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

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 4

FHL1, CK1δ, and Smad proteins form complexes.

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FHL1, CK1δ, and Smad proteins form complexes. (A) HepG2 cells were cotra...
(A) HepG2 cells were cotransfected with wwp-Luc, FHL1, and the indicated kinases, including TRIB3, DYRK2, AURKA, TESK1, and CK1δ. Twenty four hours after transfection, cells were assayed for luciferase activity. Values shown are mean ± SD of triplicate measurements. (B) Purified GST or GST-CK1δ was incubated with purified His-FHL1, and bound proteins were subjected to SDS-PAGE and Western blot with anti-His. (C) HepG2 cells were immunoprecipitated with anti-FHL1 or preimmune control serum, and precipitates were analyzed by immunoblot with anti-CK1δ. (D) HepG2 cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG, followed by immunoblotting with the indicated antibodies. The immune complexes were eluted with FLAG peptide and reimmunoprecipitated (Re-IP) using anti-HA or normal mouse serum. The resulting precipitates were analyzed by immunoblotting with the indicated antibodies. (E) FLAG-tagged CK1δ or TβR-I and GFP-tagged full-length Smad2 or its deletion mutants were cotransfected into 293T cells. Cell lysates were immunoprecipitated with anti-FLAG and precipitates were immunoblotted with anti-GFP. The molecular weights were as follows: GST-CK1δ, ~75 kDa; His-FHL1, ~33 kDa; CK1δ, ~49 kDa; HA-FHL1, ~33 kDa; FLAG-CK1δ, ~50 kDa; FLAG–TβR-I, ~61 kDa; GFP-Smad2, ~85 kDa; GFP-MH1, ~52 kDa; GFP-linker, ~40 kDa; GFP-MH2, ~52 kDa.