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. 2020 Sep 3;3(3):63.
doi: 10.3390/mps3030063.

Investigating the Role of Telomere and Telomerase Associated Genes and Proteins in Endometrial Cancer

Alice Bradfield  1 , Lucy Button  2 , Josephine Drury  1 , Daniel C Green  3 , Christopher J Hill  1 , Dharani K Hapangama  1   4
Affiliations

Investigating the Role of Telomere and Telomerase Associated Genes and Proteins in Endometrial Cancer

Alice Bradfield et al. Methods Protoc. .

Abstract

Endometrial cancer (EC) is the commonest gynaecological malignancy. Current prognostic markers are inadequate to accurately predict patient survival, necessitating novel prognostic markers, to improve treatment strategies. Telomerase has a unique role within the endometrium, whilst aberrant telomerase activity is a hallmark of many cancers. The aim of the current in silico study is to investigate the role of telomere and telomerase associated genes and proteins (TTAGPs) in EC to identify potential prognostic markers and therapeutic targets. Analysis of RNA-seq data from The Cancer Genome Atlas identified differentially expressed genes (DEGs) in EC (568 TTAGPs out of 3467) and ascertained DEGs associated with histological subtypes, higher grade endometrioid tumours and late stage EC. Functional analysis demonstrated that DEGs were predominantly involved in cell cycle regulation, while the survival analysis identified 69 DEGs associated with prognosis. The protein-protein interaction network constructed facilitated the identification of hub genes, enriched transcription factor binding sites and drugs that may target the network. Thus, our in silico methods distinguished many critical genes associated with telomere maintenance that were previously unknown to contribute to EC carcinogenesis and prognosis, including NOP56, WFS1, ANAPC4 and TUBB4A. Probing the prognostic and therapeutic utility of these novel TTAGP markers will form an exciting basis for future research.

Keywords: TCGA; bioinformatics analysis; endometrial cancer; prognosis; telomerase; telomere; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of telomeres and the main components of telomerase, adapted from Hapangama et al. [12]. Telomerase is a holoenzyme comprising three core components: human telomerase reverse transcriptase (hTERT), human telomeric RNA component (hTERC) and dyskerin (DKC1). hTERT is a catalytic protein with transcriptase activity and hTERC provides the RNA template from which new telomeric DNA is synthesized [12]. NHP2, NOP10 and GAR1, in addition to DKC1, bind the H/ACA snoRNA motif at the 3′ end of hTERC and stabilise newly transcribed telomeric RNA. The H/ACA region also binds telomerase Cajal body protein 1 (TCAB1). The shelterin complex is made up of telomeric repeat binding factors 1 and 2 (TERF1 and TERF2), repressor/activator protein 1 (RAP1), protection of telomeres 1 (POT1), TERF1 interacting nuclear factor 2 (TINF2) and TPP1 (encoded by the gene ACD). POT1 binds directly to the single stranded 3′ end of the telomere and forms a heterodimer with TPP1. TERF1 and TERF2 bind to the double-stranded telomeric sequence [11]. (Created with BioRender.com).
Figure 2
Figure 2
(a) Workflow diagram illustrating the in silico procedures. (b) Database searches for compiling the list of TTAGPs. The table displays the number of interactors identified for each protein within the telomerase and shelterin complex. WRAP53 and TERF2IP are the official gene symbols for TCAB1 and RAP1, respectively. TPP1 is encoded by the ACD gene.
Figure 3
Figure 3
Differentially expressed genes (DEGs) identified between endometrial cancer (EC) and healthy endometrium. (a) Volcano plot of DEGs amongst cancer (n = 234) and healthy samples (n = 11). Significant DEGs are coloured; red dots represent upregulated genes, and blue dots represent downregulated genes. Cut-off criteria: │log2FC > 1│and false discovery rate (FDR) < 0.01. (b) Heatmap displaying the expression of 568 DEGs. Red denotes upregulated genes and green denotes downregulated genes.
Figure 4
Figure 4
Volcano plots of DEGs between (a) endometrioid and healthy endometrium, (b) serous and healthy, and (c) carcinosarcoma and healthy. Significant DEGs are coloured; red dots represent upregulated genes, and blue dots represent downregulated genes. Cut-off criteria: │log2FC > 1│and FDR < 0.01. Venn diagrams displaying common (d) upregulated and (e) downregulated genes between each subtype.
Figure 5
Figure 5
DEGs associated with tumour grade and clinical stage. Volcano plots of DEGs between (a) grade 1 and grade 3 endometrioid, and (b) stage I and IV EC. Significant DEGs are coloured; red dots represent upregulated genes, and blue dots represent downregulated genes. Cut-off criteria: │log2FC > 1│and FDR < 0.01.
Figure 6
Figure 6
Functional Enrichment and Pathway Analysis of DEGs. GO terms and Kyoto Gene and Genome Encyclopaedia (KEGG) pathways were identified using Enrichr. The GO terms were subsequently revised into a smaller representative list using REVIGO (similarity <0.5). (a) Biological Process. (b) Molecular Function. (c) Cellular Component. (d) KEGG pathway.
Figure 7
Figure 7
Protein–Protein Interaction (PPI) network of DEGs. Upregulated and downregulated DEGs are represented by red and blue nodes, respectively. Degree ≥ 1.
Figure 8
Figure 8
(a) The module identified in the PPI network of DEGs using Molecular Complex Detection (MCODE). MCODE score = 64.171. Degree cut-off ≥ 2. (b) GO terms and (c) KEGG pathways associated with the module. Abbreviations: BP—Biological Process; CC—Cellular Component; MF—Molecular Function. GO terms and KEGG pathways were identified using Enrichr (adjusted p < 0.05). The GO terms were subsequently summarised into a smaller representative list using REVIGO (similarity < 0.5).
Figure 9
Figure 9
Top 10 hub genes of the PPI network constructed from (a) EC-specific DEGs and (b) stage I-IV DEGs, ranked according to degree. The hub genes were identified using Cytohubba. The colour of the node represents degree, with red representing a higher degree and yellow a lower degree.
Figure 10
Figure 10
Venn diagram displaying the intersections of stage I-IV DEGs, Grades 1–3 DEGs and prognostic DEGs.
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