WW Domain-containing Oxidoreductase Research Articles

Zfra Overrides WWOX in Suppressing the Progression of Neurodegeneration.

We reported that a 31-amino-acid Zfra protein (zinc finger-like protein that regulates apoptosis) blocks neurodegeneration and cancer growth. Zfra binds WW domain-containing oxidoreductase (WWOX) to both N- and C-termini, which leads to accelerated WWOX degradation. WWOX limits the progression of neurodegeneration such as Alzheimer's disease (AD) by binding tau and tau-hyperphosphorylating enzymes. Similarly, Zfra binds many protein targets and accelerates their degradation independently of ubiquitination. Furthermore, Zfra4-10 peptide strongly prevents the progression of AD-like symptoms in triple-transgenic (3xTg) mice during aging. Zfra4-10 peptide restores memory loss in 9-month-old 3xTg mice by blocking the aggregation of a protein cascade, including TPC6AΔ, TIAF1, and SH3GLB2, by causing aggregation of tau and amyloid β. Zfra4-10 also suppresses inflammatory NF-κB activation. Zfra-activated Hyal-2+ CD3- CD19- Z cells in the spleen, via Hyal-2/WWOX/Smad4 signaling, are potent in cancer suppression. In this perspective review, we provide mechanistic insights regarding how Zfra overrides WWOX to induce cancer suppression and retard AD progression via Z cells.

Unveiling the relationship between WWOX and BRCA1 in mammary tumorigenicity and in DNA repair pathway selection

Breast cancer is the leading cause of cancer-related deaths in women worldwide, with the basal-like or triple-negative breast cancer (TNBC) subtype being particularly aggressive and challenging to treat. Understanding the molecular mechanisms driving the development and progression of TNBC is essential. We previously showed that WW domain-containing oxidoreductase (WWOX) is commonly inactivated in TNBC and is implicated in the DNA damage response (DDR) through ATM and ATR activation. In this study, we investigated the interplay between WWOX and BRCA1, both frequently inactivated in TNBC, on mammary tumor development and on DNA double-strand break (DSB) repair choice. We generated and characterized a transgenic mouse model (K14-Cre;Brca1fl/fl;Wwoxfl/fl) and observed that mice lacking both WWOX and BRCA1 developed basal-like mammary tumors and exhibited a decrease in 53BP1 foci and an increase in RAD51 foci, suggesting impaired DSB repair. We examined human TNBC cell lines harboring wild-type and mutant BRCA1 and found that WWOX expression promoted NHEJ repair in cells with wild-type BRCA1. Our findings suggest that WWOX and BRCA1 play an important role in DSB repair pathway choice in mammary epithelial cells, underscoring their functional interaction and significance in breast carcinogenesis.

WWOX promotes osteosarcoma development via upregulation of Myc

Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.

Mechanistic Investigation of WWOX Function in NF-kB-Induced Skin Inflammation in Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperproliferation, aberrant differentiation of keratinocytes, and dysregulated immune responses. WW domain-containing oxidoreductase (WWOX) is a non-classical tumor suppressor gene that regulates multiple cellular processes, including proliferation, apoptosis, and migration. This study aimed to explore the possible role of WWOX in the pathogenesis of psoriasis. Immunohistochemical analysis showed that the expression of WWOX was increased in epidermal keratinocytes of both human psoriatic lesions and imiquimod-induced mice psoriatic model. Immortalized human epidermal keratinocytes were transduced with a recombinant adenovirus expressing microRNA specific for WWOX to downregulate its expression. Inflammatory responses were detected using Western blotting, real-time quantitative reverse transcription polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay. In human epidermal keratinocytes, WWOX knockdown reduced nuclear factor-kappa B signaling and levels of proinflammatory cytokines induced by polyinosinic: polycytidylic acid [(poly(I:C)] in vitro. Furthermore, calcium chelator and protein kinase C (PKC) inhibitors significantly reduced poly(I:C)-induced inflammatory reactions. WWOX plays a role in the inflammatory reaction of epidermal keratinocytes by regulating calcium and PKC signaling. Targeting WWOX could be a novel therapeutic approach for psoriasis in the future.

Abstract 17443: Wwox is a Critical Regulator of Pulmonary Artery Smooth Muscle Cell Homeostasis

Introduction: Pulmonary artery smooth muscle (PASMC) proliferation and apoptosis resistance are characteristic of pulmonary arterial hypertension (PAH). Expression loss of the tumor suppressor WW-domain containing oxidoreductase (WWOX) promotes cell survival and metabolic reprograming in cancer. We hypothesized that loss of WWOX expression in PASMC promotes pulmonary vascular remodeling by similar mechanisms. Methods and Results: mRNA and protein levels of WWOX are decreased in PASMC from patients with PAH, and in rat models of pulmonary hypertension (Sugen/ hypoxia and monocrotaline). PASMC-specific conditional Wwox knockout mice (WWOX-SMC KO) were treated with tamoxifen or vehicle for 5 days and exposed to 10% hypoxia for 4 weeks. WWOX-SMC KO animals developed worse hypoxia induced PH with increased right ventricular systolic pressure, right ventricular hypertrophy and pulmonary vascular remodeling, compared to control animals. WWOX loss via siRNA decreased mRNA and protein levels of BMPRII, upregulated HIF 1alpha expression, increased hPASMC proliferation and migration. RNAseq revealed dysregulation in multiple pathways in WWOX-silenced hPASMCs, including cell-cycle regulation, TGF-β, mTOR and STAT3 signaling. Loss of WWOX showed decreased basal and maximal respiration, ATP production, reduced glycolytic capacity (40% in relation to control) and decreased mRNA expression of glycolytic enzymes (HK2, PFK1, PKM), generating 58% less lactate, and reduced mRNA expression of lactate dehydrogenase A. These cells exhibited increased mRNA and protein expression of glutaminase-1 and citrate lyase and had higher dependency on fatty acid oxidation and glutaminolysis compared to glucose, suggesting that these pathways are used when WWOX is suppressed. We also found mitochondrial hyperpolarization (JC-1 assay), decreased Sirtuin 3 expression, increased expression of acetylated manganese-superoxide dismutase, high production of reactive oxygen species and mitochondrial fission, indicating that loss of WWOX contributes to mitochondrial dysfunction. Conclusion: Loss of WWOX promotes cell cycle progression, proliferation, and metabolic reprogramming in PASMCs. Further studies are ongoing to better understand these effects.

#6143 FAMILIAL LECITHIN: CHOLESTEROL ACYLTRANSFERASE (LCAT) DEFICIENCY AS A CAUSE OF CHRONIC KIDNEY DISEASE – A CASE REPORT

Abstract Background and Aims Genetic causes of chronic kidney disease are becoming more recognized. Familial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disorder caused by loss of function mutations in LCAT gene. Patients present with abnormal lipid profile characterized with markedly reduced HDL-C, corneal opacification, anemia, and renal disease, which eventually progresses to kidney failure. Several studies reported genetic variants in LCAT gene that are associated with FLD, and others in certain apolipoprotein genes that act as risk factors for the disease. Incomplete form of FLD, caused by certain mutation, leads to fish-eye disease characterized by progressive corneal opacification. FLD was described in studies from Europe, Latin and North America, Australia and Japan. In Jordan, genetic and clinical studies in FLD patients are absent. Method We present a 36-year-old female who presented with nephrotic-range proteinuria, high serum creatinine, hypertension and corneal opacity. Further examination showed a severely reduced HDL level, an increased triglycerides level, anemia and mild thrombocytopenia. Patient's laboratory values at presentation are shown in Table 1. Her blood film showed normocytic and normochromic red blood cells, anisopoikilocytosis and target cells. Light and electron microscopy examination of the kidney biopsy revealed intramembranous, subendothelial and mesangial lipid deposition and vacuolization. In addition, the patient was found to have a family history of corneal opacity and chronic kidney disease. Therefore, FLD was suspected. Whole exome sequencing was employed to identify FLD variants. Results The patient was found to be homozygous at 154+5delG in LCAT gene, heterozygous at 388T>C in apolipoprotein E gene (APOE) and homozygous at 1114G>C in WW domain-containing oxidoreductase gene (WWOX). The patient's chronic kidney disease was managed supportively with low salt diet, moderate protein intake, statins, angiotensin receptor blockers and blood pressure control. After one year of follow up, her serum creatinine is 220 μmol/l and estimated proteinuria is 1.2 gm/24 hrs. Conclusion FLD should be suspected in patients who present with kidney disease associated with significantly low HDL cholesterol and corneal opacity. Family members should also be screened for kidney disease and genetic mutation in LCAT gene. To date, treatment is mainly supportive and enzyme replacement therapy is not yet widely available.

WWOX Polymorphisms as Predictors of the Biochemical Recurrence of Localized Prostate Cancer after Radical Prostatectomy.

The downregulation of WW domain-containing oxidoreductase (WWOX), a tumor suppressor gene, is associated with the tumorigenesis and poor prognosis of various cancers. In this study, we investigated the associations between the polymorphisms of WWOX, clinicopathologic features of prostate cancer (PCa), and risk of postoperative biochemical recurrence (BCR). We evaluated the effects of five single-nucleotide polymorphisms (SNPs) of WWOX on the clinicopathologic features of 578 patients with PCa. The risk of postoperative BCR was 2.053-fold higher in patients carrying at least one "A" allele in WWOX rs12918952 than in those with homozygous G/G. Furthermore, patients with at least one polymorphic "T" allele in WWOX rs11545028 had an elevated (1.504-fold) risk of PCa with seminal vesicle invasion. In patients with postoperative BCR, the risks of an advanced Gleason grade and clinical metastasis were 3.317- and 5.259-fold higher in patients carrying at least one "G" allele in WWOX rs3764340 than in other patients. Our findings indicate the WWOX SNPs are significantly associated with highly aggressive pathologic features of PCa and an elevated risk of post-RP biochemical recurrence.

Population genetics of marmosets in Asian primate research centers and loci associated with epileptic risk revealed by whole-genome sequencing.

The common marmoset ( Callithrix jacchus) has emerged as a valuable nonhuman primate model in biomedical research with the recent release of high-quality reference genome assemblies. Epileptic marmosets have been independently reported in two Asian primate research centers. Nevertheless, the population genetics within these primate centers and the specific genetic variants associated with epilepsy in marmosets have not yet been elucidated. Here, we characterized the genetic relationships and risk variants for epilepsy in 41 samples from two epileptic marmoset pedigrees using whole-genome sequencing. We identified 14 558 184 single nucleotide polymorphisms (SNPs) from the 41 samples and found higher chimerism levels in blood samples than in fingernail samples. Genetic analysis showed fourth-degree of relatedness among marmosets at the primate centers. In addition, SNP and copy number variation (CNV) analyses suggested that the WW domain-containing oxidoreductase ( WWOX) and Tyrosine-protein phosphatase nonreceptor type 21 ( PTPN21) genes may be associated with epilepsy in marmosets. Notably, KCTD18-like gene deletion was more common in epileptic marmosets than control marmosets. This study provides valuable population genomic resources for marmosets in two Asian primate centers. Genetic analyses identified a reasonable breeding strategy for genetic diversity maintenance in the two centers, while the case-control study revealed potential risk genes/variants associated with epilepsy in marmosets.

WW domain-containing oxidoreductase-related epileptic encephalopathy in Two Omani Children.

Background: Autosomal recessive developmental and epileptic encephalopathy type 28 (DEE28) is a rare genetic disorder that affects children in the early months of life. It is proved to be caused by a pathogenic variance in WW domain-containing oxidoreductase (WWOX) gene. Case presentation: Here, we report a 5-year-old male patient with autosomal recessive DEE28. Whole exome sequencing (WES) test was conducted and resulted in a pathogenic result on WWOX gene pathogenic variant. To our knowledge, these are the first cases reported in Oman. Conclusion: For patients with DEE28, it is essential to take the full family history and genetic workup to assist in the diagnosis. In the future, gene therapy – which is currently being investigated - may help those patients to have a good quality of life and improve the prognosis of the disease.

Loss of tumor suppressor WWOX accelerates pancreatic cancer development through promotion of TGFβ/BMP2 signaling

Pancreatic cancer is one of the most lethal cancers, owing to its late diagnosis and resistance to chemotherapy. The tumor suppressor WW domain-containing oxidoreductase (WWOX), one of the most active fragile sites in the human genome (FRA16D), is commonly altered in pancreatic cancer. However, the direct contribution of WWOX loss to pancreatic cancer development and progression remains largely unknown. Here, we report that combined conditional deletion of Wwox and activation of KRasG12D in Ptf1a-CreER-expressing mice results in accelerated formation of precursor lesions and pancreatic carcinoma. At the molecular level, we found that WWOX physically interacts with SMAD3 and BMP2, which are known activators of the TGF-β signaling pathway. In the absence of WWOX, TGFβ/BMPs signaling was enhanced, leading to increased macrophage infiltration and enhanced cancer stemness. Finally, overexpression of WWOX in patient-derived xenografts led to diminished aggressiveness both in vitro and in vivo. Overall, our findings reveal an essential role of WWOX in pancreatic cancer development and progression and underscore its role as a bona fide tumor suppressor.

Polymorphisms in the WWOX gene are associated with brain glucose metabolism and influence cerebrospinal fluid p‐tau181 levels

AbstractBackgroundGenome‐wide association studies (GWAS) studies can be a powerful tool for identifying new single nucleotide polymorphisms (SNPs) associated with multifactorial diseases. Previous GWAS have identified more than 20 genes as potential factors of genetic susceptibility in Alzheimer’s disease (AD). One of them is the protein coding gene WW Domain Containing Oxidoreductase (WWOX), which is highly expressed in mature astrocytes and neurons. Here, we investigated the association between WWOX SNPs, 18F‐FDG PET neuroimaging and cerebrospinal fluid (CSF) levels of tau phosphorylated at threonine 181 (p‐tau181) in elderly individuals from the Alzheimer’s Disease Neuroimaging Initiative (ADNI).MethodsData was acquired from 537 participants (CU= 195; MCI= 314; AD= 28) of the ADNI database. The brain glucose metabolism was estimated using the standard uptake value ratio (SUVr, cerebellum as reference) of FDG‐PET. The presence of the SNPs were integrated to FDG PET images using voxel‐wise generalized linear regression models adjusted for age, gender, diagnostic status, and years of education (RMINC package). Through the Wilcoxon test we determined if the SNPs presence influenced p‐tau181 levels in CSF. All statistical analyzes were performed in the R statistical computing software (version 4.1.2) (significance level of p < 0.05).ResultsSixty‐seven WWOX SNPs were statistically associated with 18F‐FDG PET signal. Voxel‐wise analyses demonstrated that the top ten WWOX SNPs with the highest number of voxels altered in the FDG‐PET images were associated with cortical hypermetabolism (Figure 1, top three). Thirteen WWOX SNPs were significantly associated with lower p‐tau181 concentration in the CSF, including rs7193539 (p = 0.0004), rs7206890 (p = 0.0061), and rs11859281 (p = 0.0009), compared to the reference genotype.ConclusionSeveral SNPs of the WWOX gene were significantly correlated to brain metabolism. The presence of the SNPs can influence p‐tau181 levels, suggesting a role of WWOX in AD, as reported in other previous studies. Our findings suggest a protective effect of these WWOX gene variants. Additional studies are needed to better understand the biological implications of our findings.

Abstract 15153: Inhibition of the Tumor Suppressor Wwox Expression Alters Mitochondrial Dynamics in PASMC

Introduction: Pulmonary arterial hypertension (PAH) contributes to the morbidity and mortality of several patients with different pulmonary and cardiac diseases. Among other factors, the pathogenesis of PAH involves exaggerated pulmonary artery smoot muscle cell (PASMC) hyperproliferation, resistance to apoptosis and energy metabolism shift associated with alterations in mitochondrial metabolism and dynamics. The WW domain-containing oxidoreductase gene ( WWOX ) is a tumor suppressor gene that has the ability to interact with a large number of proteins, exerting its activity through a wide variety of molecular actions in specific cells. WWOX loss affects cell proliferation, motility, and deregulates expression of genes involved in cell cycle regulation, metabolism, and DNA damage in a variety of cancers. Considering that a similar phenotype is observed in cancer cells and PAH, we sought to investigate whether the loss of WWOX in PASMC is associated abnormalities of mitochondrial metabolism and dynamics in PAH. Methods and Results: In PASMC, inhibition of WWOX expression by short-interfering RNA (siRNA) decreased oxygen consumption by 30% when compared with siRNA control. Dysregulation of mitochondrial bioenergetics in WWOX-silenced PASMC was attributed to modulation of the oxidative state of mitochondrial electron transport chain proteins, showing that succinate dehydrogenase (SDH)(complex II) expression decreased by 15% in relation to siRNA control. JC-1 and mitotraker red CMXROS staining demonstrated that WWOX- silenced PASMC had hyperpolarized and fragmented mitochondria that generated more reactive oxygen species than the control. Changes in mitochondrial dynamics were detected with increased phosphorylation levels of the fission protein, dynamin-related protein 1 (DRP-1) and, decreased Mitofusin 2 and 1 protein levels, positive regulators of mitochondrial fusion. Conclusion: Loss of WWOX expression causes changes in PASMC mitochondrial metabolism and dynamics stimulating these cells to use different resources as a way to reprogram their metabolism and ensure their survival. These alterations may contribute, for example, to the stimulation of vascular remodeling, which consequently promote the pathogenesis of PAH.

WWOX Modulates ROS-Dependent Senescence in Bladder Cancer.

The tumor-suppressor gene, WW domain-containing oxidoreductase (WWOX), has been found to be lost in various types of cancers. ROS result as a tightly regulated signaling process for the induction of cell senescence. The aim of this study was to investigate the role of WWOX in the regulation of ROS and cell senescence, which is intriguing in terms of the possible mechanism of WWOX contributing to bladder cancer. In this study, we used the AY-27 rat bladder tumor cell line and F344 orthotopic bladder tumor models to reveal the pro-senescence effects of WWOX and the corresponding underlying mechanism in bladder cancer. WWOX-overexpressing lentivirus (LV-WWOX) remarkably stimulated cellular senescence, including increased senescence-associated secretory phenotype (SASP) formation, enlarged cellular morphology, and induced SA-β-Gal-positive staining. A further mechanism study revealed that the pro-senescence effect of LV-WWOX was dependent on increased intercellular reactive oxygen species (ROS) generation, which subsequently triggered p21/p27. Moreover, LV-WWOX significantly inhibited the tumor size by 30.49% in the F344/AY-27 rat orthotopic model (p < 0.05) by activating cellular senescence. The expression of p21 was significantly enhanced in the orthotopic bladder tumors under WWOX treatment. The orthotopic bladder tumors in the groups of rats verified the effect in vivo. Our study suggests that WWOX, an ROS-dependent senescence-induced gene, could be further studied for its therapeutic implications in bladder cancer.

Albendazole exerts an anti-hepatocellular carcinoma effect through a WWOX-dependent pathway

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related deaths. The WW-domain containing oxidoreductase (WWOX) protein suppresses carcinogenesis and its absence is closely related to aggressive HCC phenotypes. In this study, by using SPR analysis, cell viability assay and xenograft mice models, we found that albendazole (ABZ), a safe and effective anthelmintic drug, exhibited the binding affinity with WWOX protein and potential inhibition effect on HCC cells in vitro and in vivo. Overexpression and knockdown of WWOX confirmed that the suppression of HCC by ABZ. Flow cytometric analysis, western blotting analysis and Co-IP were conducted to study the mechanism of ABZ. Our data showed that ABZ regulated the interaction between WWOX and its binding proteins including p53 and C-MYC. Furthermore, ABZ triggered p53-induced intrinsic apoptosis and suppressed EMT-mediated migration by C-MYC/Fibronectin axis. In addition, ∆NP73 expression was significantly inhibited by ABZ, which further sensitized p53-induced intrinsic apoptosis and cell cycle arrest. In summary, ABZ could suppress the proliferation and migration of HCC cells by regulating WWOX-dependent signaling pathway.

WWOX inhibition by Zfra1-31 restores mitochondrial homeostasis and viability of neuronal cells exposed to high glucose.

