Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures

Proteome Science

Keith A Baggerly²  Li Shen²  Sonali Panchabhai¹  Victor A Levin¹ 

[1] Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA;Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

关键词: pancreatic cancer;    ovarian cancer;    breast cancer;    glioblastoma;   

Others  :  817625
DOI  :  10.1186/1477-5956-10-5

 received in 2011-08-29, accepted in 2012-01-25,  发布年份 2012

【 摘 要 】

Background

Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions.

Results

Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly (i.e., > 50% of tumors) elevated in 3D: FAK, AKT, Src, GSK3αβ, TSC2, p38, and NFκβp65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, β-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas (BAX, caspase 7, HIF-1α, c-JUN, MEK1, PARP 1 cleaved, Src, and VEGFR2) and none in adenocarcinomas.

Conclusions

We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter.

【 授权许可】

   
2012 Levin et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Levin VA, Panchabhai SC, Shen L, Kornblau SM, Qiu Y, Baggerly KA: Different changes in protein and phosphoprotein levels result from serum starvation of high-grade glioma and adenocarcinoma cell lines. J Proteome Res 2010, 9(1):179-191.
• [2]Höckel M, Vaupel P: Tumor Hypoxia: Definitions and Current Clinical, Biologic, and Molecular Aspects. J Natl Cancer Inst 2001, 93(4):266-276.
• [3]Sutherland R, Freyer J, Mueller-Klieser W, Wilson R, Heacock C, Sciandra J, Sordat B: Cellular growth and metabolic adaptations to nutrient stress environments in tumor microregions. Int J Radiat Oncol Biol Phys 1986, 12(4):611-615.
• [4]Fodale V, Pierobon M, Liotta L, Petricoin E: Mechanism of cell adaptation: when and how do cancer cells develop chemoresistance? CANCER JOURNAL 2011, 17(2):89-95.
• [5]Fang JS, Gillies RD, Gatenby RA: Adaptation to hypoxia and acidosis in carcinogenesis and tumor progression. Seminars in cancer biology 2008, 18(5):330-337.
• [6]Malaise EP, Fertil B, Chavaudra N, Guichard M: Distribution of radiation sensitivities for human tumor cells of specific histological types: comparison of in vitro to in vivo data. Int J Radiat Oncol Biol Phys 1986, 12(4):617-624.
• [7]Coleman CN: Hypoxia in tumors: a paradigm for the approach to biochemical and physiologic heterogeneity. J Natl Cancer Inst 1988, 80(5):310-317.
• [8]Yamada KM, Cukierman E: Modeling tissue morphogenesis and cancer in 3D. Cell 2007, 130(4):601-610.
• [9]Kim JB: Three-dimensional tissue culture models in cancer biology. Seminars in cancer biology 2005, 15(5):365-377.
• [10]Pampaloni F, Reynaud EG, Stelzer EH: The third dimension bridges the gap between cell culture and live tissue. Nature reviews Molecular cell biology 2007, 8(10):839-845.
• [11]Horning JL, Sahoo SK, Vijayaraghavalu S, Dimitrijevic S, Vasir JK, Jain TK, Panda AK, Labhasetwar V: 3-D tumor model for in vitro evaluation of anticancer drugs. Molecular pharmaceutics 2008, 5(5):849-862.
• [12]Durand RE, Olive PL: Resistance of tumor cells to chemo- and radiotherapy modulated by the three-dimensional architecture of solid tumors and spheroids. In Methods in Cell Biology. Volume 64. Edited by Darzynkiewicz Z, Crissman HA, Robinson JP. San Diego: Academic Press Inc; 2001::211-233.
• [13]Sminia P, Acker H, Eikesdal HP, Kaaijk P, Enger P, Slotman B, Bjerkvig R: Oxygenation and response to irradiation of organotypic multicellular spheroids of human glioma. Anticancer Res 2003, 23(2B):1461-1466.
• [14]Terzis AJ, Dietze A, Bjerkvig R, Arnold H: Effects of photodynamic therapy on glioma spheroids. Br J Neurosurg 1997, 11(3):196-205.
• [15]Sutherland RM, Sordat B, Bamat J, Gabbert H, Bourrat B, Mueller-Klieser W: Oxygenation and differentiation in multicellular spheroids of human colon carcinoma. Cancer Res 1986, 46(10):5320-5329.
• [16]Freyer JP, Sutherland RM: Proliferative and clonogenic heterogeneity of cells from EMT6/Ro multicellular spheroids induced by the glucose and oxygen supply. Cancer Res 1986, 46(7):3513-3520.
• [17]Chapman JD: Measurement of tumor hypoxia by invasive and non-invasive procedures: a review of recent clinical studies. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology 1991, 20(Suppl 1):13-19.
