It is now more than 20 years since the FRA16D common chromosomal fragile site was characterised and the WWOX gene spanning this site was identified. In this time, much information has been discovered about its contribution to disease; however, the normal biological role of WWOX is not yet clear. Experiments leading to the identification of the WWOX gene are recounted, revealing enigmatic relationships between the fragile site, its gene and the encoded protein. We also highlight research mainly using the genetically tractable model organism Drosophila melanogaster that has shed light on the integral role of WWOX in metabolism. In addition to this role, there are some particularly outstanding questions that remain regarding WWOX, its gene and its chromosomal location. This review, therefore, also aims to highlight two unanswered questions. Firstly, what is the biological relationship between the WWOX gene and the FRA16D common chromosomal fragile site that is located within one of its very large introns? Secondly, what is the actual substrate and product of the WWOX enzyme activity? It is likely that understanding the normal role of WWOX and its relationship to chromosomal fragility are necessary in order to understand how the perturbation of these normal roles results in disease.

• FRA16D
• common chromosomal fragile sites
• evolutionary conservation
• intragenic homozygous deletion
• megabase gene
• oxido-reductase specificity

Integration of human papilloma virus (HPV) in human genome is a random event, and fragile sites are one of the most susceptible sites for viral integrations. WWOX (WW-domain containing oxidoreductase) gene harbours the second most common fragile site, FRA16D, and can be an important candidate for HPV integration and cervical carcinogenesis. Our aim was to evaluate the potential role of WWOX in cervical carcinogenesis. Presence of HPV and its genotype was detected by PCR in normal cervix tissues and human cervical carcinoma. The expression of WWOX transcript and its protein was examined by RT-PCR, RNA in situ hybridization, and immunoblotting. Southern blotting and sequencing were used to determine the alternative transcripts of WWOX. Statistical analysis were performed by Mann Whitney U-test, Pearson correlation coefficient test at significance level of P value < 0.05. Prevalence of HPV was observed in cervicitis (40%), cervical intraepithelial neoplasia patients (50%), and invasive cervical carcinoma patients (89.6%). Clinicopathological findings suggested a correlation of reduced level of WWOX protein and progression of cervical carcinoma deciphering its role in tumorigenesis. Furthermore, we observed aberrant WWOX transcript having deleted exon 6-8 region in invasive cervical cancer tissues as well as normal cervix samples. More than 60% of cervical carcinoma samples showed reduced protein level with an increase in wild type transcript level suggesting the involvement of a negative regulator, pAck1 (activated Cdc42- associated kinase) which might ubiquitinate WWOX protein leading to its degradation. Also, nuclear retention of WWOX transcript in invasive cervical carcinoma tissues suggests its regulation at post-transcriptional level. Our findings suggest that WWOX acts as a tumor suppressor in cervical carcinoma and could act as a potential therapeutic target for the disease.

• Cervical cancer
• WWOX variants
• cervical carcinoma patients
• clinical- pathological parameters
• genotypes

• HCC
• LncRNA
• TSLD8
• WWOX

• WWOX
• aberrant transcripts
• colon and ovarian cancers
• germline mutation
• multiple primary tumors

We investigated the effect of micro‐RNA 24 (miR‐24) and WWOX on non‐small cell lung cancer (NSCLC) cell proliferation and migration in vitro and in vivo.

We performed bioinformatics analysis and 3′ untranslated region luciferase assay to investigate the direct target of miR‐24. Proliferation, apoptosis, and transwell invasion assays were employed to evaluate the effect of WWOX overexpression with pcDNA3‐WWOX and knocking down miR‐24 with miR‐24 small interfering RNA. Quantitative real‐time PCR, Western blot, and immunohistochemistry were also used to investigate miR‐24 and c‐Kit expression, and apoptosis and invasion‐related proteins. Finally, we constructed a tumor xenograft model in nude mice to confirm the effect of miR‐24 on NSCLC cell proliferation in vivo.

According to our experimental data, miR‐24 inhibition could induce apoptosis by activating caspase 3 and suppress the viability and proliferation of NSCLC cells in vitro and in vivo. MiR‐24 downregulation could reduce the invasive ability of NSCLC cells by downregulating MMP9. WWOX was identified as a functional target of miR‐24. WWOX overexpression generated the same effect with antagonizing miR‐24, while blocking WWOX counteracted the tumor suppressive effect caused by miR‐24 inhibition. MiR‐24 may function as an oncogene and play an important role in the cell growth and migration of NSCLC.

Our findings enhance understanding of the miR‐24 regulatory network and the molecular mechanism that underlies the oncogenesis and development of NSCLC. Suppressing the effect of miR‐24 on cancer cells using a miR‐24 inhibitor may be an attractive therapeutic strategy against NSCLC.

• Apoptosis
• MMP9
• WWOX
• caspase 3
• miR-24

The WW domain-containing oxidoreductase (WWOX) gene, frequently altered in breast cancer, encodes a tumor suppressor whose function is mediated through its interactions with cancer-related proteins, such as the pro-apoptotic protein p73α.

To better understand the involvement of WWOX in breast tumorigenesis, we performed a yeast two-hybrid screen and co-immunoprecipitation assays to identify novel partners of this protein. We characterized the vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) as a new regulator of WWOX. In breast cancer cells, VOPP1 sequestrates WWOX in lysosomes, impairs its ability to associate with p73α, and inhibits WWOX-dependent apoptosis. Overexpressed VOPP1 potentiates cellular transformation and enhances the growth of transplanted tumors in vivo. VOPP1 is overexpressed in breast tumors, especially in tumors that retain WWOX. Moreover, increased expression of VOPP1 is associated with reduced survival of patients with WWOX-positive, but not with WWOX-negative, tumors.

These findings emphasize the importance of the sequestration of WWOX by VOPP1 in addition to WWOX loss in breast tumors and define VOPP1 as a novel oncogene promoting breast carcinogenesis by inhibiting the anti-tumoral effect of WWOX.

The online version of this article (10.1186/s12915-018-0576-6) contains supplementary material, which is available to authorized users.

• Breast tumors
• VOPP1
• WWOX

The WWOX tumor suppressor gene is located at 16q23. 1–23.2, which covers the region of FRA16D—a common fragile sites. Deletions within the WWOX coding sequence are observed in up to 80% of breast cancer cases, which makes it one of the most common genetic alterations in this tumor type. The WWOX gene is known to play a role in breast cancer: increased expression of WWOX inhibits cell proliferation in suspension, reduces tumor growth rates in xenographic transplants, but also enhances cell migration through the basal membrane and contributes to morphological changes in 3D matrix-based cell cultures. The WWOX protein may act in several ways, as it has three functional domains—two WW domains, responsible for protein-protein interactions and an SDR domain (short dehydrogenase/reductase domain) which catalyzes conversions of low molecular weight ligands, most likely steroids. In epithelial cells, WWOX modulates gene transcription through interaction with p73, AP-2γ, and ERBB4 proteins. In steroid hormone-regulated tissues like mammary gland epithelium, the WWOX SDR domain acts as a steroid dehydrogenase. The relationship between WWOX and hormone receptors was shown in an animal model, where WWOX(C3H)+/–mice exhibited loss of both ER and PR receptors. Moreover, in breast cancer specimens, a positive correlation was observed between WWOX expression and ER status. On the other hand, decreased WWOX expression was associated with worse prognosis, namely higher relapse and mortality rates in BC patients. Recently, it was shown that genomic instability might be driven by the loss of WWOX expression. It was reported that WWOX plays role in DNA damage response (DDR) and DNA repair by regulating ATM activation through physical interaction. A genome caretaker function has also been proposed for WWOX, as it was found that WWOX sufficiency decreases homology directed repair (HDR) and supports non-homologous end-joining (NHEJ) repair as the dominant DSB repair pathway by Brca1-Wwox interaction. In breast cancer cells, WWOX was also found to modulate the expression of glycolysis pathway genes, through hypoxia-inducible transcription factor 1α (HIF1α) regulation. The paper presents the current state of knowledge regarding the WWOX tumor suppressor gene in breast cancer, as well as future research perspectives.

• WWOX
• breast cancer
• cancer progression
• carcinogenesis
• tumor suppressor

• WWOX gene
• hepatocellular carcinoma
• prognosis
• recurrence
• single nucleotide polymorphism

• WWOX
• apoptosis
• bubbling cell death
• cancer
• neurodegeneration
• phosphorylation

The long non-coding RNA PARTICLE (Gene PARTICL- ‘Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) partakes in triple helix (triplex) formation, is transiently elevated following low dose irradiation and regulates transcription of its neighbouring gene - Methionine adenosyltransferase 2A. It now emerges that PARTICLE triplex sites are predicted in many different genes across all human chromosomes. In silico analysis identified additional regions for PARTICLE triplexes at >1600 genomic locations. Multiple PARTICLE triplexes are clustered predominantly within the human and mouse tumor suppressor WW Domain Containing Oxidoreductase (WWOX) gene. Surface plasmon resonance diffraction and electrophoretic mobility shift assays were consistent with PARTICLE triplex formation within human WWOX with high resolution imaging demonstrating its enrichment at this locus on chromosome 16. PARTICLE knockdown and over-expression resulted in inverse changes in WWOX transcripts levels with siRNA interference eliminating PARTICLEs elevated transcription to irradiation. The evidence for a second functional site of PARTICLE triplex formation at WWOX suggests that PARTICLE may form triplex-mediated interactions at multiple positions in the human genome including remote loci. These findings provide a mechanistic explanation for the ability of lncRNAs to regulate the expression of numerous genes distributed across the genome.

• WW domain containing oxidoreductase gene
• common fragile site
• methylation
• monoclonal gammopathy of undetermined significance
• multiple myeloma

Epigenetic mechanisms influence molecular patterns important for the bone-metastatic process, and here we highlight the role of WW-domain containing oxidoreductase (Wwox). The tumor-suppressor Wwox lacks in almost all cancer types; the variable expression in osteosarcomas is related to lung-metastasis formation, and exogenous Wwox destabilizes HIF-1α (subunit of Hypoxia inducible Factor-1, HIF-1) affecting aerobic glycolysis. Our recent studies show critical functions of Wwox present in 1833-osteotropic clone, in the corresponding xenograft model, and in human bone metastasis from breast carcinoma. In hypoxic-bone metastatic cells, Wwox enhances HIF-1α stabilization, phosphorylation, and nuclear translocation. Consistently, in bone-metastasis specimens Wwox localizes in cytosolic/perinuclear area, while TAZ (transcriptional co-activator with PDZ-binding motif) and HIF-1α co-localize in nuclei, playing specific regulatory mechanisms: TAZ is a co-factor of HIF-1, and Wwox regulates HIF-1 activity by controlling HIF-1α. In vitro, DNA methylation affects Wwox-protein synthesis; hypoxia decreases Wwox-protein level; hepatocyte growth factor (HGF) phosphorylates Wwox driving its nuclear shuttle, and counteracting a Twist program important for the epithelial phenotype and metastasis colonization. In agreement, in 1833-xenograft mice under DNA-methyltransferase blockade with decitabine, Wwox increases in nuclei/cytosol counteracting bone metastasis with prolongation of the survival. However, Wwox seems relevant for the autophagic process which sustains metastasis, enhancing more Beclin-1 than p62 protein levels, and p62 accumulates under decitabine consistent with adaptability of metastasis to therapy. In conclusion, Wwox methylation as a bone-metastasis therapeutic target would depend on autophagy conditions, and epigenetic mechanisms regulating Wwox may influence the phenotype of bone metastasis.

• Wwox
• bone metastasis
• epigenetic reprogramming
• transcription factors

• FOR gene
• FRA16D
• WOX1
• WWOX-human gene
• Wwox- human/mouse protein
• Wwox- mouse gene
• fragile sites

• Animals
• Carcinogenesis/genetics
• Chromosome Fragile Sites/genetics
• Chromosome Fragile Sites/physiology
• Disease Models, Animal
• Humans
• Mice
• Mutation/genetics
• Mutation/physiology
• Oxidoreductases/genetics
• Oxidoreductases/physiology
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/physiology
• WW Domain-Containing Oxidoreductase

• R01 CA120516 NCI NIH HHS
• T32 OD010429 NIH HHS
• U01 CA154200 NCI NIH HHS
• 9T32 OD010429 NIH HHS

• WW domain-containing oxidoreductase
• breast cancer
• endocrine therapy
• tumorigenesis

• Animals
• Antineoplastic Agents, Hormonal/therapeutic use
• Biomarkers, Tumor/physiology
• Breast Neoplasms/etiology
• Breast Neoplasms/physiopathology
• Breast Neoplasms/therapy
• Carcinogenesis
• Disease Models, Animal
• Disease Progression
• Female
• Humans
• Mice
• Oxidoreductases/physiology
• Prognosis
• Selective Estrogen Receptor Modulators/therapeutic use
• Tamoxifen/therapeutic use
• Tumor Suppressor Proteins/physiology
• WW Domain-Containing Oxidoreductase

• Tumor suppressor
• apoptosis
• chemoresistance
• signal transduction
• transcription factor

• Chromosomal fragile site
• FRA16D
• altered metabolism
• oxidoreductase

• WWOX
• cancer
• common fragile sites
• very large genes

• Epstein–Barr virus
• Helicobactor pylori
• WW domain-containing oxidoreductase
• human T-cell leukemia virus
• infection-associated cancers

• common fragile site
• fra16d
• genomic instability
• tumor suppressor
• wwox
• atm
• hif1

• Animals
• Cell Transformation, Neoplastic/genetics
• Cell Transformation, Neoplastic/metabolism
• Cell Transformation, Neoplastic/pathology
• Chromosome Fragile Sites
• Genomic Instability
• Humans
• Neoplasms/genetics
• Neoplasms/metabolism
• Neoplasms/pathology
• Oxidoreductases/genetics
• Oxidoreductases/metabolism
• Signal Transduction
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/metabolism
• WW Domain-Containing Oxidoreductase

• WW domain-containing oxidoreductase
• glioma
• p53
• resistance

• Epilepsy
• FRA16D
• Lipid metabolism
• TGFβ
• WNT
• WWOX

• WW domain-containing oxidoreductase
• breast cancer
• hypermethylation
• lung cancer
• tumor suppressor gene

• FasL
• Glioma
• Immunosuppression
• TGF-β
• WWOX

AIM: To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721.

METHODS: Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis.

RESULTS: WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis.

CONCLUSION: Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.

• WWOX
• SMMC-7721
• Apoptosis
• Proliferation
• Hepatic carcinoma

• Base Sequence
• Breast Neoplasms/genetics
• Breast Neoplasms/pathology
• Female
• Humans
• Middle Aged
• Molecular Sequence Data
• Mutation
• Neoplasm Staging
• Oxidoreductases/genetics
• Polymerase Chain Reaction
• Tumor Suppressor Proteins/genetics
• WW Domain-Containing Oxidoreductase

• ctnna3
• lrtmm3
• alpha-t-catenin
• adherens junction
• nested gene

• Alzheimer Disease/genetics
• Animals
• Cell Adhesion/genetics
• Epigenesis, Genetic/physiology
• Gene Expression Regulation
• Humans
• Membrane Proteins/genetics
• Nerve Tissue Proteins/genetics
• Nested Genes/genetics
• Nested Genes/physiology
• alpha Catenin/genetics
• alpha Catenin/physiology

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.

• ATR checkpoint pathway
• DNA secondary structure
• RET/PTC rearrangement
• cancer-specific chromosomal translocation
• environmental mutagen
• fragile site
• stalled replication fork.

• internal rearrangements
• in-frame deletion
• cancer

• 5' Untranslated Regions
• Amino Acid Sequence
• Animals
• Antigens, Neoplasm/genetics
• Antigens, Neoplasm/immunology
• Autoantigens/genetics
• Autoantigens/immunology
• Base Sequence
• Cell Line, Tumor
• Gene Order
• Humans
• Mice
• Molecular Sequence Data
• Neoplasms/genetics
• Neoplasms/immunology
• Polymerase Chain Reaction
• RNA, Messenger/genetics
• Sequence Homology, Amino Acid

• R01 CA128920 NCI NIH HHS
• P50 CA062924 NCI NIH HHS
• CA134292 NCI NIH HHS
• CA62924 NCI NIH HHS
• P01 CA134292 NCI NIH HHS
• CA128920 NCI NIH HHS

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu–Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the ‘non-classical tumour suppressor’ behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.

