintained with DMEMsupplemented with 10% fetal bovine serum (Wisent, Inc.)and 100 mg/L penicillin/streptomycin. The commerciallypurchased normal oral epithelial cells were cultured in therecommended medium (from Celprogen). All cells weremaintained in a 37°C incubator with humidified 5% CO2.Functional analysis of differentially expressed miRNAs.

The biological effects of overexpressed miRNAs were inves-tigated by knocking down these miRNAs in HNSCC celllines using locked nucleic acid (LNA) probes containingsequence-specific antisense oligonucleotides targetingmiR-25, miR-27a, miR-93, miR-106b, or miR-423 (miR-CURY LNA miRNA knockdown probes, Exiqon). FaDu,UTSCC-8, or UTSCC-42a cells were reverse transfectedusing Lipofectamine 2000 and Opti-MEM I reduced serummedium (Invitrogen) and 40 nmol/L of LNA miRNAknockdown probes seeded in 12-well plates. A scrambledmiRNA probe (sequence: GTGTAACACGTCTATACGCC-CA) served as a negative control (Exiqon).

Clinical Cancer Research



miRNA Profile of HNSCC


Downregulated miRNAs were evaluated for biologicaleffect using pre-miR miRNA precursor molecules (30nmol/L pre-miR-10a or 10 nmol/L pre-miR-375; Ambion)transfected into each of the three HNSCC cell lines. A Pre-miR Negative Control (Ambion) with random sequencepre-miR molecules, which has been documented to bebiologically inert, was used. To examine the change inmiRNA expression, cells were removed for RNA extraction72 h after transfection and analyzed using quantitativeRT-PCR.Viability or clonogenic assays on HNSCC cell lines trans-

fected with pre-miR-375 or LNA miR-106b. Cell prolifera-tion effects of pre-miR-375 or LNA miR-106b wereexamined on HNSCC cells using the MTS cell proliferationassay according to the manufacturer's protocol (Promega).The cellular effects of pre-miR-375 or LNA miR-106b onHNSCC cells were further investigated using clonogenicassays. On the third day after transfection, HNSCC cellswere reseeded in six-well plates in triplicate and incubatedat 37°C under 5% CO2. After 10 to 12 d of incubation,plates were washed, fixed, and stained with 0.1% crystalviolet in 50% methanol, and the number of colonieswas then counted. The fraction of surviving cells was cal-culated by comparison with cells treated with scrambledLNA or pre-miR negative.Apoptosis and cell cycle effects. Cell cycle analysis of pre-

miR-375–transfected or LNA miR-106b–transfectedHNSCC cells was done as previously described (16). Brief-ly, cells were harvested and washed with fluorescence-activated cell sorting buffer (PBS/0.5% bovine serumalbumin) and then resuspended and fixed with ice-cold70% ethanol. After washing, cells were resuspended influorescence-activated cell sorting buffer and incubatedin the dark before analysis in the BD FACSCalibur usingthe FL-2A and FL-2W channels. The flow cytometry datawere analyzed using FlowJo software (TreeStar).Data pre-processing. Expression values were calculated

using the ΔΔCt method of Pfaffl (17), with a PCR ampli-fication efficiency of E = 2 for all primers, and using themean Ct of the four normal samples as reference. Ten can-didate endogenous control genes were measured (RNU6B,RNU19, RNU24, RNU38B, RNU43, RNU44, RNU48,RNU49, and Z30), from which the mean Ct of the six(RNU6B, RNU24, RNU43, RNU44, RNU48, and RNU49)with two or fewer undetermined values was used as theendogenous control. Additionally, all target miRNAs withundetermined Ct values in three of four normal samples,or 80% of tumor samples, were removed, leaving 117miRNAs for analysis. Ct values that were undeterminedor >36 were imputed to 36. Normalization and all statis-tical analyses and plotting were done in the R languageand environment for statistical computing (R Develop-ment Core Team, v2.8.1).Analysis of miRNA as a function of tumor site, tumor-

normal, or disease status. After imputing missing valuesto 36, expression values for each miRNA were clearly notnormally distributed; hence, nonparametric tests were ap-plied for all inferences. To search for miRNAs differentially

