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WW domain-containing oxidoreductase: a candidate tumor suppressor Nan-Shan Chang1,5, Li-Jin Hsu2,3, Yee-Shin Lin2,3, Feng-Jie Lai4 and Hamm-Ming Sheu4 1
Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan 70101, Republic of China Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan 70101, Republic of China 3 Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University Medical College, Tainan, Taiwan 70101, Republic of China 4 Department of Dermatology, National Cheng Kung University Medical College, Tainan, Taiwan 70101, Republic of China 5 Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840, USA 2
Common fragile site gene WWOX encodes a candidate tumor suppressor WW domain-containing oxidoreductase. Alteration of this gene, along with dramatic downregulation of WWOX protein, is shown in the majority of invasive cancer cells. Ectopic WWOX exhibits proapoptotic 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-independent growth of invasive cancer cells. Estrogen and androgen stimulate phosphorylation and nuclear translocation of WWOX, although binding of WWOX to these sex hormones is unknown. We propose that suppression 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 [1] or FOR [2], or murine Wox1 or Wwox [3], was first discovered in 2000. The large size human gene WWOX (1.1 Glossary 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 Parkinson diseases. 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
Corresponding author: Chang, N.-S. (
[email protected]). Available online 4 December 2006. www.sciencedirect.com
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 environmental 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 phosphoserineor phosphothreonine-containing motifs. WW domain-containing oxidoreductase (WWOX/Wwox): a candidate tumor suppressor and proapoptotic protein. The full-length protein, 46 kDa, possesses two N-terminal WW domains, a nuclear localization sequence (NLS) and a C-terminal short-chain alcohol dehydrogenase–reductase (SDR) domain.
1471-4914/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.11.006
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TRENDS in Molecular Medicine
million base pairs) encodes this protein. Alterations of WWOX gene in cancer cells have been thoroughly investigated 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 degradation; (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 [4] (Box 1). Database searching shows the presence of WWOX gene transcripts in monkey, mouse,
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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 Nterminal WW domains (with conserved tryptophan residues), 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. Presence 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. [5] 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 mitochondriatargeting region is shown in the SDR domain (amino acid 209–273) of murine Wwox (between Y287 and Y293) [3].
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). www.sciencedirect.com
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weight WWOX with truncations at the SDR domain, corresponding to 35-kDa WWOXD5–8, 26-kDa WWOXD6– 8, and 35.2-kDa WWOXD7–8 in colon HCT116 cells. Proteasome inhibitor MG-132 was used to stabilize these proteins, suggesting that they are unstable and might act as dominant negatives in blocking the function of WWOX [5]. Mahajan et al. [6] validated the presence of WWOXD5–8 (v3 or WWOX3) in human prostate LNCaP cells as determined 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. [7] 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 [8], breast and prostate tissues [9]. Frequently, anti-WW domain antibodies cannot effectively 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 [10] (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 domain-containing proteins represent a large protein family (1400 members) [11–13]. Functional deficiency of WW domain-containing proteins PIN1 [protein (peptidylprolyl cis/trans isomerase) NIMA-interacting 1] and WWOX might 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. nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db= t&Dopt=s&uid=34568). 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 [16]. 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 [17]. Phosphorylation at Tyr33 in the first WW domain of WWOX/Wwox has been defined both in vitro and in vivo [7– 9,18–20] (Box 1). Sex steroid hormones and stress stimuli induce Tyr33 phosphorylation of WWOX/Wwox [7–9,18– 20]. Aqeilan et al. [19] reported that tyrosine kinase SRC phosphorylates Tyr33. Mahajan et al. [6] 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. www.sciencedirect.com
