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Molecular Pathogenesis of Oral Squamous Cell Carcinoma: Implications for Therapy

Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 44, Houston, TX 77030-4009, USA

Correspondence: ^* corresponding author, jmyers{at}mdanderson.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION MOLECULAR PATHOGENESIS OF OSCC TARGETED MOLECULAR THERAPY CONCLUSION REFERENCES

 
The development of oral squamous cell carcinoma (OSCC) is a multistep process requiring the accumulation of multiple genetic alterations, influenced by a patient’s genetic predisposition as well as by environmental influences, including tobacco, alcohol, chronic inflammation, and viral infection. Tumorigenic genetic alterations consist of two major types: tumor suppressor genes, which promote tumor development when inactivated; and oncogenes, which promote tumor development when activated. Tumor suppressor genes can be inactivated through genetic events such as mutation, loss of heterozygosity, or deletion, or by epigenetic modifications such as DNA methylation or chromatin remodeling. Oncogenes can be activated through overexpression due to gene amplification, increased transcription, or changes in structure due to mutations that lead to increased transforming activity. This review focuses on the molecular mechanisms of oral carcinogenesis and the use of biologic therapy to specifically target molecules altered in OSCC. The rapid progress that has been made in our understanding of the molecular alterations contributing to the development of OSCC is leading to improvements in the early diagnosis of tumors and the refinement of biologic treatments individualized to the specific characteristics of a patient’s tumor.

Key Words: oral squamous cell carcinoma • multistep carcinogenesis • oncogene • tumor suppressor gene • molecular targeted therapy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MOLECULAR PATHOGENESIS OF OSCC TARGETED MOLECULAR THERAPY CONCLUSION REFERENCES

 
Oral squamous cell carcinoma (OSCC) is a significant public health problem in the United States; each year, 30,000 new cases are diagnosed, and an estimated 11,000 deaths are attributed to the disease (Jemal et al., 2006). Worldwide, OSCC is the sixth most common cancer; more than 300,000 new cases are diagnosed each year (Parkin et al., 1988).

Early-to-moderate-stage OSCC (American Joint Committee on Cancer stages I-III) (Greene et al., 2002) is most often treated surgically, with radiotherapy given with or without chemotherapy in the post-operative adjuvant setting for high-risk patients, those who have multiple pathologically positive lymph nodes and/or lymph nodes with metastases extending beyond the lymph node capsule (i.e., extracapsular spread) (Forastiere et al., 2001). In advanced (stage IV) disease, multidisciplinary non-surgical approaches are being used with increasing frequency to improve disease control, prolong survival, and maintain an acceptable quality of life for patients (Haraf et al., 2003; Bernier et al., 2004; Cooper et al., 2004). While these treatment approaches have been used more frequently in the laryngopharynx, the short- and long-term toxic effects of combination therapy to the oral cavity are significant. Even when the best combination of surgical and non-surgical approaches is used, more than 50% of patients with OSCC will experience relapse, whether locally, in regional lymph nodes, or at a distant site. Furthermore, recurrent and distantly metastastic OSCC carry particularly poor prognoses (Khuri et al., 2000b; Forastiere et al., 2001). Regardless of the location or stage of OSCC, however, more effective therapies are needed.

One promising strategy for the treatment of OSCC and other cancers, which has developed as a result of breakthroughs in the fields of molecular biology, cancer genetics, and cancer biology, is molecular targeted therapy. In this approach, specific molecular alterations of a cancer cell that contribute to the neoplastic phenotype are exploited as targets of antibodies, small molecules, and/or genetic constructs. This approach differs from conventional cytotoxic therapies, which inhibit the mitotic activity of all dividing cells, leading to toxic effects in all tissues that normally have rapid cell turnover, such as the oral mucosa, hair follicles, and intestinal lining (Stephenson, 2001). Targeted therapy is thought to offer a higher therapeutic index and therefore to be associated with less toxicity than cytotoxic drugs. The development of targeted approaches to OSCC requires understanding of the molecular pathogenesis of the disease, as well as further characterization of the specific molecular events involved in growth, invasion, and metastasis of this cancer. This review focuses on the molecular mechanisms of oral carcinogenesis and progression and the current status of targeted therapies directed toward critical molecular alterations in OSCC.

	MOLECULAR PATHOGENESIS OF OSCC

TOP ABSTRACT INTRODUCTION MOLECULAR PATHOGENESIS OF OSCC TARGETED MOLECULAR THERAPY CONCLUSION REFERENCES

 
OSCC arises as a result of multiple molecular events that develop from the combined influences of an individual’s genetic predisposition and exposure to environmental carcinogens (Califano et al., 1996). Chronic exposure to carcinogens such as tobacco, alcohol, oncogenic viruses, and inflammation can damage individual genes as well as larger portions of the genetic material, including chromosomes. Accumulation of such genetic alterations can lead to the development of premalignant lesions and subsequent invasive carcinoma. These genetic alterations include activating mutations or amplification of oncogenes that promote cell survival and proliferation, as well as inactivation of tumor suppressor genes involved in the inhibition of cell proliferation. From these alterations of oncogenes and tumor suppressor genes, tumor cells acquire autonomous self-sufficient growth and evade growth-inhibitory signals, resulting in uncontrolled tumor growth. Tumor cells thereby escape programmed cell death and replicate infinitely through the immortalization process by telomere lengthening. As OSCCs grow, invade, and metastasize, new blood vessel formation is critical. OSCCs, like most tumors, are able to create a blood supply by stimulating endothelial cell proliferation and new blood vessel formation. During oral carcinogenesis, there is selective disruption of this process, such that pro-angiogenic factors predominate (Hanahan and Weinberg, 2000). This angiogenesis is an essential part of solid tumor formation. The subsequent progression of OSCC includes tissue invasion and metastasis. Invasion of adjacent normal tissue requires that cellular adhesion molecules, such as integrin and cadherins, are lost, to allow cancer cells to leave their primary site. OSCCs develop through a complex process, as mentioned above. Here, we discuss those process involving genetic alteration during multistep carcinogenesis, growth regulation, apoptosis, immortalization, angiogeneis, invasion, and metastasis.

Genetic Alterations during Development of OSCC
Califano, Sidransky, and colleagues have developed a genetic progression model based on their studies of gene alterations in squamous cell carcinomas of the head and neck (SCCHN) (Fig. 1) (Sidransky, 1995; Califano et al., 1996). They found that the most common genetic alteration in SCCHN is loss of chromosomal region 9p21, which occurs in 70–80% of dysplastic lesions of the oral mucosa, suggesting that this loss is an early event in oral carcinogenesis (van der Riet et al., 1994; Califano et al., 1996; Mao et al., 1996a). This region of chromosome 9p21, known as the CDKN2A locus, encodes the tumor suppressors p16 and p14^ARF, which frequently are inactivated by promoter hypermethylation (Reed et al., 1996). Loss of the chromosome 3p region is another common early genetic alteration in oral carcinogenesis (Garnis et al., 2003; Masayesva et al., 2004). The chromosome 3p region includes FHIT (fragile histidine triad gene) and RSSFIA, tumor suppressor genes inactivated by exonic deletion and hypermethylation (Mao et al., 1996a; Kisielewski et al., 1998; Dong et al., 2003). Loss of heterozygosity (LOH) of chromosome region 17p and mutation of the p53 gene are genetic alterations that occur in the later stage of progression from dysplasia to invasive squamous carcinoma. Alterations of p53, including mutation or deletion, are associated with increased genomic instability in oral dysplasia and may accelerate the rate of genetic alterations in oral carcinogenesis. Amplification of 11q13 and overexpression of cyclin D1 have been described in 40% of cases of oral squamous dysplasia (Rousseau et al., 2001). In general, loss of chromosomal material at 9p, 3p, and 17p is observed in relatively high proportions of dysplastic lesions, indicating that those events are early markers of oral carcinogenesis, whereas losses at 13q and 8p are observed more frequently in carcinomas than in dysplasia and are associated with later stages of carcinogenesis (Califano et al., 1996).

View larger version (16K): [in this window] [in a new window]   Figure 1. Genetic progression model of multistep oral carcinogenesis. Transformation of normal epithelium by multiple genetic alterations leads to dyplasia and invasive carcinoma. The acculumated genetic changes that occur in oral carcinogenesis include activation of the epidermal growth factor receptor (EGFR), alterations of tumor suppressors p53 and p16, and cyclin D1 overexpression. Adapted from Lippman et al.(2005).

