Oncogenic Properties of the Antisense lncRNA in BRAF- and ... · Thyroid Carcinomas Roberta...

Genome and Epigenome

Oncogenic Properties of the Antisense lncRNACOMET in BRAF- and RET-Driven PapillaryThyroid CarcinomasRoberta Esposito1, Daniela Esposito1, Pierlorenzo Pallante2, Alfredo Fusco3,4,Alfredo Ciccodicola1,5, and Valerio Costa1

Abstract

RET rearrangements as well as BRAF and RAS mutationsdrive differential pathway activation in papillary thyroid car-cinomas, leading to different tumor phenotypes and progno-ses. Although The Cancer Genome Atlas Consortium hasidentified tumor subgroups based on protein-coding genesignatures, neither expression of long noncoding RNAs(lncRNA) nor their correlation with specific tumor-drivingmutations and rearrangements have been systematicallyassessed. Here, we reanalyzed our RNA-sequencing data usinga de novo discovery approach to identify lncRNAs and definetumor subtype-specific signatures of annotated lncRNAs.Among them, we identified COMET (Correlated-to-MET), anatural antisense transcript that was highly expressed in car-cinomas harboring BRAFV600E mutation or RET gene rearran-gements (i.e.,BRAF-like tumors) and induced the downstreamMAPK pathway. In papillary thyroid carcinomas, COMETwaspart of a coexpression network including different oncogenes

belonging to the MAPK pathway, and its expression highlycorrelatedwithMET expression.Depletion ofCOMET resultedin reduced expression of genes within this network, includingtheMET oncogene. COMET repression inhibited viability andproliferation of tumor cells harboring BRAFV600E somaticmutation or RET oncogene rearrangement and dramaticallyreduced motility and invasiveness of tumor cells. Moreover,silencing COMET markedly increased sensitivity to vemura-fenib, a common inhibitor of mutated B-raf. Collectively, ourresults suggest COMET as a new target to improve drug-basedcancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas.

Significance: These results highlight the oncogenic role oflncRNA COMET in thyroid and indicate it as a potential newtarget to overcome vemurafenib resistance in BRAF-mutatedand MET-addicted carcinomas.

IntroductionThe ENCODE Project has revealed a pervasive transcriptional

activity over an unexpectedly large fraction of the genome (1).Many genomic regions previously assumed to be junk DNA havebeen recently found transcribed into noncoding RNA (ncRNA)teeming with regulatory activities (1). Among them, the longnoncoding RNAs (lncRNA) are defined as transcripts >200 bplacking protein-coding potential (2, 3).

These RNA molecules can be classified into different categories,according to their genomic position comparedwith protein-codinggenes (1). Those overlapping other genes, but transcribed from theopposite strand, are classified as antisense noncoding RNAs, ornatural antisense transcripts (NAT; refs. 4, 5). NATs have beenshown to regulate in cis the expression levels of their sense gene,enhancing or inhibiting the transcription at local level or regulatingmRNA stability (6, 7), recruiting protein-coding mRNAs to activepolysomes for their translation (8), and in trans the expression ofmore distant genes through different mechanisms.

The roles of many lncRNAs have been well characterized,especially in the context of transformation and cancer progres-sion (9–11). One well-known example of NATs implicated incancer pathogenesis is the NAT zinc finger E-box binding homeo-box 2, ZEB2-AS, which regulates the alternative splicing of itssense gene, ZEB2 (12). The binding of ZEB2-AS to 50UTR of ZEBmRNAprevents correct splicing to occur, causing intron retention.This event increases the translation efficiency of ZEB2 mRNAs,leading to increased proliferation, invasion, and metastasis ofcancer cells (12). In other cases, as SAMMSON in melanoma, theknockdown of the lncRNA revealed a potent antitumorigenicactivity (13), indicating this class of ncRNAs as promising can-didate drug targets in cancer therapies. Thus, identifying cancer-related lncRNAs increases the understanding of cancer biologyand subsequently of tumor diagnosis and treatment.

Thyroid cancer is the most common endocrine-related canceraccounting for approximately 4% of all tumors (14). Its incidencehas increased by 3-fold over the last three decades. Themajority of

1Institute of Genetics and Biophysics "Adriano Buzzati-Traverso," CNR, Naples,Italy. 2Institute of Experimental Endocrinology and Oncology (IEOS), CNR,Naples, Italy. 3Institute of Experimental Endocrinology and Oncology (IEOS),CNR, Naples, Italy. 4Department of Molecular Medicine and Medical Biotech-nology, University of Naples "Federico II," Naples, Italy. 5Department of Scienceand Technology, University of Naples "Parthenope," Naples, Italy.

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

R. Esposito and D. Esposito contributed equally to this article.

Current address for R. Esposito: Departments of Medical Oncology and Bio-medical Research, Inselspital, Bern University Hospital, University of Bern, Bern,Switzerland.

Corresponding Author: Valerio Costa, National Research Council (CNR), Via P.Castellino 111, 80131, Naples, Italy. Phone/Fax: 39-081-6132617; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-2520

�2019 American Association for Cancer Research.

