RAHIMI Mol Cancer Ther 2012

Review

The Ubiquitin-Proteasome System Meets Angiogenesis

Nader Rahimi

AbstractA 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, includingVEGF, VEGFR-

2, angiogenic signaling proteins (e.g., the PLCg1 and PI3 kinase/AKT pathways), and other non-VEGFangiogenic pathways. The emerging mechanisms by which ubiquitin modification of angiogenic proteins

control angiogenesis involve bothproteolytic andnonproteolytic 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); 53848. 2012 AACR.

IntroductionAngiogenesis, the growth of new blood vessels from

preexisting vessels, is an important physiological processin thebody that is required for normalwound-healing andfemale reproduction. Pathologic angiogenesis (excessiveor insufficient) is now recognized as a common denom-inator underlying a number of deadly and debilitatinghuman diseases, including cancer, age-related maculardegeneration (AMD), diabetic retinopathy, and cardio-vascular diseases (1, 2). Although in many ways thesediseases have distinct etiologies and mechanisms ofdevelopment, they all share abnormal angiogenesis. Forexample, although cancer has nothing to do with AMD, itshares one of its characteristics, angiogenesis, with AMD(Fig. 1). Acquisition of angiogenesis by tumor cells isconsidered the most critical step in tumor growth andmetastasis. To grow beyond 2 mm in diameter, a tumormust acquire angiogenesis, which is often established byhypoxia-induced expression of VEGF-A and other angio-genesis-inducing molecules (3). To support the growth ofthe expanding tumor, an angiogenic switch is turned on,causing normally quiescent endothelial cells to proliferateand sprout (4). It is now clear that induction of VEGF-Aand other related angiogenesis inducers, and a reductionin the expression of angiogenesis inhibitors, such asthrombospondin (TSP-1), govern the tumor-inducedangiogenic switch (5, 6). Although it was initially thoughtthat angiogenesis plays a substantial role only when the

tumor mass reaches a macroscopic size, it is becomingincreasingly apparent that angiogenesis is instigated inthe early stage of tumor development (5, 7), further sup-porting angiogenesis as a vital component of tumorgrowth and metastasis.

VEGF-A also plays a central role in the development ofchoroidal neovascularization, and indeed is responsiblefor both neovascularization and vascular leakage in wetAMD (8). The hallmarks of wet AMD are drusen forma-tion [i.e., the focal deposition of debris between the retinalpigment epithelium (RPE) and Bruchs membrane], cho-roidal neovascularization, RPE cell detachment, fibrovas-cular scarring, and vitreous hemorrhage. Aberrant bloodvessel growth and blood vessel leakage subsequentlyresult in the loss of central vision (9). Many cell types inthe eye, including RPE cells, pericytes, endothelial cells,glial cells, Muller cells, and ganglion cells, synthesize andsecrete VEGF. In addition to the vital importance of VEGFin the pathology of AMD, elevated VEGF levels alsostrongly correlate with retinal ischemia-associated neo-vascularization in diabetic retinopathy and retinopathy ofprematurity (8, 10).

Once the angiogenic switch is activated, differentsequential steps take place, including the activation ofvarious proteases from activated endothelial cells result-ing in the degradation of the basement membrane sur-rounding the existing vessel, migration of the endothelialcells into the interstitial space, endothelial cell prolifera-tion, sprouting, lumen formation, generation of new base-ment membrane with the recruitment of pericytes, andfusion of the newly formed vessels (11). In general, and inmost pathologic conditions, angiogenesis starts whencells within a tissue respond to hypoxia (i.e., low oxygen)or in certain circumstances when oncogenic gene pro-ducts such as Ras and Myc induce expression of VEGFalong with other hypoxia-inducible genes (12). The VEGFfamily of growth factors includes placental growth factor

Author's Affiliation: Departments of Pathology and Ophthalmology, Bos-ton University School of Medicine, Boston, Massachusetts

Corresponding Author: Nader Rahimi, Department of Pathology, BostonUniversityMedicalCampus, 670AlbanySt., Room510,Boston,MA02118.Phone: 617-638-5011; Fax: 617-414-7914; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0555

