Abstract P5-05-03: Upregulated purinergic signaling enhances cell proliferation in human and murine breast carcinomas

Abstract

Abstract The composition of the extracellular tumor microenvironment differs from that of most other tissues and is thought to provide cancer cells with a growth and survival advantage compared to normal cells. In solid tumors, the extracellular concentration of ATP can be elevated to ~100 µm and extracellular pH can be as low as 6.5. In the current project, we investigate the consequences of purinergic signaling in human and murine breast carcinomas: we study intracellular Ca2+ signals and associated changes in cell proliferation during stimulation with extracellular nucleotides. We employ biopsies of human and murine primary breast carcinomas and compare them with matched normal breast tissue. Human biopsies are obtained with written informed consent from patients undergoing breast conserving surgery at Aarhus University Hospital or Regional Hospital Randers in Denmark. Murine biopsies are from mice overexpressing unactivated ErbB2 specifically in the breast tissue. We isolate epithelial organoids (~150 µm diameter) from tissue biopsies by partial digestion with collagenase III. Organoids loaded with the Ca2+-sensitive fluorophore Fura-2 are studied by fluorescence microscopy. In separate experiments, cell proliferation is quantified by detecting newly synthesized DNA using immunofluorescence imaging of organoids incubated with the thymidine analogue bromodeoxyuridine (BrdU). We find that intracellular Ca2+ responses during stimulation with extracellular ATP are elevated 2- to 10-fold in breast carcinomas from mice and humans, respectively, compared to matched normal breast tissue. We observe similar differences between breast cancer tissue and normal breast tissue in response to stimulation with the P2Y2/P2Y4-agonist UTP, whereas virtually no rise in the intracellular concentration of Ca2+ is observed in response to the P2X7-agonist 3'-O-(4-benzoyl)benzoyl-ATP. Application of cyclopiazonic acid – an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase – also cause exaggerated intracellular Ca2+ responses in breast cancer compared to normal breast tissue. Consistent with the elevated Ca2+ responses, stimulation with 100 µm ATP or 100 µm UTP increases the rate of cell proliferation (i.e., fraction of BrdU-positive cells) by ~2-fold in the breast cancer tissue. In conclusion, we find that purinergic signaling is upregulated in human and murine breast carcinomas compared to normal breast tissue. Activation of purinergic receptors – most likely P2Y2 and/or P2Y4 – enhances cell proliferation in breast cancer tissue. We propose that the high ATP levels in the tumor microenvironment promote breast cancer development or progression and that the associated signaling pathways represent promising targets for therapy. Citation Format: Mele M, McWhan K, Henningsen M, Vahl P, Jensen V, Johansen T, Pedersen H, Christiansen P, Bødtkjer E. Upregulated purinergic signaling enhances cell proliferation in human and murine breast carcinomas [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-03.