WW Domain-containing E3 Ubiquitin Protein Research Articles

WWP2 protects against sepsis-induced cardiac injury through inhibiting cardiomyocyte ferroptosis.

Cardiac injury plays a critical role in contributing to the mortality associated with sepsis, a condition marked by various forms of programmed cell deaths. Previous studies hinted at the WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) involving in heart failure and endothelial injury. However, the precise implications of WWP2 in sepsis-induced cardiac injury, along with the underlying mechanisms, remain enigmatic. Sepsis induced cardiac injury were constructed by intraperitoneal injection of lipopolysaccharide. To discover the function of WWP2 during this process, we designed and performed loss/gain-of-function studies with cardiac-specific vectors and WWP2 knockout mice. Combination experiments were performed to investigate the relationship between WWP2 and downstream signaling in septic myocardium injury. The protein level of WWP2 was downregulated in cardiomyocytes during sepsis. Cardiac-specific overexpression of WWP2 protected heart from sepsis induced mitochondrial oxidative stress, programmed cell death and cardiac injury, while knockdown or knockout of WWP2 exacerbated this process. The protective potency of WWP2 was predominantly linked to its ability to suppress cardiomyocyte ferroptosis rather than apoptosis. Mechanistically, our study revealed a direct interaction between WWP2 and acyl-CoA synthetase long-chain family member 4 (FACL4), through which WWP2 facilitated the ubiquitin-dependent degradation of FACL4. Notably, we observed a notable reduction in ferroptosis and cardiac injury within WWP2 knockout mice after FACL4 knockdown during sepsis. WWP2 assumes a critical role in safeguarding the heart against injury induced by sepsis via regulating FACL4 to inhibit LPS-induced cardiomyocytes ferroptosis.

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.

High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA

Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.

Down-regulation of WWP2 aggravates Type 2 diabetes mellitus-induced vascular endothelial injury through modulating ubiquitination and degradation of DDX3X

BackgroundEndothelial injury caused by Type 2 diabetes mellitus (T2DM) is considered as a mainstay in the pathophysiology of diabetic vascular complications (DVCs). However, the molecular mechanism of T2DM-induced endothelial injury remains largely unknown. Here, we found that endothelial WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) act as a novel regulator for T2DM-induced vascular endothelial injury through modulating ubiquitination and degradation of DEAD-box helicase 3 X-linked (DDX3X).MethodsSingle-cell transcriptome analysis was used to evaluate WWP2 expression in vascular endothelial cells of T2DM patients and healthy controls. Endothelial-specific Wwp2 knockout mice were used to investigate the effect of WWP2 on T2DM-induced vascular endothelial injury. In vitro loss- and gain-of-function studies were performed to assess the function of WWP2 on cell proliferation and apoptosis of human umbilical vein endothelial cells. The substrate protein of WWP2 was verified using mass spectrometry, coimmunoprecipitation assays and immunofluorescence assays. The mechanism of WWP2 regulation on substrate protein was investigated by pulse-chase assay and ubiquitination assay.ResultsThe expression of WWP2 was significantly down-regulated in vascular endothelial cells during T2DM. Endothelial-specific Wwp2 knockout in mice significantly aggravated T2DM-induced vascular endothelial injury and vascular remodeling after endothelial injury. Our in vitro experiments showed that WWP2 protected against endothelial injury by promoting cell proliferation and inhibiting apoptosis in ECs. Mechanically, we found that WWP2 is down-regulated in high glucose and palmitic acid (HG/PA)-induced ECs due to c-Jun N-terminal kinase (JNK) activation, and uncovered that WWP2 suppresses HG/PA-induced endothelial injury by catalyzing K63-linked polyubiquitination of DDX3X and targeting it for proteasomal degradation.ConclusionOur studies revealed the key role of endothelial WWP2 and the fundamental importance of the JNK-WWP2-DDX3X regulatory axis in T2DM-induced vascular endothelial injury, suggesting that WWP2 may serve as a new therapeutic target for DVCs.

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.

MORC4 plays a tumor-promoting role in colorectal cancer via regulating PCGF1/CDKN1A axis in vitro and in vivo.

