(C-85) Substrate Stiffness Regulates Focal Adhesion Kinase Alternative Splicing in Breast Cancer

Introduction:: Altered alternative splicing is considered as a hallmark of cancer. In fact, alternatively spliced protein variants can differentially influence cellular processes such as cell migration and invasion. We have shown previously that the increased matrix stiffness in tumors regulated 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 alternatively spliced isoforms that promote tumor progression. In this context, we hypothesized that matrix stiffness regulation of alternative splicing could regulate the expression of FAK spliced variants and influence tumor progression. Here, we investigated how matrix stiffness regulates alternative splicing of FAK.


Materials and Methods:: Aggressive MDA-MB-231, low invasive MCF7 breast cancer cell lines and non-tumorigenic MCF10a cells were cultured on collagen-coated polyacrylamide (PA) substrates with stiffness ranging from 0.5 to 30kPa or glass. RNAseq was performed using Oxford MinION long read RNA sequencing. Stiffness-mediated expression of the FAK splice variants mRNA was validated using RT-PCR. GFP-tagged FAK isoforms were introduced in MDA-MB-231 cells using lentiviral vectors and observed live using confocal microscopy technique. FAK splice variants expression in breast cancer patients was analyzed in publicly available RNA-seq data from The Cancer Genome Atlas project (TCGA). YAP signature is a gene signature elaborated using 14 different YAP-driven transcriptional target genes.


Results, Conclusions, and Discussions:: 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 (FAK^ΔE4) in both MDA-MB-231 and MCF10a cells. RT-PCR data from in vitro experiments confirmed that FAK ΔE4 expression was indeed modulated by substrate stiffness. Of note, FAK^ΔE4 expression at these peak stiffnesses was reduced upon inhibition of cell contractility. Interestingly, the FAK exon 4 skipping events exhibited a biphasic relationship with substrate stiffness in MDA-MB-231, MCF7 and MCF10a cells. Moreover, the ratio FAK^ΔE4 reached a maximum at 10kPa in MDA-MB-231 cells whereas the maximum was reached at around 5kPa in MCF7 cells and 1kPa in MCF10a cells (Fig. 1A). To investigate FAK^ΔE4 clinical relevance, we characterized the FAK ^ΔE4 expression as function of the tumor stage as a proxy of tumor stiffness in patients. Curiously, FAK^ΔE4 expression followed a biphasic-like curve in the normal matched tissue and in less aggressive tumor stage, while the more advanced stage exhibited high levels of exon 4 skipping. Furthermore, we quantified FAK^ΔE4 expression as function of YAP signature, a more precise proxy of tumor stiffness, in patients. Again, FAK^ΔE4 expression followed a biphasic-like curve according to the YAP signature (Fig. 1B). The biphasic-like FAK^ΔE4 expression in patients intriguingly correlates with observed FAK^ΔE4 expression in the three cell lines. Furthermore, we found that patients with higher proportions of FAK^ΔE4 had significantly worst survival odds than patients with average or low FAK^ΔE4 expression. When overexpressed in MDA-MB-231 cells, FAK^ΔE4 led to the formation of fewer and larger focal adhesion sites (Fig. 1C and D).

Our data revealed that FAK alternative splicing is regulated by matrix stiffness in MDA-MB-231, MCF7 and MCF10a cells in a biphasic manner. Moreover, the stiffness at which the maximum FAK^ΔE4 expression is observed is higher in the more invasive MDA-MB-231 cells. In addition, a biphasic-like expression of FAK^ΔE4 is also observed in patient tissues. Overall, our results shows that FAK splicing is modulated by tumor mechanics, raising the possibility of stiffness-correlated alternative splicing events playing a role in FAK-mediated mechanoregulation.

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