Cells perceive and respond to mechanical stimuli in their environment through a process known as mechanotransduction, which involves converting physical cues into biological signals. Recently, it has become clear that mechanical forces play an important role in cancer initiation and progression. However, our understanding of how these mechanical signals are translated into cellular biochemical changes remains incomplete. While mechanical cues such as extracellular matrix (ECM) stiffness and cell density are increasingly recognized as key regulators of cellular behaviour, their influence on post-transcriptional processes like alternative splicing (AS) remains poorly understood. This thesis explores the mechanosensitive regulation of alternative splicing, focusing on the splicing regulator PTBP1 and its downstream targets, particularly NUMB, in mediating cellular responses to mechanical cues. Our findings validate a proteomic screening approach that identified mechanosensitive candidates regulated by ECM stiffness, cell density, and actomyosin contractility. RNA sequencing revealed substantial changes in alternative splicing events driven by ECM stiffness and cell density, demonstrating that these mechanical stimuli orchestrate transcriptomic plasticity.
ECM stiffness was found to modulate AS patterns of key cytoskeletal and signalling genes, while high cell density reduced PTBP1’s nuclear localization, altering its splicing activity. A notable discovery is the regulation of PTBP1 subcellular localization by cell density, which influences the alternative splicing of NUMB. Specifically, we show that the inclusion of exon 9 (+E9) in NUMB generates an isoform that promotes proliferation, enabling cells to override contact inhibition of proliferation (CIP) to a certain extent. Conversely, the exclusion of exon 9 (ΔE9) produces an isoform that enforces CIP, supporting a non-proliferative state. This duality underscores the pivotal role of NUMB splicing in balancing proliferative and quiescent cellular states in response to density-related cues. Our data highlight the central role of NUMB+E9 in driving oncogenic phenotypes, such as its ability to override CIP under high-density conditions. The results suggest that PTBP1-mediated splicing regulation of NUMB integrates mechanical signals with post-transcriptional control mechanisms, linking cytoskeletal dynamics and transcriptional programs to cellular adaptation. This regulation may have significant implications for cancer progression, where contact inhibition is often disrupted, and provides a potential avenue for therapeutic intervention. By advancing our understanding of how mechanical cues regulate alternative splicing, this work establishes a foundation for targeting splicing events in diseases associated with disrupted mechanotransduction, including cancer and fibrosis.