2012 American Association for Cancer Research.

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(PlGF),VEGF-A,VEGF-B,VEGF-C,VEGF-D, andVEGF-E(13). VEGF-A isoforms result from alternative splicing ofmessenger RNA (mRNA), and VEGF-165 is considered tobe the most common VEGF-A isoform (14). Three differ-ent VEGF gene products (PlGF, VEGF-A, and VEGF-B)have been identified as ligands for VEGFR-1. VEGF-A,VEGF-D, and VEGF-C bind to and activate VEGFR-2 (13).VEGF-C and VEGF-D are also known to recognizeVEGFR-3 (also called FLT4), a receptor tyrosine kinase(RTK) that is expressed predominantly by lymphaticendothelial and hematopoietic progenitor cells (15).VEGF-E is a virally encoded VEGF-like protein that selec-tively binds to VEGFR-2 (16). VEGF family proteinsalso interact with non-RTK cell surface receptors, includ-ing neuropilin-1 and neuropilin-2, which are character-ized as coreceptors for VEGF family ligands (13, 17).Among the VEGF receptors and coreceptors, activationof VEGFR-2 by VEGF family ligands is considered themost critical event in angiogenesis (18, 19). BeyondVEGF-

mediated VEGFR-2 activation, recent studies haveshown that under certain circumstances, VEGFR-2 canalso be activated by non-VEGF family ligands, includ-ing heparan sulfate proteoglycans (20) and galectin-3, aglycan-binding protein (21). Heparan sulfate proteogly-cans have been proposed to potentiate VEGFR-2 acti-vation through cis- and trans-binding with VEGFR-2and VEGF complex (20), where galectin-3 is thought topotentiate VEGFR-2 activation by prolonging its pres-ence in the plasma membrane (21). Although VEGFfamily proteins are prominent regulators of angiogen-esis, several other growth factors and cytokines, includ-ing angiopoietin-1, Del-1, fibroblast growth factor,hepatocyte growth factor, interleukin-8 (IL-8), and lep-tin, are known to stimulate angiogenesis (22), addingfurther complexity to the regulation of angiogenesis.

The role of VEGFR-2 in angiogenesis is well estab-lished, and more-comprehensive reviews on the roleof VEGFR-2 in angiogenesis were recently published

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

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(13, 17, 19). Figure 2 summarizes VEGF superfamilyligands and their interactions with VEGF receptors (Fig.2). It is increasingly evident that angiogenic signaling isestablished through an elegant and complex system ofVEGF and non-VEGF ligands, and VEGF receptors andcoreceptors, resulting in homo- and heterodimeric acti-vation of VEGF receptors and in turn the complexprocesses of angiogenesis.

Ubiquitin-Proteasome SystemUbiquitin is an evolutionarily conserved polypeptide

that consists of 76 amino acids and was named forits ubiquitous expression in eukaryotes. Ubiquitin isactivated by a ubiquitin-activating enzyme, E1, in anATP-dependent manner and is transferred to a ubiqui-tin-conjugating enzyme, E2. Eventually, a ubiquitin-protein ligase, E3, specifically attaches the ubiquitinmolecule to a target protein through the e-amino groupof a lysine residue (Fig. 3). E3 ubiquitin ligases are a

large family of proteins (with almost 700 in the humangenome) that are known to be involved in regulating theturnover and activity of many target proteins (23). E3ligases are divided into 2 large groups: the homology tothe E6-associated protein carboxyl terminus (HECT)domain-containing E3 ligases, and the Really Interest-ing New Gene (RING) domain-containing E3 ligases.The RING-type E3 ligases include single subunit E3ligases (such as Cbl family E3 ligases) and multisubunitE3 ligases (such as Cullin-RING ubiquitin ligases). Inrecent years, additional E3 ligases have been identifiedthat use different domains to recognize E2-conjugatingenzymes, such as plant homeodomain domain-contain-ing E3 ligases and the U-box E3 ligases (23, 24).Although conjugation of ubiquitin to target proteinswas initially recognized as a signal for protein degra-dation by the 26S proteasome, it is now recognized thatubiquitination regulates a broad range of cellular func-tions, including protein processing, membrane traffick-ing, and transcriptional regulation (23, 25). Recent

