oxidoreductase: a candidate tumor
Nan-Shan Chang1,5, Li-Jin Hsu2,3, Yee-Shin Lin2,3, Feng-Jie Lai4and
1Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan 70101, Republic of China
2Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan 70101,
Republic of China
3Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University Medical College, Tainan,
Taiwan 70101, Republic of China
4Department of Dermatology, National Cheng Kung University Medical College, Tainan, Taiwan 70101, Republic of China
5Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840, USA
Common fragile site gene WWOX encodes a candidate
tumor suppressor WW domain-containing oxidoreduc-
tase. Alteration of this gene, along with dramatic down-
regulation of WWOX protein, is shown in the majority of
invasive cancer cells. Ectopic WWOX exhibits proapop-
totic and tumor inhibitory functions in vitro and in vivo,
probably interacting with growth regulatory proteins
p53, p73 and others. Hyaluronidases regulate WWOX
expression, increase cancer invasiveness and seem to
be involved in the development of hormone-indepen-
dent growth of invasive cancer cells. Estrogen and
androgen stimulate phosphorylation and nuclear trans-
location of WWOX, although binding of WWOX to these
of WWOX expression by overexpressed hyaluronidases
might contribute in part to the development of hormone
independence in invasive cancer.
WWOX is a candidate tumor suppressor
A candidate tumor suppressor WW domain-containing
oxidoreductase (see Glossary), known as human WWOX
 or FOR , or murine Wox1 or Wwox , was first
discovered in 2000. The large size human gene WWOX (1.1
TRENDS in Molecular MedicineVol.13 No.1
Apoptosis: a form of cell death in which a programmed sequence of events
leads to the elimination of cells without release of contents. Apoptosis can be
triggered by many types of stress signals. When apoptosis does not occur in
cells that should be eliminated, it might result in cancer. When apoptosis works
overly well, it might cause neurodegenerative disorders such Alzheimer’s and
Common fragile site: a region that shows site-specific gap and break on
metaphase chromosome. Common fragile sites are normally stable in somatic
cells. However, when cells are treated with replication inhibitors, fragile sites
display gaps, breaks, rearrangements and other features of unstable DNA. The
fragile sites and associated genes are frequently deleted or rearranged in many
cancer cells that are considered a hallmark of genomic instability in cancer.
Cutaneous basal cell carcinoma (BCC): a malignant neoplasm derived from
pluripotential cells in the basal layer of epidermis or follicular structures. BCC is
the most-common cancer in humans. The tumor characteristically develops on
sun-exposed skin. BCC is usually slow growing and rarely metastasizes, but it
can become invasive and cause substantial tissue damage if left untreated.
Cutaneous squamous cell carcinoma (SCC): a malignant tumor derived from
suprabasal keratinocytes of epidermis. SCC is the second most-common form
of skin cancer. Predisposing factors for SCC include precursor lesions (actinic
keratosis and Bowen’s disease), UV exposure, ionizing radiation and environ-
mental carcinogens. SCC is capable of local invasion, regional lymph-node
metastasis and distant metastasis.
Fragile histidine triad (FHIT): a member of the histidine triad gene family that
might act as a tumor suppressor. The gene encompasses the common fragile
site FRA3B on human chromosome 3, where carcinogen-induced damage can
lead to translocations and aberrant transcripts of this gene. Aberrant gene
transcripts have been found in many cancers.
Loss of heterozygosity (LOH): the loss of a single parent contribution to part of
the genome in a cell. LOH of chromosomal regions bearing mutated tumor
suppressor genes is a common event in the evolution of tumors. The remaining
copy of the tumor suppressor gene will be often inactivated by a point mutation.
LOH can arise by two ways. First, a region of a chromosome is deleted, resulting
in only one copy remaining. Second, genetic recombination leaves the cell with
two copies of the chromosomal region, but both come from the same parent.
NSYK motif: a catalytic tetrad of NSYK (Asn–Ser–Tyr–Lys) present in most of
SDR-containing protein family. SDR proteins mediate oxidation and reduction of
lipid hormones and metabolic mediators. The NSYK motif in human WWOX is
N232, S281, Y293 and K297. WWOX and its isoform WWOX2 are likely to bind to
sex steroid hormones such as androgen and estrogen via the NSYK motif.
p53: a tumor suppressor protein of 53 kDa. When DNA damage is minor, p53
halts the cell cycle until the damage is repaired. When DNA damage is major
and cannot be repaired, p53 induces the cell to suicide by apoptosis. More than
half of human cancers have p53 gene mutations and do not produce
functioning p53 protein.
Short-chain alcohol dehydrogenase–reductase (SDR): a large family of
enzymes containing >2000 protein members, most of which are NAD- or
NADP-dependent oxidoreductases. These proteins are of ?250–300 amino acid
residues, possessing at least two domains: the first domain binds to
coenzyme(s) and the second to substrate(s). The second domain determines
substrate specificity and contains amino acids that are involved in catalysis.
Tumor necrosis factor: a member of a cytokine superfamily, which might
induce tumor cell death and possess multiple proinflammatory functions.
Tumor suppressor: protein product(s) of tumor suppressor gene(s) that slows
down cell division or causes cell death to suppress tumor formation.
Alterations of tumor suppressor gene(s) might induce cancer development.
WW domain: a protein domain that is composed of a short stretch of ?40
amino acids, possessing two conserved signature tryptophan residues. This
domain folds as a stable, triple-stranded b-sheet, and can be repeated 2–4
times in proteins (Box 1). WW domain-containing proteins are normally
involved in signal transduction by binding to proteins that possess proline
motifs, PPxY (P, proline; Y, tyrosine; x, any amino acid), and/or phosphoserine-
or phosphothreonine-containing motifs.
WW domain-containing oxidoreductase (WWOX/Wwox): a candidate tumor
suppressor and proapoptotic protein. The full-length protein, 46 kDa, pos-
sesses two N-terminal WW domains, a nuclear localization sequence (NLS) and
a C-terminal short-chain alcohol dehydrogenase–reductase (SDR) domain.
