Fig. 5: YAP activation signals through ERK and mTOR to increase the cyclin D1/p27 ratio.
From: Transient proliferation by reversible YAP and mitogen control of the cyclin D1/p27 ratio
a Schematic of proposed indirect component of cyclin D1/p27 control by YAP/TEAD via mitogen receptor-activated signaling pathways. b, c Top: experimental conditions for serum-starvation of WT and CA-YAP cells followed by 24 h treatment with TEAD inhibitor for 24 h in starvation media. Bottom: Mean fold-change (FC) in protein levels of p-AKT(S473, left) and p-S6(240/244, right) (b) and Fra1 (c) (normalized to WT/DMSO condition). N = 2 (p-AKT) or n = 3 (p-S6, Fra1) independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test to DMSO: p-AKT, p = 0.97 (WT/TEADi), 0.095 (CA-YAP/DMSO), 0.99 (CA-YAP/TEADi); p-S6, p = 0.79 (WT/TEADi), 0.0050 (CA-YAP/DMSO), 0.98 (CA-YAP/TEADi); Fra1, p = 0.94 (WT/TEADi), 0.0013 (CA-YAP/DMSO), 0.38 (CA-YAP/TEADi). d Representative multiplexed images for Fra1 and p-S6(240/244) staining in CA-YAP cells treated with TEADi as in (b). Scale bar = 50 μm. Mean percent p-Rb (G1-gated) for WT and CA-YAP cells treated with PI3K (LY294002, 10 μM), AKT (MK2206, 200 nM), or mTORC1/2 (Torin2, 100 nM) inhibitors (e) or MEK (PD0325901, 100 nM) inhibitor (f) in starvation (left) or serum conditions (right). N = 3 independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple comparisons to DMSO: e −serum, p = 0.052 (WT/PI3Ki), 0.23 (WT/AKTi), 0.012 (WT/mTORi), 0.72 (CA-YAP/PI3Ki), 0.87 (CA-YAP/AKTi), 0.021 (CA-YAP/mTORi); +serum, p > 0.99 (WT/PI3Ki), 0.77 (WT/AKTi), 0.55 (WT/mTORi), >0.99 (CA-YAP/PI3Ki), >0.99 (CA-YAP/PI3Ki), 0.47 (CA-YAP/mTORi); f −serum, p = 0.90 (WT/MEKi), <1 × 10−4 (CA-YAP/DMSO), 0.92 (CA-YAP/MEKi); +serum, p = 0.056 (WT/MEKi), 0.0002 (CA-YAP/DMSO), 0.13 (CA-YAP/MEKi). g Mean FC in cyclin D1 protein levels for WT and CA-YAP cells treated with combined mTORi and MEKi relative to WT/DMSO condition as in (b). N = 2 independent experiments, where matched colors indicate values from the same experiment. h, i Top (h): experimental conditions for mTORi or MEKi time course treatment of serum-starved CA-YAP cells. Bottom (h): fold-change in p-ERK (MEKi-treated) and p-S6(240/244, mTORi-treated) following drug treatment. i Fold-change in protein levels of cyclin D1, p27, and p-Rb for MEKi and mTORi treatment. Mean ± SEM, n = 2 independent experiments, 2–3 replicate wells/experiment. Data are G1-gated.
