Multistage Carcinogenesis

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Ahallmark of carcinogenesis, the process of tumor development in an organism, is a long latent period with no clinical evidence of disease. The agedependent incidence of diagnosed cancers in humans suggests that carcinogenesis commonly proceeds via four to seven independent rate-limiting steps. Both animal carcinogenesis models and the analysis of human clinical samples support this stepwise progression of tumorigenesis. The genetic and biochemical defects that occur during this period to transform gradually a normal cell that maintains strict control over both intracellular and intercellular events to a cell mass with abnormal growth potential and an ability to invade adjacent tissues remain incompletely understood. Determining the temporal sequence of specific etiologically relevant events in tumorigenesis has been greatly aided by clinical and histopathological identification of a range of distinct stages in the progression of malignancies, i.e., from precursor lesion to metastasis. This recognizable biological progression must reflect a molecular progression within the genetic complement of the cell, which normally maintains multiple independent barriers to each stage of the malignant conversion process. During the past two decades, it has become clear that breaching these barriers depends on the gradual accumulation of irreversible alterations in an unknown number of genes.       
The molecular functions of these genes are broadly categorized as either inhibiting or promoting tumor development, i.e., tumor suppressor genes, whose functional activity is switched off or downregulated; or dominant-acting oncogenes, whose functional activity is switched on, upregulated, or otherwise deranged. Genes from both functional groups are normally involved in the homeostatic regulation of various cell processes and in coordinating communication and compatibility with neighboring cells. Current models of malignant transformation in specific tumor types are focusing on the identification of the precise genetic perturbations at each stage and elucidating the impact these molecular defects have on proliferation, differentiation, and intercellular relationships of the tumor population. Exploitation of this knowledge should result in more effective strategies for the diagnosis and treatment of the cancer patient and in the development of more specific preventive measures for the individual at high risk for developing a neoplasm.


The search for a relevant animal model of human carcinogenesis led to a series of classical experiments in the 1940s that defined how the process would be viewed for many years to come. It was observed that benign papillomas and malignant carcinomas could be induced in the skin of mice by application of a single subcarcinogenic dose of polycyclic hydrocarbons followed by a secondary treatment consisting of repeated wounding or application of an irritant such as croton oil. Neither treatment alone resulted in carcinogenesis, and reversing the order of application eliminated the effect. Surprisingly, tumor formation occurred when the secondary treatment was applied up to 1 year following the initial exposure to a carcinogen. Rous and Kidd pioneered the understanding of this latency phenomenon by conceptually dividing the process of carcinogenesis in the mouse skin model into two distinct stages: initiation and promotion.       
They defined initiation, a rapid process producing no apparent morphological change, as a priming event involving DNA damage resulting in irreversible genetic alterations that confer upon cells the ability to form tumors when subsequently exposed to a promoting agent. Promotion, characterized by clonal expansion of the initiated cells and dramatic morphological and biochemical changes, was considered to be an epigenetic phenomenon due to the finding that it could be reversed in the absence of continued treatment. Besides the application of chemical promoters such as phorbol esters, many diverse stimuli were found to have tumor-promoting effects, including UV irradiation and repeated physical abrasion. A common theme of promoting events appeared to be skin irritation.      
In 1964 Foulds described initiation and promotion as part of a larger continuous carcinogenesis process of "progression." Later investigators redefined progression as the third stage of carcinogenesis, following promotion and characterized by a higher degree of malignancy as evidenced by an increased ability to proliferate and invade local tissues and a propensity to metastasize to distant sites. The progression stage also correlates with severe genetic damage, including visible karyotypic alterations in the majority of cells. Although it was unclear whether this phenomenon was a cause or an effect of neoplastic transformation, it had clinical importance; i.e., in many tumor types, DNA aneuploidy is a poor prognostic factor. By the mid-1970s the observation that all cells in many primary tumors exhibited the same abnormal karyotype or similarities in the karyotype of marker chromosomes prompted the idea that most neoplasms arise from a single cell of origin and that genetic instability acquired during the process of neoplasia results in genetic variability within the original clone, allowing for subsequent selection of more aggressive sublines.      
As described earlier, the multiple barriers a cell must overcome to become fully malignant may explain why cancer is a relatively rare event. However, it was quickly realized that based on known mutation rates for nongermline cells (~10-7 per gene/cell division), the combination of four to six genetic events necessary for the neoplastic transformation of a cell would be mathematically so rare as to virtually preclude any spontaneous tumor development during an average human lifetime. Cancer development must therefore be a self-accelerating process in which the first mutational event or events caused genetic instability, leading to an increased mutation rate. The identification of critical genomic lesions in human carcinogenesis is complicated by the background of diverse genetic defects, including not only point mutations but also deletions, amplifications, and rearrangements of genes and chromosomes present in most biopsied tumors. Although the three-stage mouse skin model of carcinogenesis was useful, it was recognized that a more precise understanding of the molecular events in carcinogenesis was needed.


