Studies of Potential Tumor Suppressor Genes

**3**

**Chapter 1**

**Abstract**

N-Myc Downstream-Regulated

Tumor Suppressor in Multiple

*Jian Zhang, Xia Li, Liangliang Shen, Yan Li and Libo Yao*

tumor suppressor and might be a therapeutic target for cancer treatment.

**Keywords:** NDRG2, tumor suppressor, stress sensor, p53, differentiation, EMT,

The human NDRG2 sequence was first described by Deng et al. as a protein containing an acyl-carrier protein (ACP)-like domain [1, 2]. The gene was cloned from differentially expressed genes between glioblastoma and normal brain tissues using PCR-based subtractive hybridization in 2003 [2]. NDRG2, NDRG1, NDRG3, and NDRG4 comprise the NDRG gene family and share approximately 59–68% homology. Additionally, NDRG family members display over 92% homology between humans and mice [1]. We identified NDRG2 as a novel tumor suppressor gene that plays a role in regulating the proliferation, differentiation, apoptosis and metastasis of multiple types of malignant tumors [1, 2]. Consistent with this finding, NDRG2 downregulation has been observed in multiple human cancer cell lines and tumors [3–5]. Additionally, other groups later confirmed our finding [6, 7]. NDRG2 was identified as a stress sensor for hypoxia, DNA damage stimuli and endoplasmic reticulum stress (ERS), and could inhibit the proliferation and promote the differentiation of colorectal

N-myc downstream-regulated gene 2 (NDRG2) was identified as a novel tumor suppressor gene in regulating the proliferation, differentiation, apoptosis and metastasis of multiple cancer types.  We identified NDRG2 as a stress sensor for hypoxia, DNA damage stimuli and endoplasmic reticulum stress (ERS). Our recent data showed that NDRG2 could promote the differentiation of colorectal cancer cells. Interestingly, we found that reduced NDRG2 expression was a powerful and independent predictor of poor prognosis of colorectal cancer patients. Furthermore, NDRG2 can inhibit epithelial-mesenchymal transition (EMT) by positively regulating E-cadherin expression. Moreover, NDRG2-deficient mice show spontaneous development of various tumor types, including T-cell lymphomas, providing in vivo evidence that NDRG2 functions as a tumor suppressor gene. We believe that NDRG2 is a novel

Gene 2 (NDRG2) as a Novel

Human Cancers

metastasis, cancer metabolism

**1. The finding of NDRG2**

#### **Chapter 1**

## N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple Human Cancers

*Jian Zhang, Xia Li, Liangliang Shen, Yan Li and Libo Yao*

#### **Abstract**

N-myc downstream-regulated gene 2 (NDRG2) was identified as a novel tumor suppressor gene in regulating the proliferation, differentiation, apoptosis and metastasis of multiple cancer types.  We identified NDRG2 as a stress sensor for hypoxia, DNA damage stimuli and endoplasmic reticulum stress (ERS). Our recent data showed that NDRG2 could promote the differentiation of colorectal cancer cells. Interestingly, we found that reduced NDRG2 expression was a powerful and independent predictor of poor prognosis of colorectal cancer patients. Furthermore, NDRG2 can inhibit epithelial-mesenchymal transition (EMT) by positively regulating E-cadherin expression. Moreover, NDRG2-deficient mice show spontaneous development of various tumor types, including T-cell lymphomas, providing in vivo evidence that NDRG2 functions as a tumor suppressor gene. We believe that NDRG2 is a novel tumor suppressor and might be a therapeutic target for cancer treatment.

**Keywords:** NDRG2, tumor suppressor, stress sensor, p53, differentiation, EMT, metastasis, cancer metabolism

#### **1. The finding of NDRG2**

The human NDRG2 sequence was first described by Deng et al. as a protein containing an acyl-carrier protein (ACP)-like domain [1, 2]. The gene was cloned from differentially expressed genes between glioblastoma and normal brain tissues using PCR-based subtractive hybridization in 2003 [2]. NDRG2, NDRG1, NDRG3, and NDRG4 comprise the NDRG gene family and share approximately 59–68% homology. Additionally, NDRG family members display over 92% homology between humans and mice [1].

We identified NDRG2 as a novel tumor suppressor gene that plays a role in regulating the proliferation, differentiation, apoptosis and metastasis of multiple types of malignant tumors [1, 2]. Consistent with this finding, NDRG2 downregulation has been observed in multiple human cancer cell lines and tumors [3–5]. Additionally, other groups later confirmed our finding [6, 7]. NDRG2 was identified as a stress sensor for hypoxia, DNA damage stimuli and endoplasmic reticulum stress (ERS), and could inhibit the proliferation and promote the differentiation of colorectal

carcinoma cells [8]. Moreover, NDRG2-deficient mice show spontaneous development of various tumor types, providing in vivo evidence that NDRG2 functions as a tumor suppressor gene. In this chapter, we will introduce the recent findings of NDRG2 as tumor suppressor in vitro and in vivo, and also the detailed mechanism.

#### **2. NDRG2 as a hypoxia and DNA damage responder**

Our group firstly identified NDRG2 as a protein containing an acyl-carrier protein (ACP)-like domain. The gene was cloned from differentially expressed genes between glioblastoma and normal brain tissues using PCR-based subtractive hybridization in 2003. NDRG2, NDRG1, NDRG3, and NDRG4 comprise the NDRG gene family [1] and share approximately 59–68% homology. Additionally, NDRG family members display over 92% homology between humans and mice.

The expression and cellular localization of NDRG2 were altered following exposure to different stresses, supporting the role of NDRG2 as a cellular stress sensor. Wang et al. found that NDRG2 expression was markedly upregulated in several cancer cell lines exposed to hypoxic conditions or similar stresses at both the mRNA and protein levels [9]. Hypoxia-inducible factor-1α (HIF-1α) can directly bind to hypoxia response elements (HREs) in the NDRG2 promoter, thus upregulating NDRG2 expression under hypoxia. Importantly, enforcing the expression of NDRG2 can strongly increase the apoptosis of cancer cells. Alternatively, NDRG2 can translocate from the cytoplasm to the nucleus under DNA damage stress. However, no explicit nuclear localization signal (NLS) sequence has been identified in the NDRG2 protein. Although NLSs are the most common type of nuclear import elements, other mechanisms may also be involved in NDRG2 translocation. For example, Liu et al. and Cao et al. confirmed that NDRG2 was upregulated by p53 or adriamycin (ADR) treatment [10, 11]. Thus, NDRG2 can translocate into the nucleus and increase p53-dependent cell apoptosis through the DNA damage repair mechanism. Furthermore, we found that NDRG2 expression was decreased in ADR-resistant breast cancer cells. However, NDRG2 rescue could promote ADR sensitivity through inhibiting proliferation and promoting cellular damage responses and apoptosis in a p53-dependent manner. Interestingly, we found that NDRG2 upregulated Bad expression by increasing its half-life, which is associated with p53 expression in mitochondria. Thus, NDRG2 promoted the therapeutic sensitivity of breast cancer cells in a p53-dependent manner by preventing p53 from entering the nucleus to participate in DNA damage repair rather than by changing its expression [12].

We first found that NDRG2 is positively regulated by p53. The first intron of the NDRG2 gene contains a site that binds p53 directly and mediates wild-type (WT) p53-dependent transactivation [11]. In addition, NDRG2 enhances p53-mediated apoptosis, whereas overexpression of NDRG2 suppresses tumor cell growth independently of p53 mutation. NDRG2 enhances p53-mediated apoptosis of hepatocarcinoma cells by downregulating ERCC6 (also named cockayne syndrome B—CSB) expression, which is critical for nucleotide excision repair capacity [10]. Thus, excision repair cross-complementing complementation group 6 (ERCC6) is an NDRG2 inducible target gene that is involved in the p53-mediated apoptosis pathway.

#### **3. NDRG2 functions as a novel ER stress-responsive protein and unfolded protein response (UPR) modulator**

The ER is an essential organelle involved in many cellular processes, including protein folding and maturation, lipid synthesis and calcium homeostasis. When

**5**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

cells are challenged by different environmental or intracellular insults, such as energy or nutrient deficiency, hypoxia, or oxidative stresses, ER's function is disrupted, causing accumulation of unfolded or misfolded proteins in the ER, a condition which is defined as ER stress [13, 14]. This triggers integrated signaling pathways to deal with the unfolded proteins, a phenomenon known as the UPR, which operates to restore ER homeostasis or, alternatively, lead to cell death under

and the UPR in different aspects of tumorigenesis and tumor progression.

NDRG2 is a stress-responsive gene [1], and our laboratory recently reported that as such, NDRG2 is implicated in ER stress [17] in addition to the hypoxia and DNA damage response. Different ER stress inducers, including thapsigargin (Tg), tunicamycin (Tm) and dithiothreitol (DTT), can induce NDRG2 mRNA and protein expression in human hepatoma SK-Hep-1 and HepG2 cells. In NDRG2 overexpressing hepatoma cell lines and Ndrg2 knockout (KO) mouse liver tissues, among the three UPR branches, NDRG2 interacts with PERK upon ER stress and facilitates PERK pathway activity, enhancing downstream ATF4 and CHOP activity. Thus, overexpression of NDRG2 promotes ERS-induced apoptosis, while silencing or knockdown of NDRG2 does the opposite, both in cell lines and in vivo. These data suggest that NDRG2 is a novel ER stress-responsive protein and an important component of the UPRosome, acting as a PERK cofactor to facilitate PERK branch signaling and thereby contributing to ER stress-induced apoptosis [17]. Therefore, apart from its already established role, NDRG2 could be considered a component of the UPRosome and a key player in cell fate decisions during ER stress. However, whether NDRG2 regulates PERK by affecting its dimer/oligomer status or its

post-translational modification or by competing with other regulators for binding is

NDRG2 expression is mainly detected in the muscle, brain, heart, liver, colon [18]. Interestingly, NDRG2 expression is nearly undetectable in the thymus, the bone marrow, the testis, and peripheral blood leukocytes, suggesting an inverse correlation between the NDRG2 gene expression level and cell proliferation status [18–20].  Collectively findings from these studies indicate that NDRG2 expression is decreased in most tumors. Moreover, NDRG2 expression was positively correlated with tumor differentiation but negatively correlated with lymph

We used a hospital-based study cohort of 226 colorectal cancer patients to analyze the correlation of *NDRG2* mRNA levels with the tumor clinicopathologic features, disease-free survival, and overall survival of colorectal cancer patients. *NDRG2* mRNA expression was significantly correlated with differentiation status, lymph node metastasis, and tumor-node-metastasis stage [23]. Patients with reduced NDRG2 mRNA levels had significantly worse progression-free survival (PFS) and

**4. NDRG2 as a novel prognostic biomarker in cancer**

The UPR contains three branches initiated by three ER-resident transmembrane sensors: protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) [15]. ER stress and the UPR are intensively involved in not only physiological conditions but also the pathogenesis of many diseases, including cancer [14, 16]. Accumulating implicates ER stress

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

prolonged or severe ER stress [13, 14].

worthy of further investigation.

node metastasis and TNM stage (**Figure 1**).

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

cells are challenged by different environmental or intracellular insults, such as energy or nutrient deficiency, hypoxia, or oxidative stresses, ER's function is disrupted, causing accumulation of unfolded or misfolded proteins in the ER, a condition which is defined as ER stress [13, 14]. This triggers integrated signaling pathways to deal with the unfolded proteins, a phenomenon known as the UPR, which operates to restore ER homeostasis or, alternatively, lead to cell death under prolonged or severe ER stress [13, 14].

The UPR contains three branches initiated by three ER-resident transmembrane sensors: protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) [15]. ER stress and the UPR are intensively involved in not only physiological conditions but also the pathogenesis of many diseases, including cancer [14, 16]. Accumulating implicates ER stress and the UPR in different aspects of tumorigenesis and tumor progression.

NDRG2 is a stress-responsive gene [1], and our laboratory recently reported that as such, NDRG2 is implicated in ER stress [17] in addition to the hypoxia and DNA damage response. Different ER stress inducers, including thapsigargin (Tg), tunicamycin (Tm) and dithiothreitol (DTT), can induce NDRG2 mRNA and protein expression in human hepatoma SK-Hep-1 and HepG2 cells. In NDRG2 overexpressing hepatoma cell lines and Ndrg2 knockout (KO) mouse liver tissues, among the three UPR branches, NDRG2 interacts with PERK upon ER stress and facilitates PERK pathway activity, enhancing downstream ATF4 and CHOP activity. Thus, overexpression of NDRG2 promotes ERS-induced apoptosis, while silencing or knockdown of NDRG2 does the opposite, both in cell lines and in vivo. These data suggest that NDRG2 is a novel ER stress-responsive protein and an important component of the UPRosome, acting as a PERK cofactor to facilitate PERK branch signaling and thereby contributing to ER stress-induced apoptosis [17]. Therefore, apart from its already established role, NDRG2 could be considered a component of the UPRosome and a key player in cell fate decisions during ER stress. However, whether NDRG2 regulates PERK by affecting its dimer/oligomer status or its post-translational modification or by competing with other regulators for binding is worthy of further investigation.

#### **4. NDRG2 as a novel prognostic biomarker in cancer**

NDRG2 expression is mainly detected in the muscle, brain, heart, liver, colon [18]. Interestingly, NDRG2 expression is nearly undetectable in the thymus, the bone marrow, the testis, and peripheral blood leukocytes, suggesting an inverse correlation between the NDRG2 gene expression level and cell proliferation status [18–20].  Collectively findings from these studies indicate that NDRG2 expression is decreased in most tumors. Moreover, NDRG2 expression was positively correlated with tumor differentiation but negatively correlated with lymph node metastasis and TNM stage (**Figure 1**).

We used a hospital-based study cohort of 226 colorectal cancer patients to analyze the correlation of *NDRG2* mRNA levels with the tumor clinicopathologic features, disease-free survival, and overall survival of colorectal cancer patients. *NDRG2* mRNA expression was significantly correlated with differentiation status, lymph node metastasis, and tumor-node-metastasis stage [23]. Patients with reduced NDRG2 mRNA levels had significantly worse progression-free survival (PFS) and

*Genes and Cancer*

carcinoma cells [8]. Moreover, NDRG2-deficient mice show spontaneous development of various tumor types, providing in vivo evidence that NDRG2 functions as a tumor suppressor gene. In this chapter, we will introduce the recent findings of NDRG2 as tumor suppressor in vitro and in vivo, and also the detailed mechanism.

Our group firstly identified NDRG2 as a protein containing an acyl-carrier protein (ACP)-like domain. The gene was cloned from differentially expressed genes between glioblastoma and normal brain tissues using PCR-based subtractive hybridization in 2003. NDRG2, NDRG1, NDRG3, and NDRG4 comprise the NDRG gene family [1] and share approximately 59–68% homology. Additionally, NDRG

The expression and cellular localization of NDRG2 were altered following exposure to different stresses, supporting the role of NDRG2 as a cellular stress sensor. Wang et al. found that NDRG2 expression was markedly upregulated in several cancer cell lines exposed to hypoxic conditions or similar stresses at both the mRNA and protein levels [9]. Hypoxia-inducible factor-1α (HIF-1α) can directly bind to hypoxia response elements (HREs) in the NDRG2 promoter, thus upregulating NDRG2 expression under hypoxia. Importantly, enforcing the expression of NDRG2 can strongly increase the apoptosis of cancer cells. Alternatively, NDRG2 can translocate from the cytoplasm to the nucleus under DNA damage stress. However, no explicit nuclear localization signal (NLS) sequence has been identified in the NDRG2 protein. Although NLSs are the most common type of nuclear import elements, other mechanisms may also be involved in NDRG2 translocation. For example, Liu et al. and Cao et al. confirmed that NDRG2 was upregulated by p53 or adriamycin (ADR) treatment [10, 11]. Thus, NDRG2 can translocate into the nucleus and increase p53-dependent cell apoptosis through the DNA damage repair mechanism. Furthermore, we found that NDRG2 expression was decreased in ADR-resistant breast cancer cells. However, NDRG2 rescue could promote ADR sensitivity through inhibiting proliferation and promoting cellular damage responses and apoptosis in a p53-dependent manner. Interestingly, we found that NDRG2 upregulated Bad expression by increasing its half-life, which is associated with p53 expression in mitochondria. Thus, NDRG2 promoted the therapeutic sensitivity of breast cancer cells in a p53-dependent manner by preventing p53 from entering the nucleus to participate in DNA damage repair rather than by changing its expression [12].

We first found that NDRG2 is positively regulated by p53. The first intron of the NDRG2 gene contains a site that binds p53 directly and mediates wild-type (WT) p53-dependent transactivation [11]. In addition, NDRG2 enhances p53-mediated apoptosis, whereas overexpression of NDRG2 suppresses tumor cell growth independently of p53 mutation. NDRG2 enhances p53-mediated apoptosis of hepatocarcinoma cells by downregulating ERCC6 (also named cockayne syndrome B—CSB) expression, which is critical for nucleotide excision repair capacity [10]. Thus, excision repair cross-complementing complementation group 6 (ERCC6) is an NDRG2 inducible target gene that is involved in the p53-mediated apoptosis pathway.

**3. NDRG2 functions as a novel ER stress-responsive protein and**

The ER is an essential organelle involved in many cellular processes, including protein folding and maturation, lipid synthesis and calcium homeostasis. When

**unfolded protein response (UPR) modulator**

family members display over 92% homology between humans and mice.

**2. NDRG2 as a hypoxia and DNA damage responder**

**4**

#### **Figure 1.**

*The molecular working model of NDRG2. NDRG2 can be transcriptionally upregulated by p53 and KLF4, and repressed by Myc. NDRG2 inhibited cancer cells proliferation through blocking PI3K/Akt signaling, promoted colorectal cancer cells differentiation through decreasing SKP2 and increasing p21/p27 expression, inhibited EMT through Snail abrogation, and sensitized cancer cells to chemotherapy with DNA damage repair inhibition.*

overall survival (OS) than patients with preserved expression of NDRG2 mRNA. We provided the first evidence that the NDRG2 mRNA level is a novel independent prognostic biomarker for both PFS and OS in colorectal cancer patients [23].

Another study analyzed NDRG2 expression in 127 bladder cancer patients and 97 healthy controls. Similar to the findings in colorectal cancer, NDRG2 expression was significantly downregulated at both the mRNA and protein levels in the urine of patients with bladder cancer and was independently correlated with tumor grade and stage [30]. Thus, NDRG2 expression was decreased in patients with bladder cancer and might be a potential independent diagnostic biomarker for bladder cancer.

#### **5. NDRG2 and differentiation**

Differentiation deficiency is a key characteristic of cancer. Poorly differentiated cancers show high proliferation and metastasis capacities, which seriously impact patient survival and prognosis [31]. As a member of the human NDRG gene family, the involvement of NDRG2 in the regulation of cell differentiation has been fully addressed. Bioinformatics analysis of NDRG2 revealed several binding sequences for different transcription factors, which are mostly involved in growth regulation and early differentiation.

As a master switch for cell proliferation and differentiation, Myc performs its biological functions mainly through transcriptional regulation of its target genes, which are involved in cell interaction and communication with their external environment [32, 33]. We first provided the evidence that NDRG2 is transcriptionally repressed by Myc [34]. In addition, c-Myc overexpression dramatically reduced NDRG2 protein and mRNA levels. The core promoter region of NDRG2 is required for Myc-mediated repression of NDRG2 transcription, and the interaction of Myc with the core promoter region was verified both in vitro and in vivo. A mechanistic study showed that Miz-1 is involved in Myc-mediated NDRG2 repression, and is possibly through the recruitment of other epigenetic factors, such as histone deacetylases, to the promoter.

**7**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

In colorectal cancer, the vast majority of poorly differentiated cells contain constitutive activation of WNT/β-catenin signal. WNT signaling-activating truncation mutations in adenomatous polyposis coli (APC) induce the nuclear translocation of β-catenin is induced, and consequently contributes to cell-fate determination *via* β-catenin/TCF complexes [35–38]. GSK-3β phosphorylates β-catenin at critical serine and threonine residues in its N terminus, which earmarks β-catenin for ubiquitination by the SCF complex and for subsequent degradation by the proteasome pathway [39, 40]. GSK-3β inactivation by APC mutation or oncogenic PI3K/ AKT activation leads to the β-catenin/TCF complex formation, and further induced TCF target gene expressions, such as Myc, cyclin D1 [41, 42]. NDRG2 suppresses β-catenin nuclear translocation and decreases the occupancy of β-catenin/TCF complex on the promoter of E3 ligase Skp2, potentially through dephosphorylating AKT and GSK-3β. NDRG2-mediated suppression of Skp2 contributes to the induction and stabilization of p21 and p27, which are target proteins for Skp2-mediated degradation. Thus, NDRG2-meidated induction of cell differentiation is dependent on suppressing the activity of the Skp2 E3 ligase. In support of the biological significance of the reciprocal relationship between NDRG2 and Skp2, an NDRG2low/ Skp2high gene expression signature correlates with poor patient outcome and could

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

be considered as a diagnostic marker for colorectal cancers.

**6. NDRG2 inhibits EMT and cancer metastasis**

high levels of WT1 are present [54].

and Notch pathway [49].

active autocrine TGF-β production [51].

Additionally, other groups have provided evidence of NDRG2 involvement of cell differentiation induced by different transcription factors, such as Wilms' tumor gene 1 (WT1) protein, HIF-1α and glucocorticoids [33, 54, 55]. Through an oligonucleotide array approach, WT1 was found to indirectly or directly induce the expression of NDRG2 mRNA in CD34+ cells and in leukemic U937 cells through an [54]. Moreover, a novel start site for NDRG2 expression appeared to be used in WT1-transduced cells, suggesting that this promoter is utilized preferentially when

Metastasis is a unique feature of tumor cells and an important factor affecting the survival and prognosis of cancer patients; it is also an important reason that surgery cannot completely remove tumor lesions. EMT is an important process preceding tumor metastasis [43, 44]. During EMT, tumor cells change from an epithelioid morphology to a mesenchymal cell morphology. The adhesion abilities between cells were decreased [45, 46]. Various signaling pathways were found involved in the regulation of EMT, such as, TGF-β pathway [47], Wnt/β-catenin pathway [48]

Data indicate that NDRG2 is negatively regulated by TGF-β during the progression of hepatocellular carcinomas [6]. This observation may be due to impairment in the TGF-β/Smad signaling pathway or the activation of non-Smad signaling cascades (PI3K/AKT, p38MAPK and so on) in these cell lines in response to TGF-β. Accordingly, related evidence has shown that the enhancement of GSK-3β activity by NDRG2 overexpression causes proteasomal degradation of the Snail transcription factor and subsequent transcriptional regulation of EMT-related genes [50]. Thus, the tumor suppressor NDRG2 could inhibit TGF-β-induced EMT as well as cell invasion and migration in various cancers. Similarly, a study showed the inhibitory effect of NDRG2 on TGF-β-induced tumor metastasis *via* the attenuation of

In breast cancer, NDRG2 downregulated the expression of Snail, as well as the phosphorylation of signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor activated in many human malignancies, including

#### *N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

In colorectal cancer, the vast majority of poorly differentiated cells contain constitutive activation of WNT/β-catenin signal. WNT signaling-activating truncation mutations in adenomatous polyposis coli (APC) induce the nuclear translocation of β-catenin is induced, and consequently contributes to cell-fate determination *via* β-catenin/TCF complexes [35–38]. GSK-3β phosphorylates β-catenin at critical serine and threonine residues in its N terminus, which earmarks β-catenin for ubiquitination by the SCF complex and for subsequent degradation by the proteasome pathway [39, 40]. GSK-3β inactivation by APC mutation or oncogenic PI3K/ AKT activation leads to the β-catenin/TCF complex formation, and further induced TCF target gene expressions, such as Myc, cyclin D1 [41, 42]. NDRG2 suppresses β-catenin nuclear translocation and decreases the occupancy of β-catenin/TCF complex on the promoter of E3 ligase Skp2, potentially through dephosphorylating AKT and GSK-3β. NDRG2-mediated suppression of Skp2 contributes to the induction and stabilization of p21 and p27, which are target proteins for Skp2-mediated degradation. Thus, NDRG2-meidated induction of cell differentiation is dependent on suppressing the activity of the Skp2 E3 ligase. In support of the biological significance of the reciprocal relationship between NDRG2 and Skp2, an NDRG2low/ Skp2high gene expression signature correlates with poor patient outcome and could be considered as a diagnostic marker for colorectal cancers.

Additionally, other groups have provided evidence of NDRG2 involvement of cell differentiation induced by different transcription factors, such as Wilms' tumor gene 1 (WT1) protein, HIF-1α and glucocorticoids [33, 54, 55]. Through an oligonucleotide array approach, WT1 was found to indirectly or directly induce the expression of NDRG2 mRNA in CD34+ cells and in leukemic U937 cells through an [54]. Moreover, a novel start site for NDRG2 expression appeared to be used in WT1-transduced cells, suggesting that this promoter is utilized preferentially when high levels of WT1 are present [54].

#### **6. NDRG2 inhibits EMT and cancer metastasis**

Metastasis is a unique feature of tumor cells and an important factor affecting the survival and prognosis of cancer patients; it is also an important reason that surgery cannot completely remove tumor lesions. EMT is an important process preceding tumor metastasis [43, 44]. During EMT, tumor cells change from an epithelioid morphology to a mesenchymal cell morphology. The adhesion abilities between cells were decreased [45, 46]. Various signaling pathways were found involved in the regulation of EMT, such as, TGF-β pathway [47], Wnt/β-catenin pathway [48] and Notch pathway [49].

Data indicate that NDRG2 is negatively regulated by TGF-β during the progression of hepatocellular carcinomas [6]. This observation may be due to impairment in the TGF-β/Smad signaling pathway or the activation of non-Smad signaling cascades (PI3K/AKT, p38MAPK and so on) in these cell lines in response to TGF-β. Accordingly, related evidence has shown that the enhancement of GSK-3β activity by NDRG2 overexpression causes proteasomal degradation of the Snail transcription factor and subsequent transcriptional regulation of EMT-related genes [50]. Thus, the tumor suppressor NDRG2 could inhibit TGF-β-induced EMT as well as cell invasion and migration in various cancers. Similarly, a study showed the inhibitory effect of NDRG2 on TGF-β-induced tumor metastasis *via* the attenuation of active autocrine TGF-β production [51].

In breast cancer, NDRG2 downregulated the expression of Snail, as well as the phosphorylation of signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor activated in many human malignancies, including

*Genes and Cancer*

**Figure 1.**

*inhibition.*

overall survival (OS) than patients with preserved expression of NDRG2 mRNA. We provided the first evidence that the NDRG2 mRNA level is a novel independent prognostic biomarker for both PFS and OS in colorectal cancer patients [23].

*The molecular working model of NDRG2. NDRG2 can be transcriptionally upregulated by p53 and KLF4, and repressed by Myc. NDRG2 inhibited cancer cells proliferation through blocking PI3K/Akt signaling, promoted colorectal cancer cells differentiation through decreasing SKP2 and increasing p21/p27 expression, inhibited EMT through Snail abrogation, and sensitized cancer cells to chemotherapy with DNA damage repair*

Another study analyzed NDRG2 expression in 127 bladder cancer patients and 97 healthy controls. Similar to the findings in colorectal cancer, NDRG2 expression was significantly downregulated at both the mRNA and protein levels in the urine of patients with bladder cancer and was independently correlated with tumor grade and stage [30]. Thus, NDRG2 expression was decreased in patients with bladder cancer and might be a potential independent diagnostic biomarker for bladder cancer.

Differentiation deficiency is a key characteristic of cancer. Poorly differentiated cancers show high proliferation and metastasis capacities, which seriously impact patient survival and prognosis [31]. As a member of the human NDRG gene family, the involvement of NDRG2 in the regulation of cell differentiation has been fully addressed. Bioinformatics analysis of NDRG2 revealed several binding sequences for different transcription factors, which are mostly involved in growth regulation

As a master switch for cell proliferation and differentiation, Myc performs its biological functions mainly through transcriptional regulation of its target genes, which are involved in cell interaction and communication with their external environment [32, 33]. We first provided the evidence that NDRG2 is transcriptionally repressed by Myc [34]. In addition, c-Myc overexpression dramatically reduced NDRG2 protein and mRNA levels. The core promoter region of NDRG2 is required for Myc-mediated repression of NDRG2 transcription, and the interaction of Myc with the core promoter region was verified both in vitro and in vivo. A mechanistic study showed that Miz-1 is involved in Myc-mediated NDRG2 repression, and is possibly through the recruitment of other epigenetic factors, such as histone deacetylases, to the promoter.

**6**

**5. NDRG2 and differentiation**

and early differentiation.

breast cancer [24]. Further, NDRG2 overexpressing breast cancer cells showed markedly decreased Snail expression after treatment with STAT3 inhibitors. Thus, the inhibition of STAT3 signaling by NDRG2 suppresses EMT progression *via* the down-regulation of Snail expression. Moreover, high NDRG2 expression induced inactivation of NF-κB and PI3K/AKT signaling pathways *via* the dephosphorylation of the C-terminal domain of PTEN, and the inhibition of the EMT process in OSCC [7]. Therefore, NDRG2 may regulate tumor EMT *via* different regulatory mechanisms in different cancers.

#### **7. NDRG2 is involved in cancer metabolism by regulating glycolysis, glutaminolysis and fatty acid oxidation (FAO)**

A cancerous cell undergoes multiple steps to form a solid tumor entity, during which nutrient and oxygen supply insufficiencies frequently occur. In recent decades, studies have provided deep insight into cancer metabolism. In addition to glycolysis, metabolic alterations involve almost all metabolic pathways, including those of lipids, amino acids, nitrogen, and nucleic acids. Metabolic reprogramming is widely accepted to be a hallmark of cancer [52]. Cancer metabolic reprogramming has been further summarized into six hallmarks, including alterations in nutrient uptake (deregulated uptake of glucose and amino acids and the use of opportunistic modes of nutrient acquisition) and intracellular metabolic pathways (the use of glycolysis/TCA cycle intermediates for biosynthesis and NADPH production and an increased demand for nitrogen) [53]. For instance, cancer cells use glucose and glutamine as the major sources of energy and precursor intermediates, thus exhibiting enhanced glycolysis and glutaminolysis [53]. Under various stress conditions, such as, glucose deficiency, cancer cells can shift from glycolysis to FAO to maintain ATP levels and satisfy nutrient demands [54]. Not surprisingly, oncogene activation and tumor suppressor inactivation are extensively involved in these processes. For example, c-Myc, HIF-1α, and p53 can regulate the uptake of both glucose and glutamine and glycolytic flux by affecting the expression of glucose transporters and metabolic enzymes [53].

As a tumor suppressor, NDRG2 was found to regulate aerobic glycolysis and glutaminolysis in cancer cells. A previous study from our laboratory showed that NDRG2 inhibits glucose uptake by interacting with and promoting the degradation of glucose transporter 1 (GLUT1) without affecting its transcription in breast cancer cell lines [55]. Recently, Xu et al. [56] from our laboratory, using colorectal cancer cells and a xenograft model, also demonstrated that NDRG2 inhibits glucose uptake and glycolysis by suppressing the expression and activity of the glucose transporter GLUT1 and key glycolytic enzymes, including hexokinase 2 (HK2), pyruvate kinase M2 isoform (PKM2) and lactate dehydrogenase A (LDHA). In addition, NDRG2 inhibits glutaminolysis by suppressing the expression of the glutamine transporter ASC amino acid transporter 2 (ASCT2) and glutaminase 1 (GLS1) at the transcriptional level. Mechanistically, NDRG2 exerts such effects by suppressing the expression of β-catenin, leading to the repression of its target gene c-Myc. Since c-Myc is a master regulator of metabolism, additional in-depth studies on NDRG2's regulatory role in other tumor glucose catabolism pathways are needed.

Under stress conditions such as glucose limitation, FAO is always activated to preserve the supply of ATP and NADPH [54]. Interestingly, our most recent study [4] revealed that NDRG2, as a negative regulator of AMPK, suppresses glucose deprivation-induced activation of the AMPK/ACC pathway and the consequent induction of FAO genes in hepatoma cells. Thus, NDRG2 overexpression leads to dysregulation of ATP and NADPH, thereby reducing the tolerance of hepatoma

**9**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

cells to glucose limitation. Together, these data further our understanding of the tumor-suppressive mechanism of NDRG2 through its involvement in cancer metabolic reprogramming. Therefore, the application of NDRG2 alone or in combination with antiglycolytic agents such as 2-diacylglycerol (2-DG) may effectively and synergistically inhibit cancer cells, which rely heavily on either glycolysis under

Most of the evidence for the role of NDRG2 as a tumor suppressor was mainly obtained in vitro, and establishing an in vivo mouse model to confirm these findings was crucial. It is reported that *Ndrg2*-deficient mice are susceptible to spontaneous tumor formation in vivo and *Ndrg2* knockout mice developed various types of tumors, including lymphomas, hepatocellular carcinomas and bronchoalveolar carcinomas [28]. However, we did not replicate these findings in our established Ndrg2 knockout mouse model—indeed, we did not detect any tumorigenesis in mice at 24 months of age. This discrepancy might be due to the different mouse

Notably, we established intestine-specific *Ndrg2* knockout mice using a Villin-Cre; *Ndrg2*flox/flox strategy [57]. Intestinal *Ndrg2* deficiency significantly augmented colitis initiation and colitis-associated tumor development. Ndrg2 loss led to the destruction of adherens junction structure *via* E-cadherin reduction, resulting in diminished epithelial barrier function and enhanced gut permeability. We identified the novel mechanism by which NDRG2 is crucial for the interaction of the E3 ligase FBXO11 with Snail, the repressor of E-cadherin. Thus, Ndrg2 loss increased Snail protein stability and decreased E-cadherin expression (https://www.biorxiv. org/content/10.1101/473397v1). Moreover, our study revealed that NDRG2 is an essential intestinal epithelial barrier regulator and plays important roles in gut

homeostasis maintenance and colitis-associated tumor development.

**9. NDRG2 in brain tumors and other nervous system diseases**

Accumulating studies have shown that NDRG2 is associated with various nervous system diseases, including tumors, ischemic stroke, hemorrhage, trauma, and neurodegenerative disorders [1, 59]. NDRG2 was repeatedly reported to be downregulated in a variety of cerebral tumors, including glioma and meningioma [21, 60–66]. The transcription levels of human *NDRG2* are significantly reduced in human glioblastoma tissues and human glioblastoma cell lines, and exogenous overexpression of NDRG2 repressed glioblastoma cell proliferation in vitro [2]. Although direct structural alterations such as point mutations are very rare in the

Recently, we established a liver cancer metastasis model in WT and Ndrg2 knockout (Ndrg2<sup>−</sup>/<sup>−</sup>) mice and found that expression loss of the tumor suppressor Ndrg2 in the liver microenvironment significantly suppressed the growth of liver cell colonies [57, 58]. Our data highlighted the role of NDRG2 in the regulation of tumor-associated macrophage (TAM) polarization and its function in promoting cancer liver metastasis. Interestingly, a reduced metastatic burden was correlated with an increased percentage of M1-like TAMs and decreased expression of M2-associated markers in the NDRG2-deficient microenvironment [58]. In summary, our study is the first showing a crucial and unexpected role for NDRG2 in macrophage polarization and highlights the importance of investigating the function of NDRG2 in cancer cells and the tumor microenvironment differently.

non-stressful conditions or FAO under conditions of metabolic stress.

**8. NDRG2 knockout enhances tumorigenesis in vivo**

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

strains and knockout strategies.

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

cells to glucose limitation. Together, these data further our understanding of the tumor-suppressive mechanism of NDRG2 through its involvement in cancer metabolic reprogramming. Therefore, the application of NDRG2 alone or in combination with antiglycolytic agents such as 2-diacylglycerol (2-DG) may effectively and synergistically inhibit cancer cells, which rely heavily on either glycolysis under non-stressful conditions or FAO under conditions of metabolic stress.

#### **8. NDRG2 knockout enhances tumorigenesis in vivo**

Most of the evidence for the role of NDRG2 as a tumor suppressor was mainly obtained in vitro, and establishing an in vivo mouse model to confirm these findings was crucial. It is reported that *Ndrg2*-deficient mice are susceptible to spontaneous tumor formation in vivo and *Ndrg2* knockout mice developed various types of tumors, including lymphomas, hepatocellular carcinomas and bronchoalveolar carcinomas [28]. However, we did not replicate these findings in our established Ndrg2 knockout mouse model—indeed, we did not detect any tumorigenesis in mice at 24 months of age. This discrepancy might be due to the different mouse strains and knockout strategies.

Notably, we established intestine-specific *Ndrg2* knockout mice using a Villin-Cre; *Ndrg2*flox/flox strategy [57]. Intestinal *Ndrg2* deficiency significantly augmented colitis initiation and colitis-associated tumor development. Ndrg2 loss led to the destruction of adherens junction structure *via* E-cadherin reduction, resulting in diminished epithelial barrier function and enhanced gut permeability. We identified the novel mechanism by which NDRG2 is crucial for the interaction of the E3 ligase FBXO11 with Snail, the repressor of E-cadherin. Thus, Ndrg2 loss increased Snail protein stability and decreased E-cadherin expression (https://www.biorxiv. org/content/10.1101/473397v1). Moreover, our study revealed that NDRG2 is an essential intestinal epithelial barrier regulator and plays important roles in gut homeostasis maintenance and colitis-associated tumor development.

Recently, we established a liver cancer metastasis model in WT and Ndrg2 knockout (Ndrg2<sup>−</sup>/<sup>−</sup>) mice and found that expression loss of the tumor suppressor Ndrg2 in the liver microenvironment significantly suppressed the growth of liver cell colonies [57, 58]. Our data highlighted the role of NDRG2 in the regulation of tumor-associated macrophage (TAM) polarization and its function in promoting cancer liver metastasis. Interestingly, a reduced metastatic burden was correlated with an increased percentage of M1-like TAMs and decreased expression of M2-associated markers in the NDRG2-deficient microenvironment [58]. In summary, our study is the first showing a crucial and unexpected role for NDRG2 in macrophage polarization and highlights the importance of investigating the function of NDRG2 in cancer cells and the tumor microenvironment differently.

#### **9. NDRG2 in brain tumors and other nervous system diseases**

Accumulating studies have shown that NDRG2 is associated with various nervous system diseases, including tumors, ischemic stroke, hemorrhage, trauma, and neurodegenerative disorders [1, 59]. NDRG2 was repeatedly reported to be downregulated in a variety of cerebral tumors, including glioma and meningioma [21, 60–66]. The transcription levels of human *NDRG2* are significantly reduced in human glioblastoma tissues and human glioblastoma cell lines, and exogenous overexpression of NDRG2 repressed glioblastoma cell proliferation in vitro [2]. Although direct structural alterations such as point mutations are very rare in the

*Genes and Cancer*

nisms in different cancers.

breast cancer [24]. Further, NDRG2 overexpressing breast cancer cells showed markedly decreased Snail expression after treatment with STAT3 inhibitors. Thus, the inhibition of STAT3 signaling by NDRG2 suppresses EMT progression *via* the down-regulation of Snail expression. Moreover, high NDRG2 expression induced inactivation of NF-κB and PI3K/AKT signaling pathways *via* the dephosphorylation of the C-terminal domain of PTEN, and the inhibition of the EMT process in OSCC [7]. Therefore, NDRG2 may regulate tumor EMT *via* different regulatory mecha-

**7. NDRG2 is involved in cancer metabolism by regulating glycolysis,**

A cancerous cell undergoes multiple steps to form a solid tumor entity, during which nutrient and oxygen supply insufficiencies frequently occur. In recent decades, studies have provided deep insight into cancer metabolism. In addition to glycolysis, metabolic alterations involve almost all metabolic pathways, including those of lipids, amino acids, nitrogen, and nucleic acids. Metabolic reprogramming is widely accepted to be a hallmark of cancer [52]. Cancer metabolic reprogramming has been further summarized into six hallmarks, including alterations in nutrient uptake (deregulated uptake of glucose and amino acids and the use of opportunistic modes of nutrient acquisition) and intracellular metabolic pathways (the use of glycolysis/TCA cycle intermediates for biosynthesis and NADPH production and an increased demand for nitrogen) [53]. For instance, cancer cells use glucose and glutamine as the major sources of energy and precursor intermediates, thus exhibiting enhanced glycolysis and glutaminolysis [53]. Under various stress conditions, such as, glucose deficiency, cancer cells can shift from glycolysis to FAO to maintain ATP levels and satisfy nutrient demands [54]. Not surprisingly, oncogene activation and tumor suppressor inactivation are extensively involved in these processes. For example, c-Myc, HIF-1α, and p53 can regulate the uptake of both glucose and glutamine and glycolytic flux by affecting the expression of glucose

As a tumor suppressor, NDRG2 was found to regulate aerobic glycolysis and glutaminolysis in cancer cells. A previous study from our laboratory showed that NDRG2 inhibits glucose uptake by interacting with and promoting the degradation of glucose transporter 1 (GLUT1) without affecting its transcription in breast cancer cell lines [55]. Recently, Xu et al. [56] from our laboratory, using colorectal cancer cells and a xenograft model, also demonstrated that NDRG2 inhibits glucose uptake and glycolysis by suppressing the expression and activity of the glucose transporter GLUT1 and key glycolytic enzymes, including hexokinase 2 (HK2), pyruvate kinase M2 isoform (PKM2) and lactate dehydrogenase A (LDHA). In addition, NDRG2 inhibits glutaminolysis by suppressing the expression of the glutamine transporter ASC amino acid transporter 2 (ASCT2) and glutaminase 1 (GLS1) at the transcriptional level. Mechanistically, NDRG2 exerts such effects by suppressing the expression of β-catenin, leading to the repression of its target gene c-Myc. Since c-Myc is a master regulator of metabolism, additional in-depth studies on NDRG2's regulatory role in other tumor glucose catabolism pathways are needed. Under stress conditions such as glucose limitation, FAO is always activated to preserve the supply of ATP and NADPH [54]. Interestingly, our most recent study [4] revealed that NDRG2, as a negative regulator of AMPK, suppresses glucose deprivation-induced activation of the AMPK/ACC pathway and the consequent induction of FAO genes in hepatoma cells. Thus, NDRG2 overexpression leads to dysregulation of ATP and NADPH, thereby reducing the tolerance of hepatoma

**glutaminolysis and fatty acid oxidation (FAO)**

transporters and metabolic enzymes [53].

**8**

*NDRG2* gene, hypermethylation of the *NDRG2* promoter region was shown to be highly correlated with decreased *NDRG2* transcription levels in human glioblastoma [60, 61, 67, 68]. In addition to the direct impact of *NDRG2* hypermethylation *per se*, NDRG2 may control glioma cell growth by upregulating the levels of histone acetylation in glioma cells [62]. Moreover, the expression level of *NDRG2* was negatively correlated with the pathological grade of the brain tumors and positively correlated with survival in astrocytoma patients [21, 63]. Consistent with the results in glioblastoma, a decrease in the levels of *NDRG2* gene methylation and NDRG2 protein expression were detected in human meningioma [64]. In addition, the expression levels of NDRG2 were significantly further reduced in recurrent meningioma compared to that in primary meningioma [65]. The above results suggest that NDRG2 may be a potential biomarker for predicting the prognosis of human brain tumors.

NDRG family members are abundantly expressed in brain tissue; therefore, the significant functions of these NDRG2 family members in the central nervous system were anticipated and have been confirmed with *NDRG* gene knockout micebased studies [69–71]. NDRG1 deficiency leads to a progressive demyelinating in the peripheral nerves, suggesting that *NDRG1* is involved in the maintenance of and axonal survival and myelin sheath structure [69]. *Ndrg2−/−* mice exhibited typical ADHD-like behaviors, including hyperactivity, impulsivity, and inattention, as well as impaired memory [70]. *Ndrg4−/−* mice showed impaired cognition and increased susceptibility to ischemic stroke, indicating that NDRG4 has a potential neuroprotective effect [71].

In addition, NDRG2 was implicated in the ischemic stress response in several in vivo and in vitro studies [72–78]. Temporal and spatial patterns of NDRG2 expression in the rat brain were investigated after transient middle cerebral artery occlusion and reperfusion. Both the mRNA and protein levels of NDRG2 were increased following reperfusion in the ischemic penumbra, and NDRG2 was translocated from the cytoplasm to the nucleus in astrocytes. Moreover, NDRG2 expression increased in parallel with the enhancement of TUNEL signals in this ischemic animal model [73]. It is consistent with the results of the animal experiments described above, the expression of NDRG2 was also revealed to be upregulated and NDRG2 can translocate from the cytoplasm to the nucleus in C6-originated astrocytes after oxygen-glucose deprivation (OGD) treatment mimicking ischemic model in vitro [72]. Furthermore, NDRG2 was implicated in some types of cerebral ischemic preconditioning-mediated neuroprotection, including electroacupuncture (EA) [75] and sevoflurane preconditioning [74]. EA preconditioning in the Baihui acupoint was performed before transient focal cerebral ischemia and reperfusion. After EA pretreatment, the number of apoptotic cells in the ischemic penumbra and the volume of cerebral infarct were significantly decreased, and the neurological outcomes were effectively rescued. After ischemia treatment, the levels of NDRG2 expression were largely suppressed in the EA pretreatment group compared with sham group. And NDRG2 was mostly localized in the astroglial cytoplasm; only weak staining was found in the astroglial nucleus after EA pretreatment. However, NDRG2 protein was remarkably transferred from the cytoplasm into the nucleus in the sham group [75]. Recently, NDRG2 was also found to exhibit neuroprotective effects with sevoflurane preconditioning in brain ischemia models both in vivo and in vitro [74]. These results together indicate that NDRG2 takes part in the pathological process of brain ischemia-reperfusion injury and that NDRG2 may be a potential intervention target for ischemic stroke.

