ECM‐Stiffness Mediated Persistent Fibroblast Activation Requires Integrin and Formin Dependent Chromatin Remodeling

Transient activation of fibroblasts into contractile myofibroblasts is essential for extracellular matrix (ECM) production and remodeling during wound healing and tissue regeneration. While ECM-dependent mechanisms mediating transient activation is well studied, how fibroblasts switch from transient to a persistently activated state and drive fibrosis and aberrant tissue repair in diseases such as cancer is less understood. Here, we show that human cancer-associated fibroblasts (CAFs) switch from transient to persistently activated states upon prolonged exposure to stiff ECMs and stiffness-dependent secreted factors. This switch is accompanied by activation of ECM-stiffness-dependent mechanotransduction pathways and changes in the nuclear architecture and its association with chromatin. Mechanistically, we identify two pathways required for this switch- ECM ligand binding and activation of β1 integrins smoothens the nuclear lamina during prolonged exposure and reduces lamin-chromatin contacts while in parallel, exposure to the stiff ECM activates the formin mammalian Diaphanous-related formin 2 (mDia2) and independent of alterations in the nuclear architecture alters lamin-chromatin coupling, likely through its role in assembling nuclear actin. Importantly, we find that blocking either pathway prevents persistent myofibroblast activation, which is rescued by inhibition of histone deacetylases, indicating that dynamic chromatin modifications act downstream of these ECM-dependent pathways to maintain the persistently activated state. These findings link integrin-based ECM sensing to chromatin remodeling and fibroblast memory, with implications for stromal plasticity in the tumor microenvironment.