A. Oncogenes
The study of oncogenic viruses such as the Rous sarcoma virus led to the discovery of specific viral genes that were responsible for cell transformation. At the same time, investigators found that DNA isolated from human carcinomas and other tumors was able to induce neoplastic transformation at high efficiencies when transfected into transformation-sensitive "normal" mouse NIHT3 cells. In the 1970s, a group of cellular transforming genes, termed "oncogenes," was identified by homology to the transforming genes of retroviruses and by the biological activity of tumor cell DNA in transfection assays. Transfection of NIHT3 cells with mos (the normal cellular homologue of the transforming gene of the Moloney sarcoma virus) or with H-ras (the normal cellular homologue of the transforming gene of the Harvey sarcoma virus) under the control of viral transcriptional regulatory sequences resulted in cellular transformation.      
These findings suggested that oncogenesis was the result of dominant genetic alterations in which the functional activity of these genes was upregulated or expressed in an abnormal form. Protooncogenes (normal cellular homologues of transforming genes) were found to be (1) highly conserved in vertebrate evolution, (2) expressed in a highly regulated manner, and (3) key players in the growth control mechanisms of normal cells. It seemed logical that the derangement or overexpression of one or a combination of cell growth-related genes could transform cells in a "growth gone wrong" scenario. Revisiting the chemical carcinogenesis in mouse skin model, it was found that introducing the ras oncogene into keratinocytes via transducing retroviruses was tantamount to chemical initiation: subsequent application of promoting agents to infected cells resulted in papilloma formation.      
In addition, point mutations in the H-ras oncogene were invariably found in methylnitrosourea induced breast tumors in rats. It appeared as if a critical lesion in carcinogenesis had at last been identified. An important caveat in the oncogene theory of cancer was that cell transformation by transfection of a single oncogene was only observed under certain limited experimental conditions. The established rodent cell lines (such as NIHT3) used in the original transfection experiments were already phenotypically immortal and therefore partially transformed. Additionally, the results could not be duplicated in human cell lines, cautioning against oversimplification of the carcinogenic process in humans. As the number of oncogenes associated with human cancers increased, researchers were frustrated by the inability to associate a specific genetic lesion with a particular tumor type to a degree that indicated causality. Analysis of human tumors confirmed that there was no one particular oncogene that was necessary, let alone sufficient, for any given type of cancer. With the advent of transgenic mice bearing oncogenes, it was conclusively demonstrated that simply harboring oncogene mutations in a particular cell lineage is insufficient for tumor development.

B. Lessons from the Rb Gene
The examination of hereditary cancers by Knudson revealed a new paradigm important to the pathogenesis of cancer. If the stages of carcinogenesis were defined by genetic events, could a person with a hereditary disposition to cancer be already "initiated" by virtue of an inherited genetic lesion? In the case of retinoblastoma, the defective gene was localized to a band on the long arm of chromosome 13. Knudson's analysis suggested that, in contrast to oncogenes, this "antioncogene" acts in a recessive manner, with one normal allele being adequate to protect against tumorigenesis.      
Thus, two separate mutational events were needed for retinoblastoma formation, one to inactivate each copy of an antioncogene. In 1989 Weinberg refined Knudson's hypothesis by providing a more sophisticated molecular model for the process. Weinberg based his model on the insights following the molecular cloning and analysis of the retinoblastoma gene Rb. At the time, little was known about the precise function of protooncogenes in normal cells or about their regulation. When primary cell cultures (as opposed to partially transformed immortal lines such as NIHT3) are transfected with ras, only small numbers of cells acquire the oncogene and they do not proliferate to form visible foci. If, however, the transfection includes acquisition of neomycin resistance, subsequent selection results in a pure population of transformed cells whose growth proceeds in an uncontrolled manner. It had also been observed that while implantation of cells transformed by an oncogenic virus (e.g., Harvey sarcoma virus) onto the back of a mouse resulted in rapidly growing squamous carcinomas, cell proliferation resulting in tumor formation could be nearly completely prevented by implanting the transformed cells together with a fourfold excess of normal fibroblasts. Weinberg proposed that these observations could be explained if neighboring normal cells exert a constant inhibitory effect on the growth of transformed cells. Therefore, a critical event in carcinogenesis is when cells gain the ability to overcome the limiting effect of their normal tissue environment by ignoring or neutralizing the effect of inhibitory growth signals. He suggested that a number of key genes in growth regulatory pathways could contribute to carcinogenesis by suffering mutations resulting in their inactivation or downregulation. He termed this class of antioncogenes "tumor suppressor" genes.      
Weinberg and colleagues identified the Rb antioncogene, central to the development of retinoblastomas, as a putative tumor suppressor. Knudson's analysis of normal tissues and retinoblastomas from the same patients had suggested that inactivation of both alleles was the critical event in tumorigenesis. It was known that the viral E1A oncoprotein from human adenovirus type 5 formed complexes with the Rbencoded protein and that the region of E1A responsible for its ability to bind to the Rb gene product was crucial to the tumorigenic properties of E1A. The idea that loss of the Rb gene product enabled deregulation of cell growth was supported by experiments showing that introducing a cloned copy of the Rb gene into retinoblastoma cells restored normal growth control.
Subsequent work has validated the Rb gene product as an important regulator of cell growth; it is part of a cellular pathway responding to extracellular antigrowth factors such as transforming growth factor β (TGF-β) and it controls the activity of the EF2 transcription factors responsible for activation of the genes essential for progression from G1 into S phase. The pRb pathway has proved to be central to the cellular antigrowth signaling circuit and is disrupted in some manner in the majority of human cancers. The appealing notion that loss of regulation of a gene that in some manner controls cellular growth through genetic or epigenetic mechanisms was essential for cell transformation allowed carcinogenesis to be described as the net result of the combination of at least two molecular events: activation of an oncogene and inactivation of a tumor suppressor gene. This was consistent with the multistage nature of animal models of carcinogenesis. Disruption of one cellular pathway triggered proliferation, and a complimentary disruption conferred upon transformed cells the ability to overcome inhibitory effects of their normal neighbors. Tumorigenesis would be the result of sustained, uncontrolled growth that rendered the cell population susceptible to other mutagenic events.

C. The Colorectal Carcinogenesis Paradigm
Colorectal cancer was the first human tumor type in which the oncogene activation/tumor suppressor gene inactivation model was conclusively validated. In 1990, Fearon and Vogelstein published an elegant model for the development of colorectal cancer that could be broadly applied to the entire field of carcinogenesis research (Fig. 1). This tumor type was uniquely suited for the study of multistep carcinogenesis because of the availability of tissue samples representing all clinical stages of the disease (i.e., from very small adenomas to large metastatic carcinomas). During the past decade, this model has not only proven its relevance but has stimulated a wide range of important advances in the study of tumor progression.

FIGURE 1 Adaptation of Fearon and Vogelstein's pivotal model of colorectal carcinogenesis.