NDRG2 has also been repeatedly reported to be associated with other nervous system diseases, such as, neurodegeneration [79–81] and depression [82–84]. NDRG2 has been identified as one of six aberrantly phosphorylated proteins in

**11**

**Author details**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

human brains with frontotemporal lobe degeneration, and an increased phosphospectra of NDRG2 was found in these neurodegenerative tissues [85]. Accumulated NDRG2 and GFAP were detected in cortical senile plaques from the postmortem human brain tissues with Alzheimer's disease (AD) [79]. In addition, the expression levels of NDRG2 and GFAP were parallelly increased in amyloid precursor protein (APP)/presenilin (PS1) mouse, a double transgenic AD mouse model [80]. Suppressed NDRG2 expression and decreased memory impairment were detected in parallel after EA treatment to APP/PS1 transgenic mice. Furthermore, the increased reactive astrocytes andNDRG2 expression were detected in the mice which were exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a Parkinson's disease-associated neurotoxin that causes both glial activation and neurodegeneration [86]. Moreover, growing studies have demonstrated that NDRG2 is related with the function of antidepressants, which can correct depression-like behaviors and alleviate neural damages observed in depressive animals [82–84]. NDRG2 was downregulated in the rat frontal cortex after chronic use of antidepressants [84]. In contrast to the results described above antidepressants did not counteract the increase in NDRG2 expression in the hippocampus of rats with stress-induced depression-like symptoms and that antidepressants *per se* induced NDRG2 expression in normal rats [83]. Further study of the detailed mechanisms by which NDRG2 participates in these neurodegenerative or chronic psychiatric diseases

providing novel intervention strategies will thus be interesting.

in vivo data are needed to confirm its tumor suppressor function.

Jian Zhang\*, Xia Li, Liangliang Shen, Yan Li and Libo Yao

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

The State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xian, China

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

To date, both in vitro and in vivo evidence has shown that NDRG2 can inhibit cancer cell proliferation, EMT, metastasis and can promote cell differentiation and cell cycle arrest. Thus, NDRG2 might be a target for cancer treatment and therapeutic resistance. Although NDRG2 is a novel tumor suppressor, the detailed mechanism by which NDRG2 functions requires further elucidation. Moreover, additional

**10. Conclusion and perspectives**

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

human brains with frontotemporal lobe degeneration, and an increased phosphospectra of NDRG2 was found in these neurodegenerative tissues [85]. Accumulated NDRG2 and GFAP were detected in cortical senile plaques from the postmortem human brain tissues with Alzheimer's disease (AD) [79]. In addition, the expression levels of NDRG2 and GFAP were parallelly increased in amyloid precursor protein (APP)/presenilin (PS1) mouse, a double transgenic AD mouse model [80]. Suppressed NDRG2 expression and decreased memory impairment were detected in parallel after EA treatment to APP/PS1 transgenic mice. Furthermore, the increased reactive astrocytes andNDRG2 expression were detected in the mice which were exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a Parkinson's disease-associated neurotoxin that causes both glial activation and neurodegeneration [86]. Moreover, growing studies have demonstrated that NDRG2 is related with the function of antidepressants, which can correct depression-like behaviors and alleviate neural damages observed in depressive animals [82–84]. NDRG2 was downregulated in the rat frontal cortex after chronic use of antidepressants [84]. In contrast to the results described above antidepressants did not counteract the increase in NDRG2 expression in the hippocampus of rats with stress-induced depression-like symptoms and that antidepressants *per se* induced NDRG2 expression in normal rats [83]. Further study of the detailed mechanisms by which NDRG2 participates in these neurodegenerative or chronic psychiatric diseases providing novel intervention strategies will thus be interesting.

#### **10. Conclusion and perspectives**

To date, both in vitro and in vivo evidence has shown that NDRG2 can inhibit cancer cell proliferation, EMT, metastasis and can promote cell differentiation and cell cycle arrest. Thus, NDRG2 might be a target for cancer treatment and therapeutic resistance. Although NDRG2 is a novel tumor suppressor, the detailed mechanism by which NDRG2 functions requires further elucidation. Moreover, additional in vivo data are needed to confirm its tumor suppressor function.

*NDRG2* gene, hypermethylation of the *NDRG2* promoter region was shown to be highly correlated with decreased *NDRG2* transcription levels in human glioblastoma [60, 61, 67, 68]. In addition to the direct impact of *NDRG2* hypermethylation *per se*, NDRG2 may control glioma cell growth by upregulating the levels of histone acetylation in glioma cells [62]. Moreover, the expression level of *NDRG2* was negatively correlated with the pathological grade of the brain tumors and positively correlated with survival in astrocytoma patients [21, 63]. Consistent with the results in glioblastoma, a decrease in the levels of *NDRG2* gene methylation and NDRG2 protein expression were detected in human meningioma [64]. In addition, the expression levels of NDRG2 were significantly further reduced in recurrent meningioma compared to that in primary meningioma [65]. The above results suggest that NDRG2 may be a potential biomarker for predicting the prognosis of

NDRG family members are abundantly expressed in brain tissue; therefore, the significant functions of these NDRG2 family members in the central nervous system were anticipated and have been confirmed with *NDRG* gene knockout micebased studies [69–71]. NDRG1 deficiency leads to a progressive demyelinating in the peripheral nerves, suggesting that *NDRG1* is involved in the maintenance of and axonal survival and myelin sheath structure [69]. *Ndrg2−/−* mice exhibited typical ADHD-like behaviors, including hyperactivity, impulsivity, and inattention, as well as impaired memory [70]. *Ndrg4−/−* mice showed impaired cognition and increased susceptibility to ischemic stroke, indicating that NDRG4 has a potential neuropro-

In addition, NDRG2 was implicated in the ischemic stress response in several in vivo and in vitro studies [72–78]. Temporal and spatial patterns of NDRG2 expression in the rat brain were investigated after transient middle cerebral artery occlusion and reperfusion. Both the mRNA and protein levels of NDRG2 were increased following reperfusion in the ischemic penumbra, and NDRG2 was translocated from the cytoplasm to the nucleus in astrocytes. Moreover, NDRG2 expression increased in parallel with the enhancement of TUNEL signals in this ischemic animal model [73]. It is consistent with the results of the animal experiments described above, the expression of NDRG2 was also revealed to be upregulated and NDRG2 can translocate from the cytoplasm to the nucleus in C6-originated astrocytes after oxygen-glucose deprivation (OGD) treatment mimicking ischemic model in vitro [72]. Furthermore, NDRG2 was implicated in some types of cerebral ischemic preconditioning-mediated neuroprotection, including electroacupuncture (EA) [75] and sevoflurane preconditioning [74]. EA preconditioning in the Baihui acupoint was performed before transient focal cerebral ischemia and reperfusion. After EA pretreatment, the number of apoptotic cells in the ischemic penumbra and the volume of cerebral infarct were significantly decreased, and the neurological outcomes were effectively rescued. After ischemia treatment, the levels of NDRG2 expression were largely suppressed in the EA pretreatment group compared with sham group. And NDRG2 was mostly localized in the astroglial cytoplasm; only weak staining was found in the astroglial nucleus after EA pretreatment. However, NDRG2 protein was remarkably transferred from the cytoplasm into the nucleus in the sham group [75]. Recently, NDRG2 was also found to exhibit neuroprotective effects with sevoflurane preconditioning in brain ischemia models both in vivo and in vitro [74]. These results together indicate that NDRG2 takes part in the pathological process of brain ischemia-reperfusion injury and that NDRG2 may be a

NDRG2 has also been repeatedly reported to be associated with other nervous system diseases, such as, neurodegeneration [79–81] and depression [82–84]. NDRG2 has been identified as one of six aberrantly phosphorylated proteins in

**10**

potential intervention target for ischemic stroke.

Jian Zhang\*, Xia Li, Liangliang Shen, Yan Li and Libo Yao The State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xian, China

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Yao L, Zhang J, Liu X. NDRG2: A myc-repressed gene involved in cancer and cell stress. Acta Biochimica et Biophysica Sinica. 2008;**40**:625-635

[2] Deng Y, Yao L, Chau L, Ng SS, Peng Y, Liu X, et al. N-Myc downstream-regulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation. International Journal of Cancer. 2003;**106**:342-347

[3] Liu N, Wang L, Liu X, Yang Q, Zhang J, Zhang W, et al. Promoter methylation, mutation, and genomic deletion are involved in the decreased NDRG2 expression levels in several cancer cell lines. Biochemical and Biophysical Research Communications. 2007;**358**:164-169

[4] Pan T, Zhang M, Zhang F, Yan G, Ru Y, Wang Q, et al. NDRG2 overexpression suppresses hepatoma cells survival during metabolic stress through disturbing the activation of fatty acid oxidation. Biochemical and Biophysical Research Communications. 2017;**483**:860-866

[5] Liu J, Yang L, Zhang J, Zhang J, Chen Y, Li K, et al. Knock-down of NDRG2 sensitizes cervical cancer Hela cells to cisplatin through suppressing Bcl-2 expression. BMC Cancer. 2012;**12**:370

[6] Lee DC, Kang YK, Kim WH, Jang YJ, Kim DJ, Park IY, et al. Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis. Cancer Research. 2008;**68**:4210-4220

[7] Tamura T, Ichikawa T, Nakahata S, Kondo Y, Tagawa Y, Yamamoto K, et al. Loss of NDRG2 expression confers oral squamous cell carcinoma with enhanced metastatic potential. Cancer Research. 2017;**77**: 2363-2374

[8] Shen L, Qu X, Li H, Xu C, Wei M, Wang Q, et al. NDRG2 facilitates colorectal cancer differentiation through the regulation of Skp2-p21/p27 axis. Oncogene. 2018;**37**:1759-1774

[9] Wang L, Liu N, Yao L, Li F, Zhang J, Deng Y, et al. NDRG2 is a new HIF-1 target gene necessary for hypoxiainduced apoptosis in A549 cells. Cellular Physiology and Biochemistry. 2008;**21**:239-250

[10] Cao W, Zhang JL, Feng DY, Liu XW, Li Y, Wang LF, et al. The effect of adenovirus-conjugated NDRG2 on p53-mediated apoptosis of hepatocarcinoma cells through attenuation of nucleotide excision repair capacity. Biomaterials. 2014;**35**:993-1003

[11] Liu N, Wang L, Li X, Yang Q, Liu X, Zhang J, et al. N-myc downstream-regulated gene 2 is involved in p53-mediated apoptosis. Nucleic Acids Research. 2008;**36**:5335-5349

[12] Wei Y, Yu S, Zhang Y, Zhang Y, Zhao H, Xiao Z, et al. NDRG2 promotes adriamycin sensitivity through a Bad/ p53 complex at the mitochondria in breast cancer. Oncotarget. 2017;**8**:29038-29047

[13] Hetz C. The unfolded protein response: Controlling cell fate decisions under ER stress and beyond. Nature Reviews. Molecular Cell Biology. 2012;**13**:89-102

[14] Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nature Reviews. Drug Discovery. 2013;**12**:703-719

[15] Cao SS, Kaufman RJ. Unfolded protein response. Current Biology. 2012;**22**:R622-R626

[16] Wang M, Kaufman RJ. The impact of the endoplasmic reticulum

**13**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

gene 2 (NDRG2) suppresses the epithelial-mesenchymal transition (EMT) in breast cancer cells *via* STAT3/Snail signaling. Cancer Letters.

[25] Tantai J, Pan X, Hu D. RNF4 mediated SUMOylation is essential for NDRG2 suppression of lung adenocarcinoma. Oncotarget.

[26] Mordalska A, Latek J, Ferenc T, Pomorski L, Galecka E, Zygmunt A, et al. Evaluation of NDRG2 gene expression in primary papillary thyroid carcinoma and in metastases of this neoplasm to regional lymph nodes.

Thyroid Research. 2010;**3**:6

[28] Nakahata S, Ichikawa T, Maneesaay P, Saito Y, Nagai K, Tamura T, et al. Loss of NDRG2 expression activates PI3K-AKT signalling *via* PTEN phosphorylation in ATLL and other cancers. Nature Communications. 2014;**5**:3393

[29] Tschan MP, Shan D, Laedrach J, Eyholzer M, Leibundgut EO, Baerlocher GM, et al. NDRG1/2 expression is inhibited in primary acute myeloid leukemia. Leukemia Research.

[30] Zhang M, Ren B, Li Z, Niu W, Wang Y. Expression of N-myc

[31] Derwinger K, Kodeda K, Bexe-Lindskog E, Taflin H. Tumour

Oncologica. 2010;**49**:57-62

differentiation grade is associated with TNM staging and the risk of node metastasis in colorectal cancer. Acta

downstream-regulated gene 2 in bladder cancer and its potential utility as a urinary diagnostic biomarker. Medical Science Monitor. 2017;**23**:4644-4649

2008;**8**:303

2010;**34**:393-398

[27] Zhao H, Zhang J, Lu J, He X, Chen C, Li X, et al. Reduced expression of N-Myc downstream-regulated gene 2 in human thyroid cancer. BMC Cancer.

2014;**354**:33-42

2016;**7**:26837-26843

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

protein-folding environment on cancer development. Nature Reviews. Cancer.

[17] Zhang M, Liu X, Wang Q, Ru Y, Xiong X, Wu K, et al. NDRG2 acts as a PERK co-factor to facilitate PERK branch and ERS-induced cell death. FEBS Letters. 2017;**591**:3670-3681

[18] Hu XL, Liu XP, Deng YC, Lin SX, Wu L, Zhang J, et al. Expression analysis of the NDRG2 gene in mouse embryonic and adult tissues. Cell and Tissue

Research. 2006;**325**:67-76

[19] Shen L, Zhao ZY, Wang YZ, Ji SP, Liu XP, Liu XW, et al.

[20] Hou WG, Zhao Y, Shen L,

2009;**337**:257-267

oncsis.2013.48

Immunohistochemical detection of Ndrg2 in the mouse nervous system. Neuroreport. 2008;**19**:927-931

Zhao J, Liu XW, Li Z, et al. Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal

development. Cell and Tissue Research.

[21] Li W, Chu D, Chu X, Meng F, Wei D, Li H, et al. Decreased expression of NDRG2 is related to poor overall survival in patients with glioma. Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of

Australasia. 2011;**18**:1534-1537

[23] Chu D, Zhang Z, Li Y, Wu L, Zhang J, Wang W, et al. Prediction of colorectal cancer relapse and prognosis by tissue mRNA levels of NDRG2. Molecular Cancer Therapeutics. 2011;**10**:47-56

[24] Kim MJ, Lim J, Yang Y, Lee MS, Lim JS. N-myc downstream-regulated

[22] Shen L, Qu X, Ma Y, Zheng J, Chu D, Liu B, et al. Tumor suppressor NDRG2 tips the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer. Oncogene. 2014;**3**:e86. DOI: 10.1038/

2014;**14**:581-597

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

protein-folding environment on cancer development. Nature Reviews. Cancer. 2014;**14**:581-597

[17] Zhang M, Liu X, Wang Q, Ru Y, Xiong X, Wu K, et al. NDRG2 acts as a PERK co-factor to facilitate PERK branch and ERS-induced cell death. FEBS Letters. 2017;**591**:3670-3681

[18] Hu XL, Liu XP, Deng YC, Lin SX, Wu L, Zhang J, et al. Expression analysis of the NDRG2 gene in mouse embryonic and adult tissues. Cell and Tissue Research. 2006;**325**:67-76

[19] Shen L, Zhao ZY, Wang YZ, Ji SP, Liu XP, Liu XW, et al. Immunohistochemical detection of Ndrg2 in the mouse nervous system. Neuroreport. 2008;**19**:927-931

[20] Hou WG, Zhao Y, Shen L, Zhao J, Liu XW, Li Z, et al. Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal development. Cell and Tissue Research. 2009;**337**:257-267

[21] Li W, Chu D, Chu X, Meng F, Wei D, Li H, et al. Decreased expression of NDRG2 is related to poor overall survival in patients with glioma. Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australasia. 2011;**18**:1534-1537

[22] Shen L, Qu X, Ma Y, Zheng J, Chu D, Liu B, et al. Tumor suppressor NDRG2 tips the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer. Oncogene. 2014;**3**:e86. DOI: 10.1038/ oncsis.2013.48

[23] Chu D, Zhang Z, Li Y, Wu L, Zhang J, Wang W, et al. Prediction of colorectal cancer relapse and prognosis by tissue mRNA levels of NDRG2. Molecular Cancer Therapeutics. 2011;**10**:47-56

[24] Kim MJ, Lim J, Yang Y, Lee MS, Lim JS. N-myc downstream-regulated gene 2 (NDRG2) suppresses the epithelial-mesenchymal transition (EMT) in breast cancer cells *via* STAT3/Snail signaling. Cancer Letters. 2014;**354**:33-42

[25] Tantai J, Pan X, Hu D. RNF4 mediated SUMOylation is essential for NDRG2 suppression of lung adenocarcinoma. Oncotarget. 2016;**7**:26837-26843

[26] Mordalska A, Latek J, Ferenc T, Pomorski L, Galecka E, Zygmunt A, et al. Evaluation of NDRG2 gene expression in primary papillary thyroid carcinoma and in metastases of this neoplasm to regional lymph nodes. Thyroid Research. 2010;**3**:6

[27] Zhao H, Zhang J, Lu J, He X, Chen C, Li X, et al. Reduced expression of N-Myc downstream-regulated gene 2 in human thyroid cancer. BMC Cancer. 2008;**8**:303

[28] Nakahata S, Ichikawa T, Maneesaay P, Saito Y, Nagai K, Tamura T, et al. Loss of NDRG2 expression activates PI3K-AKT signalling *via* PTEN phosphorylation in ATLL and other cancers. Nature Communications. 2014;**5**:3393

[29] Tschan MP, Shan D, Laedrach J, Eyholzer M, Leibundgut EO, Baerlocher GM, et al. NDRG1/2 expression is inhibited in primary acute myeloid leukemia. Leukemia Research. 2010;**34**:393-398

[30] Zhang M, Ren B, Li Z, Niu W, Wang Y. Expression of N-myc downstream-regulated gene 2 in bladder cancer and its potential utility as a urinary diagnostic biomarker. Medical Science Monitor. 2017;**23**:4644-4649

[31] Derwinger K, Kodeda K, Bexe-Lindskog E, Taflin H. Tumour differentiation grade is associated with TNM staging and the risk of node metastasis in colorectal cancer. Acta Oncologica. 2010;**49**:57-62

**12**

2363-2374

*Genes and Cancer*

**References**

[1] Yao L, Zhang J, Liu X. NDRG2: A myc-repressed gene involved in cancer and cell stress. Acta Biochimica et Biophysica Sinica. 2008;**40**:625-635

[8] Shen L, Qu X, Li H, Xu C, Wei M, Wang Q, et al. NDRG2 facilitates colorectal cancer differentiation through the regulation of Skp2-p21/p27 axis. Oncogene. 2018;**37**:1759-1774

[9] Wang L, Liu N, Yao L, Li F, Zhang J, Deng Y, et al. NDRG2 is a new HIF-1 target gene necessary for hypoxiainduced apoptosis in A549 cells. Cellular Physiology and Biochemistry.

[10] Cao W, Zhang JL, Feng DY, Liu XW, Li Y, Wang LF, et al. The effect of adenovirus-conjugated NDRG2 on p53-mediated apoptosis of hepatocarcinoma cells through attenuation of nucleotide excision repair capacity. Biomaterials. 2014;**35**:993-1003

[11] Liu N, Wang L, Li X, Yang Q, Liu X, Zhang J, et al. N-myc

[12] Wei Y, Yu S, Zhang Y, Zhang Y, Zhao H, Xiao Z, et al. NDRG2 promotes adriamycin sensitivity through a Bad/ p53 complex at the mitochondria in breast cancer. Oncotarget.

[13] Hetz C. The unfolded protein response: Controlling cell fate decisions under ER stress and beyond. Nature Reviews. Molecular Cell Biology.

[14] Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nature Reviews. Drug Discovery. 2013;**12**:703-719

[15] Cao SS, Kaufman RJ. Unfolded protein response. Current Biology.

[16] Wang M, Kaufman RJ. The impact of the endoplasmic reticulum

Research. 2008;**36**:5335-5349

2017;**8**:29038-29047

2012;**13**:89-102

2012;**22**:R622-R626

downstream-regulated gene 2 is involved in p53-mediated apoptosis. Nucleic Acids

2008;**21**:239-250

[2] Deng Y, Yao L, Chau L, Ng SS, Peng Y, Liu X, et al. N-Myc downstream-regulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation. International Journal of

[3] Liu N, Wang L, Liu X, Yang Q, Zhang J, Zhang W, et al. Promoter methylation, mutation, and genomic deletion are involved in the decreased NDRG2 expression levels in several cancer cell lines. Biochemical and Biophysical Research Communications.

Cancer. 2003;**106**:342-347

2007;**358**:164-169

2017;**483**:860-866

2012;**12**:370

[4] Pan T, Zhang M, Zhang F, Yan G, Ru Y, Wang Q, et al. NDRG2 overexpression suppresses hepatoma cells survival during metabolic stress through disturbing the activation of fatty acid oxidation. Biochemical and Biophysical Research Communications.

[5] Liu J, Yang L, Zhang J, Zhang J, Chen Y, Li K, et al. Knock-down of NDRG2 sensitizes cervical cancer Hela cells to cisplatin through suppressing Bcl-2 expression. BMC Cancer.

[6] Lee DC, Kang YK, Kim WH, Jang YJ, Kim DJ, Park IY, et al. Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis. Cancer Research. 2008;**68**:4210-4220

[7] Tamura T, Ichikawa T, Nakahata S, Kondo Y, Tagawa Y, Yamamoto K, et al. Loss of NDRG2 expression confers oral squamous cell carcinoma with enhanced metastatic potential. Cancer Research. 2017;**77**:

[32] Vervoorts J, Luscher-Firzlaff J, Luscher B. The ins and outs of MYC regulation by posttranslational mechanisms. The Journal of Biological Chemistry. 2006;**281**:34725-34729

[33] Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annual Review of Cell and Developmental Biology. 2000;**16**:653-699

[34] Zhang J, Li F, Liu X, Shen L, Liu J, Su J, et al. The repression of human differentiation-related gene NDRG2 expression by Myc *via* Miz-1-dependent interaction with the NDRG2 core promoter. The Journal of Biological Chemistry. 2006;**281**:39159-39168

[35] Goss KH, Groden J. Biology of the adenomatous polyposis coli tumor suppressor. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 2000;**18**:1967-1979

[36] Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;**275**:1787-1790

[37] Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell. 2000;**103**:311-320

[38] Ng RC, Matsumaru D, Ho AS, Garcia-Barcelo MM, Yuan ZW, Smith D, et al. Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-betacatenin signaling and cell death of the cloaca endoderm, and anorectal malformations. Cell Death and Differentiation. 2014;**21**:978-989

[39] Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. Beta-catenin is a target for the ubiquitin-proteasome pathway. The EMBO Journal. 1997;**16**:3797-3804

[40] Marikawa Y, Elinson RP. Beta-TrCP is a negative regulator of Wnt/ beta-catenin signaling pathway and dorsal axis formation in *Xenopus* embryos. Mechanisms of Development. 1998;**77**:75-80

[41] He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, et al. Identification of c-MYC as a target of the APC pathway. Science. 1998;**281**:1509-1512

[42] Tetsu O, McCormick F. Betacatenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999;**398**:422-426

[43] Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED, et al. Epithelialmesenchymal and mesenchymalepithelial transitions in carcinoma progression. Journal of Cellular Physiology. 2007;**213**:374-383

[44] Foroni C, Broggini M, Generali D, Damia G. Epithelialmesenchymal transition and breast cancer: Role, molecular mechanisms and clinical impact. Cancer Treatment Reviews. 2012;**38**:689-697

[45] Boyer B, Valles AM, Edme N. Induction and regulation of epithelial-mesenchymal transitions. Biochemical Pharmacology. 2000;**60**:1091-1099

[46] Thiery JP. Epithelialmesenchymal transitions in tumour progression. Nature Reviews. Cancer. 2002;**2**:442-454

[47] Saitoh M, Miyazawa K. Transcriptional and posttranscriptional regulation in TGFbeta-mediated epithelial-mesenchymal transition. Journal of Biochemistry. 2012;**151**:563-571

**15**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

in colorectal cancer cells by repressing

[57] von Karstedt S. NDRG2 programs tumor-associated macrophages for tumor support. Cell Death & Disease.

[58] Li M, Lai X, Zhao Y, Zhang Y, Li M, Li D, et al. Loss of NDRG2 in liver microenvironment inhibits cancer liver metastasis by regulating tumor associate macrophages polarization. Cell Death &

[59] Lin K, Yin A, Yao L, Li Y. N-myc downstream-regulated gene 2 in the nervous system: From expression pattern to function. Acta Biochimica et Biophysica Sinica. 2015;**47**:761-766

[60] Zhou B, Tang Z, Deng Y, Hou S, Liu N, Lin W, et al. Tumor suppressor candidate gene, NDRG2 is frequently inactivated in human glioblastoma multiforme. Molecular Medicine

Reports. 2014;**10**:891-896

et al. Frequent promoter

2008;**123**:2080-2086

2012;**106**:485-492

[61] Tepel M, Roerig P, Wolter M, Gutmann DH, Perry A, Reifenberger G,

[62] Li L, Qin X, Shi M, Miao R, Wang L, Liu X, et al. Regulation of histone acetylation by NDRG2 in glioma cells. Journal of Neuro-Oncology.

[63] Li L, Wang J, Shen X, Wang L, Li X, Liu Y, et al. Expression and prognostic value of NDRG2 in human astrocytomas. Journal of the Neurological Sciences. 2011;**308**:77-82

[64] Lusis EA, Watson MA, Chicoine MR, Lyman M, Roerig P, Reifenberger G, et al. Integrative genomic analysis

hypermethylation and transcriptional downregulation of the NDRG2 gene at 14q11.2 in primary glioblastoma. International Journal of Cancer.

c-Myc expression. Oncotarget.

2015;**6**:26161-26176

Disease. 2018;**9**:248

2018;**9**:294

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

[48] Gilles C, Polette M, Mestdagt M,

Birembaut P, et al. Transactivation of vimentin by beta-catenin in human breast cancer cells. Cancer Research.

[49] Wang Z, Li Y, Kong D, Sarkar FH. The role of Notch signaling pathway in epithelial-mesenchymal transition (EMT) during development and tumor aggressiveness. Current Drug Targets.

[50] Kim YJ, Kang HB, Yim HS, Kim JH, Kim JW. NDRG2 positively regulates E-cadherin expression and prolongs overall survival in colon cancer patients. Oncology Reports. 2013;**30**:1890-1898

[51] Oh SS, Kim D, Kim DH, Chang HH, Sohn KC, Kim KH, et al. NDRG2 correlated with favorable recurrencefree survival inhibits metastasis of mouse breast cancer cells *via* attenuation of active TGF-beta production. Carcinogenesis.

Nawrocki-Raby B, Ruggeri P,

2003;**63**:2658-2664

2010;**11**:745-751

2012;**33**:1882-1888

2016;**23**:27-47

2015;**401**:213-220

[52] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;**144**:646-674

[54] Carracedo A, Cantley LC,

[53] Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism. Cell Metabolism.

Pandolfi PP. Cancer metabolism: Fatty acid oxidation in the limelight. Nature Reviews. Cancer. 2013;**13**:227-232

[55] Vriend J, Reiter RJ. The Keap1- Nrf2-antioxidant response element pathway: A review of its regulation by melatonin and the proteasome. Molecular and Cellular Endocrinology.

[56] Xu X, Li J, Sun X, Guo Y, Chu D, Wei L, et al. Tumor suppressor NDRG2 inhibits glycolysis and glutaminolysis

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

[48] Gilles C, Polette M, Mestdagt M, Nawrocki-Raby B, Ruggeri P, Birembaut P, et al. Transactivation of vimentin by beta-catenin in human breast cancer cells. Cancer Research. 2003;**63**:2658-2664

*Genes and Cancer*

2000;**16**:653-699

2006;**281**:39159-39168

2000;**18**:1967-1979

1997;**275**:1787-1790

2000;**103**:311-320

[35] Goss KH, Groden J. Biology of the adenomatous polyposis coli tumor suppressor. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology.

[36] Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science.

[37] Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell.

[38] Ng RC, Matsumaru D, Ho AS, Garcia-Barcelo MM, Yuan ZW, Smith D, et al. Dysregulation of Wnt inhibitory factor 1 (Wif1) expression resulted in aberrant Wnt-betacatenin signaling and cell death of the cloaca endoderm, and anorectal malformations. Cell Death and Differentiation. 2014;**21**:978-989

[39] Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. Beta-catenin is a target for the ubiquitin-proteasome

[32] Vervoorts J, Luscher-Firzlaff J, Luscher B. The ins and outs of MYC regulation by posttranslational

mechanisms. The Journal of Biological Chemistry. 2006;**281**:34725-34729

pathway. The EMBO Journal.

[40] Marikawa Y, Elinson RP. Beta-TrCP is a negative regulator of Wnt/ beta-catenin signaling pathway and dorsal axis formation in *Xenopus*

[41] He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, et al. Identification of c-MYC as a target of the APC pathway. Science.

[42] Tetsu O, McCormick F. Betacatenin regulates expression of cyclin D1 in colon carcinoma cells. Nature.

[43] Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams ED, et al. Epithelialmesenchymal and mesenchymalepithelial transitions in carcinoma progression. Journal of Cellular Physiology. 2007;**213**:374-383

[44] Foroni C, Broggini M, Generali D, Damia G. Epithelialmesenchymal transition and breast cancer: Role, molecular mechanisms and clinical impact. Cancer Treatment

Reviews. 2012;**38**:689-697

[45] Boyer B, Valles AM, Edme N. Induction and regulation of

Biochemical Pharmacology.

2000;**60**:1091-1099

2002;**2**:442-454

2012;**151**:563-571

[46] Thiery JP. Epithelial-

[47] Saitoh M, Miyazawa K. Transcriptional and post-

transcriptional regulation in TGFbeta-mediated epithelial-mesenchymal transition. Journal of Biochemistry.

epithelial-mesenchymal transitions.

mesenchymal transitions in tumour progression. Nature Reviews. Cancer.

embryos. Mechanisms of Development.

1997;**16**:3797-3804

1998;**77**:75-80

1998;**281**:1509-1512

1999;**398**:422-426

[33] Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annual Review of Cell and Developmental Biology.

[34] Zhang J, Li F, Liu X, Shen L, Liu J, Su J, et al. The repression of human differentiation-related gene NDRG2 expression by Myc *via* Miz-1-dependent interaction with the NDRG2 core promoter. The Journal of Biological Chemistry.

**14**

[49] Wang Z, Li Y, Kong D, Sarkar FH. The role of Notch signaling pathway in epithelial-mesenchymal transition (EMT) during development and tumor aggressiveness. Current Drug Targets. 2010;**11**:745-751

[50] Kim YJ, Kang HB, Yim HS, Kim JH, Kim JW. NDRG2 positively regulates E-cadherin expression and prolongs overall survival in colon cancer patients. Oncology Reports. 2013;**30**:1890-1898

[51] Oh SS, Kim D, Kim DH, Chang HH, Sohn KC, Kim KH, et al. NDRG2 correlated with favorable recurrencefree survival inhibits metastasis of mouse breast cancer cells *via* attenuation of active TGF-beta production. Carcinogenesis. 2012;**33**:1882-1888

[52] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;**144**:646-674

[53] Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism. Cell Metabolism. 2016;**23**:27-47

[54] Carracedo A, Cantley LC, Pandolfi PP. Cancer metabolism: Fatty acid oxidation in the limelight. Nature Reviews. Cancer. 2013;**13**:227-232

[55] Vriend J, Reiter RJ. The Keap1- Nrf2-antioxidant response element pathway: A review of its regulation by melatonin and the proteasome. Molecular and Cellular Endocrinology. 2015;**401**:213-220

[56] Xu X, Li J, Sun X, Guo Y, Chu D, Wei L, et al. Tumor suppressor NDRG2 inhibits glycolysis and glutaminolysis

in colorectal cancer cells by repressing c-Myc expression. Oncotarget. 2015;**6**:26161-26176

[57] von Karstedt S. NDRG2 programs tumor-associated macrophages for tumor support. Cell Death & Disease. 2018;**9**:294

[58] Li M, Lai X, Zhao Y, Zhang Y, Li M, Li D, et al. Loss of NDRG2 in liver microenvironment inhibits cancer liver metastasis by regulating tumor associate macrophages polarization. Cell Death & Disease. 2018;**9**:248

[59] Lin K, Yin A, Yao L, Li Y. N-myc downstream-regulated gene 2 in the nervous system: From expression pattern to function. Acta Biochimica et Biophysica Sinica. 2015;**47**:761-766

[60] Zhou B, Tang Z, Deng Y, Hou S, Liu N, Lin W, et al. Tumor suppressor candidate gene, NDRG2 is frequently inactivated in human glioblastoma multiforme. Molecular Medicine Reports. 2014;**10**:891-896

[61] Tepel M, Roerig P, Wolter M, Gutmann DH, Perry A, Reifenberger G, et al. Frequent promoter hypermethylation and transcriptional downregulation of the NDRG2 gene at 14q11.2 in primary glioblastoma. International Journal of Cancer. 2008;**123**:2080-2086

[62] Li L, Qin X, Shi M, Miao R, Wang L, Liu X, et al. Regulation of histone acetylation by NDRG2 in glioma cells. Journal of Neuro-Oncology. 2012;**106**:485-492

[63] Li L, Wang J, Shen X, Wang L, Li X, Liu Y, et al. Expression and prognostic value of NDRG2 in human astrocytomas. Journal of the Neurological Sciences. 2011;**308**:77-82

[64] Lusis EA, Watson MA, Chicoine MR, Lyman M, Roerig P, Reifenberger G, et al. Integrative genomic analysis

identifies NDRG2 as a candidate tumor suppressor gene frequently inactivated in clinically aggressive meningioma. Cancer Research. 2005;**65**:7121-7126

[65] Skiriute D, Tamasauskas S, Asmoniene V, Saferis V, Skauminas K, Deltuva V, et al. Tumor grade-related NDRG2 gene expression in primary and recurrent intracranial meningiomas. Journal of Neuro-Oncology. 2011;**102**:89-94

[66] Zhang ZG, Li G, Feng DY, Zhang J, Zhang J, Qin HZ, et al. Overexpression of NDRG2 can inhibit neuroblastoma cell proliferation through negative regulation by CYR61. Asian Pacific Journal of Cancer Prevention: APJCP. 2014;**15**:239-244

[67] Skiriute D, Vaitkiene P, Asmoniene V, Steponaitis G, Deltuva VP, Tamasauskas A. Promoter methylation of AREG, HOXA11, hMLH1, NDRG2, NPTX2 and Tes genes in glioblastoma. Journal of Neuro-Oncology. 2013;**113**:441-449

[68] Skiriute D, Steponaitis G, Vaitkiene P, Mikuciunas M, Skauminas K, Tamasauskas A, et al. Glioma malignancy-dependent NDRG2 gene methylation and downregulation correlates with poor patient outcome. Journal of Cancer. 2014;**5**:446-456

[69] Okuda T, Higashi Y, Kokame K, Tanaka C, Kondoh H, Miyata T. Ndrg1 deficient mice exhibit a progressive demyelinating disorder of peripheral nerves. Molecular and Cellular Biology. 2004;**24**:3949-3956

[70] Li Y, Yin A, Sun X, Zhang M, Zhang J, Wang P, et al. Deficiency of tumor suppressor NDRG2 leads to attention deficit and hyperactive behavior. The Journal of Clinical Investigation. 2017;**127**:4270-4284

[71] Yamamoto H, Kokame K, Okuda T, Nakajo Y, Yanamoto H, Miyata T.

NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia. The Journal of Biological Chemistry. 2011;**286**:26158-26165

[72] Li Y, Xu N, Cai L, Gao Z, Shen L, Zhang Q, et al. NDRG2 is a novel p53-associated regulator of apoptosis in C6-originated astrocytes exposed to oxygen-glucose deprivation. PLoS One. 2013;**8**:e57130

[73] Li Y, Shen L, Cai L, Wang Q, Hou W, Wang F, et al. Spatial-temporal expression of NDRG2 in rat brain after focal cerebral ischemia and reperfusion. Brain Research. 2011;**1382**:252-258

[74] Li X, Luo P, Wang F, Yang Q, Li Y, Zhao M, et al. Inhibition of N-myc downstream-regulated gene-2 is involved in an astrocyte-specific neuroprotection induced by sevoflurane preconditioning. Anesthesiology. 2014;**121**:549-562

[75] Wang F, Gao Z, Li X, Li Y, Li X, Zhong H, et al. NDRG2 is involved in anti-apoptosis induced by electroacupuncture pretreatment after focal cerebral ischemia in rats. Neurological Research. 2013;**35**:406-414

[76] Feng T, Han BH, Yang GL, Shi CJ, Gao ZW, Cao MZ, et al. Neuroprotective influence of miR-301a inhibition in experimental cerebral ischemia/ reperfusion rat models through targeting NDRG2. Journal of Molecular Neuroscience. May 2019;**68**(1):144-152

[77] Ma YL, Zhang LX, Liu GL, Fan Y, Peng Y, Hou WG. N-myc downstreamregulated gene 2 (Ndrg2) is involved in ischemia-hypoxia-induced astrocyte apoptosis: A novel target for stroke therapy. Molecular Neurobiology. 2017;**54**:3286-3299

[78] Takarada-Iemata M, Yoshikawa A, Ta HM, Okitani N, Nishiuchi T, Aida Y, et al. N-myc downstream-regulated

**17**

*N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple…*

and NDRG2 phosphorylation in frontotemporal lobar degeneration. Journal of Proteome Research.

[86] Takeichi T, Takarada-Iemata M, Hashida K, Sudo H, Okuda T, Kokame K, et al. The effect of Ndrg2 expression on astroglial activation. Neurochemistry

International. 2011;**59**:21-27

2010;**9**:6368-6379

*DOI: http://dx.doi.org/10.5772/intechopen.86798*

[79] Mitchelmore C, Buchmann-Moller S, Rask L, West MJ, Troncoso JC, Jensen NA. NDRG2: A novel Alzheimer's disease associated protein.

Neurobiology of Disease. 2004;**16**:48-58

Electroacupuncture attenuates reference memory impairment associated with astrocytic NDRG2 suppression in APP/PS1 transgenic mice. Molecular Neurobiology. 2014;**50**:305-313

[81] Rong XF, Sun YN, Liu DM, Yin HJ, Peng Y, Xu SF, et al. The pathological roles of NDRG2 in Alzheimer's disease, a study using animal models and APPwtoverexpressed cells. CNS Neuroscience & Therapeutics. 2017;**23**:667-679

[82] Nichols NR. Ndrg2, a novel gene regulated by adrenal steroids and antidepressants, is highly expressed in astrocytes. Annals of the New York Academy of Sciences.

[83] Araya-Callis C, Hiemke C, Abumaria N, Flugge G. Chronic psychosocial stress and citalopram modulate the expression of the glial proteins GFAP and NDRG2 in the hippocampus. Psychopharmacology.

[84] Takahashi K, Yamada M, Ohata H, Momose K, Higuchi T, Honda K, et al. Expression of Ndrg2 in the rat frontal cortex after antidepressant and electroconvulsive treatment. The International Journal of Neuropsychopharmacology.

[85] Herskowitz JH, Seyfried NT,

et al. Phosphoproteomic analysis reveals site-specific changes in GFAP

Duong DM, Xia Q, Rees HD, Gearing M,

2003;**1007**:349-356

2012;**224**:209-222

2005;**8**:381-389

gene 2 protects blood-brain barrier integrity following cerebral ischemia.

Glia. 2018;**66**:1432-1446

[80] Wang F, Zhong H, Li X, Peng Y, Kinden R, Liang W, et al. *N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Novel Tumor Suppressor in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.86798*

gene 2 protects blood-brain barrier integrity following cerebral ischemia. Glia. 2018;**66**:1432-1446

*Genes and Cancer*

identifies NDRG2 as a candidate tumor suppressor gene frequently inactivated in clinically aggressive meningioma. Cancer Research. 2005;**65**:7121-7126

NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia. The Journal of Biological Chemistry.

[72] Li Y, Xu N, Cai L, Gao Z, Shen L, Zhang Q, et al. NDRG2 is a novel p53-associated regulator of apoptosis in C6-originated astrocytes exposed to oxygen-glucose deprivation. PLoS One.

[73] Li Y, Shen L, Cai L, Wang Q, Hou W, Wang F, et al. Spatial-temporal expression of NDRG2 in rat brain after focal cerebral ischemia and reperfusion. Brain Research. 2011;**1382**:252-258

[74] Li X, Luo P, Wang F, Yang Q, Li Y, Zhao M, et al. Inhibition of N-myc downstream-regulated gene-2 is involved in an astrocyte-specific neuroprotection induced by sevoflurane preconditioning. Anesthesiology.

[75] Wang F, Gao Z, Li X, Li Y, Li X, Zhong H, et al. NDRG2 is involved in anti-apoptosis induced by electroacupuncture pretreatment after focal cerebral ischemia in rats. Neurological Research. 2013;**35**:406-414

[76] Feng T, Han BH, Yang GL, Shi CJ, Gao ZW, Cao MZ, et al. Neuroprotective

targeting NDRG2. Journal of Molecular Neuroscience. May 2019;**68**(1):144-152

[77] Ma YL, Zhang LX, Liu GL, Fan Y, Peng Y, Hou WG. N-myc downstreamregulated gene 2 (Ndrg2) is involved in ischemia-hypoxia-induced astrocyte apoptosis: A novel target for stroke therapy. Molecular Neurobiology.

[78] Takarada-Iemata M, Yoshikawa A, Ta HM, Okitani N, Nishiuchi T, Aida Y, et al. N-myc downstream-regulated

2017;**54**:3286-3299

influence of miR-301a inhibition in experimental cerebral ischemia/ reperfusion rat models through

2011;**286**:26158-26165

2013;**8**:e57130

2014;**121**:549-562

[65] Skiriute D, Tamasauskas S, Asmoniene V, Saferis V, Skauminas K, Deltuva V, et al. Tumor grade-related NDRG2 gene expression in primary and recurrent intracranial meningiomas.

Journal of Neuro-Oncology.

[67] Skiriute D, Vaitkiene P,

[68] Skiriute D, Steponaitis G,

Vaitkiene P, Mikuciunas M, Skauminas K, Tamasauskas A, et al. Glioma malignancy-dependent NDRG2 gene methylation and downregulation correlates with poor patient outcome. Journal of Cancer. 2014;**5**:446-456

[69] Okuda T, Higashi Y, Kokame K, Tanaka C, Kondoh H, Miyata T. Ndrg1 deficient mice exhibit a progressive demyelinating disorder of peripheral nerves. Molecular and Cellular Biology.

[70] Li Y, Yin A, Sun X, Zhang M, Zhang J, Wang P, et al. Deficiency of tumor suppressor NDRG2 leads to attention deficit and hyperactive behavior. The Journal of Clinical Investigation. 2017;**127**:4270-4284

[71] Yamamoto H, Kokame K, Okuda T, Nakajo Y, Yanamoto H, Miyata T.

2004;**24**:3949-3956

Asmoniene V, Steponaitis G, Deltuva VP, Tamasauskas A. Promoter methylation of AREG, HOXA11, hMLH1, NDRG2, NPTX2 and Tes genes in glioblastoma. Journal of Neuro-Oncology. 2013;**113**:441-449

[66] Zhang ZG, Li G, Feng DY, Zhang J, Zhang J, Qin HZ, et al. Overexpression of NDRG2 can inhibit neuroblastoma cell proliferation through negative regulation by CYR61. Asian Pacific Journal of Cancer Prevention: APJCP.

