IKKβ inhibitor in combination with bortezomib induces cytotoxicity in breast cancer cells

Hideshima,Hiromasa; Yoshida,Yasuhiro; Ikeda,Hiroshi; Hide,Maya; Iwasaki,Akinori; Anderson,Kenneth C.; Hideshima,Teru

doi:10.3892/ijo.2014.2273

IKKβ inhibitor in combination with bortezomib induces cytotoxicity in breast cancer cells

Authors:
- Hiromasa Hideshima
- Yasuhiro Yoshida
- Hiroshi Ikeda
- Maya Hide
- Akinori Iwasaki
- Kenneth C. Anderson
- Teru Hideshima
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Affiliations: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA, Department of Thoracic, Endocrine and Pediatric Surgery, Faculty of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan
Published online on: January 23, 2014 https://doi.org/10.3892/ijo.2014.2273
Pages: 1171-1176

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Abstract

Bortezomib is a proteasome inhibitor with remarkable clinical antitumor activity in multiple myeloma (MM) and is under evaluation in clinical trials in various types of cancer including breast cancer. Although the initial rationale for its use in cancer treatment was the inhibition of NF-κB activity by blocking proteasomal degradation of IκBα, direct evidence indicating inhibition of constitutive NF-κB activity by bortezomib in tumor cells in patients has not yet been reported. Moreover, recent studies have shown that bortezomib activates constitutive NF-κB activity via stimulating the canonical pathway in MM cells. In this study, we first examined protein expression of IκBα after bortezomib treatment. We observed that bortezomib upregulated the phosphorylation and downregulated IκBα protein expression in a dose- and time-dependent manner in MCF7 and T47D cells, associated with phosphorylation of IKKβ. Since IκBα is an inhibitor of nuclear translocation of NF-κB, we further examined alteration of NF-κB activity by bortezomib. Importantly, bortezomib significantly upregulates NF-κB activity in both MCF7 and T47D in a dose-dependent fashion, demonstrated by electrophoretic mobility shift analysis (EMSA). Furthermore, immunocytochemical analysis confirmed enhanced nuclear translocation of p65 NF-κB (RelA) by bortezomib treatment. Supershift assay showed supershifted bands by anti-p65 and -p50 antibodies. Taken together, these results indicate that bortezomib activates the canonical NF-κB pathway in both cell lines. Finally, we demonstrated that IKKβ inhibitor enhanced cytotoxicity, associated with inhibition of NF-κB activity induced by bortezomib in MCF7 and T47D breast cancer cells.

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1.	Jost PJ and Ruland J: Aberrant NF-κB signaling in lymphoma: mechanisms, consequences, and therapeutic implications. Blood. 109:2700–2707. 2007.
2.	Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng WJ, Van Wier S, Tiedemann R, Shi CX, Sebag M, Braggio E, Henry T, et al: Promiscuous mutations activate the noncanonical NF-κB pathway in multiple myeloma. Cancer Cell. 12:131–144. 2007.PubMed/NCBI
3.	Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, Zhan F, Lenz G, Hanamura I, Wright G, Xiao W, Dave S, Hurt EM, et al: Frequent engagement of the classical and alternative NF-κB pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell. 12:115–130. 2007.
4.	Hideshima T, Chauhan D, Kiziltepe T, Ikeda H, Okawa Y, Podar K, Raje N, Protopopov A, Munshi NC, Richardson PG, Carrasco RD and Anderson KC: Biologic sequelae of IκB kinase (IKK) inhibition in multiple myeloma: therapeutic implications. Blood. 113:5228–5236. 2009.
5.	Baldwin AS Jr: The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 14:649–683. 1996.
6.	Beg AA and Baldwin AS Jr: The IκB proteins: multifunctional regulators of Rel NF-κB transcription factors. Genes Dev. 7:2064–2070. 1993.
7.	Zandi E, Chen Y and Karin M: Direct phosphorylation of IkappaB by IKKα and IKKβ: discrimination between free and NF-κB-bound substrate. Science. 281:1360–1363. 1998.
8.	DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E and Karin M: A cytokine-responsive IκB kinase that activates the transcription factor NF-κB. Nature. 388:548–554. 1997.
9.	Hideshima T, Richardson P, Chauhan D, Palombella V, Elliott P, Adams J and Anderson KC: The proteasome inhibitor PS-341 inhibits growth, induces apoptosis and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 61:3071–3076. 2001.
10.	Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Fanourakis G, Gu X, Bailey C, Joseph M, Libermann TA, Treon SP, Munshi NC, Richardson PG, et al: Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci USA. 99:14374–14379. 2002. View Article : Google Scholar : PubMed/NCBI
11.	Hideshima T, Mitsiades C, Akiyama M, Hayashi T, Chauhan D, Richardson P, Schlossman R, Podar K, Munshi NC, Mitsiades N and Anderson KC: Molecular mechanisms mediating anti-myeloma activity of proteasome inhibitor PS-341. Blood. 101:1530–1534. 2003. View Article : Google Scholar : PubMed/NCBI
12.	Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, Rajkumar SV, Srkalovic G, Alsina M, Alexanian R, Siegel D, Orlowski RZ, et al: A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 348:2609–2617. 2003. View Article : Google Scholar : PubMed/NCBI
13.	Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, Harousseau JL, Ben-Yehuda D, Lonial S, Goldschmidt H, Reece D, San-Miguel JF, et al: Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 352:2487–2498. 2005. View Article : Google Scholar : PubMed/NCBI
14.	San Miguel JF, Schlag R, Khuageva NK, Dimopoulos MA, Shpilberg O, Kropff M, Spicka I, Petrucci MT, Palumbo A, Samoilova OS, Dmoszynska A, Abdulkadyrov KM, et al: Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. N Engl J Med. 359:906–917. 2008.
15.	Engel RH, Brown JA, Von Roenn JH, O’Regan RM, Bergan R, Badve S, Rademaker A and Gradishar WJ: A phase II study of single agent bortezomib in patients with metastatic breast cancer: a single institution experience. Cancer Invest. 25:733–737. 2007. View Article : Google Scholar : PubMed/NCBI
16.	Dees EC and Orlowski RZ: Targeting the ubiquitin-proteasome pathway in breast cancer therapy. Future Oncol. 2:121–135. 2006. View Article : Google Scholar : PubMed/NCBI
17.	Schmid P, Kuhnhardt D, Kiewe P, Lehenbauer-Dehm S, Schippinger W, Greil R, Lange W, Preiss J, Niederle N, Brossart P, Freier W, Kummel S, et al: A phase I/II study of bortezomib and capecitabine in patients with metastatic breast cancer previously treated with taxanes and/or anthracyclines. Ann Oncol. 19:871–876. 2008. View Article : Google Scholar : PubMed/NCBI
18.	Dees EC, O’Neil BH, Lindley CM, Collichio F, Carey LA, Collins J, Riordan WJ, Ivanova A, Esseltine D and Orlowski RZ: A phase I and pharmacologic study of the combination of bortezomib and pegylated liposomal doxorubicin in patients with refractory solid tumors. Cancer Chemother Pharmacol. 63:99–107. 2008. View Article : Google Scholar : PubMed/NCBI
19.	Awada A, Albanell J, Canney PA, Dirix LY, Gil T, Cardoso F, Gascon P, Piccart MJ and Baselga J: Bortezomib/docetaxel combination therapy in patients with anthracycline-pretreated advanced/metastatic breast cancer: a phase I/II dose-escalation study. Br J Cancer. 98:1500–1507. 2008. View Article : Google Scholar
20.	Cresta S, Sessa C, Catapano CV, Gallerani E, Passalacqua D, Rinaldi A, Bertoni F, Vigano L, Maur M, Capri G, Maccioni E, Tosi D, et al: Phase I study of bortezomib with weekly paclitaxel in patients with advanced solid tumours. Eur J Cancer. 44:1829–1834. 2008. View Article : Google Scholar : PubMed/NCBI
21.	Hideshima T, Ikeda H, Chauhan D, Okawa Y, Raje N, Podar K, Mitsiades C, Munshi NC, Richardson PG, Carrasco RD and Anderson KC: Bortezomib induces canonical nuclear factor-κB activation in multiple myeloma cells. Blood. 114:1046–1052. 2009.
22.	Karin M and Greten FR: NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol. 5:749–759. 2005. View Article : Google Scholar : PubMed/NCBI
23.	Karin M and Ben-Neriah Y: Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol. 18:621–663. 2000.PubMed/NCBI
24.	Dolcet X, Llobet D, Encinas M, Pallares J, Cabero A, Schoenenberger JA, Comella JX and Matias-Guiu X: Proteasome inhibitors induce death but activate NF-κB on endometrial carcinoma cell lines and primary culture explants. J Biol Chem. 281:22118–22130. 2006.
25.	Markovina S, Callander NS, O’Connor SL, Kim J, Werndli JE, Raschko M, Leith CP, Kahl BS, Kim K and Miyamoto S: Bortezomib-resistant nuclear factor-κB activity in multiple myeloma cells. Mol Cancer Res. 6:1356–1364. 2008.
26.	Chauhan D, Li G, Shringarpure R, Podar K, Ohtake Y, Hideshima T and Anderson KC: Blockade of Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells. Cancer Res. 63:6174–6177. 2003.PubMed/NCBI
27.	Hideshima T, Podar K, Chauhan D, Ishitsuka K, Mitsiades C, Tai YT, Hamasaki M, Raje N, Hideshima H, Schreiner G, Nguyen AN, Navas T, et al: p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells. Oncogene. 23:8766–8776. 2004. View Article : Google Scholar : PubMed/NCBI
28.	Oerlemans R, Franke NE, Assaraf YG, Cloos J, van Zantwijk I, Berkers CR, Scheffer GL, Debipersad K, Vojtekova K, Lemos C, van der Heijden JW, Ylstra B, et al: Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein. Blood. 112:2489–2499. 2008. View Article : Google Scholar : PubMed/NCBI
29.	Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades N, Hayashi T, Munshi N, Dang L, Castro A, Palombella V, Adams J and Anderson KC: NF-kB as a therapeutic target in multiple myeloma. J Biol Chem. 277:16639–16647. 2002. View Article : Google Scholar : PubMed/NCBI
30.	Garg AK, Hortobagyi GN, Aggarwal BB, Sahin AA and Buchholz TA: Nuclear factor-κB as a predictor of treatment response in breast cancer. Curr Opin Oncol. 15:405–411. 2003.
31.	Wu JT and Kral JG: The NF-κB/IκB signaling system: a molecular target in breast cancer therapy. J Surg Res. 123:158–169. 2005.
32.	Tapia MA, Gonzalez-Navarrete I, Dalmases A, Bosch M, Rodriguez-Fanjul V, Rolfe M, Ross JS, Mezquita J, Mezquita C, Bachs O, Gascon P, Rojo F, et al: Inhibition of the canonical IKK/NF κB pathway sensitizes human cancer cells to doxorubicin. Cell Cycle. 6:2284–2292. 2007.PubMed/NCBI