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.

CD46/CD3 costimulation induces morphological changes of human T cells and activation of Vav, Rac, and extracellular signal-regulated kinase mitogen-activated protein kinase.

Efficient T cell activation requires at least two signals, one mediated by the engagement of the TCR-CD3 complex and another one mediated by a costimulatory molecule. We recently showed that CD46, a complement regulatory receptor for C3b as well as a receptor for several pathogens, could act as a potent costimulatory molecule for human T cells, highly promoting T cell proliferation. Indeed, we show in this study that CD46/CD3 costimulation induces a synergistic activation of extracellular signal-related kinase mitogen-activated protein kinase. Furthermore, whereas T lymphocytes primarily circulate within the bloodstream, activation may induce their migration toward secondary lymphoid organs or other tissues to encounter APCs or target cells. In this study, we show that CD46/CD3 costimulation also induces drastic morphological changes of primary human T cells, as well as actin relocalization. Moreover, we show that the GTP/GDP exchange factor Vav is phosphorylated upon CD46 stimulation alone, and that CD46/CD3 costimulation induces a synergistic increase of Vav phosphorylation. These results prompted us to investigate whether CD46/CD3 costimulation induced the activation of GTPases from the Rho family. Indeed, we report that the small GTPase Rac is also activated upon CD46/CD3 costimulation, whereas no change of Rho and Cdc42 activity could be detected. Therefore, CD46 costimulation profoundly affects T cell behavior, and these results provide important data concerning the biology of primary human T cells.

Efficient syntheses of novel C2'-alkylated (+/-)-K252a analogues.

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.

Design of inhibitors of Ras--Raf interaction using a computational combinatorial algorithm.

Drugs that inhibit important protein-protein interactions are hard to find either by screening or rational design, at least so far. Most drugs on the market that target proteins today are therefore aimed at well-defined binding pockets in proteins. While computer-aided design is widely used to facilitate the drug discovery process for binding pockets, its application to the design of inhibitors that target the protein surface initially seems to be limited because of the increased complexity of the task. Previously, we had started to develop a computational combinatorial design approach based on the well-known 'multiple copy simultaneous search' (MCSS) procedure to tackle this problem. In order to identify sequence patterns of potential inhibitor peptides, a three-step procedure is employed: first, using MCSS, the locations of specific functional groups on the protein surface are identified; second, after constructing the peptide main chain based on the location of favorite locations of N-methylacetamide groups, functional groups corresponding to amino acid side chains are selected and connected to the main chain C(alpha) atoms; finally, the peptides generated in the second step are aligned and probabilities of amino acids at each position are calculated from the alignment scheme. Sequence patterns of potential inhibitors are determined based on the propensities of amino acids at each C(alpha) position. Here we report the optimization of inhibitor peptides using the sequence patterns determined by our method. Several short peptides derived from our prediction inhibit the Ras--Raf association in vitro in ELISA competition assays, radioassays and biosensor-based assays, demonstrating the feasibility of our approach. Consequently, our method provides an important step towards the development of novel anti-Ras agents and the structure-based design of inhibitors of protein--protein interactions.

Drad21, a Drosophila rad21 homologue expressed in S-phase cells.

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.

Mitochondrial FtsZ in a chromophyte alga.

A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the alpha-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.