Abstract 4893: Protooncogenic TLR4 induces NANOG-mediated oncogenic signaling via synergistic interaction with STAT3 pathway in experimental and clinical tumor-initiating cells.
Authors: Chen Chia-Lin , Liu Jian-Chang , Sher Linda , Mishra Lopa , Tsukamoto Hidekazu , Machida Keigo 
Published: August 2014

Abstract
[Background] TLR4 signaling activated by alcohol/obesity-associated endotoxemia, induces the stem cell marker Nanog to generate tumor-initiating stem-like cells (TICs) and liver tumors in the HCV nonstructural protein Ns5a transgenic (Tg) mice but not in wild type or Ns5a Tg mice deficient in TLR4 (Machida and Tsukamoto, PNAS, 2009). However, it is unknown whether the TLR4-NANOG pathway serves as a universal oncogenic signaling in the genesis of TICs and HCC in other etiological animal models and patients or how TLR4-NANOG signaling promotes oncogenesis and self-renewal Sin TICs. [Aim] We aimed to determine whether TLR4 is a putative proto-oncogene for TIC in liver oncogenesis due to different etiologies and how TLR4 is regulated at the transcriptional and epigenetic levels. [Methods] Genome-wide NANOG-binding sites were analyzed by ChIP-seq in CD133+/CD49f+ TICs isolated from HCC developed in HCV Core Tg mice fed alcohol. We generated Tg mice with liver-selective overexpression of constitutively active (ca) TLR4 using LAP promoter-driven tTA and Tet-caTLR4. TICs were also isolated from diethylnitrosamine (DEN)-treated mice and alcoholic patients with or without HCV infection. [Results] TLR4 or NANOG silencing with shRNA attenuates tumor initiating property of TICs. Hepatocyte specific expression of caTLR4 results in spontaneous development of HCC in 35% of the mice. Genome-wide NANOG-binding site analysis (ChIP-seq) in TICs shows similarity to STAT3 binding motifs. STAT3 silencing reduces NANOG promoter reporter activity in TICs. Genome-wide NANOG-binding site analysis also shows that NANOG regulates oxidative phosphorylation and β-oxidation in mitochondria. Simultaneous real time analysis quantifying mitochondrial respiration and glycolysis demonstrates that NANOG inhibits mitochondria-mediated energy production, leading to promotion of self-renewal in TICs. TLR4 expression and promoter activity are further reduced by a silencing of LIN28 or expression of Let7. Overexpression of LIN28 and NANOG transforms primary liver progentior cells through activation of TLR4-NANOG pathway. [Conclusions] These results demonstrate that CD133+/CD49f+ cells are TLR4/NANOG-dependent TICs in both experimental and clinical HCC. TLR4 is induced by LIN28 signaling and is a universal proto-oncogene responsible for the genesis of TLR4-NANOG dependent TICs through integration of NANOG pathway with STAT3 signaling, and this pathway serves as a novel therapeutic target for HCC.
Citation Format: Chia-Lin Chen, Jian-Chang Liu, Linda Sher, Lopa Mishra, Hidekazu Tsukamoto, Keigo Machida. Protooncogenic TLR4 induces NANOG-mediated oncogenic signaling via synergistic interaction with STAT3 pathway in experimental and clinical tumor-initiating 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 4893. doi:10.1158/1538-7445.AM2013-4893