2011;**102**:89-94

2014;**15**:239-244

**16**

[79] Mitchelmore C, Buchmann-Moller S, Rask L, West MJ, Troncoso JC, Jensen NA. NDRG2: A novel Alzheimer's disease associated protein. Neurobiology of Disease. 2004;**16**:48-58

[80] Wang F, Zhong H, Li X, Peng Y, Kinden R, Liang W, et al. Electroacupuncture attenuates reference memory impairment associated with astrocytic NDRG2 suppression in APP/PS1 transgenic mice. Molecular Neurobiology. 2014;**50**:305-313

[81] Rong XF, Sun YN, Liu DM, Yin HJ, Peng Y, Xu SF, et al. The pathological roles of NDRG2 in Alzheimer's disease, a study using animal models and APPwtoverexpressed cells. CNS Neuroscience & Therapeutics. 2017;**23**:667-679

[82] Nichols NR. Ndrg2, a novel gene regulated by adrenal steroids and antidepressants, is highly expressed in astrocytes. Annals of the New York Academy of Sciences. 2003;**1007**:349-356

[83] Araya-Callis C, Hiemke C, Abumaria N, Flugge G. Chronic psychosocial stress and citalopram modulate the expression of the glial proteins GFAP and NDRG2 in the hippocampus. Psychopharmacology. 2012;**224**:209-222

[84] Takahashi K, Yamada M, Ohata H, Momose K, Higuchi T, Honda K, et al. Expression of Ndrg2 in the rat frontal cortex after antidepressant and electroconvulsive treatment. The International Journal of Neuropsychopharmacology. 2005;**8**:381-389

[85] Herskowitz JH, Seyfried NT, Duong DM, Xia Q, Rees HD, Gearing M, et al. Phosphoproteomic analysis reveals site-specific changes in GFAP

and NDRG2 phosphorylation in frontotemporal lobar degeneration. Journal of Proteome Research. 2010;**9**:6368-6379

[86] Takeichi T, Takarada-Iemata M, Hashida K, Sudo H, Okuda T, Kokame K, et al. The effect of Ndrg2 expression on astroglial activation. Neurochemistry International. 2011;**59**:21-27

**19**

**Chapter 2**

**Abstract**

*Guang-Jer Wu*

as therapeutic agents to treat these cancers.

pancreatic cancer, prostate cancer, mouse models

**controlled by two sets of genes as well as CAMs**

METCAM/MUC18: A Novel Tumor

METCAM/MUC18, a component of cellular membrane, is a cell adhesion molecule (CAM) in the Ig-like gene super-family. It is capable of carrying out general functions of CAMs, such as performing intercellular interactions and interaction of cell with extracellular matrix in tumor microenvironment, interacting with various signaling pathways, and regulating social behaviors of cells. METCAM/MUC18 plays the tumor suppressor function in some cancers, such as colorectal cancer, nasopharyngeal carcinoma type I, one mouse melanoma subline K1735-9, ovarian cancer, pancreatic cancer, prostate cancer PC-3 cell line, and perhaps hemangioma. Possible mechanism in the METCAM/MUC18-mediated tumor suppression is proposed. By taking advantage of the tumor suppressor function of METCAM/MUC18, recombinant METCAM/MUC18 proteins and other derived products may be used

Suppressor for Some Cancers

**Keywords:** METCAM/MUC18, Ig-like CAM, *in vivo* tumor suppression,

colorectal cancer, nasopharyngeal carcinoma, mouse melanoma, ovarian cancer,

**1. Introduction: tumor initiation and malignant progression is mainly**

Tumor/cancer is a genetic disease due to accumulated mutations or epigenetic alterations in our genetic material, DNA [1]. 80–90% of cancer risk comes from environmental factors and the remaining 10–20% risk from hereditary factors [2]. Environment in a broad sense includes both the physical containment and the social and cultural environment and its associated effects on our lifestyle choices. The environmental factors in the physical containment include chemicals (from polluted drinking water, air and soil, and diet), physical agents (UV and environmental radiation and medical radiation), biological agents (tumor viruses, bacteria, and parasites), and the lifestyle. These agents aim to attack our DNA in the somatic cells and resulting in accumulation of mutations and epigenetic alterations in our genes throughout our life time. Hereditary factors (lineage specific cues) include both the inherited genetic mutations and epigenetic imprinting in the germ cells that pass on from generation to generation. Tumor initiation and malignant progression are mainly caused by two sets of genes, such as the tumor-promoting genes (oncogenes) and the tumor suppressor genes, thus, mutations and epigenetic alterations in these two sets of genes are doom to be responsible for the tumorigenic process [2–4].

In addition to exogenous chemical agents, physical agents, and biological agents in the environment that cause mutations in the genes, endogenous metabolic processes

#### **Chapter 2**

## METCAM/MUC18: A Novel Tumor Suppressor for Some Cancers

*Guang-Jer Wu*

#### **Abstract**

METCAM/MUC18, a component of cellular membrane, is a cell adhesion molecule (CAM) in the Ig-like gene super-family. It is capable of carrying out general functions of CAMs, such as performing intercellular interactions and interaction of cell with extracellular matrix in tumor microenvironment, interacting with various signaling pathways, and regulating social behaviors of cells. METCAM/MUC18 plays the tumor suppressor function in some cancers, such as colorectal cancer, nasopharyngeal carcinoma type I, one mouse melanoma subline K1735-9, ovarian cancer, pancreatic cancer, prostate cancer PC-3 cell line, and perhaps hemangioma. Possible mechanism in the METCAM/MUC18-mediated tumor suppression is proposed. By taking advantage of the tumor suppressor function of METCAM/MUC18, recombinant METCAM/MUC18 proteins and other derived products may be used as therapeutic agents to treat these cancers.

 Introduction: tumor initiation and malignant progression is mainly controlled by two sets of genes as well as CAMs**

Tumor/cancer is a genetic disease due to accumulated mutations or epigenetic alterations in our genetic material, DNA [1]. 80–90% of cancer risk comes from environmental factors and the remaining 10–20% risk from hereditary factors [2]. Environment in a broad sense includes both the physical containment and the social and cultural environment and its associated effects on our lifestyle choices. The environmental factors in the physical containment include chemicals (from polluted drinking water, air and soil, and diet), physical agents (UV and environmental radiation and medical radiation), biological agents (tumor viruses, bacteria, and parasites), and the lifestyle. These agents aim to attack our DNA in the somatic cells and resulting in accumulation of mutations and epigenetic alterations in our genes throughout our life time. Hereditary factors (lineage specific cues) include both the inherited genetic mutations and epigenetic imprinting in the germ cells that pass on from generation to generation. Tumor initiation and malignant progression are mainly caused by two sets of genes, such as the tumor-promoting genes (oncogenes) and the tumor suppressor genes, thus, mutations and epigenetic alterations in these two sets of genes are doom to be responsible for the tumorigenic process [2–4].

In addition to exogenous chemical agents, physical agents, and biological agents in the environment that cause mutations in the genes, endogenous metabolic processes

and chronic inflammation from our lifestyle choices produce free radicals that directly attack our DNA also resulting in mutations [5]. The major sources of free radicals are reactive oxygen species (ROS), which is a collective term for the unstable, reactive, partially reduced oxygen derivatives that are the normal by-products of our metabolic processes. They include hydrogen peroxide (H2O2), superoxide anion (O2 <sup>−</sup>), hypochlorous acid (HOCl), singlet oxygen (1 O2), and hydroxyl radical (• HO). ROS are also produced by the inflammatory macrophages and neutrophils and are spilled out to attack the DNA of bystander cells. ROS acts as the secondary messengers in cell signaling and essential for various biological processes in both normal and cancer cells and as both tumor-promoting and tumor suppressing agents. To keep the system in check, ROS is balanced by intracellular anti-oxidant enzymes, that produce a number of anti-oxidants, such as glutathione (GSH) and thioredoxin (Txn), which are also present in our foodstuffs, to remove ROS. ROS production is a mechanism shared by most chemotherapeutics to trigger cell-death in cancer cells and unfortunately also to some extent in normal cells. Thus, ROS has conflicting roles as a secondary messenger in cancer cells as well as cancer-killers during cancer chemotherapy.

Most of the mutations in the oncogenes are dominant and thus manifest obvious phenotypes of increased proliferation and survival of tumor cells (gain-of-functions). In contrast, most of mutations in the tumor suppressor genes are recessive and thus do not manifest any phenotype until both copies of the gene are mutated or altered epigenetically (loss-of-functions). Some tumor suppressor genes are gate-keepers that directly affect proliferation and death, thus directly open to tumor formation. But some tumor suppressor genes are care-takers that affect DNA repair functions and genomic stability, thus increase mutation rate of all genes and indirectly affect proliferation [2, 6].

Epigenetic alterations may change the extent of methylation (either hypo- or hyper-methylation) in the regulatory regions of both oncogenes and tumor-suppressor genes, thus affect the transcriptionally regulatory region of the genes and directly regulate transcriptional expression of the genes. Epigenetic alterations may also modify histones and non-histone proteins that affect chromosome remodeling, thus indirectly affect the transcription of the genes. Epigenetic alterations may also affect post-transcription processes (namely translational process or stability of mRNA) of the genes via microRNAs [7].

Besides the above traditional two sets of genes, other genes, such as CAMs, also contribute directly to the tumor initiation and progression or orchestrate the tumor microenvironment to affect the tumor progression [8]. CAMs are involved in several biological functions, such as tissue architecture, organ formation, blood vessel generation and angiogenesis, immune and inflammatory reactions, wound healing and social behaviors [8]. An altered expression of CAMs may have implications in tumorigenesis, since CAMs govern cellular social behaviors by directly contributing to cell adhesion and cross-talk with the intracellular signal transduction pathways [8]. As a consequence, an aberrant expression of CAMs is capable of changing mobility and invasiveness, influencing outlasting ability and proliferation of tumor cells, and altering new blood vessel formation [8]. It also affects distant organ-dissemination of carcinoma cells, because CAMs orchestrate complex interactions of tumor cells with various stromal cells in the tumor microenvironment, resulting in augmentation or reduction of the spreading potential of carcinoma cells [8]. Effects of the aberrant expression of the following CAMs on tumorigenesis and malignant progression are better studied, such as cadherin [9], integrins [10], CD44 [11], CEACAM [12], mucins [13], L1CAM [14], EpCAM [15], ALCAM [16] and METCAM/MUC18 [17]. Over the past several years, our team investigated the role of METCAM/MUC18 in several types of tumors, such as melanoma, breast, nasopharyngeal, ovarian and prostate cancers [18–36]. The resulting data showed a dual role of METCAM/MUC18 as a tumor promotor or suppressor in these cancers [17, 37].

**21**

**Figure 1.**

*METCAM/MUC18: A Novel Tumor Suppressor for Some Cancers*

**2. METCAM/MUC18: an immunoglobulin-like (Ig-like) CAM**

domain and a cytoplasmic domain of 64 residues (**Figure 1**) [38, 42].

ness, *in vitro* and *in vivo* tumorigenesis, and *in vivo* metastasis [42, 43].

*The human METCAM/MUC18 (huMETCAM/MUC18). The figure represents the protein structure of huMETCAM/MUC18 with its 3 domains: (1) a large extracellular domain showing a signal peptide (SP), the five Ig-like variables (V1 and V2) and conserved (C1, C2, C2*′ *and C2*″*) domains, each of which held together by a disulfide bond, and one X domain; six conserved N-glycosylation sites indicated as wavy lines in V1, the interdomain C2*′*/C2*″*, C2*″ *and X domains; (2) a short transmembrane domain (TM); and (3) a cytoplasmic*

*domain containing five potential phosphorylation sites (P).*

**Figure 1** shows that the N-terminal extra-cellular domain of the protein is composed of a signal peptide sequence (SP) and five immunoglobulin-like domains and one X domain [37, 42]. The intracellular cytoplasmic domain has one, three, and one protein kinase consent sequences that are potentially to be phosphorylated by PKA, PKC, and CK2, respectively [37, 38, 42]. In addition, the METCAM/MUC18 usually has an apparent molecular weight of 110–150,000 because it is heavily glycosylated in all cell types. The amino acid sequence of huMETCAM/MUC18 reveals nine possible N-glycosylation sites, of which six are conserved between human and mouse proteins, in the extracellular domain. METCAM/MUC18 is conserved in mouse, in which the amino acid sequences of mouse METCAM/MUC18 (moMETCAM/MUC18) are 72.6% identical to the huMETCAM/MUC18 [43]. Therefore, both human and mouse METCAM/MUC18's are capable of performing similar general functions of CAMs, such as controlling cellular social behaviors by impacting the adhesion status of cells and modulating signaling. Furthermore, over-expression of both human and mouse METCAM/MUC18's similarly affected tumor cells in *in vitro* motility and invasive-

Originally, METCAM/MUC18 was first demonstrated to be abundantly expressed on the cellular membrane of most malignant human melanomas, hence named as MUC18. It has been implicated to play a pivotal role in the malignant progression of human melanoma, hence was named as MCAM and Mel-CAM) [38]. However, METCAM/MUC18 was found in subsequent studies not to be exclusively expressed in melanoma, and it did not initiate the transformation of normal cutaneous melanocytes to melanoma either [39]. Instead, METCAM/MUC18 was also expressed in other epithelial tumors and it could initiate or promote the transformation of other epithelial cells into carcinomas [40]. Thus, METCAM/MUC18 also bears other names, such as S-endo1, CD146, A32, or METCAM [40, 41]. Later METCAM/MUC18 was found to be able to suppress tumorigenesis in some cancer cell lines [17, 37, 40]. The human METCAM/MUC18 is a *c*ell *a*dhesion *m*olecule (CAM) belonging to the Ig-like gene superfamily. The naked human METCAM/MUC18 is a single chain transmembrane protein of 65–72 kDa consisting in 646 amino acids with an extracellular N-terminal domain of 558 amino acids, a 24 amino acids-transmembrane

*DOI: http://dx.doi.org/10.5772/intechopen.86271*

*Genes and Cancer*

hypochlorous acid (HOCl), singlet oxygen (1

mRNA) of the genes via microRNAs [7].

as a tumor promotor or suppressor in these cancers [17, 37].

and chronic inflammation from our lifestyle choices produce free radicals that directly attack our DNA also resulting in mutations [5]. The major sources of free radicals are reactive oxygen species (ROS), which is a collective term for the unstable, reactive, partially reduced oxygen derivatives that are the normal by-products of our metabolic processes. They include hydrogen peroxide (H2O2), superoxide anion (O2

also produced by the inflammatory macrophages and neutrophils and are spilled out to attack the DNA of bystander cells. ROS acts as the secondary messengers in cell signaling and essential for various biological processes in both normal and cancer cells and as both tumor-promoting and tumor suppressing agents. To keep the system in check, ROS is balanced by intracellular anti-oxidant enzymes, that produce a number of anti-oxidants, such as glutathione (GSH) and thioredoxin (Txn), which are also present in our foodstuffs, to remove ROS. ROS production is a mechanism shared by most chemotherapeutics to trigger cell-death in cancer cells and unfortunately also to some extent in normal cells. Thus, ROS has conflicting roles as a secondary messenger

Most of the mutations in the oncogenes are dominant and thus manifest obvious phenotypes of increased proliferation and survival of tumor cells (gain-of-functions). In contrast, most of mutations in the tumor suppressor genes are recessive and thus do not manifest any phenotype until both copies of the gene are mutated or altered epigenetically (loss-of-functions). Some tumor suppressor genes are gate-keepers that directly affect proliferation and death, thus directly open to tumor formation. But some tumor suppressor genes are care-takers that affect DNA repair functions and genomic stability,

Epigenetic alterations may change the extent of methylation (either hypo- or hyper-methylation) in the regulatory regions of both oncogenes and tumor-suppressor genes, thus affect the transcriptionally regulatory region of the genes and directly regulate transcriptional expression of the genes. Epigenetic alterations may also modify histones and non-histone proteins that affect chromosome remodeling, thus indirectly affect the transcription of the genes. Epigenetic alterations may also affect post-transcription processes (namely translational process or stability of

Besides the above traditional two sets of genes, other genes, such as CAMs, also contribute directly to the tumor initiation and progression or orchestrate the tumor microenvironment to affect the tumor progression [8]. CAMs are involved in several biological functions, such as tissue architecture, organ formation, blood vessel generation and angiogenesis, immune and inflammatory reactions, wound healing and social behaviors [8]. An altered expression of CAMs may have implications in tumorigenesis, since CAMs govern cellular social behaviors by directly contributing to cell adhesion and cross-talk with the intracellular signal transduction pathways [8]. As a consequence, an aberrant expression of CAMs is capable of changing mobility and invasiveness, influencing outlasting ability and proliferation of tumor cells, and altering new blood vessel formation [8]. It also affects distant organ-dissemination of carcinoma cells, because CAMs orchestrate complex interactions of tumor cells with various stromal cells in the tumor microenvironment, resulting in augmentation or reduction of the spreading potential of carcinoma cells [8]. Effects of the aberrant expression of the following CAMs on tumorigenesis and malignant progression are better studied, such as cadherin [9], integrins [10], CD44 [11], CEACAM [12], mucins [13], L1CAM [14], EpCAM [15], ALCAM [16] and METCAM/MUC18 [17]. Over the past several years, our team investigated the role of METCAM/MUC18 in several types of tumors, such as melanoma, breast, nasopharyngeal, ovarian and prostate cancers [18–36]. The resulting data showed a dual role of METCAM/MUC18

thus increase mutation rate of all genes and indirectly affect proliferation [2, 6].

in cancer cells as well as cancer-killers during cancer chemotherapy.

