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. Consistent with this finding, we and other groups observed the decreased NDRG2 expression in multiple human cancer cell lines and tumors, including breast cancer, colorectal cancer, and cervical cancer. 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. Consistent with this finding, we and other groups observed the decreased NDRG2 expression in multiple human cancer cell lines and tumors, including breast cancer, colorectal cancer, and cervical cancer. 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]. We and other groups confirmed the pattern of decreased NDRG2 expression in tumors compared with normal tissues in cancers including glioma [2, 19, 21], colorectal cancer [8, 22, 23], breast cancer [3, 24], lung cancer [25], thyroid cancer [26, 27], myeloid leukemia [28, 29] oral squamous cell carcinoma (OSCC) and cervical cancer [5, 7]. 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]. We and other groups confirmed the pattern of decreased NDRG2 expression in tumors compared with normal tissues in cancers including glioma [2, 19, 21], colorectal cancer [8, 22, 23], breast cancer [3, 24], lung cancer [25], thyroid cancer [26, 27], myeloid leukemia [28, 29] oral squamous cell carcinoma (OSCC) and cervical cancer [5, 7]. 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: biozhangj@fmmu.edu.cn

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.

#### **Author details**

*Genes and Cancer*

human brain tumors.

tective effect [71].

*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: biozhangj@fmmu.edu.cn

© 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.

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**14**

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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.

**Keywords:** METCAM/MUC18, Ig-like CAM, *in vivo* tumor suppression, colorectal cancer, nasopharyngeal carcinoma, mouse melanoma, ovarian cancer, pancreatic cancer, prostate cancer, mouse models

#### **1. 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

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**

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

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].

organ-dissemination of melanoma [21] and breast carcinoma [45].

different cell lines in a syngenetic mouse model [21, 22].

**3.2 Nasopharyngeal carcinoma**

**22**

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 suppressor in human ovarian cancer cells.

#### **3.4 Prostate cancer**

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

#### **Figure 2.**

*Tumorigenicity of four shRNA-knockdown clones of DU145. Effect of METCAM/MUC18 expression on in vivo tumorigenicity (Left) and final tumor weight (Right). (Left) Average tumor volumes from five mice S.C. injected with each of the 46 (control), 72, 24, and 27 clones/cells, which were transfected with four corresponding shRNAs in pGIPZ vector, were plotted against time. (Right) Average final tumor weights from five mice S.C. injected with the same clones/cells and standard deviations were plotted at the end point of experiment. P values are shown in the figure by comparing the data to the control clone [51].*

#### **Figure 3.**

*Tumorigenicity of four shRNA-knockdown clones of PC-3. Effect of METCAM/MUC18 expression on in vivo tumorigenicity (Left) and final tumor weight (Right). (Left) Average tumor volumes from five mice S.C. injected with each of the 46 (control), 72, 24, and 27 clones/cells, which were transfected with the four corresponding shRNAs in pGIPZ vector, were plotted against time. (Right) Average final tumor weights from five mice S.C. injected with the same clones/cells and standard deviations were plotted at the end point of experiment. P values are shown in the figure by comparing the data to the control clone [52].*

of human prostate cancer cell line LNCaP, which was established from lymphatic lesions [31–36]. To check if the conclusion is also extended to another human prostate cancer cell line DU145, we recently tested the effect of knocking down the endogenously expressed METCAM/MUC18 on tumorigenesis in a nude mouse system, since DU145 endogenously expressed a high level of METCAM/MUC18 [51]. We found that knocking down of the endogenously expressed METCAM/ MUC18 with three shRNAs decreased the subcutaneous tumorigenesis in male nude mice in comparison to a control shRNA, as shown in **Figure 2**. We thus concluded that METCAM/MUC18 expression in DU145 cell line, which was established from brain lesions, plays a positive role in tumorigenesis (and perhaps metastasis) similar to in LNCaP cells.

In contrast, we recently used the similar knocking down strategy to test the effect of decreased the endogenous METCAM/MUC18 expression on *in vivo* tumorigenesis of another human prostate cancer cell line, PC-3, which was established from bone lesions, surprisingly we found that knocking down the endogenously expressed METCAM/MUC18 increased the tumor proliferation of PC-3 cells (which was opposite to that of DU145, as shown above in **Figure 2**), suggesting that expression of METCAM/MUC18 suppressed the tumorigenesis of the human prostate cancer cell line PC-3 [52], as shown in **Figure 3**.

**25**

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

Mouse melanoma cell lines K1735-3, K1735-10 No effect or slight

We thus conclude that METCAM/MUC18 serves as a tumor suppressor in the PC-3

*The negative role of METCAM/MUC18 in tumor formation and/or cancer metastasis of seven tumor/cancer* 

**Tumor/cancer cell lines Tumorigenesis Metastasis References**

Possible suppression

suppression

Mouse melanoma cell line K1735-9 Suppression Suppression [22]

Ovarian cancer cell lines SK-OV3, BG-1 Suppression Suppression [28, 50]

Prostate cancer human cell line PC-3 Suppression Not determined [52]

Suppression Not determined [54]

stage

Suppression Not determined [24, 25]

Suppression Suppression [55, 56]

Increasing and affecting the late

Not determined [46]

[21]

The protein METCAM/MUC18 is also expressed others cancers, such as angiosarcoma, gestational trophoblastic tumors, Kaposi's sarcoma, leiomyosarcoma, some lung squamous and small cell carcinomas, and some neuroblastoma [44]. However, its role in the development of most of these cancers is not well known. Recent meta-analysis suggests that high METCAM/MUC18 expression in many solid tumors appears to be associated with poor prognosis and patient survival [53]. However, in contrast to the conclusion, reduced expression of METCAM/MUC18 associates with the malignant progression of hemangioma [46]. Likewise, recent results of the effects of METCAM/ MUC18 expression on tumorigenesis of colorectal cancer and pancreatic cancer also appear to support the similar conclusion, as described next. Reduced expression of METCAM/MUC18 promotes tumorigenesis and stemness of colorectal cancer [54]. Targeting soluble METCAM/MUC18 with a neutralizing antibody inhibits vascularization, growth and survival of METCAM/MUC18-positive pancreatic tumors [55]. Furthermore, attenuation of METCAM/MUC18 promotes pancreatic cancer progression [56]. Thus, the possible tumor and metastasis suppressor role of METCAM/ MUC18 in solid tumors appear to extend from mouse melanoma K1735–9 subline, ovarian cancer, and NPC type I, to colorectal cancer [54] and pancreatic cancer [55, 56], and perhaps hemangioma [46]. **Table 1** summarizes the negative role of METCAM/ MUC18 in the tumor formation and/or cancer metastasis of seven tumors/cancers.

cell line, different from its role in two other prostate cancer cell lines (LNCaP and DU145), suggesting that prostate cancer cell lines established from different organs may have different intrinsic factors that modulate the function of METCAM/MUC18.

**3.5 Colorectal cancer, hemangioma and pancreatic cancer**

**4. METCAM/MUC18: a tumor promoter in most solid tumors**

In contrast to the above functions of METCAM/MUC18, recent work done on other solid tumors appears to be consistent with the meta-analysis results of solid tumors [53],

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

Colorectal cancer human cell lines HT-29, SW480, SW948, SW620, colo205, Lovo320, P6C

Hemangioma human cell lines HemEC,

Nasopharyngeal carcinoma type I cell line

Pancreatic cancer human cell lines, UACC-1273, PANC1, C81-61, KP-2, SUIT-2, MIAPaca-2, HS766T and primary CAFs

HDMEC

NPC-TW01

**Table 1.**

*cell lines.*


#### **Table 1.**

*Genes and Cancer*

**24**

**Figure 3.**

**Figure 2.**

metastasis) similar to in LNCaP cells.

prostate cancer cell line PC-3 [52], as shown in **Figure 3**.

of human prostate cancer cell line LNCaP, which was established from lymphatic lesions [31–36]. To check if the conclusion is also extended to another human prostate cancer cell line DU145, we recently tested the effect of knocking down the endogenously expressed METCAM/MUC18 on tumorigenesis in a nude mouse system, since DU145 endogenously expressed a high level of METCAM/MUC18 [51]. We found that knocking down of the endogenously expressed METCAM/ MUC18 with three shRNAs decreased the subcutaneous tumorigenesis in male nude mice in comparison to a control shRNA, as shown in **Figure 2**. We thus concluded that METCAM/MUC18 expression in DU145 cell line, which was established from brain lesions, plays a positive role in tumorigenesis (and perhaps

*Tumorigenicity of four shRNA-knockdown clones of PC-3. Effect of METCAM/MUC18 expression on in vivo tumorigenicity (Left) and final tumor weight (Right). (Left) Average tumor volumes from five mice S.C. injected with each of the 46 (control), 72, 24, and 27 clones/cells, which were transfected with the four corresponding shRNAs in pGIPZ vector, were plotted against time. (Right) Average final tumor weights from five mice S.C. injected with the same clones/cells and standard deviations were plotted at the end point of* 

*Tumorigenicity of four shRNA-knockdown clones of DU145. Effect of METCAM/MUC18 expression on in vivo tumorigenicity (Left) and final tumor weight (Right). (Left) Average tumor volumes from five mice S.C. injected with each of the 46 (control), 72, 24, and 27 clones/cells, which were transfected with four corresponding shRNAs in pGIPZ vector, were plotted against time. (Right) Average final tumor weights from five mice S.C. injected with the same clones/cells and standard deviations were plotted at the end point of* 

*experiment. P values are shown in the figure by comparing the data to the control clone [51].*

*experiment. P values are shown in the figure by comparing the data to the control clone [52].*

In contrast, we recently used the similar knocking down strategy to test the effect of decreased the endogenous METCAM/MUC18 expression on *in vivo* tumorigenesis of another human prostate cancer cell line, PC-3, which was established from bone lesions, surprisingly we found that knocking down the endogenously expressed METCAM/MUC18 increased the tumor proliferation of PC-3 cells (which was opposite to that of DU145, as shown above in **Figure 2**), suggesting that expression of METCAM/MUC18 suppressed the tumorigenesis of the human

*The negative role of METCAM/MUC18 in tumor formation and/or cancer metastasis of seven tumor/cancer cell lines.*

We thus conclude that METCAM/MUC18 serves as a tumor suppressor in the PC-3 cell line, different from its role in two other prostate cancer cell lines (LNCaP and DU145), suggesting that prostate cancer cell lines established from different organs may have different intrinsic factors that modulate the function of METCAM/MUC18.

#### **3.5 Colorectal cancer, hemangioma and pancreatic cancer**

The protein METCAM/MUC18 is also expressed others cancers, such as angiosarcoma, gestational trophoblastic tumors, Kaposi's sarcoma, leiomyosarcoma, some lung squamous and small cell carcinomas, and some neuroblastoma [44]. However, its role in the development of most of these cancers is not well known. Recent meta-analysis suggests that high METCAM/MUC18 expression in many solid tumors appears to be associated with poor prognosis and patient survival [53]. However, in contrast to the conclusion, reduced expression of METCAM/MUC18 associates with the malignant progression of hemangioma [46]. Likewise, recent results of the effects of METCAM/ MUC18 expression on tumorigenesis of colorectal cancer and pancreatic cancer also appear to support the similar conclusion, as described next. Reduced expression of METCAM/MUC18 promotes tumorigenesis and stemness of colorectal cancer [54]. Targeting soluble METCAM/MUC18 with a neutralizing antibody inhibits vascularization, growth and survival of METCAM/MUC18-positive pancreatic tumors [55]. Furthermore, attenuation of METCAM/MUC18 promotes pancreatic cancer progression [56]. Thus, the possible tumor and metastasis suppressor role of METCAM/ MUC18 in solid tumors appear to extend from mouse melanoma K1735–9 subline, ovarian cancer, and NPC type I, to colorectal cancer [54] and pancreatic cancer [55, 56], and perhaps hemangioma [46]. **Table 1** summarizes the negative role of METCAM/ MUC18 in the tumor formation and/or cancer metastasis of seven tumors/cancers.

#### **4. METCAM/MUC18: a tumor promoter in most solid tumors**

In contrast to the above functions of METCAM/MUC18, recent work done on other solid tumors appears to be consistent with the meta-analysis results of solid tumors [53],

#### **Figure 4.**

*Expression of METCAM/MUC18 in normal lung tissue (SV40-immortalized normal lung cells (WI38, lane 2) and lung type II alveolar epithelial cell carcinoma cell (A549, lane 3) and lung primary adenocarcinoma (H838, lane 4) (from Guang-Jer Wu, unpublished data).*

as described next. For example, METCAM/MUC18 expression correlates with the epithelial-mesenchymal transition (EMT) markers and a poor prognosis in gastric cancer [57]. Tumor up-take of glioma in an orthotopic xenograft mouse model correlates with the expression level of METCAM/MUC18 [58]. METCAM/MUC18 promotes metastasis and predicts poor prognosis of hepatocellular carcinoma [59]. Increased expression of METCAM/MUC18 has been found in hepatocellular carcinoma (HCC) tumor tissues as compared with the matched adjacent normal liver tissues and the METCAM/ MUC18+ cells purified from HCC tumors and cells have significantly increased colonyforming capacity consistent with the cancer stem cells or the tumor-initiating cells [60]. METCAM/MUC18 expression has been shown to express in 51% of non-small cell lung carcinoma (NSCLC) and positive expression of METCAM/MUC18 has been associated with a shorter survival of patients with adenocarcinomas and used to predict the poor overall survival in patients with lung adenocarcinomas [61–63]. METCAM/MUC18 expression mediates acquisition of cancer stemness and enhances tumor invasion and metastasis in a mouse model [64]. High expression of METCAM/MUC18 correlates with intrapulmonary metastasis of NSCLC cells in a mouse model [65]. Consistent with the results, we showed in **Figure 4** (Guang-Jer Wu, unpublished data) that METCAM/ MUC18 is expressed in a lung type II alveolar epithelial cell carcinoma cell, A549, and highly expressed in an adenocarcinoma cell line, H838, in comparison with its no expression in an immortalized normal embryonic WI38 cell line.

Furthermore, METCAM/MUC18 mediates chemoresistance of small cell lung carcinoma (SCLC) [66]. METCAM/MUC18 is expressed in osteosarcoma cell lines, but not in normal osteoblast cells [67]. Osteosarcoma is effectively treated with METCAM/MUC18 monoclonal antibodies [68, 69]. Transcription factor MEIS1 activates METCAM/MUC18 expression to promote migration of mouse pancreatic tumor cell lines [70]. METCAM/MUC18 very likely promotes the formation of angiosarcoma, as supported by our preliminary results as described next. Mouse METCAM/MUC18 was expressed in one angiosarcoma clone, SVR, which was transfected with H-Ras, at a higher level than in the control cell line, an immortalized normal endothelial cell line, MS-1 [71]. Furthermore, the tumorigenicity of the SVR cell line was higher than the control cell line, thus in direct association with the higher expression level of moMETCAM/MUC18 [40, 71]. This suggests that METCAM/MUC18 very likely promotes the formation of angiosarcoma [40, 71].

**27**

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

**Tumor/cancer tissues or cell lines Tumorigenesis Metastasis References**

Human breast cancer cell line MCF-7 Promotion Not determined [18] Human breast cancer cell line SK-BR-3 Promotion Not determined [19, 20]

Gastric cancer human tissues Promotion Not determined [57] Glioma cell lines U87MG, U251 Promotion Not Determined [58]

> No effect or slight suppression

Clinical prostate cancer human tissues Increasing Increasing and

Increasing Not determined [40, 71]

Promotion Promotion [19, 45]

Promotion Not determined [59, 60]

Promotion Promotion [61–65], our

affecting the late

Increasing and affecting the late

Promotion Not determined [24, 26]

Promotion Augmentation [67–69]

augmentation

affecting initiation in the early stage (PIN)

affecting initiation in the early stage

affecting initiation in the early stage

Promotion Not determined [66]

stage

stage

No effect Increasing and

Promotion Possible

Increasing Increasing and

Increasing Increasing and

unpublished results

[38, 72, 73]

[21]

[70]

[31]

[33]

[32, 34–36]

Hence, the positive role played by the METCAM/MUC18 in the progression of solid tumors have been extended from breast cancer, human and mouse melanoma, prostate cancer to angiosarcoma [40, 71], gastric cancer [57], glioma [58], hepatocellular carcinoma [59, 60], non-small cell lung adenocarcinoma [61–65], small cell

*The positive role of METCAM/MUC18 in tumor formation and/or cancer metastasis of various tumors/cancers.*

Human prostate cancer cell line DU145 Increasing Not determined [51]

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

Angiosarcoma human cell lines MS1,

Human breast cancer cell lines MDA-MB-231 and MDA-MB-468

Hepatocellular carcinoma human cell lines PLC/PRF/5, Huh7, MHCC97H& 97 L HepG2, SMMC-7721, focus, YY-8103, LM3, HLF and primary HCC cell lines; normal liver cell line LO2

Non-small cell lung cancer human cell lines A549, H23, H358, H460, H522, H838, HCC4006, H1650/ER, PC-9, PC9GR, and adenocarcinoma tissues

Small cell lung cancer human cell lines H69, H69AR, H82, H196, H209,

Clinical melanoma tissues and human melanoma cell lines SB-2, SK, XP-44

Mouse melanoma cell lines K1735-3,

Nasopharyngeal carcinoma type III human cell line NPC-TW04

Osteosarcoma human cell lines CR9, MNNG-HOS, OHS, KPDX, KRIB, MG-63, shYY1, SaOS, SaOS-2, TE85,

Pancreatic cancer mouse cell lines ptf1a, LSL-Kras, LSL-Trp53, Pdx1,

Human prostate cancer cell line

Prostate adenocarcinoma in TRAMP

DMS79

K1735-10

U20S

LNCaP

mice

**Table 2.**

SVR

*Genes and Cancer*

as described next. For example, METCAM/MUC18 expression correlates with the epithelial-mesenchymal transition (EMT) markers and a poor prognosis in gastric cancer [57]. Tumor up-take of glioma in an orthotopic xenograft mouse model correlates with the expression level of METCAM/MUC18 [58]. METCAM/MUC18 promotes metastasis and predicts poor prognosis of hepatocellular carcinoma [59]. Increased expression of METCAM/MUC18 has been found in hepatocellular carcinoma (HCC) tumor tissues as compared with the matched adjacent normal liver tissues and the METCAM/

*Expression of METCAM/MUC18 in normal lung tissue (SV40-immortalized normal lung cells (WI38, lane 2) and lung type II alveolar epithelial cell carcinoma cell (A549, lane 3) and lung primary adenocarcinoma* 

 cells purified from HCC tumors and cells have significantly increased colonyforming capacity consistent with the cancer stem cells or the tumor-initiating cells [60]. METCAM/MUC18 expression has been shown to express in 51% of non-small cell lung carcinoma (NSCLC) and positive expression of METCAM/MUC18 has been associated with a shorter survival of patients with adenocarcinomas and used to predict the poor overall survival in patients with lung adenocarcinomas [61–63]. METCAM/MUC18 expression mediates acquisition of cancer stemness and enhances tumor invasion and metastasis in a mouse model [64]. High expression of METCAM/MUC18 correlates with intrapulmonary metastasis of NSCLC cells in a mouse model [65]. Consistent with the results, we showed in **Figure 4** (Guang-Jer Wu, unpublished data) that METCAM/ MUC18 is expressed in a lung type II alveolar epithelial cell carcinoma cell, A549, and highly expressed in an adenocarcinoma cell line, H838, in comparison with its no

Furthermore, METCAM/MUC18 mediates chemoresistance of small cell lung carcinoma (SCLC) [66]. METCAM/MUC18 is expressed in osteosarcoma cell lines, but not in normal osteoblast cells [67]. Osteosarcoma is effectively treated with METCAM/MUC18 monoclonal antibodies [68, 69]. Transcription factor MEIS1 activates METCAM/MUC18 expression to promote migration of mouse pancreatic tumor cell lines [70]. METCAM/MUC18 very likely promotes the formation of angiosarcoma, as supported by our preliminary results as described next. Mouse METCAM/MUC18 was expressed in one angiosarcoma clone, SVR, which was transfected with H-Ras, at a higher level than in the control cell line, an immortalized normal endothelial cell line, MS-1 [71]. Furthermore, the tumorigenicity of the SVR cell line was higher than the control cell line, thus in direct association with the higher expression level of moMETCAM/MUC18 [40, 71]. This suggests that METCAM/MUC18 very likely promotes the formation of angiosarcoma [40, 71].

expression in an immortalized normal embryonic WI38 cell line.

**26**

MUC18+

**Figure 4.**

*(H838, lane 4) (from Guang-Jer Wu, unpublished data).*


#### **Table 2.**

*The positive role of METCAM/MUC18 in tumor formation and/or cancer metastasis of various tumors/cancers.*

Hence, the positive role played by the METCAM/MUC18 in the progression of solid tumors have been extended from breast cancer, human and mouse melanoma, prostate cancer to angiosarcoma [40, 71], gastric cancer [57], glioma [58], hepatocellular carcinoma [59, 60], non-small cell lung adenocarcinoma [61–65], small cell

lung cancer [66], osteosarcoma [67–69], and mouse pancreatic cancer [70]. Taken together, METCAM/MCU18 appears to be more prevalently in playing a positive role than a negative role in the tumorigenesis of solid tumors. **Table 2** summarizes the positive role of METCAM/MUC18 in the tumor formation and/or cancer metastasis of various tumors/cancers.

In conclusion, METCAM/MUC18 appears to play a dual role in the tumorigenesis and perhaps also in metastasis of solid tumors. At this point, it is not clear why METCAM/MUC18 plays a dual role in this aspect. Since METCAM/ MUC18 only plays a dual role in different cell lines from the same type of cancer or in different type of cancers, but never in the same cancer cell line. It is logical to suggest a possible explanation that the intrinsic properties of each cancer cell line may provide specific co-factors or heterophilic ligands that may positively or negatively modulate the METCAM/MUC18-mediated tumorigenesis and metastasis. This can be readily scrutinized by identifying these specific intrinsic co-factors or heterophilic ligands by using immunological co-precipitation method in the future studies. This approach is feasible as described in one of the following sections, Section 5.1.

#### **5. Putative mechanisms**

Since the huMETCAM/MUC18 was first discovered in the 1980s, three groups have worked on the role of huMETCAM/MUC18 in melanoma metastasis [38, 39, 72, 73], another group on the role of huMETCAM/MUC18 in the biology of endothelial cells [41], and our group joined in the effort to study the role of huMET-CAM/MUC18 in the progression of mouse melanoma [43] and prostate cancer [31–36, 51, 52], and later breast cancer [18–20], ovarian cancer [27–30], and NPC [23–26], as described above. Recently, more groups have participated in further exploring the possible role of METCAM/MUC18 in other solid tumors in different organs, such as colorectum [54], gastro-organ [57], glioma [58], liver [59, 60], lung [61–66], pancreas [55, 56, 70], and bone [67–69]. Preliminary work in leiomyosarcoma, esophagus squamous cell carcinoma, clear cell renal sarcoma, and gallbladder adenocarcinoma are also beginning to emerge [53].

After many decades of group effort, we are beginning to understand the biology of METCAM/MUC18-mediated tumor progression. However, the biological mechanisms describing the role of METCAM/MUC18 in tumorigenesis and malignant progression are still not well clarified such as: the protein's domain involved in cell adhesion, the domain which mediates the interactions of tumor cells with the tumor microenvironment leading to tumor progression and in the METCAM/ MUC18-mediated tumorigenesis and malignant progression, and the effects of N-glycosylation on the functions of METCAM/MUC18 in tumorigenesis. Though the huMETCAM/MUC18-mediated outside-in and inside-out signaling in endothelial cells are understood to some extent, and the METCAM/MUC18-mediated signaling, which is leading to the progression of various cancer cells, are not much known. How METCAM/MUC18 is positively or negatively regulated at the level of transcription in different cancer cells remains minimally known. As such, the following five important aspects are much needed for immediate future studies, such as different kinds or quantities of co-factors or heterophilic ligand(s) in different cancer cell lines, contributions of different domains of the protein, different signaling pathways involved, differential regulation at the transcription level in tumors of different organs, and possible different extent of N-glycosylation in different cancer cell lines, which may critically modulate the function of METCAM/MUC18 in tumor progression.

**29**

**Figure 5.**

*Putative heterophilic ligand of METCAM/MUC18 in PC-3 and DU145 cell lines.*

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

The heterophilic ligands of METCAM/MUC18 may play an important role in the cell-cell and cell-extra-cellular matrix interactions and cancer metastasis. Our preliminary results suggest that the 72 kDa protein identified by immunoprecipitation method may be one of the heterophilic ligands for METCAM/MUC18, as shown in

As shown in **Figure 5**, the putative heterophilic ligand 72 kDa is highly expressed in the PC-3 cell line, but much less in the DU145 cell line. This may reveal a possible explanation for the different role of huMETCAM/MUC18 in the tumorigenicity of

**5.2 The domains of huMETCAM/MUC18 required for tumorigenesis and** 

The relation of the protein structure of huMETCAM/MUC18 to its functions in tumorigenesis and metastasis have not been systematically defined. To begin addressing this question, we have generated mutants deleted different domains of huMETCAM/MUC18 by using a special PCR method [74] and used them to determine their contribution to tumorigenesis. Surprisingly, our results showed that the ecto-domain of huMETCAM/MUC18 induced tumorigenesis in LNCaP cells in nude mice, as well as the whole wild type of cDNA. These preliminary results suggested

**5.1 The heterophilic ligands of METCAM/MUC18**

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

the two prostate cancer cell lines [40].

**Figure 5** [40].

**metastasis**

#### **5.1 The heterophilic ligands of METCAM/MUC18**

*Genes and Cancer*

tasis of various tumors/cancers.

following sections, Section 5.1.

der adenocarcinoma are also beginning to emerge [53].

**5. Putative mechanisms**

lung cancer [66], osteosarcoma [67–69], and mouse pancreatic cancer [70]. Taken together, METCAM/MCU18 appears to be more prevalently in playing a positive role than a negative role in the tumorigenesis of solid tumors. **Table 2** summarizes the positive role of METCAM/MUC18 in the tumor formation and/or cancer metas-

In conclusion, METCAM/MUC18 appears to play a dual role in the tumorigenesis and perhaps also in metastasis of solid tumors. At this point, it is not clear why METCAM/MUC18 plays a dual role in this aspect. Since METCAM/ MUC18 only plays a dual role in different cell lines from the same type of cancer or in different type of cancers, but never in the same cancer cell line. It is logical to suggest a possible explanation that the intrinsic properties of each cancer cell line may provide specific co-factors or heterophilic ligands that may positively or negatively modulate the METCAM/MUC18-mediated tumorigenesis and metastasis. This can be readily scrutinized by identifying these specific intrinsic co-factors or heterophilic ligands by using immunological co-precipitation method in the future studies. This approach is feasible as described in one of the

Since the huMETCAM/MUC18 was first discovered in the 1980s, three groups have worked on the role of huMETCAM/MUC18 in melanoma metastasis [38, 39, 72, 73], another group on the role of huMETCAM/MUC18 in the biology of endothelial cells [41], and our group joined in the effort to study the role of huMET-CAM/MUC18 in the progression of mouse melanoma [43] and prostate cancer [31–36, 51, 52], and later breast cancer [18–20], ovarian cancer [27–30], and NPC [23–26], as described above. Recently, more groups have participated in further exploring the possible role of METCAM/MUC18 in other solid tumors in different organs, such as colorectum [54], gastro-organ [57], glioma [58], liver [59, 60], lung [61–66], pancreas [55, 56, 70], and bone [67–69]. Preliminary work in leiomyosarcoma, esophagus squamous cell carcinoma, clear cell renal sarcoma, and gallblad-

After many decades of group effort, we are beginning to understand the biology of METCAM/MUC18-mediated tumor progression. However, the biological mechanisms describing the role of METCAM/MUC18 in tumorigenesis and malignant progression are still not well clarified such as: the protein's domain involved in cell adhesion, the domain which mediates the interactions of tumor cells with the tumor microenvironment leading to tumor progression and in the METCAM/ MUC18-mediated tumorigenesis and malignant progression, and the effects of N-glycosylation on the functions of METCAM/MUC18 in tumorigenesis. Though the huMETCAM/MUC18-mediated outside-in and inside-out signaling in endothelial cells are understood to some extent, and the METCAM/MUC18-mediated signaling, which is leading to the progression of various cancer cells, are not much known. How METCAM/MUC18 is positively or negatively regulated at the level of transcription in different cancer cells remains minimally known. As such, the following five important aspects are much needed for immediate future studies, such as different kinds or quantities of co-factors or heterophilic ligand(s) in different cancer cell lines, contributions of different domains of the protein, different signaling pathways involved, differential regulation at the transcription level in tumors of different organs, and possible different extent of N-glycosylation in different cancer cell lines, which may critically modulate the function of METCAM/MUC18 in

**28**

tumor progression.

The heterophilic ligands of METCAM/MUC18 may play an important role in the cell-cell and cell-extra-cellular matrix interactions and cancer metastasis. Our preliminary results suggest that the 72 kDa protein identified by immunoprecipitation method may be one of the heterophilic ligands for METCAM/MUC18, as shown in **Figure 5** [40].

As shown in **Figure 5**, the putative heterophilic ligand 72 kDa is highly expressed in the PC-3 cell line, but much less in the DU145 cell line. This may reveal a possible explanation for the different role of huMETCAM/MUC18 in the tumorigenicity of the two prostate cancer cell lines [40].

#### **5.2 The domains of huMETCAM/MUC18 required for tumorigenesis and metastasis**

The relation of the protein structure of huMETCAM/MUC18 to its functions in tumorigenesis and metastasis have not been systematically defined. To begin addressing this question, we have generated mutants deleted different domains of huMETCAM/MUC18 by using a special PCR method [74] and used them to determine their contribution to tumorigenesis. Surprisingly, our results showed that the ecto-domain of huMETCAM/MUC18 induced tumorigenesis in LNCaP cells in nude mice, as well as the whole wild type of cDNA. These preliminary results suggested

**Figure 5.** *Putative heterophilic ligand of METCAM/MUC18 in PC-3 and DU145 cell lines.*

*Genes and Cancer*

the key role of the ecto-domain in tumorigenesis induction in prostate cancer cells *in vivo*. This may implicate a puzzling notion that the cytoplasmic domain was not essential for this process (Guang-Jer Wu, data not shown). However, the critical direct test of using only the cytoplasmic domain for inducing tumor has not been performed for LNCaP cells. From the above puzzling observation, it is very clear that a systematic study has also to be performed in other cancer cell lines before a definitive conclusion can be drawn.

#### **5.3 Signaling pathways in the METCAM/MUC18-mediated tumorigenesis and cancer metastasis**

The huMETCAM/MUC18 contains three sites which are potentially phosphorylated by PKC, PKA and CK2 in the cytoplasmic tail [38, 42]. However, these putative phosphorylation sites have not been biochemically proven. Thus, the immediate question to be answered is that how many sites in the cytoplasmic tail of the native METCAM/MUC18 protein, which are to be isolated from different cancer cell lines, are actually phosphorylated? Which protein kinase is responsible for the phosphorylation? After this is answered, then we can further study how METCAM/MUC18 mediates crosstalk and networking with different signal pathways and to see if it is similar to or different from the cytoplasmic tails of other CAMs [41, 75–77]. Knowledge learned from other CAMs seem point to one aspect that METCAM/MUC18, as an integral membrane protein and a cell adhesion molecule, should mediate inside-in, inside-out, and outside-in signals to participate in intercellular communication and interaction of cell with extra-cellular matrix, which results in impacting cell motility and invasiveness [78, 79]. Furthermore, its interaction with co-factors or cognate heterophilic ligand(s) may alter these signals, which in turn should affect intrinsic tumor proliferation or impact tumor angiogenesis and/or mediate targeting to specific organs and promoting metastasis. Moreover, METCAM/MUC18 may interact with various hormonal receptors, growth or anti-growth factors/receptors, various chemokines/receptors, and the Ca2+-mediated signaling members, which in turn affect the process of tumor progression. **Figure 6** summarizes the possible preliminary crosstalk of huMETCAM/ MUC18 with many members of signal transduction pathways that may affect its function during tumor initiation and development and malignant progression.

#### **5.4 Regulation of the huMETCAM/MUC18 gene transcription**

The mechanism of transcriptional control of METCAM/MUC18 gene is minimally studied [17]. Up to now, only the 900 bp sequences in the core promoter region of the huMETCAM/MUC18 gene are well-characterized [80]. This core promoter is rich in GC sequences but does not contain a TATA box. It includes many consensus sequences presumably as putative binding sites for various transcription regulatory factors, such as SP-1, CREB [81], AP-2 [82, 83], c-Myb [84], N-Oct2 (Brn2) [85], Ets [86], CArG [87], and Egr-1 [88]. In addition, it also contains three insulin responsive elements (one Ets and two E-box motifs) [89], suggesting that huMETCAM/MUC18 gene expression may respond to the cue of various growth signals [37, 40], as shown in **Figure 7**.

In addition, some sequences upstream of the minimal core promoter sequences should also be expected for conferring the tissue-specific expression of the huMET-CAM/MUC18 gene [90]. Recently this notion has been definitely supported by a finding that Ets sequence in the 10 kilo-bp up-stream region is involved in the regulation of the expression of huMETCAM/MUC18 gene [91]. We have also engaged in this task by searching the sequence of the up-stream region of the huMETCAM/ MUC18 promoter in the Celera or other web sites. By taking advantage of the

**31**

**Figure 6.**

**Figure 7.**

*of the huMETCAM/MUC18 gene.*

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

known sequence searched, we designed many pairs of primers to screening a genomic library and obtained several phage clones which contain at least 4 kilo-bp

*METCAM/MUC18-mediated signal transduction in tumorigenesis and malignant progression.*

The epigenetic control of the expression of huMETCAM/MUC18 gene has not been extensively studied in NPC, though it has been implicated [92]. This is because huMETCAM/MUC18 gene is located at the locus of human chromosome 11q23.3 that has been shown to be hypermethylated in NPC, suggesting that the expression of this gene may be regulated by epigenetic controls [93]. To support this notion, our preliminary results of treating NPC cell lines with 5-Aza-2′-deoxycytidine (Aza-C) showed that after the treatment with Aza-C, METCAM/MUC18 expression was somewhat elevated in the NPC-TW01 cell line, but not in the NPC-TW04 cell line (Guang-Jer Wu, unpublished data). METCAM/MUC18 has also been shown to be methylated in most of the early stage of prostate cancer [94]. Further systematic studies in this aspect should be very interesting and rewarding in the future.

*Putative transcription factor-recognized motifs in the 900 bp core promoter and 5–10 kilo bp up-stream region* 

of the gene for future studies (Guang-Jer Wu, unpublished data).

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

*METCAM/MUC18: A Novel Tumor Suppressor for Some Cancers DOI: http://dx.doi.org/10.5772/intechopen.86271*

#### **Figure 7.**

*Genes and Cancer*

definitive conclusion can be drawn.

**cancer metastasis**

the key role of the ecto-domain in tumorigenesis induction in prostate cancer cells *in vivo*. This may implicate a puzzling notion that the cytoplasmic domain was not essential for this process (Guang-Jer Wu, data not shown). However, the critical direct test of using only the cytoplasmic domain for inducing tumor has not been performed for LNCaP cells. From the above puzzling observation, it is very clear that a systematic study has also to be performed in other cancer cell lines before a

**5.3 Signaling pathways in the METCAM/MUC18-mediated tumorigenesis and** 

The huMETCAM/MUC18 contains three sites which are potentially phosphorylated by PKC, PKA and CK2 in the cytoplasmic tail [38, 42]. However, these putative phosphorylation sites have not been biochemically proven. Thus, the immediate question to be answered is that how many sites in the cytoplasmic tail of the native METCAM/MUC18 protein, which are to be isolated from different cancer cell lines, are actually phosphorylated? Which protein kinase is responsible for the phosphorylation? After this is answered, then we can further study how METCAM/MUC18 mediates crosstalk and networking with different signal pathways and to see if it is similar to or different from the cytoplasmic tails of other CAMs [41, 75–77]. Knowledge learned from other CAMs seem point to one aspect that METCAM/MUC18, as an integral membrane protein and a cell adhesion molecule, should mediate inside-in, inside-out, and outside-in signals to participate in intercellular communication and interaction of cell with extra-cellular matrix, which results in impacting cell motility and invasiveness [78, 79]. Furthermore, its interaction with co-factors or cognate heterophilic ligand(s) may alter these signals, which in turn should affect intrinsic tumor proliferation or impact tumor angiogenesis and/or mediate targeting to specific organs and promoting metastasis. Moreover, METCAM/MUC18 may interact with various hormonal receptors, growth or anti-growth factors/receptors, various chemokines/receptors, and the Ca2+-mediated signaling members, which in turn affect the process of tumor progression. **Figure 6** summarizes the possible preliminary crosstalk of huMETCAM/ MUC18 with many members of signal transduction pathways that may affect its func-

tion during tumor initiation and development and malignant progression.

The mechanism of transcriptional control of METCAM/MUC18 gene is minimally studied [17]. Up to now, only the 900 bp sequences in the core promoter region of the huMETCAM/MUC18 gene are well-characterized [80]. This core promoter is rich in GC sequences but does not contain a TATA box. It includes many consensus sequences presumably as putative binding sites for various transcription regulatory factors, such as SP-1, CREB [81], AP-2 [82, 83], c-Myb [84], N-Oct2 (Brn2) [85], Ets [86], CArG [87], and Egr-1 [88]. In addition, it also contains three insulin responsive elements (one Ets and two E-box motifs) [89], suggesting that huMETCAM/MUC18 gene expression may respond to the cue of various growth

In addition, some sequences upstream of the minimal core promoter sequences should also be expected for conferring the tissue-specific expression of the huMET-CAM/MUC18 gene [90]. Recently this notion has been definitely supported by a finding that Ets sequence in the 10 kilo-bp up-stream region is involved in the regulation of the expression of huMETCAM/MUC18 gene [91]. We have also engaged in this task by searching the sequence of the up-stream region of the huMETCAM/ MUC18 promoter in the Celera or other web sites. By taking advantage of the

**5.4 Regulation of the huMETCAM/MUC18 gene transcription**

signals [37, 40], as shown in **Figure 7**.

**30**

*Putative transcription factor-recognized motifs in the 900 bp core promoter and 5–10 kilo bp up-stream region of the huMETCAM/MUC18 gene.*

known sequence searched, we designed many pairs of primers to screening a genomic library and obtained several phage clones which contain at least 4 kilo-bp of the gene for future studies (Guang-Jer Wu, unpublished data).

The epigenetic control of the expression of huMETCAM/MUC18 gene has not been extensively studied in NPC, though it has been implicated [92]. This is because huMETCAM/MUC18 gene is located at the locus of human chromosome 11q23.3 that has been shown to be hypermethylated in NPC, suggesting that the expression of this gene may be regulated by epigenetic controls [93]. To support this notion, our preliminary results of treating NPC cell lines with 5-Aza-2′-deoxycytidine (Aza-C) showed that after the treatment with Aza-C, METCAM/MUC18 expression was somewhat elevated in the NPC-TW01 cell line, but not in the NPC-TW04 cell line (Guang-Jer Wu, unpublished data). METCAM/MUC18 has also been shown to be methylated in most of the early stage of prostate cancer [94]. Further systematic studies in this aspect should be very interesting and rewarding in the future.

#### **5.5 The possible roles of glycosylation on the protein of METCAM/MUC18 in tumorigenesis and tumor progression**

Glycosylation of a protein may affect the proper folding, stability, and/or activity of a protein [95], however, the possible roles of glycosylation in the function of MCETCAM/MUC18 protein have not been explored. The glycosylation of METCAM/MUC18 may also affect its ability in inducing/promoting or suppressing the metastasis of cancer cells [95–99]. Both huMETCAM/MUC18 and moMETCAM/MUC18 may very likely to be heavily glycosylated, sialylated, and post-translationally modified, because both have an apparent molecular weight of about 110–150 kDa, which is much more than the naked protein with a molecular weight of about 65–70 kDa [100]. To initiate the study, we subjected the huMET-CAM/MUC18, which was expressed in a human cancer cell line, to the digestion with N-glycosidase F, neuraminidase (sialidase), O-glycosidase, or endoglycosidase H, and we observed that the apparent molecular weight of the protein was decreased after digestion with N-glycosidase F and neuraminidase (sialidase), but not with O-glycosidase or endoglycosidase H [37, 40]. From this, we suggested that both sialic acid and N-glycans are probably the major carbohydrate side chains of huMETCAM/MUC18. It is also possible that glycosylation may differ depending on the type of cancers. Thus, we suggested that different N-glycans at the N-glycosylation sites of huMETCAM/MUC18 may differ in different cancer cell lines, which may have significant positive or negative impacts on their EMT abilities as well as tumorigenesis and metastasis. According to our hypothesis, a recent study described GCNT3 as an up-stream regulator of METCAM/MUC18. Moreover, GCNT3 glycosylates METCAM/MUC18 and extends its half-life which results in further elevation of S100A8/A9-mediated cellular motility in melanoma cells [101].

By searching in the primary sequence of the human huMETCAM/MUC18 protein, nine potential N-glycosylation sites (Asn-X-Ser/Thr or N-X-S/T sites) have been revealed [37, 38, 40, 42], whereas only seven sites found in the mouse METCAM/MUC18 [43]. Six N-glycosylation sites are conserved between the two proteins: 56/58 NL/FS, 418/420NRT, 449/451NLS, 467NGT/469NGS, 507NTS/509NTT, and 544/546NST [37, 38, 40, 42]. We suggest that only these six conserved N-glycosylation sites are actually glycosylated, because the apparent molecular weights of human METCAM/MUC18 and mouse METCAM/MUC18 are similar in the SDS gel. All the N-glycosylation sites are located in the external region of the protein, such as the domains of V1, C′, C″ and X. First, all these six sites should be biochemically identified before further molecular genetic task. Then, we will use genetic tools to alter the N-glycosylation sites. The mutants will be transfected back into cancer cell lines without the endogenous expression of the protein. The clones, which only express these mutated METCAM/MUC18, will be used for various *in vitro* and *in vivo* experiments to test the effect of N-glycosylation on the function huMETCAM/MUC18. They also will be used for testing effects on *in vitro* cell–cell aggregation and cell-extracellular matrix adhesion and on *in vivo* tumorigenesis and metastasis of human cancer cells. We anticipate that systematic studies on this aspect should be very informative to reveal the essential role of N-glycosylation played in the METCAM/MUC18-mediated tumor progression.

#### **6. Conclusions**

METCAM/MUC18 plays a key role in suppressing the progression of colorectal cancer, one mouse melanoma cell line, NPC type I, ovarian cancer, pancreatic cancer, prostate cancer PC-3 cell line, and perhaps hemangioma and possibly in other cancers.

**33**

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

**7. Research perspectives and clinical applications**

On the other hand, METCAM/MUC18 also play a key positive function in the progression of breast cancer, gastric cancer, hepatocellular carcinoma, lung cancer, melanoma, NPC type III, pancreatic cancer, and prostate cancer. To further understand its role in these processes, it is essential to further identify its co-factor regulators and cognate heterophilic ligands, define its functional domains, and study its crosstalk with members of various signal transduction pathways, the regulation of its expression at the level of transcription, and effects of N-glycosylation on the functions of the protein.

The current studies have laid an important biological basis for inspiring future intense investigation to further understand the detailed knowledge of METCAM/ MUC18-mediated suppression of tumorigenesis and metastasis of various cancer cell lines. For this purpose besides those have been described above, other future endeavors may include: (a) understanding three major mechanisms involved in METCAM/ MUC18-induced tumor and metastasis dormancy, such as key players participated in inhibition of intrinsic growth capability, key chemokines and cytokines participated in suppression of immunological responses, and key pro-angiogenic and antiangiogenic factors participated in the reduction of angiogenesis [102], (b) identification of possible miRNAs and non-coding RNAs participated in the process upstream and downstream of METCAM/MUC18 [103], and (c) possible clinical applications should be explored. Precaution should be taken that a complete picture may only be

possibly constructed after all the above studies are successfully executed.

The majority of the cancer-associated mortality is due to dissemination of primary tumor to distant organs (metastasis). If we are able to decrease or stop the metastatic propensity of cancer cells and keep them stayed only at the primary site, it should be a major success in cancer therapy. Alternatively, it is also a major success if we are able to control cancer cells at the state of dormancy or remaining them at the stage of micro-metastatic lesions [104]. Thus, similar to other tumor and metastasis suppressors, such as KISS1, KAI1, nm23, MAP2K4, and some micro-RNAs, METCAM/MUC18 may be used as a new therapeutic target for some clinical cancer treatments [105]. Strategically four major approaches may be taken for this purpose: (a) use gene therapeutic method to restore the functional copy of the suppressor genes or use epigenetic method to re-activate the genes. For gene therapy, the METCAM/MUC18 cDNA gene may be transported by an adenovirusassociated virus vector or a replication-defective adenovirus [106]. The human METCAM/MUC18 gene, located on 11q23-3 chromosome may be targeted with clinical reagents to reverse epigenetic repression, like Aza-C [107], or to change histone modifications to induce remodeling of the chromosome [108], (b) dispense recombinant proteins directly to the patients. For this approach, a complete copy or a partial portion of the METCAM/MUC18 recombinant protein, oligopeptides, or small molecule mimetics of METCAM/MUC18 may be directly dispensed to cancer patients, (c) target at downstream key members in the signaling pathways which are activated by the loss of the suppressor function, and (d) the co-factors or the cognate heterophilic ligand(s) of METCAM/MUC18 may be targeted. The above strategies may be used in single, or better in combination for treating the patients for the purpose of holding tumor cells at the primary sites, stopping them

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

**7.1 Research perspectives**

**7.2 Clinical applications**

On the other hand, METCAM/MUC18 also play a key positive function in the progression of breast cancer, gastric cancer, hepatocellular carcinoma, lung cancer, melanoma, NPC type III, pancreatic cancer, and prostate cancer. To further understand its role in these processes, it is essential to further identify its co-factor regulators and cognate heterophilic ligands, define its functional domains, and study its crosstalk with members of various signal transduction pathways, the regulation of its expression at the level of transcription, and effects of N-glycosylation on the functions of the protein.

### **7. Research perspectives and clinical applications**

#### **7.1 Research perspectives**

*Genes and Cancer*

**tumorigenesis and tumor progression**

**5.5 The possible roles of glycosylation on the protein of METCAM/MUC18 in** 

Glycosylation of a protein may affect the proper folding, stability, and/or activity of a protein [95], however, the possible roles of glycosylation in the function of MCETCAM/MUC18 protein have not been explored. The glycosylation of METCAM/MUC18 may also affect its ability in inducing/promoting or suppressing the metastasis of cancer cells [95–99]. Both huMETCAM/MUC18 and moMETCAM/MUC18 may very likely to be heavily glycosylated, sialylated, and post-translationally modified, because both have an apparent molecular weight of about 110–150 kDa, which is much more than the naked protein with a molecular weight of about 65–70 kDa [100]. To initiate the study, we subjected the huMET-CAM/MUC18, which was expressed in a human cancer cell line, to the digestion with N-glycosidase F, neuraminidase (sialidase), O-glycosidase, or endoglycosidase H, and we observed that the apparent molecular weight of the protein was decreased after digestion with N-glycosidase F and neuraminidase (sialidase), but not with O-glycosidase or endoglycosidase H [37, 40]. From this, we suggested that both sialic acid and N-glycans are probably the major carbohydrate side chains of huMETCAM/MUC18. It is also possible that glycosylation may differ depending on the type of cancers. Thus, we suggested that different N-glycans at the N-glycosylation sites of huMETCAM/MUC18 may differ in different cancer cell lines, which may have significant positive or negative impacts on their EMT abilities as well as tumorigenesis and metastasis. According to our hypothesis, a recent study described GCNT3 as an up-stream regulator of METCAM/MUC18. Moreover, GCNT3 glycosylates METCAM/MUC18 and extends its half-life which results in further elevation of S100A8/A9-mediated cellular motility in melanoma cells [101]. By searching in the primary sequence of the human huMETCAM/MUC18 protein, nine potential N-glycosylation sites (Asn-X-Ser/Thr or N-X-S/T sites) have been revealed [37, 38, 40, 42], whereas only seven sites found in the mouse METCAM/MUC18 [43]. Six N-glycosylation sites are conserved between the two proteins: 56/58 NL/FS, 418/420NRT, 449/451NLS, 467NGT/469NGS,

507NTS/509NTT, and 544/546NST [37, 38, 40, 42]. We suggest that only these six conserved N-glycosylation sites are actually glycosylated, because the apparent molecular weights of human METCAM/MUC18 and mouse METCAM/MUC18 are similar in the SDS gel. All the N-glycosylation sites are located in the external region of the protein, such as the domains of V1, C′, C″ and X. First, all these six sites should be biochemically identified before further molecular genetic task. Then, we will use genetic tools to alter the N-glycosylation sites. The mutants will be transfected back into cancer cell lines without the endogenous expression of the protein. The clones, which only express these mutated METCAM/MUC18, will be used for various *in vitro* and *in vivo* experiments to test the effect of N-glycosylation on the function huMETCAM/MUC18. They also will be used for testing effects on *in vitro* cell–cell aggregation and cell-extracellular matrix adhesion and on *in vivo* tumorigenesis and metastasis of human cancer cells. We anticipate that systematic studies on this aspect should be very informative to reveal the essential role of N-glycosylation played in the METCAM/MUC18-mediated tumor progression.

METCAM/MUC18 plays a key role in suppressing the progression of colorectal cancer, one mouse melanoma cell line, NPC type I, ovarian cancer, pancreatic cancer, prostate cancer PC-3 cell line, and perhaps hemangioma and possibly in other cancers.

**32**

**6. Conclusions**

The current studies have laid an important biological basis for inspiring future intense investigation to further understand the detailed knowledge of METCAM/ MUC18-mediated suppression of tumorigenesis and metastasis of various cancer cell lines. For this purpose besides those have been described above, other future endeavors may include: (a) understanding three major mechanisms involved in METCAM/ MUC18-induced tumor and metastasis dormancy, such as key players participated in inhibition of intrinsic growth capability, key chemokines and cytokines participated in suppression of immunological responses, and key pro-angiogenic and antiangiogenic factors participated in the reduction of angiogenesis [102], (b) identification of possible miRNAs and non-coding RNAs participated in the process upstream and downstream of METCAM/MUC18 [103], and (c) possible clinical applications should be explored. Precaution should be taken that a complete picture may only be possibly constructed after all the above studies are successfully executed.

#### **7.2 Clinical applications**

The majority of the cancer-associated mortality is due to dissemination of primary tumor to distant organs (metastasis). If we are able to decrease or stop the metastatic propensity of cancer cells and keep them stayed only at the primary site, it should be a major success in cancer therapy. Alternatively, it is also a major success if we are able to control cancer cells at the state of dormancy or remaining them at the stage of micro-metastatic lesions [104]. Thus, similar to other tumor and metastasis suppressors, such as KISS1, KAI1, nm23, MAP2K4, and some micro-RNAs, METCAM/MUC18 may be used as a new therapeutic target for some clinical cancer treatments [105]. Strategically four major approaches may be taken for this purpose: (a) use gene therapeutic method to restore the functional copy of the suppressor genes or use epigenetic method to re-activate the genes. For gene therapy, the METCAM/MUC18 cDNA gene may be transported by an adenovirusassociated virus vector or a replication-defective adenovirus [106]. The human METCAM/MUC18 gene, located on 11q23-3 chromosome may be targeted with clinical reagents to reverse epigenetic repression, like Aza-C [107], or to change histone modifications to induce remodeling of the chromosome [108], (b) dispense recombinant proteins directly to the patients. For this approach, a complete copy or a partial portion of the METCAM/MUC18 recombinant protein, oligopeptides, or small molecule mimetics of METCAM/MUC18 may be directly dispensed to cancer patients, (c) target at downstream key members in the signaling pathways which are activated by the loss of the suppressor function, and (d) the co-factors or the cognate heterophilic ligand(s) of METCAM/MUC18 may be targeted. The above strategies may be used in single, or better in combination for treating the patients for the purpose of holding tumor cells at the primary sites, stopping them

in a dormant state, or keeping the disseminating cancer cells at the state of micrometastases. However, the dual role of METCAM/MUC18 in cancer progression may limit the above clinical applications to only cancers exhibiting an anti-tumor activity mediated by METCAM/MUC18.

### **Acknowledgements**

I thank the support of grants from National Research Council, Taiwan.

### **Conflict of interests**

The author has no conflict of interests.

### **Author details**

Guang-Jer Wu

1 Department of Bioscience Technology, Center for Biomedical Technology, Chung Yuan Christian University, Taoyuan, Taiwan

2 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA

\*Address all correspondence to: guangj.wu@gmail.com

© 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.

**35**

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

[11] Wang Z, Zhao K, Hackert T, Zoller M. CD44/CD44v6 a reliable companion in cancer-initiating cell maintenance and tumor progression. Frontiers in Cell and Development Biology. 2018;**6**:97. DOI:

[12] Calinescu A, Turcu G, Nedelcu RI, Brinzea A, Hodorogea A, Antohe M,

[13] Bhatia P, Gautqm SK, Cannon A, Thompson C, Hall BR, Aithal A, et al. Cancer-associated mucins: Role in immune modulation and metastasis. Cancer Metastasis Review. 9 Jan 2019. 14p. DOI:10.1007/s10555-018-09775-0

[14] Altevogt P, Doberstein K, Fogel M. L1CAM in human cancer. International Journal of Cancer.

[15] Yahyazadeh Mashhadi SM, Kazemimanesh M, Arashkia A, Azadmanesh K, Meshkat Z, Golichenari B, et al. Shedding light on the EpCAM: An overview. Journal of Cellular Physiology. 2019;**234**(4):12569-12580

[16] Weidle UH, Eggle D, Klostermann S, Swart GWM. ALCAM/CD166: Cancer-related issues. Cancer Genomics and Proteomics. 2010;**7**(5):231-243

[17] Wu GJ. Chapter 13. Dual role of METCAM/MUC18 expression in the progression of cancer cells. In: Uchiumi F, editor. Gene Expression and Regulation in Mammalian Cells-Transcription from General Aspects. University Campus STeP Ri, Rijeka, Croatia: InTech Open Access Publisher; 2018. pp. 257-289.

2016;**138**:1565-1571

10.3389/fcell.2018.00097

et al. On the dual role of carcinoembryonic antigenrelated cell adhesion molecule 1 (CEACAM1) in human malignancies. Journal of Immunology Research. 2018;**2018**:ID7169081. 8p. DOI:

10.1155/2018/7169081

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

[1] Jackson M, Marks L, May GHW, Wilson JB. The genetic basis of disease. Essays in Biochemistry. 2018;**62**:643-723

[2] Klinsmith LJ. Principles of Cancer Biology. San Francisco: Pearson

[3] Weinberg RA. The Biology of Cancer. 1st ed. New York, USA and Abington,

Education Press; 2006

**References**

2016;**8**(a019505):1-35

UK: Garland Science; 2007

[4] Baylin SB, Jones PA. Epigenetic determinants in cancer. Cold Spring Harbor Perspectives in Biology.

[5] Yang H, Villani RM, Wang H, Simpson MJ, Roberts MS, Tang M, et al. The role of cellular reactive oxygen species in chemotherapy. Journal of Experimental and Clinical Cancer Research. 2018;**37**(266):1-10

[6] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;**144**:646-674. DOI: 10.1016/j.cell.2011.02.013

[8] Gkretsi V, Stylianopoulos T. Cell adhesion and matrix stiffness: Coordinating cancer cell invasion and metastasis. Frontiers in Oncology.

2018;**8**:145. DOI: 10.3389/

and cancer. Oncogene. 2018;**37**(35):4769-4780

s41568-018-0038-z

fonc.2018.00145

[7] Ramassone A, Pagotto S, Veronese A, Visone R. Epigenetics and MicroRNA in cancer. International Journal of Molecular Sciences. 2018;**19**(459):1-28

[9] Mendonsa A, Na TY, Gumbiner BM. E-cadherin in contact inhibition

[10] Hamidi H, Ivaska J. Every step of the way: Integrins in cancer progression and metastasis. Nature Reviews. Cancer. 2018;**18**(9):533-548. DOI: 10.1038/

### **References**

*Genes and Cancer*

**Acknowledgements**

**Conflict of interests**

**Author details**

Medicine, Atlanta, GA, USA

Guang-Jer Wu

activity mediated by METCAM/MUC18.

The author has no conflict of interests.

**34**

provided the original work is properly cited.

Chung Yuan Christian University, Taoyuan, Taiwan

\*Address all correspondence to: guangj.wu@gmail.com

© 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,

1 Department of Bioscience Technology, Center for Biomedical Technology,

2 Department of Microbiology and Immunology, Emory University School of

in a dormant state, or keeping the disseminating cancer cells at the state of micrometastases. However, the dual role of METCAM/MUC18 in cancer progression may limit the above clinical applications to only cancers exhibiting an anti-tumor

I thank the support of grants from National Research Council, Taiwan.

[1] Jackson M, Marks L, May GHW, Wilson JB. The genetic basis of disease. Essays in Biochemistry. 2018;**62**:643-723

[2] Klinsmith LJ. Principles of Cancer Biology. San Francisco: Pearson Education Press; 2006

[3] Weinberg RA. The Biology of Cancer. 1st ed. New York, USA and Abington, UK: Garland Science; 2007

[4] Baylin SB, Jones PA. Epigenetic determinants in cancer. Cold Spring Harbor Perspectives in Biology. 2016;**8**(a019505):1-35

[5] Yang H, Villani RM, Wang H, Simpson MJ, Roberts MS, Tang M, et al. The role of cellular reactive oxygen species in chemotherapy. Journal of Experimental and Clinical Cancer Research. 2018;**37**(266):1-10

[6] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;**144**:646-674. DOI: 10.1016/j.cell.2011.02.013

[7] Ramassone A, Pagotto S, Veronese A, Visone R. Epigenetics and MicroRNA in cancer. International Journal of Molecular Sciences. 2018;**19**(459):1-28

[8] Gkretsi V, Stylianopoulos T. Cell adhesion and matrix stiffness: Coordinating cancer cell invasion and metastasis. Frontiers in Oncology. 2018;**8**:145. DOI: 10.3389/ fonc.2018.00145

[9] Mendonsa A, Na TY, Gumbiner BM. E-cadherin in contact inhibition and cancer. Oncogene. 2018;**37**(35):4769-4780

[10] Hamidi H, Ivaska J. Every step of the way: Integrins in cancer progression and metastasis. Nature Reviews. Cancer. 2018;**18**(9):533-548. DOI: 10.1038/ s41568-018-0038-z

[11] Wang Z, Zhao K, Hackert T, Zoller M. CD44/CD44v6 a reliable companion in cancer-initiating cell maintenance and tumor progression. Frontiers in Cell and Development Biology. 2018;**6**:97. DOI: 10.3389/fcell.2018.00097

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[19] Zeng GF, Cai SX, Liu Y, Wu GJ. METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells. Gene. 2012;**492**:229-238

[20] Huang CY, Wu GJ. METCAM/ MUC18 promoted tumorigenesis of human breast cancer SK-BR-3 cells in a dosage-specific manner. Taiwanese Journal of Obstetrics and Gynecology. 2016;**55**(2):202-212. DOI: 10.1016/j. tjog.2016.02.010

[21] Wu GJ, Fu P, Wang SW, Wu MWH. Enforced expression of MCAM/MUC18 increases in vitro motility and invasiveness and in vivo metastasis of two mouse melanoma K1735 sublines in a syngeneic mouse model. Molecular Cancer Research. 2008;**6**(11):1666-1677

[22] Wu GJ. Ectopic expression of MCAM/MUC18 increases in vitro motility and invasiveness, but decreases in vivo tumorigenesis and metastasis of a mouse melanoma K1735-9 subline in a syngeneic mouse model. Clinical & Experimental Metastasis. 2016;**33**(8):817-828. DOI: 10.1007/ s10585-016-9812-z

[23] Lin JC, Chiang CF, Wang SW, Wang WY, Kwuan PC, Wu GJ. Significance and expression of human METCAM/ MUC18 in nasopharyngeal carcinoma (NPC) and metastatic lesions. Asian Pacific Journal of Cancer Prevention. 2014;**15**(1):245-252

[24] Liu YC. Putative roles of huMETCAM in modulating the development and progression of nasopharyngeal carcinoma [thesis]. Chung Yuan Christian University; 2014. Available from: http://www.lib.cycu. edu.tw/thesis

[25] Liu YC, Chen YR, Wu GJ. METCAM/MUC18 plays a tumor suppressor role in the development of nasopharyngeal carcinoma type I. 2019. (Submitted)

[26] Liu YC, Ke CC, Chen YR, Wu GJ. METCAM/MUC18 plays a tumor promoter role in the development of nasopharyngeal carcinoma type III. 2019. (Submitted)

[27] Wu GJ, Dickerson EB. Frequent and increased expression of human METCAM/MUC18 in cancer tissues and metastatic lesions associates with the clinical progression of human ovarian carcinoma. Taiwanese Journal of Obstetrics and Gynecology. 2014;**53**:509-517

[28] Wu GJ, Zeng GF. METCAM/ MUC18 is a novel tumor and metastasis suppressor for the human ovarian cancer SKOV3 cells. BMC Cancer. 2016;**16**:136. DOI: 10.1186/ S12885-016-2181-9

[29] Wu GJ. METCAM/MUC18 plays a novel tumor and metastasis suppressor role in the progression of human ovarian cancer cells. Obstetrics & Gynecology International Journal. 2017;**6**(4): 00210, pp. 1-8

[30] Wu GJ. METCAM/MUC18 decreases the malignant propensity of human ovarian carcinoma cells. International Journal of Molecular Sciences. 2018;**19**:02976

[31] Wu GJ, Varma VA, Wu MWH, Yang H, Wang SWC, Liu Z, et al. Expression of a human cell adhesion molecule, MUC18, in prostate cancer cell lines and tissues. The Prostate. 2001;**48**:305-315

**37**

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

progression of melanocytes to

[40] Wu GJ. METCAM/MUC18 expression and cancer metastasis. Current Genomics. 2005;**6**:333-349

[41] Anfosso F, Bardin N, Frances V, Vivier E, Camoin-Jau L, Sampol J, et al. Activation of human endothelial cells via S-Endo-1 antigen (CD146) stimulates the tyrosine phosphorylation of focal adhesion kinase p125FAK. The Journal of Biological Chemistry.

[42] Wu GJ, Wu MWH, Wang SW, Liu Z, Peng Q, Qu P, et al. Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cells and tissues with malignant progression. Gene.

[43] Yang H, Wang SWC, Liu Z, Wu MWH, McAlpine B, Ansel J, et al. Isolation and characterization of murine MUC18 cDNA gene, and correlation of MUC18 expression in murine melanoma cell lines with metastatic ability. Gene.

[44] Shih IM. The role of CD146 (Mel-CAM) in biology and pathology. Journal

[45] Zeng Q, Li W, Lu D, Wu Z, Duan H, Luo Y, et al. CD146, an epithelialmesenchymal transition inducer, is associated with triple-negative breast cancer. Proceedings of the National Academy of Sciences of the United States of America.

[46] Li Q, Yu Y, Bischoff J, Milliken JB, Olsen BR. Differential expression of CD146 in tissues and endothelial cells derived from infantile hemangioma and normal human skin. Journal of Pathology. 2003;**201**:296-302

of Pathology. 1999;**189**:4-11

2012;**109**(4):1127-1132

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melanoma. Experimental Dermatology.

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

[32] Wu GJ, Peng Q, Fu P, Chiang CF, Wang SWC, Dillehay DL, et al. Ectopical expression of human MUC18 increases metastasis of human prostate cancer LNCaP cells. Gene. 2004;**327**:201-213

[33] Wu GJ, Chiang CF, Fu P, Hess W, Greenberg N, Wu MWH. Increased expression of MUC18 correlates with the metastatic progression of mouse prostate adenocarcinoma in the (TRAMP) model. Journal of Urology.

[34] Wu GJ, Wu MWH, Liu Y. Enforced expression of human METCAM/ MUC18 increases the tumorigenesis of human prostate cancer cells in nude mice. Journal of Urology.

