Use of a genetically engineered mouse model as a preclinical tool for HER2 breast cancer

Abstract

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies presents a major clinical problem. Although preclinical studies have identified a number of possible mechanisms, clinical validation has been difficult. This is most likely to reflect the reliance on cell-line models that do not recapitulate the complexity and heterogeneity seen in human tumours. Here, we show the utility of a genetically engineered mouse model of HER2-driven breast cancer (MMTV-NIC) to define mechanisms of resistance to the pan-HER family inhibitor AZD8931. Genetic manipulation of MMTV-NIC mice demonstrated that loss of phosphatase and tensin homologue (PTEN) conferred de novo resistance to AZD8931, and a tumour fragment transplantation model was established to assess mechanisms of acquired resistance. Using this approach, 50% of tumours developed resistance to AZD8931. Analysis of the resistant tumours showed two distinct patterns of resistance: tumours in which reduced membranous HER2 expression was associated with an epithelial-to-mesenchymal transition (EMT) and resistant tumours that retained HER2 expression and an epithelial morphology. The plasticity of the EMT phenotype was demonstrated upon re-implantation of resistant tumours that then showed a mixed epithelial and mesenchymal phenotype. Further AZD8931 treatment resulted in the generation of secondary resistant tumours that again had either undergone EMT or retained their original epithelial morphology. The data provide a strong rationale for basing therapeutic decisions on the biology of the individual resistant tumour, which can be very different from that of the primary tumour and will be specific to individual patients.

Keywords: Breast cancer; Epithelial-to-mesenchymal transition; HER2; Resistance.

Fig. 1.

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. 2.

Transplantation of NIC-PTEN^+/− fragments generated tumours that were indistinguishable from the parental tumours. (A) Representative H&E images of fragment-derived and spontaneous tumours from NIC-PTEN^+/− mice. (B,C) Representative images of HER2 (B) and Ki67 (C) expression in fragment-derived and spontaneous tumours. Scale bar: 100 µm. (D) Growth rate of vehicle- (n=3) and paclitaxel-treated (n=3) fragment-derived tumours.

Fig. 3.

Generation of fragment-derived tumours with acquired resistance to AZD8931. (A) Representative growth curves of three independent NIC-PTEN^+/− tumour fragments treated with AZD8931. Repeated cycles of AZD8931 were administered to facilitate the selection of tumours with acquired resistance to AZD8931. Green arrows indicate the start of treatment and red arrows indicate when treatment was stopped. (B,C) Representative H&E images of AZD8931-naïve (vehicle) and AZD8931-resistant tumours. (D) AZD8931-resistant tumour phenotypically indistinguishable from AZD8931-naïve tumour. (E) AZD8931-resistant tumour consisting of spindle-shaped cells. Scale bar: 100 µm.

Fig. 4.

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. 5.

Generation of secondary resistance in NIC-PTEN^+/− tumour fragments. (A) Growth curves of NIC-PTEN^+/− AZD8931-resistant tumour fragments following treatment with repeated cycles of AZD8931. Black lines represent tumours that developed resistance to AZD8931. Red line represents tumour with growth inhibited by AZD8931. Treatment was stopped after 90 days because of lack of tumour growth. (B) Representative H&E staining of vehicle-treated tumours. (C-F) H&E staining of AZD8931-treated tumours. (C) AZD8931-responsive tumour (red line in A) with growth inhibited by AZD893. (D) AZD8931-resistant tumour that has retained the morphology of the AZD8931-naïve (vehicle) tumours. (E,F) AZD8931-resistant tumours with a spindle cell morphology. (G-P) Immunohistochemical analysis of pAkt (G-K) and pMAPK (L-P) in vehicle- and AZD8931-treated tumours. Scale bar: 50 µm.

Fig. 6.

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.

Fig. 7.

Nuclear Zeb1 expression in spindle cell AZD8931-resistant tumours. Immunohistochemical analysis of Zeb1 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.