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Biochem. J. (2013) 449, 11–23 (Printed in Great Britain)
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doi:10.1042/BJ20121323
REVIEW ARTICLE
YB-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, New
Zealand, §Department of Paediatrics and Experimental Medicine, University of British Columbia, Vancouver, 950W 28th Ave, BC V5Z 4H4, Canada, and ¶Children’s Medical Research
Institute, 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 and
persistence of tumours [Hanahan and Weinberg (2011) Cell 144,
646–674]. We have noted that many of these cancer hallmarks are
facilitated by the multifunctional protein YB-1 (Y-box-binding
protein 1). In the present review we evaluate the literature and
show how YB-1 modulates/regulates cellular signalling pathways
within each of these hallmarks. For example, we describe how
YB-1 regulates multiple proliferation pathways, overrides cell-
cycle check points, promotes replicative immortality and genomic
instability, may regulate angiogenesis, has a role in invasion and
metastasis, and promotes inflammation. We also argue that there is
strong and sufficient evidence to suggest that YB-1 is an excellent
molecular marker of cancer progression that could be used in the
clinic, and that YB-1 could be a useful target for cancer therapy.
INTRODUCTION
independent manner (comprehensively reviewed by Eliseeva
et al. [6]). In the present review we discuss the multifunctional
nature of YB-1 thereby illustrating how it facilitates the cancer
hallmarks of Hanahan and Weinberg [21].
YB-1 (Y-box-binding protein 1) encoded by the YBX1 gene, is
a member of the cold-shock protein superfamily, all of which
contain a highly conserved nucleic-acid-binding motif that binds
to both DNA and RNA. This motif is located within a region
of 65 amino acids termed the ‘cold-shock domain’ which shares
greater than 40 % identity with prokaryotic cold-shock proteins
[1]. This degree of sequence conservation supports the notion
that these proteins play an essential role in both the prokaryotic
and eukaryotic cell [2]. It is interesting to speculate whether
the ancestral nucleic-acid-binding domain of YB-1 has existed
through the evolution of prokaryotes to eukaryotes, possibly
acquiring new roles as organisms became more complex. If this
were the case, it is not surprising that YB-1 has taken on many
seemingly diverse roles.
YB-1 was originally identified as a factor that repressed gene
transcription by binding to the Y-box (an inverted CCAAT
box) of MHC class II promoters [3]. Later the same year,
protein-blotting assays using DNA probes revealed that YB-1
binds to the enhancers of the EGFR (epidermal growth factor
receptor) and the ERBB2 (HER2) genes [4]. By 1995, it
became clear that YB-1 played an important role in regulating
cellular proliferation and development [5]. Since then, YB-1
has been shown to be a transcription factor of many genes
(reviewed in [6]), but also directly affects DNA repair, RNA
splicing, exon skipping, drug resistance and cancer progression
[EMT (epithelial–mesenchymal transition)] in a transcription-
Key words: angiogenesis, genomic instability, inflammation,
invasion/metastasis, metabolism, proliferation.
The importance of YB-1 in cancer: a preamble
YB-1 rose to prominence following reports of elevated YB-1
protein levels being highly correlated with cancer progression and
poor prognosis. Initially this came from IHC (immunohistochemistry) analyses of breast tumours by Royer’s group, who showed
that levels of cytoplasmic YB-1 correlated with progression
in 27 breast cancers [7]. They also showed that the levels of
nuclear, but not cytoplasmic, YB-1 correlated with expression
of the ABC transporter (ATP-binding cassette transporter) Pglycoprotein MDR1 (multidrug resistance protein 1) in nine
cancers. These data were consistent with reports that YB-1
could transactivate the MDR1 gene [8–10] and that high MDR1
protein levels in tumours were associated with poor clinical
prognosis. The importance of the correlation between nuclear YB1 and MDR1 levels with patient prognosis was strengthened by
similar observations for osteosarcoma [11], non-small-cell lung
carcinoma [12,13], synovial sarcoma [14], prostate cancer [15],
melanoma [16] and multiple myeloma [17]. In addition, nuclear
YB-1 and MDR1 were both observed at high levels in 9/27 breast
cancers after paclitaxel treatment.
Data such as these has led to the widely accepted view that
nuclear 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 factor
receptor; EMT, epithelial–mesenchymal transition; ER, oestrogen receptor α; ERK, extracellular-signal-regulated kinase; GSK3β, glycogen synthase kinase
3β; HIF-1, hypoxia-inducible factor 1; hnRNPA, heterogeneous nuclear ribonucleoprotein A; IHC, immunohistochemistry; IL-8, interleukin 8; LEF1, lymphoid
enhancer-binding factor 1; MAPK, mitogen-activated protein kinase; MDM2, murine double minute 2; MDR1, multidrug resistance protein 1; MEF, mouse
embryonic 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, pyruvate
kinase 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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A. Lasham and others
Table 1 Cancer cell lines where YB-1 reduction has been shown to induce
apoptosis or inhibit cell proliferation
Cell location
Cancer
Cell line
Reference(s)
In vitro
Melanoma
Fibrosarcoma
Liver cancer
Lung cancer
SK-Mel-5, NZM9
B10.2, B10.5
HepG2
A549, EBC-1, PC-9,
QG56
TCCsup, KK47
GM02301, GM2132,
INA-6, MM.1
SF188
MDA-MB-231,
BT474-m1, Au565,
MDA-MD-468,
HCC1937, SUM149
HR5, HR6
MCF-7, T-47D, KPL-1
PC3
RKO HCT116
BT474-m1
A549
SF188
[18]
[18]
[18]
[18,27,52]
Bladder cancer
Multiple myeloma
Paediatric glioblastoma
Breast cancer (ER-negative)
In vivo
Breast cancer (ER-positive)
Prostate cancer
Colon cancer
Breast cancer
Lung cancer
Paediatric glioblastoma
[90]
[17,126]
[154]
[41,43,165]
[27,52,90,166]
[90]
[18,27,29]
[41]
[27]
[154]
at elevated levels, leads to increased tumour cell proliferation
and drug resistance. Then an important study in 2003 showed
that YB-1 was essential to control the growth and survival of
tumour cells of different origins in vitro [18]. A subsequent
study showed this to be dependent on the nuclear translocation
of YB-1 [19]. Consistent with this, in 2005 Royer’s laboratory
demonstrated that sustained overexpression of YBX1 in transgenic
mice invariably led to the development of invasive breast cancers,
further supporting the view that YB-1 can function as an
oncoprotein [20].
YB-1 AND THE HALLMARKS OF CANCER
In 2000, Hanahan and Weinberg proposed six regulatory
pathways that must be overcome in order for a cell to
become malignant: uncontrolled proliferative signalling, evading
growth suppressors, resisting cell death, replicative immortality,
sustained angiogenesis, and invasion and metastasis [21]. In
2011 they proposed two additional hallmarks: deregulated
metabolic pathways and avoiding immune destruction, as well
as two enabling characteristics: genomic instability and tumour
promoting inflammation [22].
The YB-1 protein is remarkably multifunctional, and in the
present review we demonstrate the significant contribution that
YB-1 makes to each of Hanahan and Weinberg’s cancer hallmarks.
We argue that the multifunctionality of YB-1 renders it a true
master-regulator of malignancy and therefore it deserves the status
given to other multi-potent oncoproteins such as Myc and Ras.
Since we focus specifically on the cancer hallmarks of Hanahan
and Weinberg, the present review is not intended to provide
a comprehensive evaluation of YB-1’s functions. For this we
recommend the excellent reviews by Eliseeva et al. [6] and Brandt
et al. [23]. In the following pages the role played by YB-1 in each
of the hallmarks of cancer will be discussed in turn.
Proliferation
The early association made between YB-1 and proliferation has
focussed research on dysregulation of proliferation and the cell
c The Authors Journal compilation c 2013 Biochemical Society
cycle by YB-1, which is arguably the most important hallmark
of a tumour cell. Reduction of YB-1 expression causes growth
inhibition or apoptosis in a broad range of cancer cells both
in vitro and in vivo (Table 1). These data suggest that YB-1 plays a
critical and non-redundant role in regulating cell proliferation. As
a consequence, several studies have investigated the mechanism
by which YB-1 regulates cell proliferation. One approach has been
to search for YB-1-binding sites or ‘Y-boxes’. However, YB-1 can
bind to a variety of DNA sequences that have little sequence
similarity [24,25], making it difficult to predict a canonical
YB-1-binding site. Thus YB-1 targets must be empirically
determined. Given this, several studies have performed largescale analyses to determine the key transcription targets of YB-1.
Using gene expression arrays, changes in transcript abundance
were determined following YB-1 knockdown with siRNAs (small
interfering RNAs) in ovarian, colorectal, lung and ER (oestrogen
receptor α)-positive breast cancer cell lines [26,27]. Another
approach has been to identify the promoters bound by YB-1 in
colorectal 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 comparison
of the transcriptional targets of YB-1 in all of these studies
showed few in common (results not shown). For example, gene
expression data of three cancer cell lines (colorectal, breast and
lung) following YB-1 siRNA treatment showed that although a
few hundred transcripts were downstream targets of YB-1, only
25 were in common in all lines [27]. Cumulatively, these results
suggest that the downstream targets of YB-1 are cell-type-specific.
This raises the possibility that any one cell type may utilize only a
subset of YB-1’s transcriptional capability, since it expresses only
a subset of YB-1’s potential transcriptional targets. This may be
due to the presence of different transcriptional co-factors or YB1-binding partners in each cell type (e.g. [31,32]). Interestingly
however, there appears to be strong common themes to YB-1’s
transcriptional targets across lineages; rather than being a random
set of genes, the downstream targets of YB-1 in different cell
types, even if only slightly overlapping, are all enriched for E2Fregulated genes [27].
The E2F pathway
A seminal paper in 2005 by Rhodes and colleagues studied gene
expression data from almost 7000 microarray experiments in
the Oncomine database [33]. They observed that transcriptional
targets of the E2F family were over-represented in many tumour
types, which led them to state, “These results reaffirm that
activation of the E2F pathway is a prevalent event in human
cancer”. As indicated, it has recently been shown that YBX1
mRNA levels are associated with the E2F1 pathway in breast,
colorectal and lung tumours [27], which was confirmed using
ChIP assays, where YB-1 bound directly to the promoters of
several canonical E2F1-regulated genes [e.g. CDC6 (cell-division
cycle 6), CCNA (cyclin A) and TOP2A (topoisomerase II α
170 kDa)]. Furthermore, bioinformatic analysis of COC data
revealed that more than 4000 of the 6000 promoters bound by
YB-1 in ER-negative breast cancer cells also have E2F1/E2Fbinding sites (see results published in [28]), and that 57 of the
88 YB-1 target genes identified by COC analysis in HCT116
colorectal cancer cells had E2F1/E2F-binding sites within their
promoters (see results published in [29]). Therefore multiple
studies suggest that YB-1 co-regulates the expression of genes
in 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
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 that
are 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 and
activates transcription of the E2F1 promoter in A549 cells. Using
different experimental systems, in the ER-negative breast cancer
cell line SUM149, YB-1 was shown to bind to the E2F2, E2F3
and E2F7 promoters (see results published in [28]), and in the
SKOV-3 ovarian cell line E2F7 expression was increased
following YB-1 knockdown (see results published in [26]). These
results suggest that the expression of ‘activator’ and ‘repressor’
members of the E2F family may be turned on and off respectively
by YB-1 in a cell-type-specific manner (Figure 1).
In summary, we suggest that YB-1 has evolved to regulate the
activity of E2F pathways through two synergistic mechanisms.
First, YB-1 transcriptionally regulates several members of the E2F
family to promote expression of the ‘activator’ E2Fs and inhibit
expression of the ‘repressor’ E2Fs. Secondly, it co-regulates
the expression of thousands of E2F target genes by binding
to their promoters. These important observations suggest that
YB-1 may in fact be an Achilles’ heel of the E2F cancer cell
proliferation pathway, which could provide an opportunity to
target 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 regulate
other cell proliferation pathways. The PI3K class I enzymes and
the pathways driven by them are dysregulated in the majority
of cancers (reviewed in [34]). Multiple cell-surface receptors, in
particular growth factor receptors (e.g. EGFR and ERBB2), relay
growth-promoting signals through the activation of this pathway
(reviewed in [35]). The PI3KCA gene encoding the catalytic
subunit p110α is frequently amplified or acquires activating
mutations in cancers to further enhance the activity of the pathway
[36]. The PI3K pathway signals through the multifunctional
protein kinase Akt which, in turn regulates mTOR to influence a
number of oncogenic functions, including proliferation, survival,
metabolism and metastasis (reviewed in [37]). The PI3K pathway
also cross-talks with the E2F pathway discussed above, by
modulating 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 transcriptionally
activates the expression of PIK3CA in basal-like breast cancer
cells [39] and YB-1 depends on Akt for its nuclear translocation
following 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 expression
is activated by YB-1 irrespective of whether the PIK3CA gene
is mutated or amplified, leading to further dysregulation of the
PI3K pathway [39]. This results in the subsequent modulation of
a number of downstream components of the PI3K pathway, which
includes molecules that can phosphorylate YB-1, phospho-RSK
13
(ribosomal S6 kinase) (Ser360 ) and phospho-Akt(Ser473 ) [16,39],
leading to further YB-1 nuclear localization and further activation
of PIK3CA transcription. It has recently been shown in melanoma
cells that inhibitors of the PI3K pathway can modestly reduce
expression from a cloned YBX1 promoter, suggesting that the
activation of this pathway can also promote transcription of YBX1
[40].
Downstream of Akt is mTOR. mTOR forms a complex with
raptor (regulatory associated protein of mTOR) called mTORC1
(mTOR complex 1), which appears to be an important hub that
controls many pathways that affect protein and lipid synthesis,
autophagy, production of inflammatory cytokines, glycolysis
and angiogenesis (reviewed in [37]). In addition to regulating
PI3K/Akt signalling, YB-1 may also regulate mTOR. Reduction
of YB-1 with siRNAs in a number of ER-negative breast cancer
cells and a paediatric glioblastoma cell line was shown to cause
a marked decrease in mTOR protein levels [41]. This was not
accompanied by a reduction in mRNA expression, suggesting
that YB-1 controls the translation or affects the mRNA stability
of mTOR.
Taken together, these results suggest that, similar to its interaction with multiple points of the E2F pathway, YB-1 interacts
with multiple points of the PI3K/Akt/mTOR pathway to increase
the activity of this pathway in cancer cells.
MAPK (mitogen-activated protein kinase) pathways
There are at least six different molecular pathways associated with
MAPKs, but only the Ras/Raf/MEK [MAPK/ERK (extracellularsignal-regulated kinase) kinase]/ERK signalling pathways will
be discussed in the present review. MAPK signalling is initiated
by growth factor receptors on the cell surface activating Ras,
and then Raf, which activates the MAPK kinases MEK and
ERK, that in turn activate several downstream pathways, which
converge to promote cellular proliferation (reviewed in [35]).
There is complex cross-talk between the Ras/Raf/MEK/ERK
and PI3K/Akt pathways (Figure 2), with both pathways able to
regulate the other at multiple points (reviewed in [35,42]).
YB-1 activates several members of the Ras/Raf/MEK/ERK
pathway (Figure 2). As described above, YB-1 is a transcriptional
activator of genes encoding the EGFR and ERBB2 cell-surface
receptors, which transmit initial signals to intracellular MAPK
pathways [4,43]. YB-1 appears to also regulate a number of genes
downstream in the MAPK pathway. COC studies in colorectal
cancer cells [29] and ER-negative breast cancer cells [28,30]
have identified that YB-1 binds to the promoters of a number
of MEK/ERK pathway genes.
The effects of this signalling pathway are modulated by a
family of dual-specific MAPK phosphatases [44]. Interestingly,
microarray analysis of cancer cell lines where YB-1 levels
were reduced by siRNA treatment showed significantly altered
expression of transcripts encoding a number of MAPK
phosphatases including MKP2 (MAPK phosphatase 2; see data
published in [27]). This suggests yet another role for YB-1 in the
regulation of this pathway.
Interestingly the Ras/Raf/MEK/ERK pathway also activates
YB-1 in a positive feedback loop. RSK1 [39] and ERK2 [45] have
been shown to phosphorylate YB-1 to promote its transcriptional
activity (Figure 2), and a MEK inhibitor has been shown to reduce
YB-1 protein abundance [46].
In summary, several pathways that promote cancer cell
proliferation are activated by YB-1. These include the E2F,
PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways. These three
pathways converge and overlap with one another [38,42,47],
and YB-1 regulates several of members of each pathway. By
c The Authors Journal compilation c 2013 Biochemical Society
14
Figure 2
A. Lasham and others
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 signalling
pathway and glycolysis via PKM2, as well as the phosphatase encoded by MKP.
doing so, YB-1 promotes cancer cell proliferation through several
parallel signalling cascades involving many effector molecules.
Again, this makes YB-1 an attractive target for therapies to control
cancer cell growth.
Evading growth suppressors and cell-cycle checkpoints
For a cancer cell to undergo sustained proliferation, the two
cell-cycle checkpoints regulated by the RB (retinoblastoma)
gatekeeper protein and the tumour suppressor p53 must be
overcome. YB-1 appears to override both of these checkpoints.
Figure 3
(a) The RB pathway
The critical role of RB as a tumour suppressor is evidenced by
the fact that RB or the RB pathway is inactivated in almost
all human tumours [48]. In normal cells RB exerts its tumour
suppressor 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 cellcycle progression. Following CDK (cyclin-dependent kinase)mediated hyperphosphorylation of RB (pRB), the activator E2Fs
are released from the RB inhibition and can transcriptionally
activate numerous genes promoting cell-cycle progression from
G1 - to S-phase (Figure 3) [50,51]. In cancers, the normal control
of cell-cycle progression by RB pathways is reduced, in part,
through YB-1.
YB-1 appears to reduce the tumour suppressive activities of
RB in several ways. First, it has been shown that YB-1 controls
the 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 and
is a transcriptional repressor of several ‘repressor’ E2Fs, as well
as a co-regulator of S-phase genes, including E2F-1 (Figure 3).
It seems possible that the elevated levels of YB-1 in cancer cells
strongly activate the expression of ‘activator’ E2Fs so that RB
binding becomes insufficient to fully inhibit these activator E2F
molecules. Further research is required to fully understand the
complex inhibition of the RB tumour suppressor pathway by
YB-1.
c The Authors Journal compilation c 2013 Biochemical Society
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 promote
hyperphosphorylation of RB leading to release of E2F1 (and activation of the transcription
factors). YB-1 also directly activates expression of S-phase genes including those encoding
E2F1, 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 as
it is inactivated more frequently in cancers than any other gene
as yet identified [53]. The p53 protein that TP53 encodes functions
as a transcription factor to control expression of a number of
genes involved in cell survival and proliferation. p53 is normally
present at very low levels in cells due to constant degradation
by the E3 ligase MDM2 (murine double minute 2) [54].
However, after stress, particularly DNA damage, p53 becomes
phosphorylated preventing interaction with MDM2, thereby
allowing p53 levels to increase dramatically [55]. When this
occurs, p53 transactivates genes to cause cell-cycle arrest allowing
DNA repair, permanent arrest of cell division (senescence) or
apoptosis, thereby preventing the accumulation of lesions that
could otherwise go on to initiate malignancy (reviewed in [55,56]).
The p53 protein is disabled in many tumours. Although this occurs
mostly by mutation, p53 can also be disabled by direct interaction
with other proteins (e.g. [57,58]).
YB-1 can disable the p53 pathway in cancers by regulating both
the activity and the expression of p53 (Figures 4A and 4B). Several
YB-1 and cancer
Figure 4
YB-1 regulates both apoptosis and proliferation pathways
YB-1 controls apoptosis and cell-cycle arrest by transcriptionally repressing the gene encoding
p53 (A) and inhibiting p53-dependent apoptosis by direct protein interaction (B). Similarly YB-1
represses expression of genes encoding both FAS and CASP7 (C) and activates transcription of
E2F1 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 transactivate
genes [59,61,63,64]. For example, YB-1 reduced the p53-driven
transcriptional activation of apoptosis-associated genes APAF1
(apoptotic peptidase activating factor 1), NOXA (NADPH oxidase
activator) and BAX (Bcl2-associated X protein), but had little
effect on the promoter of the CDKN (CDK inhibitor) 1 gene
encoding the cell-cycle inhibitory protein p21CIP1 [63]. It was
also observed that p53 had greater affinity for the promoters of
cell-cycle-associated proteins than those of apoptosis-associated
proteins, potentially making it more difficult for YB-1 to override
the regulation of cell-cycle-associated gene promoters by p53
[63]. In addition to directly affecting the activity of p53, YB-1 also
represses transcription of the TP53 gene [18]. The importance
of YB-1 in controlling this pathway was confirmed by the
observation that the reduction in YB-1 led to an increase in p53
protein levels and triggered p53-dependent apoptosis in cancer
cell lines with wild-type p53 [18,63].
In summary, YB-1 appears to help cancer cells escape cell-cycle
checkpoints by inhibiting both the RB and p53 pathways.
Resisting cell death
Cancer cells have evolved to evade the normal apoptotic pathways
that would otherwise remove damaged cells. Upon a ‘cellular cue’,
a series of proteins transduce a signal to activate an apoptosis
pathway that culminates in DNA degradation and systematic
disassembly of the cell by activation of a series of proteolytic
enzymes called caspases [65]. YB-1 is involved in protecting
tumour cells from apoptosis in several ways. p53 probably plays
the most important role here to detect damaged DNA and initiate
apoptosis if the DNA cannot be repaired. Elevated levels of
YB-1 enable cells to subvert the p53-driven apoptosis pathway
(Figures 4A and 4B).
Another apoptotic pathway involves the cell-surface death
receptor Fas (CD95). Upon binding of Fas ligand (CD95L),
an apoptotic signal is transduced from Fas into the cytoplasm
through multiple downstream effector molecules, including
the executioner caspases, CASP3 and CASP7, to promote
orderly cellular disassembly, including PARP [poly(ADP-ribose)
polymerase]-mediated cleavage of DNA and finally fragmentation
of the cell into apoptotic bodies [66]. YB-1 appears to inhibit
the Fas-mediated apoptosis pathway at several points. YB-1 is a
15
transcriptional repressor of the FAS promoter (Figure 4C) [67]
and consistent with elevated levels of YB-1 in tumours, FAS
is often down-regulated in cancers [68,69]. YB-1 also inhibits
the expression of the gene encoding the pro-apoptotic protein
BAX [18,63]. YB-1 may also repress transcription of CASP7
(Figure 4C), since ChIP analysis showed that YB-1 binds to the
CASP7 promoter (see results published in [28]) and reduction of
YB-1 levels increased CASP7 expression (see results published in
[27]). In addition to quelling the pro-apoptotic signals from death
receptors, the inhibition by YB-1 of BAX and CASP7 may also act
to suppress intrinsic apoptotic signals from DNA or mitochondrial
damage (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 promoting
the expression of pro-apoptotic genes. This has recently been the
subject of considerable interest [72,73]. In response to growth
promoting signals, such as fetal bovine serum, there is a PI3Kdependent repression of E2F1 s transcription of apoptosis genes,
whereas E2F1 continues to drive the transcription of genes
involved in proliferation [74]. As described above, YB-1 is an
integral component of the PI3K pathway, driving proliferation
by activating many pathway components and being part of
positive feedback loops. Therefore it seems probable that YB-1
promotes the expression of E2F1-dependent proliferative genes,
but not E2F1-dependent apoptotic genes. We hypothesize that, as
observed for p53-regulated cell cycle and apoptosis pathways
[63], YB-1 preferentially co-activates expression of E2F1
proliferation-associated genes and not those driving apoptosis
pathways (Figure 4D). In support of this, we have studied the
relationship between YBX1 mRNA levels and the inferred activity
of the E2F1-driven apoptotic and proliferative transcriptional
programmes [74] using PCA (principle component analysis) of
microarray data from breast tumours. The results suggest that
YBX1 mRNA levels are associated with the transcription of the
proliferative E2F1 target genes, but not apoptotic E2F1 target
genes (results not shown).
Replicative immortality and genomic instability
The immortalization of cells is stimulated by: (i) activation of
telomere maintenance mechanisms [75]; (ii) loss of the RB
checkpoint; and (iii) loss of p53 function, which together lead
to lifespan extension and genomic instability. YB-1 can promote
all three of these mechanisms.
Replicative senescence
Studies of MEFs (mouse embryonic fibroblasts) generated from
E13.5 (E is embryonic day) Ybx1 − / − mice demonstrated that these
cells prematurely senesced compared with control MEFs [76]. It
was noted that the Ybx1 − / − MEFs had increased levels of Cdkn2a
(p16Ink4a) and Cdkn1a (p21Cip1), which would promote senescence. Furthermore, Cdkn2a and Cdkn1a were also elevated at the
mRNA level, suggesting that YB-1 may be required for their transcription or mRNA stability. The involvement of these proteins
was supported as the induction of senescence could be partially
bypassed by knockdown of both of these transcripts [76]. YB-1
thus appears to play a role in controlling replicative senescence.
Genomic instability
The loss of genomic stability, leading to alterations in the genome
that include amplifications, deletions, translocations or even
c The Authors Journal compilation c 2013 Biochemical Society
16
A. Lasham and others
aneuploidy, is a characteristic of solid tumours. In hereditary
cancers, genomic instability is frequently a result of mutations
within DNA repair genes, however what promotes this in sporadic
tumours is not well understood (reviewed in [77]). The key
proteins 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 human
cancers, very few mutations in DNA repair genes have been
identified in sporadic cancers [77]. Instead the data point towards
an involvement of oncogene-driven replicative stress leading to
genomic instability.
Recently, interest has focussed on the RB/E2F pathway in
protecting the integrity of the genome, since the loss of RB
leads to genomic instability (reviewed in [78]). An elegant study,
performed in non-immortalized cells, tested the ability of both
viral and cellular oncoproteins to aberrantly activate the RB/E2F
pathway [79]. This led to enhanced cellular proliferation, but
without a concurrent increase in nucleotide metabolism, leading
to a depletion of the nucleotide pool. The outcome of this was
replicative stress, leading to incomplete progression of replication
forks and thereby DNA damage.
Given the manner in which YB-1 modulates the RB and E2F
pathways (Figure 3) it appears possible that elevated levels of
YB-1 may promote genomic instability through replicative stress,
without concurrent induction of apoptosis. Indeed, overexpression
of YBX1 does appear to promote genomic instability. In a study by
Bergmann et al. [20] the overexpression of YBX1 was associated
with genomic instability when expression was targeted to the
mammary gland of transgenic mice. All of these mice ultimately
developed mammary tumours after 52 weeks. Furthermore, in
human mammary epithelial cells, prolonged expression of YB-1
induced a loss of cell-cycle control, genomic instability and
centrosomal amplification [80].
In summary, the results of both YB-1 inactivation and
overexpression suggest that YB-1 can promote replicative
immortality and genome instability.
(v) Inducing angiogenesis
Aberrant angiogenesis is a hallmark of many solid tumours.
Once the tumour reaches a size where nutrients and oxygen
becomes limiting, a pro-angiogenic pathway is initiated. This
event is termed the ‘angiogenic switch’ [81,82]. There are
several proteins involved in this process, with perhaps the best
known 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 promoting
and regulating blood vessel development. However, in developing
tumours, angioregulatory pathways are not as tightly controlled
as occurs in normal development, so that dysfunctional vascular
beds are formed, often with irregular structure and are poorly
synchronized with the needs of the tissues they supply [84].
Because of this, the tumours become more hypoxic, driving
further aberrant angiogenesis, and resulting in decreased drug
delivery and vascular dissemination of cancer cells. It is therefore
not surprising that angiogenesis and tumour invasiveness are
closely 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 expression
of pro-angiogenic PDGF-β following thrombin treatment [85].
Studies in epithelial-derived cancer cell lines suggest that YB-1
may up-regulate the expression of other pro-angiogenic genes
c The Authors Journal compilation c 2013 Biochemical Society
Figure 5
YB-1 plays a role in the angiogenic switch
Under normoxic conditions YB-1 represses transcription of pro-angiogenic genes such as those
encoding VEGF-A, PDGF-β, IL-8 and CXCL2. Repression of VEGF-A has been shown to occur
via phosphorylation of YB-1 by activated GSK3β, which prevents binding of HIF-1 to the VEGFA
promoter. However, as oxygen levels decline, GSK3β is not activated, thus enabling HIF-1 to
access the VEGFA promoter leading to expression of pro-angiogenic factors.
in a hypoxia-dependent manner. However, under normoxic
conditions, YB-1 appears to inhibit the expression of a number of
pro-angiogenic genes. For example, in A549 lung cancer cells,
reduction of YB-1 led to increased levels of the transcripts
encoding the pro-angiogenic chemokines IL-8 (interleukin 8)
and CXCL2 [chemokine (C-X-C motif) ligand 2] (see data
published in [27]), suggesting that YB-1 is a transcriptional
repressor of these genes (Figure 5). In support of this, YB-1 has
been shown to bind to the IL-8 promoter in ER-negative breast
cancer cells (see results published in [28]). More compellingly,
YB-1 has been shown to repress transcription of VEGFA by
binding to the hypoxia-response region in the VEGFA promoter
in normoxic conditions [45,86]. The authors proposed that YB1 bound to single-stranded DNA would prevent binding of the
double-stranded DNA binding HIF-1 (hypoxia-inducible factor
1) complex. YB-1-mediated repression of the VEGFA promoter
was considerably enhanced after phosphorylation of YB-1 by
activated GSK3β (glycogen synthase kinase 3β) [45]. However,
in tumours under hypoxia GSK3β is not activated [87], potentially
reducing the binding of YB-1 to the VEGFA promoter, thereby
allowing greater access of HIF-1 to activate the expression of
VEGFA [45,86]. These tantalizing data suggest that YB-1 inhibits
angiogenesis in epithelial-derived tumour cells under normoxic
conditions, however, under hypoxia repression by YB-1 is
relieved allowing expression of pro-angiogenic factors. Therefore
the induction by YB-1 of genes encoding pro-angiogenic proteins
such as PDGF-β and VEGF-A, although requiring further
investigation, suggests that YB-1 may play an important role in
the angiogenic switch.
Invasion and metastasis
There are many steps involved in the invasion and metastasis of
tumour cells to distant sites. Tumour cells initially constrained
by BMs (basement membranes), must first dissociate from
the tumour mass and cross the BM before invading the
adjacent stromal tissue. This migratory behaviour is facilitated
by loosening the connections between adjacent cells of the BM
and also of the ECM (extracellular matrix) to allow the passage
of tumour cells into blood vessels and lymphatic system for
dissemination [88,89].
There are several lines of evidence linking YB-1 with a role
in invasion and metastasis (Figure 6). For example, in vitro
studies have shown that reducing YB-1 levels inhibits the
invasive properties of a number of cancer cell lines [39,90,91],
and overexpression of YB-1 promotes invasion of MCF-7
breast cancer cells [92] and Ras-transformed ‘normal’ mammary
epithelial cells [93]. Furthermore, analysis of tumour data showed
that high YBX1 mRNA levels are associated with lower distant
metastasis-free survival rates in breast cancer [27].
YB-1 and cancer
Figure 6
17
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 change
in 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 Notch3
receptor.
The cadherins are involved in maintaining cell–cell adhesion
within the tumour mass, particularly E-cadherin (CDH1), which
is frequently inactivated in metastatic cancers [94–96]. One
mechanism by which this can occur is via the transcriptional
repression of CDH1 by a number of transcription factors including
Snail (SNAI1), LEF1 (lymphoid enhancer-binding factor 1) and
TWIST1 [97–99]. Interestingly YB-1 has been shown to promote
the translation of the mRNAs encoding these CDH1-repressing
factors [91,93]. The tumour cell must then become motile, which
often appears to involve EMT [100]. EMT is driven by many
molecules and pathways that have been linked to YB-1. For
example, YB-1 appears to regulate the translation of TGFβ1
[101], which although a tumour suppressor in normal cells, plays
an important role in driving EMT [102]. Both the Wnt and Notch
pathways are also involved in EMT [100] and YB-1 has been
shown to bind to the promoters of a number of Wnt pathway
proteins [28]. Interestingly a potential secreted fragment of YB-1
has been identified as a ligand for Notch3 receptors [103].
The dissociation of the BM/ECM occurs via several
mechanisms, one of which is proteolysis. Multiple proteolysis
pathways appear activated in metastasis including the uPA
(urokinase-type plasminogen activator) system and the MMPs
(matrix metalloproteinases) [104,105]. uPA promotes degradation
of the BM/ECM by plasmin and also activates the MMPs [89].
YB-1 appears to be an activator of uPA as reduction of YB-1
expression led to decreased levels of uPA [39]. The MMPs are
a family of proteases with multiple specificities. Given that they
can degrade almost all proteins in the BM/ECM, their expression
is tightly regulated [105]. Several studies have shown that YB-1
transcriptionally regulates a number of MMPs, including MMP-2
[106], MMP-12 [107] and MMP-13 [108]. One of these studies
showed 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 melanoma
cells led to decreased expression of MMP2 [16]. YB-1 has also
been shown to increase the levels of the membrane-associated
MT1-MMP (membrane-type 1 MMP; also known as MMP-14),
which plays a critical role in metastasis [109,110]. In ER-positive
breast cancer cells, YB-1 performs this task by subverting the
endocytic mechanism and directing MT1-MMP back to the cell
membrane where it can interact with and degrade the ECM [92].
CD44 is another membrane-bound protein that plays an important
role in metastasis. A recent report has shown that this occurs
via interaction with MT1-MMP [111]. YB-1 is a transcriptional
activator of the CD44 gene [112], and also promotes alternate
splicing of the transcript to include exon 4 leading to the CD44v4
variant [113]. Isoforms of CD44 mRNA containing exon 4
promote 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 associated
with cell adhesion, migration, differentiation, proliferation and
cancer metastasis [115,116]. Experiments have shown that YB-1
expression 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 and
differentiation.
In conclusion, YB-1 regulates multiple proteins and pathways
involved in invasion and metastasis.
Energy metabolism
Studies on tumour cell metabolism in recent years have confirmed
that tumour cells gain a selective advantage by generating energy
not only from mitochondrial-driven oxidative respiration, but
also in the presence of oxygen, fermenting glucose into lactate
(termed the Warburg effect [117], reviewed in [118]). A number
of pathways can promote this ‘aerobic glycolysis’ to enable the
use of glucose as a fuel for cancer cells. Once again, it seems a
number of the usual players and pathways are involved, frequently
those associated with controlling tumour growth, suggesting a
close link between proliferative and metabolic pathways. Potentially the most important is the PI3K/Akt1/mTOR pathway, which
regulates many components of the glycolytic pathway. Akt1 regulates both the expression and membrane translocation of a number
of glucose transporter molecules, contributing to increased
glucose uptake [119,120]. mTOR, when part of mTORC1, acts as
a hub to link growth factor signalling with activation of metabolic
pathways for growth (reviewed in [121]). Activated mTOR has
recently been identified as a key driver of aerobic glycolysis, via
increasing the levels of the crucial enzyme in this pathway, PKM2
(pyruvate kinase M2) [122]. mTOR promotes this via the control
of HIF1 and MYC transcription factors [122–124], which then
c The Authors Journal compilation c 2013 Biochemical Society
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A. Lasham and others
increase the expression of genes encoding the splicing
proteins PTB (polypyrimidine tract-binding protein), hnRNPA
(heterogeneous nuclear ribonucleoprotein A) 1 and hnRNPA2
[122]. This results in alternative splicing of the pyruvate kinase
transcript, leading to generation of the pro-glycolytic PKM2 splice
form [125].
YB-1 may affect energy metabolism through the regulation
of PI3K/Akt1/mTOR pathways as described above (Figure 2).
Interestingly, YB-1 has also been shown to regulate the translation
of MYC mRNA via binding to an internal ribosome entry site
in the MYC 5 -untranslated region [126]. Therefore, together
with mTOR, YB-1 may increase Myc protein levels thereby
promoting glycolysis. Furthermore, reducing the expression of
YB-1 in rapidly proliferating A549 and HCT116 cells led to
a decrease in PKM2 RNA expression (see results published
in [27]).
In addition, YB-1 may affect energy metabolism through the
regulation of E2F and RB activity. A recent study of E2f1 − / − mice
showed that E2F1 is also able to influence metabolic pathways. In
conditions where energy demand was high, the RB/E2F1 pathway
blocked oxidative respiration to drive the expression of genes
involved in glycolytic metabolism [127].
Another pathway inactivated by YB-1, p53, has also been
associated with energy metabolism. p53 has been shown to
prevent aerobic glycolysis and promote oxidative phosphorylation
[128], partly through impeding the PI3K/Akt/mTOR pathway by
transcriptional activation of the Akt inhibitor PTEN (phosphatase
and tensin homologue deleted on chromosome 10) [129].
Collectively, these findings suggest that YB-1 modulates
tumour cell energy metabolism to promote aerobic glycolysis
by regulating several molecules and pathways including PI3K/
Akt1/mTOR, Myc, PKM2, the RB/E2F1 pathway and p53.
Tumour-promoting inflammation
A substantial amount of evidence now links inflammation to
the development of tumours [134]. For example, patients with
inflammatory bowel disease are prone to the development of
colorectal cancers, chronic pancreatitis with pancreatic cancer
and haemochromatosis with liver cancer (reviewed in [135]).
Furthermore, chronic inflammatory autoimmune diseases such as
rheumatoid arthritis and Sjogren’s syndrome are associated with
increased rates of lymphoma.
Many molecules regulated by YB-1, either transcriptionally
or post-transcriptionally, have been linked with promoting
inflammation 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] whose
association with YB-1 has been discussed in earlier sections
of the present review. In addition to these, YB-1 has been
studied in inflammatory diseases and found to regulate several
proteins involved in inflammatory pathways (reviewed in
[136]). For example, the chemokines CCL2 [MCP-1 (monocyte
chemoattractant protein 1)] and CCL5 [RANTES (regulated
upon activation, normal T-cell expressed and secreted)] induce
inflammatory cell infiltration, particularly macrophages, into the
tumour microenvironment [137]. This has been especially well
described in breast cancer [138]. YB-1 activates transcription
from the CCL5 promoter [139] and may also regulate CCL2 as
reduction of YB-1 led to a decrease in CCL2 mRNA levels in
ER-positive breast cancer cells (see results published in [27]).
In summary, YB-1 regulates the expression of several genes
encoding proteins known to drive inflammation, such as mTOR,
STAT3, MMP-2, CD44, CCL5 and potentially also CCL2, all of
which may contribute to tumour development.
YB-1 IN THE CLINIC
Evading immune destruction
Cancer cells have acquired several ways to evade immunosurveillance mechanisms to proliferate unhindered. These involve
both intrinsic and extrinsic mechanisms that either allow tumour
cells to avoid detection or removal by the immune cells, or by the
secretion of factors that affect immune cell function respectively
[130]. A number of molecules associated with YB-1 are involved
in intrinsic evasion. For example, to resist immune detection
and killing, MHC class II and Fas/CD95 have been observed
at lower levels in tumours than in healthy cells ([131,132] and
reviewed in [133]). The genes encoding both of these proteins are
transcriptionally repressed by YB-1 [3,67] (Figure 4C).
YB-1 also affects extrinsic mechanisms, including the TGF-β
pathway that promotes immune response evasion through multiple
mechanisms (reviewed in [130]). Although a link between YB-1
and this pathway has not been studied in cancer cells, YB-1 has
been shown to regulate translation of TGFβ (TGFB1) mRNA
in proximal tubule cells, where lower levels of YB-1 inhibit
translation [101]. This would be consistent with an established
cancer cell, where YB-1 is expressed at elevated levels, which
would promote the translation of TGFB1 leading to the activation
of the TGFβ pathway. Consistent with this model, we noted that
siRNA-mediated reduction of YB-1 expression in MCF7 breast
cancer cells reduced the levels of TGFB1 and TGFB3 mRNA (see
results published in [27]).
In summary YB-1 promotes the escape of tumour cells from the
immune system by intrinsic mechanisms such as the regulation
of the genes encoding MHC class II and Fas. YB-1 may possibly
also contribute to extrinsic mechanisms such as regulation of the
TGF-β pathway.
c The Authors Journal compilation c 2013 Biochemical Society
Given the master-regulatory role played by YB-1 in all of Hanahan
and Weinberg’s ‘hallmarks of cancer’, it is not surprising that
YB-1 is strongly associated with clinical parameters such as
tumour progression. As indicated in the opening section of the
present review, IHC and genomic studies have shown that YB-1
protein and mRNA levels are frequently elevated in advanced
breast cancer, have an inverse correlation with ER and PR
(progesterone receptor) expression, and are associated with poor
patient outcome [27,140–143]. Subsequent studies over many
years have shown that YB-1 abundance is also associated with
the outcome of a range of other human malignancies such as
glioblastoma [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 potentially
a powerful prognostic tool. Despite this the potential clinical
importance of YB-1 has been largely underplayed.
One possible reason for this is due to an historical focus
on nuclear YB-1. As described above, several studies have
suggested that it is nuclear YB-1 that is associated with the more
aggressive cancers and poor prognosis. However, a number of
studies have found that the overall YB-1 level (which is essentially
cytoplasmic) is a sufficient indicator of prognosis [17,147,148].
Indeed, it is very difficult in our opinion to discern nuclear
localization of YB-1 using IHC, and in our analyses encompassing
three different breast cancer cohorts, the proportion of tumours
or cells showing clear nuclear YB-1 is very small. In one study,
only three cells in 96 breast cancers showed unequivocal nuclear
YB-1 staining [143], and in the large cohort described by Habibi
et al. [141] only 3 % of tumours had some limited nuclear
YB-1 and cancer
19
developing standardized and properly validated (monoclonal)
antibodies against YB-1 to enable the use of YB-1 as a powerful
prognostic indicator in the clinic.
YB-1 AS A THERAPEUTIC TARGET
Figure 7 Western blot showing an example of cross-reactivity with a YB-1
antibody
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 proteins
separated by SDS/PAGE prior to Western blotting with a rabbit polyclonal antibody against
YB-1 [149]. Note that the amount of YB-1 protein (∼ 45 kDa) is considerably reduced following
transfection 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 to
be prognostic in both cases. That the absolute YB-1 level is
sufficient for prognostication is also highlighted by another study
of ∼ 400 breast cancers, which showed that abundance of the
mRNA encoding YB-1 is significantly associated with poor
clinical outcome [27].
A second reason why YB-1 has attracted less clinical and
pathological attention than expected may be due to the use of
multiple antibodies for the detection of YB-1 in tumours. This is
of critical importance since some antibodies detect both nuclear
and cytoplasmic YB-1 [7], whereas others appear to detect
essentially only cytoplasmic YB-1 [141]. This point was
emphasized in a comparative study of just two antibodies (raised
against residues 1–12 and 299–313 of YB-1), which showed clear
differences in their ability to detect nuclear YB-1 and also their
ability to show an association with tumour progression [143].
Analysis with additional antibodies against YB-1 generated yet
other patterns of staining (results not shown). Thus antibodies
raised against different regions of YB-1 may give quite different
results. It is paramount therefore that publications state which
antibodies are used to enable comparison and reproducibility
between studies.
A further issue was identified when it was shown that some
antibodies are not only poorly immunoreactive to YB-1, they
are also cross-reactive with the nuclear protein hnRNPA1 [149].
Figure 7 shows an example of this. In addition to the ∼ 45 kDa
YB-1 band, which is specifically reduced following transfection
with YB-1 siRNAs, a second protein at 37 kDa (shown to
be hnRNPA1 [149]) is also detected by this antibody and is
unchanged following YB-1 knockdown. Worryingly, hnRNPA1
is predominantly a nuclear protein, which casts doubts on studies
using these antibodies for IHC analysis of YB-1, particularly
those drawing conclusions from nuclear staining. Despite these
findings, one of the cross-reactive antibodies continues to
be used [150] (http://www.abcam.com/YB1-antibody-ab12148references.html). We propose therefore that a simple validation
of YB-1 antibodies is performed (e.g. as in Figure 7) before IHC
studies with YB-1 antibodies are published.
Collectively, the variety of antibodies with different qualities
and specificities has limited the development of antibody-based
predictive and prognostic screens utilizing YB-1. However, now
knowing the limitations of the area, the field can move on,
Given that the position of YB-1 is upstream of the molecular
pathways responsible for all nine hallmarks of cancer, YB-1 is
a very attractive therapeutic target. Several approaches to target
YB-1 have been employed, including targeting of YB-1 directly,
interfering with the activation of YB-1 or targeting the regulators
that activate YB-1.
Historically, the first of these used a ‘decoy’ YB-1-binding site
to sequester the YB-1 protein. This was successfully employed in
cultured cancer cells of many different lineages, with the result
of 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. Despite
the publication of this work almost 10 years ago, the use of
nucleic acids as therapeutics has been hampered predominantly
by issues of delivery, although rapid advances are now being made
[151,152].
Another therapeutic approach to directly target YB-1 could be
to use siRNAs. Anti-YB-1 siRNAs have been shown to suppress
tumour cell invasion, proliferation, differentiation, insensitivity to
chemotherapy and to promote apoptosis, as described previously
[17,27,41,153,154]. The challenge will be delivering siRNAs in
humans given the widely discussed problems of stability and
bioavailability and the current drive to evolve better chemistry
and delivery technologies [152].
As outlined above, the ‘transcription factor’ functions of YB-1
are activated by phosphorylation. The most widely characterized
phosphorylation site is at Ser102 , which stimulates nuclear
translocation and DNA binding [19]. Site-directed mutagenesis
showed that S102A YB-1 mutants exhibited reduced cell
proliferation [43] and an interference peptide was designed to
serve as another type of ‘molecular decoy’ that competes with
YB-1 for phosphorylation by RSK and Akt [155]. This CPP
(cell-permeable peptide) inhibited the proliferation of breast and
prostate cancer cells in cell culture [155]. Importantly, the CPP
had no effect on the growth of normal mammary epithelial cells
isolated from patients [155]. Thus peptide-based delivery systems
could be used to inhibit YB-1 therapeutically.
The use of ‘signal transduction’ inhibitors is another approach
to suppressing YB-1 activity. A side effect of blocking kinases
such as PDK-1 [156], Akt [19,157] and RSK [158] is quenching
of YB-1 phosphorylation at Ser102 and thereby reduction of the
transactivation activity of YB-1. This ‘side effect’ on YB-1 may
in fact turn out to be responsible for a significant proportion of the
activity of these inhibitors. Although Akt was originally reported
to phosphorylate and activate YB-1 [19] it was subsequently
reported that relative to other kinases such as RSK, Akt might
be a minor player in YB-1 activation [158]. In support of this,
RSK inhibition with siRNA, or small molecules such as BI-D1870
and SL0101, completely suppress the activation of YB-1, despite
the presence of activated Akt in the same cells [158]. Likewise, the
MEK inhibitor PD098059 blocks RSK and suppresses the nuclear
localization of YB-1 [159]. Moving further upstream, PDK-1
inhibition also suppresses YB-1 [156] and is known to directly
activate RSK through phosphorylation at Ser380 [160]. Given that
RSK is the most proximal kinase that activates YB-1, and given the
numerous YB-1-independent pathways that RSK also activates, it
would seem reasonable to focus on blocking RSK [161–163]. The
effect on YB-1 of other proteins associated with YB-1 pathways,
c The Authors Journal compilation c 2013 Biochemical Society
20
A. Lasham and others
Figure 8 Schematic diagram showing that YB-1 affects all of the hallmarks
of 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-permeable
inhibitory peptides, YB-1 siRNAs or oligonucleotide decoys have
shown promise in cell culture experiments. Indirect inhibition of
YB-1 is a known side effect of blocking kinases such as PDK-1,
Akt, MEK or RSK, and YB-1 blockade may potentially underlie
a significant part of the activity of these inhibitors.
CONCLUSION
The present review has described YB-1 as a master regulator
of cancer cell biology, contributing to all nine of Hanahan and
Weinberg’s ‘hallmarks of cancer’ (Figure 8) and is therefore a
bona fide oncoprotein. YB-1 is a multitasking protein that may
operate in different, but overlapping, ways in different cell types.
Moreover, due to the levels of YB-1 protein and YBX1 mRNA
being highly correlated with poor patient outcome, YB-1 should
be regarded as a useful biomarker of cancer progression and
as a novel therapeutic target. Given the strong association of
YB-1 with aggressive (basal-like) breast cancers, targeting YB-1
could, for example, be of particular value for such cancers that
are currently difficult to treat. Given the increasing consistency
with which YB-1 is being associated with cancer progression and
interest in understanding how YB-1 functions, we are confident
it will feature prominently in the cancer literature in the future
and hopefully, in time, be developed as a biomarker and target
for 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 New
Zealand [grant numbers 10/21 (to A.L. and C.G.P.) and 11/07 (to A.G.W. and A.W.B.)], the
Health Research Council of New Zealand [grant number 10/02 (to A.G.W. and A.W.B.)], the
New 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 2012
Published on the Internet 7 December 2012, doi:10.1042/BJ20121323
c The Authors Journal compilation c 2013 Biochemical Society