The Ubiquitin-Proteasome System (UPS) and the

Current Pharmaceutical Design, 2011, 17, 000-000

1

The Ubiquitin-Proteasome System (UPS) and the Mechanism of Action of Bortezomib Boris Cvek* and Zdenek Dvorak Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic Abstract: Proteasome inhibition is a modern and surprisingly successful approach how to cancer treatment. Bortezomib (Velcade®) is a first-in-class proteasome inhibitor and has been approved for first-line treatment of multiple myeloma and second-line treatment of mantle cell lymphoma. There have been almost 200 clinical trials with bortezomib, both as a single agent and in combination with other drugs, for various cancers, as listed in the US National Cancer Institute database. However, bortezomib’s mechanism of action remains elusive despite enormous progress in our knowledge of the cell biology of the ubiquitin-proteasome system (UPS) and bortezomibinduced signaling events in cancer cells. This review maps a rapidly growing and open body of research in both areas.

Keywords: Ubiquitin-proteasome system, bortezomib, mechanism of action. 1. INTRODUCTION Throughout last decade, cancer chemotherapy has largely become “targeted therapy”. The classic example of a therapeutic target is the well-known tyrosine kinase Bcr-Abl, which causes chronic myelogenous leukemia. Imatinib, a famous drug approved for disease treatment, inhibits this kinase [1]. At present, there are many key players in tumorigenesis, cancer proliferation, and metastasis that are emerging as targets for therapeutic intervention strategies including [2] cellular signal transduction pathways (e.g. receptor kinases, intracellular signaling kinases), tumor vasculature (e.g. angiogenesis and vasculature disrupting), epigenetic modulators (e.g. DNA methyltransferases and histone deacetylases), integrins, heat shock proteins, poly(ADP-ribose) polymerase, and mitotic kinases. However, one of the most surprising and attractive targets for cancer chemotherapy is the ubiquitin-proteasome (UPS) system, for which the first-in-class proteasome inhibitor bortezomib (brand name: Velcade®) is used. Bortezomib has been approved by the FDA as a first-line cancer chemotherapy for multiple myeloma since June 2008. Bortezomib, formerly known as PS-341, LDP-341, and MLM341 [3], was first reported (December 1998) to attenuate inflammation in rats through proteasomal and nuclear factor-B (NF-B) inhibition [4]. In a subsequent study (April 1999) proteasome inhibitors, including bortezomib, were a means for studying the NF-B pathway [5]. The focus of this drug shifted in an article [6] authored by Julian Adams from ProScript Inc., a biotech company started by Harvard professors A. Goldberg, T. Maniatis, M. Rosenblatt, and K. Rock in 1994 under the name Myogenics (hence, the original name MG-341 or PS-341). Adams recalled the discovery of bortezomib in an interview as follows (Myeloma Today 6 May, 2003): “Early on in its development no one believed in the molecule, or the target. It was assumed that this approach would lead to overwhelming toxicity. Many needed to be convinced. Our own employees at ProScript were skeptical, the scientific founders, the Board of Directors, all in the company. As for the outside world, it was next to impossible. David Livingston, of the Dana Farber Cancer Institute, joined our scientific board and helped me enormously in changing the views within the company. I also forged a formidable alliance with the National Cancer Institute, who was also initially skeptical, but under the guidance of Edward Sausville at the NCI, we kept building the evidence for a case to see Velcade through.” *Address correspondence to this author at the Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic; Tel: 420 585634901; Fax: 420 585634905; E-mail: [email protected] 1381-6128/11 $58.00+.00

As explained by Adams et al. [6], one of the first proteasome inhibitors [7] was MG-132, a peptide aldehyde based on calpain inhibitor I [8]. However, MG-132 is not sufficiently selective for proteasome inhibition because it also inhibits cathepsin B and calpains [9-10]. The 20S proteasome as a threonine protease [11] has been shown to be efficiently targeted by dipeptidyl boronic acids [12-15]. Consequently, the average growth inhibition value of 50% for bortezomib across the entire NCI cell panel (60 cell lines derived from multiple human tumors [16]) was 7 nM. Using the NCI COMPARE algorithm [17], it was shown that bortezomib had a unique toxicity “fingerprint” among 60,000 compounds. Such results encouraged further testing in animals, and bortezomib was found to be a potent tumor suppressor in several murine tumors and human xenografts [18]. Moreover, no adverse effects of i.v. bortezomib administered were noted by Adams et al. [6], although the proteasome was significantly inhibited in the tumor and in murine white blood cells, colon, liver, muscle, and prostate. Intravenous dosing with radiolabeled [14C]bortezomib demonstrated the highest radioactivity levels 10 min after drug administration in the adrenals, the kidney cortex, the liver, the prostate, and the spleen. On the contrary, the brain, the spinal cord, eyes, and testes had levels below the limits of detection. The majority (66%) of the radiolabeled drug was excreted in bile, and the reminder was excreted in the urine. However, neither fluid contained inhibitory activity, suggesting that only metabolized bortezomib was excreted. A full toxicological evaluation of bortezomib in rodents and primates revealed the side effects of bortezomib [6]. In primates, the side effects were anorexia, emesis, and diarrhea, and all occurred in a dose-dependent manner. Among the 40 other tissues examined, modest bortezomib effects were observed in the thymus and spleen. The drug was therefore approved for phase I clinical trials in human cancer patients [18]. During the trials, patients experienced low-grade fever and/or fatigue after several cycles of bortezomib at doses of approximately 1.0 mg/m2. Low-grade diarrhea and thrombocytopenia were also observed, but were not dose limiting. The most serious adverse effect of bortezomib, which remains true today, was peripheral neuropathy. From the phase I results, four malignancies appeared to be most susceptible to proteasome inhibition: androgen-independent prostate cancer [19], non-small cell lung cancer [20], melanoma [21], and multiple myeloma [22]. A pivotal phase II trial of bortezomib was performed in relapsed and/or refractory multiple myeloma patients who had progressed after a median of six previous therapeutic regimens [23]. The incidence of grade 4 adverse events was low, and most could be managed with standard approaches. For the 202 patients involved in the trial, the median time to progression was 7 months with bortezomib treatment, as compared with 3 months with the last

© 2011 Bentham Science Publishers Ltd.

2 Current Pharmaceutical Design, 2011, Vol. 17, No. 00

treatment before entering the trial. A positive therapeutic response to bortezomib was not influenced by the type of multiple myeloma or by the type or number of previous therapies. In May 2003, the US Food and Drug Administration granted accelerated approval for bortezomib as a third-line therapy for multiple myeloma, and a year later the European Union approved its use [24-26]. Bortezomib has subsequently been approved as a second-line [27] and a first-line1 drug for multiple myeloma treatment. Furthermore, bortezomib has been approved as a second-line therapy against mantle cell lymphoma [28]. Today, there are thousands of patients using bortezomib worldwide and hundreds of clinical trials using bortezomib for the treatment of various cancers, either as a single agent or in combination therapy. The basic principles of the UPS and proteasomal inhibition in cancer therapy were recently reviewed elsewhere [29-30]. Comprehensive knowledge of the chemical principles of the proteasome inhibition is available, for instance, in a useful study published in Chemical Reviews [31]. To avoid redundancy, this review will focus on the latest reports of the cell biology of the UPS and the mechanism of action of bortezomib. 2. UPS: MUCH MORE THAN JUST PROTEOLYSIS In his Nobel Lecture (2004), Avram Hershko explained how he and co-workers found three enzymes during the years 1970-1990, the ubiquitin-activating E1, the ubiquitin-conjugating E2 and the ubiquitin-ligating E3, that target proteins for proteasomal degradation via a chain composed of units called ubiquitin [32]. However, according to Alexander Varshavsky and colleagues, another key group in early UPS research, the role of ubiquitin is not limited to proteolysis but also includes cell cycle control, DNA repair, transcription, and other essential cellular events [33]. Today, as we increasingly understand UPS complexity, it is crucial to introduce UPS in a broader cellular context. 2.1. The Various Protein Degradation Levels For the degradation of long-lived cytosolic proteins and organelles, there is a process called autophagy (“self-eating”), which can be divided into three subtypes: chaperone-mediated autophagy, microautophagy and macroautophagy. During autophagy, which is initiated by conjugating machinery that is similar to the E1-E2-E3 ubiquitin system [34], proteins are delivered to the lysosome for degradation [35]. Intriguingly, the autophagy of membrane proteins, including diverse receptors, is ubiquitin-dependent and involves Lys-63-linked (see below) polyubiquitination (or polyubiquitylation) of proteins to be degraded [36-37]. In addition to the UPS-mediated turnover of proteins with a myriad of regulatory functions, approximately 30% of all newly synthesized proteins are defective and degraded cotranslationally or destroyed within minutes of their synthesis by the UPS [38]. This alternative process, termed endoplasmic reticulum-associated degradation (ERAD), starts when misfolded substrates are flagged by a unique glycan code that is generated by mannosidases in the ER. This signal is recognized by an E3 that is anchored in the ER membrane, and the marked protein is transported across the ER membrane to be ubiquitinated and destroyed by the proteasome [39]. Although the ERAD system focuses on defective proteins, it can have an important regulatory function as well, such as in sterol synthesis [40]. For non-native protein misfolding and aggregation, there are two cytosolic quality control compartments, the juxtanuclear quality control (JUNQ) and the insoluble protein deposit (IPOD). Misfolded and aggregated proteins are mostly ubiquitinated by the quality control machinery, which focuses them in the JUNQ, a destination where chaperones and proteasomes are concentrated in close proximity to a perinuclear ER region included in the ERAD. If the machinery needed for sorting to the JUNQ is 1

http://www.fda.gov/AboutFDA/CentersOffices/CDER/ucm094633.htm

Cvek and Dvorak

saturated, the aggregated and potentially toxic protein species are directed to the IPOD, which seems to terminally sequester protein aggregates [41]. This opens an important question in the UPS field: in addition to the machineries mentioned above, where are the proteasomes located in the cell? Here we will only show the contour of the answer: the details can be found elsewhere [42]. Within the nucleus or the cytoplasm, proteasomes rapidly diffuse, which is in contrast to their slow, unidirectional transport from the cytoplasm to the nucleus [43]. For example, proteasome subunits are generally 12 times more abundant in the cytoplasm than in the nuclear fraction of mouse liver cells [44]. The two proteasome pools, nuclear and cytoplasmic, could equilibrate during mitosis. Moreover, there could be a difference between the subtype and the subunit composition of cytoplasmic, nuclear, and microsomal proteasomes and their specific proteolytic functions [45]. In the cytoplasm, proteasomes can be tightly associated with intermediate filaments [46], actin filaments [47-48], and myofibrils [49], or they can continuously interact with proteins destined for degradation by simple collision [43]. Nuclear proteasomes have been reported to be localized to promyelocytic leukemia (PML) nuclear bodies [50-51], nucleoplasmic speckles [52] and focal clusters throughout the nucleoplasm [53]. Under some conditions, proteasomes appear in nucleoli as well [54]; however, proteasomes are not found in nucleoli under normal conditions [55-56]. 2.2. The (Immuno)Proteasomes: Structure and Function There are various proteins in all biological kingdoms, e.g. HslV protease or RNase PH in E. coli, PNPase in S. antibioticus, and archeal or eukaryotic exosomes, which appear to have evolved in a manner that is similar to proteasomes [57]. Many authoritative reviews of the composition and functions of proteasomes have been recently published [58-60]; thus, we will present only the most important features of eukaryotic proteasomes and focus on new and ongoing research in the UPS field. 2.2.1. The Core Particles The 26S proteasome, the most common proteasome type in human organisms, is a 2.4 MDa complex composed of two multisubunit subcomplexes [61-63], a barrel-like 20S core particle (CP) and a 19S regulatory particle (RP), also termed PA700. The CP contains 2 rings at the top and bottom of the barrel and 2 rings in the middle [64]. Each ring contains three catalytically active subunits 1, 2, and 5 (see Fig. 1). The active sites of the subunits are exposed in the interior of the 20S proteasome cylinder and display different peptidase activities, i.e., caspase-like or peptidylglutamyl peptide hydrolyzing-like (1), trypsin-like (2) and chymotrypsin-like (5). A cooperative multiple chaperone system is involved in the mammalian 20S proteasome assembly pathway, which is based strictly on ordered subunit incorporation [65-68]. In mammals, a number of alternative active site subunits and the proteasome maturation protein POMP are induced during the interferon- (IFN-)-mediated immune response [69]. During de novo proteasome formation, the immunosubunits 1i or LMP2, 2i or MECL1, and 5i or LMP7 are preferentially incorporated into the CP to form immunoproteasomes (iPrs) or mixed proteasomes [70]. Additionally, IFN- also induces the synthesis of the proteasome activator PA28 subunits PA28 and PA28 (see below), transporters of peptides to the ER, and a small set of aminopeptidases. The half-life of the iPrs is considerably shorter (21 hours) than that observed for the constitutive proteasomes (120 hours), and this permits cells to rapidly return to a normal state [71]. Interestingly, after IFN- stimulation, the 7 subunits are dephosphorylated, and thus, the 26S iPrs are destabilized, resulting in an increase of PA28-containing proteasome complexes [72]. Generally, IFN--induced iPrs are involved in antigen processing; however, their major function is largely an open question [73]. Immune surveillance by CD8+ T cells requires cells to display 8-10 amino

The Ubiquitin-Proteasome System (UPS)

Current Pharmaceutical Design, 2011, Vol. 17, No. 00

3

Fig. (1). The proteasomes and their composition: structure of the 20S core particle (A) and the 19S regulatory particle (B); various kinds of the proteasomes in the cell (C).