O2), and hydroxyl radical (•

<sup>−</sup>),

HO). ROS are

**20**

### **2. METCAM/MUC18: an immunoglobulin-like (Ig-like) CAM**

Originally, METCAM/MUC18 was first demonstrated to be abundantly expressed on the cellular membrane of most malignant human melanomas, hence named as MUC18. It has been implicated to play a pivotal role in the malignant progression of human melanoma, hence was named as MCAM and Mel-CAM) [38]. However, METCAM/MUC18 was found in subsequent studies not to be exclusively expressed in melanoma, and it did not initiate the transformation of normal cutaneous melanocytes to melanoma either [39]. Instead, METCAM/MUC18 was also expressed in other epithelial tumors and it could initiate or promote the transformation of other epithelial cells into carcinomas [40]. Thus, METCAM/MUC18 also bears other names, such as S-endo1, CD146, A32, or METCAM [40, 41]. Later METCAM/MUC18 was found to be able to suppress tumorigenesis in some cancer cell lines [17, 37, 40].

The human METCAM/MUC18 is a *c*ell *a*dhesion *m*olecule (CAM) belonging to the Ig-like gene superfamily. The naked human METCAM/MUC18 is a single chain transmembrane protein of 65–72 kDa consisting in 646 amino acids with an extracellular N-terminal domain of 558 amino acids, a 24 amino acids-transmembrane domain and a cytoplasmic domain of 64 residues (**Figure 1**) [38, 42].

**Figure 1** shows that the N-terminal extra-cellular domain of the protein is composed of a signal peptide sequence (SP) and five immunoglobulin-like domains and one X domain [37, 42]. The intracellular cytoplasmic domain has one, three, and one protein kinase consent sequences that are potentially to be phosphorylated by PKA, PKC, and CK2, respectively [37, 38, 42]. In addition, the METCAM/MUC18 usually has an apparent molecular weight of 110–150,000 because it is heavily glycosylated in all cell types. The amino acid sequence of huMETCAM/MUC18 reveals nine possible N-glycosylation sites, of which six are conserved between human and mouse proteins, in the extracellular domain. METCAM/MUC18 is conserved in mouse, in which the amino acid sequences of mouse METCAM/MUC18 (moMETCAM/MUC18) are 72.6% identical to the huMETCAM/MUC18 [43]. Therefore, both human and mouse METCAM/MUC18's are capable of performing similar general functions of CAMs, such as controlling cellular social behaviors by impacting the adhesion status of cells and modulating signaling. Furthermore, over-expression of both human and mouse METCAM/MUC18's similarly affected tumor cells in *in vitro* motility and invasiveness, *in vitro* and *in vivo* tumorigenesis, and *in vivo* metastasis [42, 43].

#### **Figure 1.**

*The human METCAM/MUC18 (huMETCAM/MUC18). The figure represents the protein structure of huMETCAM/MUC18 with its 3 domains: (1) a large extracellular domain showing a signal peptide (SP), the five Ig-like variables (V1 and V2) and conserved (C1, C2, C2*′ *and C2*″*) domains, each of which held together by a disulfide bond, and one X domain; six conserved N-glycosylation sites indicated as wavy lines in V1, the interdomain C2*′*/C2*″*, C2*″ *and X domains; (2) a short transmembrane domain (TM); and (3) a cytoplasmic domain containing five potential phosphorylation sites (P).*

The huMETCAM/MUC18 is expressed in at least 10 normal tissues: hair follicular cells, smooth muscle cells, endothelial cells, cerebellum, basal cells of the lung, activated T cells, intermediate trophoblasts [44], breast epithelium [18, 19], nasopharyngeal epithelium [23], and ovarian epithelium [27]. The protein is also expressed in several carcinomas, such as breast carcinoma, intermediate trophoblast tumors, melanoma, prostate adenocarcinoma, osteosarcoma, and others [17, 44]. Our studies also indicate that over-expression of METCAM/MUC18 augments tumorigenesis of breast carcinoma [18–20], nasopharyngeal carcinoma type III [24, 26], and prostate adenocarcinoma [34], but it does not have an obvious effect on tumorigenesis of most melanoma cell lines [21]. METCAM/MUC18 over-expression also initiates the distant organ-dissemination of prostate cancer [32, 33] and augments the distant organ-dissemination of melanoma [21] and breast carcinoma [45].

In contrast, over-expression of METCAM/MUC18 represses tumorigenesis of a mouse melanoma cell line, K1735-9 [22], nasopharyngeal carcinoma type I [24, 25] and perhaps hemangiomas [46]. METCAM/MUC18 over-expression also represses the distant organ-dissemination of the mouse melanoma cell line, K1735-9 [22].

#### **3. METCAM/MUC18: a tumor suppressor in several types of cancer**

#### **3.1 Mouse melanoma**

Over-expression of moMETCAM/MUC18 in one mouse melanoma cell line K1735 clone 10 (or K1735-10 subline) has no effect and that in another cell line K1735 clone 3 a slight suppression effect on subcutaneous tumorigenesis [21], but in K1735 clone 9 (or K1735-9 subline) it completely suppresses the subcutaneous tumorigenesis [22]. Thus, METCAM/MUC18 definitely acts as a tumor suppressor for the K1735-9 subline, but may have a less obvious effect on two other K1735 sublines, K1735-3 and K1735-10. In addition to its effect on tumorigenesis, overexpression of moMETCAM/MUC18 in K1735-9 also completely suppressed lung nodule formation in immunocompetent syngeneic C3H brown mouse model. In contrast, over-expression of moMETCAM/MUC18 in K1735-3 and K1735-10 subline has an opposite effect (namely promotion) on lung nodule formation. In conclusion, moMETCAM/MUC18 acts as a tumor suppressor with a different severity on different cell lines in a syngenetic mouse model [21, 22].

#### **3.2 Nasopharyngeal carcinoma**

Nasopharyngeal carcinoma (NPC) occurs in the non-lymphomatous, squamous epithelial lining of the posterior nasopharynx [24]. Histologically, three subtypes of NPC are defined according to World Health Organization (WHO) classification: WHO type I (keratinizing squamous cell carcinomas), WHO type II (non-keratinizing squamous cell carcinomas), and WHO type III (undifferentiated carcinomas) [24]. Three major risk factors suggested by epidemiological studies, such as genetic predisposition, dietary and environmental factors, and the Epstein Barr virus (EBV) infection, may cause the unusual occurrence of NPC in endemic areas [24–26]. However, the biological mechanisms of their involvement in cancer initiation, development or malignant progression are not well understood. Nevertheless, it could be hypothesized that altered cell adhesion molecules (CAMs) in NPC lead to tumorigenesis and malignant progression, since aberrant expression of CAMs, such as CD44, connexin 43, E-cadherin, and ICAM, has been associated with the progression of NPC [23]. In order to test this hypothesis, we previously studied the possible role of altered METCAM/MUC18 expression in nasopharyngeal carcinoma [23, 24].

**23**

**3.4 Prostate cancer**

*METCAM/MUC18: A Novel Tumor Suppressor for Some Cancers*

suppressor role in the development of the type I NPC [24, 25].

and promotor role in the different types of NPC.

suppressor in human ovarian cancer cells.

**3.3 Ovarian carcinoma**

Therefore, we used immunohistochemistry method to determine gene expression at the protein level in seven tissue specimens of normal nasopharynx, 97 specimens of three different types of NPC and also used immunoblot method to determine that in several cell lines established from type I and type III NPC [23]. The results showed a weak expression of the protein METCAM/MUC18 in 27% of the NPC tissues in contrast to all the normal nasopharynx tissues which exhibited high expression of the protein. According to these results, we suggested that METCAM/MUC18 may play a tumor suppressor function in the development of NPC during the progression of the disease [23]. We then tested the hypothesis by transfecting the cDNA into two NPC cell lines which weakly expressed the protein and isolated the high-expressing clones for examining the effect of METCAM/ MUC18 over-expression on *in vitro* cellular behavior and *in vivo* tumorigenesis of the two NPC cell lines in athymic nude mice. Consistent with the hypothesis, we indeed observed that METCAM/MUC18 over-expression suppressed the tumor growth of NPC-TW01 cells, which were established from type I NPC [47], as previously shown [24, 25]. We thus conclude that METCAM/MUC18 plays a tumor

Surprisingly, when a different cell line, NPC-TW04, was used for the similar set of the experiments we observed a completely opposite effect of METCAM/MUC18. We observed that over-expression of METCAM/MUC18 promoted *in vitro* and *in vivo* tumor growth of NPC-TW04 cells, which were established from type III NPC [47], as previously reported [24, 26]. We thus conclude that METCAM/MUC18 plays a tumor promoter role in the development of the type III NPC [24, 26].

Taken together we hypothesized that METCAM/MC18 plays a dual suppressor

Two independent groups showed that METCAM/MUC18 expression is correlated with the progression of ovarian cancer [27, 48], and it affects the *in vitro* behaviors of ovarian carcinoma cells [49]. However, the role of METCAM/

MUC18 in the progression of epithelial ovarian cancer has not been directly tested in animal models. To investigate this, we initiated the studies by testing the effect of over-expression of METCAM/MUC18 on the *in vitro* cellular behaviors and *in vivo* tumorigenesis and malignant progression of human ovarian cancer cell lines in nude mice. First, we used a human ovarian cell line, SK-OV-3, for testing the effects of METCAM/MUC18 expression on their *in vitro* motility and invasiveness, and *in vivo* tumor formation after subcutaneous (SC) injection and also *in vivo* progression after intraperitoneal (IP) injection in athymic nude mice. We observed that overexpression of METCAM/MUC18 reduced *in vitro* motility and invasiveness [28] and suppressed *in vivo* tumorigenesis and malignant progression of the human ovarian cancer cell line SK-OV-3 [28]. Then, we used the other human ovarian cancer cell

line, BG-1, for similar tests and also observed similar phenomenon [50].

In summary, we supplied *in vitro* and *in vivo* evidence to definitely support the conclusion that METCAM/MUC18 plays a suppressor role in the tumorigenesis and malignant progression of two human ovarian cancer cell lines [28, 50]. Our results suggest that METCAM/MUC18 is a strong candidate as a new tumor and metastasis

For the previous two decades, we have firmly established the notion that over-expression of METCAM/MUC18 promotes the tumorigenesis and metastasis

*DOI: http://dx.doi.org/10.5772/intechopen.86271*

Therefore, we used immunohistochemistry method to determine gene expression at the protein level in seven tissue specimens of normal nasopharynx, 97 specimens of three different types of NPC and also used immunoblot method to determine that in several cell lines established from type I and type III NPC [23]. The results showed a weak expression of the protein METCAM/MUC18 in 27% of the NPC tissues in contrast to all the normal nasopharynx tissues which exhibited high expression of the protein. According to these results, we suggested that METCAM/MUC18 may play a tumor suppressor function in the development of NPC during the progression of the disease [23]. We then tested the hypothesis by transfecting the cDNA into two NPC cell lines which weakly expressed the protein and isolated the high-expressing clones for examining the effect of METCAM/ MUC18 over-expression on *in vitro* cellular behavior and *in vivo* tumorigenesis of the two NPC cell lines in athymic nude mice. Consistent with the hypothesis, we indeed observed that METCAM/MUC18 over-expression suppressed the tumor growth of NPC-TW01 cells, which were established from type I NPC [47], as previously shown [24, 25]. We thus conclude that METCAM/MUC18 plays a tumor suppressor role in the development of the type I NPC [24, 25].

Surprisingly, when a different cell line, NPC-TW04, was used for the similar set of the experiments we observed a completely opposite effect of METCAM/MUC18. We observed that over-expression of METCAM/MUC18 promoted *in vitro* and *in vivo* tumor growth of NPC-TW04 cells, which were established from type III NPC [47], as previously reported [24, 26]. We thus conclude that METCAM/MUC18 plays a tumor promoter role in the development of the type III NPC [24, 26].

Taken together we hypothesized that METCAM/MC18 plays a dual suppressor and promotor role in the different types of NPC.

#### **3.3 Ovarian carcinoma**

*Genes and Cancer*

**3.1 Mouse melanoma**

The huMETCAM/MUC18 is expressed in at least 10 normal tissues: hair follicular cells, smooth muscle cells, endothelial cells, cerebellum, basal cells of the lung, activated T cells, intermediate trophoblasts [44], breast epithelium [18, 19], nasopharyngeal epithelium [23], and ovarian epithelium [27]. The protein is also expressed in several carcinomas, such as breast carcinoma, intermediate trophoblast tumors, melanoma, prostate adenocarcinoma, osteosarcoma, and others [17, 44]. Our studies also indicate that over-expression of METCAM/MUC18 augments tumorigenesis of breast carcinoma [18–20], nasopharyngeal carcinoma type III [24, 26], and prostate adenocarcinoma [34], but it does not have an obvious effect on tumorigenesis of most melanoma cell lines [21]. METCAM/MUC18 over-expression also initiates the distant organ-dissemination of prostate cancer [32, 33] and augments the distant