Cooperative Effect of Oncogenic MET and PIK3CA in an HGF-Dominant Environment in Breast Cancer

Effect of targeting PI3K and/or MET in HCC1954-derived cells in vitro and in vivo. The HCC1954-derived cells were treated as indicated with PI3K inhibitor pictilisib and/or either MET TKI tepotinib or MET antibody onartuzumab and then tested for cell survival and invasion (as described in Fig. 1 and Materials and Methods). The combined effect of tepotinib and pictilisib with variable doses on colony formation was analyzed with CalcuSyn Dose Effect Analyzer (Materials and Methods). A, Dose effect. B, Colony formation assay with tepotinib (variable doses) combined with pictilisib (fixed dose). C, Quantitative analysis of the area of total clones was performed with AlphaVIEW SA software. Data are mean ± SD of triplicates, representative of two independent experiments (*, P < 0.05; ***, P < 0.0001 vs. vehicle; #, P < 0.05; ##, P < 0.01; and ###, P < 0.001 vs. pictilisib, ANOVA). D, Invasion assay (pictilisib 0.25 μmol/L, onartuzumab 1.5 μmol/L). E, Data are mean ± SD of triplicates, representative of two independent experiments (***, P < 0.001 vs. control cells, ANOVA). In vivo studies, HCC1954-derived cells with expression of MET-WT/PIK3CA-H1047R (F) or MET-T1010I/PIK3CA-H1047R (G) were implanted into mammary fat pad of hHGF Tg/SCID female. Three days later, the mice were randomized to treatment with vehicle, onartuzumab (10 mg/kg, i.p. injection, twice weekly), pictilisib (100 mg/kg in 100 μL of 5% DMSO, daily by oral gavage), or their combination. Tumor sizes were measured with calipers twice weekly. Tumor volume was calculated with the formula V = lw²/2. Data were analyzed with two-way ANOVA (*, P < 0.0001 vs. vehicle; §, P < 0.0001 vs. onartuzumab alone; #, P < 0.01 vs. pictilisib alone).

Characterization of breast epithelial cells transfected with oncogenic PIK3CA with MET. MCF-10A–derived cells cotransfected with various MET genotypes and PIK3CA-E545K (A) or PIK3CA-H1047R (B) were seeded in 12-well plates in triplicate with a density of 2 × 10⁴ per well in modified growth medium (2.5% horse serum, withdrawn EGF, insulin, and hydrocortisone), supplemented with HGF (40 ng/mL) for 3 days. Cells were counted each day. Data are mean ± SD of triplicates, representative of two independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control cells, ANOVA). C, Effects of MET aberrations on mammary acinar morphogenesis were tested as described in the Materials and Methods. MCF-10A–derived cells with expression of PIK3CA-H1047R and various MET genes as indicated were resuspended in modified growth medium (2.5% horse serum and 5 ng/mL EGF) containing 2% Matrigel supplemented with HGF (40 ng/mL). Representative field images of acini were taken on day 8; original magnification, ×40. The clonogenic assay was analyzed as described in the Materials and Methods. The MCF-10A–derived cells (D and F) were seeded in triplicate at a density of 1,000 cells in 60-mm dishes in modified growth medium supplemented with 2.5% horse serum and 40 ng/mL HGF. The dishes were scanned on day 10. Quantitative analysis of the total number of clones was performed with AlphaVIEW SA software. Data are mean ± SD of triplicates, representative of two independent experiments (**, P < 0.01; ***, P < 0.001, vs. cells transfected with mutant PIK3CA alone, ANOVA; E and G). Cell invasion in vitro was analyzed (Materials and Methods). The MCF-10A–derived cells were induced with HGF (40 ng/mL) and fibronectin (5 μg/mL). H, Cells invading through Matrigel were photographed at ×100 magnification. I, Data are mean ± SD of triplicates, representative of two independent experiments (***, P < 0.001; ****, P < 0.0001 vs. vector, ANOVA).

Effects of combined targeting of PI3K and MET on cell growth in Matrigel or invasion in MCF-10A–derived cells. A total of 4 × 10³ MCF-10A–derived cells expressing PIK3CA-H1047R/MET-T1010I (A) or PIK3CA-H1047R/MET-WT genes (C) were resuspended in modified growth medium (2.5% horse serum and 5 ng/mL EGF) supplemented with HGF (40 ng/mL) and 2% growth factor–reduced Matrigel and treated with various drugs as indicated. The medium was exchanged every 3 days. Photographs (×40) of representative fields were taken on day 7. The figures shown are mean ± SD of triplicates, representative of two independent experiments (*, P < 0.01; **, P < 0.001; ***, P < 0.0001 vs. vehicle, ANOVA; B and D). The MCF-10A–derived cells expressing PIK3CA-H1047R/MET-T1010I (E) or PIK3CA-E545K/MET-T1010I (G) were starved for 20 hours in serum-free DMEM-F12 lacking EGF, insulin, and hydrocortisone. A total of 1 × 10⁵ cells were inoculated into the top chamber. Pictilisib, onartuzumab, or tepotinib alone or in combinations as indicated were added into both the top and bottom chambers. HGF and fibronectin were added in the bottom chamber as the inducer. Invasive cells were photographed and counted in 10 random fields. The figures shown are mean ± SD of triplicates, representative of two independent experiments (***, P < 0.0001 vs. vehicle, ANOVA; F and H).

Cooperative oncogenic effect of MET and PIK3CA on cellular signaling pathways in an environment with or without HGF. PIK3CA-H1047R/MET-T1010I–expressing MCF-10A–derived cells were starved overnight followed by stimulation with HGF (40 ng/mL) or 10% FBS for 30 minutes. Cell lysates were subject to RPPA as described in the Materials and Methods. A, Data are presented in a matrix format: each row represents an antibody target and each column a sample. In each sample, the ratio of the abundance of the molecule to its median abundance across all samples is represented by the color of the corresponding cell in the matrix (see the scale for expression levels). B, The RPPA data were further analyzed (as described in the Materials and Methods; mean ± SD; P value based on the log₂ data).

Cellular signaling response to targeting of MET and/or PIK3CA in an environment with or without HGF. PIK3CA-H1047R/MET-T1010I–expressing MCF-10A-derived cells were starved overnight followed by treatment with or without drugs for 6 hours and then were stimulated with HGF (40 ng/mL) or 10% FBS for 30 minutes. Cell lysates were subject to RPPA as described in the Materials and Methods (mean ± SD; P value based on the log₂ data). A, Cells were treated with pictilisib (P) or onartuzumab (O) alone or their combination (P+O) before stimulation with HGF or FBS. B, Cells were treated with pictilisib (P) or tepotinib (T) alone or their combination (P+T) before stimulation with HGF or FBS. C, Cells were treated with pictilisib and/or onartuzumab followed by stimulation with HGF. Data are presented in a matrix format: each row represents a sample and each column an antibody target. There are four subclusters as indicated from top to bottom: vehicle, onartuzumab, pictilisib, and their combination. Red represents high activation, and green represents low activation. D, Using tepotinib instead of onartuzumab. E, A portion of the RPPA data was validated by Western blot.