N-myc Downstream Regulated Gene 1 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.     

NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

N-myc downstream regulated gene-1 (NDRG1) has been identified as a putative metastasis suppressor gene and proved to be a key player in cancer spreading and proliferation in our previous work. However, the effects of NDRG1 on tumor invasion and the mechanisms behind it are rarely understood. Here we provided in silico evidence that NDRG1 plays a crucial role in actin reorganization in colorectal cancer (CRC). Through in vitro experiments, we next observed filopodia formation was altered in NDRG1-modified cell lines, while cell division cycle-42 (CDC42) displayed excessive activation in NDRG1-silenced cells. Mechanistically, NDRG1 loss disrupts the binding between RhoGDIα and CDC42 and triggers the activation of CDC42 and the downstream cascades PAK1/Cofilin, thereby promotes the formation of filopodia and invasiveness of CRC. The knockdown of NDRG1 led to enhanced dissemination of CRC cells in vivo and correlates with active CDC42 expression. Using clinical sample analysis, we found an elevated level of active CDC42 in patients with advanced T stage, and it was negatively related to NDRG1 expression. In sum, these results uncover a mechanism utilized by NDRG1 to regulate CDC42 activity in coordinating cytoskeleton reorganization, which was crucial in cancer invasion.     

Valproic acid inhibits the invasion of PC3 prostate cancer cells by upregulating the metastasis suppressor protein NDRG1

Valproic acid (VPA) is a clinically available histone deacetylase inhibitor with promising anticancer attributes. Recent studies have demonstrated the anticancer effects of VPA on prostate cancer cells. However, little is known about the differential effects of VPA between metastatic and non-metastatic prostate cancer cells and the relationship between the expression of metastasis suppressor proteins and VPA. In the present study, we demonstrate that inhibition of cell viability and invasion by VPA was more effective in the metastatic prostate cancer cell line PC3 than in the tumorigenic but non-metastatic prostate cell line, RWPE2. Further, we identified that the metastasis suppressor NDRG1 is upregulated in PC3 by VPA treatment. In contrast, NDRG1 was not increased in RWPE2 cells. Also, the suppressed invasion of PC3 cells by VPA treatment was relieved by NDRG1 knockdown. Taken together, we suggest that the anticancer effect of VPA on prostate cancer cells is, in part, mediated through upregulation of NDRG1. We also conclude that VPA has differential effects on the metastasis suppressor gene and invasion ability between non-metastatic and metastatic prostate cancer cells.     

抑癌候选基因NDRG2对结肠癌细胞SW620的迁移和侵袭力的影响

目的：观察NDRG2对结肠癌SW620细胞侵袭、转移等生物学行为的影响,探讨其可能的调节机制。方法：用阳离子脂质体转染方法分别转染pcDNA3.1-Ndrg2和SiRNA-Ndrg2于SW620细胞内48h,上调/下调NDRG2的表达;检测NDRG2基因mRNA及蛋白表达水平的变化;通过划痕试验及transwell细胞侵袭试验进一步对上调/下调NDRG2表达水平后的结肠癌细胞迁移和侵袭能力进行分析。结果：pcDNA3.1-Ndrg2转染SW620后,NDRG2的mRNA和蛋白表达水平明显升高,细胞的迁移和侵袭能力下降;SiRNA-Ndrg2转染SW620后,NDRG2的mRNA和蛋白表达水平明显降低,细胞的迁移和侵袭能力上升,差异具有统计学意义（P〈0.05）。结论：NDRG2作为抑癌候选基因能够降低结肠癌细胞转移和侵袭能力。     

Exploring the five different genes associated with PKCα in bladder cancer based on gene expression microarray

