Disruption Of A Tgf-Beta-Ctof Regulated Pathway Leads To Cancer Stem Cells

Background: Sporadic cancer formation from stem cells, and molecular switches leading to this remain poorly understood. Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder with an 800-fold increased risk of developing tumors. Imprinting of the IGF2/H19 and the CDKN1C/KCNQ1 loci on chromosome 11p15.5 is mediated by the chromatin insulator CCCTC-binding factor (CTCF). This regulation is lost in BWS, leading to aberrant overexpression of the growth promoting genes. Epigenetic silencing of β2-spectrin (β2SP, gene SPTBN1), a SMAD adaptor for Transforming Growth Factor-beta (TGF-β) signaling, is causally associated with BWS. With this new model for BWS, the mechanisms though which disruption of TGF-β signaling leads to tumorigenesis could be elucidated further. We further sought to determine the roles of the TGF-β-mediated β2SP/SMAD3/CTCF complex in stem cell biological function. Experimental procedures: 1. Tumorigenesis analysis in Sptbn1+/− /Smad3+/− mice. 2. Whole-transcriptome sequencing were performed in the BWS cells and Sptbn1+/−; Sptbn1−/−; Smad3+/−; and Sptbn1+/−/Smad3+/− MEFs. 3. Tumor-initiating-cell (TIC) tumorigenesis, sphere formation assays were performed for stem cell like characteristics. Results: 1. We observed that double heterozygous Sptbn1+/−/Smad3+/− mice with defective TGF-β signaling develop multiple tumors phenotypically similar to those of BWS. 2. CTCF is TGF-β-inducible and facilitates TGF-β-mediated repression of hTERT transcription via β2SP/SMAD3/CTCF interactions. This regulation is abrogated in the TGF-β defective mice and BWS, causing hTERT overexpression. 3. Whole-transcriptome RNA sequencing of MEFs isolated from these three TGF-β-deficient strains displayed increased levels of stem cell-associated genes, including PDPN, EPAS1, CXCL1 and ALDH2. 4. CTCF, β2SP, and SMAD3, and modulate stem cell like characteristics such as CD133+/CD49+ TIC tumorigenesis, sphere formation and nanog expression. 5. Further, knock-down of β2SP, Smad3, or CTCF in HepG2 cells resulted in an increase of sphere formation, further supporting a role of these elements in regulating stemness. Conclusions: Loss of CTCF-dependent imprinting of tumor promoter genes such as IGF2 and TERT is caused by a defective TGF-β pathway, giving new insight into BWS-associated tumorigenesis and sporadic human cancers that are associated with mutations in SPTBN1 and SMAD3. Dysfunction of the β2SP/SMAD3/CTCF complex increases stem-like properties and enhances TICs. Our analysis provides important insight into the switches involved in sporadic cancer formation, particularly those associated with this human stem cell disorder. Citation Format: Jian Chen, Zhixing Yao, Jiun-Sheng Chen, Young Jin Gi, Yun Seong Jeong, Wilma Jogunoori, Mitchell Belkin, Bibhuti Mishra, Jon White, Abhisek Mitra, Shulin Li, Milton Finegold, Marta Davila, Jerry Shay, Keigo Machida, Hidekazu Tsukamoto, Lopa Mishra. Disruption of a TGF-β-CTCF regulated pathway leads to cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2540.