Figure 6. Endogenous BRAF^V600E regulates p21^cip1 transcription through FOXO4 phosphorylation on the JNK target sites.

a) Characterization of WM266.4 (BRAF^V600D) cells. CHL (wt BRAF), Colo829 (BRAF^V600E) and WM266.4 (BRAF^V600D) cells were lysed and analyzed by immunoblotting. Endogenous FOXO4 expression was determined after immunoprecipitation. b) (Left panel) U0126 abrogates JNK signaling, endogenous phosphorylation of FOXO4 on Thr223+Ser226 and p21^cip1 expression in WM266.4 cells. WM266.4 cells were untreated or treated for 24hrs with 10μM U0126 and analyzed as in a). The phosphorylation status of endogenous FOXO4 was determined after immunoprecipitation. HC=Heavy Chain. (Right panel) Endogenous FOXOs regulate p21^cip1 expression in WM266.4 cells. Lysates of WM266.4 cells transfected with scrambled siRNA or siRNA against FOXO1,3a and 4 (siFOXO) and untreated or treated for 24 hrs with 20μM U0126 were analyzed by immunoblotting. d) Expression of p21^cip1, total FOXO4 and Thr223/Ser226-phosphorylated FOXO4 is elevated in neoplastic regions of BRAF^V600E-induced nevi. Top panels: Skin sections of tamoxifen treated Braf^+/LSL-V600E; Tyr::CreERT2^+/o mice were analyzed for background signal (2^nd antibody only), p21^cip1 expression, total FOXO4 and Thr223/Ser226 phosphorylated FOXO4. Higher magnifications of the nevus (arrowheads) are shown in the lower panels. The top right panel shows undifferentiated nevi. Lower right panel represents a magnification of epidermal staining from bottom left panel. Untreated tissue did not typically show positive staining. e) Model on the regulation of FOXO4 by BRAF^V600E, resulting in p21^cip1-mediated senescence. BRAF^V600E signaling activates MEK. This in turn, induces elevations in cellular ROS levels, thereby promoting activation of JNK. JNK subsequently phosphorylates FOXO4 and thereby promotes specific transcription of p21^cip1, rather than p27^kip1 or p16^ink4a, and triggers a senescence response.