Soluble Extracellular Domain Research Articles

TAM RECEPTORS IN THE PATHOLOGY OF BLADDER CANCER: APPROACHES TO PERSONALISED MEDICINE

Bladder cancer (BC) is a cancer of the lining epithelium of the bladder. In 75% of cases, it is presented as a non-muscle invasive disease (NMIBC), with a 5-year patient survival of more than 90%. However, 25% of BC patients develop an aggressive and chemoresistant muscle-invasive form of cancer (MIBC) with less than 50% 5-year survival, making BC the 13th deadliest cancer. Moreover, the treatment for MIBC, including cystectomy and bladder reconstruction, is costly and available only in developed countries, leaving patients in other countries without efficient treatment. So, there is an urgent need to develop and implement new personalised treatment schemes in Western and low – to middle-income countries with progressively ageing populations. In our work, we study the regulatory role of the TAM Receptor Tyrosine kinases (AXL, MERTK, and TYRO3) in signalling in BC. It has been shown before that TAM receptors are activated in different cancer types, where they regulate drug resistance, immune evasion and metastasis. We analyse how TAM signalling contributes to the development of chemoresistance in BC and propose that interfering with these pathways may be a part of new combination therapy approaches. We analyse TAM receptor expression and activation in primary patient materials (solid and liquid biopsies) and develop organoid cultures for testing anti-TAM inhibitors and chemotherapeutic agents. According to our recent data, soluble extracellular domains of TAMs are present in urine samples of BC patients and are suitable as urine predictive biomarkers to use in BC.

Abstract 7634: HER2 extracellular domain in serum samples from HER2+ metastatic breast cancer patients as a potential biomarker of progression-free survival

Abstract Introduction: The detection of serum biomarkers holds potential for non-invasive methods for early cancer detection, monitoring of disease progression, and assessment of treatment responses leading to earlier interventions that may significantly improve patient outcomes. Human epidermal growth factor receptor 2 (HER2) overexpression plays a key role in the prognosis of HER2+ breast cancer (BC). While HER2 is well-established as a cell surface marker, its soluble extracellular domain (ECD) in serum has gained attention as a potential prognostic biomarker. Elevated levels of HER2 ECD (>15ng/ml) have been associated with a poor outcome. However only a small percentage of primary BC patients have levels of HER2 ECD >15ng/ml. Research has suggested that HER2 targeted therapies can influence the rate of HER2 ECD shedding. Therefore, this study aimed to explore the association between circulating HER2 ECD levels with progression-free survival (PFS) and overall survival (OS) in patients with metastatic HER2+ BC. Methods: Matched Pre and post treatment serum samples (n=25) from BC patients enrolled in TH V THL (ICORG (CTRIAL-IE) 11-10/NCT01526369) clinical trial were assessed. Human ErbB2 (HER2) ELISA Kit (Invitrogen) was used to detect levels of soluble HER2 ECD in the serum. Serum levels were stratified as Low (<300pg/ml n=5), Intermediate (300-700pg/ml n=10) or High (>700pg/ml n=10). Patient data correlation was used to assess PFS and OS, Kaplan-Meier survival curves were constructed based on the serum levels of HER2 ECD. Significance p values were determined using log-rank (Mantel-Cox) test. Results: Levels of HER2 ECD detected (94-1730pg/ml) did not exceed 15ng/ml for any sample. There was no significant difference in HER2 ECD levels between treatment arms (TH vs. THL) for PFS (p=0.4685) or OS (p=0.1376). There was no change between the pre and post treatment HER2 ECD serum levels in the samples tested (p=0.344). However, when pre-treatment samples were stratified based on Low, Intermediate or High HER2 ECD levels, there was a significant increase in PFS for patients in the High cohort. Median PFS increased from 8.31 months in the Low cohort to 13.57 months in the Intermediate cohort. The median PFS increased further to 22.24 months in the High cohort (Log rank p=0.0197). The same parameters were applied for the analysis of OS, which also improved as the level of HER2 ECD increased. Median OS increased from 35.19 months (Low) to 68.7 months (Intermediate), with a Median OS not reached for the High cohort (Log rank p=0.077). Conclusions: Patients with HER2 ECD levels above 700pg/ml display a survival advantage in this limited study. Further validation and prospective studies are warranted to fully establish the clinical utility of HER2 ECD as a biomarker in metastatic HER2+ BC. Citation Format: Debbie O'Reilly, Marc Nolan, Ausra Teiserskiene, Bryan T. Hennessy, John Crown, Denis M. Collins. HER2 extracellular domain in serum samples from HER2+ metastatic breast cancer patients as a potential biomarker of progression-free survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7634.

