Emerging Role ofAngiogenin in StressResponse and Cell SurvivalUnder Adverse ConditionsSHUPING LI1,2 AND GUO-FU HU1,3*1Molecular Oncology Research Institute, Tufts Medical Center, Boston, Massachusetts2State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital,

Zhejiang University School of Medicine, Hangzhou, China3Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts

Angiogenin (ANG), also known as ribonuclease (RNASE) 5, is a member of the vertebrate-specific, secreted RNASE superfamily. ANGwas originally identified as a tumor angiogenic factor, but its biological activity has been extended from inducing angiogenesis to stimulatingcell proliferation and more recently, to promoting cell survival. Under growth conditions, ANG is translocated to nucleus where itaccumulates in nucleolus and stimulates ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. Under stressconditions, ANG is accumulated in cytoplasmic compartments and modulates the production of tiRNA, a novel class of small RNA that isderived from tRNA and is induced by stress. tiRNA suppress global protein translation by inhibiting both cap-dependent and -independenttranslation including that mediated by weak IRESes. However, strong IRES-mediated translation, a mechanism often used by genesinvolved in pro-survival and anti-apoptosis, is not affected. Thus, ANG-mediated tiRNA reprogram protein translation, save anabolicenergy, and promote cell survival. This recently uncovered function of ANGpresents a novelmechanismof action in regulating cell growthand survival.J. Cell. Physiol. 227: 2822–2826, 2012. � 2011 Wiley Periodicals, Inc.

Angiogenin

Angiogenin (ANG) belongs to a gene superfamily that encodessecreted ribonuclease (RNASE) existing only in vertebrates.This family of enzyme was previously known as pancreaticRNASE superfamily because the prototypic member of thefamily is bovine pancreatic RNASE A (Cho and Zhang, 2006).Upon the completion of human genome sequencing, it becameclear that this is the only family of enzymes that is vertebrate-specific. During the 8th International Conferences on RNASESheld in Naples, Italy in October 20–22, 2010, GiuseppeD’Alessio of University of Naples recommended the use of themore accurate and informative nomenclature ‘‘VertebrateSecreted RNASES’’ to replace the old term ‘‘PancreaticRNASES’’.

ANG is the 5th member of the human family of RNASES. Itencodes a 14 kDa protein consisting of 123 amino acid residues(Strydom et al., 1985). Although ANG is a small molecule, threedistinct function sites including a receptor-binding site, anuclear localization sequence (NLS), and a catalytic site havebeen identified. The loop region from K60 to N68 is thereceptor-binding site that interacts with a to-be-identified cellsurface receptor (Hallahan et al., 1991). Upon binding to the cellsurface receptor, ANG is internalized and translocated to thenucleus (Moroianu and Riordan, 1994b). The nucleartranslocation process is mediated by an NLS located betweenM30 and G34 (Moroianu and Riordan, 1994a). Theribonucleolytic activity of ANG, executed by the catalytic triadH13, K40, and H113 (Shapiro and Vallee, 1989), is believed tofunction in stimulating rRNA transcription after ANG islocalized in the nucleus. All three sites are essential for ANG tohave angiogenic and growth stimulating activities.

ANG-Stimulated rRNA Transcription in Angiogenesis

As a tumor angiogenic factor, the role of ANG in angiogenesishas been well documented. ANG has been shown to be

upregulated in a variety of human cancers (Li and Hu, 2010).Elevated serum level of and/or enhanced tissue expression ofANG have been noticed in all types of solid and blood cancersso far examined. A major mechanism by which ANG inducesangiogenesis is related to its activity in stimulating ribosomalRNA (rRNA) transcription (Li and Hu, 2010). ANG has beenshown to undergo nuclear translocation in endothelial cells(Moroianu and Riordan, 1994b) where it binds to the promoterregion and stimulates rRNA transcription (Xu et al., 2002,2003). rRNA transcription is the rate-limiting step in ribosomebiogenesis (Moss, 2004), a process required for cell growth aswell as maintenance and survival as proteins are required foressentially all cellular activities. ANG-stimulated rRNAtranscription has been shown to be permissive for otherangiogenic factors to induce angiogenesis (Kishimoto et al.,2005). For example, knockdown of ANG expression orinhibition of ANG activity in endothelial cells resulted in adecrease of rRNA transcription and insensitivity to growthstimuli such as VEGF and bFGF (Kishimoto et al., 2005).Compelling evidence indicates that ANG activity is necessaryfor other angiogenic factors to induce angiogenesis.ANG-induced rRNA transcription appears to be a commondownstream event of tumor angiogenesis (Kishimoto et al.,2005). Thus, ANG inhibitors have been shown to inhibit not

Contract grant sponsor: National Institutes of Health;Contract grant numbers: R01NS065237, R01CA105241.

*Correspondence to: Guo-Fu Hu, Molecular Oncology ResearchInstitute, Tufts Medical Center, 800 Washington Street, #5609,Boston, MA 02111. E-mail: ghu@tuftsmedicalcenter.org

Received 15 September 2011; Accepted 28 September 2011

Published online in Wiley Online Library(wileyonlinelibrary.com), 20 October 2011.DOI: 10.1002/jcp.23051

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only ANG-induced angiogenesis but also those induced byother angiogenic factors including VEGF, FGF, and EGF(Hirukawa et al., 2005). ANG inhibitors would therefore have aprofound effect in inhibiting tumor angiogenesis. Indeed, ANGinhibitors such as its siRNA (Ibaragi et al., 2009a), antisense(Olson et al., 2001), monoclonal antibodies (Olson et al., 2002),soluble binding proteins (Olson et al., 1995), enzymaticinhibitors (Kao et al., 2002), and nuclear translocation blockers(Hirukawa et al., 2005; Yoshioka et al., 2006; Ibaragi et al.,2009b) have all been shown to inhibit tumor angiogenesis andcancer progression in various animal models.

ANG-Mediated rRNA Transcription in CancerCell Proliferation

Sustained growth is a hallmark of cancer (Hanahan andWeinberg, 2000). Cancer cells are constantly proliferating andthus require robust ribosome biogenesis to meet the highmetabolic demand. Ribosomal biogenesis is a process involvingrRNA transcription, processing of the pre-rRNA precursor,and assembly of four mature rRNA with 79 ribosomal proteins(Nazar, 2004). Genetic, epigenetic, and environmental factorsthat cause cancers are known to enhance the production ofribosomal proteins (Ruggero and Pandolfi, 2003). However, itwas less clear how rRNAs are proportionally increased as theyneed to be incorporated with ribosomal proteins in an equalmolar level to make a functional ribosome. In order to knowwhether rRNA transcription in cancer cells is modulated byANG, a series of experiments using prostate cancer as a modelhas been performed to understand the role of ANG in rRNAtranscription, ribosome biogenesis, and cell growth (Yoshiokaet al., 2006; Ibaragi et al., 2009a,b; Li et al., 2011). Human ANG issignificantly and progressively upregulated in prostate cancer(Majumder et al., 2003; Katona et al., 2005). Mouse Ang is thehighest upregulated gene in AKT-induced prostateintraepithelial neoplasia (PIN) in murine prostate-restrictedAKT kinase transgenic (MPAKT) mice (Majumder et al., 2003).Knockdown of ANG expression in human prostate cancer cellsreduced rRNA transcription and inhibited both in vitro and invivo growth (Yoshioka et al., 2006). Knockdown ofmouse Ang1in MPAKT mice prevented PIN formation and reversedestablished PIN as a result of reduced rRNA transcription(Ibaragi et al., 2009a). ANG activity was therefore thought to bea requirement for excessive cancer cell proliferation as itprovides the extra rRNAneeded to sustain continuous growth.AKT and ANG pathways therefore mediate the production ofribosomal proteins and rRNAs, respectively, and a coordinatedaction of AKT and ANG allows ribosome biogenesis to takeplace (Li et al., 2011). Consistently, there is a crosstalk betweenAKT and ANG pathways (Fig. 1). For example, ANG activatesAKT (Trouillon et al., 2010), and activated AKT in turnstimulates nuclear translocation of ANG (Kieran et al., 2008;Ibaragi et al., 2009b). These results clearly demonstrate thatANG-stimulated rRNA transcription is a critical component inprostate cancer progression (Yoshioka et al., 2006; Ibaragi et al.,2009a). The role of ANG in other types of cancer is lessunderstood. However, ANG has also been shown to beinvolved in proliferation of HeLa cervical cancer cells, MB-435breast cancer cells, HT-29 colon adenocarcinoma cells, andA432 epidermoid carcinoma cells (Hirukawa et al., 2005; Tsujiet al., 2005). Judging from the universal upregulation of ANG invarious types of human cancer, ANG-stimulated rRNAtranscription could well be a general requirement for cancercell proliferation.

These results extended the role of ANG from inducingtumor angiogenesis to directly stimulating cancer cellproliferation. ANG seems to play a double role in cancerprogression by stimulating rRNA transcription in bothendothelial cells and cancer cell. ANG inhibitors, including

siRNA and nuclear translocation blockers, have been shown toinhibit tumor growth in animal models by inhibiting bothangiogenesis and cancer cell proliferation (Yoshioka et al., 2006;Ibaragi et al., 2009b).

ANG is Responsible for Stress-Induced tRNA Cleavage

A series of recent publications has further extended thebiological activity of ANG from enabling cell growth andproliferation to sustaining survival under adverse conditions (Fuet al., 2009; Yamasaki et al., 2009; Emara et al., 2010; Ivanovet al., 2011). In an effort to identify liver-specific miRNA,Xiaofen Zheng laboratory at Beijing Institute of RadiationMedicine cloned and profiled small RNAs (<200 nt) from fetalliver, and was somewhat surprised that 85 of the total 205clones they sequenced were from tRNA (Fu et al., 2005). Moreunexpectedly, these tRNA fragmentswere seemingly producedfrommature tRNAby a specific cleavage at the anti-codon loop.Therefore, these small tRNA fragments were named tRNAhalves. It took them a couple of years to figure out that ANG isthe endoribonuclease responsible for this cleavage (Fu et al.,2009). In vitro, ANGwas able to cleave tRNA at the anti-codonregion to produce exactly the same tRNA halves as thoseisolated from tissues. In vivo, ANG level was positivelycorrelated with the production of tRNA halves. For example,overexpression of ANG enhanced the production of tRNAhalves, whereas knockdown of ANG expression decreased

Fig. 1. Mechanism of action of Angiogenin (ANG). Growth signalsstimulate nuclear translocation of ANG, whereas stress signals directANG to stress granules. Both pathways aremediated by a cell surfacereceptor that remains to be identified. Nuclear ANG stimulatesrRNA transcription, enabling ribosome biogenesis and therefore cellgrowth and proliferation. Under stress conditions, ANG is nottranslocated into the nucleus but is rather accumulated incytoplasmic compartments such as stress granules where itmediatesthe production of tiRNA, reprograming protein translation andpromoting survival. [Color figure can be seen in the online version ofthis article, available at http://wileyonlinelibrary.com/journal/jcp]

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cellular level of tRNA halves. Another interesting finding wasthat the production of tRNA halves was induced by variousstresses including heat shock, hypothermia, hypoxia, andradiation (Fu et al., 2009). Stress-induced cleavage of tRNA hasbeen shown in Tetrahymena thermophila as a starvation responseto promote survival (Lee and Collins, 2005). Although thisparticular study did not show whether ANG expression wasconcomitantly enhanced under these stress conditions (Fuet al., 2009), ANG has already been known, as an acute-phaseprotein (Verselis et al., 1999), to be upregulated in hypoxia(Hartmann et al., 1999; Nakamura et al., 2006). Thus, a possiblerole of ANG in stress response and in cell survival of adverseconditions could be envisioned from this study.

Almost at the same time, Paul Anderson laboratory ofBrigham and Women’s Hospital reached the same conclusionfrom their study of stress-induced silencing of proteintranslation. Inmammalian cells, stress-induced phosphorylationof eIF2a inhibits global protein synthesis to conserve anabolicenergy for the repair of stress-induced damage (Yamasaki andAnderson, 2008). However, stress-induced translationalsilence is also observed in cells expressing anonphosphorylatable eIF2a mutant (S51A) (McEwen et al.,2005), indicating the existence of a phosphor-eIF2a-independent pathway of translational control. A series ofelegant experiments demonstrated that this pathway ismediated by a subset of tRNA fragments produced by a specificcleavage at the anticodon loop (Yamasaki et al., 2009). Sequenceanalyses indicated that they are identical to the tRNA halvesidentified by Zheng laboratory (Fu et al., 2009). Withoutknowing Zheng’s results, these tRNA fragments were namedtiRNA, standing for tRNA-derived, stress-induced small RNAs,to reflect the fact that they are derived from tRNAand that theyare induced by stress (Yamasaki et al., 2009). Similarly, ANGwas found to be the RNASE that is responsible for this specificcleavage. The enzymatically inactive ANG variant (H13A) lostthe ability to mediate the production of tiRNA, indicating thatthe ribonucleolytic activity of ANG is important. Consistently,overexpression of ribonuclease inhibitor (RI) completelyabolished the tiRNA-producing activity of ANG. However, amere ribonucleolytic activity is inadequate to produce tiRNA.For example, RNASE A and RNASE4, the other two membersof this superfamily which are enzymatically more active thanANG, failed to produce any tiRNA (Emara et al., 2010). P112LANG variant, which is found in amyotrophic lateral sclerosis(ALS) patients (Wu et al., 2007) and which is incapable ofnuclear localization, was unable to mediate tiRNA production,indicating that nuclear translocation of ANG, thus the cellsurface receptor of ANG, is required for ANG to cleave tRNAin vivo. This is consistent with the finding that exogenouslyadded ANG to cultured cells was able to mediate tiRNAproduction (Fu et al., 2009; Emara et al., 2010).

Role of ANG-Mediated tiRNA in ReprogramingProtein Translation

A potential role of ANG in cell survival was first implicated bythe finding that ANG-mediated tiRNA is able to stimulate theformation of stress granules (SGs) (Emara et al., 2010). SGs arecytoplasmic foci where untranslated mRNPs are transientlystored. Formation of SGs is an important mechanism of cellsurvival under adverse conditions. In response toenvironmental and genetic stresses, mammalian cellsreprogram protein translation by suppressing global proteinexpression but selectively enhancing expression of genesinvolved in pro-survival and anti-apoptosis (Yamasaki andAnderson, 2008). This process is initiated by phosphor-eIF2a-induced translational repression and is facilitated bysequestrationof untranslatedmRNAs into SGs. The finding thatANG-mediated tiRNAwas able to trigger the formation of SGs

indicated that ANGplays an active role in stress response of thecells and may promote cell survival via this mechanism. Indeed,ANG is a neuroprotective factor that prevents neuronal deathinduced by various stresses (Kieran et al., 2008; Subramanianet al., 2008; Sebastia et al., 2009). Loss-of-function mutations inthe coding region of ANGgene have recently been found inALSpatients (Greenway et al., 2006). Systemic administration ofwild type ANG protein slowed down motor neurondegeneration in SOD1G93A mice that develop ALS-likesymptoms (Kieran et al., 2008). The findings that tiRNA inducedSG formation and that ANG potentiated both oxidative stress(sodium arsenite)- and xenobiotic (pateamine)-induced SGs(Emara et al., 2010) not only shed light on the mechanism bywhich ANG mutations predispose ALS but also provided arationale for further development of ANG protein therapy inALS treatment.

The mechanism by which tiRNA inhibits protein translationwas elucidated by a recent study also fromAnderson laboratory(Ivanov et al., 2011). Among the two tRNA halves produced byANG, only the 50 halves (50-tiRNAs)were able to inhibit proteintranslation. The 30 halves (30-tiRNAs) had no activities.Moreover, not all 50-tiRNAs are active. Only a subset of50-tiRNAs such as 50-tiRNAAla and 50-tiRNACyc that contain a50-oligo-G motif (4–5 consecutive guanine residues) are able toinhibit protein translation. The reason for this structurerequirement is because this oligo-G motif is required for thetiRNA to inhibit the binding of eIF4G/A to uncappedmRNAandof eIF4E/G/A to m7G-capped mRNA. The strength ofeIF4G:RNA interaction is a critical factor to determinewhetheror not its translation initiation is inhibited by tiRNA. Forexample, translation mediated by WT EMCV-IRES (UA6) thathas a strong interaction with eIF4Gwas not inhibited by tiRNA,whereas those mediated by a mutant IRES (UA7) that has aweaker interaction with eIF4G were inhibited by tiRNA(Ivanov et al., 2011). The finding that tiRNA does not inhibitIRES-mediated translation is of significant interest andimportance as IRES is often used for translation of mRNAs thatare involved in pro-survival and anti-apoptosis. For example,the majority of the 51 human cellular IRES reported so far arefrom the genes involved in anti-apoptosis such as BAG-1(Coldwell et al., 2001), Bcl-2 (Sherrill et al., 2004), Hiap2(Warnakulasuriyarachchi et al., 2004), and XIAP (Holcik et al.,1999); angiogenesis such as HIF-1a (Lang et al., 2002), VEGF(Stein et al., 1998), Tie2 (Park et al., 2005), FGF-1 (Martineauet al., 2004), FGF-2 (Bonnal et al., 2003), and IGF1R (Giraudet al., 2001); and cell survival such as c-Myc (Stoneley et al.,1998), c-Myb (Mitchell et al., 2005), Runx1 (Pozner et al., 2000),and RunX1T1 (Mitchell et al., 2005). Thus, while ANG-mediated tiRNAs suppress global protein translation, they mayselectively enhance the translation of a subset of mRNAsinvolved in anti-apoptosis thereby promoting cell survival underadverse conditions. Consistently, ANG has been shown toupregulate the expression of Bcl-2 at both the mRNA andprotein level (Li et al., 2010). The enhancement to Bcl-2 protein(4.6-fold) is more significant than that to Bcl-2mRNA (2.6-fold),suggesting that ANG stimulates both Bcl-2 transcription andtranslation (Li et al., 2010).

Summary

ANG is an old angiogenic molecule identified from tumor-conditioned medium purely based on an in vivo angiogenesisassay (Fett et al., 1985). However, the biological role of ANGshould not be limited to induction of angiogenesis as suggestedby its widespread expression in all human organs and tissues(Weiner et al., 1987). The finding that ANG is able to directlystimulate cancer cell proliferation marked the first functionalexpansion of this molecule (Hirukawa et al., 2005; Tsuji et al.,2005; Yoshioka et al., 2006). The findings that ANG mediates

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stress-induced production of tiRNA further expanded the roleof ANG from cell growth to cell survival (Fu et al., 2009;Yamasaki et al., 2009; Emara et al., 2010; Ivanov et al., 2011).Stress-induced cleavage of tRNA is a phenomenon firstdescribed as a starvation response in Tetrahymena thermophila(Lee and Collins, 2005), and later observed in bacteria (Haiseret al., 2008), fungi (Jochl et al., 2008), and most recently inmammalian cells (Thompson et al., 2008). tiRNAs reprogramprotein translation, save anabolic energy, enhance damagerepair, and promote survival. Cellular stresses inflicted byenvironmental and genetic factors are an underlyingmechanismfor both cancers and neurodegenerative diseases, the twopathological conditions where ANG has been shown to play arole. For example, hypoxic and oxidative stresses are acommon etiology for cancer. Accumulation of misfoldedprotein aggregates that increased ER stress is a hallmark ofneurodegenerative diseases.

Based on these recent findings, a newmodeof action ofANGin mediating cell proliferation and survival can be proposed(Fig. 1). ANG has three types of target cells (endothelial cells,cancer cells, and motor neurons). These cells respond to ANGbut exhibit some difference probably due to the difference inANG receptor expression. Endothelial cells are the first type ofresponsive cells that have been used extensively for studyingANG biology. The activity of ANG in endothelial cells is strictlydependent on the cell density in vitro and proliferating status invivo (Hu et al., 1997). ANG receptor is expressed only insparsely cultured endothelial cells, and in sprouting neovesselsin vivo (Hu et al., 1997). Cancer cells are the second type ofANGresponsive cells. ANGundergoes nuclear translocation incancer cells in a cell density-independentmanner because of theconstitutive expression of ANG receptor (Tsuji et al., 2005).Constitutive nuclear translocation of ANG in cancer cells is adriving force for cancer progression (Yoshioka et al., 2006;Ibaragi et al., 2009a, b). Motor neurons are the third type ofANG responsive cells. ANG is strongly expressed in the spinalcord both during development and in adulthood (Wu et al.,2007). Loss-of-function mutations in the coding region of ANGhave been found in ALS patients (Greenway et al., 2006) andANG has been shown to enhance motor neuron survival(Kieran et al., 2008). Both growth and survival activities of ANGseem to be mediated by the same receptor. Under growthconditions, the receptor mediates internalization of ANG thatis subsequently translocated to the nucleus. Upon reaching thenucleus, ANG binds to rDNA promoter and stimulates rRNAtranscription. Under stress conditions, the receptor mediatesthe translocation of ANG into cytoplasmic compartments,most likely SG, where it may enhance tiRNA production andpromote survival. Full elucidation of the mechanism of action ofANG in regulating growth and survival would needidentification of ANG receptor and its signaling pathways.However, despite extensive search and research by manylaboratories worldwide over the past two decades, the identityof such a receptor remains elusive. It is expected that thefunctional extension of the biological activity of ANG andexpansion of its target cells will provide an alternative means tofacilitate the identification and characterization of the longoverdue ANG receptor. ANG research has entered a veryexciting period. Great discoveries are anticipated in the comingyears.

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