WW Domain-containing Oxidoreductase Expression Research Articles

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.

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.

Role of MicroRNA-134-5p in Metabolic Syndrome-Associated Pulmonary Hypertension in Heart Failure with Preserved Ejection Fraction

Background: Pulmonary hypertension in heart failure with preserved ejection fraction (PH-HFpEF) is the most common cause of PH worldwide. It is closely linked to risk factors for metabolic syndrome, including obesity and diabetes - factors known to increase proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), leading to pulmonary vascular remodeling. Qualitative studies have shown that patients with progressive vascular abnormalities develop more severe symptoms and suffer frequent hospitalization. However, underlying mechanisms involved in the regulation of pulmonary vascular remodeling in metabolic syndrome-associated PH-HFpEF are still unclear.
 Aim: We have recently observed decreased levels of the tumor suppressor WW domain-containing oxidoreductase (WWOX), which plays a housekeeping role in repressing cellular proliferation, in PASMCs isolated from rats with experimental PH-HFpEF and human subjects with obesity and diabetes. As microRNAs (miRNAs) have been shown to regulate WWOX expression in cancers, here we aimed at examining the involvement of miRNAs in WWOX-associated pulmonary vasculature regulation in metabolic syndrome-associated PH-HFpEF.
 Methods and Results: Among miRNAs that have been associated with reduced WWOX expression, including miR -134-5p, -153-3p, -29a-3b, -29b-3p and -187-5p, we found that miR-134-5p was significantly increased in PASMCs of obese and diabetic subjects. To determine the role of miR-134-5p in the regulation of WWOX in the pulmonary vasculature, we applied exogenous miR-134-5p to human PASMCs. Treatment with miR-134-5p decreased WWOX expression, increased PCNA expression (a cell proliferation marker) and enhanced cellular proliferation. Additionally, human PASMCs challenged with high concentration of glucose, palmitic acid and insulin, which mimic hyperglycemic, hyperlipidemic and hyperinsulinemic conditions, exhibited increased miR-134-5p, accompanied by elevated cellular proliferation. 
 Conclusions: These studies suggest that miR-134-5p may have a potential role in metabolic syndrome-associated PH-HFpEF through regulating WWOX in the pulmonary vasculature. These studies identify miR-134-5p as a potential therapeutic target for the treatment of metabolic syndrome-associated PH-HFpEF.
 This project was funded, in part, with support from the NIH NHLBI Short-Term Training Program in Biomedical Sciences Grant funded, in part by T35HL110854 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abstract 10677: Skeletal Muscle Sirt3 Deficiency-Mediated Loxl2 Secretion in Remote Pulmonary Vascular Remodeling and Pulmonary Hypertension in Heart Failure with Preserved Ejection Fraction

Background: Pulmonary hypertension in the context of heart failure with preserved ejection fraction (PH-HFpEF) is the most common cause of PH worldwide. However, major pathways involved in the regulation of PH-HFpEF are still not well understood. We have recently reported on a role of skeletal muscle sirtuin-3 (SIRT3) in modulating PH-HFpEF. In this study, we attempted to define the processes by which skeletal muscle SIRT3 defects affect pulmonary vascular health in PH-HFpEF. Methods and Results: Skeletal muscle-specific Sirt3 knockout mice ( Sirt3 skm-/- ) exhibited drastically reduced pulmonary vascular density accompanied by pulmonary vascular proliferative remodeling and elevated pulmonary pressures. Using global mass spectrometry-based comparative secretome analysis, we found elevated secretion of lysyl oxidase homolog 2 (LOXL2) in SIRT3-deficeint skeletal muscle cells. Elevated circulation and protein expression levels of LOXL2 were also observed in plasma and skeletal muscle of Sirt3 skm-/- mice, rats with experimental PH-HFpEF (SU5416/Obese ZSF1, Ob-Su), and patients with PH-HFpEF. Interestingly, expression levels of the tumor suppressor WW domain-containing oxidoreductase (WWOX), which plays a housekeeping role in repressing cellular proliferation, were decreased in PASMCs from Sirt3 skm-/- mice. Reduced WWOX expression levels were also detected in PASMCs from Ob-Su rats and human subjects with obesity and diabetes. Treatment with LOXL2 recombinant protein resulted in reduced WWOX expression ex vivo in precision-cut lung slices (PCLS) and in vitro in PASMCs. Media conditioned by SIRT3-deficient skeletal muscle cells reduced WWOX expression in PASMCs, concomitant with increased PCNA levels and cellular proliferation. Finally, knockdown of both SIRT3 and LOXL2 in skeletal muscle cells or suppression of LOXL2 with β-aminopropionitrile (BAPN) restored WWOX and PCNA levels in PASMCs. Conclusions: These studies indicate a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH-HFpEF. These studies also reveal a new endocrine signaling axis that links skeletal muscle SIRT3 deficiency to remote WWOX regulation in the pulmonary vasculature through myokine LOXL2.

Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes.

WW domain‐containing oxidoreductase (WWOX) is an emerging neural gene‐regulating homeostasis of the central nervous system. Germline biallelic mutations in WWOX cause WWOX‐related epileptic encephalopathy (WOREE) syndrome and spinocerebellar ataxia and autosomal recessive 12 (SCAR12), two devastating neurodevelopmental disorders with highly heterogenous clinical outcomes, the most common being severe epileptic encephalopathy and profound global developmental delay. We recently demonstrated that neuronal ablation of murine Wwox recapitulates phenotypes of Wwox‐null mice leading to intractable epilepsy, hypomyelination, and postnatal lethality. Here, we designed and produced an adeno‐associated viral vector (AAV9) harboring murine Wwox or human WWOX cDNA and driven by the human neuronal Synapsin I promoter (AAV‐SynI‐WWOX). Testing the efficacy of AAV‐SynI‐WWOX delivery in Wwox‐null mice demonstrated that specific neuronal restoration of WWOX expression rescued brain hyperexcitability and seizures, hypoglycemia, myelination deficits, and the premature lethality and behavioral deficits of Wwox‐null mice. These findings provide a proof‐of‐concept for WWOX gene therapy as a promising approach to curing children with WOREE and SCAR12.

Modeling genetic epileptic encephalopathies using brain organoids.

Developmental and epileptic encephalopathies (DEE) are a group of disorders associated with intractable seizures, brain development, and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain‐containing oxidoreductase (WWOX) are associated with a relatively mild autosomal recessive spinocerebellar ataxia‐12 (SCAR12) and a more severe early infantile WWOX‐related epileptic encephalopathy (WOREE). In this study, we generated an in vitro model for DEEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR‐engineered human ES cells and from patient‐derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt pathway and DNA damage response impairment. Furthermore, we provide a proof of concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies.

Expression of WW domain-containing oxidoreductase and its clinical implication in endometrial adenocarcinoma patients with metabolic syndrome.

Metabolic syndrome (MS) is tightly associated with the oncogenesis and prognosis of endometrioid adenocarcinoma, but the underlying mechanism is unclear. Here, we studied the relation between the expression status of WW domain-containing oxidoreductase (WWOX) and the clinicopathological features of endometrioid adenocarcinoma patients with MS. Fifty-seven samples of endometrial adenocarcinoma were chosen for detection of expression level of WWOX. Overall survival (OS) time of these patients was analyzed by univariate and multivariate analysis. Survival analysis of patients with different WWOX expression levels from the Cancer Genome Atlas (TCGA) database was also performed. The WWOX expression is significantly higher in MS group than that in non-MS group (36.4%vs 65.7%, P=.03). WWOX was closely related to MS (P=.03) and muscle invasion of tumor cells (P=.04), but age, tumor grade, status of lymphatic metastasis, and FIGO (International Federation of Gynecology and Obstetrics) stage were not significantly different between the two WWOX expression status. Univariate analysis revealed that lymphatic metastasis (P=.023) and lower stage (P=.006) are significantly associated with OS. Multivariate analysis demonstrated that stage was an independent prognostic factor for OS (hazard ratio=0.197; 95% CI, 0.043-0.896). Downregulation of WWOX was statistically associated with OS in patients from TCGA database (P=.04). WWOX may play an important role in the progression of endometrial cancer with MS.

Identification of a novel association for the WWOX/HIF1A axis with gestational diabetes mellitus (GDM).

BackgroundAlthough the WW-domain-containing oxidoreductase (WWOX)/Hypoxia-inducible factor 1 (HIF1) pathway is a well-known regulator of cellular glucose and energy metabolism in pathophysiological processes, its role in gestational diabetes mellitus (GDM), remains elusive. We undertook this study to determine the effect of WWOX/HIF1A signaling on the expression of glucose metabolism genes in GDM patients.MethodsLeukocytes were obtained from 135 pregnant women with (n = 98) or without (n = 37) GDM and, in turn, 3 months (n = 8) and 1 year (n = 12) postpartum. Quantitative RT-PCR was performed to determine gene expression profiles of the WWOX/HIF1A-related genes, including those involved in glucose transport (SLC2A1, SLC2A4), glycolytic pathway (HK2, PKM2, PFK, LDHA), Wnt pathway (DVL2, CTNNB1), and inflammatory response (NFKB1).ResultsGDM patients displayed a significant downregulation of WWOX with simultaneous upregulation of HIF1A which resulted in approximately six times reduction in WWOX/HIF1A ratio. As a consequence, HIF1A induced genes (SLC2A1, HK2, PFK, PKM) were found to be overexpressed in GDM compared to normal pregnancy and negative correlate with WWOX/HIF1A ratio. The postpartum WWOX expression was higher than during GDM, but its level was comparable to that observed in normal pregnancy.ConclusionsThe obtained results suggest a significant contribution of the WWOX gene to glucose metabolism in patients with gestational diabetes. Decreased WWOX expression in GDM compared to normal pregnancy, and in particular reduction of WWOX/HIF1A ratio, indicate that WWOX modulates HIF1α activity in normal tissues as described in the tumor. The effect of HIF1α excessive activation is to increase the expression of genes encoding proteins directly involved in the glycolysis which may lead to pathological changes in glucose metabolism observed in gestational diabetes.

WWOX promotes apoptosis and inhibits autophagy in paclitaxel‑treated ovarian carcinoma cells.

Although paclitaxel(PTX) is a first‑line chemotherapeutic agent for the treatment of epithelial ovarian cancer(EOC), its clinical use is restricted by chemoresistance. Autophagy is believed to promote drug resistance, and WW domain‑containing oxidoreductase(WWOX) has been predicted to serve an essential role in apoptosis induction and to suppress autophagy in various tumor cell types. In the present study, the role of WWOX was demonstrated using PTX‑treated EOC cells. Cell viability and apoptosis were detected using Cell Counting Kit‑8, morphological and flow cytometric analyses. WWOX and phosphorylated (p)‑WWOX were highly expressed in PTX‑treated sensitive EOC cells(A2780), which was accompanied by activation of the apoptosis‑related proteins caspase‑3 and poly(ADP‑ribose) polymerase(PARP). Conversely, PTX‑resistant EOC cells (A2780/T) were characterized by reduced WWOX expression and constant phosphorylation levels, as well as undetectable levels of activated caspase‑3 and PARP when cells were treated with PTX. The altered expression of WWOX between the two cell types was further validated by reverse transcription‑-quantitative PCR. The apoptosis‑inducing role of WWOX was also confirmed by flow cytometry after WWOX overexpression was induced in PTX‑treated A2780 cells. These findings indicated that WWOX activation may inhibit chemoresistance and trigger cancer cell death. The upregulated expression levels of the autophagy‑related protein 12‑5complex, Beclin‑1 and LC3, as well as the downregulation of P62, were also detected following PTX treatment, suggesting that PTX induced autophagic flux in both types of EOC cells. This conclusion was further supported by visualizing the accumulation of autophagosome and autolysosome vesicles, using confocal microscopy and transmission electron microscopy. PTX was also shown to inhibit mTOR signaling, indicated by a decreased level of p‑mTOR and increased expression of eukaryotic translation initiation factor 4E‑binding protein1. Finally, the interaction between WWOX, mTOR and autophagy was investigated via WWOX transfection experimentation, and indicated that WWOX activated mTOR whilst inhibiting autophagy. These data indicated that WWOX may serve a critical role in PTX‑induced apoptosis and could suppress autophagy by downregulating essential autophagic effectors in EOC cells via mTOR signaling.

Abstract 14297: Potential Role of the Tumor Suppressor WWOX in Mediating Skeletal Muscle-Lung Vasculature Crosstalk in PH-HFpEF

Introduction: Pulmonary hypertension due to left heart disease (PH-LHD; Group 2), particularly in the context of heart failure with preserved ejection fraction (HFpEF), is the most common cause of PH worldwide. At present, no specific effective therapy has been identified mainly due to the fact that major pathways involved in the regulation of PH-HFpEF are still not well understood. Results: We have recently reported on a role of skeletal muscle sirtuin-3 (SIRT3) in modulating PH-HFpEF. Using skeletal muscle-specific SIRT3 knockout mice ( Sirt3 skm-/- ), we showed that absence of SIRT3 in skeletal muscle drastically reduced the pulmonary vascular tree accompanied by vascular proliferative remodeling. Interestingly, we found that expression levels of the tumor suppressor WW domain-containing oxidoreductase (WWOX) were decreased in pulmonary arterial smooth muscle cells (PASMCs) obtained from Sirt3 skm-/- mice, while no changes in SIRT3 activation levels were detected. Reduced WWOX expression levels were also found in PASMCs isolated from SU5416/Obese ZSF1 (Ob-Su) rat model of PH-HFpEF, in which the levels of SIRT3 activation were found to be decreased in skeletal muscle, but not in the lungs and PASMCs. No changes of WWOX levels were observed in skeletal muscle of Ob-Su rats or in pulmonary artery endothelial cells (PAECs) treated with plasma obtained from Ob-Su rats. Conclusions: Since reduction of WWOX in PASMCs has been shown to promote cell proliferation, HIF1α stabilization and pulmonary arterial hypertension (PAH; Group 1), our data suggest a potential role of WWOX in mediating skeletal muscle SIRT3 deficiency-associated remote pulmonary vascular remodeling in PH-HFpEF.

Characterization of WWOX expression and function in canine mast cell tumors and malignant mast cell lines

BackgroundThe WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior.Methods/resultsWWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs.ConclusionsThese data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.

Downregulated Expression of WWOX in Cervical Carcinoma: A Case-Control Study

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.

Expression and clinical significance of WWOX, Elf5, Snail1 and EMT related factors in epithelial ovarian cancer.

Expression and clinical significance of WW domain-containing oxidoreductase (WWOX), Elf5, Snail1 and epithelial-mesenchymal transition (EMT) related factors in epithelial ovarian cancer were investigated. Ovarian cancer tissues of 300 epithelial ovarian cancer patients and the adjacent normal tissues were analyzed. Immunohistochemical method was used to detect the expressions of WWOX, Elf5, Snail1 and EMT marker molecules in the specimens. The relationship between the indicators and clinicopathological parameters, and prognosis of patients with ovarian cancer was analyzed. The relationship between WWOX, Elf5, Snail1 and EMT marker molecules E-cadherin, N-cadherin and vimentin in ovarian cancer tissues was analyzed. The expression levels of WWOX, Elf5, Snail1 and EMT marker molecules in epithelial ovarian cancer tissues were significantly different from those in adjacent normal tissues, and were related to surgical pathological stage, pathological grade and lymph node metastasis. High expressions of WWOX and Elf5 were related to the survival rate of patients. The survival rate of patients with positive expression was significantly higher than that of negative expression. FIGO stage, pathological grade, lymph node metastasis and expression of WWOX and Elf5 were all independent factors affecting postoperative prognosis in ovarian cancer patients. In conclusion, the expression levels of WWOX, Elf5, Snail1 and EMT related factors in epithelial ovarian cancer tissues are consistent and different. The expression levels of WWOX and Elf5 are related to the survival and prognosis of patients with epithelial ovarian cancer.

Wwox Deletion in Mouse B Cells Leads to Genomic Instability, Neoplastic Transformation, and Monoclonal Gammopathies.

WWOX (WW domain containing oxidoreductase) expression loss is common in various cancers and characteristic of poor prognosis. Deletions, translocations, and loss of expression affecting the WWOX gene are a common feature of various B cell neoplasms such as certain B cell lymphomas and multiple myeloma. However, the role of this common abnormality in B cell tumor initiation and/or progression has not been defined. In this study, we conditionally deleted Wwox early in B cell development by means of breeding Cd19-Cre transgenic mice crossed to Wwox floxed mice (Cd19 Wwox KO). We observed a significant reduced survival in Cd19 Wwox KO mice and the development of B cell neoplasms including B cell lymphomas, plasma cell neoplasias characterized by increased numbers of CD138+ populations as well as monoclonal gammopathies detected by serum protein electrophoresis. To investigate whether Wwox loss could play a role in genomic instability, we analyzed DNA repair functions during immunoglobulin class switch joining between DNA segments in antibody genes. While class switch recombination (CSR) was only slightly impaired, Wwox deficiency resulted in a dramatic shift of double strand break (DSB) repair from normal classical-NHEJ toward the microhomology-mediated alternative-NHEJ pathway, a pathway associated with chromosome translocations and genome instability. Consistent with this, Wwox deficiency resulted in a marked increase of spontaneous translocations during CSR. This work defines for the first time a role for Wwox for maintaining B cell genome stability during a process that can promote neoplastic transformation and monoclonal gammopathies.

Downregulation of WW domain-containing oxidoreductase leads to tamoxifen-resistance by the inactivation of Hippo signaling.

Acquired tamoxifen-resistance is an important cause of death in patients with hormone-dependent breast tumors. Therefore, understanding the molecular mechanisms underlying the development of tamoxifen-resistance is critical for successful endocrine therapy. This study aimed to define the role of WW domain-containing oxidoreductase (WWOX) in acquired tamoxifen-resistance. Our results show that low WWOX expression was significantly related to tamoxifen-resistance. Moreover, WWOX-knockdown increased resistance to tamoxifen, while WWOX overexpression decreased the resistance. Furthermore, WWOX silencing decreased Yes-associated protein (YAP) phosphorylation and increased YAP nuclear translocation. Finally, YAP silencing decreased tamoxifen-resistance in WWOX-knockdown cells. Our findings demonstrate that WWOX downregulation can lead to the development of tamoxifen-resistance by inactivating Hippo signaling. Thus, WWOX might be a valuable target and prognostic marker for tamoxifen-resistance. Impact statement Understanding the molecular pathways leading to the development of tamoxifen-resistance is an important research focus as acquired tamoxifen-resistance is the main cause of death in patients with benign primary prognosis. Although WW domain-containing oxidoreductase (WWOX) has been related to breast tumorigenesis, its role in acquired tamoxifen-resistance has not yet been demonstrated. Our findings show that WWOX might be a valuable therapeutic target and prognostic marker for tamoxifen-resistance.

Modeling WWOX Loss of Function in vivo: What Have We Learned?

The WW domain–containing oxidoreductase (WWOX) gene encompasses a common fragile sites (CFS) known as FRA16D, and is implicated in cancer. WWOX encodes a 46kDa adaptor protein, which contains two N-terminal WW–domains and a catalytic domain at its C–terminus homologous to short–chain dehydrogenase/reductase (SDR) family proteins. A high sequence conservation of WWOX orthologues from insects to rodents and ultimately humans suggest its significant role in physiology and homeostasis. Indeed, data obtained from several animal models including flies, fish, and rodents demonstrate WWOX in vivo requirement and that its deregulation results in severe pathological consequences including growth retardation, post–natal lethality, neuropathy, metabolic disorders, and tumorigenesis. Altogether, these findings set WWOX as an essential protein that is necessary to maintain normal cellular/physiological homeostasis. Here, we review and discuss lessons and outcomes learned from modeling loss of WWOX expression in vivo.

Expression of B Cell-Specific Moloney Murine Leukemia Virus Integration Site 1 (BMI-1) and WW Domain-Containing Oxidoreductase (WWOX) in Liver Cancer Tissue and Normal Liver Tissue.

BackgroundThe aim of this study was to compare the expression levels of mRNA of the B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) and the WW domain-containing oxidoreductase (WWOX) genes and their protein products in tissues from patients with liver cancer with normal liver tissues from patients without liver cancer.Material/MethodsThe liver cancer group (N=56) included patients with available tissue samples of histologically confirmed liver cancer. The control group (N=24) included histologically confirmed normal liver tissue samples. Immunofluorescence staining and Western blot were used to detect and compare protein expression of Bmi-1 and WWOX in liver tissues in the liver cancer group and the control group. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect and compare mRNA expression of BMI-1 and WWOX in liver tissues in the liver cancer group and the control group. Expression levels of the protein and mRNA levels and the clinicopathological features including patient prognosis in liver cancer were evaluated statistically using analysis of variance (ANOVA).ResultsThere were significant differences in the expression levels of protein and mRNA of BMI-1 and WWOX between the liver cancer group and the control group. BMI-1 mRNA and protein expression were significantly increased, and WWOX mRNA and protein expression were significantly reduced in liver cancer tissue, compared with normal liver tissue (p<0.05).ConclusionsIn liver cancer tissue compared with normal liver, the expression of BMI-1 and WWOX mRNA and their protein products were upregulated and down-regulated, respectively.

Abstract 5527: Characterizing the role of WWOX dysregulation in canine osteosarcoma

Abstract Introduction: The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that is frequently deleted or reduced in human osteosarcoma (OS) tumors and loss of WWOX in OS cells promotes a highly tumorigenic phenotype. The purpose of this study was to investigate the functional consequences of WWOX deficiency in canine OS, a well-established spontaneous large animal model of human OS. Methods: Real-time PCR and Western blotting was performed to evaluate WWOX expression was in primary canine OS tumors and canine OS cell lines. Full-length canine Wwox cDNA was amplified from normal canine testes and cloned into the pCDH-cop-GFP lentiviral vector (Systems Biosciences). Canine OS cell lines expressing low basal levels of WWOX were transduced with either empty or WWOX lentiviral vectors and stably transduced cells were sorted based on GFP positivity. Overexpression of canine WWOX lentiviral constructs was confirmed by Western blotting and OS cell lines were evaluated for differences in proliferative capacity, expression of bone differentiation markers, and the ability to migrate through matrigel. Transgenic mice (Tg) carrying a conditional allele of Wwox (Wwoxfl/fl-Tg) were crossed with mice expressing Cre recombinase under the osteoblast-specific Collagen1α1 promoter (Col1a1-Cre-Tg), restricting deletion of WWOX to osteoblasts. Calvarial osteoblast cultures were generated from Wwoxfl/fl and Col1a1-Cre; Wwoxfl/fl mice and the effects of WWOX deletion on osteoblast functions was evaluated using assays described above. Results and Conclusions: Western blotting demonstrated that WWOX protein is absent or reduced in primary canine OS tumors and canine OS cell lines. To assess the functional consequences of WWOX deletion on normal osteoblast behavior, the canine OSA8 and Abrams OS cell lines which express low levels of WWOX were transduced with WWOX lentiviral constructs resulting in high levels of WWOX expression as demonstrated by real time PCR and Western blotting. Restoration of WWOX in canine OS cells resulted in decreased cellular proliferation and invasion. To more critically evaluate the consequences of Wwox deficiency in normal osteoblast biology without the confounding influence of altered Wwox expression in other tissues, Col1a1-Cre; Wwoxfl/fl-Tg mice were generated in which Wwox deletion is restricted to osteoblasts. Consistent with our findings in canine OS cell lines, deletion of WWOX in osteoblasts derived from Col1a1-Cre; Wwoxfl/fl-Tg mice resulted in enhanced cellular proliferation. Studies are underway to identify WWOX binding partners and investigate regulatory pathways altered by WWOX that promote cell proliferation and invasion in normal and malignant osteoblasts. In summary, these data provide evidence supporting a role for WWOX in canine OS cell proliferation and invasion, and suggest that dysregulation of WWOX may be fundamental to the disease process in both human and canine OS. Citation Format: Justin Breitbach, Feng Xu, F. Kay Huebner, Cheryl A. London, Joelle M. Fenger. Characterizing the role of WWOX dysregulation in canine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5527.

Low expression of WW domain-containing oxidoreductase associates with hepatocellular carcinoma aggressiveness and recurrence after curative resection.

WW domain‐containing oxidoreductase (WWOX), which has a protein‐interaction domain and is regarded to be a tumor suppressor, has been known to play an important role in anti‐angiogenesis and cancer progression. This study aimed to investigate prognostic values of WWOX expression in hepatocellular carcinoma (HCC) patients after hepatectomy. Additionally, we intended to formulate a valuable prognostic nomogram for HCCs. 182 HCC patients who underwent hepatectomy from January 2009 to January 2010 were enrolled in our study. qRT‐PCR, Western blot, and immunohistochemistry on tissue microarrays were then used to determine the expression levels of WWOX. An evaluation of the role of WWOX expression levels in the prognosis and outcome of patients was established. A decrease in the expression of WWOX was found when compared to adjacent tumor‐free tissues, which led to worse overall survival (OS) and recurrence‐free survival (RFS) and, therefore, was considered as an independent negative factor in the prognosis of HCC. Two nomograms, comprising WWOX, alpha‐fetoprotein (AFP), tumor size, and γ‐glutamyltransferase (γ‐GT), were constructed to obtain superior discriminatory abilities than conventional staging systems in terms of C‐index and clinical net benefit on decision curve analysis (DCA) for OS and RFS. Our data suggest that WWOX expression is strongly related to HCC post‐resection aggressiveness and recurrence. Additional advanced and accurate predictive model through the incorporation of WWOX into nomogram could help predict OS or RFS for HCC patients.