Differential DNA Methylation of THOR and hTAPAS in the Regulation of hTERT and the Diagnosis of Cancer

SIMPLE SUMMARY: Because of its high prevalence of >45% in 9 out of 11 (82%) cancer types screened, THOR hypermethylation has been suggested to be a frequent telomerase-activating mechanism in hTERT-expressing tumor types, e.g., in cancers of the prostate, breast, blood, colon, lung, bladder, and brain. In this prime example, we present detailed DNA methylation profiles in urothelial cancer that reveal the exact positions of the most differentially methylated CpG dinucleotides within the THOR region in order to design an efficient Methylation-Specific PCR (MSPCR) approach for diagnostic and prognostic purposes. Furthermore, our data suggest an epigenetic mechanism regulating hTERT expression through the methylation status of THOR and lncRNA hTAPAS. ABSTRACT: Background: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5′ of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC). Methods: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail. Results: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT. In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated (hTAPAS), which represses hTERT. Conclusions: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided.