Abstract 5357: TLR4-dependent Nanog+ cancer stem cells exhibit defective TGF-β signaling through IGF-Akt-Yap1 pathway
Authors: Machida Keigo , Liu Jian-Chang , Kashiwabara Claudine , Feldman Douglas , Hyeongnam Jeong Joseph , Mishra Lopa , Tsukamoto Hidekazu 
Published: February 2011

[Background] We have recently identified stem cell marker Nanog as a novel target gene of Toll-like receptor-4 (TLR4) in the genesis of CD133+/CD49f+ cancer stem cells (CSC) in HCV Ns5a Tg mice fed alcohol (PNAS, 1548, 2009). Defective TGF-β pathway as seen in mice haploinsufficient for β2 spectrin (β2SP), a key adaptor protein for nuclear translocation of Smads, leads to spontaneous development of HCC (PNAS, 2445, 2008). However, the relationship between pro-oncogenic TLR4 signaling and defective TGF-β tumor suppressor pathway in liver oncogenesis is unknown. [Aim] We aimed to: 1) validate the oncogenic activity of the putative CSCs isolated from the Ns5a Tg model; and 2) determine the role of defective TGF-β signaling in the TLR4-dependent CSCs. [Methods] The CSCs and CD133-/CD49f+ (control) cells from alcohol-fed Ns5a Tg mice were examined for growth in soft agar. Their lentiviral cDNA libraries were created and tested for transformation of the oval cell line and screening for oncogenic genes. GFP-labeled CSCs were injected in nude mice to monitor tumor growth upon repetitive LPS injection by GFP imaging. Interactions of TGF-β pathway and TLR4-dependent oncogenic activity of the CSCs were also examined. [Results] Anchorage-independent growth of the CSCs but not the control cells is accelerated by LPS. The CSCs but not the control cells formed growing tumors in nude mice injected repetitively with LPS, and knockdown of Nanog abrogates this growth. Transduction of lentiviral cDNA library from the CSCs but not the control cells, transforms the oval cell line as shown by colony formation in soft agar, and known (Yap1 and Stat3) and novel oncogenic genes (IGFBP) are isolated from these colonies. The CSCs are defective in phosphorylation and nuclear translocation of Smad3, and TGF-β response element promoter activity under TLR4 activation with LPS. Further inactivation of TGF-β pathway by β2SP knockdown accelerates both spontaneous and TLR4-induced growth and Yap1 and Stat3 expression by the CSCs. IGFBP overexpression induces phosphorylation of Akt and Yap1, leading to inhibition of Smad3 nuclear translocation and Sorafenib (FDA-approved liver cancer drug)-induced cell death by cytoplasmic retension of Smad3 through interaction between Smad3 and Yap1. [Conclusions] TLR4-dependent CSCs isolated from Ns5a Tg mice are tumorigenic and have heightened IGF-Akt-Yap1 signaling and defective TGF-β signaling associated with impaired Smad3 nuclear trasnlocation under TLR4 activation. Our results suggest heightened TLR4 activation in the Nanog+ CSCs is causally associated and interactive with defective TGF-β tumor suppressor pathway for their oncogenic activity of the Nanog+ CSCs through coupling between growth signal and inhibition of tumor-killing mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5357.