acids long peptides bound to major histocompatibility complex class I (MHC I) molecules on their surface. Infected or cancerous cells exhibit new peptides derived from microbial/viral or mutated self-proteins, respectively, and they are subsequently recognized by CD8+ T cells and eliminated. The peptides are the products of a multifaceted process in the cytoplasm where the proteasomes, tripeptidyl peptidase II and other proteases degrade proteins into a mixture of peptides of variable lengths [74] that need to be further edited and customized before they yield peptide-MHC I complexes [75]. The cytotoxic T cell response to pathogens is generally directed against a number of immunodominant epitopes, while other potential epitopes are subdominant or not used at all. The iPrs, but not the constitutive proteasomes, are able to determine the subdominance of epitopes and shape the epitope hierarchy of cytotoxic T lymphocytes in vivo [76-79]. This could be the reason why viral infection tends to down-regulate iPr subunits under some circumstances [80]. Moreover, the role of the iPrs could be tissue-specific [81]; for instance, they seem to be involved in mouse susceptibility to Coxsackie virus myocarditis [82-83] and may have yet unresolved functions in T cell proliferation [84]. In addition to constitutive proteasomes and iPrs, there are unique proteasomes (thymoproteasomes) in both mouse and human cortical thymic epithelial cells. Thymoproteasomes have a novel catalytic subunit, 5t, with unusual enzymatic activity, and consequently, they are capable of presenting a set of self-peptides that are not seen in other cells and are required for positive CD8+ T cell selection [85-87]. 2.2.2. The Regulatory Particles Eukaryotic cells contain various regulatory complexes that bind directly to the outer rings (Fig. 1) of the 20S proteasome (ATPindependent 20S regulators PA28, PA200, and ATP-dependent 20S

regulator PA700) or reversibly bind to proteins whose proteasomal content is variable and often substoichiometric [88]. In contrast to PA700, the ATP-independent regulators do not bind to polyubiquitin chains. Mammals have three homologous PA28 (REG or 11S regulator) genes: PA28, PA28, and PA28. The first two are involved in IFN--induced proteasome assembly and create a heteromeric complex, whereas PA28 forms a homoheptamer. Mice with disrupted PA28 and PA28 genes have defective production of certain MHC I antigens, although nonimmunological functions of both activators also seem likely because of their wide expression and regulation under many physiological conditions [89]. Recent studies have revealed [90-92] a role of PA28 in ubiquitin-independent degradation of steroid receptor coactivator-3 and p21. However, the polymer form of PA28, interacts with p53 to promote its ubiquitination and proteasomal degradation [93]. Furthermore, proteasome-PA28 complexes are regulators of nuclear speckles [94], maintain chromosomal stability [95], and interact with the PML and a DNA damage checkpoint kinase [96]. PA200 is a nuclear protein that, following association with the 20S proteasome, enhances peptide hydrolysis after acidic residues in vitro [97-99]. In response to ionizing radiation, PA200 forms hybrid proteasomes with the 19S cap and the CP that accumulate on chromatin, resulting in increased proteolytic activity [100]. According to the same study, PA200-knockdown cells show genomic instability and reduced survival after exposure to ionizing radiation. Although proteasome configurations are highly dynamic [101], the 26 proteasome (19S/PA700 RP attached at both CP -rings, Fig. 1) remains intact during protein degradation [102]. The RP of the 26S proteasome contains six different AAA+ ATPase subunits (Rpt1-6 in yeast), which are combined with four non-ATPase


subunits (Rpn1-2, Rpn10, and Rpn13 in yeast) called the base. Rpn1 and Rpn2 form toroids, thereby physically linking the site of substrate recruitment to the site of proteolysis, as Rpn1 is stacked on Rpn2 and coupled with the CP -ring [103]. The base assembly is a rapid and orchestrated process [104-110] in which there is an opening of the gate upon ATP binding to the ATPases [111] into the CP to allow for substrate entry [112]. In contrast to the ATP binding implicated in the base assembly and the proteasome activation, ATP hydrolysis is used for 26S proteasome-dependent degradation of polyubiquitinated proteins (i.e. unfolding of substrate proteins) [113]. Not surprisingly, the function of the ATPases and the gating of the 26S proteasome are variably modulated [114-115]. The second part of the 19S RP (Fig. 1) is composed of nine non-ATPase subunits (Rpn3, Rpn5-9, Rpn11-12, and Sem1 in yeast) and is called the lid [116]. The major activity of the lid is the deubiquitination of ubiquitinated proteins by the Jab1 MPN domain metalloenzyme (JAMM) domain deubiquitinase Rpn11 (Poh1 in humans) [117-118]. The lid subunits share substantial sequence homology with the COP9 signalosome (CSN), which has been proposed to be an alternative lid of the 26S proteasome [119-120]. 2.2.3. Protein Degradation by the Proteasomes Although the proteasomes are responsible for the majority of non-lysosomal protein degradation, not all eukaryotic proteins degraded by the proteasome require ubiquitination as a flag to be recognized and digested [121]. Approximately 20% of all proteins in human cells are cleaved by the 20S or 26S proteasomes in a ubiquitin-independent fashion. Such proteins contain disordered regions that likely constitute the universal structural signal for their proteolysis [122]. The well-known examples include oxidized proteins [123-124], antizyme-1-dependent degradation of ornithine decarboxylase [125], and diubiquitin (FAT10)-flagged substrates [126-127]. Interestingly, some proteins, including p53, c-Fos, and Fra-1, are known to be degraded by both ubiquitin-dependent as well as ubiquitin-independent methods [128-129]. However, most proteins require polyubiquitin chains (polyUb) as a flag to be identified for processing by the proteasome (Fig. 2) [130]. The most important type of polyUb involved in protein degradation is linked through lysine 48 of each ubiquitin molecule (K48-linked polyUb). Nonetheless, there are a variety of ubiquitin chains, even those that are heterogenous, and they are sufficient for proteasomal substrate degradation [131-134]. Moreover, the chain can be modified and switched from a K63-linked to a K48-linked polyUb flag [135]. Still, the degradation signal may be just a multiple monoubiquitination and not necessarily a chain [136]. The ubiquitin flag is recognized and bound to the 26S proteasome (Fig. 2) by specific proteins, among which five are confirmed ubiquitin receptors (two proteasome subunits of Rpn10 with Rpn13 and three shuttle factors, Rad23, Dsk2, and Ddi1) [137-139], and others have been suggested to play such a role as well (Rpt5/S6´, Rpt1, and Rpn1) [140-141]. It was hypothesized that Rpt5 “serves as a central conduit that gathers together substrates delivered by different receptor pathways” [142]. After binding, the ubiquitin chain can be remodeled by ATP-independent deubiquitinases to allow the 26S proteasome to actively regulate the substrate commitment to degradation [143-145]. Moreover, such deubiquitinating activity, which spares ubiquitin from proteasomal degradation, is enhanced by ubiquitin deficiency within the cell [146]. In contrast, the polyubiquitinated substrate attached to the 19S RP facilitates active site access and accelerates its own degradation by the 26S proteasome [147]. Subsequently, the substrate is deubiquitinated en bloc by Poh1 (Fig. 2), and the polyubiquitin chains are cleared from the binding sites on the proteasome by other deubiquitinases [148]. However, there is an exception; monoubiquitinated substrates undergo degradation together with the ubiquitin [149-150]. Compared with K48-linked polyUb chains, cellular K63-linked polyUb chains have less proteasomal accessibility, and proteasome-bound K63

Cvek and Dvorak

chains are more rapidly deubiquitinated, which could cause inefficient degradation of K63 conjugates [151]. The unfolding [152] and degradation of previously polyubiquitinated substrate (Fig. 2) begins at an initiation site which is usually an unstructured region of the substrate [153-154]. In cases where substrate unfolding or its translocation into the CP is not the ratelimiting step, the substrate is stored in both antechambers ( rings) while the degradation is carried out within the catalytic chamber ( rings) [155]. It seems that ongoing protein degradation may enhance 26S proteasome stability and proteolysis processivity [156]. Moreover, the CP can bind two substrates, presumably one in each antechamber, to speed up their degradation [157]. The processive cleavage of the substrates continues until the products are suitably small enough to diffuse out through the axial gates [158-159] that are opened by hydrophobic peptides [160]. The human 26S and 20S proteasomes generate different pools of fragments with a similar average length [161]. These peptides are further trimmed by other peptidases into amino acids that are reusable for new protein synthesis [162] or used for antigen processing (see above). The substrate may not be completly degraded (Fig. 2). In fact, some key transcription factors, particularly NF-B proteins, are activated by the 26S proteasome in a process called regulated ubiquitin/proteasome-dependent processing [163]. Incomplete substrate digestion seems to occur due to the presence of tightly folded domains that are cleavage-resistant and/or have stretches of sequences that do not allow unfolding [164-165]. 2.2.4. Nuclear Roles of the Proteasomes In the nucleus, there are various UPS-dependent events [166] ranging from gene expression [167-168] or translation [169-170] to DNA repair [171] to protein quality control [172]. Although UPSmediated turnover is not a general requirement for function of transactivators [173], the UPS has an intrinsic role in both of the proposed general models of their modulation [174]. In yeast, the proteasome components are widely associated with chromatin [175] and interact with RNA polymerase at multiple genomic sites [176]. Moreover, the 26S proteasome silences nonspecific transcription and keeps tissue-specific genes in a transcriptionally competent state in embryonic stem cells [177]. The 19S RP and the 20S CP alter glucocorticoid and estrogen receptor-mediated transcription in the MCF-7 breast cancer cell line [178-179]. However, the 19S RP seems to be more involved in gene expression, e.g., it has critical roles in thyroid hormone receptor-mediated transactivation [180], and proteasome ATPases can either enhance transcription [181182] or actively destabilize activator-promoter complexes [183]. The ATPases are also associated with both ribosomal DNA promoters and coding regions [184]. Furthermore, Tat-binding protein1, an ATPase of the 19S RP, is able to directly bind the androgen receptor and alter its transcriptional function [185]. Another RPassociated ATPase, TRIP1 (also known as Sug1), positively regulates the transcription of major histocompatibility complex class II [186-187]. In DNA damage signaling, the UPS is responsible for degradation of mediator of DNA damage checkpoint protein 1 (MDC1), efficient assembly of BRCA1 (breast cancer 1) foci [188], and regulated histone proteolysis [189]. 2.2.5. Health Defects from Proteasome Dysfunction The proteasome is a key player in many therapeutically interesting processes such as the hypoxia response, inflammation, neurodegeneration, muscle wasting, viral infection, and carcinogenesis [190-191]. The UPS is broadly impaired in Huntington’s disease [192-193]. Under some circumstances, UPS impairment is possibly compensated by autophagy [194]. The inhibition of the 26S proteasome by misfolded prions is the reason for their neurotoxicity [195]. Thus, it is not surprising that bortezomib is able to induce peripheral neuropathy in patients. Moreover, the accumulation of non-functional proteins is linked to atherosclerosis [196], and in



5

Fig. (2). Various ways of protein recognition and degradation by the proteasome.

cotrast, proteasome inhibition could be an effective stroke therapy [197]. The 26S proteasome was also shown to regulate the human immunodeficiency virus-1 (HIV-1) promoter in both a proteolytic and non-proteolytic manner [198]. Because the activity of the UPS decreases with age, UPS dysfunction may also be a signs of aging [199]. The UPS is required for essential functions in human monocyte-derived dendritic cells [200], and as well as it is a key factor in mammalian stem and progenitor cells [201]. 2.3. The (de)Ubiquitination Machinery The basic information on ubiquitin and ubiquitination is available in many specialized reviews [202-204]. A protein destined for degradation is recognized by an E3 ligase due to a specific degradation signal or “degron” [205]. Polyubiquitination is achieved when the E3 ligase binds to both the substrate and an E2 enzyme that carries a ubiquitin molecule (Fig. 2). A ubiquitin is transferred to the substrate (the first ubiquitin) or to the substrate-bound ubiquitin chain to extend it. The ubiquitin-free E2 is substituted with a new ubiquitin-loaded E2 using a unique E1 enzyme and ubiquitination continues on to the next step; the chain is longer by one ubiquitin. However, this “canonical view” is now being modified by new discoveries. For example, recent data suggest there is only one E1 in human cells but at least two, Uba6 and Ube1, which charge different E2 cohorts [206-207]. It appears there are ubiquitin chain elongation factors, termed E4 enzymes, that are required in some cases with the E1-E2-E3 cascade for efficient ubiquitination [208]. In yeast, an AAA+ ATPase prevents the formation of excessive and useless multiubiquitin chains [209]. It was also reported that ubiquitin oligomers, which

are possibly extended by an E1 enzyme itself on an E2 enzyme [210], can directly relocate from E2 enzymes to a substrate [211]. Some E2 enzymes seem to be specific for monoubiquitination, and others are specific for chain assembly on preattached ubiquitin [212-213]. An E2 enzyme is capable of polyubiquitinating and targeting itself for proteasomal degradation by a novel mechanism [214]. In humans, there are hundreds of E3 enzymes. The two major types of human E3 ligases are defined by the presence of either a homologous to E6-associated protein C-terminus (HECT) or a really interesting new gene (RING) domain (Fig. 2) [215-216]. E3 ligases are involved in many cellular events such as oscillation of the circadian clock [217-219], nuclear processes [220], mitosis [221], and longevity in response to diet restriction [222]. In addition to K48- and K63-linked chains (see above), there are many other types of ubiquitin linkages, including K6-, K11-, K27-, K29-, and K33-linked chains; mixed linkages in which two different bonds at one ubiquitin molecule are formed and thus the chain is forked; and heterologous linkages in which ubiquitin forms chains with ubiquitin-like [223] proteins chains [224]. Some ubiquitination mechanisms lead to nondegradable forked ubiquitin chains that contain many types of linkages [225-226]. Ubiquitin has many nonproteolytic functions in cell signaling [227-228], and those that are particularly well described include those for gene expression, NF-B pathway activation, and the DNA damage response [229-235]. Ubiquitination and deubiquitination also have multiple roles in cell cycle progression and control [236238]. The structure, activity, and function of known deubiquitinases (DUBs, Fig. 2) have been summarized in current reviews [239-


242]. DUBs are divided into five families: 1. ubiquitin C-terminal hydrolases (UCHs), 2. ubiquitin-specific proteases (USPs), 3. Machado-Joseph disease protein domain proteases (MJDs), 4. ovarian tumor proteases (OTUs), and 5. JAMM motif proteases. The first four of these DUB families encode papain-like thiol proteases, and JAMM domain DUBs encode metalloproteases with a zinc atom in their active site. To date, 774 candidate interacting proteins associated with 75 DUBs have been identified [243]. According to the same study, DUBs funtion in protein turnover, transcription, RNA processing, DNA damage, and ERAD. As a therapeutic target in oncology, the JAMM domain DUB Poh1 seems to be an interesting target, as it is vital for 26S proteasome function [244]. However, future JAMM domain inhibitors would likely target other JAMM DUBs that are involved in various cellular processes such as deneddylation [245-246] and deubiquitination of K63-linked polyUb chains [247-248]. 3. 20S PROTEASOME INHIBITION: BORTEZOMIB’S MECHANISM OF ACTION Until 2010, there was no clear answer, at cellular level, for why the inhibition of proteasomes is a successful anticancer strategy. However, taking into consideration the multiple functions of proteasomes, it is clear that such pertubartion of a cell, whether cancerous or normal, must lead to multiple sequelae [249-251]. Bortezomib could also have proteasome-unrelated targets within the cell [252]. Therefore, it is not surprising that various cell lines could profoundly differ in their response to a proteasome inhibitor [253255]. For example, the downregulation of Akt signaling seems to be a major molecular determinant of bortezomib-induced apoptosis in hepatocellular carcinoma cells [256]; however, bortezomib activates pro-survival Akt in prostate cancer [257]. Cells expressing lower proteasome levels are significantly more vulnerable to a proteasome inhibitor [258]. Moreover, diverse proteasome inhibitors have assorted inhibitory effects on a variety of proteasome subtypes [259]. Interestingly, and in contrast to tumor tissues and hypertrophically growing postmitotic cells [260], healthy cells are less sensitive to proteasome inhibition (e.g., bortezomib is used in Table 1.

