NDRG1 Expression Research Articles

The Role of NDRG1 Expression in Vasculogenic Mimicry of High-grade Gliomas

The Role of NDRG1 Expression in Vasculogenic Mimicry of High-grade Gliomas

NDRG1 regulates iron metabolism and inhibits pathological cardiac hypertrophy

BackgroundCardiac pathological hypertrophy, a pathological physiological alteration in many cardiovascular diseases, can progress to heart failure. The cellular biology underlying myocardial hypertrophy remains to be fully elucidated. While NDRG1 has been reported to participate in cellular proliferation, differentiation, and cellular stress responses, its role in cardiac diseases remains unexplored. Here, we investigated the role of NDRG1 in pathological hypertrophy. MethodCardiomyocyte-specific-NDRG1 knockout (KO) transgenic mice and NDRG1-AAV9 were used in mice. Angiotensin II (AngII) stimulation were applied to induce hypertrophy. Histological, molecular, and RNA-sequencing analyses were performed, and ferroptosis markers and iron levels were studied. We used co-IP and application of iron chelator to further studied the mechanisms of NDRG1 in cardiac hypertrophy. ResultsWe found that NDRG1 expression is decreased in pathological hypertrophy induced by AngII stimulation. Conditional knockout of NDRG1 in mouse cardiomyocytes led to progressive cardiac hypertrophy and heart failure. Cardiomyocyte-specific overexpression of NDRG1 via AAV9 significantly reversed AngII-induced ventricular hypertrophy and fibrosis. Mechanistically, NDRG1-deficient cardiomyocytes exhibited iron overload and increased ferroptosis, accompanied by elevated levels of reactive oxygen species (ROS) and lipid peroxidation. Subsequently, we confirmed the involvement of NDRG1 in regulating ferroptosis and iron metabolism in myocardial cells. Finally, we identified an interaction between NDRG1 and transferrin in cells. The iron chelator Dp44mT effectively reduced myocardial iron overload and ventricular remodeling induced by NDRG1 deficiency. ConclusionThese findings highlight NDRG1's critical role in iron metabolism and ferroptosis in cardiomyocytes, suggesting that NDRG1 or iron metabolism may serve as therapeutic targets for cardiac hypertrophy.

The role of metabolic memory in diabetic kidney disease: identification of key genes and therapeutic targets.

Diabetic kidney disease (DKD) is characterized by complex pathogenesis and poor prognosis; therefore, an exploration of novel etiological factors may be beneficial. Despite glycemic control, the persistence of transient hyperglycemia still induces vascular complications due to metabolic memory. However, its contribution to DKD remains unclear. Using single-cell RNA sequencing data from the Gene Expression Omnibus (GEO) database, we clustered 12 cell types and employed enrichment analysis and a cell‒cell communication network. Fibrosis, a characteristic of DKD, was found to be associated with metabolic memory. To further identify genes related to metabolic memory and fibrosis in DKD, we combined the above datasets from humans with a rat renal fibrosis model and mouse models of metabolic memory. After overlapping, NDRG1, NR4A1, KCNC4 and ZFP36 were selected. Pharmacology analysis and molecular docking revealed that pioglitazone and resveratrol were possible agents affecting these hub genes. Based on the ex vivo results, NDRG1 was selected for further study. Knockdown of NDRG1 reduced TGF-β expression in human kidney-2 cells (HK-2 cells). Compared to that in patients who had diabetes for more than 10years but not DKD, NDRG1 expression in blood samples was upregulated in DKD patients. In summary, NDRG1 is a key gene involved in regulating fibrosis in DKD from a metabolic memory perspective. Bioinformatics analysis combined with experimental validation provided reliable evidence for identifying metabolic memory in DKD patients.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis-regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.

Merkel Cell Polyomavirus targets SET/PP2A complex to promote cellular proliferation and migration

Merkel Cell Carcinoma (MCC) is a rare neuroendocrine skin cancer. In our previous work, we decoded genes specifically deregulated by MCPyV early genes as opposed to other polyomaviruses and established functional importance of NDRG1 in inhibiting cellular proliferation and migration in MCC. In the present work, we found the SET protein, (I2PP2A, intrinsic inhibitor of PP2A) upstream of NDRG1 which was modulated by MCPyV early genes, both in hTERT-HK-MCPyV and MCPyV-positive (+) MCC cell lines. Additionally, MCC dermal tumour nodule tissues showed strong SET expression. Inhibition of the SET-PP2A interaction in hTERT-HK-MCPyV using the small molecule inhibitor, FTY720, increased NDRG1 expression and inhibited cell cycle regulators, cyclinD1 and CDK2. SET inhibition by shRNA and FTY720 also decreased cell proliferation and colony formation in MCPyV(+) MCC cells. Overall, these results pave a path for use of drugs targeting SET protein for the treatment of MCC.

Salvianolic acid B inhibits the growth and metastasis of A549 lung cancer cells through the NDRG2/PTEN pathway by inducing oxidative stress.

Salvianolic acid B (Sal B) has demonstrated anticancer activity against various types of cancer. However, the underlying mechanism of Sal B-mediated anticancer effects remains incompletely understood. This study aims to investigate the impact of Sal B on the growth and metastasis of human A549 lung cells, as well as elucidate its potential mechanisms. In this study, different concentrations of Sal B were administered to A549 cells. The effects on migration and invasion abilities were assessed using MTT, wound healing, and transwell assays. Flow cytometry analysis was employed to evaluate Sal B-induced apoptosis in A549 cells. Western blotting and immunohistochemistry were conducted to measure the expression levels of cleaved caspase-3, cleaved PARP, and E-cadherin. Commercial kits were utilized for detecting intracellular reactive oxygen species (ROS) and NAD+. Additionally, a xenograft model with transplanted A549 tumors was employed to assess the anti-tumor effect of Sal B in vivo. The expression levels of NDRG2, p-PTEN, and p-AKT were determined through western blotting. Our findings demonstrate that Sal B effectively inhibits proliferation, migration, and invasion in A549 cells while inducing dose-dependent apoptosis. These apoptotic responses and inhibition of tumor cell metastasis are accompanied by alterations in intracellular ROS levels and NAD+/NADH ratio. Furthermore, our in vivo experiment reveals that Sal B significantly suppresses A549 tumor growth compared to an untreated control group while promoting increased cleavage of caspase-3 and PARP. Importantly, we observe that Sal B upregulates NDRG2 expression while downregulating p-PTEN and p-AKT expressions. Collectively, our results provide compelling evidence supporting the ability of Sal B to inhibit both growth and metastasis in A549 lung cancer cells through oxidative stress modulation as well as involvement of the NDRG2/PTEN/AKT pathway.

Identification of proteins related to SIS3 by iTRAQ and PRM-based comparative proteomic analysis in cisplatin-induced acute kidney injury.

Cisplatin is a commonly used nephrotoxic drug and can cause acute kidney injury (AKI). In the present study, isobaric tags for relative and absolute quantification (iTRAQ) and parallel reaction monitoring (PRM)-based comparative proteomics were used to analyze differentially expressed proteins (DEPs) to determine the key molecular mechanism in mice with cisplatin-induced AKI in the presence or absence of SIS3, a specific p-smad3 inhibitor, intervention. The cisplatin-induced AKI mouse model was established and treated with SIS3. We used iTRAQ to search for DEPs, PRM to verify key DEPs and combined Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for bioinformatics analysis. We then assessed lipid deposition, malondialdehyde (MDA) and reactive oxygen species (ROS) and detected the expression of SREBF1, SCD1, CPT1A, PPARα and NDRG1 in vitro. Proteomic analysis showed that the identified DEPs were mainly enriched in energy metabolism pathways, especially in lipid metabolism. When SIS3 was applied to inhibit the phosphorylation of Smad3, the expression of NDRG1 and fatty acid oxidation key proteins CPT1A and PPARα increased, the expression of lipid synthesis related proteins SREBF1 and SCD1 decreased and the production of lipid droplets, MDA and ROS decreased. SIS3 alleviates oxidative stress, reduces lipid accumulation and promotes fatty acid oxidation through NDRG1 in cisplatin-induced AKI. Our study provides a new candidate protein for elucidating the molecular mechanisms of fatty acid metabolism disorders in cisplatin-induced acute kidney injury.

Single-cell transcriptomics reveals the aggressive landscape of high-grade serous carcinoma and therapeutic targets in tumor microenvironment

High-grade serous carcinoma (HGSC) is characterized by early abdominal metastasis, leading to a dismal prognosis. In this study, we conducted single-cell RNA sequencing on 109,573 cells from 34 tumor samples of 18 HGSC patients, including both primary tumors and their metastatic sites. Our analysis revealed a distinct S100A9+ tumor cell subtype present in both primary and metastatic sites, strongly associated with poor overall survival. This subtype exhibited high expression of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1, and C15orf48. Individual knockdown of these ten marker genes, validated through in vitro and in vivo models, significantly inhibited ovarian cancer growth and invasion. Around S100A9+ tumor cells, a population of HK2+_CAF was identified, characterized by activated glycolysis metabolism, correlating with shorter overall survival in patients. Notably, similar to CAFs, immunosuppressive tumor-associated macrophage (TAM) subtypes underwent glycolipid metabolism reprogramming via PPARgamma regulation, promoting tumor metastasis. These findings shed light on the mechanisms driving the aggressiveness of HGSC, offering crucial insights for the development of novel therapeutic targets against this formidable cancer.

Regulatory role of N-myc downregulated genes in amelogenesis in rats.

Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.

Protection of Ndrg2 deficiency on renal ischemia-reperfusion injury via activating PINK1/Parkin-mediated mitophagy.

Renal ischemia-reperfusion (R-I/R) injury is the most prevalent cause of acute kidney injury, with high mortality and poor prognosis. However, the underlying pathological mechanisms are not yet fully understood. Therefore, this study aimed to investigate the role of N-myc downstream-regulated gene 2 ( Ndrg2 ) in R-I/R injury. We examined the expression of Ndrg2 in the kidney under normal physiological conditions and after R-I/R injury by immunofluorescence staining, real-time polymerase chain reaction, and western blotting. We then detected R-I/R injury in Ndrg2-deficient ( Ndrg2-/- ) mice and wild type ( Ndrg2+/+ ) littermates in vivo , and detected oxygen and glucose deprivation and reperfusion (OGD-R) injury in HK-2 cells. We further conducted transcriptomic sequencing to investigate the role of Ndrg2 in R-I/R injury and detected levels of oxidative stress and mitochondrial damage by dihydroethidium staining, biochemical assays, and western blot. Finally, we measured the levels of mitophagy in Ndrg2+/+ and Ndrg2-/- mice after R-I/R injury or HK-2 cells in OGD-R injury. Ndrg2 was primarily expressed in renal proximal tubules and its expression was significantly decreased 24h after R-I/R injury. Ndrg2-/- mice exhibited significantly attenuated R-I/R injury compared to Ndrg2+/+ mice. Transcriptomics profiling showed that Ndrg2 deficiency induced perturbations of multiple signaling pathways, downregulated inflammatory responses and oxidative stress, and increased autophagy following R-I/R injury. Further studies revealed that Ndrg2 deficiency reduced oxidative stress and mitochondrial damage. Notably, Ndrg2 deficiency significantly activated phosphatase and tensin homologue on chromosome ten-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy. The downregulation of NDRG2 expression significantly increased cell viability after OGD-R injury, increased the expression of heme oxygenase-1, decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, and increased the expression of the PINK1/Parkin pathway. Ndrg2 deficiency might become a therapy target for R-I/R injury by decreasing oxidative stress, maintaining mitochondrial homeostasis, and activating PINK1/Parkin-mediated mitophagy.

The effect of p53 on the activity of NRF2 and NDRG2 genes through apoptotic pathway in IDH-wildtype glioblastoma

BackgroundTumor suppressor (p53) acts to integrate multiple stress signals into diverse antiproliferative responses. Its potential to transactivate or downregulate genes through apoptotic pathway in IDH-wildtype glioblastoma has never been explored. MethodsA group of twenty patients diagnosed with IDH-wildtype glioblastoma, were tested for p53 expression and NDRG2/NRF2 genes activity through protein and gene profiling assays. The connotation between these elements has been explored. ResultsThe mean patients’ age was 64-years. All tumors were IDH-wildtype. p53 was expressed in 12 tumors and absent in 8 tumors. The activity of NDRG2 gene was downregulated in all cases. The activity of NRF2 gene was upregulated in 17 tumors and downregulated in 3 tumors. There was a significant statistical difference in PFS among tumors exhibiting different levels of p53 expression and NDRG2 gene activity [p-value= 0.025], in which 12 tumors with downregulated NDRG2 expression and positive p53 expression had earlier tumor recurrence. This statistical difference in PFS was insignificant when we compared p53 expression with NRF2 gene activity [p-value= 0.079]. ConclusionsDuring cell cycle arrest at G2 phase, p53 expression in IDH-wildtype glioblastoma in elderly individuals, coupled with the downregulation of NDRG2 gene activity, led to an aberrant increase in tumor cell proliferation and accelerated tumor recurrence. However, the influence of p53 on NRF2 gene activity was found to be insignificant.

