ARTÍCULO CLÍNICO

 

Cyclooxygenase-2 (COX-2) and epidermal growth factor (EGF) in oral premalignant epithelial lesions

La ciclooxigenasa-2 (COX-2) y el factor de crecimiento epidérmico (EFG) en lesiones epiteliales orales premalignas

 

 

S. Díaz Prado¹, A. Gallego Guadalupe², J.L. López-Cedrún³, J. Ferreras Granado⁴, L. Antón Aparicio^1,5

¹ Dra. en Biología. Departamento de Medicina. Universidad de La Coruña. La Coruña. INIBIC.
² Unidad de Investigación. Estudiante de Biología.
³ Servicio Cirugía Oral y Maxilofacial. Jefe de Servicio, Dr. en Medicina.
⁴ Servicio Cirugía Oral y Maxilofacial. Médico Especialista, Licenciado en Medicina.
⁵ Departamento de Medicina. Universidad de La Coruña. La Coruña, España. Servicio Oncología Médica. Jefe de Servicio, Dr. en Medicina. C.H.U. A Coruña. La Coruña, España.

Correspondence

 

 

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ABSTRACT

Oral premalignant lesions include leukoplakia (white patch) and erythroplakia (red patch), which develop on epithelial surfaces. These lesions are markers for field cancerization because patients with oral premalignancy can develop squamous cell carcinoma at the site of the lesion(s) and at other sites in the upper aerodigestive tract. An effort is being made to identify surrogate endpoint biomarkers (SEBs) for head and neck squamous cell carcinoma (HNSCC). Candidate SEBs for invasive squamous cell carcinoma (SCC) of the upper aerodigestive tract are detectable molecular, cellular, and tissue changes that take place during tumorigenesis. Among the markers that have been proposed to date, cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) seem to be the most promising. COX-2 is overexpressed during tumor transformation from early hyperplasia to metastasic disease. EGFR is also abnormally activated in epithelial tumors, since cells of almost all these kinds of neoplasm express high levels of this receptor, a characteristic associated with poor clinical outcome. The upper aerodigestive tract provides a unique model for studying the development of squamous cell carcinoma and for investigating candidate SEBs.

Key words: Epidermal growth factor; Cyclooxygenase-2; Head and neck squamous cell carcinoma; Oral premalignant lesion; Biomarker; Field cancerization.

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RESUMEN

Las lesiones premalignas orales incluyen eritroplasias (manchas rojas) y leucoplasias (manchas blancas), las cuales se desarrollan a lo largo de superficies epiteliales. Estas lesiones son considerados marcadores en la "carcinogénesis de campo" ya que pacientes con lesiones premalignas orales pueden desarrollar carcinoma de células escamosas (CCS) en el sitio de las lesiones, así como en otros lugares de tracto aerodigestivo superior. Se está haciendo un gran esfuerzo para identificar nuevos biomarcadores SEBs (surrogate endpoint biomarkers) para el carcinoma de células escamosas de cabeza y cuello. Los SEBs candidatos para el carcinoma de células escamosas invasivo en el trato aerodigestivo superior deben ser detectables con los cambios moleculares celulares y tisulares que tienen lugar durante la formación del tumor. Entre los diferentes marcadores que se han propuesto hasta la actualidad, la ciclooxigenasa- 2 (COX-2) y el receptor del factor de crecimiento epidérmico (EGFR) parecen ser los más prometedores. COX-2 se sobre expresa durante el proceso tumoral, desde hiperplasia temprana a enfermedad metastásica. EGFR también está anormalmente activado en tumores epiteliales, pues las células de casi todas estas neoplasias expresan altos niveles de este receptor, una característica asociada con un peor pronóstico clínico. En este sentido el tracto aerodigestivo superior proporciona un sistema o modelo único para el estudio de CCS y para la investigación de nuevos candidatos SEBs.

Palabras clave: Factor de crecimiento epidérmico; Ciclooxigenasa-2; Carcinoma de células escamosas de cabeza y cuello; Lesión premaligna oral; Biomarcador; Carcinogénesis de campo.