In order to gain understanding of the different clinical stages of the disease at a molecular level, Fearon and Vogelstein analyzed data from a wide range of molecular pathological studies of colorectal cancers. By correlating the clinical stages with observed genetic derangements, they identified four key sites: ras gene mutations and deletions of chromosomes 5q, 17p, and 18q. Mutations in the ras gene were found in about half of all colorectal carcinomas and adenomas greater than 1 cm in size. Familial adenomatous polyposis, an inherited disease that predisposes patients to colorectal tumor formation, was linked to a site (now known to be the locus of the APC gene) on chromosome 5q. Allelic losses of chromosome 5q were evident in 20-50% of colorectal carcinomas. The functional inactivation of the p53 protein is seen in more than half of all human cancers studied. The p53 tumor suppressor gene maps to the common region of loss on chromosome 17p found in colorectal tumors.      
Consistent with findings in other adult tumors, Fearon and Vogelstein found that more than 75% of colorectal carcinomas exhibit the loss of a large portion of chromosome 17p. Finally, they noted that chromosome 18q was lost in more than 70% of these carcinomas and almost half of late adenomas. The DCC gene maps to the common region of loss, and DCC was recognized as a cell adhesion molecule. The nature of the prevalent genetic defects in colorectal carcinogenesis reiterated the requirement for mutational activation of an oncogene and mutational inactivation of a tumor suppressor in carcinogenesis.      
Indeed, this study supported the concept that genetic losses appear to be more important genetic gains during carcinogenesis. Consistent with the estimated four- to six-step mechanism of human carcinogenesis, a correlation between the number of genetic aberrations and the stage of the tumor was observed. While disruptions of at least four to five genes (all of the aforementioned key sites plus one additional allelic loss) appeared in colorectal carcinomas, most early adenomas contained an average of only two. They also confirmed that different, specific sets of genes were likely to be involved (but not necessarily involved) at different stages of colorectal tumorigenesis; however, they found exceptions from each stage that suggested that the process was more complex than could be explained by a particular set of genetic lesions. This led them to conclude that the total accumulation of changes is more important than their temporal order.      
Fearon and Vogelstein noted that in some cases a mutation in the p53 gene appeared to dominate the wild-type allele through oligomerization of the mutant protein with the wild-type protein, resulting in inactivation of normal p53 function. This demonstrates that a mutation in one allele of a tumor suppressor gene sometimes exerted its effect in a dominant manner. We now know that the protein encoded by the p53 gene is a central component in the biochemical circuits controlling cell proliferation and programmed cell death (apoptosis). Removal of p53 might confer a selective growth advantage via growth deregulation and insensitivity to apoptotic signals, with a concomitant increase in the mutation rate and/or chromosomal instability leading to the eventual loss of the corresponding wild-type allele through localized mutation, mitotic recombination, or chromosomal loss. This would statistically account for the formation of sporadic tumors, which would be difficult to explain using the recessive model for tumor suppressors in which two unrelated mutational events are required, one to inactivate each allele.

D. The Cutaneous Malignant Melanoma Paradigm
The colorectal model is useful for gaining a deeper understanding of neoplastic development in virtually all types of tissues. For example, the same type of analysis can be used to examine the development of cutaneous malignant melanoma in terms of successive genetic changes. Molecular analysis of the clinically evident biological phases in melanoma development shown in Fig. 2, i.e., the development of nevi displaying architectural disorder and cytologic atypia (i.e., atypical or dysplastic nevi), the unregulated proliferation of melanocytes within the epidermis in melanoma in situ, the acquired competence to invade and proliferate within the dermis in primary invasive melanoma, and the development of metastatic capacity, has proven invaluable in our present understanding of this disease.      
It appears that one of the earliest events in the malignant transformation of the melanocyte is the disruption of genetic integrity and the triggering of dynamic genetic instability. During this stage of melanoma development, DNA aneuploidy can be used to distinguish melanomas in situ from nonmalignant atypical nevi (which do not normally exhibit aneuploidy). Early melanoma cell populations with near-diploid chromosome complements are not uncommon; however, upon analysis, they can be shown to bear subtle genetic abnormalities, most probably in genes involved in maintaining genetic stability (e.g., genes critical to DNA repair, replication, cell cycle, chromosome maintenance, and mitosis).

FIGURE 2 Biological stages of development for cutaneous malignant melanoma.

Melanocytes acquire genetic disruptions via two major routes: (i) spontaneous endogenous damage due to deamination of pyrimidines, the generation of oxidative free radicals, infidelity in DNA replication, defects in DNA repair, and metabolism of toxic or mutagenic substances and (ii) exogenous damage by ultraviolet radiation (UVR). A number of efficient repair enzymes continually monitor DNA before, during, and after replication for a range of accumulated defects. Derangement of genes associated with repair of DNA damage is typically found in many types of cancer. DNA repair genes map to chromosomes that are often perturbed in melanomas, e.g., 3p and 7, possibly implicating these genes in the observed genetic instability of these lesions.      
Deregulated cell proliferation is a phase critical to the propagation of genomic disturbances. Because epidermal melanocytes rarely divide in adult skin, damage to DNA probably contributes less to the process of melanoma development than to tumorigenesis in other tissues. However, exposure of melanocytes to UVR results in a transient and limited number of cell divisions that accelerate the development of a protective skin tanning by increasing the mean density of epidermal melanocytes. Concomitantly, UVR inflicts DNA damage by provoking an increase in lipid peroxidation and free radical formation and by inducing single strand breaks and pyrimidine dimers in DNA. Thus, following sun exposure, the melanocyte is faced with two conflicting signals: (i) cease replication of DNA and repair of UVRinduced damage or (ii) proceed with UVR-initiated cell division. Normally, melanocytes will respond by arresting the cell at G1, activating DNA repair systems, and allow the cell to divide only when repairs are completed. If the damage is severe enough, the melanocyte may be driven into apoptosis and eliminated.      
However, in some instances, the intrinsic differences in repair efficiencies for a particular DNA defect or premature resumption of DNA synthesis on a damaged template results in the melanocyte repairing most, but not all, of the UVR-induced damage. One possible outcome of this incomplete repair is the formation of a premalignant melanocyte harboring a critical but biologically inert genetic lesion, which may become the first step in carcinogenesis if it is followed by a complementary lesion produced via a subsequent error in normal cell division.      
The development of deregulated proliferation of melanocytes within the epidermis is a key clinical feature that differentiates melanoma in situ from normal and atypical nevi. As in situ melanomas continue to proliferate, they can accumulate additional genetic defects. The connection between abnormal proliferation and malignant progression is underscored by the observation that any melanocytic lesion (e.g., atypical nevus, primary, or metastatic melanoma) with a disproportionately high number of cells traversing the S phase has a worse prognosis than lesions in which the S-phase fraction is similar to normal tissue. Linking genetic instability with a loss of G1/S transition control is evident from data showing that euploid melanomas have a lower percentage of cycling cells in S phase than aneuploid tumors, and this feature correlates with longer survival. How transition from the normal G0 state of the melanocyte to G1 and S phase is accomplished in a deregulated manner appears to involve the evolution of a subpopulation of cells that have lost control of the G1/S phase transition due in part to gene defects in cell cycle regulatory proteins (e.g., p16INK4A, p15INK4B, and PITSLRE proteins) and to loss of genes regulating cell senescence (several of which have been mapped to chromosomes 1,6,7,9, and 11).      
Sometime during progression, in situ melanoma cells that are restricted to growth in the epidermis spontaneously acquire an invasive phenotype and penetrate the underlying dermal layer. The clinical relevance of this new propensity is that it shows a strong positive correlation with the development of widespread metastases and increasing mortality rates. How a melanoma in situ progresses to an invasive melanoma is unknown, but it is clear from model systems that unrestrained growth of the cells alone is insufficient. Current evidence suggests that the development of melanoma cell invasion is driven by the evolution of specific biological traits, e.g., (i) melanoma-directed dysregulation of the surrounding normal tissue interactions and architecture allowing physical invasion, (ii) the ability of melanoma cells to abrogate or attenuate inhibitory growth and motility signals from the normal tissue promoting invasion, and (iii) the production by melanoma cells of paracrine and autocrine growth factors and cytokines (and their receptors) allowing altered growth and motility.      