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

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studies showed that ubiquitination can also influencecell signaling by targeting activation of proteins in aproteolysis-independent manner (2628).Multiple ubiquitin molecules can be attached to a target

proteinbymeansofmonoubiquitination (i.e., attachmentofa single ubiquitin to 1 or multiple lysine residues). Mono-ubiquitination is regarded as a signal for nonproteolyticevents suchasendocytosis,histoneregulation,DNArepair,virus budding, and nuclear export (25). Alternatively, ubi-quitin can be attached to a target protein in the form ofpolyubiquitination, where multiple ubiquitin moleculesare attached to a single lysine residue. Ubiquitin contains7 different lysine residues that potentially can be used forubiquitin-chain assembly. Lys48- and Lys29-linked poly-ubiquitination generally is associated with degradation oftarget proteins by the 26S proteasome, whereas Lys63-linkedpolyubiquitination is involved inDNArepair, signaltransduction, andendocytosis, butnotdegradation (23, 25).Clearly, the ubiquitin machinery has evolved to play aversatile role in protein functions ranging from proteinturnover to subcellular localization and kinase activation,andconsequently it ishighlyrelevant to thepathobiologyofmany human diseases.The ubiquitin-proteasome system (UPS) consists of 2

major components: substrate-recruiting enzymes (E1, E2,and E3) and substrate-degrading enzymes. E1 activates

the polypeptide ubiquitin in an ATP-dependent manner,enabling its transfer onto the ubiquitin carrier enzyme, E2.Activated ubiquitin is then transferred by the ubiquitinprotein ligase, E3, to a substrate protein (29). The sub-strate-recruiting components of UPS then catalyze theformation of an isopeptide bond between the C-terminalglycine residue of ubiquitin and the e-amino group of asubstrate protein lysine residue. Continual addition ofubiquitinmoieties onto substrate (i.e., polyubiquitination)facilitates recognition of the substrate by the proteolyticmachinery of theUPS, the 26Sproteasome (29, 30). The 26Sproteasome complex is essentially composed of 1 20S and2 19S units (Fig. 3). The 19S complex has 2 multisubunitcomponents, often described as the base and the lid. Thebase contains 6 ATPases, which belong to the TRIPLE-Afamily of ATPases, and 2 non-ATPase subunits, whichbind to the 20S catalytic core. The lid containsup to 10non-ATPase subunits (31, 32). Together, the base and lidfunction in the recognition of ubiquitinated substratesand their subsequent binding. The 20S complex is com-posed of 28 related subunits (14 different subunits) thatare arranged as 4 heptameric staggered rings. The 2 outerrings contain the a subunits (a1a7). The 2 inner ringscontain 2 copies of the b subunits (b1b7). Within the 20Sproteasome, subunits b1, b2, and b5 exhibit postglutamylpeptide hydrolyzing, trypsin-like, and chymotrypsin-like

Figure 3. Schematic of the UPS. Ubiquitin is activated by the ubiquitin-activating enzyme (E1) and then transferred to a ubiquitin-conjugating enzyme (E2). E2transfers the activated ubiquitin moieties to the protein substrate that is bound specifically to a particular ubiquitin ligase (E3). The transfer of ubiquitintakes 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 domainligases). Repeated conjugation of ubiquitinmoieties to eachother generatesapolyubiquitin chain that serves as thebinding anddegradation signal for the 26Sproteasome. The protein substrate is degraded, generating short peptides and free ubiquitin that can be further reused. Ub, ubiquitin.

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cleavage activity, respectively. More-comprehensivereviews on the function and composition of the 26Sproteasome were recently published (29, 30, 32, 33).

Regulation of VEGF expression by von Hippel-Lindau E3 ubiquitin ligase

The molecular mechanism by which hypoxia sets offexpression of VEGF has been extensively studied(12, 34). In normoxic conditions (i.e., normal oxygenlevels), VEGF expression is generally inhibited by theinteraction of von Hippel-Lindau (pVHL) E3 ubiquitinligase with hypoxia-induced transcription factor 1a(HIF-1a), leading to its ubiquitination and targetingHIF-a for degradation by 26S-proteasome. Two of the3 HIF-a isoforms, HIF-1a and HIF-2a, are closely relat-ed and can interact with hypoxia response elements toinduce VEGF expression (35, 36), whereas HIF-3aappears to be involved in the negative regulation ofhypoxia-induced gene expression (37).

