Fig. 4: A switch in cell cycle control underlies HER2-inhibitor resistance and MEK dependence of MAPK-activated HER2 + breast cancer cells.
From: HER2 + breast cancers evade anti-HER2 therapy via a switch in driver pathway
(a) Immunoblots of indicated cell cycle regulatory proteins in shRenilla control and shNF1 SKBR3 cells treated with 500 nM lapatinib (HER2i) for 0, 24, 48, 72, and 96 h. Images are representative of 3 biological replicates. Quantification normalized to beta-actin levels. (b) The cell cycle distribution of SKBR3 shRen control and shNF1 HER2i-R cells treated with DMSO, 500 nM lapatinib (HER2i), 50 nM trametinib (MEKi), or 2 uM MK2206 (AKTi) as measured by fluorescence-activated cell sorting, plotted as % of cells in S phase. Data are means± SD of three independent experiments. (p = 1.833 × 10−5, 1.03 × 10−3, 2.9 × 10−5, two-sided student’s t-tests). (c and d) Immunoblots of indicated proteins in cells from (b) treated with 50 nM trametinib (MEKi, c) or 2 uM MK2206 (AKTi, d) and collected at 0, 2, 6, 24, and 48 h. Images are representative of 3 biological repeats. (e) Inhibition of proliferation of shRenilla and shNF1 HER2i-R SKBR3 cells treated with increasing doses of indicated CDK inhibitors. Data points represent the mean of 6 biological replicates. (f) Immunoblots of phospho-Rb, cyclin E2, CDK2, and β-actin in SKBR3 shRen and shNF1 HER2i-R cells transduced with dox-inducible shRNAs against cyclin E2 or renilla, cultured in dox and 500 nM lapatinib for 48 h. (g) Graphical overview of CDK2 immunoprecipitation (IP) kinase assay. CDK2 (or IgG control) was immunoprecipitated from shRen control and shNF1 HER2i-R cells treated with DMSO or 50 nM trametinib (MEKi) for 48 h and incubated in an in vitro kinase assay using recombinant Rb1 substrate. (h) Western blot analyses of results of the IP kinase assay described in (g) and assay input. Source data for all assays are provided as a Source Data file.
