10397

NDRG1

N-myc downstream regulated 1;NDRG1;N-myc downstream regulated 1

N-myc downstream-regulated gene 1 protein|differentiation-related gene 1 protein|nickel-specific induction protein Cap43|protein NDRG1|protein regulated by oxygen-1|reducing agents and tunicamycin-responsive protein

8q24.3

protein-coding

OBJECTIVE: To investigate the relationship between NDRG1 and metastasis of breast cancer and the effects of NDRG1 overexpression on the proliferation and invasion of breast cancer cells. METHODS: NDRG1 was detected at its protein level by immunohistochemistry (IHC) and its messenger RNA (mRNA) was detected by real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) in clinical breast cancer specimens. Liposome was used to transiently transfer NDRG1 into MDA-MB-231, a highly invasive human breast cancer cell line. The proliferation of MDA-MB-231 was measured by Bromodeoxy Uridine (BrdU) incorporation assay and the transfection effect on cell cycle distribution was determined by fluorescence assisted cell sorting (FACS). The invasive ability of the transfected cells was investigated by reconstituted matrigel invasion and polycarbonate filters migration experiments. RESULTS: NDRG1 expressions at protein and mRNA levels in tumors of patients with lymph node metastases were significantly lower as compared with those with localized breast cancers (P < 0.01). The amount of NDRG1 mRNA in MCF7, a relatively non-invasive breast cancer cell line, was 10.8 times higher than that in MDA-MB-231 cells (P < 0.01). The BrdU incorporation rate declined significantly (P < 0.05) in NDRG1 overexpressing MDA-MB-231 cells. An increase of the cell population at G(0)/G(1) phase was observed 48 hours post-transfection along with a decrease of cell population at S phase. Overexpression of NDRG1 significantly retarded the invasiveness of MDA-MB-231 cells in matrigel-coated invasion chambers (P < 0.05), when compared to cells transfected with control vectors. However, the migration abilities of cells with or without the transfection were virtually identical. CONCLUSIONS: NDRG1 expression reversely correlates with breast cancer metastasis and progression, and may serve as a prognostic biomarker for predicting early metastasis. The inhibition of proliferation and invasion demonstrated by our MDA-MB-231 transfection experiments implies that NDRG1 is a tumor metastasis suppressor gene and may be a new candidate for gene therapy against human breast cancer.

tumor metastasis is an important clinical problem, contributing to the majority of cancer-related deaths. The recent discovery of metastasis suppressor genes, such as N-myc downstream-regulated gene-1 (Ndrg-1), has introduced a novel approach to treating cancer and preventing metastasis. Ndrg-1 has been identified as a protein involved in the differentiation of epithelial cells. In addition, Ndrg-1 expression can be regulated by androgens and is involved in the pathology of the disease, Hereditary Motor and Sensory Neuropathy-Lom (HMSNL). However, one of the most well documented links between Ndrg-1 and pathophysiology is its association with inhibition of tumor metastasis. The expression of Ndrg-1 was found to be significantly downregulated in a variety of different neoplasms including breast, colon and prostate cancer. Furthermore, Ndrg-1 expression was shown to be negatively correlated with tumor metastasis. Studies in vitro and in vivo have demonstrated a significant reduction in the metastatic ability of cells overexpressing Ndrg-1. The ability of these cells to invade was also compromised. The Gleason grade of prostate and breast cancers was found to correlate with Ndrg-1 expression, with more advanced and poorly differentiated tumors having lower Ndrg-1 levels. Recently, Ndrg-1 expression was demonstrated to be regulated by cellular iron levels and induced by iron chelators. These latter compounds were recently identified as potential anticancer agents as they selectively prevent cancer cell proliferation and lead to apoptosis. The discovery that iron chelators also increase Ndrg-1 expression further augments their antitumor activity and provides a novel strategy for the treatment of cancer and its metastasis.

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 (Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase (Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.

N-myc downstream-regulated gene-1 (NDRG1) has been identified as a protein involved in the differentiation of epithelial cells. As a newly metastasis suppressor gene, whether it contributes to carcinogenesis of breast cancer is still unknown. This study aimed to clarify the possible role of NDRG1 for breast cancer carcinogenesis, and further to investigate its clinicopathological significance in invasive breast cancer. We examined the expression of NDRG1 in normal epithelium of breast (n = 35), usual ductal hyperplasia (n = 22), atypical ductal hyperplasia (n = 33), atypical lobular hyperplasia (n = 8), ductal carcinoma in situ (n = 16), lobular carcinoma in situ (n = 6), invasive ductal carcinoma (n = 50), and invasive lobular carcinoma (n = 45) by immunohistochemistry and analyzed the correlation between NDRG expression and clinicopathological features of invasive breast cancer. Western blot analysis was carried out to investigate the expression of NDRG1 in 20 invasive ductal breast cancer and the paired non-tumor portion of the same case. NDRG1 expression in invasive breast cancer (70/95, 73.7%) was higher than that in noninvasive breast lesions (29/85, 34.1%; p < 0.05) which was higher than that in normal breast epithelium (5/35, 14.3%; p < 0.05). Statistical analysis revealed a significant correlation between NDRG1 expression with tumor stage in invasive breast cancer, and its expression in invasive ductal carcinoma is significantly higher than invasive lobular carcinoma (p < 0.05). It was not associated with age, menopausal status, tumor size, and lymph node metastasis. NDRG1 protein levels were significantly higher in invasive ductal breast cancer compared to the paired non-tumor portion of the same case by Western blot analysis (p < 0.05). Increased NDRG-1 expression is associated with breast atypia-to-carcinoma progression. NDRG1 expression might participate in the carcinogenesis and progression of invasive breast cancer. These findings provide further evidence that NDRG1 may serve as an important biomarker for invasive breast cancer.

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation, differentiation, invasion and metastasis of cancer cells, but its role in cervical cancer is still unclear. This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors. The NDRG1 expression in cervical tissues and cells was detected by RT-PCR. Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines. The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting, respectively. Its effects on cell proliferation, migration, invasion, cell cycle and apoptosis were detected by MTT, transwell migration assay and flow cytometry (FCM), respectively. The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P<0.001). After transfection with pEGFP-N1-NDRG1-GFP, the mRNA and protein expression of NDRG1 was up-regulated in Siha cells, which suppressed cell proliferation (P<0.001), induced cell cycle arrest (P<0.05), reduced invasion and migration of Siha cells (P<0.05), but caused no cell apoptosis. Moreover, vascular endothelial growth factor (VEGF), a tumor-induced angiogenesis factor, was markedly reduced and E-cadherin, a cell adhesion molecule, was increased in the cells transfected with pEGFP-N1-NDRG1-GFP. It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth, invasion and metastasis of human cervical cancer.

N-Myc downstream-regulated gene 1 (NDRG1) plays vital roles in tumor metastasis suppression and is frequently silenced in metastatic colon cancers. NDRG1 is silenced in a highly metastatic colon cancer cell line SW620. The objective of this study was to investigate the potential mechanisms involved in silencing of the NDRG1 gene. SW480 and SW620 are two colon cancer cell lines established from the same patient with different metastatic potentials, making them an ideal model for investigation of metastatic mechanisms. Knockdown of NDRG1 in SW480 to a level that is similar to that in SW620 also modulated cell cycle and proliferation in SW480 towards the status of the highly metastatic SW620. Epigenetic mechanisms of the transcriptional control of NDRG1 were investigated. The silencing of NDRG1 in SW620 was not due to promoter hyper-methylation as bisulfite sequencing of the NDRG1 promoter showed minimal DNA methylation in both cell lines. On the other hand, chromatin immunoprecipitation showed a significantly higher level of RNA polymerase II (Pol II) association with the NDRG1 promoter in SW480 compared to SW620, in agreement with its gene expression level. The low Pol II binding at the NDRG1 promoter in SW620 was associated with gene-wide decrease in histone H4 acetylation and increase in histone H3 serine 10 phosphorylation. Meanwhile, the NDRG1 coding region showed much higher histone H3 lysine 4 methylation in SW480. In conclusion we observed unique histone modifications in two colon cancer cell lines with different metastatic potentials, indicating possible mechanisms for the down-regulation of NDRG1 in metastatic SW620.

