Genes
Oral Cancer in the Molecular Age
Joseph A. Regezi, DDS, MS, and Richard C. K. Jordan DDS, PhD
Copyright 2001 Journal of the California Dental Association.
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Oral cancer represents an accumulation of defects in the genes that encode key proteins associated with growth and development. Dysregulation of these proteins is central to malignant conversion. This appears to involve three major changes in cell function: 1. altered cell growth, death and longevity; 2. unencumbered cell movement; and 3. development of a new blood supply (angiogenesis). Specific genes, such as p53, p27, p16, and cyclin D-1, are altered in oral cancer through mutation, amplification, or deactivation. These genes are also frequently altered in many other malignancies. In oral mucosa, etiologic agents -- especially tobacco and alcohol, and possibly some viruses -- are known to induce alterations in the genes and gene functions associated with cell cycle regulation, contributing to the development of squamous cell carcinoma and epithelial dysplasias. Identification of the specific genes/proteins and the sequence in which they appear in the transformation of a normal cell to a malignant cell is necessary for the formulation of new treatment strategies, the development of early detection methods, and the prediction of patient outcome.
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Oral cancer, like most other malignancies, represents an accumulation of molecular lesions in genes that encode for proteins that control cell cycle, cell survival, cell motility, and angiogenesis. These complex changes give the tumor cells an independent growth advantage, leading to the ability to invade and metastasize to distant sites.1,2 Understanding how oral cancers develop at the molecular level will be necessary for the development of new cancer control methods. The purposes of this paper are to briefly review the causes of oral cancer, and the molecular mechanisms that are important in oral cancer pathogenesis.
Causes of Oral Cancer
Generally, the most important cause of oral cancer is tobacco.3 The use of tobacco, including smokeless forms, is known to increase the risk of oral cancer and is directly dependent upon the amount and duration of the habit. Alcohol, either alone or with tobacco, can also lead to oral cancer. The genes that have been shown to be altered in oral cancers by tobacco carcinogens are p53 and ras genes (see below). Human papilloma viruses (particularly subtypes 16 and 18) have been associated with some oral cancers, especially verrucous forms. HPV-encoded proteins, E6 and E7, are known to block the cell cycle inhibitory effects of p53 and retinoblastoma proteins, respectively.4,5 In the herpes virus group, Epstein-Barr virus has been closely linked to carcinoma of the nasopharynx and some lymphomas. Herpes simplex -- which commonly affects perioral skin, vermilion, palate, and gingiva -- has not been convincingly linked to the etiology of oral or lip cancers.6
How the various etiologic factors (e.g., tobacco carcinogens) operate at the molecular level is under investigation and is only partially understood. They are, however, believed to effect changes in the genome. Critical growth-related genes may become mutated, amplified, or deactivated by these agents, and the encoded proteins may be dysfunctional, overexpressed, or underexpressed.
Altered Gene Expression
No two oral cancers are exactly alike. The heterogeneous nature of oral cancers is evident at all levels, from molecular to clinical. Marked differences can be seen in clinical appearances (e.g., leukoplakia, erythroplakia, verrucous), histologic patterns (e.g., good to poor differentiation, inflammatory response, invasive architecture), and biologic behaviors (e.g., prolonged precancerous state, rapidity of invasion, metastatic potential). Likewise, the molecular (genetic) alterations are variable in type and, to some degree, in the sequence in which they occur. While oral cancers do not all exhibit the same genetic patterns or profiles, certain genes are apparently more commonly affected than others. Some of the genes known to be involved in oral carcinogenesis are discussed below.
Oral cancers progress through two important biologic stages. The first stage is loss of control of cell cycle through increased proliferation and reduced apoptosis. Clinically, this is most obvious in patients with in situ carcinomas where a higher number of dividing cells are evident in all levels of the epithelium (Figures 1 and 2). The second stage is increased tumor cell motility leading to invasion and metastasis (Figures 3 and 4). Here, neoplastic epithelial cells are able to penetrate the basement membrane and invade underlying tissues, and eventually travel to regional lymph nodes. The associated genetic changes are related to the activation or up-regulation of oncogenes, and the inactivation or down-regulation of tumor-suppressor genes (anti-oncogenes).