To reconcile this finding, Braakhuis et al. proposed a progression model, in which a stem cell located in the basal cell layer of the epithelium acquires a genetic alteration and subsequently gives rise to a clonal unit, consisting of the stem cell with its daughter cells, all of which share the DNA alteration. Next, this patch of cells progresses into an expanding field as a result of additional genetic alterations (Braakhuis et al., 2004). This mucosal field pushes the normal epithelium aside and can expand to a size of several centimeters. These fields are often macroscopically undetectable, but they can also appear as oral lesions such as leukoplakia or erythroplakia. Ultimately, clonal selection leads to the development of carcinoma within this field of preneoplastic cells. The mechanism of this clonal expansion may be intra-epithelial migration of transformed cells or inoculation through saliva.

From these data, it has become clearer that the multifocality of oral carcinogenesis is an important cause of treatment failure in oral cancer. Although primary excision can completely remove an oral carcinoma, the altered field may remain, and the patient can develop a second primary tumor nearby, in the same field, that may be clinically indistinguishable from a local recurrence. Understanding of this field cancerization concept led Hong, Lippman, and other investigators to develop a strategy called ’chemoprevention’, in which systemic therapy is administered with the intent of preventing epithelium from the entire upper aerodigestive tract from progressing along the multistep pathway of carcinogenesis (Lippman et al., 2005).

Proto-oncogenes and Oncogenes
Oncogenes are genes derived through alteration of cellular proto-oncogenes, which encode proteins that mediate positive cell growth-regulatory and/or cell survival signals. When the proto-oncogene is altered and abnormally activated to become an oncogene, it can promote uncontrolled cell proliferation, leading to tumorigenesis. Some of the common mechanisms of oncogene activation include mutation, chromosomal translocation, gene amplification, and retroviral insertion (Klein and Klein, 1985; Alitalo and Schwab, 1986; Haluska et al., 1987). Oncogenes are broadly categorized into functional groups as follows: (i) growth factors or growth factor receptors (hst-1, int-2, EGFR/erbB, c-erbB-2/Her-2, sis); (ii) intracellular signal transducers (ras, raf, stat-3); (iii) transcription factors (myc, fos, jun, c-myc); (iv) cell-cycle regulators (cyclin D1); and (v) those involved in the inhibition of apoptosis (bcl-2, bax) (Sidransky, 1995). Most of these oncogenes promote aberrant cell proliferation by overriding the G/S, G/M, and M checkpoints of the cell cycle (Field, 1995). The role of these oncogenes in oral cancer development is described below.

Tumor Suppressor Genes
Tumor suppressor genes encode proteins that typically transduce negative growth-regulatory signals (Weinberg, 1991). These genes are often involved in cell-cycle regulation, including cell-cycle arrest and apoptosis. Unlike oncogenes, which can be activated by mutation of only one of the two gene copies, tumor suppressor genes are inactivated by any of several mechanisms, including point mutations and/or deletion, in both alleles of the gene, in a "two hit" fashion (Knudson, 1977; Vogelstein and Kinzler, 1993; Yokota and Sugimura, 1993). Once these genes are inactivated, the cell escapes tight cell-cycle control, predisposing it to uncontrolled growth and division, which contributes to the malignant phenotype (Levine, 1997).

Hallmarks of Cancer
Hanahan and Weinberg (2000) described 6 essential hallmarks of cancer cells that distinguish them from their normal counterparts: (i) self-sufficiency in growth signals, (ii) insensitivity to growth-inhibitory signals, (iii) evasion of programmed cell death, (iv) immortality or unlimited replicative potential, (v) sustained angiogenesis, and (vi) tissue invasion and metastasis. This conceptual framework has been very helpful for organizing the multiple genetic alterations that occur in tumorigenesis and their progression into the functional steps contributing to the malignant phenotype; this is discussed below in the context of OSCC development and progression.

Acquisition of Self-sufficient Growth-stimulatory Signaling
Normal cells require exogenous growth signals to stimulate proliferation. Growth stimuli include soluble and membrane-bound growth factors, interactions with the extracellular matrix, and cytokines (Hanahan and Weinberg, 2000). Typically, these growth signals are transduced from cell-surface receptors that subsequently activate multiple intracellular signaling pathways, resulting in cell proliferation. During oral carcinogenesis, growth signaling can become dysregulated through increases in the level of growth factor receptors and/or their ligands, to promote autocrine stimulation without exogenous factors (Todd et al., 1991). Increased expression of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGF-), can play a critical role in oral tumor development and progression (Grandis and Tweardy, 1993). Several intracellular growth signal-transducing proteins that are downstream mediators of growth factor signaling are altered frequently in OSCC and many other cancers; these are discussed in greater detail below (Cantley et al., 1991).

Epidermal Growth Factor Signaling
EGFR is a member of a membrane-bound receptor tyrosine kinase family, which is composed of 4 receptors: erbB1, erbB2, erbB3, and erbB4 (Hynes and Lane, 2005). Each family member is a single polypeptide with an extracellular ligand-binding domain, a transmembrane region that anchors the receptor within the plasma membrane, and a cytoplasmic region containing a tyrosine kinase domain. The known natural ligands of EGFR include EGF and TGF- (Tzahar et al., 1996). After binding one of its ligands, EGFR forms a dimer with another EGFR molecule, and these receptors autophosphorylate, leading to a cascade of intracellular signaling events, including activation of the Ras/Raf/mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR), Janus kinase (Jak), signal transducer and activator of transcription (STAT), and protein kinase C (PKC) pathways (Rogers et al., 2005; Kalyankrishna and Grandis, 2006). These signaling pathways, in turn, mediate multiple functions, including cell proliferation and survival, invasion, metastasis, and angiogenesis (Reuter et al., 2007).

Increased EGFR signaling activity can occur through any of several mechanisms, including receptor overexpression due to gene amplification or transcriptional up-regulation, receptor mutation, or autocrine activation by overproduction of ligands (Rogers et al., 2005; Kalyankrishna and Grandis, 2006). The most common mutation of EGFR is a truncation mutation, EGFR variant III (EGFRvIII), which is observed only in cancer cells and leads to constitutive activation of the receptor without ligand or receptor overproduction. The expression of EGFRvIII has been detected in glioma and non-small-cell lung carcinoma ( Moscatello et al., 1995; Ji et al., 2006). In SCCHN, it has been detected in 42% of 33 tumors (Sok et al., 2006). Furthermore, these mutants showed resistance to chemotherapy and targeting of EGFR. Specific blockade of EGFRvIII may increase the efficacy of EGFR targeted therapy.

Expression of EGFR and other erbB receptors can be dysregulated in many cancers, including carcinomas of the esophagus, lung, breast, colon, rectum, bladder, prostate, and ovary (Roskoski, 2004). EGFR is overexpressed in 80–100% of SCCHN (Kalyankrishna and Grandis, 2006; Reuter et al., 2007), and overexpression of EGFR increases progressively from oral premalignant lesions to invasive OSCC (Shin et al., 1994). Overexpression of the EGFR ligands is also observed frequently. Overexpression of the erbB family member erbB2 occurs occasionally in OSCC, and this finding is associated with poor prognosis (Xia et al., 1997; Werkmeister et al., 2000). Furthermore, several studies have shown that EGFR overexpression is an independent prognostic marker that correlates with increased tumor size, decreased radiation sensitivity, and increased risk of recurrence (Grandis et al., 1998a; Ang et al., 2002; Gupta et al 2002; Chen et al., 2003; Shiraki et al., 2005).

The frequently seen increase in EGFR activity in OSCC and the association of this finding with poor treatment outcomes led investigators to explore EGFR as a potential target for an OSCC-specific therapeutic strategy. The extensive and exciting progress that has been made in this area is discussed later in this article.

