The Tumor Suppressive MicroRNA miR-218 Targets the Rictor ...cancerres.aacrjournals.org/content/71/17/5765.full.pdf · The Tumor Suppressive MicroRNA miR-218 Targets the mTOR Component

Molecular and Cellular Pathobiology

The Tumor Suppressive MicroRNA miR-218 Targets themTOR Component Rictor and Inhibits AKT Phosphorylationin Oral Cancer

Atsushi Uesugi1,5, Ken-ichi Kozaki1,3, Tomohiko Tsuruta1, Mayuko Furuta1,2, Kei-ichi Morita4,5,Issei Imoto1, Ken Omura2,4,5, and Johji Inazawa1,2,3

AbstractThe incidence of oral squamous cell carcinoma (OSCC) is rising rapidly in developed countries, posing a

growing challenge due to the poor management of this type of malignancy at present. In this study, we profiledtumor suppressive microRNAs (miRNAs) that are silenced by DNA hypermethylation in OSCC using a function-based screening approach. This approach employed a cell proliferation assay for 327 synthetic miRNAs in twoOSCC cell lines. Among the 110 miRNAs identified in this set that exhibited inhibitory properties, we comparedDNA methylation and expression status in a wider panel of OSCC cell lines and primary tumor tissues, resultingin the identification ofmiR-218 andmiR-585 as functionally significant miRNA genes that are frequently silencedin OSCC by DNA hypermethylation. Ectopic expression of miR-218 and miR-585 in OSCC cells lackingendogenous expression reduced cell growth in part through caspase-mediated apoptosis. Notably, miR-218reduced levels of the rapamycin-insensitive component of mTOR, Rictor, in a manner associated with asuppression of Akt S473 phosphorylation. Together our findings definemiR-585 as a tumor suppressive functionthat is often epigenetically silenced in OSCC, and they identify Rictor as a novel target ofmiR-218, suggesting thatactivation of the mTOR-Akt signaling pathway induced by Rictor contributes centrally to oral carcinogenesis.Cancer Res; 71(17); 5765–78. �2011 AACR.

Introduction

MicroRNAs (miRNAs) are an abundant class of endogenous,small, non-coding RNAs, the products of which are smallsingle-stranded RNAs of 19 to 22 nucleotides with a primaryrole in posttranscriptional silencing generally through bindingto the 30-UTR of protein-coding transcripts, in turn triggeringmRNA degradation or translational repression (1). In humancancers, the expression of miRNAs is generally downregulatedin malignant tissues compared with the corresponding non-malignant tissues (2, 3), suggesting the deregulation of miRNAexpression and the contribution of miRNAs to the multistep

processes of carcinogenesis either as oncogenes or as tumor-suppressor genes (TSG; refs. 4, 5). Among various epigeneticmechanisms of cancer-related gene-silencing, DNA hyper-methylation of CpG sites within CpG-islands is known to leadto the inactivation of many TSGs (6) and several tumor-suppressive miRNAs (TS-miRNAs; ref. 7). In fact, DNA methy-lation-mediated downregulation of miRNAs by proximal CpG-islands has been described by a number of groups includingours (8–10).

Oral cancer, predominantly oral squamous cell carcinoma(OSCC), is the most common head and neck neoplasm,affecting approximately 270,000 people worldwide in 2002(11). In Japan, OSCC is relatively common, accounting formore than 5,500 deaths in 2003 (12). The carcinogenesis,including OSCC (13), is considered to arise through theprogressive accumulation of multiple genetic abnormalities,which may impair the functions of oncogenes or TSGs thatplay a crucial role in the development of this disease. Inaddition, evidence has emerged that epigenetic mechanisms,such as altered DNA methylation patterns, play a significantrole in the silencing of TSGs and contribute to malignanttransformation during oral carcinogenesis (14).

In this study, we identified and characterized TS-miRNAsfrequently silenced by DNA hypermethylation in OSCC andtheir targets using function-based screening with a cell pro-liferation assay for 327 synthetic miRNAs and a series ofsequential analyses of DNA methylation and expression inOSCC cell lines and primary tumors. The function-based

Authors' Affiliations: 1Department of Molecular Cytogenetics, MedicalResearch Institute and School of Biomedical Science; 2Global Center ofExcellence (GCOE) Program for International Research Center for Mole-cular Science in Tooth and Bone Diseases; 3Department of GenomeMedicine; 4Department of Advanced Molecular Diagnosis and Maxillofa-cial Surgery, Hard Tissue Genome Research Center; and 5Department ofOral and Maxillofacial Surgery, Tokyo Medical and Dental University,Tokyo, Japan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Johji Inazawa, Department of Molecular Cyto-genetics, Medical Research Institute, Tokyo Medical and Dental Univer-sity, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 03-5803-5820; Fax: 03-5803-0244; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-0368

�2011 American Association for Cancer Research.

CancerResearch

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on October 22, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 27, 2011; DOI: 10.1158/0008-5472.CAN-11-0368

http://cancerres.aacrjournals.org/

A

Analysis of growth inhibitory effects in 2 OSCC cell lines

by MTT assay (see Figure 1B and Supplementary Table S2)

Database analysis for locus and existence of CpG islands

5′′ upstream of candidate miRNAs

Database analysis for previous reports about the correlation

with DNA hypermethylation or tumor-suppressive activities

Methylation analysis in 18 OSCC cell lines

(see Figure 1C, 2A, B and Supplementary Fig. S1)

Expression analysis in 18 OSCC cell lines

(see Figure 2C and Supplementary Fig. S1)

2 miRNAs were identified as new TS-miRNAs silenced by DNA

hypermethylation (consistency of DNA hypermethylation

with downregulation in OSCC cell lines = 70%; see Table 2)

Confirmation of growth inhibitory effects in OSCC cell lines

using dsRNAs purchased from different companies (see Figure 2D)

10 miRNAs (20 CpG islands, 11 loci) that showed high-

methylation frequency in OSCC cell lines (> 50%),

were selected as possible novel TS-miRNAs (see Table 2)

25 miRNAs (35 CpG islands, 26 loci) were selected as candidates

of novel TS-miRNAs silenced by DNA hypermethylation

in OSCC cell lines (see Table 1)

51 miRNAs were selected as possible targets for DNA

hypermethylation except for miRNAs located on X-chromosome

110 miRNAs that remarkably inhibited cell growth were selected

as candidates for TS-miRNAs (growth ratio in WST < 0.5)

Functional screening of TS-miRNAs using Pre-miR miRNA

Precursor Library-Human V2 (Ambion) consisting of 327 miRNAs

Methylation and expression analyses in 15 primary OSCC cases

(see Supplementary Figures 2 and Table 2)

miR-218 was TS-miRNA frequently silenced

by CpG-island hypermethylation in primary OSCC cases

(consistency of DNA hypermethylation with down regulation

in primary OSCC cases = 70%; see Table 2)

