Advertisement

Journal of Cancer Research and Clinical Oncology

, Volume 144, Issue 7, pp 1317–1327 | Cite as

Characterization of carfilzomib-resistant non-small cell lung cancer cell lines

  • Neale T. Hanke
  • Elliot Imler
  • Marilyn T. Marron
  • Bruce E. Seligmann
  • Linda L. Garland
  • Amanda F. Baker
Original Article – Cancer Research

Abstract

Purpose

We previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines.

Methods

CFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin.

Results

The CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin.

Conclusions

Upregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

Keywords

Carfilzomib Drug resistance Lung cancer Cross-resistance Pgp Proteasome inhibitor Non-small cell lung cancer 

Notes

Acknowledgements

The authors also wish to thank Adrianna E. Pulver, BS, for her technical assistance and the University of Arizona Cancer Center/Arizona Research Laboratories Flow Cytometry Core Facility which is partially funded by P30CA023074 from the National Cancer Institute (NCI).

Funding

This work was supported by a research collaboration award by Onyx Pharmaceuticals, Inc., an Amgen subsidiary, and a Basic/Clinical Translational Partnership Pilot Grant award from the Arizona Cancer Center Support Grant P30CA023074 from the National Cancer Institute (NCI).

Compliance with ethical standards

Conflict of interest

Authors EI, MTM, and BES are employees of BioSpyder Technologies, Inc., the company that developed and now sells the TempO-Seq kits. The other authors have no conflicts to declare.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

No human participants were used in this study.

Supplementary material

432_2018_2662_MOESM1_ESM.pptx (67 kb)
Supplementary material 1 (PPTX 66 KB)
432_2018_2662_MOESM2_ESM.pptx (939 kb)
Supplemental Figure 1. Upregulation of KDM6A corresponds to SAHA sensitivity in H520 CFZ resistant cells. TempO-Seq gene expression analysis showed upregulation of an HDACi sensitizer gene, KDM6A, in H520 CFZ resistant cells compared to its parent, but not in A549 CFZ resistant cells compared to its parent. To further evaluate, cells were left untreated or treated with CFZ and/or SAHA parent 48 h IC50 doses. Detection of cleaved caspase-3 by immunoblot in total extracts of cells harvested after 24 hours of treatment as indicated. Tubulin was used as a loading control (PPTX 939 KB)

