Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Patient
4.2. Cells
4.3. Polysome Profiling
4.4. RNA Extraction
4.5. Microarray
4.6. RT-qPCR
4.7. Data Analysis
4.8. Western Blot
4.9. TMA Construction
4.10. Immunohistochemistry
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
4E-BP | eIF4E Binding Proteins |
80S | Eukaryotic ribosomes |
Akt | protein kinase B |
ECM | Extracellular matrix |
EGFR | Epidermal growth factor receptor |
eIF2α | Eukaryotic Initation Factor 2 alpha |
ERK1/2 | Extracellular Signal-Regulated Kinase 1/2 |
FCS | Fetal Calf Serum |
GBM | Glioblastoma |
HE | Hematoxylin and eosin staining |
mRNA | Messenger RNA |
mTORC1 | Mammalian Target of Rapamycin complex I |
PDGFRA | Platelet-derived growth factor receptor alpha |
PI3K | Phosphatidylinositol-4,5-bisphosphate 3-kinase |
PTEN | Phosphatase and tensin homolog |
qPCR. | Quantitative polymerase chain reaction |
RNA | Ribonucleic acid |
TGF-β | Transforming growth factor beta |
FFPE | formalin-fixed paraffin-embedded |
TMA | tissue microarray |
References
- Stupp, R.; Hegi, M.E.; Mason, W.P.; van den Bent, M.J.; Taphoorn, M.J.B.; Janzer, R.C.; Ludwin, S.K.; Allgeier, A. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009, 10, 459–466. [Google Scholar] [CrossRef]
- Lee, E.; Yong, R.L.; Paddison, P.; Zhu, J. Comparison of glioblastoma (GBM) molecular classification methods. Semin. Cancer Biol. 2018, 53, 201–211. [Google Scholar] [CrossRef]
- Verhaak, R.G.W.; Hoadley, K.A.; Purdom, E.; Wang, V.; Qi, Y.; Wilkerson, M.D.; Miller, C.R.; Ding, L.; Golub, T.; Mesirov, J.P.; et al. Integrated Genomic Analysis Identifies Clinically Relevant Subtypes of Glioblastoma Characterized by Abnormalities in PDGFRA, IDH1, EGFR and NF1. Cancer Cell 2010, 17, 98–110. [Google Scholar] [CrossRef]
- Ceccarelli, M.; Barthel, F.P.; Noushmehr, H.; Iavarone, A.; Verhaak, R.G.W. Subsets and Pathways of Progression in Diffuse Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma. Cell 2016, 164, 550–563. [Google Scholar] [CrossRef]
- Touat, M.; Idbaih, A.; Sanson, M.; Ligon, K.L. Glioblastoma targeted therapy: Updated approaches from recent biological insights. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. 2017, 28, 1457–1472. [Google Scholar] [CrossRef]
- Reifenberger, G.; Wirsching, H.-G.; Knobbe-Thomsen, C.B.; Weller, M. Advances in the molecular geneticsof gliomas—Implications for classification and therapy. Nat. Rev. Clin. Oncol. 2017, 14, 434–452. [Google Scholar] [CrossRef] [PubMed]
- Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular Biology of the Cell, 4th ed.; Garland Science: New York, NY, USA, 2002; Chapter 6. From RNA to Protein; pp. 299–369. [Google Scholar]
- Kristensen, A.R.; Gsponer, J.; Foster, L.J. Protein synthesis rate is the predominant regulator of protein expression during differentiation. Mol. Syst. Biol. 2013, 9, 689. [Google Scholar] [CrossRef]
- Schwanhausser, B.; Busse, D.; Li, N.; Dittmar, G.; Schuchhardt, J.; Wolf, J.; Chen, W.; Selbach, M. Global quantification of mammalian gene expression control. Nature 2011, 473, 337–342. [Google Scholar] [CrossRef] [Green Version]
