Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies

Abstract

IntroductionMost human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated β-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth.MethodsPre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or γ-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated β-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses.ResultsCultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated β-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism.ConclusionsOur studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential.

PIM1 kinase inhibition as a targeted therapy against triple-negative breast tumors with elevated MYC expression

“…Primary human mammary epithelial cells (HMECs) were derived from histologically normal breast tissues and cultured as previously described 7 . The cells were infected with lentivirus encoding p16 shRNA 53 and further infected with pBabe-MycER virus 8 .…”

“…This indirect treatment strategy has become known as a synthetic lethal approach 6 . To identify novel targets that are readily druggable for treating MYC-overexpressing TNBC, we conducted a kinome-wide MYC synthetic lethal shRNA screen in non-immortalized human mammary epithelial cells 7 expressing a 4-hydroxytamoxifen (TAM)-activated MycER transgene 8 (HMEC-MycER) (Fig. 1a).…”

Irradiation of Juvenile, but not Adult, Mammary Gland Increases Stem Cell Self-Renewal and Estrogen Receptor Negative Tumors

“…Compensatory regeneration is observed in irradiated bone marrow and intestine during regeneration after stem cells death . Radiation can also induce cell inactivation via senescence . Alternatively, some stem cells are thought to be resistant to radiation, and signals might increase symmetric division or self‐renewal .…”

Rapid and automated multidimensional fluorescence microscopy profiling of 3D human breast cultures

“…This is in stark contrast to the classical view and expectation of a homogenous population response. Such quantitative approaches increase our opportunity to accurately identify and investigate specific phenotypes observed after exposure to IR [30], or drug resistance observed after chemotherapeutic or kinase inhibitor treatments [31]. …”