Tissue stiffness regulates serine/arginine-rich protein-mediated splicing of the extra domain B-fibronectin isoform in tumors

Alternative splicing of proteins gives rise to different isoforms that play a crucial role in regulating several cellular processes. Notably, splicing profiles are altered in several cancer types, and these profiles are believed to be involved in driving the oncogenic process. Although the importance of alternative splicing alterations occurring during cancer is increasingly appreciated, the underlying regulatory mechanisms remain poorly understood. In this study, we use both biochemical and physical tools coupled with engineered models, patient samples, and a murine model to investigate the role of the mechanical properties of the tumor microenvironment in regulating the production of the extra domain-B (EDB) splice variant of fibronectin (FN), a hallmark of tumor angiogenesis. Specifically, we show that the amount of EDB-FN produced by endothelial cells increases with matrix stiffness both in vitro and within mouse mammary tumors. Matrix stiffness regulates splicing through the activation of serine/arginine rich (SR) proteins, the splicing factors involved in the production of FN isoforms. Activation of the SR proteins by matrix stiffness and the subsequent production of EDB-FN are dependent on intracellular contractility and PI3K-AKT signaling. Notably, matrix stiffness-mediated splicing is not limited to EDB-FN, but also affects splicing in the production of PKC beta II and the VEGF 165b splice variant. Together, these results demonstrate that the mechanical properties of the microenvironment regulate alternative splicing and establish a previously unidentified mechanism by which cells can adapt to their microenvironment.