Destructive cycles: the role of genomic instability and adaptation in carcinogenesis
Brandt L. Schneider1,3 and
Molly Kulesz-Martin2
1 Department of Cell Biology and Biochemistry, Room 5C119, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA and 2 Departments of Dermatology and Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97239, USA
3 To whom correspondence should be addressed. Tel: +1 806 743 2512; Email: brandt.schneider{at}ttmc.ttuhsc.edu
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Abstract
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Classical theories of carcinogenesis postulate that the accumulation of several somatic mutations is responsible for oncogenesis. However, these models do not explain how non-mutagenic carcinogens cause cancer. In addition, known mutation rates appear to be insufficient to account for observed cancer rates. Moreover, the current theory doesn't easily account for the long latencies observed in human cancers. Proponents of an aneuploidy-driven theory of carcinogenesis suggest that genomic instability has a causative role in carcinogenesis. In support of this theory, pre-neoplastic cells frequently display genomic instability while normal cells do not. Data obtained from a variety of model organisms have revealed that disruption of the cell cycle controls required for homeostasis results in the acquisition of genomic instability. Subsequently, this genomic instability becomes self-propagating via destructive cycles and provides a medium for cellular selection and adaptation. Genomic instability allows numerous genetic and epigenetic alterations to accumulate during carcinogenesis without markedly changing phenotype until they are qualitatively or quantitatively sufficient to be selectively advantageous in the tumor microenvironment. Observations of adaptation in tumor cell populations and application of chaos theory may help elucidate the mechanism that drives the enormous genetic heterogeneity observed in tumors and provide insights into the development of new therapeutic cancer interventions and treatments.
Abbreviations: DMBA, dimethylbenzanthracene; MNU, methylnitrosourea
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Introduction
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Why hasn't cancer research made more progress? Perhaps many of us have been challenged by a friend or a relative with this point of view. Despite the enormous progress made towards identifying the genetic and functional alterations in cancers, a complete understanding of their causes and vulnerabilities to treatment remain elusive. A major reason for this is the multitude of genetic and epigenetic changes that occur during carcinogenesis. Because of this, no two cancers are genetically or phenotypically the same. Indeed, individual cancer cells within the same tumor express a high degree of genetic and phenotypic heterogeneity. This heterogeneity may explain why many cancer therapeutic interventions fail. Because of the diversity generated by genetic heterogeneity, there is a high probability that cancer treatments inadvertently promote the selective outgrowth of resistant clones. Frequently these adapted cancer cells are highly aggressive and more malignant than the initial population of tumor cells. These observations make it increasingly clear that the war against cancer will continue to test our mettle, ingenuity and tools. Because of this, elucidation of the mechanisms responsible for tumor cell adaptation both in vivo and in vitro is a major challenge in carcinogenesis today.
Current models of carcinogenesis postulate that the accumulation of particular somatic mutations is responsible for oncogenesis. However, there is growing evidence that genomic instability and the resulting aneuploid genotypes and global gene expression changes have a significant role in the onset of cancer. In this review and commentary we examine how the role of genomic instability and aneuploidy are changing our current perspectives on carcinogenesis. We consider chaos theory and adaptation in model systems to help elucidate the causes and consequences of cancer and suggest implications of the shifting perspectives on the design of new therapeutic cancer interventions.
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Six easy pieces
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As part of the culmination of years of work on the steps required for neoplastic transformation of human cells, Hahn, Weinberg and Hanahan proposed that the pathogenesis of human cancer is dependent upon the acquisition of six capabilities: self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis (13). As part of this synthesis, specific molecular events or genetic alterations associated with each acquired capability have to some degree been delineated.
Normal cells are dependent upon mitogens and growth factors for cell growth and proliferation (Figure 1A). However, most cancer cells do not require these factors. Rather, cancer cells can achieve mitogenic growth signal autonomy through the auto-activation of downstream signal transduction pathways (Figure 1B). For example,
25% of human tumors have mutations in the growth factor-responsive Ras signal transduction pathway (2,4). In addition, loss of function of a number of tumor suppressor genes (e.g. pRB and p53) promotes insensitivity to growth inhibitory signals (reviewed in 5,6). These mutations presumably allow tumor cells to proliferate in the absence of mitogens. Moreover, many if not all cancer cells develop resistance to apoptosis. The most frequently observed genetic alteration associated with this acquired proliferative/survival capability involves mutation of the p53 tumor suppressor gene. Through mutation and a variety of other mechanisms, >50% of human tumors lack functional p53 and thus evade a number of pro-apoptotic signals (2,7). Acquisition of the above characteristics promotes tumor proliferation (13). However, in vivo studies of carcinogenesis suggest that unbridled proliferation is not sufficient for the formation of cancer (2).

