Circular RNA CpG island hypermethylation-associated silencing in human cancer

// Humberto J. Ferreira 1 , Veronica Davalos 1 , Manuel Castro de Moura 1 , Marta Soler 1 , Montserrat Perez-Salvia 1 , Alberto Bueno-Costa 1 , Fernando Setien 1 , Sebastian Moran 1 , Alberto Villanueva 2 and Manel Esteller 1, 3, 4, 5 1 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain 2 Laboratory of Translational Research, Catalan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain 3 Centro de Investigacion Biomedica en Red Cancer (CIBERONC), Madrid, Spain 4 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Catalonia, Spain 5 Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Catalonia, Spain Correspondence to: Manel Esteller, email: mesteller@idibell.cat Keywords: circular RNA; noncoding RNA; DNA methylation; cancer; epigenetics Received: February 01, 2018      Accepted: June 12, 2018      Published: June 26, 2018 ABSTRACT Noncoding RNAs (ncRNAs), such as microRNAs and long noncoding RNAs (lncRNAs), participate in cellular transformation. Work done in the last decade has also demonstrated that ncRNAs with growth-inhibitory functions can undergo promoter CpG island hypermethylation-associated silencing in tumorigenesis. Herein, we wondered whether circular RNAs (circRNAs), a type of RNA transcripts lacking 5′-3′ ends and forming closed loops that are gaining relevance in cancer biology, are also a target of epigenetic inactivation in tumors. To tackle this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in conjuction with circRNA expression microarrays. We have found that the loss of DNA methylation provokes a release of circRNA silencing. In particular, we have identified that promoter CpG island hypermethylation of the genes TUSC3 (tumor suppressor candidate 3), POMT1 (protein O-mannosyltransferase 1), ATRNL1 (attractin-like 1) and SAMD4A (sterile alpha motif domain containing 4A) is linked to the transcriptional downregulation of both linear mRNA and the hosted circRNA. Although some circRNAs regulate the linear transcript, we did not observe changes in TUSC3 mRNA levels upon TUSC3 circ104557 overexpression. Interestingly, we found circRNA-mediated regulation of target miRNAs and an in vivo growth inhibitory effect upon TUSC3 circ104557 transduction. Data mining for 5′-end CpG island methylation of TUSC3, ATRNL1, POMT1 and SAMD4A in cancer cell lines and primary tumors showed that the epigenetic defect was commonly observed among different tumor types in association with the diminished expression of the corresponding transcript. Our findings support a role for circRNA DNA methylation-associated loss in human cancer.