Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/ß-catenin signaling by stabilization of protein kinase Cα.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/ß-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased ß-catenin and downregulated glycogen synthase kinase-3ß (GSK-3ß) protein levels and its activation. However, ß-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3ß was significantly increased after NDRG1 overexpression, suggesting a GSK-3ß-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing ß-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing ß-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in ß-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and ß-catenin was identified, with the formation of a potential metabolon that promotes the latter ß-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in ß-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased ß-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling.

NDRG1 overcomes resistance to immunotherapy of pancreatic ductal adenocarcinoma through inhibiting ATG9A-dependent degradation of MHC-1.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. METHODS: Enrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. RESULTS: Here, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. CONCLUSION: Our results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.

The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining.

The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.

Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Inhibition of Aurora-A/N-Myc Protein-Protein Interaction Using Peptidomimetics: Understanding the Role of Peptide Cyclization.

Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal - is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further in silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i) the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii) the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.

Genetics in ophthalmology: molecular blueprints of retinoblastoma.

This review presents current knowledge on the molecular biology of retinoblastoma (RB). Retinoblastoma is an intraocular tumor with hereditary and sporadic forms. 8,000 new cases of this ocular malignancy of the developing retina are diagnosed each year worldwide. The major gene responsible for retinoblastoma is RB1, and it harbors a large spectrum of pathogenic variants. Tumorigenesis begins with mutations that cause RB1 biallelic inactivation preventing the production of functional pRB proteins. Depending on the type of mutation the penetrance of RB is different. However, in small percent of tumors additional genes may be required, such as MYCN, BCOR and CREBBP. Additionally, epigenetic changes contribute to the progression of retinoblastoma as well. Besides its role in the cell cycle, pRB plays many additional roles, it regulates the nucleosome structure, participates in apoptosis, DNA replication, cellular senescence, differentiation, DNA repair and angiogenesis. Notably, pRB has an important role as a modulator of chromatin remodeling. In recent years high-throughput techniques are becoming essential for credible biomarker identification and patient management improvement. In spite of remarkable advances in retinoblastoma therapy, primarily in high-income countries, our understanding of retinoblastoma and its specific genetics still needs further clarification in order to predict the course of this disease and improve therapy. One such approach is the tumor free DNA that can be obtained from the anterior segment of the eye and be useful in diagnostics and prognostics.

Transformation of A1/A2 Astrocytes Participates in Brain Ischemic Tolerance Induced by Cerebral Ischemic Preconditioning via Inhibiting NDRG2.

Cerebral ischemic preconditioning (CIP) has been shown to improve brain ischemic tolerance against subsequent lethal ischemia. Reactive astrocytes play important roles in cerebral ischemia-reperfusion. Recent studies have shown that reactive astrocytes can be polarized into neurotoxic A1 phenotype (C3d) and neuroprotective A2 phenotype (S100A10). However, their role in CIP remains unclear. Here, we focused on the role of N-myc downstream-regulated gene 2 (NDRG2) in regulating the transformation of A1/A2 astrocytes and promoting to brain ischemic tolerance induced by CIP. A Sprague Dawley rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used. Rats were divided into the following six groups: (1) sham group; (2) CIP group: left middle cerebral artery was blocked for 10 min; (3) MCAO/R group: left middle cerebral artery was blocked for 90 min; (4) CIP + MCAO/R group: CIP was performed 72 h before MCAO/R; (5) AAV-NDRG2 + CIP + MCAO/R group: adeno-associated virus (AAV) carrying NDRG2 was administered 14 days before CIP + MCAO/R; (6) AAV-Ctrl + CIP + MCAO/R group: empty control group. The rats were subjected to neurological evaluation 24 h after the above treatments, and then were sacrificed for 2, 3, 5-triphenyltetraolium chloride staining, thionin staining, immunofluorescence and western blot analysis. In CIP + MCAO/R group, the neurological deficit scores decreased, infarct volume reduced, and neuronal density increased compared with MCAO/R group. Notably, CIP significantly increased S100A10 expression and the number of S100A10+/GFAP+ cells, and also increased NDRG2 expression. MCAO/R significantly decreased S100A10 expression and the number of S100A10+/GFAP+ cells yet increased C3d expression and the number of C3d+/GFAP+ cells and NDRG2 expression, and these trends were reversed by CIP + MCAO/R. Furthermore, over-expression of NDRG2 before CIP + MCAO/R, the C3d expression and the number of C3d+/GFAP+ cells increased, while S100A10 expression and the number of S100A10+/GFAP+ cells decreased. Meanwhile, over-expression of NDRG2 blocked the CIP-induced brain ischemic tolerance. Taken together, these results suggest that CIP exerts neuroprotective effects against ischemic injury by suppressing A1 astrocyte polarization and promoting A2 astrocyte polarization via inhibiting NDRG2 expression.