Cvek and Dvorak

the clinic) [261]. Possible reasons for this are listed below [262]. First: Proteasome inhibitors are known for their ability to block the tumorigenic NF-B pathway; hence, they both suppress cancer and sensitize tumors to other anticancer drugs [263]. Growing evidence from recent years, however, paints a much more complex picture. Second: As cell cycle checkpoints are disrupted in cancer cells, the cells could depend more strongly on proteasome-mediated degradation of cell cycle regulators, particularly p21 and p27 [264]. A dysregulated cell cycle likely makes cancer cells more susceptible to most proapoptotic stimuli (e.g., p53). Some of the transcriptional regulators controlling cell cycle progression (e.g. the c-MYC oncogene) are turned on in cancer cells and may be responsible for proteasome inhibition-induced cell death. The increased protein synthesis requirement in cancer cells could result in a fatal protein aggregation during the inhibition of the proteasome [265-266]. Based on recent studies cited below, we propose a key role of the proapoptotic protein NOXA in bortezomib-mediated toxicity in most cancer cell lines (Table 1, Fig. 3). Third: In the human body, the immune and intercellular context may substantially influence the mechanism of action of bortezomib. Many cell-cell interactions were reported to be altered by proteasomal inhibition in vivo (see below). 3.1. NF-B: The Suspect Lost on the Pathway For more than 20 years, the understanding of NF-B proteins, which function as transcription factors, has become increasingly more complex [267]. In contrast to the “alternative” NF-B signaling pathway, the “canonical” or “classical” pathway is better understood and employs p50:p65 heterodimer as the transcription factor. There is substantial and compelling evidence in support of the tumorigenic activity of p50:p65 NF-B: it induces anti-apoptotic genes, it produces growth and angiogenesis factors, and it directly stimulates cell-cycle progression [268]. According to recent findings, p50:p65 NF-B is necessary for inflammation-triggered migration, invasion, and metastasis of tumor cells [269]. Because the

Bortezomib Mechanism(s) of Action: Key Proteins. Cancer

Key Proteins Published

multiple myeloma

JNK, AP-1, c-MYC, p53, Bik, synergism with TRAIL

mantle cell lymphoma

p53-independent NOXA, ATF3, ATF4

cutaneous T-cell lymphoma

p53-independent NOXA

melanoma

p53-independent NOXA, c-MYC

non-small cell lung

p53-independent NOXA, Bik, JNK, p21, synergism with TRAIL

esophageal squamous cell


hepatocellular carcinoma

JNK, p21, synergism with TRAIL

cervix adenocarcinoma

p53-independent NOXA, ATF3, ATF4

chronic lymphocytic leukemia


bladder

p21, synergism with TRAIL

prostate

p21, synergism with TRAIL

Ewing’s sarcoma

p27, p21, synergism with TRAIL

glioma

synergism with TRAIL

head&neck squamous cell

Bik, Bim



7

Fig. (3). A map of main bortezomib mechanisms of action in various cancers in vitro: Is NOXA the key?

26S proteasome is needed for p50:p65 nuclear translocation through cleavage of the p50:p65 inhibitor IB, proteasome inhibitors suppress canonical NF-B signaling in various cancers [270274]. However, the function of bortezomib on this pathway is more complex than simply blocking it, and the network dynamics of NFB pathway [275] as well as the intracellular pharmacokinetics of bortezomib are just beginning to be understood [276]. The NF-B pathway is complex, and it is able to suppress tumorigenesis as well [277-278]. It has previously been shown that NF-B essential modulator (NEMO)-mediated NF-B activation prevents spontaneous development of hepatocellular cancer in mouse liver parenchymal cells [279]. Additionally, bortezomib is frequently unable to inhibit p50:p65 NF-B in primary multiple myeloma [280] and mantle cell lymphoma [281], and there are emerging p50:p65 NF-B pathways that are proteasomeindependent or even inducible by proteasome inhibition [282]. Proteasome inhibitors activate pro-survival p50:p65 NF-B in colon cancer or hepatic cancer cells by triggering IB degradation [283284] and in multiple myeloma cells by downregulation of IB expression [285]. Thus, NF-B inhibitors could potentiate the anticancer activity of bortezomib in general. 3.2. The Cellular Responses: NOXA is Almost Everywhere According to two key publications [286-287], the apoptotic mechanism induced by bortezomib may involve three major lines in multiple myeloma cells: 1. the mitochondrial axis, 2. the c-Jun Nterminal kinase (JNK) axis, and 3. the DNA-damage axis. Because the results of the two studies do not seem fully consistent with each

other, one can speculate that bortezomib may not activate the same pathways even in multiple myeloma. However, effects similar to those in multiple myeloma were observed in medullary and anaplastic thyroid carcinoma cells [288], which is in contrast to head and neck and pancreatic cancer cell lines, where JNK appears to counteract the anticancer effects of bortezomib [289-290]. Moreover, JNK is not the only bortezomib-induced pro-survival protein. In HepG2 hepatocellular carcinoma cells, bortezomib stimulates the accumulation of heat schock protein Hsp72, which functions to prevent HepG2 cell death [291]. The mitochondrial axis is crucial for bortezomib-induced apoptosis in many cell lines (Table 1). The proapoptotic Bcl-2 protein family member NOXA, which is Latin for damage (Fig. 3), is responsible for the sensitivity of melanoma cells, versus normal melanocytes, to bortezomib [292-294]. Hypoxia-inducible factor1, E2F-1, and p53, which are key proteasome targets with binding sites in the NOXA promoter, are not essential in ultimately defining the expression of NOXA. Instead, it has been reported that the Myc protein critically impacts NOXA transcription under proteasome inhibition (Fig. 3) [295]. However, in multiple myeloma cells, NOXA-mediated apoptosis seems to be induced by cleavage of myeloid cell leukemia-1 protein (Mcl-1, Fig. 3) [296-297]. The central role of p53-independent NOXA in bortezomib antitumor activity was confirmed in mantle cell lymphoma [298], non-small cell lung cancer [299-300], esophageal squamous cell carcinoma [301], chronic lymphocytic leukemia [302], cutaneous T-cell lymphoma, and adult T-cell leukemia/lymphoma [303]. In addition, MG-132 has also been reported to induce p53-independent apopto-


sis through NOXA upregulation in chondrogenic and osteosarcoma cell lines [304]. However, there is yet another activity of the 20S proteasome inhibitors within the mitochondrial axis: they evoke a transient release of Ca2+ stores in multiple myeloma cells and mouse neurons, leading to mitochondrial Ca2+ influx and cell death [305-306]. This effect of 20S proteasome inhibition could be linked to the ability of proteasome inhibitors to cause the accumulation or upregulation of the Bik/Bim proteins in cancer cells [307-308]. If Bik is upregulated by bortezomib, it mediates calcium release and mitochondrial fragmentation [309] by possibly synergizing with NOXA (Fig. 3). Even the “guardian of the genome” p53, which is traditionally linked to the DNA damage response, may translocate to the mitochondria and induce outer membrane permeabilization. In the cytoplasm, p53 is sequestered by antiapoptotic Bcl2 proteins. A target of nuclear p53, p53 upregulated modulator of apoptosis (PUMA), functions to disrupt this interaction and release p53 to target the mitochondria [310]. Indeed, p53 activation and transcriptional induction of its target gene PUMA were shown to play a key role in the sensitivity of neuroblastoma cells and colon cancer cells to proteasome inhibitors [311-312]. The status of p53 also plays a crucial role in the response of B lymphomas to proteasome inhibition [313]. Moreover, bortezomib stabilizes a functional, nonphosphorylated p53 that is induced by proteasomal inhibition in prostate cancer cells [314]. Although bortezomib upregulates p53 in non-small cell lung cancer cells, the anticancer effects of bortezomib seem to be rather dependent on the JNK/AP-1 pathway [315]. JNK/AP-1 and p21 have also been suggested to mediate the sensitivity to bortezomib in hepatocellular carcinoma cells [316317]. In contrast, bortezomib-induced apoptosis was markedly enhanced in p21-knockout colon cancer cells [318]. It was also shown that bortezomib triggers apoptosis in multiple myeloma cells through caspase-2 activation, which is associated with ER stress and is required for the release of cytochrome c, the breakdown of mitochondrial transmembrane potential, and its downstream caspase-9 activation [319]. Because the accumulation of unfolded proteins induces ER stress, multiple myeloma cells with higher amounts of immunoglobulin subunits are more sensitive to 20S proteasome inhibition [320-322]. The bortezomibinduced ER stress is also responsible for apoptosis in pancreatic cancer cells [323] and general hypersensitivity of hypoxic tumor cells to the 20S proteasome inhibitors [324]. In squamous cell carcinoma cells, bortezomib-induced ER stress mediates apoptosis through the transcription factor ATF4 and NOXA upregulation [325]. Another study clearly demonstrated that, in various cancer cells, bortezomib activates transcription factors ATF3 and ATF4, which form a complex that is capable of binding to the NOXA promoter and triggering NOXA transcription (Fig. 3) [326]. According to a most recent report, there are 100 genes, including cMYC and NOXA, whose knockdown affects lethality in response to bortezomib and other proteasome inhibitors [327]. At the nuclear level, 20S proteasome inhibition by MG-132 and -lactacystin suppresses homologous recombination, which is indispensable for DNA double-strand break repair [328]. The suppressive effect of 20S proteasome inhibitors on homologous recombination provides us with another explanation for their strong antitumor effect. Still, bortezomib and MG-132 induce an inhibition of the general DNA damage response and the Fanconi anemia pathway at multiple levels, leading to the persistence of DNA damage and abrogation of the G1-S cell cycle checkpoint [329]. The 20S proteasome inhibitors MG-132, lactacystin, and MG-262 inhibit UV-induced translesion replication in a wide range of cancer cell lines regardless of cell origin, histological type, or p53 status; however, these inhibitors have little or no influence on normal fibroblasts or on a normal liver mesenchymal cell line [330]. In general, the mechanism of p53-independent DNA-damage-

Cvek and Dvorak

mediated apoptosis once again involves NOXA upregulation (Fig. 3) [331]. Nevertheless, further nuclear consequences of 20S proteasome inhibition could be anticipated with bortezomib-induced topoisomerase II stabilization and repression of hypoxia-inducible factor-1 (HIF-1) transcriptional activity, as previously reported [332-335]. 3.3. Immunity and Intercellular Context The anticancer activity of bortezomib could also be explained as the sensitization of the cancer cells to the immune system of the body. Some cells in the immune system express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which plays a role in both T-cell and natural killer cell-mediated tumor surveillances. Thus, TRAIL selectively induces apoptosis in a variety of tumor cells, in contrast to most normal cells [336]. However, several tumors evade TRAIL-induced apoptosis and acquire TRAIL resistance via different mechanisms. Generally, chemotherapy and radiotherapy enhance TRAIL sensitivity or reverses TRAIL resistance by regulating the downstream effectors. Because TRAIL seems to trigger mitochondrial apoptotic pathways [337], 20S proteasome inhibition should synergize with TRAIL (Tab. 1, Fig. 3). In an overnight assay, bortezomib and TRAIL were much more effective than either agent alone in promoting the apoptosis of murine myeloid leukemia and renal cancer cells [338]. Synergistic apoptotic induction occurred in prostate and bladder cancer cells, non–small cell lung cancer cells, Ewing’s sarcoma cells and primary astrocytoma cells treated with both agents [339-342]. Furthermore, bortezomib sensitizes hepatocellular carcinoma cells, but not primary human hepatocytes, to TRAIL [343-344]. Bortezomibresistant multiple myeloma cells become bortezomib-sensitive when co-treated with TRAIL [345]. Taking these data together, it is not surprising that proteasome inhibition has been shown to induce apoptosis in various cancer cells via the TRAIL death receptors DR4 and DR5 (Fig. 3), which in this way mimic the immune response [346-348]. When mice with metastatic mammary tumors were treated with bortezomib and a DR5 agonist, the median survival increased from 42 days with bortezomib alone to 180 days with both agents [349]. However, DR5 seems to be dispensable for the synergism between TRAIL and 20S proteasome inhibitors in colon cancer and prostate cancer cells [350-351]. Additionally, bortezomib overcomes TRAIL resistance in hepatocellular carcinoma cells in part through the inhibition of the Akt pathway [352]. It has been shown that natural killer cell-mediated antitumor effects can be augmented by bortezomib in mice [353], and 20S proteasome inhibition can sensitize melanoma to the lytic effects of dendritic cell-activated immune effector cells [354]. Although bortezomib sensitized breast cancer cell lines to natural killer cellmediated cell death in vitro, it failed to do so in vivo [355]. According to recent data, depletion of regulatory T cells enhances bortezomib-induced tumor sensitization to autologous natural killer cells in mice [356]. In dendritic cells (DCs), bortezomib exerts immunomodulatory effects as it reduces their phagocytic capacity, cytokine production, and immunostimulatory capacity and suppresses their phenotypic maturation [357]. In in vitro studies, bortezomib induced apoptotic cell death in immature DCs and, to a much lesser extent, in mature DCs [358]. Moreover, bortezomib was toxic for both immature and mature monocyte-derived DCs [359]. In part, bortezomib may suppress cancer by altering other cells. It interferes with angiogenesis by a direct effect on multiple myeloma patient-derived endothelial cells and their functions associated with angiogenesis [360]. Bortezomib also inhibits cell-cell adhesion and cell migration [361] and overcomes cell adhesion-mediated drug resistance [362] in vitro. Furthermore, myeloma cells within the bone marrow microenvironment exhibit an enhanced response to bortezomib [363], suggesting an important role for surrounding tissues in the sensitivity of cancer cells to bortezomib. Because multiple myeloma is a plasma cell malignancy that is characterized