Much progress has been made in understanding the mechanism of bladder cancer (BC) progression. Protein kinase C-α (PKCα) is overexpressed in many kinds of cancers. Additionally, PKCα is considered an oncogene that regulates proliferation, invasion, migration, apoptosis and cell cycle in multiple cancers. However, the mechanism underlying how these cellular processes are regulated by PKCα remains unknown. In the present study, we used PKCα siRNA to knock down PKCα gene expression and found that down-regulation of PKCα could significantly inhibit cell proliferation, migration and invasion and induce apoptosis and G1/S cell cycle arrest in vitro. Overexpression of PKCα promotes tumour growth in vivo. We applied cDNA microarray technology to detect the differential gene expression in J82 cells with PKCα knockdown and found that five key genes (BIRC2, BIRC3, CDK4, TRAF1 and BMP4) were involved in proliferation and apoptosis according to GO analysis and pathway analyses. Correlation analysis revealed a moderate positive correlation between PKCα expression and the expression of five downstream genes. BIRC2 and BIRC3 inhibit apoptosis, whereas CDK4, TRAF1 and BMP4 promote proliferation. Essentially, all five of these target genes participated in proliferation, and apoptosis was regulated by PKCα via the NF-kB signalling pathway.     

Hyperthermia-Induced NDRG2 Upregulation Inhibits the Invasion of Human Hepatocellular Carcinoma via Suppressing ERK1/2 Signaling Pathway

Hyperthermia (HT) has been proven to be able to alter the invasion capacity of cancer cells. However, the detailed mechanisms responsible for the anti-metastasis effects of HT have not been elucidated. N-myc downstream-regulated gene 2 (NDRG2), as a member of the NDRG family, has been suggested to be highly responsive to various stresses and is associated with tumor suppression. The present study aimed to investigate the biological role of NDRG2 in the invasion of human hepatocellular carcinoma (HCC) cells exposed to HT. We found that NDRG2 could be induced by HT at 45°C. In addition, NDRG2 overexpression inhibited the expression of matrix metallo proteinases-2 (MMP-2) and MMP-9 as well as the invasion of HCC cells, whereas knockingdown NDRG2 reversed the anti-invasion effect of HT in vivo. Further investigation revealed that the phosphorylation level of ERK1/2, but not that of JNK and p38MAPK, was reduced in NDRG2 overexpressing cells. Moreover, the knockdown of NDRG2 expression resulted in increased cell invasion, which was rescued by treating the HepG2 cells with the ERK1/2 inhibitor PD98059, but not with the p38MAPK inhibitor SB203580 or the JNK inhibitor SP600125. Finally, the synergistic cooperation of HT at 43°C and NDRG2 expression effectively reduced cytotoxicity and promoted the anti-invasion effect of HT at 45°C. Taken together, these data suggest that NDRG2 can be induced by HT and that it mediates the HT-caused inhibition of invasion in HCC cells by suppressing the ERK1/2 signaling pathway. The combined application of constitutive NDRG2 expression with HT may yield an optimized therapeutic benefit.     

Overexpressed MicroRNA-182 Promotes Proliferation and Invasion in Prostate Cancer PC-3 Cells by Down-Regulating N-myc Downstream Regulated Gene 1 (NDRG1)

MicroRNAs, non-coding 20–22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-182 expression was significantly upregulated in prostate cancer tissues and four cell lines, compared to benign prostatic hyperplasia tissues and normal prostatic epithelial (RWPE-1) cells. Ectopic overexpression of miR-182 significantly promotes the proliferation, increases the invasion, promotes the G1/S cell cycle transition and reduces early apotosis of PC-3 cells, while suppression of miR-182 decreased the proliferation and invasion, inhibits the G1/S cell cycle transition and increase early apotosis of PC-3 cells. Additionally, we demonstrated that miR-182 could downregulate expression of NDRG1 by directly targeting the NDRG1 3′-untranslated region. In conclusion, our results suggest that miR-182 plays an important role in the proliferation of human prostate cancer cells by directly suppressing the tumor supressor gene NDRG1. We uncovered a new epigenetic regulation of NDRG1.     

