A transposon screen identifies enhancement of NF-κB pathway as a mechanism of resistance to eribulin



Eribulin mesylate (eribulin) is an efficient microtubule inhibitor that is used for metastatic breast cancer. However, breast cancer can develop resistance to eribulin. This resistance mechanism needs to be elucidated.


A transposon mutagenesis screen was conducted using a pPB-SB-CMV-puro-SD plasmid and pCMV-PBase transposase. Viability and cytotoxicity were analyzed by MTT assay and flow cytometry, respectively. Real-time PCR and western blot were used for gene expression analysis. In addition, vivo study was also designed to analyze therapy efficiency.


TAB2, which is part of the nuclear factor-kappa B (NF-κB) pathway, was identified as a candidate eribulin-resistant gene. TAB2 down-regulation resulted in significantly lower cell viability and higher cytotoxicity of cells treated with eribulin, while TAB2 up-regulation showed opposite results. Similarly, combination of NF-κB inhibitors [Bay-117082 and QNZ (quinazoline derivative)] with eribulin showed significantly lower cell viability and higher drug cytotoxicity than single agent treatment with eribulin in MDA-MB-231 cells. However, QNZ increased NF-κB activity in MCF7 cells by up-regulating TAB2, which reduced the sensitivity to eribulin. Furthermore, combination of Bay-117082 with eribulin induced greater regression of MDA-MB-231 tumors compared to eribulin monotherapy in vivo.


These results consistently illustrated that TAB2-NF-κB pathway may increases resistance to eribulin in breast cancer models. Moreover, these results support the use of a combination strategy of eribulin with NF-κB inhibitors, and provide evidence that transposon mutagenesis screens are capable of identifying drug-resistant genes.

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  1. 1.

    Cortes J, O’Shaughnessy J, Loesch D, Blum JL, Vahdat LT, Petrakova K, et al. Eribulin monotherapy versus treatment of physician’s choice in patients with metastatic breast cancer (EMBRACE): a phase 3 open-label randomised study. Lancet. 2011;377:914–23.

    CAS  Article  Google Scholar 

  2. 2.

    Kaufman PA, Awada A, Twelves C, Yelle L, Perez EA, Velikova G, et al. Phase III open-label randomized study of eribulin mesylate versus capecitabine in patients with locally advanced or metastatic breast cancer previously treated with an anthracycline and a taxane. J Clin Oncol. 2015;33:594–601.

    CAS  Article  Google Scholar 

  3. 3.

    Pivot X, Marme F, Koenigsberg R, Guo M, Berrak E, Wolfer A. Pooled analyses of eribulin in metastatic breast cancer patients with at least one prior chemotherapy. Ann Oncol. 2016;27:1525–31.

    CAS  Article  Google Scholar 

  4. 4.

    Schmitt MW, Loeb LA, Salk JJ. The influence of subclonal resistance mutations on targeted cancer therapy. Nat Rev Clin Oncol. 2016;13:335–47.

    CAS  Article  Google Scholar 

  5. 5.

    Goto W, Kashiwagi S, Asano Y, Takada K, Takahashi K, Fujita H, et al. The effects of eribulin on breast cancer microenvironment identified using eribulin-resistant breast cancer cell lines. Anticancer Res. 2019;39:4031–41.

    CAS  Article  Google Scholar 

  6. 6.

    Sampson VB, Vetter NS, Zhang W, Patil PU, Mason RW, George E, et al. Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma. Oncotarget. 2016;7:86594–607.

    Article  Google Scholar 

  7. 7.

    Chen L, Stuart L, Ohsumi TK, Burgess S, Varshney GK, Dastur A, et al. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics. BMC Cancer. 2013;13:93.

    CAS  Article  Google Scholar 

  8. 8.

    Tsutsui M, Kawakubo H, Hayashida T, Fukuda K, Nakamura R, Takahashi T, et al. Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma. Int J Oncol. 2015;47:867–74.

    CAS  Article  Google Scholar 

  9. 9.

    Kanayama A, Seth RB, Sun L, Ea CK, Hong M, Shaito A, et al. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 2004;15:535–48.

    CAS  Article  Google Scholar 

  10. 10.