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thyroid neoplasms are differentiated forms, that is, papillary(75%–80%). Papillary thyroid carcinomas (PTC) share a subsetof genetic alterations, which includemutually exclusive activatingBRAF and RAS (H-, K-, and NRAS) mutations, accounting for40%–60% and 10%–15% of cases, respectively, and RET rear-rangements (RET/PTC oncogenes), described in approximately20%of cases (Atlas of Genetics andCytogenetics inOncology andHaematology). The large-scale study of The Cancer Genome Atlas(TCGA) Consortium (15), and our independent analysis (16),found that PTCs with somaticmutations in BRAF gene (especiallyV600E) or with rearrangements in RET oncogene (RET/PTC) havehighly overlapping expression patterns and that this subgroup ofpatients (defined BRAF-like) has a markedly distinct signaturecompared with RAS-mutated or highly similar (RAS-like) tumorsamples. TCGA also reported that MAPK and PI3K pathways aredifferentially activated in these PTC subgroups (15). The formerhas a robust activation of the ERK-mediated transcriptionalprogram, partly due to the missing sensitivity of mutant B-Rafto the inhibitory ERK-induced feedback; the latter has a markedinduction of MAPK and PI3K/AKT signaling but, unlikeBRAFV600E-mutated tumors, RAS-like PTCs have functional Rafdimers that are sensitive to ERK-induced inhibition. Thesemolec-ular differences account for distinct tumor phenotypes, mainlycharacterized by increased aggressiveness and invasiveness, higherfrequency of relapse, and onset of drug resistance in the BRAF-likePTCs. However, although subtype-specific expression patternshave been largely investigated, the noncoding fraction of thegenome has not been systematically explored in PTC.

In this work, we used a de novo discovery approach on RNA-seqdatasets of PTC to define BRAF- and RAS-like specific lncRNAs'expression signature and to identifynewcandidate lncRNAsable tomodulate, and/or interfere with, the expression of known cancerdriver genes in thyroid neoplasms. We identified a new naturalantisense Correlated-to-MET (COMET) lncRNA that is dramati-cally upregulated in BRAF-like carcinomas and has a significantpositive correlation with MET oncogene expression in the sametumors. This lncRNA is transcribed antisense toMET locus and hasa prevalent cytosolic localization. COMET-specific knockdownreduced the expression levels ofMET oncogene, as well as of otherMAPK-related genes, impairing cell proliferation, migration, andinvasiveness of BRAF-like thyroid cancer cell line. Thus, our dataconfirm the oncogene-like behavior of lncRNAs in tumors, pro-viding new evidence of a new lncRNA, COMET, which sustainstumor cell survival and proliferation and contributes to MET-driven invasive program in papillary thyroid carcinomas.

Materials and MethodsRNA-seq data analysis and identification of new lncRNAs

The workflow of the entire computational analysis is summa-rized in Supplementary Fig. S1. Cufflinks v2.0.2 (17) and Cuff-merge v2.0.2 (18) were used for ab initio reconstruction. Outputtranscripts sharing identity with exons of protein-coding geneswere filtered out. New lncRNAs were selected according to: (i)length (>200bp), (ii) lack of annotation (inGENCODEv19), (iii)presence of >2 exons, and (iv) protein-coding potential, evaluatedusing CPAT v1.2.2 (19). To select gene/lncRNA pairs, coordinatesof transcription start sites (TSS), as defined in GENCODE v19annotation, were used in ClosestBed function of BEDToolsv2.17.0A (20) to identify the nearest gene. Only pairs withdifferentially expressed protein-coding genes and differentially

expressed lncRNAs were selected. Cancer driver genes associatedwith newly identified lncRNAs were selected from a list of 114genes published by Vogelstein and colleagues (21). Peaks ofchromatin markers [H3K27Ac, H3K4me1, p300 binding, andDNase hypersensitive sites (DHS)] at gene promoters were down-loaded from ENCODE (2012) and BEDTools were used to detectpeaks (1 kb window) around the TSS of new lncRNAs. The exactexon/intron structure ofCOMET lncRNA defined by RNA-seqwasfirst validated by CAGE in different cell lines from the FANTOM5study (http://fantom.gsc.riken.jp/5/) and then experimentally bycombining RT-PCR, cloning, and direct Sanger sequencing.

Cell linesHuman papillary thyroid carcinoma cell lines, TPC-1 and

BCPAP, and normal thyroid follicular epithelial cell line, Nthy-ori3-1,were kindly provided in 2015byProf. Alfredo Fusco andProf.Massimo Santoro (Medicina Molecolare e Biotecnologie Med-iche, Universit�a degli Studi di Napoli Federico II, Napoli, Italy),respectively. Themutational statuswas confirmedbydirect SangerSequencing of PCR amplicons of the BRAF gene protein-codingregion (BCPAP) and by qPCR analysis to assess RET activation(TPC-1). Tumor cell lines, TPC-1 and BCPAP, were grown inDMEM supplemented with 10% FBS, 2 mmol/L glutamine, 100U/mL penicillin, and 100 U/mL streptomycin. Nthy-ori 3-1,instead, were cultured in RPMI1640 at the same condition. Allcell lines used in this work were maintained at 37�C and 5% CO2and all were assayed for the presence of Mycoplasma by chemilu-minescence and specific PCR-based assays (latest test in January2018). All the experiments on these cell lines were performedbetween the 4th and the 16th cell passage.

RNA fractionation and FISHNuclear and cytoplasmic RNA fractions were isolated using the

Cytoplasmic and Nuclear RNA Purification Kit (Norgen BiotekCorp, catalog no. 37400) according to themanufacturer's instruc-tions. GAPDH and U2 were used as control genes (cytosolic andnuclear fractions, respectively). Primers are listed in Supplemen-tary Table S1. For COMET cellular localization by FISH, a poolof 6 fluorescent probes targeting COMET RNA was purchased(Integrated DNA Technologies). Cells grew on slides were fixedwith 4% formaldehyde and permeabilized in 0.1% Triton. Onegram of dextran, 10% of formamide, and 2� SSC were used ashybridization buffer overnight at 37�C. DAPI and Actin Greenwere used to stain nucleus and cytoplasm, respectively. Signalswere visualized using Zeiss Axiophot (upright microscope)equipped with Nikon Coolpix995.

