Morphologically Normal-Appearing Mammary Epithelial Cells Obtained from High-Risk Women Exhibit Methylation Silencing of INK4a/ARF

Abstract

Purpose: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation.These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. Experimental Design: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. Results: INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-h2, estrogen receptor-a, and breast cancer-associated 1genes (P = 0.001). Conclusions: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation. p16(INK4a) acts to block cell cycle progression by binding to cyclin-dependent kinase-4 (CDK4) and CDK6 and inhibiting the catalytic activity of the CDK4-CDK6/cyclin D complex required for retinoblastoma protein phosphorylation (1, 2). p16(INK4a) blocks progression beyond the G 1 -S restriction point by disrupting the formation of an E2F-DB active transcriptional complexes, thereby preventing the transcription of cell cycle progression genes (3). Loss or inactivation of p16(INK4a) function has been observed in numerous tumor types (4 -6), and p16(INK4a) has been implicated to play an important role in the control of replicative senescence in fibroblasts and human mammary epithelial cells (7,8).Loss of p16(INK4a) function has been identified to be the result of both genetic and epigenetic events. Multiple mechanisms exist, including point mutation, loss of heterozygosity (LOH), small homozygous deletion (<200 kb), and promoter hypermethylation. LOH at the INK4a/ARF locus (9p21) has been reported in a number of neoplasias, including breast

The DNA Damage-Binding Protein XPC Is a Frequent Target for Inactivation in Squamous Cell Carcinomas

“…Promoter methylation could down-regulate both XPC promoters, as has been observed for the p16 gene during early stage carcinogenesis in breast epithelial cells. 32 The focal persistence of XPC expression in basal keratinocytes in SCC in situ (Figure 2, B and H) and its subsequent loss as the transformed keratinocytes ascent in the epidermis (Figure 2, B and H), would be consistent with XPC loss in SCC being the consequence of an early regulatory event.…”

Lipid Metabolism Genes in Contralateral Unaffected Breast and Estrogen Receptor Status of Breast Cancer

“…Until now rFNA samples have been used for immunohistochemistry (27,28) and DNA-based analyses (29)(30)(31)(32)(33)(34), although feasibility of gene expression measurement has been explored (35). Considering the topographic heterogeneity in the breast, rFNA sampling is a useful tool for the discovery of breast cancer risk biomarkers; it provides a predominantly epithelial sample from a large area of the breast and allows the evaluation of women with clinically normal breasts, therefore greatly expanding the population for study, and eventual application of results.…”

Origin of carcinoma associated fibroblasts

“…133 Considering the potential increased mutation rate in inflamed microenvironment, it is possible that the resident stromal cells could accumulate a random set of mutations. On the other hand, the classical working hypotheses on carcinogenesis assumes the mutation rate is affected by overall rate of proliferation, early in premalignant conditions 79 lead to an intrinsic increase in mutation rate, as well as loss of mechanisms that monitor genomic integrity and control the appropriate cellular response, such as DNA repair or cell death. Since inflammation accelerates almost all of the milestones of cancer progression, it follows that chronic inflammation is linked with increased risk of a few different cancer types.…”