E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of A 5 -integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an A 5 B 1 -integrin-blocking antibody. When E-cadherin is silenced, A 5 -integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogenactivated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/B-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of A 5 -integrin, i.p. treatment with an A 5 B 1 -integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). A 5 -Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had A 5 -integrin overexpression, and 39% had some level of A 5 -integrin expression. The median survival for patients with high A 5 -integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified A 5 -integrin upregulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. We previously showed that the Drosophila melanogaster rel family factor, Dorsal, is a substrate for Smt3 conjugation. The conjugation machinery was found to enhance Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. In this report, we show that Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, we examined the role of Smt3 conjugation in the immune response. We find that the conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo.

To identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, we employed a yeast two-hybrid screen to search for genes encoding Dorsal-interacting proteins. Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates. We have found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3. We have also identified a DmSmt3-activating enzyme, DmSAE1/DmSAE2 and found that it further potentiates Dorsal-mediated activation.Dorsoventral patterning of the Drosophila embryo is initiated by Dorsal, a member of the Rel family of transcription factors. This maternally expressed factor is distributed in a nuclear concentration gradient in the blastoderm embryo (1) and functions as an activator and repressor of transcription to establish multiple domains of zygotic gene activity, thereby subdividing the embryo into several discrete domain along its dorsoventral axis (2, 3). Dorsal-binding proteins direct the spatially regulated nuclear import of Dorsal via an evolutionarily conserved signal transduction pathway, which targets Dorsal and its cytoplasmic inhibitor Cactus (4 -6). Prior to activation of the pathway, an interaction between Cactus and Dorsal serves to retain Dorsal in the cytoplasm. Activation of the pathway by a signal originating in the ventral extraembryonic space results in phosphorylation of Cactus and Dorsal. The phosphorylation of Cactus triggers its degradation via the ubiquitin-proteasome pathway, freeing Dorsal to enter the nucleus (7), whereas the phosphorylation of Dorsal is required to render the factor competent for efficient nuclear uptake (8).A very similar pathway involving the vertebrate homolog of Cactus, IB is involved in the regulated nuclear import of vertebrate Rel family proteins, such as NFB (9, 10). In addition, recent studies suggest that the nuclear import of NFB may be influenced by the Smt3 conjugation pathway (11). Smt3 is a small ubiquitin-like protein that can be enzymatically conjugated to various protein substrates via an amide linkage between the C-terminal carboxyl group of Smt3 and a lysine ⑀-amino group on the target protein. Conjugation of mammalian SMT3C to IB is thought to stabilize IB by blocking ubiquitylation and therefore subsequent proteasomal degradation. By stabilizing IB, the vertebrate Smt3 conjugation pathway results in the down-regulation of NFB activity.In an effort to illuminate further the mechanisms by which Dorsal activity is regulated, we sought to identify novel Dorsal interacting proteins via a yeast two-hybrid screen. One of the prot...

SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substratespecific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.The regulation of protein function by reversible post-translational modification is an effective means for regulating protein stability, activity, subcellular localization, and interaction specificity. One such modification involves the covalent attachment of the small ubiquitin-like modifier protein (SUMO) 1 (also called Smt3), to its target proteins via a series of enzymatic steps that closely resemble the reactions of the ubiquitylation pathway (1-3).Despite the sequence and structural homology between SUMO and ubiquitin, it is clear that sumoylation and ubiquitylation have distinct functional consequences. Ubiquitylation most frequently results in degradation of the substrate protein through targeting to the 26 S proteasome (4). The consequences of sumoylation are diverse and target-specific, with no evidence to suggest a role in promoting protein degradation. Conjugation of SUMO to RanGAP1 is required to target it to the nuclear pore complex (NPC), where it makes a stable interaction with the Ran-binding protein RanBP2 (5-7). Sumoylation of PML is essential for its recruitment to discrete intranuclear foci called PML oncogenic domains (PODs) (8 -11). For some sumoylation targets, including Smad4 and IB␣, this modification appears to increase protein stability by blocking ubiquitin-mediated degradation (12, 13). Finally, an increasing number of DNA binding transcription factors have been identified as substrates for sumoylation (14,15). Sumoylation of transcription factors can alter their activity in multiple ways. For example, in the ca...

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