RT-PCR, qPCR, and RNA interferenceRNA was isolated using TRIzol and reverse transcribed using

High Capacity cDNA Reverse Transcription Kit (Invitrogen),according to the manufacturer's instructions. Quantitative PCRs(qPCR) were performed on the CFX Connect Real-Time PCRDetection System (Bio-Rad), using iTaq Universal SYBR GreenSupermix (Bio-Rad) as described in ref. 22. PPIA gene was used asreference for data normalization and relative gene expression wasmeasured with the 2�DDCt method. qPCR oligo pairs are listed inSupplementary Table S1.

TPC-1 and BCPAP cells were transfected with small interferingRNAs (siRNA) usingOligofectamine (Life Technologies) accordingto the manufacturer's recommendations. siRNAs targeting theCOMET 30 (2 duplex siRNAs 50-GGAAGTTTGAGTGACTCAT-30;

COMET Oncogenic Role in BRAF-like Papillary Thyroid Cancer

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50-GCTCAGAAATGACACAATT-30) were purchased from IDT.siRNAs for MET (2 specific siRNAs, Origene #SR302873), BRAF(2 specific siRNAs, Origene #300470), FOSL2 (2 specific siRNAs,Origene #SR301649), and control siRNA (Origene # SR30004)were obtained from Origene. Knockdown (KD) efficiency wasassessed by qPCR.

Viability and apoptosis assaysTPC-1 and BCPAP cells were plated in 96-well white opaque

plates. Viability was measured by luminescence assay usingCellTiter-Glo Kit (Promega) according to the manufacturer'sinstructions at 0, 24, 48, 72, and 96 hours from COMET KD.Caspase-Glo 3/7 Assay (Promega) was used to measure caspaseactivity by luminescence assay in COMET KD and control cells at16, 24, and 48 hours after silencing. Luminescence was measuredwith Victor X3 Multimode plate reader (Perkin Elmer).

Colony forming and proliferation assaysTwenty-four hours after COMET KD, 150 TPC-1 cells were

plated in 6-well plates with DMEM supplemented with FBS10%. After 8 days, cell colonies were fixed with 20% methanoland stained with 0.5% crystal violet. Cell clones were countedwith ImageJ software (NIH, Bethesda, MD).

For proliferation assay, COMET KD and control cells wereharvested and 0.1%CellTrace Violet reagent was added accordingto the manufacturer's protocol. Flow cytometry (Becton Dickin-son FACSCanto) was used to measure intracellular CellTracecontent after 24, 48, and 72 hours.

Cell migration and invasion assaysTwenty-fourhours after siRNA transfection, cellswereharvested

and seeded into the top chamber of the transwell (pore size, 8 mm;Corning). After 24 hours, migrating cells were stained in 0.1%crystal violet for 30 minutes. Crystal violet was solubilized in PBScontaining 1% SDS and its concentration was measured (absor-bance 595 nm). Invasion assay was carried out in transwellchambers precoated with 2% Matrigel (Corning). Invading cellswere counted as described above for migration assay.

Physical and chemical stimuliTo induce hypoxia, TPC-1 cells were placed in a chamber gassed

with 1% O2 at 37�C for 24 hours. Control cells were incubatedunder normoxic conditions (21% O2, 5% CO2 at 37�C) for thesame time.

Nthy-ori 3-1 cells were starvedwith RPMI1640mediumwithoutFBS for 24hours, then100ng/mLofEGF (ThermoFisher Scientific)was added to cells for the indicated times. Western blot analysis ofp-Erk was carried out to verify MAPK induction. Similarly, hepa-tocyte growth factor (HGF, Thermo Fisher Scientific) was added toNthy-ori-3-1 cells at 100 ng/mL after 24 hours of starvation.

BCPAP cells were treated with a fixed concentration of vemur-afenib (VMR; Selleckchem.com, #S1267). An equal volume ofvehicle (DMSO) was added for the same time. Viability assay andqPCR were carried out to detect viable cells and COMET expres-sion levels, respectively, after VMR treatment.

Western blot analysisTPC-1 and Nthy-ori 3-1 cell lines were treated with lysis

buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/LEDTA, 10 mmol/L Na4P2O7, 2 mmol/L Na3VO4, 100 mmol/LNaF, 30 mmol/L glycerol, 10% Triton X-100) supplemented with

protease and phosphatase inhibitor cocktail (Thermo Fisher Sci-entific). Protein concentration was evaluated using a colorimetricassay (Bradfordprotein assay, Bio-Rad). Equal amountsof proteinlysate were subjected to 6% or 10% SDS-PAGE separation andtransferred by electrophoresis to polyvinylidene difluoride mem-branes. After blocking in Tris-buffered saline–Tween 20 (TBS-T)containing 5% nonfat dried milk, membranes were probed withtargeted primary antibodies: c-Met (1:10,000; 3 hours at roomtemperature; Cell Signaling Technology, #8198), p-Erk (1:1,000;3 hours at room temperature; Cell Signaling Technology, #9101),Hsp90 (1:2,000; 1 hour at room temperature; Origene,#TA500494). Membranes were washed with TBS-T and thenincubated with appropriate horseradish peroxidase-conjugatedsecondary antibody (Bio-Rad #170-6516, #170-6515) for 1 hourat room temperature. Immunoreactive bands were detected onX-ray film using enhanced chemiluminescence method (ThermoFisher Scientific).

Statistical analysisAll the experiments were independently repeated at least three

times. Significant differences between groupswere analyzed usingtwo-tailed Student t test. A P 200 ntwith a codingpotential

Methods). Interestingly, based on these criteria, we identifiedCOMET as a new lncRNA positively correlated (r ¼ 0.7) toMET oncogene.

