Soluble Extracellular Domain Research Articles

The soluble extracellular domain of E-cadherin interferes with EPEC adherence via interaction with the Tir:intimin complex.

Enteropathogenic Escherichia coli (EPEC) causes watery diarrhea when colonizing the surface of enterocytes. The translocated intimin receptor (Tir):intimin receptor complex facilitates tight adherence to epithelial cells and formation of actin pedestals beneath EPEC. We found that the host cell adherens junction protein E-cadherin (Ecad) was recruited to EPEC microcolonies. Live-cell and confocal imaging revealed that Ecad recruitment depends on, and occurs after, formation of the Tir:intimin complex. Combinatorial binding experiments using wild-type EPEC, isogenic mutants lacking Tir or intimin, and E. coli expressing intimin showed that the extracellular domain of Ecad binds the bacterial surface in a Tir:intimin-dependent manner. Finally, addition of the soluble extracellular domain of Ecad to the infection medium or depletion of Ecad extracellular domain from the cell surface reduced EPEC adhesion to host cells. Thus, the soluble extracellular domain of Ecad may be used in the design of intervention strategies targeting EPEC adherence to host cells.-Login, F. H., Jensen, H. H., Pedersen, G. A., Amieva, M. R., Nejsum, L. N. The soluble extracellular domain of E-cadherin interferes with EPEC adherence via interaction with the Tir:intimin complex.

The Human Amyloid Precursor Protein Binds Copper Ions Dominated by a Picomolar-Affinity Site in the Helix-Rich E2 Domain.

A manifestation of Alzheimer's disease (AD) is the aggregation in the brain of amyloid β (Aβ) peptides derived from the amyloid precursor protein (APP). APP has been linked to modulation of normal copper homeostasis, while dysregulation of Aβ production and clearance has been associated with disruption of copper balance. However, quantitative copper chemistry on APP is lacking, in contrast to the plethora of copper chemistry available for Aβ peptides. The soluble extracellular protein domain sAPPα (molar mass including post-translational modifications of ∼100 kDa) has now been isolated in good yield and high quality. It is known to feature several copper binding sites with different affinities. However, under Cu-limiting conditions, it binds either Cu(I) or Cu(II) with picomolar affinity at a single site (labeled M1) that is located within the APP E2 subdomain. M1 in E2 was identified previously by X-ray crystallography as a Cu(II) site that features four histidine side chains (H313, H386, H432, and H436) as ligands. The presence of CuII(His)4 is confirmed in solution at pH ≤7.4, while Cu(I) binding involves either the same ligands or a subset. The binding affinities are pH-dependent, and the picomolar affinities for both Cu(I) and Cu(II) at pH 7.4 indicate that either oxidation state may be accessible under physiological conditions. Redox activity was observed in the presence of an electron donor (ascorbate) and acceptor (dioxygen). A critical analysis of the potential biological implications of these findings is presented.

The ProNGF/p75NTR pathway induces tau pathology and is a therapeutic target for FTLD-tau.

Tau pathology is characterized as a form of frontotemporal lobar degeneration (FTLD) known as FTLD-tau. The underlying pathogenic mechanisms are not known and no therapeutic interventions are currently available. Here, we report that the neurotrophin receptor p75NTR plays a critical role in the pathogenesis of FTLD-tau. The expression of p75NTR and the precursor of nerve growth factor (proNGF) were increased in the brains of FTLD-tau patients and mice (P301L transgenic). ProNGF-induced tau phosphorylation via p75NTR in vitro, which was associated with the AKT/glycogen synthase kinase (GSK)3β pathway. Genetic reduction of p75NTR in P301L mice rescued the memory deficits, alleviated tau hyperphosphorylation and restored the activity of the AKT/GSK3β pathway. Treatment of the P301L mice with the soluble p75NTR extracellular domain (p75ECD-Fc), which can antagonize neurotoxic ligands of p75NTR, effectively improved memory behavior and suppressed tau pathology. This suggests that p75NTR plays a crucial role in tau paGSKthology and represents a potential druggable target for FTLD-tau and related tauopathies.

Targeting the MET oncogene by concomitant inhibition of receptor and ligand via an antibody-"decoy" strategy.

