Abstract 4912: The role of long-range enhancer blocker CTCF in TGF-β/β-2 spectrin signaling: A human cancer stem cell disorder, Beckwith-Wiedemann syndrome

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy (UPD) and is characterized by overgrowth and is associated with an 800-fold increased risk of embryonal neoplasms of childhood that include Wilms’ tumors, hepatoblastomas, pancreatoblastoma, neuroblastoma, rhabdomyosarcoma, and adrenocortical carcinomas. The transcriptional insulator CTCF (CCCTC-binding factor), a highly conserved zinc finger protein, has been implicated in BWS and has diverse regulatory functions, including transcriptional activation/repression, insulation, imprinting, and X chromosome inactivation However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. TGF-β employs Smad proteins as intracellular mediators of signaling to regulate the target gene expression by activating or repressing gene transcription. The non-pleckstrin homology (PH) domain β-general-spectrin (β2SP), a scaffolding protein, functions as a potent TGF-β signaling Smad3/4 adaptor in tumor suppression and development. Yet, the role of the β2SP/TGF-β pathway in human tumor syndromes remains unclear, Our previous data have shown that β2SP is an important effector of TGF-β tumor suppressor function. Deletion of β2SP results in a dramatic and spontaneous formation of liver, gastrointestinal (GI) and pancreatic cancers. Aims: In this study, we investigated the relationship between epigenetic silencing of β-2 Spectrin/TGF-β[[Unsupported Character - ]] Signaling and loss of CTCF protein stabilization in a Human Cancer Stem Cell Disorder: Beckwith-Wiedemann Syndrome. Results show that: 1) β2SP is silenced at its promoter by DNA Methylation in human BWS non-tumor tissues and cell lines. 2) Loss of β2SP, mis-localization of Smad3 and disruption of TGF-β signaling are observed in human Beckwith-Wiedemann non-tumor tissues and cell lines 3) Ectopic expression of β2SP in human BWS cells rescues Smad3 localization and TGF-β target gene activation 4) Increased IGF2 expression in β2SP+/− and β2SP+/−/Smad3+/− mice is similar to that observed in human BWS. 5) CTCF protein levels are markedly decreased in β2SP+/− and β2SP+/−/Smad3+/− tissues. Conclusions: Our results suggest that epigenetic regulation of the TGF-β pathway results in the loss of β2SP and