Hypoxia-increased RAGE and P2X7R expression regulates tumor cell invasion through phosphorylation of Erk1/2 and Akt and nuclear translocation of NF-κB

Marco Tafani 2 Luana Schito 1 2 Laura Pellegrini 2 Lidia Villanova 2 Gabriella Marfe 0 Tahira Anwar 5 Roberta Rosa 4 Manuela Indelicato 3 Massimo Fini 3 Bruna Pucci 3 Matteo A.Russo 2 3 0 Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata" , 00133 Rome, Italy 1 Department of Medicine, Institute of Genetic Medicine, Johns Hopkins University School of Medicine, The Johns Hopkins University , Baltimore, MD 21205, USA 2 Department of Experimental Medicine, Sapienza University , 1, 00161 Rome, Italy 3 Department of Cellular and Molecular Pathology , IRCCS San Raffaele Pisana, 00163 Rome, Italy 4 Department of Molecular and Clinical Endocrinology and Oncology, University of Naples ''Federico II'' , 80138 Naples, Italy 5 Department of Biosciences Division of Biochemistry, University of Helsinki , Helsinki 00100 Finland To whom correspondence should be addressed. Tel 39 06 49970665; Fax: 39 06 49970806; Email: The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: - The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1a silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NFkB) expression. In contrast, NF-kB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1a. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-kB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-kB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-kB as well as cell invasion without affecting HIF-1a stabilization. Once in the nucleus, NF-kB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damageassociated molecular patterns receptors by HIF-1a represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype. Introduction Hypoxia occurs while transformed cells in a tumor are growing in the absence of neoangiogenesis. At a cellular level, the drop in oxygen concentration has two important consequences: activation of hypoxiainducible factor-1 (HIF-1) and necrosis of cells that are distant from blood supply. HIF-1 is a heterodimeric protein composed by two subunits a and b. Under normoxia, HIF-1a is degraded by the Abbreviations: ATP, adenosine triphosphate; DAMP, damage-associated molecular pattern; HIF, hypoxia-inducible factor; HMGB1, high mobility group box 1 protein; MMP, matrix metalloprotein; NF-jB, nuclear factor-kappaB; P2X7R, purinergic receptor P2X7; RAGE, receptor for advanced glycation end products; shRNA, short hairpin RNA. ubiquitinproteasome system, but when the intracellular oxygen concentration drops, HIF-1a is stabilized (1). Following stabilization, HIF-1a translocates to the nucleus where it binds to HIF-1b. The HIF-1a and b dimer activates the expression of vascular endothelial growth factors and their receptors (2), change in energy metabolism (3) and upregulation of RAGE (4). At the same time, necrosis causes a release of intracellular alarmins also called damage-associated molecular patterns (DAMPs) that, by binding to different receptors activates nuclear factor-kappaB (NF-jB) triggering the inflammatory response (5). RAGE and purinergic receptor P2X7 (P2X7R) represent two plasma membrane receptors able to bind DAMPs released by necrotic cells. The receptor for advanced glycation end products (RAGE) is a transmembrane receptor classified as an immunoglobulin super family member. RAGE is expressed by numerous cell types including monocytes, macrophages, T-lymphocytes, neurons, endothelial cells, osteoclasts, osteoblasts and a variety of cancer cells (611). RAGE expression is stimulated by different stressing stimuli with hypoxia being one of them (4). In fact, RAGE expression levels increased in neuron under hypoxia through the binding of the HIF-1 (4). Once expressed, RAGE binds to several ligands including advanced glycation end products, high mobility group box 1 protein (HMGB1), S100 proteins and amyloid b-fibrils. RAGE signal transduction pathways typically activate NF-jB-mediated responses involved in inflammatory gene expression and this receptor plays a significant role in the alarmin response of the innate immune system and contributes to diabetic complications and neurodegenerative disorders (12,13). Furthermore, elevated expression levels of RAGE have been detected in a large number of tumors (14,15). Therefore, RAGE is considered as a receptor capable of bridging inflammation and cancer (16,17). In fact, recent studies, starting from the fact that chronic inflammation is a major causative factor in many human malignancies, have shown a correlation between RAGE expression and tumor progression (18 20). For example, blockade of RAGE signaling by multiple strategies prevented tumor growth and metastasis formation in severe combined immune deficient mice (21). The P2X7R is an adenosine triphosphate (ATP)-sensitive, ligandgated ion channel that functions as a nonselective cation channel and, upon prolonged agonist exposure, leads to the formation of progressively enlarged cytolytic pores ( 900 Da) on the cell surface (22,23). Seven different P2X receptor subtypes have been molecularly defined arising from distinct genes (24). P2X receptors bear common topology, containing intracellular N and C termini, two transmembrane domains and a large extracellular loop. Among P2X receptors, P2X7R appears to be the most divergent member (25) since it requires high concentrations of ATP to be activated (typically present in a microevironment where cells are dying by necrosis). Furthermore, P2X7 has been shown to be specifically involved in mediating ATP-induced apoptosis of several cell lines (2629). Such cytotoxic effect of P2X7R has been widely studied and considered to be a promising therapeutic tool to activate cell death in tumorigenic cells by exposing them to increasing doses of ATP (30). However, recently, a different approach has been taken wh (...truncated)