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

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

The synergistic effect of IDH mutation and NDRG-2 dysregulation in the progression of WHO-grade 4 astrocytomas

BackgroundNDRG2 is a tumour suppressor gene involved in tumor growth inhibition. Its effect on tumour recurrence remains controversial. The aim of this study is to explore the dual effect of IDH mutation and NDRG2 dysregulation in WHO-Grade 4 astrocytoma recurrence. MethodsA group of 36 patients with WHO-Grade 4 astrocytoma were examined for NDRG2 expression using protein and gene expression assays. The relationship between IDH, NDRG2 protein and gene expressions, and recurrence-free interval [RFI] was explored. ResultsThe mean patients age in this study was 45-years with 21 males and 15 females. IDH was mutant in 22 tumors. NDRG2 protein expression was low in 23 tumors, and high in 13 tumors. NDRG2 gene expression was upregulated in 4 tumors and 32 tumors showed NDRG2 gene downregulation. The consistency between two tasting methods of NDRG2 expression was 52.8%. There was a significant statistical difference in RFI among tumors with varying NDRG2 gene expression and IDH mutation [p-value= 0.021]. IDH-mutant tumours with downregulated NDRG2 expression showed late recurrence compared to IDH-wildtype glioblastoma. ConclusionsIDH-mutant WHO Grade-4 astrocytoma with downregulated NDRG2 gene are associated with late tumor recurrence. IDH mutations cause excessive accumulation of D-2-hydroxyglutarate, that may inhibit the activity of TET proteins, potentially leading to DNA hypermethylation and gene silencing.

Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction.

Dementia is a serious complication in patients with diabetes-associated cognitive dysfunction (DACD). In this study, we aim to explore the protective effect of exercise on DACD in diabetic mice, and the role of NDRG2 as a potential guarder for reversing the pathological structure of neuronal synapses. Seven weeks of standardized exercise at moderate intensity was carried out using an animal treadmill in the vehicle+Run and STZ+Run groups. Based on quantitative transcriptome and tandem mass tag (TMT) proteome sequencing, weighted gene co-expression analysis (WGCNA) and gene set enrichment analysis (GSEA) were used to investigate the activation of complement cascades to injury neuronal synaptic plasticity. Golgi staining, Western blotting, immunofluorescence staining, and electrophysiology were used to verify the reliability of sequencing data. The role of NDRG2 was assessed by overexpressing or inhibiting the NDRG2 gene invivo. Moreover, we estimated the cognitive function in diabetic or normal patients using DSST scores. Exercise reversed the injury of neuronal synaptic plasticity and the downregulation of astrocytic NDRG2 in diabetic mice, which succeeded in attenuating DACD. The deficiency of NDRG2 aggravated the activation of complement C3 by accelerating the phosphorylation of NF-κB, ultimately leading to synaptic injury and cognitive dysfunction. Conversely, the overexpression of NDRG2 promoted astrocytic remodeling by inhibiting complement C3, thus attenuating synaptic injury and cognitive dysfunction. Meanwhile, C3aR blockade rescued dendritic spines loss and cognitive deficits in diabetic mice. Moreover, the average DSST score of diabetic patients was significantly lower than that of non-diabetic peers. Levels of complement C3 in human serum were elevated in diabetic patients compared to those in non-diabetic patients. Our findings illustrate the effectiveness and integrative mechanism of NDRG2-induced improvement of cognition from a multi-omics perspective. Additionally, they confirm that the expression of NDRG2 is closely related to cognitive function in diabetic mice and the activation of complement cascades accelerated impairment of neuronal synaptic plasticity. NDRG2 acts as a regulator of astrocytic-neuronal interaction via NF-κB/C3/C3aR signaling to restore synaptic function in diabetic mice. This study was supported by the National Natural Science Foundation of China (No. 81974540, 81801899, 81971290), the Key Research and Development Program of Shaanxi (Program No. 2022ZDLSF02-09) and Fundamental Research Funds for the Central Universities (Grant No. xzy022019020).

