THOR is a targetable epigenetic biomarker with clinical implications in breast cancer

Background Breast cancer (BC) is the most frequently diagnosed cancer and a leading cause of death among women worldwide. Early BC is potentially curable, but the mortality rates still observed among BC patients demonstrate 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 the human telomerase reverse transcriptase (h TERT ). The hypermethylation of a specific region within the h TERT promoter, termed TERT hypermethylated oncological region (THOR) has been associated with increased h TERT expression in cancer. However, its biological role and clinical potential in BC have never been studied to the best of our knowledge. Therefore, we aimed to investigate the role of THOR as a biomarker and explore the functional impact of THOR methylation status in h TERT upregulation in BC. Results THOR methylation status in BC was assessed by pyrosequencing on discovery and validation cohorts. We found that THOR is significantly hypermethylated in malignant breast tissue when compared to benign tissue (40.23% vs. 12.81%, P < 0.0001), differentiating malignant tumor from normal tissue from the earliest stage of disease. Using a reporter assay, the addition of unmethylated THOR significantly reduced luciferase activity by an average 1.8-fold when compared to the h TERT core promoter alone ( P < 0.01). To further investigate its biological impact on h TERT transcription, targeted THOR demethylation was performed using novel technology based on CRISPR-dCas9 system and significant THOR demethylation was achieved. Cells previously demethylated on THOR region did not develop a histologic cancer phenotype in in vivo assays. Additional studies are required to validate these observations and to unravel the causality between THOR hypermethylation and h TERT upregulation in BC. Conclusions THOR hypermethylation is an important epigenetic mark in breast tumorigenesis, representing a promising biomarker and therapeutic target in BC. We revealed that THOR acts as a repressive regulatory element of h TERT and that its hypermethylation is a relevant mechanism for h TERT upregulation in BC.