Corresponding author: Chang, N.-S. (firstname.lastname@example.org).
Available online 4 December 2006.
www.sciencedirect.com 1471-4914/$ – see front matter ? 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.11.006
million base pairs) encodes this protein. Alterations of
WWOX gene in cancer cells have been thoroughly investi-
gated as documented in the literature, whereas functional
properties of WWOX/Wwox are largely unknown. Here, we
update the current knowledge of WWOX/Wwox regarding:
(i) structure, subcellular distribution and functions; (ii)
phosphorylation, binding interactions and protein degra-
dation; (iii) alterations of WWOX gene; (iv) cell death and
tumor-suppressor function; (v) prosurvival role, embryonic
development and differentiation; and (vi) hyaluronidase
regulation of WWOX/Wwox expression, cancer invasion
and hormone resistance. We also discuss the areas that
have conflicting opinions.
There are at least eight human WWOX mRNA
transcripts  (Box 1). Database searching shows the
presence of WWOX gene transcripts in monkey, mouse,
dog, pig, chicken, fish, sea urchin, bee, flour beetle
(Tribolium) and fruit fly (Drosophila). The full-length
WWOX/Wwox possesses a typical C-terminal short-chain
alcohol dehydrogenase–reductase (SDR) domain, two N-
terminal WW domains (with conserved tryptophan resi-
dues), and a nuclear localization signal (NLS) between
these domains (Box 1). The first WW domain has two
conserved tryptophans, and the second WW domain has
only one [1–3].
Whether every WWOX mRNA transcript can be
successfully translated into a protein is unknown. Pre-
sence of WWOX mRNA transcripts is shown in normal
human and mouse tissues and cancer cell lines [1–3,5].
Presence of the full-length 46-kDa WWOX/Wwox protein
has been well documented in the literature. Watanabe
et al.  demonstrated the presence of low molecular
Box 1. WWOX and spliced variants
Human WWOX gene contains nine exons and encodes a full-length
WWOX. Alternative splicing of WWOX mRNA generates seven
transcripts, suggesting possible presence of protein isoforms (Figure
I). The region of WW domains is encoded by exons 1–4, and SDR
domain by exons 4–8. A nuclear localization sequence (NLS) is
identified between the two WW domains. Known phosphorylation
sites are Tyr33 (Y33) and Tyr287 (Y287). A conserved catalytic tetrad
NSYK motif for hormone- or substrate-binding is shown (N232, S281,
Y293 and K297). A conserved caspase-recognition motif QETD (amino
acid 65–68) is located in the second WW domain. A mitochondria-
targeting region is shown in the SDR domain (amino acid 209–273) of
murine Wwox (between Y287 and Y293) .
Figure I. The WWOX gene and sliced variants. v1 is the wild-type WWOX gene (46 kDa). v2 contains a partial deletion of exon 9 with a unique C-terminus (orange)
(41 kDa). v3 contains an out-of-frame deletion of exon 5–8 and frame-shift at the C-terminus (35 kDa). v4 contains an in-frame deletion of exon 6–8 (26 kDa). v5 contains
an exon 5–9 deletion (24 kDa). v6 has an amino acid sequence from the first five exons and an alternative exon 6 (22 kDa). v7 contains an exon 2–9 deletion (4 kDa). v8
contains a TG-deletion at exon 9 (red star), which results in the frame-shift at the C-terminus (59 kDa). Two protein pairs possess an identical C-terminus: v1 and v4 (last
15 amino acids, LSERLIQERLGSQSG); v3 and v8 (last 15 amino acids, EKHQQFSFFYCYRIA). The predicted hormone- or substrate-binding motif within Drosophila
WWOX protein is indicated (S231, S276, Y288 and K292).
TRENDS in Molecular MedicineVol.13 No.113
weight WWOX with truncations at the SDR domain,
corresponding to 35-kDa WWOXD5–8, 26-kDa WWOXD6–
suggesting that they are unstable and might act as
dominant negatives in blocking the function of WWOX .
Mahajan et al.  validated the presence of WWOXD5–8
(v3 or WWOX3) in human prostate LNCaP cells as deter-
mined by protein sequencing, and showed that this protein
is stable in contrast to WWOX. The differences in the
observations from these reports are unknown [5,6]. By
using specific polyclonal anti-WW domain antibodies,
Lokeshwar et al.  demonstrated the presence of WWOX
and isoform WWOX2 (v2; 41 kDa) in human prostate
DU145 cells. By using specific antibodies against the
unique C-terminus, WWOX2 is observed in hippocampal
neurons of human brains , breast and prostate tissues
. Frequently, anti-WW domain antibodies cannot effec-
tively detect the presence of small WWOX isoforms in cell
lysates probably due to low abundance and/or instability,
and potential cleavage of the N-terminal WW domain area
by a specific caspase to prevent antibody detection.
In the mouse genome, Wwox gene is located on
chromosome band 8E1  (GeneID: 80707). The encoded
Wwox protein sequence shares 93.2% identity with that of
human WWOX, where WW domain areas are identical in
both human and mouse, and the differences are at the SDR
domains. There are at least six murine Wwox mRNA
transcripts in the database, and each encodes a unique
C-terminal amino acid sequence.
WWOX: phosphorylation and degradation
WW domain was first discovered in 1996, and now the WW
(?1400 members) [11–13]. Functional deficiency of WW
domain-containing proteins PIN1 [protein (peptidylprolyl
contribute to the pathogenesis of Alzheimer’s disease
[8,14,15]. The solution structure of the second WW domain
of human WWOX has been determined (http://www.ncbi.
There are at least 2000 SDR domain-containing
proteins, which belong to a very large family of enzymes,
including nicotinamide adenine dinucleotide (NAD)- or
nicotinamide adenine dinucleotide phosphate (NADP)-
dependent oxidoreductases . Structural distortion of
SDR domain, for example, caused by binding of Ab-binding
alcohol dehydrogenase (ABAD) to amyloid-b peptide,
induces mitochondrial dysfunction in Alzheimer’s disease
Phosphorylation at Tyr33 in the first WW domain of
9,18–20] (Box 1). Sex steroid hormones and stress stimuli
induce Tyr33 phosphorylation of WWOX/Wwox [7–9,18–
20]. Aqeilan et al.  reported that tyrosine kinase SRC
phosphorylates Tyr33. Mahajan et al.  demonstrated
that polyubiquitination and proteosomal degradation of
WWOX involve phosphorylation of Tyr287 by activated cell
division cycle 42 (CDC42)-associated kinase 1 (ACK1),
which is a tyrosine kinase.
(Gln–Glu–Thr–Asp; amino acid 65–68), is located within
the second WW domain (Box 1). Whether a specific caspase
binds to and cleaves this motif (between Thr and Asp) is
unknown. In vitro translation and ectopic expression of the
full-length Wwox cDNA produced 46-kDa and 30-kDa pro-
tein bands . Whether the 30-kDa protein is a degraded
product requires verification by sequencing analysis.
WWOX: subcellular and tissue distribution
Subcellular localization of WWOX has been controversial.
We have addressed this issue in two previous reviews
[4,21], and have tried to clarify it by using electron micro-
scopy, as described below. Ectopic expression of murine
Wwox, tagged with EGFP (green fluorescence protein) or
ECFP (cyan fluorescence protein) in monkey kidney COS7
fibroblasts and lung H1299 cells, resulted in mitochondrial
mitochondria from rat liver . We have determined a
mitochondria-targeting region in the SDR domain .
Bednarek et al.  showed that ectopic human GFP–
WWOX is in the Golgi apparatus in normal breast MCF-
10F cells.These differences areprobably dueto(i) different
cell lines used, (ii) variations between human WWOX and
mouse Wwox amino acid sequences particularly at the C-
termini, and (iii) differences in culture conditions between
laboratories (e.g. sera).
endogenous WWOX/Wwox has also been shown in cell
lines, mammary gland cells and epidermal keratinocytes
[3,5,9]. Jin et al.  demonstrated that protein kinase A
(PKA)-mediated phosphorylation of ezrin is essential and
sufficient for the apical localization of WWOX protein in
parietal cells of the stomach. Also, H+–K+-ATPase recruit-
ment is needed for ezrin–WWOX interaction in the apical
cell membrane .
COS7 fibroblasts are responsive to estrogen- or
androgen-induced nuclear translocation of WWOX .
Endogenous WWOX is mostly present in perinuclear area
of breast MCF7 cells, and is refractory to undergo nuclear
translocation in response to estrogen or androgen . In
parallel, during breast-cancer progression, there is no
notable increase in nuclear localization of WWOX in
mammary gland cells at the hypertrophic and cancerous
stages . However, ?1.5-fold increases in nuclear trans-
location of WWOX at these stages are observed in prostate
By electron microscopy, cytoplasmic Wwox-positive
immunogold particles are shown on or near the surface
of nuclear membrane and rough endoplasmic reticulum
(ER) in retinal ganglion cells of normal mature rats .
Wwox is barely detectable in the mitochondria and Golgi
complex in these cells. Prolonged exposure of adult rats to
constant light for 1–2 months resulted in substantial death
of photoreceptors in the outer retinal layer . A large
number of immunogold particles of Tyr33-phosphorylated
Wwox are observed in the damaged mitochondria and
condensed nuclei of degenerating photoreceptors in the
retinal outer layer, indicating Wwox functions at these
organelles . Wwox is barely detectable in the Golgi
complex of apoptotic cells . Together, depending upon
TRENDS in Molecular Medicine Vol.13 No.1
the cell types, tissues and exogenous stimuli, WWOX/
Wwox has been shown in mitochondria, Golgi complex,
rough ER, plasma membrane and nuclei (using light, con-
focal and electron microscopy) [3,5,9,20,21–25].
Normal human tissues and organs express variable
levels of WWOX using tissue sections or microarray slides,
as determined by immunohistochemistry [5,9,26]. A gen-
eral consensus from these studies is that WWOX is mainly
expressed in epithelial cells, particularly in hormone-regu-
lated organs such as the testis, thyroid, prostate and
mammary glands. A detailed profile of WWOX/Wwox
expression in the developing and adult murine nervous
system and human nervous system has been documented
WWOX: Tyr33 phosphorylation, death signaling and
WWOX/Wwox is a proapoptotic protein and tumor
suppressor [3,6,19,22,25,27–30]. Here, we summarize the
known functional properties of human WWOX and mouse
Wwox both in vivo and in vitro (Box 2).
Functional suppression of WWOX/Wwox by antisense
mRNA, a dominant negative and small interfering RNA
(siRNA), protects cells from apoptosis by tumor necrosis
factor (TNF), staurosporine, ultraviolet (UV) light and
ectopic p53 in vitro [3,25,27]. Ectopic WWOX suppresses
growth of breast cancer cell lines MDA-MB-435 and T47D
on soft agarose, and inhibits tumorigenicity of MDA-MB-
435 in nude mice . Restoration of WWOX gene in lung
cancer cells prevents their growth both in vitro and in
vivo . Importantly, loss of WWOX protein promotes
prostate tumorigenesis in vivo . Activated ACK1
proteosomal degradation, thereby causing disappearance
of WWOX in prostate cells . Hypermethylation of
gene promoter downregulates WWOX expression in pros-
tate and lung cancer cells, and restoration with ectopic
WWOX inhibits these cells to grow both in vitro and in
vivo [29,30]. Thymocytes become refractory to undergo
apoptosis in patients with thyroid cancer, and this corre-
lates positively with alterations of common fragile site
genes WWOX and fragile histidine triad (FHIT) in these
cells , suggesting an indirect or a direct involvement
of WWOX and FHIT in the cell-death pathway.
Participation of murine Wwox in the TNF apoptotic
pathway has been reviewed [4,32]. Stable Wwox-trans-
fected L929 cells were shown to have an increased sensi-
tivity to TNF cytotoxicity . Overexpressed WW or SDR
domain or the full-length Wwox induces cell death, sug-
gesting that both domains are functional modules [3,4].
This enhancement is associated, in part, with upregulation
of proapoptotic p53 and downregulation of apoptosis inhi-
bitors B-cell CLL/lymphoma 2 (BCL2) and BCL-xL in L929
cells [3,4]. Recently, downregulation of BCL2 by overex-
pressed WWOX has been shown in certain prostate cancer
cells . In addition, Wwox enhances ectopic TNF recep-
tor-associated death domain protein (TRADD)-mediated
cell death [3,33]. TRADD is an adaptor of TNF receptors.
L929 cells are TNF sensitive. It is still unclear whether
WWOX/Wwox abolishes TNF resistance in other types of
Whether caspase activation occurs during WWOX/
Wwox-mediated cell death remains elusive. Overexpressed
N-terminal WW domains or the first WW domain of Wwox
causes death of mouse NIH/3T3 fibroblasts, and caspase
inhibitors and serine-protease inhibitors cannot block the
death event . Activation of caspase-3 (a downstream
executor of the caspase pathway) and degradation of poly
(ADP-ribose)polymerase-1 (PARP-1) is shown in WWOX-
infected lung A549, H460 and H1299 cells but not in U2020
period, disappearance of procaspase-9 and procaspase-3 is
shown in WWOX-infected LNCaP, DU145 and PC-3 pros-
tate cancer cells, but not in non-cancerous PWR-1E cells
. Thus far, there is no evidence that shows a direct link
for WWOX/Wwox induction of caspase activation.
Apoptotic or genotoxic stimuli activate WWOX/Wwox
via Tyr33 phosphorylation in the first WW domain, fol-
lowed by translocation to the mitochondria and nuclei to
induceapoptosis [4,18] (Box 3 and Box4). We haveshown a
dramatically increased presence of activated Wwox in the
damaged mitochondria and condensed or apoptotic nuclei
of degenerating retinal photoreceptors in rat eyes by elec-
tron microscopy . Tyrosine kinase SRC phosphorylates
WWOX at Tyr33  (Box 3). Whether other tyrosine
unknown. Tyr33 phosphorylation in WWOX/Wwox is cru-
cial for inducing apoptosis. Alteration of Tyr33 abolishes
WWOX/Wwox-induced apoptosis [18,19,27].
WWOX binding proteins
Both WW and SDR domains in WWOX/Wwox participate
in protein-binding interactions. First, several PPxY
Box 2. Functional properties of WWOX/Wwox
? Human WWOX and murine Wwox/Wox1 were first discovered in
year 2000 [1–3].
? WWOX/Wwox is abundant in epithelial cells of the testis, thyroid,
prostate and mammary glands [3,5,26].
? Wwox is upregulated in many organs during embryonic develop-
ment and then decreased after birth .
? Wwox is differentially expressed and distributed in developing and
adult murine nervous system .
? WWOX is expressed in many human organs (e.g. brain, skin, heart
and others) .
? Hyaluronidases PH-20, HYAL1 and HYAL2 induce WWOX/Wwox
? WWOX/Wwox is observed (by light and electron microscopy) in
mitochondria, Golgi, rough ER and nuclei [3,5,20–22,24,25].
? Tyrosine kinase SRC phosphorylates WWOX/Wwox at Tyr33 .
? Sex steroid hormones, estrogen and androgen, induce WWOX
phosphorylation at Tyr33 and nuclear translocation .
? Sex steroid hormone-induced activation of WWOX and WWOX2 is
independent of receptors for sex hormones .
? Phosphorylated-ezrin binds to and promotes apical membrane
localization of WWOX in parietal cells of stomach .
? WWOX and YAP compete for interaction with ERBB4 and other
targets and thus affect its transcriptional activity .
? WWOX binds to and triggers redistribution of nuclear AP-2g to the
cytoplasm for suppressing its transactivating function .
? WWOX binds to and triggers redistribution of nuclear p73 to the
cytoplasm and suppresses its transcriptional activity .
? Activated tyrosine kinase ACK1 phosphorylates human WWOX at
Tyr287 for polyubiquitination and degradation .
TRENDS in Molecular MedicineVol.13 No.115
motif-possessing proteins bind to the first WW domain,
including p73 , activator protein-2g (AP-2g) , v-
erb-a erythroblastic leukemia viral oncogene homolog 4
(ERBB4) , ezrin  and small integral membrane
protein of the lysosome/late endosome (SIMPLE) 
(Table 1 and Box 3). p73, AP-2g and ERBB4 are transcrip-
tion factors. Ezrin is a signal transducer located at the cell
membrane and/or cytoskeleton area . Binding of matrix
to erzin and associated proteins for regulating cell growth.
enhances the recognition of the proline-rich motif in the
above-mentioned proteins. Alteration of Tyr33 in the first
WW domain suppresses the binding [19,34,35]. Ectopic
WWOX interacts with and restricts nuclear localization of
p73 and AP-2g, thereby blocking their functions in gene
with YAP (WW domain-containing Yes-associated protein)
the transcriptional function of ERBB4 . Whether Tyr33
phosphorylation is involved in the binding of WWOX to the
PPxY motifs in ezrin and SIMPLE is unknown.
Third, the Tyr33-phosphorylated first WW domain
interacts with c-Jun N-terminal kinase 1 (JNK1) ,
p53 [3,9,18,27] and mouse double mutant 2 (MDM2) 
(Table 1). No PPxY motif is identified in these proteins.
Alteration of Tyr33 abolishes the binding interactions.
Tyr33-phosphorylated WWOX binds to p53 via phospho-
Ser46–Pro47 and an adjacent N-terminal proline-rich
region (amino acid 66–100)[3,27]. Without Ser46
Box 3. Mode of actions of WWOX/Wwox
We summarize the actions of WWOX/Wwox as follows (Figure I):
? Route 1. Stress-induced activation of WWOX/Wwox involves Tyr33
phosphorylation and translocation to the mitochondria and nuclei .
Tyrosine kinase SRC phosphorylates WWOX/Wwox at Tyr33 .
Phosphorylated WWOX/Wwox (p-WWOX) translocates to the mito-
chondria or nuclei in vitro and in vivo, or binds to Ser46-phosphory-
lated p53, followed by translocating to the mitochondria [3,4,20,21,27].
? Route 2. Ectopic WWOX (probably a Tyr33-phosphorylated form)
binds to and triggers redistribution of nuclear p73 and AP-2g to the
cytoplasm [19,34]. WWOX and YAP compete for interaction with
ERBB4 to relocate to the nuclei .
? Route 3. In neurons, WWOX/Wwox seems to prevent enzyme-
mediated Tau hyperphosphorylation .
? Route 4. Phosphorylated ezrin binds to and promotes apical mem-
brane localization of WWOX in parietal cells . WWOX phosphor-
ylation is not known.
? Route 5. WWOX interacts with SIMPLE, a small integral membrane
protein of the lysosome/late endosome . WWOX phosphorylation
is not known.
? Route 6. Activated tyrosine kinase ACK1 phosphorylates WWOX at
Tyr287 for polyubiquitination and degradation .
Figure I. Mode of actions of WWOX/Wwox.
Table 1. WWOX/Wwox binding proteins
First WW domaina
pY33 first WW domainb
pY33 first WW domainb
aThe first WW domain binds to PPxY motif in the indicated proteins. The specific
recognition sequence for each indicated protein is shown.
bY33 phosphorylation seems to enhance the binding of WWOX to p73, AP-2g or
and SIMPLE to the first WW domain depends upon Y33 phosphorylation is not
cWwox binds to the N-terminal phospho-Ser46–Pro47 and an adjacent poly-proline
segment (amino acid 66–100) in p53.
TRENDS in Molecular Medicine Vol.13 No.1
Tyr33 phosphorylation, WWOX cannot interact with p53
. Cells must be exposed to stress or apoptotic stimuli to
enable Ser46 phosphorylation in p53 and Tyr33 phosphor-
ylation in WWOX, respectively, thereby allowing the bind-
ing interaction to occur [3,9,18,27]. Aqeilan et al.  failed
to show the binding of ectopic WWOX to ectopic p53
proteins. The probable reason is that cells were not
exposed to stress stimuli.
Finally, the SDR domain of Wwox has been shown to
bind to Tau and seems to prevent Tau hyperphosphoryla-
tion . Protein motifs involved in this binding interaction
remain to be identified.
Fragile gene WWOX–FRA16D
Human WWOX gene, which encodes WWOX, spans the
common fragile site FRA16D on chromosome 16q23
[1,2,32,38–43]. Structurally, this gene is similar to fragile
genes such as FHIT and PARKIN [41–43]. Many outstand-
ing reviews have addressed the issues regarding loss of
heterozygosity (LOH), deletions and translocations of
WWOX gene in numerous types of cancers [32,38–43].
Briefly, high incidence of LOH is demonstrated in
primary tumors, including carcinomas from liver (28.7%)
, breast (81.8%) , esophagus [squamous cell carci-
noma (SCC), 38.9%] , lung (non-small cell lung cancer,
37%) , pancreas (26.7%)  and stomach (30.8%) .
Homozygous deletion of the WWOX gene in primary
tumors is rare.
Mutations in the WWOX gene are also rare. A somatic
missense mutation is found in an oral SCC, with C!T
transition at the second nucleotide of codon 329 in the SDR
domain, causing substitution of serine to phenylalanine
. In one esophageal SCC, T!C transition at the second
nucleotide of codon 291 causes leucine to proline substitu-
tion . However, genetic polymorphisms for the WWOX
gene are frequently shown in normal and clinical samples
Environmental factors contribute to genetic alterations
[25,41,52,53]. Aflatoxin B1 , UV-light exposure and
tobacco smoking  have been implicated in the altera-
carcinoma and oral SCC.
mRNA alternative splicing causes codon deletions at the
SDR domain (Box 1). Aberrant WWOX transcripts have
been noted in breast cancer (32–55%) [22,45,54], ovarian
tumors , esophageal SCC (5.6%) , oral SCC (35%)
, lung cancer (26%) , hematopoietic neoplasia (12%)
, pancreatic adenocarcinoma (6.7%) , gastric carci-
Although the presence of full-length and short mRNA tran-
scripts in cancer cells , truncated WWOX proteins have
yet to be identified and functionally tested (if present).
DNA methylation at CpG dinucleotides in promoter
regions is frequently associated with transcriptional silen-
cing of tumor suppressor genes in cancer cells. Promoter
hypermethylation of the WWOX gene at specific crucial
CpGs has been determined in two primary pancreatic
adenocarcinomas (13%) . The promoter and exon 1
regions of the WWOX gene are highly methylated in lung,
breast and bladder cancers, compared with the adjacent
non-neoplastic tissues, thus resulting in reduced protein
Finally, translational blockade of WWOX mRNA is
associated with disappearance of WWOX/Wwox in cuta-
neous SCCs in humans and hairless mice . Significant
reduction of WWOX, WWOX2 and Tyr33 phosphorylation
is observed in patients with cutaneous SCCs without
significant changes of WWOX mRNA, indicating transla-
tional blockade of WWOX mRNA into protein. In parallel,
Box 4. WWOX/Wwox: proapoptotic activity and tumor-
suppressor in vivo and in vitro [3,6,18–20,22,25,27–29].
? WWOX/Wwox enhances TNF cytotoxicity by downregulating BCL2
and BCL-xL, but upregulating p53 [3,29].
? Stress stimuli activate WWOX via phosphorylation at Tyr33 and
translocation to the mitochondria and nuclei [3,18,20,21].
? Ectopic Wwox induces caspase-independent apoptosis (cyto-
chrome c release and DNA fragmentation) in L929 cells [3,18,27].
? Adenoviral WWOX-infected prostate and lung cells have activated
caspase-3 within 72–96 hours, indicating caspase activation
? Wwox directly interacts with p53 and both induce apoptosis
? Hyaluronidase HYAL2 enhances Wwox-induced apoptosis .
activated JNK1 under stress conditions .
? Ectopic JNK1 blocks Wwox-induced inhibition of cell-cycle
progression and apoptosis .
? Tyr33 phosphorylation in WWOX/Wwox is essential for binding
and stabilizing Ser46-phosphorylated p53 .
? WWOX and p73 induce apoptosis synergistically .
? Suppression of WWOX/Wwox by antisense mRNA, dominant
negative and siRNA protects cells from apoptosis [3,18,25,27].
? Light-induced rat retinal damage involves WWOX Tyr33 phosphor-
ylation, mitochondrial and nuclear translocation .
acts asaproapoptotic proteinand tumor
nude mice .
? Restoration of WWOX gene in lung cancer cells prevents their
growth both in vitro and in vivo .
? Loss of WWOX protein promotes prostate tumorigenesis in vivo
via activated ACK1-mediated WWOX degradation .
? Inhibition of hypermethylation in lung tumor cells restores FHIT
and WWOX expression and suppresses growth in vivo .
? Restoration of WWOX expression in prostate cancer cells
suppresses growth both in vitro and in vivo .
? High level expression of WWOX mRNA is associated with better
breast-cancer survival (protein level unknown) .
? Thymocytes are refractory to death in thyroid-cancer patients with
alterations of WWOX and FHIT (indirect effect?) .
Potential prosurvival role
? WWOX is upregulated in non-invasive breast and gastric cancer
tissues, but downregulated in invasive cancers .
? WWOX, WWOX2 and Tyr33 phosphorylation are upregulated in
non-invasive breast and prostate cancer cells [9,29].
? Differentiation of skin keratinocytes is associated with upregula-
tion of WWOX/Wwox and Tyr33 phosphorylation .
? Neuronal degeneration is associated with downregulation of
WWOX, WWOX2 and Tyr33 phosphorylation .
? Oxido-reductase(FOR/WWOX)protects againstthe lethal effectsof
ionizing radiation in Drosophila .
TRENDS in Molecular Medicine Vol.13 No.117
chronic UVB-induced formation of cutaneous SCCs in
hairless mice involves dramatic reduction of Wwox and
Tyr33 phosphorylation without downregulation of WWOX
WWOX: prosurvival and role in differentiation?
Watanabe et al.  showed that WWOX protein levels are
upregulated in non-invasive breast and gastric cancer
tissues, raising the concern of whether WWOX is a typical
tumor suppressor. By contrast, WWOX is markedly down-
regulated in these tissues during metastasis . Signifi-
cantly reduced expression of WWOX protein is also shown
in invasive breast and prostate tissues [9,29,59]. High-
level expression of WWOX mRNA in estrogen receptor
(ER)-positive breast cancer cells is associated with
improved cancer survival . Additionally, the level of
WWOX mRNA is both upregulated and downregulated in
non-invasive and metastatic breast cancer cells, respec-
tively . We have examined various stages of breast-
tumor development . Breast cells at hyperplasia and
cancerous (or solid tumor) stages have upregulated
cytosolic WWOX, isoform WWOX2 and their Tyr33 phos-
phorylation, compared with those of normal controls .
These phosphorylated proteins are localized mainly in the
mitochondria , suggesting their prosurvival role in
maintaining mitochondrial homeostasis.
Another indirect evidence for its prosurvival role is that
downregulation of WWOX, WWOX2 and Tyr33 phosphor-
ylation correlates with neuronal degeneration in Alzhei-
mer’s disease, suggesting a protective effect of these
proteins . O’Keefe et al.  established DmWWOX
mutants in Drosophila. These fruit flies are viable but
exhibit an increased sensitivity to ionizing radiation.
Restoration of wild-type human or fruit fly WWOX in these
mutants reduces the radiation sensitivity, suggesting a
protective role of WWOX against environmental stress
. Nonetheless, stringent evidence is needed to verify
the prosurvival role of WWOX.
Evidence supporting its role in differentiation is that
murine Wwox is upregulated in many organs during
embryonic development and then decreased after birth
. In murine fetuses, for example, Wwox is present
prevalently in the brainstem, spinal cord and peripheral
nerve bundles, but its level is decreased shortly after birth
. Upregulation of human WWOX, WWOX2 and Tyr33
(Figure 1). Gradually increased expression of WWOX is
shown during keratinocyte differentiation. Although the
cells undergo terminal differentiation into a cornified
layer, activation of caspase-3 cannot be observed [61,62].
Activated WWOX probably contributes to the terminal
differentiation of keratinocytes, during which chromoso-
mal DNA fragmentation occurs in vivo.
How WWOX/Wwox becomes a proapoptotic protein
under environmental stress is unknown. Acute UVB-
induced skin cell hyperplasia in hairless mice is associated
with an initial upregulation and activation of Wwox
24 hours post-exposure, followed by downregulation .
This upregulation is probably needed to support the
rapidly proliferating skin keratinocytes in response to
cornification before 
UVB. Alternately, the upregulated Wwox is likely to act
as a checkpoint protein for the cell-cycle progression.
Thavathiru et al.  showed that when breast MCF7
cells were UV-irradiated and cultured for 24, 48 and
72 hours, these cells had reduced levels of both WWOX
mRNA and protein. Ishii et al.  also showed down-
regulation of Wwox and Fhit in MEF cells 24 hours after
UV exposure. Similar results were shown in cultured
primary melanoma cells . Normally, cells in culture
undergo apoptosis after UV exposure for longer than
8 hours. No upregulation of WWOX/Wwox expression in
these dying cells is expected. Also, in cell lines upregulated
expression of WWOX/Wwox and p53 normally occurs
within 1–2 hours after UV irradiation [3,18,25]. These
events are unlikely to occur in a similar fashion in vivo.
WWOX: a prognostic marker for survival and role in
most-common cancers in humans, and solar UV is the
major environmental carcinogen responsible for the devel-
opmentofBCC and SCC.In Drosophilamodel,WWOX
blocks the lethal effects of ionizing radiation . Upre-
gulation of WWOX protein in the sunburn cells commits
them to death, instead of developing into cancer .
Presumably, WWOX/Wwox-induced sunburn cell death
serves as a cancer-preventive mechanism for eliminating
pre-malignant cells against the carcinogenetic effects of
UV [64,65]. UVB cannot effectively induce skin-cell death
in the absence of WWOX .
WWOX can be regarded as a prognostic marker
[54,59,66]. Substantial studies have shown that poor prog-
nosis or unfavorable clinical outcome in patients is asso-
ciated with low or absent expression of WWOX in cancer
specimens [54,59,66]. Presence of WWOX enhances cancer
survival. Hormone-independent growth of breast, prostate
and other cancers is an unfortunate development that
makes hormone therapy ineffective. Invasive cancer cells
are frequently devoid of receptors for estrogen or androgen
that enable them to grow in a hormone-independent
manner . In certain cells, WWOX/Wwox is responsive
to estrogen- and androgen-induced Tyr33 phosphorylation
and translocation to the nuclei . Estrogen- and
androgen-induced activation of WWOX and WWOX2 is
independent of receptors for sex hormones . As
mentioned earlier, breast cancer cells are resistant to
hormone-induced WWOX nuclear translocation, whereas
prostate cancer cells are sensitive . WWOX possesses a
hormone-binding motif, although its binding to estrogen
and androgen is unknown. Conceivably, absence of WWOX
in invasive cancers increases their growth in a hormone-
Hyaluronidases regulate WWOX gene expression: the
good, the bad and the ugly
Hyaluronan and hyaluronidases are involved in embryonic
development and are overexpressed in almost every type of
cancer, and they are crucial for the progression of cancer
towards malignancy and metastasis [67–69]. Five family
proteins of hyaluronidases have been identified .
Hyaluronidases HYAL1 and HYAL2 are products of the
TRENDS in Molecular MedicineVol.13 No.1
tumor suppressor gene lung cancer (LUCA). HYAL1,
for example, might act as the promoter and suppressor of
prostate-cancer growth . Therapeutic hyaluronidase
PH-20, at high concentrations, enhances the efficacy of
chemotherapeutic drugs in penetrating into solid tumors
in vivo .
Hyaluronidase PH-20 is present in normal breast cells,
and is overexpressed in invasive and metastatic breast
cancer . Hyaluronidases PH-20, HYAL1 and HYAL2
induce WWOX/Wwox expression [3,7,73]. We cloned
murine Wwox gene by hyaluronidase induction . Con-
ceivably, PH-20 maintains a low level of WWOX in normal
breast, and optimally elevated PH-20 level increases the
level of WWOX and perhaps Tyr33 phosphorylation
during breast-cancer development before metastasis 
(Figure 2). Hyaluronan, PH-20 and other hyaluronidase
levels continue to increase during metastasis, whereas
WWOX is dramatically downregulated and the estrogen
receptor disappears . Lokeshwar et al.  provided an
outstanding model showing the dual role of HYAL1
in regulating prostate-cancer growth and correlation
with WWOX expression and phosphorylation at Tyr33.
Presumably, high levels of hyaluronidases suppress the
expression of hormone receptors and WWOX, and this
turns cancer cells to malignancy.
WWOX/Wwox has a crucial role in protection against
cancer, and probably neurodegenerative diseases .
Alterations of WWOX/Wwox gene (e.g. LOH, methylation,
etc.) are closely associated with cancer pathogenesis, pro-
gression and metastasis; however, a transgenic murine
model for Wwox knockout is still lacking. If WWOX/Wwox
has a tumor suppressor role, Wwox knockout mice are
expected to develop tumors spontaneously. Drosophila
mutants have provided an outstanding model for investi-
gating the role of WWOX/Wwox against environmental
stress  and, probably, the development of neurodegen-
erative diseases and cancer. Thus, one of the future direc-
tions is to develop a transgenic model(s) or a tissue-specific
conditional knockout model for advancing the understand-
ing of the role of WWOX/Wwox in vivo.
An additional area of research interests is the
complicatedsignaling or protein–proteininteraction
Figure 1. WWOX in normal cutaneous keratinocyte differentiation and stress-induced apoptosis. (a) As a homeostatic process, normal keratinocytes undergo
proliferation, differentiation and cornification. In the basal layer, germinative stem cells (St) proliferate and give rise to transient amplifying cells (Tac) and basal cells
(Bc), followed by differentiation into spinous cells (Sc) and granular cells (Gc) and, eventually, corneocytes (Cor). Keratins K5 and K14 and integrins are expressed in the
basal layer, which might regulate the initiation of differentiation. Morphological changes are associated with presence of distinct structural components: high molecular
weight keratin (K1 and K10), involucrin, profilaggrin/filaggrin (PF/F), loricrin, transglutaminase (TGase) 1, caspase-14, cathepsin D, neutral lipids (ceramides, cholesterol
and free fatty acids), natural moisturizing factors (NMF: amino acids, glucosamine, uric acid and ammonia) and cornified cell envelope (CCE). Unlike typical apoptosis,
terminal differentiation of keratinocytes into corneocytes and formation of a protective stratum corneum do not cause activation of caspase-3 .
