Human Molecular Genetics, 2008, Vol. 17, No. 5 642–655 doi:10.1093/hmg/ddm336 Advance Access published on November 15, 2007 Long, abundantly expressed non-coding transcripts are altered in cancer DamonS.Perez1,(cid:2),TiffanyR.Hoage2,JayR.Pritchett1,AllisonL.Ducharme-Smith1,MeredithL. Halling1, Sree C. Ganapathiraju1, Paul S. Streng3 and David I. Smith1,(cid:2) 1DivisionofExperimentalPathology,DepartmentofLaboratoryMedicineandPathology,MayoClinicandFoundation, 200FirstStreet,S.W.,Rochester,MN55905,USA,2DepartmentofBiochemistryandMolecularBiology,MayoClinic D and Foundation, 200 First Street, S.W., Rochester, MN 55905, USA and 3Advanced Genomics Technology Center, ow n Mayo Clinic and Foundation, 200 First Street, S.W., Rochester, MN 55905, USA lo a d e d ReceivedOctober19,2007;RevisedNovember7,2007;AcceptedNovember13,2007 fro m h ttp Recent studies with tiling arrays have revealed more genomic transcription than previously anticipated. s Whole new groups of non-coding transcripts (NCTs) have been detected. Some of these NCTs, including ://a c a miRNAs, can regulate gene expression. To date, most known NCTs studied have been relatively short, but d e several important regulatory NCTs, including XIST, MALAT-1, BC1 and BC200, are considerably larger in m ic length and represent a novel class of long, non-coding RNA species. Whole-genome tiling arrays were uti- .o u p lized to identify novel long NCTs across the entire human genome. Our results have identified a new .c o group of long (>400 nt), abundantly expressed NCTs and have found that a subset of these are also highly m /h evolutionarily conserved. In this report, we have begun to characterize 15 long, conserved NCTs. m g Quantitative real-time RT–PCR was used to analyze their expression in different normal human tissue and /a also in breast and ovarian cancers. We found altered expression of many of these NCTs in both cancer rtic le types. In addition, several of these NCTs have consistent mutations when sequences of normal samples -a b were compared with a panel of cancer-derived cell lines. One NCT was found to be consistently mutated s tra in a panel of endometrial cancers compared with matched normal blood. These NCTs were among the c most abundantly expressed transcripts detected. There are probably many long, conserved NCTs, albeit t/17 /5 with lower levels of expression. Although the function of these NCTs is currently unknown, our study indi- /6 4 cates that they may play an important function in both normal cells and in cancer development. 2 /5 8 6 8 8 8 b y INTRODUCTION (18–30 nt), short translational–regulatory RNAs (100–200 gu e nt), and much longer RNAs (up to 10000 nt) involved in s Resultsofseveralrecentlarge-scaletilingexperimentssuggest gene silencing (7,8). Others reported ncRNA characterized t on a significantly higher proportion of transcribed human bytheirsize(longerandshorterthan200nt),cellularlocation 29 genomic sequence than could be accounted for by existing (nuclear or cytosolic) and location in relation to gene bound- M a exon annotation, with much of the excess not encoding aries(9).Suchfindingsareindicativeofvariousundiscovered rc h protein (1–6). Genetic researches have focused on gene classes of RNA species throughout the non-coding regions of 2 0 expression with the coding portion of genes accounting for, the genome possibly associated with cellular functions and 19 at most, 2% of the entire human genome. Researchers have potentialtargetsofalterationinavarietyofdifferentdiseases. now begun to explore novel, non-coding RNA (ncRNA) Owing to the relatively recent knowledge of such species in species to characterize their potential role in regulatory pro- the 50–55% of the non-repetitive portion of the human cesses anddisease development. The humangenome includes genome corresponding to either intergenic or intronic adiversecollectionofncRNAs,suchasmiRNAsandpiRNAs sequences, there are relatively few publications on ncRNAs (cid:2)To whomcorrespondenceshouldbeaddressedat:DivisionofExperimentalPathology,DepartmentofLaboratory Medicineand Pathology,Mayo Clinic and Foundation, 200 First Street, S.W., Rochester, MN 55905, USA. Tel: þ1 5072660309; Fax: þ1 5072665193; Email: smith.david@ mayo.edu(D.I.S.)/[email protected](D.S.P.) # The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Human Molecular Genetics, 2008, Vol. 17, No. 5 643 compared with publications on the 1–2% of the genome conserved among species (26,27). The expressions of 15 containing protein-coding sequences (10–12). transcripts that originate from non-coding sequence were Some ncRNAs function to regulate and direct complex then measured in 12 different normal tissues, as well as in a pathways (13–17). An assortment of small ncRNA species panel of breast and ovarian cancers. From this study, a (e.g. miRNA, piRNA and snoRNA) are involved in critical subset of abundantly expressed, highly conserved NCTs aber- cellular processes and, in some cases, have been linked to rantlyexpressedinbreastandovariancancertissuesweredis- specific disease states including cancer (14–20). MiRNAs covered. Several of these NCTs seem to be susceptible to have been associated with initiation and progression of mutation in a panel of cancer-derived cell lines, and one cancer by acting as tumor suppressors or oncogenes and, NCT was observed to have four different mutations in thus, could be cancer therapy targets (14,15,21). Collectively, several endometrial tumors. Collectively, our results demon- transcriptome analyses comparing tumor and normal cells strate a novel group of NCTs that may have an important suggest that defects in ncRNAs, regardless of their length, impact on cancer development. D do occur in tumors (14,15). Furthermore, the deregulation of o w several ncRNAs (apart from miRNAs) have been observed n in prostate, colon, ovary, liver, breast, cervix, esophagus and RESULTS loa d t[orenvgiueewedcainnce(r1s4,)].asSucwhelrlepoasrtslienudkiecmateiasthaatntdherleymmpahyobmeaas Abundantly expressed long NCTs ed fro newclassoflongerncRNAspeciesthatareinvolvedincritical The aim of this study was to investigate the possible involve- m h regulatory processes and are targets of alteration during the mentoflong,conservedNCTsincancerdevelopment.Tothis ttp devCeulrorpemntelyn,ttohferceaanrceer9.40uniquehumanncRNAsdocumented eonfdt,hewehusmcaannnegdencoDmNeAufsrionmg ttihlienegnatirrreaynsona-nrdedfuoncduasnedtpoonrtitohne s://ac a on the RNAdb website (http://www.research.imb.uq.edu.au/ identification of only the most abundantly expressed regions de rnadb/), excluding antisense, sno, pi and miRNAs. This data- (i.e.consecutiveprobesthatexpressinthe99.5thsignalinten- mic base and other reports provide evidence of longer ncRNAs sity percentile). At this signal threshold, we found that .o u (.100 nt) within the genome. Few have been studied in there were a total of 53972 transcriptionally active regions p.c detail (13,14,22,23). A recent mapping project suggested that (TARs) in NHBE that occur, on average, every 59kb across om anappreciableportionoflong,unannotatedtranscripts(poten- the genome. In addition, we observed a substantial increase /h m tial long ncRNAs) could serve as precursors for smaller RNA in the number of TARs as we decreased the threshold to the g sspuecchieass T,S2I0X0anntdinXIleSnTg,thwh(9ic)h. Ianrespniotet porfecthuirss,olrosnfgornscmRNalAlesr i9n9ttehns(it(cid:2)y99pe0r0c0entTilAe.RsS)inacnedth9e8rteh w((cid:2)er2e10so000maTnAyRTs)ARsisgnaalt /article RNA species, have been found to be important for the regu- various lengths and expression levels, we focused our study -ab s lation of chromosome X silencing (8,18). It has also been only on the most abundantly expressed regions (i.e. TARs at tra reported that a few long ncRNAs are selectively expressed the top 0.5% of the tiling array signal), which included ct/1 in tumor cells, but not in corresponding normal cells highly expressed housekeeping genes such as b-actin. At 7 /5 (13,14,19,20). Specifically, six documented long ncRNA this threshold level, we identified 578 TARs that were /6 4 speciesrangingfrom152to8000nthavebeenshowntohave longerthan400ntinlength.Ofthese,495originatefromnon- 2 someroleincarcinogenesis,andseveral othersareimplicated protein coding sequence (intergenic or intronic regions) and /58 6 in various neurological diseases [reviewed in (14,24)]. For we bioinformatically examined these regions in order to 88 instance, the highly conserved, long non-coding 8kb choose candidate NCTs for subsequent real-time RT–PCR 8 b MALAT-1transcriptisspecificallyinvolvedinnon-smallcell (qPCR), northern blot, and sequencing experiments. y g u lung carcinoma (25). In addition, an antisense intronic tran- e s script has been found to correlate with the degree of tumor t o Conservation of long NCTs n differentiation in prostate cancer (19,20,24). NcRNAs, BC1 2 9 and BC200, are selectively expressed in tumor cells, but not Asanindicationoffunctionalimportance,sequenceconserva- M in corresponding normal tissue (19,20). Collectively, these tion of the highly expressed TARs across 17 diverse species arc studies demonstrate the importance of long ncRNAs in was analyzed using the UCSC Human Genome Browser. Of h 2 cancer. Although the functions of ncRNAs are likely to be the 578 long, abundantly expressed TARs examined, 232 dis- 01 9 diverse, both logic and evidence strongly suggest their role is playedadegreeofsequenceconservation .5%.Themajority toregulateanddirectcomplexpathways(8,13,26). (163 or 70.2%) of these conserved regions originated from In an attempt tostudy novel, longncRNA species thatmay non-coding sequences. When we examine only those TARs have importance incellular function and cancer development, that are at least 20% conserved, we found that there are 113 a tiling array platform was used to obtain unbiased transcrip- TARs, and more than half (61 TARs) of these conserved tiondataacrosstheentiregenomeinnormalhumanbronchial regionsareoriginatedfromnon-codingsequences(15intronic epithelial(NHBE)cells.Wehypothesizedthatanewgroupof and46intergenic).Inaddition,therewere27intergenicand5 long ncRNA species are involved in crucial regulatory func- intronicTARsthatdisplayedadegreeofconservation .50%. tions and, when modulated, play roles in cancer. To test this Of these, 10 were .90% conserved across 17 species. Of the hypothesis, we chose a subset of abundantly expressed long, abundantly expressed, conserved, non-coding TARs non-coding transcripts (NCTs) that are .400 nt long, which identified,wechosetoexamineadistributionof15NCTsdis- displayed a high degree of sequence conservation. DNA playing varying degrees of conservation. We studied four sequences that have functional importance are often NCTs with moderate levels of conservation (34–69%), five 644 Human Molecular Genetics, 2008, Vol. 17, No. 5 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /h m g /a rtic le -a b s tra c t/1 7 /5 /6 4 2 /5 8 6 8 8 8 b y g u e s Figure 1. Genomicregions that span two representative NCTs, (A) NC4(cid:2) located on chromosome 12 (62502932–62503496) and (B) NC26(cid:2) located on t o chromosome 4 (136186664–136187064), are displayed in both the Integrated Genome Browser (IGB) and UCSC Human Genome Browser. In the top n 2 IGBpanel,theprobesignalisrepresentedbygreenverticalbars.Thegreenhorizontalbarsbelowthesignalindicateregionsofconsecutiveprobesexpressing 9 atthetop0.5thpercentileofthearraydata.TheUCSCdisplaybelowcorrespondstotheexactregionrepresentedinIGBdisplayabove.AsdepictedontheUCSC M a HumanGenomeBrowser,eachNCTexhibitslargeregionsofhighconservationacrossmultiplespecies,noalignmentswithcodingregionsofpredictedor rc knowngenes,norepetitiveelements,andpreviouslyidentifiedTARs(3). h 2 0 1 9 with even greater levels of sequence conservation (70–98%) these regions were representative of conserved, non-coding and six that are ultraconserved (.99%). This assortment of regions. UCSC conservation graphs and corresponding tran- NCTs was selected to conduct experiments that were aimed scriptional signal from tiling arrays of two representative to examine their expression in 12 normal human tissues, NCTs are displayed in Figure 1A and B. Those NCTs that whether they are differentially expressed in breast and exhibit .90% sequence conservation across 17 species are ovarian cancer, and to determine whether these regions are denoted with an asterisk. targeted for mutation. This list of variably conserved NCTs allowed us to investigate whether there was any correlation Validation of potential NCTs between the degree of conservation and altered expression or frequency of mutation in cancers. Each NCT was analyzed To validate our tiling array observations, northern blotting comprehensively as described in detail in Materials and experiments were performed to demonstrate the lengths of Methods section for sequence conservation, and presence of detected transcripts. Representative blots are displayed in stopcodons,repetitiveregions,G-Ccontent,etc.tobecertain Figure 2. Both lanes on each image represent the same RNA Human Molecular Genetics, 2008, Vol. 17, No. 5 645 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /h m g /a rtic le -a b s tra c t/1 7 /5 /6 4 2 /5 8 6 8 8 8 b y g u e s t o Figure1.continued n 2 9 M sample run in duplicate and transferred to each membrane; Expression analysis of conserved NCTs arc hence, the two bands displayed for many NCTs in Figure 2. in normal human tissues h 2 As given in Table 1, NCT size was characterized by two 01 ways: regions of consecutive probes displaying signal in the Non-coding transcript tissue specificity was examined by 9 99.5th percentile were documented as the initial indication of analyzing expression in various normal human tissues and transcript size. This is most likely an underestimation of the cell lines using qPCR (Fig. 3A–C). Most of the NCTs were actual transcribed region. Therefore, we also documented the expressed in all samples, but at different levels. Some NCTs area surrounding this region that is also transcriptionally displayed restricted tissue expression. For instance, all 12 active, but at a level lower than the 99.5th percentile. Our tissues examined expressed most of the 15 NCTs with the northernresultsshowthattheresolvedNCTbandstendtocor- exception of NC25 (not expressed in breast), NC30(cid:2) (not respondinsizetotheentiretranscribedarearatherthantothe expressed in kidney and liver) and NC39 (not expressed in sizeofthefragmentderivedfromonlythechainofconsecutive prostate, ovary, cervix, breast, kidney and liver). The most probes expressing above the 99.5th signal intensity percentile abundantly transcribed transcripts across all 12 tissues threshold.Inadditiontonorthernblots,qPCRwasusedtopre- examined (ranked in descending order) were NC35, NC28(cid:2) ciselyquantitatetheexpressionofeachNCTindifferenthuman and NC31. Impressively, NC35 displayed expression tissuesandinapanelofbreastandovariancancers. levels 12-fold higher than b-actin in most tissues tested. 646 Human Molecular Genetics, 2008, Vol. 17, No. 5 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /h Figure2.NorthernblotsdisplayingtheexpressionofrepresentativeNCTsinNHBE.FormostNCTs,duplicatesampleswereprocuredtoconfirmreproduci- m g bility.ArrowsontherightofeachimagecorrespondtoRNAmarkerbandstoestimatetheNCTsize.Whencomparingbandssizetosignalfromthetilingarray, /a tohfecroenssueltcsustihvoewptrhoabtetheexpreressoslivnegdaNbCovTebtahned9s9te.5ntdhtsoigcnoarrleisnptoenndsitiynpsiezrecetonttihleetehnrteirsehotrladn.sNcrCibTesddaerneoattehdanwtioththaensaizsteeorifskth(e(cid:2)t)raanresc.rip9t0d%ericvoendsefrrovmed.onlythechain rticle -a b s tra Table1. NCTgenomicposition,size(accordingtothe99.5thsignalpercentileandtotalareaoftranscription)andorgininthehumangenome c t/1 7 NCT Cytoband Coordinates(start–stop) Size(nt)P99.5 Approx.lengthof Nearestgeneorexon Originoftranscript /5/6 transcription(kb) anddistance(kb) IGB RefSeq UCSCpredictions Ensembl 4 2 /5 NC04(cid:2) 12q14.2 62502932–62503496 565 2.2–2.4 TMEM5(13.8) NC NC NC NC 86 NC05(cid:2) 12p12.3 17035548–17036128 581 0.9–1.1 LMO3(384) NC NC NC NC 88 NNCC0261(cid:2) 912p2q14.41.2 761446375152538––764164735559846 443069 11..50––11..71 JFMLJJ2D525C90(3(0125)3) NNCC NI C NI C NI C 8 by NC22(cid:2) 3q24 149366637–149367000 404 0.4–1.6 AGTR1(531) NC NC NC NC gu NC25 6q13 72351607–72352009 403 1.3–1.5 C6orf155(163) NC NC NC NC es NC26(cid:2) 4q28.3 136186664–136187064 401 1.6–1.7 PCDH10(1840) NC NC NC NC t o NC28(cid:2) 8p12 34851236–34851648 413 0.7–1.6 UNC5D(665) NC NC NC NC n 2 NC29 15q21.3 54152112–54152543 432 2.4–2.7 RFXDC2(17.6) NC NC NC NC 9 NC30(cid:2) 8q21.13 81633845–81634254 410 1.3–1.8 ZBTB10(38.8) NC E NC NC M a NC31 12q23.3 103183218–103183704 487 0.6–0.7 TXNRD1(21.3) NC I I I rc h NC33 5q21.1 97703482–97704002 521 1.7–1.9 RGMB(424) NC NC NC NC 2 NC35 11p15.4 10486015–10488239 2225 2.4–3.3 AMPD3(1.4) NC NC E(60%)/NC NC/E(10%) 01 NC39 3q11.2 97818707–97820019 1313 1.3–1.4 ARL6(1145) NC NC NC NC/E(5%) 9 NC40 3q27.2 186618028–186619252 1225 1.3–1.4 MAP3K13(10.2) I I I I NCTsthatexhibit.90%sequenceconservationacross17speciesaredenotedwithanasterisk((cid:2)). P ,99.5thpercentile;IGB,IntegratedGenomeBrowser;NC,intergenic;I,intron;E,exon. 99.5 Conversely, NC21(cid:2), NC33 and NC26(cid:2) consistently displayed substantial fraction of highly proliferative lymphocytes, thus low expression levels in most samples examined. In general, indicating a possible role of these NCTs in immune response prostate, ovary and brain exhibited the lowest levels of NCT and/or cell proliferation. Collectively, these results demon- expression. On the other hand, 9 of 15 NCTs displayed strate that some NCTs exhibited tissue-specific expression higher levels of transcription in normal tonsil, spleen and whereas others were ubiquitously expressed, and that there myometrium than b-actin. One possible explanation to this was minimal correlation between tissue expression and observation in spleen and tonsil is that both contain a sequence conservation. Human Molecular Genetics, 2008, Vol. 17, No. 5 647 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /h m g /a rtic le -a b s tra c Figure3.NCTexpressionin12differentnormaltissuemeasuredbyqPCR.Expressionvalues(∆Ct)arerelativetob-actin,butwerealsocomparedwith t/1 otherhousekeepinggenesincludingGAPDH(datanotshown).Theexpressionlevelsof15NCTsinnormal(A)prostate,cervix,ovaryandbreast,(B)brain, 7 kidney,lungandliver,(C)tonsil,spleen,myometriumandkeratinocytes.MostoftheNCTswereexpressedinallsamples,butatdifferentlevels.SomeNCTs /5/6 displayedrestrictedtissueexpression,whereasothersexhibitedubiquitousexpressioninallnormalsamplestested.AllNCTsdisplayedhighlevelsofexpression 4 2 intonsil,spleenandmyometriumwhereasprostate,ovaryandbrainexhibitedthelowestlevelsofNCTexpression.NCTsdenotedwithanasterisk((cid:2))are.90% /5 conserved. 86 8 8 8 b y g Aberrant expression of several NCTs degree of sequence conservation (,90%). NC29, which u e in ovarian and breast cancer exhibited the lowest degree of conservation (34.7%), was sig- st o nificantly upregulated in all 20 ovarian cancer samplestested. n Expressionofthe15NCTswasexaminedbyqPCRinapanel 2 Thus,thechangesinthetranscriptionlevelsofthemosthighly 9 of ovarian and breast cancer samples. The breast panel was M cccoeolmmlpplirrniisseeesddaonofdfnposrhrimmoraatlr-ytceerbmlrlelaicsnutelsttu,umrbeeosnrisgo.nfTthniesosruomeva,alrciaaonnvcaeprrai-nadneelriewvpeaids- NNcraoCCtne22se62thr(cid:2)(cid:2)va,en(d9Nl6etCr.s9a2sn%-8cs(cid:2))cornieaspxentshrdvi(be.idNte9CNd03C%0aT(cid:2).csm,onOoassdneelreyrvvaetidedN)eCnnot2uc1mcbu(cid:2)ybrerNe(r9dC9oa4.f6t(cid:2)%,aoN)vlaoCrwai5nae(cid:2)dnr, arch 