In cells transfected with a null-target or control siRNA (siCTL), BzATP enhanced cancer cell invasiveness by a median factor of 1 1.95. a mesenchymal phenotype. In an immunocompetent mouse mammary cancer model, we reveal that the expression of P2X7 receptor in cancer cells, but not in the host mice, promotes tumour growth and metastasis development, which were reduced by treatment with specific P2X7 antagonists. Our results demonstrate that P2X7 receptor drives mammary tumour progression and represents a pertinent target for mammary cancer treatment. versus knock-down mice). Our results unequivocally demonstrate that P2X7R is functionally expressed AMG 548 in mammary cancer cells and its activation promotes the acquisition of a mesenchymal phenotype and enhances invadopodial activity. Furthermore, we provide compelling evidence to indicate that the P2X7R expressed in mammary cancer cells but not in the host organism plays a key role in primary tumour growth and metastatic AMG 548 development, which are significantly attenuated by treatment with specific P2X7R antagonists. These findings AMG 548 support that the P2X7R in mammary cancer cells drives mammary tumour progression and represents a pertinent target for mammary cancer treatment. 2. Results 2.1. P2X7R Expression Promotes Mammary Cancer Cell Invasiveness In this study, we aimed at assessing the potential role of P2X7R in mammary cancer progression in an immunocompetent mouse model. Therefore, we investigated the expression and activity of P2X receptors in the 4T1 mammary cancer cell line, originating from the BALB/cJ mouse strain . As shown in Figure 1a, 4T1 cells expressed mRNA transcripts for P2X2, P2X3, P2X4 and P2X7. A weak band can be visualized for P2X5. The functionality of these receptors at the plasma membrane of cancer cells were assessed using the patch-clamp recording technique. Stimulating the cells with 10 M ATP, a concentration that would activate all P2X receptors with the exception of P2X7R, did not produce any measurable current. However, exposure to 5 mM ATP triggered inward, non-desensitizing, facilitating currents (Figure S1a) that were inhibited by treatment with A438079, a specific competitive P2X7 antagonist (Figure 1b). These results suggest that 4T1 cells mainly express functional P2X7R, while the other P2X receptors (P2X2 P2X3, P2X4 and P2X5) would be either not expressed at the protein level or not functional. To further characterize the ATP-induced currents, we constructed the ATP dose-current response relationship curve (Figure 1c) that yielded the concentration evoking 50% of the maximal current response (EC50) to be 4.3 0.2 mM (= 5C6 cells), consistent with the expression of the mouse P2X7R. We further used Fura2 fluorimetry to monitor the changes in intracellular Ca2+ levels in 4T1 cells in response to ATP (Figure S1b) or BzATP stimulation (Figure 1d). Both ATP and BzATP induced a biphasic increase in intracellular Ca2+ levels in cells incubated in extracellular Ca2+-containing solutions, with a transient component followed by a long-lasting one. The long-lasting Ca2+ increase was significantly reduced in the presence of A438079 or AZ10606120, a specific non-competitive P2X7R antagonist (Figure 1e), supporting P2X7R-mediated Ca2+ entry. In addition, the long-lasting, but not the transient, component was largely abolished in extracellular Ca2+-free solutions (Figure 1d,e, Figure S1b). Under these conditions, ATP/BzATP-induced intracellular Ca2+ increases were not Rabbit Polyclonal to PARP (Cleaved-Gly215) affected by treatment with A438079 or AZ10606120, thus indicating that they are mediated by activation of G-protein coupled P2Y receptors. The P2Y11 receptor is known to be AMG 548 sensitive to both ATP and BzATP and coupled to intracellular Ca2+ release. The P2Y11 receptor was reported in cancer cells . BzATP-induced intracellular Ca2+ increase in Ca2+-free solutions was attenuated by treatment with NF340, a P2Y11 selective antagonist (Figure S1c), in support of the role of the P2Y11 receptor in mediating ATP/BzATP-induced transient Ca2+ increase in 4T1 AMG 548 cells. Open in a separate window Figure 1 P2X7R is functional in 4T1 mouse mammary cancer cells and drives cell invasiveness. (a) RT-PCR analysis of P2X mRNA expression. (b) Representative whole-cell patch clamp recordings of ATP-induced currents. Membrane potential was held at ?60 mV. While 10 s application of 10 M ATP (left) evoked no detec current, application of 5 mM ATP produced a non-desensitizing current that was reduced by treatment with 10 M A438079 (right). (c) Recordings of inward currents from one cell in response to 10 s applications of increasing concentrations of ATP (0.3, 1, 3, 5 and 10 mM) (left), and mean ATP dose-response curve, with currents expressed as a ratio of the maximum current obtained with 10 mM.