The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).

Abstract B034: CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy

Abstract Members of the tumor necrosis factor receptor superfamily (TNFRSF) are key co-stimulators of T cells. CD27, a member of the TNFRSF, is expressed only on the surface of lymphocytes, including naive and activated CD4+ and CD8+ T cells as well as NK cells. It enhances T cell activation, proliferation, and differentiation of effector and memory T cells after stimulation with its ligand, CD70. The costimulatory signal of CD27 is mediated via the NFkB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B pathways. CD27 signaling also influences the innate immune response via direct activation of NK cells and subsequent secretion of interferon-gamma (IFNg). Several published preclinical studies demonstrated that anti-CD27 agonistic monoclonal antibodies can promote T-cell activation and antitumor immunity making CD27 an attractive cancer immunotherapy target. Here we describe the characterization, preclinical development, and selection of our anti-CD27 fully human monoclonal antibody (mAb) lead candidate. We selected this candidate from a library of 147 anti-CD27 mAbs generated after immunization of humanized Trianni® mice with soluble human CD27 extracellular domain (hCD27-ECD). Anti-CD27 mAbs were tested in an accelerated stability study and showed excellent stability parameters for up to 7 days at 4 and 37°C in commonly used formulation buffers. The selected agonist anti-CD27 mAb demonstrated high affinity binding to both human and cynomolgus monkey CD27 and not to mouse CD27. It also demonstrated high specificity against CD27 with no cross-reactivity detected against other members of the TNFRSF. This lead candidate did not block the binding of CD27 natural ligand, CD70 and induced strong NFkB-mediated CD27 signaling in the absence or presence of cross-linking by Fc gamma receptors or secondary cross-linking antibodies. Moreover, this anti-CD27 mAb mediated NFkB activation is significantly potentiated by the addition of a sub-optimal amount of soluble CD70. The anti-CD27 lead mAb induced T cell proliferation and secretion of pro-inflammatory cytokines only in the presence of sub-optimal TCR stimulation in vitro using primary human T cells. It also activated NK cells demonstrated by CD69 expression induction. The anti-CD27 mAb lead candidate showed extended serum half-life in hCD27-KI mice. It also demonstrated a significant antitumor effect as a single agent in human CD27-Knockin mice (hCD27-KI) subcutaneously implanted with MC38 or in NOD-SCID mice subcutaneously implanted with Raji. These preclinical results establish that the selected anti-CD27 mAb is a promising drug candidate and we are actively pursuing its development. Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Kurt Lustig, Shawn Iadonato. CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B034.

Relationship of Soluble Lectin-Like Low-Density Lipoprotein Receptor-1 (sLOX-1) With Inflammation and Coronary Plaque Progression in Psoriasis.

Psoriasis is a chronic inflammatory condition associated with coronary artery disease risk. Uptake of oxidized low-density lipoprotein by the lectin-like low-density lipoprotein receptor-1 triggers release of the soluble extracellular domain of the receptor (sLOX-1). We sought to characterize the relationship between sLOX-1, inflammation, and coronary plaque progression in psoriasis. A total of 327 patients with psoriasis had serum sLOX-1 levels measured at baseline by an ELISA-based assay. Stratification by high-sensitivity C-reactive protein ≥4.0 mg/L (quartile 4), identified 81 participants who had coronary plaque phenotyping at baseline and were followed longitudinally by coronary computed tomography angiography. Subjects within high-sensitivity C-reactive protein quartile 4 were middle-aged (51.47±12.62 years), predominantly men (54.3%) with moderate psoriasis disease severity (6.60 [interquartile range, 3.30-13.40]). In the study cohort, participants with sLOX-1 above the median displayed increased vulnerable coronary plaque features. At baseline, sLOX-1 was associated with total burden (rho=0.296; P=0.01), noncalcified burden (rho=0.286; P=0.02), fibro-fatty burden (rho=0.346; P=0.004), and necrotic burden (rho=0.394; P=0.002). A strong relationship between sLOX-1, noncalcified burden (β=0.19; P=0.03), and fibro-fatty burden (β=0.29; P=0.003) was found in fully adjusted models at baseline and 1- and 4-year follow-up. Finally, coronary plaque features progressed over 1 year regardless of biologic or systemic treatment in subjects with high sLOX-1. Patients with psoriasis with both high sLOX-1 and high-sensitivity C-reactive protein levels have increased coronary plaque burden associated with atherosclerotic plaque progression independent of biologic and systemic treatment. Thus, sLOX-1 might be considered as a promising marker in coronary artery disease risk estimation beyond traditional risk factors. URL: https://www.clinicaltrials.gov; Unique identifier: NCT01778569.

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer.

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.

EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma

ObjectivesCetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance. Materials and methodsWe first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS). ResultsEPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner. ConclusionSoluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.

Circulating Soluble TREM2 and Cardiovascular Outcome in Cohort Study of Coronary Atherosclerosis Patients.

Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a membrane receptor in myeloid cells that mediates cellular phagocytosis and inflammation. TREM2 and its soluble extracellular domain are clearly implicated in neuroinflammation and neurodegeneration. sTREM2 is also expressed in atherosclerotic macrophages. We hypothesized that sTREM2 would predict cardiovascular mortality in patients with established coronary atherosclerosis (CAD). Consecutive patients undergoing coronary angiography with the establishment of the diagnosis of CAD (n = 230) and without CAD (n = 53) were tested for their baseline serum sTREM2 levels. All patients were followed up for 84 months or until death occurred. sTREM2 correlated with age; however, no association was found between sTREM2 and the number of atherosclerotic vessels involved (p = 0.642). After 84 months of follow-up, 68 out of the 230 CAD patients had died. After adjusting for age and other risk factors, the adjusted hazard ratio for the highest quartile of sTREM2 was 2.37 (95% confidence interval 1.17-4.83) for death. In patients with established CAD, serum sTREM2 appears to predict cardiovascular death as a potential surrogate for plaque rupture. TREM2 and its soluble extracellular form might be implicated in the fate of the atherosclerotic plaque, but corroboration within larger studies is needed.

Soluble Complement Receptor 1 Therapeutics

Human Complement Receptor 1 (CR1/CD35) is a potent negative regulator of the complement system. Its mechanism of action is through interaction with the complement activation fragments, C3b and C4b to mediate decay acceleration of the C3 and C5 convertase complexes as well as cleavage of both ligands into inactive fragments via cofactor activity. The result is inhibition of the classical, lectin, and alternative complement pathways. This article will focus on recombinant soluble forms of CR1 that have been generated as potential therapeutics for complement-mediated disorders. Specifically, we will review and contrast the in vitro and in vivo properties of: sCR1 (BRL55730/TP10/CDX-1135), the soluble full-length extracellular domain of human CR1; sCR1-sLex (TP20), a glyco-engineered version of sCR1 additionally targeted to activated endothelium; APT070 (Mirococept), a CR1 fragment conjugated to a myristoylated peptide to enhance tissue targeting; and CSL040, a soluble truncated version of the CR1 extracellular domain which exhibits altered potency and pharmacokinetic properties as compared to the parental molecule. The data obtained from studies on the effects of these CR1-based molecules in animal models of disease and their therapeutic applications will also be discussed.

The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), also known as β-secretase, is an aspartic protease. The sorting of this enzyme into Rab11-positive recycling endosomes regulates the BACE1-mediated cleavage of its substrates, however, the mechanisms underlying this targeting remain poorly understood. The neural cell adhesion molecule 2 (NCAM2) is a substrate of BACE1. We show that BACE1 cleaves NCAM2 in cultured hippocampal neurons and NCAM2-transfected CHO cells. The C-terminal fragment of NCAM2 that comprises the intracellular domain and a small portion of NCAM2’s extracellular domain, associates with BACE1. This association is not affected in cells with inhibited endocytosis, indicating that the interaction of NCAM2 and BACE1 precedes the targeting of BACE1 from the cell surface to endosomes. In neurons and CHO cells, this fragment and BACE1 co-localize in Rab11-positive endosomes. Overexpression of full-length NCAM2 or a recombinant NCAM2 fragment containing the transmembrane and intracellular domains but lacking the extracellular domain leads to an increase in BACE1 levels in these organelles. In NCAM2-deficient neurons, the levels of BACE1 are increased at the cell surface and reduced in intracellular organelles. These effects are correlated with increased levels of the soluble extracellular domain of BACE1 in the brains of NCAM2-deficient mice, suggesting increased shedding of BACE1 from the cell surface. Of note, shedding of the extracellular domain of Sez6, a protein cleaved exclusively by BACE1, is reduced in NCAM2-deficient animals. These results indicate that the BACE1-generated fragment of NCAM2 regulates BACE1 activity by promoting the targeting of BACE1 to Rab11-positive endosomes.

