Skip to main content
SpringerLink
Log in

The mTOR pathway inhibitor RAD001 (everolimus) is highly efficacious in tamoxifen-sensitive and -resistant breast cancer xenografts

  • Original Research
  • Published:
Targeted Oncology Aims and scope Submit manuscript

Abstract

The rapamycin derivative RAD001 (everolimus) is presently in clinical trials. Preclinical studies have suggested preferential activity in antiestrogen resistant breast cancer cells. We investigated the response of breast cancer xenografts with different tamoxifen (TAM) sensitivity towards RAD001 and analyzed the regulatory machinery as well as the cross-talk between different signaling pathways. The ERα-positive and TAM-sensitive patient-derived breast carcinoma model 3366, its TAM-resistant counterpart 3366/TAM and 4049, a breast cancer with inherent TAM-resistance, were transplanted to immunodeficient nude mice and treated with RAD001 or TAM or the combination of both compounds. Shock frozen tumors were prepared for Western Blot and immunohistochemical analysis to semi-quantitatively evaluate the expression of the ERα and the ERα regulated IGF-IR as well as PTEN, pAkt, mTOR, (phospho)-p70S6K, (phospho)-4E-BP1 and cyclin D1. RAD001 significantly inhibited the growth of tamoxifen responding and non-responding xenografts. The highest efficacy was found for the combined treatment with TAM and RAD001. RAD001 modified the protein expression of mTOR and its downstream molecule 4E-BP1 as well as the level of PTEN and ERα, but independent of the tumors sensitivity towards TAM. The protein kinase Akt was found in the active phosphorylated form (pAkt) only in TAM-resistant xenografts, but not detectable in the TAM-responding 3366 line. All treatment modalities down-regulated pAkt expression in the TAM-resistant tumors. p70S6K and IGF-IR proteins were not significantly influenced by RAD001 treatment. Our findings document the linkage between different growth-controlling pathways. Due to its capability to be active in a TAM-resistant in vivo model, RAD001 could potentially serve as a promising second-line therapy in breast cancer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Vignot S, Faivre S, Aguirre D, Raymond E (2005) mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol 16:525–537

    Article  PubMed  CAS  Google Scholar 

  2. Dancey JE (2005) Inhibitors of the mammalian target of rapamycin. Expert Opin Investig Drugs 14:313–328

    Article  PubMed  CAS  Google Scholar 

  3. Chan S (2004) Targeting the mammalian target of rapamycin (mTOR): a new approach to treating cancer. Br J Cancer 91:1420–1424

    Article  PubMed  CAS  Google Scholar 

  4. Hay N (2005) The Akt-mTOR tango and its relevance to cancer. Cancer Cell 8:179–183

    Article  PubMed  CAS  Google Scholar 

  5. Sarbassov dos D, Ali SM, Sabatini DM (2005) Growing roles for the mTOR pathway. Curr Opin Cell Biol 17:596–603

    Article  PubMed  CAS  Google Scholar 

  6. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, Khuri FR (2005) Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 65:7052–7058

    Article  PubMed  CAS  Google Scholar 

  7. Crowder RJ, Ellis MJ (2005) Treating breast cancer through novel inhibitors of the phosphatidylinositol 3´-kinase pathway. Breast Cancer Res 7:212–214

    Article  PubMed  CAS  Google Scholar 

  8. Carraway H, Hidalgo M (2004) New targets for therapy in breast cancer: mammalian target of rapamycin (mTOR) antagonists. Breast Cancer Res 6:219–224

    Article  PubMed  CAS  Google Scholar 

  9. Witton CJ (2004) Determining sensitivity to rapamycin and its analogues in breast cancer patients. Breast Cancer Res 7:41–42

    Article  PubMed  CAS  Google Scholar 

  10. Mita MM, Mita A, Rowinsky EK (2003) Mammalian target of rapamycin: a new molecular target for breast cancer. Clin Breast Cancer 4:126–137

    Article  PubMed  CAS  Google Scholar 

  11. deGraffenried LA, Friedrichs WE, Russell DH, Donzis EJ, Middleton AK, Silva JM, Roth RA, Hidalgo M (2004) Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant akt activity. Clin Cancer Res 10:8059–8067

