Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer


Trastuzumab-refractory, HER2 (human epidermal growth factor receptor 2)-positive breast cancer is commonly treated with trastuzumab emtansine (T-DM1), an antibody–drug conjugate of trastuzumab and the microtubule-targeting agent, DM1. However, drug response reduces greatly over time due to acquisition of resistance whose molecular mechanisms are mostly unknown. Here, we uncovered a novel mechanism of resistance against T-DM1 by combining whole transcriptome sequencing (RNA-Seq), proteomics and a targeted small interfering RNA (siRNA) sensitization screen for molecular level analysis of acquired and de novo T-DM1-resistant models of HER2-overexpressing breast cancer. We identified Polo-like kinase 1 (PLK1), a mitotic kinase, as a resistance mediator whose genomic as well as pharmacological inhibition restored drug sensitivity. Both acquired and de novo resistant models exhibited synergistic growth inhibition upon combination of T-DM1 with a selective PLK1 inhibitor, volasertib, at a wide concentration range of the two drugs. Mechanistically, T-DM1 sensitization upon PLK1 inhibition with volasertib was initiated by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, leading to caspase activation, followed by DNA damage through CDK1-dependent phosphorylation and inactivation of Bcl-2/xL. Furthermore, we showed that Ser70 phosphorylation of Bcl-2 directly regulates apoptosis by disrupting the binding to and sequestration of the pro-apoptotic protein Bim. Importantly, T-DM1 resistance signature or PLK1 expression correlated with cell cycle progression and DNA repair, and predicted a lower sensitivity to taxane/trastuzumab combination in HER2-positive breast cancer patients. Finally, volasertib in combination with T-DM1 greatly synergized in models of T-DM1 resistance in terms of growth inhibition both in three dimensional (3D) cell culture and in vivo. Altogether, our results provide promising pre-clinical evidence for potential testing of T-DM1/volasertib combination in T-DM1 refractory HER2-positive breast cancer patients for whom there is currently no treatment available.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66:7–30.

    Article  Google Scholar 

  2. 2.

    Wang Q, Li SH, Wang H, Xiao Y, Sahin O, Brady SW, et al. Concomitant targeting of tumor cells and induction of T-cell response synergizes to effectively inhibit trastuzumab-resistant breast cancer. Cancer Res. 2012;72:4417–28.

    CAS  Article  Google Scholar 

  3. 3.

    Turpin J, Ling C, Crosby EJ, Hartman ZC, Simond AM, Chodosh LA, et al. The ErbB2DeltaEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment. Oncogene. 2016;35:6053–64.

    CAS  Article  Google Scholar 

  4. 4.

    Stingl J, Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer. 2007;7:791–9.

    CAS  Article  Google Scholar 

  5. 5.

    Henjes F, Bender C, von der Heyde S, Braun L, Mannsperger HA, Schmidt C, et al. Strong EGFR signaling in cell line models of ERBB2-amplified breast cancer attenuates response towards ERBB2-targeting drugs. Oncogenesis. 2012;1:e16

    CAS  Article  Google Scholar 

  6. 6.

    Vu T, Claret FX. Trastuzumab: updated mechanisms of action and resistance in breast cancer. Front Oncol. 2012;2:62.

    Article  Google Scholar 

  7. 7.

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

    CAS  Article  Google Scholar 

  8. 8.

    Sahin O, Frohlich H, Lobke C, Korf U, Burmester S, Majety M, et al. Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance. BMC Syst Biol. 2009;3:1.

    Article  Google Scholar 

  9. 9.

    Spector NL, Blackwell KL. Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol. 2009;27:5838–47.

    CAS  Article  Google Scholar 

  10. 10.

    LoRusso PM, Weiss D, Guardino E, Girish S, Sliwkowski MX. Trastuzumab emtansine: a unique antibody–drug conjugate in development for human epidermal growth factor receptor 2-positive cancer. Clin Cancer Res. 2011;17:6437–47.

    CAS  Article  Google Scholar 

  11. 11.

    Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med. 2012;367:1783–91.

    CAS  Article  Google Scholar 

  12. 12.

    Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody–cytotoxic drug conjugate. Cancer Res. 2008;68:9280–90.

