Figure 5

MEK cooperates with Cdkn2a and Pten loss in the development of melanomas in vivo. (a) Kaplan–Meier percent survival curves for BRAF and MEK tumors. Dct::TVA; Cdkn2a^lox/lox; Pten^lox/lox mice were injected with viruses encoding Cre (raised arrow headed line, n=17 tumor incidence 0/17) or wild-type MEK+Cre (solid line, n=12, tumor incidence 0/12) or BRAF^V600E+Cre (closely dashed line, n=15, incidence 15/15) or MEK^GF+Cre (dotted line tumor incidence 22/22). Mice were also injected with MEK^V60E+Cre (wide dashed line, n=18, incidence 6/18) or MEK^C121S+Cre (dotted and dashed line, n=13, tumor incidence 5/13) or MEK^G128V+Cre (solid line, n=7 tumor incidence 0/7). At the time of publication 7 MEK^V60E & Cre and 4 MEK^C121S+Cre mice remain tumor free at just over 120 days of age and 5 MEK^V60E & Cre and 4 MEK^C121S mice remained tumor free until the experiment end point of 160 days. No significant difference was observed between MEK^C121S and MEK^V60E P=0.563. A significant difference was observed between BRAF^V600E & Cre and MEK^GF+Cre (P=0.0401). A significant increase in survival is evident between MEK^GF and MEK^V60E (P=3.19⁻⁰⁹), and MEK^GF and MEK^C121S (P=4.87⁻⁰⁵). (b) Kaplan–Meier survival curves demonstrating the effect of genetic MEK inhibition. Dct::TVA;Cdkn2a^lox/lox;Pten^lox/lox mice were injected with viruses encoding Tet-off P2A Cre+TRE-MEK^GF. Mice were monitored for tumor formation and randomized to receive a Dox diet or control diet when tumors were measured at 1.0 cm³ (Dox diet dotted line, n=4, control diet dashed line, n=4). A significant increase in survival was found between Dox-treated and control mice (P=0.0067). No difference in survival was observed between the Tet-off P2A Cre+TRE-MEK^GF control mice and MEK^GF+Cre tumors shown again in this panel for comparison (solid line, P=0.112). (c) MEK inhibition leads to tumor regression and recurrence. Plot showing the volume of four Tet-off P2A Cre+TRE-MEK^GF tumors from the first tumor measurement until death. Mice were treated with Dox when tumors were measured at 1.0 cm³ (denoted by colored arrows) and killed when tumors reached 2.5 cm³; the oldest mouse was killed at 192 days. One mouse developed stable disease before being found dead. (d) Virally delivered MEK^GF expression was detected in proteins extracted from mouse melanomas using an antibody for the HA epitope tag in tumors induced with RCAN TRE-HA-MEK^GF+RCAS Tet-off P2A Cre but absent from the recurring Dox-treated tumors and absent from untagged RCAS MEK^GF and Cre control tumors. MAPK activity was evaluated by blotting for phosphorylated and total ERK 1/2.