Single-cell analysis of p53 transitional dynamics unravels stimulus- and cell type-dependent signaling output motifs

Abstract

Background: To understand functional changes of complex biological networks, mathematical modeling of network topologies provides a quantitative measure of the way biological systems adapt to external stimuli. However, systemic network topology-based analysis often generates conflicting evidence depending on specific experimental conditions, leading to a limited mechanistic understanding of signaling networks and their differential dynamic outputs, an example of which is the regulation of p53 pathway responses to different stress stimuli and in variable mammalian cell types. Here, we employ a network motif approach to dissect key regulatory units of the p53 pathway and elucidate how network activities at the motif level generate context-specific dynamic responses. Results: By combining single-cell imaging and mathematical modeling of dose-dependent p53 dynamics induced by three chemotherapeutics of distinct mechanism-of-actions, including Etoposide, Nutlin-3a and 5-fluorouracil, and in five cancer cell types, we uncovered novel and highly variable p53 dynamic responses, in particular p53 transitional dynamics induced at intermediate drug concentrations, and identified the functional roles of distinct positive and negative feedback motifs of the p53 pathway in modulating the central p53-Mdm2 negative feedback to generate stimulus- and cell type-specific signaling responses. The mechanistic understanding of p53 network dynamics also revealed previously unknown mediators of anticancer drug actions and phenotypic variations in cancer cells that impact drug sensitivity. Conclusions: Our results demonstrate that transitional dynamics of signaling proteins such as p53, activated at intermediate stimulus levels, vary the most between the dynamic outputs of different generic network motifs and can be employed as novel quantitative readouts to uncover and elucidate the key building blocks of large signaling networks. Our findings also provide new insight on drug mediators and phenotypic heterogeneity that underlie differential drug responses.