Biological and prognostic significance of NDRG2 downregulation in oral squamous cell carcinoma.

OBJECTIVE: Downregulation of N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor gene, has been associated with poor clinical outcomes in various cancers. However, the prognostic significance of NDRG2 in oral squamous cell carcinoma (OSCC) remains unknown. This study aimed to evaluate the prognostic value of NDRG2 downregulation in OSCC and to elucidate the mechanism by which NDRG2 is downregulated and the biological role of NDRG2 in tumor progression. METHODS: Immunohistochemical and in silico analyses of NDRG2 expression were performed, and the correlation between NDRG2 expression and clinicopathological data was analyzed. The effect of NDRG2 knockdown on the biological behavior of OSCC cells was investigated and the effect of 5-aza-2'-deoxycytidine (5-aza-dC) on NDRG2 expression was determined. RESULTS: NDRG2 expression was significantly downregulated and DNA hypermethylation of NDRG2 was frequently found in head and neck SCC, including OSCC. Low NDRG2 expression was significantly correlated with adverse clinicopathological features and worse survival in OSCC. NDRG2 knockdown could enhance the oncogenic properties of OSCC cells. NDRG2 mRNA levels in OSCC cells could be restored by 5-aza-dC. CONCLUSION: Downregulation of NDRG2 promotes tumor progression and predicts poor prognosis in OSCC. Therefore, restoration of NDRG2 expression may be a potential therapeutic strategy in OSCC.

Lipid Droplets Are Beneficial for Rabies Virus Replication by Facilitating Viral Budding.

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been reported to play a role in pathogenesis of several viruses. However, their role in RABV infection remains unclear. Here, we initially found that RABV infection upregulated LD production in multiple cells and mouse brains. After treatment with atorvastatin, a specific inhibitor of LDs, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhanced the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglyceride synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication, and their biogenesis is regulated via the NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but their role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier for transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

Role of MRI radiomics for the prediction of MYCN amplification in neuroblastomas.

OBJECTIVES: We evaluate MR radiomics and develop machine learning-based classifiers to predict MYCN amplification in neuroblastomas. METHODS: A total of 120 patients with neuroblastomas and baseline MR imaging examination available were identified of whom 74 (mean age ± standard deviation [SD] of 6 years and 2 months ± 4 years and 9 months; 43 females and 31 males, 14 MYCN amplified) underwent imaging at our institution. This was therefore used to develop radiomics models. The model was tested in a cohort of children with the same diagnosis but imaged elsewhere (n = 46, mean age ± SD: 5 years 11 months ± 3 years 9 months, 26 females and 14 MYCN amplified). Whole tumour volumes of interest were adopted to extract first-order histogram and second-order radiomics features. Interclass correlation coefficient and maximum relevance and minimum redundancy algorithm were applied for feature selection. Logistic regression, support vector machine, and random forest were employed as the classifiers. Receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic accuracy of the classifiers on the external test set. RESULTS: The logistic regression model and the random forest both showed an AUC of 0.75. The support vector machine classifier obtained an AUC of 0.78 on the test set with a sensitivity of 64% and a specificity of 72%. CONCLUSION: The study provides preliminary retrospective evidence demonstrating the feasibility of MRI radiomics in predicting MYCN amplification in neuroblastomas. Future studies are needed to explore the correlation between other imaging features and genetic markers and to develop multiclass predictive models. KEY POINTS: • MYCN amplification in neuroblastomas is an important determinant of disease prognosis. • Radiomics analysis of pre-treatment MR examinations can be used to predict MYCN amplification in neuroblastomas. • Radiomics machine learning models showed good generalisability to external test set, demonstrating reproducibility of the computational models.

ETS1 regulates NDRG1 to promote the proliferation, migration, and invasion of OSCC.