• Cell Line, Tumor
• CpG Islands
• DNA Methylation
• Disease Progression
• Humans
• Loss of Heterozygosity
• Neoplasm Staging
• Oxidoreductases/genetics
• Oxidoreductases/physiology
• Stomach Neoplasms/genetics
• Stomach Neoplasms/pathology
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/physiology
• WW Domain-Containing Oxidoreductase

We have previously shown that WW domain-containing oxidoreductase (WWOX) has tumour-suppressing effects and that its expression is frequently reduced in pancreatic carcinoma. In this study, we examined WWOX expression in intraductal papillary mucinous neoplasm of the pancreas (IPMN) to assess the function of WWOX in pancreatic duct tumourigenesis using immunohistochemistry and methylation-specific polymerase chain reaction analysis. Among 41 IPMNs including intraductal papillary mucinous adenomas (IPMAs) and intraductal papillary mucinous carcinomas (IPMCs), loss or reduced WWOX immunoreactivity was detected in 3 (15%) of 20 IPMAs and 17 (81%) of 21 IPMCs. In addition, hypermethylation of the WWOX regulatory site was detected in 1 (33%) of 3 WWOX(−) IPMAs and 9 (53%) of 17 WWOX(−) IPMCs, suggesting that hypermethylation may possibly be important in the suppression of WWOX expression. Reduction of WWOX expression was significantly correlated with a higher Ki-67 labelling index but was not correlated with the ssDNA apoptotic body index. Interestingly, decreased WWOX expression was significantly correlated with loss of SMAD4 expression in these IPMNs. The results indicate that downregulation of WWOX expression by the WWOX regulatory region hypermethylation is critical for transformation of pancreatic duct.

• wwox
• hepatocellular carcinoma
• jnk
• apoptosis

• Adult
• Aged
• Aged, 80 and over
• Apoptosis/drug effects
• Carcinoma, Hepatocellular/metabolism
• Carcinoma, Hepatocellular/pathology
• Cell Line, Tumor
• Cell Proliferation/drug effects
• Chromosomes, Human, Pair 16/genetics
• Enzyme Inhibitors/pharmacology
• Female
• Gene Expression Regulation
• Humans
• Liver Neoplasms/metabolism
• Liver Neoplasms/pathology
• Loss of Heterozygosity/genetics
• Male
• Middle Aged
• Mitogen-Activated Protein Kinase 8/antagonists & inhibitors
• Mitogen-Activated Protein Kinase 8/metabolism
• Oxidoreductases/genetics
• Oxidoreductases/metabolism
• RNA, Messenger/metabolism
• Staurosporine/pharmacology
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/metabolism
• WW Domain-Containing Oxidoreductase

• CA100882 NCI NIH HHS
• R01 CA100882 NCI NIH HHS
• R56 CA100882 NCI NIH HHS
• CA82862 NCI NIH HHS
• K08 CA082862 NCI NIH HHS

• Apoptosis
• Carcinoma, Pancreatic Ductal/genetics
• Caspases/physiology
• Cell Line, Tumor
• Chromosome Fragile Sites
• CpG Islands
• DNA Methylation
• Humans
• Oxidoreductases/genetics
• Oxidoreductases/physiology
• Pancreatic Neoplasms/chemistry
• Pancreatic Neoplasms/genetics
• Pancreatic Neoplasms/mortality
• Pancreatic Neoplasms/prevention & control
• Smad4 Protein/analysis
• Smad4 Protein/genetics
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/physiology
• WW Domain-Containing Oxidoreductase

• R01 CA120516 NCI NIH HHS
• CA120516 NCI NIH HHS

• Animals
• Cells, Cultured
• Chromosome Fragile Sites
• Genomic Instability
• Humans
• MicroRNAs/genetics
• Neoplasms/genetics

• CA 43222 NCI NIH HHS