Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Abstract

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16 INK4A expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment. [Cancer Res 2009;69(12):5251-8]

TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

“…The postselection HMEC used thus far have overcome stasis, the stress-associated senescence barrier, by selecting for p16 promoter methylation (3). In that process, they also acquired additional aberrant properties, including numerous DNA methylation changes (3,27).…”

“…In that process, they also acquired additional aberrant properties, including numerous DNA methylation changes (3,27). We thus extended this work by examining normal, prestasis HMEC transduced with an shRNA targeting p16; this population does not express the aberrant methylation seen with the postselection HMEC (3).…”

“…One involves the stress-associated induction of the cyclin-dependent kinase inhibitor p16 before attaining critically short telomeres. This stasis barrier can be overcome by inhibiting p16, allowing continued proliferation, which results in agonescence, a proliferative barrier mediated by telomere depletion (3). Additionally, the ability of dysregulated oncogenic signaling to induce senescence in human cells has implicated oncogene-induced senescence (OIS) as an important tumor-suppressive barrier.…”

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

“…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.…”

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

“…Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation [4][5][6][7][8][9][10][11][12][13][14][15]16 . The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic 5,15 .…”