The Function of N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Negative Regulator in Tumor Cell Metastasis

N-myc downstream-regulated gene 2 (NDRG2) is a tumor-suppressor gene that suppresses tumorigenesis and metastasis of tumors and increases sensitivity to anti-cancer drugs. In this review, we summarize information on the clinicopathological characteristics of tumor patients according to NDRG2 expression in various tumor tissues and provide information on the metastasis inhibition-related cell signaling modulation by NDRG2. Loss of NDRG2 expression is a prognostic factor that correlates with TNM grade and tumor metastasis and has an inverse relationship with patient survival in various tumor patients. NDRG2 inhibits cell signaling, such as AKT-, NF-κB-, STAT3-, and TGF-β-mediated signaling, to induce tumor metastasis, and induces activation of GSK-3β which has anti-tumor effects. Although NDRG2 operates as an adaptor protein to mediate the interaction between kinases and phosphatases, which is essential in regulating cell signaling related to tumor metastasis, the molecular mechanism of NDRG2 as an adapter protein does not seem to be fully elucidated. This review aims to assist the research design regarding NDRG2 function as an adaptor protein and suggests NDRG2 as a molecular target to inhibit tumor metastasis and improve the prognosis in tumor patients.


Introduction
N-myc downstream-regulated gene 2 (NDRG2) belongs to the NDRG family along with NDRG1, -3, and -4, forms a homologous cluster in several species, and is characterized by an NDR protein domain consisting of an esterase-/lipase-/thioesterase-active-site serine and an α/β-hydrolase fold of approximately 220 amino acids. However, NDRG proteins have no enzymatic activity [1][2][3]. NDRG2 has been proposed as a functional gene in response to cellular stresses in cellular metabolic processes [4][5][6], hypoxia [7], and lipotoxicity [8]. Additionally, the functions of NDRG2 as a tumor suppressor contribute to tumor growth inhibition and anti-metastasis in various tumors. Tumor metastasis is known to be the main cause of death in cancer patients [9]. Metastasis is the process of moving from the primary tumor to other organs due to the limited supply of oxygen and nutrients and the epithelial-mesenchymal transition (EMT) step is a key step for metastasis [10][11][12][13]. In this review, we aim to provide useful information for tumor control studies by summarizing clinicopathological properties in various tumors and presenting the function of NDRG2 as a modulator of cell signaling for EMT.

Anti-Tumor Function of NDRG2 Based on Clinicopathological Characteristics of Various Tumor Patients
The expression of NDRG2 seems more reasonable to be suggested as a prognostic marker related to the reduction in pathological symptoms of the tumor rather than a diagnostic marker related to tumor development. NDRG2 expression is regulated at the transcriptional or the post-transcriptional step. Hypermethylation of the NDRG2 promoter region is a representative mechanism of NDRG2 transcriptional repression in various tumors [14][15][16][17][18][19]. Another mechanism regulating NDRG2 expression is mediated by microRNAs (miRNAs); miRNA-181c [20], miR-454 [21], miR-483 [22], miR-650 [23], and et al. NDRG2 expression level is positively correlated with tumor differentiation and overall survivals, but negatively correlated with tumor invasion, tumor recurrence, lymph node metastasis, and TNM staging in various tumor patients (Table 1). • NDRG2 expression is negatively correlated with favorable recurrence-free survival in breast cancer patients. [28] • Patients with high NDRG2 expression have higher disease-free survival and overall survival than patients with low NDRG expression, and NDRG2 expression was negatively correlated with GLUT1 expression. [4] Colorectal cancer • NDRG2 was downregulated in colorectal cancer compared to benign colorectal tissues. The NDRG2 expression is regulated by promoter methylation and miR-650. [23] • Methylation of NDRG2 promoter is methylated in 89% of human CRC tissues compared to the adjacent normal colonic mucosa. Higher levels of the promoter methylation are more prevalent in proximal CRC and advanced T stage. [29] • Low NDRG2 expression presents a significantly poorer 3-year overall survival rate in patients with stage IV, liver metastasis, and those receiving liver resection. [30] • NDRG2 expression levels were significantly decreased in both adenomas (p < 0.001) and colorectal cancer, and NDRG2 levels tend to decrease with increasing stages of Dukes. [31] Esophageal cancer • Low expression of NDRG2 in ESCC patients is inversely associated with clinical stage, TNM classification, histologic differentiation, and patient vital status. During the 5-year follow-up period, the survival period of ESCC patients with high NDRG2 expression was longer than that of patients with low expression. [32-34] Gallbladder cancer • Loss of NDRG2 expression is an independent predictor of decreased survival, is associated with severe progressive T stage, lymphatic invasion, and induces expression of MMP-19, which regulates Slug expression at the transcriptional level. [35] • Downregulation of NDRG2 and upregulation of CD24 show more frequent lymph node metastasis and lymphovascular invasion in gallbladder cancer patients. [36]