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A conserved caspase recognition motif, QETD (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 protein bands [3]. 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 microscopy, 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 localization [3,21]. Endogenous Wwox is present in purified mitochondria from rat liver [3]. We have determined a mitochondria-targeting region in the SDR domain [3]. Bednarek et al. [22] showed that ectopic human GFP– WWOX is in the Golgi apparatus in normal breast MCF10F cells. These differences are probably due to (i) different cell lines used, (ii) variations between human WWOX and mouse Wwox amino acid sequences particularly at the Ctermini, and (iii) differences in culture conditions between laboratories (e.g. sera). Localization to the mitochondria and nuclei of endogenous WWOX/Wwox has also been shown in cell lines, mammary gland cells and epidermal keratinocytes [3,5,9]. Jin et al. [23] 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 recruitment is needed for ezrin–WWOX interaction in the apical cell membrane [23]. COS7 fibroblasts are responsive to estrogen- or androgen-induced nuclear translocation of WWOX [9]. 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 [9]. 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 [9]. However, 1.5-fold increases in nuclear translocation of WWOX at these stages are observed in prostate cancer [9]. 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 [20]. 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 [20]. 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 [20]. Wwox is barely detectable in the Golgi complex of apoptotic cells [20]. Together, depending upon
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the cell types, tissues and exogenous stimuli, WWOX/ Wwox has been shown in mitochondria, Golgi complex, rough ER, plasma membrane and nuclei (using light, confocal 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 general consensus from these studies is that WWOX is mainly expressed in epithelial cells, particularly in hormone-regulated 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 [24,26]. WWOX: Tyr33 phosphorylation, death signaling and tumor suppression 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-MB435 in nude mice [22]. Restoration of WWOX gene in lung cancer cells prevents their growth both in vitro and in vivo [28]. Importantly, loss of WWOX protein promotes prostate tumorigenesis in vivo [6]. Activated ACK1
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 development and then decreased after birth [24]. Wwox is differentially expressed and distributed in developing and adult murine nervous system [24]. WWOX is expressed in many human organs (e.g. brain, skin, heart and others) [26]. Hyaluronidases PH-20, HYAL1 and HYAL2 induce WWOX/Wwox expression [3,7,73]. 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 [19]. Sex steroid hormones, estrogen and androgen, induce WWOX phosphorylation at Tyr33 and nuclear translocation [9]. Sex steroid hormone-induced activation of WWOX and WWOX2 is independent of receptors for sex hormones [9]. Phosphorylated-ezrin binds to and promotes apical membrane localization of WWOX in parietal cells of stomach [23]. WWOX and YAP compete for interaction with ERBB4 and other targets and thus affect its transcriptional activity [35]. WWOX binds to and triggers redistribution of nuclear AP-2g to the cytoplasm for suppressing its transactivating function [34]. WWOX binds to and triggers redistribution of nuclear p73 to the cytoplasm and suppresses its transcriptional activity [19]. Activated tyrosine kinase ACK1 phosphorylates human WWOX at Tyr287 for polyubiquitination and degradation [6]. www.sciencedirect.com
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phosphorylates WWOX for polyubiquitination and proteosomal degradation, thereby causing disappearance of WWOX in prostate cells [6]. Hypermethylation of gene promoter downregulates WWOX expression in prostate 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 correlates positively with alterations of common fragile site genes WWOX and fragile histidine triad (FHIT) in these cells [31], 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-transfected L929 cells were shown to have an increased sensitivity to TNF cytotoxicity [3]. Overexpressed WW or SDR domain or the full-length Wwox induces cell death, suggesting that both domains are functional modules [3,4]. This enhancement is associated, in part, with upregulation of proapoptotic p53 and downregulation of apoptosis inhibitors B-cell CLL/lymphoma 2 (BCL2) and BCL-xL in L929 cells [3,4]. Recently, downregulation of BCL2 by overexpressed WWOX has been shown in certain prostate cancer cells [29]. In addition, Wwox enhances ectopic TNF receptor-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 cancer cells. 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 [3]. Activation of caspase-3 (a downstream executor of the caspase pathway) and degradation of poly (ADP-ribose)polymerase-1 (PARP-1) is shown in WWOXinfected lung A549, H460 and H1299 cells but not in U2020 cells during 96 hours in culture [28]. Under a similar culture period, disappearance of procaspase-9 and procaspase-3 is shown in WWOX-infected LNCaP, DU145 and PC-3 prostate cancer cells, but not in non-cancerous PWR-1E cells [29]. 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, followed by translocation to the mitochondria and nuclei to induce apoptosis [4,18] (Box 3 and Box 4). We have shown a dramatically increased presence of activated Wwox in the damaged mitochondria and condensed or apoptotic nuclei of degenerating retinal photoreceptors in rat eyes by electron microscopy [20]. Tyrosine kinase SRC phosphorylates WWOX at Tyr33 [19] (Box 3). Whether other tyrosine kinases phosphorylate WWOX/Wwox at Tyr33 is unknown. Tyr33 phosphorylation in WWOX/Wwox is crucial 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
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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 [4]. Tyrosine kinase SRC phosphorylates WWOX/Wwox at Tyr33 [19]. Phosphorylated WWOX/Wwox (p-WWOX) translocates to the mitochondria or nuclei in vitro and in vivo, or binds to Ser46-phosphorylated 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 [35].
Route 3. In neurons, WWOX/Wwox seems to prevent enzymemediated Tau hyperphosphorylation [8]. Route 4. Phosphorylated ezrin binds to and promotes apical membrane localization of WWOX in parietal cells [23]. WWOX phosphorylation is not known. Route 5. WWOX interacts with SIMPLE, a small integral membrane protein of the lysosome/late endosome [36]. WWOX phosphorylation is not known. Route 6. Activated tyrosine kinase ACK1 phosphorylates WWOX at Tyr287 for polyubiquitination and degradation [6].
Figure I. Mode of actions of WWOX/Wwox.
motif-possessing proteins bind to the first WW domain, including p73 [19], activator protein-2g (AP-2g) [34], verb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4) [35], ezrin [23] and small integral membrane protein of the lysosome/late endosome (SIMPLE) [36] (Table 1 and Box 3). p73, AP-2g and ERBB4 are transcription factors. Ezrin is a signal transducer located at the cell membrane and/or cytoskeleton area [37]. Binding of matrix hyaluronan to membrane CD44 receptor transduces signals to erzin and associated proteins for regulating cell growth. Second, Tyr33 phosphorylation in WWOX/Wwox 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 transcription [19,34] (Box 3). Importantly, WWOX competes with YAP (WW domain-containing Yes-associated protein) for interaction with ERBB4, and this competition modulates the transcriptional function of ERBB4 [35]. 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) [18], p53 [3,9,18,27] and mouse double mutant 2 (MDM2) [27] www.sciencedirect.com
(Table 1). No PPxY motif is identified in these proteins. Alteration of Tyr33 abolishes the binding interactions. Tyr33-phosphorylated WWOX binds to p53 via phosphoSer46–Pro47 and an adjacent N-terminal proline-rich region (amino acid 66–100) [3,27]. Without Ser46 Table 1. WWOX/Wwox binding proteins
First WW domain a
pY33 first WW domain b
pY33 first WW domain b
SDR domain a
Recognition motif PPPPY PPPY PPPxY PPPPPPVY PPSY PPPPY PPPY PPPxY Poly-P/pS46P47c Unknown Unknown Unknown
Binding proteins p73 AP-2g ERBB4 Ezrin SIMPLE p73 AP-2g ERBB4 p53 JNK1 MDM2 Tau
Refs [19] [34] [35] [23] [36] [19] [34] [35] [3,27] [18] [27] [8]
The first WW domain binds to PPxY motif in the indicated proteins. The specific recognition sequence for each indicated protein is shown. b Y33 phosphorylation seems to enhance the binding of WWOX to p73, AP-2g or ERBB4. Alteration of Y33 significantly reduces the binding. Whether binding of ezrin and SIMPLE to the first WW domain depends upon Y33 phosphorylation is not known. c Wwox binds to the N-terminal phospho-Ser46–Pro47 and an adjacent poly-proline segment (amino acid 66–100) in p53.
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Box 4. WWOX/Wwox: proapoptotic activity and tumorsuppressor function Proapoptotic activity WWOX/Wwox acts as a proapoptotic protein 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 (cytochrome 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 [28,29]. Wwox directly interacts with p53 and both induce apoptosis synergistically [3,18,27]. Hyaluronidase HYAL2 enhances Wwox-induced apoptosis [73]. Tyr33-phosphorylated WWOX/Wwox directly interacts with activated JNK1 under stress conditions [18]. Ectopic JNK1 blocks Wwox-induced inhibition of cell-cycle progression and apoptosis [18]. Tyr33 phosphorylation in WWOX/Wwox is essential for binding and stabilizing Ser46-phosphorylated p53 [27]. WWOX and p73 induce apoptosis synergistically [19]. 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 phosphorylation, mitochondrial and nuclear translocation [20]. Tumor-suppressor function WWOX suppresses breast-cancer cell growth in soft agarose and in nude mice [22]. Restoration of WWOX gene in lung cancer cells prevents their growth both in vitro and in vivo [28]. Loss of WWOX protein promotes prostate tumorigenesis in vivo via activated ACK1-mediated WWOX degradation [6]. Inhibition of hypermethylation in lung tumor cells restores FHIT and WWOX expression and suppresses growth in vivo [30]. Restoration of WWOX expression in prostate cancer cells suppresses growth both in vitro and in vivo [29]. High level expression of WWOX mRNA is associated with better breast-cancer survival (protein level unknown) [54]. Thymocytes are refractory to death in thyroid-cancer patients with alterations of WWOX and FHIT (indirect effect?) [31]. Potential prosurvival role WWOX is upregulated in non-invasive breast and gastric cancer tissues, but downregulated in invasive cancers [5]. WWOX, WWOX2 and Tyr33 phosphorylation are upregulated in non-invasive breast and prostate cancer cells [9,29]. Differentiation of skin keratinocytes is associated with upregulation of WWOX/Wwox and Tyr33 phosphorylation [25]. Neuronal degeneration is associated with downregulation of WWOX, WWOX2 and Tyr33 phosphorylation [8]. Oxido-reductase (FOR/WWOX) protects against the lethal effects of ionizing radiation in Drosophila [60].
phosphorylation, p53 does not bind to WWOX [27]. Without Tyr33 phosphorylation, WWOX cannot interact with p53 [27]. Cells must be exposed to stress or apoptotic stimuli to enable Ser46 phosphorylation in p53 and Tyr33 phosphorylation in WWOX, respectively, thereby allowing the binding interaction to occur [3,9,18,27]. Aqeilan et al. [19] failed to show the binding of ectopic WWOX to ectopic p53 proteins. The probable reason is that cells were not exposed to stress stimuli. www.sciencedirect.com
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Finally, the SDR domain of Wwox has been shown to bind to Tau and seems to prevent Tau hyperphosphorylation [8]. 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 outstanding 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%) [44], breast (81.8%) [45], esophagus [squamous cell carcinoma (SCC), 38.9%] [46], lung (non-small cell lung cancer, 37%) [47], pancreas (26.7%) [48] and stomach (30.8%) [49]. 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 [50]. In one esophageal SCC, T!C transition at the second nucleotide of codon 291 causes leucine to proline substitution [46]. However, genetic polymorphisms for the WWOX gene are frequently shown in normal and clinical samples [44,47–49,51]. Environmental factors contribute to genetic alterations [25,41,52,53]. Aflatoxin B1 [44], UV-light exposure and tobacco smoking [50] have been implicated in the alterations of WWOX gene in the carcinogenesis of hepatocellular 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 [55], esophageal SCC (5.6%) [46], oral SCC (35%) [50], lung cancer (26%) [47], hematopoietic neoplasia (12%) [51], pancreatic adenocarcinoma (6.7%) [48], gastric carcinoma (12%) [49] and non-small cell lung cancer (63.6%) [56]. Although the presence of full-length and short mRNA transcripts in cancer cells [57], 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 silencing 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%) [48]. 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 expression [58]. Finally, translational blockade of WWOX mRNA is associated with disappearance of WWOX/Wwox in cutaneous SCCs in humans and hairless mice [25]. Significant reduction of WWOX, WWOX2 and Tyr33 phosphorylation is observed in patients with cutaneous SCCs without significant changes of WWOX mRNA, indicating translational blockade of WWOX mRNA into protein. In parallel,
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chronic UVB-induced formation of cutaneous SCCs in hairless mice involves dramatic reduction of Wwox and Tyr33 phosphorylation without downregulation of WWOX mRNA [25]. WWOX: prosurvival and role in differentiation? Watanabe et al. [5] 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 downregulated in these tissues during metastasis [5]. Significantly reduced expression of WWOX protein is also shown in invasive breast and prostate tissues [9,29,59]. Highlevel expression of WWOX mRNA in estrogen receptor (ER)-positive breast cancer cells is associated with improved cancer survival [54]. Additionally, the level of WWOX mRNA is both upregulated and downregulated in non-invasive and metastatic breast cancer cells, respectively [45]. We have examined various stages of breasttumor development [9]. Breast cells at hyperplasia and cancerous (or solid tumor) stages have upregulated cytosolic WWOX, isoform WWOX2 and their Tyr33 phosphorylation, compared with those of normal controls [9]. These phosphorylated proteins are localized mainly in the mitochondria [9], suggesting their prosurvival role in maintaining mitochondrial homeostasis. Another indirect evidence for its prosurvival role is that downregulation of WWOX, WWOX2 and Tyr33 phosphorylation correlates with neuronal degeneration in Alzheimer’s disease, suggesting a protective effect of these proteins [8]. O’Keefe et al. [60] 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 [60]. 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 [24]. 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 [24]. Upregulation of human WWOX, WWOX2 and Tyr33 phosphorylation occurs during normal keratinocyte growth and differentiation before cornification [25] (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 chromosomal DNA fragmentation occurs in vivo. How WWOX/Wwox becomes a proapoptotic protein under environmental stress is unknown. Acute UVBinduced skin cell hyperplasia in hairless mice is associated with an initial upregulation and activation of Wwox 24 hours post-exposure, followed by downregulation [25]. This upregulation is probably needed to support the rapidly proliferating skin keratinocytes in response to www.sciencedirect.com
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UVB. Alternately, the upregulated Wwox is likely to act as a checkpoint protein for the cell-cycle progression. Thavathiru et al. [52] 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. [53] also showed downregulation of Wwox and Fhit in MEF cells 24 hours after UV exposure. Similar results were shown in cultured primary melanoma cells [63]. 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 hormone dependence Cutaneous basal cell carcinoma (BCC) and SCC are the two most-common cancers in humans, and solar UV is the major environmental carcinogen responsible for the development of BCC and SCC [64]. In Drosophila model, WWOX blocks the lethal effects of ionizing radiation [60]. Upregulation of WWOX protein in the sunburn cells commits them to death, instead of developing into cancer [25]. 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 [25]. WWOX can be regarded as a prognostic marker [54,59,66]. Substantial studies have shown that poor prognosis or unfavorable clinical outcome in patients is associated 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 [66]. In certain cells, WWOX/Wwox is responsive to estrogen- and androgen-induced Tyr33 phosphorylation and translocation to the nuclei [9]. Estrogen- and androgen-induced activation of WWOX and WWOX2 is independent of receptors for sex hormones [9]. As mentioned earlier, breast cancer cells are resistant to hormone-induced WWOX nuclear translocation, whereas prostate cancer cells are sensitive [9]. 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 hormoneindependent way. 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 [70]. Hyaluronidases HYAL1 and HYAL2 are products of the
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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 [61]. (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 [25]. The role of WWOX in BCC development remains to be established. Abbreviations: BMZ, basement membrane zone; KHG, keratohyaline granule; LB, lamellar body.
tumor suppressor gene lung cancer (LUCA). HYAL1, for example, might act as the promoter and suppressor of prostate-cancer growth [7]. Therapeutic hyaluronidase PH-20, at high concentrations, enhances the efficacy of chemotherapeutic drugs in penetrating into solid tumors in vivo [71]. Hyaluronidase PH-20 is present in normal breast cells, and is overexpressed in invasive and metastatic breast cancer [72]. Hyaluronidases PH-20, HYAL1 and HYAL2 induce WWOX/Wwox expression [3,7,73]. We cloned murine Wwox gene by hyaluronidase induction [3]. Conceivably, 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 [9] (Figure 2). Hyaluronan, PH-20 and other hyaluronidase levels continue to increase during metastasis, whereas WWOX is dramatically downregulated and the estrogen receptor disappears [74]. Lokeshwar et al. [7] provided an outstanding model showing the dual role of HYAL1 in regulating prostate-cancer growth and correlation with WWOX expression and phosphorylation at Tyr33. www.sciencedirect.com
Presumably, high levels of hyaluronidases suppress the expression of hormone receptors and WWOX, and this turns cancer cells to malignancy. Future directions WWOX/Wwox has a crucial role in protection against cancer, and probably neurodegenerative diseases [8]. Alterations of WWOX/Wwox gene (e.g. LOH, methylation, etc.) are closely associated with cancer pathogenesis, progression 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 investigating the role of WWOX/Wwox against environmental stress [60] and, probably, the development of neurodegenerative diseases and cancer. Thus, one of the future directions is to develop a transgenic model(s) or a tissue-specific conditional knockout model for advancing the understanding of the role of WWOX/Wwox in vivo. An additional area of research interests is the complicated signaling or protein–protein interaction
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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 division? What is the environmental factor(s) responsible for breakage of common fragile sites? How many WWOX/Wwox isoforms do exist in cells? What are their functions? Cancer progression
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 Tyr33phosphorylated 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 [72]. 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 of hormone receptors [9]. WWOX possesses a hormone-binding motif, whereas its interaction with estrogen and androgen is unknown [9]. 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 hormoneindependent 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.
