Breast Cancer Research and Treatment

, Volume 146, Issue 2, pp 259–272 | Cite as

A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in HER2-overexpressing breast cancer cells through FOXO3-mediated Bim1 expression

  • Jangsoon Lee
  • Chandra Bartholomeusz
  • Oula Mansour
  • Juliane Humphries
  • Gabriel N. Hortobagyi
  • Peter Ordentlich
  • Naoto T. Ueno
Preclinical study


Although there are effective HER2-targeted agents, novel combination strategies in HER2-overexpressing breast cancers are needed for patients whose tumors develop drug resistance. To develop new therapeutic strategy, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/EGFR dual tyrosine kinase inhibitor, in HER2+ breast cancer cells. We assessed the combinational synergistic effect and its mechanism by CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time PCR, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in two xenograft mouse models (BT474 and SUM190, P < 0.001). The synergistic anti-tumor activity of the entinostat/lapatinib combination was due to downregulation of phosphorylated Akt, which activated transcriptional activity of FOXO3, resulting in induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistance-HER2-overexpressing cells to the trastuzumab/lapatinib combination and enhanced the anti-proliferation effect compare with single or double combination treatment. This study provides evidence that entinostat has enhanced anti-tumor effect in combination with HER2-targeted reagent, lapatinib, and resulting in induction of apoptosis by FOXO3-mediated Bim1 expression. Our finding justifies for conducting a clinical trial of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2-overexpressing breast cancer resistant to trastuzumab-based treatment.


Entinostat Lapatinib HER2-overexpressing breast cancer FOXO3 Bim1 



This study was supported by the Morgan Welch Inflammatory Breast Cancer Research Program (NTU), the State of Texas Rare and Aggressive Breast Cancer Research Program, and the National Institutes of Health/National Cancer Institute [through CA123318 (NTU) and MD Anderson’s Cancer Center Support Grant, P30CA016672]. We thank Dr. Mien-Chie Hung (MD Anderson Cancer Center) for providing AKT-CA plasmid constructs. We thank Sunita Patterson (Department of Scientific Publications, MD Anderson) for editorial assistance and the Flow Cytometry and Cellular Imaging Facility at MD Anderson for assistance with cell-cycle analysis.

Conflict of interest

Peter Ordentlich is an employee of Syndax Pharmaceuticals. All other authors have no conflicts of interest to disclose.

Supplementary material

10549_2014_3014_MOESM1_ESM.doc (1.9 mb)
Supplementary material 1 (DOC 1909 kb)


  1. 1.
    Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, Troester MA, Tse CK, Edmiston S, Deming SL, Geradts J, Cheang MC, Nielsen TO, Moorman PG, Earp HS, Millikan RC (2006) Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295(21):2492–2502. doi: 10.1001/jama.295.21.2492 PubMedCrossRefGoogle Scholar
  2. 2.
    Stern HM (2012) Improving treatment of HER2-positive cancers: opportunities and challenges. Sci Transl Med 4(127):127rv122. doi: 10.1126/scitranslmed.3001539 CrossRefGoogle Scholar
  3. 3.
    Molina MA, Codony-Servat J, Albanell J, Rojo F, Arribas J, Baselga J (2001) Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells. Cancer Res 61(12):4744–4749PubMedGoogle Scholar
  4. 4.
    Baselga J, Albanell J, Molina MA, Arribas J (2001) Mechanism of action of trastuzumab and scientific update. Semin Oncol 28(5 Suppl 16):4–11PubMedCrossRefGoogle Scholar
  5. 5.
    Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med 6(4):443–446. doi: 10.1038/74704 PubMedCrossRefGoogle Scholar
  6. 6.
    Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, Skarlos D, Campone M, Davidson N, Berger M, Oliva C, Rubin SD, Stein S, Cameron D (2006) Lapatinib plus capecitabine for HER2-positive advanced breast cancer. New Engl J Med 355(26):2733–2743. doi: 10.1056/NEJMoa064320 PubMedCrossRefGoogle Scholar
  7. 7.
    Spector NL, Xia W, Burris H 3rd, Hurwitz H, Dees EC, Dowlati A, O’Neil B, Overmoyer B, Marcom PK, Blackwell KL, Smith DA, Koch KM, Stead A, Mangum S, Ellis MJ, Liu L, Man AK, Bremer TM, Harris J, Bacus S (2005) Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies. J Clin Oncol 23(11):2502–2512. doi: 10.1200/JCO.2005.12.157 PubMedCrossRefGoogle Scholar
  8. 8.
    Xia W, Mullin RJ, Keith BR, Liu LH, Ma H, Rusnak DW, Owens G, Alligood KJ, Spector NL (2002) Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways. Oncogene 21(41):6255–6263. doi: 10.1038/sj.onc.1205794 PubMedCrossRefGoogle Scholar
  9. 9.
    Cadigan KM, Nusse R (1997) Wnt signaling: a common theme in animal development. Genes Dev 11(24):3286–3305PubMedCrossRefGoogle Scholar
  10. 10.
    Valabrega G, Montemurro F, Aglietta M (2007) Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol 18(6):977–984. doi: 10.1093/annonc/mdl475 PubMedCrossRefGoogle Scholar
  11. 11.
    Kim H, Kim SN, Park YS, Kim NH, Han JW, Lee HY, Kim YK (2011) HDAC inhibitors downregulate MRP2 expression in multidrug resistant cancer cells: implication for chemosensitization. Int J Oncol 38(3):807–812. doi: 10.3892/ijo.2010.879 PubMedCrossRefGoogle Scholar
  12. 12.
    Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK (2001) Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 1(3):194–202. doi: 10.1038/35106079 PubMedCrossRefGoogle Scholar
  13. 13.
    Heightman TD (2011) Therapeutic prospects for epigenetic modulation. Expert Opin Ther Targ 15(6):729–740. doi: 10.1517/14728222.2011.561786 CrossRefGoogle Scholar
  14. 14.
    Saito A, Yamashita T, Mariko Y, Nosaka Y, Tsuchiya K, Ando T, Suzuki T, Tsuruo T, Nakanishi O (1999) A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci USA 96(8):4592–4597PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Singh TR, Shankar S, Srivastava RK (2005) HDAC inhibitors enhance the apoptosis-inducing potential of TRAIL in breast carcinoma. Oncogene 24(29):4609–4623. doi: 10.1038/sj.onc.1208585 PubMedCrossRefGoogle Scholar
  16. 16.
    Yardley DA, Ismail-Khan RR, Melichar B, Lichinitser M, Munster PN, Klein PM, Cruickshank S, Miller KD, Lee MJ, Trepel JB (2013) Randomized phase II, double-blind, placebo-controlled study of exemestane with or without entinostat in postmenopausal women with locally recurrent or metastatic estrogen receptor-positive breast cancer progressing on treatment with a nonsteroidal aromatase inhibitor. J Clin Oncol 31(17):2128–2135. doi: 10.1200/JCO.2012.43.7251 PubMedCrossRefGoogle Scholar
  17. 17.
    Sabnis GJ, Goloubeva O, Chumsri S, Nguyen N, Sukumar S, Brodie AM (2011) Functional activation of the estrogen receptor-alpha and aromatase by the HDAC inhibitor entinostat sensitizes ER-negative tumors to letrozole. Cancer Res 71(5):1893–1903. doi: 10.1158/0008-5472.CAN-10-2458 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Sabnis GJ, Goloubeva OG, Kazi AA, Shah P, Brodie AH (2013) HDAC inhibitor entinostat restores responsiveness of letrozole-resistant MCF-7Ca xenografts to aromatase inhibitors through modulation of Her-2. Mol Cancer Ther 12(12):2804–2816. doi: 10.1158/1535-7163.MCT-13-0345 PubMedCrossRefGoogle Scholar
  19. 19.
    Fidock DA, Rosenthal PJ, Croft SL, Brun R, Nwaka S (2004) Antimalarial drug discovery: efficacy models for compound screening. Nat Rev Drug Discov 3(6):509–520. doi: 10.1038/nrd1416 PubMedCrossRefGoogle Scholar
  20. 20.
    Ohrt C, Willingmyre GD, Lee P, Knirsch C, Milhous W (2002) Assessment of azithromycin in combination with other antimalarial drugs against Plasmodium falciparum in vitro. Antimicrob Agents Chemother 46(8):2518–2524PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Loupakis F, Vasile E, Santini D, Masi G, Falcone A, Graziano F (2008) EGF-receptor targeting with monoclonal antibodies in colorectal carcinomas: rationale for a pharmacogenomic approach. Pharmacogenomics 9(1):55–69. doi: 10.2217/14622416.9.1.55 PubMedCrossRefGoogle Scholar
  22. 22.
    Chandarlapaty S, Sawai A, Scaltriti M, Rodrik-Outmezguine V, Grbovic-Huezo O, Serra V, Majumder PK, Baselga J, Rosen N (2011) AKT inhibition relieves feedback suppression of receptor tyrosine kinase expression and activity. Cancer Cell 19(1):58–71. doi: 10.1016/j.ccr.2010.10.031 PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Sykes SM, Lane SW, Bullinger L, Kalaitzidis D, Yusuf R, Saez B, Ferraro F, Mercier F, Singh H, Brumme KM, Acharya SS, Scholl C, Tothova Z, Attar EC, Frohling S, DePinho RA, Armstrong SA, Gilliland DG, Scadden DT (2011) AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell 146(5):697–708. doi: 10.1016/j.cell.2011.07.032 PubMedCrossRefGoogle Scholar
  24. 24.
    Santo EE, Stroeken P, Sluis PV, Koster J, Versteeg R, Westerhout EM (2013) FOXO3a is a major target of inactivation by PI3K/AKT signaling in aggressive neuroblastoma. Cancer Res 73(7):2189–2198. doi: 10.1158/0008-5472.CAN-12-3767 PubMedCrossRefGoogle Scholar
  25. 25.
    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 (2007) A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12(4):395–402. doi: 10.1016/j.ccr.2007.08.030 PubMedCrossRefGoogle Scholar
  26. 26.
    Wang L, Zhang Q, Zhang J, Sun S, Guo H, Jia Z, Wang B, Shao Z, Wang Z, Hu X (2011) PI3K pathway activation results in low efficacy of both trastuzumab and lapatinib. BMC Cancer 11:248. doi: 10.1186/1471-2407-11-248 PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Kurokawa H, Arteaga CL (2003) ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms. Clin Cancer Res 9(1 Pt 2):511S–515SPubMedGoogle Scholar
  28. 28.
    Knuefermann C, Lu Y, Liu B, Jin W, Liang K, Wu L, Schmidt M, Mills GB, Mendelsohn J, Fan Z (2003) HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells. Oncogene 22(21):3205–3212. doi: 10.1038/sj.onc.1206394 PubMedCrossRefGoogle Scholar
  29. 29.
    Huang X, Wang S, Lee CK, Yang X, Liu B (2011) HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance. Cancer Lett 307(1):72–79. doi: 10.1016/j.canlet.2011.03.019 PubMedCrossRefGoogle Scholar
  30. 30.
    Greer EL, Brunet A (2005) FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24(50):7410–7425. doi: 10.1038/sj.onc.1209086 PubMedCrossRefGoogle Scholar
  31. 31.
    Hagenbuchner J, Ausserlechner MJ (2013) Mitochondria and FOXO3: breath or die. Front Physiol 4:147. doi: 10.3389/fphys.2013.00147 PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA, Coffer PJ, Medema RH, Coombes RC, Lam EW (2003) FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 278(50):49795–49805. doi: 10.1074/jbc.M309523200 PubMedCrossRefGoogle Scholar
  33. 33.
    Weidinger C, Krause K, Mueller K, Klagge A, Fuhrer D (2011) FOXO3 is inhibited by oncogenic PI3K/Akt signaling but can be reactivated by the NSAID sulindac sulfide. J Clin Endocrinol Metab 96(9):E1361–E1371. doi: 10.1210/jc.2010-2453 PubMedCrossRefGoogle Scholar
  34. 34.
    Bean GR, Ganesan YT, Dong Y, Takeda S, Liu H, Chan PM, Huang Y, Chodosh LA, Zambetti GP, Hsieh JJ, Cheng EH (2013) PUMA and BIM are required for oncogene inactivation-induced apoptosis. Sci Signal 6(268):ra20. doi: 10.1126/scisignal.2003483
  35. 35.
    Hui RC, Gomes AR, Constantinidou D, Costa JR, Karadedou CT, Fernandez de Mattos S, Wymann MP, Brosens JJ, Schulze A, Lam EW (2008) The forkhead transcription factor FOXO3a increases phosphoinositide-3 kinase/Akt activity in drug-resistant leukemic cells through induction of PIK3CA expression. Mol Cell Biol 28(19):5886–5898. doi: 10.1128/MCB.01265-07 PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Puig O, Tjian R (2005) Transcriptional feedback control of insulin receptor by dFOXO/FOXO1. Genes Dev 19(20):2435–2446. doi: 10.1101/gad.1340505 PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Garrett JT, Olivares MG, Rinehart C, Granja-Ingram ND, Sanchez V, Chakrabarty A, Dave B, Cook RS, Pao W, McKinely E, Manning HC, Chang J, Arteaga CL (2011) Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase. Proc Natl Acad Sci USA 108(12):5021–5026. doi: 10.1073/pnas.1016140108 PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Wang S, Huang