In this study, we have investigated the interplay between genetic mutations and the establishment of global DNA methylation patterns in the development of mouse T cell lymphomas. The model used here, developed by Felsher and colleagues, uses high levels of MYC expression to mimic similar events taking place during human lymphomagenesis such as in Burkitt's lymphoma ([34
] and references therein). The clear advantage of using a mouse model to study the role of DNA methylation in cancer is that cohorts used are genetically homogeneous in all respects except on a defined set of genetic variables (i.e., overexpression of MYC). Therefore, we consider this mouse model to be suitable for studying the relationship between genetic and epigenetic changes during tumor development. We demonstrate that DNA methylation patterns in these tumors are characterized by frequent and nonrandom patterns, similar to what has been described in human malignancies. These patterns are exquisitely dependent on the genetic makeup of the tumor cell, as homozygous deletion of either p53
, or E2f2
-induced T cell lymphomas resulted in unique tumor-specific patterns of genome-wide DNA methylation. This analysis provides the first clear-cut experimental evidence showing that genetic background can drive the establishment of aberrant DNA methylation profiles during lymphoma development, offering an explanation for the tremendous epigenetic heterogeneity observed in human cancer [4
Multiple mechanisms have been proposed to explain the specificity of aberrant DNA methylation in human cancer, including the differential susceptibility of DNA sequences to methylation [8
] and the targeting of methyltransferase activity to specific promoter sequences [12
]. The fact that our RLGS analysis identified a specific signature of CpG island methylation that could only be detected late in the MYC
-induced neoplastic process suggests that MYC overexpression is insufficient to target the DNA methylation machinery to specific sites. It is unlikely that the absence of aberrant promoter methylation early in the neoplastic process was due to the notoriously slow nature of de novo methylation relative to the kinetics of gene repression, since repression was not observed at these early stages of disease, either. This view is further supported by the absence of any hypermethylation signature in cells immediately following the transformation by MYC
. Rather, specific patterns of DNA methylation could only be detected in MYC/Ras61L
-transformed MEFs that have been either cultured in vitro for an extended period of time or injected into nude mice for an equally long time. However, we cannot rule out that MYC-initiated targeting of the DNA methylation machinery could occur as an infrequent aberrant event during tumor development. Comparison of global methylation profiles between different mouse models of T cell lymphomas would not only help to answer this question but it would also address to what degree the global DNA methylation pattern is dependent on initial oncogenic insult.
The work described here provides an alternative explanation for the longstanding question of tumor-specific CpG island hypermethylation. We find that tumor-specific signatures of CpG island methylation can only be detected late in the neoplastic process, at a time when the clonal expansion of pretumor cells can be observed, suggesting that specific profiles of CpG island methylation arise from the expansion of rare neoplastic cells. While we cannot rule out the possibility that DNA methylation profiles of late-stage tumor cells may indirectly reflect the selection of cells having a unique expression profile, our observation that inactivation of different tumor suppressor pathways results in distinct aberrant DNA methylation signatures indicates that rare DNA methylation events are selected for during the course of tumor development.
The function of genes silenced by hypermethylation is expected to provide tumor cells with some selective advantage. Indeed, many of the hypermethylated genes identified by RLGS have functions consistent with a tumor suppressor role, including Id4
]. The observation that inactivation of different tumor suppressor pathways results in distinct aberrant DNA methylation signatures further supports the notion that rare DNA methylation events are selected for during the course of tumor development. We envision that the function of genes that are no longer selected for hypermethylation in each of the three tumor suppressor cohorts studied here must be somehow related or dependent on the function of the specific tumor suppressor targeted for inactivation. The inactivation of Pten
, for example, would thus diminish the selective pressure imposed during neoplastic progression for genes involved in similar or related functions. Comparison among the three tumor suppressor cohorts suggested that this selection is strongly influenced by the specific tumor suppressor inactivated in the tumor cell. Remarkably, the strength of the influence that genetics has on this selection mechanism is illustrated by the ability of unbiased statistical algorithms to faithfully predict the tumor suppressor status in each tumor. We would predict that the nature of the initiating oncogenic insult, in this case MYC, would have an equally important role in determining the CpG methylation profile of the emerging tumor.
It is clear from in vitro experiments and more recent genome-wide sequencing efforts that the transformation of both human and mouse cells to a malignant phenotype requires multiple sequential genetic lesions. In the context of an evolving tumor, it is likely that the initial genetic insult acts as a driving force for the first round of epigenetic alterations and that subsequent genetic alterations as the tumor evolves lead to further epigenetic selection. There is also evidence that epigenetic alterations influence the evolution of genetic lesions. For example, it has been shown, that the hypermethylation and silencing of the mismatch repair gene MLH1
leads to microsatellite instability in colon cancer [36
]. Similarly, the silencing induced by promoter hypermethylation of the Werner syndrome gene that occurs in several human cancer types, leads to chromosomal instability and apoptosis [38
]. Thus, the coevolution of genetic and epigenetic changes during the transformation of a normal cell may be viewed to fuel the diversity of tumor phenotypes. This interpretation is consistent with the heterogeneity of DNA methylation we observe even within a single genetic cohort of mouse lymphomas.
While the underlying mechanisms for this selection process are unknown, one could speculate that due to the loss of tumor suppressor function, gene-expression profiles are changed. These changes in gene expression may trigger a cascade of epigenetic events including nucleosome repositioning, histone tail modifications, and subsequently DNA methylation [39
]. There is indeed evidence that a similar mechanism for the specificity of CpG island methylation is also operative in human malignancies. For example, Weisenberger et al. described the tight association of BRAF
mutation with a CpG island methylator phenotype in colon cancer [19
], and it is known that in breast cancers with either BRCA1
mutations stable epigenetic changes reflect gene-expression profiles in these cells [42
]. In conclusion, our findings suggest that genetic events can determine the course in the epigenetic evolution of DNA methylation during tumor development.