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expressed in tumors from the three different anatomicsubsites, each miRNA was examined using the Kruskal-Wallis rank sum test, with the Benjamini-Hochberg adjust-ment for false discovery rate (FDR) as implemented in themulttest package (version 1.22.0; ref. 18). miRNAs differ-entially expressed between tumor and normal or betweenrelapsed and nonrelapsed tumors were identified using theMann-Whitney U test with FDR adjustment for multipletesting as described above. Hierarchical clustering for thetumor versus normal heat map was done with the hclustfunction in the R base package v2.8.1 (19), with defaultoptions and 1 minus Spearman rank correlation as the dis-tance measure, whereas the heat map was produced withthe heatmap.2 function in the package gplots v2.6.0 (20).

Results

Global miRNA profiling of HNSCC samples. Comprehen-sive miRNA profiles were generated for 51 HNSCC pa-tients using a quantitative RT-PCR approach; threepatient samples were excluded from analysis due to poorRNA quality. From a total of 322 miRNAs, 117 were de-tected in >80% of the samples, thereby serving as the poolof data for further analyses. In comparison with normaltissues, HNSCC samples contained more upregulated thandownregulated miRNAs (Supplementary Table S2). Usingan adjusted FDR of 0.3, 38 of 117 (33%) miRNAs weresignificantly differentially expressed between cancer versusnormal (Table 1), wherein 23 were upregulated and 15were downregulated. The expression pattern of these listedmiRNAs in Table 1 with >2-fold change is presented as aclustered heat map (Fig. 1), showing a distinct cluster be-tween normal and HNSCC samples. The most significantaberration was a >32-fold downregulation of miR-375,observed in 46 of 51 (91%) HNSCC samples. Other dis-tinct dysregulations included overexpression (>4-fold) ofmiR-106b, miR-142-3p, and miR-423 as well as downre-gulation of miR-125b and miR-140. Interestingly, many ofthese differentially expressed miRNAs were located on thesame chromosomal region. For example, chromosome13q31.3 harbored the largest number of upregulatedmiRNAs, including miR-20a, miR-92, miR-17-5p, andmiR-19b. Both 7q22.1 (miR-93, miR-106b, and miR-25)and Xq26.2 (miR-92, miR-19b, and miR-106a) containedthree upregulated miRNAs. On the other hand, severaldownregulated miRNAs were located on the commonlydeleted regions of chromosome 11q24.1 (miR-125b-1,let-7a, and miR-100) or 21q21.1 (miR-125b-2, miR-99a,and let-7c).Correlation of miRNA expression with clinical parameters.

The 51 HNSCC patient samples investigated in this studywere derived from the larynx, oropharynx, or hypophar-ynx. Euclidean clustering analysis showed that the globalmiRNA profiles from these three subsites were indistin-guishable; the supervised clustering in Fig. 1 also showedno apparent grouping. The expression value of individualmiRNAs was then directly compared between each tumorsubsite with that of normal (Supplementary Fig. S1),

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showing five significantly differentially expressed miRNAs(P < 0.05). However, using a FDR cutoff of 0.3, only miR-133b remained significantly differentially expressed acrosstumor subsites (FDR P = 0.01), in that it was significantlyoverexpressed in laryngeal SCCs compared with tumorsarising from either the oropharynx or the hypopharynx.In terms of miRNA expression as a function of disease

status, only miR-451 was significantly differentially ex-pressed (Supplementary Fig. S2), with a 4.7-fold higher



expression in the 9 nonrelapsed versus the 42 relapsedpatient samples (FDR < 0.3, P = 0.0009). There were 13additional differentially expressed miRNAs (using a FDRcutoff of <0.4): 1 with lower expression in nonrelapsedand 12 with higher expression between nonrelapsed andrelapsed patients (Supplementary Table S3). The patternof the other top six miRNAs with differential expressionbetween patients with or without relapse is shown in Sup-plementary Fig. S2.

