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Therapeutic blockade of the CD47/SIRPα axis by small molecules or monoclonal antibodies (mAbs) is a proven strategy to enhance macrophages-mediated anti-tumor activity. However, this strategy has been hampered by elevated on-target toxicities and rapid clearance due to the extensive CD47 expression on normal cells ("antigen sink") such as red blood cells (RBCs). To address these hurdles, we report on the development of STI-6643, an affinity-engineered fully human anti-CD47 IgG4 antibody with negligible binding to normal cells. STI-6643 exhibited no hemagglutination activity on human RBCs at concentrations up to 300 µg/mL yet specifically blocked the CD47/SIPRα interaction. Of particular interest, STI-6643 preserved T cell functionality in vitro and showed significantly lower immune cell depletion in vivo in contrast to three previously published competitor reference anti-CD47 clones Hu5F9, AO-176 and 13H3. In cynomolgus monkeys, STI-6643 was well-tolerated at the highest dose tested (300 mg/kg/week) and provided favorable clinical safety margins. Finally, STI-6643 displayed comparable anti-tumor activity to the high-affinity reference clone Hu5F9 in a RAJI-Fluc xenograft tumor model as monotherapy or in combination with anti-CD20 (rituximab) or anti-CD38 (daratumumab) mAbs. These data suggest that STI-6643 possesses the characteristics of an effective therapeutic candidate given its potent anti-tumor activity and low toxicity profile. [ABSTRACT FROM AUTHOR]

The non-canonical Wnt signalling receptor ROR1 is aberrantly expressed in numerous cancers, including ovarian and endometrial cancer. We previously reported that silencing ROR1 could inhibit the proliferation and metastatic potential of ovarian and endometrial cancer cells in vitro. Zilovertamab is an ROR1-targeting humanised monoclonal antibody, with demonstrated safety and efficacy in clinical trials of several ROR1-related malignancies. The aim of this study was to investigate the potential of zilovertamab alone, or in combination with commonly utilised gynaecological cancer therapies (cisplatin, paclitaxel and the PARP inhibitor—Olaparib) on high-grade serous ovarian cancer (HGSOC), including models of platinum resistance and homologous recombination deficiency (CaOV3, CaOV3CisR, PEO1 and PEO4) and endometrial cancer (EC) cell lines (Ishikawa and KLE). The effect of zilovertamab (at 25 µg/mL or 50 µg/mL) +/− agents was investigated using the IncuCyte S3 Live Cell imaging system. Zilovertamab alone inhibited the proliferation of HGSOC and EC cells in vitro, including in models of platinum resistance and homologous recombination deficiency. In general, the addition of commonly used chemotherapies to a fixed dose of zilovertamab did not enhance the observed anti-proliferative activity. This study supports the potential of zilovertamab, or other ROR1-targeting therapies, for treating women with HGSOC and EC. [ABSTRACT FROM AUTHOR]

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa. Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL). [ABSTRACT FROM AUTHOR]

Reported herein is that (4 S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum-sensing signaling molecule. [ABSTRACT FROM AUTHOR]

Reported herein is that (4 S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum-sensing signaling molecule. [ABSTRACT FROM AUTHOR]

Identification of cellular processes modulated by microbial organisms that undermine and disarm mammalian host defences against bacterial invaders has been the focus of significant biomedical research. In this microreview we will illustrate the role of bacterial N-acyl homoserine lactones ( AHL) as a strategy utilized by Gram-negative bacterial pathogens to enable colonization of the host through AHL-mediated inhibition of inflammation induced via innate immune receptor mechanisms. We will also highlight some of the signalling pathways in which the study of AHL-mediated effects on mammalian cells might lead to the discovery of global underlying principles linking inflammation and immunity to many chronic human diseases, including cancer and obesity. [ABSTRACT FROM AUTHOR]

Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co- expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliC's adjuvant effect and conferred robust protection against wild-type Salmonella challenge. [ABSTRACT FROM AUTHOR]

Chronic respiratory infections are a major cause of morbidity and mortality, most particularly in Cystic Fibrosis (CF) patients. The recent finding that gastro-esophageal reflux (GER) frequently occurs in CF patients led us to investigate the impact of bile on the behaviour of Pseudomonas aeruginosa and other CF-associated respiratory pathogens. Bile increased biofilm formation, Type Six Secretion, and quorum sensing in P. aeruginosa, all of which are associated with the switch from acute to persistent infection. Furthermore, bile negatively influenced Type Three Secretion and swarming motility in P. aeruginosa, phenotypes associated with acute infection. Bile also modulated biofilm formation in a range of other CF-associated respiratory pathogens, including Burkholderia cepacia and Staphylococcus aureus. Therefore, our results suggest that GER-derived bile may be a host determinant contributing to chronic respiratory infection. [ABSTRACT FROM AUTHOR]

Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 307deg;C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37°C. In addition, octanoyl-homoserine lactone (C8-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C10-HSL) and dodecanoyl-homoserine lactone (C12-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation. [ABSTRACT FROM AUTHOR]

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (∼10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1-termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak. [ABSTRACT FROM AUTHOR]

Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long- chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long- chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules. [ABSTRACT FROM AUTHOR]

Background: Quorum sensing (QS) in Sinorhizobium meliloti involves at least half a dozen different N-acyl homoserine lactone (AHL) signals. These signals are produced by SinI, the sole AHL synthase in S. meliloti Rm8530. The sinI gene is regulated by two LuxR-type transcriptional regulators, SinR and ExpR. Mutations in sinI, sinR and expR abolish the production of exopolysaccharide II (EPS II). Methodology/Principal Findings: This study investigated a new type of coordinated surface spreading of Rm8530 that can be categorized as swarming. Motility assays on semi-solid surfaces revealed that both flagella and EPS II are required for this type of motility. The production of EPS II depends on AHLs produced by SinI. Of these AHLs, only C16:1- and 3-oxo-C16:1-homoserine lactones (HSLs) stimulated swarming in an ExpR-dependent manner. These two AHLs induced the strongest response in the wggR reporter fusions. WggR is a positive regulator of the EPS II biosynthesis gene expression. The levels of the wggR activation correlated with the extent of swarming. Furthermore, swarming of S. meliloti required the presence of the high molecular weight (HMW) fraction of EPS II. Within swarming colonies, a recombinase-based RIVET reporter in the wggR gene was resolved in 30% of the cells, indicating an enhanced regulation of EPS II production in the subpopulation of cells, which was sufficient to support swarming of the entire colony. Conclusions/Significance: Swarming behavior of S. meliloti Rm8530 on semi-solid surfaces is found to be dependent on the functional QS regulatory cascades. Even though multiple AHL signals are produced by the bacterium, only two AHLs species, C16:1- and 3-oxo- C16:1-HSLs, affected swarming by up-regulating the expression of wggR. While EPS II is produced by Rm8530 as high and low molecular weight fractions, only the HMW EPS II facilitated initial stages of swarming, thus, suggesting a function for this polymer. [ABSTRACT FROM AUTHOR]

The synthesis of virulence factors by pathogenic bacteria is highly regulated and occurs in response to diverse environmental cues. An array of two component systems (TCSs) serves to link perception of different cues to specific changes in gene expression and/or bacterial behaviour. Those TCSs that regulate functions associated with virulence represent attractive targets for interference in anti-infective strategies for disease control. We have previously identified PA2572 as a putative response regulator required for full virulence of Pseudomonas aeruginosa, the opportunistic human pathogen, to Galleria mellonella (Wax moth) larvae. Here we have investigated the involvement of candidate sensors for signal transduction involving PA2572. Mutation of PA2573, encoding a probable methyl-accepting chemotaxis protein, gave rise to alterations in motility, virulence, and antibiotic resistance, functions which are also controlled by PA2572. Comparative transcriptome profiling of mutants revealed that PA2572 and PA2573 regulate expression of a common set of 49 genes that are involved in a range of biological functions including virulence and antibiotic resistance. Bacterial two-hybrid analysis indicated a REC-dependent interaction between PA2572 and PA2573 proteins. Finally expression of PA2572 in the PA2573 mutant background restored virulence to G. mellonella towards wild- type levels. The findings indicate a role for the orphan chemotaxis sensor PA2573 in the regulation of virulence and antibiotic tolerance in P. aeruginosa and indicate that these effects are exerted in part through signal transduction involving PA2572. [ABSTRACT FROM AUTHOR]

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate- contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. [ABSTRACT FROM AUTHOR]

Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-L-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria. [ABSTRACT FROM AUTHOR]

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness. [ABSTRACT FROM AUTHOR]

In Pseudomonas aeruginosa, quorum-sensing (QS) signaling regulates the expression of virulence factors and thus represents an attractive new target for anti-infective therapy. In the present study, we investigated whether solenopsin A, a venom alkaloid from the fire ant, possessed agonistic or antagonistic QS signaling activity in P. aeruginosa.We evaluated the modulation of virulence factor expression and transcriptional levels of QS-regulated genes in P. aeruginosa by solenopsinAand demonstrated that solenopsin A efficiently disrupted QS signaling. Interestingly, exogenously added C4-homoserine lactone (HSL), but not 3-oxo-C12-HSL, restored P. aeruginosa QS signaling, suggesting that solenopsin A targets the C4-HSL- dependent rhl QS system. [ABSTRACT FROM AUTHOR]

Bacteria use small diffusible molecules to exchange information in a process called quorum sensing An important class of autoinducers used by Gram-negative bacteria is the family of N-acylhomoserine lactones. Here, we report the discovery of a previously undescribed nonenzymatically formed product from N-(3-oxodo- decanoyl)-L-homoserine lactone; both the N-acylhomoserine and its novel tetramic acid degradation product, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, are potent antibacterial agents. Bactericidal activity was observed against all tested Gram-positive bacterial strains, whereas no toxicity was seen against Gram-negative bacteria. We propose that Pseudomonas aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. Additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. These findings merit new attention such that other previously identified autoinducers be reevaluated for additional biological functions. [ABSTRACT FROM AUTHOR]

Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently used pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. An alternative is to use protein-based therapeutics that can eliminate the load of cocaine, thereby attenuating its effects. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the CNS. Bacteriophage have the capacity to penetrate the CNS when administered intranasally. Here, a method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain. These antibody-displaying constructs were examined by using a locomotor activity rodent model to assess the ability of the phage-displayed proteins to block the psychoactive effects of cocaine. Results presented demonstrate a strategy in the continuing efforts to find effective treatments for cocaine addiction and suggest the application of this protein-based treatment for other drug abuse syndromes. [ABSTRACT FROM AUTHOR]

As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C12-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings. [ABSTRACT FROM AUTHOR]

Background: Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive.Methods: We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests.Results: We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001).Conclusions: Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target. [ABSTRACT FROM AUTHOR]

Background Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive. Methods We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests. Results We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001). Conclusions Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target. [ABSTRACT FROM AUTHOR]