MORC family CW-type zinc finger 4 (MORC4) possessing nuclear matrix binding domains has been observed to be involved in multiple cancer development. By digging three gene expression omnibus (GEO) gene microarrays (GSE110223, GSE110224 and GSE24514), we found that MORC4 was overexpressed in colorectal cancer (CRC) samples (log2 Fold change >1, p < 0.05). We aimed to investigate the role of MORC4 in CRC malignant behaviors, with an emphasis on polycomb group ring finger 1 (PCGF1)/cyclin-dependent kinase inhibitor 1A (CDKN1A) axis. Firstly, we confirmed MORC4 as an upregulated gene in 60 pairs of frozen CRC and adjacent normal samples. MORC4 overexpression increased proliferation and metastasis, and decreased apoptosis in SW480 and HT29 cells, which was diminished by the knockdown of PCGF1, a transcriptional repressor of CDKN1A (a potent cyclin-dependent kinase inhibitor). MORC4 was further identified as a novel molecule that interacted with PCGF1 via coimmunoprecipitation. MORC4 itself did not substantially suppress CDKN1A transcriptional activity, but it augmented PCGF1's effect on CDKN1A. Additionally, MORC4 acted as the substrate of HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2 (HECW2) and was degraded through ubiquitin-proteasome system. Collectively, our work suggested that MORC4 accelerated CRC progression via governing PCGF1/CDKN1A signaling.

WWP1 upregulation predicts poor prognosis and promotes tumor progression by regulating ubiquitination of NDFIP1 in intrahepatic cholangiocarcinoma

WW domain-containing E3 ubiquitin protein ligase1 (WWP1) is reported to be upregulated in many types of human cancers; however, its expression and function in intrahepatic cholangiocarcinoma (ICC) remain unknown. Here, in this study we investigated the expression pattern, clinical prognosis, tumor biological functions, and molecular mechanisms of WWP1 in ICC. The expression of WWP1 in patient tissues was detected by western blotting, immunohistochemistry (IHC), and immunofluorescence. CCK-8, colony formation, EdU, transwell, and xenograft models were used to explore the role of WWP1 in the proliferation and metastasis of ICC. Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation, and immunofluorescence were performed to detect the potential mechanisms. Our study revealed that WWP1 was highly expressed in ICC, and high levels of WWP1 were associated with poor prognosis. Functionally, WWP1 overexpression enhanced the proliferation and metastasis of ICC cells and vice versa. Mechanistically, MYC could be enriched in the promoter region of WWP1 to facilitate its expression. Then, WWP1 targets Nedd4 family interacting protein1 (NDFIP1) and reduces NDFIP1 protein levels via ubiquitination. Downregulation of NDFIP1 in ICC cells rescued the effects of silenced WWP1 expression. WWP1 expression was also negatively correlated with the protein level of NDFIP1 in patient tissues. In conclusion, WWP1 upregulated by MYC promotes the progression of ICC via ubiquitination of NDFIP1, which reveals that WWP1 might be a potential therapeutic target for ICC.

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.

WW Domain-Containing E3 Ubiquitin Protein Ligase 1: A Self-Disciplined Oncoprotein.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a member of C2-WW-HECT E3 ligase family. Although it may execute carcinostatic actions in some scenarios, WWP1 functions as an oncoprotein under most circumstances. Here, we comprehensively review reports on regulation of WWP1 and its roles in tumorigenesis. We summarize the WWP1-mediated ubiquitinations of diverse proteins and the signaling pathways they involved, as well as the mechanisms how they affect cancer formation and progression. According to our analysis of database, in combination with previous reports, we come to a conclusion that WWP1 expression is augmented in various cancers. Gene amplification, as well as expression regulation mediated by molecules such as non-coding RNAs, may account for the increased mRNA level of WWP1. Regulation of enzymatic activity is another important facet to upregulate WWP1-mediated ubiquitinations. Based on the published data, we conclude that WWP1 employs interactions between multiple domains to autoinhibit its polyubiquitination activity in a steady state. Association of some substrates can partially release certain autoinhibition-related domains and make WWP1 have a moderate activity of polyubiquitination. Some cancer-related mutations can fully disrupt the inhibitory interactions and make WWP1 hyperactive. High expression level or hyperactivation of WWP1 may abnormally enhance polyubiquitinations of some oncoproteins or tumor suppressors, such as ΔNp63α, PTEN and p27, and ultimately promote cell proliferation, survival, migration and invasion in tumorigenesis. Given the dysregulation and oncogenic functions of WWP1 in some cancer types, it is promising to explore some therapeutic inhibitors to tune down its activity.

High Expression of WWP1 Associates with Tumor Progression in Papillary Thyroid Cancer.

