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 January 19, 2016
Citation Information: J Clin Invest. 2016;126(2):527-542. https://doi.org/10.1172/JCI80937.
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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 8

Dysfunction of the β2SP/SMAD3/CTCF complex increases stem-like properties and promotes TIC tumorigenesis.

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Dysfunction of the β2SP/SMAD3/CTCF complex increases stem-like propertie...
(A) Increased ALDH population in β2SP, SMAD3, or CTCF knockdown cells. The positive ALDH cells were isolated from HepG2–sh-Ctrl, HepG2–sh-β2SP, HepG2–sh-SMAD3, or HepG2–sh-CTCF cells and then measured by flow cytometry. Bar graph data represent percentages of ALDH-positive cells. *P < 0.001, 1-way ANOVA with post-hoc Bonferroni’s test. (B) Knockdown of β2SP, SMAD3, or CTCF increases sphere formation. HepG2–sh-Ctrl, HepG2–sh-β2SP, HepG2–sh-SMAD3, or HepG2–sh-CTCF cells were cultured in serum-free DMEM/F12 medium with growth factors (10 ng/ml of EGF and FGF) for 6 days. Quantification of the spheres is shown. *P < 0.001, 1-way ANOVA with post-hoc Bonferroni’s test. Representative images of spheres were taken at day 6. Scale bars: 5 μm. (C) β2SP knockdown efficiency is shown in CD133+CD49f+ TICs isolated from liver tumors of alcohol-fed HCV Ns5a Tg mice. β2SP protein levels are effectively silenced by transduction of lentiviral shRNA in TICs (insets). *P < 0.01, Student’s t test. (D) β2SP knockdown increases CD133+CD49f+ TIC proliferation. Cell proliferation rates were determined by measuring 3H-uridine incorporation. *P < 0.05, Student’s t test (vs. scrambled control). (E) β2SP knockdown increases Nanog expression levels in CD133+ TICs. The mRNA expression levels of Nanog were measured by Q-PCR. *P < 0.05, Student’s t test. (F) Nanog promoter activity is higher in CD133+ TICs, while TGF-β stimulation does not significantly inhibit Nanog promoter activity in these TICs. Nanog promoter luciferase assays were performed. (G) SMAD3 knockdown enhanced subcutaneous tumor growth of TICs in a xenograft NOG mouse model. SMAD3 protein levels are effectively silenced by transduction of lentiviral shRNA in TICs (insets). Error bars are shown as SD. Each result shown is representative of 3 independent experiments (A–G).