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 5

CK1δ is required for FHL1 modulation of Smad2/3 phosphorylation, Smad2/3 and Smad4 interaction, and nuclear accumulation of Smad proteins.

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CK1δ is required for FHL1 modulation of Smad2/3 phosphorylation, Smad2/3...
(A) HepG2 cells were cotransfected with FLAG-tagged FHL1 and/or CK1δ, with or without 50 μM of the CK1δ and CK1e inhibitor, IC261. Cell lysates were immunoprecipitated with anti-FLAG, and in vitro kinase assays were performed using the indicated His-Smad2/3 or mutants (His-SmadCm or His-SmadLm) as substrates. (B) In vitro kinase assays with purified GST-CK1δ and GST-FHL1 and purified His-Smad2/3 or mutants as substrates. The molecular weight of GST-FHL1 was ~58 kDa. (C) HepG2 cells were transiently transfected with FLAG-tagged CK1δ and/or TβR-I, and cell lysates were analyzed by immunoblotting with anti–phospho-Smad2/3 or anti-Smad2/3. (D) HepG2 cells stably expressing CK1δ siRNA and FHL1 were transiently transfected with siRNA-resistant CK1δ (CK1δ-R) as indicated, and cell lysates were analyzed by immunoblotting with the indicated antibodies. Transient transfection efficiency was approximately 70%. (E and F) HepG2 cells stably expressing CK1δ siRNA and FHL1 were transiently transfected with siRNA-resistant CK1δ as in D. (E) Cells were immunoprecipitated by anti-Smad4 or preimmune control serum, followed by immunoblotting with the indicated antibodies, or (F) cells were fractionated and the lysates were probed with the indicated antibodies.