- Bhat, M.; Robichaud, N.; Hulea, L.; Sonenberg, N.; Pelletier, J.; Topisirovic, I. Targeting the translation machinery in cancer. Nat. Rev. Drug Discov. 2015, 14, 261–278. [Google Scholar] [CrossRef] [PubMed]
- Li, X.; Wu, C.; Chen, N.; Gu, H.; Yen, A.; Cao, L.; Wang, E.; Wang, L. PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma. Oncotarget 2016, 7, 33440–33450. [Google Scholar] [CrossRef] [Green Version]
- Chen, H.-Y.; Lin, L.-T.; Wang, M.-L.; Tsai, K.-L.; Huang, P.-I.; Yang, Y.-P.; Lee, Y.-Y.; Chen, Y.-W.; Lo, W.-L.; Lan, Y.-T.; et al. Musashi-1 promotes chemoresistant granule formation by PKR/eIF2alpha signalling cascade in refractory glioblastoma. Biochim. Biophys. Acta Mol. Basis Dis. 2018, 1864 Pt A, 1850–1861. [Google Scholar] [CrossRef]
- Dadey, D.Y.A.; Kapoor, V.; Khudanyan, A.; Thotala, D.; Hallahan, D.E. PERK Regulates Glioblastoma Sensitivity to ER Stress Although Promoting Radiation Resistance. Mol. Cancer Res. 2018, 16, 1447–1453. [Google Scholar] [CrossRef]
- Brennan, C.W.; Verhaak, R.G.W.; Mckenna, A.; Campos, B.; Noushmehr, H.; Salama, S.R.; Zheng, S.; Chakravarty, D.; Sanborn, J.Z.; Berman, S.H.; et al. The Somatic Genomic Landscape of Glioblastoma. Cell 2013, 155, 462–477. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Alvarenga, A.W.; Machado, L.E.; Rodrigues, B.R.; Lupinacci, F.C.S.; Sanemastu, P.; Matta, E.; Roffe, M.; Torres, L.F.B.; da Cunha, I.W.; Martins, V.R.; et al. Evaluation of Akt and RICTOR Expression Levels in Astrocytomas of All Grades. J. Histochem. Cytochem. 2017, 65, 93–103. [Google Scholar] [CrossRef]
- Sottoriva, A.; Spiteri, I.; Piccirillo, S.G.M.; Touloumis, A.; Collins, V.P.; Marioni, J.C.; Curtis, C.; Watts, C.; Tavare, S. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc. Natl. Acad. Sci. USA 2013, 110, 4009–4014. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Johnson, B.E.; Mazor, T.; Hong, C.; Barnes, M.; Aihara, K.; McLean, C.Y.; Fouse, S.D.; Yamamoto, S.; Ueda, H.; Tatsuno, K.; et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 2014, 343, 189–193. [Google Scholar] [CrossRef] [PubMed]
- Patel, A.P.; Tirosh, I.; Trombetta, J.J.; Shalek, A.K.; Gillespie, S.M.; Wakimoto, H.; Cahill, D.P.; Nahed, B.V.; Curry, W.T.; Martuza, R.L.; et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science (80-) 2014, 344, 1396–1401. [Google Scholar] [CrossRef] [Green Version]
- Szerlip, N.J.; Pedraza, A.; Chakravarty, D.; Azim, M.; McGuire, J.; Fang, Y.; Ozawa, T.; Holland, E.C.; Huse, J.T.; Jhanwar, S.; et al. Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response. Proc. Natl. Acad. Sci. USA 2012, 109, 3041–3046. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Francis, J.M.; Zhang, C.-Z.; Maire, C.L.; Jung, J.; Manzo, V.E.; Adalsteinsson, V.A.; Homer, H.; Haidar, S.; Blumenstiel, B.; Pedamallu, C.S.; et al. EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing. Cancer Discov. 2014, 4, 956–971. [Google Scholar] [CrossRef] [Green Version]
