Figure 5.

BMI1 confers resistance to the BRAF inhibitor by activation of the noncanonical Wnt pathway. (A,B) CTL and BMI1 MA2 cells were treated with the indicated levels of PLX4720 and assayed for cell number after 18 d by crystal violet staining and quantification (A) or cell death at 72 h by quantification of APC-Annexin V staining and Western blot analysis of total and cleaved PARP (B). HSP90 was used as a loading control, and P-ERK and P-MEK were used to verify drug efficacy. (C,D) sh-Ctl and sh_BMI1 MA2 cells were assessed for degree of BMI1 knockdown (by quantification of the Western blot shown in Supplemental Fig. S1D) (C, left), total accumulated cell number 18 d after culture in PLX4720 (C, right), or apoptosis in response to 48 h of culture in PLX4720 by quantification of APC-Annexin V staining and Western blot analysis of total and cleaved PARP (D). (E,G,H) CTL and BMI1 MA2 cell clones selected after long-term culture with DMSO vehicle or the indicated doses of PLX4720 were assessed for the level of BMI1 (E), Wnt5a (G), and Ror2 (H) mRNA relative to that of the CTL DMSO cells. (F) Levels of BMI1 in gene expression data sets from A375 (GSE42872 [Parmenter et al. 2014]) and WM164 (GSE54711) cells after culture in vehicle or BRAFi (left panel) or three paired biopsies from melanoma patients before and after treatment with vemurafenib (GSE50535 [Sun et al. 2014]) (right panel). (I) Levels of Wnt5a (left panel) and Ror2 (right panel) in sh-ctl and sh-Wnt5a MA2 BMI1 cells relative to sh-ctl MA2 cells. (J) Quantification of apoptosis by analysis of APC-Annexin V staining of the cell lines from I 72 h after treatment with DMSO or 2 µM PLX4720. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test, where the asterisk indicates significance versus the treated sh-Ctl samples (C,D), and one-way ANOVA with Dunnet's multiple comparisons test, where the asterisk indicates significance versus MA2 BMI1 sh-ctl treated with PLX4720 (J). See also Supplemental Fig. S5.