    CAS  Article  Google Scholar 

  13. 13.

    Barok M, Tanner M, Koninki K, Isola J. Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo. Breast Cancer Res. 2011;13:R46.

    CAS  Article  Google Scholar 

  14. 14.

    Barok M, Joensuu H, Isola J. Trastuzumab emtansine: mechanisms of action and drug resistance. Breast Cancer Res. 2014;16:209.

    Article  Google Scholar 

  15. 15.

    Marusyk A, Almendro V, Polyak K. Intra-tumour heterogeneity: a looking glass for cancer? Nat Rev Cancer. 2012;12:323–34.

    CAS  Article  Google Scholar 

  16. 16.

    Roden DM, Wilke RA, Kroemer HK, Stein CM. Pharmacogenomics: the genetics of variable drug responses. Circulation. 2011;123:1661–70.

    Article  Google Scholar 

  17. 17.

    Wright N, Rida PCG, Aneja R. Tackling intra- and inter-tumor heterogeneity to combat triple negative breast cancer. Front Biosci. 2017;22:1549–80.

    CAS  Article  Google Scholar 

  18. 18.

    Nicoletti R, Lopez S, Bellone S, Cocco E, Schwab CL, Black JD, et al. T-DM1, a novel antibody–drug conjugate, is highly effective against uterine and ovarian carcinosarcomas overexpressing HER2. Clin Exp Metastas-. 2015;32:29–38.

    CAS  Article  Google Scholar 

  19. 19.

    Hayashi T, Seiler R, Oo HZ, Jager W, Moskalev I, Awrey S, et al. Targeting HER2 with T-DM1, an antibody cytotoxic drug conjugate, is effective in HER2 over expressing bladder cancer. J Urol. 2015;194:1120–31.

    CAS  Article  Google Scholar 

  20. 20.

    Gagliato DM, Jardim DL, Marchesi MS, Hortobagyi GN. Mechanisms of resistance and sensitivity to anti-HER2 therapies in HER2+ breast cancer. Oncotarget. 2016;7:64431–46.

    Article  Google Scholar 

  21. 21.

    Gajria D, Chandarlapaty S. HER2-amplified breast cancer: mechanisms of trastuzumab resistance and novel targeted therapies. Expert Rev Anticancer Ther. 2011;11:263–75.

    CAS  Article  Google Scholar 

  22. 22.

    Oroudjev E, Lopus M, Wilson L, Audette C, Provenzano C, Erickson H, et al. Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability. Mol Cancer Ther. 2010;9:2700–13.

    CAS  Article  Google Scholar 

  23. 23.

    Lee KS, Oh DY, Kang YH, Park JE. Self-regulated mechanism of Plk1 localization to kinetochores: lessons from the Plk1-PBIP1 interaction. Cell Div. 2008;3:4.

    Article  Google Scholar 

  24. 24.

    Lindon C, Pines J. Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells. J Cell Biol. 2004;164:233–41.

    CAS  Article  Google Scholar 

  25. 25.

    Yim H. Current clinical trials with polo-like kinase 1 inhibitors in solid tumors. Anticancer Drugs. 2013;24:999–1006.

    CAS  Article  Google Scholar 

  26. 26.

    DeAngelo DJ, Sekeres MA, Ottmann OG, Sanz MA, Naoe T, Taube T, et al. Phase III randomized trial of volasertib combined with low-dose cytarabine (LDAC) versus placebo plus LDAC in patients aged > = 65 years with previously untreated, acute myeloid leukemia (AML) ineligible for intensive remission induction therapy. Clin Lymphoma Myeloma Leuk. 2015;15:S194.

    Article  Google Scholar 

  27. 27.

    Dohner H, Lubbert M, Fiedler W, Fouillard L, Haaland A, Brandwein JM, et al. Randomized, phase 2 trial of low-dose cytarabine with or without volasertib in AML patients not suitable for induction therapy. Blood. 2014;124:1426–33.

    Article  Google Scholar 

  28. 28.

    Mager PP. Structure–toxicity relationships applied to bicyclic organophosphorus poisons. Pharmazie. 1981;36:382–3.

    CAS  PubMed  Google Scholar 

  29. 29.