Oxygen-mediated posttranslational modification ofHIF-a through non-heme and iron-dependent oxyge-nases that uniquely hydroxylate specific HIF-a at prolineresidues regulates its transcriptional activity. Hydroxyl-ation of human HIF-1a at 2 proline residues (Pro402 andPro564) by prolyl hydroxylase domain (PHD) proteinscreates binding sites for the pVHL E3 ubiquitin ligasecomplex that targets HIF-1a for proteasomal degradation(38, 39). These proline hydroxylation sites contain a con-served LXXLAP (where X indicates any amino acid)motifthat is recognized by PHD proteins, leading to HIF-1a

hydroxylation by PHD (38, 40). Of interest, hydroxylationof an asparagine residue (Asn803) in the C-terminal acti-vation domain of HIF-1a by HIF asparagine hydroxylase,termed factor inhibiting HIF (FIH), inhibits HIF-1a activ-ity by blocking interaction of the HIF-1a C-terminal acti-vation domain with the transcriptional coactivator, p300(41). Under hypoxic conditions, however, VEGF is gen-erally overproduced,which leads to pathologic angiogen-esis. In response to low oxygen, pVHL E3 ubiquitin ligaseis S-nitrosylated (the covalent attachment of a nitrogenmonoxide group to the thiol side chain of cysteine), whichblocks HIF-a interaction with pVHL E3 ubiquitin ligase.HIF-1a escapes fromubiquitin-mediateddegradation as aconsequence of S-nitrosylation of pVHL (Fig. 4). Nitricoxidemediated S-nitrosylation of HIF-1a at the cysteineresidue (C800) permits interaction of HIF-a with p300,prompting its transcription activity and VEGF expression(42).

Another important aspect and additional complexity ofHIF-a regulation is the function of heat shock protein 90(Hsp90). Hsp90 is often upregulated under cellular stressconditions, such as hypoxia (43), which appears to pre-ventHIF-1adegradation in a pVHL-independentmanner(4345). Consistent with the protective role of Hsp90 inHIF-1a, agents that inhibit Hsp90 activity have also beenshown to promote ubiquitin-mediated degradation ofHIF-1a (44, 46). A recent study indicated that inhibitionof Hsp90 by hemin, a derivative of the protoporphyrincompound, increases HIF-1a ubiquitination and henceangiogenesis (47).

Figure4. Role of pVHL in expression of VEGFandangiogenesis. A, in thepresenceof oxygen, proline residues in theoxygen-dependent degradation domain ofHIF 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 isdisallowed. HIF-a stimulates expression of VEGF, which in turn stimulates angiogenesis, as manifested in cancer progression. S-nitrosylation is the covalentattachment of a nitrogen monoxide group to the thiol side chain of cysteine.

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b-Transducin repeat-containingproteinubiquitinE3ligase controls ubiquitination and degradation ofVEGFR-2Activation of VEGFR-2 by VEGF family ligands med-

iates most of the known VEGF cellular responses(13, 19, 48). Central to proper regulation of the angiogenicactivity of VEGFR-2 is the process by which VEGFR-2triggers its own internalization and degradation, conse-quently terminating its angiogenic signaling. Upon stim-ulation with VEGF family proteins, VEGFR-2 is removedfrom the cell membrane and undergoes clathrin-depen-dent endocytosis (49), which initiates its degradation (50)and recycling (51). Of interest, VEGFR-2 internalization isstabilized by cadherin-5 (52). Cadherin-5dependent sta-bilization of VEGFR-2 is established by reducing thetyrosinephosphorylation ofVEGFR-2, perhaps by recruit-ing tyrosine phosphatases to VEGFR-2 (49, 53). Ligand-mediated degradation of VEGFR-2 requires tyrosinekinase activity, and activation of the protein kinase C(PKC) pathway accelerates its degradation (50). On theother hand, activation of p38 mitogen-activated proteinkinase (MAPK) has been shown to stabilize VEGFR-2 (52),suggesting that the stability and degradation of VEGFR-2in endothelial cells are highly fine-tuned by the activitiesof the PKC and p38 MAPK pathways. Initial studiesshowed that the carboxyl terminal of VEGFR-2 plays apivotal role in VEGFR-2 stability and degradation. Pro-gressivedeletion of the carboxyl terminal of VEGFR-2wasshown to inhibit ligand-dependent degradation ofVEGFR-2 (48, 50). A recent study identified the presenceof a PEST domain in the carboxyl domain of VEGFR-2,which may account for the critical role of the carboxyldomain in VEGFR-2 degradation (52). The PEST motif[rich in proline (P), glutamic acid (E), serine (S), andthreonine (T)] is considered to be a signature of short-lived proteins that are degraded by the ubiquitin pathway(54). It is thought that PEST sequences are unstructuredregions in certain protein sequences,whichmay serve as aphosphodegron for the recruitment of F-boxcontainingubiquitin E3 ligases leading to ubiquitination and degra-dation (55, 56). Phosphorylation of Ser1188 and Ser1191 ofthe PEST domain of VEGFR-2 recruits SCF-b-transducinrepeat-containing protein 1 (Trcp1) E3 ubiquitin ligaseto VEGFR-2, leading to SCF-bTrcp1dependent ubiquiti-nation and degradation of VEGFR-2. Degradation ofVEGFR-2 is mainly attained through Lys48-linked poly-ubiquitination (52).b-TrCPs (also called FWD1) belong to a larger family of