In order for physical invasion to occur, the melanoma cell must disrupt the extracellular matrix of the dermis prior to metastatic spread. More than a simple static barrier, the extracellular matrix plays a complex role in maintaining normal homeostasis of the skin by providing structural integrity and by generating biochemical signals that control cell adhesion, growth, differentiation, and migration. Invasive tumor cells must neutralize both of these barriers to affect tissue invasion. Several mechanisms have been identified in melanoma. Derangements in the expression and/or activation of proteolytic enzymes have been found that can disrupt the physical integrity of the extracellular matrix. Melanoma cells promote changes in the expression and assembly of major matrix components. Additionally, the expression and/or function of cell surface integrins and other molecules important to cell-cell communication has been found to be altered or disrupted. Contact with the extracellular matrix during the invasive stage of tumor progression alters the expression of a wide range of genes in many cell types, including melanomas. This interaction is complex, and a fuller understanding of it is necessary for the elucidation of the molecular basis of invasiveness.      
Melanoma cells must also breach the biochemical barriers to invasion. The ability of melanoma cells to acquire invasive potential correlates with the acquisition of resistance to inhibitory factors produced by dermal fibroblasts and possibly infiltrating inflammatory cells. The mechanisms by which invasive melanoma cells become resistant to the inhibitory effect of cytokines and interleukins such as interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and TGF-β are unknown. It may involve alterations in the receptors for these molecules and/or in other genes that comprise a signal-transducing pathway that engages these molecules. Many of the growth factors, integrins, and inhibitory proteins that are perturbed in melanoma transduce signals to various cellular compartments via protein phosphorylation. Emerging data suggest a connection between defects in members of the protein tyrosine kinase family of genes and altered signal transduction in the pathogenesis of melanoma. Melanoma cells have been shown to express and secrete growth factors, providing autocrine stimulation of melanoma proliferation. In addition, the expression of a number of such factors is inducible in epidermal cells by UVR, including IL-1, IL-6, IL-8, and TNF-α. In this manner, "normal" cells within the immediate tissue environment can play a major role in the growth deregulation of transformed cells. We are beginning to appreciate that tumors are not merely collections of transformed cells. They are complex tissues in which heterogeneous mixtures of normal cells such as fibroblasts, endothelial cells, and immune cells interact continuously with cancer cells.      
The use of heterotypic organ culture systems may ultimately prove more fruitful than a traditional cell culture for elucidating the mechanisms of neoplasia and for screening potential therapeutic agents. The final stage of neoplasia, i.e., the development of metastatic competence and migration of tumor cells from the primary site to specific distant organs, is the most poorly understood in terms of relevant molecular defects. Many critical aspects are required for the cell to acquire metastatic competence, including the expression and/or dysfunction of numerous biologic pathways involved in the ability of the cell to invade tissue, survive, and proliferate within the invaded tissue, migrate through the tissue, inhibit immune defenses, orchestrate the development of new blood vessels, escape into the bloodstream, and reemerge in a distant organ. Studies are beginning to establish dominant roles for specific families of genes such as the cadherins in the metastatic process. In melanoma cells, switching cadherin expression from E-cadherin to N-cadherin results in the loss of keratinocyte control over these cells and establishment of communication with fibroblasts and endothelial cells. Disruption of growth factor signaling pathways, notably endothelin-3 and stem cell factor and their receptors, is also associated with the development of metastatic potential in melanomas.      
In addition to a series of intrinsic molecular defects within the tumor cell, the study of melanoma has also provided evidence for another critical factor that may either suppress or aid tumor development in an as yet unpredictable manner: i.e., host immune competence and responsiveness. The potentially important role of this phenomenon in tumor growth is evidenced by spontaneous tumor regression in melanoma patients, and the observation that UVR in animal model systems can have a profound stimulatory effect on the outgrowth of tumor cells by transiently suppressing host immunological defense mechanisms. At present, relatively little is known about the interplay between immune suppression and specific genetic defects in tumor cells and how this interchange impacts upon the growth and spread of tumor cells. Presumably, through a separate series of genetic defects, the tumor cell may also develop the ability to resist or abrogate host immunity.