Table 1. List of 38 differentially expressed miRNAs in HNSCC

miRNAs
Raw P FDR-adjusted P
Mean of log2

T:N ratio
Chromosomal location % Ca
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Clinical

American Associ

Described inprevious HNSCCstudies

miR-125b
0 0.12 −2.43 11q24.1/21q21.1* 78 10, 11 miR-375 0 0.12 −5.03 2q35 91 8 miR-423 0 0.12 2.33 17q11.2 73 — miR-93 0 0.12 1.95 7q22.1 75 11 let7a 0.01 0.15 −1.4 9q22.32/11q24.1/22q13.31* 65 10 miR-106b 0.01 0.16 2.5 7q22.1 82 — miR-16 0.01 0.15 1.42 13q14.3/3q26.1* 60 — miR-20a 0.01 0.15 1.86 13q31.3 78 — miR-10a 0.02 0.17 −1.72 17q21.32 69 — miR-140 0.02 0.17 −2.66 16q22.1 85 10 miR-155 0.02 0.17 1.86 21q21.3 71 9, 11 miR-193a 0.02 0.18 1.78 17q11.2 67 — miR-25 0.02 0.17 1.42 7q22.1 56 11 miR-92 0.02 0.17 0.87 13q31.3/Xq26.2* 35 — let-7i 0.03 0.18 1.23 12q14.1 47 9, 11 miR-100 0.03 0.18 −1.64 11q24.1 62 — miR-143 0.03 0.18 −1.82 5q33.1 62 — miR-17-5p 0.03 0.18 1.87 13q31.3 71 — miR-19b 0.03 0.18 1.52 13q31.3/Xq26.2* 69 — miR-223 0.03 0.18 1.74 Xq12 67 — miR-27a 0.03 0.18 1.39 19p13.12 58 — miR-99a 0.03 0.18 −1.83 21q21.1 65 10 miR-142-3 0.04 0.2 2.22 17q23.2 78 9, 10 miR-210 0.04 0.2 1.41 11p15.5 62 — miR-106a 0.05 0.2 1.34 Xq26.2 60 — miR-15a 0.05 0.2 1.35 13q14.3 58 — miR-21 0.05 0.22 1.88 17q23.1 78 8–11 miR-30c 0.05 0.2 −1.19 1p34.2 49 — miR-29b 0.06 0.24 1.11 7q32.3/1q32.3* 51 — miR-365 0.06 0.23 −1.2 16p13.12 58 — miR-127 0.07 0.26 −1.33 14q32.31 67 — miR-130b 0.07 0.25 1.3 22q11.21 56 8 let-7c 0.08 0.27 −1.06 21q21.1 49 — miR-199b 0.08 0.27 −1.17 9q34.11 58 — miR-205 0.08 0.27 1.01 1q32.2 58 — miR-422b 0.08 0.27 0.9 5q33.1 44 — let-7e 0.09 0.27 −0.96 19q13.33 44 — miR-26a 0.09 0.27 −0.91 3p22.2/12q14.1* 45 —
Abbreviations: T, tumor; N, normal.*Taqman miR primers target the same sequence of mature miRNA that originated from different stem-loop sequences.

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ation for Cancer




Functional characterization of HNSCC-associatedmiRNAs. To understand the biological roles of these differ-entially expressed miRNAs in HNSCC, the expression ofthe top 38 miRNAs (Table 1) was measured in three hu-man HNSCC cell lines: FaDu, UTSCC-8, and UTSCC-42a.From this panel of deregulated miRNAs, consistent upre-gulation of miR-25, miR-27a, miR-423, miR-93, and miR-106b as well as downregulation of miR-10a were observed



in all three cell lines (Supplementary Fig. S3). Hence, thesesix miRNAs, together with miR-375 (the most consistentaberration detected in the patient samples), were selectedfor further evaluation.Validation of the differentially expressed miRNAs on

HNSCC cells. Gain-of-function effects of miR-10a were in-vestigated in the three HNSCC cell lines using the targetedpre-miR miRNA precursor molecule. At 72 hours after

Fig. 1. Differentially expressed miRNAs in HNSCC. Supervised hierarchical clustering of 38 differentially expressed miRNAs with fold changes of >2(from Table 1) between 51 HNSCC and 4 normal samples. Tissue samples are represented in the columns, and differentially expressed miRNAs aredelineated in rows. Red indicates overexpression; green represents downregulation. Tumors (gray to black) and normals (blue) clustered separately;however, no distinct separation was observed for tumors arising from the three different subsites of larynx (black), oropharynx (darker gray), or hypopharynx(lighter gray).




