Targeting Stat3 and Smad7 to restore TGF-ß cytostatic regulation of tumor cells in vitro and in vivo.

Transforming Growth Factor-ß (TGF-ß) and Epidermal Growth Factor (EGF) signaling pathways are both independently implicated as key regulators in tumor formation and progression. Here, we report that the tumor-associated overexpression of epidermal growth factor receptor (EGFR) desensitizes TGF-ß signaling and its cytostatic regulation through specific and persistent Stat3 activation and Smad7 induction in vivo. In human tumor cell lines, reduction of TGF-ß-mediated Smad2 phosphorylation, nuclear translocation and Smad3 target gene activation were observed when EGFR was overexpressed, but not in cells that expressed EGFR at normal levels. We identified Stat3, which is activated specifically and persistently by overexpressed EGFR, as a key signaling molecule responsible for the reduced TGF-ß sensitivity. Stable knockdown of Stat3 using small hairpin RNA(shRNA) in Head and Neck (HN5) and Epidermoid (A431) tumor cell lines resulted in reduced growth compared with control shRNA-transfected cells when grown as subcutaneous tumor xenografts. Furthermore, xenografts with Stat3 knockdown displayed increased Smad3 transcriptional activity, increased Smad2 phosphorylation and decreased Smad7 expression compared with control xenografts in vivo. Consistently, Smad7 mRNA and protein expression was also significantly reduced when EGFR activity was blocked by a specific tyrosine kinase inhibitor, AG1478, or in Stat3 knockdown tumors. Similarly, Smad7 knockdown also resulted in enhanced Smad3 transcriptional activity in vivo. Importantly, there was no uptake of subcutaneous HN5 xenografts with Smad7 knockdown. Taken together, we demonstrate here that targeting Stat3 or Smad7 for knockdown results in resensitization of TGF-ß's cytostatic regulation in vivo. Overall, these results establish EGFR/Stat3/Smad7/TGF-ß signaling axis driving tumor growth, which can be targeted therapeutically.

Smad7 differentially regulates transforming growth factor beta-mediated signaling pathways.

Smad7 has been identified as a negative regulator of transforming growth factor beta (TGF-beta) signaling by interfering with the phosphorylation of other Smad proteins by TGF-beta receptor type I (TbetaRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF-beta stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF-beta signaling pathways. Consistent with previous studies, Smad7 inhibited TGF-beta-stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-beta-induced growth inhibition.

Identification of a ligand-binding site on the granulocyte colony-stimulating factor receptor by molecular modeling and mutagenesis.

Granulocyte colony-stimulating factor (G-CSF) initiates its effects on cells of the neutrophil lineage by inducing formation of a homodimeric receptor complex. The structure of the G-CSF receptor has not yet been determined, therefore we used molecular modeling to identify regions of the receptor that were likely to be involved in ligand binding. The G-CSF receptor sequence was aligned with all the available sequences of the gp130 and growth hormone receptor families and a model of the cytokine receptor homologous domain was constructed, based on the growth hormone receptor structure. Alanine substitution mutagenesis was performed on loops and individual residues that were predicted to bind ligand. Mutant receptors were expressed in factor-dependent Ba/F3 cells and assessed for proliferation response and ligand binding. Six residues were identified that significantly reduced receptor function, with Arg288 in the F'-G' loop having the greatest effect. These residues formed a binding face on the receptor model resembling the growth hormone receptor site, which suggests that the model is reasonable. However, electrostatic analysis of the model provided further evidence that the mechanism of receptor dimerization is different from that of the growth hormone receptor.

Neutralising antibodies to the granulocyte colony-stimulating factor receptor recognise both the immunoglobulin-like domain and the cytokine receptor homologous domain.

To define regions of the granulocyte colony-stimulating factor (G-CSF) receptor that are important for ligand binding, neutralising monoclonal antibodies to the human receptor have been produced. Eleven antibodies recognised six different receptor epitopes. Antibodies from three of the epitope groups were able to detect the receptor by western blotting but did not inhibit G-CSF binding. The other three antibody groups inhibited G-CSF binding either completely (groups 1 and 2) or partially (group 3). All the antibodies inhibited proliferation of BA/F3 cells expressing the G-CSF receptor to varying extents. By using human-marine chimeric receptors, the binding sites of the antibodies were mapped to the immunoglobulin-like domain (groups 1 and 3), the cytokine receptor homologous domain (group 2) or the fibronectin type III domains (groups 4 to 6). These results show that the immunoglobulin-like and cytokine receptor homologous domains of the receptor are important for ligand binding and subsequent signalling.