References

  1. Ao L et al (2012) Development of peptide-based reversing agents for p-glycoprotein-mediated resistance to carfilzomib. Mol Pharm 9:2197–2205.  https://doi.org/10.1021/mp300044b CrossRefPubMedPubMedCentralGoogle Scholar
  2. Baker AF, Hanke NT, Sands BJ, Carbajal L, Anderl JL, Garland LL (2014) Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models. J Exp Clin Can Res CR 33:111.  https://doi.org/10.1186/s13046-014-0111-8 CrossRefGoogle Scholar
  3. Bielcikova Z, Jakabova A, Pinkas M, Zemanova M, Kolostova K, Bobek V (2017) Circulating tumor cells: what we know, what do we want to know about them and are they ready to be used in clinics? Am J Transl Res 9:2807–2823PubMedPubMedCentralGoogle Scholar
  4. Chauhan D et al (2007) A novel Bcl-2/Bcl-X(L)/Bcl-w inhibitor ABT-737 as therapy in multiple myeloma Oncogene 26:2374–2380.  https://doi.org/10.1038/sj.onc.1210028 CrossRefPubMedGoogle Scholar
  5. Eckford PD, Sharom FJ (2009) ABC efflux pump-based resistance to chemotherapy drugs. Chem Rev 109:2989–3011.  https://doi.org/10.1021/cr9000226 CrossRefPubMedGoogle Scholar
  6. Frassanito MA et al (2016) Halting pro-survival autophagy by TGFbeta inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients Leukemia 30:640–648.  https://doi.org/10.1038/leu.2015.289 CrossRefPubMedGoogle Scholar
  7. Gareau C, Fournier MJ, Filion C, Coudert L, Martel D, Labelle Y, Mazroui R (2011) p21(WAF1/CIP1) upregulation through the stress granule-associated protein CUGBP1 confers resistance to bortezomib-mediated apoptosis. PLoS One 6:e20254.  https://doi.org/10.1371/journal.pone.0020254 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Grogan TM et al (1993) P-glycoprotein expression in human plasma cell myeloma: correlation with. Prior Chemother Blood 81:490–495Google Scholar
  9. Hanke NT, Garland LL, Baker AF (2016) Carfilzomib combined with suberanilohydroxamic acid (SAHA) synergistically promotes endoplasmic reticulum stress in non-small cell lung cancer cell lines. J Cancer Res Clin Oncol 142:549–560.  https://doi.org/10.1007/s00432-015-2047-6 CrossRefPubMedGoogle Scholar
  10. Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J, Anderson KC (2001) The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res 61:3071–3076PubMedGoogle Scholar
  11. Huang Z, Wu Y, Zhou X, Xu J, Zhu W, Shu Y, Liu P (2014) Efficacy of therapy with bortezomib in solid tumors: a review based on 32 clinical trials. Future Oncol 10:1795–1807.  https://doi.org/10.2217/fon.14.30 CrossRefPubMedGoogle Scholar
  12. Jain S, Diefenbach C, Zain J, O’Connor OA (2011) Emerging role of carfilzomib in treatment of relapsed and refractory lymphoid neoplasms and multiple myeloma. Core Evid 6:43–57.  https://doi.org/10.2147/CE.S13838 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Komatsu N, Kawamata N, Takeuchi S, Yin D, Chien W, Miller CW, Koeffler HP (2006) SAHA, a HDAC inhibitor, has profound anti-growth activity against non-small cell lung cancer cells. Oncol Rep 15:187–191PubMedGoogle Scholar
  14. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq. 2 Genome Biol 15:550.  https://doi.org/10.1186/s13059-014-0550-8 CrossRefGoogle Scholar
  15. Lu S et al (2008) Point mutation of the proteasome beta5 subunit gene is an important mechanism of bortezomib resistance in bortezomib-selected variants of Jurkat T cell lymphoblastic lymphoma/leukemia line. J Pharmacol Exp Ther 326:423–431.  https://doi.org/10.1124/jpet.108.138131 CrossRefPubMedGoogle Scholar
  16. Mitra AK, Stessman H, Shaughnessy J, Van Ness B (2014) Profiling bortezomib resistance in multiple myeloma: implications in personalized pharmacotherapy Resist Target Anti-C 3:117–147  https://doi.org/10.1007/978-3-319-06752-0_5 Google Scholar
  17. Mitra AK et al (2017) A gene expression signature distinguishes innate response and resistance to proteasome inhibitors in multiple myeloma. Blood Cancer J 7:e581.  https://doi.org/10.1038/bcj.2017.56 CrossRefPubMedPubMedCentralGoogle Scholar
  18. O’Connor OA et al. (2005) Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin’s lymphoma and mantle cell lymphoma J Clin Oncol 23:676–684  https://doi.org/10.1200/JCO.2005.02.050 CrossRefPubMedGoogle Scholar
  19. Oerlemans R et al (2008) Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein. Blood 112:2489–2499.  https://doi.org/10.1182/blood-2007-08-104950 CrossRefPubMedGoogle Scholar
  20. Papadopoulos KP et al (2013) A phase I/II study of carfilzomib 2–10-min infusion in patients with advanced solid tumors. Cancer Chemother Pharmacol 72:861–868.  https://doi.org/10.1007/s00280-013-2267-x [doi]CrossRefPubMedPubMedCentralGoogle Scholar
  21. Park SM et al (2016) Molecular profiling of single circulating tumor cells from lung cancer patients. Proc Natl Acad Sci USA 113:E8379-E8386.  https://doi.org/10.1073/pnas.1608461113 PubMedPubMedCentralGoogle Scholar
  22. Riz I, Hawley TS, Hawley RG (2015) KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models Oncotarget 6:14814–14831.  https://doi.org/10.18632/oncotarget.4530 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Riz I, Hawley TS, Marsal JW, Hawley RG (2016) Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic translational reprogramming Oncotarget 7:66360–66385.  https://doi.org/10.18632/oncotarget.11960 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Ruckrich T et al (2009) Characterization of the ubiquitin-proteasome system in bortezomib-adapted. Cells Leukemia 23:1098–1105.  https://doi.org/10.1038/leu.2009.8 CrossRefPubMedGoogle Scholar
  25. Rumpold H, Salvador C, Wolf AM, Tilg H, Gastl G, Wolf D (2007) Knockdown of PgP resensitizes leukemic cells to proteasome inhibitors Biochem Biophys Res Commun 361:549–554  https://doi.org/10.1016/j.bbrc.2007.07.049 CrossRefPubMedGoogle Scholar
  26. Rushworth SA, Zaitseva L, Murray MY, Shah NM, Bowles KM, MacEwan DJ (2012) The high Nrf2 expression in human acute myeloid leukemia is driven by NF-kappaB and underlies its chemo. Resist Blood 120:5188–5198.  https://doi.org/10.1182/blood-2012-04-422121 CrossRefGoogle Scholar
  27. Schmidmaier R, Baumann P, Bumeder I, Meinhardt G, Straka C, Emmerich B (2007) First clinical experience with simvastatin to overcome drug resistance in refractory multiple myeloma European. J Haematol 79:240–243.  https://doi.org/10.1111/j.1600-0609.2007.00902.x Google Scholar
  28. Siegel DS et al (2012) A phase 2 study of single-agent carfilzomib (PX-171-003-A1) in patients with relapsed and refractory multiple myeloma Blood 120:2817–2825CrossRefPubMedPubMedCentralGoogle Scholar
  29. Verbrugge SE et al (2012) Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis. J Pharmacol Exp Ther 341:174–182.  https://doi.org/10.1124/jpet.111.187542 CrossRefPubMedGoogle Scholar
  30. Vij R et al (2012) An open-label, single-arm, phase 2 (PX-171-004) study of single-agent carfilzomib in bortezomib-naive patients with relapsed and/or refractory multiple myeloma Blood 119:5661–5670.  https://doi.org/10.1182/blood-2012-03-414359 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Yeakley JM, Shepard PJ, Goyena DE, VanSteenhouse HC, McComb JD, Seligmann BE (2017) A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling. PLoS One 12:e0178302.  https://doi.org/10.1371/journal.pone.0178302 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Zhang L, Littlejohn JE, Cui Y, Cao X, Peddaboina C, Smythe WR (2010) Characterization of bortezomib-adapted I-45 mesothelioma. Cells Mol Cancer 9:110.  https://doi.org/10.1186/1476-4598-9-110 CrossRefPubMedGoogle Scholar
  33. Zheng Z, Liu T, Zheng J, Hu J (2017) Clarifying the molecular mechanism associated with carfilzomib resistance in human multiple myeloma using microarray gene expression profile and genetic interaction network. Onco Targets Ther 10:1327–1334.  https://doi.org/10.2147/OTT.S130742 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of MedicineUniversity of Arizona Cancer CenterTucsonUSA
  2. 2.BioSpyder Technologies IncCarlsbadUSA
  3. 3.Ventana Medical Systems, Inc., A Member of the Roche GroupTucsonUSA

Personalised recommendations