    Marcotte R, Sayad A, Brown KR, Sanchez-Garcia F, Reimand J, Haider M, et al. Functional genomic landscape of human breast cancer drivers, vulnerabilities, and resistance. Cell. 2016;164:293–309.

    CAS  Article  Google Scholar 

  30. 30.

    Daemen A, Griffith OL, Heiser LM, Wang NJ, Enache OM, Sanborn Z, et al. Modeling precision treatment of breast cancer. Genome Biol. 2013;14:R110.

    Article  Google Scholar 

  31. 31.

    Akbani R, Ng PK, Werner HM, Shahmoradgoli M, Zhang F, Ju Z, et al. A pan-cancer proteomic perspective on The Cancer Genome Atlas. Nat Commun. 2014;5:3887.

    CAS  Article  Google Scholar 

  32. 32.

    Gluck S, Ross JS, Royce M, McKenna EF Jr., Perou CM, Avisar E, et al. TP53 genomics predict higher clinical and pathologic tumor response in operable early-stage breast cancer treated with docetaxel-capecitabine+/− trastuzumab. Breast Cancer Res Treat. 2012;132:781–91.

    Article  Google Scholar 

  33. 33.

    Scatena CD, Stewart ZA, Mays D, Tang LJ, Keefer CJ, Leach SD, et al. Mitotic phosphorylation of Bcl-2 during normal cell cycle progression and taxol-induced growth arrest. J Biol Chem. 1998;273:30777–84.

    CAS  Article  Google Scholar 

  34. 34.

    Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol. 1999;19:8469–78.

    CAS  Article  Google Scholar 

  35. 35.

    Srivastava RK, Mi QS, Hardwick JM, Longo DL. Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci USA. 1999;96:3775–80.

    CAS  Article  Google Scholar 

  36. 36.

    Pathan N, Aime-Sempe C, Kitada S, Basu A, Haldar S, Reed JC. Microtubule-targeting drugs induce bcl-2 phosphorylation and association with Pin1. Neoplasia. 2001;3:550–9.

    CAS  Article  Google Scholar 

  37. 37.

    Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68:3421–8.

    CAS  Article  Google Scholar 

  38. 38.

    Manchado E, Guillamot M, Malumbres M. Killing cells by targeting mitosis. Cell Death Differ. 2012;19:369–77.

    CAS  Article  Google Scholar 

  39. 39.

    Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol. 2007;8:379–93.

    CAS  Article  Google Scholar 

  40. 40.

    Hain KO, Colin DJ, Rastogi S, Allan LA, Clarke PR. Prolonged mitotic arrest induces a caspase-dependent DNA damage response at telomeres that determines cell survival. Sci Rep. 2016;6:26766.

    CAS  Article  Google Scholar 

  41. 41.

    Orth JD, Loewer A, Lahav G, Mitchison TJ. Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction. Mol Biol Cell. 2012;23:567–76.

    CAS  Article  Google Scholar 

  42. 42.

    Tanner M, Kapanen AI, Junttila T, Raheem O, Grenman S, Elo J, et al. Characterization of a novel cell line established from a patient with Herceptin-resistant breast cancer. Mol Cancer Ther. 2004;3:1585–92.

    CAS  PubMed  Google Scholar 

  43. 43.

    Koninki K, Barok M, Tanner M, Staff S, Pitkanen J, Hemmila P, et al. Multiple molecular mechanisms underlying trastuzumab and lapatinib resistance in JIMT-1 breast cancer cells. Cancer Lett. 2010;294:211–9.

    Article  Google Scholar 

  44. 44.

    Barok M, Isola J, Palyi-Krekk Z, Nagy P, Juhasz I, Vereb G, et al. Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance. Mol Cancer Ther. 2007;6:2065–72.

    CAS  Article  Google Scholar 

  45. 45.

    Leow CC, Chesebrough J, Coffman KT, Fazenbaker CA, Gooya J, Weng D, et al. Antitumor efficacy of IPI-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib. Mol Cancer Ther. 2009;8:2131–41.

    CAS  Article  Google Scholar 

  46. 46.