Hepatocyte Growth Factor/c-MET
c-MET is another receptor tyrosine kinase that also has been shown to be overexpressed or mutated in a variety of human malignancies. Stimulation of c-MET via its ligand, hepatocyte growth factor/scatter factor (HGF/SF), leads to numerous biochemical and biological effects, including increased cell motility or scattering, angiogenesis, proliferation, invasion, and metastasis (Ma et al., 2003). HGF and its receptor c-Met have been found to play an important role in invasion and metastasis of OSCC through a paracrine pathway that leads to up-regulation of the expression of matrix metalloproteinases MMP-1 and MMP-9 (Hasina et al., 1999; Hanzawa et al., 2000; Uchida et al., 2001). Moreover, c-Met overexpression has been found to be independently associated with a decreased survival rate (Lo Muzio et al., 2006). While overexpression of c-MET has been identified in OSCC specimens, the activating mutations of the c-MET kinase domain that have been described in other cancers have not been observed in oral cavity cancers (Morello et al., 2001).

Ras Oncogene
Ras is one of the first proto-oncogenes found to be involved in cell growth regulation and the transduction of mitogenic cell signaling from the cell surface to the nucleus. The ras gene encodes protein p21, a guanosine triphosphatase that can be constitutively activated through mutation (Todd et al., 1997).

Members of the ras oncogene family (H-ras, K-ras, N-ras) are mutated frequently in many human cancers (Bos, 1989). In oral cancer, a high incidence of H-ras mutation has been found, mainly in Asian populations, where it has been associated with betel nut chewing (Saranath et al., 1991; Das et al., 2000). However, H-ras mutations are found much less often (less than 5%) in OSCC cases in the West (Chang et al., 1991; Clark et al., 1993), and the other ras genes are also infrequently mutated in OSCC (Anderson et al., 1994).

Cyclin D1
Cyclin D1 is a proto-oncogene encoding a positive regulator of G₁ phase progression through the cell cycle that regulates the initiation of DNA synthesis (Smith and Haffty, 1999). Cyclin D1 has been identified as the bcl-1 gene at chromosome 11q13, at the site of translocation t(11:14)(q13:q32) in B-cell malignancies (Motokura et al., 1991). Overexpression of cyclin D1 increases progression through the G₁ phase of the cell cycle in the absence of extracellular mitogen stimulation (Quelle et al., 1993). Overexpression of the cyclin D1 gene has been reported in 25–70% of oral cancers (Miyamoto et al., 2003) and a high percentage of premalignant lesions (Rousseau et al., 2001), suggesting that cyclin D1 gene amplification and consequent protein overexpression are early events during oral tumorigenesis. Moreover, cyclin D1 overexpression has been associated with more aggressive tumor behavior and a worse prognosis than tumors that do not overexpress cyclin D1 (Mineta et al., 2000a).

STAT Proteins
Members of the STAT family are latent cytoplasmic transcription factors activated by extracellular signaling proteins, such as growth factors, cytokines, hormones, and peptides (Darnell, 1997; Bromberg and Darnell, 2000). Activated STAT proteins deliver the signals by translocating into the nucleus and regulating transcription of target genes involved in normal cell functions, such as growth, apoptosis, and differentiation (Darnell, 1997; Bromberg and Darnell, 2000). There is sufficient evidence that STATs, especially STAT3 and STAT5, are involved in carcinogenesis. Activating mutations of STAT3 can lead to malignant transformation in fibroblasts (Bromberg et al., 1999). Furthermore, levels of activated STAT3 are elevated in many human cancers, including lymphomas and leukemias, breast cancer, and SCCHN (Garcia et al., 1997; Grandis et al., 1998b; Catlett-Falcone et al., 1999). Levels of activated STAT3 may be elevated in OSCC through up-regulation of signaling from EGFR, TGF-, Jak, Src, or interleukin (IL)-6 and its receptor (Bowman et al., 2000; Turkson and Jove, 2000). Constitutive activation of STAT3, in turn, can up-regulate transcription of target genes, including cell-cycle regulators, anti-apoptotic genes, and pro-angiogenic factors, resulting in uncontrolled cellular proliferation, anti-apoptotic response, and angiogenesis, all hallmarks of cancer (Leeman et al., 2006).

Studies have suggested that STATs play important roles in OSCC development and growth. Transfection of dominant-negative constructs or antisense targeting of STAT3 causes cell growth inhibition in vitro (Grandis et al., 1998b). Moreover, cells containing a dominant-active STAT3 mutant construct proliferate independently of EGFR ligand and EGFR activity, revealing that STAT3 activation can result in SCCHN growth without EGFR activity (Kijima et al., 2002). Both tumor and normal epithelia of SCCHN patients show higher levels of STAT3 expression and phosphorylated forms than in epithelium derived from individuals without cancer (Grandis et al., 2000), implicating STAT3 activation as an early step in oral carcinogenesis. Furthermore, activated STAT3 is highly expressed in poorly differentiated OSCC tumors, and is also correlated with lymph node metastasis and poor prognosis (Masuda et al., 2002). Analysis of these data suggests that STAT3 is another potential target for the treatment of OSCC.

Nuclear Factor-kappa B (NF-B)
Nuclear factor-kappa B (NF-B) is a ubiquitous nuclear transcription factor known to be involved in inflammatory and immune responses (Shishodia and Aggarwal, 2004; Nakanishi and Toi, 2005). The protein consists of a family of dimers formed by combinations of several proteins: NF-B, NF-B1 (also known as p50/p105), NF-B2 (also known as p52/p100), REL, RELA (also known as p65/NF-B3), and RELB (Nakanishi and Toi, 2005). In its inactive state, NF-B is present in the cytoplasm in a complex with an inhibitory subunit, IB ; in response to a stimulus such as a growth factor or cytokine, IB is phosphorylated, ubiquitinated, and degraded by the proteasome, resulting in NF-B being released from IB. This activated NF-B then translocates to the nucleus and regulates many target genes, including immunoregulatory and inflammatory genes, anti-apoptotic genes, and genes that positively regulate cell proliferation (Karin et al., 2002).

Recently, sufficient evidence has emerged that inappropriate NF-B activation can mediate oncogenesis and tumor progression. Previous studies demonstrated that suppression of NF-B in cancer inhibits proliferation, causes cell-cycle arrest, and leads to apoptosis, suggesting the important role of NF-B in cell proliferation and survival (Bharti and Aggarwal, 2002). Furthermore, NF-B is known to inhibit apoptosis through the induction of anti-apoptotic proteins, and to suppress the apoptotic potential of chemotherapeutic agents, leading to chemoresistance (Nakanishi and Toi, 2005). Aberrant expression of NF-B proteins has been well-documented in other cancers (Dolcet et al., 2005). Expression of NF-B has been found to be up-regulated in OSCC, the level increasing gradually from premalignant lesions to invasive cancer (Mishra et al., 2006). Moreover, NF-B1, comprised of p50 homodimers, transcriptionally regulates the anti-apoptotic protein Bcl-2, which has been shown to be overexpressed in a high proportion of oral cancer cases (Jordan et al., 1996). Analysis of these data suggests that NF-B signaling plays an important role in oral carcinogenesis.

Activating Protein-1 (AP-1)
The activating protein-1 (AP-1) family of transcription factors consists of multiple Jun (cJun, JunB, and JunD) and Fos (cFos, FosB, Fra-1, and Fra-2) members (Angel and Karin, 1991). The AP-1 complex causes multiple growth signals to converge at the transcriptional level and regulates cellular proliferation, differentiation, apoptosis, oncogene-induced transformation, and cancer cell invasion (McDonnell et al., 1990; Szabo et al., 1991; Brown et al., 1993).

Constitutive activation of AP-1 binding proteins, including the Jun family and Fra-1, can be detected in OSCC cell lines and oral dysplasia, and is associated with malignant transformation in squamous epithelial cells (Domann et al., 1994; Turatti et al., 2005). Furthermore, constitutive activation of AP-1 by transfection of c-Jun has been shown to induce malignant transformation to squamous cell carcinoma in murine models (Robinson et al., 2001). Conversely, transfection of dominant-negative c-Jun or treatment with AP-1 inhibitors blocked malignant transformation in mouse epithelial cells (Dong et al., 1994). These findings suggest that AP-1 activation induces transformation and malignant progression in OSCC.

Abnormalities in Growth-inhibitory Signals
Many tumor suppressor genes encode cell-cycle-inhibitory proteins, and when their expression is lost, progression through the cell cycle is increased (Hunter and Pines, 1994). Ultimately, both the loss of growth-inhibitory signals as well as the acquisition of self-sufficient growth signals through oncogene activation are needed for cancer development. Growth-inhibitory signals are tightly regulated by interactions of the cyclin-dependent kinase (CDK), cyclin, and the product of the retinoblastoma (Rb) gene. Furthermore, the proteins encoded by the tumor suppressor genes p16, p15, p21, and p53 also act as inhibitors of cell-cycle progression.

Retinoblastoma Gene
The Rb gene product, a key regulator of G₁/S cell cycle progression, is normally hypophosphorylated, enabling it to form a complex with the transcription factor E2F, thereby inhibiting E2F-mediated transcription of the genes that regulate DNA synthesis (Lundberg and Weinberg, 1999). Upon mitogen stimulation, Rb becomes phosphorylated and the Rb-E2F complex dissociates, freeing E2F to activate transcription of c-Myc, cyclin A, and p21^WAF-1, and cell proliferation proceeds (Goodger et al., 1997). Mutation and/or deletion of both alleles of the Rb gene leads to constitutive E2F-induced expression of these genes and promotes cell cycling.

Although Rb mutations are rare in OSCC, loss of Rb expression has been observed in 66% of OSCC cases and 64% of premalignant lesions (Pande et al., 1998). Alterations in Rb expression have been frequently observed in advanced-stage oral cancers (Pavelic et al., 1996). Other studies, however, report a low rate of Rb protein alterations (Xu et al., 1998).

p53
p53 is a tumor suppressor gene, located on chromosome 17p13.1, which plays a role in cell-cycle progression, cellular differentiation, DNA repair, and apoptosis. A major function of p53 is to serve as a guardian of the genome. Endogenous or exogenous stresses, such as DNA damage, hypoxia, and oncogene activation, increase p53 levels, leading to cell-cycle arrest that enables DNA repair to occur (Hartwell and Kastan, 1994). The induction of p53 expression can also occur through oncogenic stimulation that leads to p14^ARF activation (Vogelstein et al., 2001) or DNA double-strand breaks that activate the ATM/Chk2-dependent pathway (Levine, 1997).

p53 is the most commonly mutated gene and is altered in ~ 50% of all cancers, including 25–69% of oral cancers (Levine et al., 1991; Boyle et al., 1993; Caamano et al., 1993; Baral et al., 1998). Mutation most often occurs at a ’hot spot’ region from codon 238 to codon 248 (Somers et al., 1992; Hainaut et al., 1998; Kropveld et al., 1999) and causes defects in the binding of specific DNA sequences and the transactivation of genes whose expression is up-regulated by the wild-type protein (Vogelstein et al., 2001). Some human tumor-associated p53 mutants possess unique properties not found in the wild-type protein (Sigal and Rotter, 2000). Such "gain of function" activities include the ability to transform cells, increase tumorigenicity, and modulate the sensitivity of cancer cells to drugs (Sigal and Rotter, 2000; Song and Xu, 2007). Even in the absence of p53 mutations, p53 function can be inactivated by other mechanisms, such as infection with an "oncogenic" human papillomavirus type, such as HPV16 or HPV18. In HPV-positive SCCHN, p53 interacts with the E6 protein, which leads to increased ubiquitin-dependent proteolysis of p53 (Min et al., 1994; Nagpal et al., 2002). Another mechanism of p53 inactivation is elevation of expression of the MDM2 protein, which binds to p53 and promotes ubiquitination of the C-terminus of p53 and subsequent degradation (Oliner et al., 1993). p14^ARF interacts with MDM2, preventing association of p53 and MDM2 and thereby stabilizing p53 (Pomerantz et al., 1998). Therefore, degradation of p53 may be inappropriately stimulated by overexpression of MDM2 or by deletion or epigenetic silencing of p14^ARF.

p53 mutations commonly arise as a result of alcohol and/or tobacco exposure, and their presence is associated with the early recurrence and development of second primary tumors (Shin et al., 1996). Wild-type p53 gene therapy has been attempted in preclinical studies and in clinical trials in heavily treated patients. These studies demonstrated the feasibility of delivering the wild-type p53 gene to human tumors and yielded some clinical response and induction of apoptosis in the tumors (Clayman et al., 1998, 1999). However, difficulty in obtaining uniform delivery of the gene throughout the tumor has limited the utility of this therapeutic strategy. Other reports have demonstrated associations between p53 mutation and unfavorable responses to chemotherapy or radiation therapy (Temam et al., 2000; Warnakulasuriya et al., 2000).

p21^WAF1
p21^WAF1 is an important cell-cycle inhibitor whose expression is transactivated by wild-type, but not mutant, p53 (El Deiry et al., 1993). p21 interacts with cyclin/CDK, and this interaction leads to cell-cycle arrest. Thus, p21 plays a major role in mediating the growth-suppressing, as well as the apoptosis-promoting, functions of p53 (Sherr and Roberts, 1995). The precise role of p21 in OSCC has not been fully delineated. A recent study demonstrated that expression of p21 is increased in premalignant and malignant oral lesions with p53-dependent and -independent pathways, suggesting that alterations in p21 expression may be early events in oral carcinogenesis (Agarwal et al., 1998). Other studies have shown that reduced expression of p21 is correlated with poor prognosis (Kudo et al., 1999). In contrast to p53, p21 gene mutations have not been described in oral cancers (Heinzel et al., 1996).

p16^INK4a
The p16 tumor suppressor gene maps to chromosome 9p21–22. p16 binds to the cyclin-dependent kinases CDK4 and CDK6 to inhibit cellular proliferation by preventing entry into the S phase of the cell cycle (Serrano et al., 1993; Kamb et al., 1994). Loss of p16 is frequently observed in many human cancers, including oral cancers (Timmermann et al., 1997, 1998; Sartor et al., 1999). p16 can be inactivated through different mechanisms, including mutation, deletion, or promoter hypermethylation (Kamb et al., 1994; Merlo et al., 1995). p16 expression is lost in 83% of oral cancers and 60% of premalignant lesions (Reed et al., 1996; Pande et al., 1998; Wu et al., 1999), suggesting that p16 alteration is an early event in oral cancer progression (Loughran et al., 1996; Papadimitrakopoulou et al., 1997). Studies indicate that p16 loss in OSCC is correlated with a poorer prognosis (Bova et al., 1999).

Evasion of Apoptosis
Apoptosis is a molecularly programmed form of cell death, a tightly regulated process that eliminates senescent or altered cells that have become useless or harmful for the multicellular organism (Williams, 1991; Sen, 1992). Thus, apoptosis represents a physiologic cellular mechanism and plays an important role in cellular homeostasis. Tumor cells have been shown to exhibit increased survival and resistance to apoptosis (Oren, 1992; Manning and Patierno, 1996).

Apoptosis can be initiated by extrinsic or intrinsic cellular pathways. The extrinsic pathways involve receptor activation by ligands such as Fas ligand or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), while the intrinsic pathway is activated by BH3-containing proteins that become activated and ultimately depolarize the outer mitochondrial membrane, leading to release of several pro-apoptotic mitochondrial proteins, including cytochrome C, smac, and omi/htra2 (Suda et al., 1993; Danial and Korsmeyer, 2004; Hipfner and Cohen, 2004). After the initiation of apoptosis, cellular effector molecules, including a family of proteases known as caspases, induce proteolysis of the key proteins involved in apoptosis regulation, and this ultimately causes dissolution of the nucleus, with DNA degradation and changes in the cell membrane.

As already mentioned, p53 can be activated by DNA damage to become an important intracellular regulator of apoptosis in normal and cancer cells; its effects on the nucleus include regulation of expression of apoptosis-regulatory proteins, including Bcl-2 and Bax, and it also has direct effects on the mitochondria that lead to the release of cytochrome C (Levine et al., 1991; Miyashita et al., 1994). Thus, qualitative or quantitative alterations to p53 expression can lead to decreased apoptosis induction (Gottlieb et al., 1994; Ravi et al., 1999).

