Substrate stiffness regulates focal adhesion kinase alternative splicing in breast cancer

Altered alternative splicing is considered as a hallmark of cancer. In fact, alternatively splice protein variants can differentially influence cellular processes such as cell migration and invasion. We have shown previously that increased matrix stiffness in tumors regulates alternative splicing events through increased cell contractility. Interestingly, the focal adhesion kinase (FAK), a protein central to matrix stiffness mechanosensing in focal adhesion, has several isoforms that promote tumor progression. In this context, we hypothesized stiffness-mediated alternative splicing could regulate the expression of FAK splice variants and influence tumor progression. We first investigated FAK mRNA splicing as a function of matrix stiffness using RNAseq. Our data identified stiffness-mediated alternative splicing events in FAK exon 4 exclusion (FAK4-). RT-PCR data from in vitro experiments confirmed FAK4- expression was modulated by substrate stiffness, reaching a maximum at around 1, 5 and 10 kPa in MCF10a, MCF7 and MDA-MB-231 cells respectively. Of note, FAK4- expression at these stiffnesses was reduced upon contractility inhibition. To investigate FAK4- clinical relevance, we characterized FAK4- expression as function of the tumor stage as a proxy of tumor stiffness in patients (TCGA). Notably, we found that patients with higher proportions of FAK4- had significantly worst survival odds than patient with lower FAK4- expression. Critically, FAK4- overexpression resulted in fewer but larger FAs, suggesting a shift in FA dynamics. Taken together, our data revealed FAK alternative splicing is regulated by matrix stiffness both in patients and in vitro. Moreover, the stiffness at which the maximum FAK4- expression is observed is higher in the more invasive MDA-MB-231 cells. Overall, our results show FAK splicing is modulated by tumor mechanics, raising the possibility of an existing interplay between stiffness-driven alternative splicing and FAK-mediated mechanoregulation.