    Recondo G Jr, de la Vega M, Galanternik F, Diaz-Canton E, Leone BA, Leone JP. Novel approaches to target HER2-positive breast cancer: trastuzumab emtansine. Cancer Manag Res. 2016;8:57–65.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Zitouni S, Nabais C, Jana SC, Guerrero A, Bettencourt-Dias M. Polo-like kinases: structural variations lead to multiple functions. Nat Rev Mol Cell Biol. 2014;15:433–52.

    CAS  Article  Google Scholar 

  48. 48.

    Barr FA, Sillje HH, Nigg EA. Polo-like kinases and the orchestration of cell division. Nat Rev Mol Cell Biol. 2004;5:429–40.

    CAS  Article  Google Scholar 

  49. 49.

    van Vugt MA, Medema RH. Getting in and out of mitosis with Polo-like kinase-1. Oncogene. 2005;24:2844–59.

    Article  Google Scholar 

  50. 50.

    Gleixner KV, Ferenc V, Peter B, Gruze A, Meyer RA, Hadzijusufovic E, et al. Polo-like kinase 1 (Plk1) as a novel drug target in chronic myeloid leukemia: overriding imatinib resistance with the Plk1 inhibitor BI 2536. Cancer Res. 2010;70:1513–23.

    CAS  Article  Google Scholar 

  51. 51.

    Cheng L, Wang CC, Jing JH. Polo-like kinase 1 as a potential therapeutic target for osteosarcoma. Curr Pharm Des. 2015;21:1347–50.

    CAS  Article  Google Scholar 

  52. 52.

    Maire V, Nemati F, Richardson M, Vincent-Salomon A, Tesson B, Rigaill G, et al. Polo-like kinase 1: a potential therapeutic option in combination with conventional chemotherapy for the management of patients with triple-negative breast cancer. Cancer Res. 2013;73:813–23.

    CAS  Article  Google Scholar 

  53. 53.

    Spankuch B, Kurunci-Csacsko E, Kaufmann M, Strebhardt K. Rational combinations of siRNAs targeting Plk1 with breast cancer drugs. Oncogene. 2007;26:5793–807.

    CAS  Article  Google Scholar 

  54. 54.

    Strebhardt K, Ullrich A. Targeting polo-like kinase 1 for cancer therapy. Nat Rev Cancer. 2006;6:321–30.

    CAS  Article  Google Scholar 

  55. 55.

    Degenhardt Y, Lampkin T. Targeting Polo-like kinase in cancer therapy. Clin Cancer Res. 2010;16:384–9.

    CAS  Article  Google Scholar 

  56. 56.

    Liu X. Targeting Polo-like kinases: a promising therapeutic approach for cancer treatment. Transl Oncol. 2015;8:185–95.

    Article  Google Scholar 

  57. 57.

    Liu X, Lei M, Erikson RL. Normal cells, but not cancer cells, survive severe Plk1 depletion. Mol Cell Biol. 2006;26:2093–108.

    Article  Google Scholar 

  58. 58.

    Winkles JA, Alberts GF. Differential regulation of polo-like kinase 1, 2, 3, and 4 gene expression in mammalian cells and tissues. Oncogene. 2005;24:260–6.

    CAS  Article  Google Scholar 

  59. 59.

    Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.

    CAS  Article  Google Scholar 

  60. 60.

    Tait SW, Green DR. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol. 2010;11:621–32.

    CAS  Article  Google Scholar 

  61. 61.

    Rantanen S, Monni O, Joensuu H, Franssila K, Knuutila S. Causes and consequences of BCL2 overexpression in diffuse large B-cell lymphoma. Leuk Lymphoma. 2001;42:1089–98.

    CAS  Article  Google Scholar 

  62. 62.

    Raffo AJ, Perlman H, Chen MW, Day ML, Streitman JS, Buttyan R. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res. 1995;55:4438–45.

    CAS  PubMed  Google Scholar 

  63. 63.

    Xiao D, Yue M, Su H, Ren P, Jiang J, Li F, et al. Polo-like kinase-1 regulates Myc stabilization and activates a feedforward circuit promoting tumor cell survival. Mol Cell. 2016;64:493–506.

    CAS  Article  Google Scholar 

  64. 64.

    Kops GJ, Weaver BA, Cleveland DW. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer. 2005;5:773–85.

