Transmembrane Tight Junction Proteins Research Articles

Lactiplantibacillus plantarum ST-III and Lacticaseibacillus rhamnosus KF7 Enhance the Intestinal Epithelial Barrier in a Dual-Environment In Vitro Co-Culture Model.

Intestinal barrier hyperpermeability, which is characterised by impaired tight junction proteins, is associated with a variety of gastrointestinal and systemic diseases. Therefore, maintaining intestinal barrier integrity is considered one of the effective strategies to reduce the risk of such disorders. This study aims to investigate the potential benefits of two probiotic strains (Lactiplantibacillus plantarum ST-III and Lacticaseibacillus rhamnosus KF7) on intestinal barrier function by using a physiologically relevant in vitro model of the intestinal epithelium. Our results demonstrate that both strains increased transepithelial electrical resistance, a measure of intestinal barrier integrity. Immunolocalisation studies indicated that this improvement in barrier function was not due to changes in the co-localisation of the tight junction (TJ) proteins ZO-1 and occludin. However, we observed several modifications in TJ-related genes in response to the probiotics, including the upregulation of transmembrane and cytosolic TJ proteins, as well as TJ signalling proteins. Gene expression modulation was strain- and time-dependent, with a greater number of differentially expressed genes and higher fold-change being observed in the L. plantarum ST-III group and at the latter timepoint. Further studies to investigate how the observed gene expression changes can lead to enhanced barrier function will aid in the development of probiotic foods to help improve intestinal barrier function.

Abstract 4153: PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model

Abstract There is a critical need to develop new imaging and therapeutic strategies for pancreatic cancer. Current positron emission tomography (PET) imaging assessments of pancreatic ductal adenocarcinoma (PDAC) rely on 18F-FDG as a radiopharmaceutical. To increase the sensitivity of PDAC detection, we identified Claudin-4 (CLDN4), a tight junction transmembrane protein, as a PDAC biomarker through spatial transcriptomic and proteomic analyses. CLDN4 expression has previously been reported to be enhanced in 99% of primary and 100% of metastatic pancreatic cancer tissue samples and in 10 of 11 precancerous pancreatic intraepithelial neoplasia (PanIN) specimens. A small fragment of naturally occurring Clostridium perfringens enterotoxin (CPE) was previously reported to bind to the extracellular loop domain of CLDN4 protein (PDB:7kp4) with nanomolar (nM) affinity (Kd). A CLDN4 binding peptide (C4BP) was developed as a radioligand by modifying the n-terminus of C4BP which does not impact receptor binding. Here, we designed and synthesized a DOTA-PEG1-C4BP ligand on a rink amide resin using a microwave-assisted peptide synthesizer. Radiolabeling of DOTA-PEG1-C4BP (2 nmol) with 64CuCl2 (70 MBq) conferred 64Cu-C4BP (28 MBq/nmol) with 80% non-decay corrected yield and >99% radiochemical purity. PET/CT imaging of 64Cu-C4BP (~0.1 μg/mouse) performed in an inducible PDAC (KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato(KT);Ptf1aCreER;Trp53fl/fl) model at 84 day after tamoxifen injection resulted in ~25% injected activity per cubic centimeter (IA/cc) accumulation in metastases and at ~18% IA/cc retention in pancreatic tumors within 30 minutes of tail vein injection with minimal background accumulation. Ex vivo PET imaging confirmed the localized accumulation of 64Cu-C4BP in transgenic mice within the colon, pancreas, and liver. Blocking of 64Cu-C4BP accumulation with the cold compound in a xenograft model (NOD/SCID) bearing Capan-1 PDAC tumors demonstrated the specificity of 64Cu-C4BP binding. Here we report that CLDN4 is a promising target and 64Cu-C4BP is a promising ligand for the detection of pancreatic cancer. Citation Format: Jai Woong Seo, James Wang, Aris J. Kare, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Mallesh Pandrala, Katherine W. Ferrara. PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model [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 4153.

Cinnamic Acid and Caffeic Acid Effects on Gastric Tight Junction Proteins Analyzed in Xenopus laevis Oocytes.

Analysis of secondary plant compounds for the development of novel therapies is a common focus of experimental biomedicine. Currently, multiple health-supporting properties of plant-derived molecules are known but still information on many mechanisms is scarce. Cinnamic acid and caffeic acid are two of the most abundant polyphenols in human dietary fruits and vegetables. In this study, we investigated cinnamic acid and caffeic acid effects on the gastric barrier, which is primarily provided by members of the transmembrane tight junction protein family of claudins. The Xenopus laevis oocyte has been established, in recent years, as a heterologous expression system for analysis of transmembrane tight junction protein interactions, by performing paired oocyte experiments to identify an effect on protein-protein interactions, in vitro. In our current study, human gastric claudin-4, -5, and -18.2. were expressed and detected in the oocyte plasma membrane by freeze fracture electron microscopy and immunoblotting. Oocytes were paired and incubated with 100 µM or 200 µM cinnamic acid or caffeic acid, or Ringer's solution, respectively. Caffeic acid showed no effect on the contact area strength of paired oocytes but led to an increased contact area size. In contrast, cinnamic acid-incubated paired oocytes revealed a reduced contact area and a strengthening effect on the contact area was identified. These results may indicate that caffeic acid and cinnamic acid both show an effect on gastric barrier integrity via direct effects on tight junction proteins.

STERILE NEUTROPHILIA INDUCED VIA CXCR4 ANTAGONISM MITIGATES COLONIC INFLAMMATION BY ENHANCING IMMUNOSUPPRESSIVE REGULATORY T CELLS IN MICE

Abstract Sterile neutrophilia (SN) is commonly associated with chronic diseases including inflammatory bowel diseases (IBD), but its defined role is largely unknown. Plerixafor (AMD3100) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. We first confirmed that C57BL6/J mice administered Plerixafor (10 mg/kg/bw, i.p.,) increased their circulating neutrophils by 50% within 2.5 h. Further, to study the role of SN in IBD, we induced chronic colitis by neutralizing IL-10 signaling via αIL-10R mAb (1.0 mg/mouse, i.p., 4 weekly injections) with or without weekly twice Plerixafor. Control mice were given isotype control mAb. As expected, αIL-10R+Plerixafor-treated mice had increased peripheral and splenic neutrophils. Further, these mice displayed remarkably reduced epithelial damage, hyperplasia, immune cell infiltration, and disease activity index when compared to αIL-10R+vehicle treated mice, which had typical colitis symptoms. To delineate the mechanism for such protection by Plerixafor, we profiled immune cells in circulation, spleen and lamina propria. In circulation αIL-10R+Plerixafor-treated mice had elevated CD3+CD4+ helper T cells (Th) but reduced CD3+CD8+ cytotoxic T (Tc) cells and CD3+CD4+FoxP3+ cells (Tregs) and CD3+CD4+RORγt+ cells (Th17) remained unchanged. While in the spleens of αIL-10R+Plerixafor-treated mice displayed significant increased Tregs, but decreased Th and Tc cells. Interestingly, markedly elevated Tregs, RORγt+ Tregs and tolerogenic IL-10+ Tregs was observed in lamina propria when compared to αIL-10R+ vehicle given mice. Interestingly, the proinflammatory IL-17+ Th cells were decreased in αIL-10R+Plerixafor-treated mice which showed mild colitis. Moreover, to further elucidate the role of Plerixafor in intestinal barrier function in colitic mice, we noticed treatment with Plerixafor increased the expression of occludin and claudins (1, 2), which are the principal transmembrane tight junctions (TJ) proteins; however Plerixafor treatment reduced the expression of zonula occludens (ZO), a peripheral membrane TJ protein. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation.

Functional Analysis of Membrane-Associated Scaffolding Tight Junction (TJ) Proteins in Tumorigenic Characteristics of B16-F10 Mouse Melanoma Cells.

Tight junction (TJ) proteins (Tjps), Tjp1 and Tjp2, are tight junction-associated scaffold proteins that bind to the transmembrane proteins of tight junctions and the underlying cytoskeleton. In this study, we first analyzed the tumorigenic characteristics of B16-F10 melanoma cells, including cell proliferation, migration, invasion, metastatic potential, and the expression patterns of related proteins, after the CRISPR-Cas9-mediated knockout (KO) of Tjp genes. The proliferation of Tjp1 and Tjp2 KO cells significantly increased in vitro. Other tumorigenic characteristics, including migration and invasion, were significantly enhanced in Tjp1 and Tjp2 KO cells. Zonula occludens (ZO)-associated protein Claudin-1 (CLDN-1), which is a major component of tight junctions and functions in controlling cell-to-cell adhesion, was decreased in Tjp KO cells. Additionally, Tjp KO significantly stimulated tumor growth and metastasis in an in vivo mouse model. We performed a transcriptome analysis using next-generation sequencing (NGS) to elucidate the key genes involved in the mechanisms of action of Tjp1 and Tjp2. Among the various genes affected by Tjp KO-, cell cycle-, cell migration-, angiogenesis-, and cell-cell adhesion-related genes were significantly altered. In particular, we found that the Ninjurin-1 (Ninj1) and Catenin alpha-1 (Ctnna1) genes, which are known to play fundamental roles in Tjps, were significantly downregulated in Tjp KO cells. In summary, tumorigenic characteristics, including cell proliferation, migration, invasion, tumor growth, and metastatic potential, were significantly increased in Tjp1 and Tjp2 KO cells, and the knockout of Tjp genes significantly affected the expression of related proteins.

Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function

ABSTRACTParaprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes’ formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

Expression of tight junction proteins in smokers and non-smokers with generalized Stage III periodontitis.

This study aims to evaluate the gingival crevicular fluid (GCF) levels of tumor necrosis factor-α (TNF-α), zonula occludens-1 (ZO-1), occludin (Occ), and tricellulin (Tric) in periodontitis, as well as their alterations due to smoking. Tight junctions (TJ), which consist of transmembrane and cytoplasmic scaffolding proteins, connect the epithelial cells of the periodontium. Occ, claudins, junctional adhesion molecules, and Tric are transmembrane TJ proteins found at the cell membrane. The transmembrane TJ proteins and the intracellular cytoskeleton are directly linked by cytoplasmic scaffolding proteins such as ZO-1. Although the functions and locations of these molecules have been defined, their behavior in periodontal inflammation is unknown. The study included four groups: individuals with periodontal health without smoking (C; n = 31), individuals with generalized Stage III periodontitis without smoking (P; n = 28), individuals with periodontal health while smoking (CS; n = 22), and individuals with generalized Stage III periodontitis while smoking (PS; n = 18). Clinical periodontal parameters were recorded, and enzyme-linked immunosorbent assay (ELISA) was used to examine ZO-1, Occ, Tric, and TNF-α levels in GCF. In the periodontitis groups, clinical parameters were significantly higher (p < .001). The site-specific levels of TNF-α, ZO-1, Tric, and Occ in the P group were statistically higher than those in the other groups (p < .05). TNF-α, probing pocket depth (PPD), and bleeding on probing (BOP) exhibited positive correlations with all TJ proteins (p < .005). Smoking could potentially affect the levels of epithelial TJ proteins in the GCF, thereby potentially playing a significant role in the pathogenesis of the periodontal disease.

Evaluation of Serum Zonulin and Occludin Levels in Bipolar Disorder.

The etiology of bipolar disorder (BD) is still not fully understood. Little is currently known about the relationship between the interaction the gastrointestinal system and brain function and BD. Zonulin is the only known physiological modulator of tight junctions and is a biomarker for intestinal permeability (IP). Occludin is an integral transmembrane tight junction protein involved in the maintenance and assembly of such junctions. The current study aims to determine whether zonulin and occludin levels are altered in BD and whether they can serve as clinical biomarkers of disease. Forty-four patients with BD and 44 healthy controls were included in this study. The Young Mania Rating Scale (YMRS) was used to determine the severity of manic symptoms, while the Hamilton Depression Rating Scale (HDRS) was used to determine the severity of depressive symptoms, and the Brief Functioning Rating Scale (FAST) to assess functionality. Venous blood samples were taken from all participants and serum zonulin and occludin levels were measured. The mean serum zonulin and occludin levels of the patients were significantly higher compared to the healthy control group. There was no difference between manic, depressive, and euthymic patients in terms of zonulin and occludin levels. There was no correlation between the total number of attacks, duration of disease, YMRS, HDRS, FAST scores, and zonulin and occludin levels in the patient group. The groups were divided into three according to body mass index as normal, overweight, and obese. Zonulin and occludin levels increased as body mass index increased and were highest in the obese group. The study shows that zonulin and occludin levels in BD increase independently of the disease stage. Consideration of the role of IP in the pathogenesis of BD may be helpful in determining the appropriate treatment modality.