    Article  PubMed  CAS  Google Scholar 

  12. Mondesire WH, Jian W, Zhang H, Ensor J, Hung MC, Mills GB, Meric-Bernstam F (2004) Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells. Clin Cancer Res 10:7031–7042

    Article  PubMed  CAS  Google Scholar 

  13. Naundorf H, Fichtner I, Büttner B, Frege J (1992) Establishment and characterization of a new human oestradiol- and progesterone-receptor-positive mammary carcinoma serially transplantable in nude mice. J Cancer Res Clin Oncol 119:35–40

    Article  PubMed  CAS  Google Scholar 

  14. Naundorf H, Becker M, Lykkesfeldt AE, Elbe B, Neumann C, Büttner B, Fichtner I (2000) Development and characterization of a tamoxifen-resistant breast carcinoma xenograft. Br J Cancer 82:1844–1850

    Article  PubMed  CAS  Google Scholar 

  15. Becker M, Sommer A, Krätzschmar JR, Seidel H, Pohlenz HD, Fichtner I (2005) Distinct gene expression patterns in a tamoxifen-sensitive human mammary carcinoma xenograft and its tamoxifen-resistant subline MaCa 3366/TAM. Mol Cancer Ther 4:151–168

    PubMed  CAS  Google Scholar 

  16. Besada V, Diaz M, Becker M, Ramos Y, Castellanos-Serra L, Fichtner I (2006) Proteomics of xenografted human breast cancer indicates novel targets related to tamoxifen resistance. Proteomics 6:1038–1048

    Article  PubMed  CAS  Google Scholar 

  17. Naundorf H, Parczyl K, Zschiesche W, Reinecke S, Büttner B, Saul GJ, Sinn B, Fichtner I (1996) Relation of oestradiol-mediated growth stimulation with the expression of c-erbB-2 protein in xenotransplanted oestradiol-receptor-positive- and -negative breast carcinomas. J Cancer Res Clin Oncol 122:14–20

    Article  PubMed  CAS  Google Scholar 

  18. Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivieres S, Mercep L, Ferrari S (1998) Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol Chem 273:14424–14429

    Article  PubMed  CAS  Google Scholar 

  19. Law M, Forrester E, Chytil A, Corsino P, Green G, Davis B, Rowe T, Law B (2006) Rapamycin disrupts cyclin/cyclin-dependent kinase/p21/proliferating cell nuclear antigen complexes and cyclin D1 reverses rapamycin action by stabilizing these complexes. Cancer Res 66:1070–1080

    Article  PubMed  CAS  Google Scholar 

  20. Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM, Siderovski DP, Mak TW (1998) Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95:29–39

    Article  PubMed  CAS  Google Scholar 

  21. Testa JR, Tsichlis PN (2005) AKT signaling in normal and malignant cells. Oncogene 24:7391–7393

    Article  PubMed  CAS  Google Scholar 

  22. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H (2001) Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem 276:9817–9824

    Article  PubMed  CAS  Google Scholar 

  23. Gibbons JJ, Discafani C, Peterson R, Hernandez R, Skotnicki J, Frost J (2000) Proc Am Assoc Cancer Res 40:301

    Google Scholar 

  24. Georgakis GV, Younes A (2006) From Rapa Nui to rapamycin: targeting PI3K/Akt/mTOR for cancer therapy. Exp Rev Anticancer Ther 6:131–140

    Article  PubMed  CAS  Google Scholar 

  25. Bjornsti MA, Houghton PJ (2004) The TOR pathway: a target for cancer therapy. Nat Rev Cancer 4:335–348

    Article  PubMed  CAS  Google Scholar 

  26. Guertin DA, Sabatini DM (2005) An expanding role for mTOR in cancer. Trends Mol Med 11:353–361

    Article  PubMed  CAS  Google Scholar 

  27. Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, Cantley, LC (2004) The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6:91–99

    Article  PubMed  CAS  Google Scholar 

  28. Noh WC, Mondesire WH, Peng J, Jian W, Zhang H, Dong J, Mills GB, Hung MC, Meric-Bernstam F (2004). Determinants of rapamycin sensitivity in breast cancer cells. Clin Cancer Res 10:1013–1023

