cancers

Article Simultaneous Expression of Long Non-Coding RNA FAL1 and Extracellular Matrix 1 Defines Tumour Behaviour in Young Patients with Papillary Thyroid Cancer

Seonhyang Jeong 1,†, Seul-Gi Lee 2,†, Hyunji Kim 3, Gibbeum Lee 3, Sunmi Park 1, In-Kyu Kim 3, Jandee Lee 3,* and Young-Suk Jo 1,*

1 Department of Internal Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea; [email protected] (S.J.); [email protected] (S.P.) 2 Department of Surgery, Eulji University School of Medicine, 95 Dunsanseo-ro, Seo-gu, Daejeon 35233, Korea; [email protected] 3 Yonsei Cancer Center, Open NBI Convergence Technology Research Laboratory, Severance Hospital, Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea; [email protected] (H.K.); [email protected] (G.L.); [email protected] (I.-K.K.) * Correspondence: [email protected] (J.L.); [email protected] (Y.-S.J.); Tel.: +82-2-2228-2100 (J.L.); +82-2-2228-0752 (Y.-S.J.); Fax: +82-2-313-8289 (J.L.); +82-2-393-6884 (Y.-S.J.) † These authors contributed equally to this work.

Simple Summary: FAL1 upregulation has been reported in many types of human cancers. The   up-regulatory mechanism was identified in ovarian cancer but was not investigated in other type of cancers. Using The Cancer Genome Atlas (TCGA) database, we identified simultaneous upregulation Citation: Jeong, S.; Lee, S.-G.; Kim, of FAL1 adjacent to 1q21.3. Among 53 putative transcription factors for FAL1 and neigh- H.; Lee, G.; Park, S.; Kim, I.-K.; Lee, J.; bouring , we selected c-JUN and JUND as the best candidates. This simultaneous upregulation Jo, Y.-S. Simultaneous Expression of defines molecular biological features representing RAS-driven PTC-enriched immune-related Long Non-Coding RNA FAL1 and sets. These findings suggest that the simultaneous upregulation might be a potential diagnostic and Extracellular Matrix Protein 1 Defines therapeutic target for RAS-driven PTC. Tumour Behaviour in Young Patients with Papillary Thyroid Cancer. Cancers 2021, 13, 3223. https:// Abstract: We investigated the regulatory mechanism of FAL1 and unravelled the molecular biologi- doi.org/10.3390/cancers13133223 cal features of FAL1 upregulation in papillary thyroid cancer (PTC). Correlation analyses of FAL1 and neighbouring genes adjacent to chromosome 1q21.3 were performed. Focal amplification was Academic Editors: Chan-Kwon Jung performed using data from copy number alterations in The Cancer Genome Atlas (TCGA) database. and Andrey Bychkov To identify putative transcriptional factors, PROMO and the Encyclopaedia of DNA Elements (EN- CODE) were used. To validate c-JUN and JUND as master transcription factors for FAL1 and ECM1, Received: 25 May 2021 gene set enrichment analysis was performed according to FAL1 and ECM1 expression. Statistical Accepted: 23 June 2021 analyses of the molecular biological features of FAL1- and ECM1-upregulated PTCs were conducted. Published: 28 June 2021 FAL1 expression significantly correlated with that of neighbouring genes. Focal amplification of chromosome 1q21.3 was observed in ovarian cancer but not in thyroid carcinoma. However, PROMO Publisher’s Note: MDPI stays neutral suggested 53 transcription factors as putative common transcriptional factors for FAL1 and ECM1 with regard to jurisdictional claims in simultaneously. Among them, we selected c-JUN and JUND as the best candidates based on EN- published maps and institutional affil- FAL1- iations. CODE results. The expression of target genes of JUND simultaneously increased in and ECM1-upregulated PTCs, especially in young patients. The molecular biological features represented RAS-driven PTC and simultaneously enriched immune-related gene sets. FAL1 and ECM1 expression frequently increased simultaneously and could be operated by JUND. The simultaneous upregulation might be a potential diagnostic and therapeutic target for RAS-driven PTC. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: FAL1; ECM1; thyroid neoplasm; prognosis; RAS; immunology distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Cancers 2021, 13, 3223. https://doi.org/10.3390/cancers13133223 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3223 2 of 14

1. Introduction Papillary thyroid cancer (PTC) is the most common endocrine malignancy, and its incidence has been increasing worldwide [1,2]. The main reason for this increase has been attributed to the widespread application of highly sensitive ultrasound to health screen- ings [3]. In this context, a new approach called active surveillance is being widely studied in the clinical setting [4]. However, basic experiments and clinical studies on thyroid cancer occurring in young patients and refractory thyroid cancer relatively commonly observed in the elderly are also being conducted [5–8]. These efforts have led to the development of various tyrosine kinase inhibitors, including targeted therapy for BRAFV600E, for the clinical setting. However, owing to limited therapeutic efficacy, there remains a need for new therapeutic agents. Unfortunately, no new emerging therapeutic targets have been suggested [9]. Non-coding RNA (ncRNA), a molecule that is not translated into protein, is present abundantly and performs important biological functions [10–12]. Among various ncRNAs such as small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), small interfering RNA (siRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), piwi-interacting RNA (piRNAs), and long ncRNAs (lncRNAs) [13,14], previous studies have shown that lncRNAs act as tumour susceptible genes in PTC. For example, low expression of PTC susceptibility candidate 2 (PTSC2) was observed in PTC tumours, which, in turn, affected the expression of genes related to cell cycle and cancer [15]. The polymorphism rs944289 on PTC suscepti- bility candidate 3 (PTSC3) was reported to predispose to PTC [16]. BRAF-activated lncRNA (BANCR) is upregulated in PTC, thereby promoting cell proliferation through autophagy regulation [17]. Recently, our group reported that the oncogenic activity of f ocally amplified lncRNA on (FAL1, ENSG00000228126) is attributed to the expression of genes re- lated to the cell cycle, including transcription factor E2F transcription factor 1 (E2F1), E2F transcription factor 2 (E2F2), and cyclin D1 in thyroid cancers [18]. In this study, we investi- gated the mechanism underlying FAL1 upregulation in PTC and found that extracellular matrix protein 1 (ECM1) expression was simultaneously upregulated with FAL1 expression. In addition, we explored the biological and clinical functions of FAL1 and ECM1 and demonstrated the significance of simultaneous expression of FAL1 and ECM1.

2. Materials and Methods 2.1. Analysis of lncRNA and mRNA Expression Using Public Databases lncRNA expression values were collected using The Atlas of Noncoding RNAs in Cancer (TANRIC) derived from The Cancer Genome Atlas (TCGA) RNA-seq database [19]. TANRIC provided the quantified expression level of lncRNA as read per kilobase million (RPKM) based on the Binary Alignment/Map format File (BAM file, *.bam). Using these datasets, the expression level of the lncRNA FAL1 was confirmed in 20 types of human can- cer: colon adenocarcinoma (COAD; 157 cases), rectum adenocarcinoma (READ; 71 cases), uterine corpus endometrioid carcinoma (UCEC; 316 cases), kidney chromophobe (KICH; 66 cases), ovary serous cystadenocarcinoma (OV; 412 cases), liver hepatocellular carcinoma (LIHC; 200 cases), prostate adenocarcinoma (PRAD; 374 cases), kidney renal clear cell carci- noma (KIRC; 448 cases), stomach adenocarcinoma (STAD; 285 cases), bladder urothelial carcinoma (BLCA; 252 cases), lung squamous cell carcinoma (LUSC; 220 cases), kidney renal papillary cell carcinoma (KIRP; 198 cases), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC; 196 cases), lung adenocarcinoma (LUAD; 488 cases), skin cutaneous melanoma (SKCM; 226 cases), breast invasive carcinoma (BRCA; 837 cases), head and neck squamous cell carcinoma (HNSC; 426 cases), glioblastoma multiforme (GBM; 154 cases), thyroid carcinoma (THCA; 497 cases), and brain low grade glioma (LGG; 486 case). To compare the expression values of ECM1 and ADAMTSL4 between normal tissues and PTC, mRNA and lncRNA expression values for the normal tissue cohort were collected from the TCGA THCA RNA-seq database and TANRIC, respectively. We also se- Cancers 2021, 13, 3223 3 of 14

lected GSE127083 from the GEO (Gene Expression Omnibus) database as in vitro validation set in addition to TCGA data.

2.2. Analysis of Copy Number Alteration To determine copy number alterations in TCGA ovarian cancer (OVCA) and TCGA THCA, Genomic Identification of Significant Targets in Cancer (GISTIC) data from the Broad Firehose infrastructure were used [20]. GISTIC statistically calculated the copy number alteration occurring in many patient specimens. The data were sorted based on the genomic build hg19. The threshold used for DNA copy number amplification was 0.1, the confidence level was 0.99, and the q-value cut-off was 0.25.

2.3. Prediction and Validation of Putative Transcription Factors The binding site and transcription factors of FAL1 and ECM1 were predicted using PROMO and the Encyclopaedia of DNA Elements (ENCODE), respectively. PROMO, which used a transcription factor source from the TRANSFAC® database, predicted po- tential transcription factor-binding sites (TFBS). The upstream 1 kb sequence of FAL1 and ECM1 was input, and the dissimilarity rate was analysed using the default value (15%; 85% similarity). Information related to gene regulation based on ENCODE data was verified using the Ensembl Genome Browser. The regulatory elements of each gene were identified using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) data from EN- CODE, and each motif score was selected by applying a p-value threshold of 0.01. Gene set enrichment analysis (GSEA) was performed using software, version 4.1.0, combined with [21]. The normalised enrichment score (NES) calculated by GSEA described the correlation between the gene set and the expression dataset. Permutations were performed 1000 times according to the basic weighted enrichment statistic, and genes were ranked according to the level of differential expression between the two groups. We selected a set of important genes based on a p-value of ≤0.05 and a false discovery rate (FDR) q-value of <0.25.

2.4. Statistical Analysis Statistical analysis was performed using Prism (GraphPad Software, San Diego, CA, USA) or SPSS, version 25.0, for Windows (IBM Corp., Armonk, NY, USA). Data are pre- sented as mean ± SD. Statistical comparisons of continuous variables were performed using Student’s t-test or analysis of variance, and group comparisons were performed using χ2 test or linear association. Gene expression associations were investigated using the Pearson correlation coefficients.

