(CANCER RESEARCH 58. 3059-3065. July 15. 1998] Prevention of Brefeldin A-induced Resistance to Teniposide by the Proteasome Inhibitor MG-132: Involvement of NF-KB Activation in Drug Resistance1 Z. Ping Lin, Yoonkyung C. Boiler, Suad M. Amer, Rosalind L. Russell, Karen A. Caccili, Steven R. Patierno, and Katherine A. Kennedy2 Department of Pharmacohfi\:. The George Washington University7 Medicai Center, Washington, DC 20037 ABSTRACT Brefeldin A, an agent that disrupts protein transport from the endo- plasmic reticulum to the Golgi, induces the expression of GRP78 and the activation of nuclear factor (NF)-KB in cells. Treatment of cells with brefeldin A causes the development of resistance to topoisomerase II- directed agents, such as etoposide and doxorubicin. In this study, we show that treatment of EMT6 mouse mammary tumor cells with brefeldin A strongly induces GRP78 mRNA (8.5-fold) and resistance to teniposide (VM26). Treatment with okadaic acid causes a minor increase in GRP78 mRNA (2.1-fold) yet still induces resistance to VM26 as effectively as brefeldin A. In contrast, cells treated with castanospermine show a mod erate increase in GRP78 mRNA (3.9-fold) but no resistance to VM26. These data imply that GRP78 induction does not mediate the development of drug resistance. An alternative mechanism of drug resistance may involve activation of the transcription factor, NF-KB, and we show that both brefeldin A and okadaic acid activate NF-KB in EMT6 cells. Fur thermore, we demonstrate that treatment with the proteasome inhibitor MG-132 blocks the activation of NF-KB and prevents the development of resistance to VM26 induced by brefeldin A. Collectively, these results suggest that the resistance to VM26 in EMT6 cells treated with brefeldin A is mediated by the activation of NF-KB rather than the induction of GRP78. Our results also suggest that inhibition of NF-KB activation in tumor cells may increase the efficacy of topoisomerase II-directed agents in chemotherapy. INTRODUCTION Tumor cells present in solid tumors are often surrounded by stress ful microenvironments, such as hypoxia, low pH, and nutrient depri vation, because of inadequate vascularization ( 1). Alterations in ge netic or epigenetic properties in tumor cell subpopulations as a result of these physiological stresses may play an important role in chemo- therapeutic resistance/insensitivity found in many malignancies. In in vitro studies, stress conditions, including hypoxia and glucose depri vation, have been shown to induce resistance to the cytotoxic effects of topoisomerase II-directed chemotherapeutic agents such as etopo side and doxorubicin (2, 3). This resistance also can be induced by a variety of chemical agents that cause inhibition of protein glycosyla- tion, release of intracellular calcium stores (2, 3), or disruption in ER3-to-Golgi transport (4). Disruption of ER function by these stress conditions and agents is known to promote an accumulation of aber rantly folded proteins in the ER and induce the expression of genes for ER-resident proteins (5, 6). Received 3/5/98; accepted 5/18/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This research was supported in part by Grants CA 36946 and 41239 (to K. A. K.) from the NIH, the Elaine H. Snyder Cancer Research Award (to K. A. K. and S. R. P.), and a Faculty Research Enhancement Fund grant (to K. A. K.) from the George Wash ington University Medical Center. This research is, in part, from a dissertation to be presented to The Columbian School of Arts and Sciences of the The George Washington University by Z. P. L. in partial fulfillment of the requirements for the Ph.D. degree. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, The George Washington University Medical Center, 2300 1 Street NW, Washington, DC 20037. Phone: (202) 994-2957; Fax: (202) 994-2870. 3 The abbreviations used are: ER, endoplasmic reticulum; NF-icB, nuclear factor-xB; BFA, brefeldin A; VM26, teniposide; OA, okadaic acid; CAS, castanospermine; MG-132, carbobenzoxyl-leucinyl-leucinyl-leucinal: EMSA, electrophoretic mobility shift assay. The glucose-regulated M, 78,000 protein (GRP78), an ER-resident protein, was found to be expressed ubiquitously in mammalian cells. It binds transiently to nascent proteins in the ER to assist in their folding, assembly, and secretion under normal conditions (7). Under stress conditions, GRP78 is highly induced and stably associated with improperly folded or assembled proteins to prevent their export from the ER (8). Studies have shown that cells expressing the GRP78 antisense vector exhibited decreased survival after exposure to cal cium ionophore (9), hypoxia (10), CTLs, and tumor necrosis factor (11). These findings suggest that GRP78 is involved in alleviating ER stress and in protecting cells against certain forms of cell killing. Recent studies have shown that cells deficient in NAD+-poly(ADP) ribose polymerase metabolism and cells treated with inhibitors of poly(ADP) ribose polymerase overexpressed GRP78 and subse quently developed resistance to etoposide, suggesting that drug resist ance is independent of the pathways that trigger GRP78 induction but dependent on the level of GRP78 (12, 13). Although these accumu lated data suggest a role of GRP78 in the development of resistance to topoisomerase II-directed agents, a direct link between the resistance and GRP78 induction still remains elusive. Recent evidence reveals that a number of agents that disrupt ER function also cause activation of the transcription factor, NF-KB (14, 15). NF-KB is thought to be activated by diverse and unrelated stimuli through the production of reactive oxygen intermediates (16). The release of Ca2+ from the ER and the subsequent production of reactive oxygen intermediates have been proposed to mediate the activation of NF-KB by ER stress (15). NF-KB is a heterodimeric transcription factor usually composed of p65 (Rei A) and p50 (NF- KB1) DNA-binding subunits (17-19). In general, the NF-KB het- erodimer is bound to the inhibitory subunit IKB and retained in the cytoplasm as an inactive form (20). Upon stimulation, IKB becomes phosphorylated (21, 22) and is subsequently degraded by the protea some (23, 24). The proteolytic degradation of IKB releases NF-KB from cytoplasmic retention, leading to nuclear translocation of NF-KB (20, 23). In the nucleus, NF-KB binds to KB motifs in promoter regions and activates the transcription of many cellular and viral target genes (25). A role for NF-KB in protecting cells against apoptosis has been suggested recently. Studies have shown that inhibition of NF-KB activation by expression of a dominant-negative form of IKB en hanced apoptotic cell death induced by tumor necrosis factor a, ionizing radiation, and the chemotherapeutic agent daunorubicin (26, 27). A similar study demonstrated that expression of the NF-KB component c-rel or p65 protected cells against tumor necrosis factor- induced apoptosis (28). These findings imply that NF-KB can induce target genes, which encode proteins involved in protection against apoptotic cell death. Disruption of ER function by BFA was found to strongly induce the expression of GRP78 (29, 30) and activate NF-KB (14, 15) by distinct pathways (31, 32). It is unclear, however, whether either of these cellular responses contributes to the development of drug resistance after chemically or physiologically based stress. In the present study, the possible involvement of GRP78 induction and NF-KB activation in the development of resistance to the topoisomerase II-directed agent, VM26, was investigated in EMT6 mouse mammary tumor 3059 on March 18, 2015. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from