The origin of oncogenic mutations: where is the primary damage?

Harald B. Steen

Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, N-0310 Norway E-mail: h.b.steen{at}labmed.uio.no


    Abstract
 Top
 Abstract
 Introduction
 The model
 Discussion
 References
 
Cancer is generally believed to arise from a single cell which has become `initiated' by mutation of a few crucial genes, caused by random `hits' in its DNA, a `hit' being an error in DNA replication or a reaction of the DNA with free radicals or other chemical species of exogenous or endogenous origin. It is not obvious how the epidemiological data on cancer incidence can be interpreted within the framework of this paradigm. For example, it cannot account quantitatively for the age dependence of cancer incidence, or for the fact that the incidence of cancer in people with hereditary mutations in tumour suppressor genes is much lower than expected, or for the observation that while in some types of cancer, like colon and pancreas, certain highly oncogenic mutations, such as that of TP53, are prevalent, there is no significant increase in the incidence of these cancers in people who carry the mutations by heredity. It is argued here that although mutations in such genes appear to be of crucial importance in carcinogenesis they may not be the rate limiting events in common cancer. The epidemiological data are consistent with the hypothesis that the rate limiting processes involve large numbers of cells. Conceivably, the mutations directly underlying neoplastic transformation may be the result of a local collapse in the system of intercellular processes on which the stability of the normal genotype and phenotype depends, and thereby trigger a cascade of mutations, among them the highly oncogenic ones. This local collapse may be due to mutations of many different genes in many cells as well as to other factors affecting the integrity of tissue.


    Introduction
 Top
 Abstract
 Introduction
 The model
 Discussion
 References
 
It is generally acknowledged that mutation has a key role in carcinogenesis. During the last decade a number of genes have been associated with carcinogenesis, either as oncogenes or tumour suppressor genes. (In the following, these genes, i.e. genes which may yield oncogenic mutations, will be denoted `oncogenic genes'.) Studies of hereditary cancer seem to show that in some cases the loss of both alleles of a single tumour suppressor gene may be sufficient to cause cancer, the classical case being retinoblastoma (Rb) (1). In general, however, the connection between mutation and cancer appears to be more complex.

Epidemiological studies are consistent with the idea that cancer is due to a small number of discrete events. Armitage and Doll (2) calculated from the age dependence of the mortality of several of the most common types of cancer that the number of such events is five to seven. A similar, more recent study, based on more extensive statistics and covering a broader spectrum of cancers, found the majority of cancers in the range four to eight events with extremes of three and 12 (3). It should be emphasized that in these studies the conclusions are based on the assumption that the oncogenic events are rate limiting with regard to cancer incidence. Genetic and molecular studies have identified these events as mutations within a limited set of genes, i.e. the oncogenic genes. Thus, there is a general consensus that cancer arises from a single, neoplastic cell, and that the reason for the initiation of this cell is mutation of a few crucial genes. The oncogenic mutations may be the result of free radicals or other chemical species, of exogenic or endogenic origin, reacting with the DNA of the cell, i.e. by `direct hits' causing irreparable malfunction of the respective genes. They may also result from replication errors. Such initiating events are assumed to hit essentially at random, although some cells may be more exposed than others, and genes may differ somewhat in their susceptibility to mutation. In any case it is taken for granted that the primary damage causing mutation occurs in the cell which eventually becomes the origin of the malignant clone. In other words, that the rate limiting processes in the initiation of cancer take place in that very cell. It is essential in discussing these matters to realise that although mutations of oncogenic genes may be crucial in carcinogenesis, they are not necessarily rate limiting, as is a tacit assumption in many studies and considerations of cancer causation.

Although a great variety of mutagens, including chemical substances and different types of radiation, have been identified, the mechanisms leading to the oncogenic mutations are not fully understood. The currently accepted model implies that the oncogenic mutations, which lead to the initiation of carcinogenesis, are essentially independent of interaction with other cells, although the subsequent development of the malignant phenotype, i.e. the `promotion phase`, may depend on a variety of extracellular factors, such as hormones, immunological compatibility, stroma interactions, etc. Thus, the hypothesis that cancer originates from a single cell which has become `initiated' by mutation of a few crucial genes caused by random `hits' in its DNA, has become a paradigm of current cancer research. In the following it is shown that this paradigm does not seem to be in accordance with epidemiological data. These data rather suggest that processes involving intercellular interactions may be rate limiting also in the initiation phase of carcinogenesis.

In the present discussion I am concerned with the bulk of cancer cases, that is what may be called common cancer, which appear to arise spontaneously with no obvious reason, although exogenic and/or endogenic carcinogens are likely to be involved. My arguments do not necessarily apply to cases associated with heavy exposure to carcinogenic radiation or chemicals.


    The model
 Top
 Abstract
 Introduction
 The model
 Discussion
 References
 
As a framework for this discussion we consider the following model which contains the above paradigm as a special case. Thus, we assume a system, i.e. a tissue or organ, consisting of Nc compartments, each containing n cells, each cell with c targets, e.g. oncogenic genes. We assume further that this system is being hit at random by discrete, independent events at a rate s per target and unit time, and that a certain number, x, of such initiating events among the n·c targets in any one of the Nc compartments will result in a neoplastic cell which eventually may develop into cancer. In terms of the current paradigm each compartment is one cell, i.e. n = 1, s is the rate of mutation and c is the number of oncogenic genes per cell. (A gene in this context may be just one allele of a gene.) Assuming that the initiating events occur randomly we apply Poisson statistics to calculate the probability, p (x), that one compartment shall have x or more hits in the c targets within a period of time, t

where s is the probability of one hit per target and unit time. Since µ << 1, eqn 1Go reduces to:

The probability that none of the Nc compartments will experience x or more hits is:

The approximation being valid since Nc·p (x) < 1. Hence, the probability, P (x), that any one of the Nc compartments will experience x or more hits, i.e. that an initiated cell will occur in the system is:

To see the consequences of the current paradigm we put n = 1, which is to say that each compartment contains just one cell, and assume initially that c = x, which is to say that oncogenic transformation requires a unique combination of mutations, as seems to be the case for retinoblastoma. (This assumption is probably not valid for most types of cancer. However, it simplifies the calculations and, as we shall see, it makes essentially no difference to the conclusions as long as c is not a very large number.) If we apply this to a hypothetical organ containing Nc = 1x1010 cells with an oncogenic potential (i.e. roughly the number of cells in 10 g tissue), P(x) = 0.03, s = 3x10–7/gene/cell/year, and t = 50 years, we calculate from eqn 4Go that x {approx} 2.4. (If we assume c = 5x the estimate of x increases to 2.7.) This estimate is rather on the high side since it is assumed that every initiated cell, that is every cell with x oncogenic mutations, gives rise to an observable cancer. If the promotion phase is rate limiting at all the value of x must be smaller. But not necessarily very much; for example, if one in every 10 initiated cells gives rise to cancer, x is reduced only from 2.4 to 2.2, and if it is one in 100, x {approx} 2.0.

Of course, the values of the various parameters that go into this calculation are order of magnitude estimates which may vary significantly between different organs, etc. However, the value of x is not very sensitive to variations in these parameters. For example, changing the value of P(x) or Nc by a factor of 10 alters x by <10%, while a similar change of s, c or t gives a change in x of ~20%. Hence, we see that, even if one takes into account that the estimates of the basic parameters, i.e. rate of mutation, the number of potentially oncogenic cells, etc., may be off by an order of magnitude or more, and that the promotion phase may be rate limiting, the current paradigm leads to a number of primary oncogenic events which is incompatible with the epidemiological data referred to above (2,3).

