The Proteome of Pancreatic Cancer‐Derived Exosomes Reveals Signatures Rich in Key Signaling Pathways

Exosomes are membrane‐bound vesicles that traffic small molecular cargos. These cargos participate in cell–cell communication and contribute to the pathogenesis of many disease including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood. Performed in this study are comprehensive, quantitative comparisons of the proteomes of three PDAC cell lines to those of the exosomes they produce. Approximately 35% of whole cell proteins sort into exosomes. Analysis of composition of microbiomes (ANCOM) determined a cluster of 98 enriched pancreatic cancer exosome core proteins (ePC‐ECPs). Further, these proteins are predicted by ingenuity pathway analysis (IPA) as actively involved in signaling pathways regulating cell death and survival, cellular movement, and cell‐to‐cell signaling and interaction in particular (top three p‐value significant pathways). Significant enrichment of canonical pathways of acute phase response signaling (inflammatory response signaling pathways) and FXR and RXR activation in biosynthetic pathways are also predicted; 97 ePC‐ECPs are associated with cancer and among them, 34 are specifically associated with PDAC. In conclusion, exosomes from PDAC are enriched with cancer‐associated signaling proteins. Further assessment of these proteins as PDAC biomarkers or therapeutic targets is warranted.     

LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma

Long non-coding RNAs (lncRNAs) are a novel class of regulators in multiple cancer biological processes. However, the functions of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) remain largely unknown. In this study, we identified PWAR6 as a frequently down-regulated lncRNA in PDAC samples as well as a panel of pancreatic cancer cell lines. Down-regulated PWAR6 was associated with multiple clinical outcomes, including advanced tumour stage, distant metastasis, and overall survival of PDAC patients. In our cell-based assays, ectopic expression of PWAR6 dramatically repressed PDAC cells proliferation, invasion and migration, accelerated apoptosis, and induced cell cycle arrest at G0/G1 phase. In contrast, depletion of PWAR6 mediated by siRNA exhibited opposite effects on PDAC cell behaviours. In vivo study further validated the anti-tumour role of PWAR6 in PDAC. By taking advantage of available online sources, we also identified YAP1 as a potential PWAR6 target gene. Negative correlation between YAP1 and PWAR6 expressions were observed in both online database and our PDAC samples. Notably, rescue experiments further indicated that YAP1 is an important downstream effector involved in PWAR6-mediated functions. Mechanistically, PWAR6 could bind to methyltransferase EZH2, a core component of Polycomb Repressive Complex 2 (PRC2) in regulating gene expression, and scaffold EZH2 to the promoter region of YAP1, resulting in epigenetic repression of YAP1. In conclusion, our data manifest the vital roles of PWAR6 in PDAC tumorigenesis and underscore the potential of PWAR6 as a promising target for PDAC diagnosis and therapy.     

Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain

Background

Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown.

Results

Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.

Conclusion

Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

     

Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold

The molecular coupling of CAS and Crk in response to integrin activation is an evolutionary conserved signaling module that controls cell proliferation, survival and migration. However, when deregulated, CAS/Crk signaling also contributes to cancer progression and developmental defects in humans. Here we highlight recent advances in our understanding of how CAS/Crk complexes assemble in cells to modulate the actin cytoskeleton, and the molecular mechanisms that regulate this process. We discuss in detail the spatiotemporal dynamics of CAS/Crk assembly and how this scaffold recruits specific effector proteins that couple integrin signaling networks to the migration machinery of cells. We also highlight the importance of CAS/Crk signaling in the dual regulation of cell migration and survival mechanisms that operate in invasive cells during development and pathological conditions associated with cancer metastasis.     

Interdomain interactions in the tumor suppressor discs large regulate binding to the synaptic protein GukHolder

Multidomain scaffolding proteins are central components of many signaling pathways and are commonly found at membrane specializations. Here we have shown that multiple interdomain interactions in the scaffold Discs Large (Dlg) regulate binding to the synaptic protein GukHolder (GukH). GukH binds the Src homology 3 (SH3) and guanylate kinase-like (GK) protein interaction domains of Dlg, whereas an intramolecular interaction between the two domains inhibits association with GukH. Regulation occurs through a PDZ domain adjacent to the SH3 that allows GukH to interact with the composite SH3-GK binding site, but PDZ ligands inhibit GukH binding such that Dlg forms mutually exclusive PDZ ligand and GukH cellular complexes. The PDZ-SH3-GK module is a common feature of membrane associate guanylate kinase scaffolds such as Dlg, and these results indicate that its supramodular architecture leads to regulation of Dlg complexes.     

Regulation of G Protein-Coupled Receptor Signaling by A-Kinase Anchoring Proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

Regulation of g protein-coupled receptor signaling by a-kinase anchoring proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding ras and activating ras signaling

Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.     

CXCR2 Macromolecular Complex in Pancreatic Cancer: A Potential Therapeutic Target in Tumor Growth

The signaling mediated by the chemokine receptor CXC chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of many cancers, including pancreatic cancer, one of the most lethal human malignancies. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl termini, which might interact with potential PDZ scaffold/adaptor proteins. We have previously reported that CXCR2 PDZ motif-mediated protein interaction is an important regulator for neutrophil functions. Here, using a series of biochemical assays, we demonstrate that CXCR2 is physically coupled to its downstream effector phospholipase C-β3 (PLC-β3) that is mediated by PDZ scaffold protein Na⁺/H⁺ exchange regulatory factor 1 (NHERF1) into a macromolecular signaling complex both in vitro and in pancreatic cancer cells. We also observe that disrupting the CXCR2 complex, by gene delivery or peptide delivery of exogenous CXCR2 C-tail, significantly inhibits the biologic functions of pancreatic cancer cells (i.e., proliferation and invasion) in a PDZ motif-dependent manner. In addition, using a human pancreatic tumor xenograft model, we show that gene delivery of CXCR2 C-tail sequence (containing the PDZ motif) by adeno-associated virus type 2 viral vector potently suppresses human pancreatic tumor growth in immunodeficient mice. In summary, our results suggest the existence of a physical and functional coupling of CXCR2 and PLC-β3 mediated through NHERF1, forming a macromolecular complex that is critical for efficient and specific CXCR2 signaling in pancreatic cancer progression. Disrupting this CXCR2 complex could represent a novel and effective treatment strategy against pancreatic cancer.     

GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.     

Analysis of SHP-1-mediated down-regulation of the TRK-T3 oncoprotein identifies Trk-fused gene (TFG) as a novel SHP-1-interacting protein

SHP-1 is a cytoplasmic SH2 domain containing protein-tyrosine phosphatase (PTP) involved in the negative regulation of multiple signaling pathways in hematopoietic, nervous, and epithelial cells. The thyroid TRK-T3 oncogene consists of the NTRK1 tyrosine kinase domain fused in-frame with sequences of the TFG (TRK-fused gene), encoding a protein of unknown function. TFG contains a coiled-coil domain responsible for TRK-T3 oligomerization. In addition, recent analysis of the sequences outside of the coiled-coil domain suggested possible interactions with other proteins. Based on the presence of a putative SHP-1 SH2-binding site within the TFG sequences, we have investigated the role of the SHP-1 phosphatase in TRK-T3 oncoprotein signaling. In this study we show that SHP-1 interacts with and down-regulates TRK-T3. We provide evidence that SHP-1 SH2 and catalytic domains, respectively, associate with the TFG- and NTRK1-derived portions of TRK-T3. Our data contribute to the definition of cellular mechanisms involved in thyroid tumorigenesis. Moreover, it reveals TFG as a novel protein able to modulate SHP-1 activity.     

Identification of CrkL-SH3 binding proteins from embryonic murine brain: implications for Reelin signaling during brain development

The Crk and Crk-like (CrkL) adaptor proteins play important roles in numerous signaling pathways, bridging tyrosine kinase substrates to downstream signaling effectors by virtue of their phosphotyrosine-binding SH2 domains and their effector-binding SH3 domains. Critical to understanding the diverse roles of Crk/CrkL is the identification of tissue- and signal-specific tyrosine phosphorylated substrates to which they are recruited and the tissue-specific effector proteins they chaperone into signaling complexes. Crk and CrkL are known biochemically and genetically to be essential mediators of Reelin/Disabled-1 (Dab1) signaling, which governs proper mammalian brain development. Multimeric Reelin clusters its receptors as well as the receptor-bound intracellular scaffolding protein Dab1. Clustering induces Fyn/Src-dependent Dab1 tyrosine phosphorylation, which recruits Crk/CrkL and SH3-bound effectors. Previously, 21 Crk/CrkL-SH3 binding proteins were identified from diverse cell types. We present here the proteomic identification of 101 CrkL-SH3 binding proteins from embryonic murine brain. The identified proteins are enriched in the Crk/CrkL-SH3 binding motif and signaling activities regulating cell adhesion and motility. These results suggest Reelin-induced Dab1 tyrosine phosphorylation may generate a multifaceted signaling scaffold containing a rich array of Crk/CrkL-SH3 binding effectors and may explain a growing diversity of cellular activities suggested to be influenced by Reelin/Dab1 signaling.     

GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.     

Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins.

Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization.