WW Domain-containing Oxidoreductase Protein Research Articles

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.

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.

WWOX and Its Binding Proteins in Neurodegeneration.

WW domain-containing oxidoreductase (WWOX) is known as 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, and thereby limits 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. In this regard, the stronger complex formation between WWOX and partners may provide a better blockade of AD progression. In this review, we describe whether and how WWOX and partner proteins control inflammatory response and protein aggregation and thereby limit AD progression.

Wwox Deficiency Causes Downregulation of Prosurvival ERK Signaling and Abnormal Homeostatic Responses in Mouse Skin.

Deficiency of tumor suppressor WW domain-containing oxidoreductase (WWOX) in humans and animals leads to growth retardation and premature death during postnatal developmental stages. Skin integrity is essential for organism survival due to its protection against dehydration and hypothermia. Our previous report demonstrated that human epidermal suprabasal cells express WWOX protein, and the expression is gradually increased toward the superficial differentiated cells prior to cornification. Here, we investigated whether abnormal skin development and homeostasis occur under Wwox deficiency that may correlate with early death. We determined that keratinocyte proliferation and differentiation were decreased, while apoptosis was increased in Wwox–/– mouse epidermis and primary keratinocyte cultures and WWOX-knockdown human HaCaT cells. Without WWOX, progenitor cells in hair follicle junctional zone underwent massive proliferation in early postnatal developmental stages and the stem/progenitor cell pools were depleted at postnatal day 21. These events lead to significantly decreased epidermal thickness, dehydration state, and delayed hair development in Wwox–/– mouse skin, which is associated with downregulation of prosurvival MEK/ERK signaling in Wwox–/– keratinocytes. Moreover, Wwox depletion results in substantial downregulation of dermal collagen contents in mice. Notably, Wwox–/– mice exhibit severe loss of subcutaneous adipose tissue and significant hypothermia. Collectively, our knockout mouse model supports the validity of WWOX in assisting epidermal and adipose homeostasis, and the involvement of prosurvival ERK pathway in the homeostatic responses regulated by WWOX.

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.

Delineating WWOX Protein Interactome by Tandem Affinity Purification-Mass Spectrometry: Identification of Top Interactors and Key Metabolic Pathways Involved.

It has become clear from multiple studies that WWOX (WW domain-containing oxidoreductase) operates as a “non-classical” tumor suppressor of significant relevance in cancer progression. Additionally, WWOX has been recognized for its role in a much wider array of human pathologies including metabolic conditions and central nervous system related syndromes. A myriad of putative functional roles has been attributed to WWOX mostly through the identification of various binding proteins. However, the reality is that much remains to be learned on the key relevant functions of WWOX in the normal cell. Here we employed a Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach in order to better define direct WWOX protein interactors and by extension interaction with multiprotein complexes under physiological conditions on a proteomic scale. This work led to the identification of both well-known, but more importantly novel high confidence WWOX interactors, suggesting the involvement of WWOX in specific biological and molecular processes while delineating a comprehensive portrait of WWOX protein interactome. Of particular relevance is WWOX interaction with key proteins from the endoplasmic reticulum (ER), Golgi, late endosomes, protein transport, and lysosomes networks such as SEC23IP, SCAMP3, and VOPP1. These binding partners harbor specific PPXY motifs which directly interact with the amino-terminal WW1 domain of WWOX. Pathway analysis of WWOX interactors identified a significant enrichment of metabolic pathways associated with proteins, carbohydrates, and lipids breakdown. Thus, suggesting that WWOX likely plays relevant roles in glycolysis, fatty acid degradation and other pathways that converge primarily in Acetyl-CoA generation, a fundamental molecule not only as the entry point to the tricarboxylic acid (TCA) cycle for energy production, but also as the key building block for de novo synthesis of lipids and amino acids. Our results provide a significant lead on subsets of protein partners and enzymatic complexes with which full-length WWOX protein interacts with in order to carry out its metabolic and other biological functions while also becoming a valuable resource for further mechanistic studies.

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.

Cancerous Protein Network That Inhibits the Tumor Suppressor Function of WW Domain-Containing Oxidoreductase (WWOX) by Aberrantly Expressed Molecules.

Recent findings indicate that the WW domain-containing oxidoreductase (WWOX) is a tumor suppressor protein that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. WWOX protein mediates multiple signaling networks that suppress carcinogenesis through binding of its first WW domain to various cancer-associated proteins, i.e., p73, AP-2γ, and others. Although the tumor suppressor property of WWOX is inarguable, WWOX is not inactivated in the manner characteristic of the canonical Knudson hypothesis. Impairment of both alleles of WWOX is thought to be a rare event, only occurring in a few cancer cell lines. How is the tumor suppressor function of WWOX impaired in cancer cells? Recent advances highlight that a small transmembrane protein possessing a PPxY motif, called TMEM207, and its relatives are aberrantly expressed in various cancer cells and hinder the tumor suppressor function of WWOX through inhibiting its WW domain. Here, we review the recent findings related to the pathobiological properties of TMEM207 and its relatives based on clinicopathological and experimental pathological studies.

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.

Association of WWOX rs9926344 polymorphism with poor prognosis of hepatocellular carcinoma.

Introduction: The WW domain-containing oxidoreductase (WWOX), widely expressed in human tissues, is considered as a tumor suppressor gene and plays an important role in the incidence and progression of human cancer, HCC included. This study was to investigate the correlation between single nucleotide polymorphisms (SNPs) of the WWOX gene and the prognosis of hepatocellular carcinoma (HCC) patients.Materials and Methods: After a total of 152 HCC patients were recruited, 8 cases with tumor recurrence within 2-years after operation and 8 cases without recurrence were selected randomly for SNP genotyping and screening using Affymetrix Array 6.0. And then we confirmed candidate SNPs in the remaining 136 patients by time-of-flight mass spectrometry (TOF-MS).Results: In total, 32 SNPs were screened and identified as candidate SNPs with one SNP in particular, (rs9926344), being further verified to be valuable. We found that AA+AG genotype and A allele of WWOX rs9926344 were significantly associated with recurrent risk of HCC (p=0.002 and p=0.001, respectively). The Kaplan-Meier curve showed that patients carrying rs9926344 AA +AG genotype had poor RFS (P=0.004) and OS (P=0.005) compared to those carrying GG genotypes. The multivariate COX regression analysis showed that the AA+AG genotype were an independent prognostic factor for tumor recurrence (HR 1.787, 95% CI 1.042-3.064, P=0.035). Furthermore, IHC analysis showed that the WWOX protein down-regulation is more frequent in patients with AG genotype compared to those with GG genotype (P=0.023).Conclusion: Our findings indicate that WWOX rs9926344 polymorphism is positively correlated with tumor recurrence and can be used as an independent prognostic marker for HCC patients after operation.

RUNX2 and WWOX genes as molecular biomarkers and candidates for targeted therapy in Egyptian patients with primary conventional osteosarcoma

The conventional osteosarcoma (OS) is the commonest primary malignant, bone tumor with complex genomic profiles and poor survival. Runt-related transcription factor 2 (RUNX2) and WW domain containing oxidoreductase (WWOX) genes are implicated in normal osteogenesis as well as in the development of primary conventional OS. We retrospectively assessed protein and RNA expression of the RUNX2 and WWOX genes by quantitative real time PCR (qPCR) and immunohistochemistry (IHC) in 80 cases of primary OS and 20 normal control (NC) subjects. Proteins and RNA expression levels of both genes were correlated to clinico-pathological features of the patients, progression free and overall survival (PFS& OS) rates. In OS, RUNX2 protein was detected in 72/80 (90%) cases compared to 4/20 (20%) NC samples (p. < 0.001) and RUNX2-RNA was up regulated (up to 103.2 folds) in 60/80 (75%) (p = 0.01). WWOX protein and RNA (up to 7.2 folds) were detected in all NC samples but in 24/80 (30%) and 20/80 (20%) OS cases; respectively (p. < 0.001 for each). The concordance between the RNA and protein expressions for RUNX2 and WWOX was significantly high (X_trend^2 = 6.33; p = 0.012 and X_trend^2 = 19, p < 0.001; respectively). A significant inverse relation existed between RUNX2 and WWOX RNA and protein (p = 0.032, p = 0.008). There was significant correlation between RUNX2 RNA/protein, high tumor grade and stage (p = <0.001; each); RUNX2 RNA and male gender, tumor site and metastasis (p = 0.007, 0.041, 0.003; respectively). WWOX protein associated significantly with advanced stage and metastasis (p = 0.001& 0.024; respectively) and WWOX RNA associated with metastasis (p = 0.003). RUNX2 and WWOX play opposing roles in the development and progression of OS. They could be used as sensitive prognostic biomarkers for OS patients and RUNX2 represents a promising candidate for targeted therapy.

Deregulated WWOX is involved in a negative feedback loop with microRNA-214-3p in osteosarcoma.

WW domain-containing oxidoreductase(WWOX) is frequently inactivated in human osteosarcoma, and the restoration of its expression can suppress tumorigenicity in WWOX-negative OS cells. However, its regulatory mechanisms remain to be fully elucidated. In the present study, we demonstrate that WWOX is downregulated and that it regulates proliferation and epithelial-to-mesenchymal transition(EMT)-associated protein expression in osteosarcoma. As shown by our results, WWOX overexpression by transfection with WWOX overexpression plasmids suppressed the proliferation, migration and invasion of osteosarcoma MG63 cells(as shown by MTT and migration and invasion assays). The silencing of microRNA(miR)‑214‑3p by transfection with anti-miR‑14‑3p upregulated WWOX protein expression and also inhibited the proliferation, migration and invasion of osteosarcoma cells. Additionally, we found that WWOX negatively regulated miR‑214‑3p and miR‑10b expression. Our findings define a negative feedback pathway in control of WWOX and miR‑214‑3p expression, thus providing novel molecular targets for the treatment of osteosarcoma.

Reduced expression of the WW domain-containing oxidoreductase in human hematopoietic malignancies.

The role of the WW domain-containing oxidoreductase (WWOX) gene in multiple types of solid human cancers has been documented extensively thus far. Recently, we investigated the in vitro effects of WWOX overexpression and observed marked growth arrest in human leukemia cells; however, the clinical characterization of WWOX in leukemia remains poorly investigated. The present study evaluated the WWOX expression profiles of 182 patients with leukemia of different types and 5 leukemic cell lines, using reverse transcription-polymerase chain reaction and immunofluorescence analysis. The results found that WWOX mRNA and WWOX protein expression was significantly reduced or absent in the leukemia cases and cell lines compared with paired controls. The WWOX-positive rate was also lower in the leukemia cases compared with the rate of the normal controls. Notably, the WWOX level was reduced in newly diagnosed and relapsed cases, or in chronic myelogenous leukemia in the blastic phase, yet elevated in remission samples. Moreover, WWOX-negative cases exhibited WWOX expression restoration following induced remission. These findings suggest that WWOX may contribute to the occurrence and development of leukemia, and that it has potential to be a good biomarker or predictor for leukemia therapy.

Abstract 4952: WWOX and LOT1 genes are down-regulated in non-small cell lung cancer (NSCLC)

Abstract Lung cancer is a result of uncontrolled cell growth in lung tissue and is the most common type of cancer leading to death in men and second after breast cancer in women. Even though smoking is the main reason of lung cancer, genetic and environmental factors play an important role in the onset and prognosis of the disease. The WW domain-containing oxidoreductase (WWOX) gene is a putative tumor suppressor gene located on chromosome 16q23.3-24.1 and is composed of 9 exons. Several studies have reported the role of WWOX gene in different types of carcinomas including lung, breast, ovarian, stomach and nasopharyngeal cancer. The 46kDa WWOX protein has two WW (tryptophan) domains which can recognize and bind the proline-rich region (PPXY) of other proteins. Its main function is to inhibit tumor cell growth. WWOX also plays an important role in wnt/beta-catenin signaling pathway. The lost-on-transformation 1 (LOT1/ZAC1/PLAGL1) gene is another putative tumor suppressor gene located on chromosome 6q24-25, a common site known for loss of heterozygosity in many tumors. Deletions of the 6q region are observed in different types of cancers including breast, prostate, small cell lung cancer and mesotheliomas. The LOT1 gene codes for a 55 kDa protein with seven C2H2 type zinc finger motifs located in amino terminal half of the protein. This zinc-finger nuclear transcription factor interacts with other proteins and transcription factors exerting antiproliferative effects in cancer cells. The aim of this study was to investigate the expression levels of the WWOX and LOT1 genes by RT-PCR in tumor tissue samples from 39 patients with non small cell lung cancer (NSCLC). We observed down-regulation of WWOX and LOT1 expression in 82.1% and 48.7% of the patients, respectively. Expression levels of both genes were down-regulated simultaneously in 18 (46.1%) of the patients. When we analyzed the association between gene expression and age, sex, smoking, histology and stage, we found that WWOX expression was decreased in 28 of 31 (90.3%) patients older than 50 years (p = 0.02). Lower expression levels of both genes supports the hypothesis that WWOX and LOT1 act as tumor suppressors in NSCLC and that both genes may have a common effect on tumor progression which warrants further studies to analyze a possible cooperation between the two proteins. Citation Format: Onur Baykara, Seda Ekizoglu Eratak, Kamil Kaynak, Hikmet Koseoglu, Nur Buyru. WWOX and LOT1 genes are down-regulated in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2015-4952

Effect of the WWOX gene on the regulation of the cell cycle and apoptosis in human ovarian cancer stem cells.

In order to examine new ideas for gene therapy in ovarian cancer, the specific mechanism underlying the effects of the WW domain containing oxidoreductase (WWOX) gene on cell cycle regulation and apoptosis in human ovarian cancer stem cells was investigated. Ovarian cancer stem cells were transfected with a eukaryotic expression vector carrying the WWOX gene in vitro (recombinant plasmid) and cells transfected with the empty plasmid (empty plasmid) or untransfected cells were used as controls. Stably transfected cells were screened and amplified in culture and the WWOX protein was detected by western blot analysis in the three groups of cells. Western blot analysis was performed to detect the expression of cell cycle regulatory proteins cyclin E, cyclin-dependent kinase (CDK) 2, cyclin D1, CDK4 and apoptosis-related protein Wnt-5α and c-Jun N-terminal kinase (JNK), while polymerase chain reaction (PCR) was used to detect alterations in the mRNA expression levels of caspase-3. The results demonstrated that the WWOX protein was stably expressed in cells of the recombinant plasmid group, but was not detected in cells of the empty plasmid group and the control group. Cell proliferation at each time point decreased significantly in the recombinant plasmid group compared with the empty plasmid group and the control group. Flow cytometric analysis demonstrated that the proportion of cells in the G0/G1 phase in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. The rate of apoptosis in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. Western blot analysis demonstrated that the expression levels of cyclin E, CDK2, cyclin D1 and CDK4 in the recombinant plasmid group were significantly lower than those in the empty plasmid group and the control group; however, the expression levels of Wnt-5α and JNK were significantly higher than those in the empty plasmid group and the control group. PCR results demonstrated that the mRNA expression level of caspase-3 in the recombinant plasmid group was significantly higher than that in the empty plasmid group and the control group. In conclusion, the present study demonstrated that the WWOX gene can be stably expressed in ovarian cancer stem cells and that it inhibits the proliferation of ovarian cancer stem cells. The WWOX gene can downregulate the expression levels of cell cycle proteins cyclin E-CDK2 and cyclin D1-CDK4, which affects the cell cycle of ovarian cancer stem cells. Furthermore, the WWOX gene can upregulate the mRNA expression levels of Wnt-5α, JNK and caspase-3, thus contributing to apoptosis of ovarian cancer stem cells. The present study demonstrated that the WWOX gene may be an important molecular target for the treatment of ovarian cancer in the future.

Ectopic expression of the WWOX gene suppresses stemness of human ovarian cancer stem cells.

The present study aimed to investigate the effects of the WW domain-containing oxidoreductase (WWOX) gene on the stem cell properties of human ovarian cancer stem cells. A eukaryotic expression vector containing the WWOX gene was transfected into human ovarian cancer stem cells and Western blotting was used to assess the expression of WWOX protein in the transfected cells compared with the control cells (untransfected cells and cells transfected with the empty vector). The self-renewal abilities of these three types of stem cells was also assessed in vitro. To monitor changes in their differentiation potential, cells were cultured in medium supplemented with serum, and the expression of specific stem cell markers was determined. Drug-sensitivity tests were used to measure the sensitivity of the stem cells to cisplatin, doxorubicin, and mitoxantrone. The cells were also transplanted into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice to determine the changes in their tumorigenicity in vivo. Cells transfected with the WWOX-expressing plasmid stably expressed WWOX protein, while no WWOX protein was detected in control cells. Compared with the two types of control cells, WWOX-expressing stem cells manifested significantly reduced self-renewal ability. Compared with control cells, the expression levels of stem cell markers, including CD133, CD117, ATP-binding cassette sub-family G member 2, Nanog, octamer-binding transcription factor 4 and breast cancer resistance protein, were significantly lower in WWOX-expressing cells, while the level of the differentiation marker E-cadherin was significantly higher in WWOX-expressing cells. Furthermore, WWOX-expressing cells were more sensitive to treatment with cisplatin, doxorubicin and mitoxantrone. In NOD/SCID mice, the tumorigenicity of WWOX-expressing cells was significantly lower compared with that of control cells. The results indicate that the tumor suppressor WWOX suppresses stem cell properties in cancer stem cells, including self-renewal ability, differentiation potential, in vivo tumorigenic capability, high-level expression of stem cell genes and multidrug resistance.

Expression of WWOX and C-JUN in keloid

Objective To study the expression of WWOX and C-JUN in keloid and to approach their role and mechanism in the pathogenesis of keloid.Methods Immunohistochemical SP methods were used with computer pathological image analysis.Western blot and reverse transcription polymerase chain reaction (RT-RCR) were performed to detect the expression of WWOX and C-JUN in keloid and normal skin with statistical analysis.Results In keloid,the expression of WWOX protein was located in the cytoplasm of fibroblasts,and the expression of WWOX protein and its mRNA decreased,with significantly statistical difference (P＜0.05) compared to normal skin in the control group; the expression of C-JUN protein was located in the cell nucleus and cytoplasm of fibroblasts,with increased expression of C-JUN protein and its mRNA,with significantly statistical difference (P＜0.05) in comparison to normal skin in the control group.The expression of both was negative correlation (r=-0.626,P＜0.01).Conclusions Both WWOX with low expression and C-JUN with high expression are keloid-related genes,having significantly negative correlation between them,which may be one of the mechanisms for the keloid formation.It indicates that the WWOX protein may be an inhibitory factor to the expression of C-JUN protein,and the genes may play a major role in the pathogenesis of keloiod through fibroblasts. Key words: WW domain containing oxidoreductase (WWOX) ; Cellular Jun (C-JUN) ; Fibroblast ; Keloid

Abstract 1397: Simultaneous down-regulation of WWOX and DOK1 genes in non-small cell lung cancer (NSCLC) patients

Abstract Lung cancer is the main cause of cancer-related death worldwide and genetic, epigenetic and environmental factors affect the outcome of the disease. The WW domain-containing oxidoreductase (WWOX) gene is a putative tumor suppressor gene located on chromosome 16q23.3-24.1. It is composed of 9 exons and codes for a 414 amino acid protein. The WWOX gene has been previously reported to lose its tumor suppressor effect in several types of cancer including lung, ovarian, breast, stomach, uroepithelial and nasopharyngeal carcinomas. Docking Protein 1 (DOK1) is another putative tumor suppressor gene located on chromosome 2p13 and is known to be down-regulated and hypermethylated in various types of cancer including lung, breast or head and neck cancers and Burkitt lymphoma. WWOX activates MEK2 (MAP2K2-mitogen activated protein kinase kinase 2) which plays a crucial role in mitogen growth factor signal transduction. This activation increases ERK signaling in the MAPK/ERK pathway leading to transcription. The WWOX protein has two WW (tryptophan) domains which recognize the proline-rich region of other proteins. The DOK1 protein has a proline-rich region on its COOH terminal which may interact with the WWOX protein. DOK1 activates the RAS-GAP (GTPase activating proteins) signaling pathway leading to transcription when it is phosphorylated. Both WWOX and DOK1 also play important roles in the wnt/beta-catenin signaling pathway. The aim of this study was to investigate the relationship between WWOX and DOK genes by analyzing the expression levels of WWOX and DOK1 using RT-PCR in tumor tissue samples obtained from patients with non small cell lung cancer (NSCLC). When we analyzed expression levels by Spearman's Rank Correlation Coefficient Test, we observed down-regulation of the WWOX and DOK1 expression by 92.3% and 69.4% of the patients, respectively. Expression levels of WWOX and DOK both decreased simultaneously in 66.7% of the patients. We found a significant positive correlation between the expression levels of the WWOX and DOK1 genes when analyzed using the Wilcoxon Signed Ranks Test (p=0.033). Citation Format: Onur Baykara, Seda Ekizoglu, Esin Tuna, Ahmet Demirkaya, Kamil Kaynak, Hikmet Kose, Nur Buyru. Simultaneous down-regulation of WWOX and DOK1 genes in non-small cell lung cancer (NSCLC) patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2014-1397

Molecular alterations of the WWOX gene in nasopharyngeal carcinoma

The WW domain-containing oxidoreductase (WWOX) gene is a candidate tumor suppressor gene. However, its exact mechanism is unclear. This study aimed to investigate the role of the WWOX gene in the development of nasopharyngeal carcinoma (NPC). Tissues were collected from 65 NPC patients. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry were performed on NPC tissues to determine the expression of WWOX in NPC. The status of WWOX promoter methylation was analyzed by methylation-specific PCR. Moreover, a PCR-based loss of heterozygosity (LOH) assay was conducted to detect the presence of WWOX deletion in NPC. The expression of WWOX in NPC tissues was significantly downregulated compared with that in non-tumorous tissues (P<0.05). The low expression of WWOX was significantly correlated with clinical TNM stage (P<0.05). In addition, methylation of WWOX was detected in 27 (87%) of 31 WWOX protein negative tissues, suggesting that methylation of the WWOX promoter may regulate its expression. We found that a relatively high percentage of LOH was observed in NPC tissues. A significant inverse correlation between WWOX expression and methylation of its promoter was found in NPC tissue (rs=-0.582, P=0.001). However, LOH was not correlated with WWOX expression and methylation of its promoter. Our results show that WWOX gene alteration is an early genetic alteration and may contribute to tumorigenesis of NPC. WWOX may be an important prognostic marker in NPC.

Correlation of WWOX, RUNX2 and VEGFA protein expression in human osteosarcoma

BackgroundTo investigate associations between WW domain-containing oxidoreductase (WWOX), runt-related transcription factor 2 (RUNX2) and vascular endothelial growth factor alpha (VEGFA) in human osteosarcoma (OS).MethodsCopy number aberrations of WWOX, RUNX2and VEGFA genes were detected by microarray comparative genomic hybridization (aCGH) in 10 fresh OS tissue samples. VEGFA gene alterations were also investigated and validated by fluorescence in situ hybridization (FISH) in 54 formalin-fixed and paraffin-embedded (FFPE) OS samples. Protein expression of WWOX, RUNX2 and VEGFA were examined in 54 FFPE OS samples by immunohistochemistry (IHC).ResultsAnalysis of previously published OS aCGH data (GSE9654) and aCGH data from this study (GSE19180) identified significant deletion of WWOX in 30% (6/20) of OS samples, whilst significant increase in both RUNX2 and VEGFA gene copy numbers were detected in 55% (11/20) and 60% (12/20) of OS samples, respectively. FISH demonstrated increased VEGFA gene copy number in 65.9% (31/47) of evaluable samples, in either focal or large fragment forms. Compared with positive expression of WWOX in 38.9% of the OS samples, positive expression of RUNX2 and VEGFA protein was found in 48.1 and 75.9% of samples. Although there was no significant association between gene copy number aberration and protein expression for WWOX and RUNX2, significant positive correlation between increased VEGFA gene copy number and VEGFA protein expression was observed. Although there was no significant reverse association between WWOX and RUNX2 expression, a significantly positive relationship was observed between RUNX2 and VEGFA protein expression.ConclusionsOur data show increased RUNX2 and VEGFA gene copy numbers and elevation of their respective proteins in human OS. Positive correlation of RUNX2 and VEGFA suggests that both increased VEGFA gene copy number and RUNX2 overexpression facilitate increased expression of VEGFA.