Cellular Oncology

, Volume 41, Issue 4, pp 409–426 | Cite as

Potential of the dual mTOR kinase inhibitor AZD2014 to overcome paclitaxel resistance in anaplastic thyroid carcinoma

  • Zorica Milošević
  • Jasna Banković
  • Jelena Dinić
  • Chrisiida Tsimplouli
  • Evangelia Sereti
  • Miodrag Dragoj
  • Verica Paunović
  • Zorka Milovanović
  • Marija Stepanović
  • Nikola Tanić
  • Kostantinos Dimas
  • Milica PešićEmail author
Original Paper



Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate.


Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies.


Rho- cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho- cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth.


Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies.


Anaplastic thyroid carcinoma mTOR AZD2014 Paclitaxel Chemo-resistance Targeted therapy 



This study was supported by Grant III41031 from the Ministry of Education, Science and Technological Development, Republic of Serbia. The work was performed within the framework of COST Actions CM1106 - Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells (the first author was awarded with a STSM grant) and CM1407 - Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery.

Compliance with ethical standards

The patient samples were collected and used in the study after obtaining informed consents and approval from the Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All animals were treated according to the guidelines of the EU and Greek authorities (2010/63/EU directive and Greek PD 56/2013, respectively) governing the use and handling of experimental animals.

Conflict of interest

The authors declare that they have no conflict of interest

Supplementary material

13402_2018_380_MOESM1_ESM.pdf (403 kb)
ESM 1 (PDF 403 kb)


