Abstract 2882: Growth promoting effects of the Toll-like receptor 3 in Head and Neck carcinoma cells .

Abstract

Abstract We have previously reported an intense expression of the Toll-Like receptor 3 (TLR3) in malignant cells from EBV-associated nasopharyngeal carcinomas and other Head and Neck carcinomas (Vérillaud et al., Infectious Agents and Cancer, in press). TLR3 is involved in innate immune response triggered by the binding of double-stranded RNA (dsRNA) of viral origin. Its consistent expression by malignant epithelial cells suggests that it has some functions in cell proliferation and survival. The aim of this work was to investigate these functions using 3 cell lines derived from Head and Neck squamous cell carcinomas : CNE1 (EBV-negative nasopharyngeal carcinoma), SQ20B (laryngeal carcinoma) and Fadu (hypopharyngeal carcinoma). Cell clones conditionally knocked-down for TLR-3 were derived from each parental cell line by stable transfection of a vector encoding a microRNA targeting TLR3 transcripts and whose expression is regulated by tetracycline. Stimulation of TLR3 in parental cells was done using small concentrations (250 ng/ml) of the synthetic TLR3-ligand poly(A/U). Modest but consistent growth enhancing effects were observed in deficient culture conditions marked by low concentrations of foetal calf serum and/or low amounts of nutrients and/or hypoxia. The influence of TLR3 on growth characteristics was investigated in more details using the CNE1 cells, both parental and conditionally knocked-down for TLR3. We found that under low-serum culture conditions, cell doubling time was shortened when TLR3 was stimulated by poly(A:U). On the opposite, knocking-down TLR3 resulted in a slower growth. To better understand the mechanism underlying the growth promoting effects of TLR3 in CNE1 cells, we assessed the metabolic changes induced by poly(A/U) stimulation or TLR3 silencing . Using the Seahorse Bioscience XF Glycolysis Stress Test Kit ® we found that the glycolytic capacity of CNE1 was significantly increased under stimulation by poly(A:U), whereas it was decreased under silencing of TLR3. Finally, we investigated the impact of TLR3-stimulation on the expression of HIF1-alpha which is a key transcription factor for cell adaptation to hypoxia and a major regulator of glucid metabolism. Western blot analysis showed that HIF1-alpha could be detected even in normoxic conditions when CNE1 cells were treated by poly(A:U) for 24 hours in low serum conditions. On the other hand, knocking-down TLR3 in CNE1 cells resulted in a decrease in the cellular concentration of HIF1-alpha under hypoxia. Overall, these results suggest that TLR3-stimulation induces metabolic changes favorable to cell growth in deficient culture conditions. Future investigations will intend to investigate: 1) the in vivo functions of TLR-3 using xenografted CNE1 cells; 2) the contribution of HIF1-alpha to the metabolic changes resulting from TLR3 stimulation; 3) the nature and origin of the endogenous ligands of TLR3. Citation Format: Benjamin Vérillaud, Mélanie Gressette, Philippe Herman, Anne-Sophie Jimenez, Pierre Busson. Growth promoting effects of the Toll-like receptor 3 in Head and Neck carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2882. doi:10.1158/1538-7445.AM2013-2882