Screening of predicted targets of miR-218 (see Figure 4)

Rictor was identified as a novel target of miR-218

Ra

tio

of

co

ntr

ol a

bso

rba

nc

e

98 miRNAs

3

0

0.5

1

2

NA

Pre-miRs (327 miRNAs)

0

0.5

1

2

3

SKN3

53 miRNAs

B

1 dsRNA mimicking mature form of miR-218 showed

remarkable tumor-suppressive activities

in OSCC cell lines (see Figure 3)

Candidate

miRNA gene Locus

No. of

CpG islands in

this analysis Region

Methylation

frequency

in each

region (%)

miR-375 4.491353q2

68.93.382

2.273

1.114

3.385

miR-383 2.2715122p8

63.96.552

3

miR-218-1 9.8312313.51p4

57.9

9.832

0.053

6.554

8.775

8.776

9.887

9.888

9

miR-218-2 2.2201331.53q5

9.8311

9.8321

8.7731

miR-486 9.8812212.11p8

45.8

2

3

4

3.335

23 6

24 7

0.08

6.5952

10

9.8811

2.222162

8.7231

0.0014172

28 15

miR-326 11q13.4 14 1 27.88.722

miR-28 6.510182q3

22.20.02

4.443

miR-365-1 0.01421.31p61

22.2

0.02

0.03

miR-365-2 0.0452.11q71

1.165

2.276

miR-455 2.2217123q9

18.1

0.052

0.0381

0.0491

20 5

miR-345 0.0122.23q41

16.70.02

0.03

7.664

miR-139 0.01114.31q11

8.30.02

0.03

4

3.335

let-7i 1.11111.41q21

3.70.02

0.03

miR-191/-425 0.014313.12q3

0.00.02

0.03

miR-22 0.0193.31p71 0.00.02

miR-423 0.01612.11q71 0.00.02

miR-491 0.01923.12p9 0.00.02

miR-24-1 9q22.32 30 1

2

3

miR-24-2 19p13.12 31 4

5

miR-149 2q37.3 12 1

2

miR-208a 14q11.2 13 1

miR-409 14q32.31 7 1

2

miR-432 14q32.31 6 1

Mean of

methylation

frequency

in each

miRNA genes

(%)RT

7C

a-9

-22

HO

-1-N

-1

HO

C31

3H

OC

81

5

HS

C2

HS

C3

HS

C4

HS

C5

HS

C6

HS

C7

KO

N

KO

SC

2N

AO

M1

OM

2S

KN

3T

SU

ZA

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

miR-489 8.771533.12q7 77.8miR-483 0.0011125.51p11

75.3

2 100.0

3

0.0014

4.495

7.666

2.277

8

0.059

4.4401

0.0511

ND

ND

C

Completely digested

Partially digested

Not digested

Not determined

miR-497 17p13.1 8 1

74.44.492

0.0013

9.884

9.885

0.06

ND

Uesugi et al.

Cancer Res; 71(17) September 1, 2011 Cancer Research5766




screening presented in this paper is a powerful tool forexploring a large number of double-stranded RNAs (dsRNAs)having tumor-suppressive effects, including TS-miRNAs andsiRNAs, as therapeutic agents for several types of cancer cellsdirectly (15, 16). Through this approach, 2 epigeneticallysilenced TS-miRNAs in OSCC, miR-218 and miR-585, whichare located within the intron of SLIT2 and SLIT3, respectively,and a novel target of miR-218, Rictor, were identified in ourstudy. This study is the first to show clearly that the tumor-suppressive activity of miR-585 is epigenetically silenced inOSCC and that miR-218 targets Rictor inducing the activationof a TOR-Akt signaling pathway.

Materials and Methods

Cell lines and primary tumor samplesDerivations of and culture conditions for cell lines were

reported previously (17). OSCC cell lines were authenticated inour previous studies of array-CGH analyses (17). RT7, humanoral keratinocytes immortalized by TERT and human papillo-mavirus (HPV) type 16 E6/E7 oncogenes, was kindly providedby Dr N. Kamata (Hiroshima University Faculty of Dentistry,Japan). To analyze the restoration of genes, cells were culturedwith or without 10 mmol/L of 5-aza 20-deoxycytidine (5-aza-dCyd) for 5 days. A total of 15 frozen primary samples wereobtained from OSCC patients treated at Tokyo Medical andDentalUniversitywithwritten consent fromeach patient in theformal style and after approval by the local ethics committee.

Transfection with synthetic miRNAs and siRNAsA total of 10 nmol/L of dsRNA mimicking human mature

miRNAs or control nonspecific miRNA (Ambion; ThermoScientific Dharmacon), and 20 nmol/L of target-specific siR-NAs for PIK3CA (Dharmacon) and Rictor (Invitrogen) or theircontrol nonspecific siRNA were transfected individually intoOSCC cells using Lipofectamine RNAiMAX (Invitrogen). Thenumbers of viable cells were assessed by the colorimetricwater-soluble tetrazolium salt (WST) assay (Cell counting kit-8; Dojindo Laboratories). The cell cycle was evaluated by afluorescence-activated cell sorting (FACS) analysis asdescribed elsewhere (17).

Methylation analysisEach of gene and CpG-island was searched miRBase data-

base (release April 17, 2011; ref. 18), UCSC Genome Browser onHuman February 2009 Assembly (hg19; ref. 19) and PubMed(20). The combined bisulfite restriction analysis (COBRA) andthe bisulfite-sequencing analysis, using primer sets designed

to amplify regions of interest (Supplementary Table S1), weredone as described elsewhere (8).

Real-time reverse transcription-PCRReal-time reverse transcription PCR (RT-PCR) was done as

described elsewhere (8).

miRNA target predictions, Western blotting, andluciferase activity assay

Predicted targets for miRNAs and their target sites wereanalyzed using Microcosm Targets (21), miRanda (22), andTargetScan (23). Anti-caspase-3, anti-cleaved caspase-3, anti-cleaved PARP, anti-Akt, anti-phospho-Akt (Ser-473), anti-Rictor(Cell Signaling Technology) and anti-PIK3CA (Upstate Biotech-nology) rabbit polyclonal antibodies were used in Westernblotting. Luciferase constructs were made by ligating oligonu-cleotides containing the 30-UTR target sites downstream of theluciferase gene in thepMIR-REPORT luciferase vector (Ambion).Luciferase activity was measured as described elsewhere (8).

Statistical analysisDifferences between subgroups were tested with theMann–

Whitney U test.