On the other hand, if we accept the average epidemiological estimate of x = 6 the current paradigm leads to an excessively high rate of mutation. Thus, with n = 1 eqn 4Go yields a rate of mutation s {approx} 1.2x10–4/gene/cell/year. This is comparable with values achievable in in vitro assays with highly potent mutagens applied in concentrations significantly reducing cell survival, and therefore seems unrealistically high as a general rate of mutation in humans. Estimates of in vivo rates of `spontaneous' mutation fall in the region 1x10–7 to 5x10–6/gene/cell/year (4,5). Alternatively, the value of c would have to be quite large, i.e. in the order of c {approx} 2400, which is to say that virtually every active gene of the cell would be oncogenic, again in contradiction to the current paradigm. Hence, this paradigm (eqn 4Go with n = 1) cannot account for the age dependence of the incidence of most types of common cancer.

The discrepancy may be resolved if we assume that n >> 1, which is to say that each compartment contains not just one, but many cells. For example, if we take x = 6, in accordance with the epidemiological data, and the other parameters as above, eqn 4Go yields n·c {approx} 2400. In calculating that number we assume that the number of compartments equals that of cells (Nc = N), which implies that the compartments are overlapping, i.e. that every cell interacts with many others. (Assuming non-overlapping compartments, i.e. Nc = N/n, yields n·c {approx} 8000.) Hence, it appears that the data are consistent with a model which assumes that the events leading to a neoplastic cell need not to be confined to that very cell, but may occur within a relatively large number of its neighbours, and/or that the number of targets, i.e. oncogenic genes, is much larger than indicated by molecular studies.

Another serious discrepancy between the current paradigm and the epidemiological data appears when we consider the increase of cancer incidence in people carrying a hereditary mutation in one of the c oncogenic genes, such as Li–Fraumeni patients. From eqn 4Go we may calculate the magnitude of this increase, that is the risk ratio Q, assuming that every cell has a handicap of one event, i.e. mutation in one of its oncogenic genes, so that the number of events required to initiate cancer is reduced by one:

The current paradigm, that is, taking: n = 1, and the other parameters as above, gives: Q {approx} 7x104. Allowing for the age difference, this value is in accordance with that observed for retinoblastoma in children (1). However, it is very much higher than what is usually observed with hereditary mutations linked with common types of cancer, such as mutations in the TP53 gene or the BRCA1 gene, for which it is typically within a factor of 100 (6), and for most cancers much below that.

Again, the discrepancy can be resolved by assuming that n·c is large, i.e. in the order of several thousand. For example, assuming Q = 100 and the other parameters as above, eqn 5Go yields n·c = 4000. Hence, again we are led to conclude that the primary events may take place in other cells than the one which eventually becomes initiated, which implies that intercellular interactions may have a crucial role in the etiology of common cancer.

It may be argued that an alternative explanation for the relatively low incidence of cancer in people carrying hereditary oncogenic mutations may be that these mutations cause only a moderate increase in the general rate of mutation, i.e. a degree of genomic instability, for example mutations of genes controlling the fidelity of DNA replication, such as believed to be one function of TP53, and the mismatch repair genes, or genes for enzymes which metabolize certain carcinogenic molecules, etc. It turns out, however, that if a mutation of one of the oncogenic genes has such an effect on genomic stability the result would be rather the opposite. To illustrate this, we rewrite eqn 4Go to allow for the assumption that different genes may have different rates of mutation:

and the similar expression for the case with a hereditary mutation in one of these genes (otherwise with mutation rate s1) so that the mutation rate of the other genes are enhanced:

and take the ratio between the two:

Since s' > s, due to the genomic instability caused by the hereditary mutation, the last ratio is larger than unity and the value of Q', therefore even larger than that calculated from eqn 5Go. The same argument holds true if the mutation which increases the probability of subsequent mutations occurs later in the sequence, although its effect will be smaller. Hence, if TP53, the mismatch repair genes (7), and other tumour suppressor genes associated with genomic stability, are to be included in the set of the c oncogenic genes, and that assumption is indeed the basis for much of the current interest in this area, the possible effect of mutation of these genes on genomic stability cannot account for the observations; in contrast, it makes the discrepancy with the current paradigm even larger. This argument also applies to the case where the first, or one of the first, mutations gives rise to a clone with increased proliferation, so that partly initiated cells multiply faster than normal. Thus, in the above model (equations 68GoGo) such an increase is equivalent to an increase in the rate of mutation, s, and therefore would yield the same result as increased genomic instability, namely to increase the discrepancy with the current paradigm rather than reduce it.


    Discussion
 Top
 Abstract
 Introduction
 The model
 Discussion
 References
 
The present model encompasses, as a special case, the current paradigm, namely that cancer originates from a single cell which has become `initiated' by mutation of a few crucial genes caused by random `hits' in its DNA. The above calculations show that this paradigm does not seem to be in accordance with the epidemiological data. On the other hand, compatibility with these data is obtained if it is assumed that the primary events may take place not only in the cell which becomes initiated, but within a large number of cells, and affecting a larger number of genes.

The low number of primary events, i.e. the value of x, which results when we apply the rate of spontaneous mutation in eqn 4Go, is not only in disagreement with the estimates based on epidemiological data, it is also in contrast with the multiple mutations and chromosome aberrations seen in most cancers, although it is not known at which stage in the process of malignant transformation this damage occurs. An explanation highlighted by Loeb (8) is that the neoplastic cell acquires a `mutator phenotype' early in its development. There is ample evidence of such genomic instability in cancer, and several genes which confer such instability upon mutation have been identified (9). Mutation of these genes can be a heritable trait, which is associated with a higher than normal incidence of some types of cancer. As shown above, however, to bring this hypothesis in agreement with the epidemiological data we have to assume that the primary events are distributed over many cells and possibly many genes.

One interpretation of the observation that the relative risk ratio for people with a hereditary mutation in a tumour suppressor gene is much lower than that calculated from eqn 5Go, is that that mutation is not a primary rate limiting factor in the incidence of common cancers. However, that is not to say that this mutation cannot be crucial in the etiology of such cancers. Thus, if the rate limiting event is a process which raises the rate of oncogenic mutation radically, it will be that process, and not the ensuing oncogenic mutations, which is rate limiting, although the oncogenic mutations may still be a prerequisite for the development of the neoplastic cell. This may explain why hereditary mutations in tumour suppressor genes, like TP53, do not cause significant increase of the incidence of many cancers, like colorectal and pancreatic, although mutations of these genes are very common in such cancers, and appear to play a definite role in their etiology (10,11).

What, then, could be the nature of the rate limiting events which induce the oncogenic mutations that give rise to the neoplastic cell? The above considerations suggest that, typically, such events involve large numbers of cells, which is to say that collective, intercellular processes, or rather the interruption of such processes, may be essential in the initiation of carcinogenesis of common cancer in man. Numerous in vitro studies demonstrate that a variety of extracellular signals contribute to the control of gene expression, differentiation and proliferation. This variety includes diffusive chemical factors, like hormones and growth factors, electrochemical interactions, notably through Ca2+-mediated via gap junctions, and mechanical and structural interactions mediated through the anchorage of cells to the extracellular matrix and to adjacent cells (12). In this context it may be relevant that at least one tumour suppressor gene product, i.e. that of the APC gene, is extra-nuclear and appears to have a crucial role in transmittance of signals from the cell exterior and into the nucleus (13). Moreover, these mechanisms may be coupled. For example, it has been shown that mechanical tension applied to a cell in a confluent cell culture may trigger a wave of Ca2+ which may be transmitted over distances of many cell diameters (14,15). Vice versa, Ca2+ is known to affect the polymerization/depolymerization of cytoskeleton components responsible for the mechanical forces exerted by cells on their surroundings. Such coupling provides a basis for an extended network of feedback loops. In more general terms, the organism represents a highly complex network of information flow where proper function and stability of each element is dependent on a multitude of appropriate signals from many other elements. Thus, it may be argued that essentially every gene is part of this network, on the grounds that any gene product which is not essential to the organism, i.e. takes part in the network, would disappear in evolution. Although this may be an over-simplification, it constitutes a rationale for the assumption that a large number of genes are involved in the maintenance of the integrity of a tissue which in turn is a condition for the maintenance of genomic stability. A consequence of this assumption is that a mutation in any gene of any cell may, however little, contribute to a degradation of the integrity of the system until eventually it reaches a point where it is no longer able to keep every cell in check. The result of such degradation could be a local collapse of the system, leading to a drastic increase of the probability of an initiating oncogenic event in that locality. Thus, the rate limiting events may be mutations in virtually any of the genes which contribute to the integrity of the tissue, rather than direct `hits' in the individual oncogenic genes. This is not to say that cancer does not originate from a single cell, but that the malignant transformation of that cell may be a secondary result of many mutations and possibly other types of damage in a large number of neighbouring cells. Thus, not only mutation, but any type of damage that may degrade the cellular environment over a significant period of time, may increase the probability of an initiating event. This may be a rationale for the carcinogenic effect of some of the carcinogens which do not seem to be mutagenic.

Cancer cells are generally characterized not only by mutations of specific oncogenic genes, but a diverse spectrum of chromosome aberrations (16). Malignant tumours display a variety of major deletions and amplifications, each of which affects stretches of chromatin typically containing hundreds or thousands of genes. Although there is a tendency to clustering on certain chromosomes, these aberrations, on average six to eight per cell in cancers like breast (17), non-Hodgkin's lymphomas (18) and colon (19), are generally spread over the entire genome, with significant variation between individual tumours and also tumours of the same type. Such data demonstrate both the commonness of chromosome aberrations in cancers and the essentially random nature of the processes by which they occur. Conceivably, the `local collapse' may lead to a significant increase in the probability of chromosome aberrations. Aberrations are believed to destabilize the chromatin and further increase the probability of new aberrations (20,21). Thus, the `local collapse' may trigger a cascade of mutations, among them the highly oncogenic ones.

If the present conclusion, namely that collective effects play a significant role in the initiation phase of carcinogenesis, can be substantiated it implies new perspectives both in prevention and in treatment of cancer.