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Introduction

Carcinogenesis in the upper aerodigestive tract is a process consisting of several steps. Histologically, the process is a transition over time from normal epithelium to hyperplasia, dysplasia, and squamouscell carcinoma (SCC). This histologic transition involves a series of molecular and cellular changes, including changes in the expression of genes that encode growth receptors and growth factors. In recent decades the term "field cancerization" has been used to describe the histologic anomalies (hyperplasia, dysplasia, and SCC in situ) detected in most of the normal epithelia adjacent to a resected SCC. Among the different surrogate endpoint biomarkers (SEB), cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are interrelated. Evidence indicates that COX-2 and EGFR overexpression is an early event in epithelial carcinogenesis. It is postulated that the increase in COX-2 and EGFR levels in premalignant lesions constitutes a mechanism of "field cancerization". If these biomarkers (COX-2 and EGFR) are overexpressed in squamous-cell carcinoma (SCC), it can be deduced that the genesis, growth, and survival of a subgroup of oral premalignant lesions could depend on the intervention of the same actors in different roles and scenarios (Table 1).

 

Premalignant lesions of the oral mucosa

Erythroplakia

Erythroplakia is defined as a velvety-textured red spot that cannot be attributed clinically or pathologically to any specific disease. Some investigators consider erythroplakia as the first sign of asymptomatic SCC of the oral cavity. Erythroplakic and leukoplakic lesions can be viewed as a continuum because both experience malignant transformation. A schematic view of erythroplakia/leukoplakia defines the microscopic discoveries that may be seen in association with potential neoplastic changes of the oral mucous membrane as the tissue progresses from benign hyperkeratosis through several stages of erythroleukoplakia. Erythroplakia and leukoplakia lesions are sometimes included in the same category as mottled leukoplakia or mottled erythroleukoplakia because in many cases it is not possible to differentiate these types of lesions.

Leukoplakia

Leukoplakia is defined as a white spot or plaque on the oral mucosa. Leukoplakia has been classified into several different subtypes and approximately 6% of all oral leukoplakias become malignant. In an evaluation of patients with leukoplakia subjected to a 3-year follow-up, 31% of the lesions disappeared, 25% remained unchanged, and 30% improved.

Oral epithelial dysplasia

Epithelial dysplasia is a premalignant condition characterized clinically by an alteration in the epithelium that can make the oral mucosa turn red, white, or another color. Microscopically, dysplasia has been classified as mild, moderate, or severe. Epithelial dysplasia can develop a progressive course over a certain period, although smooth forms of dysplasia can be reversible in some cases.

Carcinogenesis of the oral mucosa

The development of SCC is a process consisting of a series of steps that involve changes related to specific genes, epigenetic events, and signal transduction in the cell (Fig. 1). The genetic alterations present very early in the course of carcinogenesis are mutations or deletions of 3p and 9p. Telomerase activation also occurs early in carcinogenesis.¹ Mutations or deletions of 17p (involving the p53 tumor suppressor gene), 13q, and 18q generally occur later in carcinogenesis. An important epigenetic event in the progression of cancer is the silencing of gene promoter regions by hypermethylation, which has been shown to affect the p16 gene suppressor gene, DAP-kinase, and E-cadherin.^2,3 The gene that encodes the beta retinoic acid receptor (RAR-beta) is silenced by methylation of its promoter.⁴ Tobacco smoke, which contains agents that can act as mutagens, activates the epidermal growth factor receptor. EGFR activation, in turn, increases the production of prostaglandins, including PGE₂, which can exercise positive feedback and stimulate EGFR signal transduction. ⁵ Cyclin-D1 generally is overexpressed in cancer of the head and neck and EGF activation is responsible for the high activity of cyclin- D1.⁶

Cyclooxygenase (COX)

COX is an enzyme that limits the rate of production of prostaglandins (PGs) and thromboxanes (TxB) from free arachidonic acid. The precursor of PG is arachidonic acid, a 20-carbon polyunsaturated fatty acid. The first step in PG synthesis is the hydrolysis of phospholipids to produce free arachidonate, a reaction catalyzed by phospholipase A₂ Molecular oxygen is added to arachidonic acid in a reaction catalyzed by the cyclooxygenase activity of COX. This reaction produces an unstable product, PGG₂, that is rapidly converted into PGH₂ by the peroxidase activity of COX (Fig. 2). PGH₂ is the common precursor of all the prostanoids (e.g., prostaglandins and thromboxanes) in reactions catalyzed by different specific synthases. The enzyme is bifunctional (Fig. 2) and has two different forms: COX-1 enzyme, which is expressed constitutively and is present in most cells and tissues, and COX-2 isoenzyme, which is inducible and expressed in response to cytokines, growth factors, oncogenes, stimuli, and tobacco carcinogens.⁷ Although COX-1 and COX-2 have similar enzymatic activities, they have different properties and different patterns of expression (Table 2). The COX-1 enzyme is expressed constitutively at low levels in most tissues, where it synthesizes the respective prostaglandin for normal physiologic functions. In contrast, COX-2 is not usually present in most cells, although strong regulation allows it to be rapidly expressed in response to growthrelated signals. This rapid increase in expression results in an increase in the synthesis of prostaglandins associated with inflammation and carcinogenesis. Tangible evidence exists that COX- 2 is overexpressed in extrastromal and intratumoral cells, as well as in the neoplastic cells of tumors. Several mechanisms explain COX-2 overexpression in these cellular types: COX-2 expression generally is regulated at the transcriptional and post-transcriptional level, although it can also be regulated by the rate of synthesis or degradation of the protein. The human COX-2 promoter contains multiple binding sites for transcriptional factors, including the element of response to cAMP and potential binding sites for Myb, the molecular factors interleukin-6 (NF-IL6) and kB (NF-kb), and Ets factors (Fig. 2). Of all these binding sites, the sites closest to the initiation of transcription are sensitive to several stimuli. Since COX-2 is a prostaglandin synthase, the most obvious consequence of COX-2 overexpression is the increase in prostaglandin production. The increase in PG synthesis can contribute to carcinogenesis in various ways, notably by stimulating cellular growth: PGE₂ and PGF₂ stimulate mitogenesis in synergy with the epidermal growth factor (EGF). COX-2 is overexpressed in a variety of premalignant and malignant conditions, including oral leukoplakia and SCC. High COX-2 levels can contribute to carcinogenesis by modulating xenobiotic metabolism, apoptosis, immune monitoring, and angiogenesis. It is believed that PGs may be important in the pathogenesis of cancer due to their effects on cell proliferation, angiogenesis, immune monitoring, and apoptosis.⁸ COX- 2 levels also are increased in the apparently normal mucosa adjacent to SCC.⁹ Evidence exists that not only demonstrates COX-2 overexpression in SCC, but also suggests the relation existing between COX-2 and tumor development.

Carcinogenesis-related COX-2 mechanisms

COX-2 can affect a number of important mechanisms in carcinogenesis. Generally speaking, COX-2 is overexpressed during the tumor process from early hyperplasia to metastatic disease (Fig. 3). In this sense, high COX-2 levels have been detected in neoplastic epithelium, inflammatory cells, and vascular cells within and adjacent to tumors. The COX-2 metabolites derived from the inflammatory cells of the infiltrate also contribute to carcinogenesis.