by a high capacity to induce osteolytic bone lesions [364], it is important to mention that bortezomib increases osteoblast differentiation in human mesenchymal cells without affecting the number of osteoblast progenitors or the viability of mature osteoblasts [365]. This appears to be confirmed by experiments performed in mice [366-367] and in clinical studies [368]. Given the similarities between the molecular events involved in the formation phase of the bone remodeling cycle and the induction of anagen in the hair cycle, it is not surprising that proteasome inhibitors are able to stimulate hair growth in humans [369]. 4. CONCLUDING REMARKS As our knowledge of the UPS is becoming increasingly complex, we should question simplistic models of the mechanism of action of bortezomib. It seems that inhibiting the proteasome has various consequences in different cancer tissues, thus explaining why the efficacy of bortezomib varies depending on the type of cancer. We need to understand what cellular events are triggered by bortezomib to suppress tumors in patients. The in vitro studies that demonstrate bortezomib efficacy in many solid tumors are rather confusing. Phase II clinical trials using bortezomib have failed in many solid cancers [370-379], suggesting that proteasome inhibition might target only blood cancers, e.g. multiple myeloma (clinically approved), mantle cell lymphoma (clinically approved), cutaneous T-cell lymphoma [380], mucosa-associated lymphoid tissue (MALT) lymphoma [381], and Waldenström’s macroglobulinemia [382-384]. Whether such blood cancer specifity is significant only for bortezomib or for all proteasome inhibitors will hopefully be uncovered in ongoing and future clinical trials [385]. ACKNOWLEDGEMENT This work was financed by the Czech Science Foundation (Grant No. 303/08/P137 and Grant No. 304/10/0149). ABBREVIATIONS AAA+ = AP-1 ATF

= =

Bcl2 Bik Bim

= = =

BRCA1 CD CP CSN DC DR4/DR5 DUB E1 E2 E3 E4 eIF2

= = = = = = = = = = = =

ER ERAD

= =

FADD

=

ATPases associated with diverse cellular activities Activator protein 1 Cyclic AMP-dependent transcription factor B-cell lymphoma 2 B-cell lymphoma 2-interacting killer One of B-cell lymphoma 2-interacting proteins Breast cancer 1 Cluster of differentiation Core particle (20S proteasome) COP9 signalosome Dendritic cells Death receptor 4/5 Deubiquitinase Ubiquitin-activating enzyme Ubiquitin-conjugating enzyme Ubiquitin-ligating enzyme Ubiquitin-chain elongation factor Eukaryotic translation initiation factor 2 Endoplasmic reticulum Endoplasmic reticulum-associated degradation Fas-associated protein with death domain


FDA HECT

= =

HIF-1 HslV IFN- IPOD IB iPr JAMM JNK JUNQ LMP2 or LMP7 MALT Mcl-1 MDC1

= = = = = = = = = = = = =

MECL1

=

MHC I

=

MJD

=

MYC NEMO NF-B OTU PA(700/28) PML PNPase Poh1 polyUb POMP PUMA

= = = = = = = = = = =

REG RING RP Rpn Rpt TRAIL

= = = = = =

TRIP1 Uba6 Ube1 UCH USP UPS

= = = = = =

9

Food and Drug Administration Homologous to E6-Associated Protein C-Terminus Hypoxia-inducible factor-1 Heat shock locus V Interferon- Insoluble protein deposit Inhibitor B Immunoproteasome JAB1/MPN/Mov34 metalloenzyme c-Jun N-terminal kinase Juxta-nuclear quality control Low mass protein 2 or 7 Mucosa-associated lymphoid tissue Myeloid cell leukemia-1 protein Mediator of DNA damage checkpoint protein 1 Multicatalytic endopeptidase complex subunit 1 Major histocompatibility complex class I Machado-Joseph disease protein domain protease Myelocytomatosis oncogene NF-B essential modulator Nuclear factor-B Ovarian tumor protease Proteasome activator Promyelocytic leukemia Polynucleotide phosphorylase Pad one homolog-1 Polyubiquitin Proteasome maturation protein p53 upregulated modulator of apoptosis Regulator Really interesting new gene (19S proteasome) regulatory particle Regulatory particle non-ATPase-like Regulatory particle ATPase-like Tumor necrosis factor-related apoptosis-inducing ligand Thyroid receptor interactor 1 Ubiquitin-activating enzyme 6 Ubiquitin-activating enzyme 1 Ubiquitin C-terminal hydrolase Ubiquitin-specific protease Ubiquitin-proteasome system

REFERENCES [1] [2] [3]

DeVita VT, Chu E. A history of cancer chemotherapy. Cancer Res 2008; 68: 8643-53. Ma WW, Adjei AA. Novel agents on the horizon for cancer therapy. CA Cancer J Clin 2009; 59: 111-37. Adams J, Kauffman M. Development of the proteasome inhihitor Velcade™ (Bortezomib). Cancer Invest 2004; 22: 304-11.

10 Current Pharmaceutical Design, 2011, Vol. 17, No. 00 [4]

[5] [6]

[7] [8]

[9]

[10] [11]

[12] [13]

[14] [15] [16]

[17] [18] [19]

[20]

[21]

[22] [23]

[24] [25]

[26] [27]

[28]

Palombella VJ, Conner EM, Fuseler JW, et al. Role of the proteasome and NF-B in streptococcal cell wall-induced polyarthritis. Proc Natl Acad Sci USA 1998; 95: 15671-6. Kalogeris TJ, Laroux FS, Cockrell A, et al. Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells. Am J Physiol 1999; 276: C856-64. Adams J, Palombella VJ, Sausville EA, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999; 59: 2615-22. Iqbal M, Chatterjee S, Kauer JC, et al. Potent inhibitors of proteasome. J Med Chem 1995; 38: 2276-7. Tsubuki S, Kawasaki H, Saito Y, Miyashita N, Inomata M, Kawashima S. Purification and characterization of a Z-Leu-Leu-LeuMCA degrading protease expected to regulate neurite formation: a novel catalytic activity in proteasome. Biochem Biophys Res Commun 1993; 196: 1195-201. Adams J, Stein R. Novel inhibitors of the proteasome and their therapeutic use in inflammation. Annu Rep Med Chem 1996; 31: 279-88. Adams J, Ma YT, Stein R, Baevsky M, Grenier L, Plamondon L. International patent application WO 96/13266, published May 9, 1996. Chem Abstr 1996; 125: 143315. Seemuller E, Lupas A, Stock D, Lowe J, Huber R, Baumeister W. Proteasome from Thermoplasma acidophilum: a threonine protease. Science 1995; 268: 579-82. Stein RL, Melandri F, Dick L. Kinetic characterization of the chymotryptic activity of the 20S proteasome. Biochemistry 1996; 35: 3899-908. Adams J, Behnke M, Chen S, et al. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg Med Chem Lett 1998; 8: 333-8. Yang W, Gao X, Wang B. Boronic acid compounds as potential pharmaceutical agents. Med Res Rev 2003; 23: 346-68. Matteson DS. -Amido boronic acids: a synthetic challenge and their properties as serine protease inhibitors. Med Res Rev 2008; 28: 233-46. Boyd MR, Paull KD. Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen. Drug Dev Res 1995; 34: 91-109. Weinstein JN, Myers TG, O’Connor PM, et al. An informationintensive approach to the molecular pharmacology of cancer. Science 1997; 275: 343-9. Adams J. Development of the proteasome inhibitor PS-341. Oncologist 2002; 7: 9-16. Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 2004; 22: 2108-21. Aghajanian C, Soignet S, Dizon DS, et al. A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 2002; 8: 2505-11. Hamilton AL, Eder JP, Pavlick AC, et al. Proteasome inhibition with bortezomib (PS-341): a phase I study with pharmacodynamic end points using a day 1 and day 4 schedule in a 14-day cycle. J Clin Oncol 2005; 23: 6107-16. Orlowski RZ, Stinchcombe TE, Mitchell BS, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol 2002; 20: 4420-7. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003; 348: 2609-17. Kane RC, Bross PF, Farrell AT, Pazdur R. Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist 2003; 8: 508-13. Bross PF, Kane R, Farrell AT, et al. Approval summary for bortezomib for injection in the treatment of multiple myeloma. Clin Cancer Res 2004; 10: 3954-64. Adams J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 2004; 5: 417-21. Kane RC, Farrell AT, Sridhara R, Pazdur R. United States Food and Drug Administration approval summary: bortezomib for the treatment of progressive multiple myeloma after one prior therapy. Clin Cancer Res 2006; 12: 2955-60. Kane RC, Dagher R, Farrell A, et al. Bortezomib for the treatment of mantle cell lymphoma. Clin Cancer Res 2007; 13: 5291-4.

Cvek and Dvorak [29] [30] [31]

[32] [33] [34] [35]

[36] [37]

[38] [39]

[40] [41] [42] [43]

[44] [45] [46]

[47]

[48] [49]

[50]

[51] [52]

[53]

[54] [55]

Myung J, Kim KB, Crews CM. The ubiquitin-proteasome pathway and proteasome inhibitors. Med Res Rev 2001;21:245-73. Meiners S, Ludwig A, Stangl V, Stangl K. Proteasome inhibitors: poisons and remedies. Med Res Rev 2008; 28: 309-27. Borissenko L, Groll M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem Rev 2007; 107: 687-717. Hershko A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ 2005; 12: 1191-7. Varshavsky A. Regulated protein degradation. Trends Biochem Sci 2005; 30: 283-6. Noda NN, Ohsumi Y, Inagaki F. ATG systems from the protein structural point of view. Chem Rev 2009; 109: 1587-98. Todde V, Veenhuis M, van der Klei IJ. Autophagy: principles and significance in health and disease. Biochim Biophys Acta 2009; 1792: 3-13. Raiborg C, Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 2009; 458: 445-452. Davies BA, Lee JR, Oestreich AJ, Katzmann DJ. Membrane protein targeting to the MVB/lysosome. Chem Rev 2009; 109: 157586. Schubert U, Anton LC, Gibbs J, Norbury CC, Yewdell JW, Bennink JR. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 2000; 404: 770-4. Hirsch C, Gauss R, Horn SC, Neuber O, Sommer T. The ubiquitylation machinery of the endoplasmic reticulum. Nature 2009; 458: 453-60. Hampton RY. Proteolysis and sterol regulation. Annu Rev Cell Dev Biol 2002; 18: 345-78. Kaganovich D, Kopito R, Frydman J. Misfolded proteins partition between two distinct quality control compartments. Nature 2008; 454: 1088-95. Wojcik C, DeMartino GN. Intracellular localization of proteasomes. Int J Biochem Cell Biol 2003; 35: 579-89. Reits EA, Benham AM, Plougastel B, Neefjes J, Trowsdale J. Dynamics of proteasome distribution in living cells. EMBO J 1997; 16: 6087-94. Foster LJ, de Hoog CL, Zhang Y, et al. A mammalian organelle map by protein correlation profiling. Cell 2006; 125: 187-199. Klare N, Seeger M, Janek K, Jungblut PR, Dahlmann B. Intermediate-type 20 S proteasomes in HeLa cells: “asymmetric” subunit composition, diversity and adaptation. J Mol Biol 2007; 373: 1-10. Olink-Coux M, Arcangeletti C, Pinardi F, et al. Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type. J Cell Sci 1994; 107: 353-66. Arcangeletti C, Sutterlin R, Aebi U, et al. Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton. J Struct Biol 1997; 119: 35-58. Bingol B, Schuman EM. Activity-dependent dynamics and sequestration of proteasomes in dendritic spines. Nature 2006; 441: 11448. Bassaglia Y, Cebrian J, Covan S, Garcia M, Foucrier J. Proteasomes are tightly associated to myofibrils in mature skeletal muscle. Exp Cell Res 2005; 302: 221-32. Fabunmi RP, Wigley WC, Thomas PJ, DeMartino GN. Interferon regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies. J Cell Sci 2001; 114: 2936. Rockel TD, von Mikecz A. Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization. J Struct Biol 2002; 140: 189-199. Chen M, Rockel TD, Steinweger G, Hemmerich P, Risch J, von Mikecz A. Subcellular recruitment of fibrillarin to nucleoplasmic proteasomes: implications for processing of a nucleolar autoantigen. Mol Biol Cell 2002; 13: 3576-87. Rockel TD, Stuhlmann D, von Mikecz A. Proteasomes degrade proteins in focal subdomains of the human cell nucleus. J Cell Sci 2005; 118: 5231-42. Arabi A, Rustum C, Hallberg E, Wright AP. Accumulation of cMyc and proteasomes at the nucleoli of cells containing elevated cMyc protein levels. J Cell Sci 2003; 116: 1707-17. Andersen JS, Lyon CE, Fox AH, et al. Directed proteomic analysis of the human nucleolus. Curr Biol 2002; 12: 1-11.

The Ubiquitin-Proteasome System (UPS) [56] [57] [58] [59]

[60] [61]

[62] [63]

[64] [65] [66]

[67] [68] [69]

[70]

[71] [72]

[73]

[74] [75]

[76]

[77]

[78]

[79]

[80]

Andersen JS, Lam YW, Leung AK, et al. Nucleolar proteome dynamics. Nature 2005; 433: 77-83. Lorentzen E, Conti E. The exosome and the proteasome: nanocompartments for degradation. Cell 2006; 125: 651-54. Pickart CM, Cohen RE. Proteasomes and their kin: proteases in the machine age. Nat Rev Mol Cell Biol 2004; 5: 177-87. Maupin-Furlow JA, Humbard MA, Kirkland PA, et al. Proteasomes from structure to function: perspectives from Archaea. Curr Top Dev Biol 2006; 75: 125-69. Marques AJ, Palanimurugan R, Matias AC, Ramos PC, Dohmen RJ. Catalytic mechanism and assembly of the proteasome. Chem Rev 2009; 109: 1509-36. Nickell S, Mihalache O, Beck F, Hegerl R, Korinek A, Baumeister W. Structural analysis of the 26S proteasome by cryoelectron tomography. Biochem Biophys Res Commun 2007;353:115-120. Nickell S, Beck F, Korinek A, Mihalache O, Baumeister W, Plitzko JM. Automated cryoelectron microscopy of “single particles” applied to the 26S proteasome. FEBS Lett 2007;581:2751-2756. Bedford L, Paine S, Sheppard PW, Mayer RJ, Roelofs J. Assembly, structure, and function of the 26S proteasome. Trends Cell Biol 2010; 20: 391-401. Sprangers R, Kay LE. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 2007; 445: 618-22. Hirano Y, Hendil KB, Yashiroda H, et al. A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes. Nature 2005; 437: 1381-5. Hirano Y, Hayashi H, Iemura S, et al. Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes. Mol Cell 2006; 24: 977-84. Hirano Y, Kaneko T, Okamoto K, et al. Dissecting -ring assembly pathway of the mammalian 20S proteasome. EMBO J 2008; 27: 2204-13. Murata S, Yashiroda H, Tanaka K. Molecular mechanisms of proteasome assembly. Nat Rev Mol Cell Biol 2009; 10: 104-15. Strehl B, Seifert U, Kruger E, Heink S, Kuckelkorn U, Kloetzel PM. Interferon-, the functional plasticity of the ubiquitinproteasome system, and MHC class I antigen processing. Immunol Rev 2005; 207: 19-30. Jayarapu K, Griffin TA. Differential intra-proteasome interactions involving standard and immunosubunits. Biochem Biophys Res Commun 2007; 358: 867-72. Heink S, Ludwig D, Kloetzel PM, Kruger E. IFN--induced immune adaptation of the proteasome system is an accelerated and transient response. Proc Natl Acad Sci USA 2005; 102: 9241-6. Bose S, Stratford FL, Broadfoot KI, Mason GG, Rivett AJ. Phosphorylation of 20S proteasome subunit C8 (7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by -interferon. Biochem J 2004; 378: 177-184. Yewdell JW. The seven dirty little secrets of major histocompatibility complex class I antigen processing. Immunol Rev 2005; 207: 8-18. Kloetzel PM. Generation of major histocompatibility complex class I antigens: functional interplay between proteasomes and TPPII. Nat Immunol 2004; 5: 661-9. Hammer GE, Kanaseki T, Shastri N. The final touches make perfect the peptide-MHC class I repertoire. Immunity 2007; 26: 397406. Basler M, Youhnovski N, Van Den Broek M, Przybylski M, Groettrup M. Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus. J Immunol 2004; 173: 3925-34. Gavioli R, Gallerani E, Fortini C, et al. HIV-1 tat protein modulates the generation of cytotoxic T cell epitopes by modifying proteasome composition and enzymatic activity. J Immunol 2004; 173: 3838-43. Ito Y, Kondo E, Demachi-Okamura A, et al. Three immunoproteasome-associated subunits cooperatively generate a cytotoxic Tlymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation. J Virol 2006; 80: 883-890. Pang KC, Sanders MT, Monaco JJ, Doherty PC, Turner SJ, Chen W. Immunoproteasome subunit deficiencies impact differentially on two immunodominant influenza virus-specific CD8+ T cell responses. J Immunol 2006; 177: 7680-8. Steers NJ, Peachman KK, McClain SR, Alving CR, Rao M. Human immunodeficiency virus type 1 Gag p24 alters the composition of


[81]

[82]

[83]

[84]

[85] [86]

[87] [88] [89]

[90] [91]

[92] [93]

[94] [95]

[96]

[97] [98]

[99] [100]

[101] [102] [103]

[104]

11

immunoproteasomes and affects antigen presentation. J Virol 2009; 83: 7049-61. Strehl B, Joeris T, Rieger M, et al. Immunoproteasomes are essential for clearance of Listeria monocytogenes in nonlymphoid tissues but not for induction of bacteria-specific CD8+ T cells. J Immunol 2006; 177: 6238-44. Szalay G, Meiners S, Voigt A, et al. Ongoing coxsackievirus myocarditis is associated with increased formation and activity of myocardial immunoproteasomes. Am J Pathol 2006; 168: 1542-52. Jakel S, Kuckelkorn U, Szalay G, et al. Differential interferon responses enhance viral epitope generation by myocardial immunoproteasomes in murine enterovirus myocarditis. Am J Pathol 2009; 175: 510-8. Caudill CM, Jayarapu K, Elenich L, Monaco JJ, Colbert RA, Griffin TA. T cells lacking immunoproteasome subunits MECL-1 and LMP7 hyperproliferate in response to polyclonal mitogens. J Immunol 2006; 176: 4075-82. Murata S, Sasaki K, Kishimoto T, et al. Regulation of CD8+ T cell development by thymus-specific proteasomes. Science 2007; 316: 1349-53. Murata S, Takahama Y, Tanaka K. Thymoproteasome: probable role in generating positively selecting peptides. Curr Opin Immunol 2008; 20: 192-6. Tomaru U, Ishizu A, Murata S, et al. Exclusive expression of proteasome subunit 5t in the human thymic cortex. Blood 2009; 113: 5186-91. Demartino GN, Gillette TG. Proteasomes: machines for all reasons. Cell 2007; 129: 659-62. Goldberg AL, Cascio P, Saric T, Rock KL. The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides. Mol Immunol 2002; 39: 147-64. Li X, Lonard DM, Jung SY, et al. The SRC-3/AIB1 coactivator is degraded in a ubiquitin- and ATP-independent manner by the REG proteasome. Cell 2006; 124: 381-92. Li X, Amazit L, Long W, Lonard DM, Monaco JJ, O’Malley BW. Ubiquitin- and ATP-independent proteolytic turnover of p21 by the REG-proteasome pathway. Mol Cell 2007; 26: 831-42. Chen X, Barton LF, Chi Y, Clurman BE, Roberts JM. Ubiquitinindependent degradation of cell-cycle inhibitors by the REGgamma proteasome. Mol Cell 2007; 26: 843-52. Zhang Z, Zhang R. Proteasome activator PA28 regulates p53 by enhancing its MDM2-mediated degradation. EMBO J 2008; 27: 852-64. Baldin V, Militello M, Thomas Y, et al. A novel role for PA28proteasome in nuclear speckle organization and SR protein trafficking. Mol Biol Cell 2008; 19: 1706-16. Zannini L, Lecis D, Buscemi G, et al. REG proteasome activator is involved in the maintenance of chromosomal stability. Cell Cycle 2008; 7: 504-12. Zannini L, Buscemi G, Fontanella E, Lisanti S, Delia D. REG/PA28 proteasome activator interacts with PML and Chk2 and affects PML nuclear bodies number. Cell Cycle 2009; 8: 2399407. Ustrell V, Hoffman L, Pratt G, Rechsteiner M. PA200, a nuclear proteasome activator involved in DNA repair. EMBO J 2002; 21: 3516-25. Kajava AV, Gorbea C, Ortega J, Rechsteiner M, Steven AC. New HEAT-like repeat motifs in proteins regulating proteasome structure and function. J Struct Biol 2004; 146: 425-30. Ortega J, Heymann JB, Kajava AV, Ustrell V, Rechsteiner M, Steven AC. The axial channel of the 20S proteasome opens upon binding of the PA200 activator. J Mol Biol 2005; 346: 1221-7. Blickwedehl J, Agarwal M, Seong C, et al. Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability. Proc Natl Acad Sci USA 2008; 105: 16165-70. Glickman MH, Raveh D. Proteasome plasticity. FEBS Lett 2005; 579: 3214-23. Kriegenburg F, Seeger M, Saeki Y, et al. Mammalian 26S proteasomes remain intact during protein degradation. Cell 2008; 135: 355-65. Rosenzweig R, Osmulski PA, Gaczynska M, Glickman MH. The central unit within the 19S regulatory particle of the proteasome. Nat Struct Mol Biol 2008; 15: 573-80. Murata S, Yashiroda H, Tanaka K. Molecular mechanisms of proteasome assembly. Nat Rev Mol Cell Biol 2009; 10: 104-15.


[106]

[107] [108]

[109] [110] [111]

[112]

[113]

[114]

[115] [116] [117] [118]

[119] [120] [121] [122]

[123] [124] [125] [126]

[127] [128] [129]

[130]

Gillette TG, Kumar B, Thompson D, Slaughter CA, DeMartino GN. Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome. J Biol Chem 2008; 283: 31813-22. Funakoshi M, Tomko RJ, Kobayashi H, Hochstrasser M. Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base. Cell 2009; 137: 887-99. Saeki Y, Toh-E A, Kudo T, Kawamura H, Tanaka K. Multiple proteasome-interacting proteins assist the assembly of the yeast 19S regulatory particle. Cell 2009; 137: 900-13. Kaneko T, Hamazaki J, Iemura S, et al. Assembly pathway of the mammalian proteasome base subcomplex is mediated by multiple specific chaperones. Cell 2009; 137: 914-25. Roelofs J, Park S, Haas W, et al. Chaperone-mediated pathway of proteasome regulatory particle assembly. Nature 2009; 459: 861-5. Park S, Roelofs J, Kim W, et al. Hexameric assembly of the proteasomal ATPases is templated through their C termini. Nature 2009; 459: 866-70. Smith DM, Kafri G, Cheng Y, Ng D, Walz T, Goldberg AL. ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. Mol Cell 2005; 20: 687-98. Smith DM, Chang SC, Park S, Finley D, Cheng Y, Goldberg AL. Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry. Mol Cell 2007; 27: 731-44. Liu CW, Li X, Thompson D, et al. ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol Cell 2006; 24: 39-50. Zhang F, Hu Y, Huang P, Toleman CA, Paterson AJ, Kudlow JE. Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6. J Biol Chem 2007; 282: 22460-71. Li X, Demartino GN. Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem J 2009; 421: 397-404. Sharon M, Taverner T, Ambroggio XI, Deshaies RJ, Robinson CV. Structural organization of the 19S proteasome lid: insights from MS of intact complexes. PLoS Biol 2006; 4: 1314-23. Yao T, Cohen RE. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 2002; 419: 403-7. Verma R, Aravind L, Oania R, et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 2002; 298: 611-5. Berndt C, Bech-Otschir D, Dubiel W, Seeger M. Ubiquitin system: JAMMing in the name of the lid. Curr Biol 2002; 12: R815-7. Li L, Deng XW. The COP9 signalosome: an alternative lid for the 26S proteasome? Trends Cell Biol 2003; 13: 507-9. Jariel-Encontre I, Bossis G, Piechaczyk M. Ubiquitin-independent degradation of proteins by the proteasome. Biochim Biophys Acta 2008; 1786: 153-77. Baugh JM, Viktorova EG, Pilipenko EV. Proteasomes can degrade a significant proportion of cellular proteins independent of ubiquitination. J Mol Biol 2009; 386: 814-27. Shringarpure R, Grune T, Mehlhase J, Davies KJ. Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J Biol Chem 2003; 278: 311-8. Jung T, Grune T. The proteasome and its role in the degradation of oxidized proteins. IUBMB Life 2008; 60: 743-52. Pegg AE. Regulation of ornithine decarboxylase. J Biol Chem 2006; 281: 14529-32. Hipp MS, Kalveram B, Raasi S, Groettrup M, Schmidtke G. FAT10, a ubiquitin-independent signal for proteasomal degradation. Mol Cell Biol 2005; 25: 3483-91. Schmidtke G, Kalveram B, Groettrup M. Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L. FEBS Lett 2009; 583: 591-4. Tsvetkov P, Reuven N, Shaul Y. Ubiquitin-independent p53 proteasomal degradation. Cell Death Differ 2010; 17: 103-8. Basbous J, Jariel-Encontre I, Gomard T, Bossis G, Piechaczyk M. Ubiquitin-independent- versus ubiquitin-dependent proteasomal degradation of the c-Fos and Fra-1 transcription factors: is there a unique answer? Biochimie 2008; 90: 296-305. Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 2009; 78: 477-513.

Cvek and Dvorak [131]

[132] [133]

[134] [135]

[136]

[137] [138] [139] [140]

[141] [142]

[143] [144]

[145] [146]

[147] [148]

[149] [150]

[151]

[152] [153]

[154] [155]

Kirkpatrick DS, Hathaway NA, Hanna J, et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat Cell Biol 2006; 8: 700-10. Matiuhin Y, Kirkpatrick DS, Ziv I, et al. Extraproteasomal Rpn10 restricts access of the polyubiquitin-binding protein Dsk2 to proteasome. Mol Cell 2008; 32: 415-25. Saeki Y, Kudo T, Sone T, et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J 2009; 28: 359-71. Xu P, Duong DM, Seyfried NT, et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 2009; 137: 133-45. Newton K, Matsumoto ML, Wertz IE, et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 2008; 134: 668-78. Kravtsova-Ivantsiv Y, Cohen S, Ciechanover A. Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-B precursor. Mol Cell 2009; 33: 496-504. Elsasser S, Finley D. Delivery of ubiquitinated substrates to protein-unfolding machines. Nat Cell Biol 2005; 7: 742-9. Husnjak K, Elsasser S, Zhang N, et al. Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 2008; 453: 481-8. Schreiner P, Chen X, Husnjak K, et al. Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction. Nature 2008; 453: 548-52. Lam YA, Lawson TG, Velayutham M, Zweier JL, Pickart CM. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 2002; 416: 763-7. Archer CT, Burdine L, Liu B, Ferdous A, Johnston SA, Kodadek T. Physical and functional interactions of monoubiquitylated transactivators with the proteasome. J Biol Chem 2008; 283: 21789-98. Verma R, Oania R, Graumann J, Deshaies RJ. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitinproteasome system. Cell 2004; 118: 99-110. Lam YA, Xu W, DeMartino GN, Cohen RE. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 1997; 385: 737-40. Hanna J, Hathaway NA, Tone Y, et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 2006; 127: 99-111. Crosas B, Hanna J, Kirkpatrick DS, et al. Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell 2006; 127: 1401-13. Hanna J, Meides A, Zhang DP, Finley D. A ubiquitin stress response induces altered proteasome composition. Cell 2007; 129: 747-59. Bech-Otschir D, Helfrich A, Enenkel C, et al. Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome. Nat Struct Mol Biol 2009; 16: 219-25. Koulich E, Li X, DeMartino GN. Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol Biol Cell 2008; 19: 1072-82. Guterman A, Glickman MH. Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome. J Biol Chem 2004; 279: 1729-38. Shabek N, Herman-Bachinsky Y, Ciechanover A. Ubiquitin degradation with its substrate, or as a monomer in a ubiquitinationindependent mode, provides clues to proteasome regulation. Proc Natl Acad Sci USA 2009; 106: 11907-12. Jacobson AD, Zhang NY, Xu P, et al. The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome. J Biol Chem 2009; 284: 35485-94. Striebel F, Kress W, Weber-Ban E. Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Curr Opin Struct Biol 2009; 19: 209-17. Prakash S, Tian L, Ratliff KS, Lehotzky RE, Matouschek A. An unstructured initiation site is required for efficient proteasomemediated degradation. Nat Struct Mol Biol 2004; 11: 830-7. Prakash S, Inobe T, Hatch AJ, Matouschek A. Substrate selection by the proteasome during degradation of protein complexes. Nat Chem Biol 2009; 5: 29-36. Sharon M, Witt S, Felderer K, Rockel B, Baumeister W, Robinson CV. 20S proteasomes have the potential to keep substrates in store for continual degradation. J Biol Chem 2006; 281: 9569-75.

The Ubiquitin-Proteasome System (UPS) [156]

[157] [158]

[159]

[160]

[161]

[162] [163]

[164] [165]

[166] [167] [168] [169]

[170] [171] [172] [173]

[174] [175] [176]

[177]

[178]

[179]

Kleijnen MF, Roelofs J, Park S, et al. Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nat Struct Mol Biol 2007; 14: 1180-8. Hutschenreiter S, Tinazli A, Model K, Tampe R. Two-substrate association with the 20S proteasome at single-molecule level. EMBO J 2004; 23: 2488-97. Kisselev AF, Akopian TN, Woo KM, Goldberg AL. The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 1999; 274: 3363-71. Kohler A, Cascio P, Leggett DS, Woo KM, Goldberg AL, Finley D. The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. Mol Cell 2001; 7: 1143-52. Kisselev AF, Kaganovich D, Goldberg AL. Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes. Evidence for peptide-induced channel opening in the -rings. J Biol Chem 2002; 277: 22260-70. Emmerich NP, Nussbaum AK, Stevanovic S, et al. The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate. J Biol Chem 2000; 275: 21140-8. Vabulas RM, Hartl FU. Protein synthesis upon acute nutrient restriction relies on proteasome function. Science 2005; 310: 19603. Rape M, Jentsch S. Productive RUPture: activation of transcription factors by proteasomal processing. Biochim Biophys Acta 2004; 1695: 209-13. Piwko W, Jentsch S. Proteasome-mediated protein processing by bidirectional degradation initiated from an internal site. Nat Struct Mol Biol 2006; 13: 691-7. Hoyt MA, Zich J, Takeuchi J, Zhang M, Govaerts C, Coffino P. Glycine-alanine repeats impair proper substrate unfolding by the proteasome. EMBO J 2006; 25: 1720-9. von Mikecz A. The nuclear ubiquitin-proteasome system. J Cell Sci 2006; 119: 1977-84. Muratani M, Tansey WP. How the ubiquitin-proteasome system controls transcription. Nat Rev Mol Cell Biol 2003; 4: 192-201. Lipford JR, Smith GT, Chi Y, Deshaies RJ. A putative stimulatory role for activator turnover in gene expression. Nature 2005; 438: 113-6. Baugh JM, Pilipenko EV. 20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3. Mol Cell 2004; 16: 575-86. Constantinou C, Elia A, Clemens MJ. Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4EBP1). Biol Cell 2008; 100: 279-89. Daulny A, Tansey WP. Damage control: DNA repair, transcription, and the ubiquitin-proteasome system. DNA Repair 2009; 8: 444-8. Gardner RG, Nelson ZW, Gottschling DE. Degradation-mediated protein quality control in the nucleus. Cell 2005; 120: 803-15. Nalley K, Johnston SA, Kodadek T. Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo. Nature 2006; 442: 1054-7. Kodadek T, Sikder D, Nalley K. Keeping transcriptional activators under control. Cell 2006; 127: 261-4. Sikder D, Johnston SA, Kodadek T. Widespread, but non-identical, association of proteasomal 19 and 20 S proteins with yeast chromatin. J Biol Chem 2006; 281: 27346-55. Auld KL, Brown CR, Casolari JM, Komili S, Silver PA. Genomic association of the proteasome demonstrates overlapping gene regulatory activity with transcription factor substrates. Mol Cell 2006; 21: 861-71. Szutorisz H, Georgiou A, Tora L, Dillon N. The proteasome restricts permissive transcription at tissue-specific gene loci in embryonic stem cells. Cell 2006; 127: 1375-88. Kinyamu HK, Archer TK. Proteasome activity modulates chromatin modifications and RNA polymerase II phosphorylation to enhance glucocorticoid receptor-mediated transcription. Mol Cell Biol 2007; 27: 4891-904. Kinyamu HK, Collins JB, Grissom SF, Hebbar PB, Archer TK. Genome wide transcriptional profiling in breast cancer cells reveals distinct changes in hormone receptor target genes and chromatin modifying enzymes after proteasome inhibition. Mol Carcinog 2008; 47: 845-85.

Current Pharmaceutical Design, 2011, Vol. 17, No. 00 [180]

[181] [182]

[183]

[184]

[185]

[186]

[187]

[188] [189]

[190] [191] [192] [193]

[194] [195]

[196] [197]

[198] [199]

[200] [201]

[202] [203] [204]

13

Satoh T, Ishizuka T, Yoshino S, et al. Roles of proteasomal 19S regulatory particles in promoter loading of thyroid hormone receptor. Biochem Biophys Res Commun 2009; 386: 697-702. Ezhkova E, Tansey WP. Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3. Mol Cell 2004; 13: 435-42. Lee D, Ezhkova E, Li B, Pattenden SG, Tansey WP, Workman JL. The proteasome regulatory particle alters the SAGA coactivator to enhance its interactions with transcriptional activators. Cell 2005; 123: 423-36. Ferdous A, Sikder D, Gillette T, Nalley K, Kodadek T, Johnston SA. The role of the proteasomal ATPases and activator monoubiquitylation in regulating Gal4 binding to promoters. Genes Dev 2007; 21: 112-23. Fatyol K, Grummt I. Proteasomal ATPases are associated with rDNA: the ubiquitin proteasome system plays a direct role in RNA polymerase I transcription. Biochim Biophys Acta 2008; 1779: 850-9. Satoh T, Ishizuka T, Tomaru T, et al. Tat-binding protein-1 (TBP1), an ATPase of 19S regulatory particles of the 26S proteasome, enhances androgen receptor function in cooperation with TBP-1interacting protein/Hop2. Endocrinology 2009; 150: 3283-90. Bhat KP, Turner JD, Myers SE, Cape AD, Ting JP, Greer SF. The 19S proteasome ATPase Sug1 plays a critical role in regulating MHC class II transcription. Mol Immunol 2008; 45: 2214-24. Koues OI, Dudley RK, Truax AD, et al. Regulation of acetylation at the major histocompatibility complex class II proximal promoter by the 19S proteasomal ATPase Sug1. Mol Cell Biol 2008; 28: 5837-50. Shi W, Ma Z, Willers H, et al. Disassembly of MDC1 foci is controlled by ubiquitin-proteasome-dependent degradation. J Biol Chem 2008; 283: 31608-16. Singh RK, Kabbaj MH, Paik J, Gunjan A. Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis. Nat Cell Biol 2009; 11: 925-33. Schwartz AL, Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 2009; 49: 73-96. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 2006; 443: 780-6. Bennett EJ, Shaler TA, Woodman B, et al. Global changes to the ubiquitin system in Huntington’s disease. Nature 2007; 448: 704-8. Seo H, Sonntag KC, Kim W, Cattaneo E, Isacson O. Proteasome activator enhances survival of Huntington's disease neuronal model cells. PLoS One 2007; 2: e238. Pandey UB, Nie Z, Batlevi Y, et al. HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature 2007; 447: 859-63. Kristiansen M, Deriziotis P, Dimcheff DE, et al. Disease-associated prion protein oligomers inhibit the 26S proteasome. Mol Cell 2007; 26: 175-88. Herrmann J, Soares SM, Lerman LO, Lerman A. Potential role of the ubiquitin-proteasome system in atherosclerosis: aspects of a protein quality disease. J Am Coll Cardiol 2008; 51: 2003-10. Wojcik C, Di Napoli M. Ubiquitin-proteasome system and proteasome inhibition: new strategies in stroke therapy. Stroke 2004; 35: 1506-18. Lassot I, Latreille D, Rousset E, et al. The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms. Mol Cell 2007; 25: 369-83. Vernace VA, Schmidt-Glenewinkel T, Figueiredo-Pereira ME. Aging and regulated protein degradation: who has the UPPer hand? Aging Cell 2007; 6: 599-606. Naujokat C, Berges C, Hoh A, et al. Proteasomal chymotrypsinlike peptidase activity is required for essential functions of human monocyte-derived dendritic cells. Immunology 2007; 120: 120-32. Naujokat C, Saric T. Concise review: role and function of the ubiquitin-proteasome system in mammalian stem and progenitor cells. Stem Cells 2007; 25: 2408-18. Pickart CM. Back to the future with ubiquitin. Cell 2004; 116: 18190. Hochstrasser M. Lingering mysteries of ubiquitin-chain assembly. Cell 2006; 124: 27-34. Ye Y, Rape M. Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 2009; 10: 755-64.


[206] [207]

[208] [209]

[210] [211]

[212] [213]

[214] [215] [216] [217]

[218] [219]

[220] [221] [222]

[223] [224] [225]

[226]

[227] [228] [229] [230] [231] [232]

Ravid T, Hochstrasser M. Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 2008; 9: 67990. Jin J, Li X, Gygi SP, Harper JW. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 2007; 447: 1135-8. Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol 2009; 10: 319-31. Hoppe T. Multiubiquitylation by E4 enzymes: “one size” doesn’t fit all. Trends Biochem Sci 2005; 30: 183-7. Richly H, Rape M, Braun S, Rumpf S, Hoege C, Jentsch S. A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell 2005; 120: 7384. Huzil JT, Pannu R, Ptak C, Garen G, Ellison MJ. Direct catalysis of lysine 48-linked polyubiquitin chains by the ubiquitin-activating enzyme. J Biol Chem 2007; 282: 37454-60. Li W, Tu D, Brunger AT, Ye Y. A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate. Nature 2007; 446: 333-7. Rodrigo-Brenni MC, Morgan DO. Sequential E2s drive polyubiquitin chain assembly on APC targets. Cell 2007; 130: 127-39. Windheim M, Peggie M, Cohen P. Two different classes of E2 ubiquitin-conjugating enzymes are required for the monoubiquitination of proteins and elongation by polyubiquitin chains with a specific topology. Biochem J 2008; 409: 723-9. Ravid T, Hochstrasser M. Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue. Nat Rev Biol 2007; 9: 422-7. Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem 2009; 78: 399-434. Rotin D, Kumar S. Physiological functions of the HECT family of ubiquitin ligases. Nat Rev Mol Cell Biol 2009; 10: 398-409. Godinho SI, Maywood ES, Shaw L, et al. The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period. Science 2007; 316: 897-900. Busino L, Bassermann F, Maiolica A, et al. SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 2007; 316: 900-4. Siepka SM, Yoo SH, Park J, et al. Circadian mutant Overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression. Cell 2007; 129: 1011-23. O’Connell BC, Harper JW. Ubiquitin proteasome system (UPS): what can chromatin do for you? Curr Opin Cell Biol 2007; 19: 20614. Sumara I, Maerki S, Peter M. E3 ubiquitin ligases and mitosis: embracing the complexity. Trends Cell Biol 2008; 18: 84-94. Carrano AC, Liu Z, Dillin A, Hunter T. A conserved ubiquitination pathway determines longevity in response to diet restriction. Nature 2009; 460: 396-9. Hochstrasser M. Origin and function of ubiquitin-like proteins. Nature 2009; 458: 422-9. Ikeda F. Dikic I. Atypical ubiquitin chains: new molecular signals. “Protein modifications: beyond the usual suspects” review series. EMBO Rep 2008; 9: 536-42. Kim HT, Kim KP, Lledias F, et al. Certain pairs of ubiquitinconjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages. J Biol Chem 2007; 282: 17375-86. Kim HT, Kim KP, Uchiki T, Gygi SP, Goldberg AL. S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains. EMBO J 2009; 28: 1867-77. Mukhopadhyay D, Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 2007; 315: 2015. Chen ZJ, Sun LJ. Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 2009; 33: 275-86. Weake VM, Workman JL. Histone ubiquitination: triggering gene activity. Mol Cell 2008; 29: 653-63. Skaug B, Jiang X, Chen ZJ. The role of ubiquitin in NF-B regulatory pathways. Annu Rev Biochem 2009; 78: 769-96. Bhoj VG, Chen ZJ. Ubiquitylation in innate and adaptive immunity. Nature 2009; 458: 430-7. Chiu YH, Zhao M, Chen ZJ. Ubiquitin in NF-B signaling. Chem Rev 2009; 109: 1549-60.

Cvek and Dvorak [233] [234]

[235] [236]

[237] [238]

[239] [240]

[241] [242]

[243] [244]

[245] [246]

[247] [248]

[249]

[250] [251]

[252]

[253] [254]

[255]

[256]

Bergink S, Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 2009; 458: 461-7. van Attikum H, Gasser SM. Crosstalk between histone modifications during the DNA damage response. Trends Cell Biol 2009; 19: 207-17. Fujii K, Kitabatake M, Sakata T, Miyata A, Ohno M. A role for ubiquitin in the clearance of nonfunctional rRNAs. Genes Dev 2009; 23: 963-74. Reddy SK, Rape M, Margansky WA, Kirschner MW. Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 2007; 446: 921-5. Stegmeier F, Rape M, Draviam VM, et al. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 2007; 446: 876-81. Wickliffe K, Williamson A, Jin L, Rape M. The multiple layers of ubiquitin-dependent cell cycle control. Chem Rev 2009; 109: 153748. Love KR, Catic A, Schlieker C, Ploegh HL. Mechanisms, biology and inhibitors of deubiquitinating enzymes. Nat Chem Biol 2007; 3: 697-705. Reyes-Turcu FE, Wilkinson KD. Polyubiquitin binding and disassembly by deubiquitinating enzymes. Chem Rev 2009; 109: 14951508. Reyes-Turcu FE, Ventii KH, Wilkinson KD. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 2009; 78: 363-97. Komander D, Clague MJ, Urbe S. Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 2009; 10: 550-63. Sowa ME, Bennett EJ, Gygi SP, Harper JW. Defining the human deubiquitinating enzyme interaction landscape. Cell 2009; 138: 389-403. Gallery M, Blank JL, Lin Y, et al. The JAMM motif of human deubiquitinase Poh1 is essential for cell viability. Mol Cancer Ther 2007; 6: 262-8. Cope GA, Suh GS, Aravind L, et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 2002; 298: 608-11. Bornstein G, Ganoth D, Hershko A. Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate. Proc Natl Acad Sci USA 2006; 103: 11515-20. Sato Y, Yoshikawa A, Yamagata A, et al. Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains. Nature 2008; 455: 358-362. Cooper EM, Cutcliffe C, Kristiansen TZ, Pandey A, Pickart CM, Cohen RE. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1. EMBO J 2009; 28: 621-31. Voorhees PM, Orlowski RZ. The proteasome and proteasome inhibitors in cancer therapy. Annu Rev Pharmacol Toxicol 2006; 46: 189-213. Choi MR, Najafi F, Safa AR, Drexler HC. Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI. Biochem Pharmacol 2008; 75: 2276-88. Weinkauf M, Zimmermann Y, Hartmann E, et al. 2-D PAGE-based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma. Electrophoresis 2009; 30: 974-86. Shin DH, Chun YS, Lee DS, Huang LE, Park JW. Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIHmediated repression of hypoxia-inducible factor-1. Blood 2008; 111: 3131-6. Codony-Servat J, Tapia MA, Bosch M, et al. Differential cellular and molecular effects of bortezomib, a proteasome inhibitor, in human breast cancer cells. Mol Cancer Ther 2006; 5: 665-75. Voortman J, Checinska A, Giaccone G. The proteasomal and apoptotic phenotype determine bortezomib sensitivity of non-small cell lung cancer cells. Mol Cancer 2007; 6: 73. Du ZX, Zhang HY, Meng X, Guan Y, Wang HQ. Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells. BMC Cancer 2009; 9: 56. Chen KF, Yeh PY, Yeh KH, Lu YS, Huang SY, Cheng AL. Downregulation of phospho-Akt is a major molecular determinant of bortezomib-induced apoptosis in hepatocellular carcinoma cells. Cancer Res 2008; 68: 6698-707.

The Ubiquitin-Proteasome System (UPS) [257]

[258] [259]

[260] [261] [262] [263] [264]

[265]

[266]

[267] [268] [269]

[270] [271]

[272] [273]

[274]

[275] [276]

[277] [278] [279] [280]

[281]

Ayala G, Yan J, Li R, et al. Bortezomib-mediated inhibition of steroid receptor coactivator-3 degradation leads to activated Akt. Clin Cancer Res 2008; 14: 7511-8. Bianchi G, Oliva L, Cascio P, et al. The proteasome load versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to proteasome inhibition. Blood 2009; 113: 3040-9. Kloss A, Meiners S, Ludwig A, Dahlmann B. Multiple cardiac proteasome subtypes differ in their susceptibility to proteasome inhibitors. Cardiovasc Res 2010; 85: 367-75. Meiners S, Dreger H, Fechner M, et al. Suppression of cardiomyocyte hypertrophy by inhibition of the ubiquitin-proteasome system. Hypertension 2008; 51: 302-8. Adams J. The proteasome: a suitable antineoplastic target. Nat Rev Cancer 2004; 4: 349-60. McConkey DJ, Zhu K. Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist Updat 2008; 11: 164-79. Nakanishi C, Toi M. Nuclear factor-B inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 2005; 5: 297-309. Strauss SJ, Higginbottom K, Juliger S, et al. The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines. Cancer Res 2007; 67: 2783-90. Hideshima T, Bradner JE, Wong J, et al. Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proc Natl Acad Sci USA 2005; 102: 8567-72. Nawrocki ST, Carew JS, Maclean KH, et al. Myc regulates aggresome formation, the induction of Noxa, and apoptosis in response to the combination of bortezomib and SAHA. Blood 2008; 112: 2917-26. Hayden MS, Ghosh S. Shared principles in NF-B signaling. Cell 2008; 132: 344-62. Karin M. Nuclear factor-B in cancer development and progression. Nature 2006; 441: 431-6. Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP. Stabilization of snail by NF-B is required for inflammation-induced cell migration and invasion. Cancer Cell 2009; 15: 416-28. Sartore-Bianchi A, Gasparri F, Galvani A, et al. Bortezomib inhibits nuclear factor-B dependent survival and has potent in vivo activity in mesothelioma. Clin Cancer Res 2007; 13: 5942-51. Galimberti S, Canestraro M, Pacini S, et al. PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells. Leuk Res 2008; 32: 103-12. Bersani F, Taulli R, Accornero P, et al. Bortezomib-mediated proteasome inhibition as a potential strategy for the treatment of rhabdomyosarcoma. Eur J Cancer 2008; 44: 876-84. Allen C, Saigal K, Nottingham L, Arun P, Chen Z, Van Waes C. Bortezomib-induced apoptosis with limited clinical response is accompanied by inhibition of canonical but not alternative nuclear factor-B subunits in head and neck cancer. Clin Cancer Res 2008; 14: 4175-85. Vu HY, Juvekar A, Ghosh C, Ramaswami S, Le DH, Vancurova I. Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of IB. Arch Biochem Biophys 2008; 475: 156-63. Ashall L, Horton CA, Nelson DE, et al. Pulsatile stimulation determines timing and specificity of NF-B-dependent transcription. Science 2009; 324: 242-6. Sung MH, Bagain L, Chen Z, et al. Dynamic effect of bortezomib on nuclear factor-B activity and gene expression in tumor cells. Mol Pharmacol 2008; 74: 1215-22. Perkins ND. Integrating cell-signalling pathways with NF-B and IKK function. Nat Rev Mol Cell Biol 2007; 8: 49-62. Chen F, Castranova V. Nuclear factor-B, an unappreciated tumor suppressor. Cancer Res 2007; 67: 11093-8. Luedde T, Beraza N, Kotsikoris V, et al. Deletion of NEMO/IKK in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 2007; 11: 119-32. Markovina S, Callander NS, O’Connor SL, et al. Bortezomibresistant nuclear factor-B activity in multiple myeloma cells. Mol Cancer Res 2008; 6: 1356-64. Yang DT, Young KH, Kahl BS, Markovina S, Miyamoto S. Prevalence of bortezomib-resistant constitutive NF-B activity in mantle cell lymphoma. Mol Cancer 2008; 7: 40.

Current Pharmaceutical Design, 2011, Vol. 17, No. 00 [282]

[283]

[284] [285]

[286] [287]

[288]

[289]

[290]

[291]

[292] [293]

[294]

[295] [296]

[297] [298]

[299]

[300]

[301]

[302] [303]

15

Cvek B, Dvorak Z. The value of proteasome inhibition in cancer. Can the old drug, disulfiram, have a bright new future as a novel proteasome inhibitor? Drug Discov Today 2008; 13: 716-22. Nemeth ZH, Wong HR, Odoms K, et al. Proteasome inhibitors induce inhibitory B (IB) kinase activation, IB degradation, and nuclear factor B activation in HT-29 cells. Mol Pharmacol 2004; 65: 342-9. Calvaruso G, Giuliano M, Portanova P, De Blasio A, Vento R, Tesoriere G. Bortezomib induces in HepG2 cells IB degradation mediated by caspase-8. Mol Cell Biochem 2006; 287: 13-9. Hideshima T, Ikeda H, Chauhan D, et al. Bortezomib induces canonical nuclear factor-B activation in multiple myeloma cells. Blood 2009; 114: 1046-52. Mitsiades N, Mitsiades CS, Poulaki V, et al. Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci USA 2002; 99: 14374-9. Hideshima T, Mitsiades C, Akiyama M, et al. Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS341. Blood 2003; 101: 1530-4. Mitsiades CS, McMillin D, Kotoula V, et al. Antitumor effects of the proteasome inhibitor bortezomib in medullary and anaplastic thyroid carcinoma cells in vitro. J Clin Endocrinol Metab 2006; 91: 4013-21. Chen Z, Ricker JL, Malhotra PS, et al. Differential bortezomib sensitivity in head and neck cancer lines corresponds to proteasome, nuclear factor-B and activator protein-1 related mechanisms. Mol Cancer Ther 2008; 7: 1949-60. Sloss CM, Wang F, Liu R, et al. Proteasome inhibition activates epidermal growth factor receptor (EGFR) and EGFR-independent mitogenic kinase signaling pathways in pancreatic cancer cells. Clin Cancer Res 2008; 14: 5116-23. Calvaruso G, Giuliano M, Portanova P, Pellerito O, Vento R, Tesoriere G. Hsp72 controls bortezomib-induced HepG2 cell death via interaction with pro-apoptotic factors. Oncol Rep 2007; 18: 447-50. Qin JZ, Ziffra J, Stennett L, et al. Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells. Cancer Res 2005; 65: 6282-93. Fernandez Y, Verhaegen M, Miller TP, et al. Differential regulation of noxa in normal melanocytes and melanoma cells by proteasome inhibition: therapeutic implications. Cancer Res 2005; 65: 6294-304. Wolter KG, Verhaegen M, Fernandez Y, et al. Therapeutic window for melanoma treatment provided by selective effects of the proteasome on Bcl-2 proteins. Cell Death Differ 2007; 14: 1605-16. Nikiforov MA, Riblett M, Tang WH, et al. Tumor cell-selective regulation of NOXA by c-MYC in response to proteasome inhibition. Proc Natl Acad Sci USA 2007; 104: 19488-93. Gomez-Bougie P, Wuilleme-Toumi S, Menoret E, et al. Noxa upregulation and Mcl-1 cleavage are associated to apoptosis induction by bortezomib in multiple myeloma. Cancer Res 2007; 67: 541824. Podar K, Gouill SL, Zhang J, et al. A pivotal role for Mcl-1 in Bortezomib-induced apoptosis. Oncogene 2008; 27: 721-31. Perez-Galan P, Roue G, Villamor N, Montserrat E, Campo E, Colomer D. The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood 2006; 107: 257-64. Voortman J, Checinska A, Giaccone G, Rodriguez JA, Kruyt FA. Bortezomib, but not cisplatin, induces mitochondria-dependent apoptosis accompanied by up-regulation of noxa in the non-small cell lung cancer cell line NCI-H460. Mol Cancer Ther 2007; 6: 1046-53. Yuan BZ, Chapman J, Reynolds SH. Proteasome inhibitors induce apoptosis in human lung cancer cells through a positive feedback mechanism and the subsequent Mcl-1 protein cleavage. Oncogene 2009; 28: 3775-86. Lioni M, Noma K, Snyder A, et al. Bortezomib induces apoptosis in esophageal squamous cell carcinoma cells through activation of the p38 mitogen-activated protein kinase pathway. Mol Cancer Ther 2008; 7: 2866-75. Baou M, Kohlhaas SL, Butterworth M, et al. Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells. Haematologica 2010; 95: 1510-8. Ri M, Iida S, Ishida T, et al. Bortezomib-induced apoptosis in mature T-cell lymphoma cells partially depends on upregulation of


[304]

[305]

[306]

[307] [308]

[309]

[310] [311] [312]

[313]

[314] [315]

[316]

[317]

[318]

[319]

[320]

[321]

[322] [323]

[324]

Noxa and functional repression of Mcl-1. Cancer Sci 2009; 100: 341-8. Jullig M, Zhang WV, Ferreira A, Stott NS. MG132 induced apoptosis is associated with p53-independent induction of pro-apoptotic Noxa and transcriptional activity of -catenin. Apoptosis 2006; 11: 627-41. Landowski TH, Megli CJ, Nullmeyer KD, Lynch RM, Dorr RT. Mitochondrial-mediated disregulation of Ca2+ is a critical determinant of Velcade (PS-341/bortezomib) cytotoxicity in myeloma cell lines. Cancer Res 2005; 65: 3828-36. Wu S, Hyrc KL, Moulder KL, Lin Y, Warmke T, Snider BJ. Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors. J Neurochem 2009; 109: 1225-36. Zhu H, Zhang L, Dong F, et al. Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS-341, Velcade) and other proteasome inhibitors. Oncogene 2005; 24: 4993-9. Li C, Li R, Grandis JR, Johnson DE. Bortezomib induces apoptosis via Bim and Bik up-regulation and synergizes with cisplatin in the killing of head and neck squamous cell carcinoma cells. Mol Cancer Ther 2008; 7: 1647-55. Fennell DA, Chacko A, Mutti L. BCL-2 family regulation by the 20S proteasome inhibitor bortezomib. Oncogene 2008; 27: 118997. Green DR, Kroemer G. Cytoplasmic functions of the tumour suppressor p53. Nature 2009; 458: 1127-30. Concannon CG, Koehler BF, Reimertz C, et al. Apoptosis induced by proteasome inhibition in cancer cells: predominant role of the p53/PUMA pathway. Oncogene 2007; 26: 1681-92. Ding WX, Ni HM, Chen X, Yu J, Zhang L, Yin XM. A coordinated action of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and apoptosis in colon cancer cells. Mol Cancer Ther 2007; 6: 1062-9. Yu D, Carroll M, Thomas-Tikhonenko A. p53 status dictates responses of B lymphomas to monotherapy with proteasome inhibitors. Blood 2007;109:4936-4943. Williams SA, McConkey DJ. The proteasome inhibitor bortezomib stabilizes a novel active form of p53 in human LNCaP-Pro5 prostate cancer cells. Cancer Res 2003; 63: 7338-44. Yang Y, Ikezoe T, Saito T, Kobayashi M, Koeffler HP, Taguchi H. Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling. Cancer Sci 2004; 95: 176-80. Lauricella M, Emanuele S, D’Anneo A, et al. JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways. Apoptosis 2006; 11: 607-25. Baiz D, Pozzato G, Dapas B, et al. Bortezomib arrests the proliferation of hepatocellular carcinoma cells HepG2 and JHH6 by differentially affecting E2F1, p21 and p27 levels. Biochimie 2009; 91: 373-82. Yu J, Tiwari S, Steiner P, Zhang L. Differential apoptotic response to the proteasome inhibitor Bortezomib [VELCADE, PS-341] in Bax-deficient and p21-deficient colon cancer cells. Cancer Biol Ther 2003; 2: 694-9. Gu H, Chen X, Gao G, Dong H. Caspase-2 functions upstream of mitochondria in endoplasmic reticulum stress-induced apoptosis by bortezomib in human myeloma cells. Mol Cancer Ther 2008; 7: 2298-307. Obeng EA, Carlson LM, Gutman DM, Harrington WJ, Lee KP, Boise LH. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood 2006; 107: 490716. Meister S, Schubert U, Neubert K, et al. Extensive immunoglobulin production sensitizes myeloma cells for proteasome inhibition. Cancer Res 2007; 67: 1783-92. Dong H, Chen L, Chen X, et al. Dysregulation of unfolded protein response partially underlies proapoptotic activity of bortezomib in multiple myeloma cells. Leuk Lymphoma 2009; 50: 974-84. Nawrocki ST, Carew JS, Dunner K, et al. Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells. Cancer Res 2005; 65: 11510-9. Fels DR, Ye J, Segan AT, et al. Preferential cytotoxicity of bortezomib toward hypoxic tumor cells via overactivation of endoplasmic reticulum stress pathways. Cancer Res 2008; 68: 9323-30.

Cvek and Dvorak [325]

[326]

[327] [328]

[329] [330]

[331] [332]

[333] [334]

[335]

[336] [337] [338] [339]

[340]

[341]

[342]

[343] [344]

[345]

[346]

[347]

Fribley AM, Evenchik B, Zeng Q, et al. Proteasome inhibitor PS341 induces apoptosis in cisplatin-resistant squamous cell carcinoma cells by induction of Noxa. J Biol Chem 2006; 281: 31440-7. Wang Q, Mora-Jensen H, Weniger MA, et al. ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3only protein NOXA in cancer cells. Proc Natl Acad Sci USA; 106: 2200-5. Chen S, Blank JL, Peters T, et al. Genome-wide siRNA screen for modulators of cell death induced by proteasome inhibitor bortezomib. Cancer Res 2010; 70: 4318-26. Murakawa Y, Sonoda E, Barber LJ, et al. Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells. Cancer Res 2007; 67: 8536-43. Jacquemont C, Taniguchi T. Proteasome function is required for DNA damage response and fanconi anemia pathway activation. Cancer Res 2007; 67: 7395-405. Takezawa J, Ishimi Y, Yamada K. Proteasome inhibitors remarkably prevent translesion replication in cancer cells but not normal cells. Cancer Sci 2008; 99: 863-71. Roos WP, Kaina B. DNA damage-induced cell death by apoptosis. Trends Mol Med 2006; 12: 440-50. Congdon LM, Pourpak A, Escalante AM, Dorr RT, Landowski TH. Proteasomal inhibition stabilizes topoisomerase II protein and reverses resistance to the topoisomerase II poison ethonafide (AMP53, 6-ethoxyazonafide). Biochem Pharmacol 2008; 75: 883-90. Birle DC, Hedley DW. Suppression of the hypoxia-inducible factor-1 response in cervical carcinoma xenografts by proteasome inhibitors. Cancer Res 2007; 67: 1735-43. Shin DH, Chun YS, Lee DS, Huang LE, Park JW. Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIHmediated repression of hypoxia-inducible factor-1. Blood 2008; 111: 3131-6. Shin DH, Li SH, Chun YS, Huang LE, Kim MS, Park JW. CITED2 mediates the paradoxical responses of HIF-1alpha to proteasome inhibition. Oncogene 2008; 27: 1939-44. Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNFfamily death receptors. Oncogene 2003; 22: 8628-33. Kim SH, Ricci MS, El-Deiry WS. Mcl-1: a gateway to TRAIL sensitization. Cancer Res 2008; 68: 2062-4. Sayers TJ, Brooks AD, Koh CY, et al. The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP. Blood 2003; 102: 303-10. Lashinger LM, Zhu K, Williams SA, Shrader M, Dinney CP, McConkey DJ. Bortezomib abolishes tumor necrosis factor-related apoptosis-inducing ligand resistance via a p21-dependent mechanism in human bladder and prostate cancer cells. Cancer Res 2005; 65: 4902-8. Voortman J, Resende TP, Abou El Hassan MA, Giaccone G, Kruyt FA. TRAIL therapy in non-small cell lung cancer cells: sensitization to death receptor-mediated apoptosis by proteasome inhibitor bortezomib. Mol Cancer Ther 2007; 6: 2103-12. Lu G, Punj V, Chaudhary PM. Proteasome inhibitor Bortezomib induces cell cycle arrest and apoptosis in cell lines derived from Ewing’s sarcoma family of tumors and synergizes with TRAIL. Cancer Biol Ther 2008; 7: 603-8. Koschny R, Holland H, Sykora J, et al. Bortezomib sensitizes primary human astrocytoma cells of WHO grades I to IV for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Clin Cancer Res 2007; 13: 3403-12. Ganten TM, Koschny R, Haas TL, et al. Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL. Hepatology 2005; 42: 588-97. Koschny R, Ganten TM, Sykora J, et al. TRAIL/bortezomib cotreatment is potentially hepatotoxic but induces cancer-specific apoptosis within a therapeutic window. Hepatology 2007; 45: 64958. Balsas P, Lopez-Royuela N, Galan-Malo P, Anel A, Marzo I, Naval J. Cooperation between Apo2L/TRAIL and bortezomib in multiple myeloma apoptosis. Biochem Pharmacol 2009; 77: 80412. Kabore AF, Sun J, Hu X, McCrea K, Johnston JB, Gibson SB. The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells. Apoptosis 2006; 11: 1175-93. Liu X, Yue P, Chen S, et al. The proteasome inhibitor PS-341 (bortezomib) up-regulates DR5 expression leading to induction of


[348] [349]

[350]

[351] [352]

[353] [354]

[355]

[356]

[357] [358]

[359]

[360] [361]

[362]

[363]

[364]

[365] [366]


apoptosis and enhancement of TRAIL-induced apoptosis despite up-regulation of c-FLIP and survivin expression in human NSCLC cells. Cancer Res 2007; 67: 4981-8. Bruning A, Burger P, Vogel M, et al. Bortezomib treatment of ovarian cancer cells mediates endoplasmic reticulum stress, cell cycle arrest, and apoptosis. Invest New Drugs 2009; 27: 543-51. Shanker A, Brooks AD, Tristan CA, et al. Treating metastatic solid tumors with bortezomib and a tumor necrosis factor-related apoptosis-inducing ligand receptor agonist antibody. J Natl Cancer Inst 2008; 100: 649-62. Zhu H, Guo W, Zhang L, Wu S, Teraishi F, Davis JJ, Dong F, Fang B. Proteasome inhibitors-mediated TRAIL resensitization and Bik accumulation. Cancer Biol Ther 2005; 4: 781-6. Li W, Zhang X, Olumi AF. MG-132 sensitizes TRAIL-resistant prostate cancer cells by activating c-Fos/c-Jun heterodimers and repressing c-FLIP(L). Cancer Res 2007; 67: 2247-55. Chen KF, Yeh PY, Hsu C, et al. Bortezomib overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells in part through the inhibition of the phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem 2009; 284: 11121-33. Hallett WH, Ames E, Motarjemi M, et al.. Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition. J Immunol 2008; 180: 163-70. Schumacher LY, Vo DD, Garban HJ, et al. Immunosensitization of tumor cells to dendritic cell-activated immune responses with the proteasome inhibitor bortezomib (PS-341, Velcade). J Immunol 2006; 176: 4757-65. Ames E, Hallett WH, Murphy WJ. Sensitization of human breast cancer cells to natural killer cell-mediated cytotoxicity by proteasome inhibition. Clin Exp Immunol 2009; 155: 504-13. Lundqvist A, Yokoyama H, Smith A, Berg M, Childs R. Bortezomib treatment and regulatory T-cell depletion enhance the antitumor effects of adoptively infused NK cells. Blood 2009; 113: 6120-7. Nencioni A, Schwarzenberg K, Brauer KM, et al. Proteasome inhibitor bortezomib modulates TLR4-induced dendritic cell activation. Blood 2006; 108: 551-8. Subklewe M, Sebelin-Wulf K, Beier C, et al. Dendritic cell maturation stage determines susceptibility to the proteasome inhibitor bortezomib. Hum Immunol 2007; 68: 147-55. Arpinati M, Chirumbolo G, Nicolini B, Agostinelli C, Rondelli D. Selective apoptosis of monocytes and monocyte-derived DCs induced by bortezomib (Velcade). Bone Marrow Transplant 2009; 43: 253-9. Roccaro AM, Hideshima T, Raje N, et al. Bortezomib mediates antiangiogenesis in multiple myeloma via direct and indirect effects on endothelial cells. Cancer Res 2006; 66: 184-91. Lorch JH, Thomas TO, Schmoll HJ. Bortezomib inhibits cell-cell adhesion and cell migration and enhances epidermal growth factor receptor inhibitor-induced cell death in squamous cell cancer. Cancer Res 2007; 67: 727-34. Noborio-Hatano K, Kikuchi J, Takatoku M, et al. Bortezomib overcomes cell-adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma. Oncogene 2009; 28: 231-42. Edwards CM, Lwin ST, Fowler JA, et al. Myeloma cells exhibit an increase in proteasome activity and an enhanced response to proteasome inhibition in the bone marrow microenvironment in vivo. Am J Hematol 2009; 84: 268-72. Giuliani N, Rizzoli V, Roodman GD. Multiple myeloma bone disease: pathophysiology of osteoblast inhibition. Blood 2006; 108: 3992-6. Giuliani N, Morandi F, Tagliaferri S, et al. The proteasome inhibitor bortezomib affects osteoblast differentiation in vitro and in vivo in multiple myeloma patients. Blood 2007; 110: 334-8. Mukherjee S, Raje N, Schoonmaker JA, et al. Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice. J Clin Invest 2008; 118: 491504.

Received: February 1, 2011

Accepted: April 19, 2011

[367]

[368]

[369] [370]

[371]

[372] [373]

[374]

[375] [376]

[377]

[378]

[379]

[380]

[381] [382]

[383]

[384]

[385]

17

Pennisi A, Li X, Ling W, Khan S, Zangari M, Yaccoby S. The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo. Am J Hematol 2009; 84: 6-14. Terpos E, Sezer O, Croucher P, Dimopoulos MA. Myeloma bone disease and proteasome inhibition therapies. Blood 2007; 110: 1098-104. Mundy G, Gutierrez G, Garrett R, et al. Proteasome inhibitors stimulate both bone formation and hair growth by similar mechanisms. Ann NY Acad Sci 2007; 1117: 298-301. Shah MH, Young D, Kindler HL, et al. Phase II study of the proteasome inhibitor bortezomib (PS-341) in patients with metastatic neuroendocrine tumors. Clin Cancer Res 2004; 10: 6111-8. Mackay H, Hedley D, Major P, et al. A phase II trial with pharmacodynamic endpoints of the proteasome inhibitor bortezomib in patients with metastatic colorectal cancer. Clin Cancer Res 2005; 11: 5526-33. Maki RG, Kraft AS, Scheu K, et al. A multicenter Phase II study of bortezomib in recurrent or metastatic sarcomas. Cancer 2005; 103: 1431-8. Markovic SN, Geyer SM, Dawkins F, et al. A phase II study of bortezomib in the treatment of metastatic malignant melanoma. Cancer 2005; 103: 2584-9. Yang CH, Gonzalez-Angulo AM, Reuben JM, et al. Bortezomib (VELCADE) in metastatic breast cancer: pharmacodynamics, biological effects, and prediction of clinical benefits. Ann Oncol 2006; 17: 813-7. Engel RH, Brown JA, von Roenn JH, et al. A phase II study of single agent bortezomib in patients with metastatic breast cancer: a single institution experience. Cancer Invest 2007; 25: 733-7. Kozuch PS, Rocha-Lima CM, Dragovich T, et al. Bortezomib with or without irinotecan in relapsed or refractory colorectal cancer: results from a randomized phase II study. J Clin Oncol 2008; 26: 2320-6. Aghajanian C, Blessing JA, Darcy KM, et al. A phase II evaluation of bortezomib in the treatment of recurrent platinum-sensitive ovarian or primary peritoneal cancer: a Gynecologic Oncology Group study. Gynecol Oncol 2009; 115: 215-20. Shah MA, Power DG, Kindler HL, et al. A multicenter, phase II study of Bortezomib (PS-341) in patients with unresectable or metastatic gastric and gastroesophageal junction adenocarcinoma. Invest New Drugs 2010; in press. Li T, Ho L, Piperdi B, et al. Phase II study of the proteasome inhibitor bortezomib (PS-341, Velcade) in chemotherapy-naïve patients with advanced stage non-small cell lung cancer (NSCLC). Lung Cancer 2010; 68: 89-93. Zinzani PL, Musuraca G, Tani M, et al. Phase II trial of proteasome inhibitor bortezomib in patients with relapsed or refractory cutaneous T-cell lymphoma. J Clin Oncol 2007; 25: 4293-7. Troch M, Jonak C, Mullauer L, et al. A phase II study of bortezomib in patients with MALT lymphoma. Haematologica 2009; 94: 738-42. Dimopoulos MA, Anagnostopoulos A, Kyrtsonis MC, Castritis E, Bitsaktsis A, Pangalis GA. Treatment of relapsed or refractory Waldenstrom’s macroglobulinemia with bortezomib. Haematologica 2005; 90: 1655-8. Chen CI, Kouroukis CT, White D, et al. National Cancer Institute of Canada Clinical Trials Group, Bortezomib is active in patients with untreated or relapsed Waldenstrom’s macroglobulinemia: a phase II study of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2007; 25: 1570-5. Treon SP, Hunter ZR, Matous J, et al. Multicenter clinical trial of bortezomib in relapsed/refractory Waldenstrom’s macroglobulinemia: results of WMCTG Trial 03-248. Clin Cancer Res 2007; 13: 3320-5. Dick LR, Fleming PE. Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy. Drug Discov Today 2010; 15: 243-9.