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It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.

The ability to rapidly respond to applied force underpins cell/tissue homeostasis. This response is mediated by mechanotransduction pathways that regulate remodeling and tension of the actomyosin cytoskeleton to counterbalance external forces. Enhanced extracellular matrix tension hyper-activates mechanotransduction and characterizes diseased states such as cancer, but is also required for normal epidermal regeneration. While the impact of extracellular matrix tension on signaling and cell biology are well appreciated, that of acute compressive force is under-studied. We show here that acute compressive force applied to cells and tissues in a native 3-dimensional context elevates RHOA-GTP levels and increases regulatory myosin phosphorylation, actomyosin contractility and tension via ROCK. In consequence, cell proliferation was increased, as was the expression of regulators of epithelial-mesenchymal transition. Pharmacological inhibition of ROCK abrogated myosin phosphorylation, but not RHOA activation. Our results strongly suggest that acute compressive stress impairs cellular homeostasis in a RHO/ROCK-dependent manner, with implications for disease states such as cancer. Refereed/Peer-reviewed

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechanotransduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano- reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3 zeta, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3 zeta(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3 zeta deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-33 zeta with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3 zeta, while cutaneous SCCs had reduced 14-3-3 zeta. These results reveal a novel 14-3-3 zeta-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. Refereed/Peer-reviewed