Results

Function-based screening of TS-miRNA in OSCC celllines

To identify TS-miRNAs in OSCC, we first examined 327synthetic miRNAs mimicking human mature miRNAs byfunction-based screening in 2 OSCC cell lines, NA andSKN3. The strategy used and partial results obtained areshown in Figure 1A. Since the proliferation-inhibitory effectwas made an index for the rating of tumor-suppressive activityin our function-based screening, relative cell growth ratios inFigure 1B and Supplementary Table S2 indicate effects of eachsynthetic miRNA on cell growth 5 days after transfection. Inthis first screening, 110 miRNAs, including known TS-miRNAs,such asmiR-34 (24),miR-124 (9, 25),miR-193a (8), andmiR-491(15), showed remarkable inhibitory effects on cell growth(relative growth ratio < 0.5). Then we selected novel miRNAswith CpG-islands in the region 50 upstream or around thesegenes and excluded miRNAs previously reported to correlatewith DNA hypermethylation and their tumor-suppressiveactivity among 110 miRNAs by means of database analyses.From the first screening, 25 mature sequences of miRNAsemerged as candidates for new TS-miRNAs silenced by DNAhypermethylation in OSCC cell lines (Table 1).

Figure 1. Strategy employed and results of a function-based screening of TS-miRNAs in OSCC cell lines. A, strategy used for the identification ofepigenetically silenced TS-miRNAs in OSCC. B, result of function-based screening of TS-miRNAs in NA and SKN3 cells using Pre-miR miRNA PrecursorLibrary-Human V2 (Ambion). Cells (1.0 � 104/well) were inoculated onto 24-well plates. Following 24 hours of incubation, 10 nmol/L of 327 dsRNAmimicking mature miRNA or control nonspecific miRNA was transfected individually into cells. The numbers of viable cells 5 days after transfection wereevaluated by the WST assay in duplicate. Relative cell growth ratios were then calculated by normalization of each result to the cell numbers in controlcells transfectedwith nonspecificmiRNA (see Supplementary Table S2). The lower solid arrow indicates the 327miRNAs examined. The top andmiddle closedarrows indicate 98 miRNAs and 53 miRNAs which showed marked growth inhibitory effects in NA and SKN3 cells, respectively (growth ratio < 0.5). C,summary of the DNA methylation status of CpG-islands around 25 mature sequences of miRNAs located at 26 loci including 35 CpG-islands in 18 OSCC celllines and RT7 as a control determined by COBRA. PCR products used for COBRAwere digested with BstUI, TaqI, orHhaI, and electrophoresed (see Figure 2Aand B, Tables 1, 2, Supplementary Fig. S2, Supplementary Table S1, and data not shown). Black, gray, white, and striped boxes indicate complete,partial, and no digestion by restriction enzymes, and not determined, respectively.

miR-218 Targeting Rictor

www.aacrjournals.org Cancer Res; 71(17) September 1, 2011 5767




Tab

le1.

Sum

maryof

25miRNAs(35CpG

island

s,26

loci)s

elec

tedas

cand

idates

forno

velT

S-m

iRNAsilenc

edbyDNAhy

permethy

latio

nin

func

tion-bas

edsc

reen

ingus

ingOSCC

celllines

andPre-m

iRmiRNAPrecu

rsor

Library

-Hum

anV2(Ambion)

Num

ber

of

cand

idate

miRNAs

miRNAs

miRNA

gen

esLo

cus

Host

gen

esClustered

miRNAs

Num

ber

of

CpG

island

inthis

analys

is

Distanc

eof

CpG

island

from

themost

50mature

miRNA

seque

nce

Methy

lation

freq

uenc

y(total%

)

1hs

a-let-7i

hsa-let-7i

12q14

.1-

-1

03.7

2hs

a-miR-345

hsa-miR-345

14q32

.2-

-2

523

16.7

3hs

a-miR-375

hsa-miR-375

2q35

--

30

68.9

4hs

a-miR-365

hsa-miR-365

-116

p13

.12

-miR-193

b4

6067

22.2

hsa-miR-365

-217

q11

.2-

miR-193

a5

15,053

5hs

a-miR-432

*hs

a-miR-432

14q32

.31

-miR-337

/665

/431

/43

3/12

7/43

2/13

66

960

ND

6hs

a-miR-409

-3p

hsa-miR-409

14q32

.31

-miR-409

-5p(m

iR-485

/453

/15

4/49

6/37

7/54

1/41

2/36

9/41

0/65

6)

7a0

ND

7hs

a-miR-409

-5p

hsa-miR-409

14q32

.31

-miR-409

-3p(m

iR-485

/453

/15

4/49

6/37

7/54

1/41

2/36

9/41

0/65

6)

7a0

ND

8hs

a-miR-497

hsa-miR-497

17p13

.1-

miR-195

84,06

078

.79

hsa-miR-22

hsa-miR-22

17p13

.3C17

orf92

-9

1,92

40.0

10hs

a-miR-28

hsa-miR-28

3q28

LPP

-10

53,421

822

.211

hsa-miR-139

hsa-miR-139

11q13

.4PDE2A

-11

27,049

8.3

12hs

a-miR-149

hsa-miR-149

2q37

.3GPC1

-12

0ND

13hs

a-miR-208

ahs

a-miR-208

a14

q11

.2MYH6

-13

1,48

2ND

14hs

a-miR-326

hsa-miR-326

11q13

.4ARRB1

-14

15,995

27.8

15hs

a-miR-383

hsa-miR-383

8p22

SGCZ

-15

61,678

163

.916

hsa-miR-423

hsa-miR-423

17q11

.2CCDC55

-16

870.0

17hs

a-miR-455

hsa-miR-455

9q32

COL2

7A1

-17

54,692

18.1

1853

,696

1953

,192

2041

,346

18hs

a-miR-483

hsa-miR-483

11p15

.5IG

F2-

213,51

375

.319

hsa-miR-486

-5p

hsa-miR-486

8p11

.21

ANK1

-22

235,31

545

.823

215,51

024

167,72

325

136,85

026

106,53

027

65,110

2841

,230

20hs

a-miR-491

hsa-miR-491

9p21

.3KIAA17

97-

2931

,468

0.0

(Con

tinue

don

thefollo

wingpag

e)

Uesugi et al.