2019 thelium, ovarian cancer cell lines and primary ovarian cancer samples with modulated expression of both NCTs tumors. The results revealed that more NCTs were aberrantly (seven samples upregulated for each NCT). Conversely, the expressed in breast tumor samples compared with the ovarian majority of NCTs were consistently downregulated in breast cancer tissues (Fig. 4A and B and Table 2, Supplementary tumors compared with normal NCTs. Of the 17 breast Material, Tables S1 and S2). We also found that several cancer samples tested, at least nine samples displayed down- NCTs displayed differential expression when comparing regulated expression in 8 of the 15 NCTs, including four of normal and cancer samples in both breast and ovary. Interest- the highest conserved NCTs (NC5(cid:2), NC21(cid:2), NC26(cid:2) and ingly, NCTs were typically upregulated in ovarian cancers in NC30(cid:2)). Thus, these data indicate that the highest conserved contrast to being downregulated in breast cancers. Three NCTs NCT are more susceptible to expression modulation in breast (NC25, NC29 and NC31) were observed to be upregulated tumors compared with ovarian cancer. It is interesting to note inmorethan halfofthe20ovarian cancersamplesexamined. that the most modulated NCTs in ovary had less altered Although these threetranscriptswere altered in moresamples expression in breast cancer and vice versa. For example, than any of the other 12 NCTs, they also displayed a lower 648 Human Molecular Genetics, 2008, Vol. 17, No. 5 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /h m g /a rtic le -a b s tra c t/1 7 /5 /6 4 2 /5 8 6 8 8 8 b y g u e s t o n 2 9 M a rc h 2 0 1 9 Figure4.ModulatedexpressionofNCTsincancerasmeasuredbyqPCR.(A)Numberofovariantumorsamples(n¼20total)withaltered(up-ordownre- gulated)NCTexpression.(B)Numberofbreasttumorsamples(n¼17)withaltered(up-ordownregulated)NCTexpression.Contrarytotheovariancancer results,manydownregulatedNCTswereobservedinthemajorityofbreasttumorsexamined.NumerousdownregulatedNCTswereobservedinatleasthalfof thebreasttumorsamplestested.Cancersamplesdisplayinganaverageexpressionchange.4-fold(aDCtofmorethan2greaterorlessthantherangeobserved inthenormalsamples)andaP-value,0.05wereconsideredsignificantlydifferentfromnormalsamples(SupplementaryMaterial,TablesS1andS2).NCTs denotedwithanasterisk((cid:2))are .90%conserved. the NCTs that had the most samples with altered expression ovarian cancer there were very few samples with modulated in ovary (NC25, NC29 and NC31) had the fewest samples expression levels for these same NCTs. This observation exhibiting altered expression in breast. In contrast, NC30(cid:2), could indicate that some of these NCTs are specific targets NC33 and NC39 displayed among the highest number of in the development of either ovarian or breast cancer, but samples with altered expression in breast cancer, but in not both. Human Molecular Genetics, 2008, Vol. 17, No. 5 649 Table2. NCTsequenceconservation,modulatedexpressionincancer,numberofmutations(incancer–derivedcelllinesandendometrialtumors)andcorrespon- dencetopreviouslyidentifiedTARsbyBertoneetal.(3) NCT Conservation No.ofbreastcancersamples No.ofovariancancersamples No.ofmutations Bertonedata Upregulated Downregulated Upregulated Downregulated Celllines Endometrium NC04(cid:2) 99.94 1 5 2 0 1 0 TAR3719,3720 NC05(cid:2) 99.78 0 16 2 1 3 0 TAR2234,2235 NC06 71.90 0 10 0 3 4 0 TAR16446 NC21(cid:2) 99.66 0 13 7 0 4 0 TAR3710,3711,3712 NC22(cid:2) 96.96 0 6 7 0 0 – – NC25 68.17 0 4 14 0 1 4 – NC26(cid:2) 99.71 0 11 6 3 1 0 TAR11287,11288,11289 NC28(cid:2) 99.57 0 6 4 2 0 – – D NC29 34.70 0 2 20 0 0 – – o NC30(cid:2) 99.33 0 12 4 5 0 – – w n NC31 86.89 0 2 14 0 0 – – lo a NC33 77.22 0 13 7 5 0 – – d e NC35 60.15 0 9 0 1 – – TAR2356,2357,2358,2359 d NC39 63.07 0 13 3 4 – – TAR9515,9516,9517 fro NC40 85.00 0 0 6 1 – – TAR10413,10414,10415 m h ttp NCTsthatexhibit.90%sequenceconservationacross17speciesaredenotedwithanasterisk((cid:2)). s://a c a d e m Sequencing analysis for mutations in NCTs Table3. MutationsfoundofsequencedNCTinvariouscancer-derivedcell ic linesandbrainxenografts .ou We next tested a panel of normal samples and cancer-derived p.c o cell lines from cancers of the stomach, breast, ovary, brain, NCT Mutationlocation Cellline Tissue m pancreas, colon, esophagus and lung to determine whether /h m anyoftheseconservedNCTswerevulnerableincancerdevel- NC4(cid:2) (274)C!CT RKO Colon g NC5(cid:2) (217)T!C SKOV3 Ovary /a opment. We found relatively few potential mutations in this (63)C!CT GBM8 Brain rtic panel; however, there were some nucleotides within some of (63)C!CT AGS Stomach le the NCTs that were consistently altered in distinct samples NC6 (68)G!GC OVCAR5 Ovary -ab from different tissues (Table 3). This observation suggests (68)G!C SU8686 Pancreas stra that such potential mutations may be selected during