N-myc Downstream-regulated Gene Family Research Articles

Clinical Diagnostic and Prognostic Potential of NDRG1 and NDRG2 in Hepatocellular Carcinoma Patients.

BackgroundPrimary liver cancer is still the most common lethal malignancy. The N-myc downstream-regulated gene family (NDRG1–4) is a group of multifunctional proteins associated with carcinogenesis. However, systematic evaluation of the diagnostic and prognostic values of NDRG1 or NDRG2 expression in liver cancer is poorly investigated.MethodThe gene expression matrix of liver hepatocellular carcinoma (LIHC) was comprehensively analyzed by the “limma” and “Dseq2” R packages. The Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) were used to identify the biological functional differences. A single-sample GSEA (ssGSEA) was conducted to quantify the extent of immune cell infiltration. Finally, the clinical and prognostic information of LIHC patients was systematically investigated using Kaplan–Meier analysis and logistic and Cox regression analysis.ResultsCompared with normal tissues, NDRG1 expression was higher, whereas NDRG2 expression was lower in tumor tissues (P <0.001). The area under the receiver operator characteristic curve (AUROC) of NDRG1 and NDRG2 for LIHC was 0.715 and 0.799, respectively. Kaplan–Meier analysis revealed that NDRG1 and NDRG2 were independent clinical prognostic biomarkers for the overall survival (OS, P = 0.001 and 2.9e−06), progression-free interval (PFI, P = 0.028 and 0.005) and disease-specific survival (DSS, P = 0.027 and P <0.001). The C-indexes and calibration plots of the nomogram suggest that NDRG1 and NDRG2 have an effective predictive performance for OS (C-index: 0.676), DSS (C-index: 0.741) and PFI (C-index: 0.630) of liver cancer patients. The mutation rate of NDRG1 in liver cancer reached up to 14%, and DNA methylation levels of NDRG1 and NDRG2 promoters correlated significantly with clinical prognosis.ConclusionsThe mRNA expression and DNA methylation of NDRG superfamily members have the potential for LIHC diagnosis and prognosis via integrative analysis from multiple cohorts.

Prognostic and biological role of the N-Myc downstream-regulated gene family in hepatocellular carcinoma

BACKGROUNDThe N-Myc downstream-regulated gene (NDRG) family is comprised of four members (NDRG1-4) involved in various important biological processes. However, there is no systematic evaluation of the prognostic of the NDRG family in hepatocellular carcinoma (HCC). AIMTo analyze comprehensively the biological role of the NDRG family in HCC.METHODSThe NDRG family expression was explored using The Cancer Genome Atlas. DNA methylation interactive visualization database was used for methylation analysis of the NDRG family. The NDRG family genomic alteration was assessed using the cBioPortal. Single-sample Gene Set Enrichment Analysis was used to determine the degree of immune cell infiltration in tumors. RESULTSNDRG1 and NDRG3 were up-regulated in HCC, while NDRG2 was down-regulated. Consistent with expression patterns, high expression of NDRG1 and NDRG3 was associated with poor survival outcomes (P < 0.05). High expression of NDRG2 was associated with favorable survival (P < 0.005). An NDRG-based signature that statistically stratified the prognosis of the patients was constructed. The percentage of genetic alterations in the NDRG family varied from 0.3% to 11.0%, and the NDRG1 mutation rate was the highest. NDRG 1-3 expression was associated with various types of infiltrated immune cells. Gene ontology analysis revealed that organic acid catabolism was the most important biological process related to the NDRG family. Gene Set Enrichment Analysis showed that metabolic, proliferation, and immune-related gene sets were enriched during NDRG1 and NDRG3 high expression and NDRG2 low expression.CONCLUSIONOverexpression of NDRG1 and NDRG3 and down-expression of NDRG2 are correlated with poor overall HCC prognosis. Our results may provide new insights into the indispensable role of NDRG1, 2, and 3 in the development of HCC and guide a promising new strategy for treating HCC.