Fbw (F-box/WD40 repeat containing) proteins that aregenerally characterized by the presence of a 4248 amino-acid F-boxmotif at the N-terminus and 7WD40 repeats atthe C-terminus (57). b-TrCPs are highly conserved acrossspecies, in particular within the F-box motif and WD40repeats motif. Human b-TrCP1 and b-TrCP2 exist inmultiple isoforms due to alternatively spliced mRNA,but all are conserved in F-box and all 7 WD40 repeats(58, 59). The most notable differences in sequencesbetween b-TrCP1 and b-TrCP2 are found in their N-

terminal regions, which are proximal to the F-box motif.It is thought that theN-terminal sequences ofb-TrCP1 andb-TrCP2 allow these proteins to undergo homo- andheterodimerization (60). It appears that both b-TrCP1 andb-TrCP2 can promote ubiquitination of VEGFR-2, whichsuggests that to some extent they may act in a redundantfashion in VEGFR-2 ubiquitination (52). A redundant roleof mammalian b-TrCP1 and b-TrCP2 in ubiquitinationand degradation of other proteins, including IkB andb-catenin, has also been suggested (61, 62).

Role of ubiquitination in phospholipase Cg1activation and angiogenesis

Activation of phospholipaseCg1 (PLCg1) in endothelialcells is considered to be one of the chief mediators of theangiogenic signaling of VEGFR-2. It catalyzes the forma-tion of inositol 1,4,5-trisphosphate (IP3) and diacylgly-cerol from phosphatidylinositol 4,5-bisphosphate (PIP2).

Phosphotyrosine 1173 on mouse VEGFR-2 (corre-sponding to Tyr1175 on human VEGFR-2) has been iden-tified as the primary site responsible for the recruitment ofPLCg1 to VEGFR-2 (26, 63, 64). Substantial informationobtained from animal models links PLCg1 to angiogene-sis. The initial evidence linking PLCg1 to endothelial cellfunction and angiogenesis was provided by targeteddeletion of PLCg1, which resulted in early embryoniclethality between embryonic days 9.5 and 10.5 due tosignificantly impaired vasculogenesis and erythrogenesis(65). Inactivation of PLCg1 in zebrafish was also shown tobe required for VEGF function and arterial development(66). Additional evidence of the importance of PLCg1 inangiogenic signaling of VEGFR-2 was obtained by phar-macological inhibition of PLCg1. U73122, a potent PLCg1inhibitor, was shown to inhibit endothelial cell tube for-mation in vitro (64) and angiogenesis in vivo in a chorio-allantoicmembrane assay (67). Silencing the expression ofPLCg1 in primary endothelial cells by an siRNA strategyalso inhibits VEGF-mediated endothelial cell tube forma-tion and proliferation (68), further underscoring theimportance of the PLCg1 pathway for angiogenic signal-ing of VEGF.

