| Smad proteins are intracellular mediators of transforming growth factor beta (TGF-beta) signaling that interact with diverse transcription factors to regulate target gene expression in response to TGF-beta. Lymphoid enhancer factor one (LEF1) is one of the Smad binding proteins. I inserted the Smad interacting motif of LEF1 into a Thioredoxin scaffold (Trx) to generate the peptide aptamer TrxLEF1D which bound to the Smads and inhibited the Smad-LEF1 interaction. TrxLEF1D also impeded the transcriptional activity of the Smad-LEF1 complex but did not affect the transcriptional activity of other Smad complexes tested, indicating that the TrxLef1D peptide aptamer could specifically disrupt a subset of responses mediated by Smads.; In an effort to characterize the role of the Smad-LEF1 complex, I established HepG2 cells stably expressing the TrxLEF1D peptide aptamer and used these cells to elucidate a new TGF-beta-independent function of Smad4. Inhibition of Smad4 function by either TrxLEF1D or Smad4 shRNAi reduced c-myc expression and the proliferation rate of HepG2 cells in the absence of TGF-beta signaling. Smad4 bound to a positive regulatory element in the c-myc promoter, stimulating c-myc transcription in the absence of TGF-beta signaling. This new function of Smad4 is in sharp contrast to its TGF-beta-dependent role, which causes the repression of c-myc transcription. Tumor-derived Smad4 mutants retained the TGF-beta-independent function while losing TGF-beta-dependent function. Expression of TrxLEF1D or Smad4 shRNAi reduced the survival of HepG2 cells in low serum.; TGF-beta induces normal mouse mammary gland (NMuMG) cells to undergo epithelial-to-mesenchymal transition (EMT), and this transition can be blocked by inhibiting Smad signaling with the peptide aptamer TrxSARA. I observed, however, that NMuMG cells expressing TrxSARA undergo EMT when those cells are surrounded by parental NMuMG cells treated with TGF-beta. The effect from the neighboring cells was also demonstrated in co-cultures of NMuMG cells and 4T1 cells, mouse mammary cancer cells. I found that beta-catenin nuclear activity is necessary in the TrxSARA or 4T1 cells for this "neighbor effect". Based on these observations, I propose that cancer cells which have lost responsiveness to TGF-beta, undergo EMT triggered by the mesenchymal transition of neighboring normal epithelial cells responding to TGF-beta. |