Oncogenes, or proto-oncogenes under normal circumstances, encode proteins that positively regulate critical cell growth functions, such as proliferation, apoptosis, cell motility, internal cell signaling, and angiogenesis. If genes in this group become altered through one of several mechanisms (e.g., mutation), protein overexpression occurs, giving rise to a clone of cells with a growth/motility advantage.
Tumor-suppressor genes encode proteins that negatively regulate or suppress proliferation and are believed to play a more important role in oral cancer development than oncogenes.7,8 Alteration of the genes in this group essentially "releases the brake" on proliferation for a clone of cells. To have a phenotypic effect, differences in or loss of both maternal and paternal gene copies (alleles) are required. This inactivation of a tumor-suppressor gene occurs in a two-step process. First, there is alteration of one allele, followed second by alteration of the other allele leading to loss of the normal maternal-paternal heterozygotic allelic combination (loss of heterozygosity).
Alterations of genes that control cell cycle seem to be of utmost importance in the malignant transition process. In fact, it has been suggested that cancer can be considered a disease of the cell cycle.9 Normally, the cell division process is divided into four phases, gap 1, DNA synthesis, gap 2, and mitosis. One of the most important events in this cycle is the progression from the gap 1 to the DNA synthesis phase. Genetic lesions, if left unrepaired in the gap 1 phase and carried into the DNA synthesis phase, can be perpetuated in subsequent cell divisions. The gap 1-DNA synthesis "checkpoint" is regulated by a complex system of proteins whose balance is critical to normal cell division (Figure 5). Overexpression of oncogenic proteins or underexpression of anti-oncogenic proteins can tip the balance in favor of proliferation and neoplastic transformation. Two important groups of intrinsic cell cycle proteins that accelerate proliferation are cyclins and their activation binding enzymes, the cyclin-dependent kinases. They are counteracted by proteins known as cyclin-dependent kinase inhibitors.
The accumulation of a number of adverse genetic lesions in oncogenes/tumor-suppressor genes may give the cell an independent growth advantage leading to a malignant phenotype. In the case of oral cancer, this series of changes would occur in a keratinocyte, ultimately creating a single clone of cells with uncontrolled proliferation and motility.
Alteration of Specific Genes/Proteins
Cell Cycle-Associated Genes and Proteins
Dysregulation of the cell cycle is a frequent finding in the development of oral cancer (Figures 6 and 7). The cell cycle-associated oncogenes cyclin D-1 and MDM-2, and the tumor-suppressor genes p53, p16, and p27 are prominent among the genes that have been confirmed as being abnormally expressed in oral cancers.10-14
p53, normally is a tumor-suppressor gene and a key negative-regulator at gap 1/DNA synthesis of the cell cycle. In about 50 percent of oral cancers, p53 is mutated; and its encoded protein is nonfunctional. Defective p53 protein allows cells to proceed into the DNA synthesis phase of the cell cycle before DNA can be repaired. The result is an accumulation of deleterious genetic defects that contribute to malignant transformation. This key protein may be dysregulated in oral precancer as well and may serve as an indicator of high-risk lesions.15-21 MDM2, which blocks the effects of p53, is also overexpressed in some oral cancers. Overexpression of cyclin D-1 appears in many oral cancers, leading to increased proliferation rate and premature transition through the gap 1-DNA synthesis checkpoint. Underexpression of the cyclin-dependent kinase inhibitors, p16 and p27, is also another important feature of oral cancer and relates to loss of control of cell cycle because of an inability to inhibit the effects of cyclins and cyclin-dependent kinases.
The antithesis of proliferation is apoptosis (genetically determined cell death). If cells live longer through the effects of anti-apoptotic proteins, they have an advantage that favors neoplasia. Some of the genes that control apoptosis, especially the Bcl-2 family, are altered in many cancers. In some oral cancers, the anti-apoptotic proteins Bcl-X and Bcl-2, are overexpressed.22,23 Moreover, expression of the proapoptotic protein, Bax, also has been positively correlated with increased sensitivity to chemotherapeutic agents in head and neck cancers.24
Cell Growth and Signaling Genes
Several other oncogenes that function in the regulation of cell growth and for the transport of signals from the cell membrane to the nucleus are also frequently altered in many oral cancers. These include genes that code for growth factors such as int-2 and hst-1 (fibroblast growth factor); growth factor receptors such as erbB1 and erbB2 (epidermal growth factor receptors); proteins involved in signal transduction such as ras (GTP binding proteins); and nuclear regulatory proteins such as myc (transcriptional activator proteins). Correlations have now been identified between growth receptor overexpression and patient outcome.25-33
Genes Associated With Cell Motility and Invasion
Many oral cancers pass through a premalignant phase (dysplasia or in situ carcinoma), while others appear to arise de novo without clinical or microscopic evidence of a pre-existing lesion. Invasive carcinomas have developed the ability to penetrate basement membrane and connective tissue, as well as enter the vascular system. These tumors are believed to have developed this invasive advantage through molecular lesions in genes and proteins associated with cell movement and extracellular matrix degradation. Changes in the phenotype of cell adhesion molecules (cadherins and integrins) release cells from their normal environment and give them the ability to move. This coupled with the enzymatic degradation of basement membrane and connective tissue provides the necessary components for invasion of the proliferating tumor.
Critical cell adhesions proteins are altered in invasive oral cancer. These proteins include intercellular adhesion molecule, e-cadherin, and the neoexpression of beta-6 integrin, a protein that assists keratinocyte motility. Matrix-related proteins produced by tumor cells and possibly by connective tissue cells (e.g., fibroblasts, macrophages) contribute to the breakdown of basement membrane and extracellular matrix proteins. Tenacin, an anti-adhesion molecule not evident in normal mucosa, is detectable in oral squamous cell carcinomas.34 Matrix metalloproteinases (MMPs 1, 2, 9, and 13) have also been demonstrated in invasive carcinomas and are believed to play a significant role in matrix degradation.35 In particular, MMP 3 and 13 are associated with advanced head and neck carcinomas.36,37 Controlling these proteins/enzymes through inhibitors or binding proteins has potential future therapeutic implications.
Genes Related to Angiogenesis
For tumors to grow much greater than 1 mm in size, a new blood supply is required (angiogenesis). This occurs through a tumor-associated process by way of induction or overexpression of angiogenic proteins (e.g., vascular endothelial growth factor [VEGF], basic fibroblastic growth factor [FGF]), and/or through the suppression of proteins that inhibit angiogenesis. VEGF, FGF, and IL-8 (proinflammatory cytokine) have been identified in head and neck cancers and are believed to be responsible, at least in part, for the angiogenesis associated with the progression of these tumors.38,39 The genetic alterations leading to the overexpression of these proteins has not been determined, but it likely involves interactions with other critical oncogenes and immunosuppressor genes. Nonetheless, identification of these abnormally expressed proteins marks another potential target for treatment of oral cancers (anti-angiogenesis).
Telomerase-Associated Tumor Cell Immortality
Another area of investigation, relative to gene-associated abnormalities in cancer, centers around telomere integrity. Telomeres are DNA-protein complexes found at the end of chromosomes and are required for chromosome stability. Normal cells have a finite life span related to telomere shortening that occurs with successive cell divisions. When a critical telomere reduction is reached, the chromosome and subsequently the cell are subject to degradation. Cancer cells may develop a mechanism that maintains telomere length and chromosome integrity and, thus, long-term viability. This mechanism is associated with the production of telomerase, an intranuclear enzyme that is not present in normal adult cells but is found in cancer cells. Most head and neck carcinomas have telomerase activity through neoexpression of this enzyme, giving the neoplastic cell extended life.40,41 This enzyme is another potential therapeutic target. Its detection in premalignant mucosal lesions (leukoplakia) may also serve as a biomarker for high-risk lesions.
Summary
Oral cancer has a complex molecular pathogenesis that is only partially understood. The cell cycle, when dysregulated, is believed to be central to malignant conversion, and with cell movement, when abnormally altered, can lead to invasion and metastasis. Angiogenesis and telomere integrity represent other facets that can contribute to cancer pathogenesis. Accumulation of defects in the genes that encode the proteins that regulate growth and development can lead to neoplastic change. In oral mucosa, etiologic agents -- especially tobacco and alcohol, and possibly some viruses -- can induce alterations in the DNA of these genes and contribute to the development of squamous cell carcinomas and dysplasias.
Further elucidation of the molecular mechanisms associated with the transition of a normal cell to a malignant cell is a vital step in oral cancer control. This new molecular era ushers in new opportunities for the development of early detection methods, novel therapeutic strategies, and outcome prediction.
Authors
Joseph A. Regezi, DDS, MS, is a professor of oral pathology and pathology at the University of California at San Francisco.
Richard C. K. Jordan, DDS, PhD, is an associate professor of oral pathology and pathology at UCSF.
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To request a printed copy of this article, please contact/Joseph A. Regezi, DDS, MS, University of California San Francisco, 513 Parnassus, S-512, San Francisco, CA 94143-0424 or at strnglv@itsa.ucsf.edu.
Legends
Figure 1. Erythroplakia of the soft palate with biopsy diagnosis of carcinoma in situ.
Figure 2. Carcinoma in situ. The abnormal epithelial changes represent hyperproliferation related to genetic alterations that led to abnormal expression of cell cycle-related proteins. It can be surmised that the cells have not acquired, as yet, the requisite genetic lesions that would facilitate basement membrane penetration and invasion.
Figure 3. Invasive squamous cell carcinoma of the floor of the mouth.
Figure 4. Invasive squamous cell carcinoma. Malignant keratinocytes have acquired the ability to degrade and move through connective tissue. Laboratory studies would show evidence (genetic lesions and altered protein expression) of impaired cell cycle, plus expression of matrix-degrading enzymes and an abnormal profile of adhesion molecules in the invading tumor cells.
Figure 5. Illustration of the cell cycle and how some critical proteins, known to be dysregulated in oral cancers, influence or control proliferation and apoptosis. Proteins that accelerate the cell cycle are in green, and proteins that retard or block proliferation are in red.
Figure 6. Diagram showing the cellular location of the protein groups that can be genetically altered or defective in oral cancer.
Figure 7. Diagram showing invasion and angiogenesis-related proteins that can be overexpressed in oral cancer due to genetic alterations.
Glossary
Adhesion molecules -- Molecules that are involved in the adherence of cells to each other or to extracellular matrix proteins.
Allele -- Alternate form of a gene that is found at the same locus of a chromosome
Angiogenesis -- The formation of new blood vessels
Apoptosis -- Physiologic or programmed cell death
Bcl-2 -- A family of genes/proteins associated with the control (both induction and inhibition) of apoptosis
Cell cycle -- The complex sequence of events associated with cell replication/proliferation
Chromosome -- A structure in the nucleus that is made up of linear strands of DNA associated with nuclear proteins and RNA
Cyclin-dependent kinase -- A cell cycle-related enzyme that, when combined with cyclin, assists in cell proliferation
Cyclin-dependent kinase inhibitor -- A protein that inhibits cell proliferation through its effects on cyclin-dependent kinase
Gene -- A region of DNA that codes for a single protein
Gene amplification -- An abnormal cellular event that results in a cell having a greater number of copies of a gene than normally present
Gene expression -- The process by which the information encoded by a gene is converted into a protein
Gene mutation -- A change in the genetic material. It can include all genetic alterations from single nucleotide substitutions to whole chromosome translocations.
Gene overexpression -- Excessive protein production due to alterations in the gene that encodes it
Gene underexpression -- Reduced protein production due to alterations in the gene that encodes it
Loss of heterozygosity -- In cancer, loss of a second allele of a gene when the first allele is already altered or lost
Matrix metalloproteinases -- A group of related proteins that promote degradation of connective tissue matrix and enhanced cell movement
Oncogene -- A mutant gene, usually related to cell cycle, that contributes to cancer development
Proto-oncogene -- A normal gene that encodes a protein usually related to cell cycle. When mutated, this gene is known as an oncogene.
Growth factor -- Extracellular peptide that signals a cell to proliferate
Growth factor receptor -- A protein on the cell surface receiving a growth factor molecule that starts a signaling cascade eventuating in proliferation
Signal transduction -- The process by which a cell transmits an external stimulus (signal) to the nucleus for a response
Telomerase -- A nuclear enzyme that is responsible for the extension and, therefore, maintenance of DNA sequences found at the end of chromosomes known as telomeres
Telomere -- DNA sequences found at the end of chromosomes that are necessary to prevent chromosome shortening and degradation
Tumor suppressor gene -- A gene whose protein product suppresses the cell cycle. It is often a target in cancer and requiring inactivation of both alleles to effect a loss of function.