PLCg1 is a multidomain protein that consists of 2 SH2domains and 1 SH3 domain between the catalyticdomains. The SH2 domains recognize phosphotyrosine1173 on VEGFR-2 (64), whereas the SH3 domain recog-nizes proline-rich sequences (PXXPmotifs). In addition toits SH domains, PLCg1 also contains a C2 domain, EFhand, and2putativePHdomains. Thepresence of bothN-and C-terminal SH2 domains is required for optimalbinding of PLCg1 to VEGFR-2 (64). PLCg1 also interactswith c-Cbl through its proline-richmotif in a noninduciblemanner (50). As a result of activation by VEGFR-2, c-Cblis recruited to VEGFR-2 and distinctly inhibits phosphor-ylation of Tyr783 on PLCg1 in an ubiquitination-depen-dent manner (26, 68). c-Cbl negatively regulates PLCg1activation in a proteolysis-independent manner. Insteadof targeting it for degradation, c-Cbl distinctivelymediates ubiquitination of PLCg1 and suppresses its

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phosphorylation on Y783 (26). How PLCg1 can escapefrom ubiquitin-mediated degradation but then enter intoa less enzymatic active state is a conundrum thatwarrantsfurther investigation.

Endothelial cells derived fromc-Cbl knockoutmice alsoshowed that loss of c-Cbl results in an increased phos-phorylation of PLCg1 with no apparent effect on its half-life (68). Overexpression of c-Cbl in endothelial cells hasalso been shown to inhibit tube formation and sproutingof endothelial cells. Conversely, overexpression of c-Cbl(70Z/3-Cbl), an E3 ligase-deficient variant form of c-Cbl,or silencing its expression by siRNA elevated sprouting ofendothelial cells (26). Recent studies showed that VEGF-and tumor-induced angiogenesis is highly elevated inc-Cbl nullizygous mouse (67, 68). It appears that the roleof c-Cbl in angiogenesis is widespread, because laser-induced angiogenesis in c-Cbl knockout mice also resultsin enhanced retinal neovascularization (67).

Role of ubiquitination in the PI3 kinase/AKTpathway

The phosphoinositide 3-kinase (PI3K) signal transduc-tion pathway is one of the main signaling routesthat VEGFR-2 uses to stimulate endothelial cell survivaland proliferation (6971). VEGFR-2 activates PI3Kthrough recruitment of p85 of PI3K involving Tyr799 andTyr1173 (67, 68). PI3K consists of an 85-kDa regulatorysubunit and a 110-kDa catalytic subunit. It is a lipidkinase that converts the plasma membrane lipid PIP2 tophosphatidylinositol-3,4,5-triphosphate (PIP3). Proteinswith pleckstrin-homology (PH) domains, such as proteinkinase B (PKB/AKT), phosphoinositide-dependentkinase-1 (PDK-1), and PDK-2, bind to PIP3. AKT is acti-vated by PIP3, PDK1, and PDK2, leading to phosphory-

lation of a host of other proteins that affect cell prolifer-ation, cell cycle progression, and cell survival (73). TheCbl-b ubiquitinE3 ligase is known to interactwith thep85-SH2 domain and catalyze p85 polyubiquitination (27,74).Of interest, the Cbl-mediated ubiquitination does not leadto degradation of p85 (27).

AKT, a serine/threonine protein kinase, is one of thekey PI3K substrates that play a central role in mediatingVEGFR-2dependent cellular events in endothelial (75,76). It was recently shown that the carboxyl terminus ofHsc-70-interacting protein (CHIP) interactswithAKTandinduces its ubiquitination (77). In addition, tetratricopep-tide repeat domain 3 (TTC3) containing E3 ligase wasrecently linked to AKT ubiquitination and degradation(78, 79). Of interest, TTC3 interacts only with active AKT,and not inactive AKT, in the nucleus (78, 79), indicatingthat perhaps TTC3-mediated AKT ubiquitination isimportant for controlling AKT signaling in the nucleus.TTC3 itself is the target of AKT and is phosphorylated atS378 by AKT, and this phosphorylation appears to benecessary for TTC3 E3 ligase activity (78, 79). BRCA1 isanother E3 ligase that also interacts with activated AKTand targets it for ubiquitination and degradation (80).Recent studies also showed that AKT is ubiquitinated byTRAF6 E3 ligase. TRAF6 directly interacts with andinduces AKT ubiquitination (28). TRAF6-mediated AKTubiquitination takes place through the K63-linked mod-ification and does not triggerAKTdegradation. K63 chainpolyubiquitination of AKT contributes to its membranelocalization, where it is phosphorylated (28). Figure 5summarizes various ubiquitin E3 ligases that are involvedin fine-tuning the abundance and activation of key angio-genic proteins. Some ubiquitin E3 ligases, such as Cblfamily proteins, target multiple angiogenic proteins,

