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. 2022 Jan;54(1):12-22.
doi: 10.1038/s12276-021-00718-4. Epub 2022 Jan 5.

The importance of enhancer methylation for epigenetic regulation of tumorigenesis in squamous lung cancer

Jae-Won Cho  1 , Hyo Sup Shim  2 , Chang Young Lee  3 , Seong Yong Park  3 , Min Hee Hong  4 , Insuk Lee  5 , Hye Ryun Kim  6
Affiliations

The importance of enhancer methylation for epigenetic regulation of tumorigenesis in squamous lung cancer

Jae-Won Cho et al. Exp Mol Med. 2022 Jan.

Abstract

Lung squamous cell carcinoma (LUSC) is a subtype of non-small cell lung cancer (NSCLC). LUSC occurs at the bronchi, shows a squamous appearance, and often occurs in smokers. To determine the epigenetic regulatory mechanisms of tumorigenesis, we performed a genome-wide analysis of DNA methylation in tumor and adjacent normal tissues from LUSC patients. With the Infinium Methylation EPIC Array, > 850,000 CpG sites, including ~350,000 CpG sites for enhancer regions, were profiled, and the differentially methylated regions (DMRs) overlapping promoters (pDMRs) and enhancers (eDMRs) between tumor and normal tissues were identified. Dimension reduction based on DMR profiles revealed that eDMRs alone and not pDMRs alone can differentiate tumors from normal tissues with the equivalent performance of total DMRs. We observed a stronger negative correlation of LUSC-specific gene expression with methylation for enhancers than promoters. Target genes of eDMRs rather than pDMRs were found to be enriched for tumor-associated genes and pathways. Furthermore, DMR methylation associated with immune infiltration was more frequently observed among enhancers than promoters. Our results suggest that methylation of enhancer regions rather than promoters play more important roles in epigenetic regulation of tumorigenesis and immune infiltration in LUSC.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overview of differentially methylated regions in lung squamous cell carcinoma (LUSC).
a Overview of our genome-wide methylation analysis: (i) Study design, (ii) Analysis of differentially methylated regions (DMRs), risk-related single nucleotide polymorphisms (SNPs) and immune infiltration, and (iii) Gene set enrichment analysis of functional DMRs. b–e Stacked bar plots for the count of promoter DMRs (pDMRs) and enhancer DMRs (eDMRs) (b), the count of target genes of pDMRs and eDMRs (c), the count of functional pDMRs (F-pDMRs) and functional eDMRs (F-eDMRs) (d), and the count of target genes of F-pDMRs and F-eDMRs (e). f–g Dimension reduction of methylation profiles for tumor and normal samples using t-stochastic neighbor embedding (tSNE) (f) and uniform manifold approximation and projection (UMAP) (g).
Fig. 2
Fig. 2. Correlation analysis of methylation with lung squamous cell carcinoma (LUSC)-specific gene expression.
a The box plot shows the correlation of methylation with the expression of LUSC-specific genes (compared to TCGA-LUAD) vs. all other genes. b The box plot shows the correlation of methylation with the expression of LUSC-specific genes (compared to 32 TCGA cancer types and normal tissue of LUSC) vs. all other genes. Significance was assessed with a two-sided Wilcoxon signed-rank test. Box plots show the median, first and third quartiles, and outliers.
Fig. 3
Fig. 3. Gene set enrichment analysis for target genes of promoter differentially methylated regions (pDMRs) and enhancer differentially methylated regions (eDMRs).
a Enrichment of cancer hallmark gene sets from the CancerSEA database for functional (F)-eDMR target genes. The dashed line indicates the significance threshold (q-value < 0.05). b Enrichment of each category of cancer-associated genes from the CancerMine database for F-pDMR and F-eDMR target genes. The dashed line indicates the significance threshold (q-value < 0.05). c Enrichment of Reactome pathway gene sets for F-pDMR and F-eDMR target genes. The bar graph indicates the number of significantly enriched pathways (q-value < 0.01) for each category. d Enrichment of cancer-testis genes for F-pDMR and F-eDMR target genes.
Fig. 4
Fig. 4. Functional analysis of super-enhancer differentially methylated region (seDMR) targets and enrichment of lung squamous cell carcinoma (LUSC) risk-related single nucleotide polymorphisms (SNPs) for DMRs.
a Stacked bar plots of the number of seDMRs, functional (F)-seDMRs, and their target genes with hypo- or hypermethylation. b Enrichment of Reactome pathway gene sets for F-seDMR target genes. c An example of seDMR and target interactions visualized with Integrative Genomic View (IGV). Methylation levels of seDMR regions and expression levels of their target genes were compared between tumor (T) and normal tissue (N). d An example of LUSC risk-related SNPs residing within DMR regions. e Pie chart for the distribution of LUSC risk-related SNPs within DMRs and outside of DMRs. The significance of SNPs within DMRs was assessed using binomial distribution.
Fig. 5
Fig. 5. Regulation of immune infiltration by DNA methylation.
a Ten immune cell types that showed abundance changes in tumors. b Schematic overview to define infiltration-associated methylation regions (IMRs) using correlation analysis between methylation and immune cell infiltration (q-value < 0.01, Spearman’s correlation). c Examples of promoters and enhancers whose methylation level correlates with immune cell infiltration. d Proportion of IMRs among differentially methylated regions (DMRs) vs. non-DMRs. e Odds ratio of DMRs to non-DMRs among IMRs. f, g Reactome pathway gene sets significantly enriched for genes regulated by IMR-pDMRs (f) or by IMR-eDMRs (g) outside partially methylated domains (PMDs) (only those with q-value < 0.001 are presented in the plot). The dashed line indicates the significance threshold (q-value < 0.001) for the presented bar plots. h Enrichment of cancer hallmark gene sets from the CancerSEA database for the genes regulated by IMR-eDMRs or IMR-pDMRs outside PMDs. The dashed line indicates the significance threshold (q-value < 0.05). i Enrichment of each category of cancer-associated genes from the CancerMine database for the genes regulated by IMR-eDMRs outside PMDs. The dashed line indicates the significance threshold (p-value < 0.05).
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References

    1. Travis WD, et al. The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification. J. Thorac. Oncol. 2015;10:1243–1260. - PubMed
    1. Desrichard A, et al. Tobacco smoking-associated alterations in the immune microenvironment of squamous cell carcinomas. J. Natl Cancer Inst. 2018;110:1386–1392. - PMC - PubMed
    1. Kenfield SA, Wei EK, Stampfer MJ, Rosner BA, Colditz GA. Comparison of aspects of smoking among the four histological types of lung cancer. Tob. Control. 2008;17:198–204. - PMC - PubMed
    1. Derman BA, Mileham KF, Bonomi PD, Batus M, Fidler MJ. Treatment of advanced squamous cell carcinoma of the lung: a review. Transl. Lung Cancer Res. 2015;4:524–532. - PMC - PubMed
    1. Darvin P, Toor SM, Sasidharan Nair V, Elkord E. Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp. Mol. Med. 2018;50:1–11. - PMC - PubMed

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