N-myc Downstream-regulated Gene 2 Expression Research Articles

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Prostate cancer invasion is promoted by the miR-96-5p-induced NDRG1 deficiency through NF-κB regulation.

The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, and prostate tissues, and validated in public databases by real-time polymerase chain reaction, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by scratch assay and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assays were used to detect the relation between miR-96-5p, NDRG1, and NF-kB pathway. Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-kB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-kB pathway.

N-myc downstream–regulated gene 1 can promote vasculogenic mimicry and angiogenesis in urothelial carcinoma

Urothelial carcinoma (UC) of the bladder is a common cause of cancer-related death worldwide. Vasculogenic mimicry (VM) is a process by which the malignant cells can generate vascular-like structures formed of periodic acid–Schiff (PAS) positive/CD31 negative extracellular matrix independent of angiogenesis and thus promotes tumor progression. N-myc downstream–regulated gene 1 (NDRG1) is a protein that can modulate tumor angiogenesis; however, its role in regulating tumor angiogenesis and VM formation has not been previously investigated in UC. This study aims to evaluate the role of intra-tumor microvessel density (MVD) (as a surrogate measure of angiogenesis), VM, and NDRG1 in UC and their correlation with different clinicopathologic features, then assess the correlation between them in UC. Sixty specimens of UC of the bladder were included. PAS-CD31 immunohistochemical double staining method was used to evaluate the intra-tumor MVD and VM. Immunohistochemical expression of NDRG1 was also examined. VM and NDRG1 expression were detected in 41.7% and 83.3% of UC specimens respectively. The mean of intra-tumor MVD, VM area, and NDRG1 was significantly higher in tumors with higher grade, lymphovascular invasion, and higher T stage. NDRG1 expression was positively correlated with MVD and VM. We can suggest that MVD, VM, and NDRG1 may serve as poor prognostic markers for UC. The positive correlation between NDRG1 and both MVD and VM may provide the first evidence that NDRG1 can induce tumor angiogenesis and VM in UC which may offer a novel pathway for further therapeutic strategies.

NDRG2 acts as a negative regulator of the progression of small-cell lung cancer through the modulation of the PTEN-AKT-mTOR signalling cascade

N-myc downstream-regulated gene 2 (NDRG2) has been recognised as a negative regulator of the progression of numerous tumours, yet its specific role in small-cell lung carcinoma (SCLC) is not fully understood. The purpose of the current study was to investigate the biological role and mechanism of NDRG2 in SCLC. Initial investigation using the Gene Expression Omnibus (GEO) dataset revealed marked downregulation of NDRG2 transcripts in SCLC. The decreased abundance of NDRG2 in SCLC was verified by examining clinical specimens. Increasing NDRG2 expression in SCLC cell lines caused significant changes in cell proliferation, cell cycle progression, colony formation, and chemosensitivity. NDRG2 overexpression decreased the levels of phosphorylated PTEN, AKT and mTOR. In PTEN-depleted SCLC cells, the upregulation of NDRG2 did not result in any noticeable impact on AKT or mTOR activation. Additionally, the reactivation of AKT reversed the antitumour effects of NDRG2 in SCLC cells. Notably, increasing NDRG2 expression retarded the growth of SCLC cell-derived xenografts in vivo. In conclusion, NDRG2 serves as an inhibitor of SCLC, and its cancer-inhibiting effects are achieved through the suppression of AKT/mTOR via the activation of PTEN. This work suggests that NDRG2 is a potential druggable target for SCLC treatment.

Inhibition of PRMT5/MEP50 Arginine Methyltransferase Activity Causes Cancer Vulnerability in NDRG2low Adult T-Cell Leukemia/Lymphoma.

N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL.

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 10min; (3) MCAO/R group: left middle cerebral artery was blocked for 90min; (4) CIP + MCAO/R group: CIP was performed 72h 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 24h 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.

NDRG2 alleviates photoreceptor apoptosis by regulating the STAT3/TIMP3/MMP pathway in mice with retinal degenerative disease.

Photoreceptor apoptosis is the main pathological feature of retinal degenerative diseases; however, the underlying molecular mechanism has not been elucidated. Recent studies have shown that N-myc downstream regulated gene 2 (NDRG2) exerts a neuroprotective effect on the brain and spinal cord. In addition, our previous studies have confirmed that NDRG2 is expressed in mouse retinal photoreceptors and counteracts N-methyl-N-nitrosourea (MNU)-induced apoptosis. However, the underlying molecular mechanism remains unclear. In this study, we observed that the expression of NDRG2 was not only significantly inhibited in photoreceptors after MNU treatment but also after hydrogen peroxide treatment, and photoreceptor apoptosis was alleviated or aggravated after overexpression or knockdown of NDRG2 in the 661W photoreceptor cell line, respectively. The apoptosis inhibitor Z-VAD-FMK rescued photoreceptor apoptosis induced by MNU after NDRG2 knockdown. Next, we screened and identified tissue inhibitor of metalloproteinases 3 (TIMP3) as the downstream molecule of NDRG2 in 661W cells by using quantitative real-time polymerase chain reaction. TIMP3 exerts a neuroprotective effect by inhibiting the expression of matrix metalloproteinases (MMPs). Subsequently, we found that signal transducer and activator of transcription 3 (STAT3) mediated the NDRG2-associated regulation of TIMP3. Finally, we overexpressed NDRG2 in mouse retinal tissues by intravitreally injecting an adeno-associated virus with mouse NDRG2 in vivo. Results showed that NDRG2 upregulated the expression of phospho-STAT3 (p-STAT3) and TIMP3, while suppressing MNU-induced photoreceptor apoptosis and MMP expression. Our findings revealed how NDRG2 regulates the STAT3/TIMP3/MMP pathway and uncovered the molecular mechanism underlying its neuroprotective effect on mouse retinal photoreceptors.

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

NDRG2 inhibition of glycolysis in liver tumor cells is regulated by SIRT1.

N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor, highly expressed in normal tissues but downregulated in many cancers. However, it has been shown to be involved in regulating glycolytic enzymes in clear cell renal cell carcinoma and colorectal cancer, although the mechanism is still not clear, and the function of NDRG2 in liver tumor glycolysis is completely unknown. Liver tumor tissues were obtained from resected tumors and confirmed by pathological review. Immunohistochemical staining was performed to assess the protein expression of NDRG2. NDRG2-overexpressed and knockdown HepG2/SMMC-7721 cell lines were infected by lentivirus and cultured, before measurement of glucose uptake, lactate production, lactase dehydrogenase activity, and oxygen consumption rate. NDRG2 and SIRT1 proteins were analyzed by western blot. Both the mRNA and protein levels of NDRG2 as a tumor suppressor were downregulated in the liver tumors and the survival rate of patients negatively correlated with the expression of NDRG2. In the NDRG2-overexpressed and knockdown cell lines, NDRG2 showed inhibition of glycolysis in liver tumor cells. Our experimental data suggested the expression of SIRT1 negatively correlated with that of NDRG2. Our study findings enrich the understanding of the role of NDRG2 in tumor growth and of the mechanism by which NDRG2 regulates glycolysis. SIRT1, a deacetylase that plays an important role in glycolysis regulation, may be negatively regulated by NDRG2 in liver tumors.

ROS promote hyper-methylation of NDRG2 promoters in a DNMTS-dependent manner: Contributes to the progression of renal fibrosis

Renal fibrosis is the common histopathological feature of chronic kidney diseases (CKD), and there is increasing evidence that epigenetic regulation is involved in the occurrence and progression of renal fibrosis. N-myc downstream-regulated gene 2 (NDRG2) is significantly down-regulated in renal fibrosis, the mechanism of which remains unclear. Previous studies have confirmed that the inhibition of NDRG2 expression in tumor cells is related to hyper-methylation, mainly regulated by DNA methyltransferases (DNMTS). Herein, we explored the expression of NDRG2 and its epigenetic regulatory mechanism in renal fibrosis. The results showed that the expression of NDRG2 was significantly inhibited in vivo and in vitro, while the overexpression of NDRG2 effectively alleviated renal fibrosis. Meanwhile, we found that the expression of DNMT1/3A/3B was significantly increased in hypoxia-induced HK2 cells and Unilateral Ureteral Obstruction (UUO) mice accompanied by hyper-methylation of the NDGR2 promoter. Methyltransferase inhibitor (5-AZA-dC) corrected the abnormal expression of DNMT1/3A/3B, reduced the methylation level of NDRG2 promoter and restored the expression of NDRG2. The upstream events that mediate changes in NDRG2 methylation were further explored. Reactive oxygen species (ROS) are important epigenetic regulators and have been shown to play a key role in renal injury due to various causes. Accordingly, we further explored whether ROS could induce DNA-epigenetic changes of the expression of NDRG2 and then participated in the development of renal fibrosis. Our results showed that mitochondria-targeted antioxidants (Mito-TEMPO) could reverse the epigenetic inhibition of NDRG2 in a DNMT-sensitive manner, showing strong ability of DNA demethylation, exhibiting epigenetic regulation and anti-fibrosis effects similar to 5-AZA-dC. More importantly, the anti-fibrotic effects of 5-AZA-dC and Mito-TEMPO were eliminated in HK2 cells with NDRG2 knockdown. These findings highlight that targeting ROS-mediated hyper-methylation of NDRG2 promoter is a potentially effective therapeutic strategy for renal fibrosis, which will provide new insights into the treatment of CKD.

Urinary NDRG2 and MCM8 Gene Expression as New Noninvasive Biomarkers for Diagnosis and Differentiation between Muscle and Non-Muscle Invasive Bladder Cancer

Objectives: Noninvasive diagnosis of cancer bladder remains a challenge. The study aimed to evaluate the urinary gene expression of NDRG-2 (N-Myc downstream-regulated gene2) and MCM8 (the mini chromosome maintenance proteins) genes and their importance as novel urinary biomarkers for bladder cancer. In addition, to assess their diagnostic value in comparison with voided urine cytology is the focus of this work. Methods: the study included twenty healthy controls and fifty patients with bladder cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) and voided urine cytology (VUC) were performed to demonstrate the NDRG2 and MCM-8 gene expression levels in the urine of healthy controls and bladder cancer patients. Results: There was a statistically significant decrease in NDRG-2 gene expression in bladder cancer group (4.38±0.66) compared to the control group (8.29±1.67). Gene expression of MCM-8 showed a statistically significant increase in bladder cancer group (5.57±0.79) in comparison to control group (4.55±1.39) with a significant negative correlation (ρ= -0.77) between NDRG-2 expression levels and tumor grade in cancer group (p<0.001), and a positive significant correlation (ρ=0.453) between MCM-8 expression levels and tumor grade in cancer group (p=0.001). NDRG-2 had the highest ability to predict bladder carcinoma (AUC of 1.0). In addition, the most precise differentiation between non–muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) with AUC of 0.814. Conclusion: Expression of NDRG-2 and MCM-8 may be novel potential noninvasive biomarkers for diagnosis and prognosis of bladder cancer and a good tool for differentiation between NMIBC and MIBC with NDRG-2 is the most precise for diagnosis and differentiation over MCM-8, VUC and combined use of NDRG-2 and MCM-8.

Targeted Overexpression of NDRG2 using Survivin Promoter Reduces Viability and Invasiveness of A549 Cell Line

Anti-tumor effects of N-myc Downstream Regulated Gene2 (NDRG2) have been demonstrated in many tumors. In the present study, NDRG2 was specifically overexpressed in lung cancer cell line using Survivin Promoter (Sur-P). Then, the effects of NDRG2 overexpression on viability, apoptosis, migration, and invasion of A549 cells were evaluated. Recombinant pAdenoVator-Sur-P-NDRG2-IRES-GFP plasmid harboring NDRG2 gene under transcriptional control of Sur-P and mock plasmid were constructed. A549 lung tumor cells and LX-2 cells (non-tumor cell line) were transfected with pAdenoVator-Sur-P-NDRG2-IRES-GFP, pAdenoVator-CMV-NDRG2-IRES-GFP, or mock plasmids. Tumor specificity of Sur-P was evaluated using fluorescent microscopy for GFP expression. The effects of NDRG2 overexpression on cell viability, apoptosis, and migration of A549 cells were measured using MTT, annexinV/7-AAD flow cytometry, and transwell migration assay, respectively. NDRG2 and matrix metalloproteinase-2 (MMP-2) expression were measured using real time-PCR. pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection resulted in a huge GFP expression in A549 cells, but not in LX-2 cells. The results of real time-PCR analysis also showed that pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection led to an abundant NDRG2 expression in A549 cells. NDRG2 overexpression decreased A549 cell viability through increasing cell apoptosis. In addition, migration, invasion, and MMP-2 expression decreased following NDRG2 overexpression in A549 cells. The findings indicate that the targeted overexpression of NDRG2 using Sur-P can reduce the viability and invasiveness of A549 cells, suggesting possible benefits of this approach in lung cancer therapy.

PKCδ promotes the invasion and migration of colorectal cancer through c-myc/NDRG1 pathway.

Colorectal cancer (CRC) is the third cause of expected cancer deaths both in men and women in the U.S. and the third most commonly diagnosed cancer in China Targeted therapy has been proven to improve overall survival for unresectable metastatic CRC. But the location of the primary tumor or the presence of various core driver gene mutations that confer resistance may limit the utility of targeted therapy. Therefore, it is of great significance to further elucidate novel mechanisms of invasion and metastasis of CRC and find potential novel therapeutic targets. Protein Kinase C Delta (PKCδ) plays an important role in various diseases, including tumors. In CRC, the function of PKCδ on proliferation and differentiation is mostly studied but various research results were reported. Therefore, the role of PKCδ in CRC needs to be further studied, especially in tumor invasion and metastasis in CRC which few studies have looked into. The expression of PRKCD was analyzed by the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases and Immunohistochemical (IHC). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) enrichment analysis were used to explore the biological functions and pathways related to PRKCD. Lentivirus transfection was used to construct CRC cell lines with overexpression and knock-down of PKCδ or N-myc Downstream Regulated Gene 1 (NDRG1). Cell invasion and migration assay, wound healing assay were used to detect the function of PKCδ and NDRG1 in the invasion and migration of cells. Flow cytometry analysis was used to detect the influence of PKCδ on the CRC cell cycles .Immunofluorescence histochemistry ,Immunoprecipitation Assay and qPCR were used to detect the relationship of PKCδ and NDRG1. Xenograft model was used to verify the role of PKCδ in vivo. PKCδ is overexpressed in CRC and could promote Epithelial-Mesenchymal Transition (EMT) and the invasion and migration of CRC in vitro. We confirmed that PKCδ and the tumor suppressor factor NDRG1 had a co-localization relationship in CRC. PKCδ inhibited NDRG1 transcription and protein expression. Overexpressing NDRG1 could inhibit the function of PKCδ in promoting tumor invasion and migration. PKCδ could regulate c-Myc, one transcription factor of NDRG1, to down-regulate NDRG1. In vivo, overexpressing PKCδ could promote xenograft growth and volume. Thus, our results showed that PKCδ reduced the expression of NDRG1 through c-Myc, promoting the invasion and migration of CRC through promoting EMT. The increased expression of PKCδ in CRC tumor tissue could promote the invasion and migration of tumor cells, and one of the mechanisms may be regulating c-Myc to inhibit the expression of NDRG1 and promote EMT.

Inhibition of PI3K/Akt/mTOR signaling by NDRG2 contributes to neuronal apoptosis and autophagy in ischemic stroke

BackgroundAstrocytic N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, has been involved in the cause of ischemic stroke (IS). However, its downstream effector in IS remains unclear. This study aimed to characterize expression of NDRG2 in IS patients and rats and to investigate the underlying mechanism. MethodsThe protein expression of NDRG2 and mammalian target of the rapamycin (mTOR) and the extent of mTOR phosphorylation in plasma of IS patients were detected by ELISA. An oxygen-glucose deprivation model was established in mouse neuronal cells CATH.a, followed by cell counting kit-8, flow cytometry, TUNEL, and western blot assays to examine cell viability, apoptosis and autophagy. Finally, the effect of NDRG2-mediated phosphatidylinositol 3-kinase/protein kinase-B/mTOR (PI3K/AKT/mTOR) pathway on neuronal apoptosis and autophagy was verified in rats treated with middle cerebral artery occlusion. ResultsNDRG2 was highly expressed in the plasma of IS patients, while the extent of mTOR phosphorylation was reduced in IS patients. NDRG2 blocked the PI3K/Akt/mTOR signaling through dephosphorylation. Depletion of NDRG2 suppressed apoptosis and autophagy in CATH.a cells, which was reversed by a dual inhibitor of PI3K and mTOR, BEZ235. In vivo experiments confirmed that NDRG2 promoted neuronal apoptosis and autophagy by dephosphorylating and blocking the PI3K/Akt/mTOR signaling. ConclusionThe present study has shown that NDRG2 impairs the PI3K/Akt/mTOR pathway via dephosphorylation to promote neuronal apoptosis and autophagy in IS. These findings provide potential targets for future clinical therapies for IS.

LncRNA XIST promotes insulin resistance in gestational diabetes mellitus via the microRNA-181b-5p/NDRG2 axis.

Many studies have explored the role of lncRNA X inactivation-specific transcript (XIST) in diabetes. This study was designed to unravel the regulatory mechanism of XIST on animal models of gestational diabetes mellitus (GDM) progression via the microRNA (miR)-181b-5p/N-myc downstream-regulated gene 2 (NDRG2) axis. XIST, miR-181b-5p, and NDRG2 expression levels in GDM mice were detected. The GDM mice were subjected to gain- and loss-of-function assays to examine the change of glucose metabolism indices (fasting blood glucose (FBG), fasting insulin (FINS) and homeostasis model assessment of insulin resistance (HOMA-IR)), serum oxidative stress factors (glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA)), serum inflammatory factors (interleukin-1 β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)), pathological changes of pancreatic tissues, and apoptotic cells in pancreatic islets in GDM mice. XIST and NDRG2 expression were elevated while miR-181b-5p expression was depleted in GDM mice. Down-regulated XIST or NDRG2 or up-regulated miR-181b-5p reduced the FBG level, HOMA-IR, and serum IL-1β, IL-6, and TNF-α, and MDA contents, elevated the FINS, GSH, and SOD level, mitigated pathological changes in pancreatic tissues, and decelerated apoptotic cells in pancreatic islets in GDM mice. Silenced XIST dampens insulin resistance in GDM mice via the modulation of the miR-181b-5p/NDRG2 axis.

SPDR-2 DEVELOPMENT OF A NOVEL THERAPEUTIC STRATEGY AGAINST MEDULLOBLASTOMA TARGETING MYC

Abstract Medulloblastoma (MB) is the most common pediatric malignant brain tumor, with aggressive behavior and central nervous system dissemination. Standard of care for medulloblastoma include surgery, chemotherapy with platinum agent, and craniospinal radiation. If patients survived with these intensive treatments, frequently faced treatment-related adverse events. Therefore, development of novel therapeutic strategy is an important issue. Driver mutation underlying MB pathogenesis have been discovered, and molecular profiling led to the identification of subgroups; Wingless (WNT), Sonic Hedgehog(SHH), Group3, and Group4. Group 3, especially the group with MYC/MYCN amplification/overexpression confer a poor prognosis. Myc is attracting as a therapeutic target, however, to date, no drugs have been developed that target myc. We had found that N-myc downstream regulated gene-1 (NDRG1) protein expression is a good prognostic factor in glioblastoma, a representative of adult malignant brain tumors, and that Differentiation inducing factor-1 (DIF-1) increased NDRG1 expression. We reported that DIF-1 crossed the Blood-Brain Barrier and could be an effective treatment for GBM in vivo. In this research process, we found that DIF-1 markedly reduced Myc expression in patient-derived glioblastoma stem cells with MYC/MYCN amplification. Based on this result, we found that DIF-1 decreased Myc expression and inhibited cell survival/proliferation even in medulloblastoma cell lines with MYC/MYCN amplification. In this presentation, we will present the results obtained in the research of a novel Myc-targeted therapy using DIF-1 for MB with MYC/MYCN amplification.

MiR-96-5p induced NDRG1 deficiency promotes prostate cancer migration and invasion through regulating the NF-κB signaling pathway.

The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, prostate tissues, and validated public databases by real-time PCR, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by wound healing and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assay was used to detect the relation between miR-96-5p, NDRG1, and NF-κB pathway. Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-κB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-κB pathway.

The Function of N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Negative Regulator in Tumor Cell Metastasis.

N-myc downstream-regulated gene 2 (NDRG2) is a tumor-suppressor gene that suppresses tumorigenesis and metastasis of tumors and increases sensitivity to anti-cancer drugs. In this review, we summarize information on the clinicopathological characteristics of tumor patients according to NDRG2 expression in various tumor tissues and provide information on the metastasis inhibition-related cell signaling modulation by NDRG2. Loss of NDRG2 expression is a prognostic factor that correlates with TNM grade and tumor metastasis and has an inverse relationship with patient survival in various tumor patients. NDRG2 inhibits cell signaling, such as AKT-, NF-κB-, STAT3-, and TGF-β-mediated signaling, to induce tumor metastasis, and induces activation of GSK-3β which has anti-tumor effects. Although NDRG2 operates as an adaptor protein to mediate the interaction between kinases and phosphatases, which is essential in regulating cell signaling related to tumor metastasis, the molecular mechanism of NDRG2 as an adapter protein does not seem to be fully elucidated. This review aims to assist the research design regarding NDRG2 function as an adaptor protein and suggests NDRG2 as a molecular target to inhibit tumor metastasis and improve the prognosis in tumor patients.

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 (GRβ) 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, GRβ, SGK1, and NDRG1 in prostate cancer and their relationship with clinicopathological and functional significance remain unknown. The association between the status of GR, GRβ, 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 GRβ and SGK1 expression. Our findings indicate that GR and NDRG1 expression status is correlated with clinicopathological features in patients with prostate cancer.

A Tumor Suppressor Gene, N-myc Downstream-Regulated Gene 1 (NDRG1), in Gliomas and Glioblastomas.

The development of potent and selective therapeutic approaches to glioblastoma (GBM) requires the identification of molecular pathways that critically regulate the survival and proliferation of GBM. Glioblastoma stem-like cells (GSCs) possess stem-cell-like properties, self-renewal, and differentiation into multiple neural cell lineages. From a clinical point of view, GSCs have been reported to resist radiation and chemotherapy. GSCs are influenced by the microenvironment, especially the hypoxic condition. N-myc downstream-regulated gene 1 (NDRG1) is a tumor suppressor with the potential to suppress the proliferation, invasion, and migration of cancer cells. Previous studies have reported that deregulated expression of NDRG1 affects tumor growth and clinical outcomes of patients with GBM. This literature review aimed to clarify the critical role of NDRG1 in tumorigenesis and acquirement of resistance for anti-GBM therapies, further to discussing the possibility and efficacy of NDRG1 as a novel target of treatment for GBM. The present review was conducted by searching the PubMed and Scopus databases. The search was conducted in February 2022. We review current knowledge on the regulation and signaling of NDRG1 in neuro-oncology. Finally, the role of NDRG1 in GBM and potential clinical applications are discussed.