The Role of N-myc Downstream-Regulated Gene Family in Glioma Based on Bioinformatics Analysis.

Glioma is the most common type of primary tumor in the central nervous system, and the molecular mechanisms remain elusive. N-myc downstream-regulated gene (NDRG) family is reported to take part in the pathogenesis of various diseases, including some preliminary exploration in glioma. However, there has been no bioinformatics analysis of NDRG family in glioma yet. Herein, we focused on the expression changes of NDRGs with their value in predicting patients' prognoses, upstream regulatory mechanisms (DNA mutation, DNA methylation, transcription factors, and microRNA regulation) and gene enrichment analysis based on co-expressed genes with data from public databases. Furthermore, the expression pattern of NDRGs was verified by the paired glioma and peritumoral samples in our institute. It was suggested that NDRGs were differentially expressed genes in glioma. In particular, the lower expression of NDRG2 or NDRG4 could serve as a predictor of higher grade tumor and poorer prognosis. Also, NDRGs might play a crucial role in signal transduction, energy metabolism, and cross-talk among cells in glioma, under the control of a complex regulatory network. This study enables us to better understand the role of NDRGs in glioma and with further research, it may contribute to the development of glioma treatment.

Characterization of the prognostic values of the NDRG family in gastric cancer.

Background:The N-myc downstream-regulated gene (NDRG) family, NDRG1-4, has been involved in a wide spectrum of biological functions in multiple cancers. However, their prognostic values remain sparse in gastric cancer (GC). Therefore, it is crucial to systematically investigate the prognostic values of the NDRG family in GC.Methods:The prognostic values of the NDRG family were evaluated by Kaplan–Meier Plotter and SurvExpress. The mRNA of the NDRG family was investigated in The Cancer Genome Atlas (TCGA). Transcription factors (TFs) and miRNAs associated with the NDRG family were predicted by NetworkAnalysis. The prognostic values of DNA methylation levels were analyzed by MethSurv. The correlation between immune cells and the NDRG family was evaluated by the Tumor Immune Estimation Resource (TIMER) database.Results:High levels of mRNA expression of NDRG2 and NDRG3 were associated with a favorable prognosis in all GCs. In HER2− GC, NDRG1 was significantly associated with a poor prognosis of GC [hazard ratio (HR) = 1.65, 95% confidence interval (CI) = 1.16–2.33, p = 0.0046]. In HER2+ GC, NDRG4 showed a poor prognosis (HR = 1.4, 95% CI: 1.06–1.85, p = 0.017). NDRG4 was an independent prognostic factor in recurrence-free survival by TCGA cohort. The low-risk NDRG-signature group displayed a significantly favorable survival outcome than the high-risk group (HR = 1.76, 95% CI: 1.2–2.59, p = 0.00385). The phosphorylated protein NDRG1 (NDRG1_pT346) displayed a favorable overall survival and was significantly associated with HER2 and phosphorylated HER2. Epidermis development was the top biological process (BP) for coexpressed genes associated with NDRG1 and NDRG4, while mitotic nuclear division and mitotic cell processes were the top BPs for NDRG2 and NDRG3, respectively. Overall, 6 CpGs of NDRG1, 4 CpGs of NDRG2, 3 CpGs of NDRG3 and 24 CpGs of NDRG4 were associated with significant prognosis. CD4+ T-cells showed the highest correlation with NDRG4 (correlation = 0.341, p = 2.14e−11). Furthermore, BCL6 in follicular helper T-cells (Tfh) cells showed the highest association with NDRG4 (correlation = 0.438, p = 00e+00).Conclusions:This study analyzed the multilevel prognostic values and biological roles of the NDRG family in GC.

NDRG2 suppression as a molecular hallmark of photoreceptor-specific cell death in the mouse retina

Photoreceptor cell death is recognized as the key pathogenesis of retinal degeneration, but the molecular basis underlying photoreceptor-specific cell loss in retinal damaging conditions is virtually unknown. The N-myc downstream regulated gene (NDRG) family has recently been reported to regulate cell viability, in particular NDRG1 has been uncovered expression in photoreceptor cells. Accordingly, we herein examined the potential roles of NDRGs in mediating photoreceptor-specific cell loss in retinal damages. By using mouse models of retinal degeneration and the 661 W photoreceptor cell line, we showed that photoreceptor cells are indeed highly sensitive to light exposure and the related oxidative stress, and that photoreceptor cells are even selectively diminished by phototoxins of the alkylating agent N-Methyl-N-nitrosourea (MNU). Unexpectedly, we discovered that of all the NDRG family members, NDRG2, but not the originally hypothesized NDRG1 or other NDRG subtypes, was selectively expressed and specifically responded to retinal damaging conditions in photoreceptor cells. Furthermore, functional experiments proved that NDRG2 was essential for photoreceptor cell viability, which could be attributed to NDRG2 control of the photo-oxidative stress, and that it was the suppression of NDRG2 which led to photoreceptor cell loss in damaging conditions. More importantly, NDRG2 preservation contributed to photoreceptor-specific cell maintenance and retinal protection both in vitro and in vivo. Our findings revealed a previously unrecognized role of NDRG2 in mediating photoreceptor cell homeostasis and established for the first time the molecular hallmark of photoreceptor-specific cell death as NDRG2 suppression, shedding light on improved understanding and therapy of retinal degeneration.

MiR-483-5p and miR-139-5p promote aggressiveness by targeting N-myc downstream-regulated gene family members in adrenocortical cancer.

Adrenocortical carcinoma (ACC) is a tumor with poor prognosis in which overexpression of a panel of microRNAs has been associated with malignancy but a very limited number of investigations on their role in ACC pathogenesis have been conducted. We examined the involvement of miR-483-5p and miR-139-5p in adrenocortical cancer aggressiveness. Using bioinformatics predictions and mRNA/miRNA expression profiles, we performed an integrated analysis to identify inversely correlated miRNA-mRNA pairs in ACC. We identified N-myc downstream-regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR-483-5p and miR-139-5p, respectively. NDRG2 and NDRG4 expressions were inversely correlated respectively with miR-483-5p and miR-139-5p levels in aggressive ACC samples from two independent cohorts of 20 and 44 ACC. Moreover, upregulation of miR-139-5p and downregulation of NDRG4 demonstrated a striking prognostic value. A direct interaction between miR-483-5p or miR-139-5p and their targets was demonstrated in reporter assays. Downregulation of miR-483-5p or miR-139-5p in the ACC cell lines NCI-H295R and SW13 increased NDRG2 or NDRG4 mRNA and protein expression, compromised adrenocortical cancer cell invasiveness and anchorage-independent growth. MiR-483-5p or miR-139-5p overexpression and NDRG2 or NDRG4 inhibition produce similar changes, which are rescued by NDRG2 or NDRG4 ectopic expression. We established that key factors mediating epithelial-to-mesenchymal transition are downstream effectors of miR-483-5p/NDRG2 and miR-139-5p/NDRG4 pathways. Collectively, our data show for the first time that miR-483-5p/NDRG2 and miR-139-5p/NDRG4 axes promote ACC aggressiveness, with potential implications for prognosis and therapeutic interventions in adrenocortical malignancies.

Potential role of the N-MYC downstream-regulated gene family in reprogramming cancer metabolism under hypoxia.

Metabolic reprogramming toward aerobic glycolysis and lactate fermentation supplies cancer cells with intermediate metabolites, which are used as macromolecule precursors. The oncogene MYC contributes to such aerobic metabolism by activating the expression of numerous genes essential for glycolysis and mitochondrial biogenesis. However, to survive and evolve in a hypoxic tumor milieu, cancer cells must revise MYC-driven metabolism because the mitochondrial respiratory chain provides free electrons to generate oxygen free radicals with inefficient production of ATP due to oxygen depletion. Instead, hypoxia-inducible transcription factor hypoxia-inducible factor 1 (HIF-1) takes over the role of MYC in glycolysis, but suppresses mitochondrial biogenesis and activity to protect cells from such threats. Recently, the N-MYC downstream-regulated gene (NDRG) family has received attention as potential biomarkers of cancer prognosis. NDRGs are repressed MYC-dependently in various cancers, but induced under hypoxia because HIF-1 directly activates their promoters and indirectly de-represses them by antagonizing MYC. In this review, we summarize the current understanding of the reprogramming of cancer metabolism via the counterbalance between MYC and HIF-1, and discuss the proven and putative roles of the NDRG family in adjusting cancer metabolism according to the ambient oxygen level.

Abundant NDRG2 Expression Is Associated with Aggressiveness and Unfavorable Patients' Outcome in Basal-Like Breast Cancer.

NDRG2, a member of the N-myc downstream-regulated gene family, is thought to be a putative tumor suppressor gene with promising clinical impact in breast cancer. Since breast cancer comprises heterogeneous intrinsic subtypes with distinct clinical outcomes we investigated the pivotal role of NDRG2 in basal-type breast cancers. Based on subtype classified tumor (n = 45) and adjacent normal tissues (n = 17) we examined NDRG2 mRNA expression and CpG-hypermethylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA). In addition, NDRG2 protein expression was evaluated immunohistochemically using a tissue micro array (TMA, n = 211). In vitro, we investigated phenotypic effects caused by NDRG2 silencing in the basal A-like HCC1806 as well as NDRG2 over-expression in basal A-like BT20 compared to luminal-type MCF7 breast cancer cells. Our tissue collections demonstrated an overall low NDRG2 mRNA expression in breast cancer subtypes compared to normal breast tissue in line with an increased CpG-hypermethylation in breast cancer tissue. Independent TCGA data sets verified a significant (P<0.001) expression loss of NDRG2 in breast tumors. Of interest, basal-like tumors more frequently retained abundant NDRG2 expression concordant with a lower CpG-hypermethylation. Unexpectedly, basal-like breast cancer revealed an association of NDRG2 expression with unfavorable patients’ outcome. In line with this observation, in vitro experiments demonstrated reduced proliferation and migration rates (~20%) in HCC1806 cells following NDRG2 silencing. In contrast, NDRG2 over-expressing luminal-type MCF7 cells demonstrated a 26% decreased proliferation rate. Until now, this is the first study investigating the putative role of NDRG2 in depth in basal-type breast cancer. Our data indicate that the described putative tumor suppressive function of NDRG2 may be confined to luminal- and basal B-type breast cancers.

N-myc Downstream Regulated Gene (NDRG): Role in Cancer Metastasis Suppression and as Drug Target in Cancer Therapeutics

N-myc downstream regulated gene (NDRG) is, a ubiquitously expressed, a family of cytosolic proteins consists of four members, NDRG1-4. It plays and important role in cancer such as aberrant expression, tumor suppressive, metastatic suppressive and oncogenic functions. In this review, we comprehensively present the expression as well as the clinical and pathological significance of NDRG in human cancers. NDRG1 one of NDRG family which is a ubiquitously expressed protein localized in different tissues of the body, especially breast. It may act as a central regulator of multiple signaling pathways that modulate tumor progression. NDRG2 is expressed in brain, gastric, pancreatic and prostate cells. NDRG3 is highly expressed in testis, prostate, muscle and ovary. Whereas, NDRG4 is highly expressed in brain, hear and colorectal cells. NDRG is down-regulated and associated with the incidence, progression and prognosis of diverse cancers. It is associated with tumor incidence, progression, and metastasis. NDRG has low expression in cancer patients, whereas, inducement of NDRG activity has metastatic suppression effect and also increased in apoptotic effect through increased p53 activity. Metastasis is the spread of a cancer from one organ or part of the body to another not directly connected with it. NDRG is negatively correlated with tumor progression in multiple neoplasms, being a promising new target for cancer treatment. Hence, a drug that has a capacity to enhance an activity of NDRG has a potential to be considered as anti-metastatic agent.