OBJECTIVE: The aim of this study was to investigate the molecular mechanism by which the transcription factor ETS1 regulates N-myc downstream regulatory gene 1 (NDRG1) to provide a new theoretical basis for the study of oral squamous cell carcinoma (OSCC). METHODS: In this study, eight human OSCC and paraneoplastic samples were collected. The expressions of NDRG1, ETS1, and Ki67 were detected by immunohistochemistry; apoptosis was detected by tdt-mediated dUTP notched end labeling; cell migration and invasion were detected by Transwell; quantitative real-time PCR was performed to detect the expression of NDRG1; RNA-binding protein immunoprecipitation (RIP) assays detected NDRG1 expression; immunofluorescence assays detected ETS1 expression. RESULTS: NDRG1 and ETS1 expression was significantly upregulated in cancer tissues and CAL-27 and SCC-6 cells. Knockdown of NDRG1 and ETS1 inhibited cell proliferation, migration, invasion, cloning, and EMT while promoting apoptosis and inhibited tumor development; ETS1 positively regulated NDRG1 expression; Finally, overexpression of NDRG1 in vivo and in vitro reversed the effect of ETS1 knockdown on CAL-27 and SCC-6 cells. CONCLUSIONS: ETS1 positively regulates the expression of NDRG1 and promotes OSCC. Therefore, ETS1 may serve as a new target for the clinical diagnosis and treatment of OSCC.

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

CD47-Dependent Regulation of Immune Checkpoint Gene Expression and MYCN mRNA Splicing in Murine CD8 and Jurkat T Cells.

Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.

mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

N-myc and STAT interactor is a novel biomarker of severity in community-acquired pneumonia: a prospective study.

OBJECTIVES: To tested the ability of N-myc and STAT interactor (NMI) levels in patients with community-acquired pneumonia (CAP) to predict the severity of the disease. METHODS: Prospective observational analysis of patients with CAP was performed. The NMI levels in serum of 394 CAP patients on admission were measured by immunoassay. Thirty-day mortality and intensive care unit (ICU) admission were set as clinical outcomes. The predicting value of NMI for clinical outcomes was determined by receiver operating characteristic curve and logistic regression analysis. The internal validity was assessed using cross-validation with bootstrap resampling. RESULTS: NMI was an independent risk factor for both 30-day mortality and admission to ICU for CAP patients. The area under curve (AUC) of NMI to predict mortality was 0.91 (95% CI: 0.86-0.96), and that to predict ICU admission was 0.92 (95% CI: 0.88-0.97), significantly higher than that of other biomarkers including procalcitonin and C-reactive protein. The proportion of clinical outcomes notably rose as NMI levels elevated (P < 0.001). The AUCs of the new score systems including NMI (N-PSI and N-CURB65 score) to predict outcomes were significantly higher than the original score systems. CONCLUSIONS: NMI is a novel biomarker for predicting CAP severity superior to former biomarkers in 30-day mortality and ICU admission.

Expression and clinicopathological significance of glucocorticoid receptor, SGK1, and NDRG1 in hormone-naïve prostate carcinoma.

Glucocorticoid receptor (GR) has been implicated in prostate carcinoma growth and progression. Glucocorticoid receptor beta (GRß) acts as an inhibitor of GR; however, its function is not well understood. Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a GR-responsive gene that phosphorylates N-myc downstream-regulated gene 1 (NDRG1) and is involved in cancer growth and invasion. However, the expression of GR, GRß, SGK1, and NDRG1 in prostate cancer and their relationship with clinicopathological and functional significance remain unknown. The association between the status of GR, GRß, SGK1, and NDRG1 immunoreactivity and clinicopathological variables was analyzed in patients with prostate carcinoma to explore their clinical significance. In prostate carcinoma cases, the relative abundance of GR and NDRG1 immunoreactivity was inversely and significantly associated with the primary tumor stage (pT), while GR immunoreactivity was inversely and significantly associated with the Ki-67 score. The relative expression status of NDRG1 was significantly associated with that of GR. However, no significant correlation was observed between any of the clinicopathological parameters and GRß and SGK1 expression. Our findings indicate that GR and NDRG1 expression status is correlated with clinicopathological features in patients with prostate cancer.

NDRG1 is expressed in human granulosa cells: An implicative role of NDRG1 in the ovary.

