Abstract
The ATP-gated receptor P2X7 is expressed in multiple malignant tumours including neuroblastoma, melanoma, prostate, lung and breast. P2X7 has a significant role in mediating diverse cell responses, which upon dysregulation are associated with tumour initiation and development. The rapid, ATP-mediated activation of P2X7 induces a fast-inward cation current in cells. However, prolonged ATP-mediated activation of P2X7 leads to formation of a pore that increases membrane permeability and eventually causes cell death. This presents a potential paradox, as the tumour microenvironment contains extracellular ATP at levels sufficient to activate the P2X7 pore and trigger cell death. However, P2X7 expression is associated with enhanced cancer cell survival, proliferation and metastatic potential. At least one distinct conformational form of P2X7, termed non-pore functional P2X7 (nfP2X7), has been described, which is not able to form a functional pore. We demonstrate for the first time in this study that exposure to a high ATP concentration, equivalent to those measured in the tumour microenvironment, drives nfP2X7 expression and also that nfP2X7 is essential for tumour cell survival. We show that monoclonal antibodies raised against a P2X7 amino acid sequence (200–216), whose conformation is distinct from that of wild-type (WT) P2X7, bind specifically to nfP2X7 expressed on the surface of tumour cells. We also show that nfP2X7 is broadly expressed in patient-derived tumour sections from a wide range of cancers. Therefore, antibodies raised against E200 provide tools that can differentiate between forms of the P2X7 receptor that have a key role in cancer.
Highlights
P2X receptors (P2Xs) are ATP-gated cation channels that form homo- and hetero-trimers at the cell membrane [1, 2]
To confirm that BIL03s does not bind to WT P2X7, we investigated the effect of two distinct small interfering RNA (siRNA) on BIL03s and L4 binding to PC3 or to PC3-P2X7 variant a (P2X7a) cells
We showed that all P2RX7-targeted siRNAs tested caused a significant increase in Caspase 3/7 activation in each cell lines indicating that non-pore functional P2X7 (nfP2X7) depletion leads to apoptosis (Fig. 6b)
Summary
P2X receptors (P2Xs) are ATP-gated cation channels that form homo- and hetero-trimers at the cell membrane [1, 2]. In the tumour microenvironment (TME), extracellular ATP (eATP) concentrations can reach hundreds of micromolar [10, 16] This is due to release of ATP through tumour cell death caused by stresses such as inflammation, hypoxia, mechanical stress and non-targeted therapies [17,18,19]. Tumour cells are exposed to ATP concentrations in the TME sufficient to activate the non-selective pore and precipitate cell death. By mimicking the high ATP concentrations present in the TME, we induce nfP2X7 and down-modulate P2X7 expression. This provides a mechanism by which tumour cells can maintain the survival advantages, while avoiding cell death induction through opening of the P2X7 pore (Fig. 8)
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