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01-10-2012 | Research Article

Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo

Authors: Yuanxi Zhu, Xiaobei Zhang, Yan Liu, Sheng Zhang, Jingjing Liu, Yi Ma, Jin Zhang

Published in: Tumor Biology | Issue 5/2012

Abstract

This study evaluated the effects of a mammalian target of mTOR inhibitor everolimus alone or in combination with trastuzumab on stem cells from HER2-overexpressing primary breast cancer cells and the BT474 breast cancer cell line in vitro and in vivo. For the in vitro studies, we sorted ESA⁺CD44⁺CD24^−/low cells as stem cells from primary breast cancer cells and BT474 cells using flow cytometry. The MTT assay was used to quantify the inhibitory effect of the drugs on total cells and stem cells specifically. Stem cell apoptosis, cell cycle distributions, and their tumorigenicity after treatment were investigated by flow cytometry or soft agar colony formation assays. For the in vivo studies, BALB/c mice were injected with BT474 stem cells, and the different treatments were administered. After necropsy, the expression of Ki67, CD31, AKT1, and phospho-AKT (Thr308) was analyzed by immunohistochemistry. For the in vitro studies, Treatment with everolimus resulted in stem cell growth inhibition in a dose-dependent manner. The combination of everolimus with trastuzumab was more effective at inhibiting cell growth (P < 0.001) and tumorigenicity (P < 0.001) compared with single-agent therapy. In addition, an increase in G1 cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either of the single-agent groups (P < 0.01). For the in vivo studies, everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single agent alone (P < 0.05). Compared with everolimus alone, the combination of everolimus and trastuzumab reduced the expression of Ki67, AKT1, and phospho-AKT (Thr308) (P < 0.05). We conclude that everolimus has effective inhibitory effects on HER2-overexpressing stem cells in vitro and vivo. Everolimus plus trastuzumab is a rational combination treatment that may be promising in human clinical trials.

Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ. Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat Clin Pract Oncol. 2006;3(5):269–80.CrossRefPubMed

Hudis CA. Trastuzumab–mechanism of action and use in clinical practice. N Engl J Med. 2007;357(1):39–51.CrossRefPubMed

Xu Y, Sun Q. Headway in resistance to endocrine therapy in breast cancer. J Thorac Dis. 2010;2(3):171–7.PubMedPubMedCentral

Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Láng I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Rüschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD, Herceptin Adjuvant (HERA) Trial Study Team. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 2005;353(16):1659–72.CrossRefPubMed

Lv YG, Yu F, Yao Q, Chen JH, Wang L. The role of survivin in diagnosis, prognosis and treatment of breast cancer. J Thorac Dis. 2010;2(2):100–10.PubMedPubMedCentral

Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev Cancer. 2006;6(9):729–34.CrossRefPubMed

Schmelzle T, Hall MN. TOR, a central controller of cell growth. Cell. 2000;103(2):253–62.CrossRefPubMed

Baldo P, Cecco S, Giacomin E, Lazzarini R, Ros B, Marastoni S. mTOR pathway and mTOR inhibitors as agents for cancer therapy. Curr Cancer Drug Targets. 2008;8(8):647–65.CrossRefPubMed

Rowinsky EK. Targeting the molecular target of rapamycin (mTOR). Curr Opin Oncol. 2004;16(6):564–75.CrossRefPubMed

10.

Gera JF, Mellinghoff IK, Shi Y, Rettig MB, Tran C, Hsu JH, Sawyers CL, Lichtenstein AK. AKT activity determines sensitivity to mammalian target of rapamycin (mTOR) inhibitors by regulating cyclin D1 and c-myc expression. J Biol Chem. 2004;279(4):2737–46.CrossRefPubMed

11.

Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100(7):3983–8.CrossRefPubMedPubMedCentral

12.

O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445(7123):106–10.CrossRefPubMed

13.

Shafee N, Smith CR, Wei S, Kim Y, Mills GB, Hortobagyi GN, Stanbridge EJ, Lee EY. Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors. Cancer Res. 2008;68(9):3243–50.CrossRefPubMedPubMedCentral

14.

Hambardzumyan D, Squatrito M, Holland EC. Radiation resistance and stem-like cells in brain tumors. Cancer Cell. 2006;10(6):454–6.CrossRefPubMed

15.

Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, Huang Y, Hu X, Su F, Lieberman J, Song E. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell. 2007;131(6):1109–23.CrossRefPubMed

16.

Setoguchi T, Taga T, Kondo T. Cancer stem cells persist in many cancer cell lines. Cell Cycle. 2004;3(4):414–5.CrossRefPubMed

17.

Magnifico A, Albano L, Campaner S, Delia D, Castiglioni F, Gasparini P, Sozzi G, Fontanella E, Menard S, Tagliabue E. Tumor-initiating cells of HER2-positive carcinoma cell lines express the highest oncoprotein levels and are sensitive to trastuzumab. Clin Cancer Res. 2009;15(6):2010–21.CrossRefPubMed

18.