There are many important regulatory molecules involved in the control of cellular apoptosis, and one critically important group of apoptosis-regulatory proteins is the Bcl-2 family, which is comprised of at least 15 proteins with either anti-apoptotic (Bcl-2, Bcl-X_L) or pro-apoptotic effects (Bax, Bak). Alteration in the expression of Bcl-2 family members or a change in ratio of pro-apoptotic to apoptosis-regulatory proteins favoring inhibition of apoptosis promotes tumor progression and can lead to resistance to cytotoxic therapies such as chemotherapy and radiation (Kroemer, 1997; Wilson et al., 2001). For example, Bcl-2 overexpression has been observed in multiple tumor types, including cancers of the lung, colon, and breast (Pezzella et al., 1993; Silvestrini et al., 1994; Sinicrope et al., 1995). Increased expression of either Bcl-2 or Bcl- X_L has been detected in oral dysplastic lesions and OSCC, as well as in carcinogen-induced papillomas in a mouse model (Popovi et al., 1996). In non-small-cell lung cancer and breast cancer, elevated Bcl-2 expression is associated with a poorer prognosis. In oral cancer, however, a clear relationship between Bcl-2 overexpression and poor prognosis has yet to be established (Pena et al., 1999). Other studies have demonstrated that high levels of Bcl-X_L expression with wild-type p53 have been linked to cisplatin resistance in SCCHN cell lines (Bauer et al. , 2005), and that inhibition of Bcl-2/x_L by non-peptide small-molecule inhibitors that bind the BH3-binding pocket of Bcl-2/x_L led to growth inhibition and apoptosis (Oliver et al., 2004). Additionally, cisplatin-resistant cells are very sensitive to this inhibitor. These findings suggest that inhibition of Bcl-x_L may be an effective therapeutic strategy for overcoming cisplatin resistance (Oliver et al., 2004; Bauer et al., 2005).

Immortalization
Normal human cells possess a limited capacity to replicate themselves, and after numerous cell doublings, they cease to proliferate, and become senescent (Harley et al., 1990; Stewart and Weinberg, 2000). For cells to become tumorigenic, they need to become immortalized, with a capacity to replicate indefinitely by lengthening their telomeres (Stewart and Weinberg, 2000). Telomeres are repetitive tandem DNA repeat sequences complexed with telomere-binding proteins that are localized at the ends of mammalian chromosomes and protect the chromosome ends from degradation (Shay and Wright, 2006). Telomeres shorten after each round of cell division, limiting the life span of the cells. The enzyme telomerase maintains telomeric repeats by elongating telomeric DNA by reverse transcription, and its activity is determined largely by the expression levels of human telomerase reverse transcriptase (hTERT), which is the protein catalytic subunit of telomerase (Shay et al., 2006). Up-regulation of telomerase is a very common finding in human cancers, whereas it is less common in normal or benign tissues (Hanahan and Weinberg, 2000).

Several study findings have suggested that the increased expression of hTERT may be an early event in oral carcinogenesis, and that a high level of hTERT expression is associated with poor treatment outcomes (Mao et al., 1996b; Kannan et al., 1997; Lee et al., 2001; Chen et al., 2007).

Angiogenesis
Angiogenesis is another important hallmark of cancer development (Hanahan and Weinberg, 2000). Since tumor growth is limited to 1–2 mm³ in the absence of adequate perfusion, solid tumors need to develop a blood supply to grow and metastasize (Folkman, 1990). They do this through a shift in the balance between pro-angiogenic and anti-angiogenic factors (Folkman, 1990; Bouck et al., 1996). Some of the major pro-angiogenic signals include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF 1 and 2), and IL-8. The major negative regulators of angiogenesis include the interferons, proteolytic fragments such as angiostatin and endostatin, and thrombospondin-1.

Vascular Endothelial Growth Factor
VEGF plays a pivotal role in the regulation of normal and pathological angiogenesis, and it also increases vessel permeability and enhances endothelial cell growth, proliferation, migration, and differentiation (Ferrara et al., 2003). So far, 6 VEGF family members have been identified. These include VEGF-A, placental growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E (Neufeld et al., 1999; Ferrara et al., 2003). These VEGF ligands have the ability to bind and activate their receptors. There are 3 VEGF receptors: VEGFR-1/Flt-1, VEGFR-2/Flk-1/KDR, and VEGFR-3/Flt-4 (Veikkola and Alitalo, 1999). Activation of VEGFR-1 promotes endothelial cell migration, but does not induce cell proliferation (Neufeld et al., 1999). VEGFR-1 may act as a decoy receptor that regulates overall VEGF concentration by binding VEGF and preventing its association with VEGFR-2 (Neufeld et al., 1999). VEGFR-2 is the major mediator of endothelial cell mitogenesis, proliferation, and survival (Cross et al., 2003). VEGFR-3 (or Flt-4) is mostly expressed in lymphatic vessels (Taipale et al., 1999). It is activated by the ligands, VEGF-C and VEGF-D, and regulates lymphangiogenesis, the growth of new lymphatic vessels (Plate, 2001; Skobe et al., 2001; Alitalo and Carmeliet, 2002; Shibuya and Claesson-Welsh, 2006). Recent studies have suggested that lymphangiogenesis can be induced by solid tumors and may promote lymphatic metastasis (Saharinen et al., 2004; Alitalo et al., 2005). Other studies have revealed that lymphangiogenesis can occur adjacent to or within human cancers, and that this correlates with metastasis to lymph nodes in some tumor types, including cutaneous melanoma and SCCHN (Beasley et al., 2002; Dadras et al., 2003). The most well-characterized signaling pathway for tumor lymphangiogenesis is through VEGF-C and/or VEGF-D stimulation of VEGFR-3 (Veikkola et al., 2001). VEGF-C expression in tumor cells may be induced by growth factors or pro-inflammatory cytokines, and some may be derived from inflammatory cells in tumors (Alitalo et al., 2005). In various animal models, the VEGF-C/VEGFR-3 axis plays a critical role in cancer metastasis by inducing lymphangiogenesis (Karpanen et al., 2001; Mandriota et al., 2001; Su et al., 2006). Because OSCC preferentially metastasizes to regional lymph nodes, the VEGF-C/VEGFR-3 signaling pathway could represent an important therapeutic target. Unlike the well-characterized VEGF-A/VEGFR-2 axis, the VEGF-C/VEGFR-3 axis may have many undefined functions and mechanisms in tumor progression. Thus, further study of this axis is needed.

Several studies have shown that tumor angiogenesis is correlated with tumor progression and aggressiveness in human cancers, including oral cancer (Weidner et al., 1991; Brawer et al., 1994; Penfold et al., 1996). Expression of VEGF was stronger in high-grade oral dysplasia than in normal or mildly dysplastic oral epithelium (Denhart et al., 1997). In contrast, some studies have reported no difference between VEGF expression in normal oral mucosa and that in epithelial dysplasia (Carlile et al., 2001). It was reported recently that VEGF-C expression was correlated with locoregional recurrence and distant failure in OSCC (Tanigaki et al., 2004). The same study reported a significant association between VEGF-C expression and overall five-year survival rate, although there was no significant correlation between survival and expression of VEGF-A, VEGFR-1, or VEGFR-3 (Tanigaki et al., 2004).

The findings of studies of VEGF in OSCC have been somewhat inconsistent. It was reported, for example, that there was no correlation between VEGF and overall survival in 156 patients with SCCHN treated with surgery and post-operative radiotherapy (Salven et al., 1997). In another study of 77 patients with OSCC treated with surgery and post-operative radiotherapy, however, VEGF was the most significant predictor of poor disease-free survival rate (Smith et al., 2000). Further studies with more uniform patient populations and treatments and quantitative methods are needed to resolve these disparate findings. Nevertheless, the relatively high expression of angiogenic factors in OSCC tissues suggests that tumor angiogenesis offers an attractive therapeutic target in the management of oral cancers.

Interleukin-8
IL-8 was originally isolated from lipopolysaccharide-stimulated peripheral blood mononuclear cells (Oppenheim et al., 1991). IL-8 is chemotactic for neutrophils as well as T-lymphocytes, and is secreted by leukocytes and tumor cells (Koch et al., 1992). The cellular response of IL-8 is known to be mediated by its 2 high-affinity cell-surface receptors, CXCR1 and CXCR2 (Brat et al., 2005). IL-8 has diverse functions in immune surveillance, inflammation, and angiogenesis. A well-known function of IL-8 in tumors is promoting angiogenesis by modulating endothelial cell proliferation and migration (Koch et al., 1992; Fujimoto et al., 2000).