Methylation and expression analyses of candidates inOSCC cell linesNext, we explored the DNA methylation status of 35 CpG-

islands around 25 miRNAs located at 26 loci in a panel of 18OSCCcell lines andanormal counterpart, RT7, an immortalizedhumanoralkeratinocyte line, byCOBRA(Fig. 1C). Sincemultiplecopies of some mature miRNAs, as listed in Table 1, aretranscribed from different loci, the number of mature formsof miRNAs is smaller than the number of genomic loci. InCOBRA, on the basis of a comparison of mean values ofmethylation frequencies in each region examined, frequentDNA hypermethylation (>50% of OSCC lines) was found in only6of25miRNAs, that is,miR-218,miR-375,miR-383,miR-483,miR-489, and miR-497, although CpG-island hypermethylation on/around these miRNAs was also detected in RT7. We nextinvestigated the consistency in the correlation between DNAmethylation status in each region of these 6 miRNA genes byCOBRA and their expression patterns in 18 OSCC cell lines andRT7 by TaqMan real-time RT-PCR (Table 2), and found thatvalues were higher in some regions ofmiR-218-1 (50.0%–77.8%)and miR-375 (100%) than other miRNA genes (0%–56.3%;Fig. 1A–C and Supplementary Fig. S1A–C). miR-218-1 andmiR-218-2 are located at 4p15.31 and 5q35.1 within the intronof SLIT2 and SLIT3, respectively (Fig. 2A and B). Interestingly,miR-585, together with miR-218-2, is also located within theintronof SLIT3, and the expressionpatternsofmiR-218 andmiR-585weresimilar to thoseofSLIT2andSLIT3, respectively (Fig. 2Band C); note that the percentage of OSCC cell lines withsignificant downregulation of miR-218, miR-585, SLIT2, SLIT3,andmiR-375 expression (<0.5-fold) was 44.4% (8/18), 55.6% (10/18), 61.1% (11/18), 77.8% (14/18), and 100% (18/18), respectively(Fig. 2C and Supplementary Fig. 1C). To determine the relation-ship betweenDNAhypermethylation inCpG-islands on/aroundmiR-218-1, miR-218-2, and miR-375 and the downregulation ofgene expression, we treated OSCC cell lines with 5-aza-dCyd,observing a remarkable restoration of the expression levels ofthese miRNAs and their host genes in 55.6% (10/18), 44.4% (8/18), 33.3% (6/18), 61.1% (11/18), and 100% (18/18) of OSCC celllines, respectively (Fig. 2C and Supplementary Fig. 1D). Con-sistentwiththese results,CpG-islandhypermethylationwasalsoconfirmed by bisulfite-sequencing in the OSCC lines lacking theexpression of those candidate miRNAs, but not in RT7 and thecell lines expressing these miRNAs (Fig. 2A and B and Supple-mentary Fig. 1E). These results strongly suggest that DNAhypermethylation of CpG sites within CpG-islands on/aroundthese genes deregulates their expressions in OSCC cell lines.

Confirmation of dsRNA-induced growth inhibition inOSCC cell linesToconfirm the growth inhibitory effects ofmiR-218,miR-585,

and miR-375 in the function-based screening, we used 2synthetic miRNAs purchased fromAmbion and Thermo Scien-tificDharmacon to take into consideration the off-target effectsof dsRNAs. The ectopic expression of these miRNAs in OSCCcell lines lacking their expressions by the transient transfectionof dsRNAwas evaluated by TaqMan real-time RT-PCR analysis(data not shown). Consistent with the results of the function-based screening (Supplementary Table S2), restoration of

Tab

le1.

Sum

maryof

25miRNAs(35CpG

island

s,26

loci)s

elec

tedas

cand

idates

forno

velT

S-m

iRNAsilenc

edbyDNAhy

permethy

latio

nin

func

tion-bas

edsc

reen

ingus

ingOSCC

celllines

andPre-m

iRmiRNAPrecu

rsor

Library

-Hum

anV2(Ambion)

(Con

t'd)

Num

ber

of

cand

idate

miRNAs

miRNAs

miRNA

gen

esLo

cus

Host

gen

esClustered

miRNAs

Num

ber

of

CpG

island

inthis

analys

is

Distanc

eof

CpG

island

from

themost

50mature

miRNA

seque

nce

Methy

lation

freq

uenc

y(total%

)

24hs

a-miR-24

hsa-miR-24-1

9q22

.32

C9o

rf3

miR-23b

/27b

300

ND

hsa-miR-24-2

19p1

3.12

-miR-23a

/27a

316,05

021

hsa-miR-218

hsa-miR-218

-14p

15.31

SLIT2

-32

273,03

057

.9hs

a-miR-218

-25q

35.1

SLIT3

miR-585

3353

2,17

022

hsa-miR-191

*hs

a-miR-191

3q21

.31

DALR

D3

miR-425

-3p

34a

184

0.0

23hs

a-miR-425

-3p

hsa-miR-425

3p21

.31

DALR

D3

miR-191

*34

a65

90.0

25hs

a-miR-489

hsa-miR-489

7q21

.3CALC

RmiR-653

3590

,755

77.8

aSam

eCpG

island

s.NOTE

:Th

esedatawerese

arch

edmiRBas

eda

tabas

e(re

leas

eApril

17,20

11),UCSC

Gen

omeBrowse

ron

Hum

anFe

brua

ry20

09Ass

embly

(hg1

9)an

dPub

Med

.






Table 2. Correlation between DNA hypermethylation status in each region of these 6 miRNA genesby COBRA and their expression patterns in OSCC cell lines and primary cases by quantitative real-timeRT-PCR analysis

OSCC cell lines Primary OSCC cases

miRNAs miRNA genes Regions inCOBRA

Methylationfrequency(%)a

Consis-tency ofmethylationwith down-regulation(%)b


Consistency ofmethylationwith downre-gulation (%)b

hsa-miR-489 hsa-miR-489 1 77.78 14 0.00 0 - -hsa-miR-483 hsa-miR-483 1 100.00 18 16.67 3 - -

2 100.00 18 16.67 3 - -3 ND - - -4 100.00 18 16.67 3 - -5 94.44 17 17.65 3 - -6 66.67 12 16.67 2 - -7 72.22 13 15.38 2 - -8 ND - - -9 50.00 9 22.22 2 - -10 44.44 8 - - -11 50.00 9 11.11 1 - -

hsa-miR-497 hsa-miR-195/497 1 ND - - -2 94.44 17 47.06 8 - -3 100.00 18 50.00 9 - -4 88.89 16 50.00 8 - -5 88.89 16 56.25 9 - -6 0.00 0 - 0 - -

hsa-miR-375 hsa-miR-375 1 88.89 16 100.00 16 - -2 83.33 15 100.00 15 - -3 72.22 13 100.00 13 - -4 11.11 2 - - -5 83.33 15 100.00 15 - -

hsa-miR-383 hsa-miR-383 1 72.22 13 7.69 1 - -2 55.56 10 10.00 1 - -3 ND - - -

hsa-miR-218 hsa-miR-218-1 1 38.89 7 - 20.00 3/15 100.00 3/32 38.89 7 - 13.33 2/15 100.00 2/23 50.00 9 77.78 7 6.67 1/15 100.00 1/14 55.56 10 70.00 7 6.67 1/15 100.00 1/15 77.78 14 50.00 7 53.33 8/15 75.00 6/86 77.78 14 57.14 8 73.33 11/15 81.82 9/117 88.89 16 50.00 8 66.67 10/15 80.00 8/108 88.89 16 50.00 8 93.33 14/15 85.71 12/149 ND - - -

hsa-miR-585/218-2 10 22.22 4 - 13.33 2/15 100.00 2/211 38.89 7 - 13.33 2/15 100.00 2/212 38.89 7 - 6.67 1/15 100.00 1/113 77.78 14 50.00 7 0 0/15 - -

hsa-miR-486-5p hsa-miR-486 1 88.89 16 31.25 5 - -2 ND - - -3 ND - - -4 ND - - -

(Continued on the following page)

Uesugi et al.





