The Ubiquitin-Proteasome System Meets Angiogenesis

Published OnlineFirst February 21, 2012; DOI: 10.1158/1535-7163.MCT-11-0555

Molecular Cancer Review Therapeutics

Nader Rahimi

Abstract A strict physiological balance between endogenous proangiogenic and antiangiogenic factors controls endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic angiogenesis, a shift occurs in the balance of regulators, favoring endothelial growth. Much of the control of angiogenic events is instigated through hypoxia-induced VEGF expression. The ubiquitin-proteasome system (UPS) plays a central role in ﬁne-tuning the functions of core proangiogenic proteins, including VEGF, VEGFR- 2, angiogenic signaling proteins (e.g., the PLCg1 and PI3 kinase/AKT pathways), and other non-VEGF angiogenic pathways. The emerging mechanisms by which ubiquitin modiﬁcation of angiogenic proteins control angiogenesis involve both proteolytic and nonproteolytic functions. Here, I review recent advances that link the UPS to regulation of angiogenesis and highlight the potential therapeutic value of the UPS in angiogenesis-associated diseases. Mol Cancer Ther; 11(3); 538–48. 2012 AACR.

Introduction tumor mass reaches a macroscopic size, it is becoming Angiogenesis, the growth of new blood vessels from increasingly apparent that angiogenesis is instigated in preexisting vessels, is an important physiological process the early stage of tumor development (5, 7), further sup- in the body that is required for normal wound-healing and porting angiogenesis as a vital component of tumor female reproduction. Pathologic angiogenesis (excessive growth and metastasis. or insufficient) is now recognized as a common denom- VEGF-A also plays a central role in the development of inator underlying a number of deadly and debilitating choroidal neovascularization, and indeed is responsible human diseases, including cancer, age-related macular for both neovascularization and vascular leakage in wet degeneration (AMD), diabetic retinopathy, and cardio- AMD (8). The hallmarks of wet AMD are drusen forma- vascular diseases (1, 2). Although in many ways these tion [i.e., the focal deposition of debris between the retinal diseases have distinct etiologies and mechanisms of pigment epithelium (RPE) and Bruch’s membrane], cho- development, they all share abnormal angiogenesis. For roidal neovascularization, RPE cell detachment, fibrovas- example, although cancer has nothing to do with AMD, it cular scarring, and vitreous hemorrhage. Aberrant blood shares one of its characteristics, angiogenesis, with AMD vessel growth and blood vessel leakage subsequently (Fig. 1). Acquisition of angiogenesis by tumor cells is result in the loss of central vision (9). Many cell types in the eye, including RPE cells, pericytes, endothelial cells, considered the most critical step in tumor growth and € metastasis. To grow beyond 2 mm in diameter, a tumor glial cells, Muller cells, and ganglion cells, synthesize and must acquire angiogenesis, which is often established by secrete VEGF. In addition to the vital importance of VEGF hypoxia-induced expression of VEGF-A and other angio- in the pathology of AMD, elevated VEGF levels also genesis-inducing molecules (3). To support the growth of strongly correlate with retinal ischemia-associated neo- the expanding tumor, an angiogenic switch is turned on, vascularization in diabetic retinopathy and retinopathy of causing normally quiescent endothelial cells to proliferate prematurity (8, 10). and sprout (4). It is now clear that induction of VEGF-A Once the angiogenic switch is activated, different and other related angiogenesis inducers, and a reduction sequential steps take place, including the activation of in the expression of angiogenesis inhibitors, such as various proteases from activated endothelial cells result- thrombospondin (TSP-1), govern the tumor-induced ing in the degradation of the basement membrane sur- angiogenic switch (5, 6). Although it was initially thought rounding the existing vessel, migration of the endothelial that angiogenesis plays a substantial role only when the cells into the interstitial space, endothelial cell proliferation, sprouting, lumen formation, generation of new basement membrane with the recruitment of pericytes, and Author's Affiliation: Departments of Pathology and Ophthalmology, Bos- fusion of the newly formed vessels (11). In general, and in ton University School of Medicine, Boston, Massachusetts most pathologic conditions, angiogenesis starts when Corresponding Author: Nader Rahimi, Department of Pathology, Boston cells within a tissue respond to hypoxia (i.e., low oxygen) University Medical Campus, 670 Albany St., Room 510, Boston, MA 02118. or in certain circumstances when oncogenic gene pro- Phone: 617-638-5011; Fax: 617-414-7914; E-mail: [email protected] ducts such as Ras and Myc induce expression of VEGF doi: 10.1158/1535-7163.MCT-11-0555 along with other hypoxia-inducible genes (12). The VEGF 2012 American Association for Cancer Research. family of growth factors includes placental growth factor

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Figure 1. Schematic presentation of tumor-induced angiogenesis and choroidal neovascularization as manifested in wet AMD. Expression of VEGF is responsible for induction of angiogenesis, which is associated with tumor progression and worsening of wet AMD.

(PlGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E mediated VEGFR-2 activation, recent studies have (13). VEGF-A isoforms result from alternative splicing of shown that under certain circumstances, VEGFR-2 can messenger RNA (mRNA), and VEGF-165 is considered to also be activated by non-VEGF family ligands, includ- be the most common VEGF-A isoform (14). Three differ- ing heparan sulfate proteoglycans (20) and galectin-3, a ent VEGF gene products (PlGF, VEGF-A, and VEGF-B) glycan-binding protein (21). Heparan sulfate proteogly- have been identiﬁed as ligands for VEGFR-1. VEGF-A, cans have been proposed to potentiate VEGFR-2 acti- VEGF-D, and VEGF-C bind to and activate VEGFR-2 (13). vation through cis-andtrans-binding with VEGFR-2 VEGF-C and VEGF-D are also known to recognize and VEGF complex (20), where galectin-3 is thought to VEGFR-3 (also called FLT4), a receptor tyrosine kinase potentiate VEGFR-2 activation by prolonging its pres- (RTK) that is expressed predominantly by lymphatic ence in the plasma membrane (21). Although VEGF endothelial and hematopoietic progenitor cells (15). family proteins are prominent regulators of angiogen- VEGF-E is a virally encoded VEGF-like protein that selec- esis, several other growth factors and cytokines, includ- tively binds to VEGFR-2 (16). VEGF family proteins ing angiopoietin-1, Del-1, ﬁbroblast growth factor, also interact with non-RTK cell surface receptors, includ- hepatocyte growth factor, interleukin-8 (IL-8), and lep- ing neuropilin-1 and neuropilin-2, which are character- tin, are known to stimulate angiogenesis (22), adding ized as coreceptors for VEGF family ligands (13, 17). further complexity to the regulation of angiogenesis. Among the VEGF receptors and coreceptors, activation The role of VEGFR-2 in angiogenesis is well estab- of VEGFR-2 by VEGF family ligands is considered the lished, and more-comprehensive reviews on the role most critical event in angiogenesis (18, 19). Beyond VEGF- of VEGFR-2 in angiogenesis were recently published

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Figure 2. VEGF superfamily ligands and receptors. A schematic of VEGF ligands and their interactions with VEGF receptors is shown.

(13, 17, 19). Figure 2 summarizes VEGF superfamily large family of proteins (with almost 700 in the human ligands and their interactions with VEGF receptors (Fig. genome) that are known to be involved in regulating the 2). It is increasingly evident that angiogenic signaling is turnover and activity of many target proteins (23). E3 established through an elegant and complex system of ligases are divided into 2 large groups: the homology to VEGF and non-VEGF ligands, and VEGF receptors and the E6-associated protein carboxyl terminus (HECT) coreceptors, resulting in homo- and heterodimeric acti- domain-containing E3 ligases, and the Really Interest- vation of VEGF receptors and in turn the complex ing New Gene (RING) domain-containing E3 ligases. processes of angiogenesis. The RING-type E3 ligases include single subunit E3 ligases (such as Cbl family E3 ligases) and multisubunit E3 ligases (such as Cullin-RING ubiquitin ligases). In Ubiquitin-Proteasome System recent years, additional E3 ligases have been identified Ubiquitin is an evolutionarily conserved polypeptide that use different domains to recognize E2-conjugating that consists of 76 amino acids and was named for enzymes, such as plant homeodomain domain-contain- its ubiquitous expression in eukaryotes. Ubiquitin is ing E3 ligases and the U-box E3 ligases (23, 24). activated by a ubiquitin-activating enzyme, E1, in an Although conjugation of ubiquitin to target proteins ATP-dependent manner and is transferred to a ubiqui- was initially recognized as a signal for protein degra- tin-conjugating enzyme, E2. Eventually, a ubiquitin- dation by the 26S proteasome, it is now recognized that protein ligase, E3, specifically attaches the ubiquitin ubiquitination regulates a broad range of cellular func- molecule to a target protein through the e-amino group tions, including protein processing, membrane traffick- of a lysine residue (Fig. 3). E3 ubiquitin ligases are a ing, and transcriptional regulation (23, 25). Recent

