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Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-β–like signaling pathway
Lihua Ding, … , Cuifen Huang, Qinong Ye
Lihua Ding, … , Cuifen Huang, Qinong Ye
Published January 12, 2009
Citation Information: J Clin Invest. 2009;119(2):349-361. https://doi.org/10.1172/JCI35930.
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Research Article Oncology

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-β–like signaling pathway

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Abstract

The four-and-a-half LIM (FHL) proteins belong to a family of LIM-only proteins that regulate cell proliferation, differentiation, and apoptosis. The exact functions of each FHL protein in cancer development and progression remain unknown. Here we report that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, in a TGF-β–independent manner. Casein kinase 1δ, but not the TGF-β receptor, was required for the FHL-mediated TGF-β–like responses, including increased phosphorylation of Smad2/3, interaction of Smad2/3 and Smad4, nuclear accumulation of Smad proteins, activation of the tumor suppressor gene p21, and repression of the oncogene c-myc. FHL1–3 inhibited anchorage-dependent and -independent growth of a human hepatoma cell line in vitro and tumor formation in nude mice. Further analysis of clinical samples revealed that FHL proteins are often downregulated in hepatocellular carcinomas and that this correlates with decreased TGF-β–like responses. By establishing a link between FHL proteins and Smad proteins, this study identifies what we believe to be a novel TGF-β–like signaling pathway and indicates that FHL proteins may be useful molecular targets for cancer therapy.

Authors

Lihua Ding, Zhaoyun Wang, Jinghua Yan, Xiao Yang, Aijun Liu, Weiyi Qiu, Jianhua Zhu, Juqiang Han, Hao Zhang, Jing Lin, Long Cheng, Xi Qin, Chang Niu, Bin Yuan, Xiaohui Wang, Cui Zhu, Yan Zhou, Jiezhi Li, Haifeng Song, Cuifen Huang, Qinong Ye

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Figure 2

FHL1 regulates TGF-β–responsive transcription and the promoter occupancy of Smad proteins.

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FHL1 regulates TGF-β–responsive transcription and the promoter occupancy...
(A and B) HepG2 cells were cotransfected with the TGF-β signaling pathway reporters wwp-Luc (A) or pHX-Luc (B) and FLAG-tagged FHL1 or CLIM-1. Cells were treated with (+) or without (–) TGF-β1 and analyzed for luciferase activity. Values are mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01 versus empty vector without TGF-β. #P < 0.05, ##P < 0.01 versus empty vector with TGF-β. (C) HepG2 cells were cotransfected with the TGF-β signaling pathway reporters p3TP-Lux, wwp-Luc, or pHX-Luc, and FHL1 siRNA (left panel). Values are mean ± SD of triplicate measurements and have been repeated 3 times with similar results. **P < 0.01 versus empty vector without TGF-β. ##P < 0.01 versus empty vector with TGF-β. The representative immunoblot with anti-FHL1 shows specific knockdown of endogenous FHL1 by FHL1 siRNA (upper-right panel). The densitometric quantitation of the FHL1 band normalized to GAPDH from 3 independent experiments is shown (lower-right panel) (mean ± SD). The molecular weight of GAPDH was ~36 kDa. (D) HepG2 cells were cotransfected with lac-Luc, lac-Smad2, Smad3, or Smad4 and FHL1 as indicated. Values shown are mean ± SD of 3 independent experiments. †P < 0.01 versus lac-Smad2, -3, or -4 alone without TGF-β. ††P < 0.05 versus lac-Smad2 alone with TGF-β. ‡P < 0.01 versus lac-Smad3 or lac-Smad4 alone with TGF-β. (E) Soluble chromatin was prepared from FHL1-transfected HepG2 cells with or without TGF-β1 and subjected to immunoprecipitation with normal serum or indicated antibodies. Immunoprecipitated DNA was PCR amplified with primers that annealed to the proximal region of the PAI-1, p21, or c-myc promoter.

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