36 (PB026) - HER2 distribution in tumour and blood of mice with xenograft human cancer

Background: HER2 is well established diagnostic and therapeutic target of the malignant tumors. It is important to precisely determine HER2 status of a tumor to achieve the best therapeutic outcome. This transmembrane receptor is shed by proteolytic cleavage and secreted as the soluble extracellular domain (ECD) of ∼97–115 kDa circulating in a patient blood. The data on HER2 protein distribution within a tumor are scarce. Therefore, our goal was to quantify the HER2 protein associated with cells and cell-free tissue fluids of tumor.

Abstract P1075: Elucidate The Mechanism Driving The Imbalance Between BMPs And Activin/Gdfs Signaling In Pulmonary Arterial Hypertension

Transforming Growth Factor-beta (TGF-β) family signaling is involved in Pulmonary Arterial Hypertension (PAH) in humans. PAH is a rare, highly morbid disorder characterized by dysregulated pulmonary vascular remodeling, elevated pulmonary vascular resistance, and the progressive obliteration of the pulmonary arteriolar circulation. The progression of PAH is largely influenced by the proliferation and migration of the pulmonary smooth muscle cells (PASMCs) and pulmonary endothelial cells (PAECs). While the exact mechanism is not understood, there is evidence that germline mutations in the BMPRII gene, one of the TGF-β family type II receptors, is present in PAH patients. Due to the functional similarity of BMPRII and type II Activin receptors ACVR2A and ACVR2B, they interact with overlapping TGF-β family ligands to activate either the SMAD 1/5/8 or SMAD 2/3 signaling. In the pulmonary vasculature, the BMP-9 ligand regulates cell proliferation by binding to type I receptors, ALK-1 and BMPRII, to activate SMAD 1/5/8 signaling. In PAH, there is a decrease in SMAD 1/5/8 signaling and an increase in SMAD 2/3 signaling. However, what causes the signaling switch is still unknown. In our lab, we found out that BMPRII and ACVR2A bind TGF-β ligands with nearly identical geometry based on our crystal structures. Both of the receptors contain a conserved hydrophobic hot spot within the ligand binding pocket. We found that the BMPRII ligand binding domain may exhibit greater flexibility than ACVR2A. We also investigated eight soluble BMPRII extracellular domain mutants and expressed them as BMPRII-Fc fusion proteins (BMPRII-Fc variants). Using surface plasmon resonance, we observed four of the BMPRII-Fc variants bind to high affinity ligands, such as BMP9, BMP10, Activin B and Activin A. In reporter gene assays, we found these BMPRII-Fc variants act as a ligand trap and attenuate BMP9 and BMP10-mediated SMAD1/5/8 signaling in HEK293 cells. Interestingly, they did not inhibit Activin B-mediated SMAD2/3 signaling. In conclusion, our findings show gain of function of specific BMPRII mutations and suggest that imbalances between SMAD1/5/8 and SMAD2/3 signaling may lead to PAH progression.

P75NTR Ectodomain Ameliorates Cognitive Deficits and Pathologies in a Rapid Eye Movement Sleep Deprivation Mice Model

The neurotrophin receptor p75 (p75NTR) is a circadian rhythm regulator and mediates cognitive deficits induced by sleep deprivation (SD). The soluble extracellular domain of p75NTR (p75ECD) has been shown to exert a neuroprotective function in Alzheimer’s disease (AD) and depression animal models. Nevertheless, the role of p75ECD in SD-induced cognitive dysfunction is unclear. In the present study we administrated p75ECD-Fc (10, 3 mg/kg), a recombinant fusion protein of human p75ECD and fragment C of immunoglobulin IgG1, to treat mice via intraperitoneal injection. The results revealed that peripheral supplementation of high-dose p75ECD-Fc (10 mg/kg) recovered the balance between Aβ and p75ECD in the hippocampus and rescued the cognitive deficits in SD mice. We also found that p75ECD-Fc ameliorated other pathologies induced by SD, including neuronal apoptosis, synaptic plasticity impairment and neuroinflammation. The current study suggests that p75ECD-Fc is a potential candidate for SD and peripheral supplementation of p75ECD-Fc may be a prospective preventive measure for cognitive decline in SD.

Sphingosine 1-phosphate receptor 1 governs endothelial barrier function and angiogenesis by upregulating endoglin signaling

BackgroundThe sphingosine 1-phosphate (S1P)/S1P receptor (S1pr) 1 signaling plays an essential role in regulating vascular integrity and angiogenesis. We have previously shown that cell-surface expression of endoglin (Eng) is sustained by S1P/S1pr1 signaling in endothelial cells (ECs). However, whether S1pr1 mediates Eng signaling, or vice versa, remains unknown.MethodsS1pr1 inhibitors were used to study whether pharmacological inhibition induces basal vascular leakage in vivo. An acute respiratory distress syndrome (ARDS) model was used to study whether S1pr1 inhibition evoked greater inflammation in lungs. A S1pr1 inhibitor, a bone morphogenetic protein 9 (BMP9) blocking antibody, or lentivirus-mediated expression of soluble extracellular domain of Eng (sEng) were used to test whether blocking both S1P/S1pr1 and BMP9/Eng signaling axes would impose any interaction in retinal angiogenesis. To clarify whether S1P and BMP9 function in a linear pathway, a study of trans-endothelial electrical resistance (TEER) measurement was carried out using a mouse islet EC line MS1; time course studies were executed to exam downstream effectors of S1P and BMP9 signaling pathways in ECs; two stable MS1 cell lines were generated, one with overexpression of human S1PR1 and the other with knockdown of Eng, to validate S1pr1 and Eng were the key players for the crosstalk. Inhibitor of extracellular regulated protein kinases (ERK) was used to check whether this signaling was involved in S1P-induced cell-surface localization of Eng.ResultsThe present study elucidated that S1pr1 and Eng are both pivotal for angiogenesis in the postnatal mouse retina, and that the activation of S1pr1 or Eng increases vascular barrier function. Activation of S1pr1 enhanced the phosphorylation of Smad family members 1, 5, and 8 (pSmad1/5/8), while the inhibition of S1pr1 reduced the levels of pSma1/5/8 induced by BMP9 treatment. Activation or loss of Eng did not affect S1pr1 signaling. Moreover, activation of ERK was involved in promoting EC-surface expression of Eng by S1pr1.ConclusionsOur data demonstrates for the first time that there exists a linear pathway of S1pr1-Eng signaling axis in ECs, which governs vascular homeostasis. Functional BMP9/Eng signaling requires S1P/S1pr1 activation, and S1pr1 signaling acts as a vascular protection mechanism upstream of Eng.

RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis.

Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gαq and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor–related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.

The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites

The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells. It is notable that CD16a is highly processed with multiple N-glycosylation sites, and preventing individual N-glycan modifications affects affinity. Furthermore, multiple groups have demonstrated that CD16a N-glycan composition is variable and composition impacts antibody binding affinity. The level of N-glycosylation at each site is not known though computational prediction indicates low to moderate potential at each site based on primary sequence (40–70%). Here we quantify occupancy of the extracellular domains using complementary mass spectrometry-based methods. All five sites of the tighter-binding CD16a V158 allotype showed 65–100% N-glycan occupancy in proteomics-based experiments. These observations were confirmed using intact protein mass spectrometry that demonstrated the predominant species corresponded to CD16a V158 with five N-glycans, with a smaller contribution from CD16a with four N-glycans. Occupancy was likewise high for the membrane-bound receptor at all detected N-glycosylation sites using CD16a purified from cultured human natural killer cells. Occupancy of the N162 site, critical for antibody binding, appeared independent of N169 occupancy based on analysis of the T171A mutant protein. The weaker-binding CD16a F158 allotype showed higher occupancy of >93% at each site.

CRIg on liver macrophages clears pathobionts and protects against alcoholic liver disease

Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg–Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.

GPNMB plays a protective role against obesity-related metabolic disorders by reducing macrophage inflammatory capacity

Obesity is a global health problem that is often related to cardiovascular and metabolic diseases. Chronic low-grade inflammation in white adipose tissue (WAT) is a hallmark of obesity. Previously, during a search for differentially expressed genes in WAT of obese mice, we identified glycoprotein nonmetastatic melanoma protein B (GPNMB), of which expression was robustly induced in pathologically expanded WAT. Here, we investigated the role of GPNMB in obesity-related metabolic disorders utilizing GPNMB-deficient mice. When fed a high-fat diet (HFD), GPNMB-deficient mice showed body weight and adiposity similar to those of wild-type (WT) mice. Nonetheless, insulin and glucose tolerance tests revealed significant obesity-related metabolic disorders in GPNMB-KO mice compared with WT mice fed with HFD. Chronic WAT inflammation was remarkably worsened in HFD-fed GPNMB-KO mice, accompanied by a striking increase in crown-like structures, typical hallmarks for diseased WAT. Macrophages isolated from GPNMB-KO mice were observed to produce more inflammatory cytokines than those of WT mice, a difference abolished by supplementation with recombinant soluble GPNMB extracellular domain. We demonstrated that GPNMB reduced the inflammatory capacity of macrophages by inhibiting NF-κB signaling largely through binding to CD44. Finally, we showed that macrophage depletion by addition of clodronate liposomes abolished the worsened WAT inflammation and abrogated the exacerbation of metabolic disorders in GPNMB-deficient mice fed on HFD. Our data reveal that GPNMB negatively regulates macrophage inflammatory capacities and ameliorates the WAT inflammation in obesity; therefore we conclude that GPNMB is a promising therapeutic target for the treatment of metabolic disorders associated with obesity.

Local adenoviral delivery of soluble CD200R-Ig enhances antitumor immunity by inhibiting CD200-β-catenin-driven M2 macrophage

CD200 is known as an immune checkpoint molecule that inhibits innate immune cell activation. Using a head and neck squamous cell carcinoma (HNSCC) model, we sought to determine whether localized delivery of adenovirus-expressing sCD200R1-Ig, the soluble extracellular domain of CD200R1, enhances antitumor immunity. Mouse-derived bone marrow cells and M1/M2-like macrophages were cocultured with tumor cells and analyzed for macrophage polarization. As an in vivo model, C57BL/6 mice were subcutaneously injected with MEER/CD200High cells, CD200-overexpressing mouse HNSCC cells. Adenovirus-expressing sCD200R1-Ig (Ad5sCD200R1) was designed, and its effect was tested. Components in the tumor-immune microenvironment (TIME) were quantified using flow cytometry. CD200 promoted tumor growth and induced the expression of immune-related genes, especially macrophage colony-stimulating factor (M-CSF). Interestingly, CD200 induced M2-like polarization both in vitro and in vivo. Consequently, CD200 recruited more regulatory T (Treg) cells and fewer CD8+ effector T cells. These effects were effectively abolished by local injection of Ad5sCD200R1. These protumor effects of CD200 were driven through the β-catenin/NF-κB/M-CSF axis. CD200 upregulated PD-L1, and the combined targeting of CD200 and PD-1 thus showed synergy. The immune checkpoint CD200 upregulated immune-related genes through β-catenin signaling, reprogrammed the TIME, and exerted protumor effects. Ad5sCD200R1 injection could be an effective targeted strategy to enhance antitumor immunoediting.

Regulated Intramembrane Proteolysis of ACE2: A Potential Mechanism Contributing to COVID-19 Pathogenesis?

Since being identified as a key receptor for SARS-CoV-2, Angiotensin converting enzyme 2 (ACE2) has been studied as one of the potential targets for the development of preventative and/or treatment options. Tissue expression of ACE2 and the amino acids interacting with the spike protein of SARS-CoV-2 have been mapped. Furthermore, the recombinant soluble extracellular domain of ACE2 is already in phase 2 trials as a treatment for SARS-CoV-2 infection. Most studies have continued to focus on the ACE2 extracellular domain, which is known to play key roles in the renin angiotensin system and in amino acid uptake. However, few also found ACE2 to have an immune-modulatory function and its intracellular tail may be one of the signaling molecules in regulating cellular activation. The implication of its immune-modulatory role in preventing the cytokine-storm, observed in severe COVID-19 disease outcomes requires further investigation. This review focuses on the regulated proteolytic cleavage of ACE2 upon binding to inducer(s), such as the spike protein of SARS-CoV, the potential of cleaved ACE2 intracellular subdomain in regulating cellular function, and the ACE2’s immune-modulatory function. This knowledge is critical for targeting ACE2 levels for developing prophylactic treatment or preventative measures in SARS-CoV infections.