Oncogene (2011) 30, 2108–2122 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE P2X7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness B Jelassi1, A Chantoˆme1, F Alcaraz-Pe´rez2, A Baroja-Mazo3, ML Cayuela2, P Pelegrin3,4, A Surprenant4 and S Roger1 1Inserm U921, Universite´ Franc¸ois Rabelais de Tours, 10 Boulevard Tonnelle´, Tours, France; 2Telomerase, Cancer and Aging Group, University Hospital ‘Virgen de la Arrixaca’-FFIS, Carretera Palmar, Murcia, Spain; 3Inflammation and Experimental Surgery Group, University Hospital ‘Virgen de la Arrixaca’-FFIS, Carretera Palmar, Murcia, Spain and 4Faculty of Life Science, Michael Smith Building D3315, University of Manchester, Manchester, UK ATP-gated P2X7 receptors (P2X7R) are unusual plasma cancer patients do not die because of local complications membrane ion channels that have been extensively studied of their primary solid tumour growth, which are in immune cells. More recently, P2X7R have been relatively well treated, but rather to the appearance of described as potential cancer cell biomarkers. However, metastases. The development of metastases consists of a mechanistic links between P2X7R and cancer cell complex series of events accomplished by tumour cells processes are unknown. Here, we show, in the highly after they acquire numerous abilities, one of which being aggressive human breast cancer cell line MDA-MB-435s, the acquisition of an invasive potency. This mainly lies that P2X7 receptor is highly expressed and fully into an enhanced migration and the ability to digest the functional. Its activation is responsible for the extension extracellular matrix (Gupta and Massague, 2006). Cell of neurite-like cellular prolongations, of the increase in migration involves dynamic cytoskeletal changes and is cell migration by 35% and in cell invasion through highly regulated by the intracellular concentration of extracellular matrix by 150%. The change in cancer cell calcium. Basement membranes underlying epithelial morphology and the increased migration appeared to be cells as well as extracellular matrices constitute barriers due to the activation of Ca2 þ -activated SK3 potassium for transformed cells to invade subjacent stroma. Their channels. The enhanced invasion through the extracellular proteolytical disruption, by extracellular proteases, matrix was related to the increase of mature forms of allows tumour cells to progress to malignancy and cysteine cathepsins in the extracellular medium, which was metastasis. In the last decade, studies mainly focused on independent of SK3 channel activity and not associated the involvement of matrix metalloproteinases in the with cell death. Pharmacological targeting of P2X7R neoplastic progression (Egeblad and Werb, 2002), but in vivo was crucial for cell invasiveness in a zebrafish recently, it appeared that cysteine cathepsins are highly model of metastases. Our results demonstrate a novel upregulated in a wide variety of cancers, including mechanistic link between P2X7R functionality in cancer breast cancer (Mohamed and Sloane, 2006). Indeed cells and invasiveness, a key parameter in tumour growth elevated cathepsin B expression correlates with poor and in the development of metastases. They also suggest a prognosis in breast cancer as in other cancers (Jedeszko potential therapeutic role for the newly developed P2X7R and Sloane, 2004) and its activity has been shown to antagonists. enhance breast cancer cells invasiveness in vitro (Gillet Oncogene (2011) 30, 2108–2122; doi:10.1038/onc.2010.593; et al., 2009). Cathepsins classical localization within published online 17 January 2011 intracellular lysosomes is found to be modified in tumours. They can be secreted both at their pro- and Keywords: P2X7 receptors; migration; SK3 channels; active forms by cancer cells (Frosch et al., 1999; Roshy cancer cell invasion; cystein cathepsins et al., 2003). However, the signalling pathway regulating their release and activation in the extracellular medium has to be clarified. Adenosine 50-triphosphate (ATP) is a well known Introduction source of free energy for cells and is found at high concentrations (5–10 mM) in intracellular compartments. Breast cancer is the most common female cancer and the In some physiological occasions, ATP can be released first cause of women death by cancer worldwide. Most extracellularly in very high concentrations. This also happens in necrotic cell death and pathological condi- tions. ATP is secreted by cancer cells into their Correspondence: Dr S Roger, Inserm U921, Universite´Franc¸ois microenvironment at relatively high concentrations Rabelais, 10 Boulevard Tonnelle´, Tours 37032, France. E-mail: [email protected] (Raffaghello et al., 