Table 2. Correlation between DNA hypermethylation status in each region of these 6 miRNA genesby COBRA and their expression patterns in OSCC cell lines and primary cases by quantitative real-timeRT-PCR analysis (Cont'd )







5 33.33 6 - - -6 ND - - -7 ND - - -8 0.00 0 0.00 0 - -9 5.56 1 - - -10 ND - - -11 88.89 16 31.25 5 - -12 22.22 4 - - -13 27.78 5 - - -14 100.00 18 27.78 5 - -15 ND - - -

hsa-miR-326 hsa-miR-326 1 ND - - -2 27.78 5 - - -

hsa-miR-28 hsa-miR-28 1 5.56 1 - - -2 0.00 0 - - -3 44.44 8 - - -

hsa-miR-365 hsa-miR-365-1 1 0.00 0 - - -2 0.00 0 - - -3 0.00 0 - - -

hsa-miR-365-2 4 0.00 0 - - -5 61.11 11 54.55 6 - -6 72.22 13 53.85 7 - -

hsa-miR-455 hsa-miR-455 1 22.22 4 - - -2 50.00 9 44.44 4 - -3 0.00 0 0.00 0 - -4 0.00 0 0.00 0 - -5 ND - - -

hsa-miR-345 hsa-miR-345 1 0.00 0 0.00 0 - -2 0.00 0 0.00 0 - -3 0.00 0 0.00 0 - -4 66.67 12 0.00 0 - -

hsa-miR-139 hsa-miR-139 1 0.00 0 - - -2 0.00 0 - - -3 0.00 0 - - -4 0.00 - - -5 33.33 6 - - -

hsa-let-7i hsa-let-7i 1 11.11 2 - - -2 0.00 0 - - -3 0.00 0 - - -

hsa-miR-191*/miR-425-3p

hsa-miR-191/miR-425 1 0.00 0 - - -

2 0.00 0 - - -3 0.00 0 - - -

(Continued on the following page)






miR-218 expression significantly reduced cell growth in theOSCC cell lines tested (Fig. 2D). In addition, a growth inhibitoryeffect of miR-585 was clearly detected in Dharmacon product,whereas Ambion product of dsRNA mimicking miR-585 wasnot on the market and could not be examined. In our in vitroanalysis, tumor-suppressive functions ofmiR-218 and miR-585were confirmed. Ambionproduct of dsRNAmimickingmiR-375induced growth inhibition whereas Dharmacon product facili-tated cell proliferation in both OSCC cell lines, and somiR-375was excluded from candidates in this study.

Methylation and expression analyses of miR-218 andmiR-585 in primary OSCCs

To determine whether the CpG-island hypermethylation ofmiR-218-1/SLIT2 and miR-218-2/miR-585/SLIT3 also occurs inprimary OSCCs in a tumor-specific manner, the correlationbetween DNA methylation status and the expression patternsof these genes in 15 primary OSCCs and corresponding non-cancerous oral mucosae was examined using COBRA andTaqMan real-time RT-PCR assay, respectively. In COBRA, thefrequency of tumor-specific DNA hypermethylation in regionsof miR-218-1 and miR-218-2/miR-585 was 6.7% to 93.4% and6.7% to 13.3%, respectively, in primary OSCCs (Table 2, Sup-plementary Fig. 2A). Although the frequency of aberrantmethy-

lation at these CpG-islands was relatively low in primarytumors, expression levels of miR-218 and miR-585 in tumorsas compared with paired normal oral mucosae were markedlyreduced in 73.3% (11/15) and 66.7% (10/15) of primary OSCCs,respectively (< 0.5-fold expression, Supplementary Fig. 2B), andthe consistencywas comparatively high in these regions ofmiR-218-1 (75.0%–100%) and miR-218-2/miR-585 (100%). AlthoughmiR-218 expression had been reported to be specificallyreduced in HPV-positive cell lines and tissues (26), all of OSCCcell lines and primary samples examined in this study wereconfirmed the negative HPV-status by genomic PCR (Supple-mentary Fig. S3). Thus, our results suggest that miR-218 andmiR-585 aremost likelyTS-miRNAs frequently silenced throughtumor-specific DNA hypermethylation in OSCC.

Tumor-suppressive effects of ectopic miR-218 andmiR-585 expression on the growth of OSCC cell lines

Weexamined tumor-suppressive effects ofmiR-218 andmiR-585 on HSC-2 and NA (Fig. 3A and B). Restoration of theexpression of these miRNAs significantly reduced cell growthin both cell lines tested. Since a large number of transfectantswere rounded and floating comparedwith the control counter-part at 5 days after transfection, we conducted a FACS analysisand a Western blotting for caspase-mediated apoptosis using

Table 2. Correlation between DNA hypermethylation status in each region of these 6 miRNA genesby COBRA and their expression patterns in OSCC cell lines and primary cases by quantitative real-timeRT-PCR analysis (Cont'd )







hsa-miR-22 hsa-miR-22 1 0.00 0 - - -2 0.00 0 - - -

hsa-miR-423 hsa-miR-423 1 0.00 0 - - -2 0.00 0 - - -

hsa-miR-491 hsa-miR-491 1 0.00 0 - - -2 0.00 0 - - -

hsa-miR-24 hsa-miR-24 1 ND - - -2 ND - - -3 ND - - -

hsa-miR-149 hsa-miR-149 1 ND - - -2 ND - - -

hsa-miR-208a hsa-miR-208a 1 ND - - -hsa-miR-409-3p/miR-409-5p

hsa-miR-409 1 ND - - -

2 ND - - -hsa-miR-432* hsa-miR-432 1 ND - - -

aFrequency of cell lines or primary cases, in which DNA hypermethylation were detected by COBRA.bFrequency of cell lines or primary cases, in which downregulation was consistent with DNA hypermethylation.ND, not done.