3. Results 3.1. Positive Correlation between FAL1 and ECM1 Expression in Human Cancers Upregulation of FAL1 expression was first reported in ovarian cancer. Focal ampli- fication of chromosome 1q21.3 is the mechanism underlying the upregulated expression of FAL1 [22]. To validate this upregulatory mechanism in PTC, we first investigated the expression status of FAL1 using pan-cancer data from TCGA. Among 20 types of human cancers, COAD, READ, and UCEC did not show any expression of FAL1. However, brain LGG, THCA, and GBM presented higher expression values than the other types of human cancers (Figure1A). Interestingly, the expression of FAL1 in OVCA was not higher than that in other types of human cancers. Because the upregulated expression of genes by focal amplification is inevitably accompanied by increased expression of genes present at the same location on the chromosome, we identified the genes located around FAL1 (Figure1B). ECM1 and ADAMTS (disintegrin and metalloproteinase with thrombospondin motifs)-like 4 (ADAMTSL4) were located before and after FAL1 on Chr1:150354111-150876737. An anal- ysis of the correlation between the expression of FAL1 and that of the neighbouring genes was performed, and the results showed that the expression of threonyl-tRNA synthetase 2, mitochondrial (TARS2), ECM1, ADAMTSL4, etc., in most tumours wherein FAL1 expres- Cancers 2021, 13, x 4 of 14

150876737. An analysis of the correlation between the expression of FAL1 and that of the Cancers 2021, 13, 3223 neighbouring genes was performed, and the results showed that the expression 4of of threo- 14 nyl-tRNA synthetase 2, mitochondrial (TARS2), ECM1, ADAMTSL4, etc., in most tumours wherein FAL1 expression was observed was positively correlated with FAL1 expression (Figuresion was 1B). observed Whereas was the positivelycorrelation correlated of FAL1 with FAL1RPRD2expression and TARS2 (Figure did1 notB). Whereasshow strong positivethe correlation correlation of FAL1 coefficientswith RPRD2 in TCGAand TARS2 THCAdid (Figure not show S1), strongas shown positive in Figure correlation 1C,D, the expressioncoefficients of inECM1 TCGA and THCA ADAMTSL4 (Figure S1), showed as shown a positive in Figure correlation1C,D, the expression with FAL1 of expressionECM1 andand wasADAMTSL4 associatedshowed with upregulated a positive correlation expression with ofFAL1 FAL1expression in PTC compared and was associated to that in the with upregulated expression of FAL1 in PTC compared to that in the normal thyroid tissues. normal thyroid tissues. Taken together, we postulated that FAL1 expression was coupled Taken together, we postulated that FAL1 expression was coupled with the upregulated withexpression the upregulated of neighbouring expression genes of such neighbouring as ECM1 and genesADAMTSL4 such as. ECM1 and ADAMTSL4.

Figure 1. Cont.

Cancers 2021, 13, 3223 5 of 14 Cancers 2021, 13, x 5 of 14

FigureFigure 1. (A 1.) (PositiveA) Positive correlation correlation of ofFAL1FAL1 expressionexpression with with the expressionexpression of of neighbouring neighbouring genes genes adjacently adjacently located located on on chromosomechromosome 1q21.3. 1q21.3. A. A.FAL1FAL1 expressionexpression statusstatus of of 20 20 types types of humanof human cancers. cancers. (B) Location (B) Location of FAL1 ofand FAL1 neighbouring and neighbouring genes geneson on chromosome chromosome 1q21.3 1q21.3 and and correlation correlation status status of FAL1 ofwith FAL1 neighbouring with neighbouring genes in termsgenesof in expression terms of expression in 17 human in cancers. 17 human cancers.(C) Positive(C) Positive correlation correlation of FAL1 ofexpression FAL1 expression and ECM1 andexpression, ECM1 expression, and upregulated and upregulated expression expression of ECM1 in tumourof ECM1 samples in tumour samplescompared compared to that to in that normal in normal tissues intissues THCA. in ( DTHCA.) Positive (D) correlation Positive correlation of FAL1 expression of FAL1 with expressionADAMTSL4 withexpression ADAMTSL4 and ex- pressionupregulated and upregulated expression expression of ADAMTSL4 of ADAMTSL4in tumour samples in tumour compared samples to that compared in normal to tissues that in in normal THCA. Abbreviations:tissues in THCA. Abbreviations:FAL1, focally FAL1 amplified, focally lncRNA amplified on chromosome lncRNA on 1; chromosome RPRD2, regulation 1; RPRD of nuclear2, regulation pre-MRNA of nuclear domain-containing pre-MRNA 2;domain-con- TARS2, tainingthreonyl-tRNA 2; TARS2, threonyl-tRNA synthetase 2, mitochondrial; synthetaseECM1 2, mitochondrial;, extracellular matrixECM1 protein, extracellular 1; ADAMTSL4 matrix-AS1, proteinADAMTSL4 1; ADAMTSL4antisense-AS1, ADAMTSL4RNA 1;MCL1, antisense myeloid RNA cell 1; MCL1, leukaemia myeloid 1; ENSA, cell endosulfine leukaemia alpha;1; ENSA, GOLPH3L, endosulfine Golgi alpha; phosphoprotein GOLPH3L, 3-like; Golgi HORMAD1, phosphopro- tein 3-like;HORMA HORMAD1, domain-containing HORMA 1; CTSS, domain-containing cathepsin S; CTSK, 1; cathepsinCTSS, cathepsin K; ARNT, S; aryl CTSK, hydrocarbon cathepsin receptor K; ARNT, nuclear aryl translocator. hydrocarbon receptorThe arrowsnuclear indicate translocator. the direction The arrows of transcription. indicate the Average direct valuesion of transcription. were compared Average using an values unpaired were t-test. compared In the scatter using an unpairedplots, t-test. data areIn the expressed scatter asplots, the meandata are± SD.expressed Correlation as the coefficient: mean ± SD. Pearson’s Correlation r. * p coefficient:< 0.05, ** p Pearson’s< 0.01. All r. p*-values p < 0.05, ** p < 0.01.are two-sided.All p-values are two-sided.

