Escape from TGF-/3 induced antiproliferative responses is a hallmark of many cancer cells. TGF-/3 signaling molecules Smads 2, 3 and 4 inhibit G1/S cell cycle progression mostly by suppression of c-Myc and cyclindependent kinases (cdks). Myc amplification, telomere maintenance, and telomerase (TERT) reactivation are common features of human foregut cancers, such as hepatocellular carcinoma (HCC). However, the specific role(s) of h-TERT and c-MYC, and their relation to the TGF-/3 pathway, in foregut cancer formation are poorly understood. We have shown that the Smad3/4 adaptor protein ELF is an important effector of TGF-(3 tumor suppressor function. Deletion of ELF results in a dramatic and spontaneous formation of liver and gastrointestinal (Gl) cancers, with exon 15 mutations in 11% of human hepatocellular (HCC) and gastric cancer cell lines tested. E/f7""""""""and elf^/SmadS^'mce develop visceromegaly and multiple Gl cancers (70% of mice), including metastatic pancreatic, HCC, intestinal adenocarcinomas and others spontaneously. This phenotype provides compelling evidence that elf''and elf'/Smad3+/~ mice are a model of the hereditary human cancer, Beckwith-Wiedemann syndrome (BWS). Tert and c-Myc are markedly elevated in elf'and elf/~/Smad3*/~ mice. Interestingly, Tert levels are far higher than can be accounted for by c-Myc levels in these tumors. Ectopic ELF and SmadS suppress Tert to a greater extent than c-Myc in the absence of TGFj3. However, ELF and SmadS associate with c-Myc in TGF-jS stimulated hepatocytes, and suppress Tert. Taken together our preliminary data suggest that divergent pathways converge on ELF and SmadS that then regulate Tert expression. We hypothesize that disruption of the TGF-(3 tumor suppressor pathway (through ELF, SmadS or Smad4) leads to a proliferative potential in cells that then acquire secondary events such as activation of pathways that include telomerase as well as c-Myc, resulting in gastrointestinal cancers. This project proposes to 1) Investiage the biochemical interactions of ELF, SmadS, c-Myc and c-Myc associated proteins, and to determine the role of these proteins in transcriptional regulation of hTERT;2) Use animal models that have predetermined mutations in elf, Smad3, Smad4 and telomerase function (mTerc) to determine their role in foregut cancer formation;3) Develop markers and targeted therapeutics to these lethal human cancers.
Our Specific Aims are to: 1. Examine the effects of ELF/SmadS on the regulation of hTERT transcription 2. Define the molecular basis for ELF/SmadS and Myc binding, and examine the effects of ELF/SmadS on Myc binding partners, towards developing new therapeutics targeted at c-Myc. 3. Investigate the collaboration between ELF, SmadS, Smad4 and telomerase dysfunction in foregut cancers by generating elf'ySmadS^'l'mTerc and elf/7Smad4+/~l mTerc mutant mice. Extend therapeutics from Project 2 and Project 4 to these models. 4. Determine whether telomerase dysfunction and c-Myc activation can be correlated temporally with loss of TGF-/3 pathway members such as ELF, SMADS and SMAD4 in BWS and human foregut cancers, from Project 1, towards developing a pathway and stage specific marker map. Inactivation of elf may greatly increase the propensity of elf'''/mTerc, elf'/SmadS^'/mTerc, and elf'' /Smad4+/'/mTerc mice to HCC and gastrointestinal tumorigenesis, compared to elf'mice. If this hypothesis is accurate, the mouse strains elf''/mTerc, elf'/SmadS^'/mTerc and elf'/SmadS^'/mTerc''will be invaluable modes for determining efficacy of anti-cancer drugs.
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