CLA and BR drafted the manuscript

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CLA and BR drafted the manuscript. immunoblotting using the indicated antibodies. The constructs include a C-terminal hemagglutinin (HA)-label bcr3601-S5.docx (227K) GUID:?77234FDB-42CB-4F99-A017-6AA7FB28572F Extra file 6: Body S2 Phosphoinositide 3-kinase (PI3K) mutation shifts the half-maximal inhibitory concentration for lapatinib inhibition of PI3K signaling. Cells expressing wild-type or mutant PI3K constructs had been treated with a variety of lapatinib concentrations (0.0016 to 5?M) and pHER2, pAkt and pS6 measured by enzyme-linked immunosorbent assay. Outcomes had been normalized to neglected cells and inhibitor response curves had been installed with GraphPad from a mean of three experiments (bars?=?SEM). bcr3601-S6.docx (73K) GUID:?98BD2FE9-9902-4C93-82BF-366BCD17E975 Additional file 7: Figure S3 Acquired E542K mutation in BT474 lapatinib resistant cells shifts the lapatinib half-maximal inhibitory concentration (IC50) for phosphoinositide 3-kinase signaling. BT474 parental or lapatinib-resistant cells cultured without lapatinib for at least 2?weeks were treated with Rifapentine (Priftin) a range of Rifapentine (Priftin) lapatinib doses and analyzed by enzyme-linked immunosorbent assay for pHER2, pAkt and pS6. The inhibitor response curves and mean IC50 values from three individual experiments are displayed (bars?=?SEM) bcr3601-S7.docx (76K) GUID:?5CE14721-DE3A-4316-8AE1-569961C087E3 Additional file 8: Figure S4 Cell lines with endogenous phosphoinositide 3-kinase (PI3K) mutations show uncoupling of PI3K signaling from HER2 inhibition by lapatinib. (A) Cell lines (wild-type or with PI3K mutations as indicated) were treated with a range of lapatinib doses and analyzed by enzyme-linked immunosorbent assay for pHER2, pAkt and pS6. The inhibitor response curves derived from a mean of three individual experiments are shown. (B) The level of pS6 remaining at the 5?M lapatinib dose, normalized to untreated cells, is shown (mean??SEM) bcr3601-S8.docx (93K) GUID:?A3AA7BF4-31EB-441C-9B91-7235E5B23C45 Additional file 9: Figure S5 Lapatinib-resistant cells utilize H1047R mutant for phosphoinositide 3-kinase (PI3K) signaling. Cells infected with E545K or H1047R mutant vectors were selected for lapatinib resistance. PI3K was immunoprecipitated from cells before and after selection for resistance, and the level of hemagglutinin (HA) expression was determined by immunoblot analysis of the immune complexes. HA band intensity was quantitated using infrared fluorescence secondary antibodies and LI-COR software. The relative intensity of HA expression in resistant cells compared with their unselected counterparts is usually shown. The mean value (SEM) of five or six replicate immunoprecipitations is usually displayed bcr3601-S9.docx (48K) GUID:?DDCFBACB-1D42-4698-A231-7A46D927C5E1 Additional file 10: Figure S6 Phosphoinositide 3-kinase (PI3K) mutation allows for emergence of resistant colonies, even to dual HER2 blockade. Cells expressing wild-type or PI3K mutations as indicated were seeded into 12-well plates and were treated with lapatinib, trastuzumab or a combination of the two 24?hours after plating as indicated. Cells were grown for 2 to 3 3?weeks in media, and drugs were replenished twice weekly. At the end of treatment, the cells were fixed and stained with crystal violet bcr3601-S10.docx (329K) GUID:?2122884F-7CED-41F7-B89D-B45352B3626E Additional Rifapentine (Priftin) file 11: Figure S7 BKM120 is required to maximally inhibit phosphoinositide 3-kinase signaling in tumor xenografts. Tumors from BT474 cells that remained after 28?days of treatment with BKM120 (B), lapatinib (L) or trastuzumab (T) in the combinations indicated were harvested, and lysates were prepared and analyzed by immunoblotting with the indicated antibodies bcr3601-S11.docx (124K) GUID:?B4D4B802-F119-4F9D-A435-FB381D07A4ED Abstract Introduction Despite multiple advances in the treatment of HER2+ breast cancers, resistance develops even to combinations of HER2 targeting agents. Inhibition of PI3K pathway signaling is critical for the efficacy of HER2 inhibitors. Activating mutations in can overlap with amplification and have been shown to confer resistance to HER2 inhibitors in preclinical studies. Methods Lapatinib-resistant cells were profiled for mutations in the PI3K pathway with the SNaPshot assay. Hotspot mutations were retrovirally transduced into mutations on the effect of HER2 and PI3K inhibitors was assayed by immunoblot, proliferation and apoptosis assays. Uncoupling of PI3K signaling from HER2 was investigated by ELISA for phosphoproteins in the HER2-PI3K signaling cascade. The combination of HER2 inhibitors with PI3K inhibition was studied in HER2-amplified xenograft models with wild-type or mutant mutation in cells selected for resistance to the HER2 tyrosine kinase inhibitor lapatinib. We also show that this gain of function conferred by these mutations partially uncouples PI3K signaling from the HER2 receptor upstream. Drug resistance conferred by this uncoupling was overcome by blockade of PI3K with the pan-p110 inhibitor Rabbit Polyclonal to PTTG BKM120. In mice bearing xenografts, dual HER2 targeting with trastuzumab and lapatinib resulted in tumor regression. The addition of a PI3K inhibitor further improved tumor regression and Rifapentine (Priftin) decreased tumor relapse after discontinuation of treatment. In a mutation and also provides benefit to HER2+ tumors with wild-type tumors. Introduction Amplification of the oncogene occurs in approximately 25% of human breast cancers and predicts response to therapies targeting human epidermal growth factor receptor 2 (HER2), including trastuzumab, a monoclonal antibody directed against HER2, and lapatinib, a tyrosine kinase inhibitor (TKI) of HER2 and epidermal growth factor receptor (EGFR) [1,2]. HER2 is usually a member of the ErbB family of receptor.