3.2.3.2. Focal Focal Amplification Amplification of of the the FAL1 FAL1 GeneGene inin Ovarian Ovarian Cancer Cancer but but Not Not In PTCIn PTC Our analysis for the expression of FAL1 and neighbouring genes suggested simul- Our analysis for the expression of FAL1 and neighbouring genes suggested simulta- taneous upregulated expression of those genes, and we investigated the amplification neousstatus upregulated of genes based expression on data ofof copythose number genes, alterationsand we investigated (CNAs) from the TCGA amplification OVCA and status of TCGAgenes based THCA. on As data previously of copy reported, number focalalterations amplification (CNAs) of from 1q21.3 TCGA was clearly OVCA observed and TCGA THCA.in OVCA As (Figurepreviously2A), butreported, no amplification focal amplification signal at the of same 1q21.3 position was wasclearly observed observed in in OVCATHCA (Figure (Figure 22A),B), suggesting but no amplification that simultaneous signal upregulated at the same expression position of wasFAL1 observedand the in THCAneighbouring (Figure 2B), genes suggesting was generated that si bymultaneous different mechanisms upregulated from expression focal amplification of FAL1 and in the neighbouringPTC. Supporting genes our was idea, generated the comparison by different of 1q arm-level mechanisms amplification from focal in TCGAamplification THCA in PTC.according Supporting to the ourFAL1 idea,expression the comparison status did notof 1q show arm-level any significant amplification difference in TCGA (Table S1THCA). accordingRecent efforts to the to FAL1 understand expression the regulatory status did mechanism not show of lncRNAany significant expression difference have empha- (Table sised the importance of transcription factors in the expression of other coding genes. In S1). Recent efforts to understand the regulatory mechanism of lncRNA expression have line with this idea, we decided to evaluate the effective transcription factors for the pro- emphasised the importance of transcription factors in the expression of other coding moter areas of FAL1 and ECM1. To achieve our goal, we identified putative TFBS in DNA genes.sequences In line defined with this in TRANSFAC, idea, we decided using PROMO to evaluate [23,24 the]. This effective virtual transcription laboratory suggested factors for the62 promoter transcription areas factors of FAL1 for andFAL1 ECM1and 63. To transcription achieve our factors goal, forweECM1 identified(Figure putative2C ). As TFBS in expected,DNA sequences most putative defined transcription in TRANSFAC, factors using overlapped PROMO in both [23,24].FAL1 Thisand virtualECM1 ( nlaboratory= 53). suggestedTaken together, 62 transcription the simultaneous factors for expression FAL1 and of FAL163 transcriptionand ECM1 might factors be for generated ECM1 (Figure by 2C).common As expected, transcription most putative factors rather transcription than by gene factors amplification. overlapped in both FAL1 and ECM1 (n = 53). Taken together, the simultaneous expression of FAL1 and ECM1 might be gener- ated by common transcription factors rather than by gene amplification.

Cancers 2021, 13, x Cancers 2021, 13, 3223 6 of 14 6 of 14

FigureFigure 2. Analysis 2. Analysis of focal of amplification focal amplification using data using of copydata numberof copy alterationsnumber alterations from TCGA from OVCA TCGA and OVCA TCGA THCA. (A) Resultsand TCGA of analysis THCA. of the(A) focalResults amplification of analysis in of the the whole focal chromosome amplification from in the TCGA whole OVCA. chromosome (B) Results of analysis of thefrom focal TCGA amplification OVCA. in (B the) Results whole chromosomeof analysis of from the TCGAfocal am THCA.plification (C)Results in the from whole PROMO chromosome analysis to identify putativefrom TFBS TCGA in DNA THCA. sequences (C)Results of the FAL1fromand PROMOECM1 analysisgenes. Abbreviations: to identify putative TF, transcription TFBS in factor; DNA TFBS, sequences transcription factor-bindingof the FAL1 sites; and TCGA, ECM1 The genes. Cancer Abbreviations: Genome Atlas; TF, THCA, transcript thyroidion carcinoma; factor; TFBS, OVCA, transcription ovarian cancer. factor- The arrows indicatebinding chromosome sites; TCGA, 1q21.3, The which Cancer was reported Genome as Atlas; a focal THCA, amplification thyroid lesion carcinoma; for FAL1 upregulation OVCA, ovarian in OVCA. can- cer. The arrows indicate chromosome 1q21.3, which was reported as a focal amplification lesion for FAL1 upregulation in OVCA.