Characterization of COMET: a new natural antisense lncRNACOMET lncRNA maps on chromosome 7q31.2 and is tran-

scribed from the antisense strand of MET oncogene (Fig. 2A),partially overlapping the GENCODE entries AC006159.3 andAC06159.4 (RefSeq LINC01510). De novo reconstruction andfurther validation by RT-PCR revealed that different transcriptsarise from this locus, one of which corresponds to LINC01510.RNA-seq also revealed the expression of a new splicing variant(with the skippingof exon 2) of the already annotated LINC01510,reported herein as LINC01510_var (Fig. 2A). More interestingly,RNA-seq data allowed us discovering longer transcripts (GenBankaccession nos. LN812953, LN812954, and LS991956) with the TSSlocated in the first intron of MET. These unannotated isoformsindicate that this lncRNA is a NAT of MET oncogene.

To support the presence of an actively transcribed locus, we firstused our RNA-seq data and then recently released FANTOM5datato precisely assess the genomic structure of the new NAT COMETand to map its TSS (Fig. 2A). Afterwards, we searched chromatinmarks in public ENCODE Consortium data (magnificationin Fig. 2A) in the genomic region surrounding COMET TSS andits putative promoter. This analysis revealed peaks of H3K4me1andH3K27Ac, characterizing active promoters, and a high enrich-ment of conserved DHS (detected in 83 cell lines out of 125analyzed by ENCODE) around COMET TSS, distinctive of openchromatin. Further supporting these data, we also mapped DHSdata from the work of Jin (GEO accession number GSE61844;ref. 23) obtained using Pico-Seq on papillary and follicularthyroid cancer samples. Many of the mapped DHSs associatedwith promoters and enhancers of cancer-related genes and,remarkably, we could identify, in more than one sample, a DHSlocated within the putative COMET promoter,

MiTranscriptome database (24) revealed that COMET is prefer-entially expressed in thyroid carcinoma samples, differently fromthe LINC01510 (Supplementary Fig. S3A), as confirmed by semi-quantitative RT-PCR on different cell lines and tumors (Supple-mentary Fig. S3B). Interestingly, some of them did not expressCOMET despite having high MET oncogene levels.

Its restricted expressionpattern and the very low codingpotential(CP ¼ 0.0248; see Materials and Methods) score compared withprotein-coding genes support COMET as a new lncRNA. Finally,analyzing the evolutionary conservation, we found that COMET isconserved in primates' genome, but not in other vertebrates.

COMET lncRNA is highly expressed in BRAF-like carcinomasIn line with the general notion that lncRNAs have low

expression, RNA-seq data revealed that COMET is expressedmarkedly less than the neighborMET oncogene. However, bothof them are overexpressed in the BRAF-like subgroup of PTCs(Fig. 2B), suggesting that the pathologic hyperactivation of Retand B-Raf proteins contributes to their increased expressionlevels. To corroborate this finding, we stratified a larger set ofindependent samples (tumors n¼ 50; healthy n¼ 11) from ourprevious work (16) according to common somatic mutations(BRAFV600E, H-, K- and N-RASmutations in codons 12, 13, and61) and RET gene rearrangements, in BRAF-like (n ¼ 32) andRAS-like (n ¼ 18) subgroups. In this independent cohort, we

confirmed by qPCR that both the NAT lncRNA COMET and itsneighbor oncogene MET are overexpressed in BRAF- versus RAS-like PTCs and control samples (Fig. 2C). Similarly, we analyzedpublic TCGA exome data of thyroid carcinoma (THCA; n ¼ 507)andwe stratifiedpatients according to thedriver genetic alteration.Then, we analyzed RNA-seq data of these patients and we con-firmed our findings even in this independent and much largercohort of PTCs (Supplementary Fig. S3C).

COMET is a new MAPK-induced cytosolic lncRNATo validate COMET expression as a transcriptional event

downstream Ret and B-Raf proteins constitutive activation, wemeasured COMET level in PTC cell lines carrying eitherBRAFV600E mutation (BCPAP) or RET gene rearrangement(TPC-1) compared with an immortalized normal thyroid cellline (Nthy-ori 3-1). As expected, COMET, as well as MET,expression was significantly higher in BRAF-like cell lines thanin normal cells (Fig. 3A); TPC-1 cell line, displaying the highestCOMET expression levels, was used as in vitromodel for most ofthe phenotypic assays in the next paragraphs.

BRAF-like PTCs are characterized by a robust activation ofERK-mediated transcriptional program (15). It promoted us toinvestigate whether COMET expression is induced downstreamMAPK activation. Thus, we stimulated RAS–RAF–MEK–ERKaxis in Nthy-ori 3-1 cells with the EGF. Induction of MAPK

Figure 2.

Genomic structure and expression analysis of the natural antisense lncRNA COMET in tumor samples.A, Schematic representation of the genomic locusencompassing the newly identified lncRNA COMET. In the top part, the exon/intron structure is depicted, with exons shown as boxes, introns as thin lines witharrows indicating the sense of transcription for each gene. The structure, defined by ab initio reconstruction from RNA-seq datasets (16), was confirmed bySanger sequencing. GENCODE (v19) and RefSeq transcriptome annotations for the neighbor genes are also depicted in the bottom part. A box containing themagnification of the genomic region containing the exon 1 of COMET is shown on the right. ENCODE tracks for H3K4Me1, H3K4Me3, and H3K27Ac mapping acrossCOMET exon 1 are represented as coverage curves (peaks); DHS from ENCODE and Pico-Seq datasets (GEO accession. no. GSE61844) are shown in black;ENCODE tracks for enhancer and heterochromatin are shown as boxes in grayscale. B and C, Boxplots showing the expression levels of COMET lncRNA and ofthe neighbor oncogeneMET from RNA-seq data (B) and measured by qPCR (C) in BRAF-, RAS-like PTCs, and healthy controls. The number of analyzed patientsper group is reported on the x-axis. COMET andMET relative expression in Cwasmeasured after normalization versus a reference gene (PPIA). �� , P < 0.001.