Uesugi et al.





both cell lines 72 hours after the transfection. In the FACSanalysis, the effect of miR-218 on the cell cycle in HSC-2 cellswasweakwhile the accumulation of NA cells in theG2/Mphasewas observed inmiR-218-transfectants. Overexpression ofmiR-585 induced the accumulation of cells in the G2/M phase andsub-G1 phase in the HSC-2 and NA cell lines, respectively.However, theWestern blotting showed that ectopic expressionofmiR-218 andmiR-585 remarkably increased protein levels ofcaspase3, cleaved caspase3 and cleaved PARP in both cell lines(Fig. 3C), miR-218- and miR-585-induced reductions of cellgrowth in these OSCC cell lines were not inhibited by caspaseinhibitors (Supplementary Fig. S4A). In contrast, the G2/M cell-cycle arrest was not confirmed by Western blotting for thephosphorylation status of Cdc2 andChk1 in these transfectants(Supplementary Fig. S4B). These results, consistent with the

previous report of miR-218-inducing apoptosis in vitro (27),suggest that miR-218 and miR-585 induce a reduction in cellgrowth at least in part through caspase-mediated apoptosis,whereas the cause of the cell death except the apoptosis inthese transfectants remains unclear.

Screening of predicted targets for miR-218 and miR-585in OSCC cell lines

While 5 genes of EGFR-coamplified and overexpressedprotein (ECOP), IkBs kinase (IKK-b), LIM and SH3 protein 1(LASP1), paxillin (PXN), and Robo1 have also been reported asdirect targets of miR-218 (28–32), the percentage of OSCC celllines with notable upregulation of these genes (>2-fold expres-sion) was 50.0% (9/18), 38.9% (7/18), 44.4% (8/18), 16.7% (3/18),and 11.1% (2/18), respectively (Fig. 4A). In addition, we also

RT

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Frequencyof methylationin each region

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Region 3

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Region 1

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miR-218-1

Region 1

CpG island

Region 2

Region 3

Region 4

Region 5

Region 6

Region 7

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Region 9

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Frequencyof methylationin each region

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(-)

Expression of

miR-218/-585

Ca9-22

HSC-2 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

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of miR-218

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Ambion Dharmacon ND, Not determined

SLIT3

100,000200,000

200400

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44.4%(8/18)

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61.1%(11/18)

77.8%(14/18)

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Figure 2.Correlation betweenmethylation and expression ofmiR-218 and the host genesSLIT2 andSLIT3 in OSCC cell lines. A and B,methylation analysis formiR-218-1/SLIT2 (A) and miR-218-2/SLIT3 (B). Top, these maps show intronic miRNAs, host genes, CpG-islands, CpG sites, and PCR products used forCOBRA and bisulfite sequencing. White boxes, exons of SLIT2 and SLIT3; gray box, CpG-island; closed arrows, PCR products (primers, SupplementaryTable S1); vertical tick marks, CpG sites; vertical arrows, restriction enzyme sites. Middle, results of COBRA in 18 OSCC cell lines and RT7. Arrows,unmethylated alleles; arrowheads, methylated alleles; stars, samples with significant restricted fragments from methylated alleles. The presence of restrictionenzyme processing is indicated with a plus or minus sign above the results of COBRA. Bottom, bisulfite sequencing of RT7 and representative OSCCcell lines with (þ) or without (�) miR-218 expression in PCR products examined by COBRA. Horizontal bars with arrowheads, PCR product; vertical arrows,restriction enzyme sites. Open and filled squares represent unmethylated and methylated CpG sites respectively, and each row represents a single clone. C,TaqMan real-time RT-PCR analysis formiR-218,miR-585,SLIT2, and SLIT3 in 18OSCC cell lines and RT7. Top, expression levels of intronic miRNAs and hostgenes were based on the amount of target message relative to RNU6B and GAPDH, respectively, to normalize the initial input of total RNA. Bar graphs showthe ratio of the expression level in each cell line to that in RT7. Bottom, restoration of the expression of these genes after treatment with 10 mmol/L 5-aza-dCydfor 5 days in 18OSCC cell lines. Bar graphs show the ratio of the expression level in treated cells to that in untreated cells. D, confirmation of tumor-suppressiveactivities of 3 candidate miRNAs in HSC-2 and NA cells using dsRNAs purchased from different companies (Ambion and Dharmacon). The numbersof viable cells 5 days after transfection with 10 nmol/L of dsRNAswere assessed byWST assay. Bar graphs show the growth ratio of cell numbers inmiR-218-,-375-, and -585-transfectants relative to those in control-transfectants. Each point represents the mean of triplicate determinations (bars, SD).






A

B

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ds-NC

ds-miR-585

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-585

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-NC

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-miR

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SubG1: 4.95%G0-G1: 76.88%S: 3.37%G2-M: 14.76%

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SubG1: 29.06%G0-G1: 47.28%S: 6.02%G2-M: 17.21%

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Figure 3. Tumor-suppressiveeffects of miR-218 and miR-585on OSCC cell lines, HSC-2 andNA, lacking their expression. Aand B, growth curves, phase-contrast micrographs, and resultsof FACS analysis in HSC-2 (A) andNA (B) cells in which 10 nmol/L ofdsRNA mimicking miR-218 ormiR-585, or control nonspecificmiRNA (ds-NC) was transfected.The numbers of viable cells aftertransfection were assessed byWST assay. Each data pointrepresents the mean of triplicatedeterminations (bars, SD) in theseexperiments. Phase-contrastmicrographs show OSCC celllines cultured for 5 days aftertransfection. The size of thepopulation in each phase of thecell cycle as assessed by FACSusing OSCC cell lines 72 hoursafter transfection. C, the results ofWestern blotting of caspase3,cleaved caspase3, and cleavedPARP in HSC-2 and NA cells48 hours after the transfection ofeach dsRNA.

Uesugi et al.





confirmed that their protein levels were decreased inmiR-218-transfectants compared with their control counterparts(Fig. 4B).To explore novel oncogenic targets of miR-218 and miR-585

in OSCC cell lines, we used the algorithms, MicrocosmTargets,miRanda, and TargetScan, and selected rapamycin-insensitivecompanion of mTOR (Rictor) as miR-218-targets and jun Bproto-oncogene (JunB) as miR-585-targets. The expression ofRictor or JunB was frequently upregulated in 38.9% (7/18) and44.4% (8/18) of OSCC cell lines, respectively, compared withRT7 (>2-fold expression; Fig. 4A and Supplementary Fig. S5A).In Western blotting of Rictor and JunB, their protein levelswere markedly reduced in miR-218- and miR-585-transfec-

tants, respectively, compared with their control counterparts(Fig. 4B and Supplementary Fig. S5B). To further determinewhether the predicted target sites of miR-218 and miR-585 inthe 30-UTR of mRNAs of Rictor and JunB, respectively (Fig. 4Band Supplementary Fig. S5C), were responsible for thetranslational regulation by dsRNA mimicking these miRNAs,we next conducted luciferase assays with vectors containingthese 30-UTR target sites downstream of the luciferasereporter gene. In this analysis, we observed a statisticallysignificant reduction of luciferase activity in a vector con-taining the target site of Rictor, but not JunB (Fig. 4B andSupplementary Fig. S5D). These findings, together with theresults of Western blotting, suggest Rictor and JunB to be a

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EGFR

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AKT

PTENPDK1 P

P

P

P

mTOR

P

mTOR

RaptorSer473 Survival

Growth

Motility

Division

Translation

PI3K

Figure 4. Identification and characterization of Rictor as a novel target of miR-218. A, expression analyses of 5 reported targets and one predicted target,Rictor, in 18 OSCC cell lines and RT7 using TaqMan real-time RT-PCR (top), and Western blotting (bottom). Expression levels of transcripts of thesetargets were based on the amount of target message relative toGAPDH to normalize the initial input of total RNA. Bar graphs show the ratio of the expressionlevel in each cell line to that in RT7. B, identification of Rictor as a novel target ofmiR-218. Top, the results of Western blotting of 5 reported targets and Rictorin HSC-2 and NA cells 48 hours after the transfection of dsRNA mimicking miR-218 or control nonspecific miRNA (ds-NC). Middle, putative bindingsites of miR-218 in the 30-UTR region of Rictor. These sites were analyzed using Microcosm Targets (21), miRanda (22), and TargetScan (23). Bottom,luciferase assays of HSC-2 and NA cells 48 hours after cotransfection of pMIR-REPORT luciferase vectors containing a 30-UTR target site of Rictor formiR-218, dsRNAmimickingmiR-218, or control nonspecific miRNA, and pRL-CMV internal control vector. C, effects ofmiR-218 on Akt activation in OSCC celllines with/without mutant forms of PIK3CA by Western blotting. Both cell lines, HSC-2 with PIK3CA mutation and NA without a genetic alteration of PIK3CA,EGFR, or PTEN, had been shown to be activated their PI3K–Akt signaling pathway (see Figure 4A; ref. 34). Top, the results ofWestern blotting in HSC-2 and NAcells 48 hours after transfection of dsRNA mimicking miR-218, control nonspecific miRNA, PIK3CA-specific siRNA, Rictor-specific siRNA, or controlnonspecific siRNA (si-NC). To determine the increased phosphorylation of Akt protein at Ser-473, levels of total Akt protein in same samples were evaluated.Bottom, relative Akt activities in these transfectants. The quantification of each protein band in the result of Western blotting was done using LAS-3000 withMultiGauge software (Fuji film). The amount of phosphorylated Akt was normalized versus that of total Akt. Bar graphs show the ratio of the Akt activitiesin ds-miR-218-, si-PIK3CA-, and si-Rictor-transfectants relative to those in control transfectants. D, schema of the negative regulation of the mTORC2-dependent Akt signaling pathway by miR-218 in OSCC.






novel direct target of miR-218 and an indirect target of miR-585, respectively. Therefore, we focused on Rictor as the mostlikely target of miR-218, and conducted further analyses toexplore the underlying molecular mechanisms of oral carci-nogenesis.

Rictor, together with the mammalian target of rapamycin(mTOR) kinase, forms mTOR complex 2 (mTORC2), and theRictor-mTOR complex directly regulates the phosphorylationof Akt at Ser-473, resulting in cell growth (33). We havepreviously reported that the phosphorylation of Akt atSer-473 and Thr-308 was markedly increased in a HSC-2 cellline harboring a missense mutation in the PIK3CA gene,A3140G in exon 20, corresponding to the amino acid changeH1047R (34). Interestingly, although NA cells without a geneticalteration of PIK3CA, EGFR, or PTEN also showed phosphory-lated Akt, it has been unknown why the phosphorylationincreased in this cell line. The phosphorylation status ofAkt at Ser-473 in 18 OSCC cell lines and RT7 were shownin Figure 4A. To investigate whether Rictor might be asso-ciated with growth inhibitory effects ofmiR-218 in OSCC cells,we analyzed the phosphorylation of Akt and cell proliferationin HSC-2 and NA cells transfected with or without dsRNAsmimicking miR-218 or specific siRNAs for Rictor and PIK3CA(Fig. 4C). The treatment with PIK3CA- or Rictor-specific siRNAsignificantly inhibited cell growth in HSC-2 and NA cellswhereas we found no noticeable differences in cell growthratio of each transfectant in both cell lines (SupplementaryFig. S6). In Western blotting, the marked inhibition of thephosphorylation of Akt at Ser-473 in HSC-2 cells was observedin the treatment of RNA interference of PIK3CA, but not inrestoration of miR-218 expression and knockdown of Rictorexpression, suggesting that phosphatidylinositol 3-kinase(PI3K) seems mainly to regulate the phospho-Akt activationin HSC-2 cells with a mutant form of PIK3CA. On the otherhand, a notable reduction in the phosphorylated Akt wasdetected in NA cells at 48 hours after the transient transfectionof dsRNAs mimicking miR-218 or specific siRNA for Rictorcompared with specific siRNA for PIK3CA, suggesting Akt tobe significantly activated and phosphorylated by the TOR-Aktsignaling pathway, not PI3K-Akt signaling pathway in NA cellswithout a mutant form of PIK3CA, EGFR, or PTEN. Our resultsclearly showed that miR-218 acts as a suppressor of the TOR-Akt signaling pathway, independently of the PI3K-Akt signal-ing pathway, in OSCC (Fig. 4D).

Discussion

Here, we clearly identified miR-218 and miR-585 as TS-miRNAs silenced through tumor-specific DNA hypermethyla-tion in oral cancer and characterized miR-218 targeting Rictorand inhibiting the phosphorylation of Akt at Ser-473 in a OSCCcell line without a mutant form of PIK3CA, EGFR, or PTEN. Inthis study, we carried out function-based screening using 327dsRNAs mimicking mature miRNAs to identify TS-miRNAshaving remarkable inhibitory effects on the growth of OSCCcell lines, although expression-based and DNA methylation-based screening had been successfully done previously inOSCC (8) and hepatocellular carcinoma (9), respectively. Since

several known TS-miRNAs, such as miR-34 (24), miR-124 (9,25), miR-193a (8), and miR-491 (15), were actually identifiedthrough this approach, function-based screening would besuitable for exploring dsRNAs, including miRNAs and siRNAs,as therapeutic agents for several types of cancer cells. Thetumor-suppressive function of candidate miRNAs eventuallyidentified in this screening was reevaluated using 2 kinds ofdsRNA purchased independently to take account of off-targeteffects, known to complicate the interpretation of phenotypiceffects in gene-silencing experiments using siRNAs (35).

