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Tumor Biology

, Volume 35, Issue 2, pp 1275–1286 | Cite as

Everolimus in combination with letrozole inhibit human breast cancer MCF-7/Aro stem cells via PI3K/mTOR pathway: an experimental study

  • Yan Liu
  • Xiaobei Zhang
  • Jingjing Liu
  • Guofang Hou
  • Sheng Zhang
  • Jin ZhangEmail author
Research Article

Abstract

This study evaluated the effects of an mTOR inhibitor everolimus alone or in combination with letrozole on MCF-7/Aro (MCF-7 cells stably transfected with CYP19) in vitro and in vivo. In vitro studies, full-length CYP19 (aromatase) was cloned in a plasmid transfer vector pH ß-Aro and then transfected into MCF-7 stem cells which were ESA+CD44+CD24−/low sorted by flow cytometry. MTT assays were used to quantify the inhibitory effect of the drugs on MCF-7/Aro stem cells (SCs) and non-stem cells (NSCs). Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumorigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In vivo studies, the BALB/c mice were injected with MCF-7/Aro SCs, and the different treatments were administered. After necropsy, the expression of KI67, CD31, AKT1, phospho-AKT (Thr308), and mTOR was analyzed by immunohistochemistry. In vitro, compared with MCF-7/Aro NSCs, there were greater resistance to the standard treatment doses of letrozole and everolimus in MCF-7/Aro SCs (17- and 15-fold, respectively). Treatment with everolimus or letrozole resulted in growth inhibition of SCs in a dose-dependent manner. Compared with single-agent therapy, the combination of everolimus with letrozole was more effective in the inhibition of cell growth (P < 0.001) and tumorigenicity (P < 0.01). In addition, an increase in G1 cell cycle arrest and increases in early apoptosis were observed in the combination treatment group compared with either single-agent group. In vivo, the xenograft tumor sizes were significantly decreased in everolimus alone group compared to control group, and everolimus plus letrozole therapy was much more effective compared with either single agent alone (P < 0.01). Compared with everolimus alone, the combination of everolimus and letrozole reduced the expression of KI67, mTOR, and phospho-AKT (Thr308; P < 0.01). Everolimus has effective inhibition on aromatase-overexpressing stem cell in vitro and in vivo. The combination everolimus and letrozole could be more effective than either drug alone.

Keywords

Breast stem cells mTOR Aromatase Everolimus Letrozole 

Notes

Acknowledgments

We thank Letitia Fulcher, Josep-Maria Peralba, and James Freeman. We thank Professor Mong-Hong Lee (Department of Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA) for his invaluable technical and intellectual assistance.

Conflicts of interest

None.

Funding

This study was supported by Major Projects of Tianjin Science and Technology (no. 09ZCZDSF04000) and Major Project of International Cooperation of China Ministry of Science (2010DFB30270).

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2013

Authors and Affiliations

  • Yan Liu
    • 1
    • 2
    • 3
  • Xiaobei Zhang
    • 1
    • 2
    • 3
  • Jingjing Liu
    • 1
    • 2
    • 3
  • Guofang Hou
    • 1
    • 2
    • 3
  • Sheng Zhang
    • 1
    • 2
    • 3
  • Jin Zhang
    • 1
    • 2
    • 3
    Email author
  1. 1.The 3rd Department of Breast Cancer, China Tianjin Breast Cancer Prevention, Treatment and Research CenterTianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of CancerTianjinPeople’s Republic of China
  2. 2.Key Laboratory of Breast Cancer Prevention and TherapyTianjin Medical University, Ministry of EducationTianjinPeople’s Republic of China
  3. 3.Key Laboratory of Cancer Prevention and TherapyTianjin Medical University, Ministry of EducationTianjinPeople’s Republic of China

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