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.

Integrative analysis of disulfidptosis and immune microenvironment in hepatocellular carcinoma: a putative model and immunotherapeutic strategies.

Hepatocellular carcinoma (HCC) is a malignant tumor with a high rate of recurrence and m metastasis that does not respond well to current therapies and has a very poor prognosis. Disulfidptosis is a novel mode of cell death that has been analyzed as a novel therapeutic target for HCC cells. This study integrated bulk ribonucleic acid (RNA) sequencing datasets, spatial transcriptomics (ST), and single-cell RNA sequencing to explore the landscape of disulfidptosis and the immune microenvironment of HCC cells. We developed a novel model to predict the prognosis of patients with HCC based on disulfidptosis. The model has good stability, applicability, and prognostic and immune response prediction abilities. N-myc downregulated gene1 (NDRG1) may contribute to poor prognosis by affecting macrophage differentiation, thus allowing HCC cells to evade the immune system. Our study explores the disulfidptosis of HCC cells through multi-omics and establishes a new putative model that explores possible targets for HCC treatment.

An electrostatically conjugated-functional MNK1 aptamer reverts the intrinsic antitumor effect of polyethyleneimine-coated iron oxide nanoparticles in vivo in a human triple-negative cancer xenograft

BackgroundTriple-negative breast cancer (TNBC) remains a difficult breast cancer subtype to treat as it exhibits a particularly aggressive behavior. The dysregulation of distinct signaling pathways underlies this aggressive behavior, with an overactivation of MAP kinase interacting kinases (MNKs) promoting tumor cell behavior, and driving proliferation and migration. Therefore, MNK1 is an excellent target to impair the progression of TNBC and indeed, an MNK1-specific aptamer has proved to be efficient in inhibiting TBNC cell proliferation in vitro. Although polyethyleneimine-coated iron oxide nanoparticles (PEI–IONPs) have been used as transfection and immunomodulating agents, no study has yet addressed the benefits of using these nanoparticles as a magnetic carrier for the delivery of a functional aptamer.ResultsHere, we tested the antitumor effect of a PEI–IONP complexed to the functional MNK1b-specific aptamer in vitro and in vivo. We demonstrated that these apMNKQ2@PEI–IONP nanoconjugates delivered three times more apMNKQ2 to MDA-MB-231 cells than the aptamer alone, and that this enhanced intracellular delivery of the aptamer had consequences for MNK1 signaling, reducing the amount of MNK1 and its target the phospho(Ser209)-eukaryotic initiation factor 4E (eIF4E). As a result, a synergistic effect of the apMNKQ2 and PEI–IONPs was observed that inhibited MDA-MB-231 cell migration, probably in association with an increase in the serum and glucocorticoid-regulated kinase-1 (SGK1) and the phospho(Thr346)-N-myc down-regulated gene 1 (NDRG1). However, intravenous administration of the apMNKQ2 alone did not significantly impair tumor growth in vivo, whereas the PEI–IONP alone did significantly inhibit tumor growth. Significantly, tumor growth was not inhibited when the apMNKQ2@PEI–IONP nanocomplex was administered, possibly due to fewer IONPs accumulating in the tumor. This apMNKQ2-induced reversion of the intrinsic antitumor effect of the PEI–IONPs was abolished when an external magnetic field was applied at the tumor site, promoting IONP accumulation.ConclusionsElectrostatic conjugation of the apMNKQ2 aptamer with PEI–IONPs impedes the accumulation of the latter in tumors, which appears to be necessary for PEI–IONPs to exert their antitumor activity.Graphical

NDRG1 in Cancer: A Suppressor, Promoter, or Both?

N-myc downregulated gene-1 (NDRG1) has been variably reported as a metastasis suppressor, a biomarker of poor outcome, and a facilitator of disease progression in a range of different cancers. NDRG1 is poorly understood in cancer due to its context-dependent and pleiotropic functions. Within breast cancer, NDRG1 is reported to be either a facilitator of, or an inhibitor of tumour progression and metastasis. The wide array of roles played by NDRG1 are dependent on post-translational modifications and subcellular localization, as well as the cellular context, for example, cancer type. We present an update on NDRG1, and its association with hallmarks of cancer such as hypoxia, its interaction with oncogenic proteins such as p53 as well its role in oncogenic and metastasis pathways in breast and other cancers. We further comment on its functional implications as a metastasis suppressor and promoter, its clinical relevance, and discuss its therapeutic targetability in different cancers.

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.

ITRAQ-Based Quantitative Proteomic Analysis of Intestines in Murine Polymicrobial Sepsis with Hydrogen Gas Treatment.

ObjectiveSepsis-associated intestinal injury has a higher morbidity and mortality in patients with sepsis, but there is still no effective treatment. Our research team has proven that inhaling 2% hydrogen gas (H2) can effectively improve sepsis and related organ damage, but the specific molecular mechanism of its role is not clear. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was used for studying the effect of H2 on intestinal injury in sepsis.MethodsMale C57BL/6J mice were used to prepare a sepsis model by cecal ligation and puncture (CLP). The 7-day survival rates of mice were measured. 4-kd fluorescein isothiocyanate-conjugated Dextran (FITC-dextran) blood concentration measurement, combined with hematoxylin-eosinstain (HE) staining and Western blotting, was used to study the effect of H2 on sepsis-related intestinal damage. iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used for studying the proteomics associated with H2 for the treatment of intestinal injury.ResultsH2 can significantly improve the 7-day survival rates of sepsis mice. The load of blood and peritoneal lavage bacteria was increased, and H2 treatment can significantly reduce it. CLP mice had significant intestinal damage, and inhalation of 2% hydrogen could significantly reduce this damage. All 4194 proteins were quantified, of which 199 differentially expressed proteins were associated with the positive effect of H2 on sepsis. Functional enrichment analysis indicated that H2 may reduce intestinal injury in septic mice through the effects of thyroid hormone synthesis and nitrogen metabolism signaling pathway. Western blot showed that H2 was reduced by down-regulating the expressions of deleted in malignant brain tumors 1 protein (DMBT1), insulin receptor substrate 2 (IRS2), N-myc downregulated gene 1 (NDRG1) and serum amyloid A-1 protein (SAA1) intestinal damage in sepsis mice.ConclusionA total of 199 differential proteins were related with H2 in the intestinal protection of sepsis. H2-related differential proteins were notably enriched in the following signaling pathways, including thyroid hormone synthesis signaling pathway, nitrogen metabolism signaling pathways, digestion and absorption signaling pathways (vitamins, proteins and fats). H2 reduced intestinal injury in septic mice by down-regulating the expressions of SAA1, NDRG1, DMBT1 and IRS2.

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.

Role of metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) in pancreatic cancer.

Metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) is a long non-coding RNA (lncRNA) that is a negative prognostic factor for patients with pancreatic cancer and several other tumors. In this study, we show that knockdown of MALAT-1 in Panc1 and other pancreatic cancer cell lines decreases cell proliferation, survival and migration. We previously observed similar results for the lncRNAs HOTTIP and HOTAIR in Panc1 cells; however, RNAseq comparison of genes regulated by MALAT-1 shows minimal overlap with HOTTIP/HOTAIR-regulated genes. Analysis of changes in gene expression after MALAT-1 knockdown shows that this lncRNA represses several tumor suppressor-like genes including N-myc downregulated gene-1 (NDRG-1), a tumor suppressor in pancreatic cancer that is also corepressed by EZH2 (a PRC2 complex member). We also observed that Specificity proteins Sp1, Sp3 and Sp4 are overexpressed in Panc1 cells and Sp knockdown or treatment with small molecules that decrease Sp proteins expression also decrease MALAT-1 expression. We also generated Kras-overexpressing p53L/L;LSL-KrasG12DL/+;p48Cre+/- (p53L/L/KrasG12D) and p53L/+;LSLKrasG12DL/+;p48Cre+/- (p53L/+/KrasG12D) mice which are p53 homo- and heterozygous, respectively. These mice rapidly develop pancreatic ductal adenocarcinoma-like tumors and were crossed with MALAT-1-/- mice. We observed that the loss of one or two MALAT-1 alleles in these Ras overexpressing mice does not significantly affect the time to death; however, the loss of MALAT-1 in the p53-/+ (heterozygote) mice slightly increases their lifespan.

Oncogenic MNK signalling regulates the metastasis suppressor NDRG1.

