Role Of N-myc Downstream-regulated Gene 1 Research Articles

NDRG1 is expressed in human granulosa cells: An implicative role of NDRG1 in the ovary.

PurposeN‐myc downstream‐regulated gene 1 (NDRG1) is expressed in various human tissues and plays a role in regulating cellular proliferation, angiogenesis, and hypoxia sensing. However, the role of NDRG1 in the ovary remains poorly understood. Therefore, we investigated NDRG1 expression and the role of NDRG1 in the human ovary.MethodsFollicular fluid (FF) and luteinized granulosa cells were collected from follicles during oocyte retrieval. KGN cells were cultured with cobalt chloride (CoCl2, a hypoxia‐mimicking agent) and/or echinomycin. mRNA, protein levels and secretion, and localization were assessed by real‐time PCR, Western blotting, ELISA, and immunohistochemical analysis, respectively. KGN cells were also transfected with NDRG1 siRNA for 72 h.ResultsNDRG1 protein was expressed in luteinized granulosa cells. NDRG1 concentration was positively correlated with vascular endothelial growth factor (VEGF) and progesterone concentrations in FF. CoCl2‐induced hypoxic stress significantly increased NDRG1 and VEGF mRNA and protein and hypoxia‐inducible factor‐1α expression compared with those in the controls. The CoCl2‐induced overexpression of NDRG1 and VEGF was suppressed by echinomycin. Transfection with NDRG1 siRNA significantly suppressed the release of progesterone in the culture medium.ConclusionsThese results indicate that ovarian NDRG1 may play important roles in follicular development, especially in the early luteinization of pre‐ovulatory follicles.

N-myc downstream-regulated gene 1 promotes oxaliplatin-triggered apoptosis in colorectal cancer cells via enhancing the ubiquitination of Bcl-2.

N-myc downstream-regulated gene1 (NDRG1) has been identified as a potent tumor suppressor gene. The molecular mechanisms of anti-tumor activity of NDRG1 involve its suppressive effects on a variety of tumorigenic signaling pathways. The purpose of this study was to investigate the role of NDRG1 in the apoptosis of colorectal cancer (CRC) cells. We first collected the clinical data of locally advanced rectal cancer (LARC) patients receiving oxaliplatin-based neoadjuvant chemotherapy in our medical center. Correlation analysis revealed that NDRG1 positively associated with the downstaging rates and prognosis of patients. Then, the effects of over-expression and depletion of NDRG1 gene on apoptosis of colorectal cancer were tested in vitro and in vivo. NDRG1 over-expression promoted apoptosis in colorectal cancer cells whereas depletion of NDRG1 resulted in resistance to oxaliplatin treatment. Furthermore, we observed that Bcl-2, a major anti-apoptotic protein, was regulated by NDRG1 at post-transcriptional level. By binding Protein kinase Cα (PKCα), a classical regulating factor of Bcl-2, NDRG1 enhanced the ubiquitination and degradation of Bcl-2, thus promoting apoptosis in CRC cells. In addition, NDRG1 inhibited tumor growth and promoted apoptosis in mouse xenograft model. In conclusion, NDRG1 promotes oxaliplatin-triggered apoptosis in colorectal cancer. Therefore, colorectal cancer patients can be stratified by the expression level of NDRG1. NDRG1-positive patients may benefit from oxaliplatin-containing chemotherapy regimens whereas those with negative NDRG1 expression should avoid the usage of this cytotoxic drug.

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-β pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

Abstract B199: Novel anti-angiogenic cancer therapeutic strategy by targeting differentiated macrophage lineage cells through N-myc downstream regulated gene 1 (NDRG1)

Abstract [Background] Angiogenesis is essential for malignant progression of cancer. Over the last couple of decades, it was highlighted that the progenitor cells derived from bone marrow (BM) differentiated, became part of the tumor stroma, and involved in tumor angiogenesis. In particular, macrophages, which differentiated into tumor-associated macrophages in tumor microenvironment, are known to play crucial roles in tumor angiogenesis. However, the specific molecules and markers of these “angiogenesis-supporting macrophages” have not been fully defined (Qian and Pollard, Cell. 2010). N-myc downstream regulated gene 1 (NDRG1), a gene responsible for a hereditary motor and sensory neuropathy (Lon-Charcot-Marie disease), plays pleiotropic roles in cell proliferation, development, differentiation, and tumorigenesis. We have previously reported that NDRG1 expression levels in cancer cells were closely correlated with tumor angiogenesis (Maruyama et al., Cancer Res. 2006; Hosoi et al., Cancer Res. 2009; Ureshino et al., PLoS One. 2012; Murakami et al., J Biol Chem. 2013). However, how NDRG1 could affect tumor angiogenesis remains unclear. In this study, we asked how NDRG1 could specifically modulate tumor angiogenesis through its differentiation control of macrophage lineage cells by using NDRG1 knock out (KO) mice. [Material and methods] NDRG1 KO mice: The NDRG1 KO mice on C57BL6 background have been kindly donated by Dr. Toshiyuki Miyata (National Cerebral and Cardiovascular Center) (Okuda et al., Mol Cell Biol., 2004). Preparation of BM derived macrophages (BMDMs): BMDMs were obtained by flushing mouse femurs from mice. Cells were seeded in dishes and washed twice 3 hours later. Adherent cells were incubated in DMEM supplemented with 10% FBS and 20ng/ml M-CSF for 7 days. Matrigel plug assay in BM suppression mice: wild type (WT) recipient mice were exposed to 3-Gy sublethal whole-body irradiation to suppress BM and temporarily deplete leukocytes. Seven days after the irradiation, the mice were injected subcutaneously with growth factor-reduced Matrigel containing B16/BL6 cells, with or without BMDMs from WT or NDRG1 KO mice. [Results] In NDRG1 KO mice as compared to their WT counterparts, [1] serum levels of M-CSF and macrophage-related cytokines were all decreased, and BM cells showed much slower growth rates in response to M-CSF in vitro; [2] trabecular bone mass and number of osteoclasts were decreased in vivo; [3] tumor growth and angiogenesis were markedly suppressed, accompanying by decreased infiltration of tumor-associated macrophages; [4] the transfer of BMDMs from KO mice into BM-eradicated WT mice induced much less tumor angiogenesis than observed in WT mice; [5] VEGF-A expression in BMDMs from KO mice were much less than those in cells from WT mice when stimulated by LPS or IL-1β. [Conclusion] We discovered the central role of NDRG1 in differentiation of macrophage lineage cells, which affected bone remodeling and tumor angiogenesis. We will discuss which molecules are specifically regulated by NDRG1 during differentiation processes of monocytes/macrophages into angiogenesis-supporting macrophages, and whether these molecules in macrophages could have potentially important target for anti-angiogenesis cancer therapeutics. Citation Format: Kosuke Watari, Tomohiro Shibata, Hiroshi Nabeshima, Ai Shinoda, Yuichi Fukunaga, Akihiko Kawahara, Kazuyuki Karasuyama, Jun-ichi Fukushi, Yuichi Murakami, Michihiko Kuwano, Mayumi Ono. Novel anti-angiogenic cancer therapeutic strategy by targeting differentiated macrophage lineage cells through N-myc downstream regulated gene 1 (NDRG1). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B199.

Abstract 1983: Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells

Abstract Hypoxia has been intensively investigated over past decades based on the observations that hypoxic tumors were more resistant to therapy and had a worse prognosis. Previously, we reported that N-myc downstream-regulated gene 1 (NDRG1) was strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuation of oxygen concentrations. However, the regulatory mechanism of NDRG1 under hypoxia remains elusive. Therefore, the purpose of this study is to identify the novel transcription factors that regulate NDRG1 and to investigate the functional roles of NDRG1 in hypoxia. In silico analysis predicted binding sites of aryl hydrocarbon receptor (AHR) existing in the NDRG1 promoter. Western blotting showed that nuclear AHR was up-regulated in the presence of cobalt and hypoxia. Immunofluorescence staining and chromatin immunoprecipitation assays showed that AHR translocated to nuclei and bound to NDRG1 promoter in hypoxia. Luciferase reporter assays further showed that the binding site of AHR at -412 ∼ -388 bp played a crucial role in regulating NDRG1 under hypoxia-mimicking conditions. Moreover, hypoxia-mimetic induction of NDRG1 by was attenuated by knockdown of AHR expression using short interfering RNA. Also, overexpression of AHR facilitated cell proliferation and migration via up-regulation of NDRG1, whereas shRNA knockdown of NDRG1 reduced cell growth and motility. In summary, these results showed for the first time that AHR positively regulates NDRG1 transcription through a AHR binding site by hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regime to prevent tumor malignancy under hypoxia. Citation Format: En-Yu Li, Wei-Yung Huang, Mong-Hsun Tsai, Eric Y. Chuang, Liang-Chuan Lai. Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1983. doi:10.1158/1538-7445.AM2015-1983

The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1.