- Stevens, M.M.; Maire, C.L.; Chou, N.; Murakami, M.A.; Knoff, D.S.; Kikuchi, Y.; Kimmerling, R.J.; Liu, H.; Haidar, S.; Calistri, N.L.; et al. Drug sensitivity of single cancer cells is predicted by changes in mass accumulation rate. Nat. Biotechnol. 2016, 34, 1161–1167. [Google Scholar] [CrossRef] [Green Version]
- Reinartz, R.; Wang, S.; Kebir, S.; Silver, D.J.; Wieland, A.; Zheng, T.; Kupper, M.; Rauschenbach, L.; Fimmers, R.; Shepherd, T.M.; et al. Functional Subclone Profiling for Prediction of Treatment-Induced Intratumor Population Shifts and Discovery of Rational Drug Combinations in Human Glioblastoma. Clin. Cancer Res. 2017, 23, 562–574. [Google Scholar] [CrossRef]
- Larsson, O.; Sonenberg, N.; Nadon, R. Anota: Analysis of Differential Translation in Genome-Wide Studies. Bioinformatics 2011, 27, 1440–1441. [Google Scholar] [CrossRef]
- Szklarczyk, D.; Franceschini, A.; Wyder, S.; Forslund, K.; Heller, D.; Huerta-Cepas, J.; Simonovic, M.; Roth, A.; Santos, A.; Tsafou, K.P.; et al. STRING v10: Protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015, 43, D447–D452. [Google Scholar] [CrossRef] [PubMed]
- Supek, F.; Bosnjak, M.; Skunca, N.; Smuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 2011, 6, e21800. [Google Scholar] [CrossRef] [PubMed]
- Park, S.Y.; Piao, Y.; Jeong, K.J.; Dong, J.; de Groot, J.F. Periostin (POSTN) Regulates Tumor Resistance to Antiangiogenic Therapy in Glioma Models. Cancer Ther. 2016, 15, 2187–2197. [Google Scholar] [CrossRef] [PubMed]
- Zhou, W.; Ke, S.Q.; Huang, Z.; Flavahan, W.; Fang, X.; Paul, J.; Wu, L.; Sloan, A.E.; McLendon, R.E.; Li, X.; et al. Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth. Nat. Cell Biol. 2015, 17, 170–182. [Google Scholar] [CrossRef] [Green Version]
- Balss, J.; Meyer, J.; Mueller, W.; Korshunov, A.; Hartmann, C.; von Deimling, A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol. 2008, 116, 597–602. [Google Scholar] [CrossRef] [PubMed]
- Ingolia, N.T.; Brar, G.A.; Rouskin, S.; McGeachy, A.M.; Weissman, J.S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protoc. 2012, 7, 1534. [Google Scholar] [CrossRef]
- Masvidal, L.; Hulea, L.; Furic, L.; Topisirovic, I.; Larsson, O. mTOR-sensitive translation: Cleared fog reveals more trees. RNA Biol. 2017, 14, 1299–1305. [Google Scholar] [CrossRef] [Green Version]
- Helmy, K.; Halliday, J.; Fomchenko, E.; Setty, M.; Pitter, K.; Hafemeister, C.; Holland, E.C. Identification of global alteration of translational regulation in glioma in vivo. PLoS ONE 2012, 7, e46965. [Google Scholar] [CrossRef]
- Gonzalez, C.; Sims, J.S.; Hornstein, N.; Mela, A.; Garcia, F.; Lei, L.; Gass, D.A.; Amendolara, B.; Bruce, J.N.; Canoll, P.; et al. Ribosome profiling reveals a cell-type-specific translational landscape in brain tumors. J. Neurosci. 2014, 34, 10924–10936. [Google Scholar] [CrossRef]
- Meyuhas, O.; Kahan, T. The race to decipher the top secrets of TOP mRNAs. Biochim. Biophys. Acta 2015, 1849, 801–811. [Google Scholar] [CrossRef] [PubMed]
- Yamashita, R.; Suzuki, Y.; Takeuchi, N.; Wakaguri, H. Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics. Nucleic Acids Res. 2008, 36, 3707–3715. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hsieh, A.C.; Liu, Y.; Edlind, M.P.; Ingolia, N.T.; Janes, M.R.; Sher, A.; Shi, E.Y.; Stumpf, C.R.; Christensen, C.; Bonham, M.J.; et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 2012, 485, 55–61. [Google Scholar] [CrossRef] [PubMed]
