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Molecular Cancer Therapeutics
Molecular Cancer Therapeutics

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Cancer Biology and Translational Studies

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

Shuying Liu, Shunqiang Li, Bailiang Wang, Wenbin Liu, Mihai Gagea, Huiqin Chen, Joohyuk Sohn, Napa Parinyanitikul, Tina Primeau, Kim-Anh Do, George F. Vande Woude, John Mendelsohn, Naoto T. Ueno, Gordon B. Mills, Debu Tripathy and Ana M. Gonzalez-Angulo
Shuying Liu
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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  • For correspondence: dtripathy@mdanderson.org sliu@mdanderson.org
Shunqiang Li
Section of Breast Oncology, Department of Internal Medicine, Washington University in St. Louis, St. Louis, Missouri.
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Bailiang Wang
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Wenbin Liu
Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Mihai Gagea
Department of Veterinary Medicine and Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Huiqin Chen
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Joohyuk Sohn
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Napa Parinyanitikul
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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  • ORCID record for Napa Parinyanitikul
Tina Primeau
Section of Breast Oncology, Department of Internal Medicine, Washington University in St. Louis, St. Louis, Missouri.
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Kim-Anh Do
Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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George F. Vande Woude
Laboratory of Molecular Oncology, Van Andel Institute, Grand Rapids, Michigan.
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John Mendelsohn
Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Naoto T. Ueno
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Gordon B. Mills
Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Debu Tripathy
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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  • For correspondence: dtripathy@mdanderson.org sliu@mdanderson.org
Ana M. Gonzalez-Angulo
Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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DOI: 10.1158/1535-7163.MCT-18-0710 Published February 2019
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    Figure 1.

    Characterization of breast cancer cells with endogenous PIK3CA-H1047R and exogenous MET. HCC1954 cells, with endogenous PIK3CA-H1047R, were transfected by WT MET or MET-T1010I genes as indicated. A, The clonogenic assay was performed as described in the Materials and Methods. The HCC1954-derived cells were seeded in triplicate at a density of 1,000 cells in 60-mm dishes in RPMI medium supplemented with 5% FBS and 40 ng/mL HGF. The dishes were scanned on day 10. B, 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 < 0.001 vs. control cells, ANOVA). C, An invasion assay was performed as described in the Materials and Methods. The HCC1954-derived cells that penetrated Matrigel to the underside of the filter were photographed and counted in 10 random fields. D, Data are mean ± SD of triplicates, representative of two independent experiments (**, P < 0.01; ***, P < 0.001 vs. control cells, ANOVA). E, A total of 1 × 107 HCC1954-derived cells were injected into the mammary fat pad of hHGF Tg/SCID female mice. Each group consisted of 5 mice. Tumor volume was calculated with the formula V = lw2/2. Differences in tumor volume between groups were analyzed using ANOVA (*, P < 0.05, vs. control group). F, Hematoxylin and eosin staining for histologic images from a representative tumor from MET-transfected groups shows tumor invasion (black arrows) to adjacent skeletal muscle (pink). G, Data show grade of tumor invasion (*, P < 0.05 vs. control group, ANOVA). The HCC1954-derived cells were seeded in 96-well plates (2,000 cells per well) in complete growth medium and were incubated at 37°C for 24 hours. The medium was changed to low-serum medium (2% FBS). Cells were incubated overnight, followed by the addition of serial dilutions of pictilisib (H) or apitolisib (I) in variable combinations for 72 hours. Growth inhibition was determined (details in the Materials and Methods). The data are mean ± SD of triplicates, representative of two independent experiments (*, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. control).

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    Figure 2.

    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).

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    Figure 3.

    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).

  • Figure 4.
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    Figure 4.

    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).

  • Figure 5.
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    Figure 5.

    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).

  • Figure 6.
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    Figure 6.

    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.

  • Figure 7.
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    Figure 7.

    Characterization of HGF/MET axis in PDXs. A, MET protein expression and activation by stimulation with HGF in PDXs. The cell lines from the PDX received starvation overnight by withdrawing serum and all growth factors, including insulin, EGF, and hydrocortisone, followed by stimulation with or without HGF (40 ng/mL) for 20 minutes. Cell lysis was collected and assessed expression and phosphorylation of MET with Western blotting. B, Quantitative densitometric analysis of the density was performed with AlphaView SA software.

Tables

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  • Table 1.

    Combination index

    Tepotinib (μmol/L)Pictilisib (μmol/L)CI
    0.6250.250.804
    1.250.250.63
    1.250.50.431
    1.251.00.101
    2.50.250.635
    5.00.250.179
    10.00.250.048

Additional Files

  • Figures
  • Tables
  • Supplementary Data

    • Table S1 - Antibody list
    • Figure S1 - MET gene constructs
    • Figure S2 - Serum HGF in hHGF Tg/SCID mice
    • Figure S3 - Establish MCF-10A derived cells expression of PIK3CA/MET genes
    • Figure S4 - Effect of targeting PI3K/MET on acini formation and cell invasion
    • Figure S5A - Effect of HGF on cell signaling
    • Figure S5B - Effect of HGF on cell signaling
    • Figure S6 - Effect of HGF on cell signaling in MET-Y1253D/PIK3CA-H1047R cells
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Molecular Cancer Therapeutics: 18 (2)
February 2019
Volume 18, Issue 2
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Cooperative Effect of Oncogenic MET and PIK3CA in an HGF-Dominant Environment in Breast Cancer
Shuying Liu, Shunqiang Li, Bailiang Wang, Wenbin Liu, Mihai Gagea, Huiqin Chen, Joohyuk Sohn, Napa Parinyanitikul, Tina Primeau, Kim-Anh Do, George F. Vande Woude, John Mendelsohn, Naoto T. Ueno, Gordon B. Mills, Debu Tripathy and Ana M. Gonzalez-Angulo
Mol Cancer Ther February 1 2019 (18) (2) 399-412; DOI: 10.1158/1535-7163.MCT-18-0710

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Cooperative Effect of Oncogenic MET and PIK3CA in an HGF-Dominant Environment in Breast Cancer
Shuying Liu, Shunqiang Li, Bailiang Wang, Wenbin Liu, Mihai Gagea, Huiqin Chen, Joohyuk Sohn, Napa Parinyanitikul, Tina Primeau, Kim-Anh Do, George F. Vande Woude, John Mendelsohn, Naoto T. Ueno, Gordon B. Mills, Debu Tripathy and Ana M. Gonzalez-Angulo
Mol Cancer Ther February 1 2019 (18) (2) 399-412; DOI: 10.1158/1535-7163.MCT-18-0710
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Molecular Cancer Therapeutics
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