2006) and is released in sites of Received 8 July 2010; revised 1 December 2010; accepted 4 December tissues damage such as perilesional regions of cancers 2010; published online 17 January 2011 (Wang et al., 2004b). Indeed, tumour sites are known P2X7R increases cancer cell invasiveness B Jelassi et al 2109 to contain much higher concentrations of ATP than detected (Figure 1c). As in cancer cells mRNA healthy tissues where it would normally be undetectable expression does not always correlate to the translation (Pellegatti et al., 2008). Extracellular ATP acts as a in functional proteins (Roger et al., 2007), we performed signalling molecule by activating plasma membrane G patch clamp experiments to assess the functionality of protein-coupled P2Y receptors and/or ligand-gated ion such receptors. We conducted experiments using 10 mM channels P2X receptors (Burnstock, 2006). Among the ATP, which is supposed to activate all P2X receptors members of the P2X receptors family, the latest cloned except P2X7R, but did not record any ATP-evoked P2X7 receptors (P2X7R) (Rassendren et al., 1997) are current from cancer cells. Only millimolar concentra- very unique by different features, some of them are: (1) tions of ATP were able to trigger the appearance of an their sensitivity to ATP (North, 2002), (2) their facilita- inward and facilitating current (Figure 1d). These are tion under successive or sustained applications of among the well-known specificities of P2X7R-evoked agonist (Hibell et al., 2000; Roger et al., 2008, 2010a) currents. Figure 1e shows representative inward currents and (3) the appearance of a large, non-selective membrane that never desensitized, recorded from a representative pore after sustained stimulations with ATP (Pelegrin cancer cell during an ATP dose-response protocol. This and Surprenant, 2006). Recently, several tumours have protocol was used to draw the ATP concentration-peak been shown to express P2X7R at unusually high levels current relationship from MDA-MB-435s cancer cells. (Adinolfi et al., 2002; Zhang et al., 2004; Slater et al., This was compared with the relationship coming from 2004a, b; Wang et al., 2004a; Raffaghello et al., 2006; HEK293 cells expressing human P2X7R (hP2X7R) Deli et al., 2007; Solini et al., 2008), however, their (Figure 1d). Fitting these relationships with sigmoidal functionality and involvement in the physiology of cancer equations gave ATP EC50 of 3.3±0.2 mM and 3.4±0.7 mM cells remain unclear. In some studies performed on breast for ATP-evoked currents coming from MDA-MB-435s and prostate cancers, it was suggested that P2X7R might and hP2X7R expressing HEK293 cells, respectively. be non-functional (Slater et al., 2004a, b), but proposed As shown in Figure 2a, multiple applications of 5 mM to serve as an early marker for cancer (Slater et al., 2005). ATP to cancer cells were responsible for a dramatic In other cancers, P2X7R activity could be responsible for increase of ATP-evoked currents. This property is a anti-apoptotic effects (Deli et al., 2007), for sustaining particularity of P2X7R and is called facilitation (Hibell cell growth (Raffaghello et al., 2006) or for the release of et al., 2000; Roger et al., 2008, 2010a). This led to a IL-6 by cancer cells (Solini et al., 2008). On the contrary, seven times increase of the current density from the first it was proposed to induce apoptosis in normal cervical to the eighth ATP application (Figure 2b). The use of epithelial cells (Wang et al., 2004a) and that its the BzATP agonist gave us an EC50 of 263±22 mM expression was decreased in cervical cancers (Li et al., (Figure 2c). This is in line with the fact that P2X7R are 2006). Therefore, in this study, we investigated the more sensitive to BzATP than to ATP (North, 2002). expression, characterized the functionality of P2X7Rin ATP-evoked currents from MDA-MB-435s cancer cells highly metastasizing breast cancer cells and tried to were inhibited by human P2X7R antagonists KN62 and clarify their involvement in oncogenic properties. A740003 (Figure 2d and supplementary Figure 1a). Current inhibition curves indicated IC50 of 16.3±2.1 nM (n ¼ 3–9 cells) and 9.8±0.9 nM (n ¼ 2–9 cells) for KN62 and A740003, respectively (Figure 2d), which are the Results classical concentrations used for antagonizing hP2X7R activity (Donnelly-Roberts and Jarvis, 2007). Sustained Human cancer cells express functional P2X7R activation of P2X7R is also known to induce membrane We first analyzed the mRNA expression of P2X7Rin permeation following the activation of pannexin-1 and human non-cancerous and cancerous mammary cells allowing the uptake of membrane-impermeant fluores- (Figure 1a). Importantly, P2X7R were not expressed in cent dyes