**2. Role of cell adhesion molecules (CAMs) in proliferation of oral cancer**

CAMs are typically single-pass transmembrane receptors [17] compounded of three stored domains: an intracellular domain that interacts with a transmembrane domain, an extracellular domain, and the cytoskeleton. These domains can interact in several different ways [18]. The first way is through homophilic binding, where the same CAMs bind to each other. They are also capable of heterophilic binding, which means a CAM on one cell will bind to different CAMs on another cell. The third type of binding is that between cells and substrate, where a mutual extracellular ligand binds two different CAMs. There are four major superfamilies, or groups, of CAMs: the immunoglobulin super family of cell adhesion molecules (IgCAMs), the cadherins, the integrins, and the c-type lectin-like domain proteins (CTLDs). Proteoglycans are also believed to be a class of CAMs. One classification system involves distinction between calcium-dependent and calcium-independent CAMs [19]. The Ig-superfamily CAMs and integrins are not dependent on calcium ions, whereas selectins and cadherins are calcium-dependent. In addition, integrins participate in cell-matrix interactions, while other CAM families play some important roles in cell-to-cell interactions [20].

#### **2.1. Neural cell adhesion molecule (NCAM)**

With the rapid global population increase and aging, the rising significance of cancer as a leading cause of death is partly correlated with a marked decline in mortality rates due to stroke and coronary heart disease in many countries. In addition, tobacco and alcohol consumption are known to be etiologically associated with carcinogenesis. Cancer is both a genetic and progressively systemic disease. In all types of cancer, somatic cells begin to divide uncontrollably and spread into surrounding tissues. Cancer cells can arise almost anywhere in the human body. Normal cells grow and divide according to the body's needs, and when they age or become damaged, they die and are replaced by new cells. However, when cancer arises, this orderly process breaks down: as cellular abnormalities increase, old or damaged cells survive rather than being removed, and new cells form when they are not needed. These extra cells can divide uncontrollably and may form malignant tumors. Although many cancers form solid tumors composed of masses of tissue, cancers of the blood, such as leukemias, generally do not form solid tumors [2–4]. Head and neck cancer is a common neoplasm that encompasses epithelial malignant tumors of the nasopharynx, larynx, and mouth, representing about 6% of all cases and accounting for an estimated over 650,000 new cancer cases and over 350,000 cancer-related deaths worldwide every year [5, 6]. Oral cancer is the most notably frequent cancer type in the head and neck region, squamous cell carcinoma being the most common single entity. Oral cancers comprise two categories: those affecting the oral cavity (lips, inner lips, cheeks, teeth, gums, the anterior two-thirds of the tongue, the floor, and palate) and those affecting the oropharynx (middle region of the throat, including the tonsils and base of the tongue). Such cancers may arise in any location, although there are certain areas that are affected more frequently, such as the tongue and gingiva. These areas represent about 90% of all malignancies of the oral cavity [7, 8]. However, despite significant advances in surgery and chemotherapy over the last few decades [9], oral cancer is still characterized by a poor prognosis and a low

84 Prevention, Detection and Management of Oral Cancer

survival rate [10]. The 5-year survival rate of those diagnosed is ~ 60%.

lance [14–16].

on its diagnosis and treatments.

In patients diagnosed with oral cancers at an advanced stage, there is a high incidence of metastasis to surrounding tissues, lymph nodes, and distant organs [5]. Metastasis is recognized as a process, whereby genetic instability in the primary tumor accelerates cell heterogeneity, allowing a few metastatic clones to eventually emerge and be positively selected to disseminate cancer at a distance [11]. This is the most annihilating stage of malignancy and the leading cause of cancer-related death. In metastasis, cancer cells break away from the primary cancer, travel through the blood or lymph system, and form new metastatic tumors elsewhere in the body. Each metastatic tumor is the same type of cancer as the primary tumor [12, 13], and the cells of each resemble each other upon microscopic observation. Moreover, they usually share common molecular features, such as the presence of specific genetic changes. It is clear that only a minority of malignant cells participate in the process of metastasis, due to interaction with host tissues and the intrinsic characteristics of the cancer cells themselves; thus, metastasis may imply an escape of these cells from the hostile environment they have created, characterized by features such as hypoxia, inflammation, and immunological surveil-

This chapter details recent findings on molecular markers that are involved in the mechanisms of proliferation and energy metabolism of oral cancer and provides new perspectives Neural cell adhesion molecule (NCAM) is a family of cell surface glycoproteins playing an important role in the development of the nervous system, fasciculation, axonal outgrowth, regulation of cell migration, and branching [21]. NCAM has several isoforms derived from alternative splicing of a single gene [22–24]. In particular, the three major isoforms with molecular weights of 120, 140, and 180 kDa have similar extracellular parts but differ in the disposition of their domains, which are cytoplasmic for the two larger polypeptides [25, 26]. Furthermore, the expression of NCAM is upregulated by transforming growth factor (TGF)-β1 [27–29]. Although NCAM was initially considered to exist only in neural tissue, it has since been observed in the human kidney, lung, fetal muscle, and colon, as well as in elements of the hemopoietic system. Furthermore, it has been described that NCAM is expressed by a variety of human tumors and associated with perineural invasion by various neoplasms, such as gallbladder cancer, melanoma, bile duct cancer, and adenoid cystic carcinoma of the head and neck [30–35]. We have also demonstrated previously that NCAM is sporadically found in the adenoid cystic carcinoma, derived from human submandibular salivary gland, *in vivo* [36]. NCAM is believed to mediate adhesion between cells through a calcium ion-independent homophilic (NCAM-NCAM) binding mechanism and to mediate adhesion between neurons and the extracellular matrix through heterophilic binding (NCAM to another ligand or counter-receptor) [21]. It has been described that exogenously added NCAM can inhibit the proliferation of cultured neonatal astrocytes and of astrocytes responding to a penetrating lesion in the adult rat brain, *in vivo* [37, 38], suggesting that these effects are mediated by homophilic binding to NCAM on the astrocyte membrane.

Adenoid cystic carcinoma (ACC) is a well-known and typical malignant salivary gland tumor. ACCs are biologically aggressive and can bring metastases even when many years have passed after excision of the primary tumor. Facial paralysis is especially frequent, causing perineural and/or neural invasion. We have attempted to examine the role of NCAM by investigating the effect of anti-NCAM antibody (MAb NCAM) and TGF-β1 in human salivary gland tumor cells. The expression and distribution of NCAM were also investigated in ACC tissues. We further found that apoptotic cell death was induced via a DNA damage signal through the mitochondria, inducing release of cytochrome *c* into the cytoplasm of salivary gland tumor cells [36]. However, MAb NCAM had no effect on human oral squamous cell carcinoma (HOSCC) cell lines, which do not express NCAM.As shown in **Figure 1**, these results indicate that the effect of MAb NCAM is specific to NCAM-expressing tumor cells, such as human salivary gland tumor cells; furthermore, blocking the ability of NCAM through MAb NCAM, as well as the homophilic (NCAM-NCAM) binding mechanism, rather than regulating a signaling pathway of cell proliferation, may in fact induce a negative signal such as apoptosis in human salivary gland tumor cells. In addition, homophilic (NCAM-NCAM) binding may activate multiple signaling pathways that differ among cell types. In view of the fact that NCAM expression on human salivary gland tumor cells is upregulated by TGF-β1, it can be hypothesized that a further homophilic (NCAM-NCAM) binding mechanism may be activated and that consequently the proliferative activity of HSG cells may also be upregulated by TGF-β1-mediated NCAM activity (**Figure 2**).

On the other hand, cimetidine, the most studied histamine type-2 receptor (H2R) antagonist used clinically, is commonly prescribed to treat gastroesophageal reflux disease as well as

gastric and duodenal ulcers [39]. Cimetidine has recently been shown to possess antitumor activity against gastric, kidney, and colon cancers, as well as melanomas [40–43]. A recent study has suggested that this behavior of cimetidine is mediated through three different effects: a direct inhibitory effect on tumor growth by blocking the cell growth activity of histamine via activation of H2 receptors and an indirect effect involving inhibition of tumorassociated angiogenesis, an immunomodulatory effect through augmentation of the host's immune response to tumor cells, and an inhibitory effect on cancer cell migration and adhesion to endothelial cells, thus inhibiting tumor angiogenesis and metastasis [44]. We have also examined the NCAM-associated impact of cimetidine on tumor growth and perineural/ neural invasion in salivary gland tumors using an in vitro cell culture system and an *in vivo* nude mouse cancer model. These experiments clearly indicated that cimetidine effectively downregulated the expression of NCAM by inhibiting NF-κB transactivation, subsequently blocking salivary gland tumor cell adhesion to neural cells, and ultimately inducing apoptosis in salivary gland tumor cells, thus preventing the growth of salivary gland tumor masses in nude mice [45]. Although malignant glandular tumors are commonly known to be resistant to chemotherapy and/or radiation, the clinical application of cimetidine as an anticancer drug might provide an integral part of future therapeutic strategies against NCAM-expressing

**Figure 2.** Schematic representation of TGF-β1-induced upregulation of NCAM expression and proliferative activity

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involved in homophilic (NCAM-NCAM) binding mechanism in NCAM-expressing cells.

tumors such as adenoid cystic carcinoma.