    Article  PubMed  CAS  Google Scholar 

  29. 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 59:3581–3587

    PubMed  CAS  Google Scholar 

  30. Bose S, Chandran S, Mirocha JM, Bose N (2006) The Akt pathway in human breast cancer: a tissue-array-based analysis. Mod Pathol 19:238–345

    Article  PubMed  CAS  Google Scholar 

  31. Yu K, Toral-Bazar L, Discafani C, Zhang WG, Skotnicki J, Frost P, Gibbons JJ (2001) mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer 8:249–258

    Article  PubMed  Google Scholar 

  32. Kim RH, Mak TW (2006) Tumours and tremors: how PTEN regulation underlies both. Br J Cancer 94:620–624

    PubMed  CAS  Google Scholar 

  33. Clark AS, West K, Streicher S, Dennis PA (2002) Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther 1:707–717

    PubMed  CAS  Google Scholar 

  34. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P (1995) Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494

    Article  PubMed  CAS  Google Scholar 

  35. Datta SR, Brunet A, Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13:2905–2927

    Article  PubMed  CAS  Google Scholar 

  36. Kurokawa H, Arteaga CL (2003). ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms. Clin Cancer Res 9:511–515

    Google Scholar 

  37. Nicholson RI, Staka C, Boyns F, Hutcheson IR, Gee JM (2004) Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy. Endocr Relat Cancer 11:623–641

    Article  PubMed  CAS  Google Scholar 

  38. Stoica GE, Franke TF, Moroni M, Mueller S, Morgan E, Iann MC, Winder AD, Reiter R, Wellstein A, Martin MB, Stoica A (2003) Effect of estradiol on estrogen receptor-alpha gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway. Oncogene 22:7998–8011

    Article  PubMed  CAS  Google Scholar 

  39. Liu M, Howes A, Lesperance J, Stallcup WB, Hauser CA, Kadoya K, Oshima RG, Abraham RT (2005) Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res 65:5325–5336

    Article  PubMed  CAS  Google Scholar 

  40. Frogne T, Jepsen JS, Larsen SS, Fog CK, Brockdorff BL, Lykkesfeldt AE (2005) Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth. Endocr Relat Cancer 12:599–614

    Article  PubMed  CAS  Google Scholar 

  41. Stewart AJ, Johnson MD, May FE, Westley BR (1990) Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells. J Biol Chem 265:21172–21178

    PubMed  CAS  Google Scholar 

  42. Wan X, Harkavy B, Shen N, Grohar P, Helman LJ (2006) Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene 26:1932–1940

    Article  PubMed  CAS  Google Scholar 

  43. Akkiprik M, Nicorici D, Cogdell D, Jia YJ, Hategan A, Tabus I, Yli-Harja O, Yu D, Sahin A, Zhang W (2006). Dissection of signalling pathways in fourteen breast cancer cell lines using reverse-phase protein lysate microarray. Technol Cancer Res Treat 6:543–552

    Google Scholar 

  44. Torres-Arzayus MI, Yuan J, DellaGatta JL, Lane H, Kung AL, Brown M (2006) Targeting the AIB1 oncogene through mammalian target of rapamycin inhibition in the mammary gland. Cancer Res 66:11381–11388

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully thank Monika Becker for excellent support concerning the animal experiments. Furthermore, we thank Heidi Lane from Novartis Pharma AG (Basel, Switzerland) for supply of RAD001 and for critical reviewing of the manuscript.

Author information

Authors and Affiliations

  1. Experimental Pharmacology, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13125, Berlin-Buch, Germany

    Diana Behrens & Iduna Fichtner

  2. Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, 2100, Copenhagen, Denmark

    Anne E. Lykkesfeldt

Authors
  1. Diana Behrens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anne E. Lykkesfeldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Iduna Fichtner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Iduna Fichtner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Behrens, D., Lykkesfeldt, A.E. & Fichtner, I. The mTOR pathway inhibitor RAD001 (everolimus) is highly efficacious in tamoxifen-sensitive and -resistant breast cancer xenografts. Targ Oncol 2, 135–144 (2007). https://doi.org/10.1007/s11523-007-0054-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11523-007-0054-5

Keywords

Search

Navigation