tumori- ((6688))GG!!CC KKYYSSEE144100 EEssoopphhaaggeeaall ct/1 genesis, rather than being random mutations in unstable NC21(cid:2) (318_321)delATGA BXPC3 Pancreas 7/5 cancercells.OuroverallresultsshowthatsixNCTshavecon- (319-322)delTGAA SKOV3 Ovary /6 4 sistent alterations (possible mutations) when sequences of (319-322)delTGAA AGS Stomach 2 normal samples are compared with cancer-derived cell lines (319-322)delTGAA OE33 Esophageal /58 from various tissues (including breast, ovary, pancreas, NNCC2225(cid:2) N(6o7n)Te!TC M–DA468 B–reast 6888 colon, esophageal, lung, stomach and brain). In particular, NC26(cid:2) (329)G!GT RKO Colon b y NC5(cid:2), NC6 and NC21(cid:2) displayed consistent alterations at a NC28(cid:2) None – – g u specific nucleotide position in at least two different cancer NC29 None – – e samples from the panel. Our results do not provide enough NC30(cid:2) None – – st o NC31 None – – n evidence to conclude that one cancer is more likely to be NC33 None – – 29 mutated; however, the cancer-derived cell lines that exhibited NC35 Didnotsequence – – M the most mutated NCTs were derived from the ovarian NC39 Didnotsequence – – arc (mutations in NC5(cid:2), NC6 and NC21(cid:2)) or esophageal cancers NC40 Didnotsequence – – h 2 (two mutations in NC6 and NC21(cid:2)). In addition, mutations 01 in NC4(cid:2) and NC26(cid:2) were observed in colon samples, and NCTsthatexhibitgreaterthan90%sequenceconservationacross17 9 mutations in NC5(cid:2) and NC21(cid:2) were observed in stomach speciesaredenotedwithanasterisk((cid:2)). cancer. As a whole, most of the mutations observed were simple substitution or addition mutations; however, we thesixNCTs(NC4(cid:2),NC5(cid:2),NC21(cid:2)andNC26(cid:2))withsequence observedasignificant4ntdeletionatthesamenucleotidepos- conservation .90% did have mutations. ition within NC21(cid:2) in cell lines derived from four different It is possible that the sequence changes presented above tissues (pancreas, ovary, stomach and esophageal). There may be polymorphic in nature. Therefore, altered regions was very little correlation between mutated NCTs and any were aligned and compared against all known SNPs from altered expression in ovary; however, mutated NC5(cid:2), NC6 human genome databases to increase the probability that the and NC21(cid:2) were, in fact, also susceptible to downregulated changes weobservedarerealmutations.Inaddition,potential expression changes in breast cancer. Overall, we did not mutations observed in cancer-derived cell lines were cross observe a strong correlation between highly conserved NCT checked against any similar alterations observed within 11 and mutational events. However, we observed that four of normal samples and found it to be certain that these changes 650 Human Molecular Genetics, 2008, Vol. 17, No. 5 Table4. Atotalof23differentendometrialtumorspecimenswereobserved and analyze the human genome. One such technology is havingmutationsinNC25 tiling arrays with probes for determining the transcriptional activity of the entire non-repetitive portion of the genome. Samplepair Positionandmutation In this study, a 35 nt probe tiling array was used to scan NHBE cells to find novel, conserved, highly transcriptionally 1/2 (251)A!AG active non-coding regions with potential cellular function 1/2 (373)G!GA 15/16 (250)A!AG and involvement in disease development. The list of 17/18 (373)G!GA (cid:2)54000 most highly expressed TARs was subsequently nar- 23/24 (251)A!AG rowed to 15 candidate regions that were then analyzed in 29/30 (250)A!AG detail. Many conserved, abundantly expressed NCTs were 31/32 (251)A!AG 31/32 (373)GA!G identified,butpotentiallymanymorewouldhavebeendiscov- 31/32 (401)A!AT eredhadwenotappliedastringentfiltertocaptureonlythose D 35/36 (382)CT!C NCTs expressed at extremely high levels. Some of the o 41/42 (249)A!AG w observednon-codingTARscouldoriginatefromtheantisense n 43/44 (249)A!AG lo strand, because our protocol converts RNA into double- a 43/44 (371)GA!G d 45/46 (248)A!AG stranded cDNA, thus the strandedness cannot be determined ed 47/48 (384)CT!C from these data. Further experimentation (e.g. 30 and 50 fro 555757///555868 (((332964999)))AGAA!!!TGG RasAtChEe)eixsacntecseizssearoyf teoacchlartirfaynstchreiiprt,praencdiseothloecrateixopneraismwenetlsl m https 59/60 (252)AG!A are also necessary to determine which strand encodes each ://a 59/60 (402)AT!A of these NCTs. c a 67/68 (370)GA!G One unique aspect of this study is our focus on only the de 69/70 (252)A!G m 69/70 (402)A!T mregoisotnshiogfhlcyonstreacnustcivriebetidllinngonp-rcoobdeisngacrsoesqsuaetncleeassts4p0a0nnnintsg. ic.o 71/72 (252)AG!A u 71/72 (402)AT!A In addition to this criterion, we also specifically chose to p.c 73/74 (252)A!AG examine those regions that were moderately to highly con- om 79/80 (248)G!A served across 17 species, rather than comparing sequences /h 79/80 (398)T!A m with two or three