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transfection with pre-miR-10a (30 nmol/L), miR-10a ex-pression was elevated by >1,000-fold in all three celllines, particularly for the FaDu cells, compared withLipofectamine 2000 or pre-miR negative control (30nmol/L; Fig. 2A). Despite this augmented miR-10a expres-sion, no significant change in cell number was observed inany of the cell lines (data not shown).The four overexpressed miRNAs (miR-25, miR-27a,

miR-93, and miR-423) were then targeted using the LNAapproach in the three HNSCC cell lines (Fig. 2B-E). De-creased expression was observed in all cell lines assayedat 72 hours after transfection, with the greatest reductionobserved for miR-25 and miR-27a by >100-fold. However,no consistent effect on cell viability or proliferation wasobserved (data not shown).Transfection of miR-375 on HNSCC cells. The most

downregulated miRNA was miR-375, with >32-fold lowerexpression in HNSCC versus normal tissues (Table 1).Transfection of miR-375 in the three HNSCC cells showedsignificant induction of miR-375 expression comparedwith pre-miR negative control–treated cells as early as 24hours, persisting until 72 hours after transfection for theFaDu cells (Fig. 3A). Similar increase in expression was ob-served for the UTSCC-8 and UTSCC-42a cells at 72 hours(Fig. 3B).



As illustrated in Fig. 3C, increased miR-375 expressioncaused a significant reduction in cell viability observed at48 hours (13.9%, P = 0.008) and 72 hours (14%, P = 0.04)after transfection, which translated to a 40% reduction inclonogenic survival (P = 0.01; Fig. 3D). However, neithercell viability nor colony-forming ability was significantlyaffected in the UTSCC-8 or UTSCC-42a cells (data notshown).Cell cycle analysis showed a modest increase (4.4%) in

the proportion of FaDu cells at sub-G1, first observed at 48hours after pre-miR-375 transfection, which increased to7.5% at 72 hours (P = 0.005; Supplementary Fig. S4A).UTSCC-8 and UTSCC-42a did not show any significantchanges in cell cycle (data not shown).Effect of miR-106b knockdown on HNSCC cells. The most

upregulated miRNA was miR-106b, being 5.56-fold higherin HNSCC versus normal tissues (Table 1). Using a 2-foldcutoff, miR-106b was overexpressed in 42 of 51 (82%) ofhead and neck tumors. Hence, knockdown of miR-106bby LNA was investigated in FaDu, UTSCC-42a, andUTSCC-8 cells, all showing significant reduction of expres-sion level compared with LNA scramble–treated cells, ob-served as early as 24 hours, persisting until 72 hours aftertransfection (Fig. 4). The greatest reduction was observedin the UTSCC-8 cells by >100-fold at 72 hours (Fig. 4C).

Fig. 2. Validation of HNSCC-associated miRNAs in cell lines. Evaluation of five differentially expressed miRNAs in HNSCC cell lines. A, FaDu, UTSCC-8,and UTSCC-42a cells were treated with Lipofectamine, negative precursor miR sequences (30 nmol/L), or pre-miR-10a (30 nmol/L). LNA sequences(40 nmol/L) were transfected into each of the three HNSCC cells for miR-25 (B), miR-27a (C), miR-93 (D), and miR-423 (E), with expression level assayedat 72 h after transfection, compared with Lipofectamine and scrambled LNA sequences (40 nmol/L). Each experiment has been conducted threeindependent times. The data are normalized to the relative expression of untreated cells, presented as mean fold change ± SE.






The knockdown in miR-106b expression was in turn as-sociated with reduced viability (Fig. 5), particularly for theFaDu and UTSCC-8 cells (Fig. 5A and C). LNA knockdownof miR-106b also reduced clonogenic survival for bothFaDu and UTSCC-42a cells, down to 60% and 75%, re-spectively, compared with scrambled LNA–treated cells(P = 0.004 and 0.01, respectively; Fig. 5D). In contrast, col-ony-forming ability was not significantly affected in theUTSCC-8 cells.Cell cycle analysis was investigated at 72 hours after

miR-106b knockdown. A modest increase in the propor-tion of cells arrested at G1 was observed for the FaDu,UTSCC-42a, and UTSCC-8 cells, by 4.7%, 4.1%, and4.3%, respectively (Supplementary Fig. S4B). In turn, thiswas associated with a slight reduction in the G2-M popu-lation; no change in the sub-G1 phase was observed.