We consider that the hypermethylation of CpG-islands on/around miRNA genes a good marker to explore novel epigen-etically silenced TS-miRNAs, similar to classic TSGs in severaltypes of cancer, and have already reported miR-137,miR-193a,miR-124, andmiR-203 as epigenetically silenced TS-miRNAs (8,9). In this study, a second screening, combining DNA methyla-tion and expression analyses in a panel of OSCC cell lines,resulted in the identification ofmiR-218 andmiR-585 as primecandidates for TS-miRNAs silenced by DNA hypermethylationin OSCC. Our study is the first to show that miR-218 and miR-585 were frequently silenced by DNA hypermethylation inOSCC. This miRNA gene is located at 4p15.31 (miR-218-1) and5q35.1 (miR-218-2) in introns of 2 host genes, SLIT2 and SLIT3,respectively, and miR-585, together with miR-218-2, is alsolocated within the intron of SLIT3 at 5q35.1. Copy numberlosses at these loci and the downregulated expressions ofmiR-218, SLIT2, and SLIT3 were reported in some types of cancer(36) although we found no homozygous loss in these regions inour previous studies of genomewide copy-number aberrationsin 39 OSCC cell lines by array-CGH analyses (17, 37). Frequentepigenetic inactivation of SLIT2 and SLIT3 was also describedin several cancers (38, 39). Although, in our study, the expres-sion levels of these miRNAs and their host genes wereremarkably restored in OSCC cell lines treated with 5-aza-dCyd, it was reported that the treatment of this inhibitor ofDNA methylation did not reactivate the downregulatedexpression of SLITs in cervical cancer (40), suggesting acomplex mechanism of inactivation in Slits in some typesof cancers. Very recently, the downreguration of miR-218 andits inhibitory effects on cell proliferation and invasion havebeen shown in several types of cancer, including HPV-positivecell lines and tissues (26–29, 31, 32, 36, 41), and in the presentstudy, we confirmed the tumor-suppressive functions and themechanism of action of this miRNA in OSCC. Regarding miR-585, however, this is the first report that this is a novel TS-miRNA frequently silenced by tumor-specific DNA hyper-methylation in OSCC, suggesting a crucial role, similar tomiR-218, in human cancers.

In this study, we successfully identified a possible directtarget of miR-218, Rictor, although ECOP, IKK-b, LASP1, PXN,and Robo1 have also been reported as direct targets of thismiRNA. Since ECOP and IKK-b are components of the NF-kBpathway, our findings of caspase-mediated apoptosis in cellsoverexpressing miR-218 strongly support early reports of acorrelation between miR-218 and targets (28, 29). LASP1,PXN, and Robo1 are known to be associated with cell adhesion,mobility, and migration, and their overexpression induced bymiR-218 suppression seem to enhance tumor invasion and

Uesugi et al.





metastasis (30–32). Interestingly, although Slit-Robo signalinghas been described to facilitate tumor cell migration, Robo1targeted by miR-218 is known to be a receptor for Slit which isthehost geneofmiR-218 and shown to formanegative feedbackloop involving Slit, miR-218, and Robo1 (27, 32). In our expres-sion analysis, the percentage of OSCCcell lineswith remarkabledownregulation of miR-218, miR-585, SLIT2, and SLIT3 (< 0.5-fold), expressionwas 44.4% (8/18), 55.6% (10/18), 61.1% (11/18),and 77.8% (14/18), respectively, whereas that with notableupregulation of these genes (>2-fold expression) was 38.9%(7/18), 11.1% (2/18), 33.3% (6/18), and 0% (0/18), respectively.Since the biological and functional significances of Slits-miR-218-Robo signaling in cancer remains largely unknown, furtherstudy is needed for the characterization of this signaling.Rictor is a component of the mTOR-containing complex

mTORC2, which directly regulates the phosphorylation of Aktat Ser-473 (33). Knockdown of Rictor ormTOR in colon cancerwas shown to inhibit cancer cell proliferation in vitro andtumor formation in vivo (42), and the selective requirement ofRictor for the development of prostate cancer induced by a lossof Pten in mice was shown (43). These results including ourfindings strongly support the notion that the silencing ofmiR-218 through the hypermethylation of CpG-islands around thismiRNA is likely to be an important mechanism of carcinogen-esis and cancer progression at least partly involving the activa-tion of mTORC2-Akt signaling in OSCC. In this study, weanalyzed the effects of miR-218 on the phosphorylation ofAkt using 2 representative OSCC cell lines; HSC-2, with amissense mutation in the PIK3CA gene and activated PI3K-Akt signaling pathway, and NA, without genetic alterations ofPIK3CA, EGFR, or PTEN (34). Although it has been unknownwhy the phosphorylation of Akt is increased in NA cells, ourfindings clearly showed miR-218 to act as a suppressor of theTOR-Akt signaling pathway, independently of the PI3K-Aktsignaling pathway, in OSCC, and that the activation of this

signaling pathway through methylation-mediated silencing ofmiR-218 may contribute to the pathogenesis of OSCC.

In conclusion, we described here the identification of 2 TS-miRNAs, miR-218 and miR-585, frequently silenced by DNAhypermethylation in OSCC, using function-based screeningand a series of sequential analyses. Moreover, we identifiedRictor as a potential target of miR-218, suggesting that theepigenetic silencing of miR-218 and consequent activation ofthe TOR-Akt signaling pathway induced by the overexpressionof Rictor contribute to oral carcinogenesis, and that targetingmiR-218 may provide a novel strategy for the treatment ofOSCC, although further studies in vivo will be needed toconfirm that dsRNA mimicking miR-218 can act as a TS-miRNA.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Ayako Takahashi and Rumi Mori for technical assistance.

Grant Support

This study was supported in part by Grant-in-Aid for Scientific Research (A),(B), and (C), and Scientific Research on Priority Areas and Innovative Areas, anda Global Center of Excellence (GCOE) Program for International ResearchCenter for Molecular Science in Tooth and Bone Diseases from the Ministryof Education, Culture, Sports, Science, and Technology, Japan; a Health andLabour Sciences Research Grant by the Ministry of Health, Labour and Welfare,Japan; and a grant from the New Energy and Industrial Technology Develop-ment Organization (NEDO).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 8, 2011; revised June 20, 2011; accepted July 13, 2011;published OnlineFirst July 27, 2011.

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2011;71:5765-5778. Published OnlineFirst July 27, 2011.Cancer Res Atsushi Uesugi, Ken-ichi Kozaki, Tomohiko Tsuruta, et al. Cancer

and Inhibits AKT Phosphorylation in OralRictorComponent Targets the mTORmiR-218The Tumor Suppressive MicroRNA

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