Multi range ERK responses shape the proliferative trajectory of single cells following oncogenic induction

Aberrant oncogene activation can promote cellular transformation and tumorigenesis, enabling cancer cells to grow and proliferate uncontrollably. For instance, activating Ras or Raf mutations bypass the need of growth factor stimulation, resulting in constitutive mitogenic signaling through the Ras or Raf to MAPK pathway. Surprisingly, in normal cells, the same oncogenic mutations typically cause cells to enter into a stable state of cell cycle arrest, a phenomenon known as oncogene-induced senescence. We hypothesized that players that mediate ERK activity-dependent cell fate decisions must themselves undergo some expression/activity changes in response to varying levels of ERK activity. To systematically identify gene expression changes associated with different levels of ERK activity, we performed deep RNA sequencing of RPE cells simultaneously treated, for varying lengths of time, with DOX (to induce BRAFV600E) and different concentrations of ERK inhibitors. The ERK inhibitor concentrations were chosen to include most dynamic ranges of proliferation response based on previous titration experiments. The time points include 0 (untreated control), 1, 2, 4, 8, 16 and 24h post DOX and drug treatment. These time points were selected based on the observation that RPE cells already showed a bell-shaped correlation between ERK activity and proliferation as early as 24h after BRaf induction. Moreover, earlier time points allow enrichment of genes that are more directly responsive to ERK activity changes. Overall design: RNA-seq of RPE cells treated with five different doses of ERK inhibitor (SCH772984) and/or induction of BrafV600E expression at baseline (0h) and six time points up to 24h post-perturbation