Mammalian cells acquire epigenetic hallmarks of human cancer during immortalization

Abstract

Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK–ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK–ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

“…32 This finding is not cancer-specific, however, since an increase in promoter CpG island DNA methylation was observed by ectopic expression of mouse Dnmt3b in normal mouse colon cells. 33 Mouse cells are capable of obtaining extensive promoter CpG island DNA hypermethylation when they are immortalized, as recently shown 29 and in this study. It is possible that this phenomenon is a result of culturing cells, as it has been observed that progressive promoter CpG island DNA hypermethylation occurs after extensive passaging of cancer cells in vitro.…”

“…26 Experiments using human cell lines have shown that genomewide DNA methylation of promoter CpG islands increases during the process of immortalization, 27,28 and most recently, this result was confirmed in mouse embryonic fibroblasts. 29 To determine whether the genome-wide DNA hypermethylation of promoter CpG islands that occurs in mouse cells during the process of immortalization is more similar to human cancers than the modest methylation changes observed in GEMMs, we created newly immortalized mouse fibroblasts by serially passaging primary cells in vitro and selecting clones that spontaneously acquired the ability to bypass replicative senescence. 30 The DAMD assay was performed on two independent newly immortalized mouse fibroblast cell lines (iMF1 and iMF2), and two well-established mouse fibroblast immortalized cell lines (3T3 and 10T1/2); an early passage culture of non-immortalized mouse primary fibroblasts served as a control.…”

The impact of age on oncogenic potential: tumor-initiating cells and the brain microenvironment

“…It has also been suggested that senescence may be a necessary step in the progression to immortality in cell culture. One recent study has shown that many pluripotency genes are hypermethylated in mouse embryonic fibroblasts during immortalization in vitro , in patterns similar to those observed in cancer cells (Tommasi et al ., 2013). Yet a direct comparison of the methylation patterns in senescent and transformed mammalian cells is still lacking.…”

Targeting the angiogenic 'splice' switch; inhibition of SRPK1 as a novel therapeutic approach in prostate cancer

“…Alternative splicing is increasingly recognized as an important process expanding proteomic and functional complexity in physiological processes and in disease, particularly in cancer [30,31] . Recent high-throughput sequencing studies indicate that more than 90% of human genes are alternatively spliced [32] .…”