Purpose: N-myc downstream-regulated gene 1 (NDRG1) is expressed in various human tissues and plays a role in regulating cellular proliferation, angiogenesis, and hypoxia sensing. However, the role of NDRG1 in the ovary remains poorly understood. Therefore, we investigated NDRG1 expression and the role of NDRG1 in the human ovary. Methods: Follicular fluid (FF) and luteinized granulosa cells were collected from follicles during oocyte retrieval. KGN cells were cultured with cobalt chloride (CoCl2, a hypoxia-mimicking agent) and/or echinomycin. mRNA, protein levels and secretion, and localization were assessed by real-time PCR, Western blotting, ELISA, and immunohistochemical analysis, respectively. KGN cells were also transfected with NDRG1 siRNA for 72 h. Results: NDRG1 protein was expressed in luteinized granulosa cells. NDRG1 concentration was positively correlated with vascular endothelial growth factor (VEGF) and progesterone concentrations in FF. CoCl2-induced hypoxic stress significantly increased NDRG1 and VEGF mRNA and protein and hypoxia-inducible factor-1α expression compared with those in the controls. The CoCl2-induced overexpression of NDRG1 and VEGF was suppressed by echinomycin. Transfection with NDRG1 siRNA significantly suppressed the release of progesterone in the culture medium. Conclusions: These results indicate that ovarian NDRG1 may play important roles in follicular development, especially in the early luteinization of pre-ovulatory follicles.

NMI Facilitates Influenza A Virus Infection by Promoting Degradation of IRF7 through TRIM21.

Acute respiratory infections caused by influenza A virus (IAV) spread widely and lead to substantial morbidity and mortality. Host cell induction of type I interferon (IFN-I) plays a fundamental role in eliminating the virus during the innate antiviral response. The potential role of N-myc and STAT interactor (NMI) and its underlying mechanisms of action during IAV infection, however, remain elusive. In this study, we found that the expression of NMI increased after IAV infection. Nmi-knockout mice infected with IAV displayed increased survival rate, decreased weight loss, lower viral replication, and attenuated lung inflammation when compared with wild-type mice. Deficiency of NMI promoted the production of IFN-I and IFN-stimulated genes in vivo and in vitro. Reduced levels of NMI also resulted in an increase of the expression of IFN regulator factor (IRF) 7. Further studies have revealed that NMI could interact with IRF7 after IAV infection, and this interaction involved its NID1 and NID2 domain. In addition, NMI facilitated ubiquitination and proteasome-dependent degradation of IRF7 through recruitment of the E3 ubiquitin ligase TRIM21 (tripartite motif-containing 21) to limit the IAV-triggered innate immunity. Our findings reveal a clearer understanding of the role of NMI in regulating the host innate antiviral response and provide a potential therapeutic target for controlling IAV infection.

Thiosemicarbazones suppress expression of the c-Met oncogene by mechanisms involving lysosomal degradation and intracellular shedding.

Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.

Astroglial N-myc downstream-regulated gene 2 protects the brain from cerebral edema induced by stroke.

Brain edema is a grave complication of brain ischemia and is the main cause of herniation and death. Although astrocytic swelling is the main contributor to cytotoxic edema, the molecular mechanism involved in this process remains elusive. N-myc downstream-regulated gene 2 (NDRG2), a well-studied tumor suppressor gene, is mainly expressed in astrocytes in mammalian brains. Here, we found that NDRG2 deficiency leads to worsened cerebral edema, imbalanced Na+ transfer, and astrocyte swelling after ischemia. We also found that NDRG2 deletion in astrocytes dramatically changed the expression and distribution of aquaporin-4 and Na+ -K+ -ATPase ß1, which are strongly associated with cell polarity, in the ischemic brain. Brain edema and astrocyte swelling were significantly alleviated by rescuing the expression of astrocytic Na+ -K+ -ATPase ß1 in NDRG2-knockout mouse brains. In addition, the upregulation of astrocytic NDRG2 by lentiviral constructs notably attenuated brain edema, astrocytic swelling, and blood-brain barrier destruction. Our results indicate a particular role of NDRG2 in maintaining astrocytic polarization to facilitate Na+ and water transfer balance and to protect the brain from ischemic edema. These findings provide insight into NDRG2 as a therapeutic target in cerebral edema.