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Identification of two novel inhibitors of mTOR signaling pathway based on high content screening

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Abstract

Purpose

Mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating cell growth, proliferation and survival. Dysregulation of mTOR signaling pathway is closely involved in cancer development and chemotherapy resistance. Inhibitors of mTOR signaling pathway have been demonstrated to be attractive therapeutics for cancer therapy. In the present study, we aim to discover novel mTOR signaling pathway inhibitors from a natural compound library.

Methods

Inhibitors of mTOR signaling pathway were discovered via high content screen assay based on the subcellular localization of eukaryotic initiation factor 4E (eIF4E) in mouse embryonic fibroblast cells. Candidate compounds were further assessed in cancer cells. Phosphorylation levels of mTOR complexes downstream targets were analyzed using Western blot. Cell cytotoxicity and apoptosis were evaluated using MTS assay and flow cytometry, respectively.

Results

Two compounds, 1,4-O-diferuloylsecoisolariciresinol (IM-1) and Pierreione B (IM-2), were identified which induced significant nuclear translocation of eIF4E in a panel of cancer cells. Both of the compounds decreased the phosphorylation levels of p70 ribosomal protein S6 kinase (S6K) and eIF4E binding protein 1 (4E-BP1), resulting in cancer cell cytotoxicity and apoptosis.

Conclusions

Via high content screen assay, two novel inhibitors of mTOR signaling, IM-1 and IM-2, were identified with strong anticancer activity. IM-1 and IM-2 could be potential candidates for anticancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.

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References

  1. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev 12(1):21–35. doi:10.1038/nrm3025

    Article  CAS  Google Scholar 

  2. Shen CX, Lancaster CS, Shi B, Guo H, Thimmaiah P, Bjornsti MA (2007) TOR signaling is a determinant of cell survival in response to DNA damage. Mol Cell Biol 27(20):7007–7017. doi:10.1128/Mcb.00290-07

    Article  PubMed  CAS  Google Scholar 

  3. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 127(3):5–19. doi:10.1016/j.cell.2006.01.016

    Google Scholar 

  4. Dufour M, Dormond-Meuwly A, Demartines N, Dormond O (2011) Targeting the mammalian target of rapamycin (mTOR) in cancer therapy: lessons from past and future perspectives. Cancers 3(2):2478–2500. doi:10.3390/cancers3022478

    Article  CAS  Google Scholar 

  5. Populo H, Lopes JM, Soares P (2012) The mTOR signalling pathway in human cancer. Int J Mol Sci 13(2):1886–1918. doi:10.3390/Ijms13021886

    Article  PubMed  CAS  Google Scholar 

  6. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14(14):1296–1302. doi:10.1016/j.cub.2004.06.054

    Article  PubMed  CAS  Google Scholar 

  7. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307(5712):1098–1101. doi:10.1126/science.1106148

    Article  PubMed  CAS  Google Scholar 

  8. Holz MK, Ballif BA, Gygi SP, Blenis J (2005) mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 123:569–580

    Article  PubMed  CAS  Google Scholar 

  9. Gingras AC, Raught B, Gygi SP, Niedzwiecka A, Miron M, Burley SK, Polakiewicz RD, Wyslouch-Cieszynska A, Aebersold R, Sonenberg N (2001) Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Gene Dev 15(21):2852–2864. doi:10.1101/gad.912401

    PubMed  CAS  Google Scholar 

  10. Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC Jr, Sonenberg N (1994) Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5′-cap function. Nature 371(6500):762–767. doi:10.1038/371762a0

    Article  PubMed  CAS  Google Scholar 

  11. Livingstone Mark, Bidinosti M (2012) Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding. Research F1000:1–11. doi:10.3410/f1000research.1-4.v1

    Google Scholar 

  12. Bhat M, Sonenberg N, Gores G (2013) The mTOR pathway in hepatic malignancies. Hepatology. doi:10.1002/hep.26323

    PubMed  Google Scholar 

  13. Culjkovic B, Tan K, Orolicki S, Amri A, Meloche S, Borden KLB (2008) The eIF4E RNA regulon promotes the Akt signaling pathway. J Cell Biol 181(1):51–63. doi:10.1083/jcb.200707018

    Article  PubMed  CAS  Google Scholar 

  14. Fischer PM (2009) Cap in hand: targeting eIF4E. Cell Cycle 8(16):2535–2541

    Article  PubMed  CAS  Google Scholar 

  15. Yuan R, Kay A, Berg WJ, Lebwohl D (2009) Targeting tumorigenesis: development and use of mTOR inhibitors in cancer therapy. J Hematol Oncol 2:45. doi:10.1186/1756-8722-2-45