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Fig. 1. Comparison of normal homeostatic cell cycles with the destructive cycles of cancer cells. (A) Homeostatic cycles. In normal cells, growth and proliferation are dependent upon mitogens and growth factors stimulating signal transduction pathways. Ultimately, these signal transduction cascades converge on the nucleus to modulate transcription. In addition, protein expression and activity is also regulated to help coordinate cell growth (e.g. the addition of cell mass) with proliferation. In so doing, cells ensure and maintain homeostasis, which is essential for maintaining cellular integrity and genomic fidelity. (B) Destructive cycles. Carcinogens damage the genetic pathways in normal cells. This damage can be direct through the mutation of genes. In this manner, mutation can activate oncogenes or inactivate tumor suppressor genes. In addition, carcinogens can apply a selective pressure to populations of cells to promote the outgrowth of resistant cells (e.g. drug resistance). In either case, the coordination of cell growth and proliferation becomes disrupted. This normally occurs through damage to cell cycle controls or signal transduction cascades. The end result is two-fold. First, cells gain the ability to proliferate in the absence of mitogens or growth factors. Second, loss of cell cycle control disrupts homeostasis and promotes genetic instability. Subsequently, this genetic instability becomes self-propagating by promoting mutagenic destructive cycles. The end result is the production of a population of cells that behave as a complex adaptive system governed by the rules of chaos. In this manner genetic instability allows numerous genetic and epigenetic alterations to accumulate during carcinogenesis without markedly changing phenotype until they are qualitatively or quantitatively sufficient to be selectively advantageous in the tumor microenvironment.
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While cancers of the epidermis may be the exception because of visual accessibility of small lesions, in general tumors must contain
108 cells to present clinically (8). This translates to
27 cell doublings. In vitro, under optimal conditions, tumor cells double every 2448 h. If a similar proliferation rate were maintained in vivo, a single tumor cell capable of unbridled proliferation would have a latency of tumor formation of <2 months. Even the most rapidly proliferating human tumors have a significantly longer latency period, and for some of the most common tumors (e.g. breast, lung and colon) the latency period can easily extend into decades (813). In addition, tumor burdens are estimated to be
1012 cells at the time of patient death (8). Again, if tumors proliferated in vivo at the same rate as cells in vitro, this number of cells would be attained in 14 more doublings, or <1 month from the initial presentation. This is not observed for any known tumor type. In fact, clinical evidence suggests that it takes on the order of 100 days for tumor volume to double (14). While the above theoretical argument discounts the role of the immune system in reducing tumor cell burden, the point it makes is still clear. Tumor mass accumulates considerably slower in vivo than in vitro. Factors and conditions innate to the in vivo tumor microenvironment (e.g. nutrient limitations, hypoxia, extracellular matrix interactions and autocrine inhibitory factors) probably contribute to these observations. Furthermore, a significant percentage of tumor cells inexplicably differentiate or lose the tumorigenic phenotype in situ (15,16). Thus, the end result is that several hundred doublings may be required for a single tumor cell to form a clinically relevant tumor.
In part because of the above argument, it has been suggested that tumor cells must acquire limitless replicative potential (13,8,17). Nearly all tumors contain a stem cell population that is immortal (discussed in 16,18). However, because the lifespan of cells in vivo is not known, the significance of an acquired limitless replicative potential is unclear. Unlike rodent cells, human cells are not easily immortalized. In addition, transfection of mutated oncogenes readily transforms rodent but not human cells. However, human cells can be immortalized and transformed by transfection with telomerase in combination with several oncogenes (e.g. SV40 large and small T antigen and Ras) (1923). This suggests that immortalization may have a causative role in carcinogenesis (19,20). In contrast, the observation that human cells can be immortalized in the absence of telomerase activity indicates that telomerase activity may be sufficient but is not always necessary for immortalization (24). Because there is no strong correlation between the length of telomeres and cancer susceptibly, the role of telomerase in carcinogenesis is not entirely clear. However, there is mounting evidence that ectopic expression of telomerase increases the rate of tumor formation even in cells where endogenous telomeres are not abnormally short (25). Moreover, up-regulation of telomerase promotes cell growth and stimulates wound healing (25). Based on this and other data, it has been suggested that up-regulation of telomerase may signal cells that the environment is conducive for proliferation (26).
Finally, in order to form a clinically relevant cancer, most tumor cells must acquire sustained angiogenesis and the ability to metastasize (13,8). Unlike the previous four acquired characteristics, the molecular and genetic pathways responsible for these phenotypes are much less thoroughly worked out. The working model of accumulation of specific somatic gene alterations detailed by Hahn, Weinberg and Hanahan is widely accepted by the scientific community and represents the current best mechanistic explanation for carcinogenesis.
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Crossroads: cancer questions without answers
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The evidence as a whole makes plain that some carcinogens induce somatic mutations whereas others do not, that some mutagenic agents fail to be carcinogenic, and that many substances closely related chemically to agents of both do neither. (Peyton Rous, 1959) (27)
The current working model for carcinogenesis is founded on the theory that somatic genetic mutations are the causative agents of carcinogenesis (13,28). Despite its innate elegance, the foundation of this view of carcinogenesis has been challenged because of its inability to easily explain a number of long-standing observations (9,2940). For example, somatic mutation theories of carcinogenesis have trouble explaining the relatively high incidence of human cancers. The current model suggests that on average four to ten somatic mutations are required for carcinogenesis (2,11,13,41). The mutation rate in normal cells is commonly assumed to be
106 per gene per cell division. Based on this rate, carcinogenesis would have a frequency of
1 in 10241060 cells. Since there are
1016 cell divisions in the course of a human lifespan, this would predict that only 1 in 1081044 people would get cancer. Clearly, this is not the case, and there is an evident need for a mechanistic explanation for this discrepancy.
The current model holds that the initiation of carcinogenesis is a mutagenic event incurred as the result of exposure to environmental carcinogens (4248). While true in many cases, this theory does not explain the tumorigenic activity of carcinogens that are not mutagens (e.g. polycyclic aromatic hydrocarbons and asbestos) (38,4953). However, it is important to note that many non-mutagenic carcinogens are converted to mutagenic compounds after metabolic activation or may indirectly mutagenize cells through the induction of reactive oxygen species. Nonetheless, many mutagens are not carcinogens (38,5053). Moreover, recent studies have demonstrated a lack of correlation between environmental carcinogens and the rate of somatic point mutations. Early observations suggested that carcinogens induce specific oncogenic mutations in cancer cells. Classic examples of these carcinogen fingerprints include reports that nearly 100% of dimethylbenzanthracene (DMBA)-initiated papillomas contained codon 61 mutations in H-Ras1 (54,55). Similarly,
85% of methylnitrosourea (MNU)-initiated mammary carcinomas showed specific codon 12 mutations in H-Ras1 (56). In addition, it has been reported that alfatoxin induces G
T transition mutations in p53 at codon 249 (57). The conclusion from these studies was that these carcinogens induced carcinogenesis through these specific mutations. However, technological advances have called into question the conclusion that carcinogen treatments were directly responsible for causing these mutations (5860). Examination of MNU-treated tissues revealed that carcinogen treatment neither increased the number of codon 12 mutations in H-Ras1 in situ nor the number of animals with mutations (61,62). Investigators concluded that the carcinogen treatment promoted the selective outgrowth of cells with pre-existing codon 12 mutations in H-Ras1 (61,62). This conclusion is supported by observations that melanoma risk increases with several sunlight-induced burns in childhood, but B-Raf, N-Ras and other melanoma-associated mutations are not signature UVB mutations (63,64). Moreover, it is unclear whether the spectrum of oncogenic mutations observed in smokers is significantly different from those observed in non-smokers (36,58,59,65). Thus, despite the observation that certain oncogenic mutations are frequently found in specific tumor types, it may be that these mutations are not directly induced by carcinogens (61,62,66). Rather, carcinogens may induce the selective outgrowth of cells that already contain these mutations.
In addition, the somatic mutation theory of carcinogenesis does not directly address the long latencies observed in the formation of human cancers. The development of clinically detectable cancers in humans frequently takes decades and in some cases can exceed 50 years (67,68). This is in sharp contrast to classical genetic studies where somatic mutations lead to a new phenotype within one to two generations. Furthermore, it is unclear why highly penetrant cancer susceptibility genes predispose individuals to only specific types of cancers (e.g. Bloom syndrome, Li-Fraumeni syndrome, neurofibromatosis and retinoblastoma) (reviewed in 69). Since these mutations are inherited and carried by all somatic cells, it is not known why tumors observed in these syndromes are tissue specific. Finally, the frequency at which oncogenic mutations are detected does not correlate well with observed cancer frequencies. In normal human skin epithelium there are 330 clones/cm2 consisting of cells containing p53 mutations (70). Because the tumor formation frequency is considerably lower than this, most of these mutations are insufficient for carcinogenesis (70). Moreover, while UV-induced p53 mutations are associated with skin cancers, patients with Li-Fraumeni syndrome, despite having germline p53 mutations, do not have elevated rates of skin cancers (71). In addition, the incidence of TEL-AML1 or AML1-ETO fusion translocations is 100-fold higher than the relative risk for the corresponding leukemia (72). In mouse skin 100% of DMBA-initiated papillomas possess mutated H-Ras oncogenes, but it is known from other studies that <10% of these papillomas progress to malignant carcinomas (54,73). From these data it can be inferred that Ras activation is insufficient for carcinoma formation. All of these observations are bolstered by a number of other reports indicating the presence of oncogenic mutations in normal non-tumorigenic tissue (7476). Taken together, these observations suggest that neither carcinogen exposure nor oncogenic mutations alone are sufficient for carcinogenesis.
In summary, the current working model for carcinogenesis has difficulty addressing several questions. Can the mutation of a small number of genes explain the formation of most human cancers? How do non-mutagenic carcinogens cause cancer? What accounts for the long latencies observed in human cancers? Finally, how is it that rodent cells are easily immortalized and transformed by the transfection of oncogenes while human cells are not?
