Skip to main content

Advertisement

Springer Nature Link
Log in

Acquisition of docetaxel resistance in breast cancer cells reveals upregulation of ABCB1 expression as a key mediator of resistance accompanied by discrete upregulation of other specific genes and pathways

  • Research Article
  • Published:
Tumor Biology

Abstract

The microtubule-targeting taxanes are important in breast cancer therapy, but no predictive biomarkers have yet been identified with sufficient scientific evidence to allow clinical routine use. The purposes of the present study were to develop a cell-culture-based discovery platform for docetaxel resistance and thereby identify key molecular mechanisms and predictive molecular characteristics to docetaxel resistance. Two docetaxel-resistant cell lines, MCF7RES and MDARES, were generated from their respective parental cell lines MCF-7 and MDA-MB-231 by stepwise selection in docetaxel dose increments over 15 months. The cell lines were characterized regarding sensitivity to docetaxel and other chemotherapeutics and subjected to transcriptome-wide mRNA microarray profiling. MCF7RES and MDARES exhibited a biphasic growth inhibition pattern at increasing docetaxel concentrations. Gene expression analysis singled out ABCB1, which encodes permeability glycoprotein (Pgp), as the top upregulated gene in both MCF7RES and MDARES. Functional validation revealed Pgp as a key resistance mediator at low docetaxel concentrations (first-phase response), whereas additional resistance mechanisms appeared to be prominent at higher docetaxel concentrations (second-phase response). Additional resistance mechanisms were indicated by gene expression profiling, including genes in the interferon-inducible protein family in MCF7RES and cancer testis antigen family in MDARES. Also, upregulated expression of various ABC transporters, ECM-associated proteins, and lysosomal proteins was identified in both resistant cell lines. Finally, MCF7RES and MDARES presented with cross-resistance to epirubicin, but only MDARES showed cross-resistance to oxaliplatin. In conclusion, Pgp was identified as a key mediator of resistance to low docetaxel concentrations with other resistance mechanisms prominent at higher docetaxel concentrations. Supporting Pgp upregulation as one major mechanism of taxane resistance and cell-line-specific alterations as another, both MCF7RES and MDARES were cross-resistant to epirubicin (Pgp substrate), but only MDARES was cross-resistant to oxaliplatin (non-Pgp substrate).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.

    Article  PubMed  Google Scholar 

  2. Mouridsen HT, Bjerre KD, Christiansen P, Jensen MB, Moller S. Improvement of prognosis in breast cancer in Denmark 1977-2006, based on the nationwide reporting to the DBCG Registry. Acta Oncol. 2008;47:525–36.

    Article  PubMed  Google Scholar 

  3. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11–30.

    Article  PubMed  Google Scholar 

  4. Gennari A, Conte P, Rosso R, Orlandini C, Bruzzi P. Survival of metastatic breast carcinoma patients over a 20-year period: a retrospective analysis based on individual patient data from six consecutive studies. Cancer. 2005;104:1742–50.

    Article  PubMed  Google Scholar 

  5. El Saghir NS, Tfayli A, Hatoum HA, Nachef Z, Dinh P, Awada A. Treatment of metastatic breast cancer: state-of-the-art, subtypes and perspectives. Crit Rev Oncol Hematol. 2011;80:433–49.

    Article  PubMed  Google Scholar 

  6. Diaz JF, Andreu JM. Assembly of purified GDP-tubulin into microtubules induced by taxol and taxotere: reversibility, ligand stoichiometry, and competition. Biochemistry. 1993;32:2747–55.

    Article  CAS  PubMed  Google Scholar 

  7. Fabbri F, Carloni S, Brigliadori G, Zoli W, Lapalombella R, Marini M. Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: a cytometric study. BMC Cell Biol. 2006;7:6.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Terrano DT, Upreti M, Chambers TC. Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis. Mol Cell Biol. 2010;30:640–56.

    Article  CAS  PubMed  Google Scholar 

  9. Trock BJ, Leonessa F, Clarke R. Multidrug resistance in breast cancer: a meta-analysis of MDR1/gp170 expression and its possible functional significance. J Natl Cancer Inst. 1997;89:917–31.

    Article  CAS  PubMed  Google Scholar 

  10. Noguchi S. Predictive factors for response to docetaxel in human breast cancers. Cancer Sci. 2006;97:813–20.

    Article  CAS  PubMed  Google Scholar 

  11. Linn SC, Pinedo HM, van Ark-Otte J, van der Valk P, Hoekman K, Honkoop AH, et al. Expression of drug resistance proteins in breast cancer, in relation to chemotherapy. Int J Cancer. 1997;71:787–95.

    Article  CAS  PubMed  Google Scholar 

  12. Carter A, Dann EJ, Katz T, Shechter Y, Oliven A, Regev R, et al. Cells from chronic myelogenous leukaemia patients at presentation exhibit multidrug resistance not mediated by either MDR1 or MRP1. Br J Haematol. 2001;114:581–90.

