TGF-β/β2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome
Jian Chen, … , Hidekazu Tsukamoto, Lopa Mishra
Jian Chen, … , Hidekazu Tsukamoto, Lopa Mishra
Published February 1, 2016; First published January 19, 2016
Citation Information: J Clin Invest. 2016;126(2):527-542. https://doi.org/10.1172/JCI80937.
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Categories: Research Article Stem cells

TGF-β/β2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome

Abstract

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of β2-spectrin (β2SP, encoded by SPTBN1), a SMAD adaptor for TGF-β signaling, is causally associated with BWS; however, a role of TGF-β deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/– Smad3+/– mice, which have defective TGF-β signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-β–defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-β inducible and facilitates TGF-β–mediated repression of TERT transcription via interactions with β2SP and SMAD3. This regulation was abrogated in TGF-β–defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-β pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Authors

Jian Chen, Zhi-Xing Yao, Jiun-Sheng Chen, Young Jin Gi, Nina M. Muñoz, Suchin Kundra, H. Franklin Herlong, Yun Seong Jeong, Alexei Goltsov, Kazufumi Ohshiro, Nipun A. Mistry, Jianping Zhang, Xiaoping Su, Sanaa Choufani, Abhisek Mitra, Shulin Li, Bibhuti Mishra, Jon White, Asif Rashid, Alan Yaoqi Wang, Milind Javle, Marta Davila, Peter Michaely, Rosanna Weksberg, Wayne L. Hofstetter, Milton J. Finegold, Jerry W. Shay, Keigo Machida, Hidekazu Tsukamoto, Lopa Mishra

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Figure 4

TGF-β/SMAD3/β2SP upregulates CTCF.

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TGF-β/SMAD3/β2SP upregulates CTCF. (A) Decreased CTCF levels in Sptbn1+/...
(A) Decreased CTCF levels in Sptbn1+/–, Smad3+/–, and Sptbn1+/– Smad3+/– mouse livers were observed. CTCF levels were detected by immunohistochemical analysis in wild-type, Sptbn1+/–, Smad3+/–, and Sptbn1+/– Smad3+/– mouse livers. Scale bars: 20 μm. (B) CTCF protein expression levels but not mRNA levels were decreased in Sptbn1+/–, Smad3+/–, and Sptbn1+/– Smad3+/– mouse livers. CTCF protein levels were detected by immunoblotting analysis. Ctcf mRNA levels were detected by Q-PCR. Error bars are shown as SD. (C and D) TGF-β increased CTCF protein expression levels in Sptbn1+/– Smad3+/– MEFs (C) and in β2SP-knockdown HepG2 cells (D). MEFs or HepG2 cells were treated with 200 pM TGF-β1 for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. The density of CTCF and the integrated optical density were measured. Asterisk designates nonspecific bands. (E) Knockdown β2SP decreased CTCF protein stability. HepG2–sh-Ctrl or HepG2–sh-β2SP cells were treated with 100 μg/ml cycloheximide (CHX) for the indicated times. The density of CTCF and the integrated optical density were measured. The turnover of CTCF is indicated graphically. (F) β2SP-mediated CTCF downregulation was proteasome dependent. HepG2–sh-Ctrl or HepG2–sh-β2SP cells were treated with or without 50 μg/ml MG132 for 6 hours. Cell lysates were immunoblotted with CTCF antibodies. Data are representative of 3 (B–E) and 2 (F) independent experiments.