Background: WWP1 (WW domain-containing E3 ubiquitin protein ligase 1) is increased in several kinds of carcinomas, but the influence of WWP1 in papillary thyroid cancer (PTC) is not well understood. Materials and Methods: The expression of WWP1 in PTC tissues and cells was detected by real-time reverse transcription PCR. The biological role of WWP1 on PTC cell growth, apoptosis, migration, and invasion ability was assessed with the Cell Counting Kit-8, colony forming, flow cytometry, wound healing, and transwell assays, respectively. Results: The expression of WWP1 mRNA and protein is increased in PTC tissue samples and cells. There is close correlation between the up expression of WWP1 and clinical parameters, such as tumor size, TNM, and distant metastasis. Knockdown of WWP1 blocks cell proliferation, migration, and invasion, causes cell cycle arrest, and induces apoptosis in PTC cells. Knockdown of WWP1 increases PTEN level and reduces p-PI3K and p-Akt level in PTC cells. Conclusions: Knockdown of WWP1 suppressed cell proliferation, migration, and invasion of PTC cell by downregulating the expression of p-PI3K and p-Akt, contributing to their understanding the pathogenesis of PTC.

Integrating HECW1 expression into the clinical indicators exhibits high accuracy in assessing the prognosis of patients with clear cell renal cell carcinoma

sBackgroundAlthough many intratumoral biomarkers have been reported to predict clear cell renal cell carcinoma (ccRCC) patient prognosis, combining intratumoral and clinical indicators could predict ccRCC prognosis more accurately than any of these markers alone. This study mainly examined the prognostic value of HECT, C2 and WW domain-containing E3 ubiquitin protein ligase 1 (HECW1) expression in ccRCC patients in combination with established clinical indicators.MethodsThe expression level of HECW1 was screened out by data-independent acquisition mass spectrometry (DIA-MS) and analyzed in ccRCC patients from the The Cancer Genome Atlas (TCGA) database and our cohort. A total of 300 ccRCC patients were stochastically divided into a training cohort and a validation cohort, and real-time PCR, immunohistochemistry (IHC) and statistical analyses were employed to examine the prognostic value of HECW1 in ccRCC patients.ResultsThe expression level of HECW1 usually decreased in human ccRCC specimens relative to control specimens in TCGA (p < 0.001). DIA-MS, Real-time PCR, and IHC analyses also showed that the majority of ccRCCs harbored decreased HECW1 expression compared with that in normal adjacent tissues (p < 0.001). Additionally, HECW1 expression was reduced in ccRCC cell lines compared with the normal renal cell line HK-2 (p < 0.001). Moreover, lower HECW1 expression was found in ccRCC patients with a higher tumor node metastasis (TNM) stage, bone metastasis, or first-line targeted drug resistance (p < 0.001). Low HECW1 expression indicated higher TNM stage, SSIGN (Stage, Size, Grade, and Necrosis) score and WHO/ISUP grade and poor prognosis in ccRCC patients (p < 0.05). Even after multivariable adjustment, HECW1, TNM stage, and SSIGN score served as independent risk factors. The c-index analysis showed that integrating intratumoral HECW1 expression into TNM stage or SSIGN score resulted in a higher c-index value than these indicators alone for predicting ccRCC patient prognosis.ConclusionHECW1 is a novel prognostic biomarker and therapeutic target in ccRCC, and integrating intratumoral HECW1 expression with established clinical indicators yields higher accuracy in assessing the postoperative prognosis of ccRCC patients.

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.

MiR-129-5p and -3p co-target WWP1 to suppress gastric cancer proliferation and migration.

Gastric cancer (GC) is a worldwide health problem. Uncovering the underlining molecular mechanisms of GC is of vital significance. Here, we identified a novel oncogene WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in GC. WWP1 could promote GC cell proliferation and migration in vitro and expedite GC growth in vivo. We also found out two microRNAs (miRNAs): miR-129-5p and -3p could both target WWP1. Interestingly, miR-129-5p bound to the CDS region of WWP1 mRNA. The miR-129 pairs (miR-129-5p and -3p) play pivotal roles in GC to suppress its proliferation and migration in vitro and slow down GC growth in vivo by repressing WWP1. In summary, we identified two tumor suppressive miRNAs which share the same precursor that could regulate the same oncogene WWP1 in GC. Our finding would add new route for GC research and treatment.

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.

WWP2 is a physiological ubiquitin ligase for phosphatase and tensin homolog (PTEN) in mice

The tumor suppressor phosphatase and tensin homolog (PTEN) plays a central role in regulating phosphatidylinositol 3-kinase (PI3K) signaling, and its gene is very frequently mutated in various human cancers. Numerous studies have revealed that PTEN levels are tightly regulated by both transcriptional and posttranslational modifications, with especially ubiquitylation significantly regulating PTEN protein levels. Although several ubiquitin ligases have been reported to mediate PTEN ubiquitylation in vitro, the ubiquitin ligase that promotes PTEN degradation in vivo has not been reported. Here we took advantage of specific knockout mouse models to demonstrate that WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) promotes PTEN degradation under physiological conditions, whereas another ubiquitin ligase, carboxyl terminus of Hsp70-interacting protein (CHIP), had no such effect. WWP2 knockout mice exhibited reduced body size, elevated PTEN protein levels, and reduced phosphorylation levels of the serine/threonine kinase and PTEN target AKT. In contrast, we observed no elevation of PTEN protein levels in CHIP knockout tissues and mouse embryonic fibroblasts. Furthermore, PTEN protein levels in CHIP/WWP2 double knockout mice were very similar to those in WWP2 single knockout mice and significantly higher than in WT and CHIP knockout mice. Our results demonstrate that WWP2, rather than CHIP, is an ubiquitin ligase that promotes PTEN degradation in vivo Considering PTEN's significant role in tumor development, we propose that WWP2 may be a potential target for fine-tuning PTEN levels in anticancer therapies.

The E3 ubiquitin ligase WWP1 sustains the growth of acute myeloid leukaemia

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27Kip1, which ultimately contributes to G0/G1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.

Abstract 2528: miR-452 inhibits migration and invasion of prostate cancer cells by targeting E3 ubiquitin ligase-1 (WWP1)

Abstract Introduction: Most prostate cancer (PCa) patients initially respond to androgen-deprivation therapy (ADT), but eventually acquire resistance to ADT and progress to castration-resistant prostate cancer (CRPC), resulting in metastasis causing PCa death. We have developed the microRNA (miRNA) expression signature of PCa using clinical specimens and determined the target genes of the tumor suppressive miRNAs, whose expressions are significantly decreased in PCa specimens. Based on the signature, our previous study revealed that miR-224 functions as a tumor suppressor, especially contributes to cancer cell metastasis directly targeting miR-224-TPD52 signaling. In human genome, miR-452 is located nearby tumor-suppressive miR-224, forming clustered miRNAs on chromosome Xq28 region. The aim of the study was to investigate the functional significance of miR-452 and to identify novel miR-452-mediated cancer pathways and responsible genes in PCa cells. Material and methods: Clinical prostate specimens were obtained from patients admitted to the Teikyo University Chiba Medical Center from 2008 to 2013. Ninety-two patients with elevated PSA levels underwent transrectal prostate needle biopsy, and three patients who died of CRPC underwent autopsies. PCa tissues (n=54), noncancerous prostate tissues (non-PCa, n=36), were used for analysis of expression levels of miRNAs. Functional studies of differentially expressed miRNAs were analyzed using PC3 and DU145 cells. The influence on CRPC-free survival of expression levels of miRNA was estimated using the Kaplan-Meier methods. In silico database and genome-wide gene expression analyses were performed to identify molecular targets regulated by the miRNAs. Results: (The miRNA expression signature of PCa specimens showed that the cluster miRNAs miR-224 was significantly downregulated, suggesting that this miRNA may act as tumor suppressor.) Clinical data using advanced PCa showed that low expression of miR-452 predicted a short duration of progression to CRPC. Restoration of miR-452 in PC3 and DU145 cells revealed significant inhibition of cancer cell migration and invasion. WW domain-containing E3 ubiquitin protein ligase-1 (WWP1) was confirmed as a direct target of miR-452 by in vitro transfection of miR-452 and Luciferase assay. Knockdown of the expression of WWP1 using siRNA showed inhibiting cell migration and invasion in PC3 and DU145 cells. Immunohistochemistry showed overexpressed WWP1 in advanced PCa. Conclusions: Loss of the tumor suppressive miR-452 enhanced migration and invasion in PCa cells. Regulation of the miR-452-WWP1 axis contributed to PCa cell migration and invasion, and elucidation of downstream signaling of this axis will provide new insights into the mechanisms of (progression to CRPC) metastasis of PCa. Citation Format: Satoko Kojima, Yusuke Goto, Akira Kurozumi, Mayuko Kato, Atsushi Okato, Takayuki Arai, Tomohiko Ichikawa, Yukio Naya, Naohiko Seki. miR-452 inhibits migration and invasion of prostate cancer cells by targeting E3 ubiquitin ligase-1 (WWP1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2528. doi:10.1158/1538-7445.AM2017-2528

Controlled and sustained delivery of siRNA/NPs from hydrogels expedites bone fracture healing.