    Li Y, Deng L, Zhao X, Li B, Ren D, Yu L, et al. Tripartite motif-containing 37 (TRIM37) promotes the aggressiveness of non-small-cell lung cancer cells by activating the NF-kappaB pathway. J Pathol. 2018;246:366–78.

    CAS  Article  Google Scholar 

  11. 11.

    Safina A, Sotomayor P, Limoge M, Morrison C, Bakin AV. TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells. Mol Cancer Res. 2011;9:1042–53.

    CAS  Article  Google Scholar 

  12. 12.

    Tobe M, Isobe Y, Tomizawa H, Nagasaki T, Takahashi H, Fukazawa T, et al. Discovery of quinazolines as a novel structural class of potent inhibitors of NF-kappa B activation. Bioorg Med Chem. 2003;11:383–91.

    CAS  Article  Google Scholar 

  13. 13.

    Pierce JW, Schoenleber R, Jesmok G, Best J, Moore SA, Collins T, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem. 1997;272:21096–103.

    CAS  Article  Google Scholar 

  14. 14.

    Xue W, Meylan E, Oliver TG, Feldser DM, Winslow MM, Bronson R, et al. Response and resistance to NF-kappaB inhibitors in mouse models of lung adenocarcinoma. Cancer Discov. 2011;1:236–47.

    CAS  Article  Google Scholar 

  15. 15.

    Christie EL, Pattnaik S, Beach J, Copeland A, Rashoo N, Fereday S, et al. Multiple ABCB1 transcriptional fusions in drug resistant high-grade serous ovarian and breast cancer. Nat Commun. 2019;10:1295.

    Article  Google Scholar 

  16. 16.

    Robinson DR, Kalyana-Sundaram S, Wu YM, Shankar S, Cao X, Ateeq B, et al. Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer. Nat Med. 2011;17:1646–51.

    CAS  Article  Google Scholar 

  17. 17.

    Zhang S, Zhang X, Sun Q, Zhuang C, Li G, Sun L, et al. LncRNA NR2F2-AS1 promotes tumourigenesis through modulating BMI1 expression by targeting miR-320b in non-small cell lung cancer. J Cell Mol Med. 2019;23:2001–11.

    CAS  Article  Google Scholar 

  18. 18.

    Bhatnagar S, Gazin C, Chamberlain L, Ou J, Zhu X, Tushir JS, et al. TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature. 2014;516:116–20.

    CAS  Article  Google Scholar 

  19. 19.

    Long J, Cai Q, Sung H, Shi J, Zhang B, Choi JY, et al. Genome-wide association study in east Asians identifies novel susceptibility loci for breast cancer. PLoS Genet. 2012;8:e1002532.

    CAS  Article  Google Scholar 

  20. 20.

    Li S, Wang L, Dorf ME. PKC phosphorylation of TRAF2 mediates IKKalpha/beta recruitment and K63-linked polyubiquitination. Mol Cell. 2009;33:30–42.

    CAS  Article  Google Scholar 

  21. 21.

    Hattori Y, Hattori S, Kasai K. Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation. Eur J Pharmacol. 2003;481:153–8.

    CAS  Article  Google Scholar 

  22. 22.

    Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature. 2001;412:346–51.

    CAS  Article  Google Scholar 

  23. 23.

    Barkett M, Gilmore TD. Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene. 1999;18:6910–24.

    CAS  Article  Google Scholar 

  24. 24.

    Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998;281:1680–3.

    CAS  Article  Google Scholar 

  25. 25.

    Wu MX, Ao Z, Prasad KV, Wu R, Schlossman SF. IEX-1L, an apoptosis inhibitor involved in NF-kappaB-mediated cell survival. Science. 1998;281:998–1001.

    CAS  Article  Google Scholar 

  26. 26.

    Kikuchi E, Horiguchi Y, Nakashima J, Kuroda K, Oya M, Ohigashi T, et al. Suppression of hormone-refractory prostate cancer by a novel nuclear factor kappaB inhibitor in nude mice. Cancer Res. 2003;63:107–10.

    CAS  PubMed  Google Scholar 

  27. 27.

    Ahmed KM, Zhang H, Park CC. NF-kappaB regulates radioresistance mediated by beta1-integrin in three-dimensional culture of breast cancer cells. Cancer Res. 2013;73:3737–48.

