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Human Cancer Biology

LINE-1 Hypomethylation, DNA Copy Number Alterations,and CDK6 Amplification in Esophageal SquamousCell Carcinoma

Yoshifumi Baba1, Masayuki Watanabe1, Asuka Murata1, Hironobu Shigaki1, Keisuke Miyake1,Takatsugu Ishimoto1, Masaaki Iwatsuki1, Shiro Iwagami1, Naoya Yoshida1, Eiji Oki3,Kentaro Sakamaki4, Mitsuyoshi Nakao2, and Hideo Baba1

AbstractPurpose: Global DNA hypomethylation plays a crucial role in genomic instability and carcinogenesis.

DNA methylation of the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good

indicator of the global DNA methylation level, and is attracting interest as a useful marker for predicting

cancer prognosis. Our previous study using more than 200 esophageal squamous cell carcinoma (ESCC)

specimensdemonstrated the significant relationship betweenLINE-1hypomethylation andpoor prognosis.

However, themechanismbywhich LINE-1 hypomethylation affects aggressive tumor behavior has yet to be

revealed.

Experimental Design: To examine the relationship between LINE-1 hypomethylation and DNA copy

number variations, we investigated LINE-1–hypomethylated and LINE-1–hypermethylated ESCC tumors

by comparative genomic hybridization array.

Results: LINE-1–hypomethylated tumors showed highly frequent genomic gains at various loci contain-

ing candidate oncogenes such as CDK6. LINE-1methylation levels were significantly associated with CDK6

mRNA and CDK6 protein expression levels in ESCC specimens. In our cohort of 129 patients with ESCC,

cases with CDK6-positive expression experienced worse clinical outcome compared with those with CDK6-

negative expression, supporting the oncogenic role of CDK6 in ESCC. In addition, we found that the

prognostic impact of LINE-1 hypomethylation might be attenuated by CDK6 expression.

Conclusion: LINE-1 hypomethylation (i.e., global DNA hypomethylation) in ESCCmight contribute to

the acquisitionof aggressive tumorbehavior through genomic gains of oncogenes such asCDK6.ClinCancer

Res; 20(5); 1114–24. �2014 AACR.

IntroductionEsophageal squamous cell carcinoma (ESCC), the major

histologic type of esophageal cancer in East Asian countries,is one of the most aggressive malignant tumors (1). Despiteremarkable advances in multimodal therapies, includingsurgery, chemotherapy, radiotherapy, and chemoradiother-apy, the prognosis of patients remains poor, even for those

whose carcinomas have been completely resected (2–4). Todevelop innovative strategies for treating ESCC, especiallythose that are molecularly targeted (5), it is of particularimportance to increase our understanding of the cellularand molecular basis of this disease. Importantly, epigeneticchanges, including alterations in DNA methylation, arereversible, and can thus be targets for cancer therapy orchemoprevention (6–8).

DNAmethylation alterations occurring inhuman cancersinclude globalDNAhypomethylation and site-specific CpGisland promoter hypermethylation (9). Global DNA hypo-methylation seems to play a crucial role in genomic insta-bility, leading to cancer development (10–12). Becauselong interspersed nucleotide element-1 (LINE-1; L1 retro-transposon) constitutes a substantial portion (approxi-mately 17%) of the human genome, the level of LINE-1methylation is regarded to be a surrogate marker of globalDNA methylation (13). In many types of human neo-plasms, LINE-1 methylation has been shown to be highlyvariable (14–16), and LINE-1 hypomethylation is stronglyassociated with a poor prognosis (17–22). In a previousstudy of 217 curatively resected ESCC specimens (23), we

Authors' Affiliations: 1Department of Gastroenterological Surgery, Grad-uate School of Medical Sciences; 2Department of Medical Cell Biology,Institute of Molecular Embryology and Genetics, Kumamoto University,Kumamoto; 3Department of Surgery and Science, Graduate School ofMedical Sciences, Kyushu University, Fukuoka; and 4Department of Bio-statistics and Epidemiology, Yokohama City University Medical Center,Yokohama, Japan

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

Corresponding Author: Hideo Baba, Graduate School of MedicalSciences, Kumamoto University, 1-1-1 Honjo, KumamotoCity, Kumamoto860-8556, Japan. Phone: 81-96-373-5213; Fax: 81-96-373-4378; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-1645

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(5) March 1, 20141114

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Published OnlineFirst January 14, 2014; DOI: 10.1158/1078-0432.CCR-13-1645

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demonstrated that LINE-1 hypomethylation is associatedwith a poor prognosis, suggesting that LINE-1 hypomethy-lation may be a biomarker that can be used to identifypatients whowill experience an inferior outcome.However,the mechanism by which LINE-1 hypomethylation (andthus global DNA hypomethylation) may confer a poorprognosis remains to be fully explored. Given the knownrelationship between genome-wide DNA hypomethylationand genomic instability (24–27), we hypothesized thatglobal DNA hypomethylationmight be an important influ-ential factor for DNA copy number variations, leading toaltered expression levels of oncogenes or tumor suppressorgenes.To test this hypothesis, we investigated LINE-1–hypo-

methylated and LINE-1–hypermethylated ESCC tumorsusing array-based comparative genomic hybridization(CGH array). LINE-1–hypomethylated tumors showedhighly frequent genomic gains at various loci containingcandidate oncogenes such asCDK6. Thus, we further exam-ined the correlation between LINE-1 methylation levels,CDK6 amplification, CDK6 protein and mRNA expression,and clinical outcome.

