Abstract
Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have attempted to identify the cellular components necessary to maintain the activity of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, using a whole genome approach involving RNAi-induced depletion of cellular polypeptides. We have used a pancreatic ductal adenocarcinoma (PDAC) cell line, Mia-PaCa for this screen; as with many pancreatic cancers, these cells exhibit constitutive activation of mTORC1. PDAC is the most common form of pancreatic cancer and the 5-year survival rate remains 3–5% despite current nonspecific and targeted therapies. Although rapamycin-related mTOR inhibitors have yet to demonstrate encouraging clinical responses, it is now evident that this class of compounds is capable of only partial mTORC1 inhibition. Identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new targets and lead to the development of beneficial therapies for pancreatic cancer.
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References
Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell.124: 471–84.
Liu Q, Thoreen C, Wang J, Sabatini D, Gray NS (2009) mTOR Mediated Anti-Cancer Drug Discovery. Drug Discov Today Ther Strateg. 6, 47–55.
Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N (2010) Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim Biophys Acta. 1804, 433–9.
Dancey J (2010) mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 7, 209–19.
Lane HA, Breuleux M (2009) Optimal targeting of the mTORC1 kinase in human cancer. Curr Opin Cell Biol. 21, 219–29.
Dowling RJ, Pollak M, Sonenberg N (2009) Current status and challenges associated with targeting mTOR for cancer therapy. BioDrugs. 23, 77–91.
Baldo P, Cecco S, Giacomin E, Lazzarini R, Ros B, Marastoni S (2008) mTOR pathway and mTOR inhibitors as agents for cancer therapy. Curr Cancer Drug Targets. 8, 647–65.
Avruch J, Hara K, Lin Y, Liu M, Long X, Ortiz-Vega S, Yonezawa K (2006) Insulin and amino-acid regulation of mTOR signaling and kinase activity through the Rheb GTPase. Oncogene. 25:6361–72.
Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, Avruch J (1998) Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem. 273, 14484–94.
Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N (2009) Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab. 296, E592–602.
Sancak Y, Sabatini DM (2009) Rag proteins regulate amino-acid-induced mTORC1 signalling. Biochem Soc Trans. 37, 289–90.
Iorns E, Lord CJ, Turner N, Ashworth A. (2007) Utilizing RNA interference to enhance cancer drug discovery. Nat Rev Drug Discov. 6, 556–68.
Gressner AM, Wool IG. (1974) The phosphorylation of liver ribosomal proteins in vivo. Evidence that only a single small subunit protein (S6) is phosphorylated. J Biol Chem. 249, 6917–25.
Bandi HR, Ferrari S, Krieg J, Meyer HE, Thomas G (1993) Identification of 40S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum. J Biol Chem. 268, 4530–3.
Ferrari S, Bandi HR, Hofsteenge J, Bussian BM, Thomas G (1991) Mitogen-activated 70KS6 kinase. Identification of in vitro 40S ribosomal S6 phosphorylation sites. J Biol Chem. 266, 22770–5.
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, 3112–24.
Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE (1992) Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science 257, 973–7.
Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J, Yonezawa K. (1999) Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J Biol Chem. 274, 34493–8.
Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J. (1998) 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol, 8, 69–81.
Weng QP, Kozlowski M, Belham C, Zhang A, Comb MJ, Avruch J. (1998) Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J Biol Chem. 273, 16621–9.
Ruvinsky I, Katz M, Dreazen A, Gielchinsky Y, Saada A, Freedman N, Mishani E, Zimmerman G, Kasir J, Meyuhas O (2009) Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit. PLoS One. 19; 4(5):e5618.
Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, Reichling LJ, Sim T, Sabatini DM, Gray NS (2009) An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem. 284, 8023–32.
Yunis AA, Arimura GK, Russin DJ (1977) Human pancreatic carcinoma (MIA PaCa-2) in continuous culture: sensitivity to asparaginase. Int J Cancer. 19, 128–35.
Sipos B, Möser S, Kalthoff H, Török V, Löhr M, Klöppel G (2003) A comprehensive characterization of pancreatic ductal carcinoma cell lines: towards the establishment of an in vitro research platform. Virchows Arch. 442, 444–52.
Grewe M, Gansauge F, Schmid RM, Adler G, Seufferlein T (1999) Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells. Cancer Res. 59, 3581–7.
Ekins S, Nikolsky Y, Bugrim A, Kirillov E, Nikolskaya T (2007) Pathway mapping tools for analysis of high content data. Methods Mol Biol. 356, 19–50.
Ganter B, Giroux CN. (2008) Emerging applications of network and pathway analysis in drug discovery and development. Curr Opin Drug Discov Devel. 11, 86–94.
Phelan MC (2006) Techniques for Mammalian Cell Culture. Curr Protocols Hum. Genet. Appendix 3.
Acknowledgments
This work is supported by NIH awards CA73818 and DK17776 and the Massachusetts General Hospital “ECOR Fund for Medical Discovery.”
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Papageorgiou, A., Avruch, J. (2012). A Genome-wide RNAi Screen for Polypeptides that Alter rpS6 Phosphorylation. In: Weichhart, T. (eds) mTOR. Methods in Molecular Biology, vol 821. Humana Press. https://doi.org/10.1007/978-1-61779-430-8_11
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DOI: https://doi.org/10.1007/978-1-61779-430-8_11
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