Figure 1.

SRC hyperactivation is a key signaling alteration in acquired trastuzumab-resistant cells. (a) MTS assay comparing cell proliferation of indicated parental breast cancer cell lines and their corresponding acquired TtzmR sublines upon treatment with freshly added trastuzumab (Ttzm, 2 µg ml⁻¹) for 4 d. (b) Tumor volume of mammary fat pad xenografts derived from either BT474 parental (P) or TtzmR subline upon treatment of IgG control or Ttzm (10 mg per kg body weight, intraperitoneally) weekly. Tumor volume at various times of treatment is presented as percentage of original tumor size at day 0 of treatment. (c) Representative histograms from flow cytometric analysis of EGFR and ERBB2 abundance in BT474 parental and TtzmR cells. (d) Relative amounts of EGFR, ERBB2, HER3 and IGF-1R in the indicated parental and TtzmR cells analyzed by flow cytometry. (e) Immunoblots comparing major cell signaling changes between the indicated parental and TtzmR sublines. P indicates phosphorylation; for example, P-EGFR-Y1068 is EFGR phosphorylated at Tyr1068 (f) Immunoblots assessing the impact of overexpression of EGFR and IGF-1R in BT474 parental (BT474.P) cells on signaling. (g) MTS assay evaluating trastuzumab resistance of BT474 cells overexpressing EGFR or IGF-1R, treated as in a. (h) Immunoblots of EGFR and phosphorylated SRC after shRNA-mediated EGFR knockdown in BT474.TtzmR cells. (i) MTS assay evaluating sensitivity of TtzmR to trastuzumab after EGFR knockdown. Cells were treated as in a. (j) Left, immunoblots of knock-down of SRC in BT474.TtzmR cells by SRC shRNA. Right, MTS assay assessing trastuzumab sensitivity of TtzmR cells after SRC knockdown. All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates.