Global Gene Expression Analysis Reveals a Link between NDRG1 and Vesicle Transport

N-myc downstream-regulated gene 1 (NDRG1) is induced by cellular stress such as hypoxia and DNA damage, and in humans, germ line mutations cause Charcot-Marie-Tooth disease. However, the cellular roles of NDRG1 are not fully understood. Previously, NDRG1 was shown to mediate doxorubicin resistance under hypoxia, suggesting a role for NDRG1 in cell survival under these conditions. We found decreased apoptosis in doxorubicin-treated cells expressing NDRG1 shRNAs under normoxia, demonstrating a requirement for NDRG1 in apoptosis in breast epithelial cells under normal oxygen pressure. Also, different cellular stress regimens, such as hypoxia and doxorubicin treatment, induced NDRG1 through different stress signalling pathways. We further compared expression profiles in human breast epithelial cells ectopically over-expressing NDRG1 with cells expressing NDRG1 shRNAs in order to identify biological pathways where NDRG1 is involved. The results suggest that NDRG1 may have roles connected to vesicle transport.     

NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cells.

The NDRG2 gene belongs to a family of N-Myc downstream-regulated genes (NDRGs) and is expressed in many normal tissues. NDRG2 gene expression has been shown to be regulated in the stress response of certain cells. However, its function is not yet fully understood. Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types. In the current study, we investigated NDRG2 involvement in hypoxia response and found that NDRG2 expression was markedly up-regulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein level. We also observed that the expression of NDRG2 was regulated by Hypoxia-inducible factor 1 (HIF-1) in tumor cells under hypoxia. Three hypoxia-responsive elements (HREs) in the NDRG2 promoter were identified. HRE1 could directly bind Hif-1 in vivo. Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase apoptosis. In addition, our data also showed that Ndrg2 was able to be translocated from the cytoplasm to the nucleus, and the segment from 101 to 178 amino acids of Ndrg2 is responsible for its translocation. Taken together, this study suggests that NDRG2 is a Hif-1 target gene and closely related with hypoxia-induced apoptosis in A549 cells.     

N-myc down-regulated gene 1 modulates the response of term human trophoblasts to hypoxic injury

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

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O(2)) than under 20% O(2) and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus, we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our (13)C NMR spectroscopic measurements indicate that (13)C-lactate is converted to (13)C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.     

N-myc downstream-regulated gene 2 controls astrocyte morphology via Rho-GTPase signaling

Astrocyte undergoes morphology changes that are closely associated with the signaling communications at synapses. N-myc downstream-regulated gene 2 (NDRG2) is specifically expressed in astrocytes and is associated with several important astrocyte functions, but its potential role(s) relating to astrocyte morphological changes remain unknown. Here, primary astrocytes were prepared from neonatal Ndrg2^+/+ and Ndrg2^−/− pups, and the drug Y27632 was used to induce stellation. We then used a variety of methods to measure the levels of NDRG2, α-Actinin4, and glial fibrillary acidic protein (GFAP), and the activity of RhoA, Rac1, and Cdc42 in Y27632-treated astrocytes as well as in Ndrg2^+/+, Ndrg2^−/−, or Ndrg2^−/− + lentivirus (restore NDRG2 expression) astrocytes. We also conducted live-imaging and proteomics studies of the cultured astrocytes. We found that induction of astrocytes stellation (characterized by cytoplasmic retraction and process outgrowth) resulted in increased NDRG2 protein expression and Rac1 activity and in reduced α-Actinin4 protein expression and RhoA activity. Ndrg2 deletion induced astrocyte flattening, whereas the restoration of NDRG2 expression induced stellation. Ndrg2 deletion also significantly increased α-Actinin4 protein expression and RhoA activity yet reduced GFAP protein expression and Rac1 activity, and these trends were reversed by restoration of NDRG2 expression. Collectively, our results showed that Ndrg2 deletion promoted cell proliferation, interrupted stellation capability, and extensively altered the protein expression profiles of proteins that function in Rho-GTPase signaling. These findings suggest that NDRG2 functions to regulate astrocytes morphology via altering the accumulation of the Rho-GTPase signaling pathway components, thereby supporting that NDRG2 should be understood as a regulator of synaptic plasticity and thus neuronal communications.     

Nitric oxide suppresses tumor cell migration through N-Myc downstream-regulated gene-1 (NDRG1) expression: role of chelatable iron

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide (^•NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to ^•NO. We demonstrate that the interaction of ^•NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological ^•NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of ^•NO. Augmenting the chelatable iron pool abolished ^•NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between ^•NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.     

HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.     

Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system

The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination.     

Microarray profiling of HepG2 cells ectopically expressing NDRG2

Previous studies have demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor that is downregulated in many human cancers and when overexpressed, can inhibit tumor growth and metastasis. However, its molecular function, its modulatory targets, and signaling pathways associated with it remain unclear. Here, in an effort to identify the genes modulated by NDRG2 expression, a microarray study was conducted to detect the expression profile of HepG2 cells overexpressing NDRG2 or LacZ. Gene Ontology (GO) biological process analysis revealed that genes related to G protein signaling pathway were upregulated. Five of them were selected and verified by real-time PCR. Gene sets related to M phase of cell cycle were downregulated. This was in agreement with cell cycle analysis. Signaling pathway analysis demonstrated apparent augmented hematopoietic cell lineage pathway and cell adhesion, but reduced glycosylphosphatidylinositol (GPI)-anchor biosynthesis, protein degradation and SNARE interactions. Furthermore, through motif analysis and experimental validation, we found that the p38 phosphorylation can be increased by NDRG2. Our research provides the molecular basis for understanding the role of NDRG2 in tumor cells and raises interesting questions about its mechanisms and potential use in cancer therapy.     

Plk3 functions as an essential component of the hypoxia regulatory pathway by direct phosphorylation of HIF-1alpha

Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1α under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1α under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1α in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1α stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.     

Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in Drosophila.

A nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is thought to play an important role in mammalian vasodilation during hypoxia. We show that Drosophila utilizes components of this pathway to respond to hypoxia. Hypoxic exposure rapidly induced exploratory behavior in larvae and arrested the cell cycle. These behavioral and cellular responses were diminished by an inhibitor of NO synthase and by a polymorphism affecting a form of cGMP-dependent protein kinase. Conversely, these responses were induced by ectopic expression of NO synthase. Perturbing components of the NO/cGMP pathway altered both tracheal development and survival during prolonged hypoxia. These results indicate that NO and protein kinase G contribute to Drosophila's ability to respond to oxygen deprivation.     

Two mechanisms involving the autophagic and proteasomal pathways process the metastasis suppressor protein,N-myc downstream regulated gene 1

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.