1. Pharmacologically inhibiting BRAF/ERK and CDK4 restores APCFZR1 E3 ligase activity.
Pharmacologically inhibiting BRAF/ERK and CDK4 restores APCFZR1 E3 ligase activity. A, Protein levels of BRAF and other APCFZR1 substrates decreased upon BRAFV600E and CDK4/6 inhibition in melanoma cells. IB analysis of BRAFV600E- and CDK4R24C-expressing SK-MEL-28 melanoma cells treated with either 1 μmol/L BRAFV600E inhibitor PLX4032 (V600Ei), 10 μmol/L pan-CDK inhibitor mimosine (pan-CDKi), 1 μmol/L CDK4/6 inhibitor PD (CDK4i), 1 μmol/L PLX μmol/L mimosine, 1 μmol/L PLX μmol/L PD , or DMSO as a negative control for 24 hours before harvesting. BRAF band intensities were quantified using ImageJ, normalized to corresponding TUBULIN band intensities, and then normalized to DMSO control lane. B, Protein levels of BRAF and other APCFZR1 substrates reduced upon MEK and CDK4/6 inhibition in melanoma cells. IB analysis of BRAFWT-expressing HBL melanoma cells treated with either 1 μmol/L MEK inhibitor PD (MEKi), 10 μmol/L pan-CDK inhibitor mimosine (pan-CDKi), 1 μmol/L CDK4/6 inhibitor PD (CDK4i), 1 μmol/L PD μmol/L mimosine, 1 μmol/L PD μmol/L PD or DMSO as a negative control for 24 hours before harvesting. BRAF band intensities were quantified using ImageJ, normalized to corresponding TUBULIN band intensities, and then normalized to DMSO control lane. C, Depletion of FZR1 in BRAFV600E-inhibited melanoma cells led to the upregulation of BRAF and PLK1. IB analysis of BRAFV600E-expressing A375 melanoma cells, which were infected with the control (shScr) or the indicated shFZR1 lentiviral shRNA constructs. The infected cells were selected with 1 μg/mL puromycin for 72 hours to eliminate the noninfected cells before harvesting. Prior to the harvest, cells were treated with DMSO (as a negative control) or 1 μmol/L BRAFV600E inhibitor PLX4032 (V600Ei) for 24 hours as indicated. BRAF band intensities were quantified using ImageJ, normalized to corresponding TUBULIN band intensities, and then normalized to DMSO control lane (top row) or normalized to the shScr + V600Ei lane (bottom row). D, A schematic illustration of the proposed model for the putative role of FZR1 in suppressing BRAF dimerization–mediated transactivation of downstream MEK/ERK signaling to bypass PLX4032-triggered BRAFV600E inhibition in melanoma cells, as well as how mechanistically hyperactive ERK and/or CYCLIN D1/CDK4-mediated phosphorylation of FZR1 inhibits APCFZR1 E3 ligase activity in BRAFV600E melanoma cells.
Lixin Wan et al. Cancer Discov 2017;7:
©2017 by American Association for Cancer Research