Diabetes has been associated with an increased risk of cognitive decline and dementia. However, the mechanisms underlying this association remain unclear and no effective therapeutic interventions exist. Accumulating evidence demonstrates that mitochondrial defects are a key feature of diabetes contributing to neurodegenerative events. It has also been demonstrated that the putative tumor suppressor WW domain-containing oxidoreductase 1 (WWOX) can interact with mitochondria in several pathological conditions. However, its role in diabetes-associated neurodegeneration remains unknown. So, this study aimed to evaluate the role of WWOX activation in high glucose-induced neuronal damage and death. Our experiments were mainly performed in differentiated SH-SY5Y neuroblastoma cells exposed to high glucose and treated (or not) with Zfra1-31, the specific inhibitor of WWOX. Several parameters were analyzed namely cell viability, WWOX activation (tyrosine 33 residue phosphorylation), mitochondrial function, reactive oxygen species (ROS) production, biogenesis, and dynamics, autophagy and oxidative stress/damage. The levels of the neurotoxic proteins amyloid β (Aβ) and phosphorylated Tau (pTau) and of synaptic integrity markers were also evaluated. We observed that high glucose increased the levels of activated WWOX. Interestingly, brain cortical and hippocampal homogenates from young (6-month old) diabetic GK rats showed increased levels of activated WWOX compared to older GK rats (12-month old) suggesting that WWOX plays an early role in the diabetic brain. In neuronal cells, high glucose impaired mitochondrial respiration, dynamics and biogenesis, increased mitochondrial ROS production and decreased mitochondrial membrane potential and ATP production. More, high glucose augmented oxidative stress/damage and the levels of Aβ and pTau proteins and affected autophagy, contributing to the loss of synaptic integrity and cell death. Of note, the activation of WWOX preceded mitochondrial dysfunction and cell death. Importantly, the inhibition of WWOX with Zfra1-31 reversed, totally or partially, the alterations promoted by high glucose. Altogether our observations demonstrate that under high glucose conditions WWOX activation contributes to mitochondrial anomalies and neuronal damage and death, which suggests that WWOX is a potential therapeutic target for early interventions. Our findings also support the efficacy of Zfra1-31 in treating hyperglycemia/diabetes-associated neurodegeneration.

Epilepsy in patients with WWOX-related epileptic encephalopathy (WOREE) syndrome

Epileptic encephalopathy (EE) is difficult to diagnose and manage. It can be caused by a variety of disorders, and its aetiology may guide management and prognosis. The human gene for WW domain-containing oxidoreductase (WWOX) has been associated with epileptic encephalopathy, which presents in infancy with seizures, psychomotor delay, microcephaly, and optic atrophy. We report nine patients with WWOX-related EE from six families. We provide detailed descriptions of clinical presentations, imaging findings, neurophysiological manifestations, and related mutations. Whole-exome sequencing (WES) was used to identify the mutations in the WWOX gene. We established correlations between genotype and phenotype in our cases and previously reported cases. Our data support previously reported findings regarding WWOX-related EE, indicating the importance of the human WWOX gene in brain development and the association between WWOX mutations and EE. Our study also highlights the power of WES, particularly in clinically challenging cases.

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

The strength of WWOX binding with protein partners correlates with cancer suppression and potentially with inhibition of Alzheimer's disease progression

Tumor suppressor WW domain‐containing oxidoreductase (WWOX) is one of the risk factors for Alzheimer's disease (AD), a neurodegenerative disease. WWOX binds Tau via its C‐terminal SDR domain and interacts with Tau phosphorylating enzymes ERK, JNK, and GSK‐3β, so as to limit AD progression. Loss of WWOX in newborns leads to severe neural diseases and early death. Gradual loss of WWOX protein in the hippocampus and cortex starting from middle age may slowly induce aggregation of a protein cascade that ultimately causes accumulation of extracellular amyloid beta plaques and intracellular tau tangles, along with reduction in inhibitory GABAergic interneurons, in AD patients over 70 years old. Age‐related increases in pS14‐WWOX accumulation in the brain promotes neuronal degeneration. Suppression of Ser14 phosphorylation by a small peptide Zfra leads to enhanced protein degradation, reduction in NF‐κB‐mediated inflammation, and restoration of memory loss in triple transgenic mice for AD. Intriguingly, tumor suppressors p53 and WWOX may counteract each other in vivo, which leads to upregulation of AD‐related protein aggregation in the brain and lung. WWOX has numerous binding proteins. We reported that the stronger the binding between WWOX and its partners, the better the suppression of cancer growth and reduction in inflammation. Thus, the stronger complex formation between WWOX and partners may provide a better blockade of AD progression.

EHBP1, TUBB, and WWOX SNPs, Gene-Gene and Gene-Environment Interactions on Coronary Artery Disease and Ischemic Stroke.

The associations among the EH domain-binding protein 1 (EHBP1), tubulin beta class I (TUBB), and WW domain-containing oxidoreductase (WWOX) single nucleotide polymorphisms (SNPs) and coronary artery disease (CAD) and ischemic stroke (IS) are not yet understood. This study aimed to detect the associations of these SNPs, gene-gene and gene-environment interactions and CAD and IS in the Guangxi Han population. A total of 1853 unrelated subjects were recruited into normal control (n = 638), CAD (n = 622), and IS (n = 593) groups. Related genotypes were determined by high-throughput sequencing. The genotypic and minor allelic frequencies of rs2278075 were different between the CAD and control groups, and those of rs2710642, rs3130685, and rs2278075 were also different between the IS and control groups. The rs2278075T allele, rs3130685-rs2222896-rs2278075, rs3130685-rs2222896-diabetes, rs3130685-rs2222896-drinking, and haplotype rs2710642A-rs10496099C-diabetes interactions were associated with increased risk, while G-T-G-C-G-A and G-T-T-T-G-T-drinking were associated with reduced risk of CAD. The rs2278075T and rs2710642G alleles, rs2710642G-rs10496099C haplotype, rs3130685-rs2278075-rs2222896, and rs2710642-rs2278075-hypertension interactions aggravated the association with IS, whereas the rs3130685T allele, rs2710642A-rs10496099C haplotype and the interactions of H1 (s2710642A-rs10496099C)-H2 (rs2710642G-rs10496099C)-drinking and I1 (A-C-G-C-A-A)-I3 (A-C-G-T-A-A)-I4 (A-C-G-T-G-A)-I5 (G-T-G-C-G-A) diminished the association with IS. Carrying WWOX rs2278075T was strongly associated with CAD or IS, while EHBP1 rs2710642 and TUBB rs3130685 might alter the association of IS by modifying the serum lipid profile. This study demonstrates that the EHBP1, TUBB, and WWOX SNPs, gene-gene and gene-environment interactions are associated with the risk of CAD and IS in the Guangxi Han population.

Effect of WWOX on epithelial-mesenchymal transition in ovarian cancer cells in vivo, and its association with Elf5/Snail-1 signal pathway

Purpose: To investigate how WW domain-containing oxidoreductase (WWOX) gene affects epithelial-mesenchymal transition (EMT) of ovarian cancer (OC) cells, and the implication of Elf5/Snail-1 pathway in the process.
 Methods: This study employed female BALB/c nude mice aged 6 weeks. The expression levels of E-cadherin, β-Catenin, N-cadherin, Vimentin, Snail1 and Elf5 were assayed using qRT-PCR. The protein levels of WW1, WW2, E-cad, β-Cat, N-cad, vimentin, Snail-1 and Elf5 were determined using western blotting (WB). Invasion changes in cells in each group were analyzed by Transwell invasion test while WWOX and Elf5 were evaluated by GST pull-down test; furthermore, WWOX and ELF5 were evaluated by co-immunoprecipitation (COIP).
 Results: Relative to control group, tumor volume and weight of WWOX high-expression mice were significantly decreased, while those of WWOX low-expression group were increased (p < 0.05). The levels of E-cad, Elf5 and β-catenin increased, but those of N-cad, Snail-1 and vimentin decreased in WWOX high expression group. In the WWOX low-expression group, the levels of E-cad, Elf5 and β-cat were down-regulated, while those of N-cad, Snail-1 and vimentin were up-regulated (p < 0.05). The expression of E-cad, Elf5 and β-cat declined; on the other hand, those of N-Cad, Snail-1 and vimentin were elevated after mutation of WW1 domain in WWOX gene (p < 0.05).
 Conclusion: Based on the in vivo results, the role of WWOX/Elf5/Snail1 signal route in the epithelial-mesenchymal transition of ovarian cancer, and its related mechanisms have been clarified. This provides a new target for gene therapy of ovarian cancer.