Zonula Occludens Proteins Signaling in Inflammation and Tumorigenesis.

Tight junction (TJ) is the barrier of epithelial and endothelial cells to maintain paracellular substrate transport and cell polarity. As one of the TJ cytoplasmic adaptor proteins adjacent to cell membrane, zonula occludens (ZO) proteins are responsible for connecting transmembrane TJ proteins and cytoplasmic cytoskeleton, providing a binding platform for transmembrane TJ proteins to maintain the barrier function. In addition to the basic structural function, ZO proteins play important roles in signal regulation such as cell proliferation and motility, the latter including cell migration, invasion and metastasis, to influence embryonic development, tissue homeostasis, damage repair, inflammation, tumorigenesis, and cancer progression. In this review, we will focus on the signal regulating function of ZO proteins in inflammation and tumorigenesis, and discuss the limitations of previous research and future challenges in ZO protein research.

Necrotizing enterocolitis in preterm infants: state of the intestinal barrier, features of vitamin D metabolism and their regulation

The priority of the national health policy is to preserve the life and a high level of quality of life for every premature baby. The clinical focus is on children born with ELBW. Among this category of children, NEC makes the main contribution to the structure of infant mortality. Based on the analysis of the literature, the authors conclude that the study of cell markers that characterize different depths of damage to enterocytes allows: to assess the likelihood of developing NEC in infants with ELMT; conduct early diagnosis of NEC; rule out NEC in neonates with similar symptoms; predict the course of NEC; propose and substantiate personalized approaches to correcting the low supply of 25(OH)D; to analyze the influence of candidate genes on the implementation of NEC, its outcomes, and 25(OH)D metabolism. To assess damage at the level of the enterocyte, the authors selected the intestinal fraction of fatty acid binding protein (I-FABP) for literature analysis. To determine the depth of damage to intercellular junctions of the intestine - the expression of transmembrane (claudin-2, claudin-3, claudin-4, occludin) and cytoplasmic (zonulin) tight junction proteins. Analysis of the results of studies on the expression of fecal calprotectin, lipocalin-2 (LCN2) and eosinophilic neurotoxin, showing the activity of local inflammation, was carried out in order to assess both the risk of NEC and its course. Intestinal damage is associated with impaired 25(OH)D metabolism, and metabolic bone disease in preterm infants with damage to the intestinal barrier up to NEC is recorded ten times more often at the stage of nursing in the NICU. A huge number of studies have shown a decrease in survival, an increase in the risk of severe complications against the background of a low supply of 25(OH)D in the preterm population. The authors analyze the relationship between 25(OH)D availability, taking into account the influence of exogenous and endogenous factors, the nature of damage to the intestinal wall and the implementation of NEC, and focus on the existing preventive and therapeutic approaches to prescribing various doses of vitamin D in preterm infants with NEC.

Application of C-Terminal Clostridium Perfringens Enterotoxin in Treatment of Brain Metastasis from Breast Cancer.

Simple SummaryBrain metastasis occurs in primary cancers, such as breast cancer, and is correlated with mortality. There are limited options available for treatment, but Clostridium perfringens Enterotoxin (CPE) and its interaction with Claudin-4, a possible diagnostic biomarker for breast cancer, can provide a molecular pathway basis for the development of treatment options for metastatic brain cancer. Analysis of the literature reveals that Claudin-4 plays an important role as a receptor for CPE, allowing for the disruption of cell membrane permeability, an influx of calcium ions, and subsequent cell death. The negligible presence of Claudin-4 in normal brain cancer cells and the high abundance of Claudin-4 in breast cancer cells metastasized to the brain, allow for the targeted binding of CPE to tumor cells in the brain. We show that the C-terminal of CPE conjugated to nanoparticles that cross the blood–brain barrier could serve as a drug delivery tool to treat metastatic cells in the brain.Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE’s high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast–brain metastatic tumors.

