Abstract
Development of novel patient stratification tools for cancer is a challenge that require advanced molecular screening and a detailed understanding of tumour signalling networks. Here, we apply phospho-specific flow cytometry for signal profiling of primary glioblastoma tumours after preservation of single-cell phosphorylation status as a strategy for evaluation of tumour signalling potential and assessment of rapamycin-mediated mTOR inhibition. The method has already enhanced insight into cancers and disorders of the immune system, and our study demonstrate a great potential to improve the understanding of aberrant signalling in glioblastoma and other solid tumours.
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Akhavan D, Cloughesy TF, Mischel PS (2010) mTOR signaling in glioblastoma: lessons learned from bench to bedside. Neuro Oncol 12:882–889
Benito R, Gil-Benso R, Quilis V, Perez M, Gregori-Romero M, Roldan P, Gonzalez-Darder J, Cerda-Nicolas M, Lopez-Gines C (2010) Primary glioblastomas with and without EGFR amplification: relationship to genetic alterations and clinicopathological features. Neuropathology 30:392–400
Chalhoub N, Baker SJ (2009) PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol 4:127–150
Choe G, Horvath S, Cloughesy TF, Crosby K, Seligson D, Palotie A, Inge L, Smith BL, Sawyers CL, Mischel PS (2003) Analysis of the phosphatidylinositol 3′-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 63:2742–2746
Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S, Hsueh T, Chen Y, Wang W, Youngkin D, Liau L, Martin N, Becker D, Bergsneider M, Lai A, Green R, Oglesby T, Koleto M, Trent J, Horvath S, Mischel PS, Mellinghoff IK, Sawyers CL (2008) Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med 5:e8
Ekstrand AJ, Longo N, Hamid ML, Olson JJ, Liu L, Collins VP, James CD (1994) Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplification. Oncogene 9:2313–2320
Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C, Chin L, DePinho RA, Cavenee WK (2007) Malignant astrocytic glioma: genetics, biology and paths to treatment. Genes Dev 21:2683–2710
Gallia GL, Rand V, Siu IM, Eberhart CG, James CD, Marie SK, Oba-Shinjo SM, Carlotti CG, Caballero OL, Simpson AJ, Brock MV, Massion PP, Carson BS, Riggins GJ (2006) PIK3CA gene mutations in pediatric and adult glioblastoma multiforme. Mol Cancer Res 4:709–714
Guertin DA, Sabatini DM (2009) The pharmacology of mTOR inhibition. Sci Signal 2:e24
Kanu OO, Mehta A, Di C, Lin N, Bortoff K, Bigner DD, Yan H, Adamson DC (2009) Glioblastoma multiforme: a review of therapeutic targets. Expert Opin Ther Targets 13:701–718
Kreisl TN, Lassman AB, Mischel PS, Rosen N, Scher HI, Teruya-Feldstein J, Shaffer D, Lis E, Abrey LE (2009) A pilot study of everolimus and gefitinib in the treatment of recurrent glioblastoma (GBM). J Neurooncol 92:99–105
Krutzik PO, Clutter MR, Trejo A, Nolan GP (2011) Fluorescent cell barcoding for multiplex flow cytometry. Curr Protoc Cytom. doi:10.1002/0471142956.cy0631s55
Krutzik PO, Nolan GP (2006) Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3:361–368
Lurje G, Lenz HJ (2009) EGFR signaling and drug discovery. Oncology 77:400–410
Masui K, Cloughesy TF, Mischel PS (2012) Review: molecular pathology in adult high-grade gliomas: from molecular diagnostics to target therapies. Neuropathol Appl Neurobiol 38:271–291
Mellinghoff IK, Wang MY, Vivanco I, Haas-Kogan DA, Zhu S, Dia EQ, Lu KV, Yoshimoto K, Huang JH, Chute DJ, Riggs BL, Horvath S, Liau LM, Cavenee WK, Rao PN, Beroukhim R, Peck TC, Lee JC, Sellers WR, Stokoe D, Prados M, Cloughesy TF, Sawyers CL, Mischel PS (2005) Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 353:2012–2024
Merlo V, Longo M, Novello S, Scagliotti GV (2011) EGFR pathway in advanced non-small cell lung cancer. Front Biosci (Schol Ed) 3:501–517
Miller TW, Rexer BN, Garrett JT, Arteaga CL (2011) Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res 13:224
Mischel PS, Shai R, Shi T, Horvath S, Lu KV, Choe G, Seligson D, Kremen TJ, Palotie A, Liau LM, Cloughesy TF, Nelson SF (2003) Identification of molecular subtypes of glioblastoma by gene expression profiling. Oncogene 22:2361–2373
Ogino S, Galon J, Fuchs CS, Dranoff G (2011) Cancer immunology-analysis of host and tumor factors for personalized medicine. Nat Rev Clin Oncol 8:711–719
Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J, Mueller M, Fumagalli S, Kozma SC, Thomas G (2004) S6K1(−/−)/S6K2(−/−) mice exhibit perinatal lethality and rapamycin-sensitive 5′-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol Cell Biol 24:3112–3124
Podsypanina K, Lee RT, Politis C, Hennessy I, Crane A, Puc J, Neshat M, Wang H, Yang L, Gibbons J, Frost P, Dreisbach V, Blenis J, Gaciong Z, Fisher P, Sawyers C, Hedrick-Ellenson L, Parsons R (2001) An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten ± mice. Proc Natl Acad Sci USA 98:10320–10325
Preusser M, de Ribaupierre S, Wohrer A, Erridge SC, Hegi M, Weller M, Stupp R (2011) Current concepts and management of glioblastoma. Ann Neurol 70:9–21
Shi Y, Gera J, Hu L, Hsu JH, Bookstein R, Li W, Lichtenstein A (2002) Enhanced sensitivity of multiple myeloma cells containing PTEN mutations to CCI-779. Cancer Res 62:5027–5034
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996
Sugawa N, Ekstrand AJ, James CD, Collins VP (1990) Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc Natl Acad Sci USA 87:8602–8606
Acknowledgments
The authors wish to thank Jorun Solheim for work on sequencing of the tumour samples. The work was supported by grants from the Norwegian Cancer Society, South-Eastern Regional Health Authority and the Research Council of Norway.
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The authors declare that they have no conflict of interest.
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Isabelle Cornez and Mrinal Joel contributed equally to the study.
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Cornez, I., Joel, M., Taskén, K. et al. EGF signalling and rapamycin-mediated mTOR inhibition in glioblastoma multiforme evaluated by phospho-specific flow cytometry. J Neurooncol 112, 49–57 (2013). https://doi.org/10.1007/s11060-012-1035-9
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DOI: https://doi.org/10.1007/s11060-012-1035-9