Functions of CancerDerived Extracellular Vesicles

Authors: Liliana Czernek Markus Düchler

Publish Date: 2017/01/18

Volume: 65, Issue: 4, Pages: 311-323

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Abstract

Extracellular vesicles including exosomes constitute an important element of intercellular communication by carrying a variety of molecules from producer to target cells The transport of mRNA and miRNA can directly modulate gene expression in the target cells The miRNA content in exosomes is characteristic for the cell from which the vesicles were derived enabling the usage of exosomes as biomarkers for the diagnosis various diseases including cancer Cancerderived exosomes support the survival and progression of tumors in many ways and also contribute to the neutralization of the anticancer immune response Exosomes participate in all known mechanisms by which cancer evades the immune system They influence the differentiation and activation of immune suppressor cells they modulate antigen presentation and are able to induce Tcell apoptosis Although cancerderived exosomes mainly suppress the immune system and facilitate tumor progression they are also important sources of tumor antigens with potential clinical application in stimulating immune responses This review summarizes how exosomes assist cancer to escape immune recognition and to acquire control over the immune systemIn the early 1980s two research groups described that the transferrin receptor of sheep reticulocytes was secreted via littleknown vesicular forms Pan and Johnstone 1984 The same researchers described the mechanism of small vesicle secretion showing that the release of membrane vesicles was preceded by inward budding of an intracellular endosome forming a multivesicular body MVB which could then fuse with the plasma membrane Pan et al 1985 Rose Johnstone used the term “exosomes” for the first time to describe small membrane vesicles formed in MVBs Johnstone et al 1987 The original function attributed to membrane vesicles was the removal of cell debris The thinking about membrane vesicles as “trash cans” of the cell was derived from the knowledge about the role of lysosomes as degradation centers Luzio et al 2007 Since the finding that exosomes can modulate the immune system extracellular vesicles gained growing interest Raposo et al 1996 The enthusiasm was further increased after the discovery of mRNA and miRNA inside exosomes Valadi et al 2007 These studies opened the door to the new research field of exosome functions in intercellular communication their biomarkers and their potential role as therapeutic toolsCells release different kinds of extracellular vesicles EVs of varying sizes and biogenesis Their classification distinguishes three main subpopulations/classes based on the vesicle’s origin The smallest vesicles are of endocytic origin exosomes with 40–150 nm in diameter Baietti et al 2012 Colombo et al 2013 Ectosomes also called shedding microvesicles with a diameter of 100–1000 nm are produced by outward protrusion or budding from the plasma membrane MuralidharanChari et al 2009 Théry et al 2009 The most heterogeneous group of vesicles ranging from 50 up to 5000 nm in diameter is apoptotic bodies Their biogenesis is based on fragmentation of apoptotic cells during programmed cell death Mathivanan et al 2010 Théry et al 2009 A common feature of all vesicle classes is their membrane structure a lipid bilayer with the same topological orientation as the plasma membrane Trajkovic et al 2008 Although the origin of microvesicles and exosomes is well known the experimental discrimination of these vesicles types is difficult and so the terms are sometimes subsumed as extracellular vesicles In this review we follow the terminology used by the authorsThe process of exosome biogenesis is not fully understood It starts within endosomes which are responsible for regulated trafficking of proteins and lipids between subcellular compartments of the secretory and endocytic pathway Lemmon and Traub 2000 The cargo of endosomes can enter recycling circuits to return membrane components back to the plasma membrane or can be sorted into lysosomes for degradation Huotari and Helenius 2011 The content of cholesterol is associated with the fate of MVBs cholesterolpoor MVBs are appointed for lysosome fusion and degradation Möbius et al 2002 Exosomes formed within MVBs are released via exocytosis into the extracellular space when cholesterolrich MVBs fuse with the plasma membrane Kalra et al 2012During vesicle formation cellular components extracellular ligands and other endocytosed molecules such as receptors are packed into the vesicles Gould and LippincottSchwartz 2009 Molecules from the early endosomes such as the tetraspanin CD63 or LAMP1 and LAMP2 are released through the vesicles Colombo et al 2014 Jaiswal et al 2002 Raposo et al 1996 The ESCRT endosomal sorting complex required for transport machinery is involved in the budding process as well as in the controlled sorting of proteins into exosomes The ESCRT machinery consists of four complexes ESCRT0 Hrs ESCRTI TSG101 and Vps28 ESCRTII Vps22 and ESCRTIII Alix and Vps2 which sort ubiquitinylated proteins to the late endosomes The ESCRTIII complex was shown to promote intraluminal budding of vesicles in endosomes which results in maturation of the cargocontaining vesicles Colombo et al 2013 Kowal et al 2014 An ESCRTindependent packaging mechanism was also proposed involving glycolipoprotein microdomains lipid rafts Trajkovic et al 2008 Besides a big range of proteins also nucleic acids like mRNA miRNA or DNA can be found in exosomes http//wwwexocartaorg