OBJECTIVE: N-myc downstream regulated gene-1 (NDRG1) is known as a differentiation-related gene that plays important roles in cell differentiation, organ formation, and embryonic development. NDRG1 was recently found to significantly down regulate in a variety of different neoplasms. Its significance in gastric cancer has not been studied. MATERIALS AND METHODS: NDRG1 was detected at its protein level by immunohistochemistry in formalin-fixed and paraffin-embedded sections with a total of 110 pair gastric cancer specimens including tumor and corresponding paraneoplastic tissues; NDRG1 mRNA was detected by real time-polymerase chain reaction. Meanwhile, the correlations between NDRG1 and clinicopathological factors were observed. Overexpression of NDRG1 has influence on the biological behavior of gastric cancer cell, which was detected by cell growth assay, apoptosis assay, and in vitro motility and invasion assay. RESULTS: NDRG1 protein was down regulated in gastric cancer tissues, and the NDRG1 low expression rate was 73.6% (79/110). Moreover, NDRG1 expression has a significant inverse correlation with tumor stromal invasion, lymph node metastasis, pathological stage, but not with distant metastasis. The patients with low NDRG1 expression had a significantly shorter survival opportunity than those with high NDRG1 expression. In addition, overexpression of NDRG1 induced early apoptosis and inhibited SGC7901 cell proliferation and its motility and invasion capability. CONCLUSIONS: NDRG1 plays a significant role in carcinogenesis and preventing the metastasis and invasion of gastric cancer cells. NDRG1 could be developed as a marker contributing to diagnosis and evaluating prognosis in gastric cancer, as well as a potential therapeutic target of gastric cancer.

Hepatocellular carcinoma is a highly lethal cancer that typically has poor prognosis. Prognostic markers can help in its clinical management and in understanding the biology of poor prognosis. Through an earlier gene expression study, we identified N-Myc downregulated gene 1 (NDRG1) to be significantly highly expressed in hepatocellular carcinoma compared to nontumor liver. As NDRG1 is a differentiation-related gene with putative metastasis suppressor activity, we investigated the clinical significance of its overexpression. Quantitative real-time polymerase chain reaction using an independent set of patient samples confirmed the significant overexpression of NDRG1 in hepatocellular carcinoma compared to nontumor liver samples (P<0.001). Additionally, high levels of NDRG1 transcript correlated with shorter overall survival (P<0.001), late tumor stage (P=0.001), vascular invasion (P=0.003), large tumor size (P=0.011), and high Edmondson-Steiner histological grade (P=0.005). Using immunohistochemistry, NDRG1 protein was found to be significantly overexpressed in hepatocellular carcinoma samples compared to nontumor liver or cirrhotic and benign liver lesions (P<0.001). Among the hepatocellular carcinoma samples, those which are moderately and poorly differentiated express higher levels of NDRG1 protein than those which are well-differentiated (P<0.005). Additionally, hepatocellular carcinomas with vascular invasion also express elevated levels of NDRG1 protein compared to those without vascular invasion (significant at P<0.005). Our results suggest NDRG1 to be a likely tumor marker for hepatocellular carcinoma, the overexpression of which is correlated with tumor differentiation, vascular invasion, and overall survival. Its significantly elevated expression in hepatocellular carcinoma could be a useful indicator of tumor aggressiveness and therefore patient prognosis.

N-myc downstream regulated gene 1 (NDRG1)/Cap43 is a 43 kDa protein that is widely distributed in the body. Its expression is regulated by nickel, cobalt, hypoxic condition and others; it is reported to be weaker in tumors than normal tissues; and NDRG1/Cap43 is considered to act suppressively to tumor metastasis. This current study immunohistochemically examined NDRG1/Cap43 expression in hepatocellular carcinoma (HCC), and analyzed its relationship to clinicopathologic factors and prognosis. The samples were 105 surgically resected HCC tissue blocks, i.e., 18 well-differentiated HCC, 61 moderately differentiated HCC, 10 poorly differentiated HCC, 9 'nodule-in-nodule' type HCC, and 7 sarcomatous HCC. In all cases, NDRG1/Cap43 was not expressed in normal liver cells. Strong expression was found in 65 of the 105 cases (62%), i.e., in 11.1% of well-differentiated HCC, 72.1% of moderately differentiated HCC, 80.0% of poorly differentiated HCC, and 71.4% of sarcomatous HCC. In the 'nodule-in-nodule' type, its expression was found in 55.6% of their well-differentiated component, and this frequency was significantly higher than that in well-differentiated HCC (11.1%). In the cases showing strong NDRG1/Cap43 expression, frequency of portal vein invasion and of intrahepatic metastasis was significantly high. No clear relationship between the expression and prognosis was observed. NDRG1/Cap43 expression that was found in advanced HCC was thought to accelerate tumor invasion and metastasis. NDRG1/Cap43 could act as a useful biomarker of HCC.

N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 (Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase (Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.

Development of resistance to therapy continues to be a serious clinical problem in lung cancer management. cancer cells undergoing epithelial-to-mesenchymal transition (EMT) have been shown to play roles in resistance to chemotherapy. Here, we utilized a proteomics-based method and identified a significant downregulation of the metastasis suppressor NDRG1 in drug resistant lung cancer cells. We showed that downregulation of DNRG1 constitutes a mechanism for acquisition of EMT phenotype and endows lung cancer cells with an increased resistance to cisplatin. We also identified a signal cascade, namely, SET--| PP2A--| c-myc--| NDRG1, in which upregulation of SET is critical for inhibition of NDRG1. We also found that blockade of SET (or reactivation of PP2A) by FTY720 reverted EMT, restored drug sensitivity, and inhibited invasiveness and growth of lung tumor xenografts. Together, our results indicated a functional link between SET-mediated NDRG1 regulation and acquisition of EMT phenotype and drug resistance, and provided an evidence that blockade of SET-driven EMT can overcome drug resistance and inhibit tumor progression.

tumor metastasis is an important clinical problem, contributing to the majority of cancer-related deaths. The recent discovery of metastasis suppressor genes, such as N-myc downstream-regulated gene-1 (Ndrg-1), has introduced a novel approach to treating cancer and preventing metastasis. Ndrg-1 has been identified as a protein involved in the differentiation of epithelial cells. In addition, Ndrg-1 expression can be regulated by androgens and is involved in the pathology of the disease, Hereditary Motor and Sensory Neuropathy-Lom (HMSNL). However, one of the most well documented links between Ndrg-1 and pathophysiology is its association with inhibition of tumor metastasis. The expression of Ndrg-1 was found to be significantly downregulated in a variety of different neoplasms including breast, colon and prostate cancer. Furthermore, Ndrg-1 expression was shown to be negatively correlated with tumor metastasis. Studies in vitro and in vivo have demonstrated a significant reduction in the metastatic ability of cells overexpressing Ndrg-1. The ability of these cells to invade was also compromised. The Gleason grade of prostate and breast cancers was found to correlate with Ndrg-1 expression, with more advanced and poorly differentiated tumors having lower Ndrg-1 levels. Recently, Ndrg-1 expression was demonstrated to be regulated by cellular iron levels and induced by iron chelators. These latter compounds were recently identified as potential anticancer agents as they selectively prevent cancer cell proliferation and lead to apoptosis. The discovery that iron chelators also increase Ndrg-1 expression further augments their antitumor activity and provides a novel strategy for the treatment of cancer and its metastasis.

The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, being a promising new target for cancer treatment. However, the precise molecular effects of NDRG1 remain unclear. Herein, we summarize recent advances in understanding the impact of NDRG1 on cancer metastasis with emphasis on its interactions with the key oncogenic nuclear factor-kappaB, phosphatidylinositol-3 kinase/phosphorylated AKT/mammalian target of rapamycin and Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathways. Recent studies demonstrating the inhibitory effects of NDRG1 on the epithelial-mesenchymal transition, a key initial step in metastasis, TGF-beta pathway and the Wnt/beta-catenin pathway are also described. Furthermore, NDRG1 was also demonstrated to regulate molecular motors in cancer cells, leading to inhibition of F-actin polymerization, stress fiber formation and subsequent reduction of cancer cell migration. Collectively, this review summarizes the underlying molecular mechanisms of the antimetastatic effects of NDRG1 in cancer cells.

