Anales RANF

S10-01 THE ROLE OF THE P2X4 RECEPTOR IN BREAST CANCER Stéphanie Chadet 2 , Lucie Brisson 2 , Stéphanie Lerondel 3 , Roseline Guibon 2 , Gaëlle Fromont 2 , Alain Le Pape 3 , Sébastien Roger 2 and Ruth Murrell-Lagnado 1 1 University of Sussex, Brighton, UK, 2 Université de Tours, Tours, France, 3 CNRS UPS44 CIPA, Orléans, France. Purinergic signalling is associated with cancer cell invasiveness and metastasis formation with P2X receptors involved on the side of the host immune cells as well as the tumour. The unusually high levels of extracellular ATP within the tumour microenvironment activate the low affinity P2X7 receptor to promote both the growth and metastatic potential of breast cancers 1,2 . P2X4 is commonly co-expressed with P2X7 and their synergistic activity is often questioned. However, unlike P2X7, P2X4 is targeted to endolysosomes 3 . In this study we assessed the role of P2X4 in mammary tumour growth and metastases, and its functional association with P2X7. At the transcript level, P2X4 is over-expressed in human breast cancers compared to normal breast tissue. We showed by immunohistochemistry, elevated P2X4 expression in >50% of human breast cancer samples compared to normal breast tissue. A combination of in vitro and in vivo approaches was carried out, utilizing highly invasive human MDA-MB-435s and murine 4T1 mammary cancer cell lines, both endogenously expressing P2X4 and P2X7. Western blot and immunocytochemistry analyses showed expression of P2X4 and its targeting to lysosomes. Knocking down the expression of P2X4 using the CRISPR/Cas9 system reduced basal invasive capacities of cells through Matrigel-coated inserts by 50%. It also inhibited the 2-fold enhancement of invasion produced by BzATP and the secretion of cathepsin D, suggesting a role for P2X4 in lysosome exocytosis triggered by P2X7 stimulation. A comparison was made of tumour growth and metastases in BALB/c mice following implantation of either P2X4-CRISPR or CTL-CRISPR cells into mammary fat pads. For two clones of the P2X4-CRISPR cells, tumour growth was inhibited by 85.5% and 91.5% compared to control cells, and metastases development was prevented. In conclusion, our results show a prominent role of P2X4 in cancer cell invasiveness, tumour growth and metastases, which could be potentiated by P2X7 stimulation. 1) Jelassi J et al . P2X(7) receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness. Oncogene, 30, 2108–2122 (2011). 2) Adinolfi E et al.. Expression of P2X7 receptor increases in vivo tumor growth. Cancer Res. 72(12):2957-69 (2012). 3) Qureshi O et al. Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. Journal of Cell Science. 120, 3838-3849 (2007)