    CAS  Article  Google Scholar 

  28. 28.

    Wang W, Cassidy J, O’Brien V, Ryan KM, Collie-Duguid E. Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells. Cancer Res. 2004;64:8167–76.

    CAS  Article  Google Scholar 

  29. 29.

    Baud V, Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov. 2009;8:33–40.

    CAS  Article  Google Scholar 

  30. 30.

    Liu B, Sun L, Liu Q, Gong C, Yao Y, Lv X, et al. A cytoplasmic NF-kappaB interacting long noncoding RNA blocks IkappaB phosphorylation and suppresses breast cancer metastasis. Cancer Cell. 2015;27:370–81.

    CAS  Article  Google Scholar 

  31. 31.

    Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov. 2004;3:17–26.

    CAS  Article  Google Scholar 

  32. 32.

    Mulligan G, Mitsiades C, Bryant B, Zhan F, Chng WJ, Roels S, et al. Gene expression profiling and correlation with outcome in clinical trials of the proteasome inhibitor bortezomib. Blood. 2007;109:3177–88.

    CAS  Article  Google Scholar 

  33. 33.

    Kumar S, Rajkumar SV. Many facets of bortezomib resistance/susceptibility. Blood. 2008;112:2177–8.

    CAS  Article  Google Scholar 

  34. 34.

    Gao Y, Xiao X, Zhang C, Yu W, Guo W, Zhang Z, et al. Melatonin synergizes the chemotherapeutic effect of 5-fluorouracil in colon cancer by suppressing PI3K/AKT and NF-kappaB/iNOS signaling pathways. J Pineal Res. 2017;62

  35. 35.

    Motwani M, Delohery TM, Schwartz GK. Sequential dependent enhancement of caspase activation and apoptosis by flavopiridol on paclitaxel-treated human gastric and breast cancer cells. Clin Cancer Res. 1999;5:1876–83.

    CAS  PubMed  Google Scholar 

  36. 36.

    Hideshima H, Yoshida Y, Ikeda H, Hide M, Iwasaki A, Anderson KC, et al. IKKbeta inhibitor in combination with bortezomib induces cytotoxicity in breast cancer cells. Int J Oncol. 2014;44:1171–6.

    CAS  Article  Google Scholar 

  37. 37.

    Gilmore TD, Herscovitch M. Inhibitors of NF-kappaB signaling: 785 and counting. Oncogene. 2006;25:6887–99.

    CAS  Article  Google Scholar 

  38. 38.

    Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004;25:280–8.

    CAS  Article  Google Scholar 

  39. 39.

    Van Laere SJ, Van der Auwera I, Van den Eynden GG, van Dam P, Van Marck EA, Vermeulen PB, et al. NF-kappaB activation in inflammatory breast cancer is associated with oestrogen receptor downregulation, secondary to EGFR and/or ErbB2 overexpression and MAPK hyperactivation. Br J Cancer. 2007;97:659–69.

    Article  Google Scholar 

  40. 40.

    Yamaguchi N, Ito T, Azuma S, Ito E, Honma R, Yanagisawa Y, et al. Constitutive activation of nuclear factor-kappaB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines. Cancer Sci. 2009;100:1668–74.

    CAS  Article  Google Scholar 

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We thank Dr. Li in kindly providing the transposon plasmid and Kazuhiro Miyao (laboratory assistant) at Keio University for assistance in the experiments.

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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by XT, TH, and TM. The first draft of the manuscript was written by XT and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Tetsu Hayashida.

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Conflict of interest

T. Hayashida and Y. Kitagawa received research grants and lecture fees from Eisai Co., Ltd. A. Nagayama’s immediate family member has a leadership position with Chugai Co., Ltd. and owns stock options of Chugai Co., Ltd. All remaining authors have no conflicts of interest to declare.

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This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of Keio university.

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Teng, X., Hayashida, T., Murata, T. et al. A transposon screen identifies enhancement of NF-κB pathway as a mechanism of resistance to eribulin. Breast Cancer (2021). https://doi.org/10.1007/s12282-021-01224-1

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  • Breast cancer
  • Drug resistance
  • Transposon mutagenesis screen
  • Eribulin
  • NF-κB