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PNAS 110 (11): 4339-4344

Copyright © 2013 by the National Academy of Sciences.

PML mediates glioblastoma resistance to mammalian target of rapamycin (mTOR)-targeted therapies

Akio Iwanamia, Beatrice Ginib,c,1, Ciro Zancab,1, Tomoo Matsutanib, Alvaro Assuncaod, Ali Naele, Julie Dangf, Huijun Yangb, Shaojun Zhug, Jun Kohyamag, Issay Kitabayashih, Webster K. Caveneeb,i, Timothy F. Cloughesyj, Frank B. Furnarib,i,k, Masaya Nakamuraa, Yoshiaki Toyamaa, Hideyuki Okanol, and Paul S. Mischelb,i,k,2

Departments of aOrthopaedic Surgery and lPhysiology, Keio University School of Medicine, Tokyo 160-8582, Japan; bLudwig Institute for Cancer Research, iMoores Comprehensive Cancer Center, and kDepartment of Pathology, University of California at San Diego, La Jolla, CA 92093; cDepartment of Neurological, Neuropsychological, Morphological and Movement Sciences, University of Verona, 37134 Verona, Italy; dUndergraduate Minor in Biomedical Research Program, and Departments of gMolecular and Medical Pharmacology and jNeurology, University of California, Los Angeles, CA 90095; eDepartment of Pathology, University of California, Irvine, CA 92697; fSchool of Pharmacy, University of California, San Francisco, CA 94104; and hDivision of Hematological Malignancy, National Cancer Center Research Institute, Tokyo 104-0045, Japan


Figure 01
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Fig. 1. PML is inversely correlated with proliferation rate and mTOR signaling in GBM clinical samples. (A and B) Tissue microarrays containing tumor samples from 87 GBM patients were stained by using PML, Ki-67, and p-S6 antibody, respectively. PML is highly expressed in 40% of GBM patients. Correlation analyses show PML significantly inversely correlate with Ki-67 (A) and p-S6 (B). (C) Immunohistochemical staining of (reddish brown) PML, Ki-67, and p-S6 from a representative GBM patient. (Magnification: 10x.) Nuclei were counterstained with hematoxylin (blue). (Scale bar: 100 μm.)

 

Figure 02
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Fig. 2. PI3K/Akt/mTOR inhibitors induce PML expression in GBM cells. (A) Western blot analysis of the effect of rapamycin treatment on PML expression in U87 cells. Cells are cultured in serum-free condition. (B) Effect of the EGFR inhibitor erlotinib and rapamycin on PML expression in GBM patient-derived cells. Cells are cultured under neurosphere conditions. (C) Immunofluorescence of PML (red) in U87 cells treated with rapamycin or control. Nuclei are stained with DAPI (blue). (Scale bar: 20 μm.)

 

Figure 03
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Fig. 3. PML overexpression decreases PI3K/Akt/mTOR signaling and slows down cell cycle. (A) Immunofluorescence in U87 control or hemagglutanin-tagged PML1 (HA-PMLI) infected cells. (Scale bar: 10 μm.) (B) Western blot analysis of PI3K/Akt/mTOR signaling pathway and cell cycle-related proteins performed on lysates from U87 control or HA-PMLI infected cells. Cells were placed in serum-free medium, cultured, and collected in each time course. (C) Proliferation of U87 control and HA-PMLI infected cells analyzed by WST assay. P value was determined by Student’s t test. (D) Effect of PMLI overexpression on cell cycle progression in U87 cells. Cell cycle distribution was performed by flow cytometric analysis. P value was determined by Student's t test. (E) Effect of treatment with rapamycin on growth of U87 control and HA-PMLI infected cells analyzed by WST assay. P value was determined by Student's t test.

 

Figure 04
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Fig. 4. PML knockdown sensitizes GBM cell lines to EGFR and mTOR targeted therapies. (A) Cell viability assays demonstrate a synergistic effect of PML knockdown and each indicated inhibitor. P values were determined by Student’s t test. (B) Effect of PML knockdown and each indicated inhibitor on multiple GBM cell lines analyzed by Trypan blue exclusion. P values were determined by Student’s t test.

 

Figure 05
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Fig. 5. As2O3 reduces PML and sensitizes GBM cells to mTOR-targeted therapies. (A) Representative images demonstrating TUNEL staining (green) to assess apoptotic effect of pp242 and As2O3 (2 μM) on U87 cells in vitro. Nuclei are stained blue. (B) Quantification of TUNEL staining. P values were determined by Student’s t test. (C) Representative photographs of U87 GBM xenografts treated daily with vehicle, pp242 (60 mg/kg per day by oral gavage), As2O3 (2.5 mg/kg intraperitoneally), or combination (n = 8 mice per condition). Images of representative PML and TUNEL stains. (D) Quantification demonstrating greater than threefold reduction in tumor size for mice treated with combined pp242 and As2O3 (P < 0.005). (E and F) Quantification of PML and TUNEL xenograft tumor staining from each treatment conditions.

 

Figure 06
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Fig. 6. Rapamycin and erlotinib treatment induces PML expression in GBM patient tumor tissues. (A) Immunohistochemical staining (reddish brown) of PML before and after treatment with rapamycin. Nuclei were counterstained with hematoxylin (blue). (B) Quantification of immunohistochemical staining from >1,000 cells from at least three representative areas of each tumor before and after rapamycin treatment. P value was determined by Wilcoxon signed-rank test. (C) Immunohistochemical staining (reddish brown) of PML before and after treatment with erlotinib. Nuclei were counterstained with hematoxylin (blue). (D) Quantification of immunohistochemical staining from >1,000 cells from at least three representative areas of each tumor before and after erlotinib treatment. P value was determined by Wilcoxon signed-rank test. (Scale bars: 50 μm.) (Magnification: 20x.)

 


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