FIGURE 3.

IGF1R, TYRO3, and PDGFRb activity is necessary to sustain signal transduction and tumor cell proliferation in response to HER2 inhibition. A, SKBR3 cells expressing TYRO3, IGF1R, or PDGFRb were treated with lapatinib (Lap) for 24 h, and then proteins were resolved by SDS-PAGE and immunoblotted using antibodies specific for the indicated signaling proteins. B, synergistic inhibition of trastuzumab-resistant cell growth by HER2 and IGF1R chemical inhibitors. rBT474 cells were cultured for 72 h in the presence of increasing concentrations of lapatinib, AEW541, or lapatinib plus AEW541 and then metabolically labeled with 2 μCi of [³H]thymidine for 16 h, after which DNA was precipitated and thymidine incorporation was measured by scintillation counting. C, rBT474 cells were cultured as in B but treated with the PDGFRb inhibitor sunitinib instead of AEW541. Error bars, S.E.; ***, p < 0.05. D and E, combined HER2 and IGF1R inhibition delays mammary tumor formation in mice. Immunodeficient BALB/c nude mice were orthotopically injected with rBT474 breast cancer cells, and, after allowing 1 week for tumor formation, were treated daily with vehicle, lapatinib, or lapatinib plus AEW541. Tumor sizes were measured twice weekly using vernier calipers until day 25, at which point mice were sacrificed, and dissected tumors were photographed (E). Mean tumor volumes ± S.E. are shown in D; ***, p < 0.05 (n = 4 mice/group).