C1orf109L binding DHX9 promotes DNA damage depended on the R-loop accumulation and enhances camptothecin chemosensitivity.

OBJECTIVES: R-loop is a three-stranded nucleic acid structure of RNA/DNA hybrid, which occurs naturally during transcription, and more R-loop accumulation can trigger serious DNA damage. There has been increasing attention to the issue of R-loop accumulation acted as a target for cancer therapy. However, the regulation of R-loop-associated proteins is poorly explored. MATERIAL AND METHOD: Quantitative real-time PCR and Western blot were used to measure the expression of C1orf109 in cell lines. In addition, C1orf109L (C1orf109 longest isoform) protein binding partner was identified and validated using immunoprecipitation-mass spectrometric (IP-MS) and immunoprecipitation assays. DNA-RNA immunoprecipitation (DR-IP) and immunofluorescence determined the C1orf109L location on R-loop. R-loop accumulation was determined by immunofluorescence. Cell cycle was determined by flow cytometry. Finally, time-lapse assay and cell counting were conducted to determined cell survival in response to camptothecin (CPT). RESULTS: We found that C1orf109L could mediate cell cycle arrest in the G2/M phase and DNA damage depended on R-loop accumulation. Meanwhile, C1orf109L could bind with DHX9 to trigger R-loop accumulation. And C1orf109L was competitive with PARP1 binding to DHX9, which would block the function of DHX9-PARP1 to prevent the R-loop accumulation. Furthermore, C1orf109L could enhance the chemosensitivity of CPT, a chemotherapeutic drug capable of promoting R-loop formation. CONCLUSIONS: Our data demonstrate that C1orf109L triggers R-loop accumulation and DNA damage to arrest cell cycle.

Evolution and extensive reassortment of H5 influenza viruses isolated from wild birds in China over the past decade.

Lethal infection of wild birds with different subtypes of H5 viruses continuously occur. To investigate the genetic evolution and pathogenicity of H5 viruses in wild birds, we performed a detailed genetic and biologic analysis of 27 viruses, including H5N1, H5N2, H5N6, and H5N8 subtypes, that were responsible for avian influenza outbreaks in wild birds in China over the past decade. We found that these 27 viruses, bearing different clades/subclades of HA, were complicated reassortants and formed 12 different genotypes. Ten of the viruses tested were highly pathogenic in chickens, but showed distinct pathotypes in ducks and mice. Five of these 10 viruses, which were all from clade2.3.4.4, could bind human-type receptors. Our findings reveal the diversity of the genetic and biologic properties of H5 viruses circulating in wild birds and highlight the need to carefully monitor and evaluate the risks these viruses pose to animal and public health.

Author Correction: Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper. The original Letter has not been corrected.

MARVELD1 interacting with catalase regulates reactive oxygen species metabolism and mediates the sensitivity to chemotherapeutic drugs in epithelial tumors of the reproductive system.

Previous investigations have found that MARVEL domain-containing 1 (MARVELD1) could inhibit tumor cell proliferation and enhance the sensitivity to chemotherapeutic drugs in hepatocellular carcinoma. Hence, it may be a valuable therapeutic target. In the study, we analyzed the responsive changes of MARVELD1 to 25 stress factors and expression of MARVELD1 in epithelial tumors of the reproductive system. We found that MARVELD1 was transferred to the cytoplasm and mitochondria under cell stress. And under cellular stress, the reactive oxygen species (ROS) levels decreased in MARVELD1 expressed cells while increased in the cells of MARVELD1-specific siRNA treatment. Meanwhile, MARVELD1 overexpression significantly promoted the inhibition of tumor cell proliferation under cellular stress via affecting ROS metabolism, not cell cycle. In xenograft tumor tissues with MARVELD1 expression, the tumor growth was inhibited and accompanied by the lower ROS levels. Furthermore, we identified that MARVELD1 could interact with catalase (CAT) to enhance latter activity and maintain stability. And the enhanced sensitivity to chemotherapeutic drugs clearly depended on the ability of MARVELD1 scavenge the ROS in carcinoma cells of the reproductive system. Our findings clearly explain that MARVELD1 may regulate tumor cell proliferation and sensitivity to chemotherapeutic drugs via reducing the exorbitant ROS. The mechanism was that MARVELD1 interacted with CAT to maintain latter stability, and then ensure continuous ROS scavenge.

MARVELD1 depletion leads to dysfunction of motor and cognition via regulating glia-dependent neuronal migration during brain development.

The establishment of functional neuronal connectivity is dependent on the neuronal migration and the accurate positioning of neurons in the developing brain. Abnormal neuronal migration can trigger neuronal maturation defects and apoptosis. However, many genetic bases remain unclear in neuronal migration disorders during brain development. In this study, we reported that MARVELD1-defected mice displayed motor and cognitive dysfunction resulting from aberrant neuronal migration during brain development. The laminar organization of the cerebral cortex and cerebellum in MARVELD1 knockout (KO) mice is disrupted, indicating impaired radial neuronal migration. Furthermore, we used the cerebellum as a model to explore the radial neuronal migration processes, and the results demonstrated that the proper neuronal migration depended on MARVELD1 expression in glial cells of the developing brain. MARVELD1 suppressed the expression of ITGB1 and FAK Tyr397 phosphorylation in glia-dependent manner. The inhibition of the MARVELD1/ITGB1/FAK signalling pathway in MARVELD1 KO mice could reverse the defects in neuronal migration in vitro. Our findings revealed that MARVELD1 regulated neuronal migration by mediating the formation of glial fibres and ITGB1/FAK signalling pathway. The depletion of MARVELD1 during mouse brain development led to the abnormity of motor and cognition functions.

SCFß-TRCP E3 ubiquitin ligase targets the tumor suppressor ZNRF3 for ubiquitination and degradation.

Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing ß-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of ß-catenin by ß-TRCP, ZNRF3 is ubiquitinated by ß-TRCP in both CKI-phosphorylation- and degron-dependent manners. Thus, our findings not only identify a novel substrate for ß-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by ß-TRCP in a context-dependent manner where ß-TRCP negatively regulates Wnt signaling by targeting ß-catenin, and positively regulates Wnt signaling by targeting ZNRF3.

Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.

Treatments that target immune checkpoints, such as the one mediated by programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating human cancers with durable clinical benefit. However, many patients with cancer fail to respond to compounds that target the PD-1 and PD-L1 interaction, and the underlying mechanism(s) is not well understood. Recent studies revealed that response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumour cells. Hence, it is important to understand the mechanistic pathways that control PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1-PD-L1 blockade in patients with cancer. Here we show that PD-L1 protein abundance is regulated by cyclin D-CDK4 and the cullin 3-SPOP E3 ligase via proteasome-mediated degradation. Inhibition of CDK4 and CDK6 (hereafter CDK4/6) in vivo increases PD-L1 protein levels by impeding cyclin D-CDK4-mediated phosphorylation of speckle-type POZ protein (SPOP) and thereby promoting SPOP degradation by the anaphase-promoting complex activator FZR1. Loss-of-function mutations in SPOP compromise ubiquitination-mediated PD-L1 degradation, leading to increased PD-L1 levels and reduced numbers of tumour-infiltrating lymphocytes in mouse tumours and in primary human prostate cancer specimens. Notably, combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumour regression and markedly improves overall survival rates in mouse tumour models. Our study uncovers a novel molecular mechanism for regulating PD-L1 protein stability by a cell cycle kinase and reveals the potential for using combination treatment with CDK4/6 inhibitors and PD-1-PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.

Cullin 3SPOP ubiquitin E3 ligase promotes the poly-ubiquitination and degradation of HDAC6.

The histone deacetylase 6 (HDAC6) plays critical roles in human tumorigenesis and metastasis. As such, HDAC6-selective inhibitors have entered clinical trials for cancer therapy. However, the upstream regulator(s), especially ubiquitin E3 ligase(s), responsible for controlling the protein stability of HDAC6 remains largely undefined. Here, we report that Cullin 3SPOP earmarks HDAC6 for poly-ubiquitination and degradation. We found that the proteasome inhibitor MG132, or the Cullin-based E3 ligases inhibitor MLN4924, but not the autophagosome-lysosome inhibitor bafilomycin A1, stabilized endogenous HDAC6 protein in multiple cancer cell lines. Furthermore, we demonstrated that Cullin 3-based ubiquitin E3 ligase(s) primarily reduced the stability of HDAC6. Importantly, we identified SPOP, an adaptor protein of Cullin 3 family E3 ligases, specifically interacted with HDAC6, and promoted its poly-ubiquitination and subsequent degradation in cells. Notably, cancer-derived SPOP mutants disrupted their binding with HDAC6 and thereby failed to promote HDAC6 degradation. More importantly, increased cellular proliferation and migration in SPOP-depleted HCT116 colon cancer cells could be partly reversed by additional depletion of HDAC6, suggesting that HDAC6 is a key downstream effector for SPOP tumor suppressor function. Together, our data identify the tumor suppressor SPOP as an upstream negative regulator for HDAC6 stability, and SPOP loss-of-function mutations might lead to elevated levels of the HDAC6 oncoprotein to facilitate tumorigenesis and metastasis in various human cancers.

Effects of Propofol on Several Membrane Characteristics of Cervical Cancer Cell Lines.

BACKGROUND: Although significant advances have been made toward understanding the molecular mechanisms underlying the effect of propofol on tumor cell metastasis, less is known regarding how cell membrane and cytoskeletal ultrastructure are affected in this process. Here, we investigated the relationship between cell morphology and cell size, which are features mainly defined by the cytoskeleton. METHODS: To confirm the effects of propofol on the migratory ability of human cervical carcinoma cells, cell migration and invasion were examined through scratch wound healing and transwell membrane assays. Furthermore, HeLa cells cultivated with different concentrations of propofol were examined by confocal microscopy and atomic force microscopy (AFM), and the mean optical density and migration ability of these cells were also assessed. In addition, cell membrane morphology was inspected using AFM. RESULTS: The results of the wound healing and transwell membrane assays indicated that propofol decreases the migratory ability of cervical carcinoma cells compared to control cells. A comparative analysis of the test results revealed that short-term (3 h) exposure to propofol induced marked changes in cell membrane microstructure and in the cytoskeleton in a dose-dependent manner. These morphological changes in the cell membrane were accompanied by cytoskeleton (F-actin) derangement. The present findings demonstrate a close relationship between changes in cell membrane ultrastructure and cytoskeletal alterations (F-actin) in propofol-treated HeLa cells. AFM scanning analysis showed that cell membrane ultrastructure was significantly changed, including a clear reduction in membrane roughness. CONCLUSION: The influence of propofol on the HeLa cell cytoskeleton can be directly reflected by changes in cellular morphology, as assessed by AFM. Moreover, the use of AFM is a good method for investigating propofol-mediated changes within cytoskeletal ultrastructure.

MARVELD1 modulates cell surface morphology and suppresses epithelial-mesenchymal transition in non-small cell lung cancer.

Integrins have been known to play pivotal roles in malignant progression and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC). We previously demonstrated that MARVELD1, a potential tumor suppressor, is epigenetically silenced in multiple cancer cells. In this study, we found MARVELD1 silencing altered cell surface ultrastructure of NSCLC cells and inhibited the formation of punctate integrin ß1/ß4 cluster in microvillus, whereas MARVELD1 overexpression suppressed TGF-ß1-induced EMT. Remarkably, the balance of integrin ß1 and ß4 was modulated by MARVELD1. MARVELD1 silencing led to imbalance of integrin ß1/ß4 and significantly reduced microvillus length, furthermore affected the localization of ß1/ß4 at microvilli tips. TGF-ß1-induced EMT was promoted by MARVELD1 silencing, while rebalance of integrin ß1/ß4 partly rescued the epithelial phenotype of MARVELD1-silenced cells. Mechanistically, we demonstrate that MARVELD1-mediated balance of integrin ß1 and ß4 regulates cell surface ultrastructure and EMT phenotype of NSCLC cells, suggesting MARVELD1 has a potential to be developed as a therapeutic target for NSCLC. © 2015 Wiley Periodicals, Inc.

Phylogenetic and pathogenic analyses of three H5N1 avian influenza viruses (clade 2.3.2.1) isolated from wild birds in Northeast China.

From April to September 2012, periodic surveillance of avian influenza H5N1 viruses from different wild bird species was conducted in Northeast China. Three highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from a yellow-browed warbler, common shoveler, and mallard. To trace the genetic lineage of the isolates, nucleotide sequences of all eight gene segments were determined and phylogenetically analyzed. The data indicated that three viruses belonged to the same antigenic virus group: clade 2.3.2.1. To investigate the pathogenicity of these three viruses in different hosts, chickens, ducks, and mice were inoculated. The results showed that chickens were susceptible to each of the three HPAI H5N1 viruses, resulting in 100% mortality within 2-6 days after infection, whereas the three isolates exhibited distinctly different virulence in ducks and mice. The results of this study demonstrated that HPAI H5N1 viruses of clade 2.3.2.1 are still circulating in wild birds through overlapping migratory flyways. Therefore, continuous monitoring of H5N1 in both domestic and wild birds is necessary to prevent a potentially wider outbreak.

Phylogenetic analysis of a novel H6N6 avian influenza virus isolated from a green peafowl in China and its pathogenic potential in mice.

To explore the ecology of the H6 subtype avian influenza viruses in Hebei Province, China, a long-term surveillance was conducted in 2012 among domestic poultry and birds in a wildlife park. In this study, we report the characterization of a novel H6N6 avian influenza virus isolated from a healthy green peafowl in Qinghuangdao Wildlife Park in 2012. A phylogenetic analysis indicated that the isolated H6N6 strain has the same gene constellation as the ST3367-like strains, which are mainly distributed in southern and eastern China. A mouse experiment showed that the isolate replicated efficiently in the lungs and turbinates of infected mice without previous adaptation, resulting in locally thickened alveolar septa and interstitial pneumonia. Further studies of the H6 subtype viruses are required to clarify their evolutionary pattern in north China, which will benefit disease control and pandemic preparedness for novel viruses.

Phylogenetic and pathogenic analysis of a novel H6N2 avian influenza virus isolated from a green peafowl in a wildlife park.

H6 subtype avian influenza virus, which has been circulating among different species, causes considerable concern for both veterinary medicine and public health. We isolated a strain of H6N2 avian influenza virus from healthy green peafowl (Pavo muticus) in Qinghuangdao Wildlife Park in Hebei Province, China, in 2012. A phylogenetic analysis indicated that the isolated H6N2 strain had the same gene constellation as southern China strains, which were predominantly isolated from waterfowl distributed in Shantou, Guangxi, and Hunan in 2001-2010. The isolate showed no and low pathogenicity in chickens and ducks, respectively. However, it replicated efficiently in the lungs and turbinate of infected mice, resulting in thickened alveolar septa and moderate interstitial pneumonia. This finding raises concerns that the H6N2 subtype maybe evolve into a novel endemic avian influenza virus. Therefore, periodical surveillance of avian influenza viruses must be undertaken to monitor the advent of novel viruses.