Materials and MethodsStudy subjectsAll samples used in this study were collected at Kuma-

moto University Hospital (Kumamoto, Japan) betweenApril 2005 and December 2011. LINE-1 methylation levelswere quantified in 40 ESCC tumors for which freshly frozenspecimens were available. Total RNA was obtained from 20ESCC tumors, matched with 15 macroscopically normalesophageal tissues from the same patients. To assess theprognostic impact of CDK6 expression, we performed

immunohistochemical staining of 129 ESCC samples. Nopatient received any preoperative treatment (chemothera-py, radiation therapy, or chemoradiotherapy). All 129patients underwent curative resection. Tumor staging wasperformed according to the American Joint Committee onCancer Staging Manual (seventh edition; ref. 28). Patientswere observed at 1- to 3-month intervals until death or untilMarch 31, 2013, whichever came first. Disease-free survivalwas defined as the period following surgical cancer treat-ment during which the patient survived with no sign ofcancer recurrence. Among the 129 patients, 48 experienceddisease recurrence during the follow-up period. This currentanalysis represents a new analysis of CDK6 on the existingESCC database that has been previously characterized forLINE-1methylation and clinical outcome (23).Wehavenotexamined CDK6 expression in any of our previous studies.Written informed consent was obtained from each subject,and the study procedureswere approved by the institutionalreview board.

Pyrosequencing to measure LINE-1 methylationGenomic DNA was extracted from frozen esophageal

cancer specimens using a QIAamp DNA Mini Kit (Qiagen)according to the manufacturer’s protocol. Genomic DNAwas modified with sodium bisulfite using an EpiTect Bisul-fite kit (Qiagen). PCR and subsequent pyrosequencing forLINE-1 were performed as previously described by Oginoand colleagues, using the PyroMark Kit (Qiagen; ref. 14).This assay amplifies a region of LINE-1 element (position305 to 331, accession no. X58075), which includes fourCpG sites. Pyrosequencing reactions were performed usingthe PyroMark Q24 System (Qiagen). Bisulfite pyrosequen-cing consists of three steps; bisulfite conversion, PCR ampli-fication, and pyrosequencing analysis. Unmethylated cyto-sine and methylated cytosine are differentiated by bisulfitetreatment followed by PCR. In the pyrosequencing step, thecytosine:methylated cytosine ratio at each CpG site is mea-sured as the ratio of thymine:cytosine. The cytosine contentrelative to the cytosine plus thymine content at each CpGsite is expressed as a percentage. In this study, the averagerelative cytosine content at the four CpG sites was used asthe overall LINE-1 methylation level in a given tumor. Inpublished literature, we have validated our LINE-1 meth-ylation pyrosequencing assay; we performed bisulfite con-version onfive differentDNA specimen aliquots and repeat-ed PCR pyrosequencing five times using four macrodis-sected cancers. Bisulfite-to-bisulfite (between-bisulfite treat-ment) SDs ranged from 1.4 to 2.9 (median, 2.3), and run-to-run (between-PCR pyrosequencing run) SDs rangedfrom 0.6 to 3.3 (median, 1.2; ref. 29).

Immunohistochemical stainingParaffin-embedded tumor sections were dewaxed in

xylene and ethanol, and antigen–epitope retrieval was per-formed using a streamer autoclave at 120�C for 15 minutesin antigen retrieval solution (pH9, Histofine; Nichirei Bios-ciences Inc.) and then allowed to cool. Endogenous perox-idase activity was blocked using 3% hydrogen peroxide.

Translational RelevanceThe level of long interspersed nucleotide element-1

(LINE-1) methylation is regarded to be a surrogatemarker of global DNAmethylation. We have previouslydemonstrated that LINE-1 hypomethylation in esoph-ageal squamous cell carcinoma (ESCC) is associatedwith a shorter survival, thus suggesting that it has poten-tial for use as a prognostic biomarker. However, themechanism by which LINE-1 hypomethylation affectsaggressive tumor behavior has yet to be revealed. In thisstudy, comparative genomic hybridization arrayrevealed that LINE-1–hypomethylated ESCC tumorspresented highly frequent genomic gains at various locicontaining CDK6. In addition, LINE-1 methylationlevels were associated with CDK6 expression in ESCCsand that the prognostic impact of LINE-1 hypomethyla-tion in patients with ESCC was attenuated by CDK6expression. Collectively, these findings may suggest thatglobal DNAhypomethylation in ESCCmight contributeto the acquisition of aggressive tumor behavior throughgenomic gains of oncogenes such as CDK6.

