The Gene

The WWOX gene spans a genomic locus of >600 kb and is composed of nine exons encoding an open reading frame of 1,245 bases; the protein sequence includes two WW domains and a short chain dehydrogenase/reductase domain that may be involved in sex-steroid metabolism, considering its sequence homology to 17β-hydroxysterol reductase 3 (Fig. 1).

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The WWOX gene also spans fragile site  FRA16D, encompasses a region involved in  Loss of heterozygosity (LOH) in cancers, is associated with  homozygous deletions in cancer-derived cell lines, with chromosome translocations in  multiple myelomas, and its promoter region is frequently hypermethylated in cancers (Fig. 2).

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Most cancer cell lines with FRA16D homozygous deletions also exhibit deletions in  FRA3B and the   FHIT gene, consistent with the finding that common fragile sites or regions are highly susceptible to  DNA damage and recombination. The mouse ortholog, Wwox, at murine chromosome region 8E1, is also fragile and is highly homologous to the human locus. Internally deleted WWOX transcripts have been observed in human breast, ovarian, and other tumor types, though bona fide point mutations have not been observed. The highest level of WWOX expression in normal tissues occurs in hormonally regulated tissues. The WWOX promoter is hypermethylated in many cancers in association with loss of Wwox expression, and detection of the  methylation status by  methylation-specific PCR (MSP) amplification may serve as a marker of cancer development or prevention.

The Protein

Wwox protein binds the proline-rich ligand PPxY and a number of proteins interacting with the first WW domain, WW1, have been identified. WW domains are grouped by binding preference for specific types of proline-rich ligands and the Wwox WW1 domain belongs to group I, that binds PPxY ligands; among these ligand-containing proteins are p73, Ap2α, Ap2γ, ErbB4, Jun, and the SIMPLE protein;  p53 has also been reported to bind Wwox or Wox1, the murine ortholog, but other studies have not confirmed this ligand. The Cytogen Corporation has developed, through informatics and binding studies, a proprietary database that lists all potential Wwox ligands among known gene products, and the first three ligands listed above were selected from the database and confirmed as Wwox-interacting proteins by in vitro analyses.

The cytoplasmic Wwox protein binds its ligands and prevents the interacting proteins from entering the nucleus, where some have roles in transcriptional activation or repression. There are thus far unconfirmed reports of Wwox subcellular location also in mitochondria and nuclei of some cells.

Biological Role

In normal cells: human tissue samples of more than 30 organs have been analyzed by immunohistochemistry for expression of Wwox; Wwox is expressed in most organs but is expressed at highest levels in secretory epithelial cells such as those of breast, ovary, testes, and prostate. Wwox is also expressed in various cells of neural origin.

 Targeted deletion of the mouse Wwox gene revealed important roles of Wwox in tumorigenesis and metabolism. Using  homologous recombination, a mouse lacking exons 2, 3, and 4 of the mouse Wwox gene was generated. Progeny from Wwox heterozygous (Wwox+/−) intercrosses resulted in offspring of all three genotypes with ratios consistent with  Mendelian distribution. At birth, homozygous Wwox-deficient (Wwox−/−) pups were indistinguishable from wild type or heterozygous littermates up to 3 days postpartum; after 3 days, homozygous pups were easily identified by their smaller size. Wwox−/− pups continued to grow more slowly than littermates, and all homozygous knockout mice died by 4 weeks after birth. Serum chemistry analysis of Wwox−/− mice showed marked hypoproteinemia, hypoalbuminemia, hypoglycemia, hypocalcemia, hypotriglyceridemia, and hypocholesterolemia, indicating that Wwox−/− pups suffered severe metabolic defects.

Macroscopic and histological examination of the organs confirmed atrophy of many organs in Wwox−/− animals without significant microscopic lesions, though Wwox−/− mice are born with gonadal abnormalities and display bone growth retardation. Juvenile Wwox−/− mice displayed impaired steroidogenesis; levels of steroid biosynthesis enzymes, including Cyp11a1 (cytochrome p450 side-chain cleavage enzyme) and Hsd3b (3-β-hydroxysteroid dehydrogenase), were reduced in mutant testis and ovary compared to wild type and heterozygous testis. Radiography and high-resolution microtomography (μCT) of limb bones showed that Wwox−/− mice develop less dense bones with slow growth rates.

Analysis of the Wwox-mutant mice demonstrated that Wwox functions as a bona fide tumor suppressor. Spontaneous  osteosarcomas in juvenile Wwox−/− and lung papillary carcinoma in adult Wwox+/− mice were observed, and Wwox+/− mice developed significantly more ethyl nitrosourea (ENU)-induced lung tumors and  lymphomas in comparison to wild type littermates. These tumors still express Wwox protein, suggesting  haploinsufficiency of Wwox is cancer-predisposing.

In cancer cells: Esophageal squamous cell carcinoma,  non-small cell lung cancer and  breast cancer showed high  LOH rates, low mutation rates, and expression of aberrant transcripts of the WWOX gene. Wwox expression is reduced in 63% of invasive breast carcinomas and is correlated with  estrogen receptor alpha level, and prognostic features; Wwox and Fhit expression is coordinately lost in breast cancers. The WWOX gene is also inactivated in breast and lung cancers by regulatory region DNA methylation; promoter methylation was also detected in tissues adjacent to breast cancer, and methylation in WWOX exon 1 distinguished breast cancer DNA from DNA of adjacent and normal tissue. Wwox restoration in lung cancer cells in vitro, and in  xenografts, suppressed growth. Wwox-deficient breast cancer cells, by treatment with  5′-Aza-2′-deoxycytidine to demethylate the WWOX promoter, was associated with effective induction of apoptosis in vitro and suppressed breast cancer xenograft growth in vivo, without affecting the Wwox-sufficient cells or causing persistent changes in global methylation levels.

 Tamoxifen is commonly used for treatment of  estrogen receptor alpha positive breast cancers, but de novo or acquired tamoxifen resistance occurs frequently. Wwox protein, which binds and retains Ap2α and γ transcription factors in the cytoplasm, may mediate tamoxifen sensitivity in vitro; Wwox loss initiates tamoxifen resistance through release of Ap2 factors to the nucleus where Ap2γ upregulates ErbB2 ( Her2) expression. Restoration of Wwox in tamoxifen-resistant breast cancer–derived cells restored tamoxifen sensitivity and abrogated ErbB2 expression.

Wwox expression was significantly (p = 0.013) reduced in tamoxifen resistant breast cancers, and a reliable marker of tamoxifen resistance, especially in premenopausal and stage 3 patients. Thus, the Wwox signaling pathway may provide new targets for therapeutic intervention in antiestrogen-resistant breast cancers.