Bio21 - Research Publications

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    Proteomic Profiling of Exosomes Secreted by Breast Cancer Cells with Varying Metastatic Potential
    Gangoda, L ; Liem, M ; Ang, C-S ; Keerthikumar, S ; Adda, CG ; Parker, BS ; Mathivanan, S (WILEY, 2017-12)
    Cancer cells actively release extracellular vesicles, including exosomes, into the surrounding microenvironment. Exosomes play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteome profile of exosomes isolated from cells with different metastatic potential and the role of these exosomes in driving metastasis remains unclear. Here, we conduct a comparative proteomic analysis of exosomes isolated from several genetically related mouse breast tumor lines with different metastatic propensity. The amount of exosomes produced and the extent of cancer-associated protein cargo vary significantly between nonmetastatic and metastatic cell-derived exosomes. Metastatic cell-derived exosomes contain proteins that promote migration, proliferation, invasion, and angiogenesis while the nonmetastatic cell-derived exosomes contain proteins involved in cell-cell/cell-matrix adhesion and polarity maintenance. The metastatic exosomes contain a distinct set of membrane proteins including Ceruloplasmin and Metadherin which could presumably aid in targeting the primary cancer cells to specific metastatic sites. Hence, it can be concluded that the exosomes contain different protein cargo based on the host cells metastatic properties and can facilitate in the dissemination of the primary tumors to distant sites.
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    Insulin Mediated Activation of PI3K/Akt Signalling Pathway Modifies the Proteomic Cargo of Extracellular Vesicles
    Liem, M ; Ang, C-S ; Mathivanan, S (WILEY, 2017-12)
    Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression.
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    Arrestin-Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles
    Anand, S ; Foot, N ; Ang, C-S ; Gembus, KM ; Keerthikumar, S ; Adda, CG ; Mathivanan, S ; Kumar, S (WILEY, 2018-09)
    Extracellular vesicles (EVs) are lipid-bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin-domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1-deficient (Arrdc1-/- ) mice. Nanoparticle tracking analysis-based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.
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    A type III effector antagonizes death receptor signalling during bacterial gut infection
    Pearson, JS ; Giogha, C ; Ong, SY ; Kennedy, CL ; Kelly, M ; Robinson, KS ; Lung, TWF ; Mansell, A ; Riedmaier, P ; Oates, CVL ; Zaid, A ; Muehlen, S ; Crepin, VF ; Marches, O ; Ang, C-S ; Williamson, NA ; O'Reilly, LA ; Bankovacki, A ; Nachbur, U ; Infusini, G ; Webb, AI ; Silke, J ; Strasser, A ; Frankel, G ; Hartland, EL (NATURE PUBLISHING GROUP, 2013-09-12)
    Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.
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    Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes
    Keerthikumar, S ; Gangoda, L ; Liem, M ; Fonseka, P ; Atukorala, I ; Ozcitti, C ; Mechler, A ; Adda, CG ; Ang, C-S ; Mathivanan, S (IMPACT JOURNALS LLC, 2015-06-20)
    Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients.
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    A novel community driven software for functional enrichment analysis of extracellular vesicles data
    Pathan, M ; Keerthikumar, S ; Chisanga, D ; Alessandro, R ; Ang, C-S ; Askenase, P ; Batagov, AO ; Benito-Martin, A ; Camussi, G ; Clayton, A ; Collino, F ; Di Vizio, D ; Falcon-Perez, J ; Fonseca, P ; Fonseka, P ; Fontana, S ; Gho, YS ; Hendrix, A ; Nolte-'t Hoen, E ; Iraci, N ; Kastaniegaard, K ; Kislinger, T ; Kowal, J ; Kurochkin, IV ; Leonardi, T ; Liang, Y ; Llorente, A ; Lunavat, TR ; Maji, S ; Monteleone, F ; Overbye, A ; Panaretakis, T ; Patel, T ; Peinado, H ; Pluchino, S ; Principe, S ; Ronquist, G ; Royo, F ; Sahoo, S ; Spinelli, C ; Stensballe, A ; Thery, C ; van Herwijnen, MJC ; Wauben, M ; Welton, JL ; Zhao, K ; Mathivanan, S (TAYLOR & FRANCIS LTD, 2017-05-26)
    Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.
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    A rigorous method to enrich for exosomes from brain tissue
    Vella, LJ ; Scicluna, BJ ; Cheng, L ; Bawden, EG ; Masters, CL ; Ang, C-S ; Willamson, N ; McLean, C ; Barnham, KJ ; Hill, AF (TAYLOR & FRANCIS LTD, 2017-07-26)
    Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.
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    Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine
    Advani, G ; Lim, YC ; Catimel, B ; Lio, DSS ; Ng, NLY ; Chueh, AC ; Tran, M ; Anasir, MI ; Verkade, H ; Zhu, H-J ; Turk, BE ; Smithgall, TE ; Ang, C-S ; Griffin, M ; Cheng, H-C (BIOMED CENTRAL LTD, 2017-08-07)
    BACKGROUND: C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. METHODS: We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. RESULTS: Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. CONCLUSIONS: SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.
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    Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth
    Samuel, M ; Chisanga, D ; Liem, M ; Keerthikumar, S ; Anand, S ; Ang, C-S ; Adda, CG ; Versteegen, E ; Jois, M ; Mathivanan, S (NATURE PORTFOLIO, 2017-07-19)
    Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.
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    Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: implications of intra-tumour heterogeneity
    Fonseka, P ; Liem, M ; Ozcitti, C ; Adda, CG ; Ang, C-S ; Mathivanan, S (TAYLOR & FRANCIS LTD, 2019-01-01)
    Neuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography-mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscopy.