Figure 5. IRS2 mutations linked to invasion in pleomorphic invasive lobular carcinoma cells.

(A) Immunoblots of cell extracts from KEP 1.11 murine pleomorphic invasive lobular carcinoma (PILC) cells stably expressing shGFP and 2 independent shRNA targeting Irs2 (shIrs2-1 and shIrs2-2). (B) Cells grown in a Matrigel/collagen I gel were scored for the distance of invasive branching (mean ± SD, n = 50 colonies from 1 of 2 representative experiments). (C) KEP 1.11 cells expressing shGFP were serum starved and pretreated with DMSO or BMS-754807 at the concentrations indicated for 4 hours and stimulated with insulin-like growth factor-1 (IGF-1; 50 ng/ml) for 30 minutes. Cell extracts containing equivalent amounts of protein were immunoblotted with antibodies specific for insulin receptor substrate 2 (IRS2), pIGF1R (Y1135/1136)/pIR (T1150/1151), IGF1R, pAkt (S473), Akt, or Tubulin. (D) Cells grown in a Matrigel/collagen I gel were treated with DMSO or BMS-754807 (BMS, 100 nM) on day 3 and scored for the distance of invasive branching on day 7 (mean ± SD, n = 50 colonies from a representative experiment). (E) Immunoblots of cell extracts from KEP 1.11 cells stably expressing empty vector (pCDH), wild-type human IRS2 (WT), or the IRS2 mutants identified in PILC. Cells grown in a Matrigel/collagen I gel were scored for (F) the extent of invasion (mean ± SD of 3 independent experiments) or (G) the distance of invasive branching (mean ± SD, n = 50 colonies from 1 of 3 representative experiments). Representative images for each cell line are shown. (H) Cell migration assay using Transwell culture chambers (mean ± SD of 3 independent experiments). (I) Glucose uptake assay (mean ± SD of 4 independent experiments). Student’s t test was performed between pCDH and WT-IRS2, and 1-way ANOVA with Bonferroni post hoc testing was performed for all other comparisons. *P < 0.05, ***P < 0.001, relative to pLKO.1 or pCDH control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, relative to WT-IRS2. Scale bar: 20 μm.