Cancers 2021, 13, x 7 of 14

Cancers 2021, 13, 3223 7 of 14

3.3. JUND as a Candidate for Common Transcription Factor for FAL1 and ECM1 To select the most reliable transcription factor from TFBS, we examined the location 3.3. JUND as a Candidate for Common Transcription Factor for FAL1 and ECM1 of TFBS on the chromosome in detail using the Encyclopaedia of DNA Elements (EN- CODE),To selectwhich the identifies most reliable functional transcription elements factor in fromthe human TFBS, we genomes examined [25]. the In location the case of of TFBS on the chromosome in detail using the Encyclopaedia of DNA Elements (ENCODE), RPRD2 and TARS, PROMO suggested 66 transcription factors for RPRD2 and 70 tran- which identifies functional elements in the human genomes [25]. In the case of RPRD2 and scription factors for TARS2. However, unfortunately, ENCODE did not have TFBS for TARS, PROMO suggested 66 transcription factors for RPRD2 and 70 transcription factors RPRD2for TARS2 (Figure. However, S2). Interestingly, unfortunately, we ENCODE observed did that not havethe TFBS TFBS of for c-JUNRPRD2 and(Figure JUND S2). was presentInterestingly, in the wepromoter observed of thatthe theECM1 TFBS gene, of c-JUN and the and TFBS JUND for was FOS present, a binding in the partner, promoter was alsoof the presentECM1 (Figuregene, and 3A). the Based TFBS foron FOSthis, findin a bindingg, we partner, hypothesised was also presentthat the (Figure expression3A). of JUNBased target on this genes finding, might we be hypothesised increased in thatFAL1- the and expression ECM1-upregulated of JUN target PTC, genes if might the expres- be sionincreased of FAL1 in andFAL1- ECM1and ECM1- was increasedupregulated by JUN PTC, transactivation. if the expression To of validateFAL1 and ourECM1 hypothesis,was weincreased performed by JUNGSEA transactivation. by dividing the To TCGA validate THCA our hypothesis,into two groups we performed according GSEA to the byFAL1 anddividing ECM1 the expression TCGA THCA status. into The two target groups genes according of c-JUN to were the FAL1 co-ordinatelyand ECM1 enrichedexpression in the highstatus. ECM1 The group target but genes not of in c-JUN the high were FAL1 co-ordinately group (Figure enriched 3B). inHowever, the high theECM1 targetgroup genes forbut JUND not in were the high co-ordinatelyFAL1 group enriched (Figure 3inB). the However, high ECM1 the targetgroup genes and showed for JUND a tendency were ofco-ordinately upregulated enriched expression in thein the high highECM1 FAL1group group and (Figure showed 3C) a tendencyeven though of upregulated JUND expres- sionexpression was not in correlated the high FAL1 withgroup FAL1 (Figure and ECM13C) even expression though JUND(Figureexpression S3). In addition, was not we FAL1 ECM1 confirmedcorrelated withthat the expressionand ofexpression the FAL1 (Figure, ECM1 S3)., and In addition, FAL1 target we confirmed genes was that also the expression of the FAL1, ECM1, and FAL1 target genes was also decreased by CRISPRi decreased by CRISPRi JUND in leukemia cells (Figure 3D). Although we did not verify JUND in leukemia cells (Figure3D). Although we did not verify these results from a virtual these results from a virtual laboratory through a wet lab-based approach, we thought that laboratory through a wet lab-based approach, we thought that these data fully supported theseour ideadata that fully simultaneous supported our expression idea that of simultaneousFAL1 and ECM1 expressionwas generated, of FAL1 and not byECM1 focal was generated,amplification not but by byfocal common amplification transcription but by factors common such transcription as JUND. factors such as JUND.

Figure 3. Cont.

Cancers 2021, 13, 3223 8 of 14 Cancers 2021, 13, x 8 of 14

Figure 3. Results of the analysis using the Encyclopaedia of DNA Elements (ENCODE) and FAL1 and ECM1 expression Figure 3. Results of the analysis using the Encyclopaedia of DNA Elements (ENCODE) and FAL1 and ECM1 expression according to patients’ age. (A) The TFBS of JUND, c-JUN, and JUN-FOS in the promoter area of ECM1 and FAL1.(B) Ex- according to patients’ age. (A)The TFBS of JUND, c-JUN, and JUN-FOS in the promoter area of ECM1 and FAL1. (B) pression of the transcriptional target genes of c-JUN according to ECM1 and FAL1 expression status in TCGA THCA. Expression of the transcriptional target genes of c-JUN according to ECM1 and FAL1 expression status in TCGA THCA. (C) Expression(C) Expression of the transcriptional of the transcriptional target genes target of genes JUND of JUNDaccording according to ECM1 to ECM1 and FAL1and FAL1 expressionexpression status status in TCGA in TCGA THCA. THCA. (D) Selected(D) Selectedtarget gene target expressions gene expressions were identified were identified in GSE127083 in GSE127083 RNA-seq RNA-seq dataset dataset for K562 for K562 cells cells treated treated with with JUNDJUND- - targeted targetedCRISPRi. CRISPRi. (E) FAL1 (E expression) FAL1 expression status according status according to patients’ to patients’ age (F age) ECM1 (F) ECM1 expressionexpression status status according according to patients’ to patients’ age. Abbreviations:age. Abbreviations: JUND, junD JUND, proto-on junD proto-oncogene;cogene; AP-1, AP-1,transcription transcription factor factor subuni subunit;t; JUN, JUN, jun proto-oncogene; jun proto-oncogene; FOS, FOS, fosfos proto-oncogene;proto-oncogene; TFBS, transcription TFBS, transcription factor-binding factor-binding sites; TCGA, sites; TCGA, The Cancer The Cancer Genome Genome Atlas; Atlas; THCA, THCA, thyroid thyroid carcinoma; carcinoma; E2F1, transcriptionE2F1, transcription factor E2F factor transcription E2F transcription factor 1; factor E2F2, 1; tran E2F2,scription transcription factor factor E2F transcripti E2F transcriptionon factor factor 2; VEGFA, 2; VEGFA, vascular vascular endothelialendothelial growth growthfactor A; factor CCND1 A; CCND1 Cyclin CyclinD1. Aver D1.age Average values values were werecompared compared using using ANOVA, ANOVA, Student’s Student’s t-test.t-test. In the In the scatter plots,scatter data plots, are data expressed are expressed as mean as ± mean SD. All± SD. p-values All p-values are two-sided. are two-sided. *p < 0.01, * p < **p 0.01, < 0.001. ** p < 0.001.

