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Roadmap of DNA methylation in breast cancer identifies novel prognostic biomarkers

Publication . Almeida, Bernardo; Apolónio, Joana; Binnie, Alexandra; Castelo-Branco, Pedro

Background Breast cancer is a highly heterogeneous disease resulting in diverse clinical behaviours and therapeutic responses. DNA methylation is a major epigenetic alteration that is commonly perturbed in cancers. The aim of this study is to characterize the relationship between DNA methylation and aberrant gene expression in breast cancer. Methods We analysed DNA methylation and gene expression profiles from breast cancer tissue and matched normal tissue in The Cancer Genome Atlas (TCGA). Genome-wide differential methylation analysis and methylation-gene expression correlation was performed. Gene expression changes were subsequently validated in the METABRIC dataset. The Oncoscore tool was used to identify genes that had previously been associated with cancer in the literature. A subset of genes that had not previously been studied in cancer was chosen for further analysis. Results We identified 368 CpGs that were differentially methylated between tumor and normal breast tissue (∆β > 0.4). Hypermethylated CpGs were overrepresented in tumor tissue and were found predominantly (56%) in upstream promoter regions. Conversely, hypomethylated CpG sites were found primarily in the gene body (66%). Expression analysis revealed that 209 of the differentially-methylated CpGs were located in 169 genes that were differently expressed between normal and breast tumor tissue. Methylation-expression correlations were predominantly negative (70%) for promoter CpG sites and positive (74%) for gene body CpG sites. Among these differentially-methylated and differentially-expressed genes, we identified 7 that had not previously been studied in any form of cancer. Three of these, TDRD10, PRAC2 and TMEM132C, contained CpG sites that showed diagnostic and prognostic value in breast cancer, particularly in estrogen-receptor (ER)-positive samples. A pan-cancer analysis confirmed differential expression of these genes together with diagnostic and prognostic value of their respective CpG sites in multiple cancer types. Conclusion We have identified 368 DNA methylation changes that characterize breast cancer tumor tissue, of which 209 are associated with genes that are differentially-expressed in the same samples. Novel DNA methylation markers were identified, of which cg12374721 (PRAC2), cg18081940 (TDRD10) and cg04475027 (TMEM132C) show promise as diagnostic and prognostic markers in breast cancer as well as other cancer types.

2019-03-12Journal article

Open access

Tert hypermethylated oncologic region (THOR) as a biomarker for breast cancer

Publication . Apolónio, Joana; Castelo-Branco, Pedro; Tabori, Uri

Breast cancer (BC) is the most frequently diagnosed cancer and a leading cause of death among women worldwide. Early BC is potentially curable, nevertheless, the mortality rates still observed among BC patients, demonstrates the urgent need of novel and more effective diagnostic and therapeutic options. Limitless self-renewal is a hallmark of cancer governed by telomere maintenance. In around 95% of BC cases, this process is achieved by telomerase reactivation through upregulation of human Telomerase Reverse Transcriptase (hTERT). The hypermethylation of a specific region within hTERT promoter, termed TERT Hypermethylated Oncological Region (THOR) has been associated with increased hTERT expression in cancer. However, its biological role and clinical potential in BC has never been studied. Therefore, we aimed to investigate the role of THOR as a biomarker, explore the functional impact of THOR in hTERT upregulation, and also identify other potential DNA methylation-based markers in BC. Firstly, we demonstrated that THOR is significantly hypermethylated in malignant breast tissue when compared to benign tissue (40.23% vs. 12.81%), representing a potential candidate biomarker for future application in BC screening and early diagnosis. Importantly, as DNA methylation marks can be determined from blood samples, assessing THOR methylation status may constitute a non-invasive assay to help in BC management. Next, using a reporter assay, we revealed that THOR acts as a repressive regulatory element of hTERT, and that THOR hypermethylation might be relevant for hTERT upregulation in BC. To further investigate its biological impact on hTERT transcription, targeted THOR demethylation was performed using the CRISPR-dCas9 system. Although, THOR demethylation was achieved, hTERT mRNA levels were not significantly reduced. Surprisingly, cells previously demethylated on THOR region led to a remarkable reduction in tumor development in vivo. Therefore, additional studies are required to validate these observations and to unravel the causality between THOR hypermethylation and hTERT upregulation in BC. Finally, through a genome-wide methylation analysis, we identified three novel DNA methylation markers, located on the PRAC2, TDRD10 and TMEM132C genes that showed diagnostic and prognostic value in BC, as well as in other cancer types. This work evidences the importance of DNA methylation in breast tumorigenesis and, more importantly, their clinical value as promising diagnostic, prognostic and therapeutic targets in BC.

2019-10-29Doctoral thesis

Open access

Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer

Publication . Leão, Ricardo; Apolónio, Joana; Lee, Donghyun; Figueiredo, Arnaldo; Tabori, Uri; Castelo-Branco, Pedro

Background Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers. Main body The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation. hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies. Conclusion Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers.

2018-03-12Journal article

Open access