Syntenin: Key player in cancer exosome biogenesis and uptake?

International audienceCancer exosomes are gaining considerable amount of attention in basic and applied clinical research for their established role in the modulation of the tumor niche, and their broad-range contribution to tumor-host cross-talk. Supporting evidence to their role in tumorigenesis comes from the observation that exosome secretion, composition and functional effects are all altered as tumors become more aggressive. At the molecular level, the mechanisms underlying exosome biogenesis and uptake are far from being understood. Recent work has highlighted the critical role for the small intracellular adaptor protein syntenin in the biogenesis of a subset of exosomes and loading of cargo. Here, we review this recent work and some unpublished data that further highlight the possible implications of syntenin and the syndecan (SDC) heparan sulphate proteoglycans during exosome uptake, suggesting a supporting role for this pathway in the entire life cycle of cancer exosomes

Phosphoinositides and PDZ domain scaffolds

The discovery that PSD-95/Discs large/ZO-1 (PDZ) domains can function as lipid-binding modules, in particular interacting with phosphoinositides (PIs), was made more than 10 years ago (Mol Cell 9(6): 1215-1225, 2002). Confirmatory studies and a series of functional follow-ups established PDZ domains as dual specificity modules displaying both peptide and lipid binding, and prompted a rethinking of the mode of action of PDZ domains in the control of cell signaling. In this chapter, after introducing PDZ domains, PIs and methods for studying protein-lipid interactions, we focus on (i) the prevalence and the specificity of PDZ-PIs interactions, (ii) the molecular determinants of PDZ-PIs interactions, (iii) the integration of lipid and peptide binding by PDZ domains, (iv) the common features of PIs interacting PDZ domains and (v) the regulation and functional significance of PDZ-PIs interactions.status: publishe

The Scribble family in cancer: twentieth anniversary

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Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells

The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.status: publishe

Syntenin-knock out reduces exosome turnover and viral transduction

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Design, synthesis and biological evaluation of pyrazolo[3,4-d] pyrimidine-based protein kinase D inhibitors

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.status: publishe

Prevalence, Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and <em>In Vitro</em> Binding Experiments

<div><h3>Background</h3><p>PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs), important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited.</p> <h3>Methodology/Principal Findings</h3><p>We screened the human proteome for PtdInsPs interacting PDZ domains by a combination of <em>in vivo</em> cell-localization studies and <em>in vitro</em> dot blot and Surface Plasmon Resonance (SPR) experiments using synthetic lipids and recombinant proteins. We found that PtdInsPs interactions contribute to the cellular distribution of some PDZ domains, intriguingly also in nuclear organelles, and that a significant subgroup of PDZ domains interacts with PtdInsPs with affinities in the low-to-mid micromolar range. <em>In vitro</em> specificity for the head group is low, but with a trend of higher affinities for more phosphorylated PtdInsPs species. Other membrane lipids can assist PtdInsPs-interactions. PtdInsPs-interacting PDZ domains have generally high pI values and contain characteristic clusters of basic residues, hallmarks that may be used to predict additional PtdInsPs interacting PDZ domains. In tripartite binding experiments we established that peptide binding can either compete or cooperate with PtdInsPs binding depending on the combination of ligands.</p> <h3>Conclusions/Significance</h3><p>Our screen substantially expands the set of PtdInsPs interacting PDZ domains, and shows that a full understanding of the biology of PDZ proteins will require a comprehensive insight into the intricate relationships between PDZ domains and their peptide and lipid ligands.</p> </div

Subcellular distribution of PDZ domains and effects of PtdInsP-modifying treatments compared to <i>in vitro</i> PtdIns(4,5)P2-binding.

<p><b>A.</b> Double reference subtracted sensorgrams of recombinant his-tagged PDZ domains injected, in a range of concentrations with the highest concentrations used indicated, over 5% PtdIns(4,5)P2 containing DOPC liposomes. <b>B–O.</b> Confocal or wide-field micrographs of MCF-7 cells transiently over-expressing selected eYFP-S1PDZ1-tagged PDZ domains (with the exception of SLC9A3R2_1* where S1PDZ1 was omitted). Sensorgrams are provided to the left of the micrographs for each PDZ domain under study. <b>B</b>. Co-expression of eYFP-S1PDZ1-MPP7 with PtdIns(4)P-5-kinase (PIPK; shown in the insets) to probe the effect of increased plasma membrane PtdIns(4,5)P2 levels on the distribution of the fluorescent protein. Note the absence of membrane enrichment of the fluorescence upon kinase over-expression. <b>C</b>. Serum stimulation of eYFP-S1PDZ1-MPP7 expressing cells to investigate the effect of increased plasma membrane PtdIns(3,4,5)P3 levels. Note the absence of membrane enrichment of the fluorescence upon serum stimulation. For control experiments of B and C, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054581#pone.0054581.s003" target="_blank">Fig. S3A</a>–B. <b>D–G</b> Ionomycin treatment (D, E), inhibition of PtdIns(4)-kinases by PAO (F) and rapamycin-induced membrane recruitment of PtdInsPs 5′ phosphatase to investigate how reducing PtdIns(4,5)P2 levels affect the membrane enrichment of eYFP-S1PDZ1-MPDZ_7 (D) and eYFP-S1PDZ1-CASK (E-G). <b>H–I.</b> Co-expression with mCherry-PTS1 identified the bright cytosolic spots enriched in eYFP-S1PDZ1-MAGI3_3 (H) and eYFP-S1PDZ1-IL16_1 (I) as peroxisomes. Co-expression with mCherry-B23 established that the subnuclear organelles enriched in eYPF-S1PDZ1-SCRIB4 (J) and eYFP-SLC9A3R2_1* (M) corresponded to nucleoli. Co-expression with a PtdIns(4,5)P2-4-phosphatase (PIP2 4 ptase, shown in the insets) or PCLΔNES, did not affect the nucleoli enrichment of eYFP-S1PDZ1-SCRIB4 (K, L) but induced a shift of eYFP-SLC9A3R2_1 (N, O) towards the nucleoplasm and the cytosol.</p

The PDZ1 of syntenin-1 function as an enhancer for PtdInsPs dependent cellular localization. A–L.

<p>Confocal micrographs illustrating the subcellular localization of different fluorescent constructs transiently over-expressed in MCF-7 cells. The eYFP-S1PDZ1 (screening construct) localizes diffusely (A). The eYFP-tagged PtdInsPs binding PDZ1 and PDZ2 domain of syntenin-2 show diffuse localization in the cytoplasm and the nucleoplasm when taken in isolation (B,C), but concentrate in subnuclear regions and at the plasma membrane when fused to S1PDZ1 (D, E). Distribution of the tandem repeat of the FYVE domain of Hrs (G), a probe for early endosomal PtIns(3)P. Fusion of eYFP-S1PDZ1 with a single FYVE domain of Hrs, that distributes diffusely when taken in isolation (F), results in the concentration of the fluorescence on vesicular structures (H) that co-localize with the endosomal marker eCFP-Rab5a (I). Treatment with the PtdIns-3-kinase inhibitors wortmannin (J) or LY294002 (K) induces release of the construct from the vesicles. The PtdIns(3)P dependent localization was further confirmed by treatment of the cells with the YM201636 PtdIns(3)P-5-kinase inhibitor inducing swollen PtdIns(3)P-rich vesicles (L). M. Scheme of the cloning strategy.</p