WW Domain-containing Research Articles

A genome-wide association study on hematopoietic stem cell transplantation reveals novel genomic loci associated with transplant outcomes.

Data on genomic susceptibility for adverse outcomes after hematopoietic stem cell transplantation (HSCT) for recipients are scarce. We performed a genome wide association study (GWAS) to identify genes associated with survival/mortality, relapse, and severe graft-versus-host disease (sGvHD), fitting proportional hazard and subdistributional models to data of n=1,392 recipients of European ancestry from three centres. The single nucleotide polymorphism (SNP) rs17154454, intronic to the neuronal growth guidant semaphorin 3C gene (SEMA3C), was genome-wide significantly associated with event-free survival (p=7.0x10-8) and sGvHD (p=7.5x10-8). Further associations were detected for SNPs in the Paxillin gene (PXN) with death without prior relapse or sGvHD, as well as for SNPs of the Plasmacytoma Variant Translocation 1 gene (PVT1, a long non-coding RNA gene), the Melanocortin 5 Receptor (MC5R) gene and the WW Domain Containing Oxidoreductase gene (WWOX), all associated with the occurrence of sGvHD. Functional considerations support the observed associations. Thus, new genes were identified, potentially influencing the outcome of HSCT.

The role of WWP1 and WWP2 in bone/cartilage development and diseases.

Bone and cartilage diseases are often associated with trauma and senescence, manifested as pain and limited mobility. The repair of bone and cartilage lesion by mesenchymal stem cells is regulated by various transcription factors. WW domain-containing protein 1 (WWP1) and WW domain-containing protein 2 (WWP2) are named for WW domain which recognizes PPXY (phono Ser Pro and Pro Arg) motifs of substrate. WWP1and WWP2 are prominentcomponents of the homologous to the E6-AP carboxyl terminus (HECT) subfamily, a group of the ubiquitin ligase. Recently, some studies have found that WWP1 and WWP2 play an important role in the pathogenesis of bone and cartilage diseases and regulate the level and the transactivation of various transcription factors through ubiquitination. Therefore, this review summarizes the distribution and effects of WWP1 and WWP2 in the development of bone and cartilage, discusses the potential mechanism and therapeutic drugs in bone and cartilage diseases such as osteoarthritis, fracture, and osteoporosis.

WWP2 binds to NKRF, enhances the NF-κB signaling, and promotes malignant phenotypes of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is one of the hematological malignancies with a high recurrence rate. WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) is identified as a pivotal regulator of tumor progression. This study aimed to assess the possible role of WWP2 in AML. Analysis of the GEPIA database indicated an elevated WWP2 expression in AML. We established stable WWP2-overexpressed or WWP2-silenced cells using lentivirus loaded with cDNA encoding WWP2 mRNA or shRNA targeting WWP2. Notably, WWP2 overexpression facilitated cell proliferation and cell cycle progression, which was manifested as the increase of colony formation number, S-phase percentage and cell cycle related protein levels. As observed, WWP2 knockdown presented opposite effects, leading to inhibition of tumorigenicity. Strikingly, WWP2 knockdown induced apoptosis, accompanied by upregulation of pro-apoptosis proteins cleaved caspase-9, Bax and cleaved caspase-3 and downregulation of anti-apoptosis protein Bcl-2. Functionally, we further confirmed that WWP2 overexpression enhanced the NF-κB signaling and upregulated the levels of downstream genes, which may contribute to aggravating the development of AML. More importantly, by co-immunoprecipitation assay, we verified that WWP2 bound to NF-κB-repressing factor (NKRF) and promoted NKRF ubiquitylation. Dramatically, NKRF overexpression abolished the role of WWP2 in facilitating the process of AML. Overall, our observations confirm that WWP2 exerts a critical role in the tumorigenicity of AML, and NKRF is regarded as an essential factor in the WWP2-mediated AML progression. WWP2 may be proposed as a promising target of AML.

Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected.These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

Systemic depletion of WWP1 improves insulin sensitivity and lowers triglyceride content in the liver of obese mice.

Obesity is a metabolic disorder associated with many diseases. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type E3 ubiquitin ligase involved in several diseases. Recently, we found that the level of WWP1 is increased in white adipose tissue in a mouse model of obesity and that obese Wwp1 knockout (KO) mice exhibit improved whole-body glucose metabolism. Here, to determine which insulin-sensitive tissues contribute to this phenotype, we investigated the levels of several insulin signaling markers in white adipose tissue, liver, and skeletal muscle of Wwp1 KO mice, which were fed a normal or high-fat diet and transiently treated with insulin. In obese Wwp1 KO mice, phosphorylated Akt levels were increased in the liver but not in white adipose tissue or skeletal muscle. Moreover, the weight and triglyceride content of the liver of obese Wwp1 KO mice were decreased. These results suggest that systemic deletion of WWP1 improves glucose metabolism via enhanced hepatic insulin signaling and suppressed hepatic fat accumulation. In summary, WWP1 participates in obesity-related metabolic dysfunction and pathologies related to hepatic steatosis via suppressed insulin signaling.

Hippo signaling and histone methylation control cardiomyocyte cell cycle re-entry through distinct transcriptional pathways.

Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low. Modifying epigenetic histone modifications or activating the Hippo-Yap pathway have been shown to promote adult CM cycling and proliferation. Whether these interventions work through common pathways or act independently is unknown. For the first time we have determined whether lysine demethylase 4D (KDM4D)-mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM cell cycle re-entry. We found that activating Yap1 in cultured neonatal rat ventricular myocytes (NRVM) through overexpressing Hippo pathway inhibitor, miR-199, preferentially increased S-phase CMs, while H3K9me3 demethylase KDM4D preferentially increased G2/M markers in CMs. Together KDM4D and miR-199 further increased total cell number of NRVMs in culture. Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducible activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Using transgenic mice over-expressing KDM4D together with Myc, we demonstrated that KDM4D/Myc significantly increased CM cell cycling but did not affect cardiac function. KDM4D effects on CM cell cycle activity are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This may have important implications for strategies that target cardiac regeneration in treating heart disease.

Abstract P1073: Pathways Regulating Cardiac Myocyte Proliferation

Introduction: Several pathways have been shown to promote cardiomyocytes (CMs) proliferation and regeneration including the Hippo signaling pathway. Our lab demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases the proliferation of adult CMs (ACMs). However, all these stimulations are insufficient to promote robust ACM proliferation. The effect of combining these interventions on CM proliferation and myocardial regeneration remains unknown. Hypothesis: KDM4D-mediated CM-specific H3K9 demethylation and Hippo pathway inhibition have additive roles in promoting CM proliferation. Methods and results: miR-199 overexpression preferentially increased S-phase CMs (EdU + ) in cultured neonatal rat ventricular cardiomyocytes (NRVMs), while H3K9me3 demethylase KDM4D preferentially increased G2/M markers (phospho-H3). The combination of KDM4D and miR-199 increased total cell number of NRVMs compared to either alone. Since the effects of miR-199 on CM proliferation are mediated through inhibition of Hippo signaling, we inhibited the pathway directly via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1). Similarly, Sav1 inhibition also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducibly activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA-mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. In vitro gene expression analysis demonstrated that KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle while miR-199 and si-Sav1 preferentially upregulated genes involved in G1/S phase. RNA-seq and promoter analysis demonstrated that KDM4D and Hippo signaling pathway regulate CMs proliferation through different transcriptional pathways. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Conclusions: KDM4D additively induces CMs proliferation with the Hippo-Yap1 pathways. This may have important implications for strategies that target cardiac regeneration in treating heart disease.

WWP2 overexpression inhibits the antitumor effects of doxorubicin in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common liver cancer that accounts for 90% of cases. Doxorubicin exhibits a broad spectrum of antitumor activity and is one of the most active agents in HCC. WW domain-containing protein 2 (WWP2) is highly expressed in HCC tissues and activates protein kinase B (AKT) signaling pathway to enhance tumor metastasis. However, the role of WWP2 in the glycolysis and antitumor effects of doxorubicin and the epigenetic alterations of WWP2 in HCC remain to be elucidated. The levels of WWP2 and N6-methyladenosine methyltransferase-like 3 (METTL3) in clinical samples and cells were investigated. WWP2 were silenced or overexpressed to study the role of WWP2 in regulating cell proliferation, colony formation, and glycolysis. RNA immunoprecipitation was performed to test m6 A levels. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot were used to measure mRNA and protein, respectively. WWP2 silencing inhibits cell proliferation, colony formation, and glycolysis, while WWP2 overexpression has the inverse effects via the AKT signaling pathway. Silencing WWP2 enhances doxorubicin's antitumor effect, while WWP2 overexpression suppresses doxorubicin's antitumor effect. Data also support that METTL3 mediates WWP2 m6A modification, and m6A reader, IGF2BP2, binds to the methylated WWP2 to promote the stability of WWP2, leading to upregulation of WWP2. METTL3 mediates WWP2 m6A modification, which can be recognized and bound by IGF2BP2 to increase the stability of WWP2, leading to WWP2 overexpression which inhibits the antitumor effects of doxorubicin through METTL3/WWP2/AKT/glycolysis axis.

Pathological Significance and Prognostic Role of WWC1 in Upper Urinary Tract Urothelial Cancer.

WW and C2 domain-containing 1 (WWC1) protein is a suppressor of malignancies. However, there is no information on the pathological significance of WWC1 in upper urinary tract cancer (UTUC). In this study, WWC1 immunoreactivity was investigated in 152 non-metastatic UTUC samples. The relationships between WWC1 expression and grade, pT stage, proliferative index (using an antibody to Ki-67), and the immunohistochemical expression of matrix metalloproteinase (MMP)-2 and -9 were evaluated. WWC1 expression was negatively associated with tumor grade and pT stage (p<0.001). Positive expression of WWC1 was a better predictor of the UTUC recurrence and subsequent metastasis, and the multivariate analysis showed that WWC1 expression was a significant predictor of subsequent metastasis (hazard ratio=0.29, p=0.020). WWC1 expression inversely correlated with the proliferative index (odds ratio=2.59, p=0.023) and expression of MMP9 (odds ratio=2.19, p=0.040) but not with MMP2 expression, by multivariate analyses. WWC1 expression was negatively associated with malignant aggressiveness via the suppression of cancer cell proliferation and MMP9 expression in patients with UTUC. This suggests WWC1 to be a useful predictor and novel therapeutic target in patients with UTUC.

LINC01588 regulates WWP2-mediated cardiomyocyte injury by interacting with HNRNPL.

Cardiomyocyte dysfunction and apoptosis induced by ischemia-hypoxia are common features of many acute and chronic heart diseases. WW domain-containing E3 ubiquitin ligase (WWP2) has been identified as an important regulator in pathogenesis of some health-threatening diseases. Although a couple of recent reports prompted the potential role of WWP2 in heart dysfunction, however, its exact role and how its expression was regulated in ischemic-hypoxic cardiomyocytes are still elusive. Here, we found that WWP2 protein level was induced in anoxia/reoxygenation (A/R) treated cardiomyocytes in a time-dependent manner, accompanied by synchronous expression of LINC01588 and HNRNPL. Knockdown of LINC01588 increased cardiomyocyte apoptosis, the level of oxidative stress, and expression of pro-inflammatory cytokine genes, down-regulated the expression of WWP2 and promoted expression of SEPT4 gene that contributed to cardiomyocyte dysfunction and was a target gene of WWP2. LINC01588 overexpression improved the functions of A/R treated cardiomyocytes, up-regulated WWP2 and reduced SEPT4 expression. In the mechanism exploration, we found that LINC01588 could directly bind with HNRNPL protein that could interact with WWP2, suggesting that WWP2 was involved in the regulation of LINC01588 in A/R treated cardiomyocytes. Moreover, WWP2 inhibition declined the protective role of LINC01588 in cardiomyocyte dysfunction induced by A/R. Finally, we demonstrated that LINC01588 overexpression improved acute myocardial infarction in mice in vivo. In conclusion, LINC01588 improved A/R-induced cardiomyocyte dysfunction by interacting with HNRNPL and promoting WWP2-mediated degradation of SEPT4.

WWP1 Deficiency Alleviates Cardiac Remodeling Induced by Simulated Microgravity.

Cardiac muscle is extremely sensitive to changes in loading conditions; the microgravity during space flight can cause cardiac remodeling and function decline. At present, the mechanism of microgravity-induced cardiac remodeling remains to be revealed. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is an important activator of pressure overload-induced cardiac remodeling by stabilizing disheveled segment polarity proteins 2 (DVL2) and activating the calcium-calmodulin-dependent protein kinase II (CaMKII)/histone deacetylase 4 (HDAC4)/myocyte-specific enhancer factor 2C (MEF2C) axis. However, the role of WWP1 in cardiac remodeling induced by microgravity is unknown. The purpose of this study was to determine whether WWP1 was also involved in the regulation of cardiac remodeling caused by microgravity. Firstly, we detected the expression of WWP1 and DVL2 in the heart from mice and monkeys after simulated microgravity using western blotting and immunohistochemistry. Secondly, WWP1 knockout (KO) and wild-type (WT) mice were subjected to tail suspension (TS) to simulate microgravity effect. We assessed the cardiac remodeling in morphology and function through a histological analysis and echocardiography. Finally, we detected the phosphorylation levels of CaMKII and HDAC4 in the hearts from WT and WWP1 KO mice after TS. The results revealed the increased expression of WWP1 and DVL2 in the hearts both from mice and monkeys after simulated microgravity. WWP1 deficiency alleviated simulated microgravity-induced cardiac atrophy and function decline. The histological analysis demonstrated WWP1 KO inhibited the decreases in the size of individual cardiomyocytes of mice after tail suspension. WWP1 KO can inhibit the activation of the DVL2/CaMKII/HDAC4 pathway in the hearts of mice induced by simulated microgravity. These results demonstrated WWP1 as a potential therapeutic target for cardiac remodeling and function decline induced by simulated microgravity.

Neuronal activation modulates enhancer activity of genes for excitatory synaptogenesis through de novo DNA methylation.

Post-mitotic neurons do exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanged. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, the occurrence of de novo DNA methylation and its impacts have been much less investigated. In the present study, we showed that neuronal activation induces de novo DNA methylation at enhancer regions, which can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 domain-containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in synaptogenesis. We found that both DNMT1 and DNMT3a were required for neuronal activity-induced de novo DNA methylation of the Wwc1 enhancer. Taken together, we concluded that neuronal activity-induced de novo DNA methylation that affects gene expression has an impact on neuronal physiology that is comparable to that of DNA demethylation. Since the different requirements of DNMTs for germ cell and embryonic development are known, our findings also have considerable implications for future studies on epigenomics in the field of reproductive biology.

Long noncoding RNA MAGI2-AS3 inhibits bladder cancer progression through MAGI2/PTEN/epithelial-mesenchymal transition (EMT) axis.

Long noncoding RNA (lncRNA) are critical regulators of tumor progression. To determine how the lncRNA membrane associated guanylate kinase, WW and PDZ domain-containing 2 (MAG12) antisense RNA 3 (MAGI2-AS3) and the phosphatase and tensin homolog (PTEN) gene function in regulating bladder cancer (Bca) progression. Total RNA from 80 Bca tissues and 30 paired para-cancerous tissues from patients was sequentially extracted, quantified, purified, and reverse transcribed using RT-PCR. A library was constructed and sequenced. Four Bca cell lines and a normal urothelial cell line were transfected with lentiviral plasmids, and cell migration and invasion were assayed in vitro. An orthotopic mouse model of Bca was created for in vivo studies. MAGI2-AS3 expression was significantly downregulated in Bca, compared with normal tissues, and negatively associated with tumor stage and a poor prognosis. MAGI2-AS3 and its sense RNA MAGI2 showed significant and positive correlation. The expression of MAGI2 and its downstream gene, PTEN, increased in Bca cells overexpressing MAGI2-AS3, and interference by MAGI2 expression reversed the migration and invasion inhibited by MAGI2-AS3 overexpression. MAGI2-AS3 overexpression inhibited Bca cell progression by regulating the MAGI2/PTEN/epithelial-mesenchymal transition, offering novel insights into the mechanism of Bca progression.

WWP1 knockout in mice exacerbates obesity-related phenotypes in white adipose tissue but improves whole-body glucose metabolism.

White adipose tissue (WAT) is important for maintenance of homeostasis, because it stores energy and secretes adipokines. The WAT of obese people demonstrates mitochondrial dysfunction, accompanied by oxidative stress, which leads to insulin resistance. WW domain‐containing E3 ubiquitin protein ligase 1 (WWP1) is a member of the HECT‐type E3 family of ubiquitin ligases and is associated with several diseases. Recently, we demonstrated that WWP1 is induced specifically in the WAT of obese mice, where it protects against oxidative stress. Here, we investigated the function of WWP1 in WAT of obese mice by analyzing the phenotype of Wwp1 knockout (KO) mice fed a high‐fat diet. The levels of oxidative stress markers were higher in obese WAT from Wwp1 KO mice. Moreover, Wwp1 KO mice had lower activity of citrate synthase, a mitochondrial enzyme. We also measured AKT phosphorylation in obese WAT and found lower levels in Wwp1 KO mice. However, plasma insulin level was low and glucose level was unchanged in obese Wwp1 KO mice. Moreover, both glucose tolerance test and insulin tolerance test were improved in obese Wwp1 KO mice. These findings indicate that WWP1 participates in the antioxidative response and mitochondrial function in WAT, but knockdown of WWP1 improves whole‐body glucose metabolism.

De Novo Coding Variants Are Strongly Associated with Tourette Syndrome

Based on the contribution of de novo coding variants to other neurodevelopmental disorders, and their utility for hypothesis-naive gene discovery, we whole-exome sequenced 325 Tourette syndrome trios (311 passed quality control) from the TIC Genetics consortium, and identified de novo sequence variants. We observed a strong association for de novo likely gene disrupting (LGD; insertion of premature stop codon, disruption of canonical splice site, or frameshift insertion/deletion) variants as well as de novo damaging variants (LGD + probably damaging missense [Mis3]) variants. We replicated these findings in 186 TS trios (173 after quality control) from the TSAICG Consortium, and performed a combined analysis strongly associating de novo LGD (RR 2.32, 95% CI 1.37-3.93, p = 0.002) and damaging (RR 1.37, 95% CI 1.11-1.69, p = 0.003) variants with TS risk. We estimate that de novo damaging variants in approximately 12% of patients contribute risk through the perturbation of approximately 400 risk genes. Finally, we leveraged recurrent de novo damaging variants and the TADA algorithm to identify one high confidence (false discovery rate (FDR) < 0.10) TS risk gene: WWC1 (WW and C2 Domain Containing 1); and three probable (FDR < 0.30) TS risk genes: CELSR3 (Cadherin EGF LAG Seven-Pass G-Type Receptor 3); NIPBL (Nipped-B-Like); and FN1 (Fibronectin 1). These findings establish the contribution of de novo variants to TS risk and highlight that de novo based gene discovery is a viable path forward for this disorder.

WW-Domain Containing Protein Roles in Breast Tumorigenesis.

Protein-protein interactions are key factors in executing protein function. These interactions are mediated through different protein domains or modules. An important domain found in many different types of proteins is WW domain. WW domain-containing proteins were shown to be involved in many human diseases including cancer. Some of these proteins function as either tumor suppressor genes or oncogenes, while others show dual identity. Some of these proteins act on their own and alter the function(s) of specific or multiple proteins implicated in cancer, others interact with their partners to compose WW domain modular pathway. In this review, we discuss the role of WW domain-containing proteins in breast tumorigenesis. We give examples of specific WW domain containing proteins that play roles in breast tumorigenesis and explain the mechanisms through which these proteins lead to breast cancer initiation and progression. We discuss also the possibility of using these proteins as biomarkers or therapeutic targets.

WW domain-containing E3 ubiquitin protein ligase 1 depletion evokes antitumor effect in cutaneous squamous cell carcinoma by inhibiting signal transducer and activator of transcription 3 signaling pathway.

ObjectivesWW domain-containing E3 ubiquitin protein ligase 1 (WWP1) has been implicated in tumor progression. We aimed to investigate the role of WWP1 in cutaneous squamous cell carcinoma (CSCC).MethodsWWP1 gene and protein levels were detected using semi-quantitative reverse transcription-polymerase chain reaction, immunohistochemistry and western blotting. The effects of WWP1 on cell cycle, apoptosis, cell migration and invasion were examined by flow cytometry, wound healing and Transwell assays, respectively. The antitumor efficacy of WWP1 small interfering RNA was determined in CSCC tumor xenografts in mice.ResultsWWP1 expression was significantly higher in CSCC tissues and cells than in normal skin and cells, respectively. WWP1 expression was significantly associated with histological grade, invasion depth and lymph node metastasis in patients with CSCC. High expression predicted metastatic potential and an unfavorable prognosis. WWP1 downregulation suppressed tumor growth in vitro and in vivo, reduced cell migration and invasion, arrested the cell cycle in G0/G1 and induced apoptosis in A431 cells. WWP1 depletion also decreased phosphorylated signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-2, cyclin D1 and Bcl-2, but did not affect total STAT3.ConclusionsWWP1 is a potential target for the diagnosis, prognosis and therapy of patients with CSCC.

WW and C2 domain-containing proteins regulate hepatic cell differentiation and tumorigenesis through the hippo signaling pathway.

Our data define an essential in vivo function for WWC proteins as regulators of canonical and noncanonical Hippo signaling in hepatic cell growth and liver tumorigenesis. Thus, expression of WWC proteins may serve as novel prognostic factors in human liver carcinoma. (Hepatology 2018;67:1546-1559).

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

The long non-coding RNA PARTICLE is associated with WWOX and the absence of FRA16D breakage in osteosarcoma patients.

Breakage of the fragile site FRA16D disrupts the WWOX (WW Domain Containing Oxidoreductase) tumor suppressor gene in osteosarcoma. However, the frequency of breakage is not sufficient to explain the rate of WWOX loss in pathogenesis. The involvement of non-coding RNA transcripts is proposed due to their accumulation at fragile sites, where they are advocated to influence specific chromosomal regions associated with malignancy. The long ncRNA PARTICLE (promoter of MAT2A antisense radiation-induced circulating long non-coding RNA) is transiently elevated in response to irradiation and influences epigenetic silencing modification within WWOX. It now emerges that elevated PARTICLE levels are significantly associated with FRA16D non-breakage in OS patients. Although not associated with overall survival, high PARTICLE levels were found to be significantly linked to metastasis free outcome. The transcription of both PARTICLE and WWOX are transiently responsive to exposure to low doses of radiation in osteosarcoma cell lines. Herein, a relationship between WWOX and PARTICLE transcription is suggested in human osteosarcoma cell lines representing alternative genetic backgrounds. PARTICLE over-expression ameliorated WWOX promoter activity in U2OS harboring FRA16D non-breakage. It can be concluded that the lncRNA PARTICLE influences the WWOX tumor suppressor and in the absence of WWOX FRA16D breakage, it is associated with OS metastasis-free survival.