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

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 3

β2SP is required for SMAD3 nuclear translocation in BWS cells.

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β2SP is required for SMAD3 nuclear translocation in BWS cells. (A) Inter...
(A) Interaction of β2SP and SMAD3 is TGF-β dependent. HepG2 cells were treated with 200 pM TGF-β1 for the indicated times. Cell lysates were immunoprecipitated with a SMAD3 antibody and immunoblotted with the indicated antibodies. Asterisk designates nonspecific bands. (B) β2SP interacts with SMAD3. SNU398 cells were cotransfected with the indicated plasmids. Cell lysates were immunoprecipitated with a V5 antibody and immunoblotted with indicated antibodies. (C) β2SP interacts with SMAD3 MH2 domain. SNU398 cells were cotransfected with indicated plasmids. Cell lysates were immunoprecipitated with a Myc antibody and immunoblotted with indicated antibodies. (D) Interaction of β2SP and p-SMAD3 in cell nucleus. Cell lysates were isolated as nuclear (N) and cytoplasmic (C) compartments. HepG2 cells were treated with 200 pM TGF-β for 2 hours. Cell lysates were immunoprecipitated with a p-SMAD3 antibody or a p-SMAD2 antibody and immunoblotted with indicated antibodies. Asterisk designates IgG heavy chain bands. (E) Knockdown of β2SP compromises TGF-β–induced SMAD3, but not SMAD2, nuclear translocation. HepG2 shRNA control cells and shRNA-β2SP stable cells were treated with 200 PM TGF-β for 1 hour. Immunofluorescent staining was performed to detect β2SP, SMAD3, or SMAD2. Scale bars: 40 μm. (F) TGFBR1 inhibitor SB431542 blocks the interaction of β2SP and SMAD3. SNU398 cells were cotransfected with indicated plasmids and were treated with 5 μM TGFBR1 inhibitor SB431542 overnight. Cell lysates were immunoprecipitated with a Flag antibody and immunoblotted with indicated antibodies. Data are representative of 3 (A, B, and D), 2 (C), and 1 (F) independent experiments.