closely related species. Seven of the 15 g 83/84 (252)AG!A /a 83/84 (402)AT!A NCTsanalyzedinthisstudyrepresentnewlydiscovered,con- rtic 85/86 (252)A!AG served and highly transcribed sequences derived from NHBE le 91/92 (251)G!A cells. The other eight NCTs have been identified previously -ab 999551///999662 (((244500001)))AAT!GT!!AAA u(Tsianbglea2)di(f3fe).reHntowtielivnegr,atrhraeyy hpalavtefornmot abnedenmcohdaerlacsteyrsitzeemd stract/1 further until now. A similar study also demonstrated that a 7 /5 group of ultraconserved ncRNAs are altered in cancer (28). /6 4 werenotpolymorphicwithinournormalsamplepanel.None- However, that study did not utilize a tiling array to discover 2 /5 theless,thepossibilitystillexiststhatthechangesweobserved these transcripts. In addition, the expression of many of 8 6 arerarepolymorphismwithinaspecificcelltype.Inattemptto those transcripts is significantly lower than the transcripts 88 8 addressthisissue,NCTsthathadthemostpotentialmutations described in this report. Our study provides additional evi- b y in cancer-derived cell lines were further sequenced in a panel dence that ncRNAs play a role in cancer development and g u of48matchedendometrialsamples(i.e.cancerversusnormal shows the alteration of NCT expression in breast and e s endometrial tissue from the same patient). No mutations of ovarian cancer. Another unique aspect of this study is our t o n NC5(cid:2), NC6 or NC21(cid:2) (the three NCTs that had the most sig- demonstration of potential consistent mutations in a panel of 2 9 nificantalterationsincancer-derivedcelllines)wereobserved cancer-derived cell lines. Unfortunately, we did not have M inendometrialcancer(Table4).However,NCT25,whichwas matched normal DNA for any of these cell lines, thus we arc onlymutatedinoneofthecancer-derivedcelllines,wasfound cannot definitively say that these are not rare polymorphisms. h 2 tobefrequentlymutatedintheendometrialcancerpanel(in23 However, when we analyzed a panel of 48 primary endo- 01 9 of 48 of the samples tested). These samples had matched metrial cancers with matched normal DNA, we found that normal DNA (from blood), and thus the observed alterations one of the NCTs was consistently mutated in almost half of are mutations and not polymorphisms. In addition, each of theendometrialcancersexamined.Theobservationofconsist- the four distinct mutations observed was found in multiple ent mutations suggests that these are not randommutations in samples. Since there was only a single possible mutation in unstable cancer cells, but could have an important functional thecancer-derivedcelllinepanel,NC25couldbeamutational role.Thus,thisprovidesfurtherevidencethattheseconserved target specifically in endometrial cancer. NCTs,inaddition tohavingaltered expressionin cancers, are also mutational targets. Most studies in cancer genetics describe mutations or other alterations(e.g.overexpression,ordownregulation)inprotein- DISCUSSION coding genes. The recent identification of new classes of Since the completion of the human genome project, many ncRNA species implicated in important steps of cancer high-throughput strategies have been developed to screen reinforces the role of these transcripts in the process of Human Molecular Genetics, 2008, Vol. 17, No. 5 651 tumorigenesis (14,15). We report that the expression of most identifying functional candidate sequences in the non-coding, NCTs is altered in ovarian and breast cancer samples. This non-repetitive regions of the genome. It is speculated that indicatesthatsomeofthe15NCTsexaminedcouldbeinvolved novel, functional ncRNAs should have high interspecies con- in the development of cancer in these tissues. Recent servation patterns similar to those of known functional publications support a similar conclusion in other cancers ncRNAs, such as miRNAs, piRNAs and snoRNAs (10,16). (14,19,20,24,29,30). Our study also demonstrates that even Forexample,thehighconservationofknownmiRNAsispre- the most conserved, long ncRNAs could be crucial targets for sumably because their sequence is constrained by functional mutation. Such an idea is somewhat counterintuitive because interaction with multiple targets (10,41,42). However, many one might assume ultraconserved sequences should be highly functionally important ncRNAs are rapidly evolving (34,43– protected from mutation. Our results suggest that all non- 45). Thus, the degree of conservation should not be the sole coding sequences, whether they are conserved or not, can be screening determinant of potential function. susceptible to mutagenesis. However,one study demonstrated The function of these NCTs is currently unknown. Their D that extreme sequence conservation does not necessarily abundance, tremendous sequence conservation and aberrant o w reflect crucial functions required for viability (31). Neverthe- expression in many ovarian and breast cancers suggest that n lo