    References
 Top
 Abstract
 Introduction
 The model
 Discussion
 References
 

  1. Knudson,A.G. (1971) Mutation and cancer: A statistical study of retinoblastoma. Proc. Natl Acad. Sci. USA, 68, 820–823.[Abstract]
  2. Armitage,P. and Doll,R. (1954) The age distribution of cancer and a multistage theory of carcinogenesis. Br. J. Cancer, 8, 1–12.[ISI]
  3. Renan,M.J. (1993) How many mutations are required for tumorigenesis? Implications from human cancer data. Mol. Carcinog., 17, 139–146.
  4. Bridges,B.A., Cole,J., Favor,J., Glickman,B.W., Mohrenweiser,H., Sankaranarayanan,K. and Skopek,T.R. (1994) Spontaneous mutation and its place in risk assessment for chemical mutagens. Report of an ICPEMC committee. Mutat. Res., 304, 3–11.[ISI][Medline]
  5. Simpson,A.J.G. (1997) The natural somatic mutation frequency and human carcinogenesis. Vande-Woude, G.F. and Klein, G. (eds) Advances in Cancer Research. Academic Press, New York, 209–240.
  6. Bishop,D.T. (1996) Genetic predisposition to cancer: an introduction. Eeles,R.A., Ponder,B.A.J., Easton,D.F., Horwich,A. and McVie,G. (eds) Genetic Predisposition to Cancer. Chapman & Hall Medical, London, pp. 3–15.
  7. Loeb,L.A. (1994) Microsatellite instability: marker of a mutator phenotype in cancer. Cancer Res., 54, 5059–5063.[ISI][Medline]
  8. Loeb,L.A. (1991) Mutator phenotype may be required for multistage carcinogenesis. Cancer Res., 51, 3075–3079.[ISI][Medline]
  9. Lengauer,C., Kinzler,K.W. and Vogelstein,B. (1998) Genetic instabilities in human cancers. Nature, 396, 643–649.[ISI][Medline]
  10. Lundin,J., Nordling,S., von Boguslawsky,K., Roberts,P.J. and Haglund,C. (1996) Prognostic value of immunohistochemical expression of p53 in patients with pancreatic cancer. Oncology, 53, 104–111.[ISI][Medline]
  11. Breivik,J., Lothe,R.A., Meling,G.I., Rognum,T.O., Børresen-Dale,A.L. and Gaudernack,G. (1997) Different genetic pathways to proximal and distal colorectal cancer influenced by sex-related factors. Int. J. Cancer, 74, 664–669.[ISI][Medline]
  12. Weaver,V.M., Petersen,O.W., Wang,F., Larabell,C.A., Briand,P., Damsky,C. and Bissell,M.J. (1997) Reversion of malignant phenotype of human breast cells in three-dimensional culture in vivo by integrin blocking antibodies. J. Cell Biol., 137, 231–245.[Abstract/Free Full Text]
  13. Ilyas,M. and Tomlinson,I.P. (1997) The interactions of APC, E-cadherin and beta-catenin in tumour development and progression. J. Pathol., 182, 128–137.[ISI][Medline]
  14. Sanderson,M.J., Charles,A.C., Boitano,S. and Dirksen,E.R. (1994) Mechanisms and function of intercellular calcium signaling. Mol. Cell. Endocrinol., 98, 173–187.[ISI][Medline]
  15. Sneyd,J., Keizer,J. and Sanderson,M.J. (1995) Mechanisms of calcium oscillations and waves: a quantitative analysis. FASEB J., 9, 1463–1472.[Abstract/Free Full Text]
  16. Mitelman,F., Mertens,F. and Johansson,B. (1997) A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nature Genet., 15, 417–474.[ISI][Medline]
  17. Tirkkonen,M., Tanner,M., Karhu,R., Kallioniemi,A., Isola,J. and Kallioniemi,O.P. (1998) Molecular cytogenetics of primary breast cancer by CGH. Genes Chromosomes Cancer, 21, 177–184.[ISI][Medline]
  18. Monni,O., Oinonen,R., Elonen,E., Franssila,K., Teerenhovi,L., Joensuu,H. and Knuutila,S. (1998) Gain of 3q and deletion of 11q22 are frequent aberrations in mantle cell lymphoma. Genes Chromosomes Cancer, 21, 298–307.[ISI][Medline]
  19. Angelis,P.M., Clausen,O.P.F. and Stokke,T. (1999) Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumor DNA ploidy, genotypes and phenotypes. Br. J. Cancer, 80, 526–535.[ISI][Medline]
  20. Cram,L.S., Bartholdi,M.F., Ray,F.A., Travis,G.L. and Kraemer,P.M. (1983) Spontaneous neoplastic evolution of chinese hamster cells in culture: multistep progression of karyotype. Cancer Res., 43, 4828–4837.[Abstract]
  21. Kuukasjarvi,T., Karhu,R., Tanner,M., Kahkonen,M., Schaffer,A., Nupponen,N., Pennanen,S., Kalliomiemi,A., Kalliomiemi,O.-P. and Isola,J. (1997) Genetic heterogeneity and clonal evolution underlying development of asynchronous metastasis in human breast cancer. Cancer Res., 57, 1597–1604.[Abstract]
Received December 16, 1999; revised May 17, 2000; accepted June 21, 2000.