Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo
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.
KeywordsBreast stem cells mTOR HER2-positive Everolimus Trastuzumab
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).
Conflicts of interest
- 4.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.CrossRefPubMedGoogle Scholar
- 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.CrossRefPubMedGoogle Scholar
- 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.CrossRefPubMedPubMedCentralGoogle Scholar
- 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.CrossRefPubMedPubMedCentralGoogle Scholar
- 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.CrossRefPubMedPubMedCentralGoogle Scholar
- 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.CrossRefPubMedGoogle Scholar
- 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.CrossRefPubMedGoogle Scholar