Thakur et al 2014 Valadi et al 2007 Interestingly the miRNA contents of exosomes do not entirely parallel the miRNA composition inside the cell indicating selective loading mechanisms Rappa et al 2013 For the selection of miRNA for exosomal export several potential routes were described one depending on neural sphingomyelinase 2 Kosaka et al 2013 a second based on uridylation versus adenylation of the 3′end of the miRNAs KoppersLalic et al 2014 a third one involving sumoylated heterogeneous nuclear ribonuleoprotein hnRNPA2B1 binding to a GGAG motif in the 3′part of miRNA sequences to be packed into exosomes VillarroyaBeltri et al 2013 and another one related to the RISC pathway Gibbings et al 2009 For the selective loading of mRNA into microvesicles a 25nucleotide sequence motif in the 3′UTR of exported mRNAs was described Bolukbasi et al 2012 In cancer cellderived exosomes also fragments of chromosomal DNA were identified Kahlert et al 2014 their sorting mechanism into the vesicles has not yet been defined The analysis of the miRNA content of exosomes allows to draw conclusions about the cell type from which the exosomes originated Thus determining the miRNA profile in extracellular vesicles derived from bodily fluids of diseased persons has a huge potential for diagnostic purposes Miller and Grunewald 2015 Verma et al 2015The immune system provides a defense against attacks of foreign invaders such as bacteria viruses and parasites or the growth of cancer cells Once it recognizes nonself antigens it activates multiple chemical and physiological processes constituting the immune response Kindt et al 2007 The immune response comprises innate and adaptive immunity The components of the innate response include antigenpresenting cells APCs like macrophages or dendritic cells DCs that are responsible for phagocytosis digestion and presentation of pathogenderived antigens on the cell surface and natural killer NK cells that directly destroy infected or transformed cells The innate immune response is followed by the adaptive one which is based on activation of specific B and T lymphocytes T cells are highly specialized cells that not only coordinate Thelper Th or suppress Tregulatory Treg the immune response but also destroy infected cells Tcytotoxic CTL B cells secrete antibodies which mark infected cells or pathogens to promote their elimination from the organism The Tcell and Bcell responses include the production of memory cells against the pathogen enabling quicker immune response in future challenges Kindt et al 2007Cancer cells have to express antigens which are recognized as nonself to elicit an immune response Such tumorassociated antigens TAA are either mutated cellular proteins or molecules with differences in posttranslational modifications Finn 2012 TAAderived peptides produced by the proteasome are presented through major histocompatibility complex MHC I complexes on the cell surface and recognized by CTLs resulting in tumor cell killing The strategies used by tumors to escape this destruction include the impairment of the executory capacity of the immune system and hiding from recognition by immune cells through the loss of target antigen expression Defective antigen presentation can be caused by the downregulation of the antigen processing machinery which may affect the MHCI pathway and other involved proteins like the proteasome subunits LMP2 latent membrane protein 2 and LMP7 the transporter associated with antigen processing and tapasin Garrido et al 1997 Hicklin et al 1999 Johnsen et al 1999 Restifo et al 1993 RotemYehudar et al 1996 When the expression of TAA is downregulated CTL no longer recognize the tumor cells Maeurer et al 1996 About 20 years ago mutations in the β2microglobulin gene have been identified in metastatic melanoma cells resulting in the absence of HLA class I antigens on the cell surface Benitez et al 1998The production of immune suppressive cytokines by cancer cells or noncancer cells in the tumor microenvironment exerts a powerful suppression of the anticancer immune response Among these cytokines are transforming growth factor TGFβ tumor necrosis factor TNFα interleukin IL1 IL6 IL8 IL10 and type I interferons IFNs Pasche 2001 Lind et al 2004 Matsuda et al 1994 Furthermore vascular endothelial growth factor VEGF has the ability to suppress proper Tcell development and function Ohm et al 2003 TGFβ and IL10 can shift the balance from a Th1 response executed by cytotoxic T cells towards an antibodybased Th2 response immune deviation Maeda and Shiraishi 1996 Induction of immune tolerance may also occur through downregulation of costimulatory molecules on APCs Engagement of the Tcell receptor TCR in the absence of costimulation induces anergy or tolerance in T cells StaveleyO’Carrol et al 1998 Tumors even eliminate tumorspecific CTLs by expressing ligands to death receptors which trigger Tcell apoptosis Bogen 1996 Advanced cancerinduced immunosuppression results in the induction and activation of immune suppressor cells like myeloidderived suppressor cells MDSCs and Treg cells Treg cells generally suppress the activity and proliferation of effector T cells Shevach 2002 fulfil an important function to maintain immune tolerance to selfantigen and are critical in the suppression of autoimmune diseases It was shown that tumorderived Tregs have comparatively higher suppressive activity than naturally occurring Tregs Yokokaw et al 2008 Gasparoto et al 2010 The induction and activation of cancerantigenspecific Treg cells seem to be the major mechanism of tumor immune escape Vinay et al 2015

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