less,thefactthatweobservedsomeconsistentmutationsissup- they not only play an important role in normal cellular a d portiveoftheconceptthatthesearenotjustrandommutations growth and immune response, but also in the development ed inunstablecancercells,butthatspecificalterationscouldplaya of cancer. Consistent potential mutations at specific nucleo- fro m functionalroleincancerdevelopment. tideswithinsomeoftheseNCTsalsosuggestthatthesealtera- h SincethefunctionofmostncRNAsiscurrentlyunknown,it tionsare notrandom, buthave an important functional rolein ttp s is difficult to speculate whether they exhibit tissue-specific cancerdevelopment.Takentogether,thereisapossibilitythat ://a roles. Several NCTs displayed expression in all tissues someofthesencRNAscouldbeusedastargetsfordrugdevel- c a (although at different levels), but others had more restricted opment and early diagnosis and prognosis. Long ncRNA de m expression. In addition, the fact that all NCTs displayed high specieshavethepotentialtobeasimportantasprotein-coding ic levels of transcription in normal tonsil and spleen is possibly genes in cancer biology. Future studies will attempt to deter- .o u because of a substantial fraction of highly proliferative lym- mine the mechanistic role of long NCTs in normal cellular p.c phocytes in both tissues, indicating a possible role in function and disease, and how they might interact with om immune response and/or cell proliferation. This idea is sup- protein-coding genes within crucial signaling pathways. /h m ported by a recent study that observed a high number of tran- g /a sfucrritbheedr,auplotsrasicbolnesceorvnendecrtieogniobnestwineenB-locenlglsN(C2T8)s.inTvoolsvuepmpeonrtt MATERIALS AND METHODS rticle inimmuneresponse,anothergroupinvestigatedtheexpression Cell culture -abs patternsofa17kbNCT,NTT,andtheirfindingsindicatethat tra the function of NNT is specifically induced upon T-cell acti- CryopreservedNHBEcellswerepurchasedfromCambrexBio ct/1 vation (32). In contrast, non-proliferative tissues such as ScienceWalkerville,Inc.(Walkerville,MD,USA)andgrown 7 /5 brain, liver and kidney had very low expression of almost in 5% CO2 at 378C in defined bronchial epithelial cell basal /6 4 all of these long, conserved NCTs. This result could indicate medium (Cambrex) containing bovine pituitary extract, 2 that these NCTs have a very limited role in these tissues. humanepidermalgrowthfactor,insulin,hydrocortisone,trans- /58 6 The very low NCT expression levels observed in brain ferrin, epinephrine, triiodothyroneine, retinoic acid (to inhibit 88 samples do contradict recent reports, suggesting that con- cell differentiation) and antibiotic, GA-1000, according to 8 b servedNCTsareassociatedwithgenesinvolvedinbrainfunc- manufacturer’s instructions. y g u tion (33,34). Nevertheless, the results suggest a correlation e s between NCT expression and degree of cell proliferation. t o Normal tissues, cancer-derived cell lines n Thus, this new group of NCTs could have a regulatory role 2 and primary tumors 9 in cellular proliferation and/or immune response. M AlthoughmostknownlongncRNAsdonotshowevolution- Liver, kidney, breast, myometrium, endometrium, prostate, arc ary conservation (35), we present a group of transcriptionally spleenandtonsiltissueswereobtainedfromtheMayoPathol- h 2 active NCTs that are, in fact, conserved. The entire regions ogy Department (Rochester, MN, USA) and were determined 01 encoding six of the NCTs (NC4(cid:2), NC5(cid:2), NC21(cid:2), NC26(cid:2), to be normal or benign by a pathologist. The brain tissue was 9 NC28(cid:2) andNC31(cid:2)) were .99%conserved across17species, purchasedfromAmbion,Inc.(Austin,TX,USA).NHBEcells which could classify them as ultraconserved sequences [pre- (Cambrex)wereusedandconsideredasnormallung.Thecan- viously defined as segments .200 nt long and having 100% cerous ovarian samples all corresponded to serous ovarian identity among orthologous regions of the human, rat and cancers, either ovarian cancer-derived cell lines or primary mouse genomes (36)]. However, the study where this defi- tumors. As a control, NCT expression in cancerous ovarian nitionwasproposeddidnotexaminewhetherornotsuchseg- tissues was compared with NCT expressionin short-term cul- ments were transcribed. As much as ten times more of the turesofnormalovarianepithelialcells.Forthebreastsamples, conserved fraction of the genome is non-coding than coding expression in breast cancer and normal breast tissues were bysomeestimates(26,37,38).DNAsequencesthathavefunc- compared.Eachcancer-derivedcelllinewasgrownusingcon- tional importance are often conserved among species because ditions recommended by their supplier. For NCT cancer of negative selection (26,33,39,40). Thus, sequence compari- expression studies, 20 primary serous ovarian tumors and 17 son between divergent species can be a useful method for breast cancer primary tumors were examined. We also