Fig 2: AZD8931 resistance is associated with EMT in a subpopulation of tumours. Analysis of AZD8931-naïve (vehicle) and AZD8931-resistant tumours showing representative images of H&E staining and immunohistochemical analysis of E-cadherin, vimentin and HER2. Scale bar: 50 µm. (A-H) AZD8931-resistant spindle cell tumour and corresponding vehicle-treated tumour. (I-P) AZD8931-resistant tumour phenotypically indistinguishable from AZD8931-naïve (vehicle) tumour and corresponding vehicle-treated tumour.

Fig 3: PTEN status determines the sensitivity to AZD8931. Cohorts of MMTV-NIC-PTEN+/+ and NIC-PTEN+/- mice were randomized to treatment with daily AZD8931 or vehicle, and the tumour response was monitored. (A) Overall survival in vehicle- (n=5) and AZD8931-treated (n=5) NIC-PTEN+/+ mice (P=0.0043, Gehan–Breslow–Wilcoxon test). (B) Overall survival in vehicle- (n=5) and AZD8931-treated (n=5) NIC-PTEN+/- mice (P=0.0039, Gehan–Breslow–Wilcoxon test). (C) Waterfall plot of percentage tumour volume change over duration of experiment in NIC-PTEN+/+ vehicle- and AZD8931-treated animals (P=0.0079, Mann–Whitney U-test). (D) Waterfall plot of percentage tumour volume change over duration of experiment in NIC-PTEN+/- vehicle- and AZD8931-treated animals (P=0.0079, Mann–Whitney U-test). Vehicle-treated mice were sacrificed when the tumour burden reached the maximal permitted size, at 10-39 days for MMTV-NIC PTEN+/+ mice and 11-24 days for MMTV-NIC PTEN+/- mice. AZD8931 treatment was stopped at 40 days, when all vehicle-treated animals were sacrificed because of tumour burden. (E) Immunohistochemical analysis was performed on paraffin-embedded sections of AZD8931- and vehicle-treated tumours with pTyr 1221/1222 HER2 and pTyr 1289 HER3 antibodies. Membranous histoscore calculated as the sum of the product of the percentage of cells stained by the intensity graded from 0 to 3, where 1=weak, 2=moderate and 3=strong staining [histoscore=(% *1)+(% *2)+(% *3)]. pTyr 1221/1222 HER2 staining: NIC-PTEN+/+, P=0.48; NIC-PTEN+/-, P=1.0; and pTyr1289 HER3 staining: NIC-PTEN+/+, P=0.20; NIC-PTEN+/-, P=0.07; Mann–Whitney U-test comparing vehicle- and AZD8931-treated tumours. Black data points represent vehicle-treated tumours. Red data points represent AZD8931-treated tumours. Bars represent mean value for each genotype. (F) Reverse-phase protein array analysis was performed on lysate from AZD8931- (red data points) and vehicle-treated (black data points) NIC-PTEN+/+ and NIC-PTEN+/- tumours. The ratio of phospho:total protein relative fluorescence intensity (RFI) value is presented and normalized to the maximal value in each data set. *P=0.05 and **P=0.01 comparing vehicle and AZD8931 for each antibody, Mann–Whitney U-test.

Fig 4: Development of EMT in AZD8931-resistant tumours. Immunohistochemical analysis of E-cadherin, vimentin and HER2 in (A) vehicle- and (B-E) AZD8931-treated tumours. (B) AZD8931-responsive tumour with growth inhibited by AZD8931. (C) AZD8931-resistant tumour that has retained the morphology of the AZD8931-naïve (vehicle) tumours. (D,E) AZD8931-resistant tumours with a spindle cell morphology. Scale bar: 50 µm.