Survivin is released from cancer cells via exosomes

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Heat shock protein 70 is secreted from endothelial cells by a non-classical pathway involving exosomes

Emerging evidence suggests that a high level of circulating heat shock protein 70 (HSP70) correlates with a lower risk of vascular disease; however, the biological significance of this inverse relationship has not been explored. Herein, we report that oxidative low density lipoprotein (Ox-LDL) and homocysteine (Hcy) induce HSP70 release from endothelial cells. In rat endothelial cells, Ox-LDL and Hcy induced robust release of HSP70, independent of the classical route of endoplasmic reticulum/Golgi protein trafficking or the formation of lipid rafts. In contrast, Ox-LDL and Hcy significantly enhanced the exosomal secretory rate and increased the HSP70 content of exosomes. Exogenous HSP70 had no impact on LPS-, Ox-LDL- and Hcy-induced activation of endothelial cells, whereas HSP70 did activate monocytes alone, resulting in monocyte adhesion to endothelial cells. These results indicate that exosome-dependent secretion of HSP70 from endothelial cells provides a novel paracrine mechanism to regulate vascular endothelial functional integrity.     

Bacterial Lipopolysaccharide Augments Febrile-Range Hyperthermia-Induced Heat Shock Protein 70 Expression and Extracellular Release in Human THP1 Cells

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.     

Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro

Failure of immune surveillance related to inadequate host antitumor immune responses has been suggested as a possible cause of the high incidence of recurrence and poor overall survival outcome of hepatocellular carcinoma. The stress-induced heat shock proteins (HSPs) are known to act as endogenous "danger signals" that can improve tumor immunogenicity and induce natural killer (NK) cell responses. Exosome is a novel secretory pathway for HSPs. In our experiments, the immune regulatory effect of the HSP-bearing exosomes secreted by human hepatocellular carcinoma cells under stress conditions on NK cells was studied. ELISA results showed that the production of HSP60, HSP70, and HSP90 was up-regulated in both cell lines in a stress-specific manner. After exposure to hepatocellular carcinoma cell-resistant or sensitive anticancer drugs (hereafter referred to as "resistant" or "sensitive" anticancer drug), the membrane microvesicles were actively released by hepatocellular carcinoma cells, differing in their ability to present HSPs on the cell surface, which were characterized as exosomes. Acting as a decoy, the HSP-bearing exosomes efficiently stimulated NK cell cytotoxicity and granzyme B production, up-regulated the expression of inhibitory receptor CD94, and down-regulated the expression of activating receptors CD69, NKG2D, and NKp44. Notably, resistant anticancer drugs enhanced exosome release and generated more exosome-carried HSPs, which augmented the activation of the cytotoxic response. In summary, our findings demonstrated that exosomes derived from resistant anticancer drug-treated HepG2 cells conferred superior immunogenicity in inducing HSP-specific NK cell responses, which provided a clue for finding an efficient vaccine for hepatocellular carcinoma immunotherapy.     

Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes

Heat shock protein (HSP)70 can be released from tumor cells and stimulate a potent antitumor immune response. However, HSP70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. We have observed HSP70 release from intact human prostate carcinoma cell lines (PC-3 and LNCaP) by a mechanism independent of de novo HSP70 synthesis or cell death. This pathway is similar to one used by the leaderless protein IL-1beta. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1beta secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system.     

Proteomics of MUC1‐containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF‐7

Apically expressed human MUC1 is known to become endocytosed and either to re‐enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi‐vesicular bodies and the release of exosomes. By using recombinant fusion‐tagged MUC1 as a bait protein we followed an anti‐myc affinity‐based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF‐7 breast cancer cells. MUC1⁺ lipid rafts were not only found to contain genuine raft proteins (flotillin‐1, prohibitin, G protein, annexin A2), but also raft‐associated proteins linking these to the cytoskeleton (ezrin/villin‐2, profilin II, HSP27, γ‐actin, β‐actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin‐dependent pathways and export via exosomes.     