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pathway, confirmed by early p-Erk increase (Fig. 3B, right), wasfollowed by a significant increase of COMET levels (Fig. 3B,left). Conversely, the inhibition of the constitutively activepathway in BRAF-mutated BCPAP cell line using chemical andgenetic approaches, either by blocking hyperactivated B-Rafprotein with vemurafenib (Fig. 3C; Supplementary Fig. S4A)or by BRAF gene siRNA-mediated KD (Fig. 3D; SupplementaryFig. S4B), significantly decreased COMET levels. Nevertheless,in cells treated with increasing VMR doses, COMET did notshow a dose-dependent downmodulation, suggesting that oth-er downstream factors contribute to COMET regulation(Fig. 3C). Thus, to better understand how this lncRNA istranscriptionally induced downstream the activation of MAPKsignaling pathway, we took advantage of publicly availableChIP-Seq ENCODE data for transcription factors bindingaround COMET TSS and/or its predicted promoter. This anal-ysis revealed enriched Fosl1:Fosl2 binding (in more than onecell line) in a DHS at about 1.5 kb from COMET TSS. Inter-estingly, FOSL2 is overexpressed in BRAF-like tumors in bothour previous RNA-seq and in TCGA datasets (Supplementary

Fig. S4CandS4D, respectively). In addition, publicly availableGTExRNA-seq data revealed this AP-1 complex member to be highlyexpressed in thyroid (https://gtexportal.org/home/gene/FOSL2).

To validate FOSL2 as potential COMET transcriptional factor,we first verified its induction in RAS–RAF–MEK–ERK–depen-dent manner in Nthy-ori 3-1. As expected, EGF-mediated stim-ulation caused a marked increase of FOSL2 expression levels(Fig. 3E). siRNA-mediated KD of this AP-1 complex member inTPC-1 cell line (Supplementary Fig. S4E) significantly reduced(up to 50%) the levels of COMET lncRNA at 12, 24, and48 hours (Fig. 3F). These data suggest that FOSL2 may con-tribute to COMET expression in PTCs, especially in tumors withmarked activation of MAPK signaling (i.e., BRAF-like patientsand tumor cell lines).

Afterwards, we evaluated COMET cellular localization. RNAfractionation coupled to qPCR revealed that, similar to the controlprotein-coding gene (GAPDH), COMET has a preferential enrich-ment in the cytosolic RNA fraction (Fig. 3G). In addition, RNAFISH further confirmed the prevalent cytoplasmic localization ofthis new NAT lncRNA (Fig. 3H).

Figure 3.

COMET is a cytosolic NAT, induced downstream MAPK pathway activation. A, Relative quantification (qPCR) of COMET and MET RNA (light and darkgray, respectively) in immortalized normal thyroid cell line (Nthy-ori 3-1) and in two papillary thyroid cancer cell lines carrying BRAFV600E mutation(BCPAP) or RET gene rearrangement (TPC-1). Data are plotted as means (n ¼ 3) � SD versus Nthy-ori 3-1 cell line. B, Left, relative quantification(qPCR) of COMET in TPC-1 cell line stimulated with 100 ng/mL of human recombinant EGF for 3 and 6 hours. Data are plotted as means (n ¼ 3) �SD versus untreated cells. Right, representative images (of three independent experiments) of Western blot assays probed for p-Erk antibody ontotal lysates from TPC-1 cells treated with 100 ng/mL of EGF (30 minutes and 1 hour) or untreated (0 hours). Hsp90 was used as loading control.C and D, Relative quantification (qPCR) of COMET in BCPAP cells treated with increased doses of VMR for 48 hours (C) or transfected with BRAF-specific siRNA oligos (D). Data are plotted as means (n ¼ 4) � SD versus untreated cells (black dotted line with arbitrary value ¼ 1; C) or cellstransfected with scrambled siRNAs at each time point (black dotted line with arbitrary value ¼ 1; D). E, Relative quantification (qPCR) of FOSL2 inNthy-ori 3-1 cell line treated with 100 ng/mL human recombinant EGF. Data are plotted as means (n ¼ 3) � SD versus untreated cells. F, Relativequantification (qPCR) of COMET in TPC-1 cells transfected with a pool of FOSL2-specific siRNAs. Data are plotted as means (n ¼ 5) � SD versus cellstransfected with scrambled siRNAs at each time point (black dotted line with arbitrary value ¼ 1). PPIA was used as reference gene for the qPCRassays reported in A–F. G, Subcellular fractionation and relative quantification (qPCR) of COMET enrichment. Barplots show COMET relativeenrichment in the nuclear (light gray bars) and the cytoplasmic (dark gray bars) fraction of TPC-1 cells. GAPDH and U2 were used as cytosolic andnuclear markers, respectively. Data are plotted as means (n ¼ 3) � SD versus the nuclear fraction. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. H,Representative fluorescence images (of three independent experiments) of RNA FISH probed for COMET in TPC-1 cells. Nuclei were stained with DAPI(blue; top left); cytoplasms were stained with Actin green (green; top right); COMET-targeting probes were conjugated with Cy3 (orange; bottomleft). Merged channels are shown in the bottom right.