    Article  CAS  PubMed  Google Scholar 

  13. Chintamani, Singh JP, Mittal MK, Saxena S, Bansal A, Bhatia A, et al. Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer—a prospective clinical study. World J Surg Oncol. 2005;3:61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987;47:936–42.

    CAS  PubMed  Google Scholar 

  15. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19:185–93.

    Article  CAS  PubMed  Google Scholar 

  16. Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004;3:Article3

  17. Smyth GK, Speed T. Normalization of cDNA microarray data. Methods. 2003;31:265–73.

    Article  CAS  PubMed  Google Scholar 

  18. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5:R80.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Hochberg Y, Benjamini Y. More powerful procedures for multiple significance testing. Stat Med. 1990;9:811–8.

    Article  CAS  PubMed  Google Scholar 

  20. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–73.

    Article  CAS  PubMed  Google Scholar 

  21. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012;40:D109–14.

    Article  CAS  PubMed  Google Scholar 

  23. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu B, Sun D, Xia W, Hung MC, Yu D. Cross-reactivity of C219 anti-p170(mdr-1) antibody with p185(c-erbB2) in breast cancer cells: cautions on evaluating p170(mdr-1). J Natl Cancer Inst. 1997;89:1524–9.

    Article  CAS  PubMed  Google Scholar 

  25. Hernandez-Vargas H, Palacios J, Moreno-Bueno G. Molecular profiling of docetaxel cytotoxicity in breast cancer cells: uncoupling of aberrant mitosis and apoptosis. Oncogene. 2007;26:2902–13.

    Article  CAS  PubMed  Google Scholar 

  26. Ajabnoor GM, Crook T, Coley HM. Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells. Cell Death Dis. 2012;3:e260.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Iseri OD, Kars MD, Gunduz U. Two different docetaxel resistant MCF-7 sublines exhibited different gene expression pattern. Mol Biol Rep. 2012;39:3505–16.

    Article  CAS  PubMed  Google Scholar 

  28. Kars MD, Iseri OD, Gunduz U. A microarray based expression profiling of paclitaxel and vincristine resistant MCF-7 cells. Eur J Pharmacol. 2011;657:4–9.

    Article  CAS  PubMed  Google Scholar 

  29. Iseri OD, Kars MD, Arpaci F, Gunduz U. Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins. Cancer Chemother Pharmacol. 2010;65:447–55.

    Article  CAS  PubMed  Google Scholar 

  30. Kars MD, Iseri OD, Gunduz U. Drug resistant breast cancer cells overexpress ETS1 gene. Biomed Pharmacother. 2010;64:458–62.

    Article  CAS  PubMed  Google Scholar 

  31. Villeneuve DJ, Hembruff SL, Veitch Z, Cecchetto M, Dew WA, Parissenti AM. cDNA microarray analysis of isogenic paclitaxel- and doxorubicin-resistant breast tumor cell lines reveals distinct drug-specific genetic signatures of resistance. Breast Cancer Res Treat. 2006;96:17–39.

    Article  CAS  PubMed  Google Scholar 

  32. Duan Z, Lamendola DE, Duan Y, Yusuf RZ, Seiden MV. Description of paclitaxel resistance-associated genes in ovarian and breast cancer cell lines. Cancer Chemother Pharmacol. 2005;55:277–85.

    Article  CAS  PubMed  Google Scholar 

  33. Wu CP, Hsieh CH, Wu YS. The emergence of drug transporter-mediated multidrug resistance to cancer chemotherapy. Mol Pharm. 2011;8:1996–2011.

    Article  CAS  PubMed  Google Scholar 

  34. Vera-Ramirez L, Sanchez-Rovira P, Ramirez-Tortosa CL, Quiles JL, Ramirez-Tortosa M, Lorente JA. Transcriptional shift identifies a set of genes driving breast cancer chemoresistance. PLoS ONE. 2013;8:e53983.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hembruff SL, Laberge ML, Villeneuve DJ, Guo B, Veitch Z, Cecchetto M, et al. Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance. BMC Cancer. 2008;8:318.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Guarneri V, Dieci MV, Conte P. Relapsed triple-negative breast cancer: challenges and treatment strategies. Drugs. 2013;73:1257–65.

    Article  CAS  PubMed  Google Scholar 

  37. Duan Z, Duan Y, Lamendola DE, Yusuf RZ, Naeem R, Penson RT, et al. Overexpression of MAGE/GAGE genes in paclitaxel/doxorubicin-resistant human cancer cell lines. Clin Cancer Res. 2003;9:2778–85.

    CAS  PubMed  Google Scholar 

  38. Cappell KM, Sinnott R, Taus P, Maxfield K, Scarbrough M, Whitehurst AW. Multiple cancer testis antigens function to support tumor cell mitotic fidelity. Mol Cell Biol. 2012;32:4131–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Materna V, Surowiak P, Kaplenko I, Spaczynski M, Duan Z, Zabel M, et al. Taxol-resistance-associated gene-3 (TRAG-3/CSAG2) expression is predictive for clinical outcome in ovarian carcinoma patients. Virchows Arch. 2007;450:187–94.