LINE-1 Methylation and CDK6 in Esophageal Cancer

www.aacrjournals.org Clin Cancer Res; 20(5) March 1, 2014 1115




Primary antibodies against CDK6 (1:50 dilution; SantaCruz Biotechnology Inc.) and cyclin D1 (1:50 dilution;Leica Biosystems Newcastle Ltd) were applied, and theslides were incubated at 4�C overnight. The secondaryantibody used was a ready-for-use anti-mouse EnVisionPeroxidase system (Dako Japan Inc.). The remaining pro-cedure was performed using a Dako EnVisionþ System(Dako Japan Inc). The stained slides were counterstainedwith hematoxylin and bluing reagent. One of the investi-gators, blinded to any other participant data, recordednuclear CDK6 and cyclin D1 expression as absent, weak,moderate, or strong. In this study, tumors with weak tostrong expression were defined as "positive," whereastumors not expressing these proteins were defined as "neg-ative." Among 129 ESCC tumors, 61 tumors (47%) and 52tumors (40%) tested positive for CDK6 and cyclin D1,respectively.

Quantitative reverse transcription PCRTotal RNA extraction, cDNA synthesis, and quantitative

reverse transcription PCR (qRT-PCR) were carried out aspreviously described (30, 31). Total cellular RNA wasextracted using the RNeasy Mini Kit (Qiagen), and cDNAwas synthesized with the SuperScript III Transcriptor FirstStrand cDNA Synthesis System for RT-PCR (Invitrogen)according to the manufacturers’ instructions. qRT-PCR wascarried out using a LightCycler 480 II instrument (Roche).To determine the differences in the gene expression levelsbetween specimens, the 2�DDCt method was used to mea-sure the fold changes among the samples. To carry out qRT-PCR, primers were designed using the Universal ProbeLibrary (Roche) following the manufacturer’s recommen-dations. Theprimer sequences andprobes used for real-timePCR were as follows: CDK6, 5- TGATCAACTAGGAAAAA-TCTTGGA-30, 5-GTCGACCGGTGCAATCTT-30, and univer-sal probe #2; glyceraldehyde-3-phosphate dehydrogenase(GAPDH), 5-AGCCACATCGCTCAGACAC-30, 5-GCCCAA-TACGACCAAATCC-30, and universal probe #60.

Array CGHArray CGH was carried out using the Agilent oligonucle-

otide array–based CGH microarray kit (SurePrint G3Human CGH Microarray Kit 2 � 400 K; Agilent Technol-ogies) according to the manufacturer’s protocol. Commer-cial male human genomic DNA was used as the controlDNA. Following hybridization, washing, and drying steps,themicroarray slideswere scanned at 3mmresolution, usingthe G2505C microarray scanner (Agilent Technologies).Features were extracted from the scanned images using theFeature Extraction software (version 1.5.1.0; Agilent Tech-nologies). The extracted features were analyzed using theAgilent Cytogenomics software (version 2.0.6.0; AgilentTechnologies).

Cell linesHuman esophageal cancer cell lines (TE-1, TE-4, TE-6, TE-

8, TE-9, TE-10, TE-11, TE-14, and TE-15) were obtainedfrom the Institute of Development, Aging, and Cancer,

Tohoku University (Sendai, Japan) and were cultured ina medium supplemented with 10% FBS in 5% CO2 atmo-sphere at 37�C.

FISHCDK6-specific FISH probe was prepared using two

human Bacterial Artificial Chromosomes (BAC) DNAclones RP11-809H24 and RP11-1102K14 by labeling withCy3. As a control, a centromeric region-specific FISH probefor chromosome 7 was prepared using RP11-90C3. Theimages were captured with the CW4000 FISH applicationprogram (Leica Microsystems Imaging Solution).

Statistical methodsFor the statistical analyses, we used the JMP software

(version 9; SAS Institute). For the survival analysis, theKaplan–Meier method was used to assess the survival timedistribution, and the log-rank test was used.We constructeda CDK6-adjusted Cox proportional hazard model to com-pute the HR according to the LINE-1 methylation status.Statistical differences were considered significant if P valueswere <0.05. All reported P values are two-sided.

ResultsPyrosequencing assay for LINE-1 methylation statususing frozen ESCC tissues

We quantified the LINE-1 methylation levels of 40 fresh-frozen esophageal cancer tissues using a bisulfite PCRpyrosequencing assay, and obtained valid results in all 40cases. Representative pyrograms for LINE-1 methylationlevels are shown in Fig. 1A. LINE-1 methylation levels inESCC fresh-frozen tissues were distributed widely andapproximately normally. The distribution was as follows:mean, 51.0; median, 52.6; SD, 14.8; range, 24.4–79.7;interquartile range, 38.6–62.5 (all in 0–100 scale; Fig. 1B).

