Genetic and epigenetic modulation of p16INK4A activity in human mammary epithelial cells provides insights into early events in breast cancer

Breast cancer is a major health concern of women worldwide. Breast cancer is typically derived from the epithelial cell component of the tissue, and is characterized by widespread chromosomal instability, as well as specific molecular lesions in known tumor suppressor genetic pathways. Human mammary epithelial cells (HMEC) isolated from healthy, disease-free women provide a physiologically relevant system in which to examine tumor suppression mechanisms.; Cultures of HMEC contain a subpopulation of variant cells with the capacity to propagate beyond an in vitro proliferation barrier. These variant HMEC (vHMEC), which contain hypermethylated and silenced p16INK4a (p16) promoters, eventually accumulate multiple chromosomal changes, many of which are similar to those detected in premalignant and malignant lesions of breast cancer. We show here that p16 is necessary in HMEC to prevent proliferation beyond the in vitro proliferation barrier. Furthermore, consistent with its role as a regulator of cell cycle progression, p16 activity is necessary for HMEC to arrest their cell cycle appropriately in response to microtubule disruption.; To determine the origin of vHMEC in vitro, we used Luria-Delbrück fluctuation analysis and found that vHMEC exist within the population prior to the proliferation barrier, thereby raising the possibility that variant HMEC exist in vivo prior to cultivation. To test this hypothesis, we examined mammary tissue from normal women for evidence of p16 promoter hypermethylation. Here we show that epithelial cells with methylation of p16 promoter sequences occur in focal patches of histologically normal mammary tissue of a substantial fraction of healthy, cancer-free women. These observations show that molecular alterations historically associated with later stages in breast cancer progression occur much earlier than previously appreciated. They also validate the use of the HMEC culture system as a tool to stimulate translational research aimed at understanding the molecular pathology of breast cancer.