Figure 5. Regulation of core angiogenic proteins by ubiquitin E3ligases. Expression of VEGF is regulated by activity of pVHL.bTrcp1 ubiquitinates VEGFR-2 and subjects it to proteasomaldegradation. c-Cbl catalyzes ubiquitination of numerousangiogenic signaling proteins, including VEGFR-1, Eph, Tie2, andPLCg1. AKT abundance and activity are regulated by TRAF6,CHIP, and TTC3. Itch regulates degradation of the Notch1receptor.

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whereas in other cases more than 1 ubiquitin E3 ligase isinvolved in the ubiquitination of an angiogenic protein, asillustrated for AKT (Fig. 5).

Role of Ubiquitination in Wnt SignalingTheWntpathway is another keyplayer in angiogenesis.

Binding of Wnt to its 7-span transmembrane receptor,Frizzled (Fz), and its coreceptor, Lrp5/6, at the cell surfaceinitiates a signaling cascade that mediates angiogenesisand other key developmental processes, including stemcell maintenance, growth, and cell-fate specification, andcell migration (81). Deregulation of the activation ofWnt/b-catenin signaling has been linked to a range of humandiseases, including cancer (81, 82).During the resting stateof canonical Wnt signaling, several key Wnt-associatedsignaling proteins, including b-catenin, are targeted viaubiquitination for degradation. Initially, the adenomatouspolyposis coli protein forms a complex with glycogensynthase kinase 3b (GSK-3b) and axin. This complex thenbinds to b-catenin in the cytoplasm, which leads to phos-phorylation of b-catenin by casein kinase 1 (CK1) andGSK-3b. Phosphorylation leads to the creation of a phos-phodegronmotif on b-catenin that allows the ubiquitin E3ligases (e.g., b-Trcp) and Jade-1 to recognize b-catenin. Asa result, b-catenin is targeted for ubiquitination, leading toits 26S-proteasomemediated degradation (83, 84).In addition, other ubiquitin E3 ligases (e.g., Siah1 and

Ozz) also target b-catenin for degradation in a cell-typeor context-specific manner (85, 86).Removal of cytosolic b-catenin through the UPS pre-

ventsb-catenin from translocating into the nucleus,whereit acts as a transcription factor for genes associated withvarious angiogenic events, such as proliferation of endo-thelial cells. In contrast, activation of the canonical Wntsignaling pathway results in inhibition of b-catenin deg-radation, leading to increased cytosolic b-catenin, whichthen translocates to the nucleus. In the nucleus, b-cateninassociates with at least one of a family of Tcf/Lef tran-scription factors and induces the expression of numerousgenes, such as cyclinD1 and c-myc (87), which are impli-cated in cellular proliferation.In addition to Wnt pathwaymediated ubiquitination

of b-catenin, the stability of the b-catenin protein is reg-ulated by ubiquitination of cadherins. For example, thec-Cblrelated ubiquitin E3 ligase Hakai associates withE-cadherin, promoting its degradation and resulting indestruction of the cadherin-b-catenin complex and itsdegradation (88).Of interest, in addition to regulationofb-cateninprotein

levels by ubiquitination, the levels and subcellular func-tions of Dvl are tightly regulated via multiple ubiquitin-dependent pathways.Bindingof theKelch-like 12 (KLHL12)E3 ligase toDvl is

regulated byWnt stimulation. Subsequent ubiquitinationof Dvl leads to its proteasomal degradation (89), suggest-ing that KLHL12 acts as a Wnt-mediated negative regu-lator of the Wnt pathway by inducing the degradation of

Dvl. Surprisingly, in neuronal cells, Dvl uniquely is ubi-quitinated by theHECT-type E3 ligaseNEDL1, andnot byKLHL12 (90), suggesting a cell-typespecific regulation ofDvl by the UPS.

The ubiquitination system as a potential target forantiangiogenesis and anticancer therapy

Given that the expression and degradation of coreproangiogenic proteins are regulated by the UPS, selec-tively targeting the different components of this pathwaymay prove to be an effective strategy for antiangiogenesistreatments. Our increasing understanding of the ubiqui-tination system and its role in angiogenesis is generatinggreat interest in the development of novel strategies toblock pathologic angiogenesis. The role of the ubiquitina-tion pathway in human diseases in general, and in angio-genesis in particular, is still unclear because themolecularmechanisms and gene products involved in the UPS arenot fully understood. Moreover, there has been no com-prehensive analysis of the functional importance of theUPS in angiogenesis-associated human diseases. TheUPShas many different components that potentially could betargeted for inhibition or stimulation in the milieu ofangiogenesis. For example, the therapeutic value of theproteasome inhibitor bortezomib (Velcade; MillenniumInc.), the first UPS-targeting drug to be approved by theU.S. Food and Drug Administration for treatment ofrelapsed or refractory multiple myeloma (91), could beexplored in angiogenesis-associated diseases.

Recent studies have linked the potential therapeuticvalue of inhibition of the proteasome pathway to angio-genesis. Indeed, treatment of endothelial cells in a cellculture system with proteasome inhibitors was shown toinhibit capillary tube formation of endothelial cells andblood vessel formation in an embryonic chick chorioal-lantoic membrane assay (92, 93). Moreover, bortezomibwas shown to inhibit tumor angiogenesis in a murinexenograft model (94). More recently, it was shown thatsmall molecules such as nutlins (Roche, Inc.) and RITAcan block p53 ubiquitination by inhibiting the activity ofMDM2 ubiquitin ligase, which is known to mediate p53ubiquitination (9597). Loss of p53 activity is linked toboth tumor growth and angiogenesis, suggesting that, inprinciple, the application of nutlins in cancer treatmentcould target both tumor cells and angiogenesis. PR-171(Proteolix, Inc.), a synthetic analog of epoxomicin, isanother proteasome inhibitor that was reported to irre-versibly inhibit the chymotryptic site of the 26S protea-some, and initial studies suggested that it hasmore potentanticancer activity than bortezomib (98).Moreover, recentpatent applications indicate that the ubiquitin activatingenzyme, E1, could also be targeted for possible therapeu-tic use (99, 100). More-comprehensive reviews on inhibi-tion of the 26S proteasome and drug discoveries wererecently published (101, 102). Numerous ubiquitin E3ligases are involved in the regulation of angiogenesis;however, it remains to bedeterminedwhether aparticularubiquitin E3 ligase can be exploited as a target for

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molecular therapeutic approaches in angiogenesis-asso-ciated diseases. Regardless of whether a particular ubi-quitinE3 ligase canbe targeted for therapeutic approachesin angiogenesis-associated diseases, broad studies areclearly required to increase our understanding of theirrole in angiogenesis. In light of the current drug-discoveryactivities involving the UPS, it is evident that by harnes-sing the UPS we may be able to design strategies fordifferent components of the UPS to selectively targetproteins for ubiquitination/degradation or inhibit proteindegradation. Hence, it is not unreasonable to expect moredrug-discovery efforts based on ubiquitin with the aim oftargeting proteins with pro- and antiangiogenesis activ-ities in the near future.

Conclusions and PerspectivesThe emerging role of the UPS in regulating angiogen-

esis highlights the importance of investigating thispathway in the milieu of angiogenesis. The reportsoutlined in this review provide examples of regulationof angiogenesis by various components of the UPS;

however, these studies represent only the beginning ofour attempt to understand how this important pathwayfunctions in the regulation of angiogenesis. Character-izing the nature of this system in angiogenesis and thecomplexity of the UPS will undoubtedly have manytherapeutic applications. Understanding the molecularbasis of the UPS and the target protein substrates inendothelial cells may also provide a foundation forlearning to stimulate or inhibit angiogenesis. Finally, itis reasonable to envision the UPS as a key component ofthe angiogenic switch in cancer and other types ofpathologic angiogenesis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Grant SupportNational Eye Institute, National Institutes of Health; Department of

Pathology, Boston University; Massachusetts Lions Foundation (toDepartment of Ophthalmology, Boston University).

Received July 29, 2011; revisedOctober 21, 2011; acceptedNovember 11,2011; published OnlineFirst February 21, 2012.

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