J, Lyu H, Lee CK, Tan J, Wang J, Liu B (2013) Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells. Cell Death Dis 4:e556. doi: 10.1038/cddis.2013.79 PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Kim YJ, Greer CB, Cecchini KR, Harris LN, Tuck DP, Kim TH (2013) HDAC inhibitors induce transcriptional repression of high copy number genes in breast cancer through elongation blockade. Oncogene 32(23):2828–2835. doi: 10.1038/onc.2013.32 PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Mateen S, Raina K, Jain AK, Agarwal C, Chan D, Agarwal R (2012) Epigenetic modifications and p21-cyclin B1 nexus in anticancer effect of histone deacetylase inhibitors in combination with silibinin on non-small cell lung cancer cells. Epigenetics 7(10):1161–1172. doi: 10.4161/epi.22070 PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Maeda T, Hanna AN, Sim AB, Chua PP, Chong MT, Tron VA (2002) GADD45 regulates G2/M arrest, DNA repair, and cell death in keratinocytes following ultraviolet exposure. J Invest Dermatol 119(1):22–26. doi: 10.1046/j.1523-1747.2002.01781.x PubMedCrossRefGoogle Scholar
  42. 42.
    Guo Y, Kyprianou N (1998) Overexpression of transforming growth factor (TGF) beta1 type II receptor restores TGF-beta1 sensitivity and signaling in human prostate cancer cells. Cell Growth Differ 9(2):185–193PubMedGoogle Scholar
  43. 43.
    Kataoka Y, Mukohara T, Shimada H, Saijo N, Hirai M, Minami H (2010) Association between gain-of-function mutations in PIK3CA and resistance to HER2-targeted agents in HER2-amplified breast cancer cell lines. Ann Oncol 21(2):255–262. doi: 10.1093/annonc/mdp304 PubMedCrossRefGoogle Scholar
  44. 44.
    Eichhorn PJ, Gili M, Scaltriti M, Serra V, Guzman M, Nijkamp W, Beijersbergen RL, Valero V, Seoane J, Bernards R, Baselga J (2008) Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235. Cancer Res 68(22):9221–9230. doi: 10.1158/0008-5472.CAN-08-1740 PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Tortora G (2011) Mechanisms of resistance to HER2 target therapy. J Natl Cancer Inst Monogr 2011(43):95–98. doi: 10.1093/jncimonographs/lgr026 PubMedCrossRefGoogle Scholar
  46. 46.
    Liu L, Greger J, Shi H, Liu Y, Greshock J, Annan R, Halsey W, Sathe GM, Martin AM, Gilmer TM (2009) Novel mechanism of lapatinib resistance in HER2-positive breast tumor cells: activation of AXL. Cancer Res 69(17):6871–6878. doi: 10.1158/0008-5472.CAN-08-4490 PubMedCrossRefGoogle Scholar
  47. 47.
    Wetterskog D, Shiu KK, Chong I, Meijer T, Mackay A, Lambros M, Cunningham D, Reis-Filho JS, Lord CJ, Ashworth A (2014) Identification of novel determinants of resistance to lapatinib in ERBB2-amplified cancers. Oncogene 33(8):966–976. doi: 10.1038/onc.2013.41 PubMedCrossRefGoogle Scholar
  48. 48.
    Srivastava RK, Kurzrock R, Shankar S (2010) MS-275 sensitizes TRAIL-resistant breast cancer cells, inhibits angiogenesis and metastasis, and reverses epithelial-mesenchymal transition in vivo. Mol Cancer Ther 9(12):3254–3266. doi: 10.1158/1535-7163.MCT-10-0582 PubMedCrossRefGoogle Scholar
  49. 49.
    Marks PA, Xu WS (2009) Histone deacetylase inhibitors: potential in cancer therapy. J Cell Biochem 107(4):600–608. doi: 10.1002/jcb.22185 PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Nagathihalli NS, Massion PP, Gonzalez AL, Lu P, Datta PK (2012) Smoking induces epithelial-to-mesenchymal transition in non-small cell lung cancer through HDAC-mediated downregulation of E-cadherin. Mol Cancer Ther 11(11):2362–2372. doi: 10.1158/1535-7163.MCT-12-0107 PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Shah P, Gau Y, Sabnis G (2014) Histone deacetylase inhibitor entinostat reverses epithelial to mesenchymal transition of breast cancer cells by reversing the repression of E-cadherin. Breast Cancer Res Treat 143(1):99–111. doi: 10.1007/s10549-013-2784-7 PubMedCrossRefGoogle Scholar
  52. 52.
    Hermanson DL, Das SG, Li Y, Xing C (2013) Overexpression of Mcl-1 confers multidrug resistance, whereas topoisomeraseII beta downregulation introduces mitoxantrone-specific drug resistance in acute myeloid leukemia. Mol Pharmacol 84(2):236–243. doi: 10.1124/mol.113.086140 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jangsoon Lee
    • 1
  • Chandra Bartholomeusz
    • 1
  • Oula Mansour
    • 1
  • Juliane Humphries
    • 1
  • Gabriel N. Hortobagyi
    • 1
  • Peter Ordentlich
    • 2
  • Naoto T. Ueno
    • 1
  1. 1.Section of Translational Breast Cancer Research, Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, Department of Breast Medical Oncology, Unit 1354The University of Texas MD Anderson Cancer CenterHoustonUSA
  2. 2.Syndax Pharmaceuticals, IncWalthamUSA

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