Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells
Chia-Lin Chen, … , Joseph H. Jeong, Keigo Machida
Chia-Lin Chen, … , Joseph H. Jeong, Keigo Machida
Published July 1, 2013; First published June 10, 2013
Citation Information: J Clin Invest. 2013;123(7):2832-2849. https://doi.org/10.1172/JCI65859.
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Category: Research Article

Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells

Abstract

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133+ TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG–dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2+/– mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.

Authors

Chia-Lin Chen, Hidekazu Tsukamoto, Jian-Chang Liu, Claudine Kashiwabara, Douglas Feldman, Linda Sher, Steven Dooley, Samuel W. French, Lopa Mishra, Lydia Petrovic, Joseph H. Jeong, Keigo Machida

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Figure 1

TLR4/NANOG–dependent TICs from liver tumors in alcohol-fed Ns5a Tg mice and patients.

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TLR4/NANOG–dependent TICs from liver tumors in alcohol-fed Ns5a Tg mice ...
(A) FACS separation of CD133+/CD49f+ cells from liver tumors of a HCV Ns5a Tg mouse. (B) Immunoblot analysis confirms induction of NANOG and OCT4 in TICs from both Core and Ns5a mice. Mouse embryonic stem cells (mESC) serve as a positive control. (C) TICs isolated from liver tumors of Ns5a Tg mice or alcoholic HCV patients express Nanog, Oct4, Sox2, and Krt19 at higher levels than CD133–/CD49f+ or CD133–/CD49f– cells, as determined by qPCR. Immunoblots reveal increased TLR4 protein levels in TICs, which are effectively silenced by transduction of lentiviral shRNA (insets). This silencing abrogates stemness gene upregulation. *P < 0.05, #P < 0.01, compared with scrambled shRNA. (D) 3H-uridine incorporation assay in culture demonstrates enhanced, TLR4-dependent cell proliferation of TICs from Ns5a Tg model (left) and patients (right). *P < 0.05, compared with scrambled shRNA. (E) TLR4 expression correlates with the stemness marker CD133. Human HCC cell line Huh7 cells were transduced by a retroviral vector expressing TLR4 (TLR4+) or shRNA for TLR4 (sh-TLR4) and examined for surface expression of CD133 by FACS, as compared with those transduced with the control vector (Vector) or scrambled shRNA (Scrambled). (F) An immunoblot confirms induced CD133 expression in Huh7 cells transduced with Tlr4 (TLR4+) and a loss of this expression by transduction of shRNA against Tlr4 (sh-TLR4). A CD133+ population of Huh7 cells (CD133+) express abundant TLR4, and CD133– Huh7 cells (CD133–) lack this expression.