The protein N-myc down-regulated gene 1 (NDRG1) represses tumour metastasis. It is phosphorylated at several sites by serum and glucocorticoid-regulated kinase 1 (SGK1). Here we show that NDRG1 is also regulated by the oncogenic MAP kinase-interacting kinase (MNK) pathway, a target for cancer therapy.Inhibiting MNKs increases the expression of NDRG1 protein and mRNA in breast cancer cells. MNK inhibition also decreases the phosphorylation of NDRG1. Phosphorylation of NDRG1 is reduced in cells lacking MNK1, but not MNK2-knockout cells, indicating that NDRG1 phosphorylation is a specific target for MNK1. However, MNK1 cannot directly phosphorylate NDRG1 in vitro, indicating that additional signalling connections are involved. Taken together, our data indicate that MNK signaling regulates NDRG1 at transcriptional and post-translational levels.We show that SGK1 phosphorylates MNK1 at a conserved site, which represses its activity. NDRG1, SGK1 and the MNKs are implicated in cell migration and metastasis. As expected, knocking-down NDRG1 promoted cell migration. However, whereas MNK inhibition impairs these processes irrespective of NDRG1 levels, SGK inhibition only did so in NDRG1-depleted cells. Thus, MNKs and SGK affect migration/invasion through distinct mechanisms.Our data reveal several novel connections between signalling pathways important for tumour biology.

The prostate metastasis suppressor gene NDRG1 differentially regulates cell motility and invasion.

Experimental and clinical evidence suggests that N‐myc downregulated gene 1 (NDRG1) functions as a suppressor of prostate cancer metastasis. Elucidating pathways that drive survival and invasiveness of NDRG1‐deficient prostate cancer cells can help in designing therapeutics to target metastatic prostate cancer cells. However, the molecular mechanisms that lead NDRG1‐deficient prostate cancer cells to increased invasiveness remain largely unknown. In this study, we demonstrate that NDRG1‐deficient prostate tumors have decreased integrin expression and reduced cell adhesion and motility. Our data indicate that loss of NDRG1 differentially affects Rho GTPases. Specifically, there is a downregulation of active RhoA and Rac1 GTPases with a concomitant upregulation of active Cdc42 in NDRG1‐deficient cells. Live cell imaging using a fluorescent sensor that binds to polymerized actin revealed that NDRG1‐deficient cells have restricted actin dynamics, thereby affecting cell migration. These cellular and molecular characteristics are in sharp contrast to what is expected after loss of a metastasis suppressor. We further demonstrate that NDRG1‐deficient cells have increased resistance to anoikis and increased invasiveness which is independent of its elevated Cdc42 activity. Furthermore, NDRG1 regulates expression and glycosylation of EMMPRIN, a master regulator of matrix metalloproteases. NDRG1 deficiency leads to an increase in EMMPRIN expression with a concomitant increase in matrix metalloproteases and thus invadopodial activity. Using a three‐dimensional invasion assay and an in vivo metastasis assay for human prostate xenografts, we demonstrate that NDRG1‐deficient prostate cancer cells exhibit a collective invasion phenotype and are highly invasive. Thus, our findings provide novel insights suggesting that loss of NDRG1 leads to a decrease in actin‐mediated cellular motility but an increase in cellular invasion, resulting in increased tumor dissemination which positively impacts metastatic outcome.

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.

Integrative functional genomics of hepatitis C virus infection identifies host dependencies in complete viral replication cycle.

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

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.

Molecule of the month: miRNA and Parkinson's disease protein PARK2.

Parkinson's disease is generally associated with aging, i.e. elderly individuals. We are gaining many deep insights into neuropathogenesis in Parkinson's disease and there are many publications on this topic; we briefly mention a few as they relate to the increasingly studied miRNAs. Parkinson's disease results from protein inclusions or Lewy bodies and destruction of (mid-brain) substantia nigra pars compacta neurons that are dopaminergic. A novel approach to investigate molecular processes in human diseases as well as in normal control and function is through the study of miRNAs. miRNAs are increasingly associated with many diseases and in Parkinson's disease [1, 2]. Up-regulation, beige Regulation, purple Co-expression, brown Physical Interaction, turquoise dotted Predicted Protein Interaction, and mauve dotted Predicted TFactor Regulation (GenePro SA Biosciences, http://www.sabiosciences.com/). Additional studies used a bioinformatics and computational approach to the analysis of gene expression using the Gene Expression Omnibus database at NIH [3]. They then predicated Parkinson's disease associated transcription factors, miRNAs, and identified 11 genes that include a new transcription factor, N-Myc down-regulated gene 1 (NDRG1) and junction plakoglobin (JUP).In Parkinson's disease, NDRG1 is regulated by miRNA-133 [4]. miRNA profile analysis indicated miR-34b and miR-34c decreased expression. These changes were found in the substantia nigra, frontal cortex, cerebellum, and amygdala. In aneuroblastoma cell culture line, depletion of miR-34b or miR- 34c resulted in cell death. In addition, depletion of these miRNAs resulted in decreased expression of Parkin or parkinson protein 2 (Park2) and parkinson protein 7 (DJ1). These cellular changes appear to result in mitochondrial dysfunction in Parkinson's disease [2]. Figure 1 shows an interactive network diagram of input neighbors to Park2 (parkin) and Figure 2 shows an interactive network diagram of input proteins from Figure 1 (Park2, CASK, SNCAIP, HSPA4, STUB1, SIM2, CBL, LIMK1, PACRG, SEPT5, PSMA7, and BAG5). It is left as a puzzle for the interested reader to identify the various genes and their functions in the figures [5–7]. Figure 1 Network of input protein (Park2) with immediate interactive proteins. In this figure, line-colors and various interactions with other genes are red Down-regulation, green Up-regulation, beige Regulation, purple Co-expression, brown Physical Interaction, ... Figure 2 Network of input proteins from Figure 1(Park2, CASK, SNCAIP, HSPA4, STUB1, SIM2, CBL, LIMK1, PACRG, SEPT5, PSMA7, BAG5) and showing additional neighbors of these proteins (up to 100 total). In this figure, line-colors and various interactions with other ...

