Mechanisms of Tumor Progression and Metastasis

**3**

**Chapter 1**

*Guang-Jer Wu*

**Abstract**

mouse models

**metastasis**

METCAM/MUC18 Promotes

in Most Human Cancers

be used as therapeutic agents to treat these cancers.

Tumor Progression and Metastasis

In addition to oncogenes and tumor suppressor genes, cell adhesion molecules (CAMs) also significantly contribute to tumor progression and metastasis. For the past two decades, we have demonstrated that METCAM/MUC18, a cell adhesion molecule in the immunoglobulin-like gene superfamily, orchestrates complex interactions of tumor cells with various stromal cells in the tumor microenvironment, resulting in augmentation or reduction of the metastatic potential of carcinoma cells. Here we show that METCAM/MUC18 plays a positive role in the tumor progression and metastasis in most human cancers, such as breast cancer, human melanoma and most mouse melanoma, nasopharyngeal carcinoma type III, prostate cancer LNCaP and DU145 cell lines, and perhaps angiosarcoma, gastric cancer, glioma, hepatocellular carcinoma, non-small cell lung adenocarcinoma, small cell lung cancer (SCLC), osteosarcoma, and human and mouse pancreatic cancer. Possible mechanisms in the METCAM/MUC18-mediated tumor progression and metastasis are proposed. Anti-METCAM/MUC18 antibodies and siRNAs may

**Keywords:** METCAM/MUC18, Ig-like CAM, tumor promotion, metastasis, breast cancer, melanoma, nasopharyngeal carcinoma, prostate cancer, many solid tumors,

**1. Introduction: cancer and CAM-mediated tumor progression and** 

Tumor/cancer is a chronic disease resulting from gradually accumulation of mutations or epigenetic alterations in our genetic material, DNA [1]. Ten to twenty percent cancer risk comes from hereditary factors and 80–90% of cancer risk from environmental factors [2]. The environmental factors in the physical containment include (a) chemically polluted drinking water, air and soil, and diet; (b) irradiation from solar UV, artificial sources, and environmental radioactive elements; (c) pathological agents (tumor viruses, bacteria, and parasites); and (d) the lifestyle (stress, chronic inflammation from obesity, and free radicals from metabolism) [3–5]. These agents aim to attack our DNA in the somatic cells resulting in slow accumulation of mutations and epigenetic alterations in our genes throughout the life span [6]. The question of "Is cancer a metabolic disease or a genetic disease?" cannot be easily answered. Prior to 1970, most cancer researchers thought cancer is a metabolic disease because of the Warburg effect. After 1970 when Warburg died and after 1971 when oncogenes

#### **Chapter 1**

## METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers

*Guang-Jer Wu*

#### **Abstract**

In addition to oncogenes and tumor suppressor genes, cell adhesion molecules (CAMs) also significantly contribute to tumor progression and metastasis. For the past two decades, we have demonstrated that METCAM/MUC18, a cell adhesion molecule in the immunoglobulin-like gene superfamily, orchestrates complex interactions of tumor cells with various stromal cells in the tumor microenvironment, resulting in augmentation or reduction of the metastatic potential of carcinoma cells. Here we show that METCAM/MUC18 plays a positive role in the tumor progression and metastasis in most human cancers, such as breast cancer, human melanoma and most mouse melanoma, nasopharyngeal carcinoma type III, prostate cancer LNCaP and DU145 cell lines, and perhaps angiosarcoma, gastric cancer, glioma, hepatocellular carcinoma, non-small cell lung adenocarcinoma, small cell lung cancer (SCLC), osteosarcoma, and human and mouse pancreatic cancer. Possible mechanisms in the METCAM/MUC18-mediated tumor progression and metastasis are proposed. Anti-METCAM/MUC18 antibodies and siRNAs may be used as therapeutic agents to treat these cancers.

**Keywords:** METCAM/MUC18, Ig-like CAM, tumor promotion, metastasis, breast cancer, melanoma, nasopharyngeal carcinoma, prostate cancer, many solid tumors, mouse models

#### **1. Introduction: cancer and CAM-mediated tumor progression and metastasis**

Tumor/cancer is a chronic disease resulting from gradually accumulation of mutations or epigenetic alterations in our genetic material, DNA [1]. Ten to twenty percent cancer risk comes from hereditary factors and 80–90% of cancer risk from environmental factors [2]. The environmental factors in the physical containment include (a) chemically polluted drinking water, air and soil, and diet; (b) irradiation from solar UV, artificial sources, and environmental radioactive elements; (c) pathological agents (tumor viruses, bacteria, and parasites); and (d) the lifestyle (stress, chronic inflammation from obesity, and free radicals from metabolism) [3–5]. These agents aim to attack our DNA in the somatic cells resulting in slow accumulation of mutations and epigenetic alterations in our genes throughout the life span [6]. The question of "Is cancer a metabolic disease or a genetic disease?" cannot be easily answered. Prior to 1970, most cancer researchers thought cancer is a metabolic disease because of the Warburg effect. After 1970 when Warburg died and after 1971 when oncogenes were discovered, most researchers shifted their thinking to view cancer as a genetic disease. After 2010–2015 when cancer was rediscovered as a metabolic disorder, the view was shifted back to "cancer is a metabolic disease." While cancer as a genetic disease looks to be impossibly complex, tumor cells are a genetic "train wreck" with an infinite number of mutations and epigenetic alterations in ~250 oncogenes and ~700 tumor suppressor genes. In contrast, cancer as a metabolic disease with only seven different "metabotypes" appears to be remarkably simple to deal with, since all the above mutations mainly affect three major metabolic pathways: aerobic glycolysis, glutaminolysis, and one-carbon metabolism [7].

Besides the traditional oncogenes and tumor suppressor genes [6], cell adhesion molecules (CAMs) also contribute directly to the tumor initiation and metastasis or orchestrate the tumor microenvironment to affect the tumor progression [8]. CAMs are involved in several biological functions, such as cellular social behaviors, tissue architecture, organ formation, blood vessel generation and angiogenesis, immune and inflammatory reactions, and wound healing [8]. An altered expression of CAMs has implications in tumor progression and metastasis, since most CAMs govern cellular social behaviors by directly contributing to cell adhesion, epithelialto-mesenchymal transition (EMT), and cross talk with the intracellular signal transduction pathways affecting other tumor progression-related processes [8]. As a consequence, the 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 25 years, our team investigated the role of METCAM/MUC18 in several types of tumors, such as melanoma and breast, nasopharyngeal, ovarian, and prostate cancers [17–37].

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

METCAM/MUC18 was first demonstrated to be abundantly expressed on the cellular membrane of most malignant human melanomas and hence was named as MUC18 [38] and MCAM [37]. It has been implicated to play a pivotal role in the malignant progression of human melanoma and hence was named as Mel-CAM [39]. However, subsequent studies showed that METCAM/MUC18 was not found to be exclusively expressed in melanoma, and furthermore, it did not initiate the transformation of normal cutaneous melanocytes to melanoma [39]. Instead METCAM/MUC18 was also expressed in endothelial cells and 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 also found to act as a suppressor in tumorigenesis and metastasis in some cancer cell lines [17, 37, 40].

The human METCAM/MUC18 (huMETCAM/MUC18) is a cell adhesion molecule (CAM) belonging to the Ig-like gene superfamily. The METCAM/MUC18 usually has an apparent molecular weight of 110–150,000 due to its high glycosylation in all cell types. The naked huMETCAM/MUC18 is a single-chain transmembrane protein of 65–72 kDa consisting of 646 amino acids with an extracellular

**5**

**Figure 1.**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

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

N-terminal domain of 558 amino acids, a transmembrane domain with 24 amino

**Figure 1** shows that the N-terminal extracellular domain of the protein is composed of a signal peptide sequence (SP) and five immunoglobulin-like domains and one X domain [37, 38, 40, 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, 40, 42]. The amino acid sequence of huMETCAM/MUC18 reveals nine potential 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 huMETCAM/ MUC18 and moMETCAM/MUC18 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, overexpression of both human and mouse METCAM/MUC18s similarly affected tumor cells in in vitro motility and invasiveness, in vitro and in vivo tumorigenesis, and in vivo

The huMETCAM/MUC18 is expressed in at least ten 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 overexpression 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 overexpression also initiates the distant organ dissemination of prostate cancer [32–33] and augments the distant organ dissemination of melanoma [21] and

In contrast, overexpression 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 overexpression also represses the distant organ dissemination of the mouse melanoma cell line, K1735-9 [22]. Thus, METCAM/MUC18 plays a dual role in some of these cancers

*The human METCAM/MUC18 (huMETCAM/MUC18). The figure represents the protein structure of huMETCAM/MUC18 with its three 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). (3) A cytoplasmic* 

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

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

metastasis [42, 43].

breast carcinoma [45].

[17, 37].

#### *METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

N-terminal domain of 558 amino acids, a transmembrane domain with 24 amino acids, and a cytoplasmic domain of 64 residues (**Figure 1**) [38, 42].

**Figure 1** shows that the N-terminal extracellular domain of the protein is composed of a signal peptide sequence (SP) and five immunoglobulin-like domains and one X domain [37, 38, 40, 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, 40, 42]. The amino acid sequence of huMETCAM/MUC18 reveals nine potential 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 huMETCAM/ MUC18 and moMETCAM/MUC18 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, overexpression of both human and mouse METCAM/MUC18s similarly affected tumor cells in in vitro motility and invasiveness, in vitro and in vivo tumorigenesis, and in vivo metastasis [42, 43].

The huMETCAM/MUC18 is expressed in at least ten 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 overexpression 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 overexpression 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, overexpression 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 overexpression also represses the distant organ dissemination of the mouse melanoma cell line, K1735-9 [22]. Thus, METCAM/MUC18 plays a dual role in some of these cancers [17, 37].

#### **Figure 1.**

*Tumor Progression and Metastasis*

glutaminolysis, and one-carbon metabolism [7].

tion or reduction of the spreading potential of carcinoma cells [8].

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

nasopharyngeal, ovarian, and prostate cancers [17–37].

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 25 years, our team investigated the role of METCAM/MUC18 in several types of tumors, such as melanoma and breast,

METCAM/MUC18 was first demonstrated to be abundantly expressed on the cellular membrane of most malignant human melanomas and hence was named as MUC18 [38] and MCAM [37]. It has been implicated to play a pivotal role in the malignant progression of human melanoma and hence was named as Mel-CAM [39]. However, subsequent studies showed that METCAM/MUC18 was not found to be exclusively expressed in melanoma, and furthermore, it did not initiate the transformation of normal cutaneous melanocytes to melanoma [39]. Instead METCAM/MUC18 was also expressed in endothelial cells and 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 also found to act as a suppressor in tumorigenesis and metastasis in some cancer cell

The human METCAM/MUC18 (huMETCAM/MUC18) is a cell adhesion molecule (CAM) belonging to the Ig-like gene superfamily. The METCAM/MUC18 usually has an apparent molecular weight of 110–150,000 due to its high glycosylation in all cell types. The naked huMETCAM/MUC18 is a single-chain transmembrane protein of 65–72 kDa consisting of 646 amino acids with an extracellular

were discovered, most researchers shifted their thinking to view cancer as a genetic disease. After 2010–2015 when cancer was rediscovered as a metabolic disorder, the view was shifted back to "cancer is a metabolic disease." While cancer as a genetic disease looks to be impossibly complex, tumor cells are a genetic "train wreck" with an infinite number of mutations and epigenetic alterations in ~250 oncogenes and ~700 tumor suppressor genes. In contrast, cancer as a metabolic disease with only seven different "metabotypes" appears to be remarkably simple to deal with, since all the above mutations mainly affect three major metabolic pathways: aerobic glycolysis,

Besides the traditional oncogenes and tumor suppressor genes [6], cell adhesion molecules (CAMs) also contribute directly to the tumor initiation and metastasis or orchestrate the tumor microenvironment to affect the tumor progression [8]. CAMs are involved in several biological functions, such as cellular social behaviors, tissue architecture, organ formation, blood vessel generation and angiogenesis, immune and inflammatory reactions, and wound healing [8]. An altered expression of CAMs has implications in tumor progression and metastasis, since most CAMs govern cellular social behaviors by directly contributing to cell adhesion, epithelialto-mesenchymal transition (EMT), and cross talk with the intracellular signal transduction pathways affecting other tumor progression-related processes [8]. As a consequence, the 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 augmenta-

**4**

lines [17, 37, 40].

*The human METCAM/MUC18 (huMETCAM/MUC18). The figure represents the protein structure of huMETCAM/MUC18 with its three 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). (3) A cytoplasmic domain containing five potential phosphorylation sites (P).*

#### **3. METCAM/MUC18: a promoter in tumor progression and metastasis of human cancers**

The protein METCAM/MUC18 is expressed in breast cancer, melanoma, nasopharyngeal carcinoma, and prostate cancer and also expressed in others cancers, such as angiosarcoma, gestational trophoblastic tumors, Kaposi's sarcoma, leiomyosarcoma, some lung adenocarcinoma and squamous and small cell carcinomas, and some neuroblastomas [44]. However, its role in the progression 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 [47]. In addition, METCAM/MUC18 expression and its possible role in other solid tumors began to emerge, such as angiosarcoma, gastric cancer, hepatocellular carcinoma, glioma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), osteosarcoma, and pancreatic cancer, as described in the following.

#### **3.1 Breast cancer**

Breast carcinomas were heterogenous with three histological subtypes (ER+, PR+, and ERBB2 receptor (HER)) ([48] for a review), with at least five distinct molecular subtypes (luminal A (ER+ and PR+), luminal B (ER+ and PR±), basal-like (ER-, PR-, AR-), HER2-enriched (HER2+), and normal-like) [49], or with ten combined genomic/transcriptomic subtypes [50]. HuMETCAM/MUC18 was found to be expressed in breast cancer cell lines and tissues of basal-like and mesenchymal subtypes at much higher levels than in luminal subtypes, which poorly or very weakly expressed the protein [18, 51]. METCAM/MUC18 was suggested by two groups to play a tumor suppressor role [52, 53] but by two other groups as a tumor promoter in the progression of human breast cancer [51, 54]. To resolve this controversy, we started separate studies to explore the real role of METCAM/MUC18 in the tumor progression of human breast cancer. We demonstrated that ectopic expression of METCAM/MUC18 in two breast cancer cell lines (MCF-7 and SK-BR-3) augmented their ability in epithelial-to-mesenchymal transition (EMT) and formation of colony in vitro and increased tumor-take and tumorigenesis (in vivo tumorigenesis) in athymic nude mice [18–20].

Treatment with an anti-METCAM/MUC18 antibody decreased the motility and invasiveness of the two basal-like cell lines, MDA-MB-231 and MDA-MB-468, which endogenously express the protein [19]. Overexpression of huMETCAM/MUC18 could also induce metastasis of the MCF7 cells in SCID/beige mice with the supplement of estrogen [45]. Furthermore, enforced expression of METCAM/MUC18 increases the metastasis of both basal-like cell lines in athymic nude mice [45]. The tumor suppression role of huMETCAM/MUC18 in tumorigenesis of human breast cancer cells previously observed by one group [52] has not been supported by evidence published later [18, 45]. The most likely reason may be due to the artifact of including fetal bovine serum in their injection mixtures, as extensively discussed in our published paper [18]. The other discrepancy may be because only in vitro experiments were done, but no in vivo animal test [51, 53, 54]. Taken together, METCAM/MUC18 plays a positive role in the tumor progression of four human breast cancer cell lines. From the results of further preliminary mechanical study, we suggest that METCAM/MUC18 promotes the progression of human breast cancer cells by increasing proliferation, angiogenesis, epithelial-to-mesenchymal transition (EMT), and switching to aerobic glycolysis [18–20]. METCAM/MUC18's downstream signaling molecules may also be used as therapeutic targets for the treatment of breast cancer.

**7**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

K1735-3 and K1735-10 subline has a minimal effect on tumor formation.

Most malignant human melanomas overly expressed huMETCAM/MUC18 on the cellular surface, suggesting that it may promote the malignant progression of human melanoma [38]. This notion is supported by the evidence that enforced expression of the huMETCAM/MUC18 increases the metastatic ability of three nonmetastatic human melanoma cell lines in the immune-incomplete mouse models [55, 56]. It is further corroborated by our results that enforced expression of moMETCAM/ MUC18 also augments the lung nodule formation ability of two low-metastatic mouse melanoma cell lines, K1735-3 and K1735-10, in a syngeneic mouse model with the complete immunity [21]. However, overexpression of moMETCAM/MUC18 in

METCAM/MUC18 enables melanoma cells to establish pulmonary metastasis only when the METCAM/MUC18-expressing melanoma cells are injected into the tail vein (experimental metastasis) [18–20, 55, 56], but not when the cells were injected subcutaneously (spontaneous metastasis) either in immune-deficient mouse models [55, 56] or in immune-competent syngeneic mouse models [21]. Thus, it bypassed the initial stages of metastasis, suggesting that METCAM/ MUC18 may promote melanoma metastasis only in the later stage of metastasis. This result is consistent with a later observation in that huMETCAM/MUC18 does not confer melanocytes the ability to initiate the tumor progression into melanoma [39]. Surprisingly when another mouse melanoma cell line, K1735-9, was used for the similar test in the syngeneic brown mouse model, a totally opposite result was obtained [22], to be described in Section 4. The exact reason for the dual role of METCAM/MUC18 in the tumor progression and metastasis is not clear, but one

Taken together, our syngeneic mouse system should be more useful than the immune-incomplete mouse system to comprehend the complex mechanisms played by METCAM/MUC18 in the malignant progression of melanoma cells. Furthermore, the knowledge learned from our syngeneic mouse systems should also be useful for testing the real efficacy of various therapeutic strategies before the treatment of clinical melanoma, because they should more closely mimic the

Most (90%) nasopharyngeal carcinoma (NPC) occurs in the non-lymphomatous, squamous epithelial lining of the posterior nasopharynx [23, 24]. Three histological subtypes of NPC are defined according to World Health Organization (WHO) classification: WHO type I (keratinizing squamous cell carcinomas), WHO type II (nonkeratinizing squamous cell carcinomas), and WHO type III (undifferentiated carcinomas) [23, 24]. Epidemiological studies suggested three major risk factors, such as genetic predisposition, dietary and environmental factors, and the Epstein-Barr virus (EBV) infection, that may induce the unusual incidence of NPC in endemic areas [23–26]. However, the biological mechanisms of their contribution to tumor initiation, development, and malignant progression remain elusive. Since aberrant expression of CAMs, such as CD44, connexin 43, E-cadherin, and ICAM, has been associated with the progression of NPC ([23] for a review), it is highly probable that these risk factors may alter cell adhesion molecule (CAM) expression and lead to tumorigenesis and malignant progression of NPC. In order to test this hypothesis, we initiated the studies on the possible role of altered METCAM/MUC18 expression in the malignant progression of nasopharyngeal carcinoma. First, we investigated if an aberrant expression of METCAM/MUC18 was associated with NPC

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

possibility is suggested in Section 5.

**3.3 Nasopharyngeal carcinoma**

clinical melanoma cases than the xenograft models.

**3.2 Melanoma**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

#### **3.2 Melanoma**

*Tumor Progression and Metastasis*

**of human cancers**

the following.

**3.1 Breast cancer**

athymic nude mice [18–20].

treatment of breast cancer.

**3. METCAM/MUC18: a promoter in tumor progression and metastasis** 

The protein METCAM/MUC18 is expressed in breast cancer, melanoma, nasopharyngeal carcinoma, and prostate cancer and also expressed in others cancers, such as angiosarcoma, gestational trophoblastic tumors, Kaposi's sarcoma, leiomyosarcoma, some lung adenocarcinoma and squamous and small cell carcinomas, and some neuroblastomas [44]. However, its role in the progression 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 [47]. In addition, METCAM/MUC18 expression and its possible role in other solid tumors began to emerge, such as angiosarcoma, gastric cancer, hepatocellular carcinoma, glioma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), osteosarcoma, and pancreatic cancer, as described in

Breast carcinomas were heterogenous with three histological subtypes (ER+, PR+, and ERBB2 receptor (HER)) ([48] for a review), with at least five distinct molecular subtypes (luminal A (ER+ and PR+), luminal B (ER+ and PR±), basal-like (ER-, PR-, AR-), HER2-enriched (HER2+), and normal-like) [49], or with ten combined genomic/transcriptomic subtypes [50]. HuMETCAM/MUC18 was found to be expressed in breast cancer cell lines and tissues of basal-like and mesenchymal subtypes at much higher levels than in luminal subtypes, which poorly or very weakly expressed the protein [18, 51]. METCAM/MUC18 was suggested by two groups to play a tumor suppressor role [52, 53] but by two other groups as a tumor promoter in the progression of human breast cancer [51, 54]. To resolve this controversy, we started separate studies to explore the real role of METCAM/MUC18 in the tumor progression of human breast cancer. We demonstrated that ectopic expression of METCAM/MUC18 in two breast cancer cell lines (MCF-7 and SK-BR-3) augmented their ability in epithelial-to-mesenchymal transition (EMT) and formation of colony in vitro and increased tumor-take and tumorigenesis (in vivo tumorigenesis) in

Treatment with an anti-METCAM/MUC18 antibody decreased the motility and invasiveness of the two basal-like cell lines, MDA-MB-231 and MDA-MB-468, which endogenously express the protein [19]. Overexpression of huMETCAM/MUC18 could also induce metastasis of the MCF7 cells in SCID/beige mice with the supplement of estrogen [45]. Furthermore, enforced expression of METCAM/MUC18 increases the metastasis of both basal-like cell lines in athymic nude mice [45]. The tumor suppression role of huMETCAM/MUC18 in tumorigenesis of human breast cancer cells previously observed by one group [52] has not been supported by evidence published later [18, 45]. The most likely reason may be due to the artifact of including fetal bovine serum in their injection mixtures, as extensively discussed in our published paper [18]. The other discrepancy may be because only in vitro experiments were done, but no in vivo animal test [51, 53, 54]. Taken together, METCAM/MUC18 plays a positive role in the tumor progression of four human breast cancer cell lines. From the results of further preliminary mechanical study, we suggest that METCAM/MUC18 promotes the progression of human breast cancer cells by increasing proliferation, angiogenesis, epithelial-to-mesenchymal transition (EMT), and switching to aerobic glycolysis [18–20]. METCAM/MUC18's downstream signaling molecules may also be used as therapeutic targets for the

**6**

Most malignant human melanomas overly expressed huMETCAM/MUC18 on the cellular surface, suggesting that it may promote the malignant progression of human melanoma [38]. This notion is supported by the evidence that enforced expression of the huMETCAM/MUC18 increases the metastatic ability of three nonmetastatic human melanoma cell lines in the immune-incomplete mouse models [55, 56]. It is further corroborated by our results that enforced expression of moMETCAM/ MUC18 also augments the lung nodule formation ability of two low-metastatic mouse melanoma cell lines, K1735-3 and K1735-10, in a syngeneic mouse model with the complete immunity [21]. However, overexpression of moMETCAM/MUC18 in K1735-3 and K1735-10 subline has a minimal effect on tumor formation.

METCAM/MUC18 enables melanoma cells to establish pulmonary metastasis only when the METCAM/MUC18-expressing melanoma cells are injected into the tail vein (experimental metastasis) [18–20, 55, 56], but not when the cells were injected subcutaneously (spontaneous metastasis) either in immune-deficient mouse models [55, 56] or in immune-competent syngeneic mouse models [21]. Thus, it bypassed the initial stages of metastasis, suggesting that METCAM/ MUC18 may promote melanoma metastasis only in the later stage of metastasis. This result is consistent with a later observation in that huMETCAM/MUC18 does not confer melanocytes the ability to initiate the tumor progression into melanoma [39]. Surprisingly when another mouse melanoma cell line, K1735-9, was used for the similar test in the syngeneic brown mouse model, a totally opposite result was obtained [22], to be described in Section 4. The exact reason for the dual role of METCAM/MUC18 in the tumor progression and metastasis is not clear, but one possibility is suggested in Section 5.

Taken together, our syngeneic mouse system should be more useful than the immune-incomplete mouse system to comprehend the complex mechanisms played by METCAM/MUC18 in the malignant progression of melanoma cells. Furthermore, the knowledge learned from our syngeneic mouse systems should also be useful for testing the real efficacy of various therapeutic strategies before the treatment of clinical melanoma, because they should more closely mimic the clinical melanoma cases than the xenograft models.

#### **3.3 Nasopharyngeal carcinoma**

Most (90%) nasopharyngeal carcinoma (NPC) occurs in the non-lymphomatous, squamous epithelial lining of the posterior nasopharynx [23, 24]. Three histological subtypes of NPC are defined according to World Health Organization (WHO) classification: WHO type I (keratinizing squamous cell carcinomas), WHO type II (nonkeratinizing squamous cell carcinomas), and WHO type III (undifferentiated carcinomas) [23, 24]. Epidemiological studies suggested three major risk factors, such as genetic predisposition, dietary and environmental factors, and the Epstein-Barr virus (EBV) infection, that may induce the unusual incidence of NPC in endemic areas [23–26]. However, the biological mechanisms of their contribution to tumor initiation, development, and malignant progression remain elusive. Since aberrant expression of CAMs, such as CD44, connexin 43, E-cadherin, and ICAM, has been associated with the progression of NPC ([23] for a review), it is highly probable that these risk factors may alter cell adhesion molecule (CAM) expression and lead to tumorigenesis and malignant progression of NPC. In order to test this hypothesis, we initiated the studies on the possible role of altered METCAM/MUC18 expression in the malignant progression of nasopharyngeal carcinoma. First, we investigated if an aberrant expression of METCAM/MUC18 was associated with NPC [23] and then the effect of METCAM/MUC18 overexpression on the tumorigenesis of two NPC cell lines in an athymic nude mouse model [24–26], as described next.

We used immunohistochemistry (IHC) method to determine the expression level of huMETCAM/MUC18 in 7 tissue specimens of normal nasopharynx and 97 specimens of three different types of NPC and also used immunoblot method to determine several cell lines established from type I to type III NPC [23]. The results showed a weak expression of the METCAM/MUC18 protein in only 27% of the NPC tissues (no expression in 73% of the NPC tissues), in contrast to all the normal nasopharynx tissues which exhibited a high expression of the protein, suggesting that METCAM/MUC18 may play a tumor suppressor role in the development of NPC during the progression of cancer [23]. Then, we further tested the hypothesis by examining the effect of ectopic METCAM/MUC18 expression on in vitro cellular behavior and in vivo tumorigenesis of the two NPC cell lines in athymic nude mice. Indeed, the predicted hypothesis was supported by the results when NPC-TW01 cells were used for the tests [24–26], as described in Section 4. Surprisingly, contrary to the hypothesis, when NPC-TW04 cell line was used for similar in vitro and in vivo tests, we observed that overexpression of METCAM/MUC18 actually promoted in vitro and in vivo tumor growth of NPC-TW04 cells [24, 26], which were established from type III NPC [57]. We thus conclude that METCAM/MUC18 plays a positive role in the tumor progression of the type III NPC [24, 26]. Overall, METCAM/MUC18 plays a dual role in the tumor progression of NPC.

#### **3.4 Prostate cancer**

For the past two decades, we have first demonstrated that METCAM/MUC18 expression in human tissues was associated with the progression of human prostate cancer [31] and also with that of mouse adenocarcinoma in a transgenic model, TRAMP [33]. We further showed that overexpression of METCAM/MUC18 promotes the progression of a human prostate cancer cell line, LNCaP, which was established from lymph node lesions [32, 34], as described next.

First, by using IHC and immunoblot assays to determine the expression of huMETCAM/MUC18 in the tissues of human normal prostate gland, patients with BPH, and patients with prostate cancer and metastatic lesions, we found that METCAM/MUC18 was highly expressed in all of the high-grade PINs and most of prostate carcinoma at advanced pathological stages and metastatic lesions, but it was not expressed in most normal prostate glands and in all BPH lesions. Thus, huMETCAM/MUC18 expression is associated with the progression of human prostate cancer [31].

Second, by using similar immunological methods to determine the expression of moMETCAM/MUC18 in the prostatic tissues of a transgenic mouse model, TRAMP, at different times of life span, we found that moMETCAM/MUC18 expression was increased with the progression of the mouse adenocarcinoma in this transgenic mouse model. Thus, moMETCAM/MUC18 overexpression is associated with the progression of mouse prostate adenocarcinoma in a transgenic mouse model, TRAMP [33].

Third, we tested the effect of overexpression of huMETCAM/MUC18 in a human prostate cancer cell line LNCaP on its tumorigenesis when the cells were injected at the non-orthotopic *SC* sites in nude mice. We observed that huMETCAM/ MUC18 overexpression promoted the tumorigenesis of the cell line at the nonorthotopic sites [34]. Then, we tested the effect of overexpression of huMETCAM/ MUC18 in the LNCaP cell line on its tumorigenesis and establishing metastatic lesions when the cells were injected at the orthotopic site (in the dorsal and lateral lobes of mouse prostate gland) in a male nude mouse model [32]. We found that

**9**

**Figure 2.**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

progression of LNCaP cells in an athymic nude mouse model [31–36].

Fourth, to check if the above 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 expresses a high level of METCAM/MUC18 [58]. 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 summary, we conclude that METCAM/MUC18 plays a positive role in the tumor progression and metastasis of two human prostate cancer cell lines, LNCaP and DU145. However, we recently observed an opposite result when the third human prostate cancer cell line PC-3 was used for the similar test, as described in Section 4, suggesting that METCAM/MUC18 also plays a dual role in the tumor

METCAM/MUC18 very likely promotes the formation of angiosarcoma, as supported by our preliminary results as described next. MoMETCAM/MUC18 was expressed at a higher level in one angiosarcoma clone, SVR, which was transfected with H-Ras, than in the control cell line, MS-1, an immortalized normal endothelial cell line [59]. 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

*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 5 mice S.C. injected with each of the 46 (control), 72, 24, and 27 clones/cells, which were transfected with 4 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 [58].*

huMETCAM/MUC18 overexpression promoted the tumorigenesis at the orthotopic prostate gland and also initiates metastatic lesions at periaortic lymph nodes and multiple distant sites (such as seminal vesicles, ureters, and the kidney). From the results, we conclude that ectopic overexpression of huMETCAM/MUC18 promotes in vivo tumorigenesis of the cells at either at non-orthotopic *SC* sites or at orthotopic prostate gland and also that it also initiates metastasis of the cells to multiple distant sites when cells were injected at the orthotopic mouse prostate gland. Taken together, we concluded that huMETCAM/MUC18 expression promotes the tumor

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

progression of human prostate cancer.

**3.5 Other solid tumors**

*3.5.1 Angiosarcoma*

#### *METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

huMETCAM/MUC18 overexpression promoted the tumorigenesis at the orthotopic prostate gland and also initiates metastatic lesions at periaortic lymph nodes and multiple distant sites (such as seminal vesicles, ureters, and the kidney). From the results, we conclude that ectopic overexpression of huMETCAM/MUC18 promotes in vivo tumorigenesis of the cells at either at non-orthotopic *SC* sites or at orthotopic prostate gland and also that it also initiates metastasis of the cells to multiple distant sites when cells were injected at the orthotopic mouse prostate gland. Taken together, we concluded that huMETCAM/MUC18 expression promotes the tumor progression of LNCaP cells in an athymic nude mouse model [31–36].

Fourth, to check if the above 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 expresses a high level of METCAM/MUC18 [58]. 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 summary, we conclude that METCAM/MUC18 plays a positive role in the tumor progression and metastasis of two human prostate cancer cell lines, LNCaP and DU145. However, we recently observed an opposite result when the third human prostate cancer cell line PC-3 was used for the similar test, as described in Section 4, suggesting that METCAM/MUC18 also plays a dual role in the tumor progression of human prostate cancer.

#### **3.5 Other solid tumors**

#### *3.5.1 Angiosarcoma*

*Tumor Progression and Metastasis*

**3.4 Prostate cancer**

prostate cancer [31].

TRAMP [33].

[23] and then the effect of METCAM/MUC18 overexpression on the tumorigenesis of two NPC cell lines in an athymic nude mouse model [24–26], as described next. We used immunohistochemistry (IHC) method to determine the expression level of huMETCAM/MUC18 in 7 tissue specimens of normal nasopharynx and 97 specimens of three different types of NPC and also used immunoblot method to determine several cell lines established from type I to type III NPC [23]. The results showed a weak expression of the METCAM/MUC18 protein in only 27% of the NPC tissues (no expression in 73% of the NPC tissues), in contrast to all the normal nasopharynx tissues which exhibited a high expression of the protein, suggesting that METCAM/MUC18 may play a tumor suppressor role in the development of NPC during the progression of cancer [23]. Then, we further tested the hypothesis by examining the effect of ectopic METCAM/MUC18 expression on in vitro cellular behavior and in vivo tumorigenesis of the two NPC cell lines in athymic nude mice. Indeed, the predicted hypothesis was supported by the results when NPC-TW01 cells were used for the tests [24–26], as described in Section 4. Surprisingly, contrary to the hypothesis, when NPC-TW04 cell line was used for similar in vitro and in vivo tests, we observed that overexpression of METCAM/MUC18 actually promoted in vitro and in vivo tumor growth of NPC-TW04 cells [24, 26], which were established from type III NPC [57]. We thus conclude that METCAM/MUC18 plays a positive role in the tumor progression of the type III NPC [24, 26]. Overall,

METCAM/MUC18 plays a dual role in the tumor progression of NPC.

established from lymph node lesions [32, 34], as described next.

For the past two decades, we have first demonstrated that METCAM/MUC18 expression in human tissues was associated with the progression of human prostate cancer [31] and also with that of mouse adenocarcinoma in a transgenic model, TRAMP [33]. We further showed that overexpression of METCAM/MUC18 promotes the progression of a human prostate cancer cell line, LNCaP, which was

First, by using IHC and immunoblot assays to determine the expression of huMETCAM/MUC18 in the tissues of human normal prostate gland, patients with BPH, and patients with prostate cancer and metastatic lesions, we found that METCAM/MUC18 was highly expressed in all of the high-grade PINs and most of prostate carcinoma at advanced pathological stages and metastatic lesions, but it was not expressed in most normal prostate glands and in all BPH lesions. Thus, huMETCAM/MUC18 expression is associated with the progression of human

Second, by using similar immunological methods to determine the expression of moMETCAM/MUC18 in the prostatic tissues of a transgenic mouse model, TRAMP, at different times of life span, we found that moMETCAM/MUC18 expression was increased with the progression of the mouse adenocarcinoma in this transgenic mouse model. Thus, moMETCAM/MUC18 overexpression is associated with the progression of mouse prostate adenocarcinoma in a transgenic mouse model,

Third, we tested the effect of overexpression of huMETCAM/MUC18 in a human prostate cancer cell line LNCaP on its tumorigenesis when the cells were injected at the non-orthotopic *SC* sites in nude mice. We observed that huMETCAM/ MUC18 overexpression promoted the tumorigenesis of the cell line at the nonorthotopic sites [34]. Then, we tested the effect of overexpression of huMETCAM/ MUC18 in the LNCaP cell line on its tumorigenesis and establishing metastatic lesions when the cells were injected at the orthotopic site (in the dorsal and lateral lobes of mouse prostate gland) in a male nude mouse model [32]. We found that

**8**

METCAM/MUC18 very likely promotes the formation of angiosarcoma, as supported by our preliminary results as described next. MoMETCAM/MUC18 was expressed at a higher level in one angiosarcoma clone, SVR, which was transfected with H-Ras, than in the control cell line, MS-1, an immortalized normal endothelial cell line [59]. 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

#### **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 5 mice S.C. injected with each of the 46 (control), 72, 24, and 27 clones/cells, which were transfected with 4 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 [58].*

moMETCAM/MUC18 [40, 59]. This suggests that METCAM/MUC18 very likely promotes the tumor progression of angiosarcoma [40, 59].

#### *3.5.2 Gastric cancer*

The expression of huMETCAM/MUC18 in gastric cancer was investigated to evaluate its clinical-pathological and prognostic significance [60]. The expression of huMETCAM/MUC18 and three EMT-related proteins (E-cadherin, β-catenin, and vimentin) was examined by IHC method in 144 gastric cancers. Forty-one percent of the gastric cancer specimens were positive for the huMETCAM/MUC18 expression. HuMETCAM/MUC18 was also correlated positively with lymph node involvement and a poor prognosis. Furthermore, the huMETCAM/MUC18 expression was directly correlated with the lost expression of the epithelial marker, E-cadherin, and the gained expression of the mesenchymal markers, nuclear β-catenin and vimentin, suggesting that huMETCAM/MUC18 promotes EMT and also tumor progression in gastric cancer. It is possibly used as an independent index for a poor prognosis in gastric cancer and as a potential therapeutic target for patients with gastric cancers [60].

#### *3.5.3 Glioblastoma*

Glioblastoma multiforme (GBM) is the most common brain malignancy, accounting for more than 45% of all primary malignant brain tumors. YY146, an anti-METCAM/MUC18 monoclonal antibody, was created and radiolabeled for the noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. 64Cu-labeled YY146 was demonstrated to be preferentially accumulated in the U87MG xenografted tumors, which permitted the obtaining of high-contrast PET images of small tumor nodules (∼2 mm). Furthermore, tumor-take of glioblastoma in an orthotopic xenograft mouse model correlates with the expression level of METCAM/MUC18 in a highly specific manner. Furthermore, YY146 can mitigate the EMT of these U87MG cells. Moreover, using YY146 as the primary antibody for histological studies of the World Health Organization, grades I through IV primary gliomas showed that there was a positive correlation between METCAM/MUC18 positive staining and high tumor grade, which concurred with the GBM data available in The Cancer Genome Atlas (TCGA). Taken together, METCAM/MUC18 appears to promote the aggressive phenotypes and hence the tumor progression of glioblastoma U87MG cells [61].

#### *3.5.4 Hepatocellular carcinoma*

Hepatocellular carcinoma (HCC) remains the fifth most common malignant cancer and as the third leading cause of cancer-related mortality [62]. Highthroughput flow cytometry (HT-FC) profiling was used to characterize the expression of METCAM/MUC18 in the tumor cells from 30 human HCC samples. Increased expression of METCAM/MUC18 expression was significantly increased in hepatocellular carcinoma (HCC) tumor tissues as compared with the matched adjacent normal liver tissues. The METCAM/MUC18+ cells purified from HCC tumors have significantly increased colony-forming capacity, consistent with the characteristics of the cancer stem cells or the tumor-initiating cells, which are considered to contribute to the pathogenesis of HCC [63]. The high expression of METCAM/MUC18 in HCC samples and in HCC cell lines isolated from HCC samples was also confirmed by RT-PCR and Western blot analyses [64]. The HCC cell lines, which stably expressed METCAM/MUC18, had been shown to promote EMT, IL8 upregulation, and STAT1 downregulation, suggesting that METCAM/

**11**

**Figure 3.**

*lane 4) [From [68]].*

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

normal embryonic WI38 cell line [68], as shown in **Figure 3**.

Furthermore, the fourth group observed that METCAM/MUC18 expression mediates acquisition of cancer stemness and enhances tumor invasion and metastasis in a mouse model [69–70]. High expression of METCAM/MUC18 correlates with intrapulmonary metastasis of NSCLC cells in a mouse model [69–70]. Taken

*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,* 

MUC18 promotes tumor progression and metastasis and predicts poor prognosis of

Lung cancer is the cancer with the highest mortality rate in the world [62], and non-small cell lung cancer (NSCLC) is the cause for about 80% of all lung cancer. The frequency of occurrence of adenocarcinoma, which is one of the major histological subtypes of NSCLC, has recently increased [65]. Eighty-five specimens of NSCLC were immunohistochemically analyzed by using an anti-METCAM/MUC18 monoclonal antibody (clone N1238) on an NSCLC tissue microarray, and the staining was semiquantitatively scored. METCAM/MUC18 has been shown to express in 51% of NSCLC, preferentially squamous cell carcinomas. Positive expression of METCAM/MUC18 has also been associated with a shorter survival of patients with adenocarcinomas and used to predict the poor overall survival in patients with lung adenocarcinomas [65, 66]. Another group also used IHC to show that METCAM/ MUC18 expression was more frequently detected in males than in females. The positive expression of METCAM/MUC18 was associated with a poorer 5-year overall survival rate according to the survival analysis, suggesting that METCAM/MUC18 may be a useful marker for predicting poor prognosis in patients with NSCLC following complete resection [65]. The third group reported that METCAM/ MUC18 protein expression was found in 46.61% of squamous cell carcinomas and 37.47% of adenocarcinomas. METCAM/MUC18 expression positively correlated with vimentin but inversely with E-cadherin, indicating a positive correlation with EMT. METCAM/MUC18 expression in surgically treated primary tumor NSCLC is clearly associated with lymph node metastasis and is a statistically significant prognostic factor [67]. Consistent with the results and also supporting the conclusion described above, we also showed 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

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

hepatocellular carcinoma [64].

*3.5.5 Non-small cell lung cancer*

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

MUC18 promotes tumor progression and metastasis and predicts poor prognosis of hepatocellular carcinoma [64].

#### *3.5.5 Non-small cell lung cancer*

*Tumor Progression and Metastasis*

*3.5.2 Gastric cancer*

*3.5.3 Glioblastoma*

glioblastoma U87MG cells [61].

*3.5.4 Hepatocellular carcinoma*

moMETCAM/MUC18 [40, 59]. This suggests that METCAM/MUC18 very likely

The expression of huMETCAM/MUC18 in gastric cancer was investigated to evaluate its clinical-pathological and prognostic significance [60]. The expression of huMETCAM/MUC18 and three EMT-related proteins (E-cadherin, β-catenin, and vimentin) was examined by IHC method in 144 gastric cancers. Forty-one percent of the gastric cancer specimens were positive for the huMETCAM/MUC18 expression. HuMETCAM/MUC18 was also correlated positively with lymph node involvement and a poor prognosis. Furthermore, the huMETCAM/MUC18 expression was directly correlated with the lost expression of the epithelial marker, E-cadherin, and the gained expression of the mesenchymal markers, nuclear β-catenin and vimentin, suggesting that huMETCAM/MUC18 promotes EMT and also tumor progression in gastric cancer. It is possibly used as an independent index for a poor prognosis in gastric cancer and as a potential therapeutic target for patients with gastric cancers [60].

Glioblastoma multiforme (GBM) is the most common brain malignancy, accounting for more than 45% of all primary malignant brain tumors. YY146, an anti-METCAM/MUC18 monoclonal antibody, was created and radiolabeled for the noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. 64Cu-labeled YY146 was demonstrated to be preferentially accumulated in the U87MG xenografted tumors, which permitted the obtaining of high-contrast PET images of small tumor nodules (∼2 mm). Furthermore, tumor-take of glioblastoma in an orthotopic xenograft mouse model correlates with the expression level of METCAM/MUC18 in a highly specific manner. Furthermore, YY146 can mitigate the EMT of these U87MG cells. Moreover, using YY146 as the primary antibody for histological studies of the World Health Organization, grades I through IV primary gliomas showed that there was a positive correlation between METCAM/MUC18 positive staining and high tumor grade, which concurred with the GBM data available in The Cancer Genome Atlas (TCGA). Taken together, METCAM/MUC18 appears to promote the aggressive phenotypes and hence the tumor progression of

Hepatocellular carcinoma (HCC) remains the fifth most common malignant cancer and as the third leading cause of cancer-related mortality [62]. Highthroughput flow cytometry (HT-FC) profiling was used to characterize the expression of METCAM/MUC18 in the tumor cells from 30 human HCC samples. Increased expression of METCAM/MUC18 expression was significantly increased in hepatocellular carcinoma (HCC) tumor tissues as compared with the matched adjacent normal liver tissues. The METCAM/MUC18+ cells purified from HCC tumors have significantly increased colony-forming capacity, consistent with the characteristics of the cancer stem cells or the tumor-initiating cells, which are considered to contribute to the pathogenesis of HCC [63]. The high expression of METCAM/MUC18 in HCC samples and in HCC cell lines isolated from HCC samples was also confirmed by RT-PCR and Western blot analyses [64]. The HCC cell lines, which stably expressed METCAM/MUC18, had been shown to promote EMT, IL8 upregulation, and STAT1 downregulation, suggesting that METCAM/

promotes the tumor progression of angiosarcoma [40, 59].

**10**

Lung cancer is the cancer with the highest mortality rate in the world [62], and non-small cell lung cancer (NSCLC) is the cause for about 80% of all lung cancer. The frequency of occurrence of adenocarcinoma, which is one of the major histological subtypes of NSCLC, has recently increased [65]. Eighty-five specimens of NSCLC were immunohistochemically analyzed by using an anti-METCAM/MUC18 monoclonal antibody (clone N1238) on an NSCLC tissue microarray, and the staining was semiquantitatively scored. METCAM/MUC18 has been shown to express in 51% of NSCLC, preferentially squamous cell carcinomas. Positive expression of METCAM/MUC18 has also been associated with a shorter survival of patients with adenocarcinomas and used to predict the poor overall survival in patients with lung adenocarcinomas [65, 66]. Another group also used IHC to show that METCAM/ MUC18 expression was more frequently detected in males than in females. The positive expression of METCAM/MUC18 was associated with a poorer 5-year overall survival rate according to the survival analysis, suggesting that METCAM/MUC18 may be a useful marker for predicting poor prognosis in patients with NSCLC following complete resection [65]. The third group reported that METCAM/ MUC18 protein expression was found in 46.61% of squamous cell carcinomas and 37.47% of adenocarcinomas. METCAM/MUC18 expression positively correlated with vimentin but inversely with E-cadherin, indicating a positive correlation with EMT. METCAM/MUC18 expression in surgically treated primary tumor NSCLC is clearly associated with lymph node metastasis and is a statistically significant prognostic factor [67]. Consistent with the results and also supporting the conclusion described above, we also showed 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 [68], as shown in **Figure 3**.

Furthermore, the fourth group observed that METCAM/MUC18 expression mediates acquisition of cancer stemness and enhances tumor invasion and metastasis in a mouse model [69–70]. High expression of METCAM/MUC18 correlates with intrapulmonary metastasis of NSCLC cells in a mouse model [69–70]. Taken

#### **Figure 3.**

*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 [68]].*

together, METCAM/MUC18 plays a positive role in the tumor progression and metastasis of NSCLC.

#### *3.5.6 Small cell lung cancer*

Small cell lung cancer (SCLC), a lung cancer subtype with an aggressive and highly metastatic nature, is the cause for about 10–20% of lung cancer incidence. The 5-year survival rate is at 7%, still very gloomy because choices of systemic treatment for SCLC patients have not been much increased. SCLC is highly responsive to chemotherapy at the start of treatment. Despite favorable responses to initial therapy, SCLC relapse occurs within a year exhibiting a multidrug-resistant phenotype, which eventually contributes strongly to poor prognosis. Through in-depth proteomic profiling, METCAM/MUC18 was identified as a markedly upregulated surface receptor in chemoresistant SCLC cell lines that exhibited a mesenchymal phenotype as well as in chemoresistant patient-derived xenografts compared to matched treatmentnaïve tumors. METCAM/MUC18 knockdown in chemoresistant cells reduced cell proliferation and decreased the IC50 inhibitory concentration of chemotherapeutic drugs. METCAM/MUC18 was found to modulate sensitivity of SCLC cells to chemotherapeutic drugs through upregulation of MRP1/ABCC1 expression and of the PI3/ AKT pathway in a SOX2-dependent manner. Metabolomic profiling revealed that METCAM/MUC18 modulates lactate production in chemoresistant cells that exhibit a distinct metabolic phenotype characterized by low oxidative phosphorylation. METCAM/MUC18 may serve as a novel therapeutic target to overcome chemoresistance in SCLC [71]. In summary, the above results point to the positive role played by METCAM/MUC18 in the tumor progression and metastasis of SCLC.

#### *3.5.7 Osteosarcoma*

Osteosarcoma is the most common primary malignant bone tumor in children. Clinically evident metastatic disease is present in 10–20% of patients at diagnosis. Despite advancements in multimodality treatment, 5-year survival rates are ~40–50% [72]. METCAM/MUC18 was widely expressed on both osteosarcoma and Ewing's sarcoma cells. METCAM/MUC18 protein and RNA are highly expressed in osteosarcoma cell lines (SaOS, MG-63, U-2OS), but not in normal osteoblast cells [72–73]. ABX-MA1, an anti-METCAM/MUC18 antibody, did not appear to inhibit the in vitro proliferation of osteosarcoma cells, and neither did it significantly inhibit the in vivo growth of KRIB human osteosarcoma cells in the tibias of nude mice. Nevertheless, after 1½ months, a noticeably fewer number of ABX-MA1-treated mice spontaneously developed pulmonary metastatic lesions than the control antibody-treated mice. Furthermore, ABX-MA1 reduced the in vitro invasiveness of osteosarcoma cells in the Matrigel-coated trans-well assay and disturbed the homotypic adhesion among osteosarcoma cells and the heterotypic interaction of them with vascular endothelial cells. Osteosarcoma is effectively treated with anti-METCAM/MUC18 monoclonal antibodies [73–74]. Taken together, METCAM/MUC18 plays a positive role in the metastasis of osteosarcoma [72–74].

#### *3.5.8 Human and mouse pancreatic cancer*

Pancreatic ductal adenocarcinoma (PDAC) is the cancer that has the fourth highest mortality rate in the western countries [62]. The 5-year survival rate of all PDAC patients is 6%. Since this tumor is highly aggressive, cancer incidence is almost equivalent to its mortality rate [62]. Most pancreatic cancer deaths are due to metastasis. Especially, the events of tumor spreading are heterogeneous,

**13**

**Table 1.**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

**Tumor/cancer tissues or cell lines Tumorigenesis Metastasis References** Angiosarcoma human cell lines MS1, SVR Increasing Not determined [40, 59] 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 [60] Glioma cell lines U87MG, U251 Promotion Not Determined [61]

> No effect or slight suppression

Clinical prostate cancer human tissues Increasing Increasing and

Human prostate cancer cell line LNCaP Increasing Increasing and

Prostate adenocarcinoma in TRAMP mice Increasing Increasing and

*The positive role of METCAM/MUC18 in the tumor progression of various solid tumors/cancers.*

Promotion Promotion [19, 45]

Promotion Not determined [63, 64]

Promotion Promotion [65–70]

Promotion Not determined [71]

stage

stage

Promotion Not determined [24, 26]

Promotion Augmentation [72–74]

augmentation

(PIN)

affecting initiation in the early stage

affecting initiation in the early stage

affecting initiation in the early stage

affecting the late

Increasing and affecting the late [38, 55, 56]

[21]

[75, 76]

[31]

[33]

[32, 34–36]

No effect Increasing and

Promotion Possible

thus limiting the therapeutic choices for patients at late stages. METCAM/MUC18 expression in cancer cells is associated with a secretion of soluble METCAM/ MUC18 (sMETCAM/MUC18) that plays an active role in tumor development. For example, sMETCAM/MUC18 causes the overproduction of its binding partner, angiomotin, in cancer cells and endothelial cells in the tumor micro-*milieu*,

Human prostate cancer cell line DU145 Increasing Not determined [58]

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

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

normal liver cell line LO2

and adenocarcinoma tissues

Hepatocellular carcinoma human cell lines PLC/PRF/5, Huh7, MHCC97H and 97L HepG2, SMMC-7721, FOCUS, YY-8103, LM3, HLF, and primary HCC cell lines;

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

Small cell lung cancer human cell lines H69, H69AR, H82, H196, H209, DMS79

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

Mouse melanoma cell lines K1735-3,

Osteosarcoma human cell lines CR9, MNNG-HOS, OHS, KPDX, KRIB, MG-63, shYY1, SaOS, SaOS-2, TE85, U20S

Pancreatic cancer human cell lines and mouse cell lines ptf1a, LSL-Kras, LSL-

Nasopharyngeal carcinoma type III human

K1735-10

Trp53, Pdx1

cell line NPC-TW04

#### *METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*


#### **Table 1.**

*Tumor Progression and Metastasis*

metastasis of NSCLC.

*3.5.7 Osteosarcoma*

metastasis of osteosarcoma [72–74].

*3.5.8 Human and mouse pancreatic cancer*

*3.5.6 Small cell lung cancer*

together, METCAM/MUC18 plays a positive role in the tumor progression and

Small cell lung cancer (SCLC), a lung cancer subtype with an aggressive and highly metastatic nature, is the cause for about 10–20% of lung cancer incidence. The 5-year survival rate is at 7%, still very gloomy because choices of systemic treatment for SCLC patients have not been much increased. SCLC is highly responsive to chemotherapy at the start of treatment. Despite favorable responses to initial therapy, SCLC relapse occurs within a year exhibiting a multidrug-resistant phenotype, which eventually contributes strongly to poor prognosis. Through in-depth proteomic profiling, METCAM/MUC18 was identified as a markedly upregulated surface receptor in chemoresistant SCLC cell lines that exhibited a mesenchymal phenotype as well as in chemoresistant patient-derived xenografts compared to matched treatmentnaïve tumors. METCAM/MUC18 knockdown in chemoresistant cells reduced cell proliferation and decreased the IC50 inhibitory concentration of chemotherapeutic drugs. METCAM/MUC18 was found to modulate sensitivity of SCLC cells to chemotherapeutic drugs through upregulation of MRP1/ABCC1 expression and of the PI3/ AKT pathway in a SOX2-dependent manner. Metabolomic profiling revealed that METCAM/MUC18 modulates lactate production in chemoresistant cells that exhibit a distinct metabolic phenotype characterized by low oxidative phosphorylation. METCAM/MUC18 may serve as a novel therapeutic target to overcome chemoresistance in SCLC [71]. In summary, the above results point to the positive role played by

METCAM/MUC18 in the tumor progression and metastasis of SCLC.

Osteosarcoma is the most common primary malignant bone tumor in children. Clinically evident metastatic disease is present in 10–20% of patients at diagnosis. Despite advancements in multimodality treatment, 5-year survival rates are ~40–50% [72]. METCAM/MUC18 was widely expressed on both osteosarcoma and Ewing's sarcoma cells. METCAM/MUC18 protein and RNA are highly expressed in osteosarcoma cell lines (SaOS, MG-63, U-2OS), but not in normal osteoblast cells [72–73]. ABX-MA1, an anti-METCAM/MUC18 antibody, did not appear to inhibit the in vitro proliferation of osteosarcoma cells, and neither did it significantly inhibit the in vivo growth of KRIB human osteosarcoma cells in the tibias of nude mice. Nevertheless, after 1½ months, a noticeably fewer number of ABX-MA1-treated mice spontaneously developed pulmonary metastatic lesions than the control antibody-treated mice. Furthermore, ABX-MA1 reduced the in vitro invasiveness of osteosarcoma cells in the Matrigel-coated trans-well assay and disturbed the homotypic adhesion among osteosarcoma cells and the heterotypic interaction of them with vascular endothelial cells. Osteosarcoma is effectively treated with anti-METCAM/MUC18 monoclonal antibodies [73–74]. Taken together, METCAM/MUC18 plays a positive role in the

Pancreatic ductal adenocarcinoma (PDAC) is the cancer that has the fourth highest mortality rate in the western countries [62]. The 5-year survival rate of all PDAC patients is 6%. Since this tumor is highly aggressive, cancer incidence is almost equivalent to its mortality rate [62]. Most pancreatic cancer deaths are due to metastasis. Especially, the events of tumor spreading are heterogeneous,

**12**

*The positive role of METCAM/MUC18 in the tumor progression of various solid tumors/cancers.*

thus limiting the therapeutic choices for patients at late stages. METCAM/MUC18 expression in cancer cells is associated with a secretion of soluble METCAM/ MUC18 (sMETCAM/MUC18) that plays an active role in tumor development. For example, sMETCAM/MUC18 causes the overproduction of its binding partner, angiomotin, in cancer cells and endothelial cells in the tumor micro-*milieu*,

which augments angiogenesis and proliferation and survival of cancer cells. These are mediated in part by the promotion and activation of c-myc in cancer cells. Dispensation of a new specific monoclonal antibody pinpointing on sMETCAM/ MUC18 represses tumor angiogenesis and growth of huMETCAM/MUC18+ pancreatic cancer cell xenografts in mice models. Taken together, sMETCAM/MUC18 secreted by METCAM/MUC18+ tumors exhibit promoting effects on tumor angiogenesis and growth. Thus, an antibody pinpointing on sMETCAM/MUC18 successfully represses vascularization, growth, and survival of METCAM/MUC18 positive pancreatic tumors [75].

The expression of a homeodomain transcription factor MEIS1 (myeloid ecotropic viral integration site) has been associated with a ductal phenotype in pancreatic tissue architecture. To investigate a possible role of MEIS1 in the malignant progression of PDAC, pancreatic cancer cell clones/lines, which overexpress MEIS1, were generated and tested for in vitro proliferation rate and motility. Overexpression of MEIS1 had no effect on in vitro proliferation rate but augmented motility. Furthermore, an upregulation of the MTCAM/MUC18 gene in the migrating cells has been found in the subsequent expression analysis. The interaction of MEIS1 with the enhancer DNA of METCAM/MUC18 is revealed by employing DNA pulldown and chromatin immunoprecipitation (ChIP) assay. Furthermore, the transcriptional activation of METCAM/MUC18 also facilitates migration of pancreatic cancer cells in vitro. Activation of METCAM/MUC18 through MEIS1 occurs in a cell type-dependent fashion, reflecting the different routes that lead to metastasis in vivo. Thus, the transcription factor MEIS1 activates METCAM/ MUC18 expression to promote migration of mouse pancreatic tumor cell lines [76].

In summary, the positive role played by the METCAM/MUC18 in the progression of solid tumors has been extended from breast cancer, human and mouse melanoma, and prostate cancer to angiosarcoma [40, 59], gastric cancer [60], glioblastoma [61], hepatocellular carcinoma [63, 64], non-small cell lung adenocarcinoma [65–70], small cell lung cancer [71], osteosarcoma [72–74], human and mouse pancreatic cancer [75, 76], and prostate cancer [32–36, 58]. Taken together, METCAM/MCU18 appears to be more prevalently playing a positive role than a negative role in the tumor formation and/or cancer metastasis of various tumors/ cancers. **Table 1** summarizes the positive role of METCAM/MUC18 in the tumor progression of many solid tumors/cancers.

#### **4. METCAM/MUC18: a tumor suppressor and metastasis suppressor in some cancers**

In contrast to the positive role played by METCAM/MUC18 in the above cancers, recent results of testing the effects of METCAM/MUC18 expression on tumorigenesis of other cancer types revealed that it also plays a negative role in the tumor progression and metastasis in some cancers, such as colorectal cancer, hemangioma, one mouse melanoma cell line K1735-9, NPC type I, ovarian cancer, human pancreatic cancer, and one human prostate cancer cell line PC-3, as described next.

#### **4.1 Colorectal cancer**

Colorectal cancer (CRC) is the third leading cause of cancer deaths in recent years [62]. Cancer stemness contributes to carcinogenesis, tumor relapse, and chemoresistance in traditional cancer therapeutics ([77] for a review). Stemness, which is the cell state with the properties of self-renewal, differentiation, and tumor-initiating potential, might be characterized by a set of more dynamic features

**15**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

the tumor progression and metastasis of colorectal cancer [77].

negative role in the progression of hemangioma [46].

**4.4 Nasopharyngeal carcinoma (NPC)**

influenced by the nature of the microenvironment. Various extrinsic cues and intrinsic signaling pathways, such as Wnt, Notch, and Hedgehog signals, are involved in the maintenance of stemness. Since METCAM/MUC18 has also been identified as a pluripotent marker for mesenchymal stem cells (MSCs), it was also hypothesized to exert potential effects on cancer cell stemness. One group of investigators has provided evidence to demonstrate that reduced expression of METCAM/MUC18 actually functions as a positive regulator of stem cell properties in colorectal cancer through augmenting the Wnt/β-catenin signaling pathway. METCAM/MUC18 may actually manifest multifaceted effects on tumor progression in a context-dependent manner. The above evidence suggests that METCAM/MUC18 expression suppresses

The expression of METCAM/M/MUC18 in hemangioma is inversely proportional to the progression of hemangioma, suggesting that METCAM/MUC18 plays a

As shown in the above section, moMETCAM/MUC18 does not have an obvious effect on the tumorigenesis of the two K1735 cell lines, such as K1735-3 and K1735- 10 [21]. In contrast, moMETCAM/MUC18 definitely acts as a tumor suppressor for the K1735-9 cell line. Overexpression of moMETCAM/MUC18 in K1735-9 also completely suppressed lung nodule formation in immunocompetent syngeneic C3H brown mouse model [22]. Thus, METCAM/MUC18 expression suppresses the tumor progression and metastasis of the mouse melanoma K1735-9 cell line [22].

According to the IHC results as shown in the above section, we suggested a hypothesis that METCAM/MUC18 may play a tumor suppressor function in the development of NPC during the progression of the cancer [23]. Then, we further tested the hypothesis by examining the effect of METCAM/MUC18 overexpression on in vitro cellular behavior and in vivo tumorigenesis of the two NPC cell lines in athymic nude mice. When the METCAM/MUC18-overexpressing NPC-TW01 clones/cells, which were established from NPC type I, were used for the animal test, indeed tumor suppression was observed [24, 25]. However, the opposite results were obtained when the METCAM/MUC18-overexpressing NPC-TW04 clones/cells, which were established from NPC type III, were used for the similar tests. Thus, METCAM/MUC18 plays a dual role in the tumor progression of NPC [23, 24–26].

Two independent groups showed that METCAM/MUC18 expression is correlated with the progression of ovarian cancer [27, 78] and it affects in vitro behaviors of ovarian carcinoma cells [79]; however, the role of METCAM/MUC18 in the progression of epithelial ovarian cancer has not been directly tested in animal models. For this purpose, we initiated testing the effect of METCAM/MUC18 overexpression 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 the in vitro and in vivo tests. We observed that overexpression of METCAM/MUC18 reduced in vitro motility and

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

**4.2 Hemangioma**

**4.3 Mouse melanoma**

**4.5 Ovarian cancer**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

influenced by the nature of the microenvironment. Various extrinsic cues and intrinsic signaling pathways, such as Wnt, Notch, and Hedgehog signals, are involved in the maintenance of stemness. Since METCAM/MUC18 has also been identified as a pluripotent marker for mesenchymal stem cells (MSCs), it was also hypothesized to exert potential effects on cancer cell stemness. One group of investigators has provided evidence to demonstrate that reduced expression of METCAM/MUC18 actually functions as a positive regulator of stem cell properties in colorectal cancer through augmenting the Wnt/β-catenin signaling pathway. METCAM/MUC18 may actually manifest multifaceted effects on tumor progression in a context-dependent manner. The above evidence suggests that METCAM/MUC18 expression suppresses the tumor progression and metastasis of colorectal cancer [77].

#### **4.2 Hemangioma**

*Tumor Progression and Metastasis*

positive pancreatic tumors [75].

progression of many solid tumors/cancers.

**some cancers**

**4.1 Colorectal cancer**

which augments angiogenesis and proliferation and survival of cancer cells. These are mediated in part by the promotion and activation of c-myc in cancer cells. Dispensation of a new specific monoclonal antibody pinpointing on sMETCAM/ MUC18 represses tumor angiogenesis and growth of huMETCAM/MUC18+ pancreatic cancer cell xenografts in mice models. Taken together, sMETCAM/MUC18 secreted by METCAM/MUC18+ tumors exhibit promoting effects on tumor angiogenesis and growth. Thus, an antibody pinpointing on sMETCAM/MUC18 successfully represses vascularization, growth, and survival of METCAM/MUC18-

The expression of a homeodomain transcription factor MEIS1 (myeloid ecotropic viral integration site) has been associated with a ductal phenotype in pancreatic tissue architecture. To investigate a possible role of MEIS1 in the malignant progression of PDAC, pancreatic cancer cell clones/lines, which overexpress MEIS1, were generated and tested for in vitro proliferation rate and motility. Overexpression of MEIS1 had no effect on in vitro proliferation rate but augmented motility. Furthermore, an upregulation of the MTCAM/MUC18 gene in the migrating cells has been found in the subsequent expression analysis. The interaction of MEIS1 with the enhancer DNA of METCAM/MUC18 is revealed by employing DNA pulldown and chromatin immunoprecipitation (ChIP) assay. Furthermore, the transcriptional activation of METCAM/MUC18 also facilitates migration of pancreatic cancer cells in vitro. Activation of METCAM/MUC18 through MEIS1 occurs in a cell type-dependent fashion, reflecting the different routes that lead to metastasis in vivo. Thus, the transcription factor MEIS1 activates METCAM/ MUC18 expression to promote migration of mouse pancreatic tumor cell lines [76]. In summary, the positive role played by the METCAM/MUC18 in the progression of solid tumors has been extended from breast cancer, human and mouse melanoma, and prostate cancer to angiosarcoma [40, 59], gastric cancer [60], glioblastoma [61], hepatocellular carcinoma [63, 64], non-small cell lung adenocarcinoma [65–70], small cell lung cancer [71], osteosarcoma [72–74], human and mouse pancreatic cancer [75, 76], and prostate cancer [32–36, 58]. Taken together, METCAM/MCU18 appears to be more prevalently playing a positive role than a negative role in the tumor formation and/or cancer metastasis of various tumors/ cancers. **Table 1** summarizes the positive role of METCAM/MUC18 in the tumor

**4. METCAM/MUC18: a tumor suppressor and metastasis suppressor in** 

recent results of testing the effects of METCAM/MUC18 expression on tumorigenesis of other cancer types revealed that it also plays a negative role in the tumor progression and metastasis in some cancers, such as colorectal cancer, hemangioma, one mouse melanoma cell line K1735-9, NPC type I, ovarian cancer, human pancreatic cancer, and one human prostate cancer cell line PC-3, as described next.

Colorectal cancer (CRC) is the third leading cause of cancer deaths in recent years [62]. Cancer stemness contributes to carcinogenesis, tumor relapse, and chemoresistance in traditional cancer therapeutics ([77] for a review). Stemness, which is the cell state with the properties of self-renewal, differentiation, and tumor-initiating potential, might be characterized by a set of more dynamic features

In contrast to the positive role played by METCAM/MUC18 in the above cancers,

**14**

The expression of METCAM/M/MUC18 in hemangioma is inversely proportional to the progression of hemangioma, suggesting that METCAM/MUC18 plays a negative role in the progression of hemangioma [46].

#### **4.3 Mouse melanoma**

As shown in the above section, moMETCAM/MUC18 does not have an obvious effect on the tumorigenesis of the two K1735 cell lines, such as K1735-3 and K1735- 10 [21]. In contrast, moMETCAM/MUC18 definitely acts as a tumor suppressor for the K1735-9 cell line. Overexpression of moMETCAM/MUC18 in K1735-9 also completely suppressed lung nodule formation in immunocompetent syngeneic C3H brown mouse model [22]. Thus, METCAM/MUC18 expression suppresses the tumor progression and metastasis of the mouse melanoma K1735-9 cell line [22].

#### **4.4 Nasopharyngeal carcinoma (NPC)**

According to the IHC results as shown in the above section, we suggested a hypothesis that METCAM/MUC18 may play a tumor suppressor function in the development of NPC during the progression of the cancer [23]. Then, we further tested the hypothesis by examining the effect of METCAM/MUC18 overexpression on in vitro cellular behavior and in vivo tumorigenesis of the two NPC cell lines in athymic nude mice. When the METCAM/MUC18-overexpressing NPC-TW01 clones/cells, which were established from NPC type I, were used for the animal test, indeed tumor suppression was observed [24, 25]. However, the opposite results were obtained when the METCAM/MUC18-overexpressing NPC-TW04 clones/cells, which were established from NPC type III, were used for the similar tests. Thus, METCAM/MUC18 plays a dual role in the tumor progression of NPC [23, 24–26].

#### **4.5 Ovarian cancer**

Two independent groups showed that METCAM/MUC18 expression is correlated with the progression of ovarian cancer [27, 78] and it affects in vitro behaviors of ovarian carcinoma cells [79]; however, the role of METCAM/MUC18 in the progression of epithelial ovarian cancer has not been directly tested in animal models. For this purpose, we initiated testing the effect of METCAM/MUC18 overexpression 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 the in vitro and in vivo tests. We observed that overexpression of METCAM/MUC18 reduced in vitro motility and

invasiveness [28] and suppressed in vivo tumorigenesis on subcutaneous (SC) sites and in intraperitoneal cavity as well as in vivo malignant progression of the human ovarian cancer cell line SK-OV-3 in intraperitoneal (IP) cavity in female athymic nude mice [28]. When the other human ovarian cancer cell line, BG-1, was similarly tested, similar results were also observed [80]. 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 [28–30, 80], suggesting that METCAM/MUC18 is a strong candidate as a new tumor and metastasis suppressor in human ovarian cancer cells.

#### **4.6 Human pancreatic cancer**

In contrast to the above results of human and mouse pancreatic cancer that METCAM/MUC18 expression plays a positive role in the malignant progression of PDAC, a group has demonstrated that METCAM/MUC18 expression in cancer-associated fibroblasts (CAFs) has been correlated with the pre-pancreatic intraepithelial neoplasia (PIN) and the invasive ductal pancreatic cancer with a low histological grade. Furthermore, the prognosis for the patients with a low METCAM/ MUC18 expression is poorer than those with a high METCAM/MUC18 expression. Suppressing METCAM/MUC18 expression in CAFs augmented tumor cell in vitro motility and invasiveness in a co-culture system that includes both tumor cells and CAFs. Knockdown of METCAM/MUC18 also augmented CAF activation, possibly via regulation of NF-kB activity, which in turn induces the yield of factors for tumorigenesis. In line with this notion, METCAM/MUC18 overexpression in CAFs decreased in vitro motility and invasiveness of the cancer cells co-cultured with CAFs. Moreover, METCAM/MUC18 expression in CAFs was decreased by interaction with cancer cells. Taken together, reduced METCAM/MUC18 expression in CAFs and reduction of METCAM/MUC18 augment tumor progression of pancreatic cancer [81]. Therefore, METCAM/MUC18 expression suppresses the tumor progression and metastasis of pancreatic cancer. In comparison with the results from Section 3.5.8, METCAM/MUC18 expression also plays a dual role in pancreatic cancer.

#### **4.7 Prostate cancer**

We recently used the knocking down strategy similar to that of DU145 cell line to test the effect of decreased 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, suggesting that expression of METCAM/MUC18 suppressed the tumorigenesis of the human prostate cancer cell line PC-3 [82]. We thus conclude that METCAM/MUC18 serves as a tumor suppressor in the PC-3 cell line. Thus, similar to mouse melanoma, NPC, and pancreatic cancer, METCAM/MUC18 expression also plays a dual role in tumor progression and metastasis in human prostate cancer.

In summary, METCAM/MUC18 may also suppress tumor progression and metastasis of the following solid tumors, such as colorectal cancer [77], mouse melanoma K1735-9 subline [22], NPC type I [24, 25], ovarian cancer [28–30], human pancreatic cancer [81], one prostate cancer cell line PC-3 [82], and perhaps hemangioma [46]. Thus, METCAM/MUC18 appears to play a negative role in tumor progression and metastasis of some solid tumors but a dual role in some other solid tumors. It is not clear why METCAM/MUC18 plays a dual role. Since METCAM/MUC18 only plays a dual role in different cell lines from the same type

**17**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

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 cofactors or heterophilic ligands by using immunological coprecipitation method in 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, 55, 56], another group on the role of huMETCAM/MUC18 in the biology of endothelial cells [41, 83], and one group on breast cancer [45], and our group joined in the effort to study the role of huMETCAM/MUC18 in the progression of mouse melanoma [43] and prostate cancer [31–36] and later breast cancer [18–20], ovarian cancer [27–30], and NPC [23–26], as described above. Recently, more research groups have participated in further exploring the possible role of METCAM/MUC18 in other solid tumors in different organs, such as the colorectum [77], gastro-organ [60], glial cells [61], liver [63, 64], lung [65–71], bone [72–74], and pancreas [75, 76, 81]. Furthermore, preliminary work in leiomyosarcoma, esophagus squamous cell carcinoma, clear cell renal sarcoma, and gallbladder adenocarcinoma are also beginning to emerge [47]. Thus, after decades of group effort, we are beginning to understand the biology of METCAM/

However, we still know very little how METCAM/MUC18 mediates or regulates tumor progression and metastasis of cancer cells. Thus, the biological mechanisms describing the role of METCAM/MUC18 in tumorigenesis and malignant progression are still not well clarified. By deducing knowledge learned from the tumorigenesis of other tumors [6, 17, 37, 40] and angiogenesis [41, 83], we may be able to find some common clues to begin understanding its mechanisms. As such, the following five important aspects are much needed for immediate future studies, such as differential regulation at the transcription level in tumors of different organs; different signaling pathways involved; contributions of different domains of the protein; possible different extent of N-glycosylation in different cancer cell lines, which may critically modulate the function of METCAM/MUC18 in tumor progression; and different kinds or quantities of cofactors or heterophilic ligand(s) in different cancer cell lines.

The mechanism of transcriptional control of METCAM/MUC18 gene is minimally studied [17]. So far, only 900 bp of the core promoter region of the huMETCAM/MUC18 gene are sequenced [84]. The core promoter reflects a typical housekeeping gene, which is rich in GC sequences but does not contain a TATA box. Nevertheless, it includes many consensus sequences presumably as putative binding sites for various transcription regulatory factors, such as SP-1, CREB [85], AP-2 [86–87], c-Myb [88], N-Oct2 (Brn2) [89], Ets [90], CArG [91], and Egr-1 [92], and three insulin-responsive elements (one Ets and two E-box motifs) [93], suggesting that transcriptional control of the huMETCAM/MUC18 gene is regulated by various growth signals [37, 40]. For example, the huMETCAM/MUC18 gene is positively regulated by PKA/CREB (cAMP-responsive element binding protein) and negatively regulated by AP-2α [94]. Having a longer DNA containing sequences for tissue-specific expression of the gene is essential for further understanding the roles

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

future studies, which is feasible as described in Section 5.5.

**5. Preliminary and possible mechanisms**

MUC18-mediated tumor progression.

**5.1 Transcriptional regulation**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

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 cofactors or heterophilic ligands by using immunological coprecipitation method in the future studies, which is feasible as described in Section 5.5.

#### **5. Preliminary and possible mechanisms**

*Tumor Progression and Metastasis*

**4.6 Human pancreatic cancer**

cancer cells.

**4.7 Prostate cancer**

invasiveness [28] and suppressed in vivo tumorigenesis on subcutaneous (SC) sites and in intraperitoneal cavity as well as in vivo malignant progression of the human ovarian cancer cell line SK-OV-3 in intraperitoneal (IP) cavity in female athymic nude mice [28]. When the other human ovarian cancer cell line, BG-1, was similarly tested, similar results were also observed [80]. 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 [28–30, 80], suggesting that METCAM/MUC18 is a strong candidate as a new tumor and metastasis suppressor in human ovarian

In contrast to the above results of human and mouse pancreatic cancer that METCAM/MUC18 expression plays a positive role in the malignant progression of PDAC, a group has demonstrated that METCAM/MUC18 expression in cancer-associated fibroblasts (CAFs) has been correlated with the pre-pancreatic intraepithelial neoplasia (PIN) and the invasive ductal pancreatic cancer with a low histological grade. Furthermore, the prognosis for the patients with a low METCAM/ MUC18 expression is poorer than those with a high METCAM/MUC18 expression. Suppressing METCAM/MUC18 expression in CAFs augmented tumor cell in vitro motility and invasiveness in a co-culture system that includes both tumor cells and CAFs. Knockdown of METCAM/MUC18 also augmented CAF activation, possibly via regulation of NF-kB activity, which in turn induces the yield of factors for tumorigenesis. In line with this notion, METCAM/MUC18 overexpression in CAFs decreased in vitro motility and invasiveness of the cancer cells co-cultured with CAFs. Moreover, METCAM/MUC18 expression in CAFs was decreased by interaction with cancer cells. Taken together, reduced METCAM/MUC18 expression in CAFs and reduction of METCAM/MUC18 augment tumor progression of pancreatic cancer [81]. Therefore, METCAM/MUC18 expression suppresses the tumor progression and metastasis of pancreatic cancer. In comparison with the results from Section

3.5.8, METCAM/MUC18 expression also plays a dual role in pancreatic cancer.

We recently used the knocking down strategy similar to that of DU145 cell line to test the effect of decreased 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, suggesting that expression of METCAM/MUC18 suppressed the tumorigenesis of the human prostate cancer cell line PC-3 [82]. We thus conclude that METCAM/MUC18 serves as a tumor suppressor in the PC-3 cell line. Thus, similar to mouse melanoma, NPC, and pancreatic cancer, METCAM/MUC18 expression also plays a dual role in tumor progression and metastasis in human prostate cancer. In summary, METCAM/MUC18 may also suppress tumor progression and metastasis of the following solid tumors, such as colorectal cancer [77], mouse melanoma K1735-9 subline [22], NPC type I [24, 25], ovarian cancer [28–30], human pancreatic cancer [81], one prostate cancer cell line PC-3 [82], and perhaps hemangioma [46]. Thus, METCAM/MUC18 appears to play a negative role in tumor progression and metastasis of some solid tumors but a dual role in some other solid tumors. It is not clear why METCAM/MUC18 plays a dual role. Since METCAM/MUC18 only plays a dual role in different cell lines from the same type

**16**

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, 55, 56], another group on the role of huMETCAM/MUC18 in the biology of endothelial cells [41, 83], and one group on breast cancer [45], and our group joined in the effort to study the role of huMETCAM/MUC18 in the progression of mouse melanoma [43] and prostate cancer [31–36] and later breast cancer [18–20], ovarian cancer [27–30], and NPC [23–26], as described above. Recently, more research groups have participated in further exploring the possible role of METCAM/MUC18 in other solid tumors in different organs, such as the colorectum [77], gastro-organ [60], glial cells [61], liver [63, 64], lung [65–71], bone [72–74], and pancreas [75, 76, 81]. Furthermore, preliminary work in leiomyosarcoma, esophagus squamous cell carcinoma, clear cell renal sarcoma, and gallbladder adenocarcinoma are also beginning to emerge [47]. Thus, after decades of group effort, we are beginning to understand the biology of METCAM/ MUC18-mediated tumor progression.

However, we still know very little how METCAM/MUC18 mediates or regulates tumor progression and metastasis of cancer cells. Thus, the biological mechanisms describing the role of METCAM/MUC18 in tumorigenesis and malignant progression are still not well clarified. By deducing knowledge learned from the tumorigenesis of other tumors [6, 17, 37, 40] and angiogenesis [41, 83], we may be able to find some common clues to begin understanding its mechanisms. As such, the following five important aspects are much needed for immediate future studies, such as differential regulation at the transcription level in tumors of different organs; different signaling pathways involved; contributions of different domains of the protein; possible different extent of N-glycosylation in different cancer cell lines, which may critically modulate the function of METCAM/MUC18 in tumor progression; and different kinds or quantities of cofactors or heterophilic ligand(s) in different cancer cell lines.

#### **5.1 Transcriptional regulation**

The mechanism of transcriptional control of METCAM/MUC18 gene is minimally studied [17]. So far, only 900 bp of the core promoter region of the huMETCAM/MUC18 gene are sequenced [84]. The core promoter reflects a typical housekeeping gene, which is rich in GC sequences but does not contain a TATA box. Nevertheless, it includes many consensus sequences presumably as putative binding sites for various transcription regulatory factors, such as SP-1, CREB [85], AP-2 [86–87], c-Myb [88], N-Oct2 (Brn2) [89], Ets [90], CArG [91], and Egr-1 [92], and three insulin-responsive elements (one Ets and two E-box motifs) [93], suggesting that transcriptional control of the huMETCAM/MUC18 gene is regulated by various growth signals [37, 40]. For example, the huMETCAM/MUC18 gene is positively regulated by PKA/CREB (cAMP-responsive element binding protein) and negatively regulated by AP-2α [94]. Having a longer DNA containing sequences for tissue-specific expression of the gene is essential for further understanding the roles

of other regulators [17]. In line with this hypothesis, recently, the Ets sequence in the 10 kilo-bp upstream region has been shown to regulate the expression of huMET-CAM/MUC18 gene [95]. We have also engaged in this task by screening in a phage library containing the human genomic sequences and obtained several phage clones which contain at least 4 kilo-bp of in the upstream region of the promoter region of the gene for future studies [96]. Thus, the regulatory mechanism of tissue-specific expression of the METCAMMUC18 gene may be forthcoming.

The epigenetic control of the expression of huMETCAM/MUC18 gene has been implicated in human cancers, because huMETCAM/MUC18 gene is located at the locus of human chromosome 11q23.3 [97] that has been shown to be hypermethylated in NPC [98], suggesting that the expression of this gene may be regulated by epigenetic controls. 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 [99]. METCAM/ MUC18 has also been shown to be methylated in the early stage of most prostate cancer [100]. Thus, it is highly possible that the gene is epigenetically controlled in other cancers.

#### **5.2 Signaling pathways**

The cytoplasmic tail of huMETCAM/MUC18 contains consensus sequences potentially to be phosphorylated by PKA, PKC, and CK2, suggesting that its functions may be mediated by these protein kinases and regulated by cross talk with various signaling pathways [17, 37, 38, 40, 42]. First, it is necessary to biochemically prove how many sites are actually phosphorylated in the cytoplasmic tail of the METCAM/MUC18 protein purified from different cancer cell lines and which protein kinase is responsible for the phosphorylation. After this is answered, then we can further study how METCAM/MUC18 mediates cross talk and networking with different signal pathways and is to be compared with the cytoplasmic tails of other CAMs [6, 8, 41]. Knowledge learned from the impact of other CAMs on tumor progression suggests that METCAM/MUC18, as an integral membrane Ig-like CAM, should mediate inside-in, inside-out, and outside-in signals to participate in intercellular communication and interaction of cell with the extracellular matrix, which results in impacting EMT [6, 8, 41, 101]. Furthermore, huMETCAM/MUC18 has been shown to express in normal mesenchymal cells (smooth muscle, endothelium, and Schwann cells) in the tissue stroma and be a marker for the mesenchymal stem cells [102]; thus, expression of METCAM/ MUC18 may augment the EMT of cancer cells and hence the progression of many cancers. Moreover, METCAM/MUC18 may affect cancer cell progression by cross talk with signaling pathways that affect apoptosis, survival and proliferation, angiogenesis, and energy metabolism of tumor cells [6, 8, 101]. This is indeed found in our preliminary mechanical studies in breast cancer [19, 20], melanoma [21, 22], NPC [24–26], ovarian cancer [28–30], and prostate cancer [34–37]. Further systematic studies by using specific RNAi's to knockdown the downstream effectors one by one in the METCAM/MUC18-expressing clones may be necessary to further understand this aspect of mechanism. Moreover, its interaction with cofactors 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. Finally, METCAM/ MUC18 may interact with various hormonal receptors, growth or anti-growth factors/receptors, various chemokines/receptors, and the Ca++-mediated signaling members and affect tumor progression [17].

**19**

functions of NK cells [117].

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

HuMETCAM/MUC18 expression in melanoma cells is reciprocally regulated by AKT, in which AKT upregulates the level huMETCAM/MUC18 and overexpression of huMETCAM/MUC18 activates endogenous AKT, which in turn inhibits apoptosis and increases survival ability [103]. A similar mechanism is also likely to be used in other cancers; however, the detailed mechanism of how AKT upregulates the expression of METCAM/MUC18 in most cancers has not been reported. After the cytoplasmic tail is phosphorylated, then it may facilitate its interaction with FAK, thus promoting cytoskeleton remodeling, which in turn augments tumor cell motility and invasiveness [83]. Alternatively, after phosphorylation, huMETCAM/MUC18 may interact with the downstream effectors of Ras, activating ERK and JNK, which in turn may transcriptionally activate the expression of AKT or other genes that promote the proliferation and angiogenesis of tumor cells. Moreover, by predicting from the relatively less selectivity of CK2 for its substrate and many CAMs which are phosphorylated by CK2, such as CD44, E-cadherin, and L1-CAM, and one of the integrin receptors in the extracellular matrix protein, vitronectin [104], huMETCAM/MUC18 is very likely to be phosphorylated by CK2 and linked to AKT to affect the proliferation, survival, and other tumorigenesis-related functions [105]. Recent findings appear to support this mechanism in that METCAM/MUC18 may promote EMT of breast cancer cells via activation of RhoA and upregulation of slug [45]. HuMETCAM/MUC18 may play an important role in regulating tumor dormancy or awakening, driving or preventing cancer cells to pre-metastatic niche, and formatting a microenvironment for favorable

HuMETCAM/MUC18 may mediate hematogenous spreading of melanoma cells, as implicated by its expression in endothelial cells and malignant melanoma cells [106] and presence in junctions of endothelial cells [107, 108], essential for tumor angiogenesis in three tumor cell lines [109] and human prostate cancer LNCaP cells [110], and it is highly likely for that in other cancers [111, 112]. HuMETCAM/MUC18 may also be implicated in promoting lymphatic metastasis of cancer cells, since it is one of the lymphatic metastasis-associated genes, which are upregulated in malignant mouse hepatocellular carcinoma [113]. However, the detailed mechanisms of huMETCAM/MUC18-mediated hematogenous and lymphatic spreading of cancer cells remain to be investigated. For this purpose, labeling cells with viable dyes and employing a newly developed non-intruding, but highly photo-penetrating imaging photoacoustic tomography (PAT) to monitor each step of the process in real time in

HuMETCAM/MUC18 may interact with the host immune system and affect tumor progression, though the immune system may have a contradictory role in the process [115]. This notion is positively supported by a recent finding that a subset of host B lymphocytes may be implicated in regulating melanoma malignant progression via interaction with huMETCAM/MUC18 [116]. Our syngeneic mouse system for mouse melanoma should be useful for exploring the role of immune T and B cells in the progression of METCAM/MUC18-expressing melanoma cells. However, the role of B and T cells in the progression of most human cancer cells may not be explored in the athymic nude mouse models since most human cancer cells can only grow as xenografts in these immunodeficient mouse models. Nevertheless, to investigate the effect of huMETCAM/MUC18 expression on mediating NK cells in metastasis may be possible in these nude mouse models, which can be tested by pretreatment of nude mice with anti-NK surface marker antibodies to deplete the NK cells prior to injection of the huMETCAM/MUC18 expressing cancer cells. This possibility is supported by the finding that the surface huMETCAM/MUC18 expressed in cancer cells may have a homophilic interaction with the NK cells, which also express huMETCAM/MUC18 and enhance cytotoxic

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

or unfavorable tumor growth in secondary sites [17, 37].

hairless nude mice may be helpful to provide some answers [114].

#### *METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

HuMETCAM/MUC18 expression in melanoma cells is reciprocally regulated by AKT, in which AKT upregulates the level huMETCAM/MUC18 and overexpression of huMETCAM/MUC18 activates endogenous AKT, which in turn inhibits apoptosis and increases survival ability [103]. A similar mechanism is also likely to be used in other cancers; however, the detailed mechanism of how AKT upregulates the expression of METCAM/MUC18 in most cancers has not been reported. After the cytoplasmic tail is phosphorylated, then it may facilitate its interaction with FAK, thus promoting cytoskeleton remodeling, which in turn augments tumor cell motility and invasiveness [83]. Alternatively, after phosphorylation, huMETCAM/MUC18 may interact with the downstream effectors of Ras, activating ERK and JNK, which in turn may transcriptionally activate the expression of AKT or other genes that promote the proliferation and angiogenesis of tumor cells. Moreover, by predicting from the relatively less selectivity of CK2 for its substrate and many CAMs which are phosphorylated by CK2, such as CD44, E-cadherin, and L1-CAM, and one of the integrin receptors in the extracellular matrix protein, vitronectin [104], huMETCAM/MUC18 is very likely to be phosphorylated by CK2 and linked to AKT to affect the proliferation, survival, and other tumorigenesis-related functions [105]. Recent findings appear to support this mechanism in that METCAM/MUC18 may promote EMT of breast cancer cells via activation of RhoA and upregulation of slug [45]. HuMETCAM/MUC18 may play an important role in regulating tumor dormancy or awakening, driving or preventing cancer cells to pre-metastatic niche, and formatting a microenvironment for favorable or unfavorable tumor growth in secondary sites [17, 37].

HuMETCAM/MUC18 may mediate hematogenous spreading of melanoma cells, as implicated by its expression in endothelial cells and malignant melanoma cells [106] and presence in junctions of endothelial cells [107, 108], essential for tumor angiogenesis in three tumor cell lines [109] and human prostate cancer LNCaP cells [110], and it is highly likely for that in other cancers [111, 112]. HuMETCAM/MUC18 may also be implicated in promoting lymphatic metastasis of cancer cells, since it is one of the lymphatic metastasis-associated genes, which are upregulated in malignant mouse hepatocellular carcinoma [113]. However, the detailed mechanisms of huMETCAM/MUC18-mediated hematogenous and lymphatic spreading of cancer cells remain to be investigated. For this purpose, labeling cells with viable dyes and employing a newly developed non-intruding, but highly photo-penetrating imaging photoacoustic tomography (PAT) to monitor each step of the process in real time in hairless nude mice may be helpful to provide some answers [114].

HuMETCAM/MUC18 may interact with the host immune system and affect tumor progression, though the immune system may have a contradictory role in the process [115]. This notion is positively supported by a recent finding that a subset of host B lymphocytes may be implicated in regulating melanoma malignant progression via interaction with huMETCAM/MUC18 [116]. Our syngeneic mouse system for mouse melanoma should be useful for exploring the role of immune T and B cells in the progression of METCAM/MUC18-expressing melanoma cells. However, the role of B and T cells in the progression of most human cancer cells may not be explored in the athymic nude mouse models since most human cancer cells can only grow as xenografts in these immunodeficient mouse models. Nevertheless, to investigate the effect of huMETCAM/MUC18 expression on mediating NK cells in metastasis may be possible in these nude mouse models, which can be tested by pretreatment of nude mice with anti-NK surface marker antibodies to deplete the NK cells prior to injection of the huMETCAM/MUC18 expressing cancer cells. This possibility is supported by the finding that the surface huMETCAM/MUC18 expressed in cancer cells may have a homophilic interaction with the NK cells, which also express huMETCAM/MUC18 and enhance cytotoxic functions of NK cells [117].

*Tumor Progression and Metastasis*

in other cancers.

**5.2 Signaling pathways**

of other regulators [17]. In line with this hypothesis, recently, the Ets sequence in the 10 kilo-bp upstream region has been shown to regulate the expression of huMET-CAM/MUC18 gene [95]. We have also engaged in this task by screening in a phage library containing the human genomic sequences and obtained several phage clones which contain at least 4 kilo-bp of in the upstream region of the promoter region of the gene for future studies [96]. Thus, the regulatory mechanism of tissue-specific

The epigenetic control of the expression of huMETCAM/MUC18 gene has been implicated in human cancers, because huMETCAM/MUC18 gene is located at the locus of human chromosome 11q23.3 [97] that has been shown to be hypermethylated in NPC [98], suggesting that the expression of this gene may be regulated by epigenetic controls. 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 [99]. METCAM/ MUC18 has also been shown to be methylated in the early stage of most prostate cancer [100]. Thus, it is highly possible that the gene is epigenetically controlled

The cytoplasmic tail of huMETCAM/MUC18 contains consensus sequences potentially to be phosphorylated by PKA, PKC, and CK2, suggesting that its functions may be mediated by these protein kinases and regulated by cross talk with various signaling pathways [17, 37, 38, 40, 42]. First, it is necessary to biochemically prove how many sites are actually phosphorylated in the cytoplasmic tail of the METCAM/MUC18 protein purified from different cancer cell lines and which protein kinase is responsible for the phosphorylation. After this is answered, then we can further study how METCAM/MUC18 mediates cross talk and networking with different signal pathways and is to be compared with the cytoplasmic tails of other CAMs [6, 8, 41]. Knowledge learned from the impact of other CAMs on tumor progression suggests that METCAM/MUC18, as an integral membrane Ig-like CAM, should mediate inside-in, inside-out, and outside-in signals to participate in intercellular communication and interaction of cell with the extracellular matrix, which results in impacting EMT [6, 8, 41, 101]. Furthermore, huMETCAM/MUC18 has been shown to express in normal mesenchymal cells (smooth muscle, endothelium, and Schwann cells) in the tissue stroma and be a marker for the mesenchymal stem cells [102]; thus, expression of METCAM/ MUC18 may augment the EMT of cancer cells and hence the progression of many cancers. Moreover, METCAM/MUC18 may affect cancer cell progression by cross talk with signaling pathways that affect apoptosis, survival and proliferation, angiogenesis, and energy metabolism of tumor cells [6, 8, 101]. This is indeed found in our preliminary mechanical studies in breast cancer [19, 20], melanoma [21, 22], NPC [24–26], ovarian cancer [28–30], and prostate cancer [34–37]. Further systematic studies by using specific RNAi's to knockdown the downstream effectors one by one in the METCAM/MUC18-expressing clones may be necessary to further understand this aspect of mechanism. Moreover, its interaction with cofactors 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. Finally, METCAM/ MUC18 may interact with various hormonal receptors, growth or anti-growth factors/receptors, various chemokines/receptors, and the Ca++-mediated signaling

expression of the METCAMMUC18 gene may be forthcoming.

**18**

members and affect tumor progression [17].

#### **5.3 Functional domain**

To begin addressing the relation of the protein structure of huMETCAM/MUC18 to its functions in tumorigenesis and metastasis, we have generated mutant-deleted different domains of huMETCAM/MUC18 by using a special PCR method [118] and used them to determine their contribution to tumorigenesis. Surprisingly, our preliminary results showed that the ecto-domain and the intact copy of huMETCAM/MUC18 cDNA equally efficiently induced tumorigenesis in LNCaP cells in nude mice, suggesting the key role of the ecto-domain in inducing tumorigenesis of prostate cancer cells in vivo. However, this stirs up a puzzling question that the cytoplasmic domain was not essential for this process [119]. Nevertheless, critical direct test of using only the cytoplasmic domain for inducing tumor should be performed. It is essential that a systematic study has also to be performed in other cancer cell lines before a definitive conclusion can be drawn.

#### **5.4 Glycosylation**

Malignant progression of cancer cells has been shown to associate with an abnormal glycosylation, resulting in expression of altered carbohydrate determinants [120]. Thus, the glycosylated status of huMETCAM/MUC18 in different cancer types may be different from normal cells and may manifest either a positive or negative effect on the progression of different cancer types, which should be very intriguing since huMETCAM/MUC18 possesses six conserved N-glycosylation sites in the extracellular domain [17, 37, 40].

Glycosylation of a protein may affect the proper folding, stability, and/or activity of a protein [121]. Both huMETCAM/MUC18 and moMETCAM/MUC18 are very likely heavily glycosylated, sialylated, and/or posttranslationally modified, because both have an apparent molecular weight of about 110–150 kDa, in comparison with the naked protein with a molecular weight of about 65–70 kDa [122]. The possible roles of METCAM/MUC18 glycosylation in inducing/promoting or suppressing the metastasis of cancer cells should be explored [123]. To initiate the study, we subjected the huMETCAM/MUC18, which was isolated from one human cancer cell line, to the digestion with N-glycosidase F, neuraminidase (sialidase), O-glycosidase, or endoglycosidase H. 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], suggesting 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. Our hypothesis is supported by a recent report that described GCNT3 as an upstream regulator of METCAM/MUC18 in that it glycosylates METCAM/ MUC18 and extends its half-life which results in further elevation of S100A8/ A9-mediated cellular motility in melanoma cells [124]. The role of glycosylation in the six N-glycosylation sites should be genetically altered to explore their effects on the functions of METCAM/MUC18 in tumor progression and metastasis.

#### **5.5 Heterophilic ligands and cofactors that modulate the function of METCAM/MUC18**

Since METCAM/MUC18 only plays a dual role in different cell lines from the same type of cancer or in different types of cancers, but never in the same cancer cell line, it

**21**

**6. Conclusions**

**7.1 Research perspectives**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

cognate heterophilic ligand(s) is critical for understanding the mechanism.

METCAM/MUC18 also plays a key positive function in the progression of angio-

sarcoma, breast cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, lung cancer, melanoma, NPC type III, osteosarcoma, pancreatic cancer, prostate cancer, and possibly other cancers. On the other hand, 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. To further understand its role in these processes, it is essential to further identify its cofactor regulators and cognate heterophilic ligands, define its functional domains, and study its cross talk 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 foundation for future intriguing investigation to further understand the detailed mechanism of METCAM/MUC18-mediated tumor progression and metastasis of various cancer cell lines. For this purpose besides those have been described above, other future endeavors may include (a) understanding the mechanisms in the METCAM/MUC18-mediated tumor progression and metastasis, such as intrinsic growth capability, key chemokines and cytokines

**7. Research perspectives and clinical applications**

is logical to suggest a possible explanation that the intrinsic properties of each cancer cell line may provide specific cofactors or heterophilic ligands that may positively or negatively modulate the METCAM/MUC18-mediated tumor progression and metastasis. This can be readily scrutinized by identifying these specific intrinsic cofactors or heterophilic ligands in different cancer cell lines by using immunological coprecipitation method. This approach appears to be feasible as shown in our preliminary result in that a putative heterophilic ligand, 72 kDa protein, is identified [17, 37, 40]. This protein is present at a higher concentration in PC-3 cells than in DU145 that may be responsible for an opposite role of METCAM/MUC18 in tumor progression of these two cell lines. Thus, it is possible that mechanisms of huMETCAM/MUC18-mediated cancer progression may be different in different cancer cell lines due to their different intrinsic properties, which possess different concentration or completely different heterophilic different ligands and/or cofactors. The heterophilic ligands and/or cofactors of METCAM/MUC18 may contribute to the cellular intrinsic properties, such as adhesion-associated signaling cascades and cytoskeleton rearrangement, leading to different EMT of these cells and modulating the huMETCAM/MUC18-mediated tumor progression and metastasis. Different intrinsic cofactors in different cancer cell lines may modulate METCAM/MUC18 and alter cell-to-cell and cell-extracellular matrix interactions in the tumor microenvironment, resulting in affecting tumor progression and metastasis in vivo. Finally, these cofactors/ligands may interact differently with METCAM/MUC18 in different cell lines and affect other host physiological factors, which may augment or suppress in vivo tumor progression and metastasis by affecting metabolic switch, pro-apoptosis/anti-apoptosis, tumor angiogenesis, and host immune system in the tumor micro-*milieu* and in various metastatic sites [17, 37, 40, 111, 112]. Thus, the identification of the cofactors and the huMETCAM/MUC18-

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

#### *METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

is logical to suggest a possible explanation that the intrinsic properties of each cancer cell line may provide specific cofactors or heterophilic ligands that may positively or negatively modulate the METCAM/MUC18-mediated tumor progression and metastasis. This can be readily scrutinized by identifying these specific intrinsic cofactors or heterophilic ligands in different cancer cell lines by using immunological coprecipitation method. This approach appears to be feasible as shown in our preliminary result in that a putative heterophilic ligand, 72 kDa protein, is identified [17, 37, 40]. This protein is present at a higher concentration in PC-3 cells than in DU145 that may be responsible for an opposite role of METCAM/MUC18 in tumor progression of these two cell lines. Thus, it is possible that mechanisms of huMETCAM/MUC18-mediated cancer progression may be different in different cancer cell lines due to their different intrinsic properties, which possess different concentration or completely different heterophilic different ligands and/or cofactors. The heterophilic ligands and/or cofactors of METCAM/MUC18 may contribute to the cellular intrinsic properties, such as adhesion-associated signaling cascades and cytoskeleton rearrangement, leading to different EMT of these cells and modulating the huMETCAM/MUC18-mediated tumor progression and metastasis. Different intrinsic cofactors in different cancer cell lines may modulate METCAM/MUC18 and alter cell-to-cell and cell-extracellular matrix interactions in the tumor microenvironment, resulting in affecting tumor progression and metastasis in vivo. Finally, these cofactors/ligands may interact differently with METCAM/MUC18 in different cell lines and affect other host physiological factors, which may augment or suppress in vivo tumor progression and metastasis by affecting metabolic switch, pro-apoptosis/anti-apoptosis, tumor angiogenesis, and host immune system in the tumor micro-*milieu* and in various metastatic sites [17, 37, 40, 111, 112]. Thus, the identification of the cofactors and the huMETCAM/MUC18 cognate heterophilic ligand(s) is critical for understanding the mechanism.

#### **6. Conclusions**

*Tumor Progression and Metastasis*

To begin addressing the relation of the protein structure of huMETCAM/MUC18 to its functions in tumorigenesis and metastasis, we have generated mutant-deleted different domains of huMETCAM/MUC18 by using a special PCR method [118] and used them to determine their contribution to tumorigenesis. Surprisingly, our preliminary results showed that the ecto-domain and the intact copy of huMETCAM/MUC18 cDNA equally efficiently induced tumorigenesis in LNCaP cells in nude mice, suggesting the key role of the ecto-domain in inducing tumorigenesis of prostate cancer cells in vivo. However, this stirs up a puzzling question that the cytoplasmic domain was not essential for this process [119]. Nevertheless, critical direct test of using only the cytoplasmic domain for inducing tumor should be performed. It is essential that a systematic study has also to be performed in other cancer cell lines before a definitive conclusion can be

Malignant progression of cancer cells has been shown to associate with an abnormal glycosylation, resulting in expression of altered carbohydrate determinants [120]. Thus, the glycosylated status of huMETCAM/MUC18 in different cancer types may be different from normal cells and may manifest either a positive or negative effect on the progression of different cancer types, which should be very intriguing since huMETCAM/MUC18 possesses six conserved N-glycosylation sites

Glycosylation of a protein may affect the proper folding, stability, and/or activity of a protein [121]. Both huMETCAM/MUC18 and moMETCAM/MUC18 are very likely heavily glycosylated, sialylated, and/or posttranslationally modified, because both have an apparent molecular weight of about 110–150 kDa, in comparison with the naked protein with a molecular weight of about 65–70 kDa [122]. The possible roles of METCAM/MUC18 glycosylation in inducing/promoting or suppressing the metastasis of cancer cells should be explored [123]. To initiate the study, we subjected the huMETCAM/MUC18, which was isolated from one human cancer cell line, to the digestion with N-glycosidase F, neuraminidase (sialidase), O-glycosidase, or endoglycosidase H. 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], suggesting 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. Our hypothesis is supported by a recent report that described GCNT3 as an upstream regulator of METCAM/MUC18 in that it glycosylates METCAM/ MUC18 and extends its half-life which results in further elevation of S100A8/ A9-mediated cellular motility in melanoma cells [124]. The role of glycosylation in the six N-glycosylation sites should be genetically altered to explore their effects on

the functions of METCAM/MUC18 in tumor progression and metastasis.

**5.5 Heterophilic ligands and cofactors that modulate the function of** 

Since METCAM/MUC18 only plays a dual role in different cell lines from the same type of cancer or in different types of cancers, but never in the same cancer cell line, it

**5.3 Functional domain**

drawn.

**5.4 Glycosylation**

in the extracellular domain [17, 37, 40].

**20**

**METCAM/MUC18**

METCAM/MUC18 also plays a key positive function in the progression of angiosarcoma, breast cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, lung cancer, melanoma, NPC type III, osteosarcoma, pancreatic cancer, prostate cancer, and possibly other cancers. On the other hand, 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. To further understand its role in these processes, it is essential to further identify its cofactor regulators and cognate heterophilic ligands, define its functional domains, and study its cross talk 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**

The current studies have laid an important foundation for future intriguing investigation to further understand the detailed mechanism of METCAM/MUC18-mediated tumor progression and metastasis of various cancer cell lines. For this purpose besides those have been described above, other future endeavors may include (a) understanding the mechanisms in the METCAM/MUC18-mediated tumor progression and metastasis, such as intrinsic growth capability, key chemokines and cytokines

participating in the evasion of immunological responses, and key pro-angiogenic and anti-angiogenic factors participating in the augmentation of angiogenesis, (b) identification of possible miRNAs and noncoding RNAs participating in the process upstream and downstream of METCAM/MUC18 [126], and (c) possible clinical applications that should be explored. Precaution should be taken that a thorough picture may be possibly revealed only after the comprehensive studies are successfully executed.

#### **7.2 Clinical applications**

Four major approaches may be taken to decrease or stop the progression and metastatic propensity of cancer cells and keep them staying at the primary site, stopping them in a dormant state or keeping the disseminating cancer cells at the state of micrometastases: (a) Dispense humanized anti-METCAM/MUC18 antibodies to the cancer patients [125]. (b) Knocking down the METCAM/MUC18 expression by siR-NAs to silence the genes [35, 36]. For knocking down therapy, the METCAM/MUC18 gene-specific siRNAs may be delivered by liposomes or other delivery methods [126]. (c) Target at downstream key members in the signaling pathways which are activated by the promotion. (d) Target at the cofactors or the cognate heterophilic ligand(s) of METCAM/MUC18. The above strategies may be used in single or better in combination for treating the patients. However, the dual role of METCAM/MUC18 in cancer progression may limit the above clinical applications to only cancers exhibiting a positive METCAM/MUC18-mediated tumor progression and metastasis.

### **Acknowledgements**

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

### **Conflict of interest**

The author has no conflict of interests.

### **Author details**

Guang-Jer Wu1,2

1 Department of Bioscience Technology and 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.

**23**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

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*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

#### **References**

*Tumor Progression and Metastasis*

**7.2 Clinical applications**

**Acknowledgements**

**Conflict of interest**

**Author details**

Guang-Jer Wu1,2

Medicine, Atlanta, GA, USA

The author has no conflict of interests.

Chung Yuan Christian University, Taoyuan, Taiwan

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

provided the original work is properly cited.

participating in the evasion of immunological responses, and key pro-angiogenic and anti-angiogenic factors participating in the augmentation of angiogenesis, (b) identification of possible miRNAs and noncoding RNAs participating in the process upstream and downstream of METCAM/MUC18 [126], and (c) possible clinical applications that should be explored. Precaution should be taken that a thorough picture may be possibly revealed only after the comprehensive studies are successfully executed.

Four major approaches may be taken to decrease or stop the progression and metastatic propensity of cancer cells and keep them staying at the primary site, stopping them in a dormant state or keeping the disseminating cancer cells at the state of micrometastases: (a) Dispense humanized anti-METCAM/MUC18 antibodies to the cancer patients [125]. (b) Knocking down the METCAM/MUC18 expression by siR-NAs to silence the genes [35, 36]. For knocking down therapy, the METCAM/MUC18 gene-specific siRNAs may be delivered by liposomes or other delivery methods [126]. (c) Target at downstream key members in the signaling pathways which are activated by the promotion. (d) Target at the cofactors or the cognate heterophilic ligand(s) of METCAM/MUC18. The above strategies may be used in single or better in combination for treating the patients. However, the dual role of METCAM/MUC18 in cancer progression may limit the above clinical applications to only cancers exhibiting a

positive METCAM/MUC18-mediated tumor progression and metastasis.

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

1 Department of Bioscience Technology and Center for Biomedical Technology,

2 Department of Microbiology and Immunology, Emory University School of

© 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,

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**29**

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tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma. International Journal of Cancer.

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[100] Brait M, Banerjee M, Maldonado L,

Ooki A, Loyo M, Guida E, et al. Promoter methylation of MCAM, ERα and ERβ in serum of early stage prostate cancer patients. Oncotarget. 2017;**8**(9):15431-15440. DOI: 10.18632/

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Falmouth, MA, USA

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340296

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contains an SF binding CArG element required for MyoD expression in skeletal myoblasts and during muscle regeneration. Molecular Biology of the

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

[93] O'Brien RM, Streeper RS, Ayala JE, Stadelmaier BT, Hornbuckle LA. Insulin-regulated gene expression. Biochemical Society Transactions.

MyoD distal regulatory region

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

Antisense to the early growth response-1 gene (Egr-1) inhibits prostate tumor development in TRAMP mice. Annals of the New York Academy

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

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2001;**29**:552-558

2010;**5**:e12452

2017;**36**(29):4150-4160

[96] Wu GJ et al. Transcriptional

regulation of METCAM/MUC18 gene in the upstream region in cancer cells. (In

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potential of human melanoma cells.

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

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potential of human melanoma cells. Oncogene. 1995;**11**:691-700

*Tumor Progression and Metastasis*

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shRNAs increasing tumorigenicity in

[83] Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J, Dignat-George F.

[84] Sers C, Kirsch K, Rothbacher U, Riethmuller G, Johnson JP. Genomic organization of the melanomaassociated glycoprotein MUC18: Implications for the evolution of the immunoglobulin domains. Proceedings of the National Academy of Sciences of the United States of America.

[85] Rummel MM, Sers C, Johnson JP. Phorbol ester and cyclic AMP-

mediated regulation of the melanomaassociated cell adhesion molecule MUC18/MCAM. Cancer Research.

[86] Jean D, Gershenwald JE, Huang S, Luca M, Hudson MJ, Tainsky MA, et al. Loss of AP-2 results in

up-regulation of CAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells. The Journal of Biological Chemistry.

[87] Ruiz M, Pettaway C, Song R, Stoeltzing O, Ellis L, Bar-Eli M. Activator protein 2α inhibits

in prostate cancer cells. Cancer Research. 2004;**64**:631-638

Acta. 1996;**1299**:F123-F139

[89] JAF T, Murphy K, Baker E,

The brn-2 gene regulates the

tumorigenicity and represses vascular endothelial growth factor transcription

[88] Ness SA. The Myb oncoprotein: Regulating a regulator. Biochem Biophys

Sutherland GR, Parsons PG, Sturm RA.

melanocytic phenotype and tumorigenic

nude mice. 2019 (submitted)

Outside-in signaling pathway linked to CD146 engagement in human endothelial cells. The Journal of Biological Chemistry.

2001;**276**:1564-1569

1993;**90**:8514-8518

1996;**56**:2218-2223

1998;**273**:16501-16508

[77] 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

[78] 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.

[79] 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

[80] 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

2006;**119**(8):1920-1926

LLC); 2019 (in press)

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

[82] 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

[81] 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.

**28**

[90] Yordy JS, Li R, Sementchenko VI, Pei H, Muise-Helmericks RC, Watson DK. S100 expression modulates ETS1 transcriptional activity and inhibits cell invasion. Oncogene. 2004;**23**:6654-6665

[91] L'honore A, Lamb NJ, Vandromme M, Turowski P, Carnac G, Fernandez A. MyoD distal regulatory region contains an SF binding CArG element required for MyoD expression in skeletal myoblasts and during muscle regeneration. Molecular Biology of the Cell. 2003;**14**:2151-2162

[92] Baron V, Duss S, Rhim J, Mercola D. Antisense to the early growth response-1 gene (Egr-1) inhibits prostate tumor development in TRAMP mice. Annals of the New York Academy of Sciences. 2003;**1002**:197-216

[93] O'Brien RM, Streeper RS, Ayala JE, Stadelmaier BT, Hornbuckle LA. Insulin-regulated gene expression. Biochemical Society Transactions. 2001;**29**:552-558

[94] Melnikova VO, Debroff AS, Zigler M, Villares GJ, Braeuer R, Wang H, et al. CREB inhibits AP-2α expression to regulate the malignant phenotype of melanoma. PLoS One. 2010;**5**:e12452

[95] Sechler M, Parrish JK, Birks DK, Jedlicka P. The histone demethylase KDM3A, and its downstream target MCAM, promote Ewing Sarcoma cell migration and metastasis. Oncogene. 2017;**36**(29):4150-4160

[96] Wu GJ et al. Transcriptional regulation of METCAM/MUC18 gene in the upstream region in cancer cells. (In press)

[97] Lung HL, Cheng Y, Kumaran MK, et al. Fine mapping of the 11Q22-23

tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma. International Journal of Cancer. 2004;**112**:628-635

[98] Wu GJ. MCAM (melanoma cell adhesion molecule). Atlas of Genetics and Cytogenetics in Oncology and Haematology. 2012. DOI: 10.4267/2042/47418. ID41314ch11q23. The manually annotated Biomax Human Genome Database Version 4.0, Biomax Informatics AG, www. biomax.com, the Biomax Solutions Inc., Falmouth, MA, USA

[99] Wu GJ et al. Epigenetic regulation of METCAM/MUC18 expression in nasopharyngeal carcinoma. (In press)

[100] Brait M, Banerjee M, Maldonado L, Ooki A, Loyo M, Guida E, et al. Promoter methylation of MCAM, ERα and ERβ in serum of early stage prostate cancer patients. Oncotarget. 2017;**8**(9):15431-15440. DOI: 10.18632/ oncotarget.14873

[101] Wong CW, Dye DE, Coombe DR. The role of immunoglobulin superfamily cell adhesion molecules in cancer metastasis. International Journal of Cell Biology. 2012;**2012**: 9 p. Article ID 340296

[102] Sorrentino A, Ferracin M, Castelli G, Biffoni M, Tomaselli G, Baiocchi M, et al. Isolation and characterization of CD146+ multipotent mesenchymal stromal cells. Experimental Hematology. 2008;**36**:1035-1046

[103] Li G, Kalabis J, Xu X, Meier F, Oka M, Bogenrieder T, et al. Reciprocal regulation of MelCAM and AKT in human melanoma. Oncogene. 2003;**22**:6891-6899

[104] Maggio F, Pinna LA. Onethousand-and-one substrates of protein kinase CK2? The FASEB Journal. 2003;**17**:349-368

[105] Datta SR, Brunet A, Greenberg ME. Cellular survival: A play in three AKTs. Genes & Development. 1999;**13**:2905-2927

[106] Sers C, Riethmuller G, Johnson JP. MUC18, a melanoma-progression associated molecule, and its potential role in tumor vascularization and hematogenous spread. Cancer Research. 1994;**54**:5689-5694

[107] Bardin N, Anfosso F, Masse J, Cramer E, Sabatier F, LeBivic A, et al. Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell adhesion. Blood. 2001;**98**:3677-3684

[108] Kang Y, Wang F, Feng J, Yang D, Yang X, Yan X. Knockdown of CD146 reduces the migration and proliferation of human endothelial cells. Cell Research. 2006;**16**:313-318

[109] Yan X, Lin Y, Tang D, Shen Y, Yuan M, Zhang Z, et al. A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth. Blood. 2003;**102**:184-191

[110] Wu GJ, Son EL. Soluble METCAM/ MUC18 blocks angiogenesis during tumor formation of human prostate cancer cells. In: Proceedings of the 97th Annual Meeting of American Association for the Cancer Research. Vol. 47. 2006. Abstract# 252

[111] Wu GJ. Chapter 7: The role of MUC18 in prostate carcinoma in Immunohistochemistry and in situ hybridization of human carcinoma. In: Hayat MA, editor. Molecular Pathology, Lung Carcinoma, Breast Carcinoma, and Prostate Carcinoma. Vol. 1. Maryland Heights, MO, USA: Elsevier Science/Academic Press; 2004. pp. 347-358

[112] Wu GJ. Chapter 11: Dual roles of the melanoma CAM (MelCAM/ METCAM) in malignant progression of melanoma. In: Research on Melanoma: A glimpse into current directions and future trends. University Campus STeP Ri, Rijeka, Croatia: InTech Open Access Publisher; 2011. pp. 229-242

[113] Song B, Tang JW, Wang B, Cui XN, Zhou CH, Hou L. Screening for lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines Hca-F and Hca-P using gene chip. Chinese Journal of Cancer. 2005;**24**(7):774-780

[114] Wang LV. Prospects of photoacoustic tomography (PAT). Medical Physics. 2008;**35**(12):5758-5767

[115] deVisser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nature Reviews. Cancer. 2006;**6**:24-37

[116] Staquicini F, Tandle A, Libutti SK, Sun J, Zigler M, Bar-Eli M, et al. A subset of host B lymphocytes controls melanoma metastasis through a melanoma cell adhesion molecule/ MUC18-dependent interaction: Evidence from mice and humans. Cancer Research. 2008;**68**(20):8419-8428

[117] Despoix N, Walzer T, Jouve N, Blot-Chabaud M, Bardin N, Paul P, et al. Mouse CD146/MCAM is a marker of natural killer cell maturation. European Journal of Immunology. 2008;**38**:2855-2864

[118] Geiser M, Cebe R, Drewello D, Schmitz R. Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzyme and DNA ligase. BioTechniques. 2001;**31**:88-92

[119] Wu GJ et al. Functional domains of METCAM/MUC18 in the tumor progression. (In press)

**31**

*METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers*

*DOI: http://dx.doi.org/10.5772/intechopen.87037*

[120] Kannagi R, Izawa M, Koike T, Miyazaki K, Kimura N. Carbohydratemediated cell adhesion in cancer metastasis and angiogenesis. Cancer

[121] Parodi A. Protein glycosylation and its role in protein folding. Annual Review of Biochemistry.

[122] Lehmann JM, Holzmann B, Breitbart EW, Schmiegelow P, Riethmuller G, Johnson JP.

Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113,000 and a protein with a molecular weight of 76,000. Cancer Research.

[123] Yamamoto H, Oviedo A, Sweeley C, Saito T, Moskal JR. α2,6-Sialylation of cell-surface N-glycans inhibits glioma formation in vivo. Cancer Research.

[125] Leslie MC, Zhao YJ, Lachman LB, Hwu P, Wu GJ, Bar-Eli M. Immunization

[126] Kasinski AL, Slack FJ. MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy. Nature Reviews. Cancer.

against MUC18/MCAM, a novel antigen that drives melanoma invasion

and metastasis. Gene Therapy.

Science. 2004;**95**(5):377-384

2000;**69**:69-93

1987;**47**:841-845

2001;**61**:6822-6829

2018;**26**(3):431-444

2007;**14**:316-323

2011;**11**:849-864

[124] Sumardika IW, Youyi C, Kondo E, Inoue Y, Ruma MW, Murata H, et al. β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-Nacetylglucosaminyltransferase 3 increases MCAM stability, which enhances S100A8/A9-mediated cancer motility. Oncology Research. *METCAM/MUC18 Promotes Tumor Progression and Metastasis in Most Human Cancers DOI: http://dx.doi.org/10.5772/intechopen.87037*

[120] Kannagi R, Izawa M, Koike T, Miyazaki K, Kimura N. Carbohydratemediated cell adhesion in cancer metastasis and angiogenesis. Cancer Science. 2004;**95**(5):377-384

*Tumor Progression and Metastasis*

2003;**17**:349-368

1999;**13**:2905-2927

1994;**54**:5689-5694

2001;**98**:3677-3684

2003;**102**:184-191

kinase CK2? The FASEB Journal.

[105] Datta SR, Brunet A, Greenberg ME. Cellular survival: A play in three AKTs. Genes & Development. [112] Wu GJ. Chapter 11: Dual roles of the melanoma CAM (MelCAM/ METCAM) in malignant progression of melanoma. In: Research on Melanoma: A glimpse into current directions and future trends. University Campus STeP Ri, Rijeka, Croatia: InTech Open Access

Publisher; 2011. pp. 229-242

2005;**24**(7):774-780

[114] Wang LV. Prospects of photoacoustic tomography (PAT). Medical Physics. 2008;**35**(12):5758-5767

[115] deVisser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nature Reviews. Cancer. 2006;**6**:24-37

[116] Staquicini F, Tandle A, Libutti SK, Sun J, Zigler M, Bar-Eli M, et al. A subset of host B lymphocytes controls melanoma metastasis through a melanoma cell adhesion molecule/ MUC18-dependent interaction:

Evidence from mice and humans. Cancer

Research. 2008;**68**(20):8419-8428

[117] Despoix N, Walzer T, Jouve N, Blot-Chabaud M, Bardin N, Paul P, et al. Mouse CD146/MCAM is a marker of natural killer cell maturation. European Journal of Immunology.

[118] Geiser M, Cebe R, Drewello D, Schmitz R. Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzyme and DNA ligase. BioTechniques. 2001;**31**:88-92

[119] Wu GJ et al. Functional domains of METCAM/MUC18 in the tumor

2008;**38**:2855-2864

progression. (In press)

[113] Song B, Tang JW, Wang B, Cui XN, Zhou CH, Hou L. Screening for lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines Hca-F and Hca-P using gene chip. Chinese Journal of Cancer.

[106] Sers C, Riethmuller G, Johnson JP. MUC18, a melanoma-progression associated molecule, and its potential role in tumor vascularization and hematogenous spread. Cancer Research.

[107] Bardin N, Anfosso F, Masse J, Cramer E, Sabatier F, LeBivic A, et al. Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell adhesion. Blood.

[108] Kang Y, Wang F, Feng J, Yang D, Yang X, Yan X. Knockdown of CD146 reduces the migration and proliferation

[109] Yan X, Lin Y, Tang D, Shen Y, Yuan M, Zhang Z, et al. A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth. Blood.

[110] Wu GJ, Son EL. Soluble METCAM/ MUC18 blocks angiogenesis during tumor formation of human prostate cancer cells. In: Proceedings of the 97th Annual Meeting of American Association for the Cancer Research.

Vol. 47. 2006. Abstract# 252

[111] Wu GJ. Chapter 7: The role of MUC18 in prostate carcinoma in Immunohistochemistry and in situ hybridization of human carcinoma. In: Hayat MA, editor. Molecular Pathology, Lung Carcinoma, Breast Carcinoma, and Prostate Carcinoma. Vol. 1. Maryland Heights, MO, USA: Elsevier Science/Academic Press; 2004.

of human endothelial cells. Cell Research. 2006;**16**:313-318

**30**

pp. 347-358

[121] Parodi A. Protein glycosylation and its role in protein folding. Annual Review of Biochemistry. 2000;**69**:69-93

[122] Lehmann JM, Holzmann B, Breitbart EW, Schmiegelow P, Riethmuller G, Johnson JP. Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113,000 and a protein with a molecular weight of 76,000. Cancer Research. 1987;**47**:841-845

[123] Yamamoto H, Oviedo A, Sweeley C, Saito T, Moskal JR. α2,6-Sialylation of cell-surface N-glycans inhibits glioma formation in vivo. Cancer Research. 2001;**61**:6822-6829

[124] Sumardika IW, Youyi C, Kondo E, Inoue Y, Ruma MW, Murata H, et al. β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-Nacetylglucosaminyltransferase 3 increases MCAM stability, which enhances S100A8/A9-mediated cancer motility. Oncology Research. 2018;**26**(3):431-444

[125] Leslie MC, Zhao YJ, Lachman LB, Hwu P, Wu GJ, Bar-Eli M. Immunization against MUC18/MCAM, a novel antigen that drives melanoma invasion and metastasis. Gene Therapy. 2007;**14**:316-323

[126] Kasinski AL, Slack FJ. MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy. Nature Reviews. Cancer. 2011;**11**:849-864

**33**

**Chapter 2**

**Abstract**

**1. Introduction**

was inadequate.

Breast Cancer

*Hülya Yazici and Beyza Akin*

Molecular Genetics of Metastatic

Breast cancer is the most common form of cancer in women. Breast cancer has a heterogeneous etiology. Genetic and environmental factors contribute to the pathogenesis and progression of breast cancer. Various genes as proliferation and nuclear factors have been identified in breast cancer. Therefore, the genetic component of patients is important in determining disease behavior, response to anticancer therapeutics, and patient survival. Prognosis of breast cancer is associated with potential metastatic properties of primary breast tumors. Metastasis is the leading cause of death in patients with breast cancer. Therefore, it is important to understand the mechanisms underlying the development of distant metastases to specific regions and has clinical value. Metastasis shows an organ-specific spread pattern and occurs with a series of complex and multistep events associated with each other, such as angiogenesis, invasion, migration-motility, extravasation, and proliferation. Breast cancer often metastasizes to the bone, liver, brain, and lungs. Metastasis may develop years after successful primary treatment. The metastatic process will become clear, as information about molecules and genes associated with metastases increases, and this is extremely important for cancer treatment.

Breast cancer, which is one of the most common malignant diseases of women worldwide, is a heterogeneous disease with unknown pathogenesis. Genetic and environmental factors contribute to the pathogenesis and progression of breast cancer. Although an improvement has recently been detected in the diagnosis and treatment of breast cancer compared with other cancers, its contribution to survival

Breast cancer-associated death or survival is associated with the potential metastatic features of the primary breast tumors. Metastatic disease is the leading cause of death in breast cancer patients. Distant metastasis develops in ~20–30% of the early-stage breast cancer patients. Approximately 90% of deaths result from the complications due to recurrent or metastatic diseases. Therefore, it is very important to understand the underlying mechanisms in the development of distant metastases to specific regions. Metastases may show an organ-specific dissemination pattern. Metastasis may develop years after successful primary treatment. Metastasis frequently develops in the bone, liver, brain, and lungs in breast cancer. Identification of the molecules, and genes associated with metastasis, and clarification of the contribution of these molecules to metastatic process are important

**Keywords:** breast cancer, metastasis, genes, pathways, organs

#### **Chapter 2**

## Molecular Genetics of Metastatic Breast Cancer

*Hülya Yazici and Beyza Akin*

#### **Abstract**

Breast cancer is the most common form of cancer in women. Breast cancer has a heterogeneous etiology. Genetic and environmental factors contribute to the pathogenesis and progression of breast cancer. Various genes as proliferation and nuclear factors have been identified in breast cancer. Therefore, the genetic component of patients is important in determining disease behavior, response to anticancer therapeutics, and patient survival. Prognosis of breast cancer is associated with potential metastatic properties of primary breast tumors. Metastasis is the leading cause of death in patients with breast cancer. Therefore, it is important to understand the mechanisms underlying the development of distant metastases to specific regions and has clinical value. Metastasis shows an organ-specific spread pattern and occurs with a series of complex and multistep events associated with each other, such as angiogenesis, invasion, migration-motility, extravasation, and proliferation. Breast cancer often metastasizes to the bone, liver, brain, and lungs. Metastasis may develop years after successful primary treatment. The metastatic process will become clear, as information about molecules and genes associated with metastases increases, and this is extremely important for cancer treatment.

**Keywords:** breast cancer, metastasis, genes, pathways, organs

#### **1. Introduction**

Breast cancer, which is one of the most common malignant diseases of women worldwide, is a heterogeneous disease with unknown pathogenesis. Genetic and environmental factors contribute to the pathogenesis and progression of breast cancer. Although an improvement has recently been detected in the diagnosis and treatment of breast cancer compared with other cancers, its contribution to survival was inadequate.

Breast cancer-associated death or survival is associated with the potential metastatic features of the primary breast tumors. Metastatic disease is the leading cause of death in breast cancer patients. Distant metastasis develops in ~20–30% of the early-stage breast cancer patients. Approximately 90% of deaths result from the complications due to recurrent or metastatic diseases. Therefore, it is very important to understand the underlying mechanisms in the development of distant metastases to specific regions. Metastases may show an organ-specific dissemination pattern. Metastasis may develop years after successful primary treatment. Metastasis frequently develops in the bone, liver, brain, and lungs in breast cancer.

Identification of the molecules, and genes associated with metastasis, and clarification of the contribution of these molecules to metastatic process are important

for the treatment of cancer. Metastasis is the dissemination of the cancer cells from their primary region to different tissues and organs in the body. Metastasis develops with a series of complex and multistep chains of events such as angiogenesis, invasion, migration-motility, extravasation, and proliferation.

The anomalies of different genes as *BRCA1*, *BRCA2*, *MYC*, *TP53*, *RB1*, *JUN*, and *CDK2A* which have roles in cell proliferation are detected in breast cancer [1]. Therefore, performing the genetic and molecular screenings of patients is important for the identification of the behavior of disease, the anticancer therapeutic response, and the survival.

Different breast cancer cellular subtypes in primary breast cancer tissue metastasize in relation to their target organ. The route of metastasis is generated with the interaction of this different subtype cells and microenvironment of the tumor and with the organ they will locate, and this is named as "organotrophic metastasis."

Understanding the molecular mechanism of organotrophic metastasis is very important for biological indicator prediction, developing the innovative therapeutic strategies, and for improving the survival. Development of metastasis in distant regions is associated with the interaction between the tumor cells and host microenvironment. Before the initiation of tumor dissemination, the host microenvironment is modified to support the tumoral growth, in other words to create a pre-metastatic niche (PMN). PMN is organized with the factors secreted from tumor cell with the changes in the host cell metabolism and microenvironment. In addition, tumor cells also interact with the extracellular matrix (ECM) of the host tissue to facilitate metastasis.

Generally, breast cancer is classified as in situ carcinoma and invasive carcinoma in a simple way, and most breast cancers are invasive. More than 80% of invasive breast cancers may be investigated in two different subgroups as invasive ductal carcinoma (IDC), and some breast cancers may be investigated as invasive lobular carcinoma (ILC). Organ preference of metastasis in ILC and IDC is significantly distinct. Invasive ductal carcinomas do metastasis to the lungs, distant lymphatic glands, and central nervous system (CNS); however, ILC is known to do threefold higher metastasis to the peritone, gastrointestinal system, and ovaries [2].

Breast cancer has a tendency to do metastasis on the bone, liver, lung, and distant lymphatic glands. The most common metastasis type is the bone metastases detected in 70% of metastatic breast cancer patients [1]. The second most common metastasis region was the liver with ~30%, and the brain was reported as the third most common metastasis region with a rate of 10–30% [1].

The most common metastatic region in all subtypes except basal-like tumors is the bone. Luminal B, HER2+/ER/PR+ and HER2+/ER/PR, tumors do more metastasis to the brain, liver, lungs, and bone than the luminal A tumors. Basal-like tumors do higher rates of the brain, lungs, and distant lymphatic node metastasis; however, the liver and bone metastases are less frequently detected in basal-like tumors [3]. Although triple negative breast cancer (TNBC) tumors show a metastatic ratio similar to non-basal tumors, TNBC tumors have less liver metastasis than the non-basal tumors [1].

Some molecules may have different roles in different metastasis regions in accordance with their content. Although transforming growth factor beta (*TGF-β*) promotes the lung metastasis of breast cancer, its interaction with Src signaling pathway may cause bone metastasis [4]. In cancer cells with insulin-like growth factor (*IGF1*) and *IGF1* receptor (*IGF1R*), bone metastasis shows higher expression than the cancer cells with brain metastasis [5]. EGFR ligands and cyclooxygenase 2 (*COX2*) were reported to be associated with lung metastasis and, however, were reported to be not associated with bone or liver metastasis [6].

*Wnt-1*-inducible-signaling pathway protein1 (*WISP-1*) and *CCN4* are heparinbinding glycoproteins of the CCN protein family that are rich in cysteine. These

**35**

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

*WISP-1* was a tumor stimulant or a tumor suppressor.

metastasis, and vitality ability of cancer cells.

**2.1 p38/MAPK pathway**

tumorigenic factors.

proteins are expressed in various inner organs such as the lung, kidney, and spleen. *WISP-1* binds to *BMP-2* and increases the mesenchymal cellular proliferation and osteoblastic differentiation. *WISP-1* was reported to be associated with the increased metastasis risk among early-stage ER-positive lymphatic node-negative breast cancer patients [7]. Therefore, future studies will demonstrate whether genetic factors associated with WISP-1 and EXT1 genes may show metastasis risk or may be used in identification of metastasis. In addition, the increase of WISP-1 expression was proven to be associated with the pathogenesis of the primary lung cancers. Although the possibility of *WISP-1* to be used as a prognostic indicator for lung metastasis of breast cancer was suggested, it was not clarified yet whether

Breast cancer cells are detected to highly express the chemokine receptors *CXCR4* and *CCR7* genes in the studies investigating the contribution of chemokine receptors to organ-specific metastases. Chemokine receptor-specific ligands *CXCL12* and *CCL21* were demonstrated to be highly expressed in the organs to which breast tumors do metastasis such as the lymph nodes, lungs, liver, and bone marrow [8]. In addition, the blockade of *CXCR4* gene in experimental animal models was demonstrated to inhibit the metastasis of breast cancer cells. The activation of the RAS/mitogen-activated protein kinase (MAPK) with chemokine signaling pathway causes changes in primary cancer cells such as changes in the intracellular actin molecule polymerization, development of pseudopodia, and increased cellular motility, cellular migration, and tissue invasion. Any of these changes contribute to the development of organ-specific metastasis by contributing to the survival,

**2. Metastasis-associated signal transduction pathways and genes**

p38/MAPK signal transduction pathway increases the breast carcinoma vascularization and growth by promoting the expression and accumulation of pro-

sion of the kinase-inactive mutant (dn-p38) of p38/MAPK14 in metastatic breast cancer cells in the studies, and with the deterioration of the tumor p38/MAPK signal, the development of breast cancer and metastasis ability was shown to decrease in breast carcinoma xenografts [9]. The conducted kinase-inactive mutant significantly decreased the dn-p38, tumor blood vessel density, and lumen dimensions. p38 controls the expression of the pro-angiogenic extracellular factors such as matrix protein fibronectin, cytokine, vascular endothelial growth factor A (*VEGFA*), and *IL8*. p38/ MAPK signal transduction was demonstrated to increase the tumoral growth, and vascularization in addition to increasing the expressions of tumor-associated fibroblasts, and pro-angiogenic factors. All these effects were suppressed by the dn-p38 kinase-inactive mutant. The data analyses showed that p38 had higher expression in breast cancers which was an indicator of recurrence and poor prognosis. The activation of the p38/MAPK signaling pathway in the tumor increased the development of breast cancer and metastasis. *p38* contributes to the vascularization of carcinoma by facilitating the expression and accumulation of the pro-angiogenic factors. In conclusion, all these results suggested that all the genes which have a role in p38/MAPK pathway might be a therapeutic target against tumor vascularization and metastasis. Tumor microenvironment (TME) is an important factor in cancer progression, recurrence, and response to treatment. TME blood vessels consist of stromal

The inactivation of the p38/MAPK signaling pathway was provided by the expres-

#### *Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

*Tumor Progression and Metastasis*

response, and the survival.

tissue to facilitate metastasis.

for the treatment of cancer. Metastasis is the dissemination of the cancer cells from their primary region to different tissues and organs in the body. Metastasis develops with a series of complex and multistep chains of events such as angiogenesis, inva-

The anomalies of different genes as *BRCA1*, *BRCA2*, *MYC*, *TP53*, *RB1*, *JUN*, and *CDK2A* which have roles in cell proliferation are detected in breast cancer [1]. Therefore, performing the genetic and molecular screenings of patients is important for the identification of the behavior of disease, the anticancer therapeutic

Different breast cancer cellular subtypes in primary breast cancer tissue metastasize in relation to their target organ. The route of metastasis is generated with the interaction of this different subtype cells and microenvironment of the tumor and with the organ they will locate, and this is named as "organotrophic metastasis." Understanding the molecular mechanism of organotrophic metastasis is very important for biological indicator prediction, developing the innovative therapeutic strategies, and for improving the survival. Development of metastasis in distant regions is associated with the interaction between the tumor cells and host microenvironment. Before the initiation of tumor dissemination, the host microenvironment is modified to support the tumoral growth, in other words to create a pre-metastatic niche (PMN). PMN is organized with the factors secreted from tumor cell with the changes in the host cell metabolism and microenvironment. In addition, tumor cells also interact with the extracellular matrix (ECM) of the host

Generally, breast cancer is classified as in situ carcinoma and invasive carcinoma in a simple way, and most breast cancers are invasive. More than 80% of invasive breast cancers may be investigated in two different subgroups as invasive ductal carcinoma (IDC), and some breast cancers may be investigated as invasive lobular carcinoma (ILC). Organ preference of metastasis in ILC and IDC is significantly distinct. Invasive ductal carcinomas do metastasis to the lungs, distant lymphatic glands, and central nervous system (CNS); however, ILC is known to do threefold

The most common metastatic region in all subtypes except basal-like tumors is the bone. Luminal B, HER2+/ER/PR+ and HER2+/ER/PR, tumors do more metastasis to the brain, liver, lungs, and bone than the luminal A tumors. Basal-like tumors do higher rates of the brain, lungs, and distant lymphatic node metastasis; however, the liver and bone metastases are less frequently detected in basal-like tumors [3]. Although triple negative breast cancer (TNBC) tumors show a metastatic ratio similar to non-basal tumors, TNBC tumors have less liver metastasis than the non-basal tumors [1]. Some molecules may have different roles in different metastasis regions in accordance with their content. Although transforming growth factor beta (*TGF-β*) promotes the lung metastasis of breast cancer, its interaction with Src signaling pathway may cause bone metastasis [4]. In cancer cells with insulin-like growth factor (*IGF1*) and *IGF1* receptor (*IGF1R*), bone metastasis shows higher expression than the cancer cells with brain metastasis [5]. EGFR ligands and cyclooxygenase 2 (*COX2*) were reported to be associated with lung metastasis and, however, were

*Wnt-1*-inducible-signaling pathway protein1 (*WISP-1*) and *CCN4* are heparinbinding glycoproteins of the CCN protein family that are rich in cysteine. These

higher metastasis to the peritone, gastrointestinal system, and ovaries [2]. Breast cancer has a tendency to do metastasis on the bone, liver, lung, and distant lymphatic glands. The most common metastasis type is the bone metastases detected in 70% of metastatic breast cancer patients [1]. The second most common metastasis region was the liver with ~30%, and the brain was reported as the third

most common metastasis region with a rate of 10–30% [1].

reported to be not associated with bone or liver metastasis [6].

sion, migration-motility, extravasation, and proliferation.

**34**

proteins are expressed in various inner organs such as the lung, kidney, and spleen. *WISP-1* binds to *BMP-2* and increases the mesenchymal cellular proliferation and osteoblastic differentiation. *WISP-1* was reported to be associated with the increased metastasis risk among early-stage ER-positive lymphatic node-negative breast cancer patients [7]. Therefore, future studies will demonstrate whether genetic factors associated with WISP-1 and EXT1 genes may show metastasis risk or may be used in identification of metastasis. In addition, the increase of WISP-1 expression was proven to be associated with the pathogenesis of the primary lung cancers. Although the possibility of *WISP-1* to be used as a prognostic indicator for lung metastasis of breast cancer was suggested, it was not clarified yet whether *WISP-1* was a tumor stimulant or a tumor suppressor.

Breast cancer cells are detected to highly express the chemokine receptors *CXCR4* and *CCR7* genes in the studies investigating the contribution of chemokine receptors to organ-specific metastases. Chemokine receptor-specific ligands *CXCL12* and *CCL21* were demonstrated to be highly expressed in the organs to which breast tumors do metastasis such as the lymph nodes, lungs, liver, and bone marrow [8]. In addition, the blockade of *CXCR4* gene in experimental animal models was demonstrated to inhibit the metastasis of breast cancer cells. The activation of the RAS/mitogen-activated protein kinase (MAPK) with chemokine signaling pathway causes changes in primary cancer cells such as changes in the intracellular actin molecule polymerization, development of pseudopodia, and increased cellular motility, cellular migration, and tissue invasion. Any of these changes contribute to the development of organ-specific metastasis by contributing to the survival, metastasis, and vitality ability of cancer cells.

#### **2. Metastasis-associated signal transduction pathways and genes**

#### **2.1 p38/MAPK pathway**

p38/MAPK signal transduction pathway increases the breast carcinoma vascularization and growth by promoting the expression and accumulation of protumorigenic factors.

The inactivation of the p38/MAPK signaling pathway was provided by the expression of the kinase-inactive mutant (dn-p38) of p38/MAPK14 in metastatic breast cancer cells in the studies, and with the deterioration of the tumor p38/MAPK signal, the development of breast cancer and metastasis ability was shown to decrease in breast carcinoma xenografts [9]. The conducted kinase-inactive mutant significantly decreased the dn-p38, tumor blood vessel density, and lumen dimensions. p38 controls the expression of the pro-angiogenic extracellular factors such as matrix protein fibronectin, cytokine, vascular endothelial growth factor A (*VEGFA*), and *IL8*. p38/ MAPK signal transduction was demonstrated to increase the tumoral growth, and vascularization in addition to increasing the expressions of tumor-associated fibroblasts, and pro-angiogenic factors. All these effects were suppressed by the dn-p38 kinase-inactive mutant. The data analyses showed that p38 had higher expression in breast cancers which was an indicator of recurrence and poor prognosis. The activation of the p38/MAPK signaling pathway in the tumor increased the development of breast cancer and metastasis. *p38* contributes to the vascularization of carcinoma by facilitating the expression and accumulation of the pro-angiogenic factors. In conclusion, all these results suggested that all the genes which have a role in p38/MAPK pathway might be a therapeutic target against tumor vascularization and metastasis.

Tumor microenvironment (TME) is an important factor in cancer progression, recurrence, and response to treatment. TME blood vessels consist of stromal cells (fibroblasts, adipocytes) and infiltrating immune cells. Myeloid cells stimulate the tumor vascularization and metastasis by secreting metalloproteinase *MMP9*/*gelatinase-B* cells which increase the gathering of endothelial cells and pericytes. In addition to myeloid cells, *MMP9* is also produced by the breast carcinoma cells, and the *MMP9* destruction in carcinoma cells significantly decreases tumor vascularization [10]. Therefore, all three cellular components of the TME of breast contribute to the tumor vascularization by interacting with *MMP9*. p38/MAPK signal contributes to the development of breast cancer and metastasis by increasing the tumor cell invasiveness and tumor vascularization.

*MMP9* that has a role in tumor angiogenesis and intratumoral vascularizationassociated *ICAM1* works correlated with p38/MAPK signal. *ICAM1* is also suggested as a target in triple negative breast cancer (TNBC) [11]. The inhibition of p38/ MAPK signal affects the TNF-induced *ICAM1* expression or the induction of *MMP9* by the cytokines *TGF-β* and TNF.

The deterioration of p38/MAPK signal causes no decrease in the expression of *MMP9* and *ICAM1* that are secreted by tumor cells. p38/MAPK signal contributes to fibronectin expression by responding to cytokines and tumor-fibroblast interactions [9].

p38/MAPK induces the expression of pro-angiogenic cytokines that include *VEGFA*, *IL8*, and *HBEGF* in addition to inducing an extracellular matrix protein fibronectin. *TAK1* controls the expression of MMP9 which releases *VEGF* and activates the *IL8* (**Figure 1**). Pro-angiogenic cytokines increase the tumoral growth by stimulating the tumoral vascularization.

p38/MAPK affects the development and metastasis of breast cancer by changing the tumor microenvironment of p38/MAPK signal. The inactivation of p38/MAPK signal in breast cancer cells decreases the growth of tumor xenografts and metastasis. Tumoral and stromal cells in breast TME stimulate the cytokine-mediated p38/ MAPK signal which increases the expression of the pro-angiogenic and pro-invasive factors such as *VEGFA*, *IL8*, *IL6*, *HBEGF*, and fibronectin. p38/MAPK which affects

**37**

**Figure 2.**

*The effect of TEM8 in breast cancer metastasis.*

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

**2.2 Tumor endothelial marker 8 (TEM8)**

therapeutic target in inhibiting the metastasis.

the vascular structure and stroma of tumor is detected to be definitely a potential target for anticancer treatment. Researchers suggested that anti-p38 drugs were a new therapeutic option in the treatment of breast cancer including metastatic disease [9].

Tumor endothelial marker (*TEM8*) was first discovered in the human tumor endothelial and was associated with tumor angiogenesis. TEM8 also known as Anthrax toxin receptor 1 (*ANTXR1*) is highly regulated in tumor endothelial and is expressed in breast cancer. *TEM8* was demonstrated to be required for tumoral growth and angiogenesis [12]. The role of *TEM8* in angiogenesis is organized with the regulation of downstream VEGF signal with its interaction with vascular endothelial growth factor receptor 2 (*VEGFR2*). Primary tumor development and metastasis are highly dependent on angiogenesis. Because the tumor cannot grow more than a few millimeters unless new blood vessels that will provide the oxygen and nutrients to tumor tissue are generated. The extravasation and dissemination of metastatic cells out of the vessel are facilitated and accelerated due to the leaky structure of the rapid developing tumor vessel network during tumor angiogenesis. Therefore, treatments targeting *TEM8* can differentiate the physiologic and pathological angiogenesis and can prevent the cancer progression without causing serious adverse effects. Due to this feature, *TEM8* is suggested to be a new possible

The destruction of *TEM8* in osteosarcoma cells causes the decrease of the cell proliferation [13]. *TEM8* interacts with the lipoprotein receptor associated protein 6 (*LRP6*) and regulates the downstream signaling of Wnt which is a protein that induces both the cellular proliferation and migration. *TEM8* was reported to regulate metastasis and a new molecule specific for metastasis by contributing the breast cancer stem cells (BCSC) and tumor growth with activating the Wnt signal with collagen VI [14]. *TEM8* is associated with invasive and aggressive phenotype in breast cancer. In addition, *TEM8* expression was demonstrated to be highly expressed in the tumor tissues of breast cancer patients compared to the normal tissues [14]. TEM8 expressed by cancer cells causes the development of angiogenesis by affecting the cancer cell proliferation and endothelial cell migration. *TEM8* knockout (KO) cells were generated using CRISPR/Cas9, *TEM8* expression was demonstrated to significantly disappear, and *TEM8* was inhibited in the studies investigating the association of *TEM8* with metastasis (**Figure 2**). Thus, angiogenesis decreased in tumor cells, and metastasis ability of *TEM8* significantly

**Figure 1.** *The role of p38/MAPK in the regulation of tumor angiogenesis in breast cancer.*

*Tumor Progression and Metastasis*

by the cytokines *TGF-β* and TNF.

by stimulating the tumoral vascularization.

*The role of p38/MAPK in the regulation of tumor angiogenesis in breast cancer.*

tions [9].

cells (fibroblasts, adipocytes) and infiltrating immune cells. Myeloid cells stimulate the tumor vascularization and metastasis by secreting metalloproteinase *MMP9*/*gelatinase-B* cells which increase the gathering of endothelial cells and pericytes. In addition to myeloid cells, *MMP9* is also produced by the breast carcinoma cells, and the *MMP9* destruction in carcinoma cells significantly decreases tumor vascularization [10]. Therefore, all three cellular components of the TME of breast contribute to the tumor vascularization by interacting with *MMP9*. p38/MAPK signal contributes to the development of breast cancer and metastasis by increasing

*MMP9* that has a role in tumor angiogenesis and intratumoral vascularizationassociated *ICAM1* works correlated with p38/MAPK signal. *ICAM1* is also suggested as a target in triple negative breast cancer (TNBC) [11]. The inhibition of p38/ MAPK signal affects the TNF-induced *ICAM1* expression or the induction of *MMP9*

The deterioration of p38/MAPK signal causes no decrease in the expression of *MMP9* and *ICAM1* that are secreted by tumor cells. p38/MAPK signal contributes to fibronectin expression by responding to cytokines and tumor-fibroblast interac-

p38/MAPK induces the expression of pro-angiogenic cytokines that include *VEGFA*, *IL8*, and *HBEGF* in addition to inducing an extracellular matrix protein fibronectin. *TAK1* controls the expression of MMP9 which releases *VEGF* and activates the *IL8* (**Figure 1**). Pro-angiogenic cytokines increase the tumoral growth

p38/MAPK affects the development and metastasis of breast cancer by changing the tumor microenvironment of p38/MAPK signal. The inactivation of p38/MAPK signal in breast cancer cells decreases the growth of tumor xenografts and metastasis. Tumoral and stromal cells in breast TME stimulate the cytokine-mediated p38/ MAPK signal which increases the expression of the pro-angiogenic and pro-invasive factors such as *VEGFA*, *IL8*, *IL6*, *HBEGF*, and fibronectin. p38/MAPK which affects

the tumor cell invasiveness and tumor vascularization.

**36**

**Figure 1.**

the vascular structure and stroma of tumor is detected to be definitely a potential target for anticancer treatment. Researchers suggested that anti-p38 drugs were a new therapeutic option in the treatment of breast cancer including metastatic disease [9].

#### **2.2 Tumor endothelial marker 8 (TEM8)**

Tumor endothelial marker (*TEM8*) was first discovered in the human tumor endothelial and was associated with tumor angiogenesis. TEM8 also known as Anthrax toxin receptor 1 (*ANTXR1*) is highly regulated in tumor endothelial and is expressed in breast cancer. *TEM8* was demonstrated to be required for tumoral growth and angiogenesis [12]. The role of *TEM8* in angiogenesis is organized with the regulation of downstream VEGF signal with its interaction with vascular endothelial growth factor receptor 2 (*VEGFR2*). Primary tumor development and metastasis are highly dependent on angiogenesis. Because the tumor cannot grow more than a few millimeters unless new blood vessels that will provide the oxygen and nutrients to tumor tissue are generated. The extravasation and dissemination of metastatic cells out of the vessel are facilitated and accelerated due to the leaky structure of the rapid developing tumor vessel network during tumor angiogenesis. Therefore, treatments targeting *TEM8* can differentiate the physiologic and pathological angiogenesis and can prevent the cancer progression without causing serious adverse effects. Due to this feature, *TEM8* is suggested to be a new possible therapeutic target in inhibiting the metastasis.

The destruction of *TEM8* in osteosarcoma cells causes the decrease of the cell proliferation [13]. *TEM8* interacts with the lipoprotein receptor associated protein 6 (*LRP6*) and regulates the downstream signaling of Wnt which is a protein that induces both the cellular proliferation and migration. *TEM8* was reported to regulate metastasis and a new molecule specific for metastasis by contributing the breast cancer stem cells (BCSC) and tumor growth with activating the Wnt signal with collagen VI [14]. *TEM8* is associated with invasive and aggressive phenotype in breast cancer. In addition, *TEM8* expression was demonstrated to be highly expressed in the tumor tissues of breast cancer patients compared to the normal tissues [14].

TEM8 expressed by cancer cells causes the development of angiogenesis by affecting the cancer cell proliferation and endothelial cell migration. *TEM8* knockout (KO) cells were generated using CRISPR/Cas9, *TEM8* expression was demonstrated to significantly disappear, and *TEM8* was inhibited in the studies investigating the association of *TEM8* with metastasis (**Figure 2**). Thus, angiogenesis decreased in tumor cells, and metastasis ability of *TEM8* significantly

**Figure 2.** *The effect of TEM8 in breast cancer metastasis.*

degraded with the deletion in cancer cells. Cancer cell proliferation, angiogenesis, and metastases are blocked with the prevention of cell cycle and the expression of the kinetochore-associated genes with the inhibition of *TEM* [15]. Cancer cells are known to secrete the pro-angiogenic signals such as *VEGFA* and open the angiogenic lock by affecting the tumor microenvironment. *TEM8* is known to work in cooperation with other factors such as *VEGF* for promoting endothelial cell migration and angiogenesis. In conclusion, *TEM8* expression is higher in tumor cells than in normal cells. Studies conducted using *TEM8* knockout metastatic breast cancer cell lines designed with CRISPR/Cas9 emphasize the role of *TEM8* in cancer development, tumor angiogenesis, and local metastasis. All these studies reveal the potential of *TEM8* as a therapeutic target for combating the disease; however, more clinical studies are required for developing the *TEM8*-targeted therapies [15].

#### **2.3 APOBEC3B gene**

Another important molecule in the development of metastatic potential of breast cancer is *APOBEC3B*. High level of *APOBEC3B* mRNA expression was demonstrated to be a significant prognostic biological indicator demonstrating the poor prognosis of breast cancer in ER-positive primary breast cancer cases. In addition, this molecule in distant metastasis regions was demonstrated to be highly expressed than the levels in regional lymph node metastases. This showed that *APOBEC3B* not only in the primary tumor stage has a role in the development of different metastatic stages of breast cancer. In conclusion, *APOBEC3B* causes the progression of metastatic breast cancer [16]. Therefore, the identification of different expression levels of *APOBEC3B* suggests that it carries a biological marker feature that may show a different metastatic stage and may be used in the identification of the metastasis stages in future.

#### **3. Metastasis of breast cancer to different organs**

#### **3.1 Lymph node metastasis**

Lymph node metastasis shows that distant metastasis risk is higher. The absence of lymph node metastases is associated with lower metastasis risk; however, the presence of more than four lymph node metastases is the precursor that distant metastasis risk is significantly higher. Distant tumor metastasis develops through axillary lymphoid nodes (ALD) and blood circulation. Therefore, lymph nodes are used as an indicator of the metastasis ability of tumor cells. There is an association between the tumor size and the rate of lymph node metastasis.

CCN proteins which have oncogenic functions in breast cancer mainly consist of *CCN1* and *CCN2*. *CCN1* protein is expressed in ~30% of breast cancers particularly in estrogen receptor (ER)-positive HER-2-negative tumors compared with the normal breast tissues. Higher *CCN1* expression is associated with lymph node metastasis and poor prognosis in breast cancer patients. *CCN1* increases the breast tumor vascularization and causes metastasis with Hg signaling [17]. In addition, *CCN1* has a regulatory role in fibroblast production by affecting MMP-1 for increasing the breast cancer cell migration and invasion. CCN4 expression is associated with lymph node metastasis and poor prognosis.

#### **3.2 Bone metastasis**

The common cause of morbidity and mortality in most advanced stage breast cancer patients is the development of osteolytic bone metastasis. The most

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*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

through *HIF-lα* and *TGF-β* signal.

frequently detected area of metastasis in metastatic breast cancer is the bone and constitutes 70% of the metastases. Most bone metastases detected in breast cancer are associated with osteolytic-type metastatic lesions owing to the osteoclast-mediated bone resorption. Although all subtypes of breast cancer have a tendency of bone metastases, luminal subtype tumors develop higher bone metastases (80.5%)

Tumor cells demonstrate different reactions in accordance with the environment

Some cancer cells in the primary tumor accumulate additional genetic changes which lead to bone metastasis. This causes invasion and colonization of tumor cells to the bone matrix. The destruction of the bone matrix with tumor cells facilitates the metastasis by the TGF and metastasis genes responding to TGF causing the increase of *CTGF* and *IL11* expression. *IL11*, *CTGF*, *CXCR4*, and *MMP-1* are the four most effective genes that are overexpressed in bone metastasis. Another effective gene is the protein osteopontin (OPN) which has various functions including the stimulating ability of the bone matrix to attach to the osteoclast. This protein is continuously overexpressed in metastatic cells. The genes effective in bone metastasis affect the tumor microenvironment toward metastasis. The overexpression of these genes develops the osteolytic bone metastasis. *IL11* is a strong osteoclast inducer which is synthesized by the progenitor cells in the bone marrow [17]. The in vivo testing of *IL11*-transfected MDA-MB-231 for metastatic activity of metastatic breast cancer cell line showed that the single expression of *IL11* did not significantly increase the metastasis. Therefore the presence of other genes in cooperation with IL11 in bone metastasis and their investigation were suggested [17]. *IL11* and *OPN* significantly increased the osteolytic bone metastasis by increasing the osteoclast function. *MMP-1* alone or in combination with *IL11* and

*OPN* is another important molecule in the development of bone metastasis.

each promoting the formation of osteolytic bone lesions.

and IL11 genes which have a role in osteoclastogenesis.

Because *TGF-β* is abundantly stored in the bone matrix, *TGF-β* that is secreted during osteolysis stimulates the metastatic breast cancer. *TGF-β* increases the *IL11* and *CTGF* expressions which are already higher in metastasis. The significantly overexpressed genes in bone metastasis encode the cell surface and secreting proteins which have functions that could possibly change the host tissue environment,

**Figure 3** demonstrates the functioning between the *CXCR4* gene responsible in bone marrow extravasation, *MMP-1* and *ADAMTS1* genes having roles of proteolysis and also *FGF5* and *CTGF* genes that are known to be expressed in angiogenesis,

Primary breast tumor develops with the accumulation of oncogenic mutations

from normal breast epithelium. The increased expression of gene classes that

in the new organ such as gene expression, growth ability, and response to treatment. Therefore, any of the breast cancer cell reaching to the bone may promote the excessive growth in molecular interaction with osteoblasts and osteoclasts. The molecules produced by cancer cells or with the parathyroid hormone-associated protein in the bone microenvironment and converting growth factor β (*TGF-β*) mediate this growth. The elimination of the tumor suppressor feature of *TGF-β* is suggested to stimulate the tumor invasion and metastasis [19]. Cytokines, chemokines, and other growth factors support the development of bone metastasis. Prometastatic cytokine *TGF-β*, osteolytic angiogenic factors interleukin-11 (*IL11*), and CTGF expression are accepted as the molecules that increase the osteolytic metastatic activity. Although *SMAD4* is a tumor suppressor which inhibits the tumor cell proliferation, it is an osteolytic metastasis promoter which binds the *TGF-β* signal to the following *IL11* induction [20]. *SMAD4* activates *VEGF* and *CXC* chemokine receptor 4 (*CXCR4*) to induce the bone metastasis in breast cancer

than the basal-like (41.7%) and HER2+ tumors (55.6%) [18].

#### *Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

*Tumor Progression and Metastasis*

**2.3 APOBEC3B gene**

**3.1 Lymph node metastasis**

degraded with the deletion in cancer cells. Cancer cell proliferation, angiogenesis, and metastases are blocked with the prevention of cell cycle and the expression of the kinetochore-associated genes with the inhibition of *TEM* [15]. Cancer cells are known to secrete the pro-angiogenic signals such as *VEGFA* and open the angiogenic lock by affecting the tumor microenvironment. *TEM8* is known to work in cooperation with other factors such as *VEGF* for promoting endothelial cell migration and angiogenesis. In conclusion, *TEM8* expression is higher in tumor cells than in normal cells. Studies conducted using *TEM8* knockout metastatic breast cancer cell lines designed with CRISPR/Cas9 emphasize the role of *TEM8* in cancer development, tumor angiogenesis, and local metastasis. All these studies reveal the potential of *TEM8* as a therapeutic target for combating the disease; however, more clinical studies are required for developing the *TEM8*-targeted therapies [15].

Another important molecule in the development of metastatic potential of breast cancer is *APOBEC3B*. High level of *APOBEC3B* mRNA expression was demonstrated to be a significant prognostic biological indicator demonstrating the poor prognosis of breast cancer in ER-positive primary breast cancer cases. In addition, this molecule in distant metastasis regions was demonstrated to be highly expressed than the levels in regional lymph node metastases. This showed that *APOBEC3B* not only in the primary tumor stage has a role in the development of different metastatic stages of breast cancer. In conclusion, *APOBEC3B* causes the progression of metastatic breast cancer [16]. Therefore, the identification of different expression levels of *APOBEC3B* suggests that it carries a biological marker feature that may show a different metastatic stage and may be used in the identification of the metastasis stages in future.

Lymph node metastasis shows that distant metastasis risk is higher. The absence of lymph node metastases is associated with lower metastasis risk; however, the presence of more than four lymph node metastases is the precursor that distant metastasis risk is significantly higher. Distant tumor metastasis develops through axillary lymphoid nodes (ALD) and blood circulation. Therefore, lymph nodes are used as an indicator of the metastasis ability of tumor cells. There is an association

CCN proteins which have oncogenic functions in breast cancer mainly consist of *CCN1* and *CCN2*. *CCN1* protein is expressed in ~30% of breast cancers particularly in estrogen receptor (ER)-positive HER-2-negative tumors compared with the normal breast tissues. Higher *CCN1* expression is associated with lymph node metastasis and poor prognosis in breast cancer patients. *CCN1* increases the breast tumor vascularization and causes metastasis with Hg signaling [17]. In addition, *CCN1* has a regulatory role in fibroblast production by affecting MMP-1 for increasing the breast cancer cell migration and invasion. CCN4 expression is associated

The common cause of morbidity and mortality in most advanced stage breast

cancer patients is the development of osteolytic bone metastasis. The most

**3. Metastasis of breast cancer to different organs**

between the tumor size and the rate of lymph node metastasis.

with lymph node metastasis and poor prognosis.

**38**

**3.2 Bone metastasis**

frequently detected area of metastasis in metastatic breast cancer is the bone and constitutes 70% of the metastases. Most bone metastases detected in breast cancer are associated with osteolytic-type metastatic lesions owing to the osteoclast-mediated bone resorption. Although all subtypes of breast cancer have a tendency of bone metastases, luminal subtype tumors develop higher bone metastases (80.5%) than the basal-like (41.7%) and HER2+ tumors (55.6%) [18].

Tumor cells demonstrate different reactions in accordance with the environment in the new organ such as gene expression, growth ability, and response to treatment. Therefore, any of the breast cancer cell reaching to the bone may promote the excessive growth in molecular interaction with osteoblasts and osteoclasts. The molecules produced by cancer cells or with the parathyroid hormone-associated protein in the bone microenvironment and converting growth factor β (*TGF-β*) mediate this growth. The elimination of the tumor suppressor feature of *TGF-β* is suggested to stimulate the tumor invasion and metastasis [19]. Cytokines, chemokines, and other growth factors support the development of bone metastasis. Prometastatic cytokine *TGF-β*, osteolytic angiogenic factors interleukin-11 (*IL11*), and CTGF expression are accepted as the molecules that increase the osteolytic metastatic activity. Although *SMAD4* is a tumor suppressor which inhibits the tumor cell proliferation, it is an osteolytic metastasis promoter which binds the *TGF-β* signal to the following *IL11* induction [20]. *SMAD4* activates *VEGF* and *CXC* chemokine receptor 4 (*CXCR4*) to induce the bone metastasis in breast cancer through *HIF-lα* and *TGF-β* signal.

Some cancer cells in the primary tumor accumulate additional genetic changes which lead to bone metastasis. This causes invasion and colonization of tumor cells to the bone matrix. The destruction of the bone matrix with tumor cells facilitates the metastasis by the TGF and metastasis genes responding to TGF causing the increase of *CTGF* and *IL11* expression. *IL11*, *CTGF*, *CXCR4*, and *MMP-1* are the four most effective genes that are overexpressed in bone metastasis. Another effective gene is the protein osteopontin (OPN) which has various functions including the stimulating ability of the bone matrix to attach to the osteoclast. This protein is continuously overexpressed in metastatic cells. The genes effective in bone metastasis affect the tumor microenvironment toward metastasis. The overexpression of these genes develops the osteolytic bone metastasis. *IL11* is a strong osteoclast inducer which is synthesized by the progenitor cells in the bone marrow [17]. The in vivo testing of *IL11*-transfected MDA-MB-231 for metastatic activity of metastatic breast cancer cell line showed that the single expression of *IL11* did not significantly increase the metastasis. Therefore the presence of other genes in cooperation with IL11 in bone metastasis and their investigation were suggested [17]. *IL11* and *OPN* significantly increased the osteolytic bone metastasis by increasing the osteoclast function. *MMP-1* alone or in combination with *IL11* and *OPN* is another important molecule in the development of bone metastasis.

Because *TGF-β* is abundantly stored in the bone matrix, *TGF-β* that is secreted during osteolysis stimulates the metastatic breast cancer. *TGF-β* increases the *IL11* and *CTGF* expressions which are already higher in metastasis. The significantly overexpressed genes in bone metastasis encode the cell surface and secreting proteins which have functions that could possibly change the host tissue environment, each promoting the formation of osteolytic bone lesions.

**Figure 3** demonstrates the functioning between the *CXCR4* gene responsible in bone marrow extravasation, *MMP-1* and *ADAMTS1* genes having roles of proteolysis and also *FGF5* and *CTGF* genes that are known to be expressed in angiogenesis, and IL11 genes which have a role in osteoclastogenesis.

Primary breast tumor develops with the accumulation of oncogenic mutations from normal breast epithelium. The increased expression of gene classes that

facilitate metastasis to different organs among tumor cells enables the invasion of the bone matrix, colonization of metastatic tumor cell, and destruction of the bone matrix [21].

CCN protein family consists of six members as *CCN1* (*Cyr61*), *CCN2* (*CTGF*), *CCN3* (*Nov*), *CCN4* (*WISP-1*), *CCN5* (*WISP2*), and *CCN6* (*WISP3*) which have central roles in development, inflammation, and tissue repair [22]. In addition, CCN proteins have roles in various pathological cases by organizing the extracellular signals in the cellular environment. In MDA-MB-231 metastatic breast cancer cell line, *CCN3* reorganizes the actin cytoskeleton and increases the cell trafficking by activating the GTPase *Rac1* [23]. *CCN3* was demonstrated to increase the bone metastasis in the studies conducted in metastatic breast cancer cell line [23]. This significant effect of *CCN3* in metastasis was reported to deteriorate the osteoblast differentiation and provided a favorable environment for osteolytic breast cancer bone metastasis owing to supporting the osteoclastogenesis [23].

One of the overexpressed genes in bone-specific metastasis is the *NAT1* (N-acetyltransferase-1) and is a potential biological indicator for breast cancer.

#### **3.3 Liver metastasis**

The liver is the most common metastatic region for cancers and represents the second organ where breast cancer metastasis occurs. The development of liver metastasis in breast cancer patients is associated with Wnt signal and Ki67 signal independent of beta-catenin and an indicator of poor prognosis.

*CXCR4* is the most common chemokine receptor that mediates the initiation of liver metastases. In addition, the dysregulation of cell adhesion molecules *N-cadherin* and *E-cadherin* was demonstrated to contribute to liver metastases in breast cancer (**Figure 4**). Breast cancer cells with higher *N-cadherin* level develop liver metastasis. *E-cadherin* which inhibits the metastasis was found lower in breast cancer cells with liver metastasis [24].

Although *N-cadherin* increases the liver metastasis, in normal conditions *E-cadherin* suppresses the development of liver metastasis. In addition, *IL-6*

*The molecular mechanisms that are mediated by the genes effective in breast cancer-associated bone metastasis.*

**41**

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

expression in liver metastasis of breast cancer facilitates the development of liver

*Regulation of cell adhesion molecules in liver metastasis with N-cadherin and E-cadherin.*

Metastasis is a multistep procedure which is responsible for most cancer-associated deaths and is affected by both cell-cell or cell-matrix interactions and tumor

Clinically, low oxygen level (hypoxia) is known to be associated with metastasis [17]. Lysyl oxidase (*LOX*) expression is both associated with tumor suppression and tumor progression, and its role in tumorigenesis changes in accordance with the cellular location, cell type, and transformation. *LOX* expression is regulated by the hypoxia-inducible factor (*HIF*). Mostly distant metastasis is detected, and overall survival is poor in patients who have tumors which highly express the *LOX*. The *LOX* inhibition eliminates metastasis in breast cancer patients. *LOX* is required in metastatic growth to form a niche. *LOX* is required for hypoxia-associated metastasis. Although LOX inhibition has no significant effect on primary tumor growth, *LOX* was associated to significantly decrease the lung metastases and inhibited the liver metastasis [25]. *LOX* molecule is suggested to be a good therapeutic target in

Brain/CNS (central nervous system) metastasis develops in 10–30% of metastatic breast cancer patients. Brain metastasis (BM) is detected as a complication that generally develops in the late stages of disease. Brain metastases develop after systemic emergence of metastases in the lungs, liver, and bone [26]. Two main primary tumors that do metastasis to the brain are lung and breast adenocarcinomas [18]. Brain metastases are associated with neurological disorders by affecting both the cognitive and sensory functions in addition to their association with highly

Breast cancer is the most common cancer type where brain metastasis develops after lung metastasis. Lung and breast cancer-associated brain metastasis is more frequently detected than the primary brain tumors. Brain metastasis incidence has gradually been increasing in breast cancer patients. Due to the development of systemic therapies, many breast cancer patients live longer, but still in a way brain metastases may develop. Various factors were described for increased brain

metastasis by inhibiting the *E-cadherin* expression [24].

microenvironment (vascularization, etc.).

prevention and elimination of metastasis [25].

**3.4 Brain metastasis**

**Figure 4.**

poor prognosis.

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

#### **Figure 4.**

*Tumor Progression and Metastasis*

bone matrix [21].

**3.3 Liver metastasis**

cancer cells with liver metastasis [24].

facilitate metastasis to different organs among tumor cells enables the invasion of the bone matrix, colonization of metastatic tumor cell, and destruction of the

bone metastasis owing to supporting the osteoclastogenesis [23].

independent of beta-catenin and an indicator of poor prognosis.

One of the overexpressed genes in bone-specific metastasis is the *NAT1* (N-acetyltransferase-1) and is a potential biological indicator for breast cancer.

The liver is the most common metastatic region for cancers and represents the second organ where breast cancer metastasis occurs. The development of liver metastasis in breast cancer patients is associated with Wnt signal and Ki67 signal

*CXCR4* is the most common chemokine receptor that mediates the initiation of liver metastases. In addition, the dysregulation of cell adhesion molecules *N-cadherin* and *E-cadherin* was demonstrated to contribute to liver metastases in breast cancer (**Figure 4**). Breast cancer cells with higher *N-cadherin* level develop liver metastasis. *E-cadherin* which inhibits the metastasis was found lower in breast

Although *N-cadherin* increases the liver metastasis, in normal conditions *E-cadherin* suppresses the development of liver metastasis. In addition, *IL-6*

*The molecular mechanisms that are mediated by the genes effective in breast cancer-associated bone metastasis.*

CCN protein family consists of six members as *CCN1* (*Cyr61*), *CCN2* (*CTGF*), *CCN3* (*Nov*), *CCN4* (*WISP-1*), *CCN5* (*WISP2*), and *CCN6* (*WISP3*) which have central roles in development, inflammation, and tissue repair [22]. In addition, CCN proteins have roles in various pathological cases by organizing the extracellular signals in the cellular environment. In MDA-MB-231 metastatic breast cancer cell line, *CCN3* reorganizes the actin cytoskeleton and increases the cell trafficking by activating the GTPase *Rac1* [23]. *CCN3* was demonstrated to increase the bone metastasis in the studies conducted in metastatic breast cancer cell line [23]. This significant effect of *CCN3* in metastasis was reported to deteriorate the osteoblast differentiation and provided a favorable environment for osteolytic breast cancer

**40**

**Figure 3.**

*Regulation of cell adhesion molecules in liver metastasis with N-cadherin and E-cadherin.*

expression in liver metastasis of breast cancer facilitates the development of liver metastasis by inhibiting the *E-cadherin* expression [24].

Metastasis is a multistep procedure which is responsible for most cancer-associated deaths and is affected by both cell-cell or cell-matrix interactions and tumor microenvironment (vascularization, etc.).

Clinically, low oxygen level (hypoxia) is known to be associated with metastasis [17]. Lysyl oxidase (*LOX*) expression is both associated with tumor suppression and tumor progression, and its role in tumorigenesis changes in accordance with the cellular location, cell type, and transformation. *LOX* expression is regulated by the hypoxia-inducible factor (*HIF*). Mostly distant metastasis is detected, and overall survival is poor in patients who have tumors which highly express the *LOX*. The *LOX* inhibition eliminates metastasis in breast cancer patients. *LOX* is required in metastatic growth to form a niche. *LOX* is required for hypoxia-associated metastasis. Although LOX inhibition has no significant effect on primary tumor growth, *LOX* was associated to significantly decrease the lung metastases and inhibited the liver metastasis [25]. *LOX* molecule is suggested to be a good therapeutic target in prevention and elimination of metastasis [25].

#### **3.4 Brain metastasis**

Brain/CNS (central nervous system) metastasis develops in 10–30% of metastatic breast cancer patients. Brain metastasis (BM) is detected as a complication that generally develops in the late stages of disease. Brain metastases develop after systemic emergence of metastases in the lungs, liver, and bone [26]. Two main primary tumors that do metastasis to the brain are lung and breast adenocarcinomas [18]. Brain metastases are associated with neurological disorders by affecting both the cognitive and sensory functions in addition to their association with highly poor prognosis.

Breast cancer is the most common cancer type where brain metastasis develops after lung metastasis. Lung and breast cancer-associated brain metastasis is more frequently detected than the primary brain tumors. Brain metastasis incidence has gradually been increasing in breast cancer patients. Due to the development of systemic therapies, many breast cancer patients live longer, but still in a way brain metastases may develop. Various factors were described for increased brain

metastasis risk in breast cancer patients. These factors may be reported as early age, poorly differentiated tumor histology (high grade), hormone receptor negativity, and metastasis in more than four lymph nodes. These factors were associated with the brain metastasis risk [26]. HER2-positive and TNBC patients have a higher risk of brain metastasis than the luminal-type breast cancer patients. Brain metastasis is detected in 30–40% of HER2-positive and triple negative breast cancer patients [26]. Brain metastasis in lung cancer generally develops within 2 years after the diagnosis of primary lung cancer, and brain metastasis in breast cancer is generally associated with the metastatic stage of the disease and develops 10 years after the primary diagnosis and after a successful treatment. However, brain metastasis in triple negative breast cancer patients develops in earlier periods. The development of brain metastasis in breast cancer was detected to be associated with Wnt, Notch, and EGFR pathways [27]. *CXCL12* that is expressed in the brain and *CXCR4* receptor located in the surface of the breast tumor cells block the cell signaling pathway together with *CXCR4* in brain metastasis. Breast cancer-associated brain metastasis generally develops in ~20–30% of breast cancer patients. Breast cancer-associated metastasis shows poor prognosis due to the lack of molecular therapeutic targets. The rate of detection of brain metastasis in HER2+ and triple negative breast cancer subtypes is 20–50%.

HER2 amplifications and mutations were frequently demonstrated in breast cancer and in breast cancers with brain metastasis [27]. There are no target-specific treatment options in the clinical practice generally in breast cancers that carry *BRCA1* and *BRCA2* gene mutation and triple negative brain metastasis. New molecular targets HER2, *EGFR*, *VEGFR*, *PARP*, *mTOR*, and *CDK-4/6* were discovered in the treatment of breast cancer with metastasis to the brain.

Brain metastasis is a multistep procedure with migration, intravasation, circulation, adhesion, extravasation, and brain microenvironment. Particularly the blood-brain barrier (BBB) is highly selective in the entrance of tumor cells and therapeutics to the brain microenvironment. In compliance with that, the cells to make a metastatic lesion in the brain have a specific clonal origin. This shows that a brain metastasis shared the common abnormalities with a metastasis ancestor cell, and the further abnormalities could only be present in only brain metastatic subclones. More frequent detection of *TP53* mutations in breast cancer with brain metastasis compared with the other breast cancers is an example. *COX2*, *EGFR*, and *HBEGF* were described as the extravasation stimulating factors through colonization in breast cancers with metastases to the brain and lung. The higher expression of the genes *CXCR4*, *PLLP*, *TNFSF4*, *VCAM1*, *SLC8A2*, and *SLC7A11* facilitates the development of brain metastases. In addition, the majority of snoRNAs and snRNAs have higher expression in breast cancer metastasizing to the brain [28].

#### **3.5 Lung metastasis**

Luminal breast tumors have the tendency to do metastasis to the bone; however, basal-like breast tumors mainly do metastasis to the lungs. The genes that are effective in the emergence of lung metastasis are generally associated with poor prognosis [29]. An epidermal growth factor receptor-ligand epiregulin (*EPR*) and the genes such as *COX2*, *MMP-1*, and *MMP-2* affect the tumor angiogenesis and facilitate the lung metastasis by reaching to the lung capillary vessels. The inhibition of *EGFR* and *COX2* minimizes the lung metastasis [30]. Protein deacetylase *SIRT7* was demonstrated to inhibit the development of lung metastasis of breast cancer cells by antagonizing the *TGF-β* signal [31]. An increased expression was reported in the genes *DSC2*, *TFCP2L1*, *UGT8*, *ITGB8*, *ANP32E*, and *FERMT1* that are associated with cell involvement and signal transduction in patients with lung metastasis of breast cancer [31].

**43**

**Table 1.**

Angiogenesis, EMT Formation of lung niches Development of lung metastasis

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

underlying mechanisms in lung metastasis.

regulating the EMT and angiogenesis.

Other genes except *PTEN* were detected to be overexpressed in the studies investigating the mechanism of lung metastasis. Although none of the described genes were found to be associated with previous metastasis, some of the encoded molecules were detected to have significant roles in the acquisition of proliferative and invasive characteristics to epithelial cells. The regulation of the epithelialmesenchymal transition (EMT) is highly important in metastatic process. Integrins regulate the EMT by mediating the *TGF-β* signal activation [32]. *FERMT1* gene is known to be an effective gene in *TGF*-mediated epithelial-mesenchymal transition.

Therefore, *FERMT1* gene is suggested to be associated with lung metastasis.

The decrease of the expression of a tumor suppressor gene *PTEN* was found to be associated with lung metastasis in a study [33]. *PTEN* is one of the main molecules which regulates the signaling pathways associated with reproduction, growth, cell viability, and cell migration and was detected to mutate in various different tumors. In addition, *PTEN* regulates the *EMT* in lung metastasis by affecting the cell viability and *CXCR4* chemotaxis. The biological indicators *EGFR* and *FOXC1* were demonstrated to be associated with each other and controlled the lung metastasis in breast cancer [33]. The survival rate of breast cancer patients with lung metastasis is very low despite the treatment options as chemotherapy, radiotherapy, and target-specific treatment against lung metastasis. Therefore, the development of new therapeutic strategies is significantly important for understanding the

A Notch signaling pathway receptor *Notch-1* was demonstrated to have a critical role in cell renewal, reproduction, and apoptosis of BCSC by regulating the epithelial-mesenchymal transition in breast cancer [34]. The abnormal activation of notch signaling pathway contributes to the breast cancer metastasis by primarily

Wnt/β-catenin signaling has a significant role in the embryonic induction and tumorigenesis of the breast gland [35]. The nuclear localization and overexpression of β-catenin are an indicator of Wnt/β-catenin signal activation. Various clinical and laboratory studies showed that the abnormal activation of Wnt/β-catenin signaling was associated with poor prognosis in breast cancer patients and mainly increased in triple negative cancer subtype [36]. In addition, the Wnt-helper receptor *LRP6* was commonly overexpressed in highly aggressive triple negative breast cancer. Wnt/β-catenin signaling pathway contributes to the EMT and breast cancer metastases in addition to controlling the cell proliferation in breast cancer (**Table 1**). Hedgehog (Hg) signaling pathway has a significant role in the development of ducts of the breast. In addition, Hg regulates the breast cancer stem cells and has a significant role in cancerogenesis [37]. Hg proteins regulate the breast cancer cell migration. Hg, Notch, and Wnt signaling pathways demonstrate joint behavior in tumor development and metastasis in cancer. These signaling pathways have significant roles in the development of breast cancer and lung metastasis.

**Notch pathway Wnt pathway Hedgehog** 

The self-renewal of breast cancer stem cells EMT *CXCL12-CXC4*

Uncontrolled growth The self-renewal of breast cancer stem cells *TGF-β*

*The functioning of signaling pathways in breast cancer-associated lung metastasis.*

**pathway**

#### *Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

*Tumor Progression and Metastasis*

metastasis risk in breast cancer patients. These factors may be reported as early age, poorly differentiated tumor histology (high grade), hormone receptor negativity, and metastasis in more than four lymph nodes. These factors were associated with the brain metastasis risk [26]. HER2-positive and TNBC patients have a higher risk of brain metastasis than the luminal-type breast cancer patients. Brain metastasis is detected in 30–40% of HER2-positive and triple negative breast cancer patients [26]. Brain metastasis in lung cancer generally develops within 2 years after the diagnosis of primary lung cancer, and brain metastasis in breast cancer is generally associated with the metastatic stage of the disease and develops 10 years after the primary diagnosis and after a successful treatment. However, brain metastasis in triple negative breast cancer patients develops in earlier periods. The development of brain metastasis in breast cancer was detected to be associated with Wnt, Notch, and EGFR pathways [27]. *CXCL12* that is expressed in the brain and *CXCR4* receptor located in the surface of the breast tumor cells block the cell signaling pathway together with *CXCR4* in brain metastasis. Breast cancer-associated brain metastasis generally develops in ~20–30% of breast cancer patients. Breast cancer-associated metastasis shows poor prognosis due to the lack of molecular therapeutic targets. The rate of detection of brain metastasis in HER2+ and triple negative breast cancer subtypes is 20–50%. HER2 amplifications and mutations were frequently demonstrated in breast cancer and in breast cancers with brain metastasis [27]. There are no target-specific treatment options in the clinical practice generally in breast cancers that carry *BRCA1* and *BRCA2* gene mutation and triple negative brain metastasis. New molecular targets HER2, *EGFR*, *VEGFR*, *PARP*, *mTOR*, and *CDK-4/6* were discov-

ered in the treatment of breast cancer with metastasis to the brain.

Brain metastasis is a multistep procedure with migration, intravasation, circulation, adhesion, extravasation, and brain microenvironment. Particularly the blood-brain barrier (BBB) is highly selective in the entrance of tumor cells and therapeutics to the brain microenvironment. In compliance with that, the cells to make a metastatic lesion in the brain have a specific clonal origin. This shows that a brain metastasis shared the common abnormalities with a metastasis ancestor cell, and the further abnormalities could only be present in only brain metastatic subclones. More frequent detection of *TP53* mutations in breast cancer with brain metastasis compared with the other breast cancers is an example. *COX2*, *EGFR*, and *HBEGF* were described as the extravasation stimulating factors through colonization in breast cancers with metastases to the brain and lung. The higher expression of the genes *CXCR4*, *PLLP*, *TNFSF4*, *VCAM1*, *SLC8A2*, and *SLC7A11* facilitates the development of brain metastases. In addition, the majority of snoRNAs and snRNAs have higher expression in breast cancer metastasizing to the brain [28].

Luminal breast tumors have the tendency to do metastasis to the bone; however,

basal-like breast tumors mainly do metastasis to the lungs. The genes that are effective in the emergence of lung metastasis are generally associated with poor prognosis [29]. An epidermal growth factor receptor-ligand epiregulin (*EPR*) and the genes such as *COX2*, *MMP-1*, and *MMP-2* affect the tumor angiogenesis and facilitate the lung metastasis by reaching to the lung capillary vessels. The inhibition of *EGFR* and *COX2* minimizes the lung metastasis [30]. Protein deacetylase *SIRT7* was demonstrated to inhibit the development of lung metastasis of breast cancer cells by antagonizing the *TGF-β* signal [31]. An increased expression was reported in the genes *DSC2*, *TFCP2L1*, *UGT8*, *ITGB8*, *ANP32E*, and *FERMT1* that are associated with cell involvement and signal transduction in patients with lung metastasis

**42**

**3.5 Lung metastasis**

of breast cancer [31].

Other genes except *PTEN* were detected to be overexpressed in the studies investigating the mechanism of lung metastasis. Although none of the described genes were found to be associated with previous metastasis, some of the encoded molecules were detected to have significant roles in the acquisition of proliferative and invasive characteristics to epithelial cells. The regulation of the epithelialmesenchymal transition (EMT) is highly important in metastatic process. Integrins regulate the EMT by mediating the *TGF-β* signal activation [32]. *FERMT1* gene is known to be an effective gene in *TGF*-mediated epithelial-mesenchymal transition. Therefore, *FERMT1* gene is suggested to be associated with lung metastasis.

The decrease of the expression of a tumor suppressor gene *PTEN* was found to be associated with lung metastasis in a study [33]. *PTEN* is one of the main molecules which regulates the signaling pathways associated with reproduction, growth, cell viability, and cell migration and was detected to mutate in various different tumors. In addition, *PTEN* regulates the *EMT* in lung metastasis by affecting the cell viability and *CXCR4* chemotaxis. The biological indicators *EGFR* and *FOXC1* were demonstrated to be associated with each other and controlled the lung metastasis in breast cancer [33]. The survival rate of breast cancer patients with lung metastasis is very low despite the treatment options as chemotherapy, radiotherapy, and target-specific treatment against lung metastasis. Therefore, the development of new therapeutic strategies is significantly important for understanding the underlying mechanisms in lung metastasis.

A Notch signaling pathway receptor *Notch-1* was demonstrated to have a critical role in cell renewal, reproduction, and apoptosis of BCSC by regulating the epithelial-mesenchymal transition in breast cancer [34]. The abnormal activation of notch signaling pathway contributes to the breast cancer metastasis by primarily regulating the EMT and angiogenesis.

Wnt/β-catenin signaling has a significant role in the embryonic induction and tumorigenesis of the breast gland [35]. The nuclear localization and overexpression of β-catenin are an indicator of Wnt/β-catenin signal activation. Various clinical and laboratory studies showed that the abnormal activation of Wnt/β-catenin signaling was associated with poor prognosis in breast cancer patients and mainly increased in triple negative cancer subtype [36]. In addition, the Wnt-helper receptor *LRP6* was commonly overexpressed in highly aggressive triple negative breast cancer. Wnt/β-catenin signaling pathway contributes to the EMT and breast cancer metastases in addition to controlling the cell proliferation in breast cancer (**Table 1**).

Hedgehog (Hg) signaling pathway has a significant role in the development of ducts of the breast. In addition, Hg regulates the breast cancer stem cells and has a significant role in cancerogenesis [37]. Hg proteins regulate the breast cancer cell migration. Hg, Notch, and Wnt signaling pathways demonstrate joint behavior in tumor development and metastasis in cancer. These signaling pathways have significant roles in the development of breast cancer and lung metastasis.


#### **Table 1.**

*The functioning of signaling pathways in breast cancer-associated lung metastasis.*

Breast cancer is characterized with a separate metastatic pattern including the regional lymph nodes, bone marrow, lung, and liver. Chemokines are a group of small-molecular-weight protein which bind to chemokine receptors attached to G protein. Chemokines have a significant role in various pathological conditions such as cell migration, development, and inflammation. Binding of chemokines to receptors causes a structural change which activates the signaling pathways and promotes the migration. Chemokine and chemokine receptors have a critical role in identification of metastatic targets of tumor cells. Chemokines are divided into two groups in accordance with their functions as inflammatory chemokines and homeostatic chemokines. Inflammatory chemokines are induced by inflammation, and homeostatic chemokines are structurally expressed and have a role in homeostatic immune regulation [38].

Chemokines have a significant role in the progression of cancers [38] and have functions in tumoral growth, aging, angiogenesis epithelial-mesenchymal transition, and metastasis. The expression of chemokines and their receptors changes in malignity and then causes abnormal chemokine receptor signaling. This change stems from the inactivation of the tumor-suppressive genes or from the structural activation of oncogenes that have a role in the regulation of chemokines [38].

Chemokine receptors *CXCR4* and *CCR7* are highly expressed in human breast cancer cells, malignant breast tumors, and metastases [38]. In breast cancer cells, *CXCR4* or *CCR7* signaling mediates the actin polymerization and pseudopodia and then induces the chemotaxis and invasion.

The in vivo inactivation of *CXCL12*/*CXCR4* interactions significantly inhibits the metastasis of breast cancer cells to the regional lymph nodes and lungs [38]. *CXCL12*/*CXCR4* interactions also cause bone marrow metastasis of breast cancer cells.

Tumor cell migration and metastasis have various similarities with the leukocyte trafficking that are regulated by chemokines and their receptors. Cell traffickingassociated ligands *CXCL12/SDF-1α* and *CCL21/6Ckine* are highly expressed in the organs representing the first targets of metastatic breast cancer [38]. Malignant melanoma which has high skin metastasis and has a similar metastatic characteristic with breast cancer has high *CCR10* expression in addition to *CXCR4* and *CCR7* [38]. Therefore, both *CXCR4* and *CCR7* are highly critical molecules for cell trafficking and tissue homeostasis.

*CXCL12* is the only ligand known for *CXCR4*. Metastatic breast cancers were demonstrated to selectively express CXCR4 and migrated to organs which highly express the ligand *CXCL12* that is also known as *SDF-1* [38]. *CXCR4* expression is known to be higher in malignant breast tumors than the levels in healthy breast tissues. *CXCL12* was highly expressed in organs such as the lung, bone, liver, and lymph nodes where the breast cancer cells preferred to do metastasis [38]. This showed that metastatic breast tumor cells selectively expressed *CXCR4*, and thus breast cancer cells which reached to organs have high *CXCL12* expression levels. In addition, the in vivo inhibition of *CXCR4*-*CXCL12* interactions was demonstrated to significantly decrease the metastasis of breast tumor cells to the lymph node and lungs [38]. Therefore, *CXCL12*-*CXCR4* signaling is suggested to be an important therapeutic target for metastatic breast cancer treatment.

*CXCR4*-*CXCL12* receptor-ligand interactions in breast cancer allow the invading of tumor cells of neighboring tissues and for successful metastasis. The receptorligand interaction triggers the actin polymerization and facilitates the formation of pseudopodia. Thus, the invading of breast tumor cells of the neighboring tissues or distant tissue is induced or facilitated [39]. Chemokine *CXCL12* activates the chemokine receptor *CXCR4* in endothelial cell which supports the endothelial cell migration and growth [39]. The high expression of *CXCL12* in the lung, liver, and lymph nodes showed that these chemokines have a role in the metastasis of breast cancer cells for these anatomic regions.

**45**

type and HER2+ molecular subtypes [1].

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

survival [38].

metastasis.

protocols.

*CCL21* and its receptor *CCR7* have critical importance in the settlement of lymphocytes to secondary lymphoid organs. The primary breast cancer cells in lymph nodes and most metastatic cancer cells express *CCR7*, and there is an association between *CCR7* expression and lymph node metastasis. In addition, higher *CCR7* expression was demonstrated to be associated with poor prognosis and shorter

Extracellular matrix (ECM) proteins tenascin-C (TNC), periostin (POSTN), and versican (VCAN) are highly important molecules in the formation of metastasis and have a critical role in the formation of breast cancer colonization in the lung tissue that has a tendency for metastasis. Tenascin-C, which is normally produced by fibroblasts, is also secreted by breast cancer stem cells. This abnormal expression of tenascin-C by breast cancer stem cells forms a niche in lung colonization and creates a metastasis-initiating effect. Periostin is a stromal factor that may bind to

Cancer-associated fibroblasts (*CAFs*) have a significant role in breast cancer metastasis by expressing the *Tiam1* and osteopontin in breast cancer tissue [40]. In addition, the expression of a *CAF*-associated protein thrombocyte-associated growth factor receptor (*PDGFRβ*) is highly associated with lung metastasis in breast cancer. In addition, CAFs increase the primary tumor growth through *TGF-β* and contribute to the development of lung metastasis-associated fibrous tissue in breast cancer [41]. Therefore, *CAF* is suggested to be a potential anticancer therapeutic target. The development of strategies targeting the microenvironment may be effective in the treatment or inhibition of breast cancer

Because the lungs have a unique histological feature, cancer cell meets with high interstitial fluid pressure and thus supports the *PDGFRβ* expression when a cancer cell does metastasis to a small interstitial tissue between the alveoles. Lung metasta-

As conclusion, the expression changes in these genes in breast cancer cells may be detected in bone, lung, brain, liver, and lymph node metastases. The studies revealed that there were important differences in metastatic behavior between breast cancer subtypes (**Table 2**). Therefore, the treatment of metastatic breast cancer must be performed by targeting the organ with metastasis, and the development of target molecules will form the future treatment

Luminal B, HER2+/ER/PR+ and HER2+/ER/PR, tumors do more metastasis to the brain, liver, lung, and bone than the luminal A tumors. Basal-like tumors do higher rates of brain and lung metastases. As demonstrated in **Table 2**, breast cancer cells do metastasis to the lung through triple negative breast cancer, basal, luminal B, HER2 molecular subtypes, the genes activated by growth factor receptors, matrix metalloproteinases, and the pathways of *COX2* and *LOX2* genes. Breast cancer cells with HER2+, luminal-HER2, triple negative breast cancer, and basal histologies primarily have a tendency to do metastasis to the brain. These molecular subtypes do metastasis to the brain with the effect of genes activated by growth factor receptors, matrix metalloproteinases, COX2, and chemokinesis. Clarifying the association of these signalings and genes with molecular subtypes suggests the significant new therapeutic targets for metastatic breast cancer treatment. The bone metastasis of luminal and HER2 breast cancer molecular subtypes is caused by growth factor genes and interleukins. Chemokine and integrin molecules that cause liver metastasis are more frequently detected in HER2+, ER+, luminal B, and luminal-HER2 molecular subtypes. BCR pathway proteins and CCN proteins, the genes responsible in Hg signaling pathway, cause lymph node metastasis in luminal

Wnt ligands and is effective in breast cancer metastasis [40].

sis is known to be associated with triple negative breast cancer.

*Tumor Progression and Metastasis*

then induces the chemotaxis and invasion.

and tissue homeostasis.

Breast cancer is characterized with a separate metastatic pattern including the regional lymph nodes, bone marrow, lung, and liver. Chemokines are a group of small-molecular-weight protein which bind to chemokine receptors attached to G protein. Chemokines have a significant role in various pathological conditions such as cell migration, development, and inflammation. Binding of chemokines to receptors causes a structural change which activates the signaling pathways and promotes the migration. Chemokine and chemokine receptors have a critical role in identification of metastatic targets of tumor cells. Chemokines are divided into two groups in accordance with their functions as inflammatory chemokines and homeostatic chemokines. Inflammatory chemokines are induced by inflammation, and homeostatic chemokines are structurally expressed and have a role in homeostatic immune regulation [38]. Chemokines have a significant role in the progression of cancers [38] and have functions in tumoral growth, aging, angiogenesis epithelial-mesenchymal transition, and metastasis. The expression of chemokines and their receptors changes in malignity and then causes abnormal chemokine receptor signaling. This change stems from the inactivation of the tumor-suppressive genes or from the structural activation of oncogenes that have a role in the regulation of chemokines [38].

Chemokine receptors *CXCR4* and *CCR7* are highly expressed in human breast cancer cells, malignant breast tumors, and metastases [38]. In breast cancer cells, *CXCR4* or *CCR7* signaling mediates the actin polymerization and pseudopodia and

The in vivo inactivation of *CXCL12*/*CXCR4* interactions significantly inhibits the metastasis of breast cancer cells to the regional lymph nodes and lungs [38]. *CXCL12*/*CXCR4* interactions also cause bone marrow metastasis of breast cancer cells. Tumor cell migration and metastasis have various similarities with the leukocyte trafficking that are regulated by chemokines and their receptors. Cell traffickingassociated ligands *CXCL12/SDF-1α* and *CCL21/6Ckine* are highly expressed in the organs representing the first targets of metastatic breast cancer [38]. Malignant melanoma which has high skin metastasis and has a similar metastatic characteristic with breast cancer has high *CCR10* expression in addition to *CXCR4* and *CCR7* [38]. Therefore, both *CXCR4* and *CCR7* are highly critical molecules for cell trafficking

*CXCL12* is the only ligand known for *CXCR4*. Metastatic breast cancers were demonstrated to selectively express CXCR4 and migrated to organs which highly express the ligand *CXCL12* that is also known as *SDF-1* [38]. *CXCR4* expression is known to be higher in malignant breast tumors than the levels in healthy breast tissues. *CXCL12* was highly expressed in organs such as the lung, bone, liver, and lymph nodes where the breast cancer cells preferred to do metastasis [38]. This showed that metastatic breast tumor cells selectively expressed *CXCR4*, and thus breast cancer cells which reached to organs have high *CXCL12* expression levels. In addition, the in vivo inhibition of *CXCR4*-*CXCL12* interactions was demonstrated to significantly decrease the metastasis of breast tumor cells to the lymph node and lungs [38]. Therefore, *CXCL12*-*CXCR4* signaling is suggested to be an important

*CXCR4*-*CXCL12* receptor-ligand interactions in breast cancer allow the invading of tumor cells of neighboring tissues and for successful metastasis. The receptorligand interaction triggers the actin polymerization and facilitates the formation of pseudopodia. Thus, the invading of breast tumor cells of the neighboring tissues or distant tissue is induced or facilitated [39]. Chemokine *CXCL12* activates the chemokine receptor *CXCR4* in endothelial cell which supports the endothelial cell migration and growth [39]. The high expression of *CXCL12* in the lung, liver, and lymph nodes showed that these chemokines have a role in the metastasis of breast

therapeutic target for metastatic breast cancer treatment.

cancer cells for these anatomic regions.

**44**

*CCL21* and its receptor *CCR7* have critical importance in the settlement of lymphocytes to secondary lymphoid organs. The primary breast cancer cells in lymph nodes and most metastatic cancer cells express *CCR7*, and there is an association between *CCR7* expression and lymph node metastasis. In addition, higher *CCR7* expression was demonstrated to be associated with poor prognosis and shorter survival [38].

Extracellular matrix (ECM) proteins tenascin-C (TNC), periostin (POSTN), and versican (VCAN) are highly important molecules in the formation of metastasis and have a critical role in the formation of breast cancer colonization in the lung tissue that has a tendency for metastasis. Tenascin-C, which is normally produced by fibroblasts, is also secreted by breast cancer stem cells. This abnormal expression of tenascin-C by breast cancer stem cells forms a niche in lung colonization and creates a metastasis-initiating effect. Periostin is a stromal factor that may bind to Wnt ligands and is effective in breast cancer metastasis [40].

Cancer-associated fibroblasts (*CAFs*) have a significant role in breast cancer metastasis by expressing the *Tiam1* and osteopontin in breast cancer tissue [40]. In addition, the expression of a *CAF*-associated protein thrombocyte-associated growth factor receptor (*PDGFRβ*) is highly associated with lung metastasis in breast cancer. In addition, CAFs increase the primary tumor growth through *TGF-β* and contribute to the development of lung metastasis-associated fibrous tissue in breast cancer [41]. Therefore, *CAF* is suggested to be a potential anticancer therapeutic target. The development of strategies targeting the microenvironment may be effective in the treatment or inhibition of breast cancer metastasis.

Because the lungs have a unique histological feature, cancer cell meets with high interstitial fluid pressure and thus supports the *PDGFRβ* expression when a cancer cell does metastasis to a small interstitial tissue between the alveoles. Lung metastasis is known to be associated with triple negative breast cancer.

As conclusion, the expression changes in these genes in breast cancer cells may be detected in bone, lung, brain, liver, and lymph node metastases. The studies revealed that there were important differences in metastatic behavior between breast cancer subtypes (**Table 2**). Therefore, the treatment of metastatic breast cancer must be performed by targeting the organ with metastasis, and the development of target molecules will form the future treatment protocols.

Luminal B, HER2+/ER/PR+ and HER2+/ER/PR, tumors do more metastasis to the brain, liver, lung, and bone than the luminal A tumors. Basal-like tumors do higher rates of brain and lung metastases. As demonstrated in **Table 2**, breast cancer cells do metastasis to the lung through triple negative breast cancer, basal, luminal B, HER2 molecular subtypes, the genes activated by growth factor receptors, matrix metalloproteinases, and the pathways of *COX2* and *LOX2* genes. Breast cancer cells with HER2+, luminal-HER2, triple negative breast cancer, and basal histologies primarily have a tendency to do metastasis to the brain. These molecular subtypes do metastasis to the brain with the effect of genes activated by growth factor receptors, matrix metalloproteinases, COX2, and chemokinesis. Clarifying the association of these signalings and genes with molecular subtypes suggests the significant new therapeutic targets for metastatic breast cancer treatment. The bone metastasis of luminal and HER2 breast cancer molecular subtypes is caused by growth factor genes and interleukins. Chemokine and integrin molecules that cause liver metastasis are more frequently detected in HER2+, ER+, luminal B, and luminal-HER2 molecular subtypes. BCR pathway proteins and CCN proteins, the genes responsible in Hg signaling pathway, cause lymph node metastasis in luminal type and HER2+ molecular subtypes [1].

#### *Tumor Progression and Metastasis*


#### **Table 2.**

*The organ-specific genes and signaling pathways effective in metastatic breast cancer.*

Individualized target-specific appropriate treatment methods will be developed for metastatic breast cancer owing to the knowledge of the association of genes with each other that cause metastasis and the follow-up of the pathways where these genes gained function. There is an association between genomic differences and various gene expressions that cause poor prognosis in breast cancer. The gene expression profiles of primary tumors must be compared and associated with metastasis for describing and clarifying the tumor factors of metastatic breast cancer. The better understanding of the functioning of these genes will help to develop specific therapeutic approaches for metastatic breast cancer.

The molecules and genes on the pathways will be used in the diagnosis, prognosis and treatment response of metastatic breast cancer in the future. These effective molecules will be used as a tumor-specific indicator, and also detected in different biological materials like tissue, saliva, blood, serum, and urine in metastatic breast cancer. In addition, these genes may be used as therapeutic targets. The inactivation of these genes by inhibition or with biological antibodies through apoptosis is significantly important to resolve the tumor and metastasis. Different therapeutic strategies will be developed, and these molecules will be used in individualized treatment for inhibiting the tumor metastasis considering the associations between these genes, and chemokines, and integrins. The breast cancer molecular subtypes will be treated, and a progress will be enabled in the treatment of metastatic breast cancer with the development of molecular drugs which inhibit the active pathways or eliminate the pathway transition of the genes effective in metastatic breast cancer.

**47**

**Author details**

Istanbul, Turkey

Hülya Yazici\* and Beyza Akin

provided the original work is properly cited.

Division of Cancer Genetics, Istanbul University, Institute of Oncology,

© 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,

\*Address all correspondence to: hulyayazici67@gmail.com

*Molecular Genetics of Metastatic Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.86674*

the University of Istanbul for their language corrections.

The authors declare that they have no conflict of interests.

The authors thank Kadriye Yilmaz from the Department of Foreign Languages at

**Acknowledgements**

**Conflict of interest**

### **Acknowledgements**

*Tumor Progression and Metastasis*

TNBC Basal Luminal B HER2+

**Growth factors** TGF-β EGFR VEGF **Matrix**

**Metalloproteinases**

MMP-1 MMP-2 **Chemokines** CXCL12 CXCR4 **BMP inhibitors Other factors** COX-2 LOX

Molecular subtypes of breast cancer

Molecular pathways and genes

**Table 2.**

Individualized target-specific appropriate treatment methods will be developed for metastatic breast cancer owing to the knowledge of the association of genes with each other that cause metastasis and the follow-up of the pathways where these genes gained function. There is an association between genomic differences and various gene expressions that cause poor prognosis in breast cancer. The gene expression profiles of primary tumors must be compared and associated with metastasis for describing and clarifying the tumor factors of metastatic breast cancer. The better understanding of the functioning of these genes will help to develop

**Tissue Lung Brain Bone Liver Lymph node**

Luminal HER2

**Growth factors** IGF1 PGE2 TGF-β **Other factors** PDGF FGF2 IL11 IL-6 IL-1 OPN

HER2+ ER+ Luminal B Luminal-HER2

**Chemokines** CXCR4 CXCL12 CCR7 CCL21 **Other factors** IL-6 N-cadherin E-cadherin LOX OPN VEGF TWIST WNT pathway ECM

Luminal HER2+

**CCN proteins BCR pathways Hedgehog (Hg) pathway**

HER2+ Luminal-HER2 TNBC Basal

MMP-9 MMP-1 **Chemokines** CXCR4 CXCL12 CCR7 CCL21 **Cytokines** CK5

**Growth factors** VEGF HBEGF **Matrix**

**Metalloproteinases**

**Notch pathways Wnt pathways Hg pathways Other factors** COX-2 LOX IL-8 COX-2 ICAM1 PTEN CAF

The molecules and genes on the pathways will be used in the diagnosis, prognosis and treatment response of metastatic breast cancer in the future. These effective molecules will be used as a tumor-specific indicator, and also detected in different biological materials like tissue, saliva, blood, serum, and urine in metastatic breast cancer. In addition, these genes may be used as therapeutic targets. The inactivation of these genes by inhibition or with biological antibodies through apoptosis is significantly important to resolve the tumor and metastasis. Different therapeutic strategies will be developed, and these molecules will be used in individualized treatment for inhibiting the tumor metastasis considering the associations between these genes, and chemokines, and integrins. The breast cancer molecular subtypes will be treated, and a progress will be enabled in the treatment of metastatic breast cancer with the development of molecular drugs which inhibit the active pathways or eliminate the pathway transition of the genes

specific therapeutic approaches for metastatic breast cancer.

*The organ-specific genes and signaling pathways effective in metastatic breast cancer.*

**46**

effective in metastatic breast cancer.

The authors thank Kadriye Yilmaz from the Department of Foreign Languages at the University of Istanbul for their language corrections.

### **Conflict of interest**

The authors declare that they have no conflict of interests.

### **Author details**

Hülya Yazici\* and Beyza Akin Division of Cancer Genetics, Istanbul University, Institute of Oncology, Istanbul, Turkey

\*Address all correspondence to: hulyayazici67@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.

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pathological angiogenesis and potentiates tumoricidal responses against multiple Cancer types. Cancer Cell. 14 Feb 2012;**21**(2):212-226

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2017;**28**(1):27-35

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[36] Khramtsov AI, Khramtsova GF, Tretiakova M, Huo D, Olopade OI, Goss KH. Wnt/β-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. The American Journal of Pathology. Jun 2010;**176**(6):2911-2920

[37] Flemban A, Qualtrough D. The potential role of hedgehog Signaling in the luminal/basal phenotype of breast epithelia and in breast Cancer invasion and metastasis. Cancers (Basel). 16 Sep 2015;**7**(3):1863-1884

[38] Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature. 1 Mar 2001;**410**(6824):50-56

[39] Baetselier HV, Verschueren H, Van der Taelen I, Dewit J, De Braekeleer J, De Baetselier P. Metastatic competence of BW5147 T-lymphoma cell lines is correlated with in vitro invasiveness, motility and F-actin content. Journal of Leukocyte Biology. Apr 1994;**55**(4):552-556

**Chapter 3**

**Abstract**

selection of individual patients.

**1. Introduction**

**51**

metabolism, epithelial-mesenchymal transition

Protein Tyrosine Phosphatases in

Promoter or Protection?

*Helon Guimarães Cordeiro and Erica Akagi*

*Carmen V. Ferreira-Halder, Stefano Piatto Clerici,*

Tumor Progression and Metastasis:

*Alessandra V. Sousa Faria, Patrícia Fernandes de Souza Oliveira,*

Reversible phosphorylation of proteins, executed by kinases and phosphatases, is the major posttranslational protein modification in eukaryotic cells, causing them to become activated or deactivated. This intracellular event represents a critical regulatory mechanism of several signaling pathways and can be related to a broad number of diseases, including cancer. Few decades ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors. However, nowadays, accumulating evidence demonstrates that a misregulation of PTP activities plays a crucial and decisive role in cancer progression and metastasis. In this chapter, we will focus on the molecular aspects that support the crucial role of PTPs in cancer and in turn make them promising for prediction, monitoring, and rational appropriate therapy

**Keywords:** protein tyrosine phosphatases, cancer hallmarks, tumor suppressor,

Protein tyrosine phosphorylation plays a key role in cellular biology, once it can create a new recognition site for protein-protein interactions, control protein stability, and specify the protein location, and, more importantly, regulates enzymatic activity. Therefore, this intracellular event represents a critical regulatory mechanism of several signaling pathways and, once it is dysregulated, can be related to a broad number of diseases, including tumor development. Reversible phosphorylation of proteins is controlled reciprocally by both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). These phosphatases are hydrolases that preferentially act on phosphotyrosine residue of a wide range of proteins, having as products dephosphorylated protein at tyrosine residue and inorganic phosphate. PTPs consist of a large protein superfamily with 107 members that can be divided into four families (class I, II, III, and IV) according to differences in the amino acid sequence at their catalytic domains and the amino acid used in the catalytic reaction, cysteine-based PTPs (class 1, 2, and 3) and aspartate-based PTPs (class 4) [1, 2]. So far, most of PTPs have been reported to act as tumor suppressors;

[40] Xu K, Tian X, Oh SY, Movassaghi M, Naber SP, Kuperwasser C, et al. The fibroblast Tiam1-osteopontin pathway modulates breast cancer invasion and metastasis. Breast Cancer Research. 2016;**14**

[41] Kim HM, Jung WH, Koo JS. Expression of cancer-associated fibroblast related proteins in metastatic breast cancer: An immunohistochemical analysis. Journal of Translational Medicine. 2015;**13**:222

#### **Chapter 3**

*Tumor Progression and Metastasis*

Experimental Oncology. Jun

Communications. 2017;**318**

[32] Margadant C, Sonnenberg

A. Integrin–TGF-β crosstalk in fibrosis, cancer and wound healing. EMBO Reports. Feb 2010;**11**(2):97-105

[33] Y J, Han B, Chen J, Wiedemeyer R, Orsulic S, Bose S, et al. FOXC1 is a critical mediator of EGFR function in human basal-like breast cancer. Annals of Surgical Oncology. Dec 2014;**21**(Suppl 4):S758-S766

[34] Damodaran DP, Kolluru V, Chandrasekaran B, Baby BV, Aman M, Suman S, et al. Targeting aberrant expression of Notch-1 in ALDH+ cancer stem cells in breast cancer. Molecular Carcinogenesis. Mar

[35] MacDonald BT, Tamai K. Wnt/βcatenin signaling: Components, mechanisms, and diseases.

Developmental Cell. Jul 2009;**17**(1):9-26

[36] Khramtsov AI, Khramtsova GF, Tretiakova M, Huo D, Olopade OI, Goss KH. Wnt/β-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. The American Journal of Pathology. Jun

[37] Flemban A, Qualtrough D. The potential role of hedgehog Signaling in the luminal/basal phenotype of breast epithelia and in breast Cancer invasion and metastasis. Cancers (Basel). 16 Sep

[38] Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature. 1 Mar

2017;**56**(3):1127-1136

2010;**176**(6):2911-2920

2015;**7**(3):1863-1884

2001;**410**(6824):50-56

[31] Tang X, Shi L, Xie N, Liu Z, Qian M, Fanbiao Meng QX-G. SIRT7 antagonizes TGF-β signaling and inhibits breast cancer metastasis. Nature [39] Baetselier HV, Verschueren H, Van der Taelen I, Dewit J, De Braekeleer J, De Baetselier P. Metastatic competence of BW5147 T-lymphoma cell lines is correlated with in vitro invasiveness,

[40] Xu K, Tian X, Oh SY, Movassaghi M, Naber SP, Kuperwasser C, et al. The fibroblast Tiam1-osteopontin pathway modulates breast cancer invasion and metastasis. Breast Cancer Research.

[41] Kim HM, Jung WH, Koo JS. Expression of cancer-associated fibroblast related proteins in metastatic breast cancer: An immunohistochemical

analysis. Journal of Translational

Medicine. 2015;**13**:222

motility and F-actin content. Journal of Leukocyte Biology. Apr

1994;**55**(4):552-556

2016;**14**

2008;**30**(2):91-95

**50**

## Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?

*Carmen V. Ferreira-Halder, Stefano Piatto Clerici, Alessandra V. Sousa Faria, Patrícia Fernandes de Souza Oliveira, Helon Guimarães Cordeiro and Erica Akagi*

#### **Abstract**

Reversible phosphorylation of proteins, executed by kinases and phosphatases, is the major posttranslational protein modification in eukaryotic cells, causing them to become activated or deactivated. This intracellular event represents a critical regulatory mechanism of several signaling pathways and can be related to a broad number of diseases, including cancer. Few decades ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors. However, nowadays, accumulating evidence demonstrates that a misregulation of PTP activities plays a crucial and decisive role in cancer progression and metastasis. In this chapter, we will focus on the molecular aspects that support the crucial role of PTPs in cancer and in turn make them promising for prediction, monitoring, and rational appropriate therapy selection of individual patients.

**Keywords:** protein tyrosine phosphatases, cancer hallmarks, tumor suppressor, metabolism, epithelial-mesenchymal transition

#### **1. Introduction**

Protein tyrosine phosphorylation plays a key role in cellular biology, once it can create a new recognition site for protein-protein interactions, control protein stability, and specify the protein location, and, more importantly, regulates enzymatic activity. Therefore, this intracellular event represents a critical regulatory mechanism of several signaling pathways and, once it is dysregulated, can be related to a broad number of diseases, including tumor development. Reversible phosphorylation of proteins is controlled reciprocally by both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). These phosphatases are hydrolases that preferentially act on phosphotyrosine residue of a wide range of proteins, having as products dephosphorylated protein at tyrosine residue and inorganic phosphate. PTPs consist of a large protein superfamily with 107 members that can be divided into four families (class I, II, III, and IV) according to differences in the amino acid sequence at their catalytic domains and the amino acid used in the catalytic reaction, cysteine-based PTPs (class 1, 2, and 3) and aspartate-based PTPs (class 4) [1, 2]. So far, most of PTPs have been reported to act as tumor suppressors;

capacity in reprogramming their metabolism through genetic or epigenetic changes in order to get survival, proliferation, migration, invasiveness, and resistance to death stimuli [5]. In recent years, it has been demonstrated that PTPs display a key

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?*

Otto Warburg showed that tumor cells substantially metabolize glucose to lactate, even with the availability of oxygen. Under normal conditions, glucose is metabolized to pyruvate by a series of enzymatic steps in the glycolytic pathway, which is subsequently oxidized by the TCA and respiratory chain, generating CO2, H2O, and 32 or 34 molecules of ATP per glucose molecule, while in glycolysis, 2 ATPs/glucose are produced. This alteration in glucose metabolism depends on increased transcription of GLUTs, glycolytic enzymes, and oncogenes and increased

Until a few years ago, the importance of protein kinases for the Warburg effect had been focused on several studies. However, recently, in the discovery that PTPs also have relevance in tumor onset and progression, attention has been given to the

**Cdc25A**—Until 2016 it was believed that the relevance of Cdc25A in cancer was due to its positive effect on CDK. However, Liang and collaborators [7] performed an elegant study showing the Cdc25A as a positive regulator of PKM2 in human glioblastoma specimens. PKM2 catalyzes the conversion of phosphoenolpyruvate to pyruvate, the last step of glycolysis pathway. These authors described that the EGFR activation triggers the phosphorylation of Cdc25A at Y59 residue, mediated by Src. Consequently, the interaction between Cdc25A and PKM2 is favored at a nuclear compartment, leading to PKM2 dephosphorylation at S37, and in turn induces PKM2-dependent β-catenin transactivation and c-Myc-upregulated expres-

**LMWPTP**—Our group demonstrated that, in chemoresistant chronic myeloid leukemia cells, the LMWPTP was overactivated and cooperated to Warburg effect. A downregulation of mitochondrial proteins—PDH1, SDHA, and VDAC— was also observed, while GLUT 1 expression and production of lactate were increased [8]. Later on, Lori and colleagues performed a phosphoproteomic analysis of A375 melanoma cells with silenced LMWPTP. These authors identified six possible substrates, of which four, PKM2, GAPDH, α-enolase, and triose phosphate isomerase, take part in the glycolytic pathway. In contrast to the findings reported by Faria and coworkers, it was observed that the inhibition of LMWPTP leads to an inactivation of PKM2, which causes a decrease in glycolytic flux and increase of GLUT1 and

**PRL-3**—It was reported that when colorectal cancer cells (LoVo cell line) overexpress, this phosphatase had an increase of glucose consumption and lactate production in comparison to LoVo cell line wild type. Accordingly, high amount of HK2, PKM2, and LDH were detected when PRL-3 is overexpressed [10]. Importantly, these authors also reported similar results when patient colorectal carcinoma samples were screened. PRL-3 displays a lower expression level in adjacent normal tissue but was overexpressed in colorectal carcinoma lesions. Furthermore, there was a positive correlation between the expression of glycolytic enzymes (GLUT1,

**PTEN**—In different models (MEFs, prostate cancer cell lines, xenografts, genetically modified mouse and patient prostate cancer samples), the loss of PTEN specifically increases the expression of HK2 [11]. More recently, it was reported that

demand of mitochondrial metabolism for biosynthetic processes [4–6].

role of these phosphatases in tumor metabolism, as it is the case of Cdc25A,

sion of the glycolytic genes *GLUT1*, *PKM2*, and *LDHA* [7].

role in favoring cancer cell metabolic plasticity.

*DOI: http://dx.doi.org/10.5772/intechopen.87963*

**2.1 PTPs and Warburg effect**

LMWPTP, PRL-3, and PTEN.

hexokinase 2 [8, 9].

**53**

HK2, PKM2, LDHA) and PRL-3.

#### **Figure 1.**

*Schematic overview of the role of PTPs in tumor plasticity. During tumor progression, cells acquire extra mutations and reprogram their metabolism in order to sustain proliferation, migration, and survival. These capacities are in part sustained by key signaling pathways in which PI3K, AKT, MAPK, and mTOR have central roles. In this context, hyperactivation and loss of specific PTPs are crucial for keeping these kinases active.*

however, some PTPs can also act as oncogenes depending on the tumor stages or the expression of their interacting partners.

Along human tumor development, cells acquired biological plasticities that were firstly defined by Hanahan and Weinberg, as hallmarks of cancer. These authors proposed some capabilities of cancer cells that contribute for the disease complexity, aggressiveness, and invasiveness: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, deregulating cellular energetics, avoiding immune destruction, and activating invasion and metastasis [3]. Surprisingly, in the last decade, some reports have shown the relevance of PTPs for tumor cell plasticities. In this chapter we aim to draw an organized picture of the molecular mechanisms by which PTPs take part on tumor biological plasticity acquisition (**Figure 1**).

#### **2. PTPs modulate energetic metabolism in tumors**

Under normal conditions, cell metabolism depends on a tightly coordinated regulation of key regulatory enzymes and, consequently, metabolic pathways responsible for converting nutrients into building blocks for synthetic macromolecules, energy production, and biomass. However, cancer cells display efficiency

capacity in reprogramming their metabolism through genetic or epigenetic changes in order to get survival, proliferation, migration, invasiveness, and resistance to death stimuli [5]. In recent years, it has been demonstrated that PTPs display a key role in favoring cancer cell metabolic plasticity.

#### **2.1 PTPs and Warburg effect**

Otto Warburg showed that tumor cells substantially metabolize glucose to lactate, even with the availability of oxygen. Under normal conditions, glucose is metabolized to pyruvate by a series of enzymatic steps in the glycolytic pathway, which is subsequently oxidized by the TCA and respiratory chain, generating CO2, H2O, and 32 or 34 molecules of ATP per glucose molecule, while in glycolysis, 2 ATPs/glucose are produced. This alteration in glucose metabolism depends on increased transcription of GLUTs, glycolytic enzymes, and oncogenes and increased demand of mitochondrial metabolism for biosynthetic processes [4–6].

Until a few years ago, the importance of protein kinases for the Warburg effect had been focused on several studies. However, recently, in the discovery that PTPs also have relevance in tumor onset and progression, attention has been given to the role of these phosphatases in tumor metabolism, as it is the case of Cdc25A, LMWPTP, PRL-3, and PTEN.

**Cdc25A**—Until 2016 it was believed that the relevance of Cdc25A in cancer was due to its positive effect on CDK. However, Liang and collaborators [7] performed an elegant study showing the Cdc25A as a positive regulator of PKM2 in human glioblastoma specimens. PKM2 catalyzes the conversion of phosphoenolpyruvate to pyruvate, the last step of glycolysis pathway. These authors described that the EGFR activation triggers the phosphorylation of Cdc25A at Y59 residue, mediated by Src. Consequently, the interaction between Cdc25A and PKM2 is favored at a nuclear compartment, leading to PKM2 dephosphorylation at S37, and in turn induces PKM2-dependent β-catenin transactivation and c-Myc-upregulated expression of the glycolytic genes *GLUT1*, *PKM2*, and *LDHA* [7].

**LMWPTP**—Our group demonstrated that, in chemoresistant chronic myeloid leukemia cells, the LMWPTP was overactivated and cooperated to Warburg effect. A downregulation of mitochondrial proteins—PDH1, SDHA, and VDAC— was also observed, while GLUT 1 expression and production of lactate were increased [8]. Later on, Lori and colleagues performed a phosphoproteomic analysis of A375 melanoma cells with silenced LMWPTP. These authors identified six possible substrates, of which four, PKM2, GAPDH, α-enolase, and triose phosphate isomerase, take part in the glycolytic pathway. In contrast to the findings reported by Faria and coworkers, it was observed that the inhibition of LMWPTP leads to an inactivation of PKM2, which causes a decrease in glycolytic flux and increase of GLUT1 and hexokinase 2 [8, 9].

**PRL-3**—It was reported that when colorectal cancer cells (LoVo cell line) overexpress, this phosphatase had an increase of glucose consumption and lactate production in comparison to LoVo cell line wild type. Accordingly, high amount of HK2, PKM2, and LDH were detected when PRL-3 is overexpressed [10]. Importantly, these authors also reported similar results when patient colorectal carcinoma samples were screened. PRL-3 displays a lower expression level in adjacent normal tissue but was overexpressed in colorectal carcinoma lesions. Furthermore, there was a positive correlation between the expression of glycolytic enzymes (GLUT1, HK2, PKM2, LDHA) and PRL-3.

**PTEN**—In different models (MEFs, prostate cancer cell lines, xenografts, genetically modified mouse and patient prostate cancer samples), the loss of PTEN specifically increases the expression of HK2 [11]. More recently, it was reported that

however, some PTPs can also act as oncogenes depending on the tumor stages or the

*Schematic overview of the role of PTPs in tumor plasticity. During tumor progression, cells acquire extra mutations and reprogram their metabolism in order to sustain proliferation, migration, and survival. These capacities are in part sustained by key signaling pathways in which PI3K, AKT, MAPK, and mTOR have central roles. In this context, hyperactivation and loss of specific PTPs are crucial for keeping these kinases*

Under normal conditions, cell metabolism depends on a tightly coordinated regulation of key regulatory enzymes and, consequently, metabolic pathways responsible for converting nutrients into building blocks for synthetic macromolecules, energy production, and biomass. However, cancer cells display efficiency

Along human tumor development, cells acquired biological plasticities that were firstly defined by Hanahan and Weinberg, as hallmarks of cancer. These authors proposed some capabilities of cancer cells that contribute for the disease complexity, aggressiveness, and invasiveness: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, deregulating cellular energetics, avoiding immune destruction, and activating invasion and metastasis [3]. Surprisingly, in the last decade, some reports have shown the relevance of PTPs for tumor cell plasticities. In this chapter we aim to draw an organized picture of the molecular mechanisms by which PTPs take part

expression of their interacting partners.

*Tumor Progression and Metastasis*

**Figure 1.**

*active.*

**52**

on tumor biological plasticity acquisition (**Figure 1**).

**2. PTPs modulate energetic metabolism in tumors**

the knockdown of PTEN in prostate cancer cells (DU145 cell line) leads to an increase of lactate, pyruvic acid, succinic acid, citric acid, fumaric acid, malic acid, and 2-ketoglutarate, in comparison to DU145 wild type [12]. These findings indicate that glycolysis and glutaminolysis pathways are active in prostate cancer DU-145 cells when PTEN is not functional. Accordingly, it was demonstrated that the PTEN higher expression compromises the proliferation and Warburg effect, in melanoma and breast tumor, by dropping the expression of HIF1 and increasing the mitochondrial function, which are, at least in part, caused by decreasing glucose uptake and inhibiting PI3K/mTOR pathway [13–16].

**LMWPTP**—In normal cells, the increase of LMWPTP expression was associated with a reduction of PDGFR phosphorylation, consequently dropping in the mitogenic capacity [30]. However, later on, LMWPTP was described as a positive modulator of Ras-MAPK, FGF, and Eph receptors [31, 32]. It was also reported that the overexpression of LMWPTP contributes for invasive profile and primary sarcoma formation in nude mice [33]. In this context, higher LMWPTP amount (mRNA and protein) in primary human prostate cancer in relation to normal adjacent tissue was found. Interestingly, the high level of mRNA of LMWPTP was detected in lymph nodes, an indication that this phosphatase takes part in the metastasis process [34]. In the same study, 147 patients out of 481 with prostate cancer presented higher expression of LMWPTP and worse clinical outcome [34]. Accordingly, the LMWPTP has been categorized as a potential biomarker for recurrence prediction for prostate cancer [35]. The importance of the LMWPTP in cancer progression was also reported in colorectal cancer. It was demonstrated that the LMWPTP overexpression in colorectal cancer correlated to a higher potential to liver metastasis [36]. Importantly, it was also demonstrated that the LMWPTP knockdown decreases CRC cell survival and sensitizes them to chemotherapy [36]. **PTP1B**—**PTP1B** is overexpressed in several cancers, such prostate, ovarian, stomach, and colorectal [37–40]. For instance, in esophagus squamous cell carcinoma, this phosphatase overexpression is directly related to invasion and metastasis [38]. Similar effect was described in lung cancer, which was due to Src and Erk activation. Interestingly, the PTP1B knockdown in colorectal cancer cells decreases proliferation rate by blocking β-catenin signaling, a pathway responsible for

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?*

*DOI: http://dx.doi.org/10.5772/intechopen.87963*

supporting the cancer secondary site colonization [39, 41].

inhibition of cell adhesion and migration [45].

chemotherapeutic agents.

**55**

**SHIP2**—**SHIP2** positively affects tumor cell proliferation and migration. For instance, it was observed that the overexpression of SHIP2 in colorectal cancer was associated with migration and invasive profile through AKT activation [42]. **SHP2 (PTPN11)**—**SHP2 (PTPN11) p**ropiciates activation of Ras and MAPK triggered by mitogens (insulin, EGF, and lysophosphatidic acid) and cell adhesion. Notably, it has been shown that this phosphatase controls cell shape by contributing to cytoskeletal organization. In addition, SHP2 also regulates integrin-mediated cell adhesion, spreading, and migration. Also, inhibition of SHP2 is accompanied by expressive increase in the numbers of actin stress fibers and focal adhesion contacts. In contrast, overexpression of the SHP2 mutant also increased the strength of cellsubstratum adhesion [43]. SHP2 has been considered as a proto-oncogene in several human cancers such as leukemia, glioblastoma, gastric carcinoma, lung cancer, and breast cancer. This phosphatase improves cancer progression and poor prognostic by activation of Ras/Raf/ERK and PI3K/Akt/mTOR pathways [44]. In hepatocellular carcinoma, the overexpression of SHP2 correlates with malignant cancer profile. Accordingly, it was reported that the inhibition of SHP2 diminishes metastasis by

During cell transformation to malignancy, tumor cells became expert in overcoming a broad diversity of stresses, such as uncontrolled signaling regulation, starvation, DNA damage, hypoxia, and also anticancer therapy. In this aspect, different researchers have shown that PTPs are involved in tumor cells resistant to

**DUSP1 or PTPN10—**It was shown that DUSP1 inhibits the MAPK (JNK) by dephosphorylation and in turn blocks apoptosis process. This effect might be one of the explanations in which DUSP1 promotes cancer cells escaping from apoptosis. Indeed, it has been reported that DUSP1 is involved in many cancers: gastric intes-

**LMWPTP**—Our group has reported that in chemoresistant human chronic myeloid leukemia cells (Lucena-1), LMWPTP is around 20-fold more active than in

tinal, lung, breast, squamous cell carcinoma, and head and neck [46].

#### **2.2 PTPs and glutamine/lipid metabolism**

Some tumor cells become "addicted" to glutamine, once this amino acid can provide energy and substrates necessary for cell division. As a consequence, the tumor increases the mass of tumor cells and controls the potential redox through the synthesis of NADPH [17]. PTEN knockdown, in prostate cancer, reduces the protein level of GLS, enzyme involved in the glutaminolysis pathway, and increases the FASN expression [12]. Tumor cells also exhibit substantial alterations in lipid metabolism. During fast growth and aggressive progression, tumor cells required many metabolic intermediates and coordinate the activation of lipid synthesis leading to membrane formation, energy storage, and second messenger production [17, 18].

#### **3. PTPs favor tumor growth through survival positive regulation, and cell death resistance**

While normal cells tightly control the synthesis, secretion of growth factors, and proliferative signaling pathways, in order to ensure cellular homeostasis, cancer cells carry one or more defects along the signaling pathways from extracellular compartment, for example, growth ligands and their receptors, to intracellular mediators, such as PI3K, MAPK, and Akt, which give them survival advantages [19, 20]. In this context, PTPs' overexpression through gene amplification, loss, or inhibition contributes for aberrant signaling and, in turn, promoting tumor cell survival as exemplified below:

**CDC25A, CDC25B, and CDC25C**—CDC25A regulates cell cycle transition, from G1 to S phase, where it activates the cyclin E/CDK2 complex, whereas the phosphatases CDC25B and CDC25C act in the G2/M phase progression [21, 22]. Deregulations of these enzymes are correlated with imbalance in the cell cycle, genetic instability, and uncontrolled proliferation. In addition, the high expression level of these proteins is related to tumorigenesis [23, 24]. For instance, the overexpression of CDC25A was related to proliferation of breast, colon, hepatocellular, ovarian, lung, and nonmelanoma cancers [25]. Besides propitiating cancer cell proliferation, it was reported that CDC25A modulates Foxo1, consequently activating the expression of matrix metalloproteinase (MMP)-1, key mediator of cell dissemination. Moreover, the CDC25B overexpression was associated with gastric cancer, and its knockdown reduces the proliferation rate of gastric cells [26].

**EYA**—EYA dephosphorylates tyrosine residues of H2AX, a protein involved with DNA repair that prevents cell death caused by damage to the DNA molecule. Chemical inhibition of EYA phosphatase diminished angiogenesis and tumor growth [27]. WD-repeat-containing protein 1 (WDR1) is a specific substrate of EYA3; thus, this PTP can modulate cytoskeletal reorganization [28]. Another identified substrate of EYA is ERβ, which its dephosphorylation decreases the antitumor potential [29].

#### *Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection? DOI: http://dx.doi.org/10.5772/intechopen.87963*

**LMWPTP**—In normal cells, the increase of LMWPTP expression was associated with a reduction of PDGFR phosphorylation, consequently dropping in the mitogenic capacity [30]. However, later on, LMWPTP was described as a positive modulator of Ras-MAPK, FGF, and Eph receptors [31, 32]. It was also reported that the overexpression of LMWPTP contributes for invasive profile and primary sarcoma formation in nude mice [33]. In this context, higher LMWPTP amount (mRNA and protein) in primary human prostate cancer in relation to normal adjacent tissue was found. Interestingly, the high level of mRNA of LMWPTP was detected in lymph nodes, an indication that this phosphatase takes part in the metastasis process [34]. In the same study, 147 patients out of 481 with prostate cancer presented higher expression of LMWPTP and worse clinical outcome [34]. Accordingly, the LMWPTP has been categorized as a potential biomarker for recurrence prediction for prostate cancer [35]. The importance of the LMWPTP in cancer progression was also reported in colorectal cancer. It was demonstrated that the LMWPTP overexpression in colorectal cancer correlated to a higher potential to liver metastasis [36]. Importantly, it was also demonstrated that the LMWPTP knockdown decreases CRC cell survival and sensitizes them to chemotherapy [36].

**PTP1B**—**PTP1B** is overexpressed in several cancers, such prostate, ovarian, stomach, and colorectal [37–40]. For instance, in esophagus squamous cell carcinoma, this phosphatase overexpression is directly related to invasion and metastasis [38]. Similar effect was described in lung cancer, which was due to Src and Erk activation. Interestingly, the PTP1B knockdown in colorectal cancer cells decreases proliferation rate by blocking β-catenin signaling, a pathway responsible for supporting the cancer secondary site colonization [39, 41].

**SHIP2**—**SHIP2** positively affects tumor cell proliferation and migration. For instance, it was observed that the overexpression of SHIP2 in colorectal cancer was associated with migration and invasive profile through AKT activation [42].

**SHP2 (PTPN11)**—**SHP2 (PTPN11) p**ropiciates activation of Ras and MAPK triggered by mitogens (insulin, EGF, and lysophosphatidic acid) and cell adhesion. Notably, it has been shown that this phosphatase controls cell shape by contributing to cytoskeletal organization. In addition, SHP2 also regulates integrin-mediated cell adhesion, spreading, and migration. Also, inhibition of SHP2 is accompanied by expressive increase in the numbers of actin stress fibers and focal adhesion contacts. In contrast, overexpression of the SHP2 mutant also increased the strength of cellsubstratum adhesion [43]. SHP2 has been considered as a proto-oncogene in several human cancers such as leukemia, glioblastoma, gastric carcinoma, lung cancer, and breast cancer. This phosphatase improves cancer progression and poor prognostic by activation of Ras/Raf/ERK and PI3K/Akt/mTOR pathways [44]. In hepatocellular carcinoma, the overexpression of SHP2 correlates with malignant cancer profile. Accordingly, it was reported that the inhibition of SHP2 diminishes metastasis by inhibition of cell adhesion and migration [45].

During cell transformation to malignancy, tumor cells became expert in overcoming a broad diversity of stresses, such as uncontrolled signaling regulation, starvation, DNA damage, hypoxia, and also anticancer therapy. In this aspect, different researchers have shown that PTPs are involved in tumor cells resistant to chemotherapeutic agents.

**DUSP1 or PTPN10—**It was shown that DUSP1 inhibits the MAPK (JNK) by dephosphorylation and in turn blocks apoptosis process. This effect might be one of the explanations in which DUSP1 promotes cancer cells escaping from apoptosis. Indeed, it has been reported that DUSP1 is involved in many cancers: gastric intestinal, lung, breast, squamous cell carcinoma, and head and neck [46].

**LMWPTP**—Our group has reported that in chemoresistant human chronic myeloid leukemia cells (Lucena-1), LMWPTP is around 20-fold more active than in

the knockdown of PTEN in prostate cancer cells (DU145 cell line) leads to an increase of lactate, pyruvic acid, succinic acid, citric acid, fumaric acid, malic acid, and 2-ketoglutarate, in comparison to DU145 wild type [12]. These findings indicate that glycolysis and glutaminolysis pathways are active in prostate cancer DU-145 cells when PTEN is not functional. Accordingly, it was demonstrated that the PTEN higher expression compromises the proliferation and Warburg effect, in melanoma and breast tumor, by dropping the expression of HIF1 and increasing the mitochondrial function, which are, at least in part, caused by decreasing glucose uptake

Some tumor cells become "addicted" to glutamine, once this amino acid can provide energy and substrates necessary for cell division. As a consequence, the tumor increases the mass of tumor cells and controls the potential redox through the synthesis of NADPH [17]. PTEN knockdown, in prostate cancer, reduces the protein level of GLS, enzyme involved in the glutaminolysis pathway, and increases the FASN expression [12]. Tumor cells also exhibit substantial alterations in lipid metabolism. During fast growth and aggressive progression, tumor cells required many metabolic intermediates and coordinate the activation of lipid synthesis leading to membrane formation, energy storage, and second messenger production [17, 18].

**3. PTPs favor tumor growth through survival positive regulation, and**

While normal cells tightly control the synthesis, secretion of growth factors, and proliferative signaling pathways, in order to ensure cellular homeostasis, cancer cells carry one or more defects along the signaling pathways from extracellular compartment, for example, growth ligands and their receptors, to intracellular mediators, such as PI3K, MAPK, and Akt, which give them survival advantages [19, 20]. In this context, PTPs' overexpression through gene amplification, loss, or inhibition contributes for aberrant signaling and, in turn, promoting tumor cell

**CDC25A, CDC25B, and CDC25C**—CDC25A regulates cell cycle transition, from G1 to S phase, where it activates the cyclin E/CDK2 complex, whereas the phosphatases CDC25B and CDC25C act in the G2/M phase progression [21, 22]. Deregulations of these enzymes are correlated with imbalance in the cell cycle, genetic instability, and uncontrolled proliferation. In addition, the high expression level of these proteins is related to tumorigenesis [23, 24]. For instance, the overexpression of CDC25A was related to proliferation of breast, colon, hepatocellular, ovarian, lung, and nonmelanoma cancers [25]. Besides propitiating cancer cell proliferation, it was reported that CDC25A modulates Foxo1, consequently activating the expression of matrix metalloproteinase (MMP)-1, key mediator of cell dissemination. Moreover, the CDC25B overexpression was associated with gastric cancer, and its knockdown reduces the proliferation rate of gastric cells [26]. **EYA**—EYA dephosphorylates tyrosine residues of H2AX, a protein involved with DNA repair that prevents cell death caused by damage to the DNA molecule. Chemical inhibition of EYA phosphatase diminished angiogenesis and tumor growth [27]. WD-repeat-containing protein 1 (WDR1) is a specific substrate of EYA3; thus, this PTP can modulate cytoskeletal reorganization [28]. Another identified substrate of EYA is ERβ, which its dephosphorylation decreases the antitumor

and inhibiting PI3K/mTOR pathway [13–16].

*Tumor Progression and Metastasis*

**2.2 PTPs and glutamine/lipid metabolism**

**cell death resistance**

survival as exemplified below:

potential [29].

their sensitive counterpart (K562). Importantly, the knockdown of LMWPTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate and culminated in inactivation of Src kinase and Bcr-Abl. Both kinases are well known to have a relevant contribution in leukemogenesis [47].

cadherin-catenin complex on different residues, resulting in a loss of cell adhesion [60]. For instance, PTP1B regulates cadherin-based adhesion by dephosphorylating β-catenin at Tyr654 [61]. In addition to β-catenin, p120-catenin phosphorylation increases binding and affinity to E-cadherin, and PTPμ appears to be a regulator of p120-catenin phosphorylation status, also acts as a scaffold, and recruits similar and regulatory molecules to sites of cell adhesion [61, 62]. SHP2 is also able to bind to

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?*

Cell migration through ECM requires integrin-mediated adhesion as well as turnover of focal adhesions [63]. A decrease in tyrosine phosphorylation by PTPs is involved in the formation and disassembly of focal adhesions. For instance, PTPα is required for the activation of Src kinase following integrin interaction [64], and the dephosphorylation of p130 CRK-associated substrate, by PTP-PEST, is necessary for disassembly of focal adhesions, enabling cell migration [64]. The relation between PTPs and upstream regulators of cell matrix adhesion and Rho family of small GTPases has also been shown [65]. Most Rho proteins have intrinsic GTPase activity which is stimulated by GTPase-activating proteins (GAPs), and these GAPs are modulated by phosphorylation at tyrosine sites. Consequently, PTPs can influence Rho protein activation through regulating the phosphorylation state of GAPs. Sastry and colleagues showed that PTP-PEST overexpression reduces Rac1 (a kind of G protein) activity resulting in protrusion and retraction during cell migration [66]. On the other hand, SHP2 seems to have some contradictory action, while some literatures reported a RhoA activity inhibition by SHP2 [67] and others suggested a stimulation [43]. In addition, p190RhoGAP, a GAP for RhoA, is a target for SHP-2

and LMWPTP and, in turn, regulating cytoskeletal rearrangement [68].

the treatment of MCF-7 breast cancer cells with BVT948 (a PTP inhibitor)

**57**

Metalloproteinases (MMPs) are one of the most important ECM-remodeling enzymes produced by tumoral cells, which are linked to tumorigenesis and metastasis [69]. More recently, it was reported that MMPs promote cell survival, angiogenesis [69], and induction of EMT [70]. Hwang and coworkers [71] observed that

decreases invasion through suppression of NF-κB-mediated MMP-9 expression. On the converse side, PTPμ knockdowns resulted in elevated adhesion, invasion, and proliferation of breast cancer cells due to activation of ERK and JNK signaling pathway and consequent elevated MMP-9 activity [72]. It was demonstrated that the overexpression of PRL-3 increased the migration and invasion capacity of DLD-1 colorectal cancer cells, which was dependent on the expression of MMP-7 [73]. Maacha and coworkers demonstrated that the contribution of the PTP4A3 for malignancy of uveal melanoma is related to MMP-14 [74]. Yuan and colleagues found that overexpression of PTPN9 reduces invasion and decreases MMP-2 gene expression in MDA-MB-231 cells through inhibition of STAT3 downregulation [75]. Interestingly, still in the context of breast cancer, William Du and his team [76] analyzed the levels of microRNA-24 in patients with breast carcinoma and found higher content of this microRNA in breast carcinoma samples than in benign breast tissue. They also generated constructs expressing miRNA-24 and studied their functions in vivo and in vitro. In vivo experiments in mice indicated that the expression of miRNA-24 enhanced tumor growth, invasion, and metastasis to the lung and decreased survival. Molecularly, in vitro and in vivo experiments showed high EGFR phosphorylation but repressed expression of PTPN9 and PTPRF due to direct target of these phosphatases by miRNA-24. Consistently, they found in patients with metastatic breast carcinoma a higher phosphorylation of EGFR but lower levels of PTPN9 and PTPRF. Another confirmation was the upregulation of MMP-2 and MMP-11 but downregulation of MMP inhibitor (TIMP-2) which supports the roles of miRNA-24 in tumor invasion and metastasis in breast cancer suggesting miRNA-24 as a potential target for cancer intervention. In another

cadherin-catenin complex and integrin molecules [62].

*DOI: http://dx.doi.org/10.5772/intechopen.87963*

**PTPN3**—Wang and collaborators [40] performed a very elegant study, in which they found somatic mutations in six PTPs, including PTPN3, in colorectal, lung, breast, and gastric cancers. Later on, it was reported that PTPN3 induces drug resistance (cisplatin and doxorubicin) in ovarian cancer [48].

**SHP2**—A study using a RNA interference-based genetic screen in BRAF-mutant colon cancer cells identified the SHP2 as one of the key mediators of intrinsic and acquired resistance. Once this phosphatase maintains the receptor tyrosine kinases activated, even in the presence of BRAF inhibitor, it is still possible to have activation of cell proliferation and survival through involvement of ERK [49].

#### **4. PTPs contribute for metastasis through extracellular matrix remodeling and epithelial-mesenchymal transition**

In this chapter subtitle, we will focus on strategies for migration and invasion as part of the metastasis process.

PTPs activate the extracellular matrix remodeling and epithelial-mesenchymal transition. ECM is a three-dimensional noncellular scaffold crucial for life in multicellular organisms which is dynamically and continuously remodeled. ECM is mainly composed of water and almost 300 proteins, for example, collagens (fibrillar forms such as I–III, V, XI and non-fibrillar forms), proteoglycan (aggrecan and glycosaminoglycan such as heparin sulfate and hyaluronic acid), and glycoproteins (especially elastin, laminins, and fibronectin) [50, 51]. This essential component is considered an extremely organized meshwork in a strict contact with cells providing both biochemical and biomechanical support. It is well known that despite the physical support to cells, ECM also modulates cell differentiation, migration, and proliferation [50, 52]. Therefore, abnormal ECM remodeling (exacerbate deposition or degradation) can be observed during pathological conditions such as fibrosis and cancer [50, 52]. In tumor microenvironment, much of the ECM proteins are produced not only by stroma cells, e.g., cancer-associated fibroblasts [52], but also tumor cells can produce ECM proteins [53]. Malignant transformation is characterized by changes in the organization of cytoskeleton resulting in abnormal cell signaling related to cell-cell and to cell-ECM adhesion, a phenomenon termed epithelial-mesenchymal transition (EMT). EMT consists of the loss of epithelial cell characteristics to possess properties of mesenchymal cells. Several studies have shown that the EMT contributes to tumor progression, invasion, metastasis, and acquisition of therapeutic resistance. During the EMT process, the cancer cells acquire a fibroblastic morphology with a positive regulation of mesenchymal markers (N-cadherin, vimentin, and α-actin) and a negative regulation of epithelial cell markers (E-cadherin, ZO-1, claudins, occludins, and cytokeratin) as well as a regulation of transcription factors that are associated with increased migratory capacity (Slug, ZEB1/ZEB2, Twist1/Twist2). These factors bind to the E-cadherin gene promoter and repress it [54–56]. EMT requires a rupture of basement membrane permitting invasion and migration of cancer cells through the ECM, then causing remodeling, and creating a tumorpermissive environment [57].

Characteristic loss of E-cadherin-mediated cell-cell adhesion is commonly found during malignant transformation [58] in which process kinases and phosphatases have key roles [59, 60]. Several PTKs, including SRC and EGFR, phosphorylate the

#### *Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection? DOI: http://dx.doi.org/10.5772/intechopen.87963*

cadherin-catenin complex on different residues, resulting in a loss of cell adhesion [60]. For instance, PTP1B regulates cadherin-based adhesion by dephosphorylating β-catenin at Tyr654 [61]. In addition to β-catenin, p120-catenin phosphorylation increases binding and affinity to E-cadherin, and PTPμ appears to be a regulator of p120-catenin phosphorylation status, also acts as a scaffold, and recruits similar and regulatory molecules to sites of cell adhesion [61, 62]. SHP2 is also able to bind to cadherin-catenin complex and integrin molecules [62].

Cell migration through ECM requires integrin-mediated adhesion as well as turnover of focal adhesions [63]. A decrease in tyrosine phosphorylation by PTPs is involved in the formation and disassembly of focal adhesions. For instance, PTPα is required for the activation of Src kinase following integrin interaction [64], and the dephosphorylation of p130 CRK-associated substrate, by PTP-PEST, is necessary for disassembly of focal adhesions, enabling cell migration [64]. The relation between PTPs and upstream regulators of cell matrix adhesion and Rho family of small GTPases has also been shown [65]. Most Rho proteins have intrinsic GTPase activity which is stimulated by GTPase-activating proteins (GAPs), and these GAPs are modulated by phosphorylation at tyrosine sites. Consequently, PTPs can influence Rho protein activation through regulating the phosphorylation state of GAPs. Sastry and colleagues showed that PTP-PEST overexpression reduces Rac1 (a kind of G protein) activity resulting in protrusion and retraction during cell migration [66]. On the other hand, SHP2 seems to have some contradictory action, while some literatures reported a RhoA activity inhibition by SHP2 [67] and others suggested a stimulation [43]. In addition, p190RhoGAP, a GAP for RhoA, is a target for SHP-2 and LMWPTP and, in turn, regulating cytoskeletal rearrangement [68].

Metalloproteinases (MMPs) are one of the most important ECM-remodeling enzymes produced by tumoral cells, which are linked to tumorigenesis and metastasis [69]. More recently, it was reported that MMPs promote cell survival, angiogenesis [69], and induction of EMT [70]. Hwang and coworkers [71] observed that the treatment of MCF-7 breast cancer cells with BVT948 (a PTP inhibitor) decreases invasion through suppression of NF-κB-mediated MMP-9 expression. On the converse side, PTPμ knockdowns resulted in elevated adhesion, invasion, and proliferation of breast cancer cells due to activation of ERK and JNK signaling pathway and consequent elevated MMP-9 activity [72]. It was demonstrated that the overexpression of PRL-3 increased the migration and invasion capacity of DLD-1 colorectal cancer cells, which was dependent on the expression of MMP-7 [73]. Maacha and coworkers demonstrated that the contribution of the PTP4A3 for malignancy of uveal melanoma is related to MMP-14 [74]. Yuan and colleagues found that overexpression of PTPN9 reduces invasion and decreases MMP-2 gene expression in MDA-MB-231 cells through inhibition of STAT3 downregulation [75]. Interestingly, still in the context of breast cancer, William Du and his team [76] analyzed the levels of microRNA-24 in patients with breast carcinoma and found higher content of this microRNA in breast carcinoma samples than in benign breast tissue. They also generated constructs expressing miRNA-24 and studied their functions in vivo and in vitro. In vivo experiments in mice indicated that the expression of miRNA-24 enhanced tumor growth, invasion, and metastasis to the lung and decreased survival. Molecularly, in vitro and in vivo experiments showed high EGFR phosphorylation but repressed expression of PTPN9 and PTPRF due to direct target of these phosphatases by miRNA-24. Consistently, they found in patients with metastatic breast carcinoma a higher phosphorylation of EGFR but lower levels of PTPN9 and PTPRF. Another confirmation was the upregulation of MMP-2 and MMP-11 but downregulation of MMP inhibitor (TIMP-2) which supports the roles of miRNA-24 in tumor invasion and metastasis in breast cancer suggesting miRNA-24 as a potential target for cancer intervention. In another

their sensitive counterpart (K562). Importantly, the knockdown of LMWPTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate and culminated in inactivation of Src kinase and Bcr-Abl. Both kinases are well known

**PTPN3**—Wang and collaborators [40] performed a very elegant study, in which they found somatic mutations in six PTPs, including PTPN3, in colorectal, lung, breast, and gastric cancers. Later on, it was reported that PTPN3 induces drug

**SHP2**—A study using a RNA interference-based genetic screen in BRAF-mutant colon cancer cells identified the SHP2 as one of the key mediators of intrinsic and acquired resistance. Once this phosphatase maintains the receptor tyrosine kinases activated, even in the presence of BRAF inhibitor, it is still possible to have activa-

In this chapter subtitle, we will focus on strategies for migration and invasion as

PTPs activate the extracellular matrix remodeling and epithelial-mesenchymal

remodeling (exacerbate deposition or degradation) can be observed during pathological conditions such as fibrosis and cancer [50, 52]. In tumor microenvironment, much of the ECM proteins are produced not only by stroma cells, e.g., cancer-associated fibroblasts [52], but also tumor cells can produce ECM proteins [53]. Malignant transformation is characterized by changes in the organization of cytoskeleton resulting in abnormal cell signaling related to cell-cell and to cell-ECM adhesion, a phenomenon termed epithelial-mesenchymal transition (EMT). EMT consists of the loss of epithelial cell characteristics to possess properties of mesenchymal cells. Several studies have shown that the EMT contributes to tumor progression, invasion, metastasis, and acquisition of therapeutic resistance. During the EMT process, the cancer cells acquire a fibroblastic morphology with a positive regulation of mesenchymal markers (N-cadherin, vimentin, and α-actin) and a negative regulation of epithelial cell markers (E-cadherin, ZO-1, claudins,

occludins, and cytokeratin) as well as a regulation of transcription factors that are associated with increased migratory capacity (Slug, ZEB1/ZEB2, Twist1/Twist2). These factors bind to the E-cadherin gene promoter and repress it [54–56]. EMT requires a rupture of basement membrane permitting invasion and migration of cancer cells through the ECM, then causing remodeling, and creating a tumor-

Characteristic loss of E-cadherin-mediated cell-cell adhesion is commonly found during malignant transformation [58] in which process kinases and phosphatases have key roles [59, 60]. Several PTKs, including SRC and EGFR, phosphorylate the

transition. ECM is a three-dimensional noncellular scaffold crucial for life in multicellular organisms which is dynamically and continuously remodeled. ECM is mainly composed of water and almost 300 proteins, for example, collagens (fibrillar forms such as I–III, V, XI and non-fibrillar forms), proteoglycan (aggrecan and glycosaminoglycan such as heparin sulfate and hyaluronic acid), and glycoproteins (especially elastin, laminins, and fibronectin) [50, 51]. This essential component is considered an extremely organized meshwork in a strict contact with cells providing both biochemical and biomechanical support. It is well known that despite the physical support to cells, ECM also modulates cell differentiation, migration, and proliferation [50, 52]. Therefore, abnormal ECM

to have a relevant contribution in leukemogenesis [47].

resistance (cisplatin and doxorubicin) in ovarian cancer [48].

tion of cell proliferation and survival through involvement of ERK [49].

**4. PTPs contribute for metastasis through extracellular matrix**

**remodeling and epithelial-mesenchymal transition**

part of the metastasis process.

*Tumor Progression and Metastasis*

permissive environment [57].

study, Liu and collaborators [41] observed that PTP1B promotes the aggressiveness of brain cancer through decreasing PTEN levels and, consequently, promoting AKT activation and increasing of MMP-2 and MMP-7. Previously, it was reported that PTP1B promotes gastric cancer cell invasiveness through modulating the expression of MMP-2, MMP-9, and MMP-14 [77]. Another interesting study shows the relationship of PTP1B and interruption of cell adhesion and induction of the *anoikis* effect in cancer cells. Inhibition of PTP1B in breast cancer cells leads to cell death and loss of extracellular matrix fixation, leading to negative regulation of cell adhesion proteins and interrupted actin polymerization. They saw that with the inhibition of PTP1B the activity of Src is consequently decreased by the adhesion pathway and motility is impaired [78].

**PRL-1**—This phosphatase causes activation of AKT, and inhibition of GSK3β, consequently, contributes for elevated levels of Snail expression and decreased Ecadherin expression. In agreement, the high level of this enzyme was associated with more aggressive phenotype and poorer prognosis in hepatocellular carcinoma

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?*

**PRL-3**—**PRL-3** activates the PI3K/PKB pathway and promotes EMT by decreasing PTEN protein expression [23]. In addition, it was demonstrated that expression of PRL-3 in hepatocellular carcinoma patients was positively correlated

**PTRB**—Overexpression of PTRB has an opposite effect on EMT markers: decreased the expression of E-cadherin and increased the amount of vimentin [91].

Tumor suppressors operate in different ways and compartments to limit cell growth and proliferation. Besides the important contribution of PTPs in cancer progression, some PTPs that act as tumor suppressors are described below:

**PTEN—**Is a central tumor suppressor, mainly due to its negative effect on key pathways related to cell proliferation, survival, and metastasis: PI3K-Akt–mTOR, NF-κB, and HIF [92, 93]. Therefore, the loss of PTEN, which occurs in the major of the tumors, is correlated with tumor aggressiveness and low response to therapy. In prostate cancer studies, PTEN has been shown to ameliorate the malignant phenotype by dephosphorylating the activator residue of PTK6 (Tyr 342), a kinase related to a cancer aggressive phenotype [94]. In addition, other oncogenic kinases, such as PDGFR and FAK, have been reported as a substrate of PTEN [95, 96]. Although the molecular mechanisms by which PTEN acts as a tumor suppressor are well known, until few years ago, there were not a lot of information about the posttranslational regulation of PTEN. Recently, Park and collaborators [97] have reported two mechanisms of PTEN regulation that directly are connected to its tumor suppressor property: (a) deubiquitination by ubiquitin-specific protease 11 (USP11), responsible for increasing the stability of nuclear and cytosolic PTEN; (b) the level and activity of PTEN are also autoregulated by this phosphatase via PI3K-forkhead

**SHP1 (PTPN6)**—Has been described as a major negative regulator of MAPK, JAK/STAT, and NF-κB signaling pathways [98, 99]. Therefore, SHP1 activity is inversely related to cancer development. Indeed, the SHP1 expression in stomach cancer is very weak. Accordingly, the overexpression of SHP1 in stomach cancer cell lines induces a decrease of proliferation, migration, and invasion [100]. In addition, Chen and colleagues showed that SHP1 dephosphorylates and inhibits PKM2, a kinase that stimulates proliferation in hepatocellular carcinoma [46].

**SHIP1**—Is expressed only in hematopoietic-derived cells and acts as a negative modulator of PI3K pathway [101]. It was described that the PTEN and SHIP1 loss is

**PTPN9 (PTP-MEG2)**—Low expression of this phosphatase predicted poor survival in patients with hepatocellular carcinoma. It was observed that PTPN9 indirectly inhibits activity of STAT3 and STAT5 through direct dephosphorylation of EGFR and HER2, in breast cancer [46]. In addition, the overexpression of PTPN9 decreases the phosphorylation of AKT protein at its activatory residue, which cul-

**PTPN12 (PTP-PEST)**—Regulates oncogenic tyrosine kinases such as HER2 and EGFR and has a role in modulating EMT. Not surprisingly, it has been decreased or lost in human hepatocellular carcinoma tissues, and by using this carcinoma cell

with the expression of MMPs 1, 9, 10, and 12 [46].

**5. PTPs that act as tumor suppressors**

*DOI: http://dx.doi.org/10.5772/intechopen.87963*

transcription factor (FOXO)-USP11 cascade [97].

deeply related to lymphoma survival [102].

**59**

minated in diminishing the EMT process efficiency [103].

patients [46].

Besides being involved in ECM remodeling by modulating MMP activities, PTPs (PTEN, SHP2, PTP1B, PRL3, PTP1B, PTRB, and PTPN9) have a key role in signaling cascades that promote expression of EMT markers.

**DUSP1**—It has been reported that the knockdown of DUSP1 culminates in low migratory and invasive efficiency of non-small cell lung cancer (NSCLC). Similar effect was also observed in in vivo model [79].

**PTEN**—It has been reported that the loss of PTEN or its negative modulation by phosphorylation or microRNA propiciates EMT. For instance, in lung cancer cells, the inactivation of PTEN stimulated the nuclear translocation of β-catenin and transcription factors snail and slug [80]. The authors also observed that the PI3K/ AKT/GSK-3β pathway is essential for inducing EMT in PTEN-knocked-down cells. The relation between PTEN and negative regulation of AKT/β-catenin pathway was also described by Li and colleagues in squamous cell carcinoma of the esophagus [81]. It was observed that the glycan-1, a cell surface proteoglycan, promotes cell proliferation by regulating the PTEN/AKT/β-catenin pathway, which culminates in a positive regulation of N-cadherin and β-catenin and a negative regulation of Ecadherin. In colorectal cancer cells, the loss of PTEN is associated with a change in E-cadherin protein expression which was linked to EMT [82]. Wang and co-authors [83] reported that tetraspanin 1 induced liver cancer cell EMT via the PI3K/AKT/ GSK-3β pathway. These authors also show that the PTEN repression was fundamental for this process. In addition to the effects reported above, one event that is associated with PTEN induction of EMT is the dysregulation of microRNAs. Studies have shown that PTEN is a target of some microRNAs. Wu and collaborators [84] showed that MiR-616-3p is upregulated in metastatic gastric cancer cells during angiogenesis process, and PTEN was one of the targets of this microRNA. Li [85] also showed that MiR-181-a is associated with lung cancer cell EMT through inhibition of PTEN protein expression. Another strategy to inhibit PTEN is via TGF-β cascade. The phosphorylation of the PTEN C-terminus leads to a conformational change, consequently provoking the loss of membrane binding and downregulation of PTEN phosphatase activity [86].TGF-β derived from the tumor microenvironment induces malignant phenotypes such as EMT and aberrant cell motility in lung cancers, by at least in part, due to inhibition of PTEN by phosphorylation [87].

**SHP2**—Sun and coworkers reported that IL-6 induces SHP2 activation by phosphorylation, which was required for breast cancer cell EMT stimulation in response to IL-6 [88]. This phosphatase also has a positive connection in lung cancer cell EMT triggered by TGF-β1 [89]. In addition, these authors identified the protein Hook1 as an interactor of SHP2 and classified this protein as an endogenous negative regulator of SHP2. The expression of Snail and Twist1, key mediators of EMT process, has been positively modulated by SHP2 in oral cancer, via its interaction with ERK1/ERK2 [89].

**PTP1B**—Hiraga and colleagues reported that PTP1B is one of the mediators of pancreatic cancer cell EMT induced by TGF-β [90].

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection? DOI: http://dx.doi.org/10.5772/intechopen.87963*

**PRL-1**—This phosphatase causes activation of AKT, and inhibition of GSK3β, consequently, contributes for elevated levels of Snail expression and decreased Ecadherin expression. In agreement, the high level of this enzyme was associated with more aggressive phenotype and poorer prognosis in hepatocellular carcinoma patients [46].

**PRL-3**—**PRL-3** activates the PI3K/PKB pathway and promotes EMT by decreasing PTEN protein expression [23]. In addition, it was demonstrated that expression of PRL-3 in hepatocellular carcinoma patients was positively correlated with the expression of MMPs 1, 9, 10, and 12 [46].

**PTRB**—Overexpression of PTRB has an opposite effect on EMT markers: decreased the expression of E-cadherin and increased the amount of vimentin [91].

#### **5. PTPs that act as tumor suppressors**

study, Liu and collaborators [41] observed that PTP1B promotes the aggressiveness of brain cancer through decreasing PTEN levels and, consequently, promoting AKT activation and increasing of MMP-2 and MMP-7. Previously, it was reported that PTP1B promotes gastric cancer cell invasiveness through modulating the expression of MMP-2, MMP-9, and MMP-14 [77]. Another interesting study shows the relationship of PTP1B and interruption of cell adhesion and induction of the *anoikis* effect in cancer cells. Inhibition of PTP1B in breast cancer cells leads to cell death and loss of extracellular matrix fixation, leading to negative regulation of cell adhesion proteins and interrupted actin polymerization. They saw that with the inhibition of PTP1B the activity of Src is consequently decreased by the adhesion pathway

Besides being involved in ECM remodeling by modulating MMP activities, PTPs (PTEN, SHP2, PTP1B, PRL3, PTP1B, PTRB, and PTPN9) have a key role in signal-

**DUSP1**—It has been reported that the knockdown of DUSP1 culminates in low migratory and invasive efficiency of non-small cell lung cancer (NSCLC). Similar

**PTEN**—It has been reported that the loss of PTEN or its negative modulation by phosphorylation or microRNA propiciates EMT. For instance, in lung cancer cells, the inactivation of PTEN stimulated the nuclear translocation of β-catenin and transcription factors snail and slug [80]. The authors also observed that the PI3K/ AKT/GSK-3β pathway is essential for inducing EMT in PTEN-knocked-down cells. The relation between PTEN and negative regulation of AKT/β-catenin pathway was also described by Li and colleagues in squamous cell carcinoma of the esophagus [81]. It was observed that the glycan-1, a cell surface proteoglycan, promotes cell proliferation by regulating the PTEN/AKT/β-catenin pathway, which culminates in a positive regulation of N-cadherin and β-catenin and a negative regulation of Ecadherin. In colorectal cancer cells, the loss of PTEN is associated with a change in E-cadherin protein expression which was linked to EMT [82]. Wang and co-authors [83] reported that tetraspanin 1 induced liver cancer cell EMT via the PI3K/AKT/ GSK-3β pathway. These authors also show that the PTEN repression was fundamental for this process. In addition to the effects reported above, one event that is associated with PTEN induction of EMT is the dysregulation of microRNAs. Studies have shown that PTEN is a target of some microRNAs. Wu and collaborators [84] showed that MiR-616-3p is upregulated in metastatic gastric cancer cells during angiogenesis process, and PTEN was one of the targets of this microRNA. Li [85] also showed that MiR-181-a is associated with lung cancer cell EMT through inhibition of PTEN protein expression. Another strategy to inhibit PTEN is via TGF-β cascade. The phosphorylation of the PTEN C-terminus leads to a conformational change, consequently provoking the loss of membrane binding and downregulation of PTEN phosphatase activity [86].TGF-β derived from the tumor microenvironment induces malignant phenotypes such as EMT and aberrant cell motility in lung cancers, by at least in part, due to inhibition of PTEN by phosphorylation [87].

**SHP2**—Sun and coworkers reported that IL-6 induces SHP2 activation by phosphorylation, which was required for breast cancer cell EMT stimulation in response to IL-6 [88]. This phosphatase also has a positive connection in lung cancer cell EMT triggered by TGF-β1 [89]. In addition, these authors identified the protein Hook1 as an interactor of SHP2 and classified this protein as an endogenous negative regulator of SHP2. The expression of Snail and Twist1, key mediators of EMT process, has been positively modulated by SHP2 in oral cancer, via its interaction

**PTP1B**—Hiraga and colleagues reported that PTP1B is one of the mediators of

and motility is impaired [78].

*Tumor Progression and Metastasis*

with ERK1/ERK2 [89].

**58**

pancreatic cancer cell EMT induced by TGF-β [90].

ing cascades that promote expression of EMT markers.

effect was also observed in in vivo model [79].

Tumor suppressors operate in different ways and compartments to limit cell growth and proliferation. Besides the important contribution of PTPs in cancer progression, some PTPs that act as tumor suppressors are described below:

**PTEN—**Is a central tumor suppressor, mainly due to its negative effect on key pathways related to cell proliferation, survival, and metastasis: PI3K-Akt–mTOR, NF-κB, and HIF [92, 93]. Therefore, the loss of PTEN, which occurs in the major of the tumors, is correlated with tumor aggressiveness and low response to therapy. In prostate cancer studies, PTEN has been shown to ameliorate the malignant phenotype by dephosphorylating the activator residue of PTK6 (Tyr 342), a kinase related to a cancer aggressive phenotype [94]. In addition, other oncogenic kinases, such as PDGFR and FAK, have been reported as a substrate of PTEN [95, 96]. Although the molecular mechanisms by which PTEN acts as a tumor suppressor are well known, until few years ago, there were not a lot of information about the posttranslational regulation of PTEN. Recently, Park and collaborators [97] have reported two mechanisms of PTEN regulation that directly are connected to its tumor suppressor property: (a) deubiquitination by ubiquitin-specific protease 11 (USP11), responsible for increasing the stability of nuclear and cytosolic PTEN; (b) the level and activity of PTEN are also autoregulated by this phosphatase via PI3K-forkhead transcription factor (FOXO)-USP11 cascade [97].

**SHP1 (PTPN6)**—Has been described as a major negative regulator of MAPK, JAK/STAT, and NF-κB signaling pathways [98, 99]. Therefore, SHP1 activity is inversely related to cancer development. Indeed, the SHP1 expression in stomach cancer is very weak. Accordingly, the overexpression of SHP1 in stomach cancer cell lines induces a decrease of proliferation, migration, and invasion [100]. In addition, Chen and colleagues showed that SHP1 dephosphorylates and inhibits PKM2, a kinase that stimulates proliferation in hepatocellular carcinoma [46].

**SHIP1**—Is expressed only in hematopoietic-derived cells and acts as a negative modulator of PI3K pathway [101]. It was described that the PTEN and SHIP1 loss is deeply related to lymphoma survival [102].

**PTPN9 (PTP-MEG2)**—Low expression of this phosphatase predicted poor survival in patients with hepatocellular carcinoma. It was observed that PTPN9 indirectly inhibits activity of STAT3 and STAT5 through direct dephosphorylation of EGFR and HER2, in breast cancer [46]. In addition, the overexpression of PTPN9 decreases the phosphorylation of AKT protein at its activatory residue, which culminated in diminishing the EMT process efficiency [103].

**PTPN12 (PTP-PEST)**—Regulates oncogenic tyrosine kinases such as HER2 and EGFR and has a role in modulating EMT. Not surprisingly, it has been decreased or lost in human hepatocellular carcinoma tissues, and by using this carcinoma cell

lines as models, it was demonstrated that PTPN12 downregulation stimulated cell migration [46].

suppressors, resisting cell death, deregulating cellular energetics, and activating invasion and metastasis). This connection might explain, at least in part, the great

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?*

Our research on this field has been supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant 2015/20412-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 303900/

130 CRK p130 Crk-associated substrate (member of an adapter

phosphorylated proteins)

AKT also known as *protein kinase B (PKB)*, is a

DUSP1 (PTPN10) dual-specificity protein *phosphatase* 1 DUSP dual-specificity protein *phosphatase*

EGFR epidermal growth factor receptor EMT epithelial-mesenchymal transition

ERK extracellular signal-regulated kinase

proteins

GSK-3beta glycogen synthase kinase 3 beta H2AX H2A histone family member X HIF *hypoxia-inducible factor*

GAPDH glyceraldehyde 3-phosphate dehydrogenase GAPs GTPase-activating proteins or GTPase-accelerating

known as ACP1

MAPK mitogen-activated protein kinase

LMWPTP low-molecular-weight protein tyrosine phosphatase, also

protein family that binds to several tyrosine-

serine/threonine-specific protein kinase

capacity of tumor cells' plasticity.

*DOI: http://dx.doi.org/10.5772/intechopen.87963*

**Acknowledgements**

**Conflict of interest**

The authors declare no conflict of interest.

Bcr-Abl tyrosine-protein kinase Cdc25 dual-specificity phosphatase CDK2 *cyclin-dependent kinases*

ECM extracellular matrix

ERbeta estrogen receptor beta

FAK focal adhesion kinase FGF fibroblast growth *factor* FOXO-1 forkhead box protein O1

GLS glutamine synthetase GLUT glucose transporter 1

LDHA lactate dehydrogenase A

Eph ephrin

Eya eyes absent

HK hexokinase JAK Janus kinase 2

**61**

**Appendices and nomenclature**

2017-2).

**FAP-1 (PTPN13)**—Downregulates Src-ERK pathway by inhibiting EphrinB1 [104]. FAP-1 can also interact and dephosphorylate Her2, thus reducing the aggressive potential of tumors that have high expression of this receptor [105]. It was also demonstrated that overexpression of this phosphatase caused an upregulation of the epithelial marker, E-cadherin, and downregulated mesenchymal markers such as Snail, Slug, and MMP-9, which are a strong indication that FAP-1 inhibits EMT in hepatocellular carcinoma progression [46].

**DUSP2**—It has been shown that DUSP2 is involved in P53-induced cell apoptosis; however, this phosphatase is dramatically reduced in different solid tumors compared to their normal counterparts. Accordingly, it was reported that the diminished DUSP2 leads to prolonged ERK phosphorylation, increased drug resistance, as well as an inflammatory response due to overproduction of prostaglandin in colorectal cancer [106]. It was also reported that DUSP2 knockdown in xenograft tumors promotes higher vessel density and metastasis events from colorectal cancer to the liver [107].

**PTPRT (PTPρ)**—Is commonly mutated in several types of cancer, including colorectal cancer [40]. Many studies have reported the tumor suppressor potential of this PTP, and among the possible substrates of this phosphatase are paxilin and STAT3 [108, 109].

**PTPRH**—This phosphatase interacts with Grb2 and then modulates Ras pathway activity. Studies have reported that PTPRH blocks cell growth and migration by dephosphorylating proteins associated with focal adhesion, such as p130 [110].

**PTPRD**—It has been shown that patients with the high level of PTPRD display better long-term survival rate and low chance of liver cancer recurrence. However, the mechanisms underlying this action are not elucidated.

**PTP receptor type F (PTPRF)**—Is involved in Src kinase inactivation; therefore, it is not surprising that this enzyme is frequently downmodulated in hepatocellular carcinoma patients and upregulation of PTPRF is associated with better prognosis [46].

**PTP receptor type O (PTPRO)**—Plays as a chronic lymphocytic leukemia, lung, and breast tumor suppressor by inhibiting proliferation and stimulating apoptosis, at least in part, due to STAT3 dephosphorylation [46].

**PTP receptor S (PTPσ)**—Is an important negative modulator of EGFR. Therefore, the downregulation of this phosphatase has been connected to decreased overall survival and high risk of postoperative recurrence in HCC patients [46].

#### **6. Conclusions**

Over the past two decades of research on PTPs, the field has achieved a great progress in understanding the immense role of these phosphatases in cancer progression. Here, we presented an organized picture that clearly shows the participation/contribution of PTPs as key mediators of cancer plasticity, due to their loss of function or overexpression. In summary the above compendium highlights the importance of PTPs not only in cancer progression but also as potential targets for therapeutic interventions. Indeed, during the transition from good to poor outcome of different cancer subtypes, PTPs are extremely plastic, with the capacity to readjust themselves across a wide spectrum of stimuli. This plasticity of PTPs together with the loss of function of PTP suppressors provides tumor cells with all conditions for growth, proliferation, and survival. Illustrative examples are PTEN (loss), LMWPTP, PRL-3, and PTP1B serving as "signaling hubs" that connect different hallmarks (such as sustaining proliferative signaling, evading growth

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection? DOI: http://dx.doi.org/10.5772/intechopen.87963*

suppressors, resisting cell death, deregulating cellular energetics, and activating invasion and metastasis). This connection might explain, at least in part, the great capacity of tumor cells' plasticity.

#### **Acknowledgements**

lines as models, it was demonstrated that PTPN12 downregulation stimulated cell

upregulation of the epithelial marker, E-cadherin, and downregulated mesenchymal markers such as Snail, Slug, and MMP-9, which are a strong indication that

**DUSP2**—It has been shown that DUSP2 is involved in P53-induced cell apoptosis; however, this phosphatase is dramatically reduced in different solid tumors compared to their normal counterparts. Accordingly, it was reported that the diminished DUSP2 leads to prolonged ERK phosphorylation, increased drug resistance, as well as an inflammatory response due to overproduction of prostaglandin in colorectal cancer [106]. It was also reported that DUSP2 knockdown in xenograft tumors promotes higher vessel density and metastasis events from colorectal cancer

**PTPRT (PTPρ)**—Is commonly mutated in several types of cancer, including colorectal cancer [40]. Many studies have reported the tumor suppressor potential of this PTP, and among the possible substrates of this phosphatase are paxilin and

**PTPRH**—This phosphatase interacts with Grb2 and then modulates Ras pathway activity. Studies have reported that PTPRH blocks cell growth and migration by dephosphorylating proteins associated with focal adhesion, such as p130 [110]. **PTPRD**—It has been shown that patients with the high level of PTPRD display better long-term survival rate and low chance of liver cancer recurrence. However,

**PTP receptor type F (PTPRF)**—Is involved in Src kinase inactivation; therefore, it is not surprising that this enzyme is frequently downmodulated in hepatocellular carcinoma patients and upregulation of PTPRF is associated with better

**PTP receptor type O (PTPRO)**—Plays as a chronic lymphocytic leukemia, lung, and breast tumor suppressor by inhibiting proliferation and stimulating apo-

fore, the downregulation of this phosphatase has been connected to decreased overall survival and high risk of postoperative recurrence in HCC patients [46].

**PTP receptor S (PTPσ)**—Is an important negative modulator of EGFR. There-

Over the past two decades of research on PTPs, the field has achieved a great progress in understanding the immense role of these phosphatases in cancer progression. Here, we presented an organized picture that clearly shows the participation/contribution of PTPs as key mediators of cancer plasticity, due to their loss of function or overexpression. In summary the above compendium highlights the importance of PTPs not only in cancer progression but also as potential targets for therapeutic interventions. Indeed, during the transition from good to poor outcome of different cancer subtypes, PTPs are extremely plastic, with the capacity to readjust themselves across a wide spectrum of stimuli. This plasticity of PTPs together with the loss of function of PTP suppressors provides tumor cells with all conditions for growth, proliferation, and survival. Illustrative examples are PTEN (loss), LMWPTP, PRL-3, and PTP1B serving as "signaling hubs" that connect different hallmarks (such as sustaining proliferative signaling, evading growth

[104]. FAP-1 can also interact and dephosphorylate Her2, thus reducing the aggressive potential of tumors that have high expression of this receptor [105]. It

was also demonstrated that overexpression of this phosphatase caused an

FAP-1 inhibits EMT in hepatocellular carcinoma progression [46].

the mechanisms underlying this action are not elucidated.

ptosis, at least in part, due to STAT3 dephosphorylation [46].

**FAP-1 (PTPN13)**—Downregulates Src-ERK pathway by inhibiting EphrinB1

migration [46].

*Tumor Progression and Metastasis*

to the liver [107].

STAT3 [108, 109].

prognosis [46].

**6. Conclusions**

**60**

Our research on this field has been supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant 2015/20412-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 303900/ 2017-2).

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Appendices and nomenclature**



**Author details**

**63**

Carmen V. Ferreira-Halder\*, Stefano Piatto Clerici, Alessandra V. Sousa Faria, Patrícia Fernandes de Souza Oliveira, Helon Guimarães Cordeiro and Erica Akagi Laboratory of Oncobiomarkers, Department of Biochemistry and Tissue Biology,

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection?*

*DOI: http://dx.doi.org/10.5772/intechopen.87963*

© 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,

Biology Institute, University of Campinas, São Paulo, Brazil

\*Address all correspondence to: carmenv@unicamp.br

provided the original work is properly cited.

*Protein Tyrosine Phosphatases in Tumor Progression and Metastasis: Promoter or Protection? DOI: http://dx.doi.org/10.5772/intechopen.87963*

### **Author details**

MEF mouse embryonic fibroblast MMP-1 matrix metalloproteinase-1 mTOR mammalian target of rapamycin

*Tumor Progression and Metastasis*

cells PI3K phosphatidyl inositol-3-kinase PKM2 pyruvate kinase isozymes M2

PTKs protein tyrosine kinases

PTPN13 PTP also referred to as FAP1

PTPs protein tyrosine phosphatases

Raf serine/threonine-specific *protein* kinase Ras class of protein called small GTPase Rho Ras homologue of small *GTPase*

RhoA Ras homologue of small *GTPase* member A SHIP1 Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 SHIP2 Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 2

SHP1 Src homology 2 (SH2) domain-containing

SHP2 Src homology 2 (SH2) domain-containing

Slug *SNAI2,* a zinc finger transcription factor Src proto-oncogene tyrosine-protein *kinase*

TCA tricarboxylic acid cycle

Twist *Twist*-related *protein*

**62**

TGFbeta transforming growth factor beta

WDR1 WD-repeat-containing protein 1 ZEB 1/2 zinc finger E-box-binding homeobox ½

STAT3 signal transducer and activator of transcription type 3

as PTP4A3 PTEN phosphatase and tensin homologue

PTP σ protein tyrosine phosphatase sigma PTP μ protein tyrosine phosphatase

threonine rich)

NFKB nuclear factor kappa-light-chain-enhancer of activated B

PRL-3 phosphatase of regenerating the liver-3, also recognized

PTP-PEST (PTPN12) PTP-PEST (PTP—proline, glutamic acid, serine, and

PTP1B tyrosine-protein phosphatase non-receptor type 1 *PTPN3* protein tyrosine phosphatase non-receptor type 3 PTPN11 protein tyrosine phosphatase non-receptor type 11

PTPN9 tyrosine-protein phosphatase non-receptor type 9 PTPRδ protein tyrosine phosphatase receptor delta PTPRF protein tyrosine phosphatase receptor type F PTPRH receptor-type protein tyrosine phosphatase H, also

phosphatase-1 (SAP-1) PTPRO protein tyrosine phosphatase receptor type O PTPρ protein tyrosine phosphatase receptor T

referred to as stomach cancer-associated protein tyrosine

phosphotyrosine phosphatase, also known as PTPN6

phosphotyrosine phosphatase 2, also known as PTPN11

Carmen V. Ferreira-Halder\*, Stefano Piatto Clerici, Alessandra V. Sousa Faria, Patrícia Fernandes de Souza Oliveira, Helon Guimarães Cordeiro and Erica Akagi Laboratory of Oncobiomarkers, Department of Biochemistry and Tissue Biology, Biology Institute, University of Campinas, São Paulo, Brazil

\*Address all correspondence to: carmenv@unicamp.br

© 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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[107] Lin SC, Hsiao KY, Chang N, Hou PC, Tsai SJ. Loss of dual-specificity phosphatase-2 promotes angiogenesis and metastasis via up-regulation of

interleukin-8 in colon cancer. The Journal of Pathology. 2017;**241**(5): 638-648. DOI: 10.1002/path.4868

[108] Zhao Y, Zhang X, Guda K, et al. Identification and functional characterization of paxillin as a target of protein tyrosine phophatase receptor T. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**(6):2592-2597. DOI: 10.1073/pnas.0914884107

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**73**

Section 2

Tumorigenesis Risk

Factors and Role of

Neuroimmune Regulations

Section 2
