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Integrative CAGE and DNA Methylation Profiling Identify Epigenetically Regulated Genes in NSCLC

Integrative CAGE and DNA Methylation Profiling Identify Epigenetically Regulated Genes in NSCLC Supplementary Figure S1 TCGA LUSC TCGA LUAD ARL14 CT83 MYEOV ARL14 CT83 MYEOV 10 10 Rho = -0.68 Rho = -0.33 Rho = -0.48 Rho = -0.72 Rho = -0.67 Rho = -0.53 14 12 12 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 12 8 8 6 6 6 4 4 4 4 2 2 2 2 2 2 0 0 0 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 β value (cg24147596) β value (cg17155263) β value (cg17155263) β value (cg01638792) β value (cg01638792) β value (cg24147596) GS1-179L18.1 LINC00857 RP11-371I1.2 GS1-179L18.1 LINC00857 RP11-371I1.2 Rho = -0.34 Rho = -0.38 3 3 5 Rho = -0.52 Rho = -0.52 4 4 Rho = -0.78 Rho = -0.80 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 2.5 P < 0.0001 P < 0.0001 3 4 3 3 2 2 1.5 2 1 1 1 1 0.5 0 0 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 β value (cg00347856) β value (cg11647108) β value (cg16662846) β value (cg00347856) β value (cg11647108) β value (cg16662846) AL022344.4 PCAT7 RP1-90G24.10 AL022344.4 PCAT7 RP1-90G24.10 Rho = -0.45 Rho = -0.33 Rho = -0.33 Rho = -0.32 Rho = -0.12 Rho = -0.47 P < 0.0001 5 P < 0.0001 3 P < 0.0001 P = 0.02 P < 0.0001 3 P < 0.0001 2 4 1 2 0.5 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 β value (cg02780400) β value (cg13647892) β value (cg25125450) β value (cg02780400) β value (cg13647892) β value (cg25125450) TCGA methylation normal vs tumor TCGA expression normal vs tumor ARL14 CT83 MYEOV ARL14 CT83 MYEOV * * * * * * * 10 * 1 1 1 * * * 12 14 8 12 0.8 0.8 0.8 8 6 0.6 0.6 0.6 4 6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 0 0 N T N T N T N T N T N T N T N T N T N T N T N T LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC RP11-371I1.2 GS1-179L18.1 LINC00857 RP11-371I1.2 GS1-179L18.1 LINC00857 * 4 * * 5 * * * 4 * 1 1 1 * * 3 0.8 0.8 0.8 0.6 0.6 0.6 1 0.4 1 0.4 0.4 0.2 0 0 0 0.2 0.2 N T N T N T N T N T N T 0 0 0 N T N T LUAD LUSC N T N T N T N T LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC RP1-90G24.10 1.5 * RP1-90G24.10 * * 1 1 0.8 0.5 0.6 0.4 N T N T 0.2 LUAD LUSC 0 N T N T LUAD LUSC GSE81089 AL022344.4 PCAT7 RP1-90G24.10 14 16 2.5 8 1.5 0.5 0 0 NSCLC Normal NSCLC Normal NSCLC Normal Methylation and expression levels of genes in lung cancer and normal lung tissues. A. Correlations between expression and methylation in TCGA LUAD (left, N = 426) and TCGA LUSC (right, N = 359). The y-axis: log (1+RPKM). The x-axis: β values. The results of 3 coding genes (ARL14, CT83, and MYEOV), 3 lncRNAs (GS1-179L18.1, LINC00857, and RP11-371l1.2), and 3 lncRNAs whose promoters are within the REP522 repeat family (AL022344.4, PCAT7, and RP1-90G24.10) are shown. B. Methylation and expression levels of 3 coding genes (ARL14, CT83, and MYEOV), 4 lncRNAs (GS1-179L18.1, LINC00857, RP11-371l1.2, and RP1-90G24.10) are shown. Left: RNA-seq of 515 LUAD vs. 59 normal lung, and 501 LUSC vs. 49 normal lung. Right: Methylation data of 431 LUAD vs. 31 normal lung, and 359 LUSC vs. 42 normal lung. N: normal lung. T: tumor tissue. *P < 0.05. C. Expression of lncRNAs in NSCLC (N = 199) and normal lung tissues (N = 19) in GSE81089 dataset. *P < 0.05. Log (1+RPKM) Log (1+RPKM) 2 Log2 (1+RPKM) Log (1+RPKM) 2 Log2 (1+RPKM) Log (1+RPKM) F PKM Log2 (1+RPKM) Log (1+RPKM) Log (1+RPKM) Log2 (1+RPKM) Log (1+RPKM) F PKM Log (1+RPKM) Log (1+RPKM) Log (1+RPKM) Log2 (1+RPKM) Log (1+RPKM) 2 F PKM β value (cg24147596) β value (cg25125450) β value (cg00347856) Log2 (1+RPKM) Log2 (1+RPKM) Log (1+RPKM) β value (cg17155263) β value (cg11647108) Log (1+RPKM) Log (1+RPKM) 2 Log (1+RPKM) β value (cg01638792) β value (cg16662846) Log (1+RPKM) Log (1+RPKM) Log2 (1+RPKM) 2 Supplementary Figure S2 GS1.179L18.1 RP11.371I1.2 PCAT7 AL022344.4 3.0 2.5 0% 20.7% 0% 26.6% 0% 43.6% 0% 33% 2.5 2.0 2.0 3 3 1.5 1.5 2 2 1.0 1.0 1 1 0.5 0.5 0.0 0 0.0 0 normal tumor normal tumor normal tumor normal tumor LINC00857 RP1.90G24.10 RP11.564D11.3 ADPRHL1 1.2 3.0 0% 74.2% 0% 15.8% 0% 85.9% 0% 23.2% 1.0 2.5 3 10 0.8 2.0 2 0.6 1.5 0.4 1.0 0.2 0.5 0.0 0.0 0 2 normal tumor normal tumor normal tumor normal tumor AIM2 ARL14 CEP55 CT83 0% 33.6% 0% 30.1% 0% 77.9% 0% 49% 10 10 0 0 normal tumor normal tumor normal tumor normal tumor IGF2BP3 KAZALD1 LRP1B MAGEA1 0% 45.5% 0% 21.9% 0% 7.6% 0% 17.6% 0 0 normal tumor normal tumor normal tumor normal tumor MAGEC2 MKRN3 MYEOV PLD1 12 14 11 0% 20.9% 0% 23.2% 0% 62.7% 0% 23.6% 6 10 4 7 0 0 0 normal tumor normal tumor normal tumor normal tumor PON1 PRAME 0% 27% 0% 52% 6 8 0 0 normal tumor normal tumor Activation frequencies in 22 epigenetically regulated genes in LUAD. Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Supplementary Figure S3 GS1.179L18.1 RP11.371I1.2 PCAT7 AL022344.4 3.5 0% 27.3% 0% 44.9% 0% 0% 71.9% 85.8% 3.0 3 2.5 2.0 1.5 1.0 0.5 0 0 0.0 0 normal tumor normal tumor normal tumor normal tumor LINC00857 RP1.90G24.10 RP11.564D11.3 ADPRHL1 1.5 3.0 3.0 0% 13.8% 0% 17.4% 0% 58.9% 0% 16.4% 2.5 2.5 1.0 2.0 2.0 7 1.5 6 1.5 0.5 1.0 1.0 0.5 0.5 0.0 0.0 0.0 normal tumor normal tumor normal tumor normal tumor AIM2 ARL14 CEP55 CT83 0% 16.4% 0% 39.1% 0% 97.8% 0% 32.1% 0 0 normal tumor normal tumor normal tumor normal tumor IGF2BP3 KAZALD1 LRP1B MAGEA1 0% 83.8% 0% 18.4% 0% 37.1% 0% 44.5% 0 0 normal tumor normal tumor normal tumor normal tumor MAGEC2 MKRN3 MYEOV PLD1 0% 36.3% 0% 66.3% 0% 0% 76.4% 12 49.5% 10 12 0 0 normal tumor normal tumor normal tumor normal tumor PON1 PRAME 0% 2.6% 0% 86.4% 6 8 0 0 normal tumor normal tumor Activation frequencies in 22 epigenetically regulated genes in LUSC. Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) CEP55 ADPRHL1 PLD1 IGF2BP3 LRP1B MAGEC2 GS1−179L18.1 AL022344.4 MKRN3 MAGEA1 RP1−90G24.10 PRAME PCAT7 RP11−564D11.3 RP11−371I1.2 PON1 KAZALD1 CT83 LINC00857 ARL14 MYEOV AIM2 HALLMARK_G2M _CHECKPOINT GO_CELL_CYCLE MYEOV PLD1 PLD1 AIM2 ARL14 MYEOV LINC00857 AIM2 LINC00857 ARL14 MYEOV ARL14 LINC00857 MAGEC2 RP11−371I1.2 CT83 GS1−179L18.1 CT83 PRAME CT83 AIM2 AL022344.4 RP11−371I1.2 LRP1B MAGEC2 KAZALD1 GS1.179L18.1 KAZALD1 AL022344.4 MKRN3 RP1−90G24.10 PON1 MAGEA1 PCAT7 RP11.564D11.3 PCAT7 PRAME CEP55 IGF2BP3 MAGEC2 IGF2BP3 LRP1B GS1−179L18.1 RP11.371I1.2 PLD1 AL022344.4 LRP1B RP1−90G24.10 KAZALD1 MAGEA1 PRAME ADPRHL1 MKRN3 RP11−564D11.3 RP1.90G24.10 PON1 CEP55 PCAT7 ADPRHL1 MAGEA1 PON1 RP11−564D11.3 ADPRHL1 IGF2BP3 MKRN3 CEP55 Supplementary Figure S4 A TCGA LUAD RNA-seq expression correlation 　 B TCGA LUSC RNA-seq expression correlation PON1 AIM2 PCAT7 MYEOV cluster1 cluster3 RP1−90G24.10 ARL14 LINC00857 ADPRHL1 KAZALD1 CT83 LRP1B KAZALD1 RP11−371I1.2 PON1 RP11−371I1.2 IGF2BP3 CEP55 RP11−564D11.3 PCAT7 RP11−564D11.3 cluster1 MAGEA1 PRAME MKRN3 RP1−90G24.10 GS1−179L18.1 MAGEA1 cluster2 MAGEC2 MKRN3 AL022344.4 AL022344.4 cluster2 PRAME GS1−179L18.1 MAGEC2 CT83 LRP1B LINC00857 MYEOV IGF2BP3 cluster3 PLD1 AIM2 ARL14 ADPRHL1 CEP55 PLD1 correlation correlation −0.2 0 0.2 0.4 0.6 0.8 1 −0.2 0 0.2 0.4 0.6 0.8 1 C Top correlated EpiFactors in TCGA LUAD (RNA-seq expression) MBIP MAP3K12 binding inhibitory protein 1 HJURP Holliday junction recognition protein BUB1 BUB1 mitotic checkpoint serine/threonine kinase correlation histogram TTK TTK protein kinase RAD54L RAD54−like (S. cerevisiae) TOP2A topoisomerase (DNA) II alpha 170kDa CDC6 cell division cycle 6 AURKA aurora kinase A AURKB aurora kinase B PBK PDZ binding kinase CDK2 cyclin−dependent kinase 2 GSG2 germ cell associated 2 (haspin) DNMT3B DNA (cytosine−5−)−methyltransferase 3 beta PRDM9 PR domain containing 9 −1.0 −0.5 0.0 0.5 1.0 correlation D Top correlated EpiFactors in TCGA LUSC (RNA-seq expression) correlation histogram PHC2 polyhomeotic homolog 2 (Drosophila) ACTB actin, beta VDR vitamin D (1,25− dihydroxyvitamin D3) receptor BRCA1 breast cancer 1, early onset CBX1 chromobox homolog 1 CBX2 chromobox homolog 2 SUZ12 SUZ12 polycomb repressive complex 2 subunit TADA2A transcriptional adaptor 2A MSH6 mutS homolog 6 SCML2 sex comb on midleg−like 2 (Drosophila) ACTL6A actin−like 6A YEATS2 YEATS domain containing 2 −1.0 −0.5 0.0 0.5 1.0 BRD4 bromodomain containing 4 IKBKAP inhibitor of kappa light polypeptide gene enhancer in B−cells, kinase complex−associated protein KEAP1 kelch−like ECH−associated protein 1 RNF168 ring finger protein 168, E3 ubiquitin protein ligase correlation The signature of 22 epigenetically regulated genes in non-small cell lung cancer. A. The correlation heatmap between expression levels of 22 epigenetically regulated genes in TCGA LUAD dataset (RNA-seq data). B. The correlation heatmap between expression levels of 22 epigenetically regulated genes in TCGA LUSC dataset (RNA-seq data). C and D. The correlations between 22 epigenetically regulated genes and correlated EpiFactors (Medvedeva et al., 2015), selected by absolute Pearson’s correlation > 0.3 with at least 5 out of 22 genes in the signature. C. In LUAD, the correlations were skewed towards positive values and the genes were very strongly enriched for cell cycle genes (12 out of 14 genes, GO_CELL_CYCLE, FDR q-value: 5.6 e-14) and more specifically in the G2/M checkpoint genes (8 out of 14 genes, HALLMARK_G2M_CHECKPOINT, FDR q-value: 4.44 e-13). The overlapping genes included TOP2A, which is up-regulated across multiple cancer types (Kaczkowski et al., 2016; Rhodes et al., 2004) and 6 kinases including AURKA and AURKB that are implicated in multiple cancer types. D. In LUSC, no clear enrichments were observed. 0.8 0.6 0.4 0.2 0 −0.2−0.4 0.4 0.2 0 −0.2 −0.4 Frequency Frequency 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 Supplementary Figure S5 RP1-90G24.10 hg19 chr22 32595612..32605296+ [len 9.7kb ] p13 p12 p11.2 p11.1 q11.1 q11.21 q11.22 q11.23 q12.1 q12.2 q12.3 q13.1 q13.2 q13.31 q13.32 q13.33 32596000 32597000 32598000 32599000 32600000 32601000 32602000 32603000 32604000 32605000 Gencode v19 ENST00000400236.3 ENST00000248983.4 ENST00000400237.1 RP1-90G24.10 Infinium HumanMethylation450 BeadChip probe cg18555433 cg01124132 cg22404498 cg01840965 cg08143133 cg12906381 cg24848192 cg03477302 cg02347904 cg26494441 