Discussion

A global miRNA profiling was conducted on 51 archivalFFPE samples of locally advanced stage HNSCC arisingfrom the larynx, oropharynx, and hypopharynx. Approxi-mately one third (38 of 117) of consistently detected



miRNAs were aberrantly expressed. Some of these miRNAshave been previously described in HNSCC studies, such asmiR-21 (8–11). Upregulation of miR-21 is among thecommonest miRNA alterations described for human can-cers (21), reported to be antiapoptotic (22), cause cell pro-liferation (23–25), and promote invasion and metastases(24, 26). Several mRNA targets of miR-21 have been iden-tified, including PTEN, RECK, NFIB, TPM-1, PDCD4, andMaspin (24–27). Specific to HNSCC, knocking down miR-21 has been shown to increase cytochrome c release withsubsequent apoptosis (9). Identification of miR-21 overex-pression (by >3.5-fold) in 78% of our samples further cor-roborated the importance of miR-21 in HNSCC.In the present study, the most significantly downregu-

lated miRNA was miR-375, being underexpressed by 32-fold compared with normal tissues (Table 1). miR-375was first noted in pancreatic islet cells (28) and is involvedin pancreatic islet development (29) and insulin secretion(30). In cancers, miR-375 is downregulated in hepatocel-lular carcinoma, associated with β-catenin mutations (31).A previous HNSCC study reported consistent downregula-tion of miR-375 and suggested that the ratio of high miR-221 to low miR-375 could distinguish cancer from normal

Fig. 3. Transfection of miR-375. The effects of miR-375 transfection in HNSCC cells. A, FaDu cells were treated with Lipofectamine, pre-miR negativecontrol (10 nmol/L), or pre-miR-375 (10 nmol/L), with quantitative RT-PCR analysis conducted 24, 48, or 72 h after transfection. B, quantitative RT-PCRanalysis of miR-375 expression level on UTSCC-8 and UTSCC-42a cells treated with pre-miR negative control (10 nmol/L) or pre-miR-375 (10 nmol/L)at 72 h after transfection. C, MTS assays were conducted on FaDu cells at 24, 48, and 72 h after treatment with Opti-MEM, pre-miR negative control(10 nmol/L), or pre-miR-375 (10 nmol/L). D, clonogenic assay of FaDu cells done 10 to 12 d after transfection with pre-miR negative control (10 nmol/L) orpre-miR-375 (10 nmol/L). Each experiment has been conducted three independent times; the data are normalized to cells treated with Lipofectaminealone, plotted as the mean fold change ± SE.




10 Unpublished data.

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tissues (8). In our current study, miR-221 was not signifi-cantly overexpressed; hence, we were unable to corrobo-rate this observation. Functional analyses showed thattransfection of miR-375 decreased cell proliferation andclonogenicity of FaDu cells (Fig. 3), along with an increasein the sub-G1 population (Supplementary Fig. S4A). Thesefinding are consistent with a previous report documentingthat miR-375–deficient mice are hyperglycemic, with de-creased pancreatic α-cell mass suggestive of impaired pro-liferation (30). Using a combinatorial analysis of putativetargets and microarray expression data, these authorsshowed that candidate miR-375 targets included genes in-volved in apoptosis, normal development, and regulationof cell growth and proliferation (30). Hence, underexpres-sion of miR-375 in HNSCC would result in dysregulatedproliferation and apoptosis, leading to uncontrolled cellgrowth.We have also identified a group of novel HNSCC-asso-

ciated miRNAs, wherein 5 of the top 10 differentially ex-pressed miRNAs are new to HNSCC, such as upregulatedmiR-423, miR-106b, miR-16, and miR-20a as well asdownregulated miR-10a (Table 1). The dysregulation ofthese novel miRNAs is recapitulated in the HNSCC cell