- Machado, L.E.; Alvarenga, A.W.; da Silva, F.F.; Roffe, M.; Begnami, M.D.; Torres, L.F.B.; da Cunha, I.W.; Martins, V.R.; Hajj, G.N.M. Overexpression of mTOR and p(240-244)S6 in IDH1 Wild-Type Human Glioblastomas Is Predictive of Low Survival. J. Histochem. Cytochem. 2018, 66, 403–414. [Google Scholar] [CrossRef] [PubMed]
- Guo, X.; Xue, H.; Shao, Q.; Wang, J.; Guo, X.; Chen, X.; Zhang, J.; Xu, S.; Li, T.; Zhang, P.; et al. Hypoxia promotes glioma-associated macrophage infiltration via periostin and subsequent M2 polarization by upregulating TGF-beta and M-CSFR. Oncotarget 2016, 7, 80521–80542. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Montoya, M. Translation under hypoxia. Nat. Struct. Mol. Biol. 2012, 19, 602. [Google Scholar] [CrossRef]
- Louis, D.N.; Perry, A.; Reifenberger, G.; von Deimling, A.; Figarella, D.; Webster, B.; Hiroko, K.C.; Wiestler, O.D.; Kleihues, P.; Ellison, D.W. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: A summary. Acta Neuropathol. 2016, 131, 803–820. [Google Scholar] [CrossRef]
- Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef] [PubMed]
- Ritchie, M.E.; Phipson, B.; Wu, D.; Hu, Y.; Law, C.W.; Shi, W.; Smyth, G.K. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015, 43, e47. [Google Scholar] [CrossRef] [PubMed]
- Langfelder, P.; Horvath, S. WGCNA: An R package for weighted correlation network analysis. BMC Bioinform. 2008, 9, 559. [Google Scholar] [CrossRef] [PubMed]
- Johnson, W.E.; Li, C.; Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 2007, 8, 118–127. [Google Scholar] [CrossRef] [PubMed]
- Alvarenga, A.W.; Coutinho-Camillo, C.M.; Rodrigues, B.R.; Rocha, R.M.; Torres, L.F.B.; Martins, V.R.; da Cunha, I.W.; Hajj, G.N.M. A comparison between manual and automated evaluations of tissue microarray patterns of protein expression. J. Histochem. Cytochem. 2013, 61, 272–282. [Google Scholar] [CrossRef] [PubMed]
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Lupinacci, F.C.S.; Kuasne, H.; Roffé, M.; Vassalakis, J.A.; da Silva, F.F.; Santos, T.G.; Andrade, V.P.; Sanematsu, P.; Martins, V.R.; Rogatto, S.R.; et al. Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity. Int. J. Mol. Sci. 2019, 20, 2177. https://doi.org/10.3390/ijms20092177
Lupinacci FCS, Kuasne H, Roffé M, Vassalakis JA, da Silva FF, Santos TG, Andrade VP, Sanematsu P, Martins VR, Rogatto SR, et al. Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity. International Journal of Molecular Sciences. 2019; 20(9):2177. https://doi.org/10.3390/ijms20092177
Chicago/Turabian StyleLupinacci, Fernanda Cristina Sulla, Hellen Kuasne, Martin Roffé, Julia Avian Vassalakis, Fernanda Ferreira da Silva, Tiago Góss Santos, Victor Piana Andrade, Paulo Sanematsu, Vilma Regina Martins, Silvia Regina Rogatto, and et al. 2019. "Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity" International Journal of Molecular Sciences 20, no. 9: 2177. https://doi.org/10.3390/ijms20092177