P44 Investigating the effects of YAP–TEAD inhibition on cancer-associated fibroblasts in cutaneous squamous cell carcinoma

Abstract Introduction and aims Cancer-associated fibroblasts (CAFs) are highly heterogeneous activated fibroblasts, which promote or supress tumour growth in different contexts. Yes-associated protein (YAP)–TEA domain (TEAD) signalling is often dysregulated in cancers, promoting proliferation, survival and migration. It has therefore become a target for therapeutic development; however, little work has been done to characterize YAP–TEAD signalling in CAFs or understand how inhibiting this pathway would affect their phenotype. Therefore, the aim of this project is to address this knowledge gap in cutaneous squamous cell carcinoma (cSCC) using isogenic fibroblast lines isolated in our lab from normal skin tissue, primary tumour, and recurrent tumour. Methods YAP–TEAD signalling pathway components were quantified in these fibroblast lines using Western blotting analysis, and the effects of three different TEAD inhibitors on proliferation of these cells were characterized using a CyQuant cell proliferation plate assay and immunofluoresence microscopy. These inhibitors allosterically inhibit YAP–TEAD binding, attenuating downstream transcription, and similar inhibitors are already in clinical trials for cancers. Results Single-cell RNA sequencing analysis of cSCC tumour and normal skin tissue had revealed increased mRNA expression of YAP and TEAD in CAFs vs. normal fibroblasts. While our Western blotting analysis revealed that their total protein levels trended lower in CAFs than normal fibroblasts, YAP nuclear localization (where it exerts downstream effects) was elevated. TEAD inhibition had little effect on cell number as assessed by CyQuant assay, but Ki67 positivity as a marker of proliferation was strongly reduced in CAFs by all three inhibitors with less effect in control fibroblasts. We are currently investigating how TEAD inhibition influences CAF–cancer cell crosstalk in colony forming and three-dimensional organotypic assays. Conclusions These results suggest YAP–TEAD signalling may be altered in CAFs compared with normal fibroblasts and TEAD inhibitors could differentially influence CAFs and normal fibroblasts, which is promising for their future clinical applications in cSCC.