Journal of Oncology and Cancer Research (JOCR) The Epigenetic Theory of Carcinogenesis: p53 as the Guardian of The Epigenome

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INTRODUCTION Basic Properties of Malignancy
The necessary and sufficient cellular properties conferring malignancy are proliferative capacity and the ability to transgress tissue boundaries.In animals these migratory properties exist in embryonic cells and are essential for development.Normally these attributes are expressed in a controlled manner and are switched off during the developmental process to yield the morphology of the fully developed organism.Given this migratory requirement, there are three ways by which malignancy can arise: (1) If transmigration is not controlled during embryogenesis, it leads to developmental malignancies such as retinoblastoma, neuroblastoma, and nephroblastoma; (2) In cell lineages that retain the transmigratory capacity, such as leucocytes, fully differentiated white cells are prevented from proliferating outside the confines of the hemopoietic compartments.A fault in the normal restriction of proliferation will lead to hematological malignancies.Since this route involves mechanisms that regulate proliferative capacity rather than transmigration it may account for the fact that the genesis of hematological cancers differs in some essential respects from that of other malignancies; (3) In adult tissues the acquisition of transmigratory property by proliferating stem cells results in cancer.This route accounts for over 90% of human malignancies.

Adult cancers
If we confine our attention to adult malignancies, we may enquire how the acquisition of the transmigratory property might occur.Clearly one possibility is that it is the result of one or more mutations influencing the expression of a set of genes involved in the control of cellular migration, for example the reexpression of migratory genes operative during embryogenesis.This might be expected to be a rare occurrence given the relatively low mutation rate in normal tissues, but there is much evidence to suggest that cancer cells and their precursors exhibit a high degree of variability.

Diagnostic features of cancer cells indicate a profound disturbance of genetic control
Among the most notable diagnostic features of cancer cells is their cytological abnormality.This manifests itself as a chaotic assortment of abnormal structural and functional features including bizarre morphology, altered cytoplasmic staining patterns, and chromosomal anomalies.The cytological features of malignant neoplasms include: • Variation in nuclear or cell size (pleomorphism).
• Increased nuclear size with an increased nuclear/cytoplasmic ratio.The general appearance, therefore, seems to be of a broad range of mutations producing a wide variety of cells with abnormal gene expression.Indeed, the range of genetic abnormalities associated with cancer has been shown to be extremely extensive [13] .
The generally accepted view hitherto has been that this is the outcome of somatic evolution involving hypermutability and/or clonal selection, but the extent and range of the cellular abnormalities point to a much more profound disturbance generating a chaotic assemblage of gene expression.
Were a raised mutation rate responsible for the multiple structural and functional abnormalities of cancer, it would be anticipated that the immune system would readily recognize the abnormal gene products and eliminate the aberrant cells.Yet a remarkable feature of cancer biology is that the immune system fails to react to the presence of the grossly deranged cells and special measures are required to elicit a response.However, if the gene products expressed were 'self' components it would account for the lack of intrinsic immune response.
The proposed alternative explanation for the multiple abnormalities of cancer cells is that they arise from a defect in the control of the pattern of gene expression, i.e. that they result from a somatically inherited epigenetic derangement.In brief, the basis of the Theory of Epigenetic Carcinogenesis is that the process of epigenetic control is deranged in affected cells and that the process of initiation involves mutations in stem cells which generate progeny with faulty epigenetic transmission [11] .
The epigenetic pattern governs the expression of genes and is responsible for maintaining the stable differentiation of cell populations with different characteristic properties.Derangement of the transmission of the epigenetic pattern will result in anomalous gene expression and will permit increasing disorder of gene expression in the progeny, some combinations of which endow the cells with malignant properties.

Mechanistic considerations
If we accept the argument that the multiple defects detectable in malignant and pre-malignant cells are the result of errors in transmission of the epigenetic pattern, it would be anticipated that the primary lesion in carcinogenesis involves mutations of genes involved in this process.The mechanisms implicated in epigenetic pattern transfer involve a wide range of activities including DNA methylation and modification of histone structure.A substantial number of genes control these functions and a detailed survey of possible epigenetic transmission genes has identified a number of candidates that have been found to be mutated in cancer cells [8] .Also, it is argued that if the primary defect affects epigenetic mechanisms it would be associated with a wide range of epigenetic abnormalities, as observed [3,4,14] .

p53 'The Guardian of the Epigenome'
Given the catastrophic consequences in multicellular organisms of the expression of an anomalous genetic pattern by tissue stem cells, it is highly probable that an authentication mechanism is in place to safeguard the accurate transfer of the epigenetic pattern.A prime candidate for such a role is p53.The p53 system might be viewed as fulfilling the role of "guardian of the epigenome" -to paraphrase the expression coined by David Lane [6] -If its function is to detect differences between the genomes of mitotic division products and to initiate the elimination by apoptosis of cells that are detected as abnormal.Therefore, it would be expected that, provided the p53 mechanism was functional, epigenetic defects would be eliminated.Consequently, there is a strong argument to suggest that a crucial abnormality leading to malignancy is the failure of p53 or some equivalent system.Consistent with this is the strong correlation between cancer and p53 mutations [9] .
The process whereby the p53 system might execute this crucial role may be envisaged as follows: During DNA replication in differentiated stem cells the pattern of gene silencing is retained by a DNA methyltransferase 1 (DNMT1) complex, which restores the pre-existing bilateral methylation pattern at hemi-methylated sites at the replication forks [5,7] .This permits the replicated DNA to retain its location and functionality in the re-associated chromatin so that the epigenetic pattern is transmitted to the division products and the differentiated pattern of gene expression and silencing retained.This state of affairs is illustrated in Scheme 1(A).

Schematic comparison of segments of methylated DNA (the strands being distinguished by red and black backbones) to show: (A) the normal copying of methylated DNA strands during replication; (B) defective copying of the epigenetic pattern giving rise to dissimilar DNA copies, the mismatch being detected by p53 and the cells eliminated by apoptosis; and (C) defective copying of the epigenetic pattern in cells devoid of normal p53 activity resulting in dissimilar products.
The existence of a defect in this mechanism resulting in failure of the of bilateral DNA methylation will generate asymmetry of the methylation patterns of the division products resulting in anomalous gene expression.This proposed abnormality in epigenetic transmission is indicated in Scheme 1(B) in which there is a failure of perpetuation of the epigenetic pattern resulting in dissimilar methylation patterns in the replicated DNA.This difference, it is argued, is detected by p53 and the cell arrested in S phase and apoptosis initiated.In the absence JOCR Volume-1 | Issue-1 March, 2017 of p53 activity (Scheme 1(C)) epigenetically dissimilar products are not eliminated, leading to the epigenetic chaos characteristic of cells destined to be malignant.

Mathematical Model: factors and age-specific incidence
The process of carcinogenesis envisaged gives a biphasic model in which, for each tissue stem cell population, the three possible compartments consist of normal (N), pre-malignant (P) and malignant (M) cells.The initial transfer probability (p1) refers to the likelihood of initiation which results in an N → P transfer, and the secondary transfer probability (p2) refers to the P → M transfer.The first phase is treated as a set of mutations affecting the relevant genes and the second phase as error-prone epigenetic copying, a process that might be referred to as 'epimutation'.Given small values of μ and θ, the transfer probabilities may be written as: p1 = (μ)^(gt_1), and p2 = θrt_2, where μ = the mutation rate, g = the number of mutations necessary to cause the defective epigenetic copying, r = the proliferation rate of the relevant stem cells, θ = the proportion of epigenetically deviant cells exhibiting the malignant phenotype, and t1 + t2 = elapsed time (t).
The time-dependent cumulative incidence (I(t)) is then given as the product of the total stem cell numbers (N) and the successive transfer probabilities, which gives the equation: I(t)=αt^((g+2) ) , where α= Nθrμ^g [g!/(g+2)!].This is equivalent to the familiar power law for the age-specific cancer incidence embodied in the Armitage-Doll model [1,2] .The value of the constant (α) includes the size and turnover rates of tissue stem cells and is consistent with the correlation shown by Tomasetti & Vogelstein (2015) [12] .Also, the inclusion of the stem cell proliferation rate in the proportionality constant enables modifications to the incidence resulting from alterations in the stem cell proliferation rate such as reduction with age [10] , and permits the risk to be adjusted to take account of tissue responses to stimuli such as hormonal status etc. e.g.HRT.

CONCLUSION
The proposed model accounts for many of the known features of cancer.These include: (1) Its failure to occur in non-proliferating cells (e.g.CNS); (2) The cancer incidence in susceptible tissues is a function of the relative proliferation rate and malignancy is comparatively rare in slowly proliferating cell populations (e.g.muscle); (3) Malignant and pre-malignant cells manifest abnormalities of DNA methylation; (4) Malignant and pre-malignant cells exhibit chromatin abnormalities; (5) Malignant and pre-malignant cells exhibit divergent clonal evolution; (6) Malignant and pre-malignant cells exhibit uncoordinated and deranged metabolism; (7) As the abnormally expressed genes code for 'self' components, malignant and pre-malignant cells do not generally elicit an immune response.

Possible diagnostic and therapeutic strategy
If the mechanistic proposal is in essence correct, then the major diagnostic and therapeutic approach to cancer would seem to involve the identification of those cells that possess the epigenetic transmission defect and to use this to target malignant and pre-malignant cells.In the simplest view the target might be the presumed existence of S-phase cells with sections of duplicated DNA in which matching cytosine methylation is absent or deranged.The existence of such mismatched methylated DNA duplication products act as specific diagnostic markers of affected cells.This suggests that agents capable of their recognition will enable the selective elimination of cells exhibiting these structures.As these cells, according to the epigenetic hypothesis, represent the category bearing the crucial initiating lesions, their elimination should halt the progress to malignancy.It does not seem beyond the realms of possibility that suitable reagents can be devised to bring about this desirable outcome.