51741

WWOX

D16S432E|EIEE28|FOR|FRA16D|HHCMA56|PRO0128|SCAR12|SDR41C1|WOX1;WW domain containing oxidoreductase;WWOX;WW domain containing oxidoreductase

WW domain-containing oxidoreductase|WW domain-containing protein WWOX|fragile site FRA16D oxidoreductase|short chain dehydrogenase/reductase family 41C member 1|short chain dehydrogenase/reductase family 41C, member 1

16q23

protein-coding

We previously reported the construction of a P1-derived artificial chromosome (PAC) contig encompassing a set of homozygous deletions of chromosome 16q23-24.1 found in primary ovarian tumor material and several tumor cell lines. Using these PAC clones in a cDNA selection experiment, we have isolated a Sau3A fragment homologous to the WWOX transcript (GenBank accession no. ) from normal human ovarian surface epithelial (HOSE) cells. We demonstrate the homozygous deletion of WWOX exons from ovarian cancer cells and three different tumor cell lines. We also identify an internally deleted WWOX transcript from a further primary ovarian tumor. In three of these samples the deletions result in frameshifts, and in each case the resulting WWOX transcripts lack part, or all, of the short chain dehydrogenase domain and the putative mitochondrial localization signal. Sequencing revealed several missense polymorphisms in tumor cell lines and identified a high level of single nucleotide polymorphism (SNP) within the WWOX gene. This evidence strengthens the case for WWOX as a tumor suppressor gene in ovarian cancer and other tumor types.

The WWOX (WW domain containing oxidoreductase) gene was recently identified as a candidate tumor suppressor gene at 16q23.3-24.1, a chromosome region that spans the common fragile site FRA16D. To evaluate the potential role of the WWOX gene in esophageal squamous cell carcinomas, we examined 36 tumors for genetic alterations of the WWOX gene. Loss of heterozygosity (LOH) at the WWOX locus was observed in 14 (39%) tumors. A tumor-specific missense mutation was found in one tumor, and LOH analysis had shown that the other allele was missing. Furthermore, we detected aberrant WWOX gene transcripts with absence of exons 6-8 in two tumors, and complete absence of transcript in one tumor. These results indicate that alteration and inactivation of the WWOX gene may play a role in esophageal squamous cell carcinogenesis.

WW domain-containing oxidoreductase (WWOX) is a candidate tumor suppressor gene. Because mutation or deletion in the coding region of WWOX is rarely found, it is speculated that the appearance of aberrant transcripts affects progression of various cancers. However, little is known about the role in these cancers of the WWOX protein. To characterize endogenous WWOX proteins, we analyzed WWOX expression using newly generated monoclonal antibodies. In immunoblot analysis of 49 cancer cell lines, only the normal form of the protein was detectable, although some of cell lines exhibited aberrant WWOX RNA transcripts. Accumulation of truncated proteins was observed by inhibiting proteasomal degradation with MG-132, whereas expression level of normal protein did not change, suggesting truncated proteins may be subjected to rapid degradation through proteasomal machinery. Immunohistochemistry for cancer cells demonstrated that WWOX protein levels are not decreased but rather elevated in gastric and breast carcinoma, challenging the notion of WWOX as a classical tumor suppressor. In noncancerous cells, WWOX was observed only in epithelial cells, including hormone-regulated cells such as Leydig cells, follicular cells, prostate epithelium, and mammary glands. Interestingly, restricted staining in nuclei was observed in some mammary gland cells while other epithelial cells exhibited localization of WWOX in cytoplasm. Nuclear localization of WWOX was also confirmed in confluent human fibroblast KMS-6, whereas WWOX was associated mainly with mitochondria before reaching confluence, indicating that WWOX shuttles between cytoplasm and nuclei. These findings provide novel insights into aspects of human WWOX function in both normal and malignant cells.

PURPOSE: WWOX (WW domain containing oxidoreductase) is a tumor suppressor gene that maps to the common fragile site FRA16D. We showed previously that WWOX is frequently altered in human lung and esophageal cancers. The purpose of this study was to delineate more precisely the role of WWOX in pancreatic carcinogenesis. EXPERIMENTAL DESIGN: We analyzed 15 paired pancreatic adenocarcinoma samples and 9 pancreatic cancer cell lines for WWOX alterations. Colony assay and cell cycle analysis were also performed to evaluate the role of the WWOX as a tumor suppressor gene. RESULTS: Loss of heterozygosity at the WWOX locus was observed in 4 primary tumors (27%). Methylation analysis showed that site-specific promoter hypermethylation was detected in 2 cell lines (22%) and treatment with the demethylating agent 5-aza-2-deoxycytidine demonstrated an increase in the expression of WWOX. In addition, 2 primary tumor samples (13%) showed promoter hypermethylation including the position of site-specific methylation. Transcripts missing WWOX exons were detected in 4 cell lines (44%) and in 2 tumor samples (13%). Real-time reverse transcription PCR revealed a significant reduction of WWOX expression in all of the cell lines and in 6 primary tumors (40%). Western blot analysis showed a significant reduction of the WWOX protein in all of the cell lines. Furthermore, transfection with WWOX inhibited colony formation of pancreatic cancer cell lines by triggering apoptosis. CONCLUSION: These results indicate that the WWOX gene may play an important role in pancreatic tumor development.

The WWOX gene encodes a tumor suppressor WW domain-containing protein, Wwox. Alterations of WWOX have been demonstrated in multiple types of cancer, and introduction of Wwox into Wwox-negative tumor cells has resulted in tumor suppression and apoptosis. The Wwox protein contains two WW domains that typically bind proline-rich motifs and mediate protein-protein interactions. Recently, we have described functional cross-talk between the Wwox protein and the p53 homologue, p73. To further explore the biological function of Wwox, we investigated other interacting candidates. In this report, we demonstrate a physical and functional association between AP-2gamma transcription factor and the Wwox protein. AP-2gamma at 20q13.2 encodes a transcription factor and is frequently amplified in breast carcinoma. We show that Wwox binds to the PPPY motif of AP-2gamma via its first WW domain. Alterations of tyrosine 33 in the first WW domain of Wwox or the proline-rich motif in AP-2gamma dramatically reduce this interaction. In addition, our results demonstrate that Wwox expression triggers redistribution of nuclear AP-2gamma to the cytoplasm, hence suppressing its transactivating function. Our results suggest that Wwox tumor suppressor protein inhibits AP-2gamma oncogenic activity by sequestering it in the cytoplasm.

Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, representing 90% of all oral carcinomas and accounting for 3-5% of all malignancies. The WWOX gene (WW-domain containing oxidoreductase) is a candidate tumor suppressor gene located at 16q23.3-24.1, spanning the second most common fragile site, FRA16D. In this report, the role of the WWOX gene was investigated in 20 tumors and 10 normal oral mucosas, and we demonstrated an altered WWOX gene in 50% (10/20) of OSCCs. Using nested RT-PCR, mRNA transcription was altered in 35% of the tumors, with the complete absence of transcripts in 2 samples as well as absence of exons 6-8 (2 tumors), exon 7 (1 tumor), exon 7 and exon 6-8 (1 tumor) and partial loss of exons 8 and 9 (1 tumor). To determine if the aberrant transcripts were translated, Western blots were performed in all samples; however, only the normal protein was detected. By immunohistochemistry, a reduction in Wwox protein expression was observed, affecting 40% of the tumors when compared with normal mucosa. In addition, a novel somatic mutation (S329F) was found. The presence of alterations in mRNA transcription correlated with the reduced expression of Wwox protein in the tumors. These results show that the WWOX gene is frequently altered in OSCC and may contribute to the carcinogenesis processes in oral cancer.

Aberrant activation of tyrosine kinases is linked causally to human cancers. Activated Cdc42-associated kinase (Ack1), an intracellular tyrosine kinase, has primarily been studied for its signaling properties but has not been linked to specific pathologic conditions. Herein, we report that expression of activated Ack1 in LNCaP cells, while minimally increasing growth in culture, enhanced anchorage-independent growth in vitro and dramatically accelerated tumorigenesis in nude mice. Molecular chaperone heat shock protein 90beta (Hsp90beta)-bound Ack1 and treatment of cells with geldanamycin, a Hsp90 inhibitor, inhibited Ack1 kinase activity and suppressed tumorigenesis. Further, we identify the tumor suppressor WW domain containing oxidoreductase (Wwox) as an Ack1-interacting protein. Activated Ack1 tyrosine phosphorylated Wwox, leading to rapid dissociation of the Ack1-Wwox complex and concomitant Wwox polyubiquitination followed by degradation. Tyrosine phosphorylation of Wwox was critical for its degradation, as splice variant WwoxDelta5-8 that was not phosphorylated by Ack1 failed to undergo polyubiquitination and degradation. It has been reported that phosphorylation of Wwox at Tyr33 stimulated its proapoptotic activity. We observed that Y33F Wwox mutant was still tyrosine phosphorylated and polyubiquitinated by Ack1 action. Site-directed mutagenesis revealed that activated Ack1 primarily phosphorylated Wwox at Tyr287, suggesting that phosphorylation of distinct tyrosine residues activate or degrade Wwox. Primary androgen-independent prostate tumors but not benign prostate showed increased tyrosine-phosphorylated Ack1 and decreased Wwox. Taken together, these data indicate that Ack1 stimulated prostate tumorigenesis in part by negatively regulating the proapoptotic tumor suppressor, Wwox. Further, these findings suggest that Ack1 could be a novel therapeutic target for prostate cancer.

expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues.

WWOX is a tumor suppressor that functions as a modular protein partner of transcription factors. WWOX contains two WW domains that mediate protein-protein interactions. In this report, we show that WWOX, via its first WW domain, specifically associates with the proline-rich motif of c-Jun proto-oncogene. Our data show that phosphorylation of c-Jun caused by overexpression of mitogen-activated protein kinase kinase kinase 1 (Mekk1), an upstream activator of c-Jun, enhances the interaction of c-Jun with WWOX. Furthermore, exposure of HaCaT keratinocytes to UVC radiation resulted in the association of endogenous WWOX and c-Jun. The WWOX-c-Jun complexes mainly occur in the cytoplasm. expression of WWOX attenuates the ability of MEKK1 to increase the activity of a c-Jun-driven activating protein-1 (AP-1)-luciferase reporter plasmid. In contrast, a point mutation in the first WW domain of WWOX has no effect on transactivation of AP-1 when coexpressed with c-Jun protein. Our findings reveal a novel functional cross-talk between c-Jun transcription factor and WWOX tumor suppressor protein.

The WW domain-containing oxidoreductase (WWOX) spans the second most common fragile site of the human genome, FRA16D, located at 16q23, and its expression is altered in several types of human cancer. We have previously shown that restoration of WWOX expression in cancer cells suppresses tumorigenicity. To investigate WWOX tumor suppressor function in vivo, we generated mice carrying a targeted deletion of the Wwox gene and monitored incidence of tumor formation. Osteosarcomas in juvenile Wwox(-/-) and lung papillary carcinoma in adult Wwox(+/-) mice occurred spontaneously. In addition, Wwox(+/-) mice develop significantly more ethyl nitrosourea-induced lung tumors and lymphomas in comparison to wild-type littermate mice. Intriguingly, these tumors still express Wwox protein, suggesting haploinsuffiency of WWOX itself is cancer predisposing. These results indicate that WWOX is a bona fide tumor suppressor.

WWOX is a putative tumor suppressor gene encoded within common chromosomal fragile site region FRA16D, in chromosome band 16q23. Multiple studies have demonstrated that WWOX expression is often reduced or lost in various tumor types. WWOX tumor suppressor activity was suggested by re-expressing WWOX in breast, ovarian, and lung tumor cell lines leading to tumor growth inhibition in vivo. To determine whether loss of Wwox gene expression has a role in tumorigenesis, we generated a mouse strain containing a Wwox gene mutated by a gene-trap vector. Homozygous Wwox gene-trap mice (Wwox(gt/gt)) had no detectable Wwox protein in most tissues examined, although, a low level could be detected in a minority of tissues. Because of these observations, we concluded that these mice are Wwox hypomorphs. Remarkably, Wwox hypomorphic mice are viable in contrast to the recently reported postnatal lethality of Wwox knockout mice. Testes from Wwox(gt/gt) males had high numbers of atrophic seminiferous tubules and reduced fertility when compared with wild-type counterparts. We observed that the Wwox(gt/gt) mice had a significantly shorter lifespan, and female hypomorphs had a higher incidence of spontaneous B-cell lymphomas. In conclusion, we describe a novel Wwox hypomorphic mouse model that overcomes postnatal lethality that was recently observed in Wwox knockout mice. Therefore, tumorigenesis studies using this model more closely recapitulates the loss of WWOX expression observed in human cancers. Importantly, our observation that Wwox hypomorphs had an increased incidence of B-cell lymphomas supports a role of Wwox as a tumor suppressor.

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.

The WW domain-containing oxidoreductase (WWOX) gene encodes a tumor suppressor. We have previously shown that targeted ablation of the Wwox gene in mouse increases the incidence of spontaneous and chemically induced tumors. To investigate WWOX function in vivo, we examined Wwox-deficient (Wwox(-/-)) mice for phenotypical abnormalities. Wwox(-/-) mice are significantly reduced in size, die at the age of 2-3 weeks, and suffer a metabolic disorder that affects the skeleton. Wwox(-/-) mice exhibit a delay in bone formation from a cell autonomous defect in differentiation beginning at the mineralization stage shown in calvarial osteoblasts ex vivo and supported by significantly decreased bone formation parameters in Wwox(-/-) mice by microcomputed tomography analyses. Wwox(-/-) mice develop metabolic bone disease, as a consequence of reduced serum calcium, hypoproteinuria, and hypoglycemia leading to increased osteoclast activity and bone resorption. Interestingly, we find WWOX physically associates with RUNX2, the principal transcriptional regulator of osteoblast differentiation, and on osteocalcin chromatin. We show WWOX functionally suppresses RUNX2 transactivation ability in osteoblasts. In breast cancer MDA-MB-242 cells that lack endogenous WWOX protein, restoration of WWOX expression inhibited Runx2 and RUNX2 target genes related to metastasis. Affymetrix mRNA profiling revealed common gene targets in multiple tissues. In Wwox(-/-) mice, genes related to nucleosome assembly and cell growth genes were down-regulated, and negative regulators of skeletal metabolism exhibited increased expression. Our results demonstrate an essential requirement for the WWOX tumor suppressor in postnatal survival, growth, and metabolism and suggest a central role for WWOX in regulation of bone tissue formation.

BACKGROUND/AIMS: The FRA16D fragile site gene WWOX is a tumor suppressor that participates in p53-mediated apoptosis. The c-jun N-terminal kinase JNK1 interacts with WWOX and inhibits apoptosis. We investigated the function of WWOX in human hepatocellular carcinoma (HCC) and the effect of JNK inhibition on WWOX-mediated apoptosis. METHODS: Allelic imbalance on chromosome 16 was analyzed in 73 HCCs using 53 microsatellite markers. WWOX mRNA in HCC cell lines and primary HCCs was measured by real-time RT-PCR. Effects of WWOX on proliferation and apoptosis and the interaction between WWOX and JNK inhibition were examined. RESULTS: Loss on chromosome 16 occurred in 34 of 73 HCCs. Of 11 HCC cell lines, 2 had low, 7 intermediate, and 2 had high WWOX mRNA. Of 51 primary tumors, 23 had low WWOX mRNA. Forced expression of WWOX in SNU387 cells decreased FGF2-mediated proliferation and enhanced apoptosis induced by staurosporine and the JNK inhibitor SP600129. Conversely, knockdown of WWOX in SNU449 cells using shRNA targeting WWOX increased proliferation and resistance to SP600129-induced apoptosis. CONCLUSIONS: WWOX induces apoptosis and inhibits human HCC cell growth through a mechanism enhanced by JNK inhibition.

The WW domain-containing oxidoreductase (WWOX) gene encodes a 46-kDa tumor suppressor. The Wwox protein contains two N-terminal WW domains that interact with several transcriptional activators containing proline-tyrosine motifs and a central short-chain dehydrogenase/reductase domain that has been suggested to play a role in steroid metabolism. Recently, we have shown that targeted deletion of the Wwox gene in mice leads to postnatal lethality and defects in bone growth. Here, we report that Wwox-deficient mice display impaired steroidogenesis. Mutant homozygous mice are born with gonadal abnormalities, including failure of Leydig cell development in testis and reduced theca cell proliferation in ovary. Furthermore, Wwox(-/-) mice displayed impaired gene expression of key steroidogenesis enzymes. Affymetrix microarray gene analysis revealed differentially expressed related genes in steroidogenesis in knockout mice testis and ovary as compared with control mice. These results demonstrate the essential requirement for the Wwox tumor suppressor in proper steroidogenesis.

BACKGROUND: Prostate cancer linkage studies have been used to localize rare and presumably highly penetrant cancer susceptibility genes. Underlying genetic heterogeneity, as well as the high sporadic background of the disease, has resulted in many signals that are often not reproducible between research studies. METHODS: We conducted a SNP-based genome wide linkage scan on 131 Caucasian prostate cancer families participating in the University of Michigan Prostate cancer genetics Project (PCGP). RESULTS: The strongest evidence for linkage was detected at 16q23 (LOD = 2.70 at rs1079635). Prostate cancer linkage to the same region of 16q23 has been observed by others and the region contains several strong candidate genes including the known prostate cancer tumor suppressor genes ATBF1 and WWOX. This linkage signal was not detected in our prior linkage study on 175 PCGP families, illustrating the genetic heterogeneity underlying prostate cancer susceptibility. CONCLUSIONS: Further linkage studies in combination with tumor analyses from linked families are in progress to identify the putative hereditary prostate cancer gene at 16q23.

The WWOX gene encodes a candidate tumor suppressor protein (WWOX) implicated in a variety of human diseases such as cancer. To better understand the molecular mechanisms of WWOX action, we investigated novel partners of this protein. Using the two-hybrid system and a coimmunoprecipitation assay, we observed a physical association between WWOX and the Dishevelled protein (Dvl) family signaling elements involved in the Wnt/beta-catenin pathway. We found that enforced WWOX expression inhibited, and inhibition of endogenous WWOX expression stimulated the transcriptional activity of the Wnt/beta-catenin pathway. Inhibition of endogenous WWOX expression also enhanced the effect of Wnt-3a on beta-catenin stability. Moreover, we observed the sequestration of Dvl-2 wild type and Dvl-2NESm, a mutated form of Dvl-2 predominantly localized in the nucleus, in the cytoplasm compartment by WWOX. Our results indicate that WWOX is a novel inhibitor of the Wnt/beta-catenin pathway. WWOX would act, at least in part, by preventing the nuclear import of the Dvl proteins.

BACKGROUND: Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells. METHODOLOGY/PRINCIPAL FINDINGS: DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. CONCLUSIONS/SIGNIFICANCE: We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.

WWOX, the gene that spans the second most common human chromosomal fragile site, FRA16D, is inactivated in multiple human cancers and behaves as a suppressor of tumor growth. Since we are interested in understanding WWOX function in both normal and cancer tissues we generated mice harboring a conditional Wwox allele by flanking Exon 1 of the Wwox gene with LoxP sites. Wwox knockout (KO) mice were developed by breeding with transgenic mice carrying the Cre-recombinase gene under the control of the adenovirus EIIA promoter. We found that Wwox KO mice suffered from severe metabolic defect(s) resulting in growth retardation and all mice died by 3 wk of age. All Wwox KO mice displayed significant hypocapnia suggesting a state of metabolic acidosis. This finding and the known high expression of Wwox in kidney tubules suggest a role for Wwox in acid/base balance. Importantly, Wwox KO mice displayed histopathological and hematological signs of impaired hematopoiesis, leukopenia, and splenic atrophy. Impaired hematopoiesis can also be a contributing factor to metabolic acidosis and death. Hypoglycemia and hypocalcemia was also observed affecting the KO mice. In addition, bone metabolic defects were evident in Wwox KO mice. Bones were smaller and thinner having reduced bone volume as a consequence of a defect in mineralization. No evidence of spontaneous neoplasia was observed in Wwox KO mice. We have generated a new mouse model to inactivate the Wwox tumor suppressor gene conditionally. This will greatly facilitate the functional analysis of Wwox in adult mice and will allow investigating neoplastic transformation in specific target tissues.

OBJECTIVE: To observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action. METHODS: Attachment assay was used to assess the adhesion of wwox-transfected PEO1 cells and vector-transfected PEO1 cells that were constructed, as well as PEO1 parent cells. Alpha/beta integrin-mediated cell adhesion assays were designed to identify cells surface integrins in PEO1 clone cells. Integrin function blocking experiments were designed to further determine integrins in PEO1 clone cells according to the integrin that was selected in integrin expression profiling. FACS analysis was used to further detect the level of integrin alpha3 on the cell membrane. RESULTS: Attachment assay showed that adhesion of WWOX-transfected PEO1 cells to fibronectin was significantly slower than that in vector-transfected controls or PEO1 parent cells, cultured on the pre-coated fibronectin for 2 hours (P<0.01). The level of membranous integrins alpha2 and alpha3 in the WWOX-transfected PEO1 cells was significantly decreased, as compared with that in vector-transfected controls (P<0.05), but there was no association with the level of functioning integrins betal or beta2 in clone cells (P>0.05). The attachment assays were repeated after pre-incubating the cells with integrin alpha2 or alpha3 function-blocking antibodies. These results showed that blocking integrin alpha3 significantly reduced the binding to fibronectin of all the PEO1 clonal lines, as compared with cells pre-incubated with a non-specific IgG antibody (P<0.05). In contrast, preincubation with alpha2 blocking antibody had very little effect on fibronectin binding in these cells (P>0.05). FACS analysis showed that membranous integrin alpha3 expression revealed a marked reduction in WWOX-transfected cells than that in vector-transfected cells. CONCLUSION: WWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix, decreasing integrin activity and adhesion of tumor cells to fibronectin. This suggests an important role for loss of WWOX tumor suppressor in promoting attachment and adhesion of ovarian cancer cells on locoregional peritoneum, and further resulting in enhancing locoregional peritoneal tumor spread.

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P < 0.0001). Compared with the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorigenicity in nude mice. expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas RUNX2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease.

Mortality from lung cancer is important worldwide. Recently, epigenetic aberration of lung cancer, not only genomic DNA methylation but also chromatin modification, has become an important target for lung cancer research, although previous research has demonstrated that lung cancer develops as a result of both environmental and genetic factors. Here, we demonstrated that an epigenetic regulator/polycomb group protein Bmi1 is more highly expressed in small-cell lung cancer (SCLC) than in non-small-cell lung cancer by immunohistochemical analysis. In vitro experiments indicated that Bmi1 reduction by lentivirus-derived shRNA significantly suppressed proliferation, colony formation and in vivo tumor formation. Importantly, apoptosis was induced by Bmi1 depletion in small-cell lung cancer cells. Furthermore, a tumor suppressor WWOX was identified as a Bmi1 target in the cells by a chromatin immunoprecipitation assay and a quantitative real-time PCR assay; WWOX had a role as a tumor suppressor in SCLC cells; therefore, the Bmi1/WWOX pathway could be a new candidate for a new therapeutic approach for SCLC.

We report a hypothesis-driven study aimed to detect genetic markers of susceptibility to differentiated thyroid carcinomas (DTC). A large number of candidate genes were first selected through literature search (genome-wide studies were also included). To restrict the analysis to single nucleotide polymorphisms (SNPs) with a high likelihood to be associated with increased risk, each SNP must comply with several a priori hypotheses. Only one SNP, the rs3764340 encoding for the aminoacidic substitution proline-to-alanine at codon 282 of the tumor suppressor gene WWOX, passed the selection. A case-control association study was carried out, involving a total of 1,741 cases and 1,042 controls. The logistic regression analysis revealed an increased risk of DTC for the carriers of the G-allele (crude odds ratio, OR = 1.53; 95% confidence interval, CI = 1.18-1.99; p = 1.38 x 10(-3) ). When we controlled for covariates, the adjusted OR was 1.48 with a 95% CI of 1.08-2.03 (p = 8.0 x 10(-3) ). The association was confirmed after stratification for histology (for papillary thyroid carcinoma the adjusted OR was 1.43; 95% CI 1.02-2.00; p = 0.037), incident cases and smokers, but was also at the limit of statistical significance in all the other categories considered. In silico analyses showed that when alanine substitutes proline, subtle changes of the proteic structure can be predicted. These findings together with other observations from literature on human cancers and the fact that the proline at codon 282 is extremely conserved in phylogenetically distant organisms (including Drosophila) suggest that the variant allele-282 could affect the biological function of WWOX, thereby predisposing individuals to thyroid cancer.

Primary effusion lymphoma (PEL) is a diffuse-large B-cell lymphoma with poor prognosis. One hundred percent of PELs carry the genome of Kaposi sarcoma-associated herpesvirus and a majority are coinfected with Epstein-Barr virus (EBV). We profiled genomic aberrations in PEL cells using the Affymetrix 6.0 SNP array. This identified for the first time individual genes that are altered in PEL cells. Eleven of 13 samples (85%) were deleted for the fragile site tumor suppressors WWOX and FHIT. Alterations were also observed in the DERL1, ETV1, RASA4, TPK1, TRIM56, and VPS41 genes, which are yet to be characterized for their roles in cancer. Coinfection with EBV was associated with significantly fewer gross genomic aberrations, and PEL could be segregated into EBV-positive and EBV-negative clusters on the basis of host chromosome alterations. This suggests a model in which both host genetic aberrations and the 2 viruses contribute to the PEL phenotype.