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COMET lncRNAknockdown impairs the expression of differentoncogenes

To predict a function for COMET and to define its potentialbiomedical significance in the context of papillary thyroid cancer,a guilt-by-association analysis was carried out. Taking advantageof the TCGA-based lncRNA atlas TANRIC (25), we assessedwhether COMET expression in thyroid carcinoma samplescorrelated with the expression of other genes, using Pearsoncorrelation (Supplementary Table S2). In line with our initialfinding, the highest correlation value was measured with theneighbor oncogene MET (r ¼ 0.88). Moreover, pathway analysiscarried out on genes having significant correlation with COMET(FDR

siRNAs displayed a higher silencing efficiency, in line withCOMET cytosolic localization, and all further knockdownexperiments were carried out using siRNAs pool. As shownin Fig. 4B, COMET KD significantly impaired the expressionlevels of most of the genes within the network, and especially ofAKT3, CREB5, and DUSP5. A significant drop in MET mRNAand protein levels upon COMET KDwas measured (Fig. 4C). Toavoid any potential confounding effect due to siRNAs' off-target activity on MET expression, we assessed silencing spec-ificity transfecting HEK293 cell line, which expresses MET butdoes not express COMET, with the same COMET-specific poolof siRNAs. No variation in MET oncogene levels was measuredin this cell line upon COMET KD (Supplementary Fig. S5D),indicating that COMET-specific siRNAs do not affect METoncogene levels. We further tested whether a reciprocal regu-lation may occur, that is, whether MET KD could affect thelevels of COMET and other genes belonging to COMET coex-pression network. Remarkably, upon MET KD (up to 80%) inTPC-1 cells, we could not measure any significant variation ofCOMET levels as well as of COMET-correlated genes (Fig. 4B),suggesting that the reduction of their expression levels observedupon COMET KD is MET-independent.

Furthermore, to explore a putative common regulation ofCOMET lncRNA and its neighbor oncogene MET, we evaluatedwhether MET-inducing stimuli can also trigger COMET lncRNAexpression. Thus, we focused on two stimuli known to induceMET expression, that is, hypoxia and hepatocyte growth factor(HGF) treatment. TPC-1 grew in hypoxic condition displayed2-fold increase in COMET level compared with cells grown innormoxic conditions (Fig. 4D), revealing that hypoxia-mediatedtranscriptional response also impacts COMET expression. Simi-larly, the acute stimulation of Nthy-ori 3-1 with the membranec-Met receptor ligand HGF also induced an increase of COMETlncRNA levels (Fig. 4E). Notably, treatment with HGF alsoinduced FOSL2 expression (Supplementary Fig. S6A), furthersuggesting its contribution in the regulation of COMET.

COMET knockdown inhibits cell proliferation and inducesapoptosis

Because COMET KD caused a relevant drop in the levels ofc-Met as well as a significant decrease in the expression levels ofother MAPK-related oncogenes in thyroid cancer cells, we furtherinvestigated the phenotypic effects of COMET KD on tumor cells.To this aim, wemeasured cell viability and proliferation of TPC-1cell line following COMET KD at different time points. Interest-ingly, the percentage of viable cells significantly decreased up toapproximately 40% (at 72 hours) inCOMET KD versus scramble-transfected cells (Fig. 5A). Similarly, COMET KD markedlyreduced cell proliferation, as measured by cell proliferation assay(Fig. 5B).

In addition, to further understand how COMET regulates cellgrowth of thyroid cancer cells, we verified whether COMET KDwas able to induce apoptosis, measuring the activity of caspase-3and -7. As shown in Fig. 5C,COMETKD significantly increased thenumber of apoptotic TPC-1 cells after 24 hours of silencing.

Finally, to verify whether COMET KD affects in vitro the tumor-igenic potential of thyroid tumor cells, the colony-forming abilityof COMET KD TPC-1 and control cells was assayed. As shownin Fig. 5D, COMET silencing significantly affected the oncogeniccapacity, in terms of number and size of colonies, of thyroidcancer cells.

COMET silencing affects migration and invasiveness of thyroidcancer cells

Considering the effect of COMET silencing on c-Met proteinand the primary role of the latter in the invasive growth programin tumors,we explored the effects ofCOMETKDonmigration andinvasiveness of thyroid cancer cells. Data summarized in Fig. 6Aand B show that COMET knockdown significantly reduces (upto 40%) both the motility and invasiveness of TPC-1 cells.Furthermore, upon COMET KD, tumor cells displayed a lowerexpression of vimentin (mRNAandprotein),marker of epithelial-to-mesenchymal transition (EMT), without any variation ofN-cadherin (Fig. 6C).

Taken together, these data support an oncogenic role in vitro forthe newly identified COMET lncRNA in thyroid, strongly suggest-ing that such a property relies, at least in part, on its modulatoryactivity on the expression levels of MET and of MAPK-relatedoncogenes.

In vitro assessment of the therapeutic potential of COMETtargeting

Activation of receptor tyrosine kinases, and especially of c-Metreceptor, has been reported as a mechanism for tumor cells toescape vemurafenib-mediated inhibition of mutant B-Raf pro-tein (26, 27). As our data supportCOMET as a newMET regulator,we askedwhether itmight also contribute to vemurafenib respon-siveness in BRAF-mutated tumor cells. First, we assessed that,similar to TPC-1 cells, COMET silencing had a strong effect onviability (up to 50%) of BRAFV600E-mutated BCPAP cell line(Fig. 6D), with a concomitant drop inMET levels (SupplementaryFig. S6B). A similar effect (up to 60% of cell viability reduction;Supplementary Fig. S6C) was also measured when BCPAP weretreated with VMR. Therefore, we exposed BCPAP cells knocked-down for COMET to VMR to assess whether the combinedtreatment may exert an additive effect. Interestingly, data plottedin Fig. 6D demonstrate that COMET KD BCPAP cells had amarkedly improved VMR sensitivity compared with control cells(i.e., cells treated only with VMR and/or transfected with controlsiRNAs, Fig. 6D), indicating COMET as a new target to improvechemotherapy in BRAFV600E-mutated tumors.

DiscussionDifferential signaling consequences upon genetic alteration of

BRAF, RET, and RAS genes have been reported by TCGA (15) andthen by our group (16) in papillary thyroid carcinomas, especiallyon MAPK signaling activation. One of the most relevant discov-eries, with intriguing clinical perspectives, was the finding thatBRAF-like tumors exhibit a very robust activation of ERK-medi-ated transcriptional program, partially explained by the missingsensitivity of mutant B-Raf proteins to ERK-induced inhibitoryfeedback loop. Conversely,RAS-like tumors have amarked induc-tionofbothMAPKandPI3K/AKT signaling, but aremore sensitiveto ERK-mediated inhibition due to the presence of functional Rafdimers. These findings have highlighted the need to reconsidertheir classification scheme,moving thyroid cancer therapy towardthe route of precision medicine. However, much of the effortshave been directed to explain the role of protein-coding genes intumor etiology, with a consequent poor knowledge aboutlncRNAs' roles in neoplastic transformation and response tochemotherapy. Inmore recent years, a growing number of studiesare shifting the focus on this class of ncRNAs, because they






represent very attractive therapeutic targets, even in PTC. One ofthe most recent and convincing work is the identification oflncRNA AB074169 (lncAB) as tumor suppressor in PTC tumor-igenesis (28). In this study, we specifically focused our attentiontoward the identification of lncRNAs that are activated by specificmutated oncogenes in papillary thyroid carcinoma.

Coupling ab initio transcriptome reconstruction and tar-geted resequencing of somatic mutations to the analysis ofpublicly available TCGA omics data and ENCODE tracks forepigenetic marks, we identified COMET as a new naturalantisense transcript highly expressed in BRAF-like PTC sub-type. Noteworthy, the identification of lncRNA profiles asso-ciated with specific somatic alterations in PTCs also consti-tutes a step toward the understanding of BRAF-, RET-, andRAS-mediated tumorigenesis and progression. In this regard,few recent reports have suggested some PTC-deregulatedlncRNAs, enriched in BRAF-mutated patients, as new potentialdiagnostic and therapeutic targets in this tumor subtype (29).

More recently, the BRAF-activated lncRNA (BANCR) has beeninvestigated for its potential role as oncogene or oncosup-pressor, with opposite results in the context of PTC (30–32).Likewise, Orilnc1 lncRNA was recently identified in BRAF-mutant melanoma as regulated by RAS–RAF–MEK–ERK sig-naling pathway through AP-1 transcriptional complex (33).

Our study identifies COMET as a new cytosolic lncRNA withlow expression, in line with other lncRNAs, such as VELUCT (34)that, despite its extremely low abundance, leads to strong loss-of-function phenotype. COMET is markedly overexpressed in BRAF-like versus RAS-driven tumors and healthy thyroid, is induceddownstream MAPK activation and is required for tumor cellsurvival and growth. Our study did not definitely demonstratehow COMET is regulated. Nevertheless the presence of a consen-sus site for Fosl2 upstream COMET TSS and the drop of COMETexpression upon FOSL2 KD provide strong suggestion aboutthe contribution of Fosl2 to COMET regulation and a possiblemolecular explanation of its induction in BRAF-like tumors with

Figure 5.

COMET silencing inhibits cell proliferation and induces apoptosis. A, Line chart displaying the number (thousands) of viable TPC-1 cells following COMET KD,at different time points. Data are plotted as means (n¼ 3 for each time point)� SD. B, Representative images (of three independent experiments) of FITC-Aemission spectra measured by flow cytometry in control and COMET KD TPC-1 cells (dark and light gray, respectively). Peaks represent live cells stained withCellTrace and cultured for the days indicated in the top left corner of each subpanel. C, Line chart displaying caspase-3/7 quantification (absorbance) followingCOMET KD at different time points in TPC-1 cells. Data are plotted as means (n¼ 3 for each time point)� SD. D, Left, a representative image (of threeindependent experiments) of colony formation assay. Right, number of colonies (%) after 7 days of COMET KD in TPC-1 cells. Data are reported as mean (n¼ 3)� SD versus cells transfected with scrambled siRNAs; the number of colonies for TPC-1 cells transfected with scrambled siRNAs was arbitrarily set to 100%.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Esposito et al.





RAF–MEK–ERK pathway constitutive activation. In addition,FOSL2 expression is rapidly induced upon HGF treatment con-comitantly with COMET lncRNA increase. However, we cannotexclude the contribution of other transcription factors and/orcoactivators to this process. Indeed, as reported by Grossman andcolleagues in a very recent large-scale analysis (35), Fosl2 belongsto a cluster of transcriptional activators that, compared with otherclasses of transcription factors (TF), binds more frequently othercoactivators (p300 and CREB-binding protein) and tend to inter-act with each other, suggesting a cooperative role in activatinggene transcription. In addition, as the transcription factors form-ing AP-1 complex are generally required to maintain accessiblechromatin, it allows the binding of other stimulus-regulatedTFs (36). Therefore, it is likely that Fosl2 is one of the MAPK-induced TFs involved in COMET regulation.