Identification of potential genes/proteins regulated by Tiam1 in colorectal cancer by microarray analysis and proteome analysis

Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1), a guanine nucleotide exchange factor that activates Rac, is a colorectal cancer metastasis-related gene. In this study, we aimed to better understand the mechanism underlying Tiam1-mediated metastasis. We applied gene microarray and proteome analysis and compared expression of genes and proteins in a stable Tiam1-silencing colorectal cancer cell line and in a control cell line. Our analysis identified three genes, high-mobility group box1 (HMGB1), annexin IV (ANXA4) and phosphoglycerate mutase 1 (PGAM1) that were associated with Tiam1. Analysis of these proteins, which may be directly or indirectly regulated by Tiam1, may provide insight into the role and mechanism of Tiam1 in colorectal cancer metastasis.     

Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer

Pancreatic cancer is a highly lethal disease with a poor prognosis; the molecular mechanisms of the development of this disease have not yet been fully elucidated. N-myc downstream regulated gene 2 (NDRG2), one of the candidate tumor suppressor genes, is frequently downregulated in pancreatic cancer, but there has been little information regarding its expression in surgically resected pancreatic cancer specimens. We investigated an association between NDRG2 expression and prognosis in 69 primary resected pancreatic cancer specimens by immunohistochemistry and observed a significant association between poor prognosis and NDRG2-negative staining (P = 0.038). Treatment with trichostatin A, a histone deacetylase inhibitor, predominantly up-regulated NDRG2 expression in the NDRG2 low-expressing cell lines (PANC-1, PCI-35, PK-45P, and AsPC-1). In contrast, no increased NDRG2 expression was observed after treatment with 5-aza-2′ deoxycytidine, a DNA demethylating agent, and no hypermethylation was detected in either pancreatic cancer cell lines or surgically resected specimens by methylation specific PCR. Our present results suggest that (1) NDRG2 is functioning as one of the candidate tumor-suppressor genes in pancreatic carcinogenesis, (2) epigenetic mechanisms such as histone modifications play an essential role in NDRG2 silencing, and (3) the expression of NDRG2 is an independent prognostic factor in pancreatic cancer.     

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

Macrophages are a major cellular component of innate immunity and are mainly known to have phagocytic activity. In the tumor microenvironment (TME), they can be differentiated into tumor-associated macrophages (TAMs). As the most abundant immune cells in the TME, TAMs promote tumor progression by enhancing angiogenesis, suppressing T cells and increasing immunosuppressive cytokine production. N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene, whose expression is down-regulated in various cancers. However, the effect of NDRG2 on the differentiation of macrophages into TAMs in breast cancer remains elusive. In this study, we investigated the effect of NDRG2 expression in breast cancer cells on the differentiation of macrophages into TAMs. Compared to tumor cell-conditioned medium (TCCM) from 4T1-mock cells, TCCM from NDRG2-overexpressing 4T1 mouse breast cancer cells did not significantly change the morphology of RAW 264.7 cells. However, TCCM from 4T1-NDRG2 cells reduced the mRNA levels of TAM-related genes, including MR1, IL-10, ARG1 and iNOS, in RAW 264.7 cells. In addition, TCCM from 4T1-NDRG2 cells reduced the expression of TAM-related surface markers, such as CD206, in peritoneal macrophages (PEM). The mRNA expression of TAM-related genes, including IL-10, YM1, FIZZ1, MR1, ARG1 and iNOS, was also downregulated by TCCM from 4T1-NDRG2 cells. Remarkably, TCCM from 4T1-NDRG2 cells reduced the expression of PD-L1 and Fra-1 as well as the production of GM-CSF, IL-10 and ROS, leading to the attenuation of T cell-inhibitory activity of PEM. These data showed that compared with TCCM from 4T1-mock cells, TCCM from 4T1-NDRG2 cells suppressed the TAM differentiation and activation. Collectively, these results suggest that NDRG2 expression in breast cancer may reduce the differentiation of macrophages into TAMs in the TME.     

应用DNA芯片鉴定HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制

高危型人乳头瘤病毒（human papillomavirus,HPV）的E6基因在宫颈癌的发生中起关键作用,特异siRNA能有效抑制宫颈癌HeLa 细胞内HPV18 E6基因的表达,诱导肿瘤细胞凋亡.为进一步探讨HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制,针对HPV18-E6基因设计siRNA序列,利用人源U6启动子为模板,经PCR表达框架法体外扩增,转染宫颈癌HeLa细胞抑制HPV18 -E6基因表达,从而诱导肿瘤细胞凋亡.对转染前后HeLa细胞总RNA样品进行荧光标记后,与Agilent Human 1A寡核苷酸芯片杂交、扫描、数据分析及标准化处理,确定表达差异的基因并经荧光定量PCR对部分基因进行验证,结合PANTHER数据分析系统,将这些基因按照生物学功能进行归类,查阅GenBank数据库及相关文献,对其结果进行深入分析及讨论.在检测的18 716个基因和EST中,共筛出差异表达基因359个,其中307个基因表达上调,52个基因表达下调,主要包括细胞周期相关基因CCNG1、p21;凋亡相关基因CASP4、CASP6、IGFBP3、DFFA;泛素蛋白酶解途径相关基因E6-AP、UBE2C;角化细胞分化相关基因KRT4、KRT6E、KRT18;抑癌基因RECK、VHL等.研究结果表明,HPV18 -E6基因抑制引起的细胞凋亡效应主要是通过P53信号途径和泛素蛋白酶解信号途径调节细胞周期相关基因和凋亡相关基因的表达,从而抑制HeLa细胞增殖、促进细胞凋亡.同时,抑癌基因的激活,角化细胞分化和免疫相关基因的表达上调,都说明了E6抑制后肿瘤细胞恶性转化程度的下降.     

Inhibition of endothelial cell proliferation and tumor angiogenesis by up-regulating NDRG2 expression in breast cancer cells

The N-myc downstream-regulated gene 2 (NDRG2) is involved in tumor cell differentiation and apoptosis, but its function in tumor angiogenesis remains to be established. Here, we employed adenovirus overexpressing NDRG2 (Ad-NDRG2) to efficiently up-regulate target gene expression in the NDRG2-low-expressing, breast cancer cell line MCF-7. Moreover, VEGF secretion was decreased in MCF-7 cells infected by Ad-NDRG2, and medium conditioned by these infected cells could significantly inhibit the proliferation, tube formation and invasion of human umbilical vein endothelial cells (HUVECs). Further study indicated that the angiogenesis promoting factors VEGF and HIF-1α were down-regulated, whereas the angiogenesis suppressing factors p53 and VHL were up-regulated in MCF-7 cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for 20 days significantly inhibited the growth and angiogenesis of xenografted MCF-7 tumors. In summary, these data indicate that NDRG2 may be involved in angiogenesis by impacting the expression of angiogenesis related factors. Thus, specific overexpression of NDRG2 by adenovirus represents a promising approach for the treatment of tumor angiogenesis.     

Hypermethylation-mediated silencing of NDRG4 promotes pancreatic ductal adenocarcinoma by regulating mitochondrial function

The N-myc downstream regulated gene (NDRG) family members are dysregulated in several tumors. Functionally, NDRGs play an important role in the malignant progression of cancer cells. However, little is known about the potential implications of NDRG4 in pancreatic ductal adenocarcinoma (PDAC). The aim of the current study was to elucidate the expression pattern of NDRG4 in PDAC and evaluate its potential cellular biological effects. Here, we firstly report that epigenetic-mediated silencing of NDRG4 promotes PDAC by regulating mitochondrial function. Data mining demonstrated that NDRG4 was significantly down-regulated in PDAC tissues and cells. PDAC patients with low NDRG4 expression showed poor prognosis. Epigenetic regulation by DNA methylation was closely associated with NDRG4 down-regulation. NDRG4 overexpression dramatically suppressed PDAC cell growth and metastasis. Further functional analysis demonstrated that up-regulated NDRG4 in SW1990 and Canpan1 cells resulted in attenuated mitochondrial function, including reduced ATP production, decreased mitochondrial membrane potential, and increased fragmented mitochondria. However, opposite results were obtained for HPNE cells with NDRG4 knockdown. These results indicate that hypermethylation-driven silencing of NDRG4 can promote PDAC by regulating mitochondrial function and that NDRG4 could be as a potential biomarker for PDAC patients.     

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.     

Impaired OXPHOS complex III in breast cancer

We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.