Xenobiotic metabolism

COX-2, a bifunctional enzyme, has peroxidase and cyclooxygenase activity; its peroxidase activity catalyzes the conversion of procarcinogen into carcinogen. Oxidative reactions are catalyzed mainly by the P-450S cytochrome. Tissues like those of the upper aerodigestive tract generally have low concentrations of P-450S. Consequently, significant amounts of xenobiotics can be oxidized to mutagens through the peroxidase activity of COX-2. In addition to catalyzing mutagen synthesis, COX-2 can also be induced by procarcinogens. For example, benzo [a] pyrene (B[a] P), a procarcinogen of tobacco smoke, stimulates COX-2 transcription. COX-2 overexpression, in particular, can condition an increase in mutagen production: malondialdehyde (MDA) can be produced by PGH₂ isomerization via enzymatic and nonenzymatic pathways. In addition, other types of carcinogens can be formed by the oxidation of aromatic amines, heterocyclic amines, and dihydrodiol derivatives of polycyclic hydrocarbons. Through these mechanisms, COX-2 overexpression can lead to DNA damage, thus contributing to carcinogenesis.

Apoptosis

It has been shown that apoptosis, or programmed cell death, diminishes during carcinogenesis and that COX-2 overexpression in epithelial cells leads to a reduction in apoptosis. This effect has been attributed, at least in part, to an increase in the levels of the anti-apoptotic protein Bcl-2. COX- 2 overexpression may possibly prolong the survival of abnormal cells, thus favoring the sequential accumulation of genetic changes that increase the risk of carcinogenesis.

Inflammation

Chronic inflammation is a recognized risk factor in epithelial carcinogenesis. Cytokine-mediated COX-2 overexpression contributes to the increase in PG synthesis in inflamed tissues, providing an explanation of the cause-effect relation between chronic inflammation and carcinogenesis by means of COX-2 overexpression.

Angiogenesis

Any significant increase in tumor mass must be preceded by an increase in vascular development to channel nutrients and oxygen to the tumor. It has been found recently that COX-2 levels correlate with VEGF expression and tumor vascularization in SCC.¹⁰

Cell mobility and adherence

The invasive capacity fostered by COX-2 is mediated by CD44, a cell surface receptor for hyaluronate. COX-2 overexpression enhances cellular invasive capacity and also increases CD44 expression. The biochemical changes associated with these tumor cell dynamics include increased expression and alteration of metalloprotease-2 and diminished expression of E-cadherin.

 

Epidermal growth factor receptor (EGFR)

The EGF receptor was the first receptor to be identified in a family of receptors known as ErbB receptors. This family consists of the following related receptors: EGFR (ErbB1/EGFR/HER1), ErbB2 (HER2/neu), Erb3 (HER3), and ErbB4 (HER4). Under physiologic conditions, a variety of ligands of the EGFR family induce the formation of homodimer or heterodimer complexes between the four types of ErbB receptor. These complexes maintain signal diversification and amplification.¹¹ In tumor cells these receptors can be activated by additional mechanisms and receptor overexpression in tumors can lead to dimerization of the independent ligand receptor. In some tumors, mutant forms of EGFR, which arise from genetic changes, reduce the constitutive expression of the dependent ligand receptor. These phenomena indicate that tumor cells have other mechanisms of EGFR activation in addition to receptor overexpression, mutations, and production of the autocrine ligand.

Structure, function, and signal transduction

Epidermal growth factor receptor (EGFR), also known as HER1 or erbB1, is a ubiquitous glycoprotein that crosses the cell membrane. It is made up of an extracellular ligand- binding part (amino-terminal), a hydrophobic transmembrane region, and a cytoplasmic part containing the tyrosine kinase domain and the carboxy-terminal region, which has critical tyrosine residues and receptor regulator motifs. The cell surface is where the initial ligand-receptor and receptor-receptor interactions occur. The binding of a specific set of ligands (EGF, TGF-a, amphiregulin, betacellulin, or epiregulin) to the extracellular domain activates the cytoplasmic catalytic function, promoting EGFR dimerization and autophosphorylation of the receptor at tyrosine residues.¹² These phosphorylated tyrosines serve as binding sites for a number of adaptor and signal transducer molecules that initiate a number of signaling pathways that result in cellular proliferation, differentiation, migration, adhesion, protection against apoptosis, and transformation, among other events. The cytoplasmic signal transduction pathways activated by active EGFR include PLC-gamma-1, Ras-Raf-MEK-MAPKs, phosphatidylinositol- 3 kinase (PI3K), Akt, Src (SAPKs, stress-activated protein kinases), PAK-JNKK-JNK, and signal transducers and transcription activators (STATs).¹³ The major signaling pathway of the ErbB family is via the Ras-Raf-Map-kinase pathway. Ras activation initiates a cascade of phosphorylations in multiple steps that lead to the activation of MAPKs, ERK1, and ERK2, which are regulators of the transcription of molecules related to cellular proliferation, survival, and transformation. Another important EGFR signaling pathway is phosphatidylinositol-3 kinase (PI3K) and the serine/threonine kinase Akt protein.¹⁴ Akt transduces signals that trigger a chain of responses from proliferation and cell growth to survival and motility.¹⁴ Another signaling pathway is the protein kinase pathway activated under conditions of stress, in which protein kinase C and Jak/Start participate. The activation of these pathways is translated in the nucleus into different transcriptional programs that mediate a variety of cellular responses, including cell division, survival (or death), mobility, invasion, adhesion, and repair.

Baseline expression of EGF-R and TGF-α in premalignant lesions

The levels of TGF-α and EGFR expression are higher in leukoplakia than in adjacent normal mucosa. TGF-α expression levels in the normal mucosa adjacent to dysplastic leukoplakia are higher on average than the levels detected in the oral mucosa of volunteers without leukoplakia (Fig. 3). In contrast, immunohistochemical study shows that the expression of TGF-α and EGFR is significantly elevated in patients with SCC and in the adjacent normal mucosa but not in the oral mucosa of patients without SCC. The mechanisms by which TGF-α and EGFR expression increases in leukoplakia and SCC have not been determined, but they may include alterations in genetic controls, SCC cell lines, and the mechanisms by which increased TGF-α and EGFR mRNA levels initially induce an increment in the rate of gene transcription but not in the number of gene copies or alterations in messenger stability.

EGFR in cancer of the head and neck

The evidence that TGF-α and EGFR participate in carcinogenesis comes from studies of transfection and analysis of RNA and proteins in experiments with human tumor cell lines, including SCC. In humans, it has been observed that TGF-α and EGFR levels are elevated in SCC and in the adjacent normal mucosa. Epidermal growth factor receptor (EGFR) is abnormally activated in epithelial tumors; the cells of almost all these neoplasms express high EGFR levels, which is a characteristic associated with a less favorable clinical prognosis. Detectable levels of EGFR are found in the tumors analyzed, although with noticeable differences between patients. Some investigators have not found significant differences in EGFR expression between either different anatomic sites or different states of tumor differentiation. Significant differences have not been found in the distribution of EGFR levels between tumors T1-2 and T3-4. In contrast, a significant difference is observed in the distribution of EGFR levels between tumor stages I and II and tumor stages III and IV.

Based on this threshold value, EGFR overexpression has been associated with a lower recurrence-free rate and overall survival rate with respect to patients who exhibit lower EGFR levels. Univariate analysis of survival shows that EGFR and tumor stage are significant variables, tumor stage being the most significant variable associated. For recurrence-free survival, EGFR has been the only significant parameter. In other studies, the frequency of SCC tumors with high EGFR levels generally is lower than previously published data^15,16 and the correlation of well or moderately differentiated tumors and small tumors does not coincide with previous studies. In this sense, EGFR expression correlates with well differentiated and moderately differentiated SCC; the size of the tumor correlates with an increase in EGFR expression, particularly in T1-2 tumors. In contrast, no correlation is found between tumor size and the degree of histologic differentiation. A possible explanation for the contradictory results obtained in different published studies is the lack of consensus on the visualization of the receptor and the definition of cutoff points. In addition, the intensity of staining may be dependent on tissue fixation parameters. Other possible explanations for the differences observed could be related to differences in EGFR expression in different points of the tumor and the association between the presence of human papilloma virus infection and expression of the receptor.¹⁷ Other studies have demonstrated that 80-90% of SCCs overexpress EGFR and its ligand, TGF-α, compared to levels in the normal mucosa of patients without cancer, with mean increases of 69-fold and 5-fold, respectively. A later study by the same group of investigators revealed that EGFR mRNA and protein also are overexpressed in patients with SCC in dysplastic lesions and even in the adjacent normal mucosa. These observations indicate that EGFR overexpression is an early event in SCC development.