FAL1 wasFAL1 firstwas investigated first investigated in ovarian in ovarian high-grade high-grade serous serous carcinoma carcinoma (HGSC), (HGSC), show- showing ing a differenta different correlation correlation pattern pattern compared compared to toTCGA TCGA THCA THCA (Figure (Figure 1B).1B). We We thought thought this this differencedifference might mightindicate indicate a diff aerent different regulatory regulatory mechanism mechanism of ofFAL1FAL1 upregulationupregulation accord- according FAL1 ing to cancerto cancer types. types. As As described, described, in in HGSC, HGSC, focal focal amplification amplification increased increased FAL1 upregulation.upregula- In the case of GBM, the regulatory mechanism was not investigated. To understand the tion. In the case of GBM, the regulatory mechanism was not investigated. To understand different regulatory mechansim in HGSC and GBM, we performed GSEA using c-JUN and the different regulatory mechansim in HGSC and GBM, we performed GSEA using c-JUN JUND target genes in HGSC and GBM. High ECM1 expression was related to coordinately and JUNDenrichment target genes of c-JUN in HGSC and JUND and targetGBM. genes High butECM1 high expressionFAL1 expression was related was not to (Figure coordi- S4). nately Thisenrichment data indicated of c-JUN that an JUNDd JUND was target not involved genes inbut simutaneous high FAL1 upexpression regulation was of ECM1not (Figureand S4).FAL1 Thisin data HGSC indicated and GBM. that JUND was not involved in simutaneous up regulation of ECM1 and FAL1 in HGSC and GBM. 3.4. Clinical Relevance of the Simultaneous Expression of FAL1 and ECM1 3.4. Clinical ToRelevance determine of the the Simult clinicalaneous implication Expression of theof FAL1 simultaneous and ECM1 expression of FAL1 and ToECM1 determine, we analysed the clinical the clinicopathological implication of the features simultaneous of high FAL1expressionand ECM1 of FAL1expression and ECM1, we analysed the clinicopathological features of high FAL1 and ECM1 expression groups. Among the results, the most interesting finding was that FAL1 and ECM1 expres- sion was upregulated in young patients aged <45 years (Figure 3D,E). In line with this

Cancers 2021, 13, 3223 9 of 14 Cancers 2021, 13, x 9 of 14

groups. Among the results, the most interesting finding was that FAL1 and ECM1 expres- finding, we performed GSEA to understand the differences in gene expression patterns sion was upregulated in young patients aged <45 years (Figure3D,E). In line with this according to age (young vs. old age) and found that KRAS- and immune-related gene sets finding, we performed GSEA to understand the differences in gene expression patterns were co-ordinately enriched in the young age group (<45 years old), whereas metabolism- according to age (young vs. old age) and found that KRAS- and immune-related gene sets and epithelial–mesenchymal transition-related gene sets were co-ordinately enriched in were co-ordinately enriched in the young age group (<45 years old), whereas metabolism- the old age group (≥55 years old) (Figure 4A,B). According to high FAL1 or ECM1 expres- and epithelial–mesenchymal transition-related gene sets were co-ordinately enriched in the sion, KRAS- and immune-related gene sets were co-ordinately enriched (Figure 4C,D). old age group (≥55 years old) (Figure4A,B). According to high FAL1 or ECM1 expression, Taken together, FAL1 and ECM1 upregulated expression was frequently observed in the KRAS- and immune-related gene sets were co-ordinately enriched (Figure4C,D). Taken young patient group, and this upregulation might contribute to defining tumour behav- together, FAL1 and ECM1 upregulated expression was frequently observed in the young iourpatient in the group, young and patient this upregulation group. To validate might our contribute hypothesis to defining that simultaneous tumour behaviour expression in ofthe FAL1 young and patient ECM1 group. might Tobe validaterelated to our RAS-driven hypothesis PTC, that simultaneouswe compared expressionthe molecular of FAL1 bio- logicaland ECM1 featuresmight of be PTC related according to RAS-driven to FAL1 PTC,and ECM1 we compared expression the molecular(Tables 1 and biological 2). FAL1 fea-- upregulatedtures of PTC PTC according presented to FAL1 fewerand mRNAECM1 clusexpressionters but (Tablesmore miRNA1 and2). clusters.FAL1-upregulated RAS muta- tionsPTC presentedwere frequently fewer detected mRNA clustersin FAL1-upregulated but more miRNA PTC, whereas clusters. the RAS frequency mutations of BRAF were mutationsfrequently was detected relatively in FAL1 low.-upregulated According PTC,to the whereas frequent the RAS frequency mutation of in BRAF FAL1 mutations-upregu- latedwas relatively PTC, the low.RAS/RAF, According ERK, to and the differen frequenttiation RAS mutationscores were in FAL1 compatible-upregulated with RAS- PTC, driventhe RAS/RAF, PTC. However, ERK, and serine differentiation 473 phosphorylation scores were of PKB/AKT compatible increased with RAS-driven in FAL1-upreg- PTC. ulatedHowever, PTC. serine In the 473 analysis phosphorylation of ECM1-upregulated of PKB/AKT PTC, increased frequent in RASFAL1 mutations-upregulated and a PTC. rel- ativelyIn the analysislow frequency of ECM1 of-upregulated BRAF mutations PTC, were frequent also observed RAS mutations as FAL1 and-upregulated a relatively PTC. low However,frequency the of BRAFfrequency mutations of TERT were promoter also observed mutation as FAL1increased,-upregulated and the PTC.ERK score However, also increasedthe frequency compared of TERT to that promoter in ECM1 mutation-downregulated increased, PTC. and In the summary, ERK score all alsothese increased molecu- larcompared biological to that features in ECM1 might-downregulated be related to PTC. the Inaggressive summary, behaviou all theser molecular of RAS-driven biological tu- mours.features might be related to the aggressive behaviour of RAS-driven tumours.