There is compelling evidence that IL-8 is a potent angiogenic factor in tumors. In a xenograft model of human non-small-cell lung carcinoma, passive immunization with neutralizing antibodies to IL-8 resulted in decreased tumor size, and this finding was associated with a decline in tumor-associated vascular density and angiogenic activity (Arenberg et al., 1996). Other studies showed that IL-8 mRNA expression was correlated with vascularization and metastasis in tumor specimens (Singh et al., 1994; Kitadai et al., 1998). Some human OSCC cell lines have been found to produce IL-8 constitutively in conditioned medium, and IL-8 production by these cell lines is up-regulated by the presence of cytokines, such as IL-1, IL-1β, TGF-β, EGF, and TNF- (Cohen et al., 1995; Maruyama et al., 1995). Moreover, IL-8 has been found in OSCC cells within tumor specimens, and its expression correlated with neovascularity (Richards et al., 1997; Chen et al., 1999).

Invasion and Metastasis
OSCC is characterized by local invasion and a propensity for dissemination to cervical lymph nodes. The ability of malignant cells to invade surrounding tissues is one of the major hallmarks that distinguish them from normal cells. Cancer cell invasion and metastasis represent complex, multistep processes involving cell adhesion, cytoskeletal rearrangements, cell migration and dissolution of the basement membrane, intravasation, survival in the bloodstream, extravasation at a distant site, and growth of metastatic cells in the distant site, with stimulation of neo-angiogenesis (Liotta, 1986; Chambers et al., 2002). During invasive and metastatic progression, the first step is characterized by increased cell motility and invasiveness, and it has been hypothesized that these processes may be associated with an epithelial-mesenchymal transition (Thiery, 2002). Epithelial-mesenchymal transition is a process in which epithelial cells change from a polarized, epithelial phenotype to a fibroblast-like mesenchymal phenotype, leading to dissolution of epithelial integrity, increased migration, local invasion, and, ultimately, metastasis. Loss of epithelial cell polarity and acquisition of motility require loss of cell-cell adhesion, reorganization of the cytoskeleton, and redistribution of organelles, including alterations in the gene expression profiles of cancer cells (Thiery, 2002; Thiery and Sleeman, 2006). Cadherins, a key target in epithelial-mesenchymal transition, are cell adhesion molecules that mediate cell-cell adhesions between normal mucosal cells that maintain epithelial integrity. Alterations in E-cadherin expression and subcellular localization occur during tumor progression, as cells change shape and become more motile and invasive, like mesenchymal cells. Cell-surface receptors, such as the integrins, bind to extracellular matrix components and play a major role in alteration of the cell attachment required for cell motility and invasion, and they can also transduce cell survival signals. Finally, the process of oral cancer invasion and dissemination requires proteolysis of the basement membrane by enzymes, including the matrix metalloproteinases.

E-cadherin
As already mentioned, E-cadherin plays an important role in maintaining the tight cell-to-cell contacts in normal oral epithelia. Decreased expression of E-cadherin during epithelial-mesenchymal transition leads to a decrease in cell-cell adhesion, and thereby contributes to cell dissociation and increased motility and invasion (Takeichi, 1991; Gumbiner, 1996). Thus, E-cadherin helps to suppress tumor cell motility, invasion, and metastasis (Birchmeier and Behrens, 1994). Decreased or complete loss of E-cadherin expression has been associated with lymph node metastasis and poor prognosis in OSCC (Diniz-Freitas et al., 2006). In one of these studies, E-cadherin expression was inversely correlated with cervical lymph node metastasis in stages I and II tongue cancer (Lim et al., 2004). During tumor progression, E-cadherin can be inactivated by different mechanisms, including promoter hypermethylation and transcriptional repression (Peinado et al., 2004), which may result from the activation of repressors such as Snail, Slug, SIP1, and Twist (Larue and Bellacosa, 2005). Snail is abnormally expressed in many epithelial tumor types and is associated with aggressive behavior and loss of E-cadherin expression (De Craene et al., 2005). Furthermore, down-regulation of E-cadherin can result from Twist repression of the E-cadherin promoter via E-box elements (Yang et al., 2004). In OSCC, transcriptional repressors Snail and Sip1 and promoter hypermethylation have all been implicated in E-cadherin down-regulation (Maeda et al., 2005).

Integrins
Integrins are heterodimeric, cation-dependent transmembrane glycoproteins that mediate cell-cell and cell-matrix interactions. These proteins play a role in the maintenance of tissue integrity and in the regulation of cell proliferation, growth, differentiation, and migration (Thomas and Speight, 2001).

In OSCC, immunohistochemical evaluation of integrin ₆ β₄ expression has shown this cell-surface protein to be associated with early recurrence and metastasis (Cortesina et al., 1995). Expression of the _v subunit is also altered in OSCC, since expression of _vβ₅ is lower in OSCC than in normal epithelium (Jones et al., 1993, 1997). In contrast to _vβ₅, _vβ₆ expression is up-regulated in oral dysplasia, where its expression is correlated with progression to malignant disease (Hamidi et al., 2000). It has been suggested that _v β₆ may be useful in predicting malignant transformation (Thomas et al., 2006). Studies with cell lines deficient in expression of the _v subunit have demonstrated the importance of this protein in the inhibition of terminal differentiation and increased anchorage-independent growth (Jones et al., 1996). Analysis of these data suggests that integrins may play varied and complex roles in the progression of oral cavity tumors, and that these roles may depend on their subunit composition.

Matrix Metalloproteinases
MMPs are a family of zinc metalloenzymes that are involved in extracellular matrix remodeling (Birkedal-Hansen et al., 1993). The family of MMPs is subdivided into collagenases, gelatinases, stromelysins, stromelysin-like MMPs, membrane-type MMPs, and new MMPs (Kuropkat et al., 2002). Malignant cells can use MMPs to help break down the basement membrane and degrade interstitial stroma, thus facilitating tumor invasion and/or metastases. MMP expression is elevated in several different malignancies, and its expression has been correlated with aggressive tumor behavior and poor patient prognosis (Jones and Walker, 1997).

Many studies have found that MMP-2 expression was higher in cell nests of metastatic tumors than in those of non-metastatic tumors in oral cancer, suggesting that MMP-2 is a predictive marker for tumor metastasis (Kawamata et al., 1998; Kurahara et al., 1999). MMP-3, -10, and -11 have also been implicated in the progression of oral cancer. One report found significant expression of MMP-3 at the invasive front, and correlations between this finding and tumor size, thickness, and mode of invasion (Kusukawa et al., 1995). Overexpression of MMP-10 and -11 has been linked to local invasiveness, although no clear correlation has been made with nodal metastasis (Muller et al., 1993). Tissue inhibitors of metalloproteinase (TIMP) can inhibit the action of MMPs, and a recent study showed that overexpression of TIMP is correlated with regional and distant metastasis, and poor prognosis (Katayama et al., 2004). The relationship of these inhibitors and MMP expression with clinical features is not fully understood. These results indicate, however, that many MMPs appear to have an important role in the progression of OSCC.

	TARGETED MOLECULAR THERAPY

TOP ABSTRACT INTRODUCTION MOLECULAR PATHOGENESIS OF OSCC TARGETED MOLECULAR THERAPY CONCLUSION REFERENCES

 
With improved understanding of the major hallmarks of OSCC and some of the molecules that are critical in mediating one or more of these hallmarks, it has become apparent that treatments directed against these molecular targets might be effective therapeutic strategies. The principles of targeted molecular therapy have been proved in preclinical models with several different types of agents, and clinical trials have shown the tremendous potential of these new types of drugs to augment the benefits of conventional therapy significantly without expanding treatment-related toxicity. However, the difficult decision is which target to select for any given patient with OSCC. The ideal target should be found in cancer cells specifically and involve multiple pathways of carcinogenesis. So, a molecule that is involved in cell proliferation, apoptosis, invasion, and angiogenesis is a better target than a molecule that is involved in mediating only a single cancer hallmark. For OSCC, potential targets include those related to growth regulation (EGFR), angiogenesis (VEGF), apoptosis (p53), and inflammation (cyclooxygenase-2 pathway) (Fig. 2). These molecules have been extensively studied as markers to predict clinical outcomes in OSCC. Here, we discuss results of promising preclinical and clinical studies of targeted therapy of OSCC using the abovementioned targets.

View larger version (30K): [in this window] [in a new window]   Figure 2. Schematic representation of selected target agents in oral cancer. Signaling from growth factor receptor tyrosine kinases such as EGFR and VEGFR can be blocked by monoclonal antibodies or intracellular small-molecule tyrosine kinase inhibitors. In addition, other agents inhibiting Ras, Raf, mTOR, nuclear proteins, and intracytoplasmic proteins represent promising anti-cancer drugs. Adapted from Le Tourneau et al.(2007).

Monoclonal Antibodies
Cetuximab (Erbitux) is one of the first EGFR monoclonal antibodies to be studied in OSCC. Preclinical data showed that cetuximab is synergistic with chemotherapy and radiotherapy in mediating antitumor effects. In human OSCC cell lines, this drug induces cell accumulation in the G₁ phase through increased expression of p27Kip1 and p15ink4b (Bonner et al., 2000; Harari and Huang, 2001; Kiyota et al., 2002). In clinical studies, cetuximab has shown encouraging effects as a single agent and when combined with radiation or chemotherapy (Bonner et al., 2006). In a phase III multicenter trial that compared radiotherapy alone with radiotherapy plus cetuximab, the combination yielded significant improvement of locoregional control and overall survival in patients with locoregionally advanced SCCHN (Bonner et al., 2006).

Given the encouraging results with cetuximab, several new anti-EGFR monoclonal antibodies are being developed and evaluated in preclinical and clinical studies. Panitumimab is a fully humanized antibody to EGFR, and thus differs from cetuximab, whose murine sequences may contribute to the infrequent, though potentially severe, hypersensitivity reactions to this agent (Alekshun and Garrett, 2005; Kim and Erlichman, 2007). Matuzumab, another humanized IgG1 anti-EGFR monoclonal antibody, has been tested in a phase I trial in SCCHN (Vanhoefer et al., 2004). Further studies of these newer anti-EGFR antibodies are needed to determine their role in treating OSCC.

Small-molecule Tyrosine Kinase Inhibitors
Gefitinib (Iressa) and erlotinib (Tarceva) are small-molecule EGFR tyrosine kinase inhibitors (TKI) that target the intracellular portion of the receptor. In SCCHN cell lines, gefitinib treatment inhibits EGFR phosphorylation, which leads to a decrease in phosphorylation of AKT, STATs, and MAPK and increased expression of p27Kip 1 and p21, with arrest at G₀/G₁ (Shintani et al., 2004). Preclinical studies have demonstrated synergistic effects when these EGFR TKI are combined with radiotherapy or chemotherapy (Sirotnak et al., 2000; Al-Hazzaa et al., 2005). In phase II studies, erlotinib has shown modest effects in the treatment of recurrent or metastatic SCCHN (Soulieres et al., 2004). On the basis of these encouraging findings, there is a great deal of interest in incorporating these agents into the treatment of OSCC in numerous disease settings, including (i) chemoprevention, (ii) neoadjuvant approaches, (iii) combinations with cytotoxic treatments in untreated patients, (iv) post-operative adjuvant treatment, and (v) treatment of recurrent and metastatic disease. The findings of studies with these agents to date suggest that EGFR-targeted therapy will play a major role in the treatment of OSCC in different settings.

p53 Status and Novel Therapeutic Strategies
The p53 protein has multiple functions, including DNA repair, apoptosis, and induction of cell cycle arrest (Vogelstein et al., 2001). Alterations in p53 structure and expression play important roles in OSCC development and tumor progression (Boyle et al., 1993; Koch et al., 1996). p53 mutations occur in 45 to 70% of HNSCC patients; alcohol and tobacco use are associated with these mutations (Boyle et al., 1993). In tumors with a normal p53 gene sequence, loss of p53 function can occur through p53 protein inhibition or degradation. Furthermore, p53 has been associated with both carcinogenesis and overall prognosis in OSCC (Cabelguenne et al., 2000). Therefore, strategies targeting the p53 gene may reverse or decrease the process of tumorigenesis and metastasis.

Gene therapy to replace wild-type p53 in OSCC tumors in which this gene is mutated or lost is an appealing strategy. Gene therapy involves using adenoviral or retroviral vectors, or even DNA directly, to deliver a functionally normal gene to replace a mutated gene within a tumor. In a phase I/II clinical trial, adenoviral vectors were used to carry the wild-type gene into cancer cells of patients with recurrent disease, and this approach was well-tolerated and feasible, and demonstrated modest antitumor activity in several patients (Clayman et al., 1998, 1999). Another p53-targeted approach uses the genetically engineered ONYX-015 virus, an adenovirus with the 55K EIB gene deleted, engineered to replicate selectively in p53-deficient cells and leave cells with normal p53 unaffected (Bischoff et al., 1996). The results of phase I/II clinical trials suggest that administration of ONYX-015 is associated with biological activity in patients with refractory SCCHN (Ganly et al., 2000; Nemunaitis et al., 2000, 2001). In particular, the combination of intratumoral ONYX-015 injection with cisplatin and 5-fluorouracil has produced substantial and durable responses (Khuri et al., 2000a; Lamont et al., 2000). In a trial of ONYX-015 used as a mouthwash for chemoprevention in patients with oral dysplasia, reversible responses were seen (Rudin et al., 2003).

Targeting Vascular Endothelial Growth Factor Receptor
Tumor growth and metastasis are regulated, in part, by the development of new blood vessels in the tumor through angiogenesis regulated by both pro-angiogenic and anti-angiogenic factors. Of the angiogenic factors, VEGF is a particularly appealing target pathway for cancer therapy, due to its role in both physiologic and pathologic angiogenesis. VEGF is involved in vessel proliferaton, invasion, and survival, and plays an important role in the angiogenesis associated with tumor growth and the metastatic process. In OSCC, VEGF levels are elevated and may be related to poor prognosis (Mineta et al., 2000b). The expression of VEGF and VEGFR-2 has been correlated with a higher proliferation index and worse survival in patients with OSCC (Kyzas et al., 2005a,b). In addition, traditional cytotoxic agents, including cisplatin and carboplatin, have increased VEGF expression in OSCC (Riedel et al., 2004). Thus, targeting VEGF is an appealing concept. As discussed earlier, VEGF pathways have multiple isoforms and multiple transmembrane receptors: VEGFR-1, VEGFR-2, VEGFR-3. In humans, VEGF-A is the most powerful mediator of angiogenesis. VEGF binds to the external membrane domain of VEGFR-2 and induces intracellular signaling, resulting in cellular proliferation and migration of endothelial cells. Therefore, therapeutically targeted angiogenesis has mostly focused on the VEGFA ligand and VEGFR-2.

Strategies to inhibit VEGF include monoclonal antibodies that bind VEGF or the VEGFRs and small-molecule inhibitors of VEGFR. Bevacizumab is a recombinant humanized monoclonal antibody against VEGF that has been studied extensively in many human cancers. Bevacizumab has been approved by the US Food and Drug Administration for colon cancer and is under active study in SCCHN. Combined targeting of important signaling pathways increases the therapeutic effects (O’Reilly, 2002). There is sufficient evidence that combined targeting of the VEGF and EGFR pathways is more effective than the targeting of either pathway alone. EGFR induces angiogenesis via VEGF expression, and EGFR inhibition can lead to decreased VEGF secretion and tumor microvessel production in vivo (Ciardiello et al., 2001). Furthermore, analysis of preclinical data suggests that resistance to EGFR inhibitors is associated with increased levels of VEGF (Viloria-Petit et al., 2001). Thus, combined inhibition of the EGFR and VEGFR signaling pathways is an appealing therapeutic strategy; exciting preclinical data have led to several clinical trials based upon this approach (Yigitbasi et al., 2004; Prichard et al., 2007). A phase I/II trial to evaluate the effect of bevacizumab in combination with erlotinib in patients with recurrent or metastatic SCCHN recently completed accrual and has shown encouraging responses (Vokes et al., 2005). The dual small-molecule EGFR TKI ZD6474 (AstraZeneca, Macclesfield, UK) is now being tested in phase II clinical trials for patients with SCCHN (Gilbert and Argiris, 2006).

AZD2171 (AstraZeneca), a highly potent oral VEGFR TKI, has also demonstrated dose-dependent growth inhibition and decreased microvessel density in an in vivo xenograft model (Wedge et al., 2005; Gomez-Rivera et al., 2007). A phase I trial of the combination of AZD2171 and gefitinib is under way in patients with refractory metastatic SCCHN.

As mentioned previously, there is considerable evidence suggesting that tumor lymphangiogenesis is correlated with metastatic spread to regional lymph nodes; the VEGF-C/VEGFR-3 signaling system is a key regulator of tumor lymphangiogenesis. Therefore, preclinical studies are under way to determine whether inhibition of VEGFR-3 activation might be an effective therapeutic strategy for the suppression of tumor growth and regional metastasis, and several methods of VEGF-C pathway inhibition are currently being evaluated. These include soluble VEGFR-3 protein constructs, neutralizing monoclonal antibodies to VEGFR-3 and VEGF-D, and small-molecule inhibitors of VEGFR-3 kinase (Lin et al., 2005; Pytowski et al., 2005). Some of these reagents also may block the contributions of VEGF-C and VEGF-D to tumor angiogenesis via VEGFR-2 (Jimenez et al., 2005). While the effects of VEGFR-3 inhibitors in OSCC are currently being tested in xenograft models, there have been few reports on the therapeutic role of VEGF-C/VEGFR-3-targeted agents in OSCC. Thus, further studies are needed to determine whether targeting VEGF-C/VEGFR-3 signaling is an effective strategy to prevent metastasis to the cervical lymph nodes in OSCC.

Other Novel Targets and Drugs in Development
COX-2 Inhibitors in Combination Therapy
Cyclo-oxygenase 2 (COX-2) is the rate-limiting enzyme in the synthesis of prostaglandin E₂, which plays well-known roles in tumorigenesis, including the inhibition of apoptosis, and the modulation of angiogenesis and invasiveness (Dannenberg et al., 2005). COX-2 inhibition has been demonstrated to decrease tumor growth, survival, metastasis, and angiogenesis in many cancers (Sheng et al., 1997; Sawaoka et al., 1998). COX-2 is overexpressed in 86% of the OSCC and DNA aneuploid oral dysplastic lesions that confer a high risk of developing biologically aggressive oral cancer, but not in normal oral mucosa (Chan et al., 1999; Lin et al., 2002; Lippman et al., 2005). Moreover, a 150-fold increase in COX-2 mRNA expression has been noted in invasive HNSCC compared with normal controls, in addition to a 50-fold increase in COX-2 mRNA expression in adjacent normal-appearing mucosa of HNSCC patients (Chan et al., 1999). Given that COX-2 is up-regulated during malignant transformation, non-steroidal anti-inflammatory drugs (NSAIDS) that inhibit COX-2 activity represent a promising approach for managing high-risk oral dysplasia patients. A COX-2 selective NSAID, celecoxib, was clinically tested as a preventive modality in familial adenomatous polyposis (Steinbach et al., 2000). However, these COX-2 selective agents have some adverse cardiovscular effects. Non-selective COX-2 inhibitors, such as some of the NSAIDs, may not be as toxic from a cardiovascular standpoint and may be preferable agents for the chemoprevention of cancer (White et al., 2002).

In a preclinical model, COX-2 inhibitors prevented tongue carcinoma progression (Shiotani et al., 2001). Interestingly, COX-2 inhibitors have also demonstrated promising results when used in combination with other targeted agents or in cytotoxic chemotherapy. In a study in SCCHN cell lines, the combination of an EGFR inhibitor and celecoxib, a COX-2 inhibitor, led to decreased cell growth (Chen et al., 2004). In preclinical studies in SCCHN, combination therapy with gefinitib and celecoxib significantly delayed tumor progression (Zhang et al., 2005). A phase I trial of gefitinib and celecoxib in recurrent or metastatic SCCHN yielded a 22% response rate (Wirth et al., 2005). These results suggest that further investigations into the inhibition of signaling through the COX-2 pathway in SCCHN may be worthwhile.

Signal Transducers: Ras, Raf, mTOR
The intracellular molecules Ras, Raf, and mammalian target of rapamycin (mTOR) are part of the downstream signaling cascades of both the EGFR and c-Met receptor tyrosine kinases. As discussed earlier, Ras is the prototypic oncogene and has multiple functions in carcinogenesis, including cell survival, proliferation, and apoptosis. Ras is dependent on the post-translational modification known as ’farnesylation’ for its full functional activity. The fact that Ras farnesylation is critical for its activation, together with the well-known role of Ras as an oncogene, led to development of the farnesyl transferase inhibitors (FTI). Farnesyl transferase inhibitors (FTI) were originally designed to inhibit post-translational activation of Ras by blocking farnesylation (Kohl et al., 1993), but may actually work by targeting the non-Ras protein Rho-B (Prendergast, 2001). From preclinical data, FTI have been found to be radiosensitizers and showed great potency against HNSCC cells (Brunner et al., 2003). A phase I trial with the FTI L-778,123 used in combination with radiation in SCCHN demonstrated a complete clinical response in two of three patients, with good tolerability (Hahn et al., 2002). In a phase I trial, patients with newly diagnosed advanced HNSCC and who were scheduled for surgery received FTI SCH66336 (lonafarnib). Although there was a short eight- to 14-day window for FTI administration prior to surgery, potent inhibition of protein farnesylation was demonstrated in 2 target proteins (DNA-J and prelamin A), and four patients experienced tumor reduction. A recently completed phase I/II study of lonafarnib and paclitaxel has shown evidence of clinical activity in SCCHN (Khuri et al., 2004), and phase II studies of the combination in head and neck cancers are planned or ongoing.

Raf is frequently overexpressed in SCCHN (Riva et al., 1995; Lyons et al., 2001). Sorafenib is an orally available small-molecule inhibitor of Raf-1 and BRAF kinases that is currently being evaluated for its activity against SCCHN in phase II clinical studies (Lee and McCubrey, 2003).

The mTOR is a serine-threonine kinase that regulates cell growth, proliferation and apoptosis through modulation of cell cycle progression, and ribosomal function (Schmelzle and Hall, 2000). mTOR regulates protein translation downstream of PI3K and AKT and is up-regulated in 57–82% of SCCHN (Mandal et al., 2006). In preclinical studies, rapamycin has significantly inhibited tumor growth (Nathan et al., 2007). Currently, there are at least 3 mTOR inhibitors (CCI-779, RAD001, and AP23573) in early clinical trials in patients with SCCHN. Buck and colleagues demonstrated, in multiple xenograft models, synergistic activity with rapamycin and erlotinib (Buck et al., 2006). Therefore, disease-specific evaluation of mTOR inhibitors in HNSCC would be of interest, especially in combination with EGFR inhibitors.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MOLECULAR PATHOGENESIS OF OSCC TARGETED MOLECULAR THERAPY CONCLUSION REFERENCES

 
During the past few decades, a great deal of progress has been made in our understanding of the molecular pathogenesis of oral cavity cancer. The development of OSCC is a multistep process requiring the accumulation of genetic alterations, influenced by tobacco, alcohol, and possibly viruses, against a background of heritable susceptibility to mutagens (Renan, 1993). Two key targets of these genetic alterations are inactivation of tumor suppressor genes and activation of oncogenes. These events result in the increased production of growth factors or cell-surface receptors and the activation of intracellular messenger signaling, leading to autonomous growth of tumor cells without extracellular growth stimuli. This, along with evasion of growth-inhibitory signals by the inactivation of tumor suppressors and the inhibition of apoptosis, leads normal oral epithelial cells to acquire the malignant phenotype.

The rapid progress that has been made in our understanding of the molecular biology of OSCC has led to the development of some promising new treatment strategies involving agents with specific molecular targets. Preclinical studies of some of these agents have shown promising results that have led to clinical trials. Encouraging results have emerged from studies of patients with OSCC, particularly when EGFR-targeted therapies have been used. It is likely that targeted therapy will play an important role in the management of OSCC in the coming years.

Received for publication September 18, 2007. Accepted for publication October 15, 2007.

	REFERENCES

TOP ABSTRACT INTRODUCTION MOLECULAR PATHOGENESIS OF OSCC TARGETED MOLECULAR THERAPY CONCLUSION REFERENCES

 

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Journal of Dental Research, Vol. 87, No. 1, 14-32 (2008)
DOI: 10.1177/154405910808700104


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