  1. 1.
    J.L. Pasiekal, Anaplastic thyroid cancer. Curr. Opin. Oncol. 15, 78–83 (2003)CrossRefGoogle Scholar
  2. 2.
    S. Edge, D.R. Byrd, C.C. Compton, A.G. Fritz, F. Greene, A. Trotti, AJCC Cancer Staging Manual, 7th edn. (Springer, New York, 2010), pp. 1–646Google Scholar
  3. 3.
    C. Are, A.R. Shaha, Anaplastic thyroid carcinoma: Biology, pathogenesis, prognostic factors, and treatment approaches. Ann. Surg. Oncol. 13, 453–464 (2006)CrossRefPubMedGoogle Scholar
  4. 4.
    R.O. Wein, R.S. Weber, Anaplastic thyroid carcinoma: Palliation or treatment? Curr Opin Otolaryngol. Head. Neck. Surg. 19, 113–118 (2011)CrossRefPubMedGoogle Scholar
  5. 5.
    R.I. Haddad, W.M. Lydiatt, D.W. Ball, N.L. Busaidy, D. Byrd, G. Callender, P. Dickson, Q.Y. Duh, H. Ehya, M. Haymart, C. Hoh, J.P. Hunt, A. Iagaru, F. Kandeel, P. Kopp, D.M. Lamonica, J.C. McCaffrey, J.F. Moley, L. Parks, C.D. Raeburn, J.A. Ridge, M.D. Ringel, R.P. Scheri, J.P. Shah, R.C. Smallridge, C. Sturgeon, T.N. Wang, L.J. Wirth, K.G. Hoffmann, M. Hughes, Anaplastic tyroid carcinoma, Version 2. 2015. J. Natl. Compr. Canc. Netw. 13, 1140–1150 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    N. Smith, C. Nucera, Personalized therapy in patients with anaplastic thyroid cancer: Targeting genetic and epigenetic alterations. J. Clin. Endocrinol. Metab. 100, 35–42 (2015)CrossRefPubMedGoogle Scholar
  7. 7.
    Z. Liu, P. Hou, M. Ji, H. Guan, K. Studeman, K. Jensen, V. Vasko, A.K. El-Naggar, M. Xing, 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. 93, 3106–3116 (2008)CrossRefPubMedGoogle Scholar
  8. 8.
    M.C. Mendoza, E.E. Er, J. Blenis, The Ras-ERK and PI3K-mTOR pathways: Cross-talk and compensation. Trends. Biochem. Sci. 36, 320–328 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    J.A. McCubrey, L.S. Steelman, S.L. Abrams, J.T. Lee, F. Chang, F.E. Bertrand, P.M. Navolanic, D.M. Terrian, R.A. Franklin, A.B. D'Assoro, J.L. Salisbury, M.C. Mazzarino, F. Stivala, M. Libra, Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Adv. Enzym. Regul. 46, 249–279 (2006)CrossRefGoogle Scholar
  10. 10.
    Z. Milosevic, M. Pesic, T. Stankovic, J. Dinic, Z. Milovanovic, J. Stojsic, R. Dzodic, N. Tanic, J. Bankovic, Targeting RAS-MAPK-ERK and PI3K-AKT-mTOR signal transduction pathways to chemosensitize anaplastic thyroid carcinoma. Transl. Res. 164, 411–423 (2014)CrossRefPubMedGoogle Scholar
  11. 11.
    D.A. Guertin, D.M. Sabatini, Defining the role of mTOR in cancer. Cancer Cell 12, 9–22 (2007)CrossRefPubMedGoogle Scholar
  12. 12.
    J. Copp, G. Manning, T. Hunter, TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): Phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res. 69, 1821–1827 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Y. Alvarado, M.M. Mita, S. Vemulapalli, D. Mahalingam, A.C. Mita, Clinical activity of mammalian target of rapamycin inhibitors in solid tumors. Target. Oncol. 6, 69–94 (2011)CrossRefPubMedGoogle Scholar
  14. 14.
    S.A. Wander, B.T. Hennessy, J.M. Slingerland, Next-generation mTOR inhibitors in clinical oncology: How pathway complexity informs therapeutic strategy. J. Clin. Invest. 121, 1231–1241 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    M. Hardt, N. Chantaravisoot, F. Tamanoi, Activating mutations of TOR (target of rapamycin). Genes Cells 16, 141–151 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    M. Saji, M.D. Ringel, The PI3K-Akt-mTOR pathway in initiation and progression of thyroid tumors. Mol. Cell. Endocrinol. 321, 20–28 (2010)CrossRefPubMedGoogle Scholar
  17. 17.
    M.S. Petrulea, T.S. Plantinga, J.W. Smit, C.E. Georgescu, R.T. Netea-Maier, PI3K/Akt/mTOR: A promising therapeutic target for non-medullary thyroid carcinoma. Cancer. Treat. Rev. 41, 707–713 (2015)CrossRefPubMedGoogle Scholar
  18. 18.
    N.N. Bennani, B.R. LaPlant, S.M. Ansell, T.M. Habermann, D.J. Inwards, I.N. Micallef, P.B. Johnston, L.F. Porrata, J.P. Colgan, S.N. Markovic, G.S. Nowakowski, W.R. Macon, C.B. Reeder, J.R. Mikhael, D.W. Northfelt, I.M. Ghobrial, T.E. Witzig, Efficacy of the oral mTORC1 inhibitor everolimus in relapsed or refractory indolent lymphoma. Am. J. Hematol. 92, 448–453 (2017)CrossRefPubMedGoogle Scholar
  19. 19.
    D.X. Assad, S.T. Elias, A.C. Melo, C.G. Ferreira, G. De Luca Canto, E.N. Guerra, Potential impact of mTOR inhibitors on cervical squamous cell carcinoma: A systematic review. Oncol. Lett. 12, 4107–4116 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    R. Zoncu, A. Efeyan, D.M. Sabatini, mTOR: From growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell. Biol. 12, 21–35 (2011)CrossRefPubMedGoogle Scholar
  21. 21.