Expression of other ErbB markers

Her2 expression is detected in 20% of cases and its overexpression seems to be associated with a high risk of lymph node metastasis and lower survival in patients with oral carcinoma. Approximately half of the carcinomas of the oral cavity express Her3 and Her3 overexpression, like Her2 overexpression, is associated with a higher incidence of lymph node dissemination. ¹⁸ Her4 expression also has been detected in half of the carcinomas of the oral cavity.¹⁹

 

Clinical perspectives and therapeutic trends

Chemopreventive agents Prevention using drugs, or chemoprevention, is a strategy that has been evaluated and found to hold some promise of reducing the morbidity and mortality associated with cancer. Diverse agents have been evaluated as possible chemopreventive therapies. Among them are vitamins (A, E, and C) and minerals (selenium). As for the search for new molecular targets, other studies have included COX-2 inhibitors and EGFR inhibitors (Fig. 4) as possible therapeutic agents.

Molecular targets

Evidence exists that demonstrates that prostaglandins are involved in carcinogenesis; prostaglandin E2 is responsible for the activation of EGFR, which leads to stimulation of cell proliferation (Fig. 5). In this sense, COX-2 can be a determinant of EGFR expression in oral premalignant epithelial lesions as well as in SCC (Fig. 6), which is why the inhibition of the COX-2 and/or EGFR pathway would be an ideal target for molecular therapy of premalignant lesions of the oral cavity. The implication of EGFR in squamous cell carcinoma has inspired the clinical use of several agents to block the function of this protein. In particular, the effectiveness of two monoclonal antibodies, cetuximab and gefitinib, as therapeutic agents for oral carcinoma has already been demonstrated.

Gefitinib arrests cell proliferation and the cell cycle in the G1 phase and reduces regional lymph node metastases in mice with oral squamous-cell carcinoma (OSCC).²⁰ Similarly, cetuximab promotes the detention of OSCC cell cultures in the G1 phase of the cell cycle.²¹ Exposure to gefitinib also enhances the radiosensitivity of OSCC cells in vitro and in vivo.²² Recently, a randomized clinical trial was conducted of the EGFR inhibitor EKB-569 alone, celecoxib (400 mg twice a day) alone, the combination of the two drugs, or placebo in patients who presented oral aneuploid dysplastic leukoplakia.⁵

 

Conclusions

Oral premalignant lesions develop on the epithelial surfaces of the aerodigestive tract. These lesions can lead to the development of SCC in the same area of the lesion. In this sense, field cancerization of the aerodigestive tract involves a multiple-step histologic transition from normal epithelium to SCC. This process involves a series of changes not only at the cellular and molecular level, but also changes in the expression of genes that encode different growth factors. For that reason concerted efforts are being made to identify new markers of premalignant lesions of the mucosa of the upper aerodigestive tract. Among the markers that have been proposed so far, cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) appear to be the most promising. Real evidence that COX-2 and EGFR are overexpressed in SCC of the head and neck suggests that it would be useful to study whether these two markers can be used as SEBs in premalignant lesions (leukoplakia type) of the oral mucosa.

 

Acknowledgments

We thank A. Carro Ramos for technical assistance. S. Diaz Prado received a postdoctoral research contract from the Isidro Parga Pondal Program of the Regional Executive of Galicia (Corunna, Galicia, Spain).

 

 

Correspondence:
L.M. Antón Aparicio
Servicio Oncología Médica
C.H.U. A Coruña. Hospital Materno Infantil Teresa Herrera
C/ As Xubias S/N
15006 A Coruña. España
E-mail: lantapa@canalejo.org

Recibido: 08.10.08
Aceptado: 02.03.09

 

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