Auriemma, Margherita (2025) The two-faced role of Syndesmos: riboregulation of DNA damage response. [Tesi di dottorato]

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Abstract

Breast cancer is the most frequently diagnosed cancer and the second leading cause of death in women. Two main breast cancer-associated genes, BRCA1 and BRCA2, have been identified, which can be exploited for therapy, being BRCA mutations synthetically lethal with PARP inhibitor treatment. Protein Syndesmos (SDOS, aka NUDT16L1 or TIRR) has been identified as a key regulator of 53BP1-mediated DNA damage response (DDR): its overexpression prevents 53BP1 function, leading to PARP inhibitor resistance in BRCA1-mutated tumors. However, SDOS is also an RNA-binding protein, which associates with polysomes and regulates protein synthesis. Remarkably, SDOS-mediated 53BP1 inhibition is decreased by SDOS-interacting RNAs, clearly indicating that the two SDOS functions are not separated. In support of this hypothesis, our preliminary observation obtained by the OOPs technique shows that global SDOS-RNA binding is reduced by exposure to etoposide. Among the transcripts bound by SDOS in unstressed conditions, we identified the selective autophagy regulator p62/SQSTM1. Polysome profiling coupled with RT-qPCR showed that SQSTM1 mRNA translation is enhanced upon SDOS knock-down. SQSTM1 mRNA translation is significantly reduced by etoposide in shSDOS cells, while sustained in nonsilencing controls, suggesting that SDOS acts as a translational repressor in resting conditions, to then release protected mRNA for stress-responsive translation. Interestingly, the protein SQSTM1 is also an SDOS interactor, suggesting a multiple-level regulation of SQSTM1 activity by SDOS. Indeed, by employing RIP assays, we show that SDOS expression also influences the riboregulation exerted by the small noncoding vault-RNAs on SQSTM1 activity. In fact, binding between the SQSTM1 protein and its riboregulators following etoposide is reduced, and autophagy is increased upon SDOS silencing. Etoposide treatment in MCF7 cells also induced a dramatic increase in ISG15 and IRF1, thus suggesting the presence of DNA damage-induced interferon response. Such increase is enhanced upon SDOS silencing, thereby confirming the capacity of low SDOS expressing cells to better respond to DNA damage. On the contrary, IL6 expression remains low upon etoposide treatment, suggesting that specifically the interferon pathway is active in this setting. According to this model, SDOS participates in the DDR at multiple levels by both translation regulation of transcripts encoding stress-responsive proteins, and riboregulation of their activity. These molecular mechanisms account at least in part for the clinical relevance of SDOS expression in breast tumors: high SDOS expression correlates with better patient survival and enhanced response to therapy. We propose a model in which SDOS overexpression has a positive impact on tumorigenesis by inducing genomic instability, while representing a vulnerability in advanced tumors undergoing chemotherapy for its inhibitory effects on DDR and pro-survival mechanisms like autophagy.

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