STEM-26. BLOOD-TUMOR BARRIER IS COMPOSED OF MECHANOSENSING TUMOR CELLS THAT MASK THERAPEUTIC VULNERABILITY

Two major obstacles in brain cancer treatment are the blood-tumor barrier (BTB), which restricts delivery of most therapeutic agents, and the quiescent brain tumor-initiating cells (BTICs), which evade cell cycle-targeting chemotherapy. Mechanosensation, the transduction of mechanical cues into cellular signaling, underlies physiological processes such as touch, pain, proprioception, hearing, respiration, epithelial homeostasis, and vascular and lymphatic development. We report that medulloblastoma (MB) BTICs are mechanosensing, a property conferred by force-activated ion channel Piezo2. In contrast to the prevailing view that astrocytes function as a physical barrier in BTB, BTICs project endfeet to ensheathe capillaries. MB develops a tissue stiffness gradient as a function of distance to capillaries. Piezo2 senses substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion at BTIC growth cones, which allow BTICs to mechanically interact with their substrate and sequester β-Catenin to prevent WNT/β-Catenin signaling in BTICs. Our single cell analysis uncovers a two-branched BTIC trajectory that progresses from a deep quiescent state to two cycling states. Tumor cell-specific Piezo2 knockout reverses the off-on WNT/β-Catenin signaling states in BTICs and endothelial cells, collapses the BTB, reduces quiescence depth of BTICs, and markedly enhances MB response to chemotherapy. Our study reveals that BTICs co-opt astrocytic mechanism to contribute to the BTB and provides the first evidence that BTB depends on mechanochemical signaling to mask tumor chemosensitivity. Targeting Piezo2 addresses BTB and BTIC properties that underlie therapy failures in brain cancer.

Fibronectin: Molecular Structure, Fibrillar Structure and Mechanochemical Signaling

The extracellular matrix (ECM) plays a key role as both structural scaffold and regulator of cell signal transduction in tissues. In times of ECM assembly and turnover, cells upregulate assembly of the ECM protein, fibronectin (FN). FN is assembled by cells into viscoelastic fibrils that can bind upward of 40 distinct growth factors and cytokines. These fibrils play a key role in assembling a provisional ECM during embryonic development and wound healing. Fibril assembly is also often upregulated during disease states, including cancer and fibrotic diseases. FN fibrils have unique mechanical properties, which allow them to alter mechanotransduction signals sensed and relayed by cells. Binding of soluble growth factors to FN fibrils alters signal transduction from these proteins, while binding of other ECM proteins, including collagens, elastins, and proteoglycans, to FN fibrils facilitates the maturation and tissue specificity of the ECM. In this review, we will discuss the assembly of FN fibrils from individual FN molecules; the composition, structure, and mechanics of FN fibrils; the interaction of FN fibrils with other ECM proteins and growth factors; the role of FN in transmitting mechanobiology signaling events; and approaches for studying the mechanics of FN fibrils.

Fibronectin: Molecular Structure, Fibrillar Structure, and Mechanochemical Signaling

The extracellular matrix (ECM) plays a key role as both structural scaffold and regulator of cell signal transduction in tissues. In times of ECM assembly and turnover, cells upregulate assembly of the ECM protein, fibronectin (FN). FN is assembled by cells into viscoelastic fibrils that can bind upward of 40 distinct growth factors and cytokines. These fibrils play a key role in assembling a provisional ECM during embryonic development and wound healing. Fibril assembly is also often upregulated during disease states, including cancer and fibrotic diseases. FN fibrils have unique mechanical properties, which allow them to alter mechanotransduction signals sensed and relayed by cells. Binding of soluble growth factors to FN fibrils alters signal transduction from these proteins, while binding of other ECM proteins, including collagens, elastins, and proteoglycans, to FN fibrils facilitates the maturation and tissue specificity of the ECM. In this review, we will discuss the assembly of FN fibrils from individual FN molecules; the composition, structure, and mechanics of FN fibrils; the interaction of FN fibrils with other ECM proteins and growth factors; the role of FN in transmitting mechanobiology signaling events; and approaches for studying the mechanics of FN fibrils.

Asymmetric Contraction of Adherens Junctions arises through RhoA and E-cadherin feedback

Tissue morphogenesis often arises from the culmination of discrete changes in cell-cell junction behaviors, namely ratcheted junction contractions that lead to collective cellular rearrangements. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically as one highly motile vertex contracts toward a relatively less motile tricellular vertex. The underlying mechanisms driving asymmetric vertex movement remains unknown. Here, we use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. We find that both local and global RhoA activation leads to increases in junctional tension, thereby facilitating vertex motion. RhoA activation occurs in discrete regions along the junction and is skewed towards the less-motile vertex. At these less-motile vertices, E-cadherin acts as an opposing factor to limit vertex motion through increased frictional drag. Surprisingly, we uncover a feedback loop between RhoA and E-cadherin, as regional optogenetic activation of specified junctional zones pools E-cadherin to the location of RhoA activation. Incorporating this circuit into a mathematical model, we find that a positive feedback between RhoA-mediated tension and E-cadherin-induced frictional drag on tricellular vertices recapitulates experimental data. As such, the location of RhoA determines which vertex is under high tension, pooling E-cadherin and increasing the frictional load at the tricellular vertex to limit its motion. This feedback drives a tension-dependent intercellular "clutch" at tricellular vertices which stabilizes vertex motion upon tensional load.

The “Stressful” Life of Cell Adhesion Molecules: On the Mechanosensitivity of Integrin Adhesome

Cells have evolved into complex sensory machines that communicate with their microenvironment via mechanochemical signaling. Extracellular mechanical cues trigger complex biochemical pathways in the cell, which regulate various cellular processes. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes, also known as the integrin adhesome, that link the extracellular matrix (ECM) to the actin cytoskeleton, and are part of powerful intracellular machinery orchestrating mechanotransduction pathways. As forces are transmitted across FAs, individual proteins undergo structural and functional changes that involve a conversion of chemical to mechanical energy. The local composition of early adhesions likely defines the regional stress levels and determines the type of newly recruited proteins, which in turn modify the local stress distribution. Various approaches have been used for detecting and exploring molecular mechanisms through which FAs are spatiotemporally regulated, however, many aspects are yet to be understood. Current knowledge on the molecular mechanisms of mechanosensitivity in adhesion proteins is discussed herein along with important questions yet to be addressed, are discussed.

Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell–ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression.