Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors

Abstract Context: Adrenocortical carcinoma (ACC) is a rare tumor with poor prognosis. The expression of innate immunity receptor Toll-like receptor (TLR)-4 was recently reported in various human tumors, and TLR4 was shown to regulate tumor immune escape processes, proliferation, and resistance to chemotherapeutical agents. Objective: The aim of this study was to investigate TLR4 expression, signaling, and function in the process of tumorigenesis in the human adrenal cortex. Measurements and Main Results: Real-time PCR analysis of human ACC (n = 8), adenoma (n = 8), and ACC cell lines (SW13, NCI-H295R, and HAC15) revealed a significant down-regulation of TLR4, MD2, and CD14 mRNA compared with normal human adrenal cortex and adrenocortical cells in primary culture. Furthermore, immunohistochemistry revealed an abrogation of TLR4 and CD14 expression in ACC but not adenoma tissues. Western blot analysis of MAPK, AKT, activator protein-1, and nuclear factor-κB signaling revealed that the ACC cell lines are unresponsive to lipopolysaccharide action. Restoration of TLR4 signaling by stable transfection of TLR4-CD14 plasmid into NCI-H295R cells sensitized them to lipopolysaccharide incubation as shown by nuclear factor-κB activation and decreased cell viability and induced apoptosis in these cells. Conclusion: Our results demonstrate a significant reduction in the expression of TLR4 and CD14 and an inactivation of TLR4 signaling in ACCs. Furthermore, our data show that reintroduction of TLR4 expression in ACCs may provide a novel therapeutic strategy for adrenal cancer.