Recent progress in the emerging role of exosome in hepatocellular carcinoma

Abstract Exosomes are small membrane vesicles 50‐150 nm in diameter released by a variety of cells, which contain miRNAs, mRNAs and proteins with the potential to regulate signalling pathways in recipient cells. Exosomes deliver nucleic acids and proteins to participate in orchestrating cell‐cell communication and microenvironment modulation. In this review, we summarize recent progress in our understanding of the role of exosomes in hepatocellular carcinoma (HCC). This review focuses on recent studies on HCC exosomes, considering biogenesis, cargo and their effects on the development and progression of HCC, including chemoresistance, epithelial‐mesenchymal transition, angiogenesis, metastasis and immune response. Finally, we discuss the clinical application of exosomes as a therapeutic agent for HCC.


| INTRODUC TI ON
Since the first observation of exosomes as "trash cans" that simply allows cells to dispose of unwanted proteins, 1 the further functions of exosomes have recently been explored. It has been proven that exosomes could be secreted by most cell types. 2 With regard to the liver, exosomes mainly released from three types of cells: hepatocytes, non-parenchymal immune cells (such as Kupffer cells, natural killer cells, T cells and B cells) and non-parenchymal liver cells (eg, liver stellate cells). 3 As for a subtype of the extracellular vesicle, they implicated in many normal and pathological processes. 4 Especially in tumours, they play a vital role in tumour chemoresistance, angiogenesis, epithelial-mesenchymal transition (EMT) and metastasis by modulating extracellular communication. On the one hand, tumour cells impact adjacent cells through exosomes and establish tumorigenic microenvironment. On the other hand, the stroma cells (such as stellate cells and MSCs) and immune cells could influence tumour cells to promote or prevent tumorigenesis through exosomes. 5 Importantly, the versatile roles of exosomes are mostly determined by their donor cells and their contents including lipids, nucleic acids and proteins 6,7 (Figure 1). The information has been deposited in ExoCarta (www.exocarta.org).
Furthermore, exosomes have the potential to be utilized in therapeutic tools due to their numerous characteristics, which we will discuss as follow.

| E XOSOME S B I OG ENE S IS
Recently, there has been a great interest in the study of exosomes as the major regulator in tumorigenesis. Based on recent studies, endosomal sorting complexes required for transport (ESCRT) is considered as the main mechanism of exosomes production 8 (Figure 2), which was first defined as a ubiquitin-dependent protein sorting pathway in yeast. 9 Vps4, one of the compositions of ESCRT complex, is known as a multimeric mechanoenzyme with an ATP-binding domain which binds to ESCRT-III subunits then provides energy through dehydrating ATP to disassociate them from the cell membrane. [10][11][12] Surprisingly, Wei et al 13 found that the downregulation of Vps4 is an independent risk factor for recurrence-free survival of hepatocellular carcinoma (HCC) patients. Their study showed that Vps4A is associated with inhibition of biological activity of HCC cell-derived exosomes and the recipient cells' response to exosomes. PI3K/Akt signalling pathway might be a candidate mechanism due to its inactivation occurrence while Vps4 overexpressed in HCC cells. 13 This study extends our knowledge that the exosomes production is associated with tumour progression, metastasis and worse prognosis.
Numerous studies demonstrated that exosomal cargo sorting is an active process. 9,14 The content of exosomes is determined by their donor cells. Up to now, a bunch of molecules have been found in exosomes such as heat shock proteins (eg, Hsp90 and Hsp70), 15,16 cytoskeletal proteins (actin, tubulin, cofilin, etc), lipids and enzymes, along with RNAs, including microRNAs, mRNAs, and other non-coding RNAs (ncRNAs), and mitochondrial DNA (mtDNA) and single strand DNA (ssDNA). 17

| E XOSOME CONTENTS
It has been reported that cancer cells produce and secrete an increased amount of exosomes as tumour-inducing agents compared to non-cancer cells. 18 Exosomes play a critical role in manipulating the microenvironment that favours cancer cells 19 by transferring oncogene, 20 inducing angiogenesis, 21 establishing pre-metastasis niche 22 and inducing EMT in recipient cells. 23 Importantly, it has been demonstrated that the functions of exosomes are mainly determined by their cargoes 24 which are different in various situations. F I G U R E 2 The ESCRT complex promotes the formation of exosomes. A, EXCRT-0 recognizes ubiquitinated cargo and then initiates the budding of exosomes. B, EXCRT-0 recruits EXCRT-I; then, EXCRT-II is recruited by EXCRT-I and may contribute to cargo clustering. C, EXCRT-III degrades EXCRT-0, EXCRT-I and EXCRT-II to promote the exosomes budding. This process is accompanied by the deubiquitinated of cargoes. D, EXCRT-III is disassembled by Vps-4, resulting in the exosomes budding These results indicated that there is a possibility to reveal the mechanisms that altered by exosomes uptake. 25 Thus, we summarize the molecules found in exosomes in patients with HCC including proteins and RNAs (miRNA, lncRNA), and the purpose is to clarify the mechanism by which exosomes promote HCC progression.

| Proteins
According to Vesiclepedia database, the number of proteins in exosomes is at ~1800 levels, and in HCC cell line-derived exosomes, 213 unique proteins were found by mass spectrometry analysis. 26 Exosomal proteins include cargo proteins and membrane proteins, depending on location in exosomes. Membrane proteins are associated with exosomal internalization by recipient cells and target organ selection. Cargo proteins composition is different in exosomes during tumour progression in different cells. 27

| Nucleic acids
Considering that liver biopsy, a gold-standard method for monitoring and evaluating liver disease, has the risk of bleeding and infection, noninvasive diagnostic tools are urgently needed. 18 Thus, a "Liquid biopsy" which implements early diagnosis and prognostic prediction of HCC through serum exosomes becomes more attractive. 18,28 However, "Liquid biopsy" is based on markers for HCC development and progression. In addition to protein, it has been demonstrated that nucleic acids, particularly miRNAs, are also one of the compositions of exosomes 29 (Table 1) Sugimachi et al have shown that miR-718 can serve as a preoperative biomarker for the prediction of HCC recurrence after surgery. Their study showed that the expression level of miR-718 in exosomes collected in patients with HCC recurrence after liver transplantation was significantly lower than those without HCC recurrence. Furthermore, a validated cohort study showed that decreased expression of miR-718 and overexpression of the potential target gene HOXB8 were associated with tumour aggressiveness and poor prognosis. 32 These results show the potent value of selecting patients who need liver transplantation, and therefore use donor organs properly. In addition, Liu et al 33 have reported that exosomal miR-125b could serve as a prognostic marker due to miR-125b level in exosomes was an independent factor for time to recurrence and overall survival of HCC patients.
Although exosomal miRNAs might be useful tools to reflect their donor cells feature that can be used as biomarkers for tumour cell, the extent to which exosomal miRNAs play a role in HCC remains poorly understood. Furthermore, there are controversial results of miRNA expression level and functions under specific conditions, and some cohort studies did not include healthy participants, due to the conveniences of collecting serum sample from patients with liver disease compared with healthy people. 31,[33][34][35][36][37][38][39][40][41][42] Recently, increased studies have focused on a role of long noncoding RNA in exosome in addition to miRNA. Long non-coding RNAs (lncRNAs) are defined as non-coding RNAs more than 200 nucleotides in length. [43][44][45][46] Lnc-ROR and lnc-LVDR which expressed in HCC-derived exosome had widely explored. [47][48][49] It has recently been found that the ultraconserved lncRNA (ucRNA) expression is dramatically altered within extracellular vesicles as compared to donor cells. 50,51 For instance, the ucRNA named TUC339 is mostly enriched in HCC cell-derived exosomes and promotes HCC growth and spread. Above all, these studies explored the nucleic acids that transferred within cells via exosome that modulate tumour cells and function as an intracellular signalling mediators.

| MECHANIS MS OF INTER AC TION B E T WEEN E XOSOME S AND RECIPIENT CELL S
Recently, dynamic regulation of exosomes uptake by recipient cells extensively explored. There are several models considered as a possible mechanism of exosomes internalization by recipient cells, the receptor-mediated endocytosis, and classic fluid-phase endocytosis 52 ( Figure 3). The latter one is considered to be a common approach for microvesicle internalization that lacks the specificity. However, Schneider et al 53 documented that the mechanism of exosomes update by alveolar epithelial cells is similar, but not same, to classic macropinocytosis depending on dynamin function and actin polymerization.
In contrast, receptor-mediated endocytosis attracted more interest for its cell-specific feature that allows further modifications of exosomes for therapeutic use. 53 Integrins are one of the receptors commonly expressed on exosomes membrane. It has been found that exosomal integrins have the ability to predict metastatic organ.
For instance, exosomes expressing ITGα v β 5 specifically bind to Kupffer cells, mediating liver tropism whereas exosomal ITGα 6 β 4 and ITGα 6 β 1 bind lung-resident fibroblasts and epithelial cells governing lung tropism. 54 Thus, targeting exosomal integrins has a potential to prevent tumour metastasis. 54 Furthermore, the blockade of Scavenger Receptor Class A family (SR-A), a novel monocyte/macrophage uptake receptor for exosomes, with dextran sulphate in vivo enhances tumour accumulation by reducing exosomes clearance in mice liver. 55  Furthermore, an oncogenic transformation of the recipient cells was observed following exposure of exosomes isolated from serum of cancer patients. 57 This phenomenon has a synergy when combined with mutations in tumour suppression gene in recipient cells. 57,58 Collectively, these results indicate a hypothesis that the migration of cancer cells might not be necessary for metastasis and that this can be achieved by exosomal transport.

| THE ROLE S OF E XOSOME S IN H CC PROG RE SS I ON
Intercellular communication is essential in liver physiology and pathology including tumorigenesis since liver is a multicellular organ.   30 In this part, we summarize the recent studies on the progress of HCC involving exosomes.