    Article  PubMed  Google Scholar 

  16. Huang S, Houghton PJ (2003) Targeting mTOR signaling for cancer therapy. Curr Opin Pharmacol 3(4):371–377

    Article  PubMed  CAS  Google Scholar 

  17. Petroulakis E, Mamane Y, Le Bacquer O, Shahbazian D, Sonenberg N (2006) mTOR signaling: implications for cancer and anticancer therapy. Brit J Cancer 94(2):195–199. doi:10.1038/sj.bjc.6602902

    Article  PubMed  CAS  Google Scholar 

  18. Dobashi Y, Suzuki S, Kimura M, Matsubara H, Tsubochi H, Imoto I, Ooi A (2011) Paradigm of kinase-driven pathway downstream of epidermal growth factor receptor/Akt in human lung carcinomas. Hum Pathol 42(2):214–226. doi:10.1016/j.humpath.2010.05.025

    Article  PubMed  CAS  Google Scholar 

  19. Burris HA 3rd (2013) Overcoming acquired resistance to anticancer therapy: focus on the PI3K/AKT/mTOR pathway. Cancer Chemother Pharmacol. doi:10.1007/s00280-012-2043-3

    Google Scholar 

  20. Zaytseva YY, Valentino JD, Gulhati P, Evers BM (2012) mTOR inhibitors in cancer therapy. Cancer Lett 319(1):1–7. doi:10.1016/j.canlet.2012.01.005

    Article  PubMed  CAS  Google Scholar 

  21. Nicholson KM, Anderson NG (2002) The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 14(5):381–395

    Article  PubMed  CAS  Google Scholar 

  22. Avellino R, Romano S, Parasole R, Bisogni R, Lamberti A, Poggi V, Venuta S, Romano MF (2005) Rapamycin stimulates apoptosis of childhood acute lymphoblastic leukemia cells. Blood 106(4):1400–1406. doi:10.1182/blood-2005-03-0929

    Article  PubMed  CAS  Google Scholar 

  23. Zaytseva YY, Valentino JD, Gulhati P, Evers BM (2012) mTOR inhibitors in cancer therapy. Cancer Lett 319(1):1–7. doi:10.1016/j.canlet.2012.01.005

    Article  PubMed  CAS  Google Scholar 

  24. Zhou H, Luo Y, Huang S (2010) Updates of mTOR inhibitors. Anticancer Agents Med Chem 10(7):571–581. doi:10.2174/187152010793498663

    Article  PubMed  CAS  Google Scholar 

  25. Punt CJ, Boni J, Bruntsch U, Peters M, Thielert C (2003) Phase I and pharmacokinetic study of CCI-779, a novel cytostatic cell-cycle inhibitor, in combination with 5-fluorouracil and leucovorin in patients with advanced solid tumors. Ann Oncol 14(6):931–937

    Article  PubMed  CAS  Google Scholar 

  26. Sessa C, Tosi D, Vigano L, Albanell J, Hess D, Maur M, Cresta S, Locatelli A, Angst R, Rojo F, Coceani N, Rivera VM, Berk L, Haluska F, Gianni L (2010) Phase Ib study of weekly mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) with weekly paclitaxel. Ann Oncol 21(6):1315–1322

    Google Scholar 

  27. Amato RJ, Jac J, Giessinger S, Saxena S, Willis JP (2009) A phase 2 study with a daily regimen of the oral mTOR inhibitor RAD001 (everolimus) in patients with metastatic clear cell renal cell cancer. Cancer 115(11):2438–2446

    Article  PubMed  CAS  Google Scholar 

  28. Eynott PR, Salmon M, Huang TJ, Oates T, Nicklin PL, Chung KF (2003) Effects of cyclosporin A and a rapamycin derivative (SAR943) on chronic allergic inflammation in sensitized rats. Immunology 109(3):461–467

    Article  PubMed  CAS  Google Scholar 

  29. Fernandes J, Castilho RO, da Costa MR, Wagner-Souza K, Kaplan MAC, Gattass CR (2003) Pentacyclic triterpenes from Chrysobalanaceae species: cytotoxicity on multidrug resistant and sensitive leukemia cell lines. Cancer Lett 190(2):165–169. doi:10.1016/S0304-3835(02)00593-1

    Article  PubMed  CAS  Google Scholar 

  30. Beevers CS, Chen L, Liu L, Luo Y, Webster NJG, Huang S (2009) Curcumin disrupts the mammalian target of rapamycin-raptor complex. Cancer Res 69(3):1000–1008. doi:10.1158/0008-5472.Can-08-2367