Gijsen M, King P, Perera T, Parker PJ, Harris AL, Larijani B, Kong A. HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer. PLoS Biol. 2010;8(12):e1000563.CrossRefPubMedPubMedCentral

19.

Shah C, Miller TW, Wyatt SK, McKinley ET, Olivares MG, Sanchez V, Nolting DD, Buck JR, Zhao P, Ansari MS, Baldwin RM, Gore JC, Schiff R, Arteaga CL, Manning HC. Imaging biomarkers predict response to anti-HER2 (ErbB2) therapy in preclinical models of breast cancer. Clin Cancer Res. 2009;15(14):4712–21.CrossRefPubMedPubMedCentral

20.

Miller TW, Forbes JT, Shah C, Wyatt SK, Manning HC, Olivares MG, Sanchez V, Dugger TC, de Matos Granja N, Narasanna A, Cook RS, Kennedy JP, Lindsley CW, Arteaga CL. Inhibition of mammalian target of rapamycin is required for optimal antitumor effect of HER2 inhibitors against HER2-overexpressing cancer cells. Clin Cancer Res. 2009;15(23):7266–76.CrossRefPubMedPubMedCentral

21.

Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414(6859):105–11.CrossRefPubMed

22.

Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, Sherlock G, Lewicki J, Shedden K, Clarke MF. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med. 2007;356(3):217–26.CrossRefPubMed

23.

Xu CX, Li Y, Yue P, Owonikoko TK, Ramalingam SS, Khuri FR, Sun SY. The combination of RAD001 and NVP-BEZ235 exerts synergistic anticancer activity against non-small cell lung cancer in vitro and in vivo. PLoS One. 2011;6(6):e20899.CrossRefPubMedPubMedCentral

24.

Advani SH. Targeting mTOR pathway: a new concept in cancer therapy. Indian J Med Paediatr Oncol. 2010;31(4):132–6.CrossRefPubMedPubMedCentral

25.

Noh WC, Mondesire WH, Peng J, Jian W, Zhang H, Dong J, Mills GB, Hung MC, Meric-Bernstam F. Determinants of rapamycin sensitivity in breast cancer cells. Clin Cancer Res. 2004;10(3):1013–23.CrossRefPubMed

26.

Dragowska WH, Weppler SA, Qadir MA, Wong LY, Franssen Y, Baker JH, Kapanen AI, Kierkels GJ, Masin D, Minchinton AI, Gelmon KA, Bally MB. The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity. BMC Cancer. 2011;11:420.CrossRefPubMedPubMedCentral

27.

Mosley JD, Poirier JT, Seachrist DD, Landis MD, Keri RA. Rapamycin inhibits multiple stages of c-Neu/ErbB2 induced tumor progression in a transgenic mouse model of HER2-positive breast cancer. Mol Cancer Ther. 2007;6(8):2188–97.CrossRefPubMedPubMedCentral

28.

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. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006;66(3):1500–8.CrossRefPubMedPubMedCentral

29.

Wan X, Harkavy B, Shen N, Grohar P, Helman LJ. Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene. 2007;26(13):1932–40.CrossRefPubMed

30.

Okuzumi T, Fiedler D, Zhang C, Gray DC, Aizenstein B, Hoffman R, Shokat KM. Inhibitor hijacking of Akt activation. Nat Chem Biol. 2009;5(7):484–93.CrossRefPubMedPubMedCentral

31.

Papademetriou K, Ardavanis A, Kountourakis P. Neoadjuvant therapy for locally advanced breast cancer: focus on chemotherapy and biological targeted treatments’ armamentarium. J Thorac Dis. 2010;2(3):160–70.PubMedPubMedCentral

32.

Berns K, Horlings HM, Hennessy BT, Madiredjo M, Hijmans EM, Beelen K, Linn SC, Gonzalez-Angulo AM, Stemke-Hale K, Hauptmann M, Beijersbergen RL, Mills GB, van de Vijver MJ, Bernards R. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell. 2007;12(4):395–402.CrossRefPubMed

33.

Hill MM, Hemmings BA. Inhibition of protein kinase B/Akt. implications for cancer therapy. Pharmacol Ther. 2002;93(2–3):243–51.CrossRefPubMed

34.

Andre F, Campone M, O’Regan R, Manlius C, Massacesi C, Sahmoud T, Mukhopadhyay P, Soria JC, Naughton M, Hurvitz SA. Phase I study of everolimus plus weekly paclitaxel and trastuzumab in patients with metastatic breast cancer pretreated with trastuzumab. J Clin Oncol. 2010;28(34):5110–5.CrossRefPubMed

Title: Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo
Authors: Yuanxi Zhu
Xiaobei Zhang
Yan Liu
Sheng Zhang
Jingjing Liu
Yi Ma
Jin Zhang
Publication date: 01-10-2012
Publisher: Springer Netherlands
Published in: Tumor Biology / Issue 5/2012
Print ISSN: 1010-4283
Electronic ISSN: 1423-0380
DOI: https://doi.org/10.1007/s13277-012-0383-6

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