A Novel ECM Stiffness-driven FAK isoform Tunes Cell Mechanosensing in Breast Cancer

Introduction: : The stiffness of the extracellular matrix (ECM) progressively increases during tumor progression. In breast cancer, the ECM stiffens from 1 kPa to over 30 kPa in advanced tumor stages. Tumor cells are able to sense and respond to mechanical changes of their surrounding matrix through mechanotransduction, a process that converts mechanical cues to biochemical cues. Notably, increased ECM stiffness promotes biological processes such as cell migration, invasion and metastasis. Our previous work uncovered that ECM stiffness regulates alternative splicing. Alternative splicing can alter protein structure and functions. Interestingly, the focal adhesion kinase (FAK), a central mediator of mechanosensing in focal adhesions, has several alternatively spliced isoforms that promote tumor progression. In this context, we hypothesized that stiffness-mediated alternative splicing could regulate the expression of a FAK splice variant, and influence tumor cell behavior through altered mechanosensing. Notably, we identified a previously uncharacterized stiffness-regulated FAK isoform. Here, we investigated the impact of this stiffness-regulated FAK isoform on focal adhesion mediated-mechanosensing and cell migration leading to cancer metastasis.

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 substrates with stiffnesses ranging from 0.5 to 30 kPa 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. FAK splice variants expression in breast cancer patients was analyzed in publicly available RNA-seq data from The Cancer Genome Atlas project (TCGA). GFP-tagged FAK isoforms (canonical FAK and FAK∆e4) were introduced in MDA-MB-231 cells using lentiviral vectors. GFP-positive cells were imaged to assess cell spreading using epifluorescence microscopy. Live imaging was used to track cell migration every 15 minutes for 12 hours. FRAP experiment and YAP nucleus/cytoplasm ratio assay were performed using confocal microscopy. Focal adhesion dynamics and maturation assays were obtained using TIRF microscopy technique.

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∆e4) in both MDA-MB-231 and MCF10a cells, confirmed by RT-PCR. Interestingly, FAK∆e4 expression exhibits a biphasic relationship with substrate stiffness in MDA-MB-231, MCF7 and MCF10a cells with a maximum expression at 10 kPa in MDA-MB-231 cells, 5 kPa in MCF7 cells and 1 kPa in MCF10a cells (Fig.1A). To investigate FAK∆e4 clinical relevance, we found that patients with high FAK∆e4 expression had significantly worst survival odds (Fig.1B). We generated MDA-MB-231 cell lines overexpressing GFP-tagged canonical FAK (cFAK) and GFP-tagged FAK∆e4. To study the impact of FAK∆e4 on mechanotransduction, we cultured the cells on a broad range of stiffnesses (from 0.5 kPa to 50 kPa). We observed that FAK∆e4 downshifts optimal ECM stiffness for cell spreading from 10 kPa to 5 kPa. FAK∆e4 downshifts optimal 2D cell migration from 10-18 kPa to 5 kPa. Also, FAK∆e4 expression decreases cell migration speed at 10 kPa and higher stiffnesses (Fig.1C). Similarly, FAK∆e4 downshifts optimal ECM stiffness for YAP nuclear translocation from 10 kPa to 5 kPa (Fig.1D). Using stiffness gradient gels ranging from 1 kPa to 30 kPa, FAK∆e4 expression reduces optimal ECM stiffness for cell durotaxis from 18 kPa to 10 kPa (Fig.1E). In vivo experiments are ongoing to investigate whether FAK∆e4 has a role in tumor metastasis.
We then investigated the impact of FAK∆e4 on focal adhesion structures. Using fluorescence recovery after photobleaching (FRAP) experiment, we observed that FAK∆e4 strongly weakens focal adhesion turnover (Fig.2A). Using live TIRF experiment, we found that FAK∆e4 decreases focal adhesion assembly and disassembly speeds compared to cFAK (Fig.2B-C). Finally, we showed that FAK∆e4 decreases colocalization with vinculin and zyxin compared with cFAK, while talin was not impacted, indicating impaired focal adhesion maturation (Fig.2D-E).
Overall, our results shows that FAK∆e4 expression is modulated by substrate mechanics and, in turn, FAK∆e4 regulates global focal adhesion dynamics and stiffness-driven cell mechanosensing. Additionally, the newly discovered FAK isoform has a plausible clinical relevance considering the ongoing clinical trials for FAK inhibitors.

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