Dynamic regulation of p53 signaling and chemoresistance in three-dimensional (3D) cancer models

Cellular behaviors in tissues/organs critically depend on the three-dimensional (3D) architectures and microenvironments, and cannot be recapitulated by traditional 2D cell lines. The development of 3D spheroid and organoid models have enabled the study of 3D-specific cellular mechanisms using human-derived cells, providing a much needed alternative complementary to animal models. One important research area where cellular
behaviors differ significantly in 3D vs. 2D is anticancer drug response of solid tumors. Extensive effort has been made to unravel 3D-specific drug mechanisms and targets using 3D culture tumor models. However, our quantitative and mechanistic
understanding of the 3D-specific tumor signaling response to drug treatment is still limited, especially at the dynamic level.

We previously developed single-cell microscopy assays and mathematical models to characterize drug responses mediated by the p53 signaling pathway in 2D cancer cells. Given the significant 3D vs. 2D variations of cancer, we set out to further examine how
p53 mediated-drug response dynamics are altered in 3D. We found drug-induced p53 signaling and subsequent cancer cell death were significantly attenuated in 3D cancer spheroids. Intriguingly, this signaling attenuation was not due to the well-known decrease in drug uptake, but unique 3D-specific activation of an upstream kinase, Casein Kinase 1a (CK1a), which inhibited p53 upregulation and p53-mediated cell death response. Moreover, p53 induction in 3D exhibited a long lag time (12-20 hours) upon drug treatment, which was not observed in 2D cells. Building on these novel data, in this proposed project we will combine 3D single-cell imaging and computational analysis of
p53 pathway models to elucidate the quantitative mechanism underlying the 3D-specific signaling dynamics of p53 that we uncovered and the resulting chemoresistance in 3D cancer spheroid models. The key and previously unresolved questions that our study will address include: (1) spatiotemporal characteristics of drug-induced p53 dynamics in 3D and their regulation by the 3D-specific signaling activity of CK1a; (2) cancer cell typedependent variations of the p53 signaling dynamics and CK1a-p53 control in 3D; and (3) p53 pathway motif and rate-limiting parameters that govern the observed 3D
signaling responses.

Overall, results from our proposed study will provide novel insight into the dynamic control over p53 signaling in 3D and reveal new mechanistic angles to identify combinatorial targets to combat chemoresistance in solid tumors. The quantitative methodology that we develop, integrating 3D single-cell microscopy and mathematical modeling of pathway dynamics, is readily extendable for study of other single-cell behaviors in 3D.