Stimulation of the toll-like receptor 3 promotes metabolic reprogramming in head and neck carcinoma cells

// Mathieu Veyrat 1 , Sylvere Durand 2 , Marion Classe 3 , Tanja Matijevic Glavan 4 , Natalie Oker 1, 5 , Nikiforos-Ioannis Kapetanakis 1 , Xiaojun Jiang 1 , Aurore Gelin 1 , Philippe Herman 5 , Odile Casiraghi 6 , David Zagzag 7 , David Enot 2 , Pierre Busson 1 , Benjamin Verillaud 1, 5 1 University Paris-Sud (Paris 11), CNRS-UMR 8126, Gustave Roussy, Villejuif, France 2 Equipe 11 Labelisee par la Ligue Nationale Contre le Cancer, INSERM U1138, Centre de Recherche des Cordeliers, Paris, France, Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France 3 Department of Pathology, Lariboisiere Hospital, AP-HP, University Paris-Diderot Paris 7, Paris, France 4 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 5 Department of Head and Neck surgery, Lariboisiere Hospital, AP-HP, University Paris-Diderot Paris 7, Paris, France 6 Department of Biopathology, Gustave Roussy, Villejuif, France 7 Department of Neuropathology, New York University School of Medicine, New York, NY, USA Correspondence to: Benjamin Verillaud, email: benjamin.verillaud@gmail.com Keywords: toll-like receptor 3, innate immunity, warburg effect, HIF, metabolomics Received: February 12, 2015      Accepted: October 19, 2016      Published: October 25, 2016 ABSTRACT In this study, a possible link between the innate immune recognition receptor TLR3 and metabolic reprogramming in Head and Neck carcinoma (HNC) cells was investigated. The effects of TLR3 stimulation/knock-down were assessed under several culture conditions in 4 HNC cell-lines by cell growth assays, targeted metabolomics, and glycolysis assays based on time-resolved analysis of proton release (Seahorse analyzer). The stimulation of TLR3 by its synthetic agonist Poly(A:U) resulted in a faster growth of HNC cells under low foetal calf serum conditions. Targeted analysis of glucose metabolism pathways demonstrated a tendency towards a shift from tricarboxylic acid cycle (Krebs cycle) to glycolysis and anabolic reactions in cells treated with Poly(A:U). Glycolysis assays confirmed that TLR3 stimulation enhanced the capacity of malignant cells to switch from oxidative phosphorylation to extra-mitochondrial glycolysis. We found evidence that HIF-1α is involved in this process: addition of the TLR3 agonist resulted in a higher cell concentration of the HIF-1α protein, even in normoxia, whereas knocking-down TLR3 resulted in a lower concentration, even in hypoxia. Finally, we assessed TLR3 expression by immunohistochemistry in a series of 7 HNSCC specimens and found that TLR3 was detected at higher levels in tumors displaying a hypoxic staining pattern. Overall, our results demonstrate that TLR3 stimulation induces the Warburg effect in HNC cells in vitro , and suggest that TLR3 may play a role in tumor adaptation to hypoxia.