Figure 2.

TKI resistance is associated with increased expression of αvβ3 integrin. (A) Cell surface expression of the αv and β3 integrin subunits was analysed in TBCP-1 or TBCP-1NR cells by flow cytometry. Black dashed line = isotype control, green = TBCP-1 and red = TBCP-1NR. (B) Expression levels of integrin β3 were compared in mouse or human TKI-sensitive and resistant pairs by Western blot analysis of whole cell lysates and were normalised relative to GAPDH. The data show representative blots and mean fold increase relative to matched sensitive pairs ± SD from three independent lysates (n = 3). The intensity ratio of each band relative to GAPDH is indicated in red. (C) Sensitivity of the indicated human brain metastatic HER2-positive breast cancer cell lines to neratinib was determined in a 3-day SRB colourimetric assay. The data show a representative curve and mean IC50 values ± SD for each cell line from three independent experiments (n = 3). (D) Western blot analysis of integrin β3 and HER2 expression in whole cell lysates of human brain metastatic lines. GAPDH was used as the loading control. The intensity ratio of each band relative to GAPDH is indicated in red. (E) The association between TKI resistance and integrin β3 expression in breast cancer cell models was analysed using Cancer Dependency Map (DepMap) analysis tool. The data show the association of ITGB3 copy number (log2 relative to ploidy +1, in 21Q1 dataset) with neratinib (AUC values from CTRP: 418,038 dataset) or lapatinib sensitivity (AUC values from CTRP:634309 dataset) for a panel of 38 breast cancer cell lines. The strength of the linear association was determined by measurement of Pearson coefficient and linear regression t-test was used to determine the slope of the regression and statistical significance, p < 0.05 was considered significant. The uncropped blots are shown in Supplementary File S1.