• [18]Vaupel P, Schlenger K, Knoop C, Hockel M: Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res 1991, 51(12):3316-3322.
• [19]Kunz M, Ibrahim SM: Molecular responses to hypoxia in tumor cells. Mol Cancer 2003, 2:23. BioMed Central Full Text
• [20]Harris AL: Hypoxia--a key regulatory factor in tumour growth. Nature reviews Cancer 2002, 2(1):38-47.
• [21]Koh MY, Darnay BG, Powis G: Hypoxia-associated factor, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible factor 1alpha, leading to its oxygen-independent degradation. Mol Cell Biol 2008, 28(23):7081-7095.
• [22]Sano K, Hayakawa A, Piao JH, Kosaka Y, Nakamura H: Novel SH3 protein encoded by the AF3p21 gene is fused to the mixed lineage leukemia protein in a therapy-related leukemia with t(3;11) (p21;q23). Blood 2000, 95(3):1066-1068.
• [23]Fukuoka M, Suetsugu S, Miki H, Fukami K, Endo T, Takenawa T: A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42. J Cell Biol 2001, 152(3):471-482.
• [24]Lim CS, Kim SH, Jung JG, Kim JK, Song WK: Regulation of SPIN90 phosphorylation and interaction with Nck by ERK and cell adhesion. J Biol Chem 2003, 278(52):52116-52123.
• [25]Ronty M, Taivainen A, Heiska L, Otey C, Ehler E, Song WK, Carpen O: Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling. Exp Cell Res 2007, 313(12):2575-2585.
• [26]Kim Y, Chang S: Ever-expanding network of dynamin-interacting proteins. Mol Neurobiol 2006, 34(2):129-136.
• [27]Kim DJ, Kim SH, Lim CS, Choi KY, Park CS, Sung BH, Yeo MG, Chang S, Kim JK, Song WK: Interaction of SPIN90 with the Arp2/3 complex mediates lamellipodia and actin comet tail formation. J Biol Chem 2006, 281(1):617-625.
• [28]Mendez O, Zavadil J, Esencay M, Lukyanov Y, Santovasi D, Wang SC, Newcomb EW, Zagzag D: Knock down of HIF-1alpha in glioma cells reduces migration in vitro and invasion in vivo and impairs their ability to form tumor spheres. Mol Cancer 2010, 9:133. BioMed Central Full Text
• [29]Zagzag D, Esencay M, Mendez O, Yee H, Smirnova I, Huang Y, Chiriboga L, Lukyanov E, Liu M, Newcomb EW: Hypoxia- and vascular endothelial growth factor-induced stromal cell-derived factor-1alpha/CXCR4 expression in glioblastomas: one plausible explanation of Scherer's structures. Am J Pathol 2008, 173(2):545-560.
• [30]Zagzag D, Lukyanov Y, Lan L, Ali MA, Esencay M, Mendez O, Yee H, Voura EB, Newcomb EW: Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion. Lab Invest 2006, 86(12):1221-1232.
• [31]Wouters BG, Koritzinsky M: Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nature reviews Cancer 2008, 8(11):851-864.
• [32]Kreisl TN, Lassman AB, Mischel PS, Rosen N, Scher HI, Teruya-Feldstein J, Shaffer D, Lis E, Abrey LE: A pilot study of everolimus and gefitinib in the treatment of recurrent glioblastoma (GBM). J Neurooncol 2009, 92(1):99-105.
• [33]Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S, Hsueh T, Chen Y, Wang W, Youngkin D, et al.: Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med 2008, 5(1):e8.
• [34]Galanis E, Buckner JC, Maurer MJ, Kreisberg JI, Ballman K, Boni J, Peralba JM, Jenkins RB, Dakhil SR, Morton RF, et al.: Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 2005, 23(23):5294-5304.
• [35]Chang SM, Wen P, Cloughesy T, Greenberg H, Schiff D, Conrad C, Fink K, Robins HI, De Angelis L, Raizer J, et al.: Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme. Invest New Drugs 2005, 23(4):357-361.
• [36]Levin VA, Tada K, Mircean C: Lysate Array Analyses of Signal Transduction Inhibitors in Tumor Cell Lines. Clin Proteomics 2006, 2:33-44.
• [37]Kornblau SM, Tibes R, Qiu Y, Chen W, Kantarjian HM, Andreeff M, Coombes KR, Mills GB: Functional proteomic profiling of AML predicts response and survival. Blood 2008. blood-2007-2010-119438
• [38]Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, Kornblau SM: Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Mol Cancer Ther 2006, 5(10):2512-2521.
• [39]Hu J, He X, Baggerly KA, Coombes KR, Hennessy BT, Mills GB: Non-parametric quantification of protein lysate arrays. Bioinformatics 2007, 23(15):1986-1994.