Investigating the roles of lncRNA TERRA in telomerase-to-ALT transition using cancer cell lines and multicellular organisms

Telomeres protect eukaryotic chromosomes from fusions and degradations and progressively shorten over generations due to the “end replication problem” in human somatic cells, leading to cell cycle arrest, senescence and ultimately cell death. To achieve replicative immortality, tumors elongate their telomeres in two possible ways: by reactivating telomerase, in about 85-90% of cancers, or through homologous recombination (HR) by the mechanism of alternative lengthening of telomeres (ALT), in about 10-15% of cancers. Even though anti-telomerase treatments are currently being developed, there is a number of tumors that can change from one mechanism to the other with a switch named telomerase-to-ALT, about which almost nothing is known to date. One of the ALT hallmarks is the presence of high levels of telomeric lncRNA TERRA, which is thought to play a role in HR during ALT. However, little is known about the involvement of TERRA in the onset of ALT. In this PhD project, we aim at investigating the roles of TERRA in ALT and during telomerase-to-ALT transition. In particular, we employed an ALT-positive human cancer cell line in which we downregulated TERRA through antisense oligonucleotide (ASO) and measured changes in the ALT phenotype. In addition, we employed C. elegans mutant strains to study the dynamics of TERRA in worms lacking telomerase and elongating their telomeres using an ALT-like mechanism. We also took advantage of a zebrafish brain cancer model that was characterized to be ALT-positive and expressing high TERRA levels. With this model, we performed micro-injections of ASO targeting TERRA with the aim of downregulating TERRA and studying its effects on the development of the tumor and ALT. The results obtained helped us to shed light on the involvement of TERRA in the ALT mechanisms both in a non-cancerous context using C. elegans and in cancer using human cells and the zebrafish model.