Abstract 2919: Different ROS control mechanisms and mutagenic consequences in primitive subsets of normal human mammary cells.

Reactive oxygen species (ROS) are known mediators of DNA damage and likely contributors to oncogenesis. However, very little is known about the mechanisms controlling ROS in the cells that make up the normal human mammary gland, in spite of its being a major site of cancer development. To investigate how ROS are generated and their potential role in two functionally distinct, primitive normal human mammary epithelial cell compartments (luminal and basal), we isolated these subsets at high purities by FACS and compared the levels within them of ROS, components that positively and negatively regulate ROS, their responses to oxidative stressors and evidence of ROS-associated DNA damage. The results show that purified progenitors of the cells that line the gland lumen (defined by their EpCAM+ CD49f+ phenotype) contain significantly higher levels of superoxide (O2*) anions and H2O2 than the basal cells (defined as EpCAMlow/- CD49fhigh cells and highly enriched in bipotent progenitors and stem cells). We also find that the elevated levels of these ROS elements in the luminal progenitors are associated with a higher content of mitochondria and higher levels of all 3 superoxide dismutases (SOD-1, 2 and 3). The luminal progenitors are also highly resistant to glutathione depletion and express higher levels of both non-canonical non-glutathione anti-oxidant enzymes and multiple enzymes that control ROS-induced nucleotide damage (i.e., OGG-1, MTH-1, MUTYH) providing a likely explanation for their ability to survive following GSH depletion. Interestingly, we found the mitochondrial antioxidant glutathione peroxidase (GPX)-2 enzyme to be expressed almost exclusively and at high levels in basal mammary cells and its depletion, using a shRNA lentivirus, resulted in loss of progenitor viability/activity by basal but not luminal cells. Luminal progenitors also displayed greater resistance to acute oxidative insults (H2O2 and X-radiation) and displayed an increased accrual of oxidative damage-induced (genomic 8-oxo-dGTP) mutations. Our findings reveal a major difference in the molecular machinery that control ROS levels in normal human basal and luminal mammary progenitors. These correlate with an ability of the luminal progenitors to maintain and tolerate elevated levels of ROS and the continuous acquisition of unrepaired ROS-induced DNA damage, thus suggesting a novel and previously unanticipated vulnerability of these cells to undergo oncogenic transformation. (Supported by Canadian Breast Cancer Foundation/BC-Yukon and the Canadian Cancer Society) Citation Format: Nagarajan Kannan, Maisam Makarem, Long Nguyen, Jeff Dong, Peter Eirew, Connie Eaves. Different ROS control mechanisms and mutagenic consequences in primitive subsets of normal human mammary cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2919. doi:10.1158/1538-7445.AM2013-2919