Cleavage Of E-cadherin Research Articles

Prolactin-induced tyrosyl phosphorylation of PAK1 facilitates epithelial-mesenchymal transition.

PAK1 and prolactin (PRL) regulate breast cancer. Prolactin-activated JAK2 tyrosyl phosphorylates PAK1 (pTyr-PAK1). We demonstrate here that pTyr-PAK1 regulates epithelial-mesenchymal transition (EMT) in breast cancer cells. PRL treatment of T47D PAK1 WT cells leads to downregulation of E-cadherin surface expression and "ectodomain shedding" (extracellular cleavage of E-cadherin). pTyr-PAK1 increases mRNA levels of Snail, Slug, and Twist2, transcriptional factors implicated in E-cadherin repression. pTyr-PAK1 also significantly increases PRL-dependent Slug activity leading to expression of vimentin, a hallmark of EMT. Thus, our current data on pTyr-PAK1 regulation of EMT bring insight into the role of PAK1 and PRL in human breast cancer.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma

ObjectiveChronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting...

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin.

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

A single-nucleotide polymorphism in Helicobacter pylori promotes gastric cancer development

Single-nucleotide polymorphisms (SNPs) in various human genes are key factors in carcinogenesis. However, whether SNPs in bacterial pathogens are similarly crucial in cancer development is unknown. Here, we analyzed 1,043 genomes of the stomach pathogen Helicobacter pylori and pinpointed a SNPin the serine protease HtrA (position serine/leucine 171) that significantly correlates with gastric cancer. Our functional studies reveal that the 171S-to-171L mutation triggers HtrA trimer formation and enhances proteolytic activity and cleavage of epithelial junction proteins occludin and tumor-suppressor E-cadherin. 171L-type HtrA, but not 171S-HtrA-possessing H.pylori, inflicts severe epithelial damage, enhances injection of oncoprotein CagA into epithelial cells, increases NF-κB-mediated inflammation and cell proliferation through nuclear accumulation of β-catenin, and promotes host DNA double-strand breaks, collectively triggering malignant changes. These findings highlight the 171S/L HtrA mutation as a unique bacterial cancer-associated SNP and as a potential biomarker for risk predictions in H.pylori infections.

Amprenavir inhibits pepsin-mediated laryngeal epithelial disruption and E-cadherin cleavage in vitro.

Laryngopharyngeal reflux (LPR) causes chronic cough, throat clearing, hoarseness, and dysphagia and can promote laryngeal carcinogenesis. More than 20% of the US population suffers from LPR and there is no effective medical therapy. Pepsin is a predominant source of damage during LPR which disrupts laryngeal barrier function potentially via E-cadherin cleavage proteolysis and downstream matrix metalloproteinase (MMP) dysregulation. Fosamprenavir (FDA-approved HIV therapeutic and prodrug of amprenavir) is a pepsin-inhibiting LPR therapeutic candidate shown to rescue damage in an LPR mouse model. This study aimed to examine amprenavir protection against laryngeal monolayer disruption and related E-cadherin proteolysis and MMP dysregulation in vitro. Laryngeal (TVC HPV) cells were exposed to buffered saline, pH 7.4 or pH 4 ± 1 mg/mL pepsin ± amprenavir (10-60 min). Analysis was performed by microscopy, Western blot, and real time polymerase chain reaction (qPCR). Amprenavir (1 μM) rescued pepsin acid-mediated cell dissociation (p < .05). Pepsin acid caused E-cadherin cleavage indicative of regulated intramembrane proteolysis (RIP) and increased MMP-1,3,7,9,14 24-h postexposure (p < .05). Acid alone did not cause cell dissociation or E-cadherin cleavage. Amprenavir (10 μM) protected against E-cadherin cleavage and MMP-1,9,14 induction (p < .05). Amprenavir, at serum concentrations achievable provided the manufacturer's recommended dose of fosamprenavir for HIV, protects against pepsin-mediated cell dissociation, E-cadherin cleavage, and MMP dysregulation thought to contribute to barrier dysfunction and related symptoms during LPR. Fosamprenavir to amprenavir conversion by laryngeal epithelia, serum and saliva, and relative drug efficacies in an LPR mouse model are under investigation to inform development of inhaled formulations for LPR.

Topical Alginate Protection against Pepsin-Mediated Esophageal Damage: E-Cadherin Proteolysis and Matrix Metalloproteinase Induction.

Epithelial barrier dysfunction is a hallmark of gastroesophageal reflux disease (GERD) related to symptom origination, inflammatory remodeling and carcinogenesis. Alginate-based antireflux medications were previously shown to topically protect against peptic barrier disruption, yet the molecular mechanisms of injury and protection were unclear. Herein, Barrett's esophageal (BAR-T) cells were pretreated with buffered saline (HBSS; control), dilute alginate medications (Gaviscon Advance or Gaviscon Double Action, Reckitt Benckiser), a viscosity-matched placebo, or ADAM10 and matrix metalloproteinase (MMP) inhibitors before exposure to HBSS pH7.4 or pH4 ± 1 mg/mL pepsin for 10-60 min. Cell viability was assessed by ATP assay; mediators of epithelial integrity, E-cadherin, ADAM10, and MMPs were examined by Western blot and qPCR. Alginate rescued peptic reduction of cell viability (p < 0.0001). Pepsin-pH4 yielded E-cadherin fragments indicative of regulated intramembrane proteolysis (RIP) which was not rescued by inhibitors of known E-cadherin sheddases. Transcriptional targets of E-cadherin RIP fragments were elevated at 24 h (MMP-1,2,9,14; p < 0.01). Alginate rescued E-cadherin cleavage, ADAM10 maturation, and MMP induction (p < 0.01). Results support RIP as a novel mechanism of peptic injury during GERD. Alginate residue after wash-out to mimic physiologic esophageal clearance conferred lasting protection against pepsin-induced molecular mechanisms that may exacerbate GERD severity and promote carcinogenesis in the context of weakly acidic reflux.

The Protease Inhibitor Amprenavir Protects against Pepsin-Induced Esophageal Epithelial Barrier Disruption and Cancer-Associated Changes.

Gastroesophageal reflux disease (GERD) significantly impacts patient quality of life and is a major risk factor for the development of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Proton pump inhibitors (PPIs) are the standard-of-care for GERD and are among the most prescribed drugs in the world, but do not protect against nonacid components of reflux such as pepsin, or prevent reflux-associated carcinogenesis. We recently identified an HIV protease inhibitor amprenavir that inhibits pepsin and demonstrated the antireflux therapeutic potential of its prodrug fosamprenavir in a mouse model of laryngopharyngeal reflux. In this study, we assessed the capacity of amprenavir to protect against esophageal epithelial barrier disruption in vitro and related molecular events, E-cadherin cleavage, and matrix metalloproteinase induction, which are associated with GERD severity and esophageal cancer. Herein, weakly acidified pepsin (though not acid alone) caused cell dissociation accompanied by regulated intramembrane proteolysis of E-cadherin. Soluble E-cadherin responsive matrix metalloproteinases (MMPs) were transcriptionally upregulated 24 h post-treatment. Amprenavir, at serum concentrations achievable given the manufacturer-recommended dose of fosamprenavir, protected against pepsin-induced cell dissociation, E-cadherin cleavage, and MMP induction. These results support a potential therapeutic role for amprenavir in GERD recalcitrant to PPI therapy and for preventing GERD-associated neoplastic changes.

Abstract 1232: Gastroesophageal reflux disease promotes E-cadherin cleavage and activates EMT via APE1-redox function in esophageal adenocarcinoma

Abstract The incidence of esophageal adenocarcinoma (EAC) has increased rapidly over the past four decades. Chronic gastroesophageal reflux disease (GERD), where acidic bile salts (ABS) abnormally refluxate into the esophagus, is the leading risk factor for the development of a metaplastic condition known as Barrett’s esophagus (BE) and its progression to EAC. We used 3D organotypic culture, mouse and human tissue samples to assess the role of ABS in Epithelial-to-Mesenchymal Transition (EMT) in EAC. Analysis of public databases revealed significant enrichment of EMT signaling in EAC progression. RNA-seq analysis of EAC cells showed activation of EMT pathway in response to ABS exposure. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of Vimentin, activation of ß-catenin and EMT-transcription factors (EMT-TFs), cell morphological changes, and enhancement of cell migration and invasion capabilities. Mechanistically, we discovered that ABS induced E-cadherin cleavage via MMP14 proteolytic cascade. APE1 silencing, or APE1-redox-specific inhibitor (E3330), abrogated the ABS-induced EMT process and signaling by downregulating MMP14. Furthermore, APE1 and MMP14 co-expression levels were inversely correlated with E-cadherin expression in gastroesophageal junctions of human EAC tissues, and the L2-IL1ß transgenic mouse model of BE/EAC. EAC patients with high APE1 and EMT signatures had worse relapse free survival than those with low signature. In a summary, this study demonstrates the role of ABS in promoting EMT via the redox-sensitive signaling axis of APE1/MMP14/E-cadherin/ß-catenin. Pharmacological inhibition of APE1-redox function could be a potential therapeutic approach to effectively reduce the risk of Barrett’s carcinogenesis. Citation Format: Heng Lu, Longlong Cao, Farah Ballout, Abbes Belkhiri, Dunfa Peng, Lei Chen, Mohammed Soutto, Oliver McDonald, Alexander Zaika, Jianwen Que, Wael El-Rifai. Gastroesophageal reflux disease promotes E-cadherin cleavage and activates EMT via APE1-redox function in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1232.

Alpha-1 antitrypsin limits neutrophil extracellular trap disruption of airway epithelial barrier function.

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.

Geometric tumor embolic budding characterizes inflammatory breast cancer.

Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli especially within dermal lymphatics. The explanation remains a mystery. This study combines experimental studies with two different IBC xenografts with image algorithmic studies utilizing human tissue microarrays (TMAs) of IBC vs non-IBC cases to support a novel hypothesis to explain IBC's sina qua non signature of florid lymphovascular emboli. In the human TMAs, compared to tumor features like nuclear grade (size), mitosis and Ki-67 immunoreactivity which show that IBC is only modestly more proliferative with larger nuclei than non-IBC, what really sets IBC apart is the markedly greater number of tumor emboli and distinctly smaller emboli whose numbers indicate geometric or exponential differences between IBC and non-IBC. In the experimental xenograft studies, Mary-X gives rise to tight spheroids in vitro which exhibit dynamic budding into smaller daughter spheroids whereas Karen-X exhibits only loose non-budding aggregates. Furthermore Mary-X emboli also bud dramatically into smaller daughter emboli in vivo. The mechanism that regulates this involves the generation of E-cad/NTF1, a calpain-mediated cleavage 100kDa product of 120kDa full length membrane E-cadherin. Inhibiting this calpain-mediated cleavage of E-cadherin by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides that both penetrate the cell membrane and mimic either the cleavage site or the binding site on E-cadherin, inhibits the generation of E-cad/NTF1 in a dose-dependent manner, reduces spheroid compactness and decreases budding. Since E-cad/NFT1 retains the p120ctn binding site but loses the α-and β-catenin sites, promoting its 360° distribution around the cell's membrane, the vacilating levels of this molecule trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of emboli seen in IBC.

The UBC9/SUMO pathway affects E-cadherin cleavage in HPV-positive head and neck cancer.

Functional loss of E-cadherin is frequent during tumor progression and occurs through a variety of mechanisms, including proteolytic cleavage. E-cadherin downregulation leads to the conversion of a more malignant phenotype promoting Epithelial to Mesenchymal Transition (EMT). The UBC9/SUMO pathway has been also shown to be involved in the regulation of EMT in different cancers. Here we found an increased expression of UBC9 in the progression of Head and Neck Cancer (HNC) and uncovered a role for UBC9/SUMO in hampering the HPV-mediated E-cadherin cleavage in HNC.

Genomic erosion and horizontal gene transfer shape functional differences of the ExlA toxin in Pseudomonas spp.

SummaryTwo-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlAPch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.

The Hu2G10 mAb targets the cleaved-activated form of Trop-2 and exploits vulnerability of multiple human cancers.

e14548 Background: FDA granted accelerated approval to the anti-Trop-2 sacituzumab govitecan-hziy (TRODELVY) antibody-drug conjugate (ADC), for patients with metastatic triple-negative breast cancer. Skin rash and mucosal inflammation were, though, dose-limiting toxicities of other high-effectiveness anti-Trop-2 antibodies, and impeded their development. These adverse events appeared as on-target/off-cancer effects, related to the expression of Trop-2 by normal tissues. Trop-2 is a transmembrane glycoprotein (E. Guerra et al., The Trop-2 signalling network in cancer growth. Oncogene 32, 1594-1600 (2013)), that is cleaved and activated by ADAM10 to induce cancer growth and metastatic diffusion (M. Trerotola et al., Trop-2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis. Neoplasia 23, 415-428 (2021); E. Guerra et al., Trop-2 induces ADAM10-mediated cleavage of E-cadherin and drives EMT-less metastasis in colon cancer. Neoplasia 23, 898-911 (2021)). This cleavage causes a spatial rearrangement of the extracellular portion of Trop-2 and exposes hidden, activation-specific epitopes. We succeeded in generating monoclonal antibodies (mAbs) capable of recognizing these cancer-exposed sites. Methods: Most Trop-2-targeted approaches are based on anti-Trop-2 mAbs that recognize a single immunodominant epitope. Such mAbs have limited therapeutic index. We removed the immunodominant epitope by deletion mutagenesis, and optimized breadth of immune response, by immunizing mice with recombinant proteins with diverse end-glycosylation patterns. Screening for cancer-specific mAbs was performed on mutagenized Trop-2, by cell-based ELISA assays, bio-layer interferometry, flow cytometry and fluorescence microscopy. Results: mAbs were obtained that efficiently bound Trop-2 expressing cancer cells and were able to inhibit cell growth in vitro. We humanized the 2G10 mAb into the Hu2G10 IgG1, with an affinity for recombinant, cancer-specific, cleaved Trop-2 that is several orders of magnitude higher than existing competitor mAbs. In vivo the naked Hu2G10 antibody was effective at inhibiting the growth of breast, colon, ovary and prostate cancers. In the ADC setting Hu2G10 drove high anticancer specificity and effectiveness. Conclusions: Hu2G10 shows unprecedented specificity and efficacy, for best-in-class anti-Trop-2 anticancer therapy.

Inhibiting ADAM10‐MMP14 Complex Blocks Cystogenesis of Kidney Epithelial Cells

BackgroundPolycystic kidney disease (PKD) is the most common genetic kidney disorder. It is characterized by the aberrant renal tubule epithelial cells proliferation leads to the formation of multiple fluid‐filled cysts, and it causes renal failure. Currently, kidney transplant or dialysis is the only effective treatment. A disintegrin‐like metalloproteinase 10 (ADAM10) and matrix metalloproteinase 14 (MMP14) are two major members in the superfamily of metalloproteinases that primarily cleave several cell membrane proteins and growth factors. Inhibition of ADAM10 activity reduces cystic growth of renal epithelial cells, and inhibition of MMP14 also reduces the number of cysts in the kidneys of rats with PKD. In our study, we propose to elucidate the unclear relationship between ADAM10 and MMP14 in the development of cystogenesis in kidney.MethodsMadin‐Darby Canine Kidney (MDCK) cells with stable expression of MMP14 and its mutants were developed and used in our study. MTT proliferation assay was used to measure cell growth. Immunoprecipitation and Western blots were used to determine physical association and proteinase activity. 3‐dimensional (3D) culture of MDCK cells was used to examine cell growth patterns.ResultsWe found that a unique ADAM10‐MMP14 complex forms in cell membrane. The hemopexin domain of MMP14 is essential for its association with ADAM10. Their interaction causes differential cleavage of cell membrane proteins, E‐cadherin and L1 cell adhesion molecule protein (L1CAM). In MDCK cells, only ADAM10 cleaves E‐cadherin but its cleavage is dependent on the active form of MMP14. In addition, both ADAM10 and MMP14 cleave L1CAM but inactive MMP14 blocks the ADAM10 cleavage of L1CAM. We tested the effects of ADAM10 and MMP14 interaction on cell proliferation and cystic growth. Ectopic expression of MMP14 increases cell proliferation and cystic growth of MDCK cells in a 3D culture matrix. Further, use of MMP14‐specific inhibitor NSC405020 or ectopic expression of a dominant‐negative form of MMP14 mutant also results in proper contact inhibition, and further promotes tubular growth of MDCK cells in 3D culture. Inhibition of ADAM10 with siRNA or enzymatic inhibitor GI254023X significantly reduces cell proliferation and promotes tubular growth. Combined use of GI254023X and NSC405020 results in a 73.4 ±8.23% decrease in cell proliferation compared to GI254023X, and 68.6 ± 6.84% decrease compared to NSC405020 alone. In 3D culture, MDCK‐MMP14 (ectopic expression of MMP14) cells form cystic growth only, but addition of both ADAM10 and MMP14 inhibitors leads to tubular growth only.ConclusionOur data identified the solid physical association of ADAM10 and MMP14. Their functional interaction causes differential cleavage of cell membrane proteins, E‐cadherin and L1CAM. Inhibition of ADAM10‐MMP14 complex blocks proliferation and the cystogenesis of of kidney epithelial cells. Individual ADAM10 or MMP14 inhibitors have been used in clinical trials for certain tumors, however, they have never been used together nor have been used in PKD treatments. Our results suggest the combination of ADAM10 and MMP14 inhibitors could be served as a novel effective therapeutic strategy of PKD.

Bacteroides fragilis Toxin Induces Intestinal Epithelial Cell Secretion of Interleukin-8 by the E-Cadherin/β-Catenin/NF-κB Dependent Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) has emerged as a gut microbiome pathogen that can promote colitis associated cancer in humans. ETBF secretes the metalloprotease, B. fragilis toxin (BFT), which can induce ectodomain cleavage of E-cadherin and IL-8 secretion through the β-catenin, NF-κB, and MAPK pathways in intestinal epithelial cells. However, it is still unclear whether E-cadherin cleavage is required for BFT induced IL-8 secretion and the relative contribution of these signaling pathways to IL-8 secretion. Using siRNA knockdown and CRISPR knockout studies, we found that E-cadherin cleavage is required for BFT mediated IL-8 secretion. In addition, genetic ablation of β-catenin indicates that β-catenin is required for the BFT induced increase in transcriptional activity of NF-κB, p65 nuclear localization and early IL-8 secretion. These results suggest that BFT induced β-catenin signaling is upstream of NF-κB activation. However, despite β-catenin gene disruption, BFT still activated the MAPK pathway, suggesting that the BFT induced activation of the MAPK signaling pathway is independent from the E-cadherin/β-catenin/NF-κB pathway. These findings show that E-cadherin and β-catenin play a critical role in acute inflammation following ETBF infection through the inflammatory response to BFT in intestinal epithelial cells.

E-Cadherin Orthologues as Substrates for the Serine Protease High Temperature Requirement A (HtrA).

Helicobacter pylori (H. pylori) expresses the serine protease and chaperone High temperature requirement A (HtrA) that is involved in periplasmic unfolded protein stress response. Additionally, H. pylori-secreted HtrA directly cleaves the human cell adhesion molecule E-cadherin leading to a local disruption of intercellular adhesions during pathogenesis. HtrA-mediated E-cadherin cleavage has been observed in response to a broad range of pathogens, implying that it is a prevalent mechanism in humans. However, less is known whether E-cadherin orthologues serve as substrates for bacterial HtrA. Here, we compared HtrA-mediated cleavage of human E-cadherin with murine, canine, and simian E-cadherin in vitro and during bacterial infection. We found that HtrA targeted mouse and dog E-cadherin equally well, whereas macaque E-cadherin was less fragmented in vitro. We stably re-expressed orthologous E-cadherin (Cdh1) in a CRISPR/Cas9-mediated cdh1 knockout cell line to investigate E-cadherin shedding upon infection using H. pylori wildtype, an isogenic htrA deletion mutant, or complemented mutants as bacterial paradigms. In Western blot analyses and super-resolution microscopy, we demonstrated that H. pylori efficiently cleaved E-cadherin orthologues in an HtrA-dependent manner. These data extend previous knowledge to HtrA-mediated E-cadherin release in mammals, which may shed new light on bacterial infections in non-human organisms.

Trop-2, Na+/K+ ATPase, CD9, PKCα, cofilin assemble a membrane signaling super-complex that drives colorectal cancer growth and invasion.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca2+ signal. We reveal here that Trop-2 binds the cell membrane Na+/K+-ATPase, and that clustering of Trop-2 induces an intracellular Ca2+ rise followed by membrane translocation of PKCα, which in turn phosphorylates the Trop-2 cytoplasmic tail. This feed-forward signaling is promoted by the binding of Trop-2 to the PKCα membrane-anchor CD9. CRISPR-based inactivation of CD9 in CRC cells shows that CD9 is required by Trop-2 for recruiting PKCα and cofilin-1 to the cell membrane. This induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the β-actin cytoskeleton, and activation of Akt and ERK. The interaction between Trop-2 and CD9 was validated in vivo in murine models of CRC growth and invasion. Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of CRC patients, supporting a pivotal relevance in CRC malignant progression. Our findings demonstrate a previously unsuspected layer of cancer growth regulation, which is dormant in normal tissues, and is activated by Trop-2 in cancer cells.

Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans.

Pneumonia is a life-threatening disease often caused by infection with Streptococcus pneumoniae and Pseudomonas aeruginosa. Many of the mediators (e.g., TNF, IL-6R) and junction molecules (e.g., E-cadherin) orchestrating inflammatory cell recruitment and loss of barrier integrity are proteolytically cleaved through a disintegrin and metalloproteinases (ADAMs). We could show by Western blot, surface expression analysis and measurement of proteolytic activity in cell-based assays, that ADAM10 in epithelial cells is upregulated and activated upon infection with Pseudomonas aeruginosa and Exotoxin A (ExoA), but not upon infection with Streptococcus pneumoniae. Targeting ADAM10 by pharmacological inhibition or gene silencing, we demonstrated that this activation was critical for cleavage of E-cadherin and modulated permeability and epithelial integrity. Stimulation with heat-inactivated bacteria revealed that the activation was based on the toxin repertoire rather than the interaction with the bacterial particle itself. Furthermore, calcium imaging experiments showed that the ExoA action was based on the induction of calcium influx. Investigating the extracellular vesicles and their proteolytic activity, we could show that Pseudomonas aeruginosa triggered exosomal release of ADAM10 and proteolytic cleavage in trans. This newly described mechanism could constitute an essential mechanism causing systemic inflammation in patients suffering from Pseudomonas aeruginosa-induced pneumonia stimulating future translational studies.

Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression.

Simple SummaryColorectal cancer is the second leading cause of cancer-related death in the world. Upregulation of fatty acid metabolism is a hallmark of cancer and recent studies demonstrate that blocking fatty acid uptake is a promising therapeutic strategy. We have previously shown that CD36, a transporter of fatty acid, promotes colorectal cancer tumor growth. We have also demonstrated that high expression of CD36 is associated with cancer cells that are prone to metastasis. Here, in studying the role of CD36 in colorectal cancer, we found that CD36 promotes colorectal cancer invasion in vitro and metastasis in vivo and that overexpression of CD36 upregulates expression of the matrix metalloproteinase MMP28. Our data demonstrates that MMP28 cleaves and decreases expression of E-cadherin, a marker for epithelial-to-mesenchymal transition in colorectal cancers. This newly defined CD36–MMP28–E-cadherin axis provides new therapeutic targets for the treatment of colorectal cancer.Altered fatty acid metabolism continues to be an attractive target for therapeutic intervention in cancer. We previously found that colorectal cancer (CRC) cells with a higher metastatic potential express a higher level of fatty acid translocase (CD36). However, the role of CD36 in CRC metastasis has not been studied. Here, we demonstrate that high expression of CD36 promotes invasion of CRC cells. Consistently, CD36 promoted lung metastasis in the tail vein model and GI metastasis in the cecum injection model. RNA-Seq analysis of CRC cells with altered expression of CD36 revealed an association between high expression of CD36 and upregulation of MMP28, a novel member of the metallopeptidase family of proteins. Using shRNA-mediated knockdown and overexpression of CD36, we confirmed that CD36 regulates MMP28 expression in CRC cells. siRNA-mediated knockdown of MMP28 decreases invasion of CRC cells, suggesting that MMP28 regulates the metastatic properties of cells downstream of CD36. Importantly, high expression of MMP28 leads to a significant decrease in active E-cadherin and an increase in the products of E-cadherin cleavage, CTF1 and CTF2. In summary, upregulation of CD36 expression promotes the metastatic properties of CRC via upregulation of MMP28 and an increase in E-cadherin cleavage, suggesting that targeting the CD36–MMP28 axis may be an effective therapeutic strategy for CRC metastasis.

Matrix metalloproteinase-7 induces E-cadherin cleavage in acid-exposed primary human pharyngeal epithelial cells via the ROS/ERK/c-Jun pathway

Laryngopharyngeal reflux disease (LPRD) is caused by pharyngeal mucosal damage due to the reflux of gastric contents, including acid, pepsin, and bile juice. Our previous study has demonstrated that LPRD is associated with the cleavage of E-cadherin, which is facilitated by the acid-activated matrix metalloproteinase-7 (MMP-7); however, the mechanism by which the acid activates MMP-7 remains unclear. The purpose of this study was to investigate the mechanism by which MMP-7 is activated in the pharyngeal epithelial cells that are exposed to acid. The levels of reactive oxygen species (ROS) were measured in the epithelial cells exposed to acid. To investigate the signaling mechanism of ROS in the expression of MMP-7, the mechanism of action of the mitogen-activated protein kinase was examined. The expression of various signaling factors was determined, according to the presence or absence of each inhibitor in the acid-exposed pharyngeal epithelial cells. To identify changes in the cleavage of E-cadherin, the integrity of the mucosal membrane was assessed using a transepithelial permeability test. We found that acid exposure increased the levels of ROS, phosphorylated-extracellular signal-regulated kinase (p-ERK) 1/2, and phosphorylated-c-Jun (p–c-Jun) in pharyngeal epithelial cells. The ROS inhibitor reduced the expression of p-ERK and MMP-7, while the ERK inhibitor reduced the expression of p–c-Jun and MMP-7. Moreover, the c-Jun inhibitor reduced the expression of MMP-7 and blocked the degradation of E-cadherin. In addition, decrease in the levels of immunostained E-cadherin and increase in transepithelial permeability after acid exposure were collectively alleviated by the inhibitors of ROS, ERK, and c-Jun. The degradation of E-cadherin that occurs after human mucosal cells are exposed to acid appears to be caused by an increase in the expression of MMP-7 via the ROS/ERK/c-Jun pathway, which is thought to be an important mechanism associated with the development of LPRD.Key messages• ROS is triggered when reflux occurs.• ROS regulates the transcription factor c-Jun via the ERK pathway.• The increase in MMP-7 that induces LPRD is induced via the ROS/ERK/c-Jun pathway.• This study revealed for the first time the expression mechanism of MMP-7, which is one of the causes of LPRD.