Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells
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
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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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 7

Inhibition of the TGF-β pathway promotes TLR4-mediated oncogenesis.

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Inhibition of the TGF-β pathway promotes TLR4-mediated oncogenesis. (A) ...
(A) Subcutaneous tumor growth by TICs from Ns5a Tg mouse tumors in NOG mice is attenuated by a gain-of-function approach for TGF-β signaling using adenoviral transduction of the constitutively active TβRI (caALK5), while it is promoted by overexpression of Smad7 or shRNA against Spnb2. (B) SPNB2 knockdown induces TLR4 and tumor-initiating property of Huh7 cells, which do not cause spontaneous xenograft growth. This growth by SPNB2 knockdown is largely prevented by knockdown of TLR4 with shRNA. Representative pictures of NOG mice bearing tumors at day 88 are shown. (C) Heterozygosity of Spnb2 induces TLR4 expression and downstream signaling (TAK1/TRAF6 association), and these changes are accentuated by alcohol feeding for 12 months. (D) The spontaneous liver tumor incidence in the Spnb2+/– mice, but not in Spnb2+/–Tlr4 mice, is increased by alcohol feeding. (E) Knockdown of SPNB2 with shRNA induces TLR4, while overexpression of caSMAD reduces TLR4 in Huh7 cells. (F) Knockdown of SPNB2 in Huh7 cells equally promotes LPS-induced activity of the TLR4 promoter containing 3 proximal SMAD-responsive elements (SRE). (G) Expression of CA-SMAD3 inhibits LPS-mediated TLR4 promoter (–4121/+180 nt) activity in Huh7 cells. *P < 0.05, **P < 0.01.