Differences in Barriers to Immortalization and Oncogenic Transformation in Normal Breast Epithelial Cells from Donors with and without Mutations in Breast Cancer Susceptibility Genes

Presenter

Gaia Taig

Campus

UMass Amherst

Sponsor

D Joseph Jerry, Department of Veterinary and Animal Sciences, UMass Amherst

Schedule

Session 1, 10:30 AM - 11:15 AM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A74, Campus Center Auditorium, Row 4 (A61-A80) [Poster Location Map]

Abstract

Background: With breast cancer affecting one in eight women, understanding the factors driving its development and exploring preventative measures are imperative. Senescence limits 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: This study selected donors without familial breast cancer risk (Average Risk) and those with germline mutations in BRCA1, BRCA2, or TP53 (High Risk). Lentiviral constructs infected breast epithelial cells to express CDK4 and TERT genes, bypassing senescence via p16/INK4A and preventing telomere shortening, respectively. Estrogen receptor alpha (ERα) was inducibly expressed and tagged with GFP for selection via fluorescence-activated cell sorting. 

Results: CDK4+TERT treatment immortalized all cell lines, while TERT alone immortalized three out of ten lines across Average-Risk and High-Risk categories. Cells kept epithelial morphology and surpassed 40 population doublings with CDK4+TERT. Average-Risk cells reached PD15 in 34 days on average, while High-Risk cells were slower, averaging 63.7 days. The cells remained mostly euploid and preserved P53 protein expression. Additionally, a portion showed estrogen receptor alpha (ERα) expression, which, once inducibly expressed, allowed continued cell proliferation.

Conclusion: These cells act as donor avatars, helping to explore immortalization regulation and individual variations in breast cancer susceptibility mutations. Estrogen receptors play a key role in gene and protein expression in breast development. Our goal is to further investigate ERα signaling's role in cancer transformation by targeting p53 and other genes using CRISPR-Cas9.

Keywords

Breast cancer, Senescence, Immortalization, CDK+TERT, Estrogen Receptor

Research Area

Cancer Studies

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