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 interactions. Elucidation of the signaling network in normal and cancer cells advances the understanding of how WWOX loses control of cancer growth. Concluding remarks 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 elevated expression of WWOX in cancer specimens. Thus, a 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? WWcontaining E3 ligase ITCH regulates the stability of p63 in keratinocytes [75]. 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? www.sciencedirect.com
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? Apoptosis Caspase-3 activation is absent in the process of keratinocyte cornification. What is the precise role of WWOX/Wwox in keratinocyte terminal differentiation? Do activated WWOX/Wwox and caspases participate in the apoptosis of sunburn cells? Transcription factors 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 [75]. Can WWOX/Wwox do the same? 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 expression. Also, we might be able to restrain cancer progression by tightly monitoring the expression of hyaluronidases, hyaluronan and WWOX (Figure 2). Elevated hyaluronidases increase cancer invasiveness and concurrently suppress 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 presumably hormone-independence. Acknowledgements 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.
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57 Gourley, C. et al. (2005) WWOX mRNA expression profile in epithelial ovarian cancer supports the role of WWOX variant 1 as a tumour suppressor, although the role of variant 4 remains unclear. Int. J. Oncol. 26, 1681–1689 58 Iliopoulos, D. et al. (2005) Fragile genes as biomarkers: epigenetic control of WWOX and FHIT in lung, breast and bladder cancer. Oncogene 24, 1625–1633 59 Guler, G. et al. (2004) The fragile genes FHIT and WWOX are inactivated coordinately in invasive breast carcinoma. Cancer 100, 1605–1614 60 O’Keefe, L.V. et al. (2005) FRA16D common chromosomal fragile site oxido-reductase (FOR/WWOX) protects against the effects of ionizing radiation in Drosophila. Oncogene 24, 6590–6596 61 Lippens, S. et al. (2005) Death penalty for keratinocytes: apoptosis versus cornification. Cell Death Differ. 12, 1497–1508 62 Candi, E. et al. (2005) The cornified envelope: a model of cell death in the skin. Nat. Rev. Mol. Cell Biol. 6, 328–340 63 Yang, G. et al. (2006) Expression profiling of UVB response in melanocytes identifies a set of p53-target genes. J. Invest. Dermatol. 126, 2490–2506 64 Diepgen, T.L. and Mahler, V. (2002) The epidemiology of skin cancer. Br. J. Dermatol. 61, 1–6 65 Van Laethem, A. et al. (2005) The sunburn cell: regulation of death and survival of the keratinocyte. Int. J. Biochem. Cell Biol. 37, 1547–1553
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66 Nunez, M.I. et al. (2005) WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome. BMC Cancer 5, 64 67 Csoka, T.B. et al. (1997) Hyaluronidases in tissue invasion. Invasion Metastasis 17, 297–311 68 Toole, B.P. (2004) Hyaluronan: from extracellular glue to pericellular cue. Nat. Rev. Cancer 4, 528–539 69 Stern, R. (2005) Hyaluronan metabolism: a major paradox in cancer biology. Pathol. Biol. (Paris) 53, 372–382 70 Csoka, A.B. et al. (2001) The six hyaluronidase-like genes in the human and mouse genomes. Matrix Biol. 20, 499–508 71 St Croix, B. et al. (1998) Reversal of intrinsic and acquired forms of drug resistance by hyaluronidase treatment of solid tumors. Cancer Lett. 131, 35–44 72 Beech, D.J. et al. (2002) Expression of PH-20 in normal and neoplastic breast tissue. J. Surg. Res. 103, 203–207 73 Chang, N-S. (2002) Transforming growth factor-b1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts. BMC Cell Biol. 3, 8 74 Nunez, M.I. et al. (2005) Frequent loss of WWOX expression in breast cancer: correlation with estrogen receptor status. Breast Cancer Res. Treat. 89, 99–105 75 Rossi, M. et al. (2006) The E3 ubiquitin ligase Itch controls the protein stability of p63. Proc. Natl. Acad. Sci. U. S. A. 103, 12753–12758
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