Abstract
Mammalian target of rapamycin (mTOR) signaling is often aberrantly activated, particularly when genetically altered, in human cancers. mTOR inhibitors targeting the activated mTOR signaling are highly promising anti-cancer drugs. Knowing the activating genetic change in mTOR can help guide the use of mTOR inhibitors for cancer treatment. This study was conducted to identify and characterize novel oncogenic mTOR mutations that can potentially be therapeutic targets in human cancer. We sequenced 30 exons of the mTOR gene in 12 thyroid cancer cell lines, 3 melanoma cell lines, 20 anaplastic thyroid cancer (ATC) tumors, and 23 melanoma tumors and functionally characterized the identified novel mTOR mutations in vitro and in vivo. We identified a novel point mutation A1256G in ATC cell line and G7076A in melanoma tumor in exon 9 and exon 51 of the mTOR gene, respectively. Over-expression of the corresponding mTOR mutants H419R and G2359E created through induced mutagenesis showed markedly elevated protein kinase activities associated with the activation of mTOR/p70S6K signaling in HEK293T cells. Stable expression of the two mTOR mutants in NIH3T3 cells strongly activated the mTOR/p70S6K signaling pathway and induced morphologic transformation, cell focus formation, anchorage-independent cell growth, and invasion. Inoculation of these mutant-expressing cells in athymic nude mice induced rapid tumor development, showing their driving oncogenicity. We also demonstrated that transfection with the novel mutants conferred cells high sensitivities to the mTOR inhibitor temsirolimus. We speculate that human cancers harboring these mTOR mutations, such as ATC and melanoma, may be effectively treated with inhibitors targeting mTOR.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93.
Paquette M, El-Houjeiri L, Pause A. mTOR pathways in cancer and autophagy. Cancers. 2018;10:E18.
Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005;307:1098–101.
Karbowniczek M, Spittle CS, Morrison T, Wu H, Henske EP. mTOR is activated in the majority of malignant melanomas. J Invest Dermatol. 2008;128:980–7.
Souza EC, Ferreira AC, Carvalho DP. The mTOR protein as a target in thyroid cancer. Expert Opin Ther Targets. 2011;15:1099–112.
Janku F, Yap TA, Meric-Bernstam F. Targeting the PI3K pathway in cancer: are we making headway? Nat Rev Clin Oncol. 2018;15:273–91.
Sekulić A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res. 2000;60:3504–13.
Urano J, Sato T, Matsuo T, Otsubo Y, Yamamoto M, Tamanoi F. Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. Proc Natl Acad Sci USA. 2007;104:3514–9.
Ohne Y, Takahara T, Hatakeyama R, Matsuzaki T, Noda M, Mizushima N, et al. Isolation of hyperactive mutants of mammalian target of rapamycin. J Biol Chem. 2008;283:31861–70.
Edinger AL, Thompson CB. An activated mTOR mutant supports growth factor-independent, nutrient-dependent cell survival. Oncogene. 2004;23:5654–63.
Sato T, Nakashima A, Guo L, Coffman K, Tamanoi F. Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer. Oncogene. 2010;29:2746–52.
Murugan AK, Alzahrani A, Xing M. Mutations in critical domains confer the human mTOR gene strong tumorigenicity. J Biol Chem. 2013;288:6511–21.
Xing M. Genetic alterations in the phosphatidylinositol-3 kinase/Akt pathway in thyroid cancer. Thyroid. 2010;20:697–706.
Berven LA, Crouch MF. Cellular function of p70S6K: a role in regulating cell motility. Immunol Cell Biol. 2000;78:447–51.
Kong Y, Si L, Li Y, Wu X, Xu X, Dai J, et al. Analysis of mTOR gene aberrations in melanoma patients and evaluation of their sensitivity to PI3K-AKT-mTOR pathway inhibitors. Clin Cancer Res. 2016;22:1018–27.
Jiao Q, Bi L, Ren Y, Song S, Wang Q, Wang YS. Advances in studies of tyrosine kinase inhibitors and their acquired resistance. Mol Cancer. 2018;17:36.
Grabiner BC, Nardi V, Birsoy K, Possemato R, Shen K, Sinha S, et al. A diverse array of cancer-associated MTOR mutations are hyperactivating and can predict rapamycin sensitivity. Cancer Discov. 2014;4:554–63.
Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, et al. Response and acquired resistance to everolimus in anaplastic thyroid cancer. N Engl J Med. 2014;371:1426–33.
Li YY, Hanna GJ, Laga AC, Haddad RI, Lorch JH, Hammerman PS. Genomic analysis of metastatic cutaneous squamous cell carcinoma. Clin Cancer Res. 2015;21:1447–56.
Kunstman JW, Juhlin CC, Goh G, Brown TC, Stenman A, Healy JM, et al. Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing. Hum Mol Genet. 2015;24:2318–29.
Murugan AK, Humudh EA, Qasem E, Al-Hindi H, Almohanna M, Hassan ZK, et al. Absence of somatic mutations of the mTOR gene in differentiated thyroid cancer. Meta Gene. 2015;6:69–71.
Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci USA. 2005;102:802–7.
Davies H, Hunter C, Smith R, Stephens P, Greenman C, Bignell G, et al. Somatic mutations of the protein kinase gene family in human lung cancer. Cancer Res. 2005;65:7591–5.
Loch-head PA. Protein kinase activation loop autophosphorylation in cis: overcoming a Catch-22 situation. Sci Signal. 2009;2:pe4.