Despite great potential, delivery remains as the most significant barrier to the widespread use of siRNA therapeutics. siRNA has delivery limitations due to susceptibility to RNase degradation, low cellular uptake, and poor tissue-specific localization. Here, we report the development of a hybrid nanoparticle (NP)/hydrogel system that overcomes these challenges. Hydrogels provide localized and sustained delivery via controlled release of entrapped siRNA/NP complexes while NPs protect and enable efficient cytosolic accumulation of siRNA. To demonstrate therapeutic efficacy, regenerative siRNA against WW domain-containing E3 ubiquitin protein ligase 1 (Wwp1) complexed with NP were entrapped within poly(ethylene glycol) (PEG)-based hydrogels and implanted at sites of murine mid-diaphyseal femur fractures. Results showed localization of hydrogels and controlled release of siRNA/NPs at fractures for 28 days, a timeframe over which fracture healing occurs. siRNA/NP sustained delivery from hydrogels resulted in significant Wwp1 silencing at fracture callus compared to untreated controls. Fractures treated with siRNA/NP hydrogels exhibited accelerated bone formation and significantly increased biomechanical strength. This NP/hydrogel siRNA delivery system has outstanding therapeutic promise to augment fracture healing. Owing to the structural similarities of siRNA, the development of the hydrogel platform for invivo siRNA delivery has myriad therapeutic possibilities in orthopaedics and beyond.

Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer

BackgroundMicroRNAs are endogenously expressed, small non-coding RNAs that modulate gene expression by targeting specific mRNAs, resulting in translational repression or mRNA degradation. Although miR-584-5p has been reported to play a vital role in various malignancies, its role and the molecular mechanisms underlying the effects of miR-584-5p in gastric cancer (GC) remain to be clarified. In this study, we investigated the role of miR-584-5p in GC.MethodsThe expression of miR-584-5p and its specific target gene were determined in human GC specimens and cell lines by microRNA real-time polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR) and Western blot. The effects of miR-584-5p depletion or ectopic expression on GC proliferation were evaluated in vitro using CCK-8 proliferation assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, colony formation assays and cell-cycle assays and the in vivo effects were investigated using a mouse tumorigenicity model. Cell apoptosis was evaluated by in vitro flow cytometric analysis, cell viability assays and in vivo TUNEL assays. Luciferase reporter assays were employed to identify interactions between miR-584-5p and its specific target gene.ResultsA series of in vitro and in vivo gain- and loss-of-function assays revealed that miR-584-5p inhibited GC cell proliferation, while apoptosis was induced. Luciferase reporter assays and Western blot analysis revealed WWP1 to be a direct target of miR-584-5p. The effects of miR-584-5p-mimic were rescued by WWP1 overexpression. In contrast, the effects of the miR-584-5p-inhibitor were impaired by WWP1-shRNA. Furthermore, miR-584-5p expression levels correlated negatively with WWP1 protein expression in GC tissues and GC cell lines. A series of investigations indicated that miR-584-5p promoted senescence and activated the TGFβ signaling pathway by downregulation of WWP1.ConclusionTaken together, these results suggest that downregulation of miR-584-5p contributes to tumor progression by downregulation of WWP1, thus, highlighting the potential of miR-584-5p as a therapeutic target for human GC.

Regulation of E3 ubiquitin ligase-1 (WWP1) by microRNA-452 inhibits cancer cell migration and invasion in prostate cancer.

Background:MicroRNA-224 (miR-224) and microRNA-452 (miR-452) are closely located on the human chromosome Xq28 region. miR-224 functions as a tumour suppressor by targeting tumour protein D52 (TPD52) in prostate cancer (PCa). Here, we aimed to investigate the functional significance of miR-452 in PCa cells.Methods:Functional studies of PCa cells were performed using transfection with mature miRNAs or siRNAs. Genome-wide gene expression analysis, in silico analysis, and dual-luciferase reporter assays were applied to identify miRNA targets. The association between miR-452 levels and overall patient survival was estimated by the Kaplan–Meier method.Results:Expression of miR-452 was significantly downregulated in PCa tissues. Transfection with mature miR-452 inhibited the migration and invasion of PCa cells. Kaplan–Meier survival curves showed that low expression of miR-452 predicted a short duration of progression to castration-resistant PCa. WW domain-containing E3 ubiquitin protein ligase-1 (WWP1) was a direct target of miR-452, and knockdown of WWP1 inhibited the migration and invasion of PCa cells. WWP1 was upregulated in PCa clinical specimens.Conclusions:Regulation of the miR-452–WWP1 axis contributed to PCa cell migration and invasion, and elucidation of downstream signalling of this axis will provide new insights into the mechanisms of PCa oncogenesis and metastasis.