Structure Composition and Intracellular Transport of Clathrin-Mediated Intestinal Transmembrane Tight Junction Protein.

Tight junctions (TJs) are located in the apical region of the junctions between epithelial cells and are widely found in organs such as the brain, retina, intestinal epithelium, and endothelial system. As a mechanical barrier of the intestinal mucosa, TJs can not only maintain the integrity of intestinal epithelial cells but also maintain intestinal mucosal permeability by regulating the entry of ions and molecules into paracellular channels. Therefore, the formation disorder or integrity destruction of TJs can induce damage to the intestinal epithelial barrier, ultimately leading to the occurrence of various gastrointestinal diseases, such as inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), and irritable bowel syndrome (IBS). However, a large number of studies have shown that TJs protein transport disorder from the endoplasmic reticulum to the apical membrane can lead to TJs formation disorder, in addition to disruption of TJs integrity caused by external pathological factors and reduction of TJs protein synthesis. In this review, we focus on the structural composition of TJs, the formation of clathrin-coated vesicles containing transmembrane TJs from the Golgi apparatus, and the transport process from the Golgi apparatus to the plasma membrane via microtubules and finally fusion with the plasma membrane. At present, the mechanism of the intracellular transport of TJ proteins remains unclear. More studies are needed in the future to focus on the sorting of TJs protein vesicles, regulation of transport processes, and recycling of TJ proteins, etc.

O005 Evaluation of occludin, a tight junction transmembrane protein in human lung cancer and its potential role in metastatic disease

Abstract Introduction Tight junctions (TJs) maintain cell adhesion. Modulation can promote cell dissociation/motility/ metastasis. Occludin is a transmembrane protein within TJs and may play a role in TJ modulation. The aim of this research was to identify the expression of Occludin in human lung cancer and its effect in cell motility/dissociation. Methods qPCR was used to quantify Occludin transcripts in a human lung cancer cohort (n=80) (Peking University Cancer Hospital Research Ethics Committee). In vitro experiments on A549/COR-L23 lung cancer cell lines included immunofluorescence staining, cell attachment/migration using ECIS Z-Theta. Cells were treated with Hepatocyte Growth Factor (HGF, 50ng/μl) and/or cell migration inhibitors N-WASPi (Wiskostatin) and ROCKi (Y-27632). Results qPCR analysis revealed reduced expression of Occludin in patients with metastasis (no bone mets 21.7+/- 12 v bone mets 3.23+/- 3 (p=0.42) v boneActv 0.263+/-0.096 (p=0.077). A549 cells showed a similar cell migration pattern with HGF, ROCKi/HGF+ROCKi. In COR-L23, HGF increased cell migration; ROCKi/HGF+ROCKi showed suppression. N-WASPi/HGF+N-WASPi prevented all cell migration. Immunofluorescence demonstrated that HGF caused loss of Occludin from cell-to-cell membranes with single cells and increased pseudopodia in COR-L23 and A549. Conclusion This preliminary study suggests an association between Occludin and bone metastasis in lung cancer. HGF promoted Occludin loss from cell membranes, cell dissociation and increased pseudopodia/increased cell motility. This suggests that loss of Occludin from cell membranes is linked to reduced TJ function leading to cell dissociation/increased motility. NWASPi may provide pharmacological value in future lung carcinoma treatment.

Cellular Distribution Pattern of tjp1 (ZO-1) in Xenopus laevis Oocytes Heterologously Expressing Claudins

Epithelial barriers constitute a fundamental requirement in every organism, as they allow the separation of different environments and set boundaries against noxious and other adverse effectors. In many inflammatory and degenerative diseases, epithelial barrier function is impaired because of a disturbance of the paracellular seal. Recently, the Xenopus laevis oocyte has been established as a heterologous expression model for the analysis of transmembrane tight junction protein interactions and is currently considered to be a suitable screening model for barrier effectors. A prerequisite for this application is a physiological anchoring of claudins to the cytoskeleton via the major scaffolding protein tjp1 (tight junction protein 1, ZO-1). We have analyzed the oocyte model with regard to the interaction of heterologously expressed claudins and tjp1. Our experiments have revealed endogenous tjp1 expression in protein and mRNA analyses of unfertilized Xenopus laevis oocytes expressing human claudin 1 (CLDN1) to claudin 5 (CLDN5). The amphibian cell model can therefore be used for the analysis of claudin interactions.Graphical

Abstract 5558: Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation

Abstract Background Claudin 6 (CLDN6) is a tight junction transmembrane protein normally only expressed during embryonic development, but is aberrantly expressed in a variety of tumors, including ovarian cancer, testicular cancer, hepatocellular and lung adenocarcinoma. Here, we generated a CLDN6 X 4-1BB bispecific antibody (BsAb) which could conditionally activate T cells through 4-1BB stimulation upon CLDN6 engagement, positioning it as a potential novel immunotherapy for ovarian cancer and other CLDN6 positive tumors. Methods CLDN6 X 4-1BB BsAb was evaluated for its antigen binding through cell-based binding assay. In vitro function of the antibody was confirmed in a 4-1BB signaling reporter assay and primary human PBMC assay. In vivo efficacy of the antibody was further demonstrated in 4-1BB humanized syngeneic mouse model. Ex vivo analysis of tumor infiltrating and peripheral immune cells was conducted by FACS. Safety parameters such as liver enzymes and pathology upon antibody administration were also assessed. Results CLDN6 X 4-1BB BsAb showed strong binding to several ovarian cancer cell lines with different levels of CLDN6 expression. Functional evaluation of CLDN6X4-1BB BsAb indicated that the activation of 4-1BB signaling was dependent on CLDN6 expression on tumor cells and the activity was stronger than reference 4-1BB monoclonal antibody urelumab. In a humanized 4-1BB mouse model, CLDN6X4-1BB BsAb showed tumor growth inhibition superior to the combination of anti-CLDN6 and anti-4-1BB monospecific antibodies, and the treatment effect was associated with an increase in tumor infiltrating CD45 and CD8 cells as well as CD8/Treg ratio. From the safety perspective, there were no significant changes in liver enzymes or liver histopathology following repeated BsAb administration, suggesting little risks for liver toxicity commonly induced by other 4-1BB agonist antibodies. Conclusions We have successfully generated a novel CLDN6-targeted 4-1BB bispecific antibody which could induce potent 4-1BB stimulation and anti-tumor activity in a CLDN6-dependent manner while minimizing the risk of liver toxicity. Taken together, these data support further development of CLDN6 X 4-1BB bispecific antibody towards IND and clinical trial in 2022. Citation Format: Jian Li, Wenqing Jiang, Yan Liu, Zhengyi Wang, Xi Chen, Taylor B. Guo. Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5558.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Aberrant expression of claudin-6 contributes to malignant potentials and drug resistance of cervical adenocarcinoma.

Recent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin‐6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins and drug metabolism‐associated proteins (aldo‐keto reductase [AKR] family proteins) were significantly increased in CLDN6‐overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell‐cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell‐cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6‐targeting therapy, against cervical ADC.

The ACE2 Receptor for Coronavirus Entry Is Localized at Apical Cell-Cell Junctions of Epithelial Cells.

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell—Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.

Expression of tight junction transmembrane protein Claudin-1 in gastric carcinoma and effects on tumor cell proliferation, invasion and migration

Abstract Background: Claudin-1 is involved in various cancers, but its expression and role in gastric carcinoma remain unclear. Materials and Methods: Gastric carcinoma and adjacent normal tissues were harvested from 60 patients. Claudin-1 expression was detected by RT-qPCR. The expressions in human gastric carcinoma MKN45, SGC7901 and MKN28 cells and immortalized human gastric epithelium GES-1 cells were determined by RT-qPCR and Western blotting. Claudin-1 was overexpressed in SGC7901 cells by lentiviral transfection, and they were divided into Control (untransfected), normal control (NC) (transfected with lentiviral vector) and Claudin-1 (transfected with Claudin-1 overexpression lentivirus) groups. The proliferation, invasion and migration of gastric carcinoma cells were detected through cell counting kit-8, Transwell and wound healing assays, respectively. The effects of Claudin-1 on the expressions of epithelial-mesenchymal transition (EMT) marker proteins E-cadherin and N-cadherin were detected by Western blotting. Ten 4-week-old male BALB/c nude mice were subcutaneously injected with lentivirus-treated SGC7901 cells to establish the transplanted tumor model, and the effect of overexpression of Claudin-1 was explored. Results: The expression of Claudin-1 in gastric carcinoma tissues was significantly lower than that in adjacent tissues (P&lt;0.05). Overexpression of Claudin-1 significantly inhibited the proliferation, invasion and migration of SGC7901 cells, increased the expression of E-cadherin, and decreased that of N-cadherin (P&lt;0.05). Overexpression of Claudin-1 in the mouse model significantly inhibited the growth of subcutaneous transplanted tumors (P&lt;0.05). Conclusion: Claudin-1 has low expression in gastric carcinoma tissues. Overexpression of Claudin-1 inhibits the proliferation, invasion, migration, and EMT of gastric carcinoma cells, and subcutaneous tumorigenesis in nude mice.

Abstract PS4-44: Expression of the claudin transmembrane tight junction protein family (CLDN) and the prediction value of a claudin subset to the clinical outcome of patients with breast cancer

Abstract Background: Tight Junction (TJ) proteins are directly or indirectly involved in breast cancer progression and metastasis where the mutual adhesiveness of cancer cells is significantly weaker than normal cells and so reduced cell-cell interaction results in a loss of normal tissue architecture (1). This adhesiveness is in addition to the role of the proteins in the control of the barrier functions of epithelial and endothelial cells. The claudin family (CLDN) of transmembrane proteins, the largest protein family of the tight junctions, are located in the TJ of cells of epithelial and endothelial origin where they are key in the maintenance of TJ function and have been increasingly shown to have important roles during key steps in the progression of cancer. We have previously demonstrated that the expression of some TJ molecules (occludin, claudins -5, -16, -20) are associated with disease progression in breast cancer (2-5). This study sought to evaluate the possible clinical and prognostic value of the entire CLDN family, in patients with breast cancer. Methods: Members of the claudin family of transmembrane TJ proteins were assessed and correlated with clinical and pathological parameters at the messenger level in a Cardiff breast cancer cohort. Breast cancer primary tumours (n=114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR CLDN-1 to -24. The expression profile was analysed against the clinical and pathological information and most importantly the clinical outcome of the patients. Results. Mammary tissues expressed varying levels of CLDNs. However, the levels of CLDN1, CLDN3, CLDN4, CLDN11, CLDN19, and CLDN22 were found to be significantly aberrant in breast cancer tissues compared with normal tissues. Members of the family, namely CLDN10, CLDN11, and CLDN18 were found to have a significant predictive value to the prognosis when using the Nottingham Prognostic Index. Of the claudin family, we identified eight members whose expression was negatively correlated with long term survival, and three members positively correlated with the survival. A comprehensive bioinformatic analyses of these CLDN members revealed a highly significant signature in predicting the clinical outcome. A favourable CLDN signature predicted a subset of the patients who all survived the ten-year followup, compared with those with unfavourable signature (69.1%), p&amp;lt;0.0001 (Log ranked). Likewise, the CLDN signature also successfully predicted the occurrence of breast cancer related incidence (97.6% vs 61.8%, p&amp;lt;0.0001). Multivariate analyses confirmed that the CLDN signature has a highly significant value as an independent predictor for both overall survival (p=0.001) and disease-free survival (p=0.001). Moreover, the predictive power was more profound for patients with ER negative and Her2 negative tumours. Discussion: Claudins, key tight junctional transmembrane proteins, have important prognostic value in human breast cancer. Together with their pivotal roles in controlling the adhesiveness and permeability of the cell layers, this family is a key player in the development and progression of breast cancer.