**Figure 1.** Schematic representation of MAb NCAM-induced apoptotic signal transduction pathways via the DNA damage signal through the mitochondria involved in apaf-1 and caspase activation in NCAM-expressing cells.

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after excision of the primary tumor. Facial paralysis is especially frequent, causing perineural and/or neural invasion. We have attempted to examine the role of NCAM by investigating the effect of anti-NCAM antibody (MAb NCAM) and TGF-β1 in human salivary gland tumor cells. The expression and distribution of NCAM were also investigated in ACC tissues. We further found that apoptotic cell death was induced via a DNA damage signal through the mitochondria, inducing release of cytochrome *c* into the cytoplasm of salivary gland tumor cells [36]. However, MAb NCAM had no effect on human oral squamous cell carcinoma (HOSCC) cell lines, which do not express NCAM.As shown in **Figure 1**, these results indicate that the effect of MAb NCAM is specific to NCAM-expressing tumor cells, such as human salivary gland tumor cells; furthermore, blocking the ability of NCAM through MAb NCAM, as well as the homophilic (NCAM-NCAM) binding mechanism, rather than regulating a signaling pathway of cell proliferation, may in fact induce a negative signal such as apoptosis in human salivary gland tumor cells. In addition, homophilic (NCAM-NCAM) binding may activate multiple signaling pathways that differ among cell types. In view of the fact that NCAM expression on human salivary gland tumor cells is upregulated by TGF-β1, it can be hypothesized that a further homophilic (NCAM-NCAM) binding mechanism may be activated and that consequently the proliferative activity of

86 Prevention, Detection and Management of Oral Cancer

HSG cells may also be upregulated by TGF-β1-mediated NCAM activity (**Figure 2**).

On the other hand, cimetidine, the most studied histamine type-2 receptor (H2R) antagonist used clinically, is commonly prescribed to treat gastroesophageal reflux disease as well as

**Figure 1.** Schematic representation of MAb NCAM-induced apoptotic signal transduction pathways via the DNA damage signal through the mitochondria involved in apaf-1 and caspase activation in NCAM-expressing cells.

**Figure 2.** Schematic representation of TGF-β1-induced upregulation of NCAM expression and proliferative activity involved in homophilic (NCAM-NCAM) binding mechanism in NCAM-expressing cells.

gastric and duodenal ulcers [39]. Cimetidine has recently been shown to possess antitumor activity against gastric, kidney, and colon cancers, as well as melanomas [40–43]. A recent study has suggested that this behavior of cimetidine is mediated through three different effects: a direct inhibitory effect on tumor growth by blocking the cell growth activity of histamine via activation of H2 receptors and an indirect effect involving inhibition of tumorassociated angiogenesis, an immunomodulatory effect through augmentation of the host's immune response to tumor cells, and an inhibitory effect on cancer cell migration and adhesion to endothelial cells, thus inhibiting tumor angiogenesis and metastasis [44]. We have also examined the NCAM-associated impact of cimetidine on tumor growth and perineural/ neural invasion in salivary gland tumors using an in vitro cell culture system and an *in vivo* nude mouse cancer model. These experiments clearly indicated that cimetidine effectively downregulated the expression of NCAM by inhibiting NF-κB transactivation, subsequently blocking salivary gland tumor cell adhesion to neural cells, and ultimately inducing apoptosis in salivary gland tumor cells, thus preventing the growth of salivary gland tumor masses in nude mice [45]. Although malignant glandular tumors are commonly known to be resistant to chemotherapy and/or radiation, the clinical application of cimetidine as an anticancer drug might provide an integral part of future therapeutic strategies against NCAM-expressing tumors such as adenoid cystic carcinoma.

Finally, it was suggested that NCAM might be associated with not only a cell-to-cell adhesion mechanism but also tumorigenesis, including the occurrence, development, and perineural/ neural invasion of human salivary gland tumors.

Further studies will be required to identify the signal transduction pathways by which treatment with cimetidine suppresses the growth of salivary gland tumors and to establish a strategy for cimetidine-based therapy for those tumors.

### **2.2. Coxsackievirus and adenovirus receptor (CAR/CXADR)**

Coxsackievirus and adenovirus receptor (CAR/CXADR), a transmembrane glycoprotein, was initially characterized as a viral attachment site on the surface of epithelial cells (**Figure 3**) [46]. Later it was identified as a component of the tight junction (TJ) complex, an interacting partner for a number of other TJ proteins and a regulator of TJ formation [47–52]. Furthermore, CAR is known to be a cell-cell adhesion molecule [53, 54]. In terms of function, loss of CAR has been considered to diminish intercellular adhesion, increase proliferation, and promote the migration as well as invasion of cancer cells [55, 56]. On the basis of these observations, a tumor-suppressive role of CAR in human cancers has speculated. Although it has recently been described [55–58] that CAR is observed in various organs, it is still unclear whether it is expressed in oral cancer. Therefore, we examined the role of CAR in SCC in the oral cavity (data not shown). This revealed that CAR was constitutively expressed in five oral SCC cell lines. To analyze the function of CAR, we then examined the proliferative activity of SAS cells

after *CAR* gene knockdown. However, *CAR* knockdown did not promote the proliferative activity of SAS cells. Although the expression level of CAR was decreased by *CAR* knockdown, that of NF-κB p65 (RelA) showed little change. Furthermore, SAS cell numbers were notably reduced by CAR overexpression. Finally, it was suggested that the overexpression of CAR in SAS cells led to apoptosis via activation of caspase-9. In addition, the localizations of CAR and RelA in 40 samples of HOSCC at various stages were investigated using immunohistochemistry. A positive reaction for polyclonal antibody (PAb) CAR was weakly observed on the membrane of carcinoma cells in 19 of 40 cases (47.5%) of HOSCC. The immunoreactivity for CAR further tended to fade at the invasive front of oral SCC (**Figure 4**). In the meantime, RelA immunoreactivity was strongly positive, particularly on the nucleus of carcinoma cells at the invasive front, in 30 of 40 cases (75%) of HOSCC. These observations suggest that CAR

**Figure 4.** (A) The positive reaction for PAb CAR was observed on the membrane of tumor cells in 19 of 40 cases (47.5%) of SCCs. (B) NF-κB immunoreactivity were clearly detected in 30 of 40 cases (100%) in the HOSCC tissues. The immunoreactivity for CAR especially tended to fade away in the invasive front of oral SCC tissues. NF-kappaB (+)/CAR

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Cytokines are composed of a large family of secreted proteins that bind to and signal through defined cell surface receptors on a wide variety of target cells, playing an important role in

plays a significant role in the inhibition of oral cancer cell growth.

(−), 15/21 cases (71.4%).

**3. Role of cytokines in oral cancer cell proliferation**

**Figure 3.** Schematic representation of coxsackievirus and adenovirus receptor on the tumor cells.

**Figure 4.** (A) The positive reaction for PAb CAR was observed on the membrane of tumor cells in 19 of 40 cases (47.5%) of SCCs. (B) NF-κB immunoreactivity were clearly detected in 30 of 40 cases (100%) in the HOSCC tissues. The immunoreactivity for CAR especially tended to fade away in the invasive front of oral SCC tissues. NF-kappaB (+)/CAR (−), 15/21 cases (71.4%).

after *CAR* gene knockdown. However, *CAR* knockdown did not promote the proliferative activity of SAS cells. Although the expression level of CAR was decreased by *CAR* knockdown, that of NF-κB p65 (RelA) showed little change. Furthermore, SAS cell numbers were notably reduced by CAR overexpression. Finally, it was suggested that the overexpression of CAR in SAS cells led to apoptosis via activation of caspase-9. In addition, the localizations of CAR and RelA in 40 samples of HOSCC at various stages were investigated using immunohistochemistry. A positive reaction for polyclonal antibody (PAb) CAR was weakly observed on the membrane of carcinoma cells in 19 of 40 cases (47.5%) of HOSCC. The immunoreactivity for CAR further tended to fade at the invasive front of oral SCC (**Figure 4**). In the meantime, RelA immunoreactivity was strongly positive, particularly on the nucleus of carcinoma cells at the invasive front, in 30 of 40 cases (75%) of HOSCC. These observations suggest that CAR plays a significant role in the inhibition of oral cancer cell growth.
