Abstract 1812 Vinculin Expression and Substrate Stiffness Regulate Proliferation, Activin-A Secretion and Mechanosensory Gene Expression in Mesenchymal Stem Cells Article Swipe

Mesenchymal stem cells (MSCs) are multipotent fibroblast-like stem cells that can differentiate into muscle, bone, cartilage and fat cells. In response to injury, MSCs home to wounded tissue and secrete growth factors, cytokines, extracellular vesicles and matrix proteins that mediate a host of physiological processes including cell division, immune-regulation and angiogenesis. However, MSCs can also be exploited by cancer cells to potential metastasis and therapy resistance in solid tumors. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is a cytoskeleton-associated kinase that has been shown to integrate growth factor and ECM-integrin signaling crosstalk in fibroblast cell types and is part of the integrin adhesion complexes (IACs). Through combined mass spectrometry, interactomics and clinical database mining methods, we discovered that localization of Vinculin (VCL), a focal adhesion mechanotransduction protein, is depleted from MSC IACs when PEAK1 is silenced. While MSCs are known to invoke senescence associated secretory phenotypes (SASPs), little is known about whether cell substrate stiffness may regulate the expression of SASP and mechanosensory genes, or how PEAK1 and/or VCL may control these effects. Here, we first established optimal conditions for primary MSC spreading and demonstrated that activin-A stimulation potentiates focal adhesion length. Stable VCL knockdown (KD) in primary MSCs decreased cell proliferation and activin-A secretion. Interestingly, mining Cancer BioPortal data revealed that total VCL and phospho-VCL (S159) levels are elevated in breast cancer patients expressing PEAK1 and INHBA (activin-A) transcripts. Since S159 is a predicted NIMA-related kinase 2 (NEK2) substrate, we evaluated whether phospho-serine levels in VCL were altered in PEAK1 knockdown MSCs or MSCs treated with a NEK2 inhibitor. Notably, we observed that both PEAK1 and NEK2 signaling are necessary for serine phosphorylation in VCL. In agreement with these data, we also observed diminished focal adhesion length in MSCs treated with the PLK1/NEK2 inhibitor volasertib. Using silicon substrates that vary in stiffness from 0.2 – 64 kPa, we observed that expression levels of CD73, ACTA2, VEGF and NAT10 were maximal when primary MSCs were exposed to medium substrates (8 kPa). Taken together, this work identifies PEAK1/NEK2 as a mediator of VCL and substrate stiffness dependencies in primary MSCs that control proliferation, activin-A secretion and gene expression, and which may be leveraged to improve tissue regeneration strategies and treatment of cancers.