Hardt M, Chantaravisoot N, Tamanoi F. Activating mutations of TOR (target of rapamycin). Genes Cells. 2011;16:141–51.
Sturgill TW, Hall MN. Activating mutations in TOR are in similar structures as oncogenic mutations in PI3KCα. ACS Chem Biol. 2009;4:999–1015.
Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–92.
Yamaguchi H, Kawazu M, Yasuda T, Soda M, Ueno T, Kojima S, et al. Transforming somatic mutations of mammalian target of rapamycin kinase in human cancer. Cancer Sci. 2015;106:1687–92.
Ghosh AP, Marshall CB, Coric T, Shim EH, Kirkman R, Ballestas ME, et al. Point mutations of the mTOR-RHEB pathway in renal cell carcinoma. Oncotarget. 2015;6:17895–910.
Xu J, Pham CG, Albanese SK, Dong Y, Oyama T, Lee CH, et al. Mechanistically distinct cancer-associated mTOR activation clusters predict sensitivity to rapamycin. J Clin Invest. 2016;126:3526–40.
Neff RL, Farrar WB, Kloos RT, Burman KD. Anaplastic thyroid cancer. Endocrinol Metab Clin North Am. 2008;37:525–38.
Sadow PM, Priolo C, Nanni S, Karreth FA, Duquette M, Martinelli R, et al. Role of BRAFV600E in the first preclinical model of multifocal infiltrating myopericytoma development and microenvironment. J Natl Cancer Inst. 2014;106:dju182. https://doi.org/10.1093/jnci/dju182.
Prete A, Lo AS, Sadow PM, Bhasin SS, Antonello ZA, Vodopivec DM, et al. Pericytes elicit resistance to vemurafenib and sorafenib therapy in thyroid carcinoma via the TSP-1/TGFβ1 axis. Clin Cancer Res. 2018;24:6078–97.
Liu J, Brown RE. Morphoproteomics demonstrates activation of mTOR pathway in anaplastic thyroid carcinoma: a preliminary observation. Ann Clin Lab Sci. 2010;40:211–7.
Saji M, Ringel MD. The PI3K-Akt-mTOR pathway in initiation and progression of thyroid tumors. Mol Cell Endocrinol. 2010;321:20–8.
Liu Z, Hou P, Ji M, Guan H, Studeman K, Jensen K, et al. Highly prevalent genetic alterations in receptor tyrosine kinases and phosphatidylinositol 3-kinase/akt and mitogen-activated protein kinase pathways in anaplastic and follicular thyroid cancers. J Clin Endocrinol Metab. 2008;93:3106–16.
Liu D, Hou P, Liu Z, Wu G, Xing M. Genetic alterations in the phosphoinositide 3-kinase/Akt signaling pathway confer sensitivity of thyroid cancer cells to therapeutic targeting of Akt and mammalian target of rapamycin. Cancer Res. 2009;69:7311–9.
Schweppe RE, Klopper JP, Korch C, Pugazhenthi U, Benezra M, Knauf JA. Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab. 2008;93:4331–41.
Murugan AK, Dong J, Xie J, Xing M. MEK1 mutations, but not ERK2 mutations, occur in melanomas and colon carcinomas, but none in thyroid carcinomas. Cell Cycle. 2009;8:2122–4.
Völkers M, Toko H, Doroudgar S, Din S, Quijada P, Joyo AY, et al. Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1. Proc Natl Acad Sci USA. 2013;110:12661–6.
Murugan AK, Xing M. Anaplastic thyroid cancers harbor novel oncogenic mutations of the ALK gene. Cancer Res. 2011;71:4403–11.
Fukui Y, Ihara S. A mutant of SWAP-70, a phosphatidylinositoltrisphosphate binding protein, transforms mouse embryo fibroblasts, which is inhibited by sanguinarine. PLoS ONE. 2010;5:e14180.
Agrawal N, Akbani R, Aksoy BA, Ally A, Arachchi H, Asa SL, et al. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014;159:676–90.
Landa I, Ibrahimpasic T, Boucai L, Sinha R, Knauf JA, Shah RH, et al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest. 2016;126:1052–66.
Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013;6:pl1.
Acknowledgements
We thank Drs. N. E. Heldin, K.B. Ain, N. Onoda, M. Santoro, D. Wynford Thomas, G. Brabant, A.P. Dackiw, R. Schweppe, and B. Haugen for kindly providing us the accessibility to the cell lines used in this study.
Funding
This study was supported by U.S.A. National Institutes of Health (NIH) grants R01CA215142 and R01CA189224 to M. Xing.
Author information
Authors and Affiliations
Contributions
Research design: AKM and MX. Research performance: AKM, LR and MX. Data analysis: AKM, LR, and MX. Manuscript writing: AKM and MX. Final approval of manuscript: AKM, LR, and MX.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Murugan, A.K., Liu, R. & Xing, M. Identification and characterization of two novel oncogenic mTOR mutations. Oncogene 38, 5211–5226 (2019). https://doi.org/10.1038/s41388-019-0787-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41388-019-0787-5
This article is cited by
-
The multifaced role and therapeutic regulation of autophagy in ovarian cancer
Clinical and Translational Oncology (2022)
-
Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK
BMC Cancer (2021)
-
Mutational drivers of cancer cell migration and invasion
British Journal of Cancer (2021)
-
Contemporary Management of Anaplastic Thyroid Cancer
Current Treatment Options in Oncology (2020)