Abstract
EGF-dependent ERK signaling pulses influence whether a cell enters S phase.
Major finding: EGF-dependent ERK signaling pulses influence whether a cell enters S phase.
Clinical relevance: Targeted therapies modify ERK signaling frequency and amplitude in different ways.
Impact: Signal-response curves can indicate the level of pathway inhibition needed to block proliferation.
Quantitative analyses of oncogenic signaling pathways have the potential to uncover relationships between cellular signals and responses and the effect of therapeutic agents on signaling dynamics. Albeck and colleagues sought to define the relationship between EGF-dependent ERK activation and cellular proliferation in human cells at steady-state conditions to model chronic EGF signaling in cancer cells. ERK signaling and cellular proliferation were measured across a range of extracellular EGF concentrations in live cells with a fluorescence energy transfer–based ERK activity reporter, an ERK target that fluoresced upon phosphorylation, and a fluorescent geminin-based S-phase indicator. Intermittent pulses of high and low ERK activity were observed, with the frequency of the pulses dependent on the EGF concentration. At higher EGF concentrations, the pulses lasted longer and were closer together, resulting in a higher average amount of time in which ERK was “on.” Single-cell comparisons captured signaling dynamics that could not be observed by population-level analyses and indicated that this fraction of time in the ERK-on state, not the number of ERK signaling pulses, was a key determinant of whether a cell entered S phase. Interestingly, inhibitors of different parts of the pathway had different effects on ERK dynamics: EGFR inhibition with gefitinib reduced the frequency of ERK pulses, whereas MEK inhibition with PD0325901 affected the intensity, or amplitude, of the pulses. Plotting ERK pathway output against proliferation rate also predicted that at least 90% ERK inhibition would be necessary for a significant decrease in proliferation, which was validated in multiple cell lines and consistent with clinical findings. Together, these quantitative studies reveal a potential benefit for combined inhibition of multiple ERK pathway nodes and may provide a framework for evaluating the capacity of targeted agents to affect signaling output and cell fate.