NDRG1 facilitates self-renewal of liver cancer stem cells by preventing EpCAM ubiquitination.

Portal vein tumour thrombus (PVTT) is the main pathway of HCC intrahepatic metastasis and is responsible for the poor prognosis of patients with HCC. However, the molecular mechanisms underlying PVTT vascular metastases have not been fully elucidated. NDRG1 expression was assessed by immunohistochemistry and immunoblotting in clinical specimens obtained from curative surgery. The functional relevance of NDRG1 was evaluated using sphere formation and animal models of tumorigenicity and metastasis. The relationship between NDRG1 and EpCAM was explored using molecular biological techniques. NDRG1 protein was upregulated in HCC samples compared to non-tumorous tissues. Furthermore, NDRG1 expression was enhanced in the PVTT samples. Our functional study showed that NDRG1 was required for the self-renewal of tumour-initiating/cancer stem cells (CSCs). In addition, NDRG1 knockdown inhibited the proliferation and migration of PVTT-1 cells in vitro and in vivo. NDRG1 was found to stabilise the functional tumour-initiating cell marker EpCAM through protein-protein interactions and inhibition of EpCAM ubiquitination. Our findings suggest that NDRG1 enhances CSCs expansion, PVTT formation and growth capability through the regulation of EpCAM stability. NDRG1 may be a promising target for the treatment of patients with HCC and PVTT.

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.

Analysis of the Prognosis Prediction Ability of a Necroptosis-Related Gene Signature and its Relationship With the Hepatocellular Carcinoma Immune Microenvironment Using Bioinformatics Analysis and Experimental Validation.

Background: Hepatocellular carcinoma (HCC) is one of the most malignant cancers and has a poor prognosis. The immune microenvironment is closely related to the drug sensitivity of a tumor. Necroptosis was reported to be a key factor for HCC. The prognostic value of necroptosis-related genes and their association with the tumor immune microenvironment are still unknown. Methods: Necroptosis-related genes that could comprise a signature for predicting the prognosis of HCC cases were identified using univariate analysis and least absolute shrinkage and selection operator Cox regression analysis. The association between this prognosis prediction signature and HCC immune microenvironment was analyzed. The immunological activities and drug sensitivities were compared between different risk score groups identified using the prognosis prediction signature. The expression levels of the five genes comprising the signature were validated using RT-qPCR. Results: A prognosis prediction signature consisting of five necroptosis-related genes was constructed and validated. Its risk score was = (0.1634 × PGAM5 expression) + (0.0134 × CXCL1 expression) - (0.1007 × ALDH2 expression) + (0.2351 × EZH2 expression) - (0.0564 × NDRG2 expression). The signature was found to be significantly associated with the infiltration of B cells, CD4+ T cells, neutrophils, macrophages, and myeloid dendritic cells into the HCC immune microenvironment. The number of infiltrating immune cells and the expression levels of immune checkpoints in the immune microenvironment of high-risk score patients were higher. Sorafenib and immune checkpoint blockade were determined to be ideally suited for treating high-risk score patients and low-risk score patients, respectively. Finally, RT-qPCR results confirmed that the expression levels of EZH2, NDRG2, and ALDH2 were significantly down-regulated in HuH7 and HepG2 cells compared to those in LO2 cells. Conclusion: The necroptosis-related gene signature developed herein can classify patients with HCC according to prognosis risk well and is associated with infiltration of immune cells into the tumor immune microenvironment.

Abstract PR012: Spatial transcriptomics reveals racial disparities in the transcriptional substructure of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is an aggressive and clinically challenging disease that disproportionately affects African-American (AA) women. Despite this well-known disparity, AA women have historically been underrepresented in large clinical and genomic studies of TNBC. Furthermore, while a genetic association of disease risk with African ancestry has been established, the tumor biology of AA TNBC has not been well characterized. We therefore sought to investigate the contribution of transcriptional substructure and tumor heterogeneity to TNBC disparities. We applied 10x Genomics Visium spatial transcriptomics (ST) technology to 28 samples representing primary TNBC tumors from 14 patients, 7 of whom were AA and 7 who were Caucasian. ST allows collection of whole transcriptome information from up to 5,000 spatially resolved coordinates within a tissue sample, providing remarkable insight into cellular organization within the larger framework of tissue architecture. Normalization of our ST expression data followed by clustering analysis confirmed extensive intra-tumoral subclonality, consistent with the genetic and molecular heterogeneity of TNBC. Most samples contained several transcriptionally distinct tumor clusters, characterized by distinctive activation of oncogenic and immunogenic signaling pathways revealed by gene set enrichment analysis (GSEA). For direct comparison of AA and Caucasian ST data, we performed an integration of all samples to consolidate the variation across genetic backgrounds. The integrated dataset was subject to clustering analysis to produce nine integrated clusters (ICs) representing shared cell populations that were present across all samples in the cohort. Interestingly, while all nine ICs were present in all individual samples, the representation of ICs varied by race. Molecular annotation of ICs using tools such as GSEA, ESTIMATE, and CIBERSORTx revealed that ICs enriched in Caucasian samples were immune-rich, while those enriched in AA samples were immunodeficient and distinctly hypoxic. These results support previous studies that have posited a unique tumor microenvironment in AA TNBC. Furthermore, investigation of the spatial organization of ICs using join count analysis revealed consistent non-random spatial incompatibility of hypoxic regions with immune infiltrate, which is consistent with established roles for hypoxia in immunosuppression. To examine the clinical significance of IC representation in TNBC, we used top marker genes of relevant ICs to perform survival analysis in publicly available breast cancer datasets (4929 samples). We found that high expression of IGKC, which was associated with Caucasian samples in our cohort, is significantly protective in basal-like breast cancer. Conversely, high expression of NDRG1, a hypoxia-related gene overrepresented in AA samples in our cohort, was significantly associated with poor survival. Thus, our study utilizes advanced molecular profiling to provide novel characterizations of AA TNBC with clinical implications in TNBC outcomes disparities. Citation Format: Rania Bassiouni, Michael O. Idowu, Lee D. Gibbs, Valentina Robila, Pamela J. Grizzard, Michelle G. Webb, Jiarong Song, Ashley Noriega, David W. Craig, John D. Carpten. Spatial transcriptomics reveals racial disparities in the transcriptional substructure of triple negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR012.

Lactate attenuates astrocytic inflammation by inhibiting ubiquitination and degradation of NDRG2 under oxygen–glucose deprivation conditions

BackgroundBrain lactate concentrations are enhanced in response to cerebral ischemia and promote the formation of reactive astrocytes, which are major components of the neuroinflammatory response and functional recovery, following cerebral ischemia. NDRG2 is upregulated during reactive astrocyte formation. However, its regulation and function are unclear. We studied the relationship between lactate and NDRG2 in astrocytes under conditions of ischemia or oxygen–glucose deprivation (OGD).MethodsWe examined astrocytic NDRG2 expression after middle cerebral artery occlusion (MCAO) using western blot and immunofluorescence staining. Under hypoxia conditions, we added exogenous L-lactate sodium (lactate) to cultured primary astrocytes to explore the effects of lactate on the ubiquitination modification of NDRG2. We profiled the transcriptomic features of NDRG2 silencing in astrocytes after 8 h of OGD conditions as well as exogenous lactate treatment by performing RNA-seq. Finally, we evaluated the molecular mechanisms of NDRG2 in regulating TNFα under OGD conditions using western blot and immunohistochemistry.ResultsReactive astrocytes strongly expressed NDRG2 in a rat model of MCAO. We also showed that lactate stabilizes astrocytic NDRG2 by inhibiting its ubiquitination. NDRG2 inhibition in astrocytes increased inflammation and upregulated immune-associated genes and signaling pathways. NDRG2 knockdown induced TNFα expression and secretion via c-Jun phosphorylation.ConclusionsWe revealed that under OGD conditions, lactate plays an important anti-inflammatory role and inhibits TNFα expression by stabilizing NDRG2, which is beneficial for neurological functional recovery. NDRG2 may be a new therapeutic target for cerebral ischemia.

NDRG1 Signaling Is Essential for Endothelial Inflammation and Vascular Remodeling.

NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1β (interleukin-1β) and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.