Human N-myc downstream-regulated gene 1 (NDRG1) is a metastasis suppressor gene with several potential functions, including cell differentiation, cell cycle regulation and response to hormones, nickel and stress. The purpose of this study was to investigate the immunoexpression of NDRG1 in oral and oropharyngeal squamous cell carcinomas searching for its role in the clinical course of these tumors. We investigated immunohistochemical expression of NDRG1 protein in 412 tissue microarray cores of tumor samples from 103 patients with oral and oropharyngeal squamous cell carcinomas and in 110 paraffin-embedded surgical margin sections. The results showed NDRG1 up-regulation in 101/103 (98.1 %) tumor samples, but no expression in any normal tissue sample. Western blot assays confirmed the immunohistochemical findings, suggesting that lower levels of NDRG1 are associated with a high mortality rate. NDRG1 overexpression was related to long-term specific survival (HR = 0.38; p = 0.009), whereas the presence of lymph-node metastasis showed the opposite association with survival (HR = 2.45; p = 0.013). Our findings reinforce the idea that NDRG1 plays a metastasis suppressor role in oral and oropharyngeal squamous cell carcinomas and may be a useful marker for these tumors.

N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.

Cap43 has been identified as a nickel- and calcium-induced gene, and is also known as N-myc downstream-regulated gene 1 (NDRG1), Drg-1 and rit42. It is also reported that overexpression of Cap43 suppresses metastasis of some malignancies, but its precise role remains unclear. In this study, we asked how Cap43 could modulate the tumor growth of pancreatic cancer. Stable Cap43 cDNA transfectants of pancreatic cancer cells with Cap43 overexpression showed similar growth rates in culture as their control counterparts with low Cap43 protein level. By contrast, Cap43 overexpression showed a marked decrease in tumor growth rates in vivo. Moreover, a marked reduction in tumor-induced angiogenesis was observed. Gelatinolytic activity by matrix metalloproteinase-9 and invasive ability in Matrigel invasion activity were markedly decreased in pancreatic cancer cell lines with high Cap43 expression. Cellular expression of matrix metalloproteinase-9 and two major angiogenic factors, vascular endothelial growth factor and interleukin-8, were also significantly decreased in cell lines with Cap43 overexpression as compared with their parental counterparts. Immunohistochemical analysis of specimens from 65 patients with pancreatic ductal adenocarcinoma showed a significant association between Cap43 expression and tumor microvascular density (P = 0.0001) as well as depth of invasion (P = 0.0003), histopathologic grading (P = 0.0244), and overall survival rates for patients with pancreatic cancer (P = 0.0062). Thus, Cap43 could play a key role in the angiogenic on- or off-switch of tumor stroma in pancreatic ductal adenocarcinoma.

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 (Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase (Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.

AIMS: The metastasis suppressor gene, N-myc downstream regulated gene-1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, including pancreatic cancer. Moreover, NDRG1 is an iron-regulated gene that is markedly upregulated by cellular iron-depletion using novel antitumor agents such as the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), in pancreatic cancer cells. However, the exact function(s) of NDRG1 remain to be established and are important to elucidate. RESULTS: In the current study, using gene-array analysis along with NDRG1 overexpression and silencing, we identified the molecular targets of NDRG1 in three pancreatic cancer cell lines. We demonstrate that NDRG1 upregulates neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) and GLI-similar-3 (GLIS3). Further studies examining the downstream effects of NEDD4L led to the discovery that NDRG1 affects the transforming growth factor-beta (TGF-beta) pathway, leading to the upregulation of two key tumor suppressor proteins, namely phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and mothers against decapentaplegic homolog-4 (SMAD4). Moreover, NDRG1 inhibited the phosphatidylinositol 3-kinase (PI3K) and Ras oncogenic pathways. INNOVATION: This study provides significant insights into the mechanisms underlying the antitumor activity of NDRG1. For the first time, a role for NDRG1 is established in regulating the key signaling pathways involved in oncogenesis (TGF-beta, PI3K, and Ras pathways). CONCLUSION: The identified target genes of NDRG1 and their effect on the TGF-beta signaling pathway reveal its molecular function in pancreatic cancer and a novel therapeutic avenue.

NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFkappaB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade.