E-cadherin is a Smad4 target in colon carcinoma cells: Evidence for novel TGF-β independent functions of the tumor suppressor Smad4

Abstract

The most frequent genetic alteration that is associated with human malignancies, such as colorectal or gastric cancer, is a mutation in the p53 gene. Most of these p53 mutations result in the high accumulation of a non-functional p53 protein that no longer binds to its cognate DNA motif and therefore does not perform its normal function as a transcriptional activator. To correct this defect, it has been attempted to restore the DNA binding activity of mutant p53 by drugs that change the conformation of the protein. So far, however, this was not efficient enough to be applied in cancer therapy. In another attempt to eliminate tumor cells, wild type p53 was expressed through adenovirus vectors, p53 induces cell death and increases the sensitivity of cells to chemotherapy and radiation. However, this strategy is not specific for tumor cells and might cause damage to normal cells. In a strategy to combine efficiency with specificity, we have developed an adaptor protein that specifically restores the transcriptional activity of mutant p53. p53 activity was tested in transient transfections of cultured cells, followed by reporter assays. The adaptor protein was also expressed by a recombinant adenovirus, and p53-responsive gone products, such as p21 and mdm2, were quantified by immunoblot analysis. An adaptor protein was engineered to form a bridge between p53-responsive promoter DNA and mutant p53. The central and C-terminal portions of the p53-homologue p73 were linked to the oligomerization domain of p53. This adaptor binds to the DNA through the p73-derived portions, and it binds to mutant p53 through the oligomerization domain. Thereby, mutant p53 is titersd to the DNA of p53-responsive promoters, and transcription is activated. When the adaptor was expressed in tumor cells that contain mutant p53, p53-responsive genes were strongly activated. No such activation was observed in cells that contain wild type p53 or no p53 at all. The expression of the adaptor through an adenovirus vector, alone or in combination with chemotherapy and radiation, may provide a tool to selectively eliminate tumor cells that contain mutant p53.