The identification of a network of coexpressed genes whoseexpression is strongly perturbed only in tumors driven by specificsomatic alterations, that is, BRAFV600E mutation and rearrange-ment of RET oncogene, defines amore complex scenario in whichCOMET is a new candidate nonprotein effector of MAPK signal-ing. Interestingly, silencing of COMET in BRAF-like tumor cells issufficient to downmodulate cancer-related genes belonging toMAPK and PI3K signaling pathways (such as AKT3, MAPK1,DUSP5, and CREB5). More interestingly, a much relevant repres-sing effect was measured on MET oncogene. This finding iscompletely in line with a very recent report from Cen andcolleagues (37), published at the time of writing, showing thatLINC01510, a shortest isoform of COMET lncRNA, promotesproliferation of colorectal cancer cells, at least in part, by mod-ulating MET oncogene expression. Here we have demonstrated

Figure 6.

COMET silencing rescues the invasive tumor cell phenotype and potentiates the activity of vemurafenib. A and B,Migration and invasion assays of TPC-1 cellsfollowing COMET KD (transwell chambers) in absence (A) or presence (B) of Matrigel.A and B, Left, a representative image of three independent experimentsper condition. Right, barplots reporting crystal violet absorbance (percentage) of migrating (A) and invading (B) cells upon COMET KD. Data are presented asmean (n¼ 3)� SD versus cells transfected with scrambled siRNAs. The number of crystal violet-stained cells transfected with scrambled siRNAs was arbitrarilyset to 100%. C, Top, relative quantification (qPCR) of VIM and CDH2 genes in TPC-1 cells following COMET KD (72 hours). Data are plotted as means (n¼ 5)� SDversus cells transfected with scrambled siRNAs. PPIAwas used as reference gene. Bottom, representative images (of three independent experiments) ofWestern blot assay probed for vimentin (left) and N-cadherin (right) on total lysates from TPC-1 cells following COMET KD and transfected with scrambledsiRNAs (72 hours upon transfection). Gapdh and b-actin were used as loading controls. D, Line chart showing cell viability (percentage) of BCPAP cells followingCOMET KD, or treated with VMR or with the combination of both. For the combined treatment, BCPAP cells were first transfected with COMET (or scrambledsiRNAs) for 24 hours and then treated with VMR (5 mmol/L) at the indicated time points. Data are plotted as means (n¼ 3 for each condition)� SE. Asterisksindicate significant variation compared with cells transfected with scrambled siRNAs; braces refer to the comparison between specific conditions. � , P < 0.05;�� , P < 0.01; �� , P < 0.001.






that repressing COMET expression inhibited viability and prolif-eration of tumor cells harboring either RET oncogene rearrange-ment orBRAFV600E somaticmutation.Moreover, compatiblewiththe finding that COMET KD impairs mRNA and protein levels ofthe oncogeneMET, key driver of the invasive program in tumors,we also demonstrated that COMET repression dramaticallyreduces motility and invasiveness of tumor cells. Our findingsare particularly relevant especially for tumors carrying BRAFV600E

mutation. Indeed, despite patients of this subgroup, representingthe most abundant fraction of PTC as well as ATC cases, can beeffectively treated with B-Raf inhibitors targeting constitutionallyactive mutated B-Raf, they often develop drug resistance (38, 39).Recently, the activation of c-Met oncogene has been reported asone of the mechanisms that BRAF-mutated cells adopt to escapeVMR-induced blockade of MAPK signaling (40). Nonetheless,c-Met inhibitors have providedonly partial benefits due to the lowspecificity of the drugs and to the off-target effects (41, 42).AlthoughMET oncogene overexpression has been reported morethan 20 years ago in thyroid carcinomas, only recently it hasbeen proposed as one of the possible contributors to VMRresistance. In line with this, the analysis of independent cohortsof PTC samples revealed that BRAF-like tumors, unlike RAS-likeand healthy thyroids, overexpress both MET oncogene and thenewly identified COMET lncRNA. The finding that COMET KD iscapable to impair the levels of MET and of other MAPK-relatedoncogenes and that tumors with repressed COMET stronglyrespond to VMR exposure, render this lncRNA a very attractivetarget to be tested in vivo experiments.

Our results warrant further investigation on the noncodingeffectors downstream BRAF signaling and suggest that COMET

repression may represent a new therapeutic tool to overcometumor resistance to BRAF inhibition.

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

Authors' ContributionsConception and design: R. Esposito, D. Esposito, A. Ciccodicola, V. CostaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. CostaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Esposito, D. Esposito, A. Ciccodicola, V. CostaWriting, review, and/or revision of the manuscript: R. Esposito, D. Esposito,A. Ciccodicola, V. CostaStudy supervision: V. Costa

AcknowledgmentsThe authors acknowledge Project SATIN - POR Campania FESR 2014/2020

(to V. Costa) and Associazione Italiana per la Ricerca sul Cancro (AIRC IG 2013-14689 to A. Ciccodicola). The authors thank Prof. Alfredo Fusco and Dr.Pierlorenzo Pallante for kindly providing RNA samples from thyroid biopsiesand human papillary thyroid carcinoma cell lines TPC-1 and BCPAP, and Prof.Massimo Santoro for kindly providing normal thyroid follicular epithelial cellline Nthy-ori 3-1. The authors also thank the FACS and Integrated MicroscopyFacilities of the IGB-CNR for technical support.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 14, 2018; revised January 10, 2019; accepted March 6, 2019;published first March 12, 2019.

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2019;79:2124-2135. Published OnlineFirst March 12, 2019.Cancer Res Roberta Esposito, Daniela Esposito, Pierlorenzo Pallante, et al.

-Driven Papillary Thyroid CarcinomasRETand -BRAF in COMETOncogenic Properties of the Antisense lncRNA

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