    Article  CAS  PubMed  Google Scholar 

  40. Grigoriadis A, Caballero OL, Hoek KS, da Silva L, Chen YT, Shin SJ, et al. CT-X antigen expression in human breast cancer. Proc Natl Acad Sci U S A. 2009;106:13493–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Chen YT, Ross DS, Chiu R, Zhou XK, Chen YY, Lee P, et al. Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers. PLoS ONE. 2011;6:e17876.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hellman M, Tossavainen H, Rappu P, Heino J, Permi P. Characterization of intrinsically disordered prostate associated gene (PAGE5) at single residue resolution by NMR spectroscopy. PLoS ONE. 2011;6:e26633.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Aoudjit F, Vuori K. Integrin signaling in cancer cell survival and chemoresistance. Chemother Res Pract. 2012;2012:283181.

    PubMed  PubMed Central  Google Scholar 

  44. Groth-Pedersen L, Jaattela M. Combating apoptosis and multidrug resistant cancers by targeting lysosomes. Cancer Lett. 2013;332:265–74.

    Article  CAS  PubMed  Google Scholar 

  45. Nomura T, Katunuma N. Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells. J Med Invest. 2005;52:1–9.

    Article  PubMed  Google Scholar 

  46. Zhang XJ, Chen S, Huang KX, Le WD. Why should autophagic flux be assessed? Acta Pharmacol Sin. 2013;34:595–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Vibeke Jensen and Signe Lykke Nielsen for excellent technical assistance.This work was supported by The Harboe Foundation, A Race Against Breast Cancer; Danish National Research Foundation: Sino-Danish Breast Cancer Research Centre; Danish Centre for Translational Breast Cancer Research; The Danish Cancer Society and The Danish Council for Independent Research: Medical Sciences.

Conflict of interest

None.

Author information

Author notes

    Authors and Affiliations

    1. Sino-Danish Breast Cancer Research Center, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark

      Stine Ninel Hansen, David Westergaard, Mathilde Borg Houlberg Thomsen, Mette Vistesen, Khoa Nguyen Do, Louise Fogh, Kirstine C. Belling, Jun Wang, Huanming Yang, Ramneek Gupta, Henrik J. Ditzel, José Moreira, Nils Brünner, Jan Stenvang & Anne-Sofie Schrohl

    2. Section for Molecular Disease Biology, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark

      Stine Ninel Hansen, Mathilde Borg Houlberg Thomsen, Mette Vistesen, Louise Fogh, José Moreira, Nils Brünner, Jan Stenvang & Anne-Sofie Schrohl

    3. Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kemitorvet building 208, DK-2800, Lyngby, Denmark

      David Westergaard, Khoa Nguyen Do, Kirstine C. Belling & Ramneek Gupta

    4. Beijing Genomics Institute, Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China

      Jun Wang & Huanming Yang

    5. Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloewsvej 25.3, DK-5000, Odense C, Denmark

      Henrik J. Ditzel

    Authors
    1. Stine Ninel Hansen
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. David Westergaard
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Mathilde Borg Houlberg Thomsen
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Mette Vistesen
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Khoa Nguyen Do
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Louise Fogh
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Kirstine C. Belling
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Jun Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Huanming Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Ramneek Gupta
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Henrik J. Ditzel
      View author publications

      You can also search for this author in PubMed Google Scholar

    12. José Moreira
      View author publications

      You can also search for this author in PubMed Google Scholar

    13. Nils Brünner
      View author publications

      You can also search for this author in PubMed Google Scholar

    14. Jan Stenvang
      View author publications

      You can also search for this author in PubMed Google Scholar

    15. Anne-Sofie Schrohl
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Jan Stenvang.

    Additional information

    Jan Stenvang and Anne-Sofie Schrohl contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    ESM 1

    (XLSX 8395 kb)

    ESM 2

    (XLSX 66 kb)

    Online Resource 3

    Identification of altered molecular networks in docetaxel-resistant cell lines. Significant functional annotation clusters and pathways were identified in MCF7RES and MDARES by gene set enrichment analysis, with a KEGG pathway false discovery rate (FDR) ≤0.4 considered significantly enriched. The normalized enrichment score (NES) describes the level of downregulation (yellow) and upregulation (red) (EPS 172 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Hansen, S.N., Westergaard, D., Thomsen, M.B.H. et al. Acquisition of docetaxel resistance in breast cancer cells reveals upregulation of ABCB1 expression as a key mediator of resistance accompanied by discrete upregulation of other specific genes and pathways. Tumor Biol. 36, 4327–4338 (2015). https://doi.org/10.1007/s13277-015-3072-4

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s13277-015-3072-4

    Keywords

    Search

    Navigation