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Journal of Clinical Oncology, Vol 23, No 21 (July 20), 2005: pp. 4776-4789 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.05.081
The Ubiquitin-Proteasome Pathway and Its Role in CancerFrom the Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC Address reprint requests to E. Gelmann, MD, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3800 Reservoir Rd NW, Washington, DC 20007-2197; e-mail: Gelmanne{at}georgetown.edu
Critical cellular processes are regulated, in part, by maintaining the appropriate intracellular levels of proteins. Whereas de novo protein synthesis is a comparatively slow process, proteins are rapidly degraded at a rate compatible with the control of cell cycle transitions and cell death induction. A major pathway for protein degradation is initiated by the addition of multiple 76–amino acid ubiquitin monomers via a three-step process of ubiquitin activation and substrate recognition. Polyubiquitination targets proteins for recognition and processing by the 26S proteasome, a cylindrical organelle that recognizes ubiquitinated proteins, degrades the proteins, and recycles ubiquitin. The critical roles played by ubiquitin-mediated protein turnover in cell cycle regulation makes this process a target for oncogenic mutations. Oncogenes of several common malignancies, for example colon and renal cell cancer, code for ubiquitin ligase components. Cervical oncogenesis by human papillomavirus is also mediated by alteration of ubiquitin ligase pathways. Protein degradation pathways are also targets for cancer therapy, as shown by the successful introduction of bortezomib, an inhibitor of the 26S proteasome. Further work in this area holds great promise toward our understanding and treatment of a wide range of cancers.
Ubiquitin is an abundant and essential cellular 9-kd protein that is conserved across evolution from yeast to man. Ubiquitin is used by cells as a covalent modifier of other proteins both to activate their function and to target them for degradation, depending on the degree of ubiquitin ligation.1 Addition of long polyubiquitin chains targets proteins for degradation by the 26S proteasome. Because ubiquitin has a broad range of biochemical interactions and effects, the control of ubiquitination is complex and is regulated by a wide array of ubiquitin ligase enzymatic complexes whose assembly is triggered by diverse signals in the cell. The relevance of ubiquitination to cancer arises from oncogenic mutations that disrupt ubiquitination of proteins that control cell growth and death. In addition, recent development and approval of bortezomib, a drug that inhibits the 26S proteasomal degradation machinery, has underscored the importance of this pathway to cancer treatment strategies.
The role of the ubiquitin molecule in cell physiology has been elucidated in the last 25 years but had its roots in experiments performed in the 1930s, which showed that proteins had a finite life span and were continually synthesized, degraded, and resynthesized in a constant state of flux.2 Subsequent studies showed that degradation assumed equal weight as synthesis in determining the intracellular content of proteins.3-5 It was found that proteins had different half-lives that were largely determined by the rate of degradation.6,7 Although lysosomal enzymes were implicated in protein degradation,8-10 the notion was abandoned when it was discovered that lysosomal inhibitors like leupeptin, antipain, and chymostatin were inactive as inhibitors of basal protein breakdown and that protein turnover occurred in cells that lacked lysosomes.11,12 It was first shown more than half a century ago that enzyme degradation is adenosine triphosphate (ATP) –dependent, implying that an active, energy-requiring process is responsible for protein turnover.13,14 Exploiting the rabbit reticulocyte that synthesized and degraded essentially only hemoglobin,15 Ciechanover et al16 identified a 9-kd peptide component of the protein degradation machinery that was essential for degradation but lacked any proteolytic activity on its own. It was subsequently shown that this 9-kd factor was ligated to larger proteins and targeted them for ATP-dependent degradation.17,18 Because of its widespread distribution in tissues and across species, the protein was renamed ubiquitin.1,19,20 The discovery of ubiquitin and the understanding of its role in protein degradation was recognized by the 2004 Nobel Prize for Chemistry awarded to Avram Hershko, Aaron Ciechanover, and Irwin Rose. Polyubiquitination was shown to target proteins to a cellular organelle known as the 26S proteasome.21 Proteasomal degradation, which is regulated by ubiquitination, is a critical component of numerous cellular processes including cell cycle regulation,22 induction of the inflammatory response,23 and antigen presentation.24 Therefore, it is not surprising that aberrations of ubiquitination have been linked to a wide range of pathologic states including cancer.
The ubiquitin-proteasome protein degradation pathway is comprised of ubiquitin, a three-enzyme ubiquitination complex, the intracellular protein ubiquitination targets, and the proteasome that is the organelle of protein degradation. The ubiquitination machinery is present both in the cytosol and the nucleus. We will review the ubiquitination and protein degradation process. The reader is referred to other reviews for more extensive descriptions of the biochemistry and comprehensive citation lists.25-29 The ubiquitin-activating enzyme, E1, initiates ubiquitin ligation by adenylating ubiquitin (Fig 1). 30 One ATP molecule is expended for each E1-ubiquitin linkage. The ubiquitin molecule is transferred to the ubiquitin-conjugating enzyme E2, which transiently carries ubiquitin. E2 works in conjunction with the ubiquitin ligase E3, which is responsible for conferring substrate specificity on the reaction. E3 mediates the transfer of ubiquitin to an internal lysine of the target protein. The first conjugated ubiquitin serves as the nidus for the formation of a polyubiquitin chain, which constitutes the protein degradation signal. The polyubiquitinated target protein undergoes proteolytic cleavage by the proteasome, which is a large, cylindrical multisubunit complex. Byproducts of proteolysis are cleaved fragment peptides of the target protein and intact, recyclable ubiquitin molecules. The hierarchical organization26 of the ubiquitin-proteasome enzymatic conjugation cascade places a single E1 enzyme at the top of the cascade that activates ubiquitin for all subsequent downstream reactions and interacts with all E2s.31 At the next stage, there are many E2 ubiquitin-conjugating enzymes that have defined but broad specificity. The last phase of ubiquitination is mediated by the E3 family of ubiquitin ligases, which is largely responsible for target protein specificity. Each E2 interacts with several E3s, and each E3 targets several substrates based on shared recognition motifs. Each E3 can interact with more than one E2, and some substrates can be targeted by more than one E3.
Eukaryotic ubiquitins share an identical sequence.32 Although absent in most prokaryotes including Escherichia coli, ubiquitin has been identified in a eubacterium, the cyanobacterium Anabaena variabilis (blue-green algae).33 This stringent evolutionary conservation of ubiquitin underscores the fundamental importance of the ubiquitin-proteasome pathway in basic cellular physiology. Ubiquitin is a 76–amino acid protein folded into a tightly packed globular conformation and is found either as a free monomer in the cytosol or covalently linked to itself and other proteins. Thermal stability of this small protein is conferred by a pronounced hydrophobic core and extensive hydrogen bonding.34 The amino acid glycine 76 (G76), at the extreme C-terminal end of the peptide, protrudes from the protein core to serve as the site for covalent amide conjugation.34 All known linkages of ubiquitin to other proteins involve peptide bonds with this C-terminal G76.35 Addition of the first ubiquitin monomer to a protein occurs between the  -carboxyl group of G76 and the  -amino group in the side chain of a target protein lysine residue. Ubiquitination is a steady-state process that can be reversed by deubiquitinating enzymes. Deubiquitination can occur at any time during the addition of ubiquitin moieties to a protein, underscoring the complexity of the balance between protein survival and degradation. Thus, subsequent to the addition of a single ubiquitin monomer, a protein can be immediately deubiquitinated, undergo further rounds of ubiquitination, or remain monoubiquitinated. Depending on the nature of the ubiquitin modification, the target protein may be destined for degradation or alternative nonproteolytic fates, highlighting the functional diversity of the ubiquitin signal as a key post-translational modification similar to phosphorylation. Ubiquitin itself has seven lysine residues, and ubiquitin polymers are formed when the C-terminal G76 of each ubiquitin unit is linked to a specific lysine residue of the previous ubiquitin. G76-K48–linked chains are the principal targeting signal for proteasomal degradation.36 A chain of at least four ubiquitin adducts is the minimal length that will target a protein for efficient proteasomal degradation.37 Alternative chain elongation schemes may also determine alternative metabolic fates.38 For example, K63-G76–linked chains are implicated in nonproteolytic signaling such as DNA repair.39 Monoubiquitination is a unique signal that regulates protein activities ranging from membrane transport to transcriptional regulation.40
A single enzyme, E1, initiates ubiquitination by activating the ubiquitin peptide monomer.31,41 Two isoforms of human E1 enzyme arise by translation from alternate protein start sites on the same mRNA42 and have been found in both the cytoplasm and the nucleus.43,44 The essential nature of the protein is indicated by the finding that inactivation of the yeast E1 gene, UBA1, is lethal.45 Ubiquitin activation begins with the formation of a ubiquitin-adenylate intermediate that serves as the donor of ubiquitin to a conserved cysteine residue in the E1 active site, where it is exchanged for adenosine monophosphate.46 In a subsequent transthiolation reaction, the activated ubiquitin moiety is passed from the E1 cysteine residue to the active-site cysteine of the E2 ubiquitin-conjugating enzyme. The complexity of ubiquitination in cellular physiology is manifest, in part, by the fact that there are at least 25 mammalian genes for ubiquitin-conjugating enzymes or E2s, which are the first determinants of substrate specificity in the ubiquitin-proteasome pathway. All E2 enzymes share a conserved approximately 150–amino acid core UBC domain.47 Centrally located within the UBC domain is the conserved ubiquitin-binding cysteine residue that is essential for E2 activity.47-49 Although smaller members of the E2 family of proteins consist almost entirely of the conserved UBC domain, other larger members have significant amino- or C-terminal extensions that may facilitate specific interactions with E3s.50 E2 enzymes may attach ubiquitin either to target proteins directly or to E3 ubiquitin ligases, depending on the kind of E3 in the interaction. However, most ubiquitination seems to require both E2 and E3 enzymes, although in some cases, E2 enzymes can autoubiquitinate.51 Further complexity in the ubiquitination process is conferred by the large array of E3 ubiquitin ligases that, together, comprise the third component of the ubiquitin enzymatic cascade. These ligases comprise a large and heterogeneous family of proteins that work by a variety of different mechanisms and vary in their interactions with E2 enzymes. E3 enzymes are the components that bind to specific protein substrates and promote the transfer of ubiquitin from a thioester intermediate to amide linkages with proteins or polyubiquitin chains.52,53 E3 ubiquitin ligases include the following two main classes of enzymes: homologous to E6-associated protein (E6-AP) C terminus (HECT) E3 ligases and really interesting new gene (RING) E3 ligases. E2 enzymes are not restricted in their interactions and may complex with E3 ligases from both families.54,55 To mediate transfer of ubiquitin from E2s to protein substrates, the E3 enzymes are sensitive to distinct degradation signals in the substrate. The HECT E3 ligases actively take up E2-bound ubiquitin via thioester linkages and then transfer the ubiquitin to the substrate. RING E3 ligases mediate transfer of ubiquitin bound to E2 directly to the substrate without forming a thioester bond with ubiquitin. HECT E3 ligases have a modular structure consisting of several specialized domains. All HECT E3s share a 350–amino acid C-terminal HECT domain that is homologous to the C terminus of E6-AP, which is the prototypical member of the HECT family of E3 ubiquitin ligases that is recruited by the E6 oncoprotein of human papillomavirus (HPV).56 A conserved cysteine within the HECT domain serves as the site for ubiquitin transfer from E2 via a transthiolation reaction catalyzed by the HECT E3 ligase.57,58 Isolated HECT domain peptides interact strongly with E2, and deletion of the HECT domain does not affect substrate binding.59 The highly variable N-terminal domain is responsible for specific substrate recognition and binding.59 Some HECT E3s have N-terminal WW domains, which are peptide motifs characterized by two conserved tryptophans that form hydrophobic pockets. The WW domain may bind phosphoserine- or phosphothreonine-containing peptides in target proteins whose ubiquitination is triggered by phosphorylation.60,61 The WW domain can also bind proline-rich sequences.62 For example, the WW domain of the E3 ubiquitin ligase NEDD4, binds proline-rich subunits of the epithelial sodium channel. Mutations of the proline-rich regions of the epithelial sodium channel predispose to an inherited hypertensive disorder called Liddle syndrome, in which the activity of the channel is enhanced because proteasomal degradation is compromised.63-67
RING finger E3s contain a characteristic structure composed of conserved histidine and cysteine residues in complex with two central Zn2+ ions that is conserved across the family of RING E3 ligases.68,69 The RING E3s serve as docking sites that bring together the target substrate and E2 enzymes to mediate transfer of the ubiquitin moiety but do not form thioester bonds with ubiquitin. RING domains of E3 enzymes bind E2 ligases.55 The RING finger proteins can be categorized into two distinct groups, single- and multisubunit proteins. Examples of single-subunit RING E3s are the MDM2 oncoprotein, the E3 ligase of P5370,71 and CBL, the E3 ligase of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor.72,73 Other RING E3s include the Skp1-cullin1-F-box protein family (SCF), which consists of multisubunit complexes that include a RING finger domain as one of several components.74 In this case, substrate recognition and E2 binding are executed by separate subunits. In the SCF E3 ligases, the RING finger component Rbx1 functions to recruit the E2.75,76 The cullins act as scaffolds between Rbx1 and proteins involved in substrate selection. Substrate recognition is mediated through substrate-specific F-box proteins that are, themselves, recognized and recruited to the SCF E3 complex by the Skp1 adaptor protein. Skp1 binds to cullins and thereby juxtaposes the substrate and ubiquitination machinery.77,78 F-box proteins are characterized by an approximately 40–amino acid motif that was first identified in cyclin F. There are a large number of human F-box proteins that can be subcategorized as Fbws if they contain WD40 repeats, as Fbls if they contain leucine motifs, and as Fbxs if they are not in the other two categories.79 In many cases, the F-box proteins bind phosphoproteins. SCF E3s regulate the cell cycle by causing rapid degradation of p2780,81 and cdc25,82 which are two important cell cycle–regulatory proteins. Some examples of F-box proteins are ßTrCP, which recognizes phosphorylated ß-catenin and the inhibitor of nuclear factor
A large subcellular organelle, the proteasome, which is a multisubunit protein complex, is the site for ATP-dependent degradation of ubiquitin-tagged proteins.21,87,88 The structure and function of the proteasome are highly conserved from archaebacteria to eukaryotes, and the proteasome is essential for cell and organism viability in eukaryotes.89-92 Proteasomes are not found in eubacteria. Although the subunit number and complexity of the proteasome has evolved, the proteasome is essentially a hollow cylinder-shaped particle that is deployed to different sites in the cytosol or nucleus.93 The 26S proteasome is composed of two major subunits that can assemble in an ATP-dependent manner.93,94 The 20S catalytic component contains multiple proteolytic sites, and the 19S regulatory component contains multiple ATPases and a binding site for ubiquitin concatemers (Fig 2). 95
The 20S subunit is the core of the 26S proteasome and is made up of four heptameric protein rings stacked like four doughnuts. The two inner beta rings harbor catalytic sites that face into the hollow center of the ring structure.92,96 The 20S catalytic ß-subunits contain trypsin, chymotrypsin, and postglutamyl-like hydrolytic activities.97 The two alpha rings sandwich the beta rings. The amino terminus of the -subunits blocks access to the proteolytic chamber.98 Thus, the inner cavity of the proteasome is only accessed through the narrow pores on either end of the cylinder.99 The 19S regulatory components assemble at each pore of the 20S subunit to form the 26S proteasome. The 19S regulatory subunit acts as a gate agent to limit entry to the proteasome to targeted proteins. The 19S subunit is also essential for proteolytic activity because the 20S subunit alone is inactive.37,100 The 19S regulatory particle is composed of two substructures, a lid and base, and is involved in substrate selection, preparation, and protein translocation into the catalytic 20S chamber for degradation.100-102 Each 19S particle is composed of numerous subunits, including six ATPases, that most likely provide the energy necessary for substrate unfolding that is required before entry into the 20S chamber.103 The outer-lid subcomplex of the 19S component is involved in the recognition and ubiquitin chain processing before substrate translocation and degradation.104,105 Proteins are degraded in a processive manner by the proteasome; thus, a single protein is hydrolyzed to final products before the next substrate enters.106 This ordered process contrasts to the activity of cytosolic proteases that cleave proteins once before dissociating from their substrates. Various protein components of the 26S proteasome have differential ability to bind multiubiquitin chains in protein conjugates and may confer an additional degree of specificity in the targeting of proteins for degradation.107 Cleavage products in the proteasome average six to 10 amino acids in length, and eventual hydrolysis to individual amino acids occurs in the cytosol.108
Ubiquitination requires binding of the target protein to the appropriate E3 ubiquitin ligase. Several modes of recognition by E3 ubiquitin ligases are well characterized. One mode of recognition is governed by the N-end rule, based on the finding that the in vivo half-life of a protein is related to the properties of its amino-terminal residue. Short-lived proteins commonly have basic or bulky hydrophobic residues at their N terminus, and more stable proteins have one of the amino acids of cysteine, alanine, serine, threonine, glycine, valine, or methionine at the N terminus.109 The E3 ubiquitin ligase E3 /Ubr1 is responsible for targeting N-end rule substrates.110,111 However, most proteins are targeted for ubiquitination by more complex mechanisms than recognition of an N-terminal amino acid. For example, post-translational modifications, such as phosphorylation, are common signals for ubiquitination. A number of important transcription factors are affected by phosphorylation-dependent ubiquitination. Nuclear factor-kappa B is activated after its inhibitory chaperone I B is phosphorylated and consequently ubiquitinated.112 ß-Catenin, which is part of the T-cell factor/lymphoid enhancer factor (TCF/LEF) heterodimeric transcription complex, is regulated by ubiquitination and targeted for ubiquitin ligation by phosphorylation at N-terminal serine residues.113 Some short-lived proteins contain a PEST sequence, which is a phosphorylation site enriched in the four amino acids of proline, glutamic acid, serine, and threonine that regulates ubiquitination.114 Ubiquitination can also be regulated by activation of some E3 ligases, which themselves may be synthesized as inactive enzymes and undergo post-translational modification as the activation step. The anaphase-promoting complex/cyclosome E3 ligase is phosphorylated late in mitosis to initiate degradation of cyclin B and progression of the cell cycle.115-118 Cyclins are cell cycle–regulatory proteins that are rapidly activated and degraded to control progression through the different phases of the cell cycle.115,116,119,120 These critical cell cycle–control processes are susceptible to interference early during viral infection. Because of the critical role that ubiquitin ligation has in regulating the cell cycle, viruses have evolved mechanisms to sustain cell division after infection and thus assure viral replication. An excellent example of this is the role of the HPV in cell cycle control of cervical epithelium as discussed in the Cervical Cancer section.
The notion that malfunction of proteasomal degradation could either enhance the effect of oncoproteins or reduce the amount of suppressor proteins was first conceived when a number of oncogene and suppressor gene products were found to be targets of ubiquitination.121-123 Since then, a number of oncogenic mutations and suppressor gene disruptions have been shown to affect ubiquitination and proteasomal degradation. We will describe the human malignancies for which this is known to be the case.
The VHL gene is the gatekeeper for clear cell carcinoma of the kidney and also the locus for the inherited von Hippel-Lindau syndrome. VHL is a component of an E3 ubiquitin ligase and, as such, binds both elongin B and elongin C at a region of the VHL protein frequently altered by germline von Hippel-Lindau mutations.124,125 CUL2, a member of the multigene family of cullins, associates with the VHL-elongin B-C complex (VBC).126 In addition, the VBC complex further associates with Rbx1, an evolutionarily conserved protein that contains a RING-H2 finger-like motif and interacts with cullins.127 The VBC-CUL2-Rbx1 complex acts as an SCF-like RING E3 ubiquitin ligase (Fig 3). 128-130
The VHL ubiquitin ligase complex targets members of the hypoxia-inducible transcription factor family (HIF) for degradation under normoxic conditions. Hypoxia-inducible factors mediate a physiologic response to hypoxia by activating the expression of genes that promote angiogenesis, such as vascular endothelial growth factor (VEGF). As a component of a multiprotein ubiquitin E3 ligase complex, VHL interacts directly with the oxygen-dependent destruction domain of the hypoxia-inducible factors.131-137 The HIF transcription factor is a heterodimer of - and ß-subunits. Under normoxic conditions, the HIFs are hydroxylated at each of two conserved proline residues within the -subunit, thus predisposing them to recognition by VHL.138-140 Mutations in VHL prevent the degradation of HIF-subunits under normoxic conditions and predispose to the formation of hypervascular lesions and renal tumors.141 HIF upregulation in renal cell carcinoma is correlated with enhanced expression of VEGF and other hypoxia-inducible genes. This is one reason why therapeutic trials have focused on the use of VEGF receptor antagonists as potential therapeutic agents for renal cell carcinoma.142-145
The gatekeeper gene for colonic carcinoma that is associated with polyp formation is adenomatous polyposis coli (APC), which is the same gene on chromosome 5q21 that is affected in hereditary familial adenomatous polyposis syndromes.146-150 A significant fraction of the adenomas of familial adenomatous polyposis will progress to cancer if either chemoprophylaxis or surgical interventions are not applied. APC functions as a classic tumor suppressor gene and is mutated in more than 70% of all colorectal cancers.151,152 Although, mutations are found throughout the APC gene, most cluster in a central region.151 Most APC mutations introduce a premature stop codon in the gene and, thus, result in C-terminally truncated proteins.151 The APC gene product binds to and regulates cellular levels of ß-catenin,153,154 a 95-kd protein that functions in cell surface adhesion complexes and as a heterodimeric transcription complex with TCF-4/LEF to activate transcription of genes like cyclin D1, MYC, and matrilysin (Fig 4). 155-158 Truncation of APC disrupts its interaction with axin/conductin, glycogen synthase kinase-3 beta, and ß-catenin. Axin/conductin serves as a scaffold that stabilizes the complex. APC is phosphorylated by glycogen synthase kinase-3 beta, which allows it to bind ß-catenin.159-161 APC presents ß-catenin to casein kinase I (CKI) for phosphorylation at the N terminus and, thus, targets it for ubiquitination and degradation, which serves to regulate cellular levels of the protein.113,162,163 The F-box protein, ßTrCP/Slimb is a component of the SCF E3 ubiquitin ligase that ubiquitinates phosphorylated ß-catenin.164 APC truncation affects the complex formation and increases levels of intracellular ß-catenin.153 The critical nature of this molecular lesion in colon cancer pathogenesis is illustrated by the fact that, in a minority of colon cancer tissues where APC is not mutated, the ß-catenin gene CTNN1B is subject to point mutations at an N-terminal six–amino acid sequence, containing three essential serines and a threonine phosphorylation target.165 Mutations that alter any one of these three serines or the threonine disrupt N-terminal phosphorylation that signals ubiquitination and increases the activity of ß-catenin in the cell.165
The importance of the ubiquitin-proteasome system through evolution is underscored by the observation that viruses manipulate the cellular ubiquitin-proteasome pathway to infect and replicate. Notably, HPV early region genes E6 and E7 target key cell cycle–control proteins to facilitate persistent viral infection and replication. The E6 proteins of HPV types 16 and 18 mediate binding of the E6-AP ubiquitin ligase and the P53 tumor suppressor protein (Fig 5). 166 E7 binds to the RB retinoblastoma tumor suppressor gene product to mediate its degradation.167,168
The P53 gene, which is located on chromosome 17p13.1, is the most common genetic target in human cancers.169,170 Biallelic disruption of P53 is a critical event in the molecular programs of both solid tumors and leukemias. The P53 protein is a sequence-specific DNA-binding transcription factor that responds to DNA damage and stress and activates cell cycle arrest and apoptosis to prevent the propagation of damaged cells.171 Inactivation of P53 permits the accumulation of genetic damage leading to transformation and tumorigenesis. HPV E6 protein binds to E6-AP, a cellular E3 ubiquitin ligase, to form a dimer that binds P53.56 The ternary complex results in E6-AP–mediated ubiquitination and degradation of P53.56 E6-AP is a complex protein with multiple functions, as suggested by the fact that inherited mutations in the E6-AP gene result in inactivation of its ubiquitin ligase properties and that E6-AP has been tied to Angelman syndrome, a hereditary neurologic disorder.172 The N-terminal domain of E6-AP, as in other HECT domain E3s, mediates substrate recognition. E6 binds to E6-AP within this N-terminal domain.173 Because formation of the E6-E6-AP complex precedes association with P53, this effectively redirects the substrate specificity of the E6-AP N-terminal domain toward P53.173 It has been shown that only the high-risk HPV strains have E6 proteins that induce the ubiquitin-proteasome degradation of P53.174,175 The E6 proteins of high-risk HPV strains efficiently bind the core region of P53, which is an interaction that is critical for P53 degradation.176 Furthermore, E6-AP can interact with E6 proteins from high-risk but not nononcogenic HPV strains.177
Regulation of P53 ubiquitination is also affected by endogenous E3 ligases that are the target of oncogenic activation. P53 mediates its own downregulation by activating expression of MDM2, a RING finger E3 ubiquitin ligase. MDM2 induces the ubiquitination of P53 and targets it for proteasomal degradation (Fig 5).71,178,179 Overexpression of MDM2 resulting from gene amplification causes downregulation of P53 and represents a mechanism by which this important suppressor gene is inactivated in a subset of cancers. The overall frequency of MDM2 amplification in human tumors is 7%. The human MDM2 gene maps to chromosome 12q13-14 and was found to be amplified in up to one third of sarcomas, including Ewing’s sarcoma, leiomyosarcomas, lipomas, liposarcomas, malignant fibrous histiocytomas, malignant Schwannomas, and rhabdomyosarcomas.180 MDM2 amplification and P53 mutation are mutually exclusive in sarcomas, consistent with the notion that either genetic alteration accomplishes the same result in the malignant program.181 However, overexpression of MDM2 does not always correlate with gene amplification, just as overexpression of P53 does not always correlate with mutations, so there may be other mechanisms underlying the activation of both of these genes.182 Many therapeutic strategies have focused on the restoration of wild-type P53 function. Preclinical studies of CP-31398, a small molecule that binds to and stabilizes P53, have shown that this agent induces cell death.183-185 CP-31398, a styrylquinazoline, was selected in a high-throughput drug screen for agents that restored a wild-type DNA-binding conformation of mutant P53.186 The drug works by altering conformation of P53 to prevent ubiquitination (but not phosphorylation, which is important for P53 activation).187
BRCA1 is a large protein encoded by the breast cancer susceptibility locus on chromosome 17p. The BRCA1 gene undergoes heterozygous mutation in families predisposed to breast cancer, ovarian cancer, and, to a lesser degree, other malignancies. This protein plays a role in the DNA damage response and interacts with members of the Fanconi anemia protein family.188 Structural analysis suggested that BRCA1 is also a RING finger protein that harbors E3 ubiquitin ligase activity.189,190 BRCA1 heterodimerizes with another RING domain protein, BARD1, to form a more potent E3 ligase.191,192 In fact, although BARD1 enhances the activity of BRCA1, it is BRCA1 that harbors the sole binding site to the E2 ligase UbcH5c.193 The importance of the ubiquitin ligase activity to the tumor suppressor properties of BRCA1 is underscored by the observations that missense mutations that predispose to breast cancer cluster in the Zn2+-binding residues of the BRCA1 RING finger domain critical to the ubiquitin ligase function.194-196 Mutations that predispose to breast cancer affect the phosphopeptide-binding pocket of BRCA1 and, thus, affect its intermolecular interactions.197 The BRCA1/BARD1 complex also catalyzes autoubiquitination via polymerization at lysine-6 of the ubiquitin molecule, which is an unusual polymerization site that may mediate protein stability rather than target a protein for degradation.198,199 BRCA1/BARD1 targets histone 2A/2AX as a substrate, suggesting that chromatin structure may be modified by the complex.200 In addition, the BRCA1/BARD1 complex ubiquitinates itself and, thus, increases its catalytic activity nearly 20-fold.201 The full range of functions of the BRCA1/BARD1 complex is not completely understood. The heterodimer associates with other proteins that influence its function. For example, BAP1, a ubiquitin hydrolase, complexes with BRCA1 but not with mutant BRCA1 protein products associated with breast cancer susceptibility.202 BAP1 also cooperates with BRCA1 in controlling growth of breast cancer cell lines and is likely another component of a multiprotein complex that mediates the complex effects of BRCA1.
Cell cycle regulation is frequently targeted by genetic disruption in cancer. The protein p27Kip-1 inhibits the G1 phase of the cell cycle by interacting with the cyclin-dependent kinase (CDK) 2/cyclin E and CDK2/cyclin A complexes. Regulation of p27Kip-1 levels is achieved post-transcriptionally through ubiquitin-mediated degradation.203,204 p27Kip-1 levels themselves may influence malignant transformation because haploinsufficiency of P27 can predispose to cancer in genetically altered mice.205 Low levels of p27Kip-1 protein have been identified and associated with tumor progression and poor prognosis in various cancers including colon, breast, prostate, ovarian, and brain cancer.206-209 Although mutation or loss of one P27 allele may have occurred, the loss of both alleles is extremely rare.210,211 Instead, it seems that the low levels of p27Kip-1 protein observed in these cases is the result of an abnormal overactivation of ubiquitin-mediated proteolysis.209,212 The F-box protein Skp2 has been identified as the substrate recognition component that binds and targets p27Kip-1 for ubiquitination and degradation.80,85,213 Several studies have found that, in a number of tumor types, Skp2 levels correlate with tumor grade and inversely correlate with prognosis and p27Kip-1 levels.214-218 Moreover, SKP2 may be a target for gene amplification, thus adding it to the list of ubiquitin ligases that are bona fide human oncogenes.219 A putative mechanism for transformation by Skp2 overexpression could be via p27Kip-1 degradation.
The EGFR family of tyrosine kinases plays an important role in the pathogenesis of a number of cancers, most notably gliomas and breast cancer. Approximately 40% of glioblastomas harbor mutations in EGFR, the most common of which results in a constitutively activated protein with lower levels of tyrosine autophosphorylation. The glioblastoma deletion (  EGFR) removes exons 2 to 7 and results in an 801–amino acid deletion that affects a large part of the extracellular ligand-binding domain.220,221 Although this mutant protein has a slightly lower level of phosphorylation, the overall effect of the mutation is to cause constitutive activation of EGFR because EGFR does not undergo downregulation by the CBL E3 ligase.222 Activated EGFR and its family members human EGFR (HER) 2, HER3, and HER4 undergo C-terminal phosphorylation of cytoplasmic tyrosine residues after receptor dimerization. The receptor signals are downregulated when E3 RING finger ubiquitin ligases called CBL bind specific phosphotyrosine residues.72,73,223-225 Ubiquitinated receptors are internalized in clathrin-coated endosomes, which then fuse with lysosomes and lead to eventual EGFR degradation by lysosomal proteases.226,227 In the absence of CBL-mediated ubiquitination, EGFR family members are recycled back to the cell surface, where they are available for reactivation.226 Attenuated ubiquitination may also play a role in the effects of amplified HER2 in human breast cancer and other cancers that have HER2 gene amplification. Overexpression of HER2 favors the formation of EGFR/HER2 heterodimers228 that recruit CBL to a lesser degree than EGFR homodimers and are, therefore, recycled to the cell surface.228,229 The effects of trastuzumab are, at least in part, a result of enhancing the recruitment of CBL to HER2 to enhance receptor degradation.230
Cell cycle–regulatory proteins are important targets for mutation in human cancer. Genetic alterations in cell cycle–control genes are found in almost all human carcinomas. Levels of cell cycle–regulatory proteins are controlled by the ubiquitin-proteasome pathway. Proper progression through the cell cycle depends on the periodic accumulation of cyclins and their timed interplay with their catalytic partners, cyclin-dependent kinases (CDKs). For example, the level of cyclin E is low in early G1 and increases to a peak at the G1-S transition, at which point cyclin E activates CDK2. Formation of the cyclin E-CDK2 complex commits the cell to S-phase genome duplication. Levels of cyclin E are controlled by both transcription and ubiquitin-mediated degradation. The importance of tight regulation of cyclin E levels is underscored by experimental data that shows that aberrant accumulation and overabundance of cyclin E leads to premature S-phase entry, chromosome instability, and tumor formation.231-233 Moreover, overexpression of cyclin E is an indicator of poor prognosis in several human cancers.234-239 In some cases, cyclin E overexpression is caused by a defect in ubiquitin-mediated degradation. A multisubunit SCF ubiquitin ligase is responsible for the ubiquitination and proteasomal degradation of G1/S cyclins, including cyclin E. Association with CDK2 results in the autophosphorylation of cyclin E.240,241 Once phosphorylated, cyclin E is recognized and bound by the SCF-associated F-box protein Fbw7 (also designated hCdc4).240-242 The gene encoding this F-box protein is mutated in some breast and ovarian cancer cell lines that express high levels of cyclin E and 16% of human endometrial tumors.242-244 In fact, the gene for the Fbw7 protein is localized to a region of chromosome 4q32, which is deleted in more than 30% of human tumors.245 These findings characterize the gene for Fbw7 as a putative tumor suppressor.
It was not intuitively obvious that inhibition of the 26S proteasome would result in selective inhibition of malignant cell growth, but a number of agents have been identified that inhibit proteasomal degradation and have been shown to induce apoptosis,246,247 kill tumor cells,248 overcome drug resistance,249,250 and enhance radiation sensitivity.248 Bortezomib, which is a small molecule with a boronate moiety linked to a dipeptide, has recently been approved by the US Food and Drug Administration for treatment of relapsed multiple myeloma.251 Bortezomib has been shown to have a high degree of specificity to inhibit the chymotryptic activity of the proteasome. The effects on a cell seem to be mediated through activation of apoptosis pathways, including both a decrease in expression of antiapoptotic proteins and increased activity of cell death pathways.246,247,252 Some of the effects on cell death activation may be mediated by stabilization of I B, the cellular chaperone and inhibitor of the nuclear factor-kappa B transcription factor.253,254 However, the degree to which IB stabilization is responsible for the effects of bortezomib on multiple myeloma is unknown. Mechanisms of resistance to bortezomib are just being elucidated. One resistance mechanism seems to involve increased expression of heat shock protein-27, the inhibition of which overcomes drug resistance.255,256
The authors indicated no potential conflicts of interest.
We thank Eric Rubin for reviewing the manuscript before publication. We would also like to thank Anna T. Riegel and Emma T. Bowden for their advice and support throughout this project.
Supported in part by US Public Health Service grant Nos. ES09888 and CA96854 (E.P.G.). Authors’ disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
