Exploring the role of the ESCRT-111 proteins in response to telomeric replication stress in ALT cancer cells

• Autor(a) principal: Justo, Inês Vaz Fernandes Teixeira
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/164719
• topic: ESCRT-III PROTEINS; TELOMERIC REPLICATION STRESS; ALT CANCER CELLS; Ciências Médicas

Abstract Due to the end-replication problem, normal somatic cells gradually shorten their telomeres at each cell division. This progressive shortening eventually triggers a DNA damage response (DDR), cellular senescence or apoptosis. Cancer cells must counteract telomere shortening to enable continuous cell division. These cells can elongate their telomeres by reactivating telomerase or by homology-directed repair (HDR) mechanisms, called Alternative Lengthening of Telomeres (ALT). ALT cells have distinct molecular features, such as accumulation of telomeres at ALT-associated PML bodies (APBs), and abundant extrachromosomal telomeric repeats (ECTRs) that includes partially single-stranded circles (C- and G-circles). ALT cancers perpetuate telomere maintenance based on telomeric replication stress and HDR pathways. This ALT-specific Telomeric Replication Stress (ATRS) must be carefully regulated, otherwise, it can result in cell cycle arrest and death when in excess. However, the full nature of the mechanisms underlying the response to exacerbated ATRS remains elusive. Preliminary data in the Azzalin lab showed that induction of ATRS by depletion of FANCM, a replication stress alleviator, results in the recruitment of several proteins to ALT-telomeric DNA, among them ESCRT-III proteins. Here, I explore the involvement of two subunits of the ESCRT-III complex, CHMP4B and CHMP7 in the response to telomeric replication stress in ALT cancer cells. In this work, I confirmed that both CHMPs are involved in the regulation of telomeric replication stress in ALT and telomerase-positive cells (U2OS and HOS, respectively). The findings show that CHMP4B and CHMP7 act as suppressors of ATRS and are not essential for cell cycle progression in U2OS. When CHMP4B and CHMP7 are depleted in U2OS, ATRS increases and, consequently, APB formation is induced. On the other hand, no alterations in C-circles levels are detected upon loss of CHMP4B and CHMP7, but the distribution of C-rich telomeric single-stranded DNA within the cytoplasm and the nucleus is perturbed. Thus, I propose that ESCRT-III complex may facilitate the clearance of by-products of telomere elongation from the nucleus, such as C-circles. On the other hand, CHMP4B induces telomeric replication stress in HOS cells through a mechanism that remains unidentified. Nevertheless, CHMP4B has a dual role towards telomeric replication stress depending on the existing telomere maintenance mechanism. Overall, these results suggest that CHMP4B and CHMP7 are implicated in the regulation of telomeric replication stress in ALT cells. Further studies should focus on elucidating the precise mechanisms through which CHMP4B and CHMP7 contribute to the regulation of telomeric replication stress in ALT and telomerase-positive cancer cells.