WWOX Protein Expression Research Articles

WWOX-rs13338697 genotype predicts therapeutic efficacy of ADI-PEG 20 for patients with advanced hepatocellular carcinoma.

Previous studies have identified three single nucleotide polymorphisms (SNPs): GALNT14-rs9679162, WWOX-rs13338697 and rs6025211. Their genotypes are associated with therapeutic outcomes in hepatocellular carcinoma (HCC). Herein, we examined whether these SNP genotypes could predict the clinical outcome of HCC patients treated with ADI-PEG 20. Totally 160 patients with advanced HCC, who had previously been enrolled in clinical trials, including 113 receiving ADI-PEG 20 monotherapy (cohort-1) and 47 receiving FOLFOX/ADI-PEG 20 combination treatment (cohort-2), were included retrospectively. The WWOX-rs13338697-GG genotype was associated with favorable overall survival in cohort-1 patients (P = 0.025), whereas the rs6025211-TT genotype was associated with unfavorable time-to-tumor progression in cohort-1 (P = 0.021) and cohort-1 plus 2 patients (P = 0.008). As ADI-PEG 20 can reduce plasma arginine levels, we examined its pretreatment levels in relation to the WWOX-rs13338697 genotypes. Pretreatment plasma arginine levels were found to be significantly higher in patients carrying the WWOX-rs13338697-GG genotype (P = 0.006). We next examined the association of the WWOX-rs13338697 genotypes with WWOX tissue protein levels in 214 paired (cancerous/noncancerous) surgically resected HCC tissues (cohort-3). The WWOX-rs13338697-GG genotype was associated with decreased tissue levels of WWOX and ASS1. Mechanistic studies showed that WWOX and ASS1 levels were downregulated in hypoxic HCC cells. Silencing WWOX to mimic low WWOX protein expression in HCC in patients with the WWOX-rs13338697-GG genotype, enhanced HIF1A increment under hypoxia, further decreased ASS1, and increased cell susceptibility to ADI-PEG 20. In summary, the WWOX-rs13338697 and rs6025211 genotypes predicted treatment outcomes in ADI-PEG 20-treated advanced HCC patients. The WWOX-rs13338697-GG genotype was associated with lower tissue WWOX and ASS1 levels and higher pretreatment plasma arginine levels, resembling an arginine auxotrophic phenotype requires excessive extracellular arginine supply. Silencing WWOX to mimic HCC with the WWOX-rs13338697-GG genotype further stimulated HCC cell response to hypoxia through increased HIF1A expression, leading to further reduction of ASS1 and thus increased cell susceptibility to ADI-PEG 20.

Interaction of Wwox with Brca1 and associated complex proteins prevents premature resection at double-strand breaks and aberrant homologous recombination

Down regulation of Wwox protein expression occurs in many cancers, contributing to insensitivity to ionizing radiation (IR) and platinum drug treatments. Patients with reduced Wwox expression in their cancer tissue show decreased overall survival following these treatments, in accord with our earlier finding that reduced Wwox protein expression in cancers is associated with changes in choice of DNA double-strand break (DSB) repair pathway. Our current investigation of mechanisms underlying the initial choice of repair by homologous recombination/single-strand annealing (HR/SSA) in Wwox-deficient cells, showed immediate DNA end-resection at DSBs following IR, abrogating initial repair by the expected non-homologous end-joining (NHEJ) pathway. Mechanisms supporting the expected choice of DSB repair by NHEJ in Wwox-sufficient cells are: 1) direct recruitment of Wwox protein binding to Brca1 through the Brca1 981PPLF984 Wwox-binding motif; 2) possible Wwox blocking of Brca1-Rad50 interaction and of Brca1 activation by Chk2 phosphorylation of Brca1 S988; 3) Wwox suppression of Brca1 interaction with the B and C complex proteins, Brip1 and CtIP, thereby delaying the process of DSB end-resection post-IR. Wwox deficiency, instead, leads to early formation of the Brca1-CtIP/MRN complex at induced DSBs, stimulating immediate post-IR end-resection. This premature resection at DNA DSBs leads to inappropriate HR/SSA repair not restricted to late S/G2 cell cycle phases, and increases mutations in genomes of radiation or platinum-resistant colonies. Prevention of premature initiation of end-resection, by combining Chk2 inhibition with IR or carboplatin treatment, successfully sensitized IR and platinum-resistant Wwox-deficient cells by synthetic lethality, but did not alter response of Wwox-sufficient cells. Our results establish Wwox as a biomarker for treatment response and provide potential targets, such as Chk2, for reversal of treatment resistance.

Cellular Expression and Subcellular Localization of Wwox Protein During Testicular Development and Spermatogenesis in Rats.

A well-known putative tumor suppressor WW domain-containing oxidoreductase (Wwox) is highly expressed in hormonally regulated tissues and is considered important for the normal development and function of reproductive organs. In this study, we investigated the cellular and subcellular localization of Wwox in normal testes during postnatal days 0-70 using Western blotting and immunohistochemistry. Wwox is expressed in testes at all ages. Immunohistochemistry showed that fetal-type and adult-type Leydig cells, immature and mature Sertoli cells, and germ cells (from gonocytes to step 17 spermatids) expressed Wwox except peritubular myoid cells, step 18-19 spermatids, and mature sperm. Wwox localized diffusely in the cytoplasm with focal intense signals in all testicular cells. These signals gradually condensed in germ cells with their differentiation and colocalized with giantin for cis-Golgi marker and partially with golgin-97 for trans-Golgi marker. Biochemically, Wwox was detected in isolated Golgi-enriched fractions. But Wwox was undetectable in the nucleus. This subcellular localization pattern of Wwox was also confirmed in single-cell suspension. These findings indicate that Wwox is functional in most cell types of testis and might locate into Golgi apparatus via interaction with Golgi proteins. These unique localizations might be related to the function of Wwox in testicular development and spermatogenesis.

Abstract 16915: Potential Role of Endothelial Cell WWOX in the Pulmonary Vascular Response to Hypoxia

Introduction: Pulmonary arterial hypertension (PAH) is characterized by abnormal proliferation and dysfunction of pulmonary artery smooth muscle and endothelial cells (PASMC, PAEC). The WWOX/ Wwox gene encodes a tumor suppressor WW domain-containing oxidoreductase, known as WWOX. WWOX has been linked to several protein networks, highlighting its functions in cell homeostasis, survival and proliferation. Loss of WWOX gene expression is observed in human cancer cells, as lung cancer. We hypothesize that during hypoxia, WWOX expression is modulated in PAECs, regulating cell proliferation and selecting apoptosis-resistant cells, thus causing vascular remodeling. Methods and Results: Endothelial cell specific Wwox knockout mice were developed by a conditional ablation of Wwox gene expression using the Cre -Lox site-specific recombination system. We crossed Wwox flox/flox mice with CDH5 Cre+ mice, generating Wwox floxed, CDH-Cre+ specific mice that are tamoxifen-inducible (EC- WWOX KO). EC-WWOX KO mice were treated daily with tamoxifen or vehicle for 5 days and exposed to 10% hypoxia. After four weeks of hypoxia exposure, EC-WWOX KO mice showed increased right ventricular systolic pressure (RVSP) (P=0.0003) and Fulton index (RV (LV+S)) ( P=0.049), when compared to controls, suggesting PH development. In vitro , PAECs exposure to 3% hypoxia for 6 hours showed an increase of WWOX mRNA (P=0.0154) and protein (P<0.05) expression levels, decreased of cell proliferation detected by CyQUANT assay and PCNA protein expression (P=0.0024). These effects did not extend to cells exposed to 3% hypoxia for 24h. Immunoblots showed activation of apoptotic markers, such as cleaved caspase 3, PARP and Bax, suggesting that in hypoxia there is a potential controlled balance between increased WWOX expression, decreased cell proliferation and stimulation of apoptosis in these cells. Conclusion: Loss of EC WWOX expression plays a significant role in the development of PH in mice exposed to chronic hypoxia. We speculate that increased WWOX expression with acute hypoxia exposure could contribute to the selection of proliferative and pro-apoptotic PAECs, characteristic of PAH. Studies seeking to understand the mechanisms surrounding WWOX expression in PAECs are ongoing.

Abstract LB-091: Tumor suppressor WW domain-containing oxidoreductase controls epidermal keratinocyte growth

Abstract Human fragile WWOX gene encodes a candidate tumor suppressor WW domain-containing oxidoreductase. Ectopically overexpressed WWOX protein induces apoptosis in cancer cells and suppresses tumor growth both in vitro and in vivo. Functional suppression of WWOX by antisense mRNA, dominant negatives, and small interfering RNA protects cells from apoptosis induced by tumor necrosis factor, anticancer drugs, UV light, and ectopic p53 in vitro. Interestingly, substantial studies have revealed that progression of various cancers to a pre-metastatic stage positively correlates with upregulation of WWOX protein expression, suggesting that WWOX may act more than a tumor suppressor. In normal human epidermis, WWOX is abundant in proliferating epithelial cells of the skin, and its expression is increased in intensity toward the superficial layers. In this study, we determined a reduced number of Ki67-positive proliferating keratinocytes in the epidermis of our newly developed Wwox gene knockout (Wwox-/-) mice. A marked reduction in the basal (K5-positive) and suprabasal (K10-positive) keratinocyte numbers was examined in the keratinized squamous epithelium of the Wwox-/- mouse skin. Apparent decreases in Wwox-/- epidermal and dermal thickness were found. To further verify whether WWOX regulates keratinocyte growth, we detected significantly decreased cell proliferation in both Wwox-/- primary keratinocyte cultures and WWOX-knockdown HaCaT cells as compared to the control cells. No significant increases in the number of TUNEL-positive cells were observed in the Wwox-/- epidermal tissues. Taken together, our results clearly demonstrated a crucial role of tumor suppressor WWOX in regulating normal keratinocyte proliferation for maintaining skin homeostasis. Note: This abstract was not presented at the meeting. Citation Format: Li-Jin Hsu. Tumor suppressor WW domain-containing oxidoreductase controls epidermal keratinocyte growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-091.

WW Domain‐containing Oxidoreductase Regulates Keratinocyte Growth for Maintaining Epidermal Homeostasis

Human fragile WWOX gene encodes a candidate tumor suppressor WW domain‐containing oxidoreductase. Previous studies have shown that ectopically overexpressed WWOX induces apoptosis in cancer cells and suppresses tumor growth both in vitro and in vivo. Functional suppression of WWOX by antisense mRNA, dominant negatives, and small interfering RNA protects cells from apoptosis by tumor necrosis factor, anticancer drugs, UV light, and ectopic p53 in vitro. Interestingly, substantial evidence revealed that progression of various cancers to a pre‐metastatic stage positively correlates with upregulation of WWOX protein expression, suggesting that WWOX may act more than a tumor suppressor. In normal human epidermis, WWOX is abundant in proliferating epithelial cells of the skin, and its expression is increased in intensity toward the superficial layers. In this study, we determined a reduced number of Ki67‐positive proliferating keratinocytes in the epidermis of our newly developed Wwox gene knockout (Wwox−/−) mice. A marked reduction in the basal (K5‐positive) and suprabasal (K10‐positive) keratinocyte numbers was examined in the keratinized squamous epithelium of the Wwox−/− mouse skin. Apparent decreases in Wwox−/− epidermal and dermal thickness were found. To further verify whether WWOX regulates keratinocyte growth, we detected significantly decreased cell proliferation in both ex vivo Wwox−/− primary keratinocyte cultures and WWOX‐knockdown HaCaT cells as compared to the controls. No significant increase in the number of TUNEL‐positive cells was observed in the Wwox−/− epidermis. Moreover, our recent data showed that keratinocyte differentiation was slightly enhanced in WWOX‐knockdown HaCaT cells. Together, our results clearly demonstrated a crucial role of WWOX in regulating normal keratinocyte proliferation for maintaining skin homeostasis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

WW Domain‐containing Oxidoreductase Induces Cellular Senescence in Response to Replication Stress

The induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for the maintenance of cellular genome integrity during DNA replication. Under replication stress, cellular senescence is a DDR mechanism that prevents the proliferation of cells with DNA damage to avoid mitotic anomalies and inheritance of the damage over cell generations. A reduction in the ability of cells to trigger senescence has been suggested to be associated with cancer initiation and progression. Human WWOX gene resides within a common chromosomal fragile site FRA16D and encodes a tumor suppressor WW domain‐containing oxidoreductase. Downregulation of WWOX protein expression has been frequently found in various human cancers. Increased genome instability has been shown in the cells null for WWOX. However, the role of WWOX in DDR remains largely unclear. In this study, we demonstrated that Wwox gene knockout (Wwox−/−) mouse embryonic fibroblasts (MEFs) failed to undergo replication‐induced cellular senescence after multiple passages in culture. Strikingly, accelerated cell cycle progression and increased apoptosis were detected in late‐passage Wwox−/− MEFs. Increased γH2AX‐positive cells were observed in late‐passage Wwox−/− MEFs, indicating massive accumulation of DNA damages in these cells. The p16INK4A/Rb‐ and p53/p21cip1/waf1‐mediated signaling pathways have been suggested to control cellular senescence. We determined significantly reduced expression of p16INK4A and p21cip1/waf1 proteins in late‐passage Wwox−/− MEFs. Treatment of Wwox−/− MEFs with DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine reversed p16INK4A and p21cip1/waf1 expression, and ectopic expression of p16INK4A and p21cip1/waf1 increased cellular senescence in late‐passage Wwox−/− MEFs. These results suggest that hypermethylation of p16INK4A and p21cip1/waf1 promoters may lead to the failure of senescence induction in Wwox−/− MEFs. Moreover, inhibition of ROS production by N‐acetyl‐L‐cysteine during cell replication in vitro increased protein expression of p16INK4A and p21cip1/waf1 and the induction of cellular senescence in Wwox−/− MEFs. In conclusion, our results support a crucial role for WWOX in DDR for maintaining genome integrity via the induction of cellular senescence in response to replication stress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correlation between osteosarcoma and the expression of WWOX and p53.

The objective of this study was to analyze the effect of the expression of WWOX and p53 on the growth of MG-63 osteosarcoma cells and to explore the correlation between osteosarcoma and the expression of WWOX and p53. WWOX and p53-overexpressing MG-63 osteosarcoma cell lines were established by transfection and named the MW and MP cell lines, respectively. Untransfected MG-63 cells (blank control) were used as control. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of WWOX and wild-type p53 mRNA and protein, respectively. The effects of WWOX and p53 (wild-type) on the activity of MG-63 cells were determined by MTT assay and flow cytometry. The expression of mutant p53 protein in 65 cases of osteosarcoma was detected by immunohistochemistry to analyze the correlation between p53 and the development of osteosarcoma. qPCR showed that WWOX and p53 mRNA was overexpressed in MW and MP cells, respectively. Western blot analysis showed that the levels of WWOX and p53 protein in MW and MP cells were higher than in the blank control group. MTT assay showed that the cell proliferation ability of MW and MP cells was significantly lower than in the blank control group. Flow cytometry showed that 78.49% of MW and 66.76% of MP cells were arrested in the G0/G1 phase. Immunohistochemistry showed that mutant p53 was highly expressed in osteosarcoma, with a positive expression rate of 47.7%. The expression rate was positively correlated with the pathological grade of cancer. In conclusion, WWOX can affect the cell cycle of MG-63 osteosarcoma cells to inhibit cell proliferation, which provides new insights into gene therapy for osteosarcoma. The two types of the p53 gene have different functions in the development of osteosarcoma. Wild-type p53 acts as a tumor suppressor, while mutant p53, which is overexpressed in malignant osteosarcoma, has a carcinogenic effect associated with the degree of osteosarcoma.

Wwox\u2013Brca1 interaction: role in DNA repair pathway choice

In this study, loss of expression of the fragile site-encoded Wwox protein was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. WWOX gene deletions occur in a variety of human cancer types, and reduced Wwox protein expression can be detected early during cancer development. We found that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox-knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). Cancer cells that survive radiation recur more rapidly in a xenograft model of irradiated breast cancer cells; Wwox-deficient cells exhibited significantly shorter tumor latencies vs Wwox-expressing cells. This Wwox effect has important consequences in human disease: in a cohort of cancer patients treated with radiation, Wwox deficiency significantly correlated with shorter overall survival times. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR) and decreased non-homologous end-joining (NHEJ) repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. Upon silencing of Rad51, a protein critical for HDR, Wwox-deficient cells were resensitized to radiation. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for WWOX, in which Brca1–Wwox interaction supports NHEJ as the dominant DSB repair pathway in Wwox-sufficient cells. Taken together, the experimental results suggest that reduced Wwox expression, a common occurrence in cancers, dysregulates DSB repair, enhancing efficiency of likely mutagenic repair, and enabling radiation and cisplatin treatment resistance.

Fhit and Wwox loss-associated genome instability: A genome caretaker one-two punch.

Expression of Fhit and Wwox protein is frequently lost or reduced in many human cancers. In this report, we provide data that further characterizes the molecular consequences of Fhit loss in the initiation of DNA double-strand breaks (DSBs), and of Wwox loss in altered repair of DSBs. We show that loss of Fhit initiates mild genome instability in early passage mouse kidney cells, confirming that DNA damage associated with Fhit-deficiency is not limited to cancer cells. We also demonstrate that the cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation in early passage mouse kidney cells before extensive genome instability occurs. As for consequences of Wwox loss in cancer, we show in a small panel of breast cancer cells and mouse embryonic fibroblasts that Wwox expression predicts response to radiation and mitomycin C, all agents that cause DSBs. In addition, loss of Wwox significantly reduced progression free survival in a cohort of ovarian cancer patients treated with platin-based chemotherapies. Finally, stratification of a cohort of squamous lung cancers by Fhit expression reveals that Wwox expression is significantly reduced in the low Fhit-expressing group, suggesting that loss of Fhit is quickly succeeded by loss of Wwox. We propose that Fhit and Wwox loss work synergistically in cancer progression and that DNA damage caused by Fhit could be targeted early in cancer initiation for prevention, while DNA damage caused by Wwox loss could be targeted later in cancer progression, particularly in cancers that develop resistance to genotoxic therapies.

Effects of 5-Aza-2′-deoxycytidine on the methylation state and function of the WWOX gene in the HO-8910 ovarian cancer cell line

The aim of this study was to explore the effects of 5-Aza-2′-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor, on the methylation state and function of the WWOX gene in the HO-8910 ovarian cancer cell line. The HO-8910 cells were divided into two groups, a control group and a 5-Aza-CdR-treated group. The methylation state of the WWOX gene was evaluated using a methylation-specific PCR assay. The effect of 5-Aza-CdR on the HO-8910 cells was analyzed using MTT and cell invasion assays, as well as flow cytometry. The animal models were established by intraperitoneal transplantation of the cells into nude mice. Following treatment with 5-Aza-CdR, a demethylation state was detected in the HO-8910 cells. WWOX protein expression was significantly higher in the 5-Aza-CdR-treated group compared with that in the control group. The cell growth rate at each tested time point and the number of invasive cells were lower in the 5-Aza-CdR-treated group compared with that in the control group. Flow cytometry revealed that 67.13% of the cells were arrested at the G0/G1 stage in the 5-Aza-CdR-treated group. The tumorigenic ability of the 5-Aza-CdR-treated group was lower compared with that of the control group. In conclusion, the methylation state of the WWOX gene in HO-8910 cells may be reversed using 5-Aza-CdR, which may also inhibit the growth of these cells.

Hypoxia inducible factor-1 is activated by transcriptional co-activator with PDZ-binding motif (TAZ) versus WWdomain-containing oxidoreductase (WWOX) in hypoxic microenvironment of bone metastasis from breast cancer

The hypoxic microenvironment of bone marrow favours the bone metastasis process. Hypoxia inducible factor (HIF)-1α is hallmark for hypoxia, correlating with poor prognosis and radio/chemotherapy resistance of primary-breast carcinoma. For bone metastasis, the molecular mechanisms involved in HIF-1α expression and HIF-1 (α/β heterodimer)-transcription factor activity are scarcely known. We studied the role played by HIF-1 in the cross-talk between neoplastic and supportive-microenvironmental cells. Also, WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ) were taken into consideration evaluating whether these Hippo-pathway effectors affect bone-metastatic phenotype through HIF-1 activity. Considering bone-metastasis specimens, nuclear HIF-1α–TAZ co-localisation occurred in neoplastic and supportive cells, such as fibroblasts and endotheliocytes. Based on these data, the functional importance was verified using 1833-bone metastatic clone under hypoxia: nuclear HIF-1α and TAZ expression increased and co-immunoprecipitated, activating HIF-1-DNA binding and transactivation. In contrast, Wwox localised at perinuclear level in neoplastic cells of bone metastasis, being almost absent in supportive cells, and Wwox-protein expression diminished in hypoxic-1833 cells. Thus, TAZ regulation of HIF-1 activity might be important for bone-secondary growth, participating in metastasis-stroma cross-talk. Further, TAZ and HIF-1α-protein levels seemed correlated. In fact, blocking cyclooxygenase-2 with NS398 in hypoxic-1833 cells, not only HIF-1α decreased but also molecular-mechanism(s) upstream of the Hippo pathway were triggered: LATS-dependent TAZ phosphorylation seemed responsible for TAZ nucleus/cytoplasm translocation and degradation. In the 1833-xenograft model, NS398 largely prevented the outgrowth of bone-metastatic cells, probably related to remarkable-extracellular matrix assembly. We gained clinical insight into HIF-1α and TAZ as candidate biomarkers for bone avidity, relevant for early-therapeutic intervention against bone metastasis.

Expression of BRCA1 and WWOX and their clinicopathologic implication in breast carcinomas occurring in young women

To explore the clinicopathologic characteristics and biological markers of breast carcinomas in young women. Immunohistochemical SP method was used to study breast cancer susceptibility gene (BRCA1) and WWOX in breast carcinomas of patient ≤ 35 years of age (107 cases) and ≥ 60 years of age (112 cases). The findings were correlated with clinicopathological features. In addition, PCR amplification and direct sequencing were performed to detect the BRCA1 gene mutation of exons 2 and 20 using fresh frozen tissue samples in other 10 patients who were ≤ 35 years of age. The positive rate of BRCA1 protein expression was higher in the young age group [65.4% (70/107)] than that of the old age group [35.7% (40/112)]. ER, PR, HER2, and WWOX protein expression and proliferation marker Ki-67 were no statistically different in the two groups (all P > 0.05). BRCA1 expression was significantly correlated with pTNM and axillary lymph node metastasis (both P < 0.05), but not with ER, PR, HER2 and WWOX protein expression (all P > 0.05). Ki-67 and histological grading showed no statistical correlation (P > 0.05). WWOX protein expression showed no correlation with clinicopathologic characteristics (all P > 0.05). Mutation of exons 2 and 20 of the BRCA1 gene was not detected in any of 10 cases studied. BRCA1 cytoplasmic expression statistically correlates with the development and prognosis of breast cancer of young patients.

WWOX Protein Expression Varies Among RCC Histotypes and Downregulation of WWOX Protein Correlates with Less-Favorable Prognosis in Clear RCC

WWOX has been shown to be a candidate tumor suppressor gene in numerous human cancers. The objective of this study is to determine the expression of WWOX in human renal cell carcinoma tumor cells and its possible correlation with clinical outcome. The WWOX protein expressions of human renal cell carcinoma (RCC) tumor and of matched normal renal parenchyma were examined, and its correlation with clinical cancer-specific survival was investigated. Downregulation of WWOX only in clear cell type RCC was demonstrated in our results including immunohistochemistry, Western blot, and RT-PCR assay. For the remnant cell types of RCC, sample sizes were insufficient to draw any conclusion of WWOX protein expression. The decreased expression of WWOX in clear cell RCC tumor compared with matched normal renal parenchyma was also correlated with clinical cancer-specific survival (Kaplan-Meier, p=0.0482). We proved that the expression level of WWOX is downregulated in human clear cell RCC. Moreover, the decreased expression of WWOX in clear cell RCC tumor compared with matched normal renal parenchyma was also correlated with clinical cancer-specific survival. Since clear cell RCC is a special human cancer using unique molecular pathogenesis, further investigation will provide more linking intracellular signaling of WWOX and novel therapeutic targets of human renal cell carcinoma.

Effect of transfection of WWOX gene on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism

Objective To investigate the effect of transfection of WWOX gene on apoptosis of human cholangiocarcinoma QBC939 cells in vitro andin vivoand its possible mechanism.Methods The recombinant WWOX eukaryotic expression plasmid was introduced into QBC939 cells by liposome-mediated transfection.The mRNA and protein expression levels in QBC939 cells stably transfected with WWOX were detected by using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting before and after transfection.Cell apoptosis was assessed by flow cytometry (FCM).The alteration of mitochondria membrane potential ( △ Ψm) was assayed by JC-l staining method.The expression change of bcl-2 mRNA and protein was examined by using quantitative RT-PCR and Western blotting.All the experimental subjects were divided into three groups:QBC939 cells were cultured in natural status (nature control group) ; QBC939 cells were transfected with blank plasmid (blank control group),and QBC939 cells were transfected with WWOX/pmCherry-N1 (experimental group).The three groups were subcutaneously inoculated into nude mice and the growth of xenografted tumor was observed.TUNEL was used to evaluate the apoptosis.Results QBC939 cells with stable transfection of WWOX were established.QuantitativeRT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western blotting demonstrated that WWOX protein expression was markedly increased.FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.24 ±0.35)％ vs (1.73 ±0.48)％ vs (21.40 ±2.35)％,P＜0.01].JC-1 staining method indicated that the experimental group was loss of △Ψm [ (4.27 ± 0.64) ％ vs (4.96 ± 0.52 ) ％ vs (28.60 ± 3.94 ) ％,P ＜ 0.01 ].Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased (P ＜O.05 ).The growth of implanted tumor of WWOX/pmCherry-N1 cells was significantly slowed down (P ＜0.05 ).Number of apoptotic cells was significantly increased in the experimental group [ (4.1 ± 0.6) ％ vs (4.4±0.5)％ vs (13.6 ± 1.5)％,P＜0.01].Conclusion WWOX gene can promote apoptosis of cholangiocarcinoma cells,which may be associated with downregulation of bcl-2 expression,and activation of the mitochondrial apoptosis pathway. Key words: Cholangiocarcinoma; Gene transfection; Apoptosis

Wwox inactivation enhances mammary tumorigenesis

Breast cancer is the leading cause of cancer-related death in women worldwide. Expression of the WWOX tumor suppressor is absent or reduced in a large proportion of breast tumors suggesting that loss of WWOX may contribute to breast tumorigenesis. Wwox-deficient mice die by 3-4 weeks of age precluding adult tumor analysis. To evaluate the effect of WWOX-altered expression on mammary tumor formation, the Wwox-heterozygous allele was back crossed onto the C3H mammary tumor-susceptible genetic background (Wwox(C3H)+/-) and incidence of mammary tumor formation was evaluated. Although 50% of the female Wwox(C3H)+/- mice developed mammary carcinomas, only 7% of Wwox(C3H)+/+ mice did. Intriguingly, mammary tumors in Wwox(C3H)+/- mice frequently lost WWOX protein expression suggesting a genetic predisposition toward mammary tumorigenesis. Immunohistochemical staining of hormone receptors revealed loss of estrogen receptor-α (ER) and progesterone receptor in the majority of these tumors. In vitro, depletion of WWOX in MCF7 ER-positive cells led to reduced ER expression and reduced sensitivity to tamoxifen and estrogen treatment and was associated with enhanced survival and anchorage-independent growth. Finally, cDNA array analyses of murine normal mammary epithelial cells and mammary tumors identified 163 significantly downreguated and 129 upregulated genes in the tumors. The majority of differentially expressed genes were part of pathways involved in cellular movement, cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation and cell death. These changes in gene expression of mouse mammary tumors in Wwox(C3H)+/- mice resemble, at least in part, human breast cancer development. Our findings demonstrate the critical role that the WWOX tumor suppressor gene has in preventing tumorigenesis in breast cancer.

871 Deficiency of the WWOX Fragile Gene Impairs DNA Damage Response

Oral leukoplakia is the most prevalent and potentially malignant disorder of the oral mucosa. Previous studies have demonstrated that molecular changes of the WWOX gene (WW-domain containing oxidoreductase), a candidate tumor suppressor gene located at 16q23.3–24.1 that spans FRA16D, the second most common fragile site, are present in several malignant neoplasias, including oral squamous cell carcinoma. In this report, the role of the WWOX gene was investigated in 23 cases of oral leukoplakias. Using nested RT-PCR and immunohistochemistry, altered mRNA transcription and/or reduced Wwox protein expression was observed in 35% of the lesions when compared with normal mucosa. The majority of lesions (4/6) with altered transcripts had a reduction in the expression of Wwox protein. Although normal WWOX expression was found in some lesions with dysplasia, all lesions with WWOX mRNA and/or protein expression showed histological evidence of dysplasia and none of the cases without dysplasia presented this alteration. These results show that the WWOX gene alteration is an early genetic alteration and may contribute to oral carcinogenesis.

Abstract 2150: Genetic alterations in the major pathways in human osteosarcoma and their clinical significance

Abstract Background Osteosarcoma (OS) is a genetically unstable malignancy that most frequently occurs in children and young adults. It has high incidence of metastasis and recurrence after treatment. The lack of progress in clinical management of this devastating disease prompts us to take a systems approach to profile key genetic alterations at the level of gene copy number aberration on a genome-wide scale and to interrogate the pathways that are critical for the hallmarks of OS. Our goal is to gain insight into potentially novel targets for therapeutics. Methods We conducted a high-density genome-wide array comparative genomic hybridization (aCGH) profiling with genomic DNA isolated from 10 fresh osteosarcoma tissues obtained from the Tumor Tissue Bank of the Tianjin Medical University Cancer Institute and Hospital (GSE19180). In addition, we downloaded aCGH data of an independent investigation from the GEO database (GSE9654). The patterns of the two independent aCGH data showed marked similarities. The pooled aCGH data were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to identify altered pathways. Protein expression of specific genes was examined by immunohistochemistry (IHC) on 58 archival formalin fixed, paraffin embedded (FFPE) tissues and correlated with clinical follow-up information. Results Genome-wide aCGH analysis revealed that 1p35-1p35, 1q23.1-1q21.1, 6p22.1-6p21.31, and 19p13.11-p13.2 were significantly amplified, whereas 5q12.3-5q13.2, 5q14.3-5q22.2, and 13q13.2-13p14.3 were significantly deleted. These regions included 1,162 genes with amplifications and 146 genes with deletions. Pathway analysis showed that a number of cancer related pathways were significantly altered. These pathways included VEGF, cell adhesion, adherins junction, non-homologous end-joining, hedgehog signaling, and Wnt signaling pathways. Among the VEGF pathway genes, 13 genes including VEGFA itself were amplified. Detailed analysis showed that VEGFA protein expression (74.1%) was associated with microvascular density (MVD) (20.81±2.04 per mm2) and was an independent predictor for metastasis and/or recurrence (p=0.01). The aCGH analysis also led to the discovery that the tumor suppressor gene WWOX was deleted in 30% of OS. IHC analysis showed that loss of WWOX protein expression was even more frequent (61.8%), suggesting deletion of WWOX gene and ablated expression might be an important early event in OS pathogenesis. In addition, we found that the APEX1 gene was amplified in 60% in OS and the abundant expression of APEX1 protein (64.9%) was an independent predictor for the local recurrence and/or metastasis (p=0.04). Conclusions The systematic analysis of the genetic alterations has revealed involvement of multiple pathways in the pathogenesis and progression of OS suggesting that multi-pronged combinational therapy is needed for effective therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2150.

Study of FHIT and WWOX expression in mucoepidermoid carcinoma and adenoid cystic carcinoma of salivary gland

Mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma (ACC) are salivary gland neoplasms with divergent morphological features and clinical behavior. ACC is a basaloid tumor whereas MEC is a glandular epithelial neoplasm. FHIT and WWOX are tumor suppressor genes that encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3, respectively. In previous studies, we have shown concordant loss of Fhit and Wwox expression in breast cancer, with significantly more frequent loss in cancers of basal-like phenotype. To determine if there is a similar association in salivary gland neoplasms, we designed a study of MEC and ACC of salivary gland on tissue microarrays (TMA). TMAs were constructed from 25 MEC and 19 ACC of salivary gland. Fhit and Wwox protein expression was assessed by immunohistochemical staining of cores on TMAs. Correlations among immunohistochemical markers and histological type were determined by statistical analyses. Significantly reduced Fhit and Wwox expression was observed in ACC ( p = 0.002 and p < 0.001, respectively). The results suggest that, as for breast cancer, loss of Fhit and Wwox expression might have a role in the pathogenesis of basaloid differentiation in salivary gland neoplasms; alternatively, differences in chromatin structure at chromosome fragile regions might make fragile genes more accessible to DNA damage and rearrangement early during preneoplastic stages of basaloid cancers. Studies of basaloid tumors of other organ systems may show similar results and these findings may have implications for treatment modalities designed for basal-like tumors.

Expression of WWOX in hepatocellular carcinoma and resection margin

Objective To investigate the clinical significance of WWOX expression in hepatocellular carcinoma(HCC) and resection margin and explore its relation to postoperative recurrence and prognosis.Methods The expression of WWOX protein was detected with immunohistochemical method-SP in 76 cases of HCC and resection margin and 20 cases of normal liver tissues,while the apoptosis rate was detected with TUNEL method in 76 cases of HCC.The relationship between these data and clinicopathological features was analyzed with SPSS software.Results Abnormal nuclear localization of WWOX protein in HCC and nuclear/ cytoplasm localization in resection margin were detected.The WWOXLI in HCC was 35.2 %±20.1%,lower than those of resection margin and normal liver tissues (P<0.05).The lower level of WWOX expression in HCC was significantly related to the lower apoptosis rate,higher PCNALI,huge tumor,cancerous thrombus in branches of portal vein,lower differentiation,lower survival rate and postoperative recurrence etc.(P<0.05).The WWOX expression level in resection margin was significantly related to postoperative recurrence rate(P<0.05) but not the distance of resection margin.Conclusion The lower expression and abnormal cellular location of WWOX gene in HCC can be a frequent event,which can be related to some biological characteristics of HCC to influence its progress and prognosis.The expression level of WWOX gene in HCC and resection margin may be one of useful indicators in forecasting the progress and prognosis and be used as a safe biomarker of molecular margin. Key words: Carcinoma,hepatocellular; WWOX; Immunohistochemistry; Resection margin; Prognosis