Downregulation of stromal syntenin sustains AML development.

The crosstalk between cancer and stromal cells plays a critical role in tumor progression. Syntenin is a small scaffold protein involved in the regulation of intercellular communication that is emerging as a target for cancer therapy. Here, we show that certain aggressive forms of acute myeloid leukemia (AML) reduce the expression of syntenin in bone marrow stromal cells (BMSC). Stromal syntenin deficiency, in turn, generates a pro-tumoral microenvironment. From serial transplantations in mice and co-culture experiments, we conclude that syntenin-deficient BMSC stimulate AML aggressiveness by promoting AML cell survival and protein synthesis. This pro-tumoral activity is supported by increased expression of endoglin, a classical marker of BMSC, which in trans stimulates AML translational activity. In short, our study reveals a vicious signaling loop potentially at the heart of AML-stroma crosstalk and unsuspected tumor-suppressive effects of syntenin that need to be considered during systemic targeting of syntenin in cancer therapy.

PDZ scaffolds regulate extracellular vesicle production, composition, and uptake.

Extracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators. We reveal that the PDZ proteome (ca. 150 proteins in human) establishes a discrete number of direct interactions with the tetraspanins CD9, CD63, and CD81, well-known EV constituents. Strikingly, PDZ proteins interact more extensively with syndecans (SDCs), ubiquitous membrane proteins for which we previously demonstrated an important role in EV biogenesis, loading, and turnover. Nine PDZ proteins were tested in loss-of-function studies. We document that these PDZ proteins regulate both tetraspanins and SDCs, differentially affecting their steady-state levels, subcellular localizations, metabolism, endosomal budding, and accumulations in EVs. Importantly, we also show that PDZ proteins control the levels of heparan sulfate at the cell surface that functions in EV capture. In conclusion, our study establishes that the extensive networking of SDCs, tetraspanins, and PDZ proteins contributes to EV heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication.

The Human PDZome 2.0: Characterization of a New Resource to Test for PDZ Interactions by Yeast Two-Hybrid.

PSD95-disc large-zonula occludens (PDZ) domains are globular modules of 80-90 amino acids that co-evolved with multicellularity. They commonly bind to carboxy-terminal sequences of a plethora of membrane-associated proteins and influence their trafficking and signaling. We previously built a PDZ resource (PDZome) allowing us to unveil human PDZ interactions by Yeast two-hybrid. Yet, this resource is incomplete according to the current knowledge on the human PDZ proteome. Here we built the PDZome 2.0 library for Yeast two-hybrid, based on a PDZ library manually curated from online resources. The PDZome2.0 contains 305 individual clones (266 PDZ domains in isolation and 39 tandems), for which all boundaries were designed based on available PDZ structures. Using as bait the E6 oncoprotein from HPV16, a known promiscuous PDZ interactor, we show that PDZome 2.0 outperforms the previous resource.

Differential proteomics argues against a general role for CD9, CD81 or CD63 in the sorting of proteins into extracellular vesicles.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.

Discovery of a PDZ Domain Inhibitor Targeting the Syndecan/Syntenin Protein-Protein Interaction: A Semi-Automated "Hit Identification-to-Optimization" Approach.

The rapid identification of early hits by fragment-based approaches and subsequent hit-to-lead optimization represents a challenge for drug discovery. To address this challenge, we created a strategy called "DOTS" that combines molecular dynamic simulations, computer-based library design (chemoDOTS) with encoded medicinal chemistry reactions, constrained docking, and automated compound evaluation. To validate its utility, we applied our DOTS strategy to the challenging target syntenin, a PDZ domain containing protein and oncology target. Herein, we describe the creation of a "best-in-class" sub-micromolar small molecule inhibitor for the second PDZ domain of syntenin validated in cancer cell assays. Key to the success of our DOTS approach was the integration of protein conformational sampling during hit identification stage and the synthetic feasibility ranking of the designed compounds throughout the optimization process. This approach can be broadly applied to other protein targets with known 3D structures to rapidly identify and optimize compounds as chemical probes and therapeutic candidates.

A Journey on Extracellular Vesicles for Matrix Metalloproteinases: A Mechanistic Perspective.

Matrix metalloproteinases (MMPs) are key players in matrix remodeling and their function has been particularly investigated in cancer biology. Indeed, through extracellular matrix (ECM) degradation and shedding of diverse cell surface macromolecules, they are implicated in different steps of tumor development, from local expansion by growth to tissue invasion and metastasis. Interestingly, MMPs are also components of extracellular vesicles (EVs). EVs are membrane-limited organelles that cells release in their extracellular environment. These "secreted" vesicles are now well accepted players in cell-to-cell communication. EVs have received a lot of interest in recent years as they are also envisioned as sources of biomarkers and as potentially outperforming vehicles for the delivery of therapeutics. Molecular machineries governing EV biogenesis, cargo loading and delivery to recipient cells are complex and still under intense investigation. In this review, we will summarize the state of the art of our knowledge about the molecular mechanisms implicated in MMP trafficking and secretion. We focus on MT1-MMP, a major effector of invasive cell behavior. We will also discuss how this knowledge is of interest for a better understanding of EV-loading of MMPs. Such knowledge might be of use to engineer novel strategies for cancer treatment. A better understanding of these mechanisms could also be used to design more efficient EV-based therapies.

[Tetraspanins and syndecans: Partners in crime for 'dealing' exosomes?] / Tétraspanines et syndécanes - Complices dans le « trafic ¼ des exosomes ?

Exosomes are small extracellular vesicles derived from endosomal compartments. The molecular mechanisms supporting the biology of exosomes, from their biogenesis to their internalization by target cells, rely on 'dedicated' membrane proteins. These mechanisms of action need to be further clarified. This will help to better understand how exosome composition and heterogeneity are established. This would also help to rationalize their use as source of biomarkers and therapeutic tools. Here we discuss how syndecans and tetraspanins, two families of membrane scaffold proteins, cooperate to regulate different steps of exosome biology.

TITLE: Tétraspanines et syndécanes - Complices dans le « trafic ¼ des exosomes ? ABSTRACT: Les exosomes sont de petites vésicules extracellulaires qui sont produites dans des compartiments endosomaux. Les mécanismes moléculaires sur lesquels reposent la biologie des exosomes, de leur biogenèse à leur internalisation par les cellules cibles, font notamment appel à des protéines membranaires particulières. Ces mécanismes méritent d'être clarifiés, afin de mieux comprendre la complexité de la composition des exosomes et de rationaliser leur utilisation comme biomarqueurs ou comme outils thérapeutiques. Nous discutons ici comment les syndécanes et les tétraspanines, deux familles de protéines d'échafaudage, coopèrent pour réguler les différentes étapes de la biologie des exosomes.

Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread.

Syntenin stimulates exosome production and its expression is upregulated in many cancers and implicated in the spread of metastatic tumor. These effects are supported by syntenin PDZ domains interacting with syndecans. We therefore aimed to develop, through a fragment-based drug design approach, novel inhibitors targeting syntenin-syndecan interactions. We describe here the optimization of a fragment, 'hit' C58, identified by in vitro screening of a PDZ-focused fragment library, which binds specifically to the syntenin-PDZ2 domain at the same binding site as the syndecan-2 peptide. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 45 and 57 (IC50 = 33 µM and 47 µM; respectively), two representatives of syntenin-syndecan interactions inhibitors, that selectively affect the syntenin-exosome release. These findings demonstrate that it is possible to identify small molecules inhibiting syntenin-syndecan interaction and exosome release that may be useful for cancer therapy.

Identification of PDZ Interactions by Yeast Two-Hybrid Technique.

The yeast two-hybrid technique is a powerful method to detect direct protein-protein interactions. Due to its accessibility, speed, and versatility, this technique is easy to set up in any laboratory and suitable for small and large scale screenings. Here we describe the implementation of an array-based screening that allows for the probing of the entire human PDZ ORFeome (or hPDZome) by yeast two-hybrid technique. With this approach, one can rapidly identify the PDZ domains that are able to interact (up to KD in the high µmolar range) with any candidate protein among a panel of 266 individual clones, thereby comprehensively identifying its PDZ interactome.

Study of PDZ-Peptide and PDZ-Lipid Interactions by Surface Plasmon Resonance/BIAcore.

Surface plasmon resonance (SPR)/BIAcore technology enables the characterization of molecular interactions, including determination of affinities and kinetics. In BIAcore, one of the interaction partners (the ligand) is immobilized on a chip and the other (the analyte) is provided in solution. BIAcore allows to study association and dissociation rates in real time without the use of labeling. BIAcore can be applied to molecular interactions involving small compounds and biological macromolecules such as proteins, lipids, nucleic acids, or carbohydrates. Here we describe protocols for the measurements of PDZ domain-peptide (oriented biotinylated peptides), PDZ domain-liposomes (lipid membranes), and PDZ-lipid-peptide tripartite interactions.

Syntenin-knock out reduces exosome turnover and viral transduction.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

A High-Affinity Peptide Ligand Targeting Syntenin Inhibits Glioblastoma.

Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.

Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo.

Exosomes support cell-to-cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF-7 breast carcinoma cells, this compound is non-toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin-exosomal pathway, of potential interest for exosome research and oncology.

Syndecan 4 Upregulation on Activated Langerhans Cells Counteracts Langerin Restriction to Facilitate Hepatitis C Virus Transmission.

Sexually transmitted Hepatitis C virus (HCV) infections and high reinfections are a major concern amongst men who have sex with men (MSM) living with HIV-1 and HIV-negative MSM. Immune activation and/or HIV-1 coinfection enhance HCV susceptibility via sexual contact, suggesting that changes in immune cells or external factors are involved in increased susceptibility. Activation of anal mucosal Langerhans cells (LCs) has been implicated in increased HCV susceptibility as activated but not immature LCs efficiently retain and transmit HCV to other cells. However, the underlying molecular mechanism of transmission remains unclear. Here we identified the Heparan Sulfate Proteoglycan Syndecan 4 as the molecular switch, controlling HCV transmission by LCs. Syndecan 4 was highly upregulated upon activation of LCs and interference with Heparan Sulfate Proteoglycans or silencing of Syndecan 4 abrogated HCV transmission. These data strongly suggest that Syndecan 4 mediates HCV transmission by activated LCs. Notably, our data also identified the C-type lectin receptor langerin as a restriction factor for HCV infection and transmission. Langerin expression abrogated HCV infection in HCV permissive cells, whereas langerin expression on the Syndecan 4 expressing cell line strongly decreased HCV transmission to a target hepatoma cell line. These data suggest that the balanced interplay between langerin restriction and Syndecan 4 transmission determines HCV dissemination. Silencing of langerin enhanced HCV transmission whereas silencing Syndecan 4 on activated LCs decreased transmission. Blocking Heparan Sulfate Proteoglycans abrogated HCV transmission by LCs ex vivo identifying Heparan Sulfate Proteoglycans and Syndecan 4 as potential targets to prevent sexual transmission of HCV. Thus, our data strongly suggest that the interplay between receptors promotes or restricts transmission and further indicate that Syndecan 4 is the molecular switch controlling HCV susceptibility after sexual contact.

The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis.

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.

Heparanase Involvement in Exosome Formation.

Exosomes are secreted vesicles involved in signaling processes. The biogenesis of a class of these extracellular vesicles depends on syntenin, and on the interaction of this cytosolic protein with syndecans. Heparanase, largely an endosomal enzyme, acts as a regulator of the syndecan-syntenin-exosome biogenesis pathway. The upregulation of syntenin and heparanase in cancers may support the suspected roles of exosomes in tumor biology.

Tetraspanin-6 negatively regulates exosome production.

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4-TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine.

Lipids in Exosome Biology.

Extracellular vesicles (EVs), and exosomes in particular, were initially considered as "garbage bags" for secretion of undesired cellular components. This view has changed considerably over the last two decades, and exosomes have now emerged as important organelles controlling cell-to-cell signaling. They are present in biological fluids and have important roles in the communication between cells in physiological and pathological processes. They are envisioned for clinical use as carriers of biomarkers, therapeutic targets, and vehicles for drug delivery. Important efforts are being made to characterize the contents of these vesicles and to understand the mechanisms that govern their biogenesis and modes of action. This chapter aims to recapitulate the place given to lipids in our understanding of exosome biology. Besides their structural role and their function as carriers, certain lipids and lipid-modifying enzymes seem to exert privileged functions in this mode of cellular communication. By extension, the use of selective "lipid inhibitors" might turn out to be interesting modulators of exosomal-based cell signaling.

Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo.

Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes.

Phospholipase D and phosphatidic acid in the biogenesis and cargo loading of extracellular vesicles.

Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made toward characterizing the contents of these vesicles and the mechanisms that govern their biogenesis. It has been recently demonstrated that the lipid modifying enzyme, phospholipase D (PLD)2, is involved in exosome production and acts downstream of the small GTPase, ARF6. This review aims to recapitulate our current knowledge of the role of PLD2 and its product, phosphatidic acid, in the biogenesis of exosomes and to propose hypotheses for further investigation of a possible central role of these molecules in the biology of these organelles.