[Cell Cycle 8:13, 2014-2018; 1 July 2009]; ©2009 Landes Bioscience
Review
Microvesicles Messengers and mediators of tumor progression Khalid Al-Nedawi, Brian Meehan and Janusz Rak* Montreal Children’s Hospital Research Institute; McGill University; Montreal, QC CA
Abbreviations: EGFR, epidermal growth factor receptor; EGFRvIII, mutant EGFR (variant III); GBM, glioblastoma multiforme; IL-6, interleuklin 6; IL-8, interleukin 8; mRNA, messenger ribonucleic acid; MVs, microvesicles; PS, phosphatidylserine; RTK, receptor tyrosine kinase; VEGF, vascular endothelial growth factor; VEGFR-2, VEGF receptor 2; TF, tissue factor Key words: cancer, oncogenes, microvesicles, exosomes, receptors, angiogenesis, glioma, EGFRvIII
Cellular interactions play a crucial role in progression, angiogenesis and invasiveness of tumors, including glioma. The traditionally accepted view is that medium and long-range cellular communications occur primarily through gradients of soluble ligands, recognizable by the cell-associated receptors. Recent findings, however, suggest the existence of another mode of intercellular communication, where the ‘units’ of information are microvesicles containing a multitude of biologically active protein and RNA species, including oncogenic receptors, such as EGFRvIII. Moreover, microvesicles can be retrieved from the circulating blood of cancer patients, and reveal the presence of oncogenes in their tumors, thereby potentially serving as information-rich prognostic and predictive biomarkers.
Microvesicles For over a century the search and discovery of biochemically identifiable ‘factors’ that regulate growth, development, metabolism, angiogenic activity and virtually all other discernable cellular functions has been a potent organizing paradigm in biology and medicine. While the basic tenets of the ‘factor theory’ remain biologically correct and useful, questions could be raised, as to its ultimate and sufficient nature. The case in point is the accumulating evidence that in addition to molecular messages, whether soluble or immobilized, cells may also compose more complex multi-molecular ‘texts’ packaged as specialized extracellular organelles known as microvesicles, or exosomes.1-5 Indeed, microvesicles emerge as new complex ‘units’ of biological information in cancer, with *Correspondence to: Janusz Rak; Montreal Children’s Hospital Research Institute; McGill University; 4060 Ste Catherine West; Montreal, QC H3Z 2Z3 CA; Email:
[email protected] Submitted: 05/08/09; Accepted: 05/11/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8988
2014
implications for processes of tumor growth, angiogenesis; metastasis and immune responses,2,4,6,7 as well as a novel and unique potential source of disease-related biomarkers.4,6,8 Among the most recent developments in this exciting field is the series of papers that have demonstrated the emission of microvesicles from brain tumor cells (glioma), and pointed to the pathogenetically and prognostically important cargo of these organelles, including mRNA, micro RNA and proteins, even active oncogenes.4,6 To fully appreciate the significance and promise of these6 and preceding findings,4,9 it may be useful to consider in more detail what is known about the genesis, intercellular trafficking and the cargo of cellular microvesicles, and how these properties may affect their role in cancer.
Cellular Microvesiculation and Cancer Progression It is well established that cells possess the capacity to encapsulate some of their molecular constituents in spherical structures (organelles) surrounded by the plasma membrane and generally referred to as vesicles or microvesicles. This process, which allows a selective release of the cellular content into the surrounding milieu was first noticed by Wolf in 1967, as formation of a procoagulant particulate matter (‘dust’) around activated blood platelets.10 Subsequently, similar organelles (exosomes) were implicated by Johnstone in the removal of transferrin receptors from differentiating red blood cells,3 and various forms of vesiculation were noticed to accompany cancer cells.11 These intriguing observations were then extended to a number of biological events,1-3 and it is surprising that the nature and significance of the underlying process have long remained obscure, something that still resonates in controversies related to the nomenclature, biogenesis, roles and cargo of various microvesicles.3 Notably, microvesicle production occurs during biological processes of considerable diversity, including: cellular differentiation, stress, activation, senescence,12 stimulation with cytokines or shear force,2 exposure to ATP,13 or apoptotic cellular breakdown.14 This also includes malignant transformation,11 for instance due
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Oncogenic microvesicles
to the action of mutant oncogenes, such as K-ras15 and epidermal growth factor receptor (EGFR),4 as well as activation,16 or loss15 of p53 tumor suppressor in various tumor settings. Accordingly, the properties and biological roles of microvesicles generated in these different contexts may be very different. For instance, microvesicles could serve as a rapid release mechanism, e.g., to remove spare proteins,3 to release cytokines,13 or other biological transmitters.2 They could also participate in a defensive shedding of complement attack complexes,1 or in deployment of immunomodulating activities.17 The known emission of membrane-anchored receptors, adhesion molecules, enzymes and signaling proteins1,2,4 via microvesicles has recently been complemented by the finding of functional mRNA and micro RNA species in their cargo.2,9 It has also come to light that the microvesicle composition is not a random sample of the cellular content, but rather is assembled through a highly selective process,9 the nature of which remains unclear. Implicitly, the microvesicle content could be determined by their cellular source and pathway of their generation. Indeed, microvesicles studied in different experimental settings have been distinguished by their different molecular and morphological features, which also led to their elaborate nomenclature, including terms such as: exosomes, ectosomes, promininosomes, prostasomes, epididimosomes, argosomes, archeosomes, oncosomes or microparticles.1,3,4,18 On the other extreme of this spectrum lies the view that all types of microvesicles result from a fundamentally similar process, and therefore the related terms, especially microvesicles and exosomes, can be used interchangeably.3 Perhaps the most informative way of approaching this question, however, is to recognize that it is extremely unlikely that identical organelles containing a similar cargo could be produced by the fundamentally different mechanisms of cellular microvesiculation, of which there are presently at least three known varieties: apoptotic breakdown, plasma membrane blebbing and endocytosis-related formation of exosomes.1-3 Thus, a terminal consequence of membrane blebbing and fragmentation of apoptotic cells is the formation of microvesicles.19 As this process effectively ends the existence of their cellular source, it is of a more circumscribed interest in the context of this article (e.g., as indicator of anticancer drug effects). On the other hand, microvesicles could also emanate from viable cells through the outward blebbing of their plasma membranes.1,3,20 These entities, mostly referred to simply as microvesicles or microparticles, are usually relatively large in size (>100 nm up to 1,000 nm in diameter) and often contain elements of the membrane lipid rafts, such as flotilin-1, tissue factor,15,21 lineage markers,22 and, in some cases, oncogenic growth factor receptors, such as EGFR.4 The mechanism of this form of (exo)vesiculation is related to rearrangements in symmetry of membrane phospholipids by the enzymatic machinery that includes: aminophospholipid translocase, scramblase flippase, floppase and calpain.22 Recently, an elegant study demonstrated a mechanism involvoving ATP stimulation, mobilization of acidic sphingomyelinase (A-SMAse) and resulting membrane phospholipid rearrangement as a mode of microvesicular release of IL-1b from cultured glial cells.13 Such perturbations result in changes in the phospholipid bilayer organization and physical ‘bend’, exposure of phosphatidylserine www.landesbioscience.com
(PS) on the cell surface, followed by localized changes in the structure of the cellular membrane and cytoskeleton, blebbing and formation of a microvesicle.22 Interestingly, a genetic defect in the activity of the lipid scramblase leads to the inability of blood platelets to expose PS, or produce procoagulant microparticles, resulting in a rare bleeding disorder known as Scott syndrome.22 There are no published data as to how this, or similar defects in microvesiculation may affect progression, angiogenesis or treatment of solid tumors, but agents that block PS have been already shown to possess anticancer activities,5,23 possibly due to inhibition of microvesicular interactions4 required for angiogenesis and other processes. Generation of microvesicles may also occur via a pathway of endocytosis.24 In this case, the budding of the plasma membrane occurs inward, often as a consequence of signaling cues from the receptor tyrosine kinases (RTKs), in the recycling of which this pathway plays a central role.24 The major steps of this cascade are controlled by the ‘endosomal sorting complexes required for transport’ (ESCRT system), whereby the invaginated plasma membrane containing ubiquitinated molecular cargo develops into intracellular vesicle known as early endosome, which is later transformed into more complex multivesicular body (MVB).24 The fate of endosomes includes their fusion with lysosomes resulting in destruction of their cargo, or entail a return (recycling) of this material to the plasma membrane for reutilization.1,24 Alternatively, the secondary inward budding of endosomal membranes may lead to formation of much smaller inner microvesicles (within MVBs) with the outsideout membrane orientation and destined for extracellular release rather than destruction.1,25 In this case the ESCRT processing for lysosomal destruction is aborted, phospholipids surrounding the inner micovesicles spontaneously rearrange,25 MVBs are directed to the plasma membrane and release their vesicular content (bona fide exosomes) into the cellular exterior.1,25 Exosomes differ markedly from microvesicles derived by the external budding of the plasma membrane,1,26 in that they are much smaller in size (
