ABSTRACT
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.
Subject(s)
Neoplasms/metabolism , STAT3 Transcription Factor/metabolism , Smad7 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cell Line, Tumor , ErbB Receptors/biosynthesis , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , STAT3 Transcription Factor/genetics , Signal Transduction , Smad7 Protein/genetics , TransfectionABSTRACT
Recent efforts in our laboratories have resulted in a synthetic approach toward C2'-alkylated K252a analogues via extension of a K252a cyclofuranosylation strategy. The bis-indole-N-glycosidic coupling of 6-N-(3,4-dimethoxybenzyl)-staurosporinone (21) with a number of highly functionalized carbohydrates has given access to previously unattainable, biologically relevant analogues.
Subject(s)
Carbazoles/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Protein Kinase C/antagonists & inhibitors , Alkylation , Indicators and Reagents , Indole Alkaloids , Molecular Conformation , StereoisomerismABSTRACT
Cohesin is an evolutionarily conserved multiprotein complex required to establish and maintain sister chromatid cohesion. Here, we report the cloning and initial characterization of the Drosophila homologue of the fission yeast rad21 cohesin subunit, called Drad21. The Drad21 coding region was localized to centromeric heterochromatin and encodes a 715 amino acid (aa) protein with 42% aa identity to vertebrate Rad21p-homologues, 25% with Scc1p/Mcd1p (S. cerevisiae) and 28% with Rad21p (S. pombe). Sequences with similarity to the sites of proteolytic cleavage identified in Scc1p/Mcd1p are not evident in DRAD21. Northern blot and mRNA in-situ studies show that Drad21 is developmentally regulated, with high levels of expression in early embryogenesis, in S-phase cells of proliferating imaginal tissues, and in the early endocycling cells of the embryonic gut.
