WWOX,large common fragile site genes,and cancer

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.     

Common fragile genes

Common chromosome fragile sites show susceptibility to DNA damage, leading to alterations that contribute to cancer development. The cloning and characterization of fragile sites have demonstrated that fragile sites are associated with genes that relate to tumorigenesis. Identification of the basis of instability at fragile sites and the related genes provides an entree to understanding of important aspects of chromosomal instability, a prominent feature of neoplastic genomes. FHIT/FRA3B and WWOX/FRA16D, the most sensitive common fragile genes in the human genome, function as tumor suppressor genes. The common features of these two common fragile genes are summarized, and suggest clues to understanding the relation between genomic instability and tumor biology.     

WWOX, a new potential tumor suppressor gene

BACKGROUND: WWOX (WW domain-containing oxidoreductase) gene, located on chromosome 16q 23.3-24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various cancers: breast, ovarian, prostate, esophageal, lung, pancreatic, gastric and hepatic. The aim of this study was to describe (i) putative protein interactions of WWOX (ii) the molecular mechanisms of tumor suppressor activity (iii) present an overview of WWOX in relation to nervous system and breast, prostate and ovarian cancers. METHODS AND RESULTS: WWOX expression is up-regulated in endocrine organs indicating its importance in these tissues. In many cancers WWOX expression is down-regulated and low WWOX expression is related to poor prognosis. CONCLUSION: All the evidence suggest that WWOX can be considered as a new tumor suppressor gene and target for gene therapy due to the association of high WWOX expression with improved disease free survival.     

129 The molecular mechanism of breakage at fragile site FRA16D

Replication stress induces physical breakage at discrete loci in chromosomes, which can be visualized on a metaphase chromosome spread. These common fragile sites (CFS) are conserved across species and are hotspots for sister chromatid recombination, viral integration, rearrangements, translocations, and deletions (Glover et al 2005). Despite multiple theories, the molecular mechanisms of CFS expression and genomic instability are still not well understood. The fragile site FRA16D is of special interest because it is the second most highly expressed fragile site and is located within the WWOX tumor suppressor gene. Previous data identified a polymorphic AT repeat within a FRA16D subregion called F1 that causes chromosome fragility and replication fork stalling in a yeast model (Zhang and Freudenreich 2007). Recently, we have found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeat in the context of F1 forms a secondary structure, making the region more vulnerable to breakage.     

WWOX at the crossroads of cancer,metabolic syndrome related traits and CNS pathologies

WWOX was cloned as a putative tumor suppressor gene mapping to chromosomal fragile site FRA16D. Deletions affecting WWOX accompanied by loss of expression are frequent in various epithelial cancers. Translocations and deletions affecting WWOX are also common in multiple myeloma and are associated with worse prognosis. Metanalysis of gene expression datasets demonstrates that low WWOX expression is significantly associated with shorter relapse-free survival in ovarian and breast cancer patients. Although somatic mutations affecting WWOX are not frequent, analysis of TCGA tumor datasets led to identifying 44 novel mutations in various tumor types. The highest frequencies of mutations were found in head and neck cancers and uterine and gastric adenocarcinomas.     

WWOX in biological control and tumorigenesis

The WW domain-containing oxidoreductase (WWOX) gene is located at 16q23.1-16q23.2, a region that spans the second most common human fragile site, FRA16D. The WWOX protein contains two N-terminal WW domains and a central short chain oxidoreductase-like domain. In the last few years, considerable amount of data have shown inactivation of WWOX in a variety of human malignancies. Moreover, interacting partners have been identified biochemically that define, at least in part, the molecular mechanism of WWOX action. Recently, we demonstrated that targeted deletion of the Wwox gene in the mouse led to increased incidence of spontaneous and chemically induced tumor formation, thereby providing the first in vivo evidence that WWOX is a bona fide tumor suppressor. This review focuses on the most recent progress in understanding WWOX function as a tumor suppressor.     

DMD and IL1RAPL1: two large adjacent genes localized within a common fragile site (FRAXC) have reduced expression in cultured brain tumors

Common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. Spanning the center of the two most frequently expressed CFS regions, FRA3B (3p14.3) and FRA16D (16q23.2), are the 1.5 Mb FHIT gene and the 1.0 Mb WWOX gene. These genes are frequently deleted and/or altered in many different cancers. Both FHIT and WWOX have been demonstrated to function as tumor suppressors, both in vitro and in vivo. A number of other large CFS genes have been identified and are also frequently inactivated in multiple cancers. Based on these data, several additional very large genes were tested to determine if they were derived from within CFS regions, but DCC and RAD51L1 were not. However, the 2.0 Mb DMD gene and its immediately distal neighbor, the 1.8 Mb IL1RAPL1 gene are CFS genes contained within the FRAXC CFS region (Xp21.2-->p21.1). They are abundantly expressed in normal brain but were dramatically underexpressed in every brain tumor cell line and xenograft (derived from an intracranial model of glioblastoma multiforme) examined. We studied the expression of eleven other large CFS genes in the same panel of brain tumor cell lines and xenografts and found reduced expression of multiple large CFS genes in these samples. In this report we show that there is selective loss of specific large CFS genes in different cancers that does not appear to be mediated by the relative instability within different CFS regions. Further, the inactivation of multiple large CFS genes in xenografts and brain tumor cell lines may help to explain why this type of cancer is highly aggressive and associated with a poor clinical outcome.     

WWOX protein expression in normal human tissues

WWOX is a putative tumor suppressor gene that spans approximately a 1 Mb genomic region and is the site for the second most common chromosomal fragile site, FRA16D at 16q23. Various studies have focused on the expression of WWOX in human cancer mostly at the RNA level, but little is known about the normal pattern of WWOX protein expression in non-neoplastic tissues. In this study, a comprehensive analysis of WWOX protein expression in normal tissues was performed by means of immunohistochemistry utilizing a very specific anti-WWOX polyclonal antibody. We analyzed tissue cores of human samples representing more than 30 organs, using various tissue microarray (TMA) slides. Due to the potential role of WWOX in sex-steroid metabolism, whole sections from hormonally regulated organs like breast, ovaries, testes and prostate were also analyzed. The results from our study indicate that WWOX is preferentially highly expressed in secretory epithelial cells of reproductive, endocrine and exocrine organs, as well as in ductal epithelial cells from specific segments of the urinary system. Interestingly, we also observed significant WWOX protein expression in various cell types of neural origin including neurons, ependymal cells and astrocytes. No expression of WWOX was detected in adipose, connective, and lymphoid tissues, myelinized structures and blood vessels. By better defining the topographic distribution of WWOX in normal tissues this study provides some insight on the potential physiological role of this novel protein.     

Components of DNA damage checkpoint pathway regulate UV exposure-dependent alterations of gene expression of FHIT and WWOX at chromosome fragile sites

Common chromosome fragile sites are highly recombinogenic and susceptible to deletions during the development of environmental carcinogen-induced epithelial tumors. Previous studies showed that not only genetic but also epigenetic alterations in cancerous cells are involved in inactivation of the genes FHIT and WWOX at chromosome fragile sites, reported to be potential tumor suppressor genes. Here we investigated the effect of UV light on the gene expression. After exposure to UV, the mRNA and protein of the two genes in murine embryonic fibroblasts (MEF) were unstable, apparently at the G1-S phase of the cell cycle, which was consistent with nuclear run-on assay. A study of MEFs synchronized via a double thymidine block indicated that, after the exposure, the expression of Fhit and Wwox was reduced in E2f-1-deficient cells and markedly in wild-type cells, whereas the reduction was partially inhibited in Trp53-deficient cells; cells at the S phase seemed to be sensitive to exogenous FHIT, suggesting a role of the checkpoint at the G1-S phase in the stability of gene expression and a possible involvement of FHIT function at the S phase. The transfection experiment showed that the UV-induced decrease in expression was partially inhibited by transfection of kinase-dead Atr (ataxia telangiectasia mutated and Rad3 related), which is a sensor of UV-induced damage. Taken together, the present study showed that UV-induced alterations of the fragile site gene expression are involved at least partially in the checkpoint function, suggesting the role in the process of carcinogenesis after exposure to UV.     

A mouse model of the fragile gene FHIT: From carcinogenesis to gene therapy and cancer prevention

Mouse models of tumor suppressors are increasingly useful to investigate biomedical aspects of cancer genetics. Some tumor suppressor genes are located at common fragile sites that are specific chromosomal regions highly susceptible to DNA lesions. The tumor suppressor gene FHIT, at the fragile site FRA3B, is the first fragile gene with a developed and characterized mouse knockout model. The human gene FHIT is frequently deleted in cancers and cancer cell lines of many epithelial tissues, and Fhit protein is absent or reduced in most cancers. The mouse Fhit ortholog is also located at a common fragile site, Fra14A2 on murine chromosome 14, and sustains homozygous deletions in murine cancer cell lines. The Fhit knockout mouse is, therefore, an adequate model to study human FHIT function. To establish an animal model and to explore the role of FHIT in tumorigenesis, we have developed a mouse strain carrying one or two inactivated Fhit alleles. Insights into Fhit mouse genetics that have emerged in the last 7 years, and are reviewed in the present article, allowed for development of new tools in carcinogenesis and gene delivery studies.     

Human chromosome fragility

Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites. From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.     

WWOX oxidoreductase--substrate and enzymatic characterization

WWOX is a tumour suppressor gene that spans the common fragile site FRA16D. Analysis of the WWOX expression pattern in normal human tissues showed the highest expression in testis, prostate, and ovary. Its altered expression has been demonstrated in different tissues and tumour types. The WWOX gene encodes a 414-amino acids protein, which is the first discovered protein with a short-chain dehydrogenase/reductase (SDR) central domain and two WW domains at the NH2 terminus. Due to its potential role in sex-steroid metabolism, using two bacterial expression systems, we have cloned WWOX fusion proteins showing oxidoreductase activity in a crude extract, defined a course of enzymatic reactions for selected steroid substrates, and determined related Km values. Our results show that the SDR domain of the WWOX protein has dehydrogenase activity and is reactive both in the presence of NAD+ and NADP+ for all examined steroid substrates. On the other hand, with the same substrates and reduced cofactors (NADH and NADPH) reduction activity was not observed.     

Conditional inactivation of the mouse Wwox tumor suppressor gene recapitulates the null phenotype

WW domain‐containing oxidoreductase (WWOX) is highly conserved in both human and murine. WWOX spans the second most common human chromosomal fragile site, FRA16D, and is commonly inactivated in multiple human cancers. Modeling WWOX inactivation in mice revealed a complex phenotype including postnatal lethality, defects in bone metabolism and steroidogenesis and tumor suppressor function resulting in osteosarcomas. For better understanding of WWOX roles in different tissues at distinct stages of development and in pathological conditions, Wwox conditional knockout mice were generated in which loxp sites flank exon 1 in the Wwox allele. We demonstrated that Cre‐mediated recombination using EIIA‐Cre, a Cre line expressed in germline, results in postnatal lethality by age of 3 weeks and decreased bone mineralization resembling total ablation of WWOX as in conventional null mice. This animal model will be useful to study distinct roles of WWOX in multiple tissues at different ages. J. Cell. Physiol. 228: 1377–1382, 2013. © 2012 Wiley Periodicals, Inc.     

Common Chromosomal Fragile Site Gene WWOX in Metabolic Disorders and Tumors

WWOX, a gene that spans the second most common chromosomal fragile site (FRA16D), often exhibits homozygous deletions and translocation breakpoints under multiple cellular stresses induced by extrinsic or intrinsic factors, such as hypoxia, UV, and DNA damage regents. Loss of WWOX is closely related to genomic instability, tumorigenesis, cancer progression and therapy resistance. WWOX heterozygous knockout mice show an increased incidence of spontaneous or induced tumors. WWOX can interact via the WW domain with proteins that possess proline PPxY motifs and is involved in a variety of cellular processes. Accumulating evidence has shown that WWOX that contains a short-chain dehydrogenase/reductase (SDR) domain is involved in steroid metabolism and bone development. Reduced or lost expression of WWOX will lead to development of metabolic disease. In this review, we focus on the roles of WWOX in metabolic disorders and tumors.     

The fragile site WWOX gene and the developing brain

WWOX was cloned as a tumor suppressor gene mapping to chromosomal fragile site FRA16D. Loss of WWOX is closely related to tumorigenesis, cancer progression, and therapy resistance. Recent studies demonstrate the growing role of WWOX gene in other human pathologies such as metabolic and nervous system-related conditions. The neurologic phenotype of WWOX mutation includes seizures, ataxia, developmental delay, and spasticity of variable severity. WWOX is a ubiquitous protein with high expression in many tissues including brain, cerebellum, brain stem, and spinal cord. WWOX is highly expressed in different brain regions during murine fetal development and remained unchanged in the cortex and the corpus callosum in adult mice. The mechanism or the putative role of WWOX in the nervous system is still unclear but may include abnormal signaling protein, disruption of neuronal pathways, neuronal differentiation, mitochondrial dysfunction, or apoptosis. Homozygous mutations affecting WWOX in humans are likely to be more described in the future using exome sequencing. The described findings highlight that WWOX plays a critical role in normal central nervous system development and disease.The aim of this review is to summarize the roles of WWOX in the developing brain.