NDRG2-mediated Modulation of SOCS3 and STAT3 Activity Inhibits IL-10 Production

이은별,임종석,김애영,강경아,김혜리 대한면역학회 2010 Immune Network Vol.10 No.6

Background: N-myc downstream regulated gene 2 (NDRG2) is a member of the NDRG gene family. Our previous report indicated a possible role for NDRG2 in regulating the cytokine, interleukin-10 (IL-10), which is an important immunosuppressive cytokine. Several pathways, including p38-MAPK, NF-κB, and JAK/STAT, are used for IL-10 production, and the JAK/STAT pathway can be inhibited in a negative feedback loop by the inducible protein, SOCS3. In the present study, we investigated the effect of NDRG2 gene expression on IL-10 signaling pathway that is modulated via SOCS3 and STAT3. Methods: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 in IL-10 production. U937 cells were also transfected with SOCS3- or NDRG2-specific siRNAs to examine whether the knockdown of SOCS3 or NDRG2 influenced IL-10 expression. Lastly, STAT3 and SOCS3 induction was measured to identify the signaling pathway that was associated with IL-10 production. Results: RT-PCR and ELISA assays showed that IL-10 was increased in U937-mock cells upon stimulation with PMA, but IL-10 was inhibited by overexpression NDRG2. After PMA treatment, STAT3 phosphorylation was decreased in a time-dependent manner in U937- mock cells, whereas it was maintained in U937-NDRG2 cells. SOCS3 was markedly reduced in U937-NDRG2 cells compared with U937-mock cells. IL-10 production after PMA stimulation was reduced in U937 cells when SOCS3 was inhibited, but this effect was less severe when NDRG2 was inhibited. Conclusion: NDRG2 expression modulates SOCS3 and STAT3 activity, eventually leading to the inhibition of IL-10 production.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells

Kang, Kyeong-Ah,Jung, Hye-Youn,Nam, So-Rim,Lim, Jong-Seok The Korean Association of Immunobiologists 2011 Immune Network Vol.11 No.6

Background: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMAstimulated U937 cells. Methods: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. Results: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. Conclusion: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells

강경아,임종석,정혜윤,남소림 대한면역학회 2011 Immune Network Vol.11 No.6

Background: N-myc downstream-regulated gene 2 (NDRG2),a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMAstimulated U937 cells. Methods: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. Results: NDRG2 overexpression in U937cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. Conclusion: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

NDRG2-mediated Modulation of SOCS3 and STAT3 Activity Inhibits IL-10 Production

Lee, Eun-Byul,Kim, Ae-Yung,Kang, Kyeong-Ah,Kim, Hye-Ree,Lim, Jong-Seok The Korean Association of Immunobiologists 2010 Immune Network Vol.10 No.6

Background: N-myc downstream regulated gene 2 (NDRG2) is a member of the NDRG gene family. Our previous report indicated a possible role for NDRG2 in regulating the cytokine, interleukin-10 (IL-10), which is an important immunosuppressive cytokine. Several pathways, including p38-MAPK, NF-${\kappa}B$, and JAK/STAT, are used for IL-10 production, and the JAK/STAT pathway can be inhibited in a negative feedback loop by the inducible protein, SOCS3. In the present study, we investigated the effect of NDRG2 gene expression on IL-10 signaling pathway that is modulated via SOCS3 and STAT3. Methods: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 in IL-10 production. U937 cells were also transfected with SOCS3- or NDRG2-specific siRNAs to examine whether the knockdown of SOCS3 or NDRG2 influenced IL-10 expression. Lastly, STAT3 and SOCS3 induction was measured to identify the signaling pathway that was associated with IL-10 production. Results: RT-PCR and ELISA assays showed that IL-10 was increased in U937-mock cells upon stimulation with PMA, but IL-10 was inhibited by overexpression NDRG2. After PMA treatment, STAT3 phosphorylation was decreased in a time-dependent manner in U937-mock cells, whereas it was maintained in U937-NDRG2 cells. SOCS3 was markedly reduced in U937-NDRG2 cells compared with U937-mock cells. IL-10 production after PMA stimulation was reduced in U937 cells when SOCS3 was inhibited, but this effect was less severe when NDRG2 was inhibited. Conclusion: NDRG2 expression modulates SOCS3 and STAT3 activity, eventually leading to the inhibition of IL-10 production.

A Study for Expression and Biological Function of N-myc Downstream Regulated Gene 2 in Breast Cancer

정성후 한국유방암학회 2007 Journal of breast cancer Vol.10 No.3

Purpose: It is important to identify a potential tumor marker that is associated with pathophysiologic processes of breast cancer. N-Myc downstream regulated genes (NDRG) are composed of four subtypes (NDRG 1-4) and NDRG2 gene has been reported as a specifically expressed gene in the human solid tumor including breast cancer. Although NDRG2 inhibits cell proliferation and promote differentiation, the molecular basis of the tumor-suppressor activity of NDRG2 in breast cancer is unknown. Herein, we tried to reveal the correlations between the expression of NDRG2 and the various clinicopathologic prognostic factors and evaluate its functional and pathophysiological roles in tumorigenesis of breast cancer. Methods: We were obtained the 67 breast cancers and paired normal tissue samples from patients who operated for breast cancer between June 2002 and June 2004. The expression of NDRG2 were measured with immmunohistochemistry using monoclonal antibody and it was used eukaryotic transfection to manipulate the expression in MDAMB-231 breast cancer cell line. Cell proliferation analysis were evaluated with trypan blue stain and status of differentially-expressed genes by NDRG2 overexpression were investigated with oligo microarray chip analysis. Results: Significant difference of NDRG2 mRNA expression between breast cancer and normal tissue was not detected. However, NDRG2 was significantly down-regulated in breast cancer tissue, compared to normal tissue (p<0.0001). It was a inverse-correlation between the NDRG2 expression and tumor size, histologic grade although other clinicopathological parameters such as axillary lymph node metastasis were not correlated. Overexpression of NDRG2 in MDA-MB-231 cell showed a decrease of cell proliferation compared with Mock control. Of the 24,000 genes, 64 genes were increased in expression while 256 genes including cyclin D1 were repressed by NDRG2 overexpression. Conclusion: Our results suggest that NDRG2 can function as a regulator of cell differentiation and cell cycle (as a tumor suppressor gene) in the early stage of breast cancer. In addition, NDRG2 protein indicates a prognostic tumor marker for breast cancer.

SOCS1 induced by NDRG2 expression negatively regulates STAT3 activation in breast cancer cells

Park, Yongjin,Shon, Soo-Kyung,Kim, Aeyung,Kim, Keun Il,Yang Young,Cho, Dae Ho,Lee, Myeong-Sok,Lim, Jong-Seok Research Institute of Women's Health Sookmyung Wom 2007 WOMEN And HEALTH Vol.3 No.1

Although NDRG2 inactivation has recently been found to have an important role in some tumorigenesis, its role in intracellular signal transduction pathways remains poorly defined. In the present study, we demonstrate that NDRG2 overexpression in malignant breast cancer cells specifically inhibits Akt phosphorylation and induces phosphorylation of p38 MAP kinase and SAPK/JHK. In addition, we investigated whether NDRG2 expression affects JAK/STAT- or mitogen-activated protein kinase-mediated signal activation. JAK2 or STAT3 activation in both resting and IGF-stimulating cells was remarkably inhibited by NDRG2 expression. Furthermore, NDRG2 has been found to highly up-regulate the expression level of SOCS1 mRNA and protein. We have found that NDRG2 was able to regulate cytokine signaling in breast cancer cells through the regulation of SOCS1 expression. Finally, inhibition of p38 MAPK activity blocked the induction of SOCS1 expression by NDRG2, resulting in the recovery of STAT3 phosphorylation leve.l Together, these data demonstrate that NDRG2 expression in breast cancer cells is able to inhibit STAT3 activation via SOCS1 induction in a p38 MAPK dependent manner, implicating NDRG2 as a growth inhibitory gene in signal transduction pathways of breast tumor cells.

NDRG2 Controls COX-2/PGE₂-Mediated Breast Cancer Cell Migration and Invasion

김명진,김학수,이수환,양영,이명석,임종석 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.10

N-myc downstream-regulated gene 2 (NDRG2), which is known to have tumor suppressor functions, is frequently down-regulated in breast cancers and potentially involved in preventing the migration and invasion of malignant tumor cells. In the present study, we examined the inhibitory effects of NDRG2 overexpression, specifically focusing on the role of cyclooxygenase-2 (COX-2) in the migration of breast cancer cells. NDRG2 overexpression in MDA-MB-231 cells inhibited the expression of the COX-2 mRNA and protein, the transcriptional activity of COX-2, and prostaglandin E2 (PGE2) production, which were induced by a treatment with phorbol-12-myristate-13-acetate (PMA). Nuclear transcription factor-κB (NF-κB) signaling attenuated by NDRG2 expression resulted in a decrease in PMA-induced COX-2 expression. Interestingly, the inhibition of COX-2 strongly suppressed PMA-stimulated migration and invasion in MDA-MB-231-NDRG2 cells. Moreover, siRNA-mediated knockdown of NDRG2 in MCF7 cells increased the COX-2 mRNA and protein expression levels and the PMA-induced COX-2 expression levels. Consistent with these results, the migration and invasion of MCF7 cells treated with NDRG2 siRNA were significantly enhanced following treatment with PMA. Taken together, our data show that the inhibition of NF-κB signaling by NDRG2 expression is able to suppress cell migration and invasion through the down-regulation of COX-2 expression.

NDRG2 Controls COX-2/PGE₂-Mediated Breast Cancer Cell Migration and Invasion

Kim, Myung-Jin,Kim, Hak-Su,Lee, Soo-Hwan,Yang, Young,Lee, Myeong-Sok,Lim, Jong-Seok Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.10

N-myc downstream-regulated gene 2 (NDRG2), which is known to have tumor suppressor functions, is frequently down-regulated in breast cancers and potentially involved in preventing the migration and invasion of malignant tumor cells. In the present study, we examined the inhibitory effects of NDRG2 overexpression, specifically focusing on the role of cyclooxygenase-2 (COX-2) in the migration of breast cancer cells. NDRG2 overexpression in MDA-MB-231 cells inhibited the expression of the COX-2 mRNA and protein, the transcriptional activity of COX-2, and prostaglandin $E_2$ ($PGE_2$) production, which were induced by a treatment with phorbol-12-myristate-13-acetate (PMA). Nuclear transcription factor-${\kappa}B$ (NF-${\kappa}B$) signaling attenuated by NDRG2 expression resulted in a decrease in PMA-induced COX-2 expression. Interestingly, the inhibition of COX-2 strongly suppressed PMA-stimulated migration and invasion in MDA-MB-231-NDRG2 cells. Moreover, siRNA-mediated knockdown of NDRG2 in MCF7 cells increased the COX-2 mRNA and protein expression levels and the PMA-induced COX-2 expression levels. Consistent with these results, the migration and invasion of MCF7 cells treated with NDRG2 siRNA were significantly enhanced following treatment with PMA. Taken together, our data show that the inhibition of NF-${\kappa}B$ signaling by NDRG2 expression is able to suppress cell migration and invasion through the down-regulation of COX-2 expression.

Loss of NDRG2 promotes epithelial-mesenchymal transition of gallbladder carcinoma cells through MMP-19-mediated Slug expression

Lee, D.G.,Lee, S.H.,Kim, J.S.,Park, J.,Cho, Y.L.,Kim, K.S.,Jo, D.Y.,Song, I.C.,Kim, N.,Yun, H.J.,Park, Y.J.,Lee, S.J.,Lee, H.G.,Bae, K.H.,Lee, S.C.,Shim, S.,Kim, Y.M.,Kwon, Y.G.,Kim, J.M.,Lee, H.J.,Mi Elsevier Science Publishers 2015 Journal of hepatology Vol.63 No.6

Background & Aims: Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract and one of the most lethal forms of human cancer. However, there is limited information about the molecular pathogenesis of GBC. Here, we examined the functional role of the tumor suppressor N-myc downstream-regulated gene 2 (NDRG2) and the underlying molecular mechanisms of disease progression in GBC. Methods: Clinical correlations between NDRG2 expression and clinicopathological factors were determined by immunohistochemical analysis of tumor tissues from 86 GBC patients. Biological functions of NDRG2 and NDRG2-mediated signaling pathways were determined in GBC cell lines with NDRG2 knockdown or overexpression. Results: Loss of NDRG2 expression was an independent predictor of decreased survival and was significantly associated with a more advanced T stage, higher cellular grade, and lymphatic invasion in patients with GBC. GBC cells with loss of NDRG2 expression showed significantly enhanced proliferation, migration, and invasiveness in vitro, and tumor growth and metastasis in vivo. Loss of NDRG2 induced the expression of matrix metalloproteinase-19 (MMP-19), which regulated the expression of Slug at the transcriptional level. In addition, MMP-19-induced Slug, increased the expression of a receptor tyrosine kinase, Axl, which maintained Slug expression through a positive feedback loop, and stabilized epithelial-mesenchymal transition of GBC cells. Conclusions: The results of our study help to explain why the loss of NDRG2 expression is closely correlated with malignancy of GBC. These results strongly suggest that NDRG2 could be a favorable prognostic indicator and promising target for therapeutic agents against GBC.

Regulation of tumor-associated macrophage (TAM) differentiation by NDRG2 expression in breast cancer cells

이소연,이아람,Jihyun Lim,임종석 생화학분자생물학회 2022 BMB Reports Vol.55 No.2

Macrophages are a major cellular component of innate immunityand are mainly known to have phagocytic activity. In the tumormicroenvironment (TME), they can be differentiated into tumorassociatedmacrophages (TAMs). As the most abundant immunecells in the TME, TAMs promote tumor progression by enhancingangiogenesis, suppressing T cells and increasing immunosuppressivecytokine production. N-myc downstream-regulated gene 2(NDRG2) is a tumor suppressor gene, whose expression is downregulatedin various cancers. However, the effect of NDRG2on the differentiation of macrophages into TAMs in breast cancerremains elusive. In this study, we investigated the effect ofNDRG2 expression in breast cancer cells on the differentiationof macrophages into TAMs. Compared to tumor cell-conditionedmedium (TCCM) from 4T1-mock cells, TCCM from NDRG2-overexpressing4T1 mouse breast cancer cells did not significantlychange the morphology of RAW 264.7 cells. However, TCCMfrom 4T1-NDRG2 cells reduced the mRNA levels of TAM-relatedgenes, including MR1, IL-10, ARG1 and iNOS, in RAW 264.7cells. In addition, TCCM from 4T1-NDRG2 cells reduced theexpression of TAM-related surface markers, such as CD206, inperitoneal macrophages (PEM). The mRNA expression of TAMrelatedgenes, including IL-10, YM1, FIZZ1, MR1, ARG1 andiNOS, was also downregulated by TCCM from 4T1-NDRG2cells. Remarkably, TCCM from 4T1-NDRG2 cells reduced theexpression of PD-L1 and Fra-1 as well as the production ofGM-CSF, IL-10 and ROS, leading to the attenuation of T cellinhibitoryactivity of PEM. These data showed that comparedwith TCCM from 4T1-mock cells, TCCM from 4T1-NDRG2 cellssuppressed the TAM differentiation and activation. Collectively,these results suggest that NDRG2 expression in breast cancermay reduce the differentiation of macrophages into TAMs inthe TME.