Article Text
Abstract
Introduction RAF kinases activate the ERK1/2 cascade, a key pathway involved in cardiac remodelling and cytoprotection. Since activating mutations in BRAF cause cancer, small molecule inhibitors of RAF have been developed. However, a paradoxical effect is observed with some inhibitors which activate rather than inhibit ERK1/2. Therefore, ‘paradox breaker’ inhibitors have been developed as new-generation cancer therapies void of this effect (e.g. PLX8394). Here, we determined the effects of PLX8394 on endothelial cell (EC) ERK1/2 signalling and the heart in vivo.
Methods Murine ECs were incubated with PLX8394 and effects on ERK1/2 activity determined by western blotting for the phosphorylated (i.e. activated) kinases. Effects on gene expression were determined by qPCR. The effects of PLX8394 on the heart in vivo were determined by infusing male wildtype C57Bl/6J mice (10-12wks, n= 6/group) with PLX8394 (5mg/kg/d, 7d) using osmotic minipumps. Cardiac function/dimensions were assessed using echocardiography; effects on cardiac morphology were assessed by histological staining. mRNA expression was assessed by qPCR. Statistical tests used 1-way ANOVA with Holm-Sidak’s post-test (in vitro studies) and unpaired t-tests (in vivo studies).
Results PLX8394 activated ERK1/2 in ECs in a time (7.4±2.3-fold at 5 min; p=0.0365; n=5) and concentration (>1uM; p=0.0625; n=3) dependent manner. This was associated with significant increases in expression of mRNAs encoding the immediate early gene Fos (6.1±2.6-fold; p<0.0001; n=4) and the vasoconstrictor peptide endothelin-1 (Edn1) (2.7±0.9-fold; p=0.0038; n=4). In vivo, PLX8394 decreased cardiac output (p=0.0092), predominantly through reduced stroke volume (p=0.0103). Structurally, PLX8394 promoted cardiac hypertrophy, with increased diastolic left ventricular (LV) posterior wall thickness (p=0.0425) and decreased LV internal diameter (p=0.0463) at 7 d. Cardiac hypertrophy resulted from increased cardiomyocyte cross-sectional area (p=0.0002) despite no changes in Myh7, Nppa or Nppb mRNAs. Moreover, PLX8394-induced cardiac remodelling was not due to increased fibrosis, with no change in mRNA expression of collagens1-4 and using histological assessment.
Conclusion Despite being developed as a ‘paradox breaker’ for cancer, PLX8394 promoted ERK1/2 signalling in murine ECs and cardiac remodelling in vivo. These preliminary findings suggest that such inhibitors, currently in Phase 3 trials for RAF-mutant cancers, have potential to modulate cardiac function in patients.
Conflict of Interest N/A