Abstract 4864: Aroma terpenes prevent UVB-induced sunburn in Skh1 mouse skin.

Abstract Understanding how aroma terpenes prevent sunburn and/or skin cancer in mice could lead to more effective and safer ways of blocking sun damage to human skin. In our previous study, β-damascenone, an aroma terpene, protected against sunburn by activating both keratinocyte and sebaceous gland pathways that fortified and thickened the cornified envelope and sebum layers of mouse epidermis. In the current study, the sunburn preventive activity of d-limonene was examined to confirm that these related compounds work similarly. In addition to sunburn, immunohistochemical analyses of proliferation (PCNA) and a damage-response gene N-Myc Down Regulated Gene 1 (NDRG1) were investigated in control versus d-limonene-treated Skh-1 mice. The mice were treated either topically on the dorsum or orally with different concentrations of d-limonene in liponate (100%, 10%, 1% and 0%) followed by exposure of the dorsum to solar simulation ultraviolet radiation (UVR) of 1.5 kJ/m2 from fluorescent ‘sunlamps’. Either 4 topical 5 μL doses or a single dose of 20 μL (0.95 μg/g body weight) of 100% d-limonene provided complete protection from UVR-induced sunburn. The protection was less but still significant at 10% d-limonene but was absent at 1% d-limonene in comparison to UVR alone. PCNA-labeling showed a marked proliferation increase of epidermal basal cells, outer root sheath cells of hair follicles and sebaceous gland cells by separate exposures to d-limonene and UVR relative to controls. The combined treatment of d-limonene followed by UVR produced fewer PCNA-positive cells in epithelial tissue relative to UVR-only mice. The NDRG1 protein (an indirect measure of DNA damage) was overwhelmingly (∼85% of keratinocytes) induced by UVR indicating heavy DNA damage. The UVR-induced NDRG1 index remained at ∼85% at 1% d -limonene, but was significantly reduced at 10% d-limonene to ∼24% and to less than 1% at 100% d -limonene indicating nearly complete elimination of cellular damage as a pure compound; a finding that is consistent with complete sunburn prevention. The thickness of the cornified envelope plus sebum layers nearly doubled within 5 days after administration of the d-limonene corresponding to the time of UVR application. Overall the results indicate that d-limonene protected against sunburn by activating keratinocyte/sebaceous gland-based pathways that fortified and thickened the cornified envelope plus sebum layers on the skin surface by releasing elevated levels of UVR-absorbing proteins that decreased UVR dose to underlying cutaneous tissues. Citation Format: Ahmed N. Uddin, Ivica Labuda, Feng Wu, KamMeng Tchou-Wong, Fredric J. Burns. Aroma terpenes prevent UVB-induced sunburn in Skh1 mouse skin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2013-4864

Identification of Regulatory Relationships in Parkinson's Disease

Parkinson's disease is a complex chronic neurodegenerative disease common in elderly people and greatly affects the quality of their life. However, the pathogenesis of Parkinson's disease is still incompletely understood to date. The purpose of this present study is to explore the pathogenesis of Parkinson's disease using a computational bioinformatics analysis of gene expression. We downloaded gene expression profiles on Parkinson's disease from the Gene Expression Omnibus database and predicted the miRNAs and transcription factors of differentially expressed genes in Parkinson's disease. A total of 11 genes associated with Parkinson's disease initiation were identified, including junction plakoglobin (JUP). Besides, we identified a new transcription factor, N-Myc down-regulated gene 1 (NDRG1), which is regulated by miRNA-133 in Parkinson's disease. Furthermore, we proposed a hypothesis that there may be two kinds of regulatory relationships among miRNA-133, NDRG1, and JUP: direct regulatory relationship and indirect relationship. The results presented in this work confirmed the role of miRNA-133 in Parkinson's disease and substantiated our understanding of miRNA-related neurodegenerative states in general.

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.

Down-Regulation of NDRG1 Promotes Migration of Cancer Cells during Reoxygenation

One characteristic of tumor microenvironment is oxygen fluctuation, which results from hyper-proliferation and abnormal metabolism of tumor cells as well as disorganized neo-vasculature. Reoxygenation of tumors can induce oxidative stress, which leads to DNA damage and genomic instability. Although the cellular responses to hypoxia are well known, little is known about the dynamic response upon reoxygenation. In order to investigate the transcriptional responses of tumor adaptation to reoxygenation, breast cancer MCF-7 cells were cultured under 0.5% oxygen for 24 h followed by 24 h of reoxygenation in normoxia. Cells were harvested at 0, 1, 4, 8, 12, and 24 h during reoxygenation. The transcriptional profile of MCF-7 cells upon reoxygenation was examined using Illumina Human-6 v3 BeadChips. We identified 127 differentially expressed genes, of which 53.1% were up-regulated and 46.9% were down-regulated upon reoxygenation. Pathway analysis revealed that the HIF-1-alpha transcription factor network and validated targets of C-MYC transcriptional activation were significantly enriched in these differentially expressed genes. Among these genes, a subset of interest genes was further validated by quantitative reverse-transcription PCR. In particular, human N-MYC down-regulated gene 1 (NDRG1) was highly suppressed upon reoxygenation. NDRG1 is associated with a variety of stress and cell growth-regulatory conditions. To determine whether NDRG1 plays a role in reoxygenation, NDRG1 protein was overexpressed in MCF-7 cells. Upon reoxygenation, overexpression of NDRG1 significantly inhibited cell migration. Our results revealed the dynamic nature of gene expression in MCF-7 cells upon reoxygenation and demonstrated that NDRG1 is involved in tumor adaptation to reoxygenation.

Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.

Abstract 2208: Overexpression of NDRG1 inhibits cell migration of breast cancer cells via reactive oxygen species during reoxygenation

Abstract One characteristic of tumor microenvironment is oxygen fluctuation, which results from hyper-proliferation and abnormal metabolism of tumor cells as well as disorganized neo-vasculature. Reoxygenation in tumor microenvironment can induce oxidative stress, which further leads to DNA damage and genomic instability. Although multiple cellular responses are activated in order to survive under this microenvironment, little is known about the dynamic response upon reoxygenation. Therefore, in order to investigate the transcriptional response of tumor adaptation to reoxygenation, breast cancer cells MCF-7 was cultured under 0.5% of hypoxia for 24h followed by 24h of reoxygenation in normoxia. Cells were harvested respectively at 0, 1, 4, 8, 12, and 24h during reoxygenation. The genomic profiling of MCF-7 upon reoxygenation was examined using Illumina Human-6 v3 BeadChips. We identified 127 differentially expressed genes in which 53.1% were up-regulated and 46.9% were down-regulated upon reoxygenation. Pathway analysis revealed that HIF-1-alpha transcription factor network was most significantly (P=2.02×10-3) enriched in these differentially expressed genes. Among these genes, a group of selected genes of interest were further validated by quantitative real-time PCR. In particular, NDRG1 was highly suppressed upon reoxygenation. NDRG1 (human N-myc down-regulated gene 1) is associated with a variety of stress and cell growth-regulatory conditions. To characterize whether NDRG1 play a role in reoxygenation, NDRG1 protein was overexpressed in MCF-7 cells. Upon reoxygenation, overexpression of NDRG1 inhibited cell migration, and this inhibition could be reversed by adding reactive oxygen species scavengers. Our results revealed dynamic expression profiling of MCF-7 upon reoxygenation, and demonstrated that NDRG1 is involved in tumor adaptation to reoxygenation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2011-2208