lines, suggesting their potential role in disease progression(Fig. 2). The potential significance of the miR-106b-25cluster was suggested by our knockdown studies of miR-25, miR-93, and miR-106b, all resulting in significant re-duction in their respective miRNA expression levels in theHNSCC cell lines (Figs. 2 and 4). miR-106b is the miRNAwith the highest (5.6-fold) overexpression in our cohort ofpatient samples. Functional analyses further showed thatknocking down miR-106b decreased proliferation and clo-nogenicity of HNSCC cells (Fig. 5), along with G1 arrest(Supplementary Fig. S4B), similar to another study onesophageal cancer (32). The specific downstream targetsof miR-106b in HNSCC remain to be dissected, but pre-liminary results from our in vitro mRNA profiling studysuggest that cell cycle and cell proliferation genes are likelyrelevant.10 In other cancers, miRNAs located on the miR-106b-25 cluster are involved in deregulating E2F1 activity,resulting in transforming growth factor-β (TGF-β) resis-tance (33). The mechanisms are multileveled, but this

Fig. 4. Extent of miR-106b knockdown. Reduction in miR-106b expression in HNSCC cells transfected with LNA targeting miR-106b (40 nmol/L) comparedwith scrambled miR sequence (40 nmol/L). Quantitative RT-PCR was done at 24, 48, and 72 h after transfection in FaDu (A), UTSCC-42a (B), andUTSCC-8 (C) cells. Each experiment has been conducted three independent times; the data are normalized to cells treated with Lipofectamine alone,plotted as the mean fold change ± SE.






miR-106b-25 cluster could be activated by E2F1, in paral-lel with its host gene Mcm7, which in turn interferes withTGF-β signaling via suppression of p21 mediated by miR-106b and miR-93 as well as silencing of Bim via miR-25(34). As an added level of complexity, miR-106b and miR-93 can independently regulate E2F1 expression, leading toa negative feedback loop, which is probably important inpreventing E2F1 self-activation and apoptosis (34). Simi-lar observations have been reported for the miR-106b-25cluster in neoplastic transformation of Barrett's esophagusmediated by suppression of p21 and Bim (32).The role of deregulated TGF-β signaling in HNSCC pro-

gression is complex (35, 36), wherein both low levels TGF-β expression and loss of TGF-β RII have been described(37, 38). Similarly, overexpression of E2F1 and MCM7has been observed (39), correlated with increased inva-siveness (40, 41) and more aggressive disease (42), andsuggested to be predictive markers for nodal metastases(43). Our current study strongly suggests the biologicalsignificance of the miR-106b-25 cluster in HNSCC, indi-cating that overexpression of this cluster might play an on-cogenic role, perhaps mediated by E2F1 activation andimpaired TGF-β signaling, ultimately resulting in uncon-trolled proliferation, dysregulated cell cycling, and in-creased invasiveness.



The mechanisms leading to aberrant miRNA expressionin human cancers remain unclear. Previous studies haveidentified that most (53%) of the dysregulated miRNAsare located in cancer-associated genomic regions or fragilesites (44). In this current study, we have also observednonrandom distributions of aberrant miRNA, whichmap to chromosomal regions that have been previouslydescribed in association with HNSCC. Specifically,miRNAs located on the oncogenic miR-17-92 polycistronwere consistently upregulated in our investigated samples(Table 1), including increased expression of miR-20a,miR-92, miR-17-5p, and miR-19b-1, all located in themiR-17-92 cistron at the c13ORF25 of 13q31. Similarly,overexpression of miR-92a, miR-19b, miR-106a, andmiR-20b (Supplementary Table S2), which belong to thehomologue of miR-106a-92 cluster, located on chromo-some Xq26 (Table 1). In addition, upregulation of theaforementioned miR-106b-25 cluster, comprising miR-106b, miR-93, and miR-25, which are all located in intron13 of the MCM7 gene. These three regions are frequentlyamplified in HNSCC (42, 45–49). Alternatively, we iden-tified underexpressed miRNAs mapping to two frequentlydeleted regions in HNSCC (45, 47, 49–51), such as11q24.1 (miR-125b, let-7a, and miR-100) and 21q21.1(miR-99a and let-7c). These results collectively show that

Fig. 5. Viability and clonogenicity after miR-106b knockdown. MTS assays were conducted on HNSCC cells at 24, 48, and 72 h after transfection withOpti-MEM, scrambled LNA sequences (40 nmol/L), or LNA targeting miR-106b (40 nmol/L). The data are all normalized to viability on day 0. A, FaDu cells.B, UTSCC-42a cells. C, UTSCC-8 cells. D, clonogenic survival of HNSCC cells conducted 10 to 12 d after transfection with either scrambled LNAsequences (40 nmol/L) or LNA targeting miR-106b (40 nmol/L). Each experiment has been conducted three independent times; the data are plotted asthe mean ± SE.




