Covalent Modification of the Transcriptional Repressor Tramtrack by the Ubiquitin-Related Protein Smt3 in Drosophila Flies

The ubiquitin-related SUMO-1 modifier can be covalently attached to a variety of proteins. To date, four substrates have been characterized in mammalian cells: RanGAP1, IκBα, and the two nuclear body-associated PML and Sp100 proteins. SUMO-1 modification has been shown to be involved in protein localization and/or stabilization and to require the activity of specialized E1-activating and E2 Ubc9-conjugating enzymes. SUMO-1 homologues have been identified in various species and belong to the so-called Smt3 family of proteins. Here we have characterized the Drosophila homologues of mammalian SUMO-1 and Ubc9 (termed dSmt3 and dUbc9, respectively). We show that dUbc9 is the conjugating enzyme for dSmt3 and that dSmt3 can covalently modify a number of proteins in Drosophila cells in addition to the human PML substrate. The dSmt3 transcript and protein are maternally deposited in embryos, where the protein accumulates predominantly in nuclei. Similar to its human counterpart, dSmt3 protein is observed in a punctate nuclear pattern. We demonstrate that Tramtrack 69 (Ttk69), a repressor of neuronal differentiation, is a bona fide in vivo substrate for dSmt3 conjugation. Finally, we show that both the modified and unmodified forms of Ttk69 can bind to a Ttk69 binding site in vitro. Moreover, dSmt3 and Ttk69 proteins colocalize on polytene chromosomes, indicating that the dSmt3-conjugated Ttk69 species can bind at sites of Ttk69 action in vivo. Altogether, these data indicate a high conservation of the Smt3 conjugation pathway and further suggest that this mechanism may play a role in the transcriptional regulation of cell differentiation in Drosophila flies

Regulation of Pax3 transcriptional activity by SUMO-1-modified PML

International audiencePax3 is an evolutionarily conserved transcription factor that plays a major role in a variety of developmental processes. Mutations in Pax3 lead to severe malformations as seen in human Waardenburg syndrome and in the Splotch mutant mice. The transcriptional activity of Pax3 was recently shown to be repressed by Daxx whereas the oncogenic fusion protein Pax3-FKHR is unresponsive to this repressive action. Here we demonstrate that Daxx-mediated repression of Pax3 can be inhibited by the nuclear body (NB)-associated protein PML. Interestingly, this suppression of Daxx properties correlates with its recruitment to the NBs. Factors such as arsenicals and interferons that enhance NB formation, trigger both the targeting of Daxx to these nuclear structures and the relief of the repressive activity of Daxx. Conversely, lack of structurally intact NBs profoundly impairs Pax3 transcriptional activity, likely by increasing the pool of available nucleoplasmic Daxx. Moreover, a PML mutant that can not be modified by the ubiquitin-related SUMO-1 modifier is no more able to interact with Daxx. Consistently, such a mutant fails both to inhibit the Daxx repressing effect on Pax3 and to induce its accumulation into the NBs. Taken together, these results argue that SUMO-1 modified PML can derepress Pax3 transcriptional activity through sequestration of the Daxx repressor into the NBs and suggest a role for these nuclear structures in the transcriptional control by Pax proteins

c-Jun and p53 Activity Is Modulated by SUMO-1 Modification

International audienceThe ubiquitin-related SUMO-1 molecule has been shown recently to modify covalently a number of cellular proteins including IkappaBalpha. SUMO-1 modification was found to antagonize IkappaBalpha ubiquitination and protect it from degradation. Here we identify the transcription factors c-Jun and p53, two well known targets of ubiquitin, as new substrates for SUMO-1 both in vitro and in vivo. In contrast to ubiquitin, SUMO-1 preferentially targets a single lysine residue in c-Jun (Lys-229), and the abrogation of SUMO-1 modification does not compromise its ubiquitination. Activation of Jun NH(2)-terminal kinases, which induces a reduction in c-Jun ubiquitination, similarly decreases SUMO-1 modification. Accordingly, loss of the two major Jun NH(2)-terminal kinase phosphorylation sites in c-Jun, Ser-63 and Ser-73, greatly enhances conjugation by SUMO-1. A SUMO-1- deficient c-JunK229R mutant shows an increased transactivation potential on an AP-1-containing promoter compared with wild-type c-Jun, suggesting that SUMO-1 negatively regulates c-Jun activity. As with c-Jun, SUMO-1 modification of p53 is abrogated by phosphorylation but remains unaltered upon chemical damage to DNA or Mdm2-mediated ubiquitination. The SUMO-1 attachment site in p53 (Lys-386) resides within a region known to regulate the DNA binding activity of the protein. A p53 mutant, defective for SUMO-1 conjugation, shows unaltered ubiquitination but has a slightly impaired apoptotic activity, indicating that modification by SUMO-1 might be important for the full biological activity of p53. Taken together, these data provide a first link between the SUMO-1 conjugation pathway and the regulation of transcription factors

The SUMO Pathway Is Essential for Nuclear Integrity and Chromosome Segregation in Mice

Comment in Mouse Ubc9 knockout: many path(way)s to ruin. [Dev Cell. 2005]International audienceCovalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals

A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo

Antivascular Therapy for Multidrug-Resistant Ovarian Tumors by Macitentan, a Dual Endothelin Receptor Antagonist1

Endothelin receptors (ETRs) are often overexpressed in ovarian tumors, which can be resistant to conventional therapies. Thus, we investigated whether blockage of the ETR pathways using the dual ETR antagonist macitentan combined with taxol or cisplatinum can produce therapy for orthotopically growing multidrug-resistant (MDR) human ovarian carcinoma. In several studies, nude mice were injected in the peritoneal cavity with HeyA8-MDR human ovarian cancer cells. Ten days later, mice were randomized to receive vehicle (saline), macitentan (oral, daily), taxol (intraperitoneal, weekly), cisplatinum (intraperitoneal, weekly), macitentan plus taxol, or macitentan plus cisplatinum. Moribund mice were killed, and tumors were collected, weighed, and prepared for immunohistochemical analysis. The HeyA8-MDR tumors did not respond to taxol, cisplatinum, or macitentan administered as single agents. In contrast, combination therapy with macitentan and taxol or macitentan and cisplatinum significantly decreased the tumor incidence and weight and significantly increased the survival of mice and their general condition. Multiple immunohistochemical analyses revealed that treatment with macitentan and macitentan plus taxol or cisplatinum inhibited the phosphorylation of ETRs, decreased the levels of pVEGFR2, pAkt, and pMAPK in tumor cells after 2 weeks of treatment and induced a first wave of apoptosis in tumor-associated endothelial cells followed by apoptosis in surrounding tumor cells. Our study shows that ovarian cancer cells, which express the endothelin axis and are multidrug resistant, are exquisitely sensitive to treatment with a dual ET antagonist and can be resensitized to both taxol and cisplatinum. This combined therapy led to a significant reduction in tumor weight

Macitentan (ACT-064992), a Tissue-Targeting Endothelin Receptor Antagonist, Enhances Therapeutic Efficacy of Paclitaxel by Modulating Survival Pathways in Orthotopic Models of Metastatic Human Ovarian Cancer

Potential treatments for ovarian cancers that have become resistant to standard chemotherapies include modulators of tumor cell survival, such as endothelin receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1, growing orthotopically in nude mice. Mice with established disease were treated with vehicle (control), paclitaxel (weekly, intraperitoneal injections), macitentan (daily oral administrations), or a combination of paclitaxel and macitentan. Treatment with paclitaxel decreased tumor weight and volume of ascites. Combination therapy with macitentan and paclitaxel reduced tumor incidence and further reduced tumor weight and volume of ascites when compared with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but alone had no effect on tumor incidence or ascites. Immunohistochemical analyses revealed that treatment with macitentan and macitentan plus paclitaxel inhibited the phosphorylation of ETRs and suppressed the survival pathways of tumor cells by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan necessary for inhibition of phosphorylation correlated with the dose required to increase antitumor efficacy of paclitaxel. Treatment with macitentan enhanced the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis in tumor cells and tumor-associated endothelial cells. Collectively, these results show that administration of macitentan in combination with paclitaxel prevents the progression of ovarian cancer in the peritoneal cavity of nude mice in part by inhibiting survival pathways of both tumor cells and tumor-associated endothelial cells

Hepatocyte growth factor induces cell scattering through MAPK/Egr-1-mediated upregulation of Snail

Hepatocyte growth factor/scatter factor (HGF) exerts several functions in physiological and pathological processes, among them the induction of epithelial cell scattering and motility. Its pivotal role in angiogenesis, tumor progression, and metastasis is evident; however, the underlying molecular mechanisms are still poorly understood. Here, we demonstrate that HGF induces scattering of epithelial cells by upregulating the expression of Snail, a transcriptional repressor involved in epithelial–mesenchymal transition (EMT). Snail is required for HGF-induced cell scattering, since shRNA-mediated ablation of Snail expression prevents this process. HGF-induced upregulation of Snail transcription involves activation of the mitogen-activated protein kinase (MAPK) pathway and requires the activity of early growth response factor-1 (Egr-1). Upon induction by Egr-1, Snail represses the expression of E-cadherin and claudin-3 genes. It also binds to the Egr-1 promoter and represses Egr-1 transcription, thereby establishing a negative regulatory feedback loop. These findings indicate that Snail upregulation by HGF is mediated via the MAPK/Egr-1 signaling pathway and that both Snail and Egr-1 play a critical role in HGF-induced cell scattering, migration, and invasion