YB-1: oncoprotein, prognostic marker and therapeutic target? · to cover the biological changes required for the development and persistence of tumours [Hanahan and Weinberg (2011)

Biochem. J. (2013) 449, 11–23 (Printed in Great Britain) doi:10.1042/BJ20121323 11

REVIEW ARTICLEYB-1: oncoprotein, prognostic marker and therapeutic target?Annette LASHAM*1, Cristin G. PRINT*†, Adele G. WOOLLEY‡, Sandra E. DUNN§ and Antony W. BRAITHWAITE‡¶*Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, †Bioinformatics Institute,University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, ‡Department of Pathology, School of Medicine, University of Otago, PO Box 913, Dunedin, 9054, NewZealand, §Department of Paediatrics and Experimental Medicine, University of British Columbia, Vancouver, 950W 28th Ave, BC V5Z 4H4, Canada, and ¶Children’s Medical ResearchInstitute, University of Sydney, Locked Bag 23, Wentworthville, NSW 2145, Australia

Hanahan and Weinberg have proposed the ‘hallmarks of cancer’to cover the biological changes required for the development andpersistence of tumours [Hanahan and Weinberg (2011) Cell 144,646–674]. We have noted that many of these cancer hallmarks arefacilitated by the multifunctional protein YB-1 (Y-box-bindingprotein 1). In the present review we evaluate the literature andshow how YB-1 modulates/regulates cellular signalling pathwayswithin each of these hallmarks. For example, we describe howYB-1 regulates multiple proliferation pathways, overrides cell-

cycle check points, promotes replicative immortality and genomicinstability, may regulate angiogenesis, has a role in invasion andmetastasis, and promotes inflammation. We also argue that there isstrong and sufficient evidence to suggest that YB-1 is an excellentmolecular marker of cancer progression that could be used in theclinic, and that YB-1 could be a useful target for cancer therapy.

Key words: angiogenesis, genomic instability, inflammation,invasion/metastasis, metabolism, proliferation.

INTRODUCTION

YB-1 (Y-box-binding protein 1) encoded by the YBX1 gene, isa member of the cold-shock protein superfamily, all of whichcontain a highly conserved nucleic-acid-binding motif that bindsto both DNA and RNA. This motif is located within a regionof 65 amino acids termed the ‘cold-shock domain’ which sharesgreater than 40% identity with prokaryotic cold-shock proteins[1]. This degree of sequence conservation supports the notionthat these proteins play an essential role in both the prokaryoticand eukaryotic cell [2]. It is interesting to speculate whetherthe ancestral nucleic-acid-binding domain of YB-1 has existedthrough the evolution of prokaryotes to eukaryotes, possiblyacquiring new roles as organisms became more complex. If thiswere the case, it is not surprising that YB-1 has taken on manyseemingly diverse roles.

YB-1 was originally identified as a factor that repressed genetranscription by binding to the Y-box (an inverted CCAATbox) of MHC class II promoters [3]. Later the same year,protein-blotting assays using DNA probes revealed that YB-1binds to the enhancers of the EGFR (epidermal growth factorreceptor) and the ERBB2 (HER2) genes [4]. By 1995, itbecame clear that YB-1 played an important role in regulatingcellular proliferation and development [5]. Since then, YB-1has been shown to be a transcription factor of many genes(reviewed in [6]), but also directly affects DNA repair, RNAsplicing, exon skipping, drug resistance and cancer progression[EMT (epithelial–mesenchymal transition)] in a transcription-

independent manner (comprehensively reviewed by Eliseevaet al. [6]). In the present review we discuss the multifunctionalnature of YB-1 thereby illustrating how it facilitates the cancerhallmarks of Hanahan and Weinberg [21].

The importance of YB-1 in cancer: a preamble

YB-1 rose to prominence following reports of elevated YB-1protein levels being highly correlated with cancer progression andpoor prognosis. Initially this came from IHC (immunohistochem-istry) analyses of breast tumours by Royer’s group, who showedthat levels of cytoplasmic YB-1 correlated with progressionin 27 breast cancers [7]. They also showed that the levels ofnuclear, but not cytoplasmic, YB-1 correlated with expressionof the ABC transporter (ATP-binding cassette transporter) P-glycoprotein MDR1 (multidrug resistance protein 1) in ninecancers. These data were consistent with reports that YB-1could transactivate the MDR1 gene [8–10] and that high MDR1protein levels in tumours were associated with poor clinicalprognosis. The importance of the correlation between nuclear YB-1 and MDR1 levels with patient prognosis was strengthened bysimilar observations for osteosarcoma [11], non-small-cell lungcarcinoma [12,13], synovial sarcoma [14], prostate cancer [15],melanoma [16] and multiple myeloma [17]. In addition, nuclearYB-1 and MDR1 were both observed at high levels in 9/27 breastcancers after paclitaxel treatment.

Data such as these has led to the widely accepted view thatnuclear YB-1 can function as an oncoprotein which, when present

Abbreviations used: BAX, Bcl2-associated X protein; BM, basement membrane; CASP, caspase; CDK, cyclin-dependent kinase; CDKN, CDK inhibitor;ChIP, chromatin immunoprecipitation; COC, ChIP on chip; CPP, cell-permeable peptide; ECM, extracellular matrix; EGFR, epidermal growth factorreceptor; EMT, epithelial–mesenchymal transition; ER, oestrogen receptor α; ERK, extracellular-signal-regulated kinase; GSK3β, glycogen synthase kinase3β; HIF-1, hypoxia-inducible factor 1; hnRNPA, heterogeneous nuclear ribonucleoprotein A; IHC, immunohistochemistry; IL-8, interleukin 8; LEF1, lymphoidenhancer-binding factor 1; MAPK, mitogen-activated protein kinase; MDM2, murine double minute 2; MDR1, multidrug resistance protein 1; MEF, mouseembryonic fibroblast; MEK, MAPK/ERK kinase; MKP, MAPK phosphatase; MMP, matrix metalloproteinase; MT1-MMP, membrane-type 1 MMP; mTOR,mammalian target of rapamycin; mTORC1, mTOR complex 1; PDGF-β, platelet-derived growth factor β; PI3K, phosphoinositide 3-kinase; PKM2, pyruvatekinase M2; RB, retinoblastoma; RSK, ribosomal S6 kinase; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; TGF-β,transforming growth factor β; TP53, tumour protein p53; uPA, urokinase-type plasminogen activator; VEGF-A, vascular endothelial growth factor A; YB-1,Y-box-binding protein 1.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2013 Biochemical Society

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12 A. Lasham and others

Table 1 Cancer cell lines where YB-1 reduction has been shown to induceapoptosis or inhibit cell proliferation

Cell location Cancer Cell line Reference(s)

In vitro Melanoma SK-Mel-5, NZM9 [18]Fibrosarcoma B10.2, B10.5 [18]Liver cancer HepG2 [18]Lung cancer A549, EBC-1, PC-9,

QG56[18,27,52]

Bladder cancer TCCsup, KK47 [90]Multiple myeloma GM02301, GM2132,

INA-6, MM.1[17,126]

Paediatric glioblastoma SF188 [154]Breast cancer (ER-negative) MDA-MB-231,

BT474-m1, Au565,MDA-MD-468,HCC1937, SUM149HR5, HR6

[41,43,165]

Breast cancer (ER-positive) MCF-7, T-47D, KPL-1 [27,52,90,166]Prostate cancer PC3 [90]Colon cancer RKO HCT116 [18,27,29]

In vivo Breast cancer BT474-m1 [41]Lung cancer A549 [27]Paediatric glioblastoma SF188 [154]

at elevated levels, leads to increased tumour cell proliferationand drug resistance. Then an important study in 2003 showedthat YB-1 was essential to control the growth and survival oftumour cells of different origins in vitro [18]. A subsequentstudy showed this to be dependent on the nuclear translocationof YB-1 [19]. Consistent with this, in 2005 Royer’s laboratorydemonstrated that sustained overexpression of YBX1 in transgenicmice invariably led to the development of invasive breast cancers,further supporting the view that YB-1 can function as anoncoprotein [20].

YB-1 AND THE HALLMARKS OF CANCER

In 2000, Hanahan and Weinberg proposed six regulatorypathways that must be overcome in order for a cell tobecome malignant: uncontrolled proliferative signalling, evadinggrowth suppressors, resisting cell death, replicative immortality,sustained angiogenesis, and invasion and metastasis [21]. In2011 they proposed two additional hallmarks: deregulatedmetabolic pathways and avoiding immune destruction, as wellas two enabling characteristics: genomic instability and tumourpromoting inflammation [22].

The YB-1 protein is remarkably multifunctional, and in thepresent review we demonstrate the significant contribution thatYB-1 makes to each of Hanahan and Weinberg’s cancer hallmarks.We argue that the multifunctionality of YB-1 renders it a truemaster-regulator of malignancy and therefore it deserves the statusgiven to other multi-potent oncoproteins such as Myc and Ras.Since we focus specifically on the cancer hallmarks of Hanahanand Weinberg, the present review is not intended to providea comprehensive evaluation of YB-1’s functions. For this werecommend the excellent reviews by Eliseeva et al. [6] and Brandtet al. [23]. In the following pages the role played by YB-1 in eachof the hallmarks of cancer will be discussed in turn.

Proliferation

The early association made between YB-1 and proliferation hasfocussed research on dysregulation of proliferation and the cell

cycle by YB-1, which is arguably the most important hallmarkof a tumour cell. Reduction of YB-1 expression causes growthinhibition or apoptosis in a broad range of cancer cells bothin vitro and in vivo (Table 1). These data suggest that YB-1 plays acritical and non-redundant role in regulating cell proliferation. Asa consequence, several studies have investigated the mechanismby which YB-1 regulates cell proliferation. One approach has beento search for YB-1-binding sites or ‘Y-boxes’. However, YB-1 canbind to a variety of DNA sequences that have little sequencesimilarity [24,25], making it difficult to predict a canonicalYB-1-binding site. Thus YB-1 targets must be empiricallydetermined. Given this, several studies have performed large-scale analyses to determine the key transcription targets of YB-1.Using gene expression arrays, changes in transcript abundancewere determined following YB-1 knockdown with siRNAs (smallinterfering RNAs) in ovarian, colorectal, lung and ER (oestrogenreceptor α)-positive breast cancer cell lines [26,27]. Anotherapproach has been to identify the promoters bound by YB-1 incolorectal cancer or ER-negative breast cancer cells using ChIP(chromatin immunoprecipitation) promoter arrays [called COC(ChIP on chip)] [28,29] and ChIP sequencing [30]. A comparisonof the transcriptional targets of YB-1 in all of these studiesshowed few in common (results not shown). For example, geneexpression data of three cancer cell lines (colorectal, breast andlung) following YB-1 siRNA treatment showed that although afew hundred transcripts were downstream targets of YB-1, only25 were in common in all lines [27]. Cumulatively, these resultssuggest that the downstream targets of YB-1 are cell-type-specific.This raises the possibility that any one cell type may utilize only asubset of YB-1’s transcriptional capability, since it expresses onlya subset of YB-1’s potential transcriptional targets. This may bedue to the presence of different transcriptional co-factors or YB-1-binding partners in each cell type (e.g. [31,32]). Interestinglyhowever, there appears to be strong common themes to YB-1’stranscriptional targets across lineages; rather than being a randomset of genes, the downstream targets of YB-1 in different celltypes, even if only slightly overlapping, are all enriched for E2F-regulated genes [27].

The E2F pathway

A seminal paper in 2005 by Rhodes and colleagues studied geneexpression data from almost 7000 microarray experiments inthe Oncomine database [33]. They observed that transcriptionaltargets of the E2F family were over-represented in many tumourtypes, which led them to state, “These results reaffirm thatactivation of the E2F pathway is a prevalent event in humancancer”. As indicated, it has recently been shown that YBX1mRNA levels are associated with the E2F1 pathway in breast,colorectal and lung tumours [27], which was confirmed usingChIP assays, where YB-1 bound directly to the promoters ofseveral canonical E2F1-regulated genes [e.g. CDC6 (cell-divisioncycle 6), CCNA (cyclin A) and TOP2A (topoisomerase II α170 kDa)]. Furthermore, bioinformatic analysis of COC datarevealed that more than 4000 of the 6000 promoters bound byYB-1 in ER-negative breast cancer cells also have E2F1/E2F-binding sites (see results published in [28]), and that 57 of the88 YB-1 target genes identified by COC analysis in HCT116colorectal cancer cells had E2F1/E2F-binding sites within theirpromoters (see results published in [29]). Therefore multiplestudies suggest that YB-1 co-regulates the expression of genesin the E2F1/E2F pathway.

Not only does YB-1 co-regulate E2F target genes, importantly,it also controls expression of different E2F family members, in


YB-1 and cancer 13

Figure 1 Multiple E2F family members are downstream targets of YB-1

Microarray data suggest that YB-1 promotes transcription of activator E2Fs (e.g. E2F1–E2F3)and inhibits transcription of repressor E2Fs (e.g. E2F5 and E2F7). The E2F family members thatare regulated by YB-1 appear to be cell-line-specific.

what appears to be a cell-type-specific manner [27]. For example,YB-1 binds to the E2F2 and E2F5 promoters in MCF7 cells andactivates transcription of the E2F1 promoter in A549 cells. Usingdifferent experimental systems, in the ER-negative breast cancercell line SUM149, YB-1 was shown to bind to the E2F2, E2F3and E2F7 promoters (see results published in [28]), and in theSKOV-3 ovarian cell line E2F7 expression was increasedfollowing YB-1 knockdown (see results published in [26]). Theseresults suggest that the expression of ‘activator’ and ‘repressor’members of the E2F family may be turned on and off respectivelyby YB-1 in a cell-type-specific manner (Figure 1).

In summary, we suggest that YB-1 has evolved to regulate theactivity of E2F pathways through two synergistic mechanisms.First, YB-1 transcriptionally regulates several members of the E2Ffamily to promote expression of the ‘activator’ E2Fs and inhibitexpression of the ‘repressor’ E2Fs. Secondly, it co-regulatesthe expression of thousands of E2F target genes by bindingto their promoters. These important observations suggest thatYB-1 may in fact be an Achilles’ heel of the E2F cancer cellproliferation pathway, which could provide an opportunity totarget this pathway by therapeutically targeting YB-1.

PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin)pathway

In addition to the E2F pathway YB-1 also appears to regulateother cell proliferation pathways. The PI3K class I enzymes andthe pathways driven by them are dysregulated in the majorityof cancers (reviewed in [34]). Multiple cell-surface receptors, inparticular growth factor receptors (e.g. EGFR and ERBB2), relaygrowth-promoting signals through the activation of this pathway(reviewed in [35]). The PI3KCA gene encoding the catalyticsubunit p110α is frequently amplified or acquires activatingmutations in cancers to further enhance the activity of the pathway[36]. The PI3K pathway signals through the multifunctionalprotein kinase Akt which, in turn regulates mTOR to influence anumber of oncogenic functions, including proliferation, survival,metabolism and metastasis (reviewed in [37]). The PI3K pathwayalso cross-talks with the E2F pathway discussed above, bymodulating the pro-apoptotic functions of E2F1 [38].

YB-1 appears to be linked to, and plays an integral role within,the PI3K/Akt/mTOR pathway (Figure 2). It transcriptionallyactivates the expression of PIK3CA in basal-like breast cancercells [39] and YB-1 depends on Akt for its nuclear translocationfollowing phosphorylation at Ser102 in a number of cell lines,including basal-like breast cancer cells [19], ovarian cancer cells[26] and melanoma cells [40]. Interestingly, PIK3CA expressionis activated by YB-1 irrespective of whether the PIK3CA geneis mutated or amplified, leading to further dysregulation of thePI3K pathway [39]. This results in the subsequent modulation ofa number of downstream components of the PI3K pathway, whichincludes molecules that can phosphorylate YB-1, phospho-RSK

(ribosomal S6 kinase) (Ser360) and phospho-Akt(Ser473) [16,39],leading to further YB-1 nuclear localization and further activationof PIK3CA transcription. It has recently been shown in melanomacells that inhibitors of the PI3K pathway can modestly reduceexpression from a cloned YBX1 promoter, suggesting that theactivation of this pathway can also promote transcription of YBX1[40].

Downstream of Akt is mTOR. mTOR forms a complex withraptor (regulatory associated protein of mTOR) called mTORC1(mTOR complex 1), which appears to be an important hub thatcontrols many pathways that affect protein and lipid synthesis,autophagy, production of inflammatory cytokines, glycolysisand angiogenesis (reviewed in [37]). In addition to regulatingPI3K/Akt signalling, YB-1 may also regulate mTOR. Reductionof YB-1 with siRNAs in a number of ER-negative breast cancercells and a paediatric glioblastoma cell line was shown to causea marked decrease in mTOR protein levels [41]. This was notaccompanied by a reduction in mRNA expression, suggestingthat YB-1 controls the translation or affects the mRNA stabilityof mTOR.

Taken together, these results suggest that, similar to its inter-action with multiple points of the E2F pathway, YB-1 interactswith multiple points of the PI3K/Akt/mTOR pathway to increasethe activity of this pathway in cancer cells.

MAPK (mitogen-activated protein kinase) pathways

There are at least six different molecular pathways associated withMAPKs, but only the Ras/Raf/MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathways willbe discussed in the present review. MAPK signalling is initiatedby growth factor receptors on the cell surface activating Ras,and then Raf, which activates the MAPK kinases MEK andERK, that in turn activate several downstream pathways, whichconverge to promote cellular proliferation (reviewed in [35]).There is complex cross-talk between the Ras/Raf/MEK/ERKand PI3K/Akt pathways (Figure 2), with both pathways able toregulate the other at multiple points (reviewed in [35,42]).

YB-1 activates several members of the Ras/Raf/MEK/ERKpathway (Figure 2). As described above, YB-1 is a transcriptionalactivator of genes encoding the EGFR and ERBB2 cell-surfacereceptors, which transmit initial signals to intracellular MAPKpathways [4,43]. YB-1 appears to also regulate a number of genesdownstream in the MAPK pathway. COC studies in colorectalcancer cells [29] and ER-negative breast cancer cells [28,30]have identified that YB-1 binds to the promoters of a numberof MEK/ERK pathway genes.

The effects of this signalling pathway are modulated by afamily of dual-specific MAPK phosphatases [44]. Interestingly,microarray analysis of cancer cell lines where YB-1 levelswere reduced by siRNA treatment showed significantly alteredexpression of transcripts encoding a number of MAPKphosphatases including MKP2 (MAPK phosphatase 2; see datapublished in [27]). This suggests yet another role for YB-1 in theregulation of this pathway.

Interestingly the Ras/Raf/MEK/ERK pathway also activatesYB-1 in a positive feedback loop. RSK1 [39] and ERK2 [45] havebeen shown to phosphorylate YB-1 to promote its transcriptionalactivity (Figure 2), and a MEK inhibitor has been shown to reduceYB-1 protein abundance [46].

In summary, several pathways that promote cancer cellproliferation are activated by YB-1. These include the E2F,PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways. These threepathways converge and overlap with one another [38,42,47],and YB-1 regulates several of members of each pathway. By



Figure 2 YB-1 regulates multiple growth signalling pathways

YB-1 transcriptionally activates the gene encoding PI3K and also downstream targets of PI3K such as those encoding RSK and Akt, which provide a positive feedback loop by phosphorylating YB-1(indicated by P) leading to enhanced PI3K activation. YB-1 may also regulate mTOR at a translational level (shown as a broken line), components of the Ras/Raf/MEK/ERK arm of the MAPK signallingpathway and glycolysis via PKM2, as well as the phosphatase encoded by MKP.

doing so, YB-1 promotes cancer cell proliferation through severalparallel signalling cascades involving many effector molecules.Again, this makes YB-1 an attractive target for therapies to controlcancer cell growth.

Evading growth suppressors and cell-cycle checkpoints

For a cancer cell to undergo sustained proliferation, the twocell-cycle checkpoints regulated by the RB (retinoblastoma)gatekeeper protein and the tumour suppressor p53 must beovercome. YB-1 appears to override both of these checkpoints.

(a) The RB pathway

The critical role of RB as a tumour suppressor is evidenced bythe fact that RB or the RB pathway is inactivated in almostall human tumours [48]. In normal cells RB exerts its tumoursuppressor activity by interacting with multiple proteins [49].Arguably the most important of these proteins are ‘activator’members of the E2F family, which RB inhibits to suppress cell-cycle progression. Following CDK (cyclin-dependent kinase)-mediated hyperphosphorylation of RB (pRB), the activator E2Fsare released from the RB inhibition and can transcriptionallyactivate numerous genes promoting cell-cycle progression fromG1- to S-phase (Figure 3) [50,51]. In cancers, the normal controlof cell-cycle progression by RB pathways is reduced, in part,through YB-1.

YB-1 appears to reduce the tumour suppressive activities ofRB in several ways. First, it has been shown that YB-1 controlsthe expression of upstream regulators of RB, namely cyclin D1[17,27,52], CDK1 and CDK2 [52]. Secondly, as described above,YB-1 is a transcriptional activator of several ‘activator’ E2Fs andis a transcriptional repressor of several ‘repressor’ E2Fs, as wellas a co-regulator of S-phase genes, including E2F-1 (Figure 3).It seems possible that the elevated levels of YB-1 in cancer cellsstrongly activate the expression of ‘activator’ E2Fs so that RBbinding becomes insufficient to fully inhibit these activator E2Fmolecules. Further research is required to fully understand thecomplex inhibition of the RB tumour suppressor pathway byYB-1.

Figure 3 YB-1 modulates RB tumour suppressor activity

The diagram illustrates the regulation of RB function and how YB-1 affects this process.YB-1 transactivates the upstream regulators of RB, cyclin D1 and CDK1/2, which promotehyperphosphorylation of RB leading to release of E2F1 (and activation of the transcriptionfactors). YB-1 also directly activates expression of S-phase genes including those encodingE2F1, cyclin E and cyclin A. Both of these processes promote cell-cycle progression. P,phosphorylation.

The p53 pathway

TP53 (tumour protein p53) is renowned as a tumour suppressor asit is inactivated more frequently in cancers than any other geneas yet identified [53]. The p53 protein that TP53 encodes functionsas a transcription factor to control expression of a number ofgenes involved in cell survival and proliferation. p53 is normallypresent at very low levels in cells due to constant degradationby the E3 ligase MDM2 (murine double minute 2) [54].However, after stress, particularly DNA damage, p53 becomesphosphorylated preventing interaction with MDM2, therebyallowing p53 levels to increase dramatically [55]. When thisoccurs, p53 transactivates genes to cause cell-cycle arrest allowingDNA repair, permanent arrest of cell division (senescence) orapoptosis, thereby preventing the accumulation of lesions thatcould otherwise go on to initiate malignancy (reviewed in [55,56]).The p53 protein is disabled in many tumours. Although this occursmostly by mutation, p53 can also be disabled by direct interactionwith other proteins (e.g. [57,58]).

YB-1 can disable the p53 pathway in cancers by regulating boththe activity and the expression of p53 (Figures 4A and 4B). Several


YB-1 and cancer 15

Figure 4 YB-1 regulates both apoptosis and proliferation pathways

YB-1 controls apoptosis and cell-cycle arrest by transcriptionally repressing the gene encodingp53 (A) and inhibiting p53-dependent apoptosis by direct protein interaction (B). Similarly YB-1represses expression of genes encoding both FAS and CASP7 (C) and activates transcription ofE2F1 growth-associated gene targets, thus enhancing cell proliferation (D).

studies have demonstrated that YB-1 interacts directly with p53[59–62] and interferes with the ability of p53 to transactivategenes [59,61,63,64]. For example, YB-1 reduced the p53-driventranscriptional activation of apoptosis-associated genes APAF1(apoptotic peptidase activating factor 1), NOXA (NADPH oxidaseactivator) and BAX (Bcl2-associated X protein), but had littleeffect on the promoter of the CDKN (CDK inhibitor) 1 geneencoding the cell-cycle inhibitory protein p21CIP1 [63]. It wasalso observed that p53 had greater affinity for the promoters ofcell-cycle-associated proteins than those of apoptosis-associatedproteins, potentially making it more difficult for YB-1 to overridethe regulation of cell-cycle-associated gene promoters by p53[63]. In addition to directly affecting the activity of p53, YB-1 alsorepresses transcription of the TP53 gene [18]. The importanceof YB-1 in controlling this pathway was confirmed by theobservation that the reduction in YB-1 led to an increase in p53protein levels and triggered p53-dependent apoptosis in cancercell lines with wild-type p53 [18,63].

In summary, YB-1 appears to help cancer cells escape cell-cyclecheckpoints by inhibiting both the RB and p53 pathways.

Resisting cell death

Cancer cells have evolved to evade the normal apoptotic pathwaysthat would otherwise remove damaged cells. Upon a ‘cellular cue’,a series of proteins transduce a signal to activate an apoptosispathway that culminates in DNA degradation and systematicdisassembly of the cell by activation of a series of proteolyticenzymes called caspases [65]. YB-1 is involved in protectingtumour cells from apoptosis in several ways. p53 probably playsthe most important role here to detect damaged DNA and initiateapoptosis if the DNA cannot be repaired. Elevated levels ofYB-1 enable cells to subvert the p53-driven apoptosis pathway(Figures 4A and 4B).

Another apoptotic pathway involves the cell-surface deathreceptor Fas (CD95). Upon binding of Fas ligand (CD95L),an apoptotic signal is transduced from Fas into the cytoplasmthrough multiple downstream effector molecules, includingthe executioner caspases, CASP3 and CASP7, to promoteorderly cellular disassembly, including PARP [poly(ADP-ribose)polymerase]-mediated cleavage of DNA and finally fragmentationof the cell into apoptotic bodies [66]. YB-1 appears to inhibitthe Fas-mediated apoptosis pathway at several points. YB-1 is a

transcriptional repressor of the FAS promoter (Figure 4C) [67]and consistent with elevated levels of YB-1 in tumours, FASis often down-regulated in cancers [68,69]. YB-1 also inhibitsthe expression of the gene encoding the pro-apoptotic proteinBAX [18,63]. YB-1 may also repress transcription of CASP7(Figure 4C), since ChIP analysis showed that YB-1 binds to theCASP7 promoter (see results published in [28]) and reduction ofYB-1 levels increased CASP7 expression (see results published in[27]). In addition to quelling the pro-apoptotic signals from deathreceptors, the inhibition by YB-1 of BAX and CASP7 may also actto suppress intrinsic apoptotic signals from DNA or mitochondrialdamage (reviewed [70]).

As well as playing a role in activating cellular proliferation,under the control of the PI3K/Akt [38] and MAPK pathways[71], E2F1 can also initiate an apoptotic pathway, by promotingthe expression of pro-apoptotic genes. This has recently been thesubject of considerable interest [72,73]. In response to growthpromoting signals, such as fetal bovine serum, there is a PI3K-dependent repression of E2F1′s transcription of apoptosis genes,whereas E2F1 continues to drive the transcription of genesinvolved in proliferation [74]. As described above, YB-1 is anintegral component of the PI3K pathway, driving proliferationby activating many pathway components and being part ofpositive feedback loops. Therefore it seems probable that YB-1promotes the expression of E2F1-dependent proliferative genes,but not E2F1-dependent apoptotic genes. We hypothesize that, asobserved for p53-regulated cell cycle and apoptosis pathways[63], YB-1 preferentially co-activates expression of E2F1proliferation-associated genes and not those driving apoptosispathways (Figure 4D). In support of this, we have studied therelationship between YBX1 mRNA levels and the inferred activityof the E2F1-driven apoptotic and proliferative transcriptionalprogrammes [74] using PCA (principle component analysis) ofmicroarray data from breast tumours. The results suggest thatYBX1 mRNA levels are associated with the transcription of theproliferative E2F1 target genes, but not apoptotic E2F1 targetgenes (results not shown).

Replicative immortality and genomic instability

The immortalization of cells is stimulated by: (i) activation oftelomere maintenance mechanisms [75]; (ii) loss of the RBcheckpoint; and (iii) loss of p53 function, which together leadto lifespan extension and genomic instability. YB-1 can promoteall three of these mechanisms.

Replicative senescence

Studies of MEFs (mouse embryonic fibroblasts) generated fromE13.5 (E is embryonic day) Ybx1− / − mice demonstrated that thesecells prematurely senesced compared with control MEFs [76]. Itwas noted that the Ybx1− / − MEFs had increased levels of Cdkn2a(p16Ink4a) and Cdkn1a (p21Cip1), which would promote senes-cence. Furthermore, Cdkn2a and Cdkn1a were also elevated at themRNA level, suggesting that YB-1 may be required for their tran-scription or mRNA stability. The involvement of these proteinswas supported as the induction of senescence could be partiallybypassed by knockdown of both of these transcripts [76]. YB-1thus appears to play a role in controlling replicative senescence.

Genomic instability

The loss of genomic stability, leading to alterations in the genomethat include amplifications, deletions, translocations or even



aneuploidy, is a characteristic of solid tumours. In hereditarycancers, genomic instability is frequently a result of mutationswithin DNA repair genes, however what promotes this in sporadictumours is not well understood (reviewed in [77]). The keyproteins driving the response to DNA damage, p53 and ATM(ataxia telangiectasia mutated), are frequently mutated in cancers,but despite the volume of sequence information on humancancers, very few mutations in DNA repair genes have beenidentified in sporadic cancers [77]. Instead the data point towardsan involvement of oncogene-driven replicative stress leading togenomic instability.

Recently, interest has focussed on the RB/E2F pathway inprotecting the integrity of the genome, since the loss of RBleads to genomic instability (reviewed in [78]). An elegant study,performed in non-immortalized cells, tested the ability of bothviral and cellular oncoproteins to aberrantly activate the RB/E2Fpathway [79]. This led to enhanced cellular proliferation, butwithout a concurrent increase in nucleotide metabolism, leadingto a depletion of the nucleotide pool. The outcome of this wasreplicative stress, leading to incomplete progression of replicationforks and thereby DNA damage.

Given the manner in which YB-1 modulates the RB and E2Fpathways (Figure 3) it appears possible that elevated levels ofYB-1 may promote genomic instability through replicative stress,without concurrent induction of apoptosis. Indeed, overexpressionof YBX1 does appear to promote genomic instability. In a study byBergmann et al. [20] the overexpression of YBX1 was associatedwith genomic instability when expression was targeted to themammary gland of transgenic mice. All of these mice ultimatelydeveloped mammary tumours after 52 weeks. Furthermore, inhuman mammary epithelial cells, prolonged expression of YB-1induced a loss of cell-cycle control, genomic instability andcentrosomal amplification [80].

In summary, the results of both YB-1 inactivation andoverexpression suggest that YB-1 can promote replicativeimmortality and genome instability.

(v) Inducing angiogenesis

Aberrant angiogenesis is a hallmark of many solid tumours.Once the tumour reaches a size where nutrients and oxygenbecomes limiting, a pro-angiogenic pathway is initiated. Thisevent is termed the ‘angiogenic switch’ [81,82]. There areseveral proteins involved in this process, with perhaps the bestknown being VEGF-A (vascular endothelial growth factor A),but also includes PDGF-β (platelet-derived growth factor β),ANG-1 (angiopoietin 1), PGF (placental growth factor), TGF-β(transforming growth factor β), Notch and Wnt pathway proteins[83]. Each of these proteins plays a different role in promotingand regulating blood vessel development. However, in developingtumours, angioregulatory pathways are not as tightly controlledas occurs in normal development, so that dysfunctional vascularbeds are formed, often with irregular structure and are poorlysynchronized with the needs of the tissues they supply [84].Because of this, the tumours become more hypoxic, drivingfurther aberrant angiogenesis, and resulting in decreased drugdelivery and vascular dissemination of cancer cells. It is thereforenot surprising that angiogenesis and tumour invasiveness areclosely linked [83]. The association between YB-1 and TGF-β,the Notch and Wnt pathways will be discussed below.

In endothelial cells, YB-1 has been shown to activate expressionof pro-angiogenic PDGF-β following thrombin treatment [85].Studies in epithelial-derived cancer cell lines suggest that YB-1may up-regulate the expression of other pro-angiogenic genes

Figure 5 YB-1 plays a role in the angiogenic switch

Under normoxic conditions YB-1 represses transcription of pro-angiogenic genes such as thoseencoding VEGF-A, PDGF-β , IL-8 and CXCL2. Repression of VEGF-A has been shown to occurvia phosphorylation of YB-1 by activated GSK3β , which prevents binding of HIF-1 to the VEGFApromoter. However, as oxygen levels decline, GSK3β is not activated, thus enabling HIF-1 toaccess the VEGFA promoter leading to expression of pro-angiogenic factors.

in a hypoxia-dependent manner. However, under normoxicconditions, YB-1 appears to inhibit the expression of a number ofpro-angiogenic genes. For example, in A549 lung cancer cells,reduction of YB-1 led to increased levels of the transcriptsencoding the pro-angiogenic chemokines IL-8 (interleukin 8)and CXCL2 [chemokine (C-X-C motif) ligand 2] (see datapublished in [27]), suggesting that YB-1 is a transcriptionalrepressor of these genes (Figure 5). In support of this, YB-1 hasbeen shown to bind to the IL-8 promoter in ER-negative breastcancer cells (see results published in [28]). More compellingly,YB-1 has been shown to repress transcription of VEGFA bybinding to the hypoxia-response region in the VEGFA promoterin normoxic conditions [45,86]. The authors proposed that YB-1 bound to single-stranded DNA would prevent binding of thedouble-stranded DNA binding HIF-1 (hypoxia-inducible factor1) complex. YB-1-mediated repression of the VEGFA promoterwas considerably enhanced after phosphorylation of YB-1 byactivated GSK3β (glycogen synthase kinase 3β) [45]. However,in tumours under hypoxia GSK3β is not activated [87], potentiallyreducing the binding of YB-1 to the VEGFA promoter, therebyallowing greater access of HIF-1 to activate the expression ofVEGFA [45,86]. These tantalizing data suggest that YB-1 inhibitsangiogenesis in epithelial-derived tumour cells under normoxicconditions, however, under hypoxia repression by YB-1 isrelieved allowing expression of pro-angiogenic factors. Thereforethe induction by YB-1 of genes encoding pro-angiogenic proteinssuch as PDGF-β and VEGF-A, although requiring furtherinvestigation, suggests that YB-1 may play an important role inthe angiogenic switch.

Invasion and metastasis

There are many steps involved in the invasion and metastasis oftumour cells to distant sites. Tumour cells initially constrainedby BMs (basement membranes), must first dissociate fromthe tumour mass and cross the BM before invading theadjacent stromal tissue. This migratory behaviour is facilitatedby loosening the connections between adjacent cells of the BMand also of the ECM (extracellular matrix) to allow the passageof tumour cells into blood vessels and lymphatic system fordissemination [88,89].

There are several lines of evidence linking YB-1 with a rolein invasion and metastasis (Figure 6). For example, in vitrostudies have shown that reducing YB-1 levels inhibits theinvasive properties of a number of cancer cell lines [39,90,91],and overexpression of YB-1 promotes invasion of MCF-7breast cancer cells [92] and Ras-transformed ‘normal’ mammaryepithelial cells [93]. Furthermore, analysis of tumour data showedthat high YBX1 mRNA levels are associated with lower distantmetastasis-free survival rates in breast cancer [27].


YB-1 and cancer 17

Figure 6 YB-1 regulates genes involved in invasion and metastasis

YB-1 regulates SNAI1, LEF1 and TWIST1 that transcriptionally repress the gene encoding E-cadherin (CDH1), which normally maintains cell adhesion. This loss of adhesion leads to a changein cell phenotype (EMT) and the cell then becomes invasive. YB-1 also regulates the translation of TGFB1 mRNA, which drives EMT and binds to Wnt pathway proteins and the Notch3receptor.

The cadherins are involved in maintaining cell–cell adhesionwithin the tumour mass, particularly E-cadherin (CDH1), whichis frequently inactivated in metastatic cancers [94–96]. Onemechanism by which this can occur is via the transcriptionalrepression of CDH1 by a number of transcription factors includingSnail (SNAI1), LEF1 (lymphoid enhancer-binding factor 1) andTWIST1 [97–99]. Interestingly YB-1 has been shown to promotethe translation of the mRNAs encoding these CDH1-repressingfactors [91,93]. The tumour cell must then become motile, whichoften appears to involve EMT [100]. EMT is driven by manymolecules and pathways that have been linked to YB-1. Forexample, YB-1 appears to regulate the translation of TGFβ1[101], which although a tumour suppressor in normal cells, playsan important role in driving EMT [102]. Both the Wnt and Notchpathways are also involved in EMT [100] and YB-1 has beenshown to bind to the promoters of a number of Wnt pathwayproteins [28]. Interestingly a potential secreted fragment of YB-1has been identified as a ligand for Notch3 receptors [103].

The dissociation of the BM/ECM occurs via severalmechanisms, one of which is proteolysis. Multiple proteolysispathways appear activated in metastasis including the uPA(urokinase-type plasminogen activator) system and the MMPs(matrix metalloproteinases) [104,105]. uPA promotes degradationof the BM/ECM by plasmin and also activates the MMPs [89].YB-1 appears to be an activator of uPA as reduction of YB-1expression led to decreased levels of uPA [39]. The MMPs area family of proteases with multiple specificities. Given that theycan degrade almost all proteins in the BM/ECM, their expressionis tightly regulated [105]. Several studies have shown that YB-1transcriptionally regulates a number of MMPs, including MMP-2[106], MMP-12 [107] and MMP-13 [108]. One of these studiesshowed that whether YB-1 is an MMP activator or repressor,perhaps not surprisingly, depended on the cellular context [106].However, reduction of YB-1 expression in invasive melanomacells led to decreased expression of MMP2 [16]. YB-1 has alsobeen shown to increase the levels of the membrane-associatedMT1-MMP (membrane-type 1 MMP; also known as MMP-14),which plays a critical role in metastasis [109,110]. In ER-positivebreast cancer cells, YB-1 performs this task by subverting theendocytic mechanism and directing MT1-MMP back to the cellmembrane where it can interact with and degrade the ECM [92].

CD44 is another membrane-bound protein that plays an importantrole in metastasis. A recent report has shown that this occursvia interaction with MT1-MMP [111]. YB-1 is a transcriptionalactivator of the CD44 gene [112], and also promotes alternatesplicing of the transcript to include exon 4 leading to the CD44v4variant [113]. Isoforms of CD44 mRNA containing exon 4promote increased invasion of cancer cells (e.g. [114]).

YB-1 can also regulate a member of the integrin family.These proteins are adhesion receptors classically associatedwith cell adhesion, migration, differentiation, proliferation andcancer metastasis [115,116]. Experiments have shown that YB-1expression is linked to that of integrin α6 (also known as CD49f)in both mammary progenitor and breast cancer cells [28,112]and may be involved in the modulation of proliferation anddifferentiation.

In conclusion, YB-1 regulates multiple proteins and pathwaysinvolved in invasion and metastasis.

Energy metabolism

Studies on tumour cell metabolism in recent years have confirmedthat tumour cells gain a selective advantage by generating energynot only from mitochondrial-driven oxidative respiration, butalso in the presence of oxygen, fermenting glucose into lactate(termed the Warburg effect [117], reviewed in [118]). A numberof pathways can promote this ‘aerobic glycolysis’ to enable theuse of glucose as a fuel for cancer cells. Once again, it seems anumber of the usual players and pathways are involved, frequentlythose associated with controlling tumour growth, suggesting aclose link between proliferative and metabolic pathways. Poten-tially the most important is the PI3K/Akt1/mTOR pathway, whichregulates many components of the glycolytic pathway. Akt1 regu-lates both the expression and membrane translocation of a numberof glucose transporter molecules, contributing to increasedglucose uptake [119,120]. mTOR, when part of mTORC1, acts asa hub to link growth factor signalling with activation of metabolicpathways for growth (reviewed in [121]). Activated mTOR hasrecently been identified as a key driver of aerobic glycolysis, viaincreasing the levels of the crucial enzyme in this pathway, PKM2(pyruvate kinase M2) [122]. mTOR promotes this via the controlof HIF1 and MYC transcription factors [122–124], which then



increase the expression of genes encoding the splicingproteins PTB (polypyrimidine tract-binding protein), hnRNPA(heterogeneous nuclear ribonucleoprotein A) 1 and hnRNPA2[122]. This results in alternative splicing of the pyruvate kinasetranscript, leading to generation of the pro-glycolytic PKM2 spliceform [125].

YB-1 may affect energy metabolism through the regulationof PI3K/Akt1/mTOR pathways as described above (Figure 2).Interestingly, YB-1 has also been shown to regulate the translationof MYC mRNA via binding to an internal ribosome entry sitein the MYC 5′-untranslated region [126]. Therefore, togetherwith mTOR, YB-1 may increase Myc protein levels therebypromoting glycolysis. Furthermore, reducing the expression ofYB-1 in rapidly proliferating A549 and HCT116 cells led toa decrease in PKM2 RNA expression (see results publishedin [27]).

In addition, YB-1 may affect energy metabolism through theregulation of E2F and RB activity. A recent study of E2f1− / − miceshowed that E2F1 is also able to influence metabolic pathways. Inconditions where energy demand was high, the RB/E2F1 pathwayblocked oxidative respiration to drive the expression of genesinvolved in glycolytic metabolism [127].

Another pathway inactivated by YB-1, p53, has also beenassociated with energy metabolism. p53 has been shown toprevent aerobic glycolysis and promote oxidative phosphorylation[128], partly through impeding the PI3K/Akt/mTOR pathway bytranscriptional activation of the Akt inhibitor PTEN (phosphataseand tensin homologue deleted on chromosome 10) [129].

Collectively, these findings suggest that YB-1 modulatestumour cell energy metabolism to promote aerobic glycolysisby regulating several molecules and pathways including PI3K/Akt1/mTOR, Myc, PKM2, the RB/E2F1 pathway and p53.

Evading immune destruction

Cancer cells have acquired several ways to evade immunosur-veillance mechanisms to proliferate unhindered. These involveboth intrinsic and extrinsic mechanisms that either allow tumourcells to avoid detection or removal by the immune cells, or by thesecretion of factors that affect immune cell function respectively[130]. A number of molecules associated with YB-1 are involvedin intrinsic evasion. For example, to resist immune detectionand killing, MHC class II and Fas/CD95 have been observedat lower levels in tumours than in healthy cells ([131,132] andreviewed in [133]). The genes encoding both of these proteins aretranscriptionally repressed by YB-1 [3,67] (Figure 4C).

YB-1 also affects extrinsic mechanisms, including the TGF-βpathway that promotes immune response evasion through multiplemechanisms (reviewed in [130]). Although a link between YB-1and this pathway has not been studied in cancer cells, YB-1 hasbeen shown to regulate translation of TGFβ (TGFB1) mRNAin proximal tubule cells, where lower levels of YB-1 inhibittranslation [101]. This would be consistent with an establishedcancer cell, where YB-1 is expressed at elevated levels, whichwould promote the translation of TGFB1 leading to the activationof the TGFβ pathway. Consistent with this model, we noted thatsiRNA-mediated reduction of YB-1 expression in MCF7 breastcancer cells reduced the levels of TGFB1 and TGFB3 mRNA (seeresults published in [27]).

In summary YB-1 promotes the escape of tumour cells from theimmune system by intrinsic mechanisms such as the regulationof the genes encoding MHC class II and Fas. YB-1 may possiblyalso contribute to extrinsic mechanisms such as regulation of theTGF-β pathway.

Tumour-promoting inflammation

A substantial amount of evidence now links inflammation tothe development of tumours [134]. For example, patients withinflammatory bowel disease are prone to the development ofcolorectal cancers, chronic pancreatitis with pancreatic cancerand haemochromatosis with liver cancer (reviewed in [135]).Furthermore, chronic inflammatory autoimmune diseases such asrheumatoid arthritis and Sjogren’s syndrome are associated withincreased rates of lymphoma.

Many molecules regulated by YB-1, either transcriptionallyor post-transcriptionally, have been linked with promotinginflammation in cancer. These include EGFR, ERBB2, STAT3(signal transducer and activator of transcription 3) and mTOR[4,41], as well as MMP-2 and CD44 [106,112] whoseassociation with YB-1 has been discussed in earlier sectionsof the present review. In addition to these, YB-1 has beenstudied in inflammatory diseases and found to regulate severalproteins involved in inflammatory pathways (reviewed in[136]). For example, the chemokines CCL2 [MCP-1 (monocytechemoattractant protein 1)] and CCL5 [RANTES (regulatedupon activation, normal T-cell expressed and secreted)] induceinflammatory cell infiltration, particularly macrophages, into thetumour microenvironment [137]. This has been especially welldescribed in breast cancer [138]. YB-1 activates transcriptionfrom the CCL5 promoter [139] and may also regulate CCL2 asreduction of YB-1 led to a decrease in CCL2 mRNA levels inER-positive breast cancer cells (see results published in [27]).

In summary, YB-1 regulates the expression of several genesencoding proteins known to drive inflammation, such as mTOR,STAT3, MMP-2, CD44, CCL5 and potentially also CCL2, all ofwhich may contribute to tumour development.

YB-1 IN THE CLINIC

Given the master-regulatory role played by YB-1 in all of Hanahanand Weinberg’s ‘hallmarks of cancer’, it is not surprising thatYB-1 is strongly associated with clinical parameters such astumour progression. As indicated in the opening section of thepresent review, IHC and genomic studies have shown that YB-1protein and mRNA levels are frequently elevated in advancedbreast cancer, have an inverse correlation with ER and PR(progesterone receptor) expression, and are associated with poorpatient outcome [27,140–143]. Subsequent studies over manyyears have shown that YB-1 abundance is also associated withthe outcome of a range of other human malignancies such asglioblastoma [144], melanoma [16], multiple myeloma [17],osteosarcoma [11], synovial sarcoma [14], prostate cancer [15],colorectal cancer [145], ovarian cancer [26,146] and lung cancer[12]. Thus the detection and quantitation of YB-1 is potentiallya powerful prognostic tool. Despite this the potential clinicalimportance of YB-1 has been largely underplayed.

One possible reason for this is due to an historical focuson nuclear YB-1. As described above, several studies havesuggested that it is nuclear YB-1 that is associated with the moreaggressive cancers and poor prognosis. However, a number ofstudies have found that the overall YB-1 level (which is essentiallycytoplasmic) is a sufficient indicator of prognosis [17,147,148].Indeed, it is very difficult in our opinion to discern nuclearlocalization of YB-1 using IHC, and in our analyses encompassingthree different breast cancer cohorts, the proportion of tumoursor cells showing clear nuclear YB-1 is very small. In one study,only three cells in 96 breast cancers showed unequivocal nuclearYB-1 staining [143], and in the large cohort described by Habibiet al. [141] only 3% of tumours had some limited nuclear


YB-1 and cancer 19

Figure 7 Western blot showing an example of cross-reactivity with a YB-1antibody

A549 cells were transfected with two different siRNAs to YB-1 (si-YB-1#1 and si-YB-1#2)or a control siRNA [27]. Cell lysates were collected at 72 h post-transfection and proteinsseparated by SDS/PAGE prior to Western blotting with a rabbit polyclonal antibody againstYB-1 [149]. Note that the amount of YB-1 protein (∼45 kDa) is considerably reduced followingtransfection with the YB-1 siRNAs, but a 37 kDa (hnRNPA1) protein band is unchanged.M. molecular mass markers (masses are shown in kDa on the left-hand side).

YB-1 staining. However, the overall YB-1 level was found tobe prognostic in both cases. That the absolute YB-1 level issufficient for prognostication is also highlighted by another studyof ∼400 breast cancers, which showed that abundance of themRNA encoding YB-1 is significantly associated with poorclinical outcome [27].

A second reason why YB-1 has attracted less clinical andpathological attention than expected may be due to the use ofmultiple antibodies for the detection of YB-1 in tumours. This isof critical importance since some antibodies detect both nuclearand cytoplasmic YB-1 [7], whereas others appear to detectessentially only cytoplasmic YB-1 [141]. This point wasemphasized in a comparative study of just two antibodies (raisedagainst residues 1–12 and 299–313 of YB-1), which showed cleardifferences in their ability to detect nuclear YB-1 and also theirability to show an association with tumour progression [143].Analysis with additional antibodies against YB-1 generated yetother patterns of staining (results not shown). Thus antibodiesraised against different regions of YB-1 may give quite differentresults. It is paramount therefore that publications state whichantibodies are used to enable comparison and reproducibilitybetween studies.