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Figure 3. Schematic of the UPS. Ubiquitin is activated by the ubiquitin-activating enzyme (E1) and then transferred to a ubiquitin-conjugating enzyme (E2). E2 transfers the activated ubiquitin moieties to the protein substrate that is bound speciﬁcally to a particular ubiquitin ligase (E3). The transfer of ubiquitin takes place either directly (in the case of RING ﬁnger ligases) or via an additional thiol-ester intermediate on the ligase (in the case of HECT domain ligases). Repeated conjugation of ubiquitin moieties to each other generates a polyubiquitin chain that serves as the binding and degradation signal for the 26S proteasome. The protein substrate is degraded, generating short peptides and free ubiquitin that can be further reused. Ub, ubiquitin.

studies showed that ubiquitination can also inﬂuence the polypeptide ubiquitin in an ATP-dependent manner, cell signaling by targeting activation of proteins in a enabling its transfer onto the ubiquitin carrier enzyme, E2. proteolysis-independent manner (26–28). Activated ubiquitin is then transferred by the ubiquitin Multiple ubiquitin molecules can be attached to a target protein ligase, E3, to a substrate protein (29). The sub- protein bymeansofmonoubiquitination (i.e.,attachment of strate-recruiting components of UPS then catalyze the a single ubiquitin to 1 or multiple lysine residues). Mono- formation of an isopeptide bond between the C-terminal ubiquitination is regarded as a signal for nonproteolytic glycine residue of ubiquitin and the e-amino group of a eventssuchasendocytosis,histoneregulation,DNArepair, substrate protein lysine residue. Continual addition of virus budding, and nuclear export (25). Alternatively, ubi- ubiquitin moieties onto substrate (i.e., polyubiquitination) quitin can be attached to a target protein in the form of facilitates recognition of the substrate by the proteolytic polyubiquitination, where multiple ubiquitin molecules machinery of the UPS, the 26S proteasome (29, 30). The 26S are attached to a single lysine residue. Ubiquitin contains proteasome complex is essentially composed of 1 20S and 7 different lysine residues that potentially can be used for 2 19S units (Fig. 3). The 19S complex has 2 multisubunit ubiquitin-chain assembly. Lys48- and Lys29-linked poly- components, often described as the base and the lid. The ubiquitination generally is associated with degradation of base contains 6 ATPases, which belong to the TRIPLE-A target proteins by the 26S proteasome, whereas Lys63- family of ATPases, and 2 non-ATPase subunits, which linked polyubiquitination is involvedin DNA repair, signal bind to the 20S catalytic core. The lid contains up to 10 non- transduction, and endocytosis, but not degradation (23, 25). ATPase subunits (31, 32). Together, the base and lid Clearly, the ubiquitin machinery has evolved to play a function in the recognition of ubiquitinated substrates versatile role in protein functions ranging from protein and their subsequent binding. The 20S complex is com- turnover to subcellular localization and kinase activation, posed of 28 related subunits (14 different subunits) that andconsequentlyit ishighly relevanttothe pathobiology of are arranged as 4 heptameric staggered rings. The 2 outer many human diseases. rings contain the a subunits (a1–a7). The 2 inner rings The ubiquitin-proteasome system (UPS) consists of 2 contain 2 copies of the b subunits (b1–b7). Within the 20S major components: substrate-recruiting enzymes (E1, E2, proteasome, subunits b1, b2, and b5 exhibit postglutamyl and E3) and substrate-degrading enzymes. E1 activates peptide hydrolyzing, trypsin-like, and chymotrypsin-like

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cleavage activity, respectively. More-comprehensive hydroxylation by PHD (38, 40). Of interest, hydroxylation reviews on the function and composition of the 26S of an asparagine residue (Asn803) in the C-terminal acti- proteasome were recently published (29, 30, 32, 33). vation domain of HIF-1a by HIF asparagine hydroxylase, termed factor inhibiting HIF (FIH), inhibits HIF-1a activ- Regulation of VEGF expression by von Hippel- ity by blocking interaction of the HIF-1a C-terminal acti- Lindau E3 ubiquitin ligase vation domain with the transcriptional coactivator, p300 The molecular mechanism by which hypoxia sets off (41). Under hypoxic conditions, however, VEGF is gen- expression of VEGF has been extensively studied erally overproduced, which leads to pathologic angiogen- (12, 34). In normoxic conditions (i.e., normal oxygen esis. In response to low oxygen, pVHL E3 ubiquitin ligase levels), VEGF expression is generally inhibited by the is S-nitrosylated (the covalent attachment of a nitrogen interaction of von Hippel-Lindau (pVHL) E3 ubiquitin monoxide group to the thiol side chain of cysteine), which ligase with hypoxia-induced transcription factor 1a blocks HIF-a interaction with pVHL E3 ubiquitin ligase. (HIF-1a), leading to its ubiquitination and targeting HIF-1a escapes from ubiquitin-mediated degradation as a HIF-a for degradation by 26S-proteasome. Two of the consequence of S-nitrosylation of pVHL (Fig. 4). Nitric 3HIF-a isoforms, HIF-1a and HIF-2a, are closely relat- oxide–mediated S-nitrosylation of HIF-1a at the cysteine ed and can interact with hypoxia response elements to residue (C800) permits interaction of HIF-a with p300, induce VEGF expression (35, 36), whereas HIF-3a prompting its transcription activity and VEGF expression appears to be involved in the negative regulation of (42). hypoxia-induced gene expression (37). Another important aspect and additional complexity of Oxygen-mediated posttranslational modiﬁcation of HIF-a regulation is the function of heat shock protein 90 HIF-a through non-heme and iron-dependent oxyge- (Hsp90). Hsp90 is often upregulated under cellular stress nases that uniquely hydroxylate speciﬁc HIF-a at proline conditions, such as hypoxia (43), which appears to pre- residues regulates its transcriptional activity. Hydroxyl- vent HIF-1a degradation in a pVHL-independent manner ation of human HIF-1a at 2 proline residues (Pro402 and (43–45). Consistent with the protective role of Hsp90 in Pro564) by prolyl hydroxylase domain (PHD) proteins HIF-1a, agents that inhibit Hsp90 activity have also been creates binding sites for the pVHL E3 ubiquitin ligase shown to promote ubiquitin-mediated degradation of complex that targets HIF-1a for proteasomal degradation HIF-1a (44, 46). A recent study indicated that inhibition (38, 39). These proline hydroxylation sites contain a con- of Hsp90 by hemin, a derivative of the protoporphyrin served LXXLAP (where X indicates any amino acid) motif compound, increases HIF-1a ubiquitination and hence that is recognized by PHD proteins, leading to HIF-1a angiogenesis (47).

Figure 4. Role of pVHL in expression of VEGF and angiogenesis. A, in the presence of oxygen, proline residues in the oxygen-dependent degradation domain of HIF are hydroxylated. This allows HIF-a to interact with pVHL. The interaction between HIF and pVHL causes degradation of HIF through ubiquitination. B, in response to low oxygen (i.e., hypoxia), pVHL is S-nitrosylated, preventing HIF-a from interacting with pVHL, and the degradation of HIF-a is disallowed. HIF-a stimulates expression of VEGF, which in turn stimulates angiogenesis, as manifested in cancer progression. S-nitrosylation is the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine.

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b-Transducin repeat-containing protein ubiquitin E3 terminal regions, which are proximal to the F-box motif. ligase controls ubiquitination and degradation of It is thought that the N-terminal sequences of b-TrCP1 and VEGFR-2 b-TrCP2 allow these proteins to undergo homo- and Activation of VEGFR-2 by VEGF family ligands med- heterodimerization (60). It appears that both b-TrCP1 and iates most of the known VEGF cellular responses b-TrCP2 can promote ubiquitination of VEGFR-2, which (13, 19, 48). Central to proper regulation of the angiogenic suggests that to some extent they may act in a redundant activity of VEGFR-2 is the process by which VEGFR-2 fashion in VEGFR-2 ubiquitination (52). A redundant role triggers its own internalization and degradation, conse- of mammalian b-TrCP1 and b-TrCP2 in ubiquitination quently terminating its angiogenic signaling. Upon stim- and degradation of other proteins, including IkB and ulation with VEGF family proteins, VEGFR-2 is removed b-catenin, has also been suggested (61, 62). from the cell membrane and undergoes clathrin-dependent endocytosis (49), which initiates its degradation (50) Role of ubiquitination in phospholipase Cg1 and recycling (51). Of interest, VEGFR-2 internalization is activation and angiogenesis stabilized by cadherin-5 (52). Cadherin-5–dependent sta- Activation of phospholipase Cg1 (PLCg1) in endothelial bilization of VEGFR-2 is established by reducing the cells is considered to be one of the chief mediators of the tyrosine phosphorylation of VEGFR-2, perhaps by recruit- angiogenic signaling of VEGFR-2. It catalyzes the forma- ing tyrosine phosphatases to VEGFR-2 (49, 53). Ligand- tion of inositol 1,4,5-trisphosphate (IP3) and diacylgly- mediated degradation of VEGFR-2 requires tyrosine cerol from phosphatidylinositol 4,5-bisphosphate (PIP2). kinase activity, and activation of the protein kinase C Phosphotyrosine 1173 on mouse VEGFR-2 (corre- (PKC) pathway accelerates its degradation (50). On the sponding to Tyr1175 on human VEGFR-2) has been iden- other hand, activation of p38 mitogen-activated protein tified as the primary site responsible for the recruitment of kinase (MAPK) has been shown to stabilize VEGFR-2 (52), PLCg1 to VEGFR-2 (26, 63, 64). Substantial information suggesting that the stability and degradation of VEGFR-2 obtained from animal models links PLCg1 to angiogene- in endothelial cells are highly fine-tuned by the activities sis. The initial evidence linking PLCg1 to endothelial cell of the PKC and p38 MAPK pathways. Initial studies function and angiogenesis was provided by targeted showed that the carboxyl terminal of VEGFR-2 plays a deletion of PLCg1, which resulted in early embryonic pivotal role in VEGFR-2 stability and degradation. Pro- lethality between embryonic days 9.5 and 10.5 due to gressive deletion of the carboxyl terminal of VEGFR-2 was significantly impaired vasculogenesis and erythrogenesis shown to inhibit ligand-dependent degradation of (65). Inactivation of PLCg1 in zebrafish was also shown to VEGFR-2 (48, 50). A recent study identified the presence be required for VEGF function and arterial development of a PEST domain in the carboxyl domain of VEGFR-2, (66). Additional evidence of the importance of PLCg1in which may account for the critical role of the carboxyl angiogenic signaling of VEGFR-2 was obtained by phar- domain in VEGFR-2 degradation (52). The PEST motif macological inhibition of PLCg1. U73122, a potent PLCg1 [rich in proline (P), glutamic acid (E), serine (S), and inhibitor, was shown to inhibit endothelial cell tube for- threonine (T)] is considered to be a signature of short- mation in vitro (64) and angiogenesis in vivo in a chorio- lived proteins that are degraded by the ubiquitin pathway allantoic membrane assay (67). Silencing the expression of (54). It is thought that PEST sequences are unstructured PLCg1 in primary endothelial cells by an siRNA strategy regions in certain protein sequences, which may serve as a also inhibits VEGF-mediated endothelial cell tube forma- phosphodegron for the recruitment of F-box–containing tion and proliferation (68), further underscoring the ubiquitin E3 ligases leading to ubiquitination and degra- importance of the PLCg1 pathway for angiogenic signal- dation (55, 56). Phosphorylation of Ser1188 and Ser1191 of ing of VEGF. the PEST domain of VEGFR-2 recruits SCF-b-transducin PLCg1 is a multidomain protein that consists of 2 SH2 repeat-containing protein 1 (Trcp1) E3 ubiquitin ligase domains and 1 SH3 domain between the catalytic to VEGFR-2, leading to SCF-bTrcp1–dependent ubiquiti- domains. The SH2 domains recognize phosphotyrosine nation and degradation of VEGFR-2. Degradation of 1173 on VEGFR-2 (64), whereas the SH3 domain recog- VEGFR-2 is mainly attained through Lys48-linked poly- nizes proline-rich sequences (PXXP motifs). In addition to ubiquitination (52). its SH domains, PLCg1 also contains a C2 domain, EF b-TrCPs (also called FWD1) belong to a larger family of hand, and 2 putative PH domains. The presence of both N- Fbw (F-box/WD40 repeat containing) proteins that are and C-terminal SH2 domains is required for optimal generally characterized by the presence of a 42–48 amino- binding of PLCg1 to VEGFR-2 (64). PLCg1 also interacts acid F-box motif at the N-terminus and 7 WD40 repeats at with c-Cbl through its proline-rich motif in a noninducible the C-terminus (57). b-TrCPs are highly conserved across manner (50). As a result of activation by VEGFR-2, c-Cbl species, in particular within the F-box motif and WD40 is recruited to VEGFR-2 and distinctly inhibits phosphor- repeats motif. Human b-TrCP1 and b-TrCP2 exist in ylation of Tyr783 on PLCg1 in an ubiquitination-depen- multiple isoforms due to alternatively spliced mRNA, dent manner (26, 68). c-Cbl negatively regulates PLCg1 but all are conserved in F-box and all 7 WD40 repeats activation in a proteolysis-independent manner. Instead (58, 59). The most notable differences in sequences of targeting it for degradation, c-Cbl distinctively between b-TrCP1 and b-TrCP2 are found in their N- mediates ubiquitination of PLCg1 and suppresses its

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phosphorylation on Y783 (26). How PLCg1 can escape lation of a host of other proteins that affect cell prolifer- from ubiquitin-mediated degradation but then enter into ation, cell cycle progression, and cell survival (73). The a less enzymatic active state is a conundrum that warrants Cbl-b ubiquitin E3 ligase is known to interact with the p85- further investigation. SH2 domain and catalyze p85 polyubiquitination (27,74). Endothelial cells derived from c-Cbl knockout mice also Of interest, the Cbl-mediated ubiquitination does not lead showed that loss of c-Cbl results in an increased phos- to degradation of p85 (27). phorylation of PLCg1 with no apparent effect on its half- AKT, a serine/threonine protein kinase, is one of the life (68). Overexpression of c-Cbl in endothelial cells has key PI3K substrates that play a central role in mediating also been shown to inhibit tube formation and sprouting VEGFR-2–dependent cellular events in endothelial (75, of endothelial cells. Conversely, overexpression of c-Cbl 76). It was recently shown that the carboxyl terminus of (70Z/3-Cbl), an E3 ligase-deficient variant form of c-Cbl, Hsc-70-interacting protein (CHIP) interacts with AKT and or silencing its expression by siRNA elevated sprouting of induces its ubiquitination (77). In addition, tetratricopep- endothelial cells (26). Recent studies showed that VEGF- tide repeat domain 3 (TTC3) containing E3 ligase was and tumor-induced angiogenesis is highly elevated in recently linked to AKT ubiquitination and degradation c-Cbl nullizygous mouse (67, 68). It appears that the role (78, 79). Of interest, TTC3 interacts only with active AKT, of c-Cbl in angiogenesis is widespread, because laser- and not inactive AKT, in the nucleus (78, 79), indicating induced angiogenesis in c-Cbl knockout mice also results that perhaps TTC3-mediated AKT ubiquitination is in enhanced retinal neovascularization (67). important for controlling AKT signaling in the nucleus. TTC3 itself is the target of AKT and is phosphorylated at Role of ubiquitination in the PI3 kinase/AKT S378 by AKT, and this phosphorylation appears to be pathway necessary for TTC3 E3 ligase activity (78, 79). BRCA1 is The phosphoinositide 3-kinase (PI3K) signal transduc- another E3 ligase that also interacts with activated AKT tion pathway is one of the main signaling routes and targets it for ubiquitination and degradation (80). that VEGFR-2 uses to stimulate endothelial cell survival Recent studies also showed that AKT is ubiquitinated by and proliferation (69–71). VEGFR-2 activates PI3K TRAF6 E3 ligase. TRAF6 directly interacts with and through recruitment of p85 of PI3K involving Tyr799 and induces AKT ubiquitination (28). TRAF6-mediated AKT Tyr1173 (67, 68). PI3K consists of an 85-kDa regulatory ubiquitination takes place through the K63-linked mod- subunit and a 110-kDa catalytic subunit. It is a lipid ification and does not trigger AKT degradation. K63 chain kinase that converts the plasma membrane lipid PIP2 to polyubiquitination of AKT contributes to its membrane phosphatidylinositol-3,4,5-triphosphate (PIP3). Proteins localization, where it is phosphorylated (28). Figure 5 with pleckstrin-homology (PH) domains, such as protein summarizes various ubiquitin E3 ligases that are involved kinase B (PKB/AKT), phosphoinositide-dependent in fine-tuning the abundance and activation of key angio- kinase-1 (PDK-1), and PDK-2, bind to PIP3. AKT is acti- genic proteins. Some ubiquitin E3 ligases, such as Cbl vated by PIP3, PDK1, and PDK2, leading to phosphory- family proteins, target multiple angiogenic proteins,

Figure 5. Regulation of core angiogenic proteins by ubiquitin E3 ligases. Expression of VEGF is regulated by activity of pVHL. bTrcp1 ubiquitinates VEGFR-2 and subjects it to proteasomal degradation. c-Cbl catalyzes ubiquitination of numerous angiogenic signaling proteins, including VEGFR-1, Eph, Tie2, and PLCg1. AKT abundance and activity are regulated by TRAF6, CHIP, and TTC3. Itch regulates degradation of the Notch1 receptor.

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whereas in other cases more than 1 ubiquitin E3 ligase is Dvl. Surprisingly, in neuronal cells, Dvl uniquely is ubi- involved in the ubiquitination of an angiogenic protein, as quitinated by the HECT-type E3 ligase NEDL1, and not by illustrated for AKT (Fig. 5). KLHL12 (90), suggesting a cell-type–speciﬁc regulation of Dvl by the UPS.

Role of Ubiquitination in Wnt Signaling The ubiquitination system as a potential target for The Wnt pathway is another key player in angiogenesis. antiangiogenesis and anticancer therapy Binding of Wnt to its 7-span transmembrane receptor, Given that the expression and degradation of core Frizzled (Fz), and its coreceptor, Lrp5/6, at the cell surface proangiogenic proteins are regulated by the UPS, selec- initiates a signaling cascade that mediates angiogenesis tively targeting the different components of this pathway and other key developmental processes, including stem may prove to be an effective strategy for antiangiogenesis cell maintenance, growth, and cell-fate specification, and treatments. Our increasing understanding of the ubiqui- cell migration (81). Deregulation of the activation of Wnt/ tination system and its role in angiogenesis is generating b-catenin signaling has been linked to a range of human great interest in the development of novel strategies to diseases, including cancer (81, 82). During the resting state block pathologic angiogenesis. The role of the ubiquitina- of canonical Wnt signaling, several key Wnt-associated tion pathway in human diseases in general, and in angio- signaling proteins, including b-catenin, are targeted via genesis in particular, is still unclear because the molecular ubiquitination for degradation. Initially, the adenomatous mechanisms and gene products involved in the UPS are polyposis coli protein forms a complex with glycogen not fully understood. Moreover, there has been no com- synthase kinase 3b (GSK-3b) and axin. This complex then prehensive analysis of the functional importance of the binds to b-catenin in the cytoplasm, which leads to phos- UPS in angiogenesis-associated human diseases. The UPS phorylation of b-catenin by casein kinase 1 (CK1) and has many different components that potentially could be GSK-3b. Phosphorylation leads to the creation of a phos- targeted for inhibition or stimulation in the milieu of phodegron motif on b-catenin that allows the ubiquitin E3 angiogenesis. For example, the therapeutic value of the ligases (e.g., b-Trcp) and Jade-1 to recognize b-catenin. As proteasome inhibitor bortezomib (Velcade; Millennium a result, b-catenin is targeted for ubiquitination, leading to Inc.), the first UPS-targeting drug to be approved by the its 26S-proteasome–mediated degradation (83, 84). U.S. Food and Drug Administration for treatment of In addition, other ubiquitin E3 ligases (e.g., Siah1 and relapsed or refractory multiple myeloma (91), could be Ozz) also target b-catenin for degradation in a cell-type– explored in angiogenesis-associated diseases. or context-specific manner (85, 86). Recent studies have linked the potential therapeutic Removal of cytosolic b-catenin through the UPS pre- value of inhibition of the proteasome pathway to angio- vents b-catenin from translocating into the nucleus, where genesis. Indeed, treatment of endothelial cells in a cell it acts as a transcription factor for genes associated with culture system with proteasome inhibitors was shown to various angiogenic events, such as proliferation of endo- inhibit capillary tube formation of endothelial cells and thelial cells. In contrast, activation of the canonical Wnt blood vessel formation in an embryonic chick chorioal- signaling pathway results in inhibition of b-catenin deg- lantoic membrane assay (92, 93). Moreover, bortezomib radation, leading to increased cytosolic b-catenin, which was shown to inhibit tumor angiogenesis in a murine then translocates to the nucleus. In the nucleus, b-catenin xenograft model (94). More recently, it was shown that associates with at least one of a family of Tcf/Lef tran- small molecules such as nutlins (Roche, Inc.) and RITA scription factors and induces the expression of numerous can block p53 ubiquitination by inhibiting the activity of genes, such as cyclinD1 and c-myc (87), which are impli- MDM2 ubiquitin ligase, which is known to mediate p53 cated in cellular proliferation. ubiquitination (95–97). Loss of p53 activity is linked to In addition to Wnt pathway–mediated ubiquitination both tumor growth and angiogenesis, suggesting that, in of b-catenin, the stability of the b-catenin protein is reg- principle, the application of nutlins in cancer treatment ulated by ubiquitination of cadherins. For example, the could target both tumor cells and angiogenesis. PR-171 c-Cbl–related ubiquitin E3 ligase Hakai associates with (Proteolix, Inc.), a synthetic analog of epoxomicin, is E-cadherin, promoting its degradation and resulting in another proteasome inhibitor that was reported to irre- destruction of the cadherin-b-catenin complex and its versibly inhibit the chymotryptic site of the 26S protea- degradation (88). some, and initial studies suggested that it has more potent Of interest, in addition to regulation of b-catenin protein anticancer activity than bortezomib (98). Moreover, recent levels by ubiquitination, the levels and subcellular func- patent applications indicate that the ubiquitin activating tions of Dvl are tightly regulated via multiple ubiquitin- enzyme, E1, could also be targeted for possible therapeu- dependent pathways. tic use (99, 100). More-comprehensive reviews on inhibi- Binding of the Kelch-like 12 (KLHL12) E3 ligase to Dvl is tion of the 26S proteasome and drug discoveries were regulated by Wnt stimulation. Subsequent ubiquitination recently published (101, 102). Numerous ubiquitin E3 of Dvl leads to its proteasomal degradation (89), suggest- ligases are involved in the regulation of angiogenesis; ing that KLHL12 acts as a Wnt-mediated negative regu- however, it remains to be determined whether a particular lator of the Wnt pathway by inducing the degradation of ubiquitin E3 ligase can be exploited as a target for

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molecular therapeutic approaches in angiogenesis-asso- however, these studies represent only the beginning of ciated diseases. Regardless of whether a particular ubi- our attempt to understand how this important pathway quitin E3 ligase can be targeted for therapeutic approaches functions in the regulation of angiogenesis. Character- in angiogenesis-associated diseases, broad studies are izing the nature of this system in angiogenesis and the clearly required to increase our understanding of their complexity of the UPS will undoubtedly have many role in angiogenesis. In light of the current drug-discovery therapeutic applications. Understanding the molecular activities involving the UPS, it is evident that by harnes- basis of the UPS and the target protein substrates in sing the UPS we may be able to design strategies for endothelial cells may also provide a foundation for different components of the UPS to selectively target learning to stimulate or inhibit angiogenesis. Finally, it proteins for ubiquitination/degradation or inhibit protein is reasonable to envision the UPS as a key component of degradation. Hence, it is not unreasonable to expect more the angiogenic switch in cancer and other types of drug-discovery efforts based on ubiquitin with the aim of pathologic angiogenesis. targeting proteins with pro- and antiangiogenesis activities in the near future. Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed. Conclusions and Perspectives Grant Support The emerging role of the UPS in regulating angiogen- National Eye Institute, National Institutes of Health; Department of esis highlights the importance of investigating this Pathology, Boston University; Massachusetts Lions Foundation (to Department of Ophthalmology, Boston University). pathway in the milieu of angiogenesis. The reports outlined in this review provide examples of regulation Received July 29, 2011; revised October 21, 2011; accepted November 11, of angiogenesis by various components of the UPS; 2011; published OnlineFirst February 21, 2012.

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548 Mol Cancer Ther; 11(3) March 2012 Molecular Cancer Therapeutics

The Ubiquitin-Proteasome System Meets Angiogenesis

Nader Rahimi

Mol Cancer Ther 2012;11:538-548. Published OnlineFirst February 21, 2012.

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