Hui et al.

1138


one of the common mechanisms of aberrant miRNA ex-pression in HNSCC is a consequence of genomic amplifi-cations or deletions.In relation to clinical parameters, our current study shows

no distinct differences in the global miRNA profiles be-tween squamous cell cancers arising from the larynx, oro-pharynx, or hypopharynx. The only exception was miR-133b, which was more highly expressed in laryngeal versusthe other two subsites; the significance of which remains tobe defined. This lack of distinct miRNA expression patternas a function of subsite would be concordant with previoussimilar studies wherein no differences in expression profileswere observed among tongue, oropharyngeal, and laryn-geal cancer cell lines (12) or differential expression ofmiR-21 and miR-494 among these subsites (9). Given ouraforementioned hypothesis that aberrant miRNA expres-sion is likely arising from genomic aberrations, this wouldbe consistent with reports on similar patterns of chromo-somal abnormalities among laryngeal, oropharyngeal,and hypopharyngeal squamous cancers (50).Disappointingly, we were unable to identify a distinct

miRNA pattern between relapsed and nonrelapsed pa-tients in this study, which might be due to the limitedsample size and imbalance between relapsed (42 samples)and nonrelapsed (9 samples) patients. It should be men-tioned that the treatment delivered in the study in whichthese patients were enrolled would be considered of lesserintensity as compared with current standards deliveringhigher doses with or without concurrent chemotherapy.This and the fact that the technical aspects of treatmentplanning and delivery for these patients were differentfrom current practice may account for the relatively highrates of relapse. Disease relapse due to potential under-treatment by technical or dose intensity factors may over-shadow the influence of biological factors and hinder theiridentification. It remains to be seen therefore if therewould exist a correlation between miRNA profiles andoutcome in a more contemporary patient population.The only significant differential expression between these



groups was downregulation of miR-451 in relapsed pa-tients. miR-451 seems to be a key molecule in normal ery-throid differentiation (52) and has also been shown toregulate the multidrug resistance 1 gene in cervix, ovarian,and breast cancer cell lines (53, 54). Underexpression ofmiR-451 has been associated with worse prognosis in gas-tric cancer, with macrophage inhibitor factor suggested asits potential mRNA target (55). Hence, downregulation ofmiR-451 could be a viable candidate marker for HNSCC;we are currently collecting a larger cohort of FFPE samplesto determine the potential role of miR-451 in HNSCC.In conclusion, global miRNA profiling of archival for-

malin-fixed HNSCC samples has identified that approxi-mately one third of the miRNAs are dysregulated in thisdisease, with “anatomic” chromosomal aberrations asone mechanism leading to their abnormal expression.The miR-106b-25 cluster and miR-375 likely mediateHNSCC development and progression; miR-451 couldbe a potential prognostic marker. Further understandingof miRNA biology in HNSCC is a fruitful avenue to derivenovel insights into this complex disease.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Ontario Institute for Cancer Research, Canadian Institutes of Health Re-search, and Dr. Mariano Elia Chair in Head and Neck Cancer Research. Weacknowledge the philanthropic support of the Wharton family, Joe's Team,and Gordon Tozer. Computational analysis was supported by the CanadaResearch Chair Program, Canada Foundation for Innovation (grants 12301and 203383), and IBM. Support also provided from the Campbell FamilyInstitute for Cancer Research, and the Ministry of Health and Long-TermPlanning.

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 toindicate this fact.

Received 8/14/09; revised 12/2/09; accepted 12/4/09; publishedOnlineFirst 2/9/10.

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2010;16:1129-1139. Published OnlineFirst February 15, 2010.Clin Cancer Res Angela B.Y. Hui, Michelle Lenarduzzi, Tiffaney Krushel, et al. Squamous Cell CarcinomasComprehensive MicroRNA Profiling for Head and Neck

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