Abstract A23: Human mammary luminal progenitor cells use cKIT-H2O2 interactions to regulate their growth

Abstract

Abstract Hydrogen peroxides (H2O2) are known to activate multiple cell signaling pathways but the mechanisms involved and how they are differentially regulated in specific normal mammary cell types is unknown. The luminal progenitor (LP) fraction of cells of the normal human mammary gland are of particular interest in this regard because, compared to the basal cells (BCs), these cells consume more O2, sustain higher levels of ROS, and are more resistant to H2O2 levels by virtue of their repertoire of enzymes that reduce both ROS and oxidized nucleotide products of ROS. However, these features of normal human LPs are also accompanied by their accumulation of more DNA damage. Here we examine the idea that the greater tolerance of LPs to ROS may be associated with a previously unknown intracellular signaling role of ROS in these cells. Using an optimized quantitative twin-photon and confocal-reflectance imaging system, we have found that the size of the spherical 3D structures produced in Matrigel cultures by freshly isolated, FACS-purified normal human LPs is increased in the presence of exogenous H2O2 at concentrations that are toxic to BCs. In addition to LPs, co-purified non-clonogenic luminal cells (LCs) display elevated levels of peroxiredoxin-1 peroxidase, a negative regulator of H2O2 action, as compared to BCs. Both of the luminal cell types (but not BCs) also showed tyrosine phosphorylation of peroxiredoxin-1 peroxidase (a biomarker of H2O2 action) when exposed for 10 minutes to exogenous H2O2, but LPs only showed marked inactivation of peroxiredoxin-1 in the absence of an external stimulus. Western blot analysis revealed a parallel and dramatic H2O2-induced pan-tyrosine phosphorylation response selectively in both luminal subsets, and their analysis at the single cell level by mass cytometry using a CyTOF identified multiple activated signaling intermediates. From FACS, immunohistochemistry, Western blot, microarray and RNA-Seq data analyses, we identified cKIT as the most differentially and highly expressed (albeit trypsin-sensitive) tyrosine kinase in LPs. Epigenetic analysis of the cKIT promoter showed it to be in an ‘open’ state exclusively in human LPs, and H2O2 treatment alone was sufficient to rapidly activate auto-phosphorylation of the cKIT Y719 residue, a site known to bind and thereby lead to the activation of PI3 kinase. Taken together, these findings reveal a new, ligand-independent function of a cell surface receptor in mediating a potent, lineage-specific signaling function of H2O2 that, in normal human mammary cells influences cell growth. Citation Format: Nagarajan Kannan, Kingsley Shih, Yifei Dong, Peter Eirew, David Knapp, Davide Pellacani, Hequn Wang, Haishan Zeng, Connie Eaves. Human mammary luminal progenitor cells use cKIT-H2O2 interactions to regulate their growth. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A23.