Gastric cancer
• NDRG2 expression is regulated by DNA methylation and the downregulation of NRG2 expression is related to lymph node metastasis, tumor invasion, Borrmann classification and TNM stage in gastric cancer patients. [37] • Loss of NDRG2 expression is an important and independent prognostic indicator by multivariate analysis, which affects survival rate in gastric cancer patients. [38] • NDRG2 promoter methylation is triggered by H. pylori infection and downregulation of NDRG2 expression is associated with worse prognosis, which is an independent prognostic factor for the disease-free survival of gastric tumor patients.
[15] Glioma • A Kaplan-Meier analysis of glioma patients showed improved survival time in the patients with mRNA and protein expression, and NDRG2 downregulation may affect glioma tumor progression to be higher malignancy. [19] • NDRG2 gene transcript is expressed in normal bran and low-grade glioma but is very low in glioblastoma, the most severe form of brain glioma. [39] • NDRG2 expression is inversely associated with lower survival rates and is an independent prognostic factor for overall survival in glioma patients. [40] Liver cancer • Reduction of NDRG2 is associated with its promoter hypermethylation and significantly correlated with tumor-node-metastasis stage, differentiation grade, portal vein thrombi, infiltrative growth pattern, nodal/distant metastasis, recurrent tumor, and shorter survival rates in liver cancer patients. [41] • Low expression of NDRG2 is strongly associated with tumor metastasis and high expression of NDRG2 is correlated with higher survival rates of hepatoblastoma patients. [17] • Downregulation of NDRG2 is strongly correlated with CD24 overexpression and is observed in hepatocarcinoma patients with elevated AFP serum level, late TNM stage, poor differentiation grade, tumor invasion, and recurrence. [42] • NDRG2 inhibits cholangiocarcinoma cell proliferation, chemoresistance, and metastasis, NDRG2 is a target of miR-181c which can be activated by leukemia inhibitory factor. [20] Lung cancer • Lung adenocarcinoma patients showing NDRG2-low/CD24-high expression have the lowest survival rate, and NDRG2-high/CD24-low and NDRG2-low/CD24-high expression patterns are independent prognostic indicators of lung adenocarcinoma. [43] Renal cancer • Loss of NDRG2 expression is an independent poor prognostic factor for renal cell carcinoma patients. [44] • NDRG2 expression is downregulated in clear cell renal cell carcinoma, which is negatively associated with aggressive tumor behaviors such as TNM stage, Fuhrman grade, tumor invasion, shorter patient survival, and tumor recurrence. In addition, NDRG2 can inhibit cancer cell invasion by regulating MMP-9 expression and activity. [45]

Epithelial-Mesenchymal Transition (EMT) and NDRG2
Cancers have the potential to metastasize and most cancer patients die from uncontrolled metastasis [46,47]. Therefore, establishing a strategy to control or inhibit tumor metastasis is very important for increasing the survival rate and treatment of tumor patients. Metastasis is a multi-step process initiated by the escape of tumor cells from primary tumor tissue and ending with the colonization of secondary tumors at distant tissues from the primary site. The steps of metastasis can be subdivided into the escape of cancer cells from the primary tumor, intravascular invasion, survival, extravasation, and colonization [48][49][50][51]. EMT is a key mechanism for promoting cancer metastasis, whereby cell types with a mesenchymal phenotype generally become invasive [52]. EMT is regulated by activation of the SNAI, ZEB, and TWIST transcription factor network (EMT-TF) families, which directly repress epithelial marker genes involved in cell adhesion, polarity, and cytoskeletal organization [53]. Various cellular signaling pathways, such as TGF-β signaling and AKT, NF-κB, and STAT3 activation, can induce the expression of EMT-TFs and metastatic effector proteins [54][55][56][57][58][59]. NDRG2 expression correlates inversely with TNM grade and metastasis in tumor patients (Table 1) and inhibits invasion and metastasis of tumor cells by modulating metastatic cell signaling pathways.

AKT and NDRG2
Bone is a common metastasis site in various cancer patients [60][61][62]. The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway has been implicated in bone metastasis in several tumors. pAKT levels are elevated in 81.8% of primary HER2-positive breast cancers with bone metastasis, suggesting that the HER2/CXCR4/AKT pathway plays a role in bone metastasis [57]. The function of the CXCL12/CXCR4 axis in promoting metastasis is mediated in part by AKT activation [63,64]. In addition, bone metastasis is more frequently induced by breast cancer cells with hypoxia-inducing factor 1α (HIF-1α), which responds to hypoxia and is stabilized by AKT, resulting in CXCR4 upregulation [65]. Further, overexpression of AKT1 in prostate cancers upregulates CXCR4 expression and loss of PTEN in the cells enhances AKT-mediated expression of CXCL12 and CXCR4 [66]. Low NDRG2 expression is associated with pAKT and XIAP upregulation, while overexpression of NDRG2 suppresses AKT/XIAP signaling pathway and EMT in esophageal cancer cells [34]. NDRG2 is considered a PTEN-binding protein recruiting protein phosphatase 2A (PP2A) to PTEN, which inhibits PI3K-AKT pathway via PTEN activation by PP2A in adult T-cell leukemia-lymphoma (ATLL) (Figure 1) [67]. Therefore, it is possible NDRG2 inhibits metastasis mediated by AKT activation pathway by acting as a bridge between PP2A and PTEN to inhibit PI3K activation. PTEN is a dual protein/lipid phosphatase using phosphatidylinositol, 3,4,5 triphosphate (PIP3), a product of PI3K, as a substrate. An increase in PIP3 recruits AKT to the membrane, which is activated by other PIP3-dependent kinases. NDRG2 acts as a bridge mediating the interaction between PP2A and PTEN, thus the NDRG2-PP2A complex recruits PTEN and triggers its activation. PTEN can inhibit AKT activity by inducing dephosphorylation of PIP3, thereby inhibiting AKT-dependent tumor cell metastasis.

Nuclear Factor-κB (NF-κB) and NDRG2
NF-κB is a key transcription factor contributing to pathogenesis of inflammation and cancer [68]. NF-κB forms a complex with IκB in the cytoplasm, and when IκBα is ubiquitinated and subsequently degraded by the 26S proteasome, NF-κB is released and migrates to the nucleus to act as a transcription factor. Specifically, NF-κB activation is a potential mechanism explaining the relationship between inflammation and tumors [69]. Metastatic breast cancer cells overexpress the chemokine receptor CXCR4, and metastatic tissues express a large amount of CXCL12, a ligand of CXCR4 [48]. Further, NF-κB activation induces expression of matrix metalloproteinases, urokinase-type plasminogen activators, and cytokines in metastatic breast cancer cell lines [58]. NF-κB p65 (RelA) is constitutively activated in gastric cancer, which correlated with cancer invasion-associated clinicopathological features, lymphoid invasion, depth of invasion, and peritoneal metastasis [70]. Activation of NF-κB induces osteolytic bone metastasis by inducing CSF2 (GM-CSF) expression [71]. Loss of NDRG2 expression is associated with tumor metastasis by inducing EMT via NF-κB activation [72]. NDRG2 attenuated NF-κB activation in PMA-treated human breast cancer cell (MDA-MB-231), which suppresses migration and invasion by inhibiting COX-2 expression [73]. Furthermore, NDRG2 suppresses tumor invasion by attenuating matrix metalloproteinase 9 (MMP9) expression via NF-κB inhibition [74].

