Abstract 1723 Differences in barriers to immortalization in normal breast epithelial cells from donors with and without mutations in breast cancer susceptibility genes

Background: With breast cancer affecting one in eight women, understanding the factors driving its development and exploring potential preventative measures are imperative. Senescence is a major barrier limiting the replicative lifespan of cells and is a key hurdle in preventing the growth of cancer cells. This study investigates the key factors necessary to bypass replicative senescence and achieve immortalization in primary cultures of breast epithelial cells from normal tissue. Methods: Donors with no known familial risk of breast cancer (designated Average Risk) and those with germline mutations in breast cancer susceptibility genes BRCA1, BRCA2, or TP53 (designated High Risk) were selected for this study. Lentiviral constructs were employed to infect primary cultures of breast epithelial cells and express CDK4 and TERT genes. CDK4 was used to overcome senescence signals by p16/INK4A; TERT was used to prevent telomere shortening. Results: Immortalization of cells from High-Risk donors was delayed significantly compared to Average-Risk honors. The immortalized cell lines consistently maintained an epithelial cell morphology and demonstrated a remarkable capacity to proliferate beyond 40 population doublings with the CDK4+TERT treatment. Expression of TERT-alone immortalized cells from only 30% of donors, while CDK4+TERT immortalized cells from all donors tested. Using this method, immortalized cells have been obtained from 16 donors. The cells are largely euploid and retain expression of the P53 protein. A subset of cell lines expressed detectable levels of estrogen receptor alpha (ERα). Conclusion: These cells provide avatars for the donors, which can be used to identify mechanisms regulating immortalization and the variable penetrance of pathogenic mutations in breast cancer susceptibility among individuals. Estrogen receptors play a key role in orchestrating gene and protein expression necessary for normal breast development. To explore the role of differences in ERα signaling among individuals, we will use a system to inducibly express ERα in the immortalized breast epithelial cells. The induced ERα cells will be evaluated for their ability to undergo estrogen-dependent cell proliferation and induce the transcription of ERα target genes through qPCR. We will also determine whether ERα signaling enhances oncogenic transformation in these cells. This poster's research was partly funded by the NIH's National Institute of Environmental Health Sciences (award U01ES026140, DJJ, SSS), a University of Massachusetts fellowship (JP) under the Chemistry-Biology Interface Training Program (NRSA T32 GM139789), Rays of Hope Center for Breast Cancer Research grants, and an Honors Research Grant from UMass Amherst.