    CAS  Article  Google Scholar 

  65. 65.

    Vogel C, Kienitz A, Muller R, Bastians H. The mitotic spindle checkpoint is a critical determinant for topoisomerase-based chemotherapy. J Biol Chem. 2005;280:4025–8.

    CAS  Article  Google Scholar 

  66. 66.

    Yamada HY, Gorbsky GJ. Spindle checkpoint function and cellular sensitivity to antimitotic drugs. Mol Cancer Ther. 2006;5:2963–9.

    CAS  Article  Google Scholar 

  67. 67.

    Trakala M, Partida D, Salazar-Roa M, Maroto M, Wachowicz P, de Carcer G, et al. Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1-deficient mice. Blood. 2015;126:1707–14.

    CAS  Article  Google Scholar 

  68. 68.

    Yuan J, Sanhaji M, Kramer A, Reindl W, Hofmann M, Kreis NN, et al. Polo-box domain inhibitor poloxin activates the spindle assembly checkpoint and inhibits tumor growth in vivo. Am J Pathol. 2011;179:2091–9.

    CAS  Article  Google Scholar 

  69. 69.

    Lee GY, Kenny PA, Lee EH, Bissell MJ. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods. 2007;4:359–65.

    CAS  Article  Google Scholar 

  70. 70.

    Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–78.

    CAS  Article  Google Scholar 

  71. 71.

    Mutlu M, Saatci O, Ansari SA, Yurdusev E, Shehwana H, Konu O, et al. miR-564 acts as a dual inhibitor of PI3K and MAPK signaling networks and inhibits proliferation and invasion in breast cancer. Sci Rep. 2016;6:32541.

    CAS  Article  Google Scholar 

  72. 72.

    Raza U, Saatci O, Uhlmann S, Ansari SA, Eyupoglu E, Yurdusev E, et al. The miR-644a/CTBP1/p53 axis suppresses drug resistance by simultaneous inhibition of cell survival and epithelial–mesenchymal transition in breast cancer. Oncotarget. 2016;7:49859–77.

    PubMed  PubMed Central  Google Scholar 

  73. 73.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001;25:402–8.

    CAS  Article  Google Scholar 

  74. 74.

    Sahin O, Lobke C, Korf U, Appelhans H, Sultmann H, Poustka A, et al. Combinatorial RNAi for quantitative protein network analysis. Proc Natl Acad Sci USA. 2007;104:6579–84.

    CAS  Article  Google Scholar 

  75. 75.

    Sonntag J, Schluter K, Bernhardt S, Korf U. Subtyping of breast cancer using reverse phase protein arrays. Expert Rev Proteom. 2014;11:757–70.

    CAS  Article  Google Scholar 

Download references


This project was supported by TUBITAK-BMBF Bilateral Grant numbers: TUBITAK, 214Z130 (to ÖŞ) and BMBF WTZ, 01DL16003 (to SW). ÖŞ further acknowledges the support from EMBO Installation Grant Number 2791. Special thanks to Genentech, USA Inc. for providing us with T-DM1 under Material Transfer Agreement with MTA number OR-213615. We thank the DKFZ Genomics and Proteomics Core Facility for providing sequencing excellent services. We also thank Deniz Atasoy and Pelin Dilsiz from Istanbul Medipol University for their help with confocal microscopy. ÖK acknowledges support from Baskent University and The Science Academy.

Authors contributions

ÖS designed and performed experiments; acquired, analyzed and interpreted data; and prepared the manuscript. SB, ÖA and SDE performed experiments; acquired and analyzed data; UR contributed to in vivo experimental design and data collection; EE and CA performed the transcriptome data analyses; AA performed the immunohistochemical stainings of xenografts and contributed to data interpretation; ÖK performed the co-immunoprecipitation experiments and contributed to data interpretation; SW designed experiments, interpreted data and critically read and edited the manuscript; ÖŞ designed the study, oversaw experiments and data analysis, and prepared the manuscript. All authors reviewed and commented on the manuscript.

Author information



Corresponding author

Correspondence to Özgür Şahin.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Saatci, Ö., Borgoni, S., Akbulut, Ö. et al. Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer. Oncogene 37, 2251–2269 (2018).

Download citation

Further reading