A. Simplifying Complexity
The two tumor models discussed earlier clearly show that the evolution of genetic instability, deregulated proliferation, and invasive and metastatic competence are complicated events developing, not as the result of a single genetic defect, but driven by an accumulation of molecular alterations in an overlapping succession of cell subpopulations. This disruption of the cellular genome results in defects in numerous regulatory mechanisms that control normal cell homeostasis, and because these mechanisms are complex, it is not surprising that an impressive diversity of cancer genotypes and phenotypes exists. Since the 1980s, more than 100 oncogenes and tumor suppressor genes have been identified, including representatives from virtually all of the major regulatory circuits of the cell. Components of the pathways regulating the cell cycle (e.g., Rb, E2Fs, p21INK4A, p15INK4B), apoptosis (e.g., p53, Bax, Bcl-2), inflammatory response (cyclooxygenase-2), cell communication (e.g., integrins, cadherins), and genetic stability (e.g., mismatch repair-associated proteins, telomerase) are disrupted, damaged, or abnormally regulated in neoplasia.      
Nonnuclear genetic defects may also play a role in carcinogenesis. The occurrence of somatic mutations of the mitochondrial genome in human colorectal cancer has been examined. Mutations of this type have the potential to interfere with normal oxidative phosphorylation, a disruption possibly accompanied by an increase in the level of cellular reactive oxygen species (which are known to affect DNA damage leading to mutations). Due to the nature of mitochondrial replication, it is conceivable that the entire mitochondrial population within a clonal cell population may become homogeneous if the end result of the mutated mitochondrial genome is to confer a selective growth advantage.      
The carcinogenic effects of epigenetic events further complicate the picture, such as the binding of chemical promoters of carcinogenesis to cellular receptors to regulate certain gene products. Cancer cells display a variety of epigenetic mechanisms by which they circumvent normal barriers to neoplasia, e.g., the disruption of the FAS death signal pathway by upregulation of a nonsignaling decoy receptor that titrates signals away from the apoptosis pathway. Hypermethylation of the promoter regions of cancerassociated genes is another important epigenetic mechanism of carcinogenesis. Methylation of CpG islands in the promoter region of a gene can block its expression; aberrant hypermethylation of cancerrelated gene promoters has been observed in several types of cancers.     
There have been frequent attempts to simplify and codify this apparent diversity of mechanisms through categorization. The most recent and intellectually satisfying systems group cancer-associated genes into categories representing their functional activities. Classical tumor suppressor genes such as p53, Rb, and APC, which prevent cancer through direct control of growth have been termed "gatekeepers." Genes that suppress neoplasia in an indirect manner by maintaining the fidelity of the genome, including DNA mismatch repair genes, spindle checkpoint genes such as BUB1 and MAD1, or DNA damage checkpoint genes that include ATM, Brca1, and Brca2, have been called "caretakers."      
A third pathway to cancer (carcinogenesis via "landscaper" defects) has been proposed based on the observation that, under certain conditions, normal cells proximal to a rapidly proliferating defective cell population are at an increased risk of neoplastic transformation due to the biochemical factors present in their abnormal microenvironment.      
Although these classification systems have proven useful, they fail to address the cancer problem in a manner that connects them in a meaningful way to cell phenotype and biological behavior and that would lead to a unified understanding of the malignant process. Such an understanding is crucial to the eventual development of comprehensive therapeutic or preventive strategies. We have been fortunate in having available for study the colorectal cancer and malignant melanoma paradigms, which are amenable to analysis at multiple clinical stages, in the pursuit of a molecular mechanism of multistage carcinogenesis.      
Both these models focus on the genetic and biological characteristics a cell must acquire to overcome cellular and tissue barriers to oncogenesis. This theme is reflected in current models of carcinogenesis. During the next few years, aided in part by the sequencing of the human genome and recent technical advances in sophisticated and sensitive analysis of global expression of genes and proteins, such models will be greatly refined as we continue to link the phenotype and biological behavior of a tumor cell with specific genes and signaling circuits.

B. Current Models
Recent models of tumorigenesis have attempted to synthesize our understanding of the molecular events underlying the stages of neoplasia with our increasing knowledge of the central molecular circuitry of the cell. Examination of tissues from various types of cancers has shown that when cells progress from a preneoplastic state through advanced malignancy, they acquire a set of characteristics that are the hallmarks of cancer. In a current model proposed by Hanahan and Weinberg, carcinogenesis can be viewed as a process in which disruption of each key cellular circuit results in the acquisition by the cell of a new capability, enabling the cell to successfully breach one of the anticancer defense mechanisms of the organism. Malignancy is thus achieved through genetic and epigenetic disruptions, resulting in the acquisition of a set of six acquired capabilities essential for fully malignant neoplastic transformation (Table 1). They argue that virtually all genetic and epigenetic disruptions that contribute to carcinogenesis result in the acquisition of one or more of these capabilities and they reiterate Fearon and Vogelstein's observation that the precise order and nature of genetic and epigenetic derangements may not always be important for carcinogenesis.

TABLE I Hanahan and Weinberg's Model of Multistage Carcinogenesis

Acquired capabilitya Common mechanisms Specific examples
Self-sufficiency in growth signals Deregulation of growth signal pathways via disruption of extracellular signals, signal transducers, and corresponding intracellular circuits Production of PDGF, bFGF, or TGFα (autocrine stimulation), upregulation or truncation of EGF-R/erbB, upregulation of HER2/neu, SOSRas-Raf-MAP kinase mitogenic cascade mutants, defects in herterotypic signaling
Insensitivity to antigrowth Derangement of the pathways that recognize and signals respond to the antigrowth signals that normally force proliferating cells into the G0 stage or direct them to terminally differentiate and lose their proliferative, capacity Rb gene mutation or pRb sequestration. Other disruptions of the pRb signaling circuit, including downregulation and/or mutation of TGFβ receptors, p16INK4A, p15INK4B and CDK4. Overexpression of c-myc, erbA
Evading apoptosis Defects in sensors directing apoptosis or effectors carrying it out p53 tumor suppressor gene abnormalities, upregulation of bcl-2, disruption of FAS deathsignaling, IGF-2 gene expression
Limitless replicative potential Circumvention of telomere shortening Upregulation of telomerase expression, induction of alternate mechanisms of telomere maintenance
Sustained angiogenesis Disregulation of angiogenic factors, altered expression, or proteolytic modification of pro- and anti-angiogenic signaling factors Activation of ras, upregulation of VEGF, FGF1/2, downregulation of thrombospondin-1, β-interferon
Tissue invasion and metastasis Changes in expression of cell-cell adhesion molecules, integrins, growth factors, and extracellular proteases Switch in expression from E-cadherin to Nmetastasis cadherin, preferential expression of α3β1 and αVβ3 integrins, downregulation of endothelin-3

aThe process is viewed as the gradual acquisition of six key capabilities.

The expansion of our understanding of the molecular circuitry of the cell has facilitated an in-depth exploration of how different mutational and epigenetic routes can be followed that lead to the same end and how phenotypically identical cancers can have very different genotypes. We now recognize that knowledge of the unique molecular signature of a given tumor, rather than its histological identity, may prove to be the pivotal factor in designing the most effective therapeutic regimen. It has also become clear why some mutations are more oncogenic than others. The study of multifunctional proteins such as p53 and pRb indicates that it is because the gene product involved is central to more than one circuit, hence deregulation at one key point allows the cell to breach multiple barriers to neoplasia simultaneously.


The recent refinements of the carcinogenesis model should result in superior prognostic and diagnostic modeling as well as improved clinical management of the patient with cancer. By focusing on biological characteristics common to most forms of neoplasms, new therapies can be developed that target the acquisition of these cancer-associated biological phenomena and the underlying genetic abnormalities.      
Antiangiogenic therapies, for example, hold the promise of being applicable to a wide variety of cancers. Through definition and elucidation of the specific molecular pathways that can lead to cancer and understanding how a cell interconnects and organizes signaling pathways in a hierarchical system to control the behavior of a cell, we may be able to identify pharmacological targets that mitigate or attenuate those circuits that often become permuted and/or rerouted during cancer development. The great strides made in large-scale gene expression analyses of cells and tissues should materially and quickly provide targets for novel therapies. The ongoing genetic mapping studies of polymorphisms in putative cancer susceptibility genes will further allow exciting opportunities to not only tailor cancer treatment, but also to identify those who may be at increased risk for a primary or secondary cancer. These individuals are potential candidates for cancer chemoprevention clinical trials.

Anthony P. Albino
Ellen D. Jorgensen
The American Health Foundation, Valhalla, New York

See Also

apoptosis Programmed cell death via biochemical circuits responding to aberrations or defects in the cell. A normal defense against the propagation of mutant or damaged cells.

carcinogenesis The process of tumor development in an organism.

clonal expansion The selective replication of a mutated cell within a population resulting in the eventual genetic homogeneity of the cell population.

initiation The first step in the three-step model of multistage carcinogenesis in which an irreversible genetic alteration occurs, sensitizing the cell to promoting agents.

kryotypic instability The inability to maintain genomic integrity, as evidenced by chromosome rearrangement, truncation, and loss.

oncogene Implicated in cancer development when its expression is upregulated, activated, or deranged.

progression Third step in the three-step model of multistage carcinogenesis in which accumulated genetic alterations result in the ability of affected cells to invade local tissues or to metastasize to distant sites.

promotion Second step in the three-step model of multistage carcinogenesis in which initiated cells are stimulated to proliferate by promoting agents.

protooncogene Normal cellular homologue of an oncogene.

transformation Process by which normal cells become neoplastic.

tumor suppressor Gene implicated in cancer development when its expression is turned off, downregulated, or disrupted.

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