cg21235359 cg12427303 cg25125450 cg13405775 Repeatmasker AluY MLT1D CT-rich MLT1D REP522 REP522 REP522 CAGE peak PC-14 PolII ChIP-seq PC-14 H3K4me3 ChIP-seq PC-14 H3K27ac ChIP-seq AL022344.4 Chr10 43184715 - 43188929 p15.3 p15.2 p15.1p14 p13 p12.33 p12.32 p12.31 p12.2 p12.1 p11.23 p11.22p11.21 p11.1 q11.1 q11.21 q11.22 q11.23 q21.1 q21.2 q21.3 q22.1 q22.2q22.3 q23.1 q23.2 q23.31q23.32 q23.33 q24.1 q24.2q24.31 q24.32 q24.33 q25.1 q25.2 q25.3 q26.1q26.12 1 q26.13 q26.2 q26.3 43185000 43186000 43187000 43188000 Gencode v19 AL022344.4 Infinium HumanMethylation450 BeadChip probe cg11519260 cg02314702 cg17575113 cg26988283 cg10989871 cg02780400 cg05042728 Repeatmasker GA-rich L1PA8 AluY (CAA)n AluSg4 AluSx3 REP522 REP522 REP522 CAGE peak PC-14 PolII ChIP-seq PC-14 H3K4me3 ChIP-seq PC-14 H3K27ac ChIP-seq ZENBU genome browser view of RP1-90G24.10 and AL022344.4. Genomic coordinate, transcript, Infinium 450K methylation array probe, REP522 elements, CAGE peaks, and ChIP peaks (H3K4me3, H3K27ac, and PolII) in PC-14 cells. Supplementary Figure S6 MYEOV Chr11 69060828 - 69065539 p15.5 p15.4 p15.3 p15.2 p15.1 p14.3 p14.2 p14.1 p13 p12 p11.2 p11.12 p11.1q 111 q12.1 q12.2 q12.3 q13.1 q13.2 q13.3 q13.4 q13.5 q14.1 q14.2 q14.3 q21 q22.1 q22.2 q22.3 q23.1q23.2 q23.3 q24.1 q24.2 q24.3 q25 69061000 69062000 69063000 69064000 69065000 RefSeq MYEOV CAGE peak DPI clusters p1@MYEOV p3@MYEOV p5@MYEOV Infinium HumanMethylation450 BeadChip probe cg26548122 cg24776407 cg01638792 cg22779330 cg08759026 cg20363347 ZENBU genome browser view of MYEOV. Three CAGE-defined promoters for MYEOV (p1, p3, and p5) exhibited up-regulation and hypomethylation in NSCLC cell lines. The MYEOV gene is located at chromosome 11q13, and the major transcript of MYEOV which codes a long isoform has three exons. MYEOV gene methylation was evaluated by cg24776407, a probe that corresponds to the 5’ untranslated region (UTR) and the first exon. Supplementary Figure S7 adenocarcinoma Hazard Ratio Study (227342_s_at) n HR 95%-CI GSE3141 58 1.15 [0.83; 1.60] GSE31210 204 1.35 [1.10; 1.65] GSE37745 106 0.89 [0.70; 1.13] GSE30219 85 1.43 [1.10; 1.86] GSE50081 127 1.33 [1.03; 1.72] E-MTAB-923 90 1.04 [0.75; 1.43] Fixed effect model 1.20 [1.08; 1.33] Random effects model 1.19 [1.02; 1.39] Fixed effect model P = 0.0007 Random effects model P = 0.0278 0.75 1 1.5 squamous cell carcinoma Hazard Ratio Study (227342_s_at) n HR 95%-CI GSE3141 53 1.17 [0.78; 1.75] GSE37745 66 1.40 [1.02; 1.93] GSE30219 61 1.19 [0.85; 1.69] GSE50081 43 1.12 [0.77; 1.62] E-MTAB-1727 93 1.11 [0.90; 1.38] Fixed effect model 1.18 [1.03; 1.35] Random effects model 1.18 [1.03; 1.35] P = 0.0017 Fixed effect model Random effects model P = 0.0017 0.75 1 1.5 Higher MYEOV expression is associated with poor prognosis in NSCLC. A. Meta-analysis of 6 lung adenocarcinoma microarray datasets (GSE50081, GSE3141, GSE31210, GSE30219, GSE37745, and E-MTAB-923). The probe 227342_s_at is evaluated. The result of fixed effect and random effects model is represented by a diamond, the center of the diamond indicates the pooled estimate, and the dimensions correspond to the 95% CI. B. Meta-analysis of 5 lung squamous cell carcinoma datasets (GSE3141, GSE37745, GSE30219, GSE50081, and E-MTAB-1727). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Cancer Research Unpaywall

Integrative CAGE and DNA Methylation Profiling Identify Epigenetically Regulated Genes in NSCLC

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1541-7786
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10.1158/1541-7786.mcr-17-0191
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Abstract

Supplementary Figure S1 TCGA LUSC TCGA LUAD ARL14 CT83 MYEOV ARL14 CT83 MYEOV 10 10 Rho = -0.68 Rho = -0.33 Rho = -0.48 Rho = -0.72 Rho = -0.67 Rho = -0.53 14 12 12 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 12 8 8 6 6 6 4 4 4 4 2 2 2 2 2 2 0 0 0 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 β value (cg24147596) β value (cg17155263) β value (cg17155263) β value (cg01638792) β value (cg01638792) β value (cg24147596) GS1-179L18.1 LINC00857 RP11-371I1.2 GS1-179L18.1 LINC00857 RP11-371I1.2 Rho = -0.34 Rho = -0.38 3 3 5 Rho = -0.52 Rho = -0.52 4 4 Rho = -0.78 Rho = -0.80 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 2.5 P < 0.0001 P < 0.0001 3 4 3 3 2 2 1.5 2 1 1 1 1 0.5 0 0 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 β value (cg00347856) β value (cg11647108) β value (cg16662846) β value (cg00347856) β value (cg11647108) β value (cg16662846) AL022344.4 PCAT7 RP1-90G24.10 AL022344.4 PCAT7 RP1-90G24.10 Rho = -0.45 Rho = -0.33 Rho = -0.33 Rho = -0.32 Rho = -0.12 Rho = -0.47 P < 0.0001 5 P < 0.0001 3 P < 0.0001 P = 0.02 P < 0.0001 3 P < 0.0001 2 4 1 2 0.5 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 β value (cg02780400) β value (cg13647892) β value (cg25125450) β value (cg02780400) β value (cg13647892) β value (cg25125450) TCGA methylation normal vs tumor TCGA expression normal vs tumor ARL14 CT83 MYEOV ARL14 CT83 MYEOV * * * * * * * 10 * 1 1 1 * * * 12 14 8 12 0.8 0.8 0.8 8 6 0.6 0.6 0.6 4 6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 0 0 N T N T N T N T N T N T N T N T N T N T N T N T LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC RP11-371I1.2 GS1-179L18.1 LINC00857 RP11-371I1.2 GS1-179L18.1 LINC00857 * 4 * * 5 * * * 4 * 1 1 1 * * 3 0.8 0.8 0.8 0.6 0.6 0.6 1 0.4 1 0.4 0.4 0.2 0 0 0 0.2 0.2 N T N T N T N T N T N T 0 0 0 N T N T LUAD LUSC N T N T N T N T LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC LUAD LUSC RP1-90G24.10 1.5 * RP1-90G24.10 * * 1 1 0.8 0.5 0.6 0.4 N T N T 0.2 LUAD LUSC 0 N T N T LUAD LUSC GSE81089 AL022344.4 PCAT7 RP1-90G24.10 14 16 2.5 8 1.5 0.5 0 0 NSCLC Normal NSCLC Normal NSCLC Normal Methylation and expression levels of genes in lung cancer and normal lung tissues. A. Correlations between expression and methylation in TCGA LUAD (left, N = 426) and TCGA LUSC (right, N = 359). The y-axis: log (1+RPKM). The x-axis: β values. The results of 3 coding genes (ARL14, CT83, and MYEOV), 3 lncRNAs (GS1-179L18.1, LINC00857, and RP11-371l1.2), and 3 lncRNAs whose promoters are within the REP522 repeat family (AL022344.4, PCAT7, and RP1-90G24.10) are shown. B. Methylation and expression levels of 3 coding genes (ARL14, CT83, and MYEOV), 4 lncRNAs (GS1-179L18.1, LINC00857, RP11-371l1.2, and RP1-90G24.10) are shown. Left: RNA-seq of 515 LUAD vs. 59 normal lung, and 501 LUSC vs. 49 normal lung. Right: Methylation data of 431 LUAD vs. 31 normal lung, and 359 LUSC vs. 42 normal lung. N: normal lung. T: tumor tissue. *P < 0.05. C. Expression of lncRNAs in NSCLC (N = 199) and normal lung tissues (N = 19) in GSE81089 dataset. *P < 0.05. Log (1+RPKM) Log (1+RPKM) 2 Log2 (1+RPKM) Log (1+RPKM) 2 Log2 (1+RPKM) Log (1+RPKM) F PKM Log2 (1+RPKM) Log (1+RPKM) Log (1+RPKM) Log2 (1+RPKM) Log (1+RPKM) F PKM Log (1+RPKM) Log (1+RPKM) Log (1+RPKM) Log2 (1+RPKM) Log (1+RPKM) 2 F PKM β value (cg24147596) β value (cg25125450) β value (cg00347856) Log2 (1+RPKM) Log2 (1+RPKM) Log (1+RPKM) β value (cg17155263) β value (cg11647108) Log (1+RPKM) Log (1+RPKM) 2 Log (1+RPKM) β value (cg01638792) β value (cg16662846) Log (1+RPKM) Log (1+RPKM) Log2 (1+RPKM) 2 Supplementary Figure S2 GS1.179L18.1 RP11.371I1.2 PCAT7 AL022344.4 3.0 2.5 0% 20.7% 0% 26.6% 0% 43.6% 0% 33% 2.5 2.0 2.0 3 3 1.5 1.5 2 2 1.0 1.0 1 1 0.5 0.5 0.0 0 0.0 0 normal tumor normal tumor normal tumor normal tumor LINC00857 RP1.90G24.10 RP11.564D11.3 ADPRHL1 1.2 3.0 0% 74.2% 0% 15.8% 0% 85.9% 0% 23.2% 1.0 2.5 3 10 0.8 2.0 2 0.6 1.5 0.4 1.0 0.2 0.5 0.0 0.0 0 2 normal tumor normal tumor normal tumor normal tumor AIM2 ARL14 CEP55 CT83 0% 33.6% 0% 30.1% 0% 77.9% 0% 49% 10 10 0 0 normal tumor normal tumor normal tumor normal tumor IGF2BP3 KAZALD1 LRP1B MAGEA1 0% 45.5% 0% 21.9% 0% 7.6% 0% 17.6% 0 0 normal tumor normal tumor normal tumor normal tumor MAGEC2 MKRN3 MYEOV PLD1 12 14 11 0% 20.9% 0% 23.2% 0% 62.7% 0% 23.6% 6 10 4 7 0 0 0 normal tumor normal tumor normal tumor normal tumor PON1 PRAME 0% 27% 0% 52% 6 8 0 0 normal tumor normal tumor Activation frequencies in 22 epigenetically regulated genes in LUAD. Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Supplementary Figure S3 GS1.179L18.1 RP11.371I1.2 PCAT7 AL022344.4 3.5 0% 27.3% 0% 44.9% 0% 0% 71.9% 85.8% 3.0 3 2.5 2.0 1.5 1.0 0.5 0 0 0.0 0 normal tumor normal tumor normal tumor normal tumor LINC00857 RP1.90G24.10 RP11.564D11.3 ADPRHL1 1.5 3.0 3.0 0% 13.8% 0% 17.4% 0% 58.9% 0% 16.4% 2.5 2.5 1.0 2.0 2.0 7 1.5 6 1.5 0.5 1.0 1.0 0.5 0.5 0.0 0.0 0.0 normal tumor normal tumor normal tumor normal tumor AIM2 ARL14 CEP55 CT83 0% 16.4% 0% 39.1% 0% 97.8% 0% 32.1% 0 0 normal tumor normal tumor normal tumor normal tumor IGF2BP3 KAZALD1 LRP1B MAGEA1 0% 83.8% 0% 18.4% 0% 37.1% 0% 44.5% 0 0 normal tumor normal tumor normal tumor normal tumor MAGEC2 MKRN3 MYEOV PLD1 0% 36.3% 0% 66.3% 0% 0% 76.4% 12 49.5% 10 12 0 0 normal tumor normal tumor normal tumor normal tumor PON1 PRAME 0% 2.6% 0% 86.4% 6 8 0 0 normal tumor normal tumor Activation frequencies in 22 epigenetically regulated genes in LUSC. Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) Log2 (1+RPKM) CEP55 ADPRHL1 PLD1 IGF2BP3 LRP1B MAGEC2 GS1−179L18.1 AL022344.4 MKRN3 MAGEA1 RP1−90G24.10 PRAME PCAT7 RP11−564D11.3 RP11−371I1.2 PON1 KAZALD1 CT83 LINC00857 ARL14 MYEOV AIM2 HALLMARK_G2M _CHECKPOINT GO_CELL_CYCLE MYEOV PLD1 PLD1 AIM2 ARL14 MYEOV LINC00857 AIM2 LINC00857 ARL14 MYEOV ARL14 LINC00857 MAGEC2 RP11−371I1.2 CT83 GS1−179L18.1 CT83 PRAME CT83 AIM2 AL022344.4 RP11−371I1.2 LRP1B MAGEC2 KAZALD1 GS1.179L18.1 KAZALD1 AL022344.4 MKRN3 RP1−90G24.10 PON1 MAGEA1 PCAT7 RP11.564D11.3 PCAT7 PRAME CEP55 IGF2BP3 MAGEC2 IGF2BP3 LRP1B GS1−179L18.1 RP11.371I1.2 PLD1 AL022344.4 LRP1B RP1−90G24.10 KAZALD1 MAGEA1 PRAME ADPRHL1 MKRN3 RP11−564D11.3 RP1.90G24.10 PON1 CEP55 PCAT7 ADPRHL1 MAGEA1 PON1 RP11−564D11.3 ADPRHL1 IGF2BP3 MKRN3 CEP55 Supplementary Figure S4 A TCGA LUAD RNA-seq expression correlation 　 B TCGA LUSC RNA-seq expression correlation PON1 AIM2 PCAT7 MYEOV cluster1 cluster3 RP1−90G24.10 ARL14 LINC00857 ADPRHL1 KAZALD1 CT83 LRP1B KAZALD1 RP11−371I1.2 PON1 RP11−371I1.2 IGF2BP3 CEP55 RP11−564D11.3 PCAT7 RP11−564D11.3 cluster1 MAGEA1 PRAME MKRN3 RP1−90G24.10 GS1−179L18.1 MAGEA1 cluster2 MAGEC2 MKRN3 AL022344.4 AL022344.4 cluster2 PRAME GS1−179L18.1 MAGEC2 CT83 LRP1B LINC00857 MYEOV IGF2BP3 cluster3 PLD1 AIM2 ARL14 ADPRHL1 CEP55 PLD1 correlation correlation −0.2 0 0.2 0.4 0.6 0.8 1 −0.2 0 0.2 0.4 0.6 0.8 1 C Top correlated EpiFactors in TCGA LUAD (RNA-seq expression) MBIP MAP3K12 binding inhibitory protein 1 HJURP Holliday junction recognition protein BUB1 BUB1 mitotic checkpoint serine/threonine kinase correlation histogram TTK TTK protein kinase RAD54L RAD54−like (S. cerevisiae) TOP2A topoisomerase (DNA) II alpha 170kDa CDC6 cell division cycle 6 AURKA aurora kinase A AURKB aurora kinase B PBK PDZ binding kinase CDK2 cyclin−dependent kinase 2 GSG2 germ cell associated 2 (haspin) DNMT3B DNA (cytosine−5−)−methyltransferase 3 beta PRDM9 PR domain containing 9 −1.0 −0.5 0.0 0.5 1.0 correlation D Top correlated EpiFactors in TCGA LUSC (RNA-seq expression) correlation histogram PHC2 polyhomeotic homolog 2 (Drosophila) ACTB actin, beta VDR vitamin D (1,25− dihydroxyvitamin D3) receptor BRCA1 breast cancer 1, early onset CBX1 chromobox homolog 1 CBX2 chromobox homolog 2 SUZ12 SUZ12 polycomb repressive complex 2 subunit TADA2A transcriptional adaptor 2A MSH6 mutS homolog 6 SCML2 sex comb on midleg−like 2 (Drosophila) ACTL6A actin−like 6A YEATS2 YEATS domain containing 2 −1.0 −0.5 0.0 0.5 1.0 BRD4 bromodomain containing 4 IKBKAP inhibitor of kappa light polypeptide gene enhancer in B−cells, kinase complex−associated protein KEAP1 kelch−like ECH−associated protein 1 RNF168 ring finger protein 168, E3 ubiquitin protein ligase correlation The signature of 22 epigenetically regulated genes in non-small cell lung cancer. A. The correlation heatmap between expression levels of 22 epigenetically regulated genes in TCGA LUAD dataset (RNA-seq data). B. The correlation heatmap between expression levels of 22 epigenetically regulated genes in TCGA LUSC dataset (RNA-seq data). C and D. The correlations between 22 epigenetically regulated genes and correlated EpiFactors (Medvedeva et al., 2015), selected by absolute Pearson’s correlation > 0.3 with at least 5 out of 22 genes in the signature. C. In LUAD, the correlations were skewed towards positive values and the genes were very strongly enriched for cell cycle genes (12 out of 14 genes, GO_CELL_CYCLE, FDR q-value: 5.6 e-14) and more specifically in the G2/M checkpoint genes (8 out of 14 genes, HALLMARK_G2M_CHECKPOINT, FDR q-value: 4.44 e-13). The overlapping genes included TOP2A, which is up-regulated across multiple cancer types (Kaczkowski et al., 2016; Rhodes et al., 2004) and 6 kinases including AURKA and AURKB that are implicated in multiple cancer types. D. In LUSC, no clear enrichments were observed. 0.8 0.6 0.4 0.2 0 −0.2−0.4 0.4 0.2 0 −0.2 −0.4 Frequency Frequency 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 Supplementary Figure S5 RP1-90G24.10 hg19 chr22 32595612..32605296+ [len 9.7kb ] p13 p12 p11.2 p11.1 q11.1 q11.21 q11.22 q11.23 q12.1 q12.2 q12.3 q13.1 q13.2 q13.31 q13.32 q13.33 32596000 32597000 32598000 32599000 32600000 32601000 32602000 32603000 32604000 32605000 Gencode v19 ENST00000400236.3 ENST00000248983.4 ENST00000400237.1 RP1-90G24.10 Infinium HumanMethylation450 BeadChip probe cg18555433 cg01124132 cg22404498 cg01840965 cg08143133 cg12906381 cg24848192 cg03477302 cg02347904 