800 TGF-β Pathway Members ELF and SMAD3 Are Key Tumor Suppressors in Human Beckwith-Wiedemann Syndrome

The Smad3/4 adaptor protein ELF is emerging as a potent regulator of tumorigenesis by its ability to affect TGF-β tumor suppressor function (Science, 2003, 299:574-577, Science, 2005, 310:68-71). However, to date, the role of the TGF-β pathway at specific stages in gastrointestinal (GI) tumor development such as metaplasia, dysplasia and carcinoma remains unclear. A surprising and dramatic discovery by us is that elf+/and elf+/-/Smad3+/mice develop multiple GI cancers, with a phenotype strongly suggestive of Beckwith Wiedemann syndrome (BWS), a hereditary human cancer syndrome with no known causal mutations, and associated with loss of imprinting (LOI) at multiple loci on chromosome 11p15 including IGF2. Methods and Results: 1. Elf+/and Elf+/-/Smad3+/mice develop visceromegaly, multiple liver lobes, abnormal ear folds, and 70% of the mice develop multiple tumors that include metastatic pancreatic carcinoma, hepatocellular carcinoma, and small bowl adenocarcinoma. 2. A dramatic decrease in elf mRNA (200-2000 fold) was observed in elf+/-/Smad3+/tumor tissues without significant decrease of Smad3 or p53 compared to normal tissues. 3.We observed a loss of the 200 Kda ELF protein expression 4 out of 11 human cancer cell lines (HCC, gastric and pancreatic) by Western Blotting and in 19/20 human HCC and gastric cancer tissues by IHC labeling using a N-terminal peptide specific anti-ELF Ab. 4.We identified a missense mutation Arg 928 to Ile (G→A) in elf exon15 in 2 of the human GI cancer cell lines with loss of ELF expression. 5. Importantly we observed a loss of “C” to “U” RNA editing at elf Exon 15 in GI cancer cell lines with loss of ELF expression. 6. All 4/4 Human BWS patient tissues displayed a marked decrease in ELF RNA (by 50%), and a complete loss of ELF protein expression (western blot and IHC). Smad3 RNA and protein expression were not significantly altered. 7. In all 4 BWS tissues, we observed a loss of “C” to “U” RNA editing at elf Exon 15 with loss of ELF expression. 8 Array, proteomic analyses and IHC revealed a markedly high expression (about 4-6 fold) of IGF2, β-catenin, CDK4, and hTERT in Elf+/and Elf+/-/Smad3+/tissues. Conclusions: 1.The TGF-β pathway through elf+/and elf+/-/Smad3+/phenotype provides a novel and strongly compelling model for Beckwith-Wiedemann (BWS) a human cancer overgrowth syndrome. 2. Alterations of ELF/ SMAD3 expression at the mRNA level are the major genetic disposition of human BWS. 3. In BWS patient samples, aberrant elf RNA editing may be a predominant mechanism for loss of ELF protein expression observed in BWS.