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

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Tumor Biology

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.

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Acknowledgments

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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Authors and Affiliations

  1. 3rd Department of Breast Cancer, China Tianjin Breast Cancer Prevention, Treatment and Research Center, Tianjin Medical University Cancer Institute and Hospital, West Beihuanhu Rd, Tianjin, 300060, People’s Republic of China

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

  2. Key Laboratory of Breast Cancer Prevention and Therapy of Ministry of Education, Tianjin, People’s Republic of China

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

  3. Key Laboratory of Cancer Prevention and Therapy, Tianjin, People’s Republic of China

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

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  1. Yuanxi Zhu
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  2. Xiaobei Zhang
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  3. Yan Liu
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  4. Sheng Zhang
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  7. Jin Zhang
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Corresponding author

Correspondence to Jin Zhang.

Additional information

Y. Zhu and X. Zhang contributed equally to this study.

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Zhu, Y., Zhang, X., Liu, Y. et al. Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo. Tumor Biol. 33, 1349–1362 (2012). https://doi.org/10.1007/s13277-012-0383-6

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  • DOI: https://doi.org/10.1007/s13277-012-0383-6

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