Signal Transducer and Activator of Transcription 3 (STAT3) and NDRG2
Activation of STAT proteins is typically induced by Janus-Kinase (JAK) family proteins which are receptor-associated tyrosine kinases [81]. Activation of STAT3 occurs in a variety of tumors and aberrant STAT3 activation is associated with tumorigenesis and invasion promotion [82][83][84][85]. Metastatic spread of tumor cells requires cell motility, extracellular matrix infiltration, and angiogenesis. STAT3 activation contributes to enhancement of integrin 6 expression that promotes tumor cell motility, acquisition of invasive traits such as MUC1, Bcl6, cathepsins, and UPA, and increases expression of MMPs [86][87][88]. Furthermore, expression of E-cadherin, a representative anti-metastatic phenotype, is inhibited by STAT3 activation. STAT3 regulates ZEB1 expression, which can participate in STAT3-induced cell invasion and E-cadherin downregulation in colorectal cancer [89]. TWIST, an EMT-TF, is transcriptionally induced by STAT3 activation and plays a role as a transcriptional repressor of E-cadherin [59,90]. STAT3 also contributes to angiogenesis through the action of a transcription factor inducing vascular endothelial growth factor (VEGF) expression [91,92]. In unstimulated cells, STAT3 is regulated by negative regulators, protein inhibitors of activated STATs (PIAS), members of the cytokine signaling inhibitor (SOCS) family, protein tyrosine phosphatases (SHP1, SHP2, PTPN1, PTPN2 PTPRD, and PTPRT), and ubiquitin enzymes, to remain inactive in the cytoplasm [93,94]. SOCS expression can be induced by STAT3 activation, after which SOCS becomes a STAT3 regulator [95]. Overexpression of NDRG2 can inhibit STAT3 activation by inducing SOCS1 [96] and inhibition of STAT3 by NDRG2 suppresses EMT by reducing SNAIL expression (Figure 3) [97]. The transcription factor MYB activates STAT3 and AKT pathways by inhibiting NDRG2 expression via miR-130a expression in salivary adenoid cystic carcinoma [98]. Although NDRG2 regulates on STAT3 and SOCS1 induction in NDRG2-expressed cells, which is considered a mechanism for NDRG2-mediated STAT3 inhibition, the molecular mechanism is still unclear. Further analysis into the molecular mechanisms is needed, such as (1) how does NDRG2 induce SOCS1 expression, and (2) can NDRG2 play a role as a bridge to allow JAKs or STAT3 to interact with protein tyrosine phosphatases? Figure 2. Inhibition of NF-κB-dependent tumor cell metastasis by NDRG2. The PI3K/AKT/ IKKα/NF-κB pathway contributes to tumor cell metastasis by inducing related genes, such as CSF2, COX-2, and MMP9. IKKα is also a substrate of NIK. NDRG2 inhibits the canonical NF-κB pathway via AKT inhibition to inhibit IKKα activation. Activated NIK induces phosphorylation of IKKα, which phosphorylates p100 to process p100 to p52 in a proteasome-dependent manner. The NDRG2-PP2A complex dephosphorylates NIK to inhibit the non-canonical NF-κB pathway.

TGF-β and NDRG2
Although TGF-β plays a tumor-suppressor function, in the later stages of cancer progression, cancer cells remain responsive to TGF-β but become resistant to the cell proliferation inhibitory effects by TGF-β. Here, TGF-β acts directly on the cancer cells, leading to EMT through induction of E-cadherin transcriptional repressors, such as SNAIL, ZEB, and TWIST [99,100]. TGF-β binds to the receptor and induces formation of a complex with Smad2, Smad3, and Smad4, which regulates the expression of various target genes [101]. Further, TGF-β-induced EMT is closely related to proteolytic activity of MMPs [102]. Among the MMP family, MMP2 plays an important role in basement membrane remodeling by degrading collagen I, IV, V, plasminogen, and laminin-5 [103][104][105]. Further, NDRG2 can antagonize TGF-β-mediated liver cancer cell invasion by downregulating expression of MMP2 and laminin 332 pathway components [41]. Meanwhile, integrin complexes, integrin αν and integrin β6, induce conversion from latent TGF-β into its active form [106]. In a mouse breast cancer cell model, overexpression of NDRG2 disrupts TGF-β-mediated gene expression and inhibits conversion from latent TGF-β by reducing of integrin β6 expression, which inhibits tumor cell invasion (Figure 4) [28]. Despite growing cell biological and clinical evidence on the relationship between NDRG2 expression and inhibition of TGF-β-mediated tumor metastasis, its molecular mechanism is yet to be identified.

Conclusions and Outlook
Although expression of NDRG2 is not abolished in all carcinomas and tumor tissues, low expression of NDRG2 is closely related to poor prognosis, such as increased metastasis, higher TNM stage, and reduced survival. specifically, metastasis of tumor cells can worsen patient prognosis. Therefore, tumor metastasis inhibition may be a therapeutic strategy to improve prognosis and improve patient survival rate. Studies on NDRG2-mediated inhibition of tumor metastasis can provide useful scientific information on development of tumor therapeutics.
Although NDRG2 is a protein without enzymatic activity, it is involved in many cellular signaling pathways. We propose the role of NDRG2 bridging in mediating interactions between different proteins is crucial in modulating NDRG2-mediated cellular signaling. As an adapter protein, NDRG2 is already known to mediate AKT inhibition, NF-κB inhibition, and GSK3 activation by mediating PP2A/PTEN, PP2A/NIK and PP2A/GSK3 interactions, respectively. However, to clarify the molecular mechanism of NDRG2 related to the anti-tumor phenotype, further analysis of the interactions between kinase and NDRG2 located upstream of cell signaling, as well as interaction between NDRG2 and various phosphatases, such as serine/threonine phosphatases and tyrosine phosphatases, is needed. In particular, although a direct inhibitory mechanism for receptor tyrosine kinases (RTK) by NDRG2 has not been reported so far, we suggest research on the NDRG2-tyrosine phosphatase complex is necessary as a regulatory mechanism for RTK.