Studio dei ruoli non-canonici della proteina di riparazione del DNA APE1 con un focus sul metabolismo dell'RNA e sulla secrezione tramite vescicole extracellulari utilizzando diversi modelli tumorali

APE1, traditionally known for its pivotal role in the Base Excision Repair (BER) pathway, exhibits non-canonical functions that extend beyond DNA repair. Indeed, APE1 has been implicated in the decay of oxidized and abasic RNA, and in miRNA processing, contributing to the post-transcriptional regulation of gene expression. Interestingly, APE1 overexpression in various cancer types suggests its potential role as a key player in tumorigenesis and chemoresistance, impacting the response of cancer cells to genotoxic stress due to its activity in DNA repair and in the regulation of genes involved in tumor progression. Notably, APE1 secretion through extracellular vesicles (EVs) and its high levels in cancer patients’ serum underscore its potential as a prognostic biomarker for several malignancies. Hence, understanding the non-canonical functions of APE1, with attention to its protein-protein interaction network, provides valuable insights into the complex molecular landscape of cancer. Therefore, this thesis investigated the following topics: APE1 in RNA damage response. The RNA nuclear exosome, along with its cofactor MTR4, is a critical component of RNA quality control. Since APE1 is also involved in RNA processing/decay, we wondered whether the interaction between APE1 and the nuclear exosome could be involved in the processing of RNA damaged by chemotherapeutic agents. Through the use of human cancer cell lines, we presented compelling evidence that APE1 actively participates in the elimination of damaged RNA induced by cisplatin (CDDP) and 5-fluorouracil (5-FU) treatments, and notably, in an MTR4-independent manner. Furthermore, we demonstrated that the interaction between APE1 and MTR4 is stimulated under CDDP and 5-FU treatments, and that the depletion of APE1 and MTR4 leads to R-loop formation, activating the DNA damage response (DDR) pathway. These findings shed light on the previously unexplored role of MTR4 in DDR, and underscore APE1 function in RNA quality control during genotoxic treatments, with potential implications in chemoresistance. APE1 in miRNA processing and sorting into EVs. Expanding our exploration into APE1 involvement in microRNA (miRNA) processing, we found several miRNAs, whose expression is dependent on APE1, that increase in response to genotoxic treatment in different cancer cell lines. Interestingly, these miRNAs have oncogenes and tumor-suppressor genes as targets, further supporting APE1 role in regulating cell responses to chemotherapy drugs in a transcriptional and post-transcriptional manner. Additionally, we showed that APE1 cooperates with its interacting protein hnRNPA2B1 for the sorting into EVs of specific miRNAs, including the oncogenic miRNA miR-1246. This discovery contributes to a novel post-transcriptional role for APE1 in miRNA secretion mechanisms and, possibly, miRNA quality control processes in the extracellular environment. Characterization of secreted APE1 (sAPE1) intracellular pathway. Building on revelations that sAPE1 is secreted through EVs in an enzymatically active form and is involved in the sorting of miRNAs in EVs, potentially contributing to chemoresistance and premetastatic niche formation, we focused on the secretory pathway of sAPE1, unknown so far, in response to genotoxic stress. The absence of an endoplasmic reticulum (ER)-targeting signal peptide on APE1, led us to explore the unconventional protein secretion (UCPS) pathway, highlighting the central role of endosomes and multivesicular bodies (MVBs) in this process. Notably, the same pathway is triggered in the secretion of sAPE1 by genotoxic stress, with CDDP and MMS inducing autophagy-based UCPS routes in cancer cell lines, thus offering new perspectives on the biological relevance of sAPE1 in the tumor context. Collectively, this thesis sheds new light on the many functions of APE1, that still have an undiscovered dark side, especially regarding its contribution to cancer development and progression.