    K. Yu, L. Toral-Barza, C. Shi, W.G. Zhang, J. Lucas, B. Shor, J. Kim, J. Verheijen, K. Curran, D.J. Malwitz, D.C. Cole, J. Ellingboe, S. Ayral-Kaloustian, T.S. Mansour, J.J. Gibbons, R.T. Abraham, P. Nowak, A. Zask, Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin. Cancer Res. 69, 6232–6240 (2009)CrossRefPubMedGoogle Scholar
  22. 22.
    T. Powles, M. Wheater, O. Din, T. Geldart, E. Boleti, A. Stockdale, S. Sundar, A. Robinson, I. Ahmed, A. Wimalasingham, W. Burke, S.J. Sarker, S. Hussain, C. Ralph, A randomised phase 2 study of AZD2014 versus Everolimus in patients with VEGF-refractory metastatic clear cell renal Cancer. Eur. Urol. 69, 450–456 (2016)CrossRefPubMedGoogle Scholar
  23. 23.
    S.M. Guichard, J. Curwen, T. Bihani, C.M. D'Cruz, J.W. Yates, M. Grondine, Z. Howard, B.R. Davies, G. Bigley, T. Klinowska, K.G. Pike, M. Pass, C.M. Chresta, U.M. Polanska, R. McEwen, O. Delpuech, S. Green, S.C. Cosulich, AZD2014, an inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast Cancer when administered using intermittent or continuous schedules. Mol. Cancer Ther. 14, 2508–2518 (2015)CrossRefPubMedGoogle Scholar
  24. 24.
    L. Doyle, D.D. Ross, Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 22, 7340–7358 (2003)CrossRefPubMedGoogle Scholar
  25. 25.
    R.L. Juliano, V. Ling, A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta 455, 152–162 (1976)CrossRefPubMedGoogle Scholar
  26. 26.
    M.M. Gottesman, V. Ling, The molecular basis of multidrug resistance in cancer: The early years of P-glycoprotein research. FEBS Lett. 580, 998–1009 (2006)CrossRefPubMedGoogle Scholar
  27. 27.
    R.G. Deeley, C. Westlake, S.P. Cole, Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol. Rev. 86, 849–899 (2006)CrossRefPubMedGoogle Scholar
  28. 28.
    J.H. Hooijberg, N.A. de Vries, G.J. Kaspers, R. Pieters, G. Jansen, G.J. Peters, Multidrug resistance proteins and folate supplementation: Therapeutic implications for antifolates and other classes of drugs in cancer treatment. Cancer Chemother. Pharmacol. 58, 1–12 (2006)CrossRefPubMedGoogle Scholar
  29. 29.
    B. Sarkadi, L. Homolya, G. Szakacs, A. Varadi, Human multidrug resistance ABCB and ABCG transporters: Participation in a chemoimmunity defense system. Physiol. Rev. 86, 1179–1236 (2006)CrossRefPubMedGoogle Scholar
  30. 30.
    E.E. Pakos, J.P. Ioannidis, The association of P-glycoprotein with response to chemotherapy and clinical outcome in patients with osteosarcoma. A meta-analysis Cancer 98, 581–589 (2003)PubMedGoogle Scholar
  31. 31.
    A.J. Zurita, J.E. Diestra, E. Condom, X. Garcia Del Muro, G.L. Scheffer, R.J. Scheper, J. Perez, J.R. Germa-Lluch, M.A. Izquierdo, Lung resistance-related protein as a predictor of clinical outcome in advanced testicular germ-cell tumours. Br. J. Cancer 88, 879–886 (2003)CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    T.C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. 22, 27–55 (1984)CrossRefGoogle Scholar
  33. 33.
    W. Kang, O. Nielsen, C. Fenger, G. Leslie, U. Holmskov, K.B. Reid, Induction of DMBT1 expression by reduced ERK activity during a gastric mucosa differentiation-like process and its association with human gastric cancer. Carcinogenesis 26, 1129–1137 (2005)CrossRefPubMedGoogle Scholar
  34. 34.
    L.G. Mahaira, C. Tsimplouli, N. Sakellaridis, K. Alevizopoulos, C. Demetzos, Z. Han, P. Pantazis, K. Dimas, The labdane diterpene sclareol (labd-14-ene-8, 13-diol) induces apoptosis in human tumor cell lines and suppression of tumor growth in vivo via a p53-independent mechanism of action. Eur. J. Pharmacol. 666, 173–182 (2011)CrossRefPubMedGoogle Scholar
  35. 35.
    G. Nagaiah, A. Hossain, C.J. Mooney, J. Parmentier, S.C. Remick, Anaplastic thyroid cancer: A review of epidemiology, pathogenesis, and treatment. J. Oncol. 2011, 542358 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    K.L. Fung, M.M. Gottesman, A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function. Biochim. Biophys. Acta 1794, 860–871 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    J.Y. Yun, Y.A. Kim, J.Y. Choe, H. Min, K.S. Lee, Y. Jung, S. Oh, J.E. Kim, Expression of cancer stem cell markers is more frequent in anaplastic thyroid carcinoma compared to papillary thyroid carcinoma and is related to adverse clinical outcome. J. Clin. Pathol. 67, 125–133 (2014)CrossRefPubMedGoogle Scholar
  38. 38.
    X. Zheng, D. Cui, S. Xu, G. Brabant, M. Derwahl, Doxorubicin fails to eradicate cancer stem cells derived from anaplastic thyroid carcinoma cells: Characterization of resistant cells. Int. J. Oncol. 37, 307–315 (2010)CrossRefPubMedGoogle Scholar
  39. 39.
    V. Carina, G. Zito, G. Pizzolanti, P. Richiusa, A. Criscimanna, V. Rodolico, L. Tomasello, M. Pitrone, W. Arancio, C. Giordano, Multiple pluripotent stem cell markers in human anaplastic thyroid cancer: The putative upstream role of SOX2. Thyroid 23, 829–837 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    M. Saiselet, S. Floor, M. Tarabichi, G. Dom, A. Hebrant, W.C. van Staveren, C. Maenhaut, Thyroid cancer cell lines: An overview. Front. Endocrinol. 3, 133 (2012)Google Scholar
  41. 41.
    F. Marcucci, P. Ghezzi, C. Rumio, The role of autophagy in the cross-talk between epithelial-mesenchymal transitioned tumor cells and cancer stem-like cells. Mol. Cancer 16, 3 (2017)CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    U. Schumacher, N. Nehmann, E. Adam, D. Mukthar, I.N. Slotki, H.P. Horny, M.J. Flens, B. Schlegelberger, D. Steinemann, MDR-1-overexpression in HT 29 colon cancer cells grown in SCID mice. Acta Histochem. 114, 594–602 (2012)CrossRefPubMedGoogle Scholar
  43. 43.
    M. Kavallaris, Microtubules and resistance to tubulin-binding agents. Nat. Rev. Cancer 10, 194–204 (2010)CrossRefPubMedGoogle Scholar
  44. 44.
    H. Zou, L. Li, I. Garcia Carcedo, Z.P. Xu, M. Monteiro, W. Gu, Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis. Int. J. Nanomedicine 11, 1947–1958 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    C.J. Guigon, L. Fozzatti, C. Lu, M.C. Willingham, S.Y. Cheng, Inhibition of mTORC1 signaling reduces tumor growth but does not prevent cancer progression in a mouse model of thyroid cancer. Carcinogenesis 31, 1284–1291 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    D. Giuffrida, H. Gharib, Anaplastic thyroid carcinoma: Current diagnosis and treatment. Ann. Oncol. 11, 1083–1089 (2000)CrossRefPubMedGoogle Scholar
  47. 47.
    V. Kolsch, P.G. Charest, R.A. Firtel, The regulation of cell motility and chemotaxis by phospholipid signaling. J. Cell. Sci. 121, 551–559 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    F.M. Vega, A.J. Ridley, Rho GTPases in cancer cell biology. FEBS Lett. 582, 2093–2101 (2008)CrossRefPubMedGoogle Scholar
  49. 49.
    P. Gulhati, K.A. Bowen, J. Liu, P.D. Stevens, P.G. Rychahou, M. Chen, E.Y. Lee, H.L. Weiss, K.L. O'Connor, T. Gao, B.M. Evers, mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res. 71, 3246–3256 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    H. Liao, Y. Huang, B. Guo, B. Liang, X. Liu, H. Ou, C. Jiang, X. Li, D. Yang, Dramatic antitumor effects of the dual mTORC1 and mTORC2 inhibitor AZD2014 in hepatocellular carcinoma. Am. J. Cancer Res. 5, 125–139 (2015)PubMedGoogle Scholar
  51. 51.
    K.G. Pike, K. Malagu, M.G. Hummersone, K.A. Menear, H.M. Duggan, S. Gomez, N.M. Martin, L. Ruston, S.L. Pass, M. Pass, Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: The discovery of AZD8055 and AZD2014. Bioorg. Med. Chem. Lett. 23, 1212–1216 (2013)CrossRefPubMedGoogle Scholar
  52. 52.
    A. Shafer, C. Zhou, P.A. Gehrig, J.F. Boggess, V.L. Bae-Jump, Rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and induction of apoptosis. Int. J. Cancer 126, 1144–1154 (2010)PubMedPubMedCentralGoogle Scholar
  53. 53.
    X.F. Le, W.N. Hittelman, J. Liu, A. McWatters, C. Li, G.B. Mills, R.C. Bast Jr., Paclitaxel induces inactivation of p70 S6 kinase and phosphorylation of Thr421 and Ser424 via multiple signaling pathways in mitosis. Oncogene 22, 484–497 (2003)CrossRefPubMedGoogle Scholar
  54. 54.
    C.K. Ip, A.S. Wong, Exploiting p70 S6 kinase as a target for ovarian cancer. Expert. Opin. Ther. Targets 16, 619–630 (2012)CrossRefPubMedGoogle Scholar
  55. 55.
    T. Corbett, L. Polin, P. LoRusso, F. Valeriote, C. Panchapor, S. Pugh, K. White, J. Knight, L. Demchik, J. Jones, L. Jones, L. Lisow, In Vivo Methods for Screening and Preclinical Testing. (Humana Press, 2004), pp. 24Google Scholar
  56. 56.

Copyright information

© International Society for Cellular Oncology 2018

Authors and Affiliations

  • Zorica Milošević
    • 1
  • Jasna Banković
    • 1
  • Jelena Dinić
    • 1
  • Chrisiida Tsimplouli
    • 2
  • Evangelia Sereti
    • 2
  • Miodrag Dragoj
    • 1
  • Verica Paunović
    • 3
  • Zorka Milovanović
    • 4
  • Marija Stepanović
    • 1
  • Nikola Tanić
    • 1
  • Kostantinos Dimas
    • 2
  • Milica Pešić
    • 1
    Email author
  1. 1.Institute for Biological Research “Siniša Stanković”University of BelgradeBelgradeSerbia
  2. 2.Department of Pharmacology, Faculty of Medicine, School of Health SciencesUniversity of ThessalyLarissaGreece
  3. 3.Institute of Microbiology and Immunology, School of MedicineUniversity of BelgradeBelgradeSerbia
  4. 4.Institute for Oncology and Radiology of SerbiaBelgradeSerbia

Personalised recommendations