MiR-483-5p and miR-139-5p promote invasion of adrenocortical carcinoma by targeting N-Myc Downstream-Regulated Gene family members

MicroRNAs have emerged as key biomarkers for diagnosis and prognosis of adrenocortical tumors. We have previously shown that miR-483-5p and miR-139-5p were upregulated in adrenocortical carcinomas (ACC) (Chabre et al., 2013). However, the role of these microRNAs in the pathogenesis of ACC is still unknown. Our aim was to identify target genes of miR-483-5p and miR-139-5p in ACC and to investigate their contribution to adrenocortical tumorigenesis. Target prediction was performed using miRWalk database combined with the previously published transcriptome of adrenocortical tumors (de Reynies et al., 2009). Tumor miR-483-5p and miR-139-5p levels were measured by RT–qPCR in 30 patients from COMETE Network and correlated with the expression of their putative targets. The 3′-untranslated regions of target genes were cloned into pMiR-Luciferase vectors to evaluate miRNA-mRNA interactions. The role of miR-483-5p or miR-139-5p in the malignant phenotype of NCI-H295R cells was investigated using miRNA inhibitors. We identified NDRG2 (N-Myc Downstream-Regulated Gene 2 and NDRG4 as targets of miR-483-5p and miR-139-5p, respectively. In ACC, expression of NDRG2 and NDRG4 was inversely correlated with miR-483-5p and miR-139-5p levels. Downregulation of miR-483-5p or miR-139-5p in NCI-H295R cells induced an increase of NDRG2 or NDRG4 mRNAs. Moreover, NDRG2 and NDRG4 transcripts were negatively regulated via specific miRNA target sites. Finally, silencing miR-483-5p or miR-139-5p did not affect proliferation or apoptosis of NCI-H295R cells but inhibited their anchorage-independent growth and invasion. Our data indicate for the first time that targeting of NDRG2 and NDRG4 tumor suppressor genes by miR-483-5p and miR-139-5p might contribute to ACC aggressiveness.

Regulation of histone acetylation by NDRG2 in glioma cells

NDRG2, a member of the N-Myc downstream-regulated gene family, was shown to be a putative tumor suppressor gene in glioblastoma and other cancers. Through a bioinformatic analysis, we found that NDRG2 protein contains an acyl carrier domain. In the current study, we therefore hypothesized that NDRG2 may play an important role in the regulation of histone acetylation. Treatment of U251 and U87 glioma cells with trichostatin A, an inhibitor of histone deacetylase, upregulated the expression of NDRG2 and acetylated forms of histones H3 and H4, reduced tumor cell viability and arrested the cell cycle at the G1/G0 phase. Overexpression of NDRG2 by transfecting glioma cells with adenovirus containing the NDRG2 gene upregulated the levels of acetylated forms of H3 and H4 whereas inhibition of NDRG2 expression by siRNA-mediated knockdown downregulated the level of histone acetylation. Furthermore, NDRG2 siRNA significantly reduced the level of histone acetylation induced by trichostatin A. Taken together, these data demonstrate that NDRG2 can regulate the level of histone acetylation to control glioma cell growth.

Expression of N-myc downstream-regulated gene 1 in primary gallbladder carcinoma and its correlation with clinicopathological features and clinical outcome

N-myc downstream-regulated gene 1 (NDRG1), a member of the N-myc downstream-regulated gene family, is induced under a wide variety of stress and cell growth-regulatory conditions. In several cancers, recent studies have shown its association with inhibition of tumor metastasis and suggested it to be a tumor suppressor gene. However, its significance in primary gallbladder carcinoma (PGC) has not been studied. Therefore, the aim of this study was to investigate NDRG1 expression in PGC and its prognostic significance. We examined NDRG1 expression in tumor specimens from 138 patients with PGC by immunohistochemistry and analyzed the correlation between NDRG1 expression and clinicopathologic factors or survival. NDRG1 was expressed in 63.8% of PGC but not in the normal epithelium of the gallbladder, remarkably at the invasive front of the tumors. In addition, NDRG1 expression was significantly associated with high histologic grade, advanced pathologic T stage and clinical stage, positive nodal metastasis and venous/lymphatic invasion. Moreover, Kaplan-Meier curves showed that NDRG1 over-expression was significantly related to poor overall and disease-free survival (both P = 0.02). Furthermore, multivariate analyses showed that NDRG1 expression (hazard ratio, 3.338; P = 0.02) and clinical stage (hazard ratio, 3.128; P = 0.03) were independent risk factors for disease-free survival. Our data demonstrate for the first time that NDRG1 expression in PGC was significantly correlated with unfavorable clinicopathologic features and an independent poor prognostic factor for disease-free survival in patients. Taken together, our findings suggest that NDRG1 expression could be used as a novel prognostic factor for patient survival and might be a potential therapeutic target in PGC.

Expression and prognostic value of NDRG2 in human astrocytomas

The pathological grading system of human astrocytoma is usually used to evaluate the outcomes of brain glioma patients. However, it is true that some astrocytoma patients with similar grades underwent obvious discrepancy in survival. Increasing evidence shows that certain tumor biomarkers are more suitable for prognosis assessment of tumors than the grading system. NDRG2, a member of the N-myc downstream-regulated gene family, plays an important role in cell proliferation and differentiation, but whether it can be used as a biomarker for prognosis assessment of astrocytomas remains unknown. Immunohistochemistry and semi-quantitative RT-PCR were performed to examine the expression profile of NDRG2 in human astrocytoma specimens. Spearman correlation coefficient was used to describe the association between NDRG2 expression and the clinical parameters of astrocytoma patients. Our results showed that both protein and mRNA expression levels of NDRG2 were significantly downregulated in astrocytomas. In the analysis of the relationship of NDRG2 expression with pathological grades of astrocytoma and with patient survival rate, we found that NDRG2 expression was negatively correlated with pathological grading but positively with the life span of astrocytoma patients. NDRG2 can serve as a potential prognostic biomarker for human astrocytoma.

Crystal Structure of the Human N-Myc Downstream-regulated Gene 2 Protein Provides Insight into Its Role as a Tumor Suppressor

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.

The N‐myc downstream regulated gene (NDRG) family: diverse functions, multiple applications

The N-myc downstream regulated gene (NDRG) family of proteins consists of 4 members, NDRG1-4, which are well conserved through evolution. The first member to be discovered and responsible for the family name was NDRG1, because its expression is repressed by the proto-oncogenes MYCN and MYC. All family members are characterized by an α/β hydrolase-fold motif; however, the precise molecular and cellular function of these family members has not been fully elucidated. Although the exact function of NDRG family members has not been clearly elucidated, emerging evidence suggests that mutations in these genes are associated with diverse neurological and electrophysiological syndromes. In addition, aberrant expression as well as tumor suppressor and oncogenic functions affecting key hallmarks of carcinogenesis such as cell proliferation, differentiation, migration, invasion, and stress response have been reported for several of the NDRG proteins. In this review, we summarize the current literature on the NDRG family members concerning their structure, origin, and tissue distribution. In addition, we review the current knowledge regarding the regulation and signaling of the NDRG family members in development and normal physiology. Finally, their role in disease and potential clinical applications (their role as detection or prognostic markers) are discussed.

Organ-specific alternatively spliced transcript isoforms of the sunflower SF21C gene

The sunflower sf21C gene is a member of a small plant gene family related to the human N-myc downstream-regulated gene family (NDRG) involved in stress and hormone responses, cell growth and differentiation. We have shown earlier that this sunflower gene is interrupted by nine introns and generates multiple mRNAs by alternative splicing of its primary transcript (Lazarescu et al. in Gene 374:77-86, 2006). In the present study, we have characterized 20 splice isoforms of this gene encoding 14 different protein variants. We show that these isoforms occur in distinct combinations in the various plant organs. Five identified variants were generated by splicing at novel sites, different from those classically recognized by U2- or U12-type spliceosomes. Twelve transcript variants contain premature termination codons (PTCs), which make them potential candidates for nonsense-mediated RNA degradation (NMD). Interestingly, all 12 transcripts are relatively abundant in at least one organ, suggesting that NMD is not a "default" mechanism for PTC-containing RNA transcripts generated by alternative splicing. These data indicate that alternative splicing of the sunflower sf21C primary transcript as well as the mRNA quality control mechanism by NMD are complex and regulated spatially and developmentally.

Expression of NDRG2 in esophageal squamous cell carcinoma

N-Myc downstream-regulated gene 2 (NDRG2), a new member of the N-Myc downstream-regulated gene family, has been found to be a differentially expressed gene involved in a variety of cancers. The present study aimed to investigate the expression of NDRG2 in esophageal squamous cell carcinoma (ESCC). Immunohistochemistry was performed in 154 samples from patients with ESCC to detect the expression level of NDRG2 and C-MYC. Results indicated that the expression level of NDRG2 in the cancer samples was significantly lower than that in normal tissues; the trend of C-MYC was the reverse. The Wilcoxon-Mann-Whitney test showed significant difference in the expression of NDRG2 in patients with different T stage, TNM stage, and differentiation degree of cancers (P = 0.036, 0.031, 0.001, respectively). Patients in stages I and II were followed up for 5 consecutive years and Kaplan-Meier survival analysis demonstrated that the survival time of ESCC patients with high expression of NDRG2 was longer than those with low expression during the 5-year follow-up period (P = 0.0018). Cox regression analysis indicated that low expression of NDRG2, cancer stage of pT1, and distant organ metastasis (pM1) were the independent poor prognostic factors of ESCC (P = 0.004, 0.019, 0.0013, respectively). Furthermore, up-regulation of NDRG2 was introduced to ESCC cell lines (EC9706 and EC109) by plasmid transfection. In vivo and in vitro studies indicated that overexpression of NDRG2 markedly reduced proliferation and promoted the apoptosis of EC9706 and EC109 cells. In summary, our results demonstrated that NDRG2 played an important role in the proliferation of ESCC cells and the expression of NDRG2 in ESCC was closely related with the prognosis.

NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1-3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133(+) (cancer stem cell (CSC)-enriched) and CD133(-) primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G(1) cell cycle arrest followed by apoptosis. The initial G(1) arrest is associated with a decrease in cyclin D1 expression and an increase in p27(Kip1) expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.

NDRG2 expression decreases with tumor stages and regulates TCF/ -catenin signaling in human colon carcinoma

NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription–polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes’ stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular β-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of β-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/β-catenin signaling for the maintenance of healthy colon tissues.

Differential Expression Patterns of NDRG Family Proteins in the Central Nervous System

The N-myc downstream-regulated gene (NDRG) family consists of four proteins: NDRG1, NDRG2, NDRG3, and NDRG4 in mammals. NDRG1 has been thoroughly studied as an intracellular protein associated with stress response, cell growth, and differentiation. A nonsense mutation in the NDRG1 gene causes hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D. We previously generated Ndrg1-deficient mice and found that they exhibited peripheral nerve degeneration caused by severe demyelination, but that the complicated motor abilities were retained. These results implied that other NDRG family proteins may compensate for the NDRG1 deficiency in the central nervous system. In this study we raised specific antibodies against each member of the NDRG protein family and examined their cellular expression patterns in the mouse brain. In the cerebrum, NDRG1 and NDRG2 were localized in oligodendrocytes and astrocytes, respectively, whereas NDRG3 and NDRG4 were ubiquitous. In the cerebellum, NDRG1 and NDRG4 were localized in Purkinje cells and NDRG2 in Bergmann glial cells. NDRG3 was detected in the nuclei in most cells. These expression patterns demonstrated the cell type-specific and ubiquitous localization of the NDRG family proteins. Each NDRG may play a partially redundant role in specific cells in the brain.