N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics.

Overexpression of N-myc downstream-regulated gene 1 inhibits human glioma proliferation and invasion via phosphoinositide 3-kinase/AKT pathways.

N-myc downstream-regulated gene 1 (NDRG1) was previously shown to exhibit low expression in glioma tissue as compared with that in normal brain tissue; however, the role of NDRG1 in human glioma cells has remained to be elucidated. The present study used the U87 MG and SHG-44 human glioma cell lines as well as the normal human astrocyte cell line 1800, which are known to have differential NDRG1 expression. Small interfering (si)RNA targeting NDRG1, and NDRG1 overexpression vectors were transfected into the SHG-44 and U87 MG glioma cells, respectively. Cell proliferation, invasion, apoptosis and cell cycle arrest were subsequently examined by MTT assay, transwell chamber assay, flow cytometry and western blot analysis, respectively. Furthermore, a subcutaneous tumor mouse model was used to investigate the effects of NDRG1 on the growth of glioma cells in vivo. Overexpression of NDRG1 was shown to inhibit cell proliferation and invasion, and induce apoptosis in the U87 MG glioma cells, whereas NDRG1 downregulation increased proliferation, suppressed apoptosis and promoted invasion of the SHG-44 glioma cells. In addition, in the subcutaneous tumor mouse model, overexpression of NDRG1 in U-87 MG cells suppressed tumorigenicity in vivo. The findings of the present study indicated that NDRG1 is required for the inhibition of gliomagenesis; therefore, targeting NDRG1 and its downstream targets may represent novel therapies for the treatment of glioma.

Dp44mT targets the AKT, TGF-β and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells

Background:Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-β (TGF-β) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression.Methods:Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA.Results:Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels.Conclusion:NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-β and ERK pathways.

Equivocal Differential Effect of NDRG1 in Human Ovarian Cancer Cells

Inactivation of tumor suppressor genes is a key factor in cancer regulation. N-myc downstream regulated gene 1 (NDRG1) is a tumor suppressor gene well known to be involved in carcinogenesis of numerous cancer types. The present study was designed to investigate the role of NDRG1 in human ovarian cancer, using SKOV-3 and SW626 (moderately and well differentiated cancer cells, respectively). Our results revealed that over-expressed NDRG1 significantly up-regulated the differentiation marker p21, in the ovarian cancer cell lines. This regulation led to decrease in cell viability and DNA synthesis rates in SW626 cells (83% and 89.5%, respectively). However, no effect on viability or on DNA synthesis was observed in SKOV-3 NDRG1-transfected cells. These findings prove that NDRG1 is indubitably functional in human ovarian cancer cells, as it up-regulated p21 expression. Nevertheless, this regulation showed differential effect on cell viability and DNA formation thus promoting the perception that downstream regulation of p21 could be inefficient in some cancer cells, a concept that needs to be further explored in order to understand its disability to play as regulator of cell cycle progression.

<i>N-myc</i> Downstream Regulated Gene 1 Increases Differentiation Factors Level in Human Prostate Cancer Cells without Affecting Cell Proliferation and Cell Cycle Profiles

N-myc downstream regulated gene 1 (NDRG1), also known as differentiation related gene 1, was previously identified as an up-regulated gene upon cellular differentiation. Even though its sequence along with its expressional pattern in cancer cell lines are evident, the functional aspects concerning cell proliferation, viability, differentiation and cell cycle regulation of NDRG1 remains vague. The aim of the present study was to elucidate the functional role of NDRG1 in human prostate cancer. Our results showed basal levels of NDRG1 expression in PC-3 (poorly differentiated, null p53), DU-145 (moderately differentiated, mutant p53) and LNCaP (well-differentiated, wiled type p53). Nevertheless, NDRG1 sequencing assay disclosed no mutations in the gene. Furthermore, human cDNA of NDRG1 from normal placenta was cloned into a eukaryotic expression vector and transfected into the three cancer cell lines. This resulted in over-expression of NDRG1, which in turn markedly up regulated two differentiation markers of the prostate tissue, p21 and cytokeratin 8/18. Unpredictably, cell cycle progression, cell proliferation and DNA synthesis were unaffected following NDRG1 expression. These results revealed that NDRG1 is functional in prostate cancer cells and able to induce expression of differentiation factors through p53 independent pathway. However, the pathway downstream NDRG1, involving p21 and c8/18, regulating cell cycle progression and DNA synthesis is unfunctional. Loss of sensitivity to p21 cell cycle control may be associated with prostatic cancer behavior. Further studies are required to clarify the intra cellular molecular pathways affecting NDRG1 function in human prostate cancer.

N-myc Downstream-regulated Gene 1 (NDRG1) a Differentiation Marker of Human Breast Cancer

N-myc downstream-regulated gene 1 (NDRG1), also called differentiation-related gene-1 (Drg1) and Cap43, is expressed in various normal tissues and suppressed in several malignancies. In this study, whether NDRG1 expression was correlated with differentiation of human breast cancer cells has been investigated. Endogenous expression level of NDRG1 was closely correlated with differentiation status of breast cancer cell lines. Furthermore, sodium butyrate (NaB), an inducer of cellular differentiation, increased the expression of β-casein, a milk-related differentiation marker, together with up-regulation of NDRG1 in breast cancer cells. In contrast, inhibition of NDRG1 by its siRNA resulted in reduced accumulation of β-casein. Immunohistochemical analysis showed co-expression of NDRG1 and β-casein or milk fat protein (MFP), another differentiation marker of breast tissue, in the mouse xenograft model of breast cancer. Furthermore, overexpression of NDRG1 expanded the differentiated area in the xenograft model of breast cancer. In human breast cancer, using samples from 45 patients, we also showed close relationship between NDRG1 and β-casein or MFP expression. Altogether, in vitro and in vivo data demonstrated a possible role of NDRG1 in differentiation of breast cancer. We concluded that NDRG1 could be used as a biomarker for differentiation of breast cancer for both diagnostic and therapeutic purposes.

Biological function of NDRG1 and its roles in tumorigenesis

N-myc downstream-regulated gene 1 ( NDRG1 ), a member of the NDRG gene family, is a cell proliferation and differentiation related gene which usually express in various human tissues, especially higher in kidney, prostate and placenta. A variety of studies have reported that NDRG1 is correlated with cell proliferation and formation of periphery nerve sheath which might be regulated by hypoxia, anti-oncogene and metallic ion metabolism. Although NDRG1 gene expresses abnormally in tumor tissue, which is probably involved in tumorigenesis, development and metastasis, the exact role of NDRG1 in tumorigenesis is not clearly defined and further studies need to be done. Key words: NDRG1 gene; Neoplasms; Cell differentiation

Correlation of N-myc downstream-regulated gene 1 overexpression with progressive growth of colorectal neoplasm

To study the function of N-myc downstream-regulated gene 1 (NDRG1) in colorectal carcinogenesis and its correlation with tumor lymph node metastasis. NDRG1 was detected at its protein level by immunohistochemistry (IHC) and image analysis (IA), and NDRG1 mRNA was detected by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded sections with a total of 190 specimens including 38 normal colorectal mucosae, 31 colorectal adenomas, 45 non-metastatic colorectal carcinomas (CRCs), 38 metastatic primary CRC and subsequently regional lymph nodes respectively. At the same time, the correlations of NDRG1 with sex, age of patients and histological types of colorectal carcinomas were observed. NDRG1 proteins were gradually increased in colorectal carcinogenesis (P<0.05 or P<0.01). There was a significant difference in the expression of NDRG1 between non-metastatic and metastatic CRCs (P<0.05), and the correlation was positive (P<0.01, r(s)=0.329). However, there was no obvious difference in the expression of NDRG1 between the primary sites of CRCs and that in the metastatic sites of corresponding regional lymph nodes, nor was there an apparent difference in sex, age, and histological types. The expression of NDRG1 mRNA was generally in concordance with that of NDRG1 protein. NDRG1 gene may play an important role in colorectal carcinogenesis. In addition, NDRG1 may be a putative tumor metastasis promoter gene and is regarded as one of the molecular biological markers that can forecast early metastasis of CRCs. NDRG1 gene in the metastatic sites of regional lymph nodes may preserve its expression characteristics in the primary sites of CRCs to some extent. The expression of NDRG1 is not affected by sex, age and histological types. The role of NDRG1 in tumor metastatic process can be demonstrated by in vivo and in vitro.