Eugene A. Volpe

Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation

Transforming growth factor-β (TGF-β) signaling members, TGF-β receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf+/− mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-β signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

Inactivation of ELF/TGF- β signaling in human gastrointestinal cancer

TGF-β/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. ELF, a β-spectrin, plays a key role in the transmission of TGF-β-mediated transcriptional response through Smads. ELF was originally identified as a key protein involved in endodermal stem/progenitor cells committed to foregut lineage. Also, as a major dynamic adaptor and scaffolding protein, ELF is important for the generation of functionally distinct membranes, protein sorting and the development of polarized differentiated epithelial cells. Disruption of elf results in the loss of Smad3/Smad4 activation and, therefore, a disruption of the TGF-β pathway. These observations led us to pursue the function of ELF in gastrointestinal (GI) epithelial cell–cell adhesion and tumor suppression. Here, we show a significant loss of ELF and reduced Smad4 expression in human gastric cancer tissue samples. Also, of the six human gastric cancer cell lines examined, three show deficient ELF expression. Furthermore, we demonstrate the rescue of E-cadherin-dependent homophilic cell–cell adhesion by ectopic expression of full-length elf. Our results suggest that ELF has an essential role in tumor suppression in GI cancers.

RING finger-dependent ubiquitination by PRAJA is dependent on TGF- β and potentially defines the functional status of the tumor suppressor ELF

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-β) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-β signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-β-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P<0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Δ-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Δ-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-β signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t1/2) and rate constant for degradation (kD) of ELF is 1.91 h and 21.72 min−1 when coupled with ectopic expression of PRAJA in cells stimulated by TGF-β, compared to PRAJA-transfected unstimulated cells (t1/2=4.33 h and kD=9.6 min−1). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.

W1964 Telomerase Reverse Transcriptase Regulation By TGF-β Signaling Through Adaptor ELF and SMAD3 That Is Independent of C-MYC

3442 Transforming Growth Factor-β (TGF-β) induced antiproliferative responses is a hallmark of many cancer cells. Smad proteins as intracellular mediators for TGF-β signaling and regulate target gene expression by activating or repressing gene transcription. Smads 2, 3 and 4 inhibit G1/S cell cycle progression mostly by suppression of c-Myc and cyclin-dependent kinases (cdks). Myc amplification, telomere maintenance, and telomerase reverse transcriptase (TERT) reactivation are common features of human foregut cancers, such as hepatocellular carcinoma (HCC). Emerging evidence indicates that ELF, a Smad3/Smad4 adaptor protein required for TGF-β signaling, is a powerful tumor suppressor [Science, 2005, 310(5745):68-71, Oncogene, 2007, 26(50):7103-10]. Elf+/-, elf+/-/Smad3+/- and elf+/-/Smad4+/-, (but not Smad3-/-) mice dramatically develop foregut cancers, including hepatocellular, pancreatic and gastric cancers. However, the specific role(s) of h-TERT, c-MYC and Elf, and their relation to the TGF-β pathway, in foregut cancer formation are poorly understood. Deletion of ELF results in a dramatic and spontaneous formation of liver and gastrointestinal cancers, with exon 15 mutations in 11% of human HCC and gastric cancer cell lines tested. Elf+/-andelf+/-/Smad3+/- mice develop visceromegaly and multiple GI cancers (70% of mice), spontaneously. This phenotype provides compelling evidence that elf+/-and elf+/-/Smad3+/- mice are a model of the hereditary human cancer syndrome Beckwith-Wiedemann (BWS). Aims: In this study, we investigated the mechanism of role of Elf, Smad3 and c-Myc in regulating human TERT gene expression by TGF-β in HepG2, PLC/PRF/5, SNU 298 cell lines and elf+/-/Smad3+/- mice tumor tissues in vitro and in vivo. Results show that: 1) TheElf+/-/Smad3+/-mice develop visceromegaly and multiple cancers, including metastatic pancreatic, hepatocellular, small bowel lymphomas, adrenocortical carcinomas, renal carcinomas and tumor tissues from elf+/-/Smad3+/- mice show a dramatic decrease of ELF mRNA; 2) TERT and c-Myc are markedly elevated in elf+/-andelf+/-/Smad3+/- mice; Interestingly, TERT levels are far higher than can be accounted for by c-Myc levels in elf+/-andelf+/-/Smad3+/- tumors; 3)Ectopic ELF and Smad3 suppress TERT greater than c-Myc in the absence of TGF-β. 4) Both ELF and Smad3 associate with c-Myc in TGF-β stimulated hepatocytes and suppress TERT. 5)Overexpression of ELF and/or Smad3 decreases hTERT RNA levels in PLC/PRF/5 and SNU 298 human cell lines. Conclusions: Taken together our preliminary data suggest that divergent pathways converge on ELF and Smad3 that then regulate TERT. Inactivation of the TGF-β signaling pathway with TERT activation provides a strong strategy for generating targeted therapeutics at TERT in these lethal human cancers.