[35] Wu GJ. Human METCAM/MUC18 as a novel biomarker to drive and its specific SiRNAs to block the malignant

[36] Wu GJ. Human METCAM/MUC18 is a new diagnostic marker of and a driver for promoting and its specific siRNAs, derived oligopeptides and antibodies be used for decreasing the malignant progression of prostate cancer. Journal of Stem Cell Research &

progression of prostate cancer. Journal of Cell Science and Therapy.

Therapeutics. 2016;**1**(5):00035

DOI: 10.1155/2012/853797

[37] Wu GJ. Dual role of METCAM in the progression of different cancers. Journal of Oncology. 2012;**2012**:853797.

[38] Lehmann JM, Reithmuller G, Johnson JP. MUC18, a marker of tumor progression in human melanoma. Proceedings of the National Academy of Sciences of the United States of America. 1989;**86**:9891-9895

[39] Meier F, Caroli U, Satyamoorthy K, Schittek B, Bauer J, Berking C, et al. Fibroblast growth factor-2 but not Mel-CAM and/or β3 integrin promotes

2005;**173**:1778-1783

2011;**185**:1504-1512

2015;**6**(5):1000227

*METCAM/MUC18: A Novel Tumor Suppressor for Some Cancers DOI: http://dx.doi.org/10.5772/intechopen.86271*

[32] Wu GJ, Peng Q, Fu P, Chiang CF, Wang SWC, Dillehay DL, et al. Ectopical expression of human MUC18 increases metastasis of human prostate cancer LNCaP cells. Gene. 2004;**327**:201-213

*Genes and Cancer*

978-953-51-3855-6

ISBN 978-953-51-3856-3, Print ISBN

development and progression of nasopharyngeal carcinoma [thesis]. Chung Yuan Christian University; 2014. Available from: http://www.lib.cycu.

[25] Liu YC, Chen YR, Wu GJ. METCAM/MUC18 plays a tumor suppressor role in the development of nasopharyngeal carcinoma type I. 2019.

[26] Liu YC, Ke CC, Chen YR, Wu GJ. METCAM/MUC18 plays a tumor promoter role in the development of nasopharyngeal carcinoma type III.

[27] Wu GJ, Dickerson EB. Frequent and increased expression of human METCAM/MUC18 in cancer tissues and metastatic lesions associates with the clinical progression of human ovarian carcinoma. Taiwanese Journal of Obstetrics and Gynecology.

[28] Wu GJ, Zeng GF. METCAM/ MUC18 is a novel tumor and

metastasis suppressor for the human ovarian cancer SKOV3 cells. BMC Cancer. 2016;**16**:136. DOI: 10.1186/

[29] Wu GJ. METCAM/MUC18 plays a novel tumor and metastasis suppressor role in the progression of human ovarian cancer cells. Obstetrics & Gynecology International Journal. 2017;**6**(4):

[30] Wu GJ. METCAM/MUC18 decreases the malignant propensity of human ovarian carcinoma cells. International Journal of Molecular

[31] Wu GJ, Varma VA, Wu MWH, Yang H, Wang SWC, Liu Z, et al. Expression of a human cell adhesion molecule, MUC18, in prostate cancer cell lines and tissues. The Prostate.

Sciences. 2018;**19**:02976

2001;**48**:305-315

edu.tw/thesis

(Submitted)

2019. (Submitted)

2014;**53**:509-517

S12885-016-2181-9

00210, pp. 1-8

Up-regulation of METCAM/MUC18 promotes motility, invasion, and tumorigenesis of human breast cancer cells. BMC Cancer. 2011;**11**:113. DOI:

[19] Zeng GF, Cai SX, Liu Y, Wu GJ. METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells. Gene.

[20] Huang CY, Wu GJ. METCAM/ MUC18 promoted tumorigenesis of human breast cancer SK-BR-3 cells in a dosage-specific manner. Taiwanese Journal of Obstetrics and Gynecology. 2016;**55**(2):202-212. DOI: 10.1016/j.

[21] Wu GJ, Fu P, Wang SW, Wu MWH. Enforced expression of MCAM/MUC18 increases in vitro motility and invasiveness and in vivo metastasis of two mouse melanoma K1735 sublines in a syngeneic mouse model. Molecular Cancer Research.

[22] Wu GJ. Ectopic expression of MCAM/MUC18 increases in vitro motility and invasiveness, but decreases in vivo tumorigenesis and metastasis of a mouse melanoma K1735-9 subline in a syngeneic mouse model. Clinical & Experimental Metastasis. 2016;**33**(8):817-828. DOI: 10.1007/

[23] Lin JC, Chiang CF, Wang SW, Wang WY, Kwuan PC, Wu GJ. Significance and expression of human METCAM/ MUC18 in nasopharyngeal carcinoma (NPC) and metastatic lesions. Asian Pacific Journal of Cancer Prevention.

[18] Zeng GF, Cai SX, Wu GJ.

10.1186/1471-2407-11-113

2012;**492**:229-238

tjog.2016.02.010

2008;**6**(11):1666-1677

s10585-016-9812-z

2014;**15**(1):245-252

[24] Liu YC. Putative roles of huMETCAM in modulating the

**36**

[33] Wu GJ, Chiang CF, Fu P, Hess W, Greenberg N, Wu MWH. Increased expression of MUC18 correlates with the metastatic progression of mouse prostate adenocarcinoma in the (TRAMP) model. Journal of Urology. 2005;**173**:1778-1783

[34] Wu GJ, Wu MWH, Liu Y. Enforced expression of human METCAM/ MUC18 increases the tumorigenesis of human prostate cancer cells in nude mice. Journal of Urology. 2011;**185**:1504-1512

[35] Wu GJ. Human METCAM/MUC18 as a novel biomarker to drive and its specific SiRNAs to block the malignant progression of prostate cancer. Journal of Cell Science and Therapy. 2015;**6**(5):1000227

[36] Wu GJ. Human METCAM/MUC18 is a new diagnostic marker of and a driver for promoting and its specific siRNAs, derived oligopeptides and antibodies be used for decreasing the malignant progression of prostate cancer. Journal of Stem Cell Research & Therapeutics. 2016;**1**(5):00035

[37] Wu GJ. Dual role of METCAM in the progression of different cancers. Journal of Oncology. 2012;**2012**:853797. DOI: 10.1155/2012/853797

[38] Lehmann JM, Reithmuller G, Johnson JP. MUC18, a marker of tumor progression in human melanoma. Proceedings of the National Academy of Sciences of the United States of America. 1989;**86**:9891-9895

[39] Meier F, Caroli U, Satyamoorthy K, Schittek B, Bauer J, Berking C, et al. Fibroblast growth factor-2 but not Mel-CAM and/or β3 integrin promotes

progression of melanocytes to melanoma. Experimental Dermatology. 2003;**12**:296-306

[40] Wu GJ. METCAM/MUC18 expression and cancer metastasis. Current Genomics. 2005;**6**:333-349

[41] Anfosso F, Bardin N, Frances V, Vivier E, Camoin-Jau L, Sampol J, et al. Activation of human endothelial cells via S-Endo-1 antigen (CD146) stimulates the tyrosine phosphorylation of focal adhesion kinase p125FAK. The Journal of Biological Chemistry. 1998;**273**:26852-26858

[42] Wu GJ, Wu MWH, Wang SW, Liu Z, Peng Q, Qu P, et al. Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cells and tissues with malignant progression. Gene. 2001;**279**:17-31

[43] Yang H, Wang SWC, Liu Z, Wu MWH, McAlpine B, Ansel J, et al. Isolation and characterization of murine MUC18 cDNA gene, and correlation of MUC18 expression in murine melanoma cell lines with metastatic ability. Gene. 2001;**265**:133-145

[44] Shih IM. The role of CD146 (Mel-CAM) in biology and pathology. Journal of Pathology. 1999;**189**:4-11

[45] Zeng Q, Li W, Lu D, Wu Z, Duan H, Luo Y, et al. CD146, an epithelialmesenchymal transition inducer, is associated with triple-negative breast cancer. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(4):1127-1132

[46] Li Q, Yu Y, Bischoff J, Milliken JB, Olsen BR. Differential expression of CD146 in tissues and endothelial cells derived from infantile hemangioma and normal human skin. Journal of Pathology. 2003;**201**:296-302

[47] Lin CT, Wong CI, Chan WY, et al. Establishment and characterization of two nasopharyngeal carcinoma cell lines. Laboratory Investigation. 1990;**62**:713-724

[48] Aldovini D, Demichelis F, Doglioni C, Di Vizio D, Galligioni E, et al. M-CAM expression as marker of poor prognosis in epithelial ovarian cancer. International Journal of Cancer. 2006;**119**(8):1920-1926

[49] Wu Z, Wu ZY, Li J, Yang X, Wang Y, et al. MCAM is a novel metastasis marker and regulates spreading, apoptosis and invasion of ovarian cancer cells. Tumor Biology. 2012;**33**:1619-1628

[50] Wu GJ. Enforced expression of METCAM/MUC18 decreases in vitro motility and invasiveness and tumorigenesis and in vivo tumorigenesis of human ovarian cancer BG-1 cells. In: Schatten H, editor. Ovarian Cancer: Molecular & Diagnostic Imaging and Treatment Strategies, Advances in Experimental Medicine and Biology. Humana Press (Springer Science + Business Media LLC); 2019. (In press)

[51] Wu G-J, Chang YR, Chu JT. METCAM/MUC18 plays a positive role in the tumorigenesis of human prostate cancer DU145 cells: Knockdown effects shRNAs decreasing tumorigenicity in nude mice. 2019. (Submitted)

[52] Wu G-J, Chang YR, Chu JT. METCAM/MUC18 plays a negative role in the tumorigenesis of human prostate cancer PC-3 cells: Knockdown effects shRNAs increasing tumorigenicity in nude mice. 2019. (Submitted)

[53] Zeng P, Li H, Lu PH, Zhou LN, Tang M, Liu CY, et al. Prognostic value of CD146 in solid tumor: A systematic review and meta-analysis. Scientific Reports. 2017;**7**(1):4223. DOI: 10.1038/ s41598-017-01061-3

[54] Liu D, Du L, Chen D, Ye Z, Duan H, Tu T, et al. Reduced CD146 expression

promotes tumorigenesis and cancer stemness in colorectal cancer through activating Wnt/β-catenin signaling. Oncotarget. 2016;**7**(26):40704-40718

[55] Stalin J, Nollet M, Garigue P, Fernandez S, Vivavancos L, Essaah A, et al. Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth, and survival of CD146 positive tumors. Oncogene. 2016;**35**:5489-5500

[56] Zheng B, Ohuchida K, Chijiiwa Y, Zhao M, Mizuuchi Y, Cui L, et al. CD146 attenuation in cancer-associated fibroblasts promotes pancreatic cancer progression. Molecular Carcinogenesis. 2016;**55**(11):1560-1572

[57] Liu WF, Ji SR, Sun JJ, Zhang Y, Liu ZY, Liang AB, et al. Gastric cancer CD146 expression correlates with epithelial-mesenchymal transition markers and a poor prognosis in gastric cancer. International Journal of Molecular Sciences. 2012;**13**:6399-6406

[58] Yang Y, Hernandez R, Rao J, Yin L, Qu Y, Wu J, et al. Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**(47):E6525-E6534

[59] Jiang G, Zhang L, Zhu Q, Bai D, Zhang C, Wang X. CD146 promotes metastasis and predicts poor prognosis of hepatocellular carcinoma. Journal of Experimental & Clinical Cancer Research. 2016;**35**:38. DOI: 10.1186/ s13046-016-0313-3

[60] Chen K, Ding A, Ding Y, Ghanekar A. High-throughput flow cytometry screening of human hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells. Biochemistry and Biophysics Reports. 2016;**8**:107-113

**39**

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

et al. A fully human anti-melanoma cellular adhesion molecule/MUC18 antibody inhibits spontaneous

pulmonary metastasis of osteosarcoma cells in vivo. Clinical Cancer Research.

[69] Westrøm S, Bønsdorff TB, Abbas N, Bruland ØS, Jonasdottir TJ, Mælandsmo GM, et al. Evaluation of CD146 as target for radioimmunotherapy against osteosarcoma. PLoS One. 2016;**11**(10):e0165382. DOI: 10.1371/

[70] von Burstin J, Bachhuber F, Paul M, Schmid RM, Rustgi AK. The TALE homeodomain transcription factor MEIS1 activates the pro-metastatic melanoma cell adhesion molecule MCAM to promote migration of pancreatic cancer cells. Molecular Carcinogenesis. 2017;**56**(3):936-944

[71] LaMontagne KR Jr, Moses MA, Wiederschain D, Mahajan S, Holden J, Ghazizadeh H, et al. Inhibition of MAP kinase causes morphological reversion and dissociation between soft agar growth and in vivo tumorigenesis in angiosarcoma cells. American Journal of Pathology. 2000;**157**:1937-1945

[72] Xie S, Luca M, Huang S, Gutman M, Reich R, Johnson JP, et al. Expression of MCAM/MCU18 by human melanoma cells leads to increased tumor growth and metastasis. Cancer Research.

[73] Schlagbauer-Wadl H, Jansen B, Muller M, Polterauer P, Wolff K, Eichler HG, et al. Influence of MUC18/ MCAM/CD146 expression on human melanoma growth and metastasis in SCID mice. International Journal of

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[62] Oka S, Uramoto H, Chikaishi Y, Tanaka F. The expression of CD146 predicts a poor overall survival in patients with adenocarcinoma of the lung. Anticancer Research.

[63] Zhang X, Wang Z, Kang Y, Li X, Ma X, Ma L. MCAM expression is associated with poor prognosis in non-small cell lung cancer. Clinical and Translational Oncology. 2014;**16**:178-183

[64] Zhang F, Wang J, Wang X, Wei N, Liu H, Zhang X. CD146-mediated acquisition of stemness phenotype enhances tumor invasion and metastasis after EGFR-TKI resistance in lung cancer. Clinical Respiratory Journal.

[65] England CG, Jiang D, Hernandez R, Sun H, Valdovinos HF, Ehlerding EB, et al. ImmunoPET imaging of CD146 in murine models of intrapulmonary metastasis of non-small cell lung cancer. Molecular Pharmaceutics.

2003;**25**:77-81

2012;**32**:861-864

2019;**13**(1):23-33

2017;**14**(10):3239-3247

[66] Tripathi SC, Fahrmann JF, Celiktas M, Aguilar M, Marini KD, Jolly MK, et al. A novel mechanism of chemoresistance in small cell lung cancer mediated by MCAM via PI3K/ AKT/SOX2 signaling pathway. Cancer Research. 2017;**77**(16):4414-44252

[67] Schiano C, Grimaldi V,

Casamassimi A, Infante T, Esposito A, Giovane A, et al. Different expression of CD146 in human normal and osteosarcoma cell lines. Medical Oncology. 2012;**29**(4):2998-3002

[68] McGary EC, Heimberger A, Mills L, Weber K, Thomas GW, Shtivelband M,

#### *METCAM/MUC18: A Novel Tumor Suppressor for Some Cancers DOI: http://dx.doi.org/10.5772/intechopen.86271*

[61] Kristiansen G, Yu Y, Schlüns K, Sers C, Dietel M, Petersen I. Expression of the cell adhesion molecule CD146/ MCAM in non-small cell lung cancer. Analytical Cellular Pathology. 2003;**25**:77-81

*Genes and Cancer*

1990;**62**:713-724

[47] Lin CT, Wong CI, Chan WY, et al. Establishment and characterization of two nasopharyngeal carcinoma cell lines. Laboratory Investigation.

promotes tumorigenesis and cancer stemness in colorectal cancer through activating Wnt/β-catenin signaling. Oncotarget. 2016;**7**(26):40704-40718

[55] Stalin J, Nollet M, Garigue P, Fernandez S, Vivavancos L, Essaah A, et al. Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth, and survival of CD146 positive tumors. Oncogene.

[56] Zheng B, Ohuchida K, Chijiiwa Y, Zhao M, Mizuuchi Y, Cui L, et al. CD146 attenuation in cancer-associated fibroblasts promotes pancreatic cancer progression. Molecular Carcinogenesis.

[57] Liu WF, Ji SR, Sun JJ, Zhang Y, Liu ZY, Liang AB, et al. Gastric cancer CD146 expression correlates with epithelial-mesenchymal transition markers and a poor prognosis in gastric cancer. International Journal of Molecular Sciences.

[58] Yang Y, Hernandez R, Rao J, Yin L, Qu Y, Wu J, et al. Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**(47):E6525-E6534

[59] Jiang G, Zhang L, Zhu Q, Bai D, Zhang C, Wang X. CD146 promotes metastasis and predicts poor prognosis of hepatocellular carcinoma. Journal of Experimental & Clinical Cancer Research. 2016;**35**:38. DOI: 10.1186/

2016;**35**:5489-5500

2016;**55**(11):1560-1572

2012;**13**:6399-6406

s13046-016-0313-3

2016;**8**:107-113

[60] Chen K, Ding A, Ding Y, Ghanekar A. High-throughput flow cytometry screening of human

hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells. Biochemistry and Biophysics Reports.

[48] Aldovini D, Demichelis F, Doglioni C, Di Vizio D, Galligioni E, et al. M-CAM expression as marker of poor prognosis in epithelial ovarian cancer. International Journal of Cancer. 2006;**119**(8):1920-1926

[49] Wu Z, Wu ZY, Li J, Yang X, Wang Y, et al. MCAM is a novel metastasis marker and regulates spreading,

apoptosis and invasion of ovarian cancer cells. Tumor Biology. 2012;**33**:1619-1628

[50] Wu GJ. Enforced expression of METCAM/MUC18 decreases in vitro motility and invasiveness and tumorigenesis and in vivo tumorigenesis of human ovarian cancer BG-1 cells. In: Schatten H, editor. Ovarian Cancer: Molecular & Diagnostic Imaging and Treatment Strategies, Advances in Experimental Medicine and Biology. Humana Press (Springer Science + Business Media LLC); 2019. (In press)

[51] Wu G-J, Chang YR, Chu JT.

nude mice. 2019. (Submitted)

[52] Wu G-J, Chang YR, Chu JT.

nude mice. 2019. (Submitted)

s41598-017-01061-3

[53] Zeng P, Li H, Lu PH, Zhou LN, Tang M, Liu CY, et al. Prognostic value of CD146 in solid tumor: A systematic review and meta-analysis. Scientific Reports. 2017;**7**(1):4223. DOI: 10.1038/

[54] Liu D, Du L, Chen D, Ye Z, Duan H, Tu T, et al. Reduced CD146 expression

METCAM/MUC18 plays a positive role in the tumorigenesis of human prostate cancer DU145 cells: Knockdown effects shRNAs decreasing tumorigenicity in

METCAM/MUC18 plays a negative role in the tumorigenesis of human prostate cancer PC-3 cells: Knockdown effects shRNAs increasing tumorigenicity in

**38**

[62] Oka S, Uramoto H, Chikaishi Y, Tanaka F. The expression of CD146 predicts a poor overall survival in patients with adenocarcinoma of the lung. Anticancer Research. 2012;**32**:861-864

[63] Zhang X, Wang Z, Kang Y, Li X, Ma X, Ma L. MCAM expression is associated with poor prognosis in non-small cell lung cancer. Clinical and Translational Oncology. 2014;**16**:178-183

[64] Zhang F, Wang J, Wang X, Wei N, Liu H, Zhang X. CD146-mediated acquisition of stemness phenotype enhances tumor invasion and metastasis after EGFR-TKI resistance in lung cancer. Clinical Respiratory Journal. 2019;**13**(1):23-33

[65] England CG, Jiang D, Hernandez R, Sun H, Valdovinos HF, Ehlerding EB, et al. ImmunoPET imaging of CD146 in murine models of intrapulmonary metastasis of non-small cell lung cancer. Molecular Pharmaceutics. 2017;**14**(10):3239-3247

[66] Tripathi SC, Fahrmann JF, Celiktas M, Aguilar M, Marini KD, Jolly MK, et al. A novel mechanism of chemoresistance in small cell lung cancer mediated by MCAM via PI3K/ AKT/SOX2 signaling pathway. Cancer Research. 2017;**77**(16):4414-44252

[67] Schiano C, Grimaldi V, Casamassimi A, Infante T, Esposito A, Giovane A, et al. Different expression of CD146 in human normal and osteosarcoma cell lines. Medical Oncology. 2012;**29**(4):2998-3002

[68] McGary EC, Heimberger A, Mills L, Weber K, Thomas GW, Shtivelband M,

et al. A fully human anti-melanoma cellular adhesion molecule/MUC18 antibody inhibits spontaneous pulmonary metastasis of osteosarcoma cells in vivo. Clinical Cancer Research. 2003;**9**:6560-6566

[69] Westrøm S, Bønsdorff TB, Abbas N, Bruland ØS, Jonasdottir TJ, Mælandsmo GM, et al. Evaluation of CD146 as target for radioimmunotherapy against osteosarcoma. PLoS One. 2016;**11**(10):e0165382. DOI: 10.1371/ journal.pone.0165382

[70] von Burstin J, Bachhuber F, Paul M, Schmid RM, Rustgi AK. The TALE homeodomain transcription factor MEIS1 activates the pro-metastatic melanoma cell adhesion molecule MCAM to promote migration of pancreatic cancer cells. Molecular Carcinogenesis. 2017;**56**(3):936-944

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melanocytic phenotype and tumorigenic potential of human melanoma cells.

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[88] Baron V, Duss S, Rhim J, Mercola D.

Cell. 2003;**14**:2151-2162

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of Sciences. 2003;**1002**:197-216

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The brn-2 gene regulates the

Oncogene. 1995;**11**:691-700

1996;**56**:2218-2223

1998;**273**:16501-16508

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Biology. 1993;**5**:819-831

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**40**

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**43**

Section 2

Genes with Dual Tumor

Suppressor and Oncogenic

Activities

### Section 2