Relationship between LINE-1 methylation levels andchromosomal aberrations

We have previously demonstrated that LINE-1 hypo-methylation is associated with a poor prognosis in a studyof 217 ESCC paraffin-embedded specimens (23). However,the biologicmechanismbywhich LINE-1 hypomethylationaffects aggressive tumor behavior is not yet fully under-stood. We hypothesized that global DNA hypomethylationmight be a crucial influential factor for DNA copy numbervariations. Thus, we carried out a CGH array analysis usingthree LINE-1–hypomethylated tumors (LINE-1 methyla-tion level, 24.4% for case #1; 31.4% for case #2; and33.5% for case #3) and three LINE-1–hypermethylatedtumors (65.3% for case #4; 65.9% for case #5; and79.2% for case #6). The analyzed cases were selected basedon sufficient amount of freshly frozen tissues and sufficientDNA quality. Their clinical and pathologic characteristicsare shown in Supplementary Table S1. Pyrograms for cases#1 and #4 are shown in Fig. 1A.

The average number of chromosomal aberrations in 6ESCC tumors was 168.2 (range, 24–423). Interestingly,LINE-1–hypomethylated tumors showed higher frequency

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Clin Cancer Res; 20(5) March 1, 2014 Clinical Cancer Research1116




of chromosomal gain or loss comparedwith LINE-1–hyper-methylated tumors. Figure 2A shows a summary of DNAcopy number aberrations for LINE-1–hypomethylatedtumors and LINE-1–hypermethylated tumors. The numberof aberrations was significantly higher in LINE-1–hypo-methylated cases (median, 310.0) than in LINE-1–hyper-methylated cases (median, 26.3; P ¼ 0.0076; Fig. 2B). Asimilar result was observed for total length of aberrations (P¼ 0.0013; Fig. 2B). Regions with an abnormal number ofchromosome copies in all hypomethylated tumors areshown in Table 1. Genomic gains were much more com-mon than losses, and genomic imbalance was not found inchromosome 13, 14, 15, or 16. Amplification of 7q21–22

containing a candidate oncogene CDK6was observed in allLINE-1–hypomethylated tumors, whereas this locus wasnot amplified in all LINE-1–hypermethylated tumors. Pre-vious studies have shown that CDK6 amplification is asso-ciated with CDK6 overexpression and poor survival inesophageal cancer, supporting that CDK6 might markmalignant esophageal cancer (32, 33). From these findings,we further hypothesize that global DNA hypomethylationin ESCC contributes to aggressive phenotype by genomicgain of oncogenes such as CDK6. Thus, the correlationbetween CDK6 aberrant expression (protein and mRNAlevels) and LINE-1 hypomethylation in ESCCs was furtherinvestigated.

Figure 1. LINE-1 methylationlevels in esophageal cancers. A,representative pyrograms forLINE-1 methylation level(cases #1 and #4). B, LINE-1methylation levels in 40 resectedesophageal cancers. Dark stainedbars indicate six selectedcases for CGH array(#1–3 as LINE-1–hypomethylatedcases and #4–6 asLINE-1–hypermethylatedcases).






Relationship between CDK6 expression and LINE-1methylation in ESCC tissues

WemeasuredCDK6mRNA expression levels by qRT-PCRin 15 ESCCs tissues andmatched normal esophagealmuco-sa. Cancer tissues showed significantly higher levels ofCDK6mRNA expression than matched esophageal mucosa(P¼ 0.0046; Fig. 3A). Then, CDK6 expression at the proteinlevel in ESCCs was evaluated by immunohistochemistry.CDK6 immunoreactivity was absent in normal squamouscell epithelium (Fig. 3B-a). Among 40 tumors, 24 tumors(60%) showed CDK6-positive expression (Fig. 3B-b), and16 tumors (40%) showed CDK6-negative expression (Fig.3B-c). These observations of CDK6 overexpression maydemonstrate that CDK6 has vital roles in ESCC tumorigen-esis. To evaluate the impact of LINE-1 hypomethylation onCDK6 expression, we assessed the association betweenCDK6 expression and LINE-1 methylation levels in ESCCtissues. In 20 ESCCs for which LINE-1 data and RNA wereavailable, CDK6 mRNA expression levels were inverselyassociated with LINE-1 methylation levels (P ¼ 0.039; Fig.3C). Tumors with CDK6-positive immunoreactivity exhib-

ited significantly lower LINE-1methylation levels (median,42.3; mean, 45.1; SD, 13.1) than tumors with negativeimmunoreactivity (median, 60.1; mean, 60.0; SD, 12.8; P¼ 0.0001 by the paired t test; Fig. 3D). These resultscollectively suggest that LINE-1 methylation level (andtherefore global DNA methylation level) might have aninfluence on CDK6 expression through genomic gain ofCDK6.