(b) Immunohistochemistry of a normal human epidermal tissue section. WWOX expression is gradually increased along the line of keratinocyte differentiation and
is most noted in the nuclei of the terminally differentiated granular cells (before cornification). (c) Keratinocytes seem to undergo typical apoptosis with WWOX
upregulation in response to acute UV exposure (sunburn), cutaneous graft-versus-host disease (GVHD), toxic epidermal necrolysis (TEN) and chemotherapy. In most
cases, caspase-3 activation occurs. Defects in apoptosis might lead to skin diseases such as cutaneous SCC and BCC. WWOX protein expression is markedly decreased in
cutaneous SCC that enables cell survival . The role of WWOX in BCC development remains to be established. Abbreviations: BMZ, basement membrane zone; KHG,
keratohyaline granule; LB, lamellar body.
TRENDS in Molecular MedicineVol.13 No.1 19
network and the underlying functional associations. Tyr33
phosphorylation of WWOX contributes to functioning in
cell survival and death. The Tyr33-phosphorylated WW
domain interacts with many more binding motifs than the
PPxY motif, indicating a broad spectrum of protein inter-
actions. Elucidation of the signaling network in normal
and cancer cells advances the understanding of how
WWOX loses control of cancer growth.
In summary, endogenous WWOX/Wwox seems to support
embryonic development and differentiation, and probably
has a homeostatic role in normal cell-cycle progression in
concert with p53, p73, ERBB4 and other transcription
factors. However, environmental stress can turn WWOX/
Wwox into a proapoptotic protein. Cancer cells, however,
are defective in producing WWOX at the invasive stage.
Favorable clinical outcome in patients is associated with
novel strategy to fight cancer is to stimulate WWOX
expression in cancer cells.
There are many questions left unanswered regarding
WWOX and its functional properties in vivo (Box 5). One
interesting area is that both p73 and p63 have an identical
PPxY motif. Does WWOX/Wwox interact with p63? WW-
containing E3 ligase ITCH regulates the stability of p63 in
keratinocytes . Can WWOX/Wwox bind to and stabilize
p63? Also, does WWOX/Wwox compete with YAP or ITCH
(all WW proteins) in stabilizing p73 (and p63) steady state
protein levels, and thus its function?
We might be able to manipulate hormone resistance in
breast and prostate cancers by controlling WWOX expres-
sion. Also, we might be able to restrain cancer progression
by tightly monitoring the expression of hyaluronidases,
hyaluronan and WWOX (Figure 2). Elevated hyaluroni-
dases increase cancer invasiveness and concurrently sup-
press WWOX expression. Disappearance of WWOX in
invasive cancer correlates with their developed hormone
resistance. WWOX provides an apparent connection for
hyaluronidase-regulated cancer growth, metastasis and
Research of N.S.C. was supported in part by the American Heart
Association, the Department of Defense (DAMD17–03–1-0736), and the
Guthrie Foundation for Education and Research. L.J.H. was supported by
the Ministry of Education, Taiwan, ROC (91-B-FA09–1-4) and National
Science Council, Taiwan (95–2320-B-006–072-MY2). We appreciate the
critical review of this article by Dr. M. Sudol of the Weis Center for
Research, Geisinger Clinic, Danville, Pennsylvania, USA.
1 Bednarek, A.K. et al. (2000) WWOX, a novel WW domain-containing
protein mapping to human chromosome 16q23.3-24.1, a region
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2 Ried, K. et al. (2000) Common chromosomal fragile site FRAD16D
sequence: identification of the FOR gene spanning FRAD16D and
Box 5. Outstanding questions
Although alterations of WWOX gene contribute, in part, to the
pathogenesis of cancer, many questions remain to be answered
regarding the functional properties of WWOX/Wwox.
Gene and protein
? How fragile is WWOX gene? What is the crucial time point for
WWOX gene breaking apart during cell-cycle progression and cell
age of common fragile sites?
? How many WWOX/Wwox isoforms do exist in cells? What are their
? Elevated hyalurondiases promote cancer invasiveness. Does this
event induce WWOX gene alteration and silencing?
? The SDR domain of WWOX/Wwox might metabolize estrogen and
androgen. If so, how does it do that?
? Does loss of WWOX/Wwox protein constitute development of
hormone resistance in breast and prostate cancers?
? Caspase-3 activation is absent in the process of keratinocyte cor-
nification. What is the precise role of WWOX/Wwox in keratinocyte
? Do activated WWOX/Wwox and caspases participate in the apop-
tosis of sunburn cells?
? Both p73 and p63 possess an identical PPxY motif. Does WWOX/
Wwox interact with p63?
? WW-containing E3 ligase ITCH regulates the stability of p63 in
keratinocytes . Can WWOX/Wwox do the same?
stabilizing p73 (and p63) steady state protein levels and, thus, its
Figure 2. A schematic model of hyaluronidase regulation of WWOX expression and
development of hormone-independent growth in breast cancer. (a) In normal
epithelial cells, WWOX level is low but significantly upregulated and Tyr33-
phosphorylated during cell differentiation and might or might not decrease
subsequently. However, during apoptosis WWOX is dramatically increased in vivo
[20,24]. (b) Normal and breast-cancer cells produce hyaluronidase PH-20 and
hyaluronan . Hyaluronidases, including PH-20, HYAL1 and HYAL2, induce
WWOX/Wwox expression [3,7,73]. Estrogen and androgen stimulate Tyr33
phosphorylation and nuclear translocation of WWOX/Wwox in various types of
cells,independently ofhormone receptors.WWOXpossessesa hormone-binding
motif, whereas its interaction with estrogen and androgen is unknown .
Hyaluronidases probably maintain hormone-dependent growth of breast-cancer
cells (estrogen-receptor positive, ER+) during early stages of cancer development by
upregulating WWOX expression (hyperplasia and solid tumor or adenocarcinoma).
High levels of hyaluronidases increase breast-cancer invasiveness and hormone-
independent growth by downregulating WWOX. Whether hyaluronidases control
the expression of receptors for estrogen (ER) and androgen (AR) remains to be
established. Abbreviations: HA, hyaluronan; HAase, hyaluronidase.
TRENDS in Molecular Medicine Vol.13 No.1
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