| Exosomes participate in HCC chemoresistance
Sorafenib is the first-line molecular targeted drug for advanced HCC approved by US Food and Drug Administration. However, after long-term treatment of sorafenib, HCC cells exhibit resistance to sorafenib. 62 Accumulating evidence has shown that exosomes are involved in HCC chemoresistance as well. Here, we summarize several possible mechanisms related to exosomes. stress through p53 dependent manner. 48 These results indicate that exosomal cargoes participate in chemical therapeutic response modulation and provide therapeutic targets that enhance the chemosensitivity of HCC cells.

| Exosomes modulate epithelial-mesenchymal transition of HCC cells
Epithelial-mesenchymal transition (EMT) is an initial step in cancer distance metastasis. [73][74][75] EMT defined as a process by which cell lose epithelial markers like E-cadherin and acquire mesenchymal cell hallmarks like N-cadherin. 76,77 EMT and the reverse process MET are the basis of the complex three-dimensional structure of the internal organs. 27 However, tumour cells achieve mobility and invasiveness through the EMT process, leading to cancer metastasis. 73 Taken together, these results support the notion that exosomes participate in EMT that associated with aggressive, invasive and metastatic potential in cancer cells. However, more research is needed to better understand the exact mechanism by which exosomes modulate EMT in HCC.

| Exosomes promote angiogenesis in HCC tissue
It has been demonstrated that cancer cells undergoing EMT capable of efficiently transferring angiogenetic proteins to the recipient endothelial cell via exosomes. 87 In addition, secretion of exosomes

| Exosomes promote HCC metastasis
Long-term survival rate is low in patients with HCC due to the high metastases and/or high post-surgical recurrence rate. 98 Tumour metastasis is a multistep process that includes invasion, intravasation and colonization of distal sites through the circulatory system. 99 EMT, the initial step of metastasis, has been described above.
It has been found that exosomes facilitate the pre-metastatic niche formation and metastasis, whether derived from cancer cells or adjacent stromal cells. 26

| Exosomes trigger immune responses
Immune tolerance, the unique immune microenvironment of the liver, is the main obstacle to immunotherapy for treating HCC. 4 It is paradoxical that exosomes trigger immune response. On the one hand, exosomes are found in a variety of known immunosuppressive mechanisms, such as activation of immune suppressor cells, antigen presentation defects and induction of T-cell apoptosis. 107,108 On the other hand, exosomes are a key source of tumour antigens exposed by tumour cells and immune cells. 109 For example, Lv et al 15  These findings demonstrated that exosomes not only present TAA from tumour cells to APCs but also are capable of presenting them to T lymphocytes that elicit an antigen-mediated anti-tumour immune response. This greatly promotes the development of HCC immunotherapy by providing cell-free vaccines.

| Exosomes are a promising agent for anticancer therapy
Cell membrane-derived nanoparticles have many properties, such as protecting their cargo, low immunogenicity and proper size through the endothelium, 88 which can be used as drug delivery agents. [111][112][113][114] For example, Lou et al 112  and MADS-box transcription factor SRF 113,114 and is correlated with poor prognosis and metastasis in human HCC patient. 113,115 MSCs are widely used due to they are the most prolific producer of exosomes among the cell types. 116 In addition to adipose-derived MSCs, 117 the bone marrow-derived exosomes are commonly used in stem cell-based therapies. 86 Furthermore it has been reported that the DC-derived exosomes are used as cancer vaccines. 110,118 Tian et al 111 suggested that it may have a potential value for clinic application that modifying exosomes by targeting ligands which used for a drug delivery vesicle. For instance, modification of exosomes membrane with Arg-Gly-Asp (RGD) peptide elicits blood vessel targeting effect, which may be a new strategy for therapeutic angiogenesis. 119 Meanwhile, exosomes have been reported to be involved in chemodrug resistance, and serveral studies indicated that inhibition of exosomes secretion has been shown to be effective in sensitize cancer cells to therapeutic drugs. 8,120 Overall, exosomes are promising agents for HCC treatment therapy.

| CON CLUS ION
Exosomes in cancer include HCC is a research hot spot over the past few years. In this review, the aim was to better understand the exosomes in HCC development. To the best of our knowledge, exosomes promote HCC progression by regulating multiple tumorigenic processes, including chemoresistance, EMT, angiogenesis, metastasis and immune response. An implication of this is the possibility that exosomes may be promising candidates for the treatment of HCC. It had been found that exosomes have several advantages as a drug delivery agent in the treatment of HCC. These findings had offered a framework for the exploration of new therapeutic tools for HCC. However, research is limited by the lack of information on the clinical safety and efficacy of exosomes. Therefor, further studies are still required to better understand the relationship between exosomes and HCC development.

ACK N OWLED G EM ENTS
The work was supported by grants from the National Natural Science Foundation of China (No.81772995 and 81472266) and the Excellent Youth Foundation of Jiangsu Province (BK20140032). We apologize to all colleagues whose relevant contributions could not be cited due to space limitations.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.