Figure 4.4. Results of GSEA according to patients’ age, FAL1 upregulation and ECM1 upregulation. ( A)Representative gene sets co-ordinatelyco-ordinately enrichedenriched in in the the young young patient patient group. group. (B )(B Representative) Representative gene gene sets sets co-ordinately co-ordinately enriched enriched in the in old-agedthe old- group.aged group. (C) Representative (C) Representative gene gene sets co-ordinately sets co-ordinately enriched enriched in the inFAL1 the FAL1-upregulated-upregulated group. group. (D) Representative(D) Representative gene gene sets co-ordinatelysets co-ordinately enriched enriched in the in theECM1 ECM1-upregulated-upregulated group. group. Red Red colour colour refers refers to to gene gene sets sets overlapping overlapping inin thethe young-agedyoung-aged group and the FAL1-- andand ECM1-upregulated-upregulated group.group. Abbreviations:Abbreviations: GSEA, gene set enrichment analysis; NES, normalised enrichment score. enrichment score.

Cancers 2021, 13, 3223 10 of 14

Table 1. Molecular features of TCGA THCA according to FAL1 expression status.

FAL1 Molecular Feature Low Expression High Expression p Value n = 124 (%) n = 124 (%) mRNA cluster number 1 12 (9.8) 59 (48.8) 2 13 (10.7) 22 (18.2) 3 32 (26.2) 5 (4.1) <0.0001 † 4 39 (32.0) 8 (6.6) 5 26 (21.3) 27 (22.3) miRNA cluster number 1 2 (1.6) 2 (1.6) 2 48 (39.0) 19 (15.4)

3 26 (21.1) 7 (5.7) † 4 16 (13.0) 58 (47.2) <0.0001 5 16 (13.0) 19 (15.4) 6 15 (12.2) 18 (14.6) RAS mutation

Absent 122 (98.4) 89 (72.4) † Present 2 (1.6) 34 (27.6) <0.0001 BRAF mutation

Absent 31 (25.0) 97 (78.9) † Present 93 (75.0) 26 (21.1) <0.0001 TERT promoter mutation

Absent 88 (87.1) 81 (89.0) † Present 13 (12.9) 10 (11.0) 0.688 RAS/RAF score −0.67 ± 0.49 0.21 ± 0.67 <0.0001 * ERK score 11.37 ± 16.27 2.14 ± 20.26 0.001 * Differentiation score −0.49 ± 0.99 0.54 ± 1.08 <0.0001 * Akt pT308 −0.02 ± 0.64 0.06 ± 0.47 0.275 * Akt pS473 −0.02 ± 0.47 0.09 ± 0.34 0.047 * TCGA, The Cancer Genome Atlas; THCA, thyroid carcinoma. † p values calculated by Student’s t test; * p values calculated by chi-square test or linear-by-linear association.

Table 2. Molecular features of TCGA THCA according to ECM1 expression status.

ECM1 Molecular Feature Low Expression High Expression p Value n = 124 (%) n = 124 (%) mRNA cluster number 1 58 (47.9) 51 (43.2) 2 7 (5.8) 21 (17.8) 3 36 (29.8) 5 (4.2) 0.190 † 4 5 (4.1) 14 (11.9) 5 15 (12.4) 27 (22.9) miRNA cluster number 1 3 (2.5) 3 (2.4) 2 16 (13.1) 22 (17.9)

3 24 (19.7) 11 (8.9) † 4 46 (37.7) 51 (41.5) 0.796 5 19 (15.6) 21 (17.1) 6 14 (11.5) 15 (12.2) RAS mutation

Absent 117 (95.1) 90 (73.2) † Present 6 (4.9) 33 (26.8) <0.0001 BRAF mutation

Absent 71 (57.7) 92 (74.8) † Present 52 (42.3) 31 (25.2) 0.005 Cancers 2021, 13, 3223 11 of 14

Table 2. Cont.

ECM1 Molecular Feature Low Expression High Expression p Value n = 124 (%) n = 124 (%) TERT promoter mutation

Absent 91 (95.8) 78 (84.8) † Present 4 (4.2) 14 (15.2) 0.011 RAS/RAF score 0.01 ± 0.81 0.07 ± 0.68 0.533 * ERK score −5.25 ± 23.26 4.92 ± 19.35 0.001 * Differentiation score 0.32 ± 1.37 0.39 ± 1.00 0.674 * Akt pT308 0.09 ± 0.54 0.06 ± 0.35 0.691 * Akt pS473 0.14 ± 0.66 0.01 ± 0.46 0.122 * TCGA, The Cancer Genome Atlas; THCA, thyroid carcinoma. † p values calculated by Student’s t test; * p values calculated by chi-square test or linear-by-linear association.

4. Discussion The incidence of PTC has been increasing since the last two decades in Korea even though there is controversy regarding the overtreatment of papillary thyroid microcar- cinoma [3,26]. Recent clinical evidence has suggested the optimal indication of active surveillance for this indolent carcinoma [27]. Refractory thyroid cancer is defined as a tumour with poor response to current treatments such as surgery and radioactive iodine therapy, and it could be managed with newly developed targeted therapy using sorafenib and vemurafenib. However, these novel therapeutics mainly target BRAFV600E-driven PTC. In fact, currently available drugs for RAS-driven PTCs are limited. LncRNA has been investigated as a novel diagnostic and therapeutic target in many types of human cancers [12]. In the case of PTCs, lncRNAs were first reported as cancer susceptible genes such as PTCSC1, PTCSC2, and PTCSC3; following this, many interesting papers reported various kinds of tumour-suppressive or oncogenic lncRNAs in human PTC, such as Cancer Susceptibility 2 (CASC2), Promoter Of CDKN1A Antisense DNA Damage Activated RNA (PANDAR), Maternally Expressed 3 (MEG3), Non-Protein-Coding RNA, Associated With MAP Kinase Pathway And Growth Arrest (NAMA), HOX Antisense Intergenic RNA (HOTAIR), Nuclear Enriched Abundant Transcript 1 (NEAT1), Metas- tasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), Antisense Noncoding RNA in the INK4 Locus (ANRIL), Plasmacytoma Variant Translocation 1 (PVT1), and BANCR [28–32]. Our group also reported LOC100507661 and FAL1 as potential oncogenic lncRNAs in PTC [18,33]. In fact, FAL1 was first investigated in ovarian high-grade serous carcinoma (HGSC). This interesting study suggested that the mechanism underlying the upregulated expression of FAL1 was focal amplification of the chromosome-harbouring FAL1 gene, as indicated by the name itself. FAL1 expression was closely related to the up- regulation of E2F1, suggesting the oncogenic function of the FAL1-promoting cell cycle [22]. First, we aimed to confirm the regulatory mechanism of FAL1 in PTC by focal amplification. However, copy number alteration data from TCGA THCA did not show any amplification signals on chromosome 1q21.3, even though FAL1 and the neighbouring genes were highly expressed in PTC with a highly positive correlation with each other. Based on this finding, we prepared a virtual laboratory for the identification of TFBS in FAL1 and ECM1 promoter sequences. Results from PROMO showed many putative transcription factors for both FAL1 and ECM1. After the analysis using ENCODE, we selected c-JUN and JUND as the best candidates for simultaneous expression of FAL1 and ECM1, supported by GSEA according to the FAL1 and ECM1 expression status. In fact, ENCODE was generated by CHIP-seq from 48 cell lines, indicating our analysis using ENCODE are based on bench work experiments. All these analyses suggested that FAL1 upregulation in PTC might be directed by transcription factors such as JUND but not by focal amplification of the FAL1-located chromosome. Cancers 2021, 13, 3223 12 of 14

In terms of clinical and biological relevance, upregulated expression of FAL1 and ECM1 was seen in the young age group and RAS-like PTC. In fact, the old-aged group showed more aggressive clinical features. In addition, our biological understanding is that BRAF-like PTC is more aggressive, and currently developed novel therapeutics have focused on BRAF-like refractory PTC [5,17]. However, in clinical settings, we have not encountered young patients with highly aggressive clinical features and RAS-like PTC harbouring TERT promoter mutation. Interestingly, our GSEA according to FAL1 and ECM1 expression status showed that the upregulated expression of these genes was func- tionally related to RAS signalling and immune-related genes. In the past, we considered epithelial-to-mesenchymal transition (EMT) as a representative mechanism of carcino- genesis [4,6,34–36]. However, in recent years, the importance of factors determining the tumour microenvironment has not been revealed, and it is understood that the relationship between tumour cells and the surrounding cells, especially immunological interaction, is an important factor in determining the prognosis of a tumour. In line with the current advances in tumour immunology, our data may suggest that RAS-like PTC with FAL1 and ECM1 upregulation proceeds from carcinogenesis to aggressive PTC by an immunological mechanism compared to classical BRAF-like PTC. Our data did not show any statistically significant difference in clinical features, except age. We believe that this is due to the characteristics of the TCGA THCA cohort, which makes it difficult to reflect the clinically significant role of FAL1 and ECM1 in a limited sample number of aggressive RAS-like PTC because the prognosis of BRAF-like PTC, which is more frequently observed, is generally poor. However, the coordinated enrichment of RAS signalling and immune-related genes was consistently observed in PTC harbouring upregulated expression of FAL1 and ECM1 and PTC in young patients; we believe that this finding will be an important reference and will serve as the basis for future studies.

5. Conclusions In conclusion, our data suggest that FAL1 upregulation might be induced by selective transcription factors such as JUND and could be a useful diagnostic and therapeutic marker in aggressive RAS-like PTC, especially in young patients. Further studies that include a large sample size should be conducted to confirm our data.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13133223/s1, Figure S1. Correlation analysis of FAL1 expression with RPRD2 and TARS2 expression in THCA. (A) Correlation of FAL1 expression with RPRD2 expression (n = 497). Figure S2. Confirmation of transcription factor binding site of RPRD2 and TARS2 promoters. (A) Results of PROMO analysis that predicted TFBS from the DNA sequence of RPRD2 gene. Figure S3. Correlation analysis of JUND expression with FAL1, ECM1 expression in THCA. (A) Correlation of JUND expression with FAL1 expression (n = 497). Table S1: Comparison of 1q arm-level amplification in TCGA THCA according to FAL1 expression status. Author Contributions: Y.-S.J. and J.L. designed the project and supervised the research; S.J., S.-G.L., H.K., G.L. and S.P. performed the analyses; S.J. and S.-G.L. performed the computational analysis; S.P. and I.-K.K. contributed to the discussion about the research; S.J., S.-G.L., Y.-S.J. and J.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: S.G.L was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (NRF-2020R1F1A1048986). J.L. was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (NRF-2020R1A2C1006047) and by the Korean Foundation for Cancer Research (2020). Y.S.J. was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (NRF-2018R1A2B6004179, NRF-2021R1H1A2012035). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Cancers 2021, 13, 3223 13 of 14

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Severance Medical Centre (IRB No. 4-2013-0546, 30 September 2013). Informed Consent Statement: Patient consent was waived due to the nature of this study using public data. Data Availability Statement: A description of the publicly archived datasets analyzed in this study is detailed in the Section2. Acknowledgments: The authors would like to thank Ji Young Kim (Severance Hospital, Yonsei Cancer Center), Hwanju Lee (Severance Hospital, Yonsei Cancer Center), Hee Chang Yu (Severance Hospital, Yonsei Cancer Center), and Hoyoung Kim (Severance Hospital, Yonsei Cancer Center) for their technical support. Conflicts of Interest: The authors declare no conflict of interest.

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