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Genomic instability and carcinogenesis
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In attempting to answer these questions, a number of scientists are applying modern technological advances to re-evaluate the role of somatic mutations in carcinogenesis. For example, cancer profiling studies have revealed that no single somatic alteration is shared by all cancers (reviewed in 77). In fact, many cancers lack genetic mutations in any known oncogene or tumor suppressor gene. Even more confounding is the observation that expression of certain oncogenes can be reduced in tumor cells as compared with normal cells (78). Thus, it appears unlikely that solid tumor cancers overall will be defined by a particular molecular signature of mutated oncogenes and tumor suppressor genes. In the course of attempting to correlate a discrete set of mutations with specific cancers, it has become apparent that tumor cells themselves are not genetically or phenotypically homogeneous (15,16). Most tumor cells are aneuploid. Moreover, genomic profiling of cancer cells has proven very difficult because nearly all human solid tumor cancer samples represent a single time point yet cancers are genetically unstable (12,7986). The significance of genomic instability was first appreciated in 1914, when Boveri postulated that cancer arose from single cells as a result of wrongly combined chromosomes (87). Recently, a number of scientists have begun to examine genomic instability in a new light (9,2940,8899). Specifically, researchers have begun to ask are these massive genetic changes the consequence or the cause of cancer?
Tumor cells frequently exhibit gross chromosomal abnormalities and are highly aneuploid (12,79,8587). In contrast to specific genetic alterations, proponents of a theory of aneuploidy-driven carcinogenesis point out that aneuploidy is the most common genetic alteration observed in solid tumors (9,39,85,95,100103). Because both genotoxic and non-genotoxic compounds can generate aneuploidy, this may explain the carcinogenic nature of some compounds that are not mutagenic. Moreover, observations suggest that the degree of aneuploidy correlates well with the severity of the malignancy (reviewed in (9)).
Aneuploidy may promote cancer formation by altering the dosages of thousands of otherwise normal genes (9,39,95,99). Indicative of the importance of gene dosage, a number of tumor suppressor genes are haploinsufficient (reviewed in 104,105). In this case, the loss of only a single tumor suppressor allele is associated with cancer formation (reviewed in 104). Moreover, it has been suggested that aneuploidy and the accompanying genetic instability may explain the rapid appearance and disappearance of multidrug resistance in tumor cells (95,96). In addition, increased gene dosage in the absence of gene mutations is strongly associated with the formation of a number of cancers (reviewed in 106). Thus, proponents of an aneuploidy-driven theory of carcinogenesis suggest that disruption of gene dosage has a significant role in the initiation of cancer formation.
Aneuploid cells are inherently unstable (88). In an aneuploidy-driven theory of carcinogenesis, aneuploidy induced by carcinogens begets more aneuploidy (88) (Figure 1B). This autocatalytic chain reaction produces heterogeneous populations of cells with varying degrees of unbalanced gene dosages. The high degree of instability of aneuploid cell genomes may account for the high degree of heterogeneity in tumors and in cancer cells.
Final stage tumor cells barely resemble normal cells genetically, but endogenous mutation rates do not seem to be high enough to account for the number of mutations observed in cancer cells (reviewed in 107,108). Based in part on the observation that bacteria under stress display a hyper-mutable phenotype, Loeb proposed that a similar phenotype, termed the mutator phenotype, could account for the higher mutation rates observed in cancer cells as compared with their normal counterparts (reviewed in 107,108). Essentially, it was proposed that cancer cells contain thousands of mutations and that these mutations were responsible for the tumorigenic phenotype. In support of this, the use of comparative genomic hybridization and loss of heterozygosity assays have demonstrated that single cancer cells may contain thousands of mutations. More direct approaches have suggested that colorectal cancer cells may have 104105 mutations per cell (100102). Importantly, the genomic instability essential for accumulation of these large number of mutations appears very early during neoplastic transformation (49,90,92,101,109). Moreover, aneuploidy in pre-neoplastic lesions frequently indicates a high risk of malignant transformation (49,88,109111). However, few mutator genes have been discovered. Thus, aneuploidy itself may be responsible for a mutator phenotype (88) (Figure 1B). Regardless of its origin, a mutator phenotype that occurs early in carcinogenesis could also explain the highly pleiotropic genetic nature of malignant tumors.
Is genetic instability a cause or consequence of carcinogenesis? As yet there is no answer. However, if genetic instability were merely a consequence of carcinogenesis, then all cancer cells should have equivalent levels of genetic instability. This is not the case. Colon cancer cells with DNA repair deficiencies are genetically unstable at the DNA sequence level, but are stable at the chromosome level (99,112,113). In contrast, colon cancer cells without DNA repair deficiencies are stable at the DNA sequence level but lose chromosomes at a very high rate (99,112,113). Interestingly, the level and type of genetic instability found in cancer cells seems to be carcinogen-specific (91). Subsequently, cells that survive carcinogen treatments in vitro become resistant to the carcinogen that induced the genetic instability (91). This reflects the realities of failed anticancer therapeutics in the clinic and suggests that carcinogens create a selective pressure that promotes the outgrowth of adapted cancer cells. Some form of genetic instability is found in nearly every solid tumor cell. Because this instability is detected early in carcinogenesis, it may be closely associated with the initiation of carcinogenesis. However, it is important to note that transfection of the combination of small T antigen, large T antigen, activated Ras and telomerase can malignantly transform normal human cells without large-scale chromosomal instability (1923). This important observation is the subject of some debate (19,20,32,114,115); its significance will be bolstered by validation in other systems of whether this observation can be generalized to most cancers, oncogenes or cells used. In addition, under some circumstances in vivo murine tumors can be induced without widespread aneuploidy (116). Thus, the role of genomic instability in the initiation of carcinogenesis needs to be more fully investigated.
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Homeostasis and genomic instability: insights from model organisms
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What causes and propagates genomic instability? Results from a variety of model systems indicate that the maintenance of homeostasis between cell growth and proliferation is essential in preventing genomic instability. In nearly all somatic cells, cell growth (e.g. the addition of cell mass) is required for cell proliferation. Despite this, the mechanism that links cell growth to proliferation is still not well understood (117119). Because of this, the dissection of the genetic pathways that link cell growth to proliferation is a key goal in cell biology today.
Oncogenes and tumor suppressor genes are integral components of the cell cycle machinery that regulate proliferation (Figure 1). Research in a number of model systems indicates that oncogenes and tumor suppressor genes also control cell growth. For example, overexpression or up-regulation of Myc, Ras, Akt or PI3K or loss of function of PTEN, TSC1 or TSC2 stimulate cell growth (reviewed in 120123). Thus, mutation of these genes disrupts homeostasis by altering both cell growth and proliferation.
Research in mice, Drosophila, Arabidopsis and yeast has revealed that the genetic pathways that regulate cell growth are dependent upon G1 phase cyclin expression and are highly conserved (119,122129). G1 phase cyclins are required for commitment to cell division and their expression is strongly induced by mitogenic signals (reviewed in 130). This requirement links environmental stimuli to cell cycle progression to ensure that proliferation in vivo is tightly regulated.
A number of cancers up-regulate G1/S phase cyclins (e.g. cyclins D and E) and this up-regulation is implicated in the initiation of carcinogenesis (130139). Data from many laboratories indicate that overexpression of G1 phase cyclins disrupts homeostasis. Moreover, recent exciting data indicate that this loss of homeostasis generates a mutator phenotype (140142). Significantly, elevated G1 phase cyclin levels promote genomic instability (140,141).
In mammalian cells G1 phase cyclins help promote cell cycle progression by phosphorylating pRb (reviewed in 130,143). Similarly, in yeast G1 phase cyclins promote DNA replication by targeting an inhibitor of S phase cyclin-dependent kinases, Sic1, for degradation (142,144147). Importantly, loss of Sic1 or elevated G1 phase cyclin levels induces genomic instability by disrupting the fidelity of DNA replication (140142,148). In addition, G1 phase cyclins modulate spindle pole body duplication. Spindle pole bodies are the yeast equivalent of mammalian centrosomes. In this light, it is intriguing that damaged centrosomes are found in conjunction with chromosome instability early in carcinogenesis (90,92). These data suggest that cells overexpressing G1 phase cyclins may incur genetic instability as the result of centrosomal defects (90,92,140,141,148). In turn, this damage may drive carcinogenesis (Figure 1B).
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Chaos and cancer
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Cancer cells are extremely genetically and phenotypically complex. Genetic differences and resultant phenotypes can be subtle or enormous. Moreover, the behavior of tumors is not easily predicted. This is evidenced by observations that in some cases oncogene activation or tumor suppressor gene inactivation are associated with a good prognosis. For example, in neuroblastomas overexpression of HRAS and TRKA correlate with an increased probability of tumor regression (149). Similarly, up-regulation of Bcl-2 is often associated with a better clinical outcome (116,150). In addition, loss of p53 in transgenic mice expressing v-Fos or v-Hras inhibits tumorigenesis (151,152). Because of these and other observations, the development of generalized models of carcinogenesis that provide reliable and meaningful predictions has proved to be very difficult. In fact, the high degree of genetic heterogeneity has led some researchers to consider the unsatisfactory possibility that nearly every cancer arises via a unique mechanism (3). Alternatively, it has been suggested that the application of mathematical models of chaos theory to carcinogenesis may offer new perspectives for explaining the behavior of tumor cell populations (31,153157).
Mathematical modeling, and in particular chaos theory, is becoming recognized as an essential stepping stone towards a better understanding of carcinogenesis and other complex biological pathways (31,153157,200). Chaos theory is a mathematical attempt to characterize non-linear dynamic systems. Specifically, this theory deals with the manner in which simple systems respond to specific stimuli to yield complex outcomes (158). Elegant illustrations of chaos theory at work have demonstrated that the generation and propagation of complexity is not dependent upon complicated initial conditions (158). Rather, chaos theory purports that small perturbations of basic initial conditions can lead to enormous complexity. A key to chaos theory lies in the observations that the complex behavior derived by a system is self-ordering, i.e. while many complex systems appear to be completely random, they are in reality subtly and intricately organized. In other words, this theory states that chaotic behavior is really a complex order disguised as disorder (158).
Chaos theory may provide new clues to elucidating and exploiting the property of genomic instability in carcinogenesis to face two critical questions: what causes cancer and, once formed, how can tumors be eradicated? Valentino Braitenberg postulates that the analysis of complex systems like cancer may follow the law of uphill analysis and downhill design (159). By this he means that the road to complexity is essentially a one-way street (159). In this regard, it is much easier for small perturbations of simple systems (e.g. the disruption of cell cycle controls and homeostasis in a non-tumorigenic cell) to generate complex behavior (e.g. cancer) than it is to work backwards and deduce how the perturbations caused cancer (Figure 1B). Despite this difficulty, chaotic behavior is not random. Rather it possesses an innate order specific to self-replicating structures. One of the most confounding features of chaos theory is its ability to demonstrate self-similarity under any perspective. In this regard, the parallels with carcinogenesis are striking. On the surface, cancer cells appear to be so genetically disordered that their behavior is almost completely unpredictable. However, the discovery that tumors may contain cancer stem cells, suggests that cancer cells possess some degree of hierarchical order. In particular, examination of the phenotypic characteristics of breast tumor cells suggests that the evolution of cancer cells may obey chaos theory (15,31,153157). Specifically, despite the fact that the breast tumors studied were found to be highly genetically and phenotypically heterogeneous, Al-Haij et al. found that small numbers of cancer stem cells could be selected and propagated (15). Remarkably, this small subset of cells was capable of generating a population of cancer cells that were as genetically diverse as the original tumor (15). In other words, by regenerating tumor heterogeneity these cancer stem cells retain the ability to self-order. It is likely that this trait and the tremendous complexity observed in cancer cells are due to genomic instability. In the simplest sense, chaos theory demonstrates that small perturbations of simple initial conditions can generate highly complex systems that nonetheless behave in a probabilistic manner. Applying this concept to carcinogenesis, one could postulate that the genetic heterogeneity observed in tumors is not due to random disorder but rather results from the accumulation of numerous genetic and epigenetic alterations that are qualitatively or quantitatively sufficient to be selectively advantageous in the tumor microenvironment.
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Cellular adaptation in cancer
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Tumor cell adaptation is the process of genetic or epigenetic change that confers new properties conducive to tumorigenesis and malignant potential. The term adaptation has been applied to the process of explanted cells acquiring properties conducive to growth in culture, to microorganisms becoming resistant to toxins and antibiotics in their microenvironment and to unicellular and multicellular organisms becoming more able to withstand physical, chemical or biological changes in their environment. Adaptation has been used to describe cold-tolerant plant species, arsenic-induced skin cancer resistance, up-regulation of cell surface receptors to compensate for scarce ligand and prey/predator population fluxes. Thus, the term adaptation has been applied broadly to explain phenomena from the cross-species jump of new viruses to the origin of the species.
Just as somatic mutations alone are likely insufficient for carcinogenesis, adaptation of an evolving tumor cannot occur in the absence of selective pressures. Genetically unstable cells in the absence of selective pressures would rapidly disappear. Likewise, in the absence of genetic heterogeneity selective pressures are mute. Genetic studies in bacteria have helped elucidate the role of genetic instability in evolution. Under stressful conditions genetically unstable clones tend to predominate (160165). This is likely because genetic instability provides a greater opportunity to produce a phenotype that is selectively advantageous. Moreover, genetic instability is more common in pathogenic than non-pathogenic bacteria, suggesting that this phenotype may correlate with parasitic adaptation (166168). Thus, if a cancer cell were to be viewed as a foreign parasite, the tumor phenotype and its ability to adapt and survive in situ depends upon intrinsic and extrinsic factors. Intrinsic events include genetic instability and epigenetic events and extrinsic factors include the tissue microenvironment (epithelialstromalcell matrix interactions such as angiogenesis and immune recruitmentinflammation) and systemic factors (body responses to tumor burden and treatments). However, cancer cells are not foreign parasites; they are host cells whose interactions with their environment have gone awry. Small fluctuations in gene expression levels or protein signaling within the evolving tumor cells could allow adaptation and selective advantage within the tumor cell environment. Subsequently, with time and selection of genetically unstable cells, these epigenetic changes could become heritable. Together these intrinsic and extrinsic forces drive adaptation in tumor cells. In this process tumor cells escape differentiation programs, gain the ability to proliferate in the absence of mitogenic signals and become insensitive to factors which inhibit proliferation or promote apoptosis. All the while, they maintain the genetic functions essential for viability and for continued adaptation to the ever changing microenvironment of a tumor.
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Adaptation in cancer cells in vitro and in vivo
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The use of cell culture models to study carcinogenesis has helped reveal the role of selective pressures and adaptation during the evolution of cancer. Numerous studies using chemical and physical carcinogens to initiate carcinogenesis in rodent cells in vitro have demonstrated that transformation is extremely efficient. In fact, several reports have concluded that the observed transformation efficiencies were too high to be solely due to the induction of somatic mutations (29,37,169). Mouse NIH3T3 fibroblasts have been used extensively to model carcinogenesis in vitro. From these studies it has become very clear that culture conditions dramatically affect transformation frequencies (29,37,169). Spontaneous transformation rates are strongly dependent upon serum concentrations, passage number and cell densities (29,37,169). The observation that these factors affect a large number of cells suggests that adaptive changes are responsible. In rat liver cells transformation efficiencies are higher in untreated cells kept at high densities as compared with carcinogen-treated cells kept at low density (36,170). In addition, autocrine factors affect carcinogenesis and may selectively promote the outgrowth of cancer cells carrying out specific genetic mutations. For example, the inclusion of epidermal growth factor in fibroblast-conditioned medium reduces the frequency of Ras mutations observed in keratinocytes (171). Moreover, extracellular factors (e.g. calcium) that promote differentiation can affect the spectrum of oncogene mutations observed during in vitro carcinogenesis (171,172). These observations indicate that culture conditions can affect both the genotype and phenotype of cancer cells. This point is extremely important in the consideration of conclusions based on frequently used cancer cell lines. Cell lines propagated continually in culture are prone to significant genetic drift (173,174). This can cause problems when results from these cell lines are generalized to predict pathobiological behavior or pharmacological response of tumors to anticancer drugs. Cultured cells are particularly useful when their clonal origin is known and homogeneous cell lots are cryopreserved for multiple experiments. In addition, culture conditions should mimic the in vivo environment as closely as possible and support the normal physiological functions and properties of cultured cells.
The origin of selective pressures in the tumor microenvironment is less clear. However, tumors are assaulted by hypoxia, nutrient limitations, autocrine inhibitory factors and immune surveillance, any of which could apply selective pressures to a tumor cell population (175). The importance of the tumor microenvironment in driving tumor cell selection is exemplified by the observation that p53/ cells are selected for when angiogenesis is inhibited (176). As discussed above, mounting evidence suggests that a number of chemical and environmental carcinogens and tumor promoters function by favoring cells with a selective proliferation advantage. Because of this, clues to uncovering the mechanisms of these selective pressures may come from the re-evaluation of in vitro and in vivo chemical carcinogenesis experiments. In addition, the recent ability to turn off mutated oncogene expression in vivo has helped elucidate the consequences of adaptation in carcinogenesis. Using the conditional expression of mutated oncogenes in vivo, targeted inactivation of oncogene expression frequently results in tumor regression (reviewed in 177,178). However, more importantly, most tumors relapse despite the absence of expression of the initiating oncogene (reviewed in 177,178). In many cases genetic alterations or instability occur concomitant with tumor relapse. This suggests that tumor cells adapt such that they can survive in the absence of the initiating oncogene. These observations are significant in the light of the development and use of chemotherapeutic drugs designed to inactivate specific oncogene products. Therefore, the propensity for tumor cells to adapt and proliferate independent of the targeted oncogene must be taken into account for molecular targeted therapies to succeed.
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Implications for therapeutic cancer interventions
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Multiple pathways to cancer implies that each cancer could be different in its etiology and molecular disease characteristics and that individualized treatment is needed for cure. This is a daunting task in human disease. However, a more optimistic perspective is that multiple pathways contributing to any given cancer implies multiple options for molecular treatments to delay onset or extend regression of disease. Thus, when eradication is not possible, treatments that induce dramatic cancer regression may result in a significantly improved quality and duration of life.
The vast majority of human cancers have damaged cell cycle controls that disrupt cellular and tissue homeostasis. It is probable that the loss of homeostatic mechanisms induces genomic instability (Figure 1B). Subsequently, this genomic instability becomes self-propagating via destructive cycles and provides a medium for cellular selection and adaptation. Under these conditions cancers behave as complex adaptive systems and carcinogenesis becomes governed by chaos theory (Figure 1B). This results in a high degree of genetic heterogeneity within a single tumor. This high degree of heterogeneity virtually ensures that within a tumor population there are cells that are resistant to every known anticancer agent. However, it is also likely that this model of cancer can be used to design new treatments for cancer intervention (Table I).
The perspective that genetic instability induces destructive cycles that drive environmental selection and cancer cell adaptation during carcinogenesis offers two specific possibilities that might be exploited in cancer treatment (Table I). First, the combination of genetic mutation and chromosomal instability-induced gene expression changes may make damaged cancer stem cells overly dependent upon interactions with or factors from the host environment. Like normal stem cells, these tumor stem cell populations could be multipotent and capable of generating both true tumor cells as well as a camouflage of heterogeneous tumor-specific cell types with limited growth potential. However, the core tumor stem cell population may have a set of discernible characteristics that make it dependent upon a specific niche in the host microenvironment. In addition, the hyper-mutable phenotype of cancer stem cells would make these cells fundamentally different from endogenous normal stem cells. Put another way, hyper-mutability provides the capacity for populations to adapt, but lessens the fitness of individual cells.
The fitness versus adaptation capability of cells can be considered globally as well as at the level of particular drug interventions. Globally, tumor cells experience stresses from adaptation to a novel tissue environment (surmounted in successful invasion and metastasis) and from host systemic factors, including nutrition, stress-induced neuropeptides and hormones, drugs and other therapies. The host experiences stresses imposed by tumor burden, toxic therapy and disease-related psychological states. Attention to the host environment at each of these levels during chemotherapy may modulate tumor cell response and cancer recurrence by affecting cellular membrane lipid content, signaling pathways and drug transport. For example, chemotherapy response and cancer metastasis were affected in animals by light/dark exposure (179) and social isolation stress (180). ATP-binding transporters affect chemotherapy (181). The lipid microenvironment, e.g. cholesterol, affects P-glycoprotein activity and chemotherapeutic drug resistance by cell membrane conditioning effects on multidrug resistance family membrane protein pumps (182). Lipid-lowering drugs have been associated with lower melanoma incidence and hint at applications in chemoprevention (183). Of major importance in the control of recurrence, the immune status is nonetheless a prime targeted bystander of chemotherapeutic toxicity. More approaches are needed to understand how subsets of immune cells recover after chemotherapy (184) and bolster tumor-specific immunocompetence by timely activation or restoration of specific immune cell subpopulations (185). All these factors, cytokine, hormonal and nutritional, could be manipulated to tip the stress balance within the tumor microenvironment to target its narrow window of fitness compared with normal cells and circumvent its ability to adapt.
At the level of particular drug interventions, cancer cells have fewer fail-safes and unlike normal cells rarely stop proliferating to repair genetic damage (186). Thus, proliferative signals can induce cancer cells but not normal cells to undergo apoptosis (reviewed in 187). Examples of this include caffeine and the PKC inhibitors UCN-01, GÖ6976 and staurosporine, which can abrogate cell cycle arrest and promote proliferation leading to lethality in genetically damaged cells (188192). Moreover, two pharmacological agents, flavopiridol and butyrolactone, have been shown to repress the Cdk inhibitor p21, which increases G1 phase Cdk activity and sensitizes these cells to apoptosis (193,194). In addition, hyper-expression of many genes that promote proliferation also promotes apoptosis, supporting the hypothesis that inappropriate proliferation can result in cell death (reviewed in 187,195). Thus, it may be possible to specifically target death of tumor stem cell populations. The above data suggest that the efficacy of DNA-damaging anticancer agents could be increased if they were administered along with agents that promote tumor cell proliferation (discussed in 191,192,196,197). In this scenario normal cells with intact cell cycle controls would not proliferate. In contrast, the combination of multiple DNA damaging agents with an agent promoting proliferation could selectively increase the sensitivity of tumor cells with irreparable DNA damage to undergo apoptosis (Table I). In addition, therapies could be designed to selectively disrupt tumor stem cell-specific niches. This could be done by using agents that target specific host factors upon which tumor cells are dependent. In either case, therapies that deliberately disrupt tumor stem cellhost interactions should effectively reduce tumor cell burdens by inducing apoptosis only in the genetically damaged cells.
In addition, we propose that adaptation by cancer cells sets them apart genetically and phenotypically from non-adapted tumor cells (Table I). For example, prolonged chemotherapy promotes the selective outgrowth of resistant tumor stem cells since non-adapted cells have been killed. While the selective pressure applied by the chemotherapeutic agent has not been successful in killing all cancer cells, it has successfully reduced the genetic and phenotypic heterogeneity of the tumor. By lowering the complexity of the tumor, the treatment has temporarily reduced the number of genetic variants and the adaptive potential of the population. Moreover, studies from experimental genetic organisms indicate that genomes are buffered by genetic redundancies. Functionally redundant genes perform similar functions such that only the loss of two or more genes results in cell death. Genomes that have lost or inactivated only one such gene or only one allele of a critical gene are less genetically buffered and become compromised. Because of this, cells with compromised genomes are more sensitive and susceptible to agents that target the second gene than are normal wild-type cells. Thus, chemotherapeutic-driven adaptation in vivo may make adapted cells more vulnerable than the initial population to challenge by a second agent. This strategy has been used in yeast to selectively kill cells with compromised genomes (198,199). In a like manner, it may be practical to discover synergistic chemotherapeutic agents (reviewed in 191,196,197). Through the use of in vitro and in vivo models of carcinogenesis, the therapeutic scheme can be developed to take advantage of the ability of tumor cells to rapidly adapt. Prolonged treatment of cancer cells with agent A will inevitably result in the selection of resistant variants. However, based on this prediction and on the loss of heterogeneity that results with the selection process, it may be possible to identify a second agent B to which the resistant cancer cells, but not normal cells, are now exquisitely sensitive, followed by C and so on. In addition, a more generalized scheme may also prove successful. Multiple anticancer agents could be delivered individually and randomly for short intervals, whereby the selective pressures vary too rapidly to allow for intervening outgrowth of resistant cells (Table I). Regardless of which perspective proves most compatible with favorable cancer outcomes, it is apparent that new ways of looking at cancer evolution will reveal numerous potentially exciting anticancer therapies on the horizon waiting to be tested.
In summary, genomic instability allows numerous genetic and epigenetic alterations to accumulate during carcinogenesis without markedly changing phenotype until they are qualitatively or quantitatively sufficient to be selectively advantageous in the tumor microenvironment. Moreover, chaos theory may be useful in understanding the causes of carcinogenesis and the steps necessary for the eradication of cancer. Importantly, recent studies of adaptation in unicellular organisms and eukaryotic cancer model systems in vivo and in vitro are broadening our perspective and opening new avenues for cancer treatment.
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Acknowledgments
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We thank Colette Schneider, Jody Markwardt, Tim Bowden, Jim Hutson and Simon Williams for helpful discussions and comments. B.L.S. is supported by grants from the American Heart Association, The CH Foundation, the Wendy Will Cancer Fund, the Houston Endowment Incorporation, the South Plains Foundation and Texas Tech University Health Sciences Center. M.K.M. is supported by grants CA98577 and CA98893 and the OHSU Cancer Institute grant CA69533.
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Received March 11, 2004;
revised May 25, 2004;
accepted May 27, 2004.