Molecular and functional consequences of Smad4 C-terminal missense mutations in colorectal tumour cells

Smad4 is an essential signal transducer of the transforming growth C factor (TGF-beta) signalling pathway and has been identified as a tumour Suppressor. being mutated in approx. 50 % of pancreatic cancers and approx. 15 % of colorectal cancers. Two missense mutations in the C-terminal domain of Smad4, D351H(Asp(351)-->His) and D537Y (Asp(537)-->Tyr), have been described recently in the human colorectal cancer cell lines CACO-2 and SW948 respectively [Woodford-Richens, Rowan, Gorman, Halford, Bicknell. Wasan. Rovlance. Bodmer and Tomlinson (2001) Proc. Natl. Acad. Sci. U.S.A. 98. 9719-9723]. Previous work in vitro suggested that only Asp-351 was required for interaction with Smad2 [Wu. Fairman. Penry and Shi (2001) J. Biol. Chem. 276, 20688-20694]. In the present study, we investigate the functional consequences of these point mutations in vivo. We demonstrate that neither of these colorectal cancer cells undergo growth arrest in response to TGF-beta. which can be explained. at least in part. by their inability to up-regulate cyclin-dependent kinase inhibitors p21(CIPI) or p15(INK4b) after TGF-beta stimulation. Although the point-mutated Smad4s are expressed at normal levels in these colorectal cancer cells, they cannot interact with either TGF-beta-induced phosphorylated Smad2 or Smad3. As a result, these Smad4 mutants do not accumulate in the nucleus after TGF-beta stimulation, are not recruited to DNA by relevant Smad-binding transcription factors and cannot generate transcriptionally active DNA-bound complexes. Therefore both these colorectal tumour cells completely lack functional Smad4 activity owing to the missense mutations. Given the location of these mutations ill the three-dimensional Structure of the Sri C-terminal domain, the results also give us significant insights into Smad complex formation.