Mechanisms of HSP72 release

Currently two mechanisms are recognized by which heat shock proteins (HSP) are released from cells; a passive release mechanism, including necrotic cell death, severe blunt trauma, surgery and following infection with lytic viruses, and an active release mechanism which involves the non classical protein release pathway. HSPs are released both as free HSP and within exosomes. This review covers recent findings on the mechanism by which stress induces the release of HSP72 into the circulation and the biological significance of circulating HSP72 to host defense against disease.     

Hsp72 release: mechanisms and methodologies

To date there are two mechanisms that are recognized by which heat shock proteins (HSP) are released from cells; a passive release mechanism, including necrotic cell death, severe blunt trauma, surgery and following infection with lytic viruses, and an active release mechanism which involves the non classical protein release pathway in which HSPs are released within highly immunologically potent exosomes and as free HSP. This chapter describes the experimental procedures that have been developed to study the mechanism by which stress induces the release of HSP72 into the circulation and addresses the biological significance of circulating HSP72 to host defense against disease.     

Heat shock protects HCT116 and H460 cells from TRAIL-induced apoptosis

Heat shock proteins have been shown to protect cells from a variety of stressful conditions, including hyperthermia, oxidative and DNA damage, serum withdrawal, and a variety of chemicals. HSP27, HSP70, and HSP90 have been shown to downregulate different aspects of apoptosome assembly. TRAIL is a member of the TNF family of ligands and is a promising anti-cancer agent. It has been shown to be nontoxic to most normal cell types, while it is a potent killer of many different cancer cells. TRAIL engages both the receptor-mediated (extrinsic) and the mitochondria-initiated (intrinsic) cascades. We tested whether heat shock affects TRAIL-induced apoptosis in different cancer cells. TRAIL treatment does not induce HSP27, HSP70, or HSP90 levels. Nonetheless, when treated with TRAIL for 3 h after release from heat shock, the human colon cancer cell line HCT116 is protected from apoptosis whereas the human colon cancer cell line SW480 is not. This pattern is consistent with the previously observed behavior of HCT116 as Type II cells that depend on mitochondrial signaling and SW480 as Type I, whose TRAIL-induced death is not sensitive to inhibition of caspase 9. Moreover, the failure of heat shock to protect SW480 cells is not due to a lack of HSP70 or HSP90 upregulation. HSP70 and HSP90 are induced 3 h after release from heat shock, whereas HSP27 is induced much later. Thus, the observed protective effect against TRAIL is probably due to the anti-apoptotic effects of HSP70 and HSP90. These results further illustrate interactions between TRAIL receptor signaling and the intrinsic cell death pathway and have practical implications for the potential use of TRAIL and hyperthermia in cancer therapy.     

HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway

The heat shock proteins (HSP) are a highly conserved family of proteins with critical functions in protein folding, protein trafficking, and cell signaling. These proteins also protect the cell against injury. HSP60 has been found in the extracellular space and has been identified in the plasma of some individuals. HSP60 is thought to be a "danger signal" to the immune system and is also highly immunogenic. Thus extracellular HSP60 is possibly toxic to the cell. The mechanism by which HSP60 is released into the extracellular space is unknown, as is whether it is released by cardiac myocytes. We investigated several different pathways controlling protein release including the classic, Golgi-mediated pathway. We found that HSP60 is released via exosomes, and that within the exosome, HSP60 is tightly attached to the exosome membrane.     

Cardioprotection mediated by exosomes is impaired in the setting of type II diabetes but can be rescued by the use of non‐diabetic exosomes in vitro

Many patients with ischaemic heart disease also have diabetes. As myocardial infarction is a major cause of mortality and morbidity in these patients, treatments that increase cell survival in response to ischaemia and reperfusion are needed. Exosomes—nano‐sized, lipid vesicles released from cells—can protect the hearts of non‐diabetic rats. We previously showed that exosomal HSP70 activates a cardioprotective signalling pathway in cardiomyocytes culminating in ERK1/2 and HSP27 phosphorylation. Here, we investigated whether the exosomal cardioprotective pathway remains intact in the setting of type II diabetes. Exosomes were isolated by differential centrifugation from non‐diabetic and type II diabetic patients, from non‐diabetic and Goto Kakizaki type II diabetic rats, and from normoglycaemic and hyperglycaemic endothelial cells. Exosome size and number were not significantly altered by diabetes. CD81 and HSP70 exosome markers were increased in diabetic rat exosomes. However, exosomes from diabetic rats no longer activated the ERK1/2 and HSP27 cardioprotective pathway and were no longer protective in a primary rat cardiomyocytes model of hypoxia and reoxygenation injury. Hyperglycaemic culture conditions were sufficient to impair protection by endothelial exosomes. Importantly, however, exosomes from non‐diabetic rats retained the ability to protect cardiomyocytes from diabetic rats. Exosomes from diabetic plasma have lost the ability to protect cardiomyocytes, but protection can be restored with exosomes from non‐diabetic plasma. These results support the concept that exosomes may be used to protect cardiomyocytes against ischaemia and reperfusion injury, even in the setting of type II diabetes.     

The human HSP70 family of chaperones: where do we stand?

The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as “chaperokines” or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5′-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target.     

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurrence of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-crystallin was observed. HSP32 represents heme oxygenase 1 (HO-1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress-induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO-1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase-1 has been linked to inflammatory processes and oxidative stress, its specific up-regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.     

Expression of heat shock proteins during development of barley

Barley heat shock proteins have been cloned, characterized by hybrid release translation and sequenced. Clones coding for proteins of 17, 18, 30, 32 and 70 kDa have been obtained. Out of these the 32 and 30 kDa proteins have been characterized as precursors to plastidic proteins of 26 kDa by posttranslational transport and by cDNA sequencing. The coding regions of these two transcribed genes are highly homologous. Accumulation of the plastid HSP as well as of HSP 70 as well as their corresponding mRNAs has been studied in 2- to 6-day old seedlings and in the 7-day old barley leaf. The mRNA for all investigated proteins were only found after a heat shock; the mRNA levels increase towards the tip of the leaf and with development. Furthermore, under the conditions used the mRNAs for all investigated heat shock proteins accumulate in parallel. Unexpectedly, both proteins, HSP 70 and HSP 26, are found by western blotting in the 2-day old control plants in the absence of any inducing heat shock. At later stages of development and in the leaf gradient only immunoreactivity with HSP 70 was observed. In contrast to the levels of their mRNAs the highest levels of HSP 30–26 and 70 have been observed in the basal segments indicating that translational control plays a role during HSP expression. Under severe heat shock a protein of 30 kDa is induced whose identity is not known but which reacts with the antibody to HSP 30–26 and might represent the accumulating precursors of the plastidic proteins.     

Degradation of AP2 during reticulocyte maturation enhances binding of hsc70 and Alix to a common site on TFR for sorting into exosomes

Reticulocytes release small membrane vesicles termed exosomes during their maturation in erythrocytes. The transferrin receptor (TfR) is completely lost from the red cell surface by its segregation in the secreted vesicles where it interacts with the heat shock cognate 70 kDa protein (hsc70). We have now determined a region of the TfR that can potentially interact with hsc70. The peptide P1 (YTRFSLARQV) from the TfR cytosolic domain: (i) binds to hsc70 (ii) with an increased affinity in oxidative conditions, (iii) competes for binding of an unfolded protein to hsc70, and (iv) inhibits the interaction of hsc70 with a recombinant protein corresponding to the cytosolic domain of the receptor. This peptide encompasses the internalization motif (YTRF) of the receptor, and accordingly an affinity column made with the immobilized peptide retains hsc70 and also the AP2 adaptor complex. On the other hand, we show that AP2 is degraded by the proteasome system during reticulocyte maturation and that the presence of the proteasome inhibitor during in vitro red cell maturation inhibits AP2 degradation and specifically decreases TfR secretion via exosomes. Finally, coimmunoprecipitation of Alix with the exosomal TfR, and binding of P1 peptide to the Alix homolog PalA suggest that Alix also interacts with the YTRF motif and contributes to exosomal TfR sorting.