Mechanisms underlying divergent relationships between Ca2+ and YAP/TAZ signalling.

Yes-associated protein (YAP) and its homologue TAZ are transducers of several biochemical and biomechanical signals, integrating multiplexed inputs from the microenvironment into higher level cellular functions such as proliferation, differentiation and migration. Emerging evidence suggests that Ca2+ is a key second messenger that connects microenvironmental input signals and YAP/TAZ regulation. However, studies that directly modulate Ca2+ have reported contradictory YAP/TAZ responses: in some studies, a reduction in Ca2+ influx increases the activity of YAP/TAZ, while in others, an increase in Ca2+ influx activates YAP/TAZ. Importantly, Ca2+ and YAP/TAZ exhibit distinct spatiotemporal dynamics, making it difficult to unravel their connections from a purely experimental approach. In this study, we developed a network model of Ca2+ -mediated YAP/TAZ signalling to investigate how temporal dynamics and crosstalk of signalling pathways interacting with Ca2+ can alter the YAP/TAZ response, as observed in experiments. By including six signalling modules (e.g. GPCR, IP3-Ca2+ , kinases, RhoA, F-actin and Hippo-YAP/TAZ) that interact with Ca2+ , we investigated both transient and steady-state cell response to angiotensin II and thapsigargin stimuli. The model predicts that stimuli, Ca2+ transients and frequency-dependent relationships between Ca2+ and YAP/TAZ are primarily mediated by cPKC, DAG, CaMKII and F-actin. Simulation results illustrate the role of Ca2+ dynamics and CaMKII bistable response in switching the direction of changes in Ca2+ -induced YAP/TAZ activity. A frequency-dependent YAP/TAZ response revealed the competition between upstream regulators of LATS1/2, leading to the YAP/TAZ non-monotonic response to periodic GPCR stimulation. This study provides new insights into underlying mechanisms responsible for the controversial Ca2+ -YAP/TAZ relationship observed in experiments. KEY POINTS: YAP/TAZ integrates biochemical and biomechanical inputs to regulate cellular functions, and Ca2+ acts as a key second messenger linking cellular inputs to YAP/TAZ. Studies have reported contradictory Ca2+ -YAP/TAZ relationships for different cell types and stimuli. A network model of Ca2+ -mediated YAP/TAZ signalling was developed to investigate the underlying mechanisms of divergent Ca2+ -YAP/TAZ relationships. The model predicts context-dependent Ca2+ transient, CaMKII bistable response and frequency-dependent activation of LATS1/2 upstream regulators as mechanisms governing the Ca2+ -YAP/TAZ relationship. This study provides new insights into the underlying mechanisms of the controversial Ca2+ -YAP/TAZ relationship to better understand the dynamics of cellular functions controlled by YAP/TAZ activity.

Macromolecular crowding tunes 3D collagen architecture and cell morphogenesis.

Collagen I is the primary extracellular matrix component of most solid tumors and influences metastatic progression. Collagen matrix engineering techniques are useful for understanding how this complex biomaterial regulates cancer cell behavior and for improving in vitro cancer models. Here, we establish an approach to tune collagen fibril architecture using PEG as an inert molecular crowding agent during gelation and cell embedding. We find that crowding produces matrices with tighter fibril networks that are less susceptible to proteinase mediated degradation, but does not significantly alter matrix stiffness. The resulting matrices have the effect of preventing cell spreading, confining cells, and reducing cell contractility. Matrix degradability and fibril length are identified as strong predictors of cell confinement. Further, the degree of confinement predicts whether breast cancer cells will ultimately undergo individual or collective behaviors. Highly confined breast cancer cells undergo morphogenesis to form either invasive networks reminiscent of aggressive tumors or gland and lobule structures reminiscent of normal breast epithelia. This morphological transition is accompanied by expression of cell-cell adhesion genes, including PECAM1 and ICAM1. Our study suggests that cell confinement, mediated by matrix architecture, is a design feature that tunes the transcriptional and morphogenic state of breast cancer cells.

3D collagen architecture induces a conserved migratory and transcriptional response linked to vasculogenic mimicry.

The topographical organization of collagen within the tumor microenvironment has been implicated in modulating cancer cell migration and independently predicts progression to metastasis. Here, we show that collagen matrices with small pores and short fibers, but not Matrigel, trigger a conserved transcriptional response and subsequent motility switch in cancer cells resulting in the formation of multicellular network structures. The response is not mediated by hypoxia, matrix stiffness, or bulk matrix density, but rather by matrix architecture-induced ß1-integrin upregulation. The transcriptional module associated with network formation is enriched for migration and vasculogenesis-associated genes that predict survival in patient data across nine distinct tumor types. Evidence of this gene module at the protein level is found in patient tumor slices displaying a vasculogenic mimicry (VM) phenotype. Our findings link a collagen-induced migration program to VM and suggest that this process may be broadly relevant to metastatic progression in solid human cancers.