Exploring The Molecular Mechanisms Underlying The Role Of Ion Channels In Cancer Development

This Ph.D. thesis focuses on elucidating the role of various ion channels in tumorigenic processes, particularly by investigating their non-canonical functions through the analysis of their interactome and the consequent modulation of associated signaling pathways. The investigation of ion channel interactomes using proximity labeling techniques based on biotin ligases, including BioID and TurboID, constituted the central approach connecting the three projects presented in this thesis. The first project aimed at investigating the molecular mechanisms by which the intermediate calcium-activated potassium channel (KCa3.1) orchestrates the development of pancreatic ductal adenocarcinoma (PDAC). TurboID-based proximity labeling analyses identified the involvement of KCa3.1 in cancer-relevant signaling cascades and its physical interaction with STIM1 and integrin β4 was further validated with co-immunoprecipitation. In addition, PDAC cells with downregulated KCa3.1 expression were characterized showing that the absence of the channel results in reduced proliferation, migration, alteration of mitochondrial physiology, and extensive gene remodeling. Furthermore, in vivo orthotopic experiments showed a significant reduction in PDAC tumor growth. These findings suggest that KCa3.1 acts not only as an ion-conducting pore but also as a signaling hub in PDAC. In the second project a similar approach was employed to explore the non-canonical functions of the voltage-gated potassium channel Kv1.3 in the context of cancer. BioID analysis revealed that the Kv1.3 channel interacts with several cancer-related signaling pathways, particularly those linked to STAT3 transcription factor. Kv1.3 knockdown melanoma cells showed a remodeled gene expression, impaired proliferation in vitro, and resulted in significantly smaller tumors with reduced metastasis in vivo. This phenotype was shown to depend on mitochondrial ROS release following either Kv1.3 deletion or its pharmacological inhibition, which also led to reduced STAT3 phosphorylation, stabilization of the p53 tumor suppressor, a metabolic switch, and altered expression of Kv1.3 putative interactors previously identified through BioID. The final project aimed at investigating the biological and clinical relevance of the volume-regulated anion channel (VRAC) and its pore-forming subunit LRRC8A in cancer. Pan-cancer analyses revealed that high LRRC8A expression correlates with poor prognosis in several cancer types, including colorectal cancer, and is associated with cancer-related signaling cascades. We then focused on elucidating the role of LRRC8A/VRAC in the cancer context and performed functional studies in human colon cancer cell lines lacking LRRC8A, which exhibited impaired proliferation and altered transcriptional profiles. To gain more insight into the molecular mechanisms responsible for this phenotype, we mapped the interactome of the LRRC8A protein using BioID proximity labeling. This analysis allowed us to identify novel LRRC8A putative interactors of the channel and to define, based on channel interactors, the contribution of VRAC to different biological processes, including cell-cell junctions and cytoskeletal organization, calcium homeostasis, and Rho GTPase signaling, suggesting that LRRC8A/VRAC acts as a regulator of cancer-related cellular processes. In conclusion, this thesis provides new insights into the non-canonical functions of ion channels in cancer, highlighting their roles as signaling hubs and potential therapeutic targets. The use of proximity labeling techniques proved to be a powerful tool in uncovering novel protein-protein interactions and signaling pathways associated with these ion channels in the context of cancer.