cg26494441 cg21235359 cg12427303 cg25125450 cg13405775 Repeatmasker AluY MLT1D CT-rich MLT1D REP522 REP522 REP522 CAGE peak PC-14 PolII ChIP-seq PC-14 H3K4me3 ChIP-seq PC-14 H3K27ac ChIP-seq AL022344.4 Chr10 43184715 - 43188929 p15.3 p15.2 p15.1p14 p13 p12.33 p12.32 p12.31 p12.2 p12.1 p11.23 p11.22p11.21 p11.1 q11.1 q11.21 q11.22 q11.23 q21.1 q21.2 q21.3 q22.1 q22.2q22.3 q23.1 q23.2 q23.31q23.32 q23.33 q24.1 q24.2q24.31 q24.32 q24.33 q25.1 q25.2 q25.3 q26.1q26.12 1 q26.13 q26.2 q26.3 43185000 43186000 43187000 43188000 Gencode v19 AL022344.4 Infinium HumanMethylation450 BeadChip probe cg11519260 cg02314702 cg17575113 cg26988283 cg10989871 cg02780400 cg05042728 Repeatmasker GA-rich L1PA8 AluY (CAA)n AluSg4 AluSx3 REP522 REP522 REP522 CAGE peak PC-14 PolII ChIP-seq PC-14 H3K4me3 ChIP-seq PC-14 H3K27ac ChIP-seq ZENBU genome browser view of RP1-90G24.10 and AL022344.4. Genomic coordinate, transcript, Infinium 450K methylation array probe, REP522 elements, CAGE peaks, and ChIP peaks (H3K4me3, H3K27ac, and PolII) in PC-14 cells. Supplementary Figure S6 MYEOV Chr11 69060828 - 69065539 p15.5 p15.4 p15.3 p15.2 p15.1 p14.3 p14.2 p14.1 p13 p12 p11.2 p11.12 p11.1q 111 q12.1 q12.2 q12.3 q13.1 q13.2 q13.3 q13.4 q13.5 q14.1 q14.2 q14.3 q21 q22.1 q22.2 q22.3 q23.1q23.2 q23.3 q24.1 q24.2 q24.3 q25 69061000 69062000 69063000 69064000 69065000 RefSeq MYEOV CAGE peak DPI clusters p1@MYEOV p3@MYEOV p5@MYEOV Infinium HumanMethylation450 BeadChip probe cg26548122 cg24776407 cg01638792 cg22779330 cg08759026 cg20363347 ZENBU genome browser view of MYEOV. Three CAGE-defined promoters for MYEOV (p1, p3, and p5) exhibited up-regulation and hypomethylation in NSCLC cell lines. The MYEOV gene is located at chromosome 11q13, and the major transcript of MYEOV which codes a long isoform has three exons. MYEOV gene methylation was evaluated by cg24776407, a probe that corresponds to the 5’ untranslated region (UTR) and the first exon. Supplementary Figure S7 adenocarcinoma Hazard Ratio Study (227342_s_at) n HR 95%-CI GSE3141 58 1.15 [0.83; 1.60] GSE31210 204 1.35 [1.10; 1.65] GSE37745 106 0.89 [0.70; 1.13] GSE30219 85 1.43 [1.10; 1.86] GSE50081 127 1.33 [1.03; 1.72] E-MTAB-923 90 1.04 [0.75; 1.43] Fixed effect model 1.20 [1.08; 1.33] Random effects model 1.19 [1.02; 1.39] Fixed effect model P = 0.0007 Random effects model P = 0.0278 0.75 1 1.5 squamous cell carcinoma Hazard Ratio Study (227342_s_at) n HR 95%-CI GSE3141 53 1.17 [0.78; 1.75] GSE37745 66 1.40 [1.02; 1.93] GSE30219 61 1.19 [0.85; 1.69] GSE50081 43 1.12 [0.77; 1.62] E-MTAB-1727 93 1.11 [0.90; 1.38] Fixed effect model 1.18 [1.03; 1.35] Random effects model 1.18 [1.03; 1.35] P = 0.0017 Fixed effect model Random effects model P = 0.0017 0.75 1 1.5 Higher MYEOV expression is associated with poor prognosis in NSCLC. A. Meta-analysis of 6 lung adenocarcinoma microarray datasets (GSE50081, GSE3141, GSE31210, GSE30219, GSE37745, and E-MTAB-923). The probe 227342_s_at is evaluated. The result of fixed effect and random effects model is represented by a diamond, the center of the diamond indicates the pooled estimate, and the dimensions correspond to the 95% CI. B. Meta-analysis of 5 lung squamous cell carcinoma datasets (GSE3141, GSE37745, GSE30219, GSE50081, and E-MTAB-1727).

Journal

Molecular Cancer Research – Unpaywall

Published: Oct 1, 2017

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