    Article  PubMed  CAS  Google Scholar 

  31. Don AS, Zheng XF (2011) Recent clinical trials of mTOR-targeted cancer therapies. Rev Recent Clin Trials 6(1):24–35

    Article  PubMed  Google Scholar 

  32. Gingras AC, Kennedy SG, O’Leary MA, Sonenberg N, Hay N (1998) 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Gene Dev 12(4):502–513

    Article  PubMed  CAS  Google Scholar 

  33. Hsieh AC, Ruggero D (2010) Targeting eukaryotic translation initiation factor 4E (eIF4E) in cancer. Clin Cancer Res 16(20):4914–4920. doi:10.1158/1078-0432.Ccr-10-0433

    Article  PubMed  CAS  Google Scholar 

  34. Rong L, Livingstone M, Sukarieh R, Petroulakis E, Gingras AC, Crosby K, Smith B, Polakiewicz RD, Pelletier J, Ferraiuolo MA, Sonenberg N (2008) Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs. RNA 14(7):1318–1327. doi:10.1261/Rna.950608

    Article  PubMed  CAS  Google Scholar 

  35. Oh N, Kim KM, Cho H, Choe J, Kim YK (2007) Pioneer round of translation occurs during serum starvation. Biochem Biophys Res Co 362(1):145–151. doi:10.1016/j.bbrc.2007.07.169

    Article  CAS  Google Scholar 

  36. Livingstone M, Larsson O, Sukarieh R, Pelletier J, Sonenberg N (2009) A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E. Chem Biol 16(12):1240–1249. doi:10.1016/j.chembiol.2009.11.010

    Article  PubMed  CAS  Google Scholar 

  37. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KL (2005) eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3′UTR. J Cell Biol 169(2):245–256. doi:10.1083/jcb.200501019

    Article  PubMed  CAS  Google Scholar 

  38. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KLB (2006) eIF4E is a central node of an RNA regulon that governs cellular proliferation. J Cell Biol 175(3):415–426. doi:10.1083/jcb.200604099

    Article  PubMed  CAS  Google Scholar 

  39. Konicek BW, Dumstorf CA, Graff JR (2008) Targeting the eIF4F translation initiation complex for cancer therapy. Cell Cycle 7(16):2466–2471

    Article  PubMed  CAS  Google Scholar 

  40. De Benedetti A, Graff JR (2004) eIF-4E expression and its role in malignancies and metastases. Oncogene 23(18):3189–3199. doi:10.1038/sj.onc.1207545

    Article  PubMed  Google Scholar 

  41. Livingstone M (2010) A nuclear role for the eukaryotic translation initiation factor 4E-binding proteins. Dissertation, McGill University

  42. O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N (2006) mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66(3):1500–1508. doi:10.1158/0008-5472.Can-05-2925

    Article  PubMed  Google Scholar 

  43. Moon SS, Rahman AA, Kim JY, Kee SH (2008) Hanultarin, a cytotoxic lignan as an inhibitor of actin cytoskeleton polymerization from the seeds of Trichosanthes kirilowii. Bioorg Med Chem 16(15):7264–7269. doi:10.1016/j.bmc.2008.06.032

    Article  PubMed  CAS  Google Scholar 

  44. Gao S, Xu YM, Valeriote FA, Gunatilaka AAL (2011) Pierreiones A–D, solid tumor selective pyranoisoflavones and other cytotoxic constituents from antheroporum pierrei. J Nat Prod 74(4):852–856. doi:10.1021/Np100763p

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This project was supported financially by the hundreds of talents program (Y.L) and the west light foundation (H.Z) of the Chinese Academy of Sciences, the Major State Basic Research Development Program of China (No. 2009CB522300), the NSFC (No. 81173076) and the project of recruited top talent of science and technology of Yunnan Province (2009C1120).

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Authors and Affiliations

  1. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China

    Juming Yan, Hongyu Zhou, Lingmei Kong, Jing Zhang, Qinshi Zhao & Yan Li

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Juming Yan, Lingmei Kong & Jing Zhang

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  1. Juming Yan
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  2. Hongyu Zhou
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  3. Lingmei Kong
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Correspondence to Yan Li.

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Yan, J., Zhou, H., Kong, L. et al. Identification of two novel inhibitors of mTOR signaling pathway based on high content screening. Cancer Chemother Pharmacol 72, 799–808 (2013). https://doi.org/10.1007/s00280-013-2255-1

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