MET, a master gene sustaining "invasive growth," is a relevant target for cancer precision therapy. In the vast majority of tumors, wild-type MET behaves as a "stress-response" gene and relies on the ligand (HGF) to sustain cell "scattering," invasive growth and apoptosis protection (oncogene "expedience"). In this context, concomitant targeting of MET and HGF could be crucial to reach effective inhibition. To test this hypothesis, we combined an anti-MET antibody (MvDN30) inducing "shedding" (i.e., removal of MET from the cell surface), with a "decoy" (i.e., the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction-and subsequent neutralization-we identified a single aminoacid in the extracellular domain of MET-lysine 842-that is critical for MvDN30 binding and engineered the corresponding recombinant decoyMET (K842E). DecoyMETK842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMETK842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell "scattering." The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In a preclinical model, built by orthotopic transplantation of a human pancreatic carcinoma in SCID mice engineered to express human HGF, concomitant treatment with antibody and decoy significantly reduces metastatic spread. The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined target therapy of MET "expedience."

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations

Background & AimsAlagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms.MethodsMice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines.ResultsJag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1.ConclusionsIn mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.

Leucine-rich repeat-containing G protein–coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics.

Soluble Extracellular Domain of Death Receptor 5 Inhibits TRAIL-Induced Apoptosis by Disrupting Receptor–Receptor Interactions

Dysregulation of tumor necrosis factor (TNF) receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand–receptor interactions, which can cause dangerous side effects by inhibiting the receptors that are not involved in the disease condition. Thus, alternate strategies that target receptor–receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of death receptor 5 (DR5) could block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5–DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of death receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF receptor signaling mechanisms that are associated in a wide range of clinical conditions.

Expression and purification of functional insulin and insulin-like growth factor 1 holoreceptors from mammalian cells

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. The current proposed models of activation are derived from structural studies using soluble extracellular domains and cytoplasmic tyrosine kinase domains. Preparations of full length IR and IGF1R have been hampered by the need for unconventional affinity chromatography resins and/or harsh eluting conditions. Here, we present a purification protocol to obtain full-length, detergent solubilized IR and IGF1R at quantities suitable for biochemical and structural characterization. We screened a panel of 24 structurally diverse detergents for optimal ligand activation. The receptors purified in n-dodecyl-β-D-maltoside showed ligand-stimulated autophosphorylation and kinase activity, suggesting an intact transmembrane signaling mechanism. This convenient purification protocol can be used to produce high quantities of IR, IGF1R, or other RTKs, and can be adapted for other challenging membrane proteins.

Abstract 33: The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis

Abstract Introduction: MCLA-128 is as an ADCC-enhanced IgG1 bispecific antibody that targets the HER2:HER3 dimer and is currently being tested in Phase I/II clinical trials. MCLA-128 demonstrates an in vitro potency superior to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG) thereby overcoming one of the resistance mechanisms of current HER2 therapies. This study investigates the binding mode of MCLA-128 and proof of concept studies in HRG-driven tumor models. Methods: Alanine scanning shotgun mutagenesis was used to map the epitopes of MCLA-128 to HER2 and HER3. Fab fragments of MCLA-128 were crystallized with the soluble extracellular domains of HER2 and HER3. SAXS analysis on the HER2-HER3-MCLA-128 complex was performed to investigate the binding mode of the bispecific antibody in solution. Ligand-induced dimer specificity was investigated with PathHunter® heterodimerization assays. Bispecific anti-HER2xHER3 antibody and its parental anti-HER3 monoclonal antibody were labelled with 64Cu to compare their biodistribution profiles. The efficacy of MCLA-128 in HRG-driven systems was shown in vitro in MDA-MB-175 cells and in vivo in an orthotopic intracranial patient-derived xenograft (PDX) model originating from a breast cancer brain metastasis Results: The shotgun mutagenesis study identified that the bispecific antibody MCLA-128 binds amino acids T144, R166, R181 in HER2 domain I and R426 in HER3 domain III. Crystallographic studies confirmed the involvement of these critical residues and suggested that MCLA-128 locks the HER3 receptor in its ligand-unbound inactive confirmation. SAXS analysis suggests that the bispecific antibody MCLA-128 forms inter-dimer rather than intra-dimer interactions. In vitro, MCLA-128 specifically blocked HRG-induced signaling of HER2:HER3 but not HER2:HER4 heterodimers. Biodistribution of MCLA-128 in a xenograft model of breast cancer showed that the penetration of MCLA-128 in JIMT-1 HER2-amplified tumors is HER2-dependent despite the high affinity of the HER3 Fab arm for its receptor. MCLA-128 efficiently blocked tumor growth of the HRG-driven HER2 (1+) breast cancer cell line MDA-MB-175 in 3D in vitro. Treatment of orthotopically transplanted HER2-amplified breast cancer brain tumors in mice led to 100% survival with MCLA-128, in contrast to 38% and 0% survival in T-DM1 and vehicle treated mice respectively. Conclusion: MCLA-128 targets HER2-positive tumors via its HER2 arm and locks HER3 in an inactive confirmation. The potent anti-proliferative activity of MCLA-128 in vitro and in vivo supports the clinical development of this bispecific HER2xHER3 antibody in HRG-driven tumors. Citation Format: David Maussang-Detaille, Camilla de Nardis, Linda Hendriks, Carina Bartelink-Clements, Eric Rovers, Tristan Gallenne, Robert Doornbos, Lex Bakker, John de Kruif, Ton Logtenberg, Piet Gros, Cecile Geuijen, Mark Throsby. The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 33. doi:10.1158/1538-7445.AM2017-33

High-efficiency expression and purification of the soluble glycoprotein extracellular domain of Rabies virus strain CTN

Objective To express and purify the glycoprotein extracellular domain (Ex-GP) of Rabies virus strain CTN in soluble form with high efficiency. Methods A recombinant expression plasmid containing the gene encoding the Ex-GP was constructed. Various expression conditions were screened to obtain an optimum prokaryotic expression system for Ex-GP in soluble form. The expressed target protein was purified using affinity chromatography and gel filtration chromatography. Results The target protein Ex-GP with high antigenicity was efficiently expressed in soluble form by using the recombinant PBCX expression system and effectively purified by using affinity and gel filtration chromatography. Conclusion The soluble form of Ex-GP is successfully expressed and purified in a simple and convenient way. This study paves the way for further researches on the biological functions of rabies virus glycoprotein, the pathogenic mechanism of rabies and the development of diagnostic reagent and vaccines for rabies virus. Key words: Rabies virus; Glycoprotein extracellular domain; Soluble protein expression

Development of Novel Monoclonal Antibodies for Evaluation of Transmembrane Prostate Androgen-Induced Protein 1 (TMEPAI) Expression Patterns in Gastric Cancer.

Transmembrane prostate androgen-induced protein 1 (TMEPAI) is a single-span membrane protein, functionally involved in transforming growth factor beta signaling pathway. The particular protein presented in cells in three isoforms, which differs in the length of the soluble N-terminal extracellular domain, making it challenging for the immunochemical recognition. By using quantitative real-time polymerase chain reaction, we identified significant upregulation of PMEPA1 gene expression in malignant tissues of patients with gastric adenocarcinoma. The main part of commercially available anti-TMEPAI antibodies are having polyclonal nature or not suitable for immunocytochemical localization of target protein in tissue specimens. Hence, we decide to generate a set of novel rat monoclonal antibodies (mAb) directed against conservative C-terminal cytoplasmic epitope. Immunoblotting analysis showed that monoclonal antibodies, 2E1, 6C6, and 10A7 were able to recognize specifically target protein in transiently transfected HEK293T and CHO-K1 cells. Especially established mAb, named 10A7, showed the excellent binding ability to target protein in immunohistochemistry. By using developed antibodies, we observed pronounced expression of TMEPAI in normal gastric epithelial cells while tumor cells from gastric adenomas, and adenocarcinoma samples were mostly negative for target protein expression. Also, we found that gastric epithelium cells lose the TMEPAI expression concurrently with severe dysplasia progression, which probably caused by a mechanism involving specific microRNA.

Preliminary results from a phase 1 trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGF-β, in advanced solid tumors.

3006 Background: M7824 (MSB0011359C) is a novel bifunctional fusion protein comprised of a fully human IgG1 monoclonal antibody against programmed death ligand 1 (PD-L1) fused to the soluble extracellular domain of transforming growth factor-β (TGF-β) receptor II, which acts as a TGF-β trap. We report preliminary data from a phase 1 trial of M7824 in patients (pts) with advanced solid tumors. Methods: NCT02517398 is a phase 1, open-label, 3+3 dose-escalation study. Eligible pts receive M7824 at 1, 3, 10, or 20 mg/kg Q2W until confirmed progressive disease, unacceptable toxicity, or trial withdrawal; treatment beyond progression is generally allowable. The primary objective is to determine the safety and maximum tolerated dose of M7824; secondary objectives include pharmacokinetics (PK), immunogenicity, and best overall response per RECIST v1.1. Results: 16 heavily pretreated pts with ECOG performance status 0-1 have received M7824. Our PK data show a dose-linear increase in exposure starting at a dose of 3 mg/kg; furthermore, M7824 saturates peripheral PD-L1 and sequesters any released plasma TGF-β1, -β2, and -β3 throughout the dosing period in a dose-dependent manner. Grade 3 drug-related treatment-emergent adverse events (TEAEs) occurred in 3 pts (skin infection secondary to grade 2 bullous pemphigoid [BP], lipase increased, and colitis with associated anemia); there were no grade 4-5 drug-related TEAEs. BP and colitis responded well to steroids. Colitis and its secondary events of anemia and rectal hemorrhage (in a previously radiated area) were considered dose limiting in 1 pt. There was preliminary evidence of efficacy across all dose levels, including 1 ongoing confirmed complete response (cervical), 1 durable partial response (pancreatic), a 25% reduction in the sum of diameters of target lesions after 2 doses of M7824 (cervical), and 2 cases of prolonged stable disease (pancreatic; carcinoid). Conclusions: Preliminary data from this phase 1 dose-escalation study suggest that M7824 has a manageable safety profile in pts with heavily pretreated advanced solid tumors. Early signs of clinical efficacy warrant further study. Clinical trial information: NCT02517398.

Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work.FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5–20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

Soluble Ectodomain of Neuroligin 1 Decreases Synaptic Activity by Activating Metabotropic Glutamate Receptor 2.

Synaptic cell adhesion molecules represent important targets for neuronal activity-dependent proteolysis. Postsynaptic neuroligins (NLs) form trans-synaptic complexes with presynaptic neurexins (NXs). Both NXs and NLs are cleaved from the cell surface by metalloproteases in an activity-dependent manner, releasing a soluble extracellular fragment and membrane-tethered C-terminal fragment. The cleavage of NL1 depresses synaptic transmission, but the mechanism by which this occurs is unknown. Metabotropic glutamate receptor 2 (mGluR2) are located primarily at the periphery of presynaptic terminals, where they inhibit the formation of cyclic adenosine monophosphate (cAMP) and consequently suppress the release of glutamate and decrease synaptic transmission. In the present study, we found that the soluble ectodomain of NL1 binds to and activates mGluR2 in both neurons and heterologous cells, resulting in a decrease in cAMP formation. In a slice preparation from the hippocampus of mice, NL1 inhibited the release of glutamate from mossy fibers that project to CA3 pyramidal neurons. The presynaptic effect of NL1 was abolished in the presence of a selective antagonist for mGluR2. Thus, our data suggest that the soluble extracellular domain of NL1 functionally interacts with mGluR2 and thereby decreases synaptic strength.

Viable Strategy for Inhibition of Death Receptor 5 Signaling by Disrupting Receptor‐Receptor Interactions

Therapeutic targeting of TNFR signaling in inflammatory diseases is a billion‐dollar industry. Current treatments for TNF‐related diseases function either by sequestering ligand or blocking ligand‐receptor interactions, which can cause adverse effects by inhibiting the receptors that are not involved in the disease condition. In the current study, we prove the viability of an alternative approach, namely the targeting of receptor‐receptor interactions. We use the soluble extracellular domain of Death Receptor 5 to block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the intracellular signaling machinery to trigger apoptosis. Such a strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.Support or Funding InformationThis work is supported by National Institutes of Health Grant R01 GM107175

Investigating the Levels of Soluble Extracellular Domain of HER2 Protein in the Sera of Meningioma Patients.

Bearing in mind the association of breast cancer and meningioma, the present study aimed to investigate the levels of the soluble extracellular domain of HER2 protein in meningioma cases and control group. Besides, in the present research, its associations with pathological features and prognostic indicators of meningioma were examined. A total of 68 meningioma patients along with 20 healthy age-sex matched individuals, as controls, were selected. Levels of HER2 in the sera were measured by a quantitative enzyme-linked immunosorbent assay (ELISA). The observations showed that Serum HER2 levels in meningioma patients were significantly lower than normal controls. However, outlier quantities were mostly observed in the cases. Furthermore, in meningiomas with higher histological grade (grade II, III), statistically significant elevated serum levels of HER2 were observed compared to patients with low-grade meningiomas (grade I). Serum HER2 levels were a poor biomarker for determination of pathological and prognostic characteristics of meningiomas and coupling serum HER2 levels with immunohistochemistry examination of HER2 in meningioma tissue samples would be helpful in future studies.

SrGAP1 mediates the migration inhibition effect of Slit2-Robo1 in colorectal cancer.

BackgroundThe neuronal guidance molecule Slit2 plays suppressive role in tumorigenesis and progression. We previously showed that Slit2-Robo1 inhibit cell migration in colorectal cancer (CRC). However, little is known about its downstream effectors in CRC. This study tries to identify whether the Slit-Robo Rho GTPase activating protein 1 (srGAP1) could mediate the inhibitory effect of Slit2-Robo1 on CRC cell migration.MethodsThe protein expression of srGAP1 in clinical CRC tissues was tested by immunohistochemistry staining. Conditioned medium was prepared from HEK293 cells stably expressing Slit2-myc, Robo1-HA or RoboN (a soluble extracellular domain of Robo1). Immunoprecipitation (IP) was applied to check the interaction between Robo1 and srGAP1, and immunofluorescence (IF) was used to observe the subcellular localization of Robo1 and srGAP1. Small GTPase pull-down assay was used to determine the activity of Cdc42. A modified wound healing assay was performed to detect cell migration.ResultsThe protein expression of srGAP1 was remarkably decreased in 47.5% of CRC tissues compared with adjacent noncancerous tissues, and the decreased srGAP1 expression was associated with lymphatic invasion, poor tumor differentiation, high TNM stage, and poor survival (P < 0.05). IP and IF assays revealed that srGAP1 was a Robo1-interacting protein and exhibited similar dynamic subcellular distribution after Slit2 treatment in CRC cells. Small GTPase pull-down assay and migration assay indicated that Slit2-Robo1 signaling inhibited Cdc42 activity and CRC cell motility through srGAP1.ConclusionDownregulation of srGAP1 in CRC was associated with tumor progression and poor prognosis. srGAP1 is an important downstream molecule of Slit2 signalling in CRC, and mediates the anti-migration function of Slit2 by inhibiting Cdc42.

Erythrocytosis in hepatocellular carcinoma portends poor prognosis by respiratory dysfunction secondary to mitochondrial DNA mutations.

These findings suggest clinical and therapeutic implications for erythrocytosis in hepatocellular carcinoma. There is an underlying link between mitochondrial function and hypoxia inducible factor alpha signaling, revealing a mechanism of erythrocytosis in a subset of hepatocellular carcinoma patients who may benefit from treatment involving EPO signaling interference. (Hepatology 2017;65:134-151).

Functional Differences of Very-Low-Density Lipoprotein Receptor Splice Variants in Regulating Wnt Signaling.

The very-low-density lipoprotein receptor (VLDLR) negatively regulates Wnt signaling. VLDLR has two major alternative splice variants, VLDLRI and VLDLRII, but their biological significance and distinction are unknown. Here we found that most tissues expressed both VLDLRI and VLDLRII, while the retina expressed only VLDLRII. The shed soluble VLDLR extracellular domain (sVLDLR-N) was detected in the conditioned medium of retinal pigment epithelial cells, interphotoreceptor matrix, and mouse plasma, indicating that ectodomain shedding of VLDLR occurs endogenously. VLDLRII displayed a higher ectodomain shedding rate and a more potent inhibitory effect on Wnt signaling than VLDLRI in vitro and in vivo. O-glycosylation, which is present in VLDLRI but not VLDLRII, determined the differential ectodomain shedding rates. Moreover, the release of sVLDLR-N was inhibited by a metalloproteinase inhibitor, TAPI-1, while it was promoted by phorbol 12-myristate 13-acetate (PMA). In addition, sVLDLR-N shedding was suppressed under hypoxia. Further, plasma levels of sVLDLR-N were reduced in both type 1 and type 2 diabetic mouse models. We concluded that VLDLRI and VLDLRII had differential roles in regulating Wnt signaling and that decreased plasma levels of sVLDLR-N may contribute to Wnt signaling activation in diabetic complications. Our study reveals a novel mechanism for intercellular regulation of Wnt signaling through VLDLR ectodomain shedding.