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Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis. [ABSTRACT FROM AUTHOR]

Purpose: Nano-drug delivery systems are designed to contain surface ligands including antibodies for "active targeting". The number of ligands on each nanoparticle, known as the valency, is considered a critical determinant of the "targeting" property. We sought to understand the correlation between valency and binding properties using antibody conjugated liposomes, i.e. immunoliposomes (ILs), as the model. Methods: Anti-CD3 Fab containing a terminal cysteine residue were conjugated to DSPE-PEG-maleimide and incubated with preformed liposomes at 60°C. The un-incorporated antibodies were removed and the obtained ILs were characterized to contain in average 2–22 copies of anti-CD3 Fabs per liposome. The Biolayer Interferometry (BLI) probe surface was coated with various densities of CD3 epsilon&delta heterodimer (CD3D/E) to imitate different CD3 expression levels on target cells. The inference wavelength shifts upon anti-CD3 liposome binding were monitored and analyzed. Results: The data indicated ILs may bind either monovalently or multivalently, determined mainly by the surface ligand density rather than the ILs antibody valency. The ILs valency indeed correlated with the dissociation rate constant (Koff), but not with the association rate constant (Kon). Their binding capabilities also did not necessarily increase with the surface anti-CD3 valency. Conclusion: We proposed a model for understanding the binding properties of ILs with different ligand valencies. The binding mode may change when the targeted surfaces had different antigen densities. The model should be important for the designing and optimization of active targeting drug delivery systems to fit different applications. [ABSTRACT FROM AUTHOR]

Purpose: Topical treatment of various skin disorders requires drug absorption and penetration through the stratum corneum (SC) into the epidermis and dermis tissues. The use of nano-drug delivery systems including liposomes and lipid nanoparticles (SLNs) have been shown to facilitate SC penetration. The goal of this work was to study the impact of liposome sizes and the resulted drug distribution inside various skin tissue. Methods: All trans retinoic acid (ATRA) was used as the model drug and loaded into gel phase HSPC/CHOL/DSPE-PEG liposomes (lipo-ATRA) with sizes ranging from 80 nm to more than 300 nm. The percutaneous drug absorption process was monitored and analyzed. Results: There were significant differences in percutaneous absorption and tissue distribution resulted from liposomes smaller than 100 nm and those bigger than 200 nm. Lipo-ATRA with a mean diameter of 83 nm can deliver the content to epidermis and dermis. But for 200 nm - 300 nm liposomes, the resulted epidermis and dermis ATRA levels were less than about one third, suggesting bigger liposomes had poor penetration through the brick and mortar structure of SC. Conclusions: Gel phase liposomes with sizes under 100 nm improved encapsulated drug absorption and distribution into the epidermis and dermis tissues. A size dependent mechanism for liposome penetration of the stratum corneum was proposed. [ABSTRACT FROM AUTHOR]

Although legumain has been found to be a prognostic factor in both breast cancer and colorectal cancer, its effects on gastric cancer are unknown. In this study, we investigated effects of legumain on gastric cancer and the correlation between legumain expression and prognosis of gastric cancer patients. SGC7901 cells were transduced with legumain cDNA (SGC7901-hLeg) for overexpression of legumain or with legumain shRNA to knock down legumain. In vitro tumor migration was examined by wound healing assay. Furthermore, a tumorigenicity and metastasis mouse model was used to examine legumain function in vivo; asparaginyl endopeptidase inhibitor (AEPI, an inhibitor of legumain) was injected to the mice (i.p.) to evaluate its therapeutic effect. Tissue microarray analysis from 112 gastric cancer patients was performed to evaluate the association between legumain expression and the cumulative survival time. Legumain was highly expressed in gastric cancer patients and some gastric cancer cell lines. Legumain promoted gastric cell migration in vitro and promoted gastric tumor growth and metastasis in vivo, and these effects were reversed by knockdown of legumain with shRNA or treated with AEPI. In gastric cancer clinical samples, legumain expression in tumor was significantly higher than in non-tumor and was negatively associated with the cumulative survival rate. In conclusion, legumain was highly expressed in gastric adenocarcinoma; legumain promoted gastric cancer tumorigenesis and metastasis in vitro and in vivo. Legumain expression in tumor was a poor prognostic factor for gastric cancer patients, and legumain could be a potential target molecule for gastric cancer therapy in clinic. [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]