Relationship between CDK6 expression and LINE-1methylation in ESCC cell lines

We then tested this correlation between LINE-1 methyl-ation levels and CDK6 expression levels using nine ESCCcell lines. LINE-1 methylation levels in nine ESCC cell lineswere widely distributed (range, 22.8–67.0). The inverserelationship between LINE-1 methylation levels and CDK6mRNA expression levels was observed (P¼ 0.028; Fig. 4A).Furthermore, to confirm the gene amplification of CDK6 inESCCs,we applied the FISHanalysis for TE11 cell line (blackdot in Fig. 4B), which demonstrated lower levels of LINE-1methylation and high levels of CDK6 mRNA expression in

Figure 2. Relationship between chromosomal aberrations and LINE-1 methylation. A, summary of DNA copy number aberrations for LINE-1–hypomethylatedcases (top) and LINE-1–hypermethylated cases (bottom). Losses (green) are displayed to the left and gains (red) plotted to the right. B, relationship ofLINE-1 methylation status with number of aberrations (top) and total length of aberrations (bottom).

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Table 1. Chromosome imbalance regions in LINE-1–hypomethylated tumors

Cytoband Aberration Start position Stop position Size Candidate gene �log10 (P)

1q21 Gain 143224322 150145845 6921524 LOC100130000 1.301029991q21 Gain 150145845 150437139 291295 ANP32E, CA14 1.301029991q21 Gain 150437139 153949859 3512721 1.301029991q21 Gain 153949859 154295703 345845 JTB, RAB13 1.301029991q21 Gain 154295703 155309112 1013410 1.301029991q22 Gain 155309112 155904332 595221 ASH1L, MIR555 1.301029991q21 Gain 155904332 156796795 892464 S100A4 1.301029991q21–22 Gain 156815942 161569102 4753161 NTRK1,INSRR 1.301029991q42 Gain 196800817 225328624 28527808 CNIH3 1.301029991q42 Gain 225334959 227316832 1981874 DNAH14,LBR 1.301029991q44 Gain 248738898 248789540 50643 OR2T10 1.301029991q21 Gain 248789540 249211884 422345 LOC100130000 1.301029992q21 Gain 50765550 52053468 1287919 FSHR, NRXN1 1.301029992q35 Loss 219296581 219500621 204041 VIL1,USP37 1.301029993q21 Loss 50733803 50889948 156146 DOCK3,p21.2 1.301029993q21 Loss 51474959 51486485 11527 VPRBP 1.301029993p14 Loss 71118465 71129430 10966 FOXP1,p13 1.301029993q13 Gain 117950181 162209974 44259794 IGSF11 1.301029993q29 Gain 193132436 193156879 24444 ATP13A4,q29 1.301029993q29 Gain 193156879 194964380 1807502 C3orf21,q29 1.301029994q25 Loss 113504214 113802089 297876 C4orf21,LARP7 1.301029994q28 Loss 128854114 129114533 260420 MFSD8 1.301029995p15 Gain 223613 256351 32739 SDHA, p15.33 1.301029995p15 Gain 822005 887520 65516 ZDHHC11 1.301029995p15 Gain 9197190 14749366 5552177 SEMA5A 1.301029995q31 Loss 125891676 125896749 5074 ALDH7A1 1.301029996p21 Loss 32542694 32565064 22371 HLA-DRB1 1.301029996q22 Gain 125315619 125934949 619331 RNF217 1.301029996q23 Gain 134625874 134958897 333024 SGK1,CpG:21 1.301029997p22 Gain 1886724 2203806 317083 MAD1L1,CpG 1.301029997p21 Gain 7283309 26893878 19610570 C1GALT1 1.301029997q31 Gain 26930268 55712859 28782592 LANCL2 1.301029997q11 Gain 69085548 70537428 1451881 AUTS2,CpG 1.301029997q21 Gain 78545354 86367244 7821891 MAGI2 1.301029997q21–22 Gain 92247365 96665717 4418353 CDK6 1.301029997q31 Gain 107825148 110714112 2888965 NRCAM 1.301029998q11 Gain 49434350 54585979 5151630 EFCAB1,SNAI2 1.301029998q21 Gain 75130234 79574535 4444302 JPH1,GDAP1 1.301029998q22 Gain 105068014 120115974 15047961 RIMS2,TM7SF4 1.301029998q24 Gain 131275342 140955982 9680641 ASAP1 1.301029999q21 Gain 82261109 94649728 12388620 TMC1,TLE4,TLE1 1.301029999q22 Gain 104105604 109937886 5832283 BAAT, MRPL50 1.301029999q33 Gain 117848315 121937952 4089638 TNC, DEC1 1.3010299910q11 Gain 52685729 63413583 10727855 PRKG1,MIR605,ZNF488,RBP3 1.3010299910q11 Gain 77139109 83286274 6147166 LOC441666,ZNF503 1.3010299910q26 Gain 124348251 124351778 3528 DMBT1,q26.13 1.3010299911p11 Gain 49170280 49714750 544471 FOLH1 1.3010299911q22 Gain 107091691 107502415 410725 CWF19L2 1.3010299912p13 Gain 2690550 2786288 95739 CACNA1C 1.3010299912p12 Gain 21613937 21622974 9038 PYROXD1 1.3010299912p11 Gain 33663998 34591668 927671 ALG10,CpG:25 1.30102999

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vitro. We counted the number of signals for CDK6 andchromosome 7 centromere (control) in 100 TE11 cells. In91 of 100 cells, the number of signals for CDK6 was largerthan that for chromosome 7 centromere (SupplementaryTable S2), confirming the presence of CDK6 amplificationin ESCC cells.

CDK6 expression, LINE-1 hypomethylation, andpatient survival

To understand the relationship between CDK6 andtumor malignant behavior in ESCC, we examined theprognostic role of CDK6 in 129 cases with availableLINE-1 methylation data and CDK6 expression data. Allcases received no preoperative treatment. The relationshipsbetween levels of LINE-1 methylation and clinical andpathologic features are summarized in SupplementaryTable S3. The median follow-up time for censored patientswas 2.7 years. Patients with tumor expressing CDK6 (n ¼

61) experienced lower disease-free survival rates thanpatients not expressing CDK6 (n ¼ 68; log-rank P ¼0.043, Fig. 5A). This result certainly implies that CDK6mayhave oncogenic roles in ESCC and contribute to ESCCprogression.

We next examined whether the influence of LINE-1hypomethylation on patient prognosis was modified byCDK6 expression status. In this current study, LINE-1hypomethylation was defined as "<55.5" according to ourprevious article (23). In Kaplan–Meier analysis, patientswith LINE-1 hypomethylation (n ¼ 31) experienced signif-icantly shorter disease-free survival compared with LINE-1–hypermethylated cases (n ¼ 98; log-rank P ¼ 0.0005; Fig.5B). In univariate Cox regression analysis, compared withLINE-1–hypermethylated cases, patients with LINE-1 hypo-methylation experienced a significantly higher diseaserecurrence rate [HR, 2.69; 95% confidence interval (CI),1.48–4.78; P¼ 0.0015; Fig. 5C]. In the CDK6-adjusted Cox

Table 1. Chromosome imbalance regions in LINE-1–hypomethylated tumors (Cont'd )

Cytoband Aberration Start position Stop position Size Candidate gene �log10 (P)

17q11 Gain 31344645 33156978 1812334 ACCN1,CCL2 1.3010299917q21 Gain 39092472 39890632 798161 KRT23,KRT39 1.3010299920p13 Gain 6343032 25493634 19150603 DEFB125,BMP2,HAO1 1.3010299920q12–13 Gain 40674508 41626812 952305 PLCG1,ZHX3,PTPRT, q12 1.3010299920q13 Gain 60895001 62070367 1175367 BMP7,SPO11,LAMA5,RPS21 1.30102999X Gain 62063537 154788597 92725061 SPIN4 1.30102999Y Loss 17972056 26066972 8094917 1.30102999Y Loss 28548426 28784095 235670 CpG:32,q11.23 1.30102999

Figure 3. Association of CDK6expression andLINE-1methylationlevels. A, CDK6 mRNA expressionlevels in esophageal cancers andmatched normal mucosa. Thecancer tissues showedsignificantly higher levels ofexpression than the matchednormal mucosa (P ¼ 0.0046 by thepaired t test). B, a, CDK6immunostaining for esophagealcancer and normal esophagealmucosa. Cancerous lesion showspositive staining, whereas normalmucosa shows negative staining.b, positive expression ofCDK6 in nuclei of esophagealcancer cells. c, negativeexpression of CDK6 in nuclei ofesophageal cancer cells. C,correlation between LINE-1methylation levels and CDK6mRNA expression levels. D,CDK6-positive tumors showedsignificantly lower levels of LINE-1methylation than CDK6-negativetumors (P¼ 0.00010 by the paired ttest).

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model, the HR of LINE-1 hypomethylation for diseaserecurrence was decreased to 2.25 (95% CI, 1.22–4.04).Thus, this result shows the proportional reduction in theregression coefficient for LINE-1 methylation due to theinclusion of CDK6 expression in the Cox regression model.In the multivariate Cox model adjusted for sex (male vs.female), age at surgery (<64vs. 65�), tumor location (uppervs. lower), tumor stage (stage I vs. II, III), histologic grade(G1 vs. G2–4), andCDK6 expression (positive vs. negative),LINE-1 hypomethylation was found to be associated with asignificantly higher recurrence rate (multivariate HR, 2.43;95% CI, 1.19–4.89; P ¼ 0.016).

Cyclin D1 expression, LINE-1 methylation level, andpatient survivalGiven that cyclin D1 is the positive regulatory partner of

CDK6,we further examined the relationship between cyclin

D1 expression, LINE-1 methylation, and patient outcome.No relationshipwas observed between cyclinD1 expressionand level of LINE-1 methylation (P ¼ 0.94; SupplementaryFig. S2A and S2B). We also found no association betweenstatus of cyclinD1 expression and disease-free survival (log-rank P ¼ 0.60; Supplementary Fig. S2C). Furthermore, theprognostic impact of LINE-1 hypomethylation was notreduced by cyclin D1 expression (cyclin D1-adjusted HR,2.74; 95% CI, 1.51–4.88).

DiscussionWe previously demonstrated the correlation between

LINE-1 hypomethylation (i.e., global DNA hypomethyla-tion) and poor prognosis in ESCCs (23). In this currentstudy, toclarify themechanismbywhichLINE-1hypomethy-lation affects ESCC aggressive behavior, we investigated

Figure 4. CDK6 mRNA expressionand CDK6 amplification in ESCCcell lines. A, relationshipbetween LINE-1 methylation levelsand CDK6 mRNA expressionlevels. Black spots indicate TE11cell lines analyzed by FISH.B, FISH images with the CDK6probe (red) and thechromosome 7 centromereprobe (green; control).

Figure 5. Survival analyses of CDK6expression and LINE-1 methylationlevel. A, Kaplan–Meier curvesaccording to CDK6 expressionstatus. B, Kaplan–Meier curvesaccording to LINE-1 methylationstatus. C, patients with LINE-1hypomethylation experienced asignificantly higher diseaserecurrence rate (HR, 2.69). In theCDK6-adjusted Cox model, the HRof LINE-1 hypomethylation fordisease recurrence was decreasedto 2.25. D, possible mechanismby which LINE-1 hypomethylationconfers a poor prognosis in ESCC.






LINE-1–hypomethylated and LINE-1–hypermethylatedtumorsusingCGHarray.We found thatLINE-1–hypomethy-lated tumors presented highly frequent genomic gains atvarious loci containing candidate oncogenes includingCDK6. The association between CDK6 amplification,CDK6 expression, and LINE-1 methylation levels inESCCs was also observed. CDK6 expression was signifi-cantly associated with unfavorable prognosis in 128patients with ESCC. Moreover, we have shown the pro-portional reduction in the regression coefficient for LINE-1 methylation due to the inclusion of CDK6 expression inthe Cox regression model. Collectively, CDK6 may be anintermediate factor explaining the relationship betweenLINE-1 hypomethylation and poor prognosis. GlobalDNA hypomethylation in ESCC might contribute to theacquisition of tumor aggressive behavior through geno-mic gains of oncogenes such as CDK6 (Fig. 5D).

LINE-1 represents a major repetitive element and occu-pies approximately 17% of the human genome. Thus, thelevel of LINE-1 methylation is regarded to be a surrogatemarker of global DNA methylation (13). Tumor LINE-1hypomethylation has been associated with inferior prog-nosis in not only ESCC but also many different humantumor types such as colon cancer, glioma, ovarian cancer,lung cancer, and gastric cancer (17–22). In addition,LINE-1 hypomethylation was associated with clinicallyaggressive disease in patients with prostate cancer andgastrointestinal stromal tumors (16, 34). Nonetheless,the mechanism by which tumoral LINE-1 hypomethyla-tion correlates with malignant phenotype or patientprognosis in human cancers remains to be fully explored.Experimental evidence supports the relationship betweenLINE-1 hypomethylation (i.e., global DNA hypomethyla-tion) and genomic instability. A study using Nf1þ/�

p53þ/� mice has shown that introduction of a hypo-morphic Dnmt1 allele causes genome-wide DNA hypo-methylation that leads to significant increases in the lossof heterozygosity rate and tumor development (35).Another study has shown that genomic hypomethylationin ApcMin/þ mice leads to increases in microadenomaformation through loss of heterozygosity at the Apc locus(36). Thus, we carried out an array CGH analysis to assessthe potential implication of LINE-1 hypomethylation ingenomic instability. Interestingly, LINE-1–hypomethy-lated tumors showed highly frequent genomic gains atvarious loci containing numerous candidate oncogenes.Our finding is likely consistent with aforementionedexperimental results.

In recent years, high-resolution array-based CGH hasbeen applied to identify target oncogenes and tumor sup-pressor genes through defining recurrent gains and losses invarious cancers (37). Two studies with CGH array analysisfor ESCC samples have revealed high-level amplifications at3q27.1, 7p11, 8q21.11, 8q24.21, 11q13.3, 11q22, 12q15–q21.1, 18q11.2, and 19q13.11–q13.12, and homozygousdeletions at 4q34.3–q35.1 and 9p21.3 (38, 39). Bandla andcolleagues conducted a comprehensive analysis of DNAcopy number abnormalities in both ESCCs and esoph-

ageal adenocarcinoma. They reported a number of onco-genes that were amplified in both histologic types,including CDK6, MCL1, EGFR, SMURF1, KRAS, ERBB2,CCNE1, VEGFA, MET, and IGF1R (32). Among thesegenes, CDK6 amplification has been associated withCDK6 overexpression and a poor survival in esophagealcancer (33), supporting that CDK6 might mark esoph-ageal cancer with aggressive biologic behavior. We alsofound that CDK6 expression was related with a poorprognosis in ESCCs. These results certainly imply thatCDK6 has oncogenic roles in ESCC and contributesto ESCC progression. Our finding of the correlationbetween LINE-1 hypomethylation, CDK6 amplification,and CDK6 aberrant expression suggests that LINE-1–hypomethylated tumors might acquire malignant aggres-sive behavior by increasing genomic amplification andby altering the expression levels of candidate oncogenesrepresented by CDK6. However, other mechanisms mayalso be involved. In addition to its role as a surrogatemarker for global DNA methylation, LINE-1 methylationstatus by itself likely has biologic effects, because retro-transposons such as LINE-1 elements can provide alter-native promoters and contribute to noncoding RNAexpression, which regulates the functions of a numberof genes.

CDK6, which belongs to a family of serine–threoninekinases, is an important regulator of G1–S-phase progres-sion. Together with its binding partner cyclin D1, CDK6forms active complexes that promote cellular prolifera-tion by phosphorylating and inactivating the retinoblas-toma protein (40). CDK6 amplification has been previ-ously reported in human neoplasms including esophage-al cancer, in which (in most cases) it is correlated with anadverse prognosis (33). Our current study revealed arelationship between CDK6 expression and LINE-1 hypo-methylation (i.e., global DNA hypomethylation) andpoor prognosis, suggesting that aberrant expression ofCDK6 might be epigenetically regulated, and mark anaggressive type of esophageal cancer. Importantly, thecurrent study found no relationship between cyclin D1expression and LINE-1 hypomethylation, suggesting that,with regard to G1/transition regulation, cyclin D1 isregulated by an entirely different mechanism from thatof CDK6.

Interestingly, CDK6 is capable of blocking cell differen-tiation apart from the classical role in promoting cellproliferation (41, 42). This uniqueness makes it an attrac-tive candidate for the development of small-molecule inhi-bitors. PD-0332991 is a recently developed specific inhib-itor targeting CDK4/6-specific phosphorylation of retino-blastoma protein, and is currently in phase I clinical trialsfor advanced cancers (43). This small-molecule inhibitorhas been shown to potently suppress proliferation andanchorage-independent growth of esophageal cancer celllines (33). Hereafter, CDK6 expression in the resectedspecimensmight attract increasing attention as a biomarkerfor patient selection. In this respect, our findings may haveclinical implications.

Baba et al.





In summary, LINE-1–hypomethylated ESCC tumors pre-sented highly frequent genomic gains at various loci con-taining CDK6. In addition, we found that LINE-1 methyl-ation levelswere associatedwithCDK6 expression in ESCCsand that the prognostic impact of LINE-1 hypomethylationin patients with ESCCmight be attenuated by CDK6 expres-sion. Collectively, these findings may suggest that globalDNA hypomethylation in ESCC might contribute to theacquisition of aggressive tumor behavior through genomicgains of oncogenes such asCDK6. Future studies are neededto confirmour findings, and also to examine other potentialmechanism(s) by which genome-wide DNA hypomethyla-tion affects tumor behavior.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Baba, H. Baba

Development of methodology: Y. Baba, H. BabaAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Y. Baba, A.Murata, H. Shigaki, K.Miyake, H. BabaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Y. Baba, M. Watanabe, T. Ishimoto, K.Sakamaki, H. BabaWriting, review, and/or revision of the manuscript: Y. Baba, M. Wata-nabe, H. BabaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. ShigakiStudy supervision: M. Watanabe, M. Iwatsuki, N. Yoshida, E. Oki, M.Nakao

Grant SupportThis work was supported in part by a Grant-in-Aid for Scientific Research

from the Japan Society for the Promotion of Science (JSPS; grant number24659617).

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 June 15, 2013; revised November 25, 2013; accepted December2, 2013; published OnlineFirst January 14, 2014.

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2014;20:1114-1124. Published OnlineFirst January 14, 2014.Clin Cancer Res Yoshifumi Baba, Masayuki Watanabe, Asuka Murata, et al. Amplification in Esophageal Squamous Cell Carcinoma

CDK6LINE-1 Hypomethylation, DNA Copy Number Alterations, and

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