A further issue was identified when it was shown that someantibodies are not only poorly immunoreactive to YB-1, theyare also cross-reactive with the nuclear protein hnRNPA1 [149].Figure 7 shows an example of this. In addition to the ∼45 kDaYB-1 band, which is specifically reduced following transfectionwith YB-1 siRNAs, a second protein at 37 kDa (shown tobe hnRNPA1 [149]) is also detected by this antibody and isunchanged following YB-1 knockdown. Worryingly, hnRNPA1is predominantly a nuclear protein, which casts doubts on studiesusing these antibodies for IHC analysis of YB-1, particularlythose drawing conclusions from nuclear staining. Despite thesefindings, one of the cross-reactive antibodies continues tobe used [150] (http://www.abcam.com/YB1-antibody-ab12148-references.html). We propose therefore that a simple validationof YB-1 antibodies is performed (e.g. as in Figure 7) before IHCstudies with YB-1 antibodies are published.

Collectively, the variety of antibodies with different qualitiesand specificities has limited the development of antibody-basedpredictive and prognostic screens utilizing YB-1. However, nowknowing the limitations of the area, the field can move on,

developing standardized and properly validated (monoclonal)antibodies against YB-1 to enable the use of YB-1 as a powerfulprognostic indicator in the clinic.

YB-1 AS A THERAPEUTIC TARGET

Given that the position of YB-1 is upstream of the molecularpathways responsible for all nine hallmarks of cancer, YB-1 isa very attractive therapeutic target. Several approaches to targetYB-1 have been employed, including targeting of YB-1 directly,interfering with the activation of YB-1 or targeting the regulatorsthat activate YB-1.

Historically, the first of these used a ‘decoy’ YB-1-binding siteto sequester the YB-1 protein. This was successfully employed incultured cancer cells of many different lineages, with the resultof inhibition of tumour cell growth and p53-mediated apoptosis[18,63]. In this case, normal cells (fibroblasts and melanocytes,results not shown) were not sensitive to YB-1 inhibition. Despitethe publication of this work almost 10 years ago, the use ofnucleic acids as therapeutics has been hampered predominantlyby issues of delivery, although rapid advances are now being made[151,152].

Another therapeutic approach to directly target YB-1 could beto use siRNAs. Anti-YB-1 siRNAs have been shown to suppresstumour cell invasion, proliferation, differentiation, insensitivity tochemotherapy and to promote apoptosis, as described previously[17,27,41,153,154]. The challenge will be delivering siRNAs inhumans given the widely discussed problems of stability andbioavailability and the current drive to evolve better chemistryand delivery technologies [152].

As outlined above, the ‘transcription factor’ functions of YB-1are activated by phosphorylation. The most widely characterizedphosphorylation site is at Ser102, which stimulates nucleartranslocation and DNA binding [19]. Site-directed mutagenesisshowed that S102A YB-1 mutants exhibited reduced cellproliferation [43] and an interference peptide was designed toserve as another type of ‘molecular decoy’ that competes withYB-1 for phosphorylation by RSK and Akt [155]. This CPP(cell-permeable peptide) inhibited the proliferation of breast andprostate cancer cells in cell culture [155]. Importantly, the CPPhad no effect on the growth of normal mammary epithelial cellsisolated from patients [155]. Thus peptide-based delivery systemscould be used to inhibit YB-1 therapeutically.

The use of ‘signal transduction’ inhibitors is another approachto suppressing YB-1 activity. A side effect of blocking kinasessuch as PDK-1 [156], Akt [19,157] and RSK [158] is quenchingof YB-1 phosphorylation at Ser102 and thereby reduction of thetransactivation activity of YB-1. This ‘side effect’ on YB-1 mayin fact turn out to be responsible for a significant proportion of theactivity of these inhibitors. Although Akt was originally reportedto phosphorylate and activate YB-1 [19] it was subsequentlyreported that relative to other kinases such as RSK, Akt mightbe a minor player in YB-1 activation [158]. In support of this,RSK inhibition with siRNA, or small molecules such as BI-D1870and SL0101, completely suppress the activation of YB-1, despitethe presence of activated Akt in the same cells [158]. Likewise, theMEK inhibitor PD098059 blocks RSK and suppresses the nuclearlocalization of YB-1 [159]. Moving further upstream, PDK-1inhibition also suppresses YB-1 [156] and is known to directlyactivate RSK through phosphorylation at Ser380 [160]. Given thatRSK is the most proximal kinase that activates YB-1, and given thenumerous YB-1-independent pathways that RSK also activates, itwould seem reasonable to focus on blocking RSK [161–163]. Theeffect on YB-1 of other proteins associated with YB-1 pathways,


http://www.abcam.com/YB1-antibody-ab12148-references.html

http://www.abcam.com/YB1-antibody-ab12148-references.html


Figure 8 Schematic diagram showing that YB-1 affects all of the hallmarksof cancer

such as mTOR or ILK (integrin-linked kinase) inhibitors [91,164],have not been fully investigated.

In summary, direct targeting of YB-1 using cell-permeableinhibitory peptides, YB-1 siRNAs or oligonucleotide decoys haveshown promise in cell culture experiments. Indirect inhibition ofYB-1 is a known side effect of blocking kinases such as PDK-1,Akt, MEK or RSK, and YB-1 blockade may potentially underliea significant part of the activity of these inhibitors.

CONCLUSION

The present review has described YB-1 as a master regulatorof cancer cell biology, contributing to all nine of Hanahan andWeinberg’s ‘hallmarks of cancer’ (Figure 8) and is therefore abona fide oncoprotein. YB-1 is a multitasking protein that mayoperate in different, but overlapping, ways in different cell types.Moreover, due to the levels of YB-1 protein and YBX1 mRNAbeing highly correlated with poor patient outcome, YB-1 shouldbe regarded as a useful biomarker of cancer progression andas a novel therapeutic target. Given the strong association ofYB-1 with aggressive (basal-like) breast cancers, targeting YB-1could, for example, be of particular value for such cancers thatare currently difficult to treat. Given the increasing consistencywith which YB-1 is being associated with cancer progression andinterest in understanding how YB-1 functions, we are confidentit will feature prominently in the cancer literature in the futureand hopefully, in time, be developed as a biomarker and targetfor therapy.

ACKNOWLEDGEMENTS

We thank Ms Sunali Mehta for critically reading the paper prior to submission.

FUNDING

The work described in the present review was supported by the Cancer Society of NewZealand [grant numbers 10/21 (to A.L. and C.G.P.) and 11/07 (to A.G.W. and A.W.B.)], theHealth Research Council of New Zealand [grant number 10/02 (to A.G.W. and A.W.B.)], theNew Zealand Breast Cancer Research Trust [grant number 3621880 (to A.L. and C.G.P.)],the University of Otago Dean’s Bequest (NZ), the Canadian Institutes for Health Research(to S.E.D.) and the Cancer Institute NSW [grant number RLP 05/01 (to A.W.B.)].

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Received 21 August 2012/2 October 2012; accepted 9 October 2012Published on the Internet 7 December 2012, doi:10.1042/BJ20121323


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YB-1: oncoprotein, prognostic marker and therapeutic target? · to cover the biological changes required for the development and persistence of tumours [Hanahan and Weinberg (2011)