(610f) The Key Roles of P53 and YAP in Mediating Cell Survival in Confinement

Intravital microscopy has established that in vivo, cells reside in and migrate through confining 3D microenvironments. Although transient exposure to confinement can activate intracellular signaling pathways and alter the modes and mechanisms of cell migration, the effects of long-term confinement on cell behavior remain unknown.

To address this gap in knowledge, we developed a novel high-throughput cell confinement assay that enabled continuous monitoring of cell behavior in physiologically relevant confined microenvironments. This device was fabricated by combining standard multilayer photolithography and photopatterning. First, PDMS-based parallel microchannels of different widths or heights were created. These microchannels were then coated with collagen-type I using light-induced patterning. Cancerous (HT-1080 fibrosarcoma cells, MDA-MB-231 breast cancer cells) or non-cancerous cells (human Fibroblasts, human aortic smooth muscle cells) were introduced into the microchannels and cell behavior was examined over a period of three days. Using this assay, we investigated how long-term confinement affected cell survival and studied the roles of actomyosin contractility, nuclear envelope rupture, P53 and YAP in confinement-induced responses.

Our research showed that confining microchannels with dimensions of (W)idth x (H)eight :10 x 3 μm² triggered higher cell death rates (~ 30%) in HT-1080 cells, fibroblasts and smooth muscle cells compared to less restrictive microenvironments (W x H =10 x 10 μm²) or 2D-like environments (W x H =400 x 50 μm²)). This outcome was not due to nutrient deprivation since cell access to nutrients was confirmed by monitoring the diffusion of a fluorescent dye in these channels. Interestingly, MDA-MB-231 breast cancer cells, which carry a mutant P53 gene, maintained high viability in 30 μm² microchannels. To investigate the role of P53 in cell death, we employed a live cell reporter that measures P53-transcriptional activity. Our results showed that P53 activity increased in 30 μm² but not in 100 μm² microchannels. Inhibiting P53-dependent gene expression using pifithrin-α or P53 knockdown reduced cell death rates in confinement. Further investigation showed that confinement promoted P53 hyperactivation by increasing nuclear envelope rupture, which in turn triggered DNA damage responses. Inhibition of actomyosin contractility via Y-27632, which reduced nuclear rupture, also decreased P53 activation and cell death. Given the fact that ~ 70% of cells were capable of surviving in confinement, we hypothesized that there should be another mechanism limiting p53 activation and enabling cell survival. In line with this, we found that HT-1080 cells that survived in 30 μm² microchannels had lower levels of YAP in the nucleus compared to those that died. Additionally, we showed that HT-1080 cells and fibroblasts treated with siYAP1 experienced fewer nuclear rupture events in confinement compared to scramble control cells. YAP1 knockdown also reduced confinement-induced P53 activation and cell death. In contrast, LATS2 knockdown, which increased YAP-dependent gene expression, exacerbated cell death in confining microchannels.

In conclusion, we used advanced bioengineering tools, molecular biology and imaging techniques to show that 3D confinement negatively affects cell survival by activating P53. Also, we demonstrated the significance of YAP in mediating cell adaptation to confinement. Our work suggests that tuning the nuclear levels of YAP may allow us to promote or inhibit cell survival in confining microenvironments.