Expression Of N-myc Down-regulated Gene 1 Research Articles

NDRG1 is a prognostic biomarker in breast cancer and breast cancer brain metastasis

AbstractBrain metastases are secondary brain tumours characterised by their aggressive nature and poor prognosis. Breast cancer is one of the most common primary tumours in women to spread to the brain. A lack of biomarkers predicting likely spread to the brain and limited therapeutic interventions represents major areas of clinical unmet need. We investigated N‐myc downregulated gene‐1 (NDRG1) as a clinically relevant biomarker in breast cancer brain metastasis patients. NDRG1 expression was investigated using immunohistochemistry on tissue microarrays of two clinical cohorts: (i) brain metastatic breast cancers (n = 48) and brain metastases (n = 64; including a subset of 39 patient‐matched breast and brain metastasis cases) and (ii) unselected primary breast cancers (n = 336). NDRG1 was highly expressed in breast‐to‐brain metastases, as well as in high‐grade primary breast cancers. High NDRG1 expression and also an absence of expression were associated with worse survival outcomes in both breast cancer and breast cancer brain metastasis patients. This establishes NDRG1 as a ‘Goldilocks’ protein, where too much or too little has a negative effect on survival. We pose that this accounts for its previous categorisation as both tumour suppressor and oncoprotein. Additionally, a shift in NDRG1 localisation with a gain of nuclear expression was seen at the brain metastasis stage. Significant survival benefit in cases expressing cytoplasmic NDRG1 was observed, whereas NDRG1 localisation in the nucleus showed a clear association with poorer survival. In vitro analyses revealed that hypoxic stress significantly elevated NDRG1 expression and resulted in its nuclear localisation. Our findings suggest NDRG1 expression and subcellular localisation are clinically relevant biomarkers for poor prognosis in breast cancer and breast cancer brain metastasis.

The metastasis suppressor NDRG1 directly regulates androgen receptor signaling in prostate cancer

N-myc-downregulated gene 1 (NDRG1) has potent anticancer effects and inhibits cell growth, survival, metastasis, and angiogenesis. Previous studies suggested that NDRG1 is linked to the androgen signaling network, but this mechanistic relationship is unclear. Considering the crucial role of the androgen receptor (AR) in prostate cancer (PCa) progression, here we examined for the first time the effect of NDRG1 on AR expression, activation, and downstream signaling in LNCaP, 22Rv1, and C4-2B PCa cell types. We demonstrate that NDRG1 effectively promotes interaction of AR with the chaperone HSP90, which in turn stabilizes the AR while decreasing its androgen-mediated activation. The expression of NDRG1 suppressed: (1) AR activation, as measured by p-ARSer213 and p-ARSer81; (2) expression of a major AR transcriptional target, prostate-specific antigen (PSA); and (3) AR transcriptional activity, probably via inhibiting the c-Jun-AR interaction by reducing c-Jun phosphorylation (p-c-JunSer63). NDRG1 was also demonstrated to inhibit multiple key molecules involved in androgen-dependent and -independent signaling (namely EGFR, HER2, HER3, PI3K, STAT3, and NF-κB), which promote the development of castration-resistant prostate cancer. We also identified the cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of NDRG1 as vital for inhibition of AR activity. Examining NDRG1 and p-NDRG1 in PCa patient specimens revealed a significant negative correlation between NDRG1 and PSA levels in prostatectomy patients that went on to develop metastasis. These results highlight a vital role for NDRG1 in androgen signaling and its potential as a key therapeutic target and biomarker in PCa.

Decreased NDRG1 expression is associated with pregnancy loss in mice and attenuates the in vitro decidualization of endometrial stromal cells.

Embryo implantation is an essential step for a successful pregnancy, and any defect in this process can lead to a range of pregnancy pathologies. The objective of this study was to explore the role of N-myc downregulated gene 1 (NDRG1) in embryo implantation. It was found that uterine NDRG1 expression has a dynamic pattern during the estrous cycle in nonpregnant mice and that uterine NDRG1 expression was elevated during the implantation process in pregnant mice. The distinct accumulation of NDRG1 protein signals was observed in the primary decidual zone adjacent to the implanting embryo during early pregnancy. Furthermore, uterine NDRG1 expression could be induced by activated implantation or artificial decidualization in mice. Decreased uterine NDRG1 expression was associated with pregnancy loss in mice and was associated with recurrent miscarriages in humans. The in vitro decidualization of both mouse and human endometrial stromal cells (ESCs) was accompanied by increased NDRG1 expression and downregulated NDRG1 expression in ESCs effectively inhibited decidualization. Collectively, these data suggest that NDRG1 plays an important role in decidualization during the implantation process, and the abnormal expression of NDRG1 may be involved in pregnancy loss.

NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability

The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.

The Metastasis Suppressor, N-myc Downregulated Gene 1 (NDRG1), Is a Prognostic Biomarker for Human Colorectal Cancer

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years’ follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.

Inhibition of N-Myc down regulated gene 1 inin vitrocultured human glioblastoma cells

To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 10(7) cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays.

Expression and biological function of N-myc down-regulated gene 1 in human cervical cancer

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation, differentiation, invasion and metastasis of cancer cells, but its role in cervical cancer is still unclear. This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors. The NDRG1 expression in cervical tissues and cells was detected by RT-PCR. Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines. The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting, respectively. Its effects on cell proliferation, migration, invasion, cell cycle and apoptosis were detected by MTT, transwell migration assay and flow cytometry (FCM), respectively. The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P<0.001). After transfection with pEGFP-N1-NDRG1-GFP, the mRNA and protein expression of NDRG1 was up-regulated in Siha cells, which suppressed cell proliferation (P<0.001), induced cell cycle arrest (P<0.05), reduced invasion and migration of Siha cells (P<0.05), but caused no cell apoptosis. Moreover, vascular endothelial growth factor (VEGF), a tumor-induced angiogenesis factor, was markedly reduced and E-cadherin, a cell adhesion molecule, was increased in the cells transfected with pEGFP-N1-NDRG1-GFP. It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth, invasion and metastasis of human cervical cancer.

Identification of High-Risk Stage II Colorectal Tumors by Combined Analysis of the NDRG1 Gene Expression and the Depth of Tumor Invasion

Experiments on cancer cell lines and animal models indicated that alteration in expression of N-myc down-regulated gene 1 (NDRG1) is associated with development of colon cancer. However, few clinical data are available to assess the role of NDRG1 in progression of human colorectal cancer. This study was undertaken to reveal the prognostic and predictive usefulness of NDRG1 expression determination in colorectal cancer. The expression of NDRG1 mRNA was investigated in 108 colorectal cancer tissues by real-time polymerase chain reaction. The level of NDRG1 protein was investigated by immunohistochemistry. Patients with lowered level of NDRG1 mRNA had a statistically significantly shorter 5-year survival rate compared with patients with unchanged expression of NDRG1 (P = .01). The overall survival time for patients with II tumor, node, metastasis system (TNM) stage disease and tumors displaying reduced expression of NDRG1 was significantly shorter compared with patients with preserved NDRG1 expression (P = .024). Moreover, the survival rate of patients with TNM stage II disease and T4 lesion was significantly lower (P = .0005) for patients with reduced level of NDRG1 expression compared with patients with unchanged NDRG1 expression. The stepwise multivariate regression analysis revealed that advanced TNM stage and lowered NDRG1 expression level were independent unfavorable prognostic factors for patient survival. The assessment of NDRG1 expression offers valuable prognostic information for patients with colorectal cancer, especially for those with stage II disease. We propose that NDRG1 expression level could be used to select patients with stage II disease who are at increased risk of unfavorable outcome, and who may benefit from adjuvant therapy.

NDRG1 Expression Is Inhibited in AML and Its Knockdown Attenuates Neutrophil Differentiation.

The N-myc down-regulated gene 1 (NDRG1) is a stressed induced protein whose expression is associated with growth arrest and differentiation of tumor cells. Although the exact function of NDRG1 protein remains unknown, various studies support its role as a suppressor of tumor metastasis. In prostate, colon and breast cancer its expression is associated with a better disease prognosis and patient survival. In hematopoietic cells, NDRG1 was identified in a differential display screen for differentiation-related genes in human myelomonocytic U937 cells. In the present study, we sought to investigate the role of NDRG1 in myeloid differentiation. To this end we first evaluated NDRG1 mRNA expression in acute myeloid leukemia (AML; n=82) patient samples as well as in CD34+ progenitor cells (n=5) and neutrophils (n=6) of healthy donors using quantitative real-time RT-PCR. We found significantly higher NDRG1 mRNA levels in granulocytes as compared to CD34+ (p=0.0043) or AML blast cells (p<0.0001), whereas no significant difference between CD34+ progenitor and AML blast cells was seen (Figure A). Moreover, we found that NDRG1 mRNA levels were increased in 4/5 APL patients upon ATRA therapy. In contrast, the closest relative of NDRG1, NDRG2, did not show significantly different expression in these primary cells, thus indicating a unique role for NDRG1 in granulocyte differentiation. Next we examined NDRG1 expression using quantitative RT-PCR and Western blotting in two different cell line models for ATRA-induced neutrophil differentiation. ATRA treatment of NB4 and HT93 acute promyelocytic leukemia (APL) cells induced NDRG1 mRNA 2.3- and 14.3- fold, respectively. Increased NDRG1 mRNA expression was paralleled by an increase of NDRG1 protein as well as a decrease in c-myc protein. Earlier reports described that NDRG1 is also suppressed by c-myc suggesting that down-regulation of c-myc in our cell line models allowed for an increase of NDRG1. In line with these observations, lentivirus-driven short hairpin (sh)RNA-mediated silencing of NDRG1 diminished ATRA-induced neutrophil differentiation of NB4 and U937 cells as measured by CD11b, CD11c and CD18 surface expression. In NB4 NDRG1 knockdown versus non-targeting shRNA expressing cells mean fluorescent intensities (MFI) for CD11b, CD11c and CD18 upon six days of ATRA-treatment were 99±17 vs 146±7, 20±2 vs 32±10 and 19±2 vs 45±6, respectively (Figure B). Similarly, U937 NDRG1 knockdown versus control cells displayed the following MFIs for CD11b and CD18 upon neutrophil differentiation: 61±1 vs 102±2 and 11±4 vs 33±13, respectively. In conclusion, we report here for the first time an association of low NDRG1 levels with an immature hematopoietic cell phenotype. Using RNAi technology we further provide evidence that NDRG1 is functionally involved in neutrophil maturation. [Display omitted]

N-Myc down-regulated gene 1 mediates proliferation, invasion, and apoptosis of hepatocellular carcinoma cells

The over-expression of N-myc down-regulated gene 1 (NDRG1) in hepatocellular carcinoma (HCC) was previously reported to correlate with vascular invasion and patient survival. Our current study aims to elucidate its functions in HCC. We found that it lacked the tumorigenic ability to promote soft agar colony formation or serum-independent growth of NIH3T3 cells. We used specific small interfering RNA (siRNA) oligos to suppress the expression of NDRG1 in human HCC (Hep3B and HepG2) cell lines, and found that this significantly reduced cell proliferation and invasion, and induced apoptosis. Additionally, NDRG1-specific siRNA inhibited the HepG2 xenograft growth in nude mice. These results are consistent with our earlier clinical observations that NDRG1 is associated with more aggressive tumor behavior, and suggest that NDRG1 may be a potential therapeutic target for HCC.

P53-Dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion

Normal intestinal mucosal growth requires polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. Our previous studies have shown that polyamine depletion stabilizes p53, resulting in inhibition of intestinal epithelial cell (IEC) proliferation, but the exact downstream targets of induced p53 are still unclear. The NDRG1 (N-myc downregulated gene-1) gene encodes a growth-related protein, and its transcription can be induced in response to stress. The current study tests the hypothesis that induced p53 inhibits IEC proliferation by upregulating NDRG1 expression following polyamine depletion. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with alpha-difluoromethylornithine not only induced p53 but also increased NDRG1 transcription as indicated by induction of the NDRG1 promoter activity and increased levels of NDRG1 mRNA and protein, all of which were prevented by using specific p53 siRNA and in cells with a targeted deletion of p53. In contrast, increased levels of cellular polyamines by ectopic expression of the ODC gene decreased p53 and repressed expression of NDRG1. Consistently, polyamine depletion-induced activation of the NDRG1-promoter was decreased when p53-binding sites within the NDRG1 proximal promoter region were deleted. Ectopic expression of the wild-type NDRG1 gene inhibited DNA synthesis and decreased final cell numbers regardless of the presence or absence of endogenous p53, whereas silencing NDRG1 promoted cell growth. However, overexpression of NDRG1 failed to directly induce cell death and to alter susceptibility to apoptosis induced by tumor necrosis factor-alpha/cycloheximide. These results indicate that NDRG1 is one of the direct mediators of induced p53 following polyamine depletion and that p53-dependent NDRG1 expression plays a critical role in the negative control of IEC proliferation.

N-Myc Down-regulated Gene 1 Modulates the Response of Term Human Trophoblasts to Hypoxic Injury

The placenta is susceptible to diverse insults during human pregnancy. The expression of the protein N-myc down-regulated gene 1 (NDRG1) is regulated during cell proliferation, differentiation, and in response to stress. Nevertheless, the function of this protein in humans remains unknown. We tested the hypothesis that NDRG1 is up-regulated in hypoxic primary human trophoblasts and that NDRG1 modulates trophoblast response to hypoxia. We initially demonstrated that the expression of NDRG1 is enhanced in primary human trophoblasts exposed to hypoxia. Importantly, we found a similar increase in NDRG1 expression in placental samples derived from either singleton gestations complicated by intrauterine growth restriction or from dizygotic twin gestation where one twin exhibited growth restriction. Having established efficient lentivirus-mediated transfection of primary human trophoblasts, we overexpressed NDRG1 in trophoblasts, which resulted in enhanced trophoblast differentiation. In contrast, lentivirus-driven short interfering RNA-mediated silencing of NDRG1 diminished trophoblast viability and differentiation. Consistent with these results, NDRG1 reduced the expression level of p53 in trophoblasts cultured in standard or hypoxic conditions. Furthermore, NDRG1 expression was regulated by the activity of SIRT1 (Sir2-like protein 1), which promotes cell survival. Together, our data indicate that NDRG1 interacts with SIRT1/p53 signaling to attenuate hypoxic injury in human trophoblasts.

Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development.

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.

Rapid detection of the hypoxia-regulated CA-IX and NDRG1 gene expression in different glioblastoma cells in vitro

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of tumor cell hypoxia. It regulates the expression of several genes related to oxygen homeostasis in response to hypoxic stress. Carbonic anhydrase IX (Ca-IX) has been found to be a stable marker of acute or chronic hypoxia. N-Myc down-regulated gene 1 (NDRG1) has been shown to possess more specific characteristics for clinical analysis and identification purposes. HIF-1 activates gene expression of the two genes and promotes tumor cell survival under hypoxic conditions. Herein, we modified a flow cytometry protocol to separate NDRG1- and CA-IX-negative and -positive cells in vitro to sort chronically hypoxic cells from glioblastoma tumors. The FITC-anti-CA-IX fluorescence differed between positive and negative cells by a factor of 60-160 in U373, U87-MG, U251 and GaMG, respectively. A clear effect of the O2 concentration on CA-IX expression was visible in GaMG and U251 cell lines whereas U373 showed a less differentiated pattern. NDRG1 expression was present in U373, U251 and GaMG with the lowest expression rate in GaMG. It was stable over 48 h of reoxygenation after 24 h of extreme hypoxia (0.1% O2). During reoxygenation NDRG1 was relatively stable in the four tumor cell lines with the lowest expression in GaMG. An oxygen- and time-dependent elevation of nuclear HIF-1alpha binding on HRE was displayed. FACS analysis of CA-IX and NDRG1 expression may be a new approach to determining the hypoxic state of tumor cells. However, an extensive analysis of other hypoxia-regulated genes in different tumors is required to identify additional markers for the detection of the oxygenation state in human tumors in order to tailor effective tumor-specific therapeutic strategies.