传染病学传染病学 (11).pdf
O role of extracellular vesicles in mediating progression,metastasis and potential treatment of hepatocellular carcinomaNaibin Yang1,Shanshan Li2,Guoxiang Li1,Shengguo Zhang2,Xinyue Tang2,Shunlan Ni2,Xiaomin Jian3,Cunlai Xu4,Jiayin Zhu5 and Mingqin Lu21 Department of Infection and Liver Diseases,Ningbo First Hospital,Ningbo,China2 Department of Infection and Liver Diseases,The First Affiliated Hospital of Wenzhou Medical University,Institute of Liver Research,Wenzhou Medical University,Wenzhou,China3 Department of The First Clinical Medical,Wenzhou Medical University,Wenzhou,China4 Department of Respiration,Lishui Peoples Hospital of Wenzhou Medical University,Lishui,China5 Laboratory Animal Center,Wenzhou Medical University,Wenzhou,ChinaCorrespondence to:Mingqin Lu,email:Keywords:extracellular vesicles,exosomes,miRNA,hepatocellular carcinoma,MSCsReceived:April 28,2016 Accepted:September 28,2016 Published:October 04,2016ABSTRACTHepatocellular carcinoma(HCC)is a major cause of cancer-related death worldwide.As vectors for intercellular information exchange,the potential role of extracellular vesicles(EVs)in HCC formation,progression and therapy has been widely investigated.In this review,we explore the current status of the researches in this field.Altogether there is undeniable evidence that EVs play a crucial role in HCC development,metastasis.Moreover,EVs have shown great potential as drug delivery systems(DDSs)for the treatment of HCC.Exosomal miRNAs derived from HCC cells can enhance transformed cell growth in recipient cells by modulating the expression of transforming growth factor-activated kinase-1(TAK1)and downstream signaling molecules.Furthermore,vacuolar protein sortin 4 homolog A(VPS4A)and insulin-like growth factor(IGF)-1 regulate exosome-mediated miRNAs transfer.Immune cells-derived EVs containing integrin M2 or CD147 may facilitate HCC metastasis.In addition,EVs-mediated shuttle of long non-coding RNAs(lncRNAs),specifically linc-VLDLR and linc-ROR promote chemoresistance of malignant cells.Heat shock proteins(HSPs)-harboring exosomes derived from HCC tumor cells increase the antitumor effect of natural killer(NK)cells,thus enhancing HCC immunotherapy.Indeed,inhibition of HCC tumor growth has been associated with tumor cell-derived exosomes(TEX)-pulsed dentritic cells(DCs).Exosomes are also essential in liver metastasis during colorectal carcinoma(CRC)and pancreatic ductal adenocarcinomas(PDAC).Therefore,as nucleic acid and drug delivery vehicles,EVs show a tremendous potential for effective treatment against HCC.INTRODUCTIONHepatocellular carcinoma(HCC)is the sixth most common incident cancer worldwide and the third leading cause of cancer death annually 1,2.In China,HCC is one out of the four leading causes of cancer-related death 3.The development of primary liver tumors,including HCC and cholangiocarcinoma are associated with hepatocyte damage.Large-scales studies have thoroughly described not only the pathogenesis of HCC development and metastasis 4-6 but also the multiple treatment options for HCC treatment,including surgical resection,orthotopic liver transplantation(OLT),transcatheter arterial chemoembolization(TACE),systemic or regional chemotherapy,and targeted immunotherapy 7,8.Recently,the emerging role of extracellular vesicles(EVs)in HCC progression and therapy attracted considerable attention.ReviewO EVs-including exosomes and microvesicles(MVs)-were previously considered as cellular debris,they are currently well-recognized vectors for intercellular exchange of information 9.EVs mediate intercellular communication by transferring biologically and functionally active proteins and RNA across cells.Exosomes with 30-120 nm diameter are formed by release of intracellular multivesicular bodies,whereas MVs with a 120-1000 nm diameter,are formed by cell membrane shedding 10.Many cells,including neurons 11,dendritic cells(DCs)12,B cells,T cells,hepatocytes 13,stem cells 14,erythrocytes 15,mast cells,epithelial cells 16,tumor cells 17,along with some multicellular parasites 18-20,have the capacity to release exosomes.Exosomes can be usually found in biological fluids such as blood,urine and ascitic fluid 21-23 and they are usually detected using presence of CD63,tumor susceptibility gene(TSG)-101,alix and absence of endoplasmic reticulum marker Grp94 and calnexin,peroxisome marker protein(PMP)70,mitochondria marker COX IV 24-27.Exosomes have been described as a means of communication between tumor cells 28.Dysregulation in this cell-to-cell communication and undesirable cellular cross-talks are considered to contribute to cancer development and progression.A growing body of evidence already described that uptake of exosomes stuffed with proteins,mRNAs,miRNAs and lipids could deliver biological information that regulate the function of target cells 17,29.This mechanism may explain how exosomes mediate tumor progression and metastasis.Exosomes interact with their target cells mainly via fusion of membranes and transfer of exosomal contents,especially miRNAs.HCC tumor cell-derived EVs have been reported to potentially contribute to local spread,intrahepatic metastases and multifocal growth of HCC 28.EVs-mediated intercellular transfer of biologically active RNA and proteins might enable HCC cells to affect the tumor microenvironment,thereby causing HCC development and metastasis 30.Experimental and clinical studies have elucidated the role of EVs in HCC development and metastasis,in order to be employed in future novel therapies against HCC including immunotherapy and chemotherapy,as biomarkers or as drug delivery systems(DDSs).Among these studies,human HCC cell lines Huh-7,HepG2,Hep3G,Hep3B,PLC/PRF/5,SMMC-7721,HKCI-8,FHCC-98,HKCI-C3,MHCC97L and mouse HCC cell lines including H22 were used to investigate the role of EVs in HCC cells in vitro.Accumulating evidence indicate that EVs are involved in tumor progression,metastasis,and treatment failure,thus showing great potential as DDSs for the treatment of HCC.In the present article,current studies investigating the mechanism on the contribution of EVs to HCC development,progression,metastasis and treatment are reviewed.CONTENTS AND FUNCTIONS OF HCC-DERIVED EVSComponents and tumorigenic mechanism of HCC-derived exosomes To rapidly and efficiently extract exosomes secreted by tumor cells,different methods were explored 31-34.Among them,sequential ultracentrifugation is the method of choice to isolate exosomes from culture supernatant of HCC cells in a consistent manner.The RNA expression profile of exosomes derived from human HCC cell lines Hep3B and PLC/PRF/5 was investigated 28.Interestingly,the exosomal was below 200 bases in size(mainly miRNAs)with a very low fraction of internal control genes,including 18S ribosomal RNA(rRNA),28S rRNA,small nuclear RNA(snRNA)U6,small nucleolar RNA(snoRNA)U38B and snoRNA U43 28.The expression of 11 miRNAs(miR-584,miR-517c,miR-378,miR-520f,miR-142-5p,miR-451,miR-518d,miR-215,miR-376a,miR-133b and miR-367)was specifically detected in Hep3B-derived exosomes,indicating selective enrichment of a specific set of miRNAs in HCC-derived exosomes 28.Similarly,a total of 20 miRNAs were detected exclusively in PLC/PRF/5-derived exosomes.There was a moderate correlation between the findings in both cell lines,indicating the existence of a sorting mechanism that guides HCC cells to secrete specific intracellular miRNAs into exosomes.The incubation of HCC cells with a neutral sphingomyelinase 2(nSMase)inhibitor GW4869 resulted in an unchanged cellular expression and reduced exosomal expression of miR-16,indicating that the release of specific miRNAs from HCC cells into exosomes might occur via a ceramide dependent manner 28,35.Interestingly,some of these miRNAs(e.g.miR-451)were also found to preferentially enter exosomes in many other cell types 36.HCC-derived exosomes mediated miRNA transfer is an important mechanism of environmental modulation of HCC growth and progress 28.While being taken up and internalized,HCC-derived exosomes transfer their miRNAs contents into recipient cells to mediate transmission of functional transgenes and genetic modulation of cellular activities.The transfer of exosomal miRNAs regulates target gene expression,cell signaling,biological behavior and transformation of recipient cells.A combinatorial analysis on 108 potential genes identified that the transforming growth factor-activated kinase-1(TAK1)pathway might be a very likely candidate pathway targeted by these miRNAs 28.TAK1 has been extensively associated with the activation of signaling cascades mediated by interleukin(IL)-1,tumor necrosis factor(TNF-)and transforming growth factor(TGF)-37.It is an upstream member of the mitogen-activated O and an essential component of cellular homeostasis,intercellular communication and tumorigenesis in the liver.Loss or downregulation of TAK1 in hepatocytes is linked to HCC 38.The modulation of TAK1 expression and associated signaling pathways in recipient cells could represent an important mechanism of exosomal miRNA mediated HCC tumor progression(Figure 1).HCC-derived exosomes can transfer their miRNA contents into recipient cells,inhibit the constitutive expression of TAK1 and downstream signaling associated with TAK1,and consequently lead to HCC development and metastasis.In this direction exosomes derived from Hep3B cells are able to both increase anchorage-independent growth of transformed cell and modestly reduce cell viability of recipient cells 28.Some exosomal miRNAs and long non-coding RNAs(lncRNAs)are involved in HCC progression and treatment failure.For the first time,Li et al.reported that enhanced expression of miR-429 in liver tissue-caused by hypomethylation may be used as a prognosis factor in HCC patients 39.The enrichment of miR-429 in HCC cells,especially in epithelial cell adhesion molecule(EPCAM)+-tumor initiating cells(T-ICs)could lead to shedding of miR-429-harboring exosomes,thus facilitating tumor formation.These exosomes could shuttle and relocate miR-429 into their surrounded target cells,targeting Rb binding protein 4(RBBP4)expression and further promoting the transcriptional activity of E2F1,finally upregulating the expression of POU class 5 homeobox 1(POU5F1)39 in recipient cells.As a result,exosomes crucially contribute to hepatocyte self-renewal,tumorigenicity,malignant proliferation,chemoresistance and progression.Furthermore,exosomal transfer of the lncRNA regulator of reprogramming(linc-RoR)may contribute to failure of anticancer therapies and HCC development by increasing HCC resistance against adverse environmental conditions,such as hypoxia 40.The levels of linc-RoR in normal hepatocytes are low 40 potentially inhibiting cell proliferation and tumorigenesis,Figure 1:HCC-derived exosomal miRNAs may mediate tumor progression through modulating the TAK1-associated signaling pathway in recipient cells.TAK1 is involved in the activation of signaling cascades mediated by IL-1,TNF-,TGF-in physiological conditions(black arrows).After uptake of HCC-derived exosomes by neighboring cells present in the tumor microenvironment,the given exosomal miRNAs can down-regulate the constitutive expression of TAK1,TAB 2/3 in recipient cells and restrain the phosphorylation of TAB1,TAB2/3 and TAK1.Activation of their three main downstream signaling pathways,including IKK/-NF-kB,MKK4/7-JNK-API and MKK3/6-p38/MAPK-CREB is then suppressed.The pathological unbalance finally causes failure of cellular homeostasis,leading to tumorigenesis in the liver and HCC tumor progression.(Abbreviation:TAB,TAK-1 binding protein;IKK,inhibitor of nuclear factor kappa-B kinase;MKK,MAPK kinase;CREB,cAMP response element binding protein).O part,by specifically increasing stability of the c-myc mRNA 41.High expression of linc-RoR in HCC decreases the expression of miR-145,a linc-RoR target,thereby increasing hypoxia inducible factor-1(HIF-1)and pyruvate dehydrogenase kinase isozyme 1(PDK1)protein expression,overall improving mitochondrial function during hypoxia via the tricarboxylic acid cycle in recipient cells.Similarly,Takayuki and collaborators demonstrated that the most highly expressed lncRNA in HCC cell-derived EVs was TUC339.Suppression of TUC339 with short interfering RNA(siRNA)significantly reduced HCC cell proliferation and adhesion.Therefore,EVs-mediated transfer of lncRNA-TUC339 is a unique signaling mechanism to promote HCC growth and metastasis 42.HCC suppressors/promoters exert effects by exosome-mediated miRNAs shuttle Based on the evidence that vacuolar protein sorting 4 homolog A(Vps4A)is frequently down-regulated in human HCC tissue and that Vps4A represses the colony formation,migration,growth and invasion of HCC cells in vitro,Vps4A was identified as a HCC suppressor 43.Vps4A utilized exosomes as mediators to modulate secretion,uptake and final profiles of miRNAs in HCC cells 43.In this study,Wei and colleagues compared SMMC-7721 cells transfected with either Vps4A(SMMC-Vps4A)or exosomes secreted by SMMC-Vps4A(SMMC-Vps4A-exo).They observed that Vps4A suppressed the bioactivity of exosomes via selectively packaging oncogenic miR-27b-3p and miR-92a-3p into exosomes and accumulating tumor-suppressive miR-193a-3p,miR-320a,and miR-132-3p in HCC cells.Moreover,they demonstrated that Vps4A decreased the recipient HCC cell response to exosomes via selective uptake of exosomal tumor-suppressive miR-122-5p,miR-33a-5p,miR-34a-5p,miR-193a-3p,miR-16-5p,and miR-29b-3p.However,insulin-like growth factor-1(IGF-1)is considered as a HCC promoter since it can override homeostasis and lead to tumor progression during the initial steps of HCC development 44.Expression of tumor suppressor miR-122,a liver-specific anti-proliferative miRNA,is usually down-regulated in HCC cells compared with that in normal hepatocytes surrounding the tumor 45.Transfer of exosomal miR-122 from healthy hepatocytes inhibits tumor progression.However,this method for the maintainance of homeostasis cannot be kept for a long time.T-ICs subsequently release IGF-1 to prevent miR-122 production in neighbouring normal hepatocytes and thereby curtail its intercellular transfer within exosomes,leading to low levels of the anti-proliferative miRNA in HCC cells.Thus eventually tumor progression and metastasis occurs 46.ROLE OF EVS IN HCC METASTASIS,CHEMORESISTANCE AND POTENTIAL IMMUNOTHERAPYImmune cells-derived EVs facilitate HCC metastasis Immune cells such as immature myeloid cells,macrophages,and mast cells are considered the roots of metastasis of tumor cells 47,48.In a recent study 49 murine innate immune cells-derived microparticles(MPs)were co-cultured with H22 tumor cells,leading to tumor cell migration,invasion,attachment to the endothelium in vitro and metastasis in vivo.Indeed,MPs mediate the acquisition of a metastatic phenotype by HCC cells via the effective relay of integrin M2(CD11b/CD18)from stimulated innate immune cells.Th
