Biological functions and clinical applications of exosomal long non‐coding RNAs in cancer

Abstract Exosomes are extracellular vesicles secreted by donor cells, and one of the important roles of exosomes is intercellular communication. Exosomes contain proteins, lipids, DNA and RNA. The components exert their functions by modulating the cellular processes of recipient cells. Exosomal long non‐coding RNAs (lncRNAs) are important components and play multiple roles in tumorigenesis and tumour development. In this review, we summarize the biological functions and clinical applications of exosomal lncRNAs in cancer. Exosomal lncRNAs regulate cell proliferation, metastasis, drug resistance and angiogenesis in human cancers. Since exosomal lncRNAs are associated with clinicopathological characteristics of cancer, these might be potentially useful biomarkers for diagnosis and prognosis of cancer. Exosomal lncRNAs participate in multiple processes of cancer progression, which makes them promising therapeutic targets for cancer treatment.


| INTRODUC TI ON
Exosomes are microvesicles that are derived from multivesicular bodies (MVBs) and released into the extracellular space upon fusion of MVBs with the plasma membrane. 1 Exosomes contain multiple components including lipids, proteins, RNA and DNA. Exosomes take part in the intercellular communication by transferring cargoes from donor cells to recipient cells.
One of the cargoes of exosomes is long non-coding RNA (lncRNA). LncRNAs are RNA transcripts longer than 200 nt and have limited protein-coding potential. 2 LncRNAs are involved in numerous cellular processes. LncRNAs participate in the pathogenesis of many diseases, including cancer. 3 Lots of studies have demonstrated that lncRNAs regulate the malignant characteristics of cancer such as metastasis and drug resistance.
Exosomal lncRNAs are RNA molecules, and exosomal lncRNAs acquired by recipient cells will exert their cancer-related roles in the recipient cells to regulate cancer progression. In this review, we summarize recent research regarding exosomal lncRNAs in cancers. We describe the biological roles of exosomal lncRNAs in cancer and discuss the potential clinical applications of exosomal lncRNAs in the future.

| E XOSOME S
Exosomes are extracellular vesicles with a diameter of 30-100 nm and are released by multiple types of cells. [4][5][6] In the 1980s, exosomes were observed during reticulocyte maturation. 7,8 The production of exosomes begins with a process called endocytosis. 9 Exosomes are derived from inward budding of the plasma membrane. The inward budding of the plasma membrane forms an endosome. Further inward budding of the membrane results in the formation of intraluminal vesicles (ILVs) inside the MVB. Then, the MVB fuses with the plasma membrane and releases the ILVs called exosomes to the extracellular milieu ( Figure 1).
Various factors take part in the formation of exosomes, such as proteins and lncRNAs. 10,11 Rab GTPases regulate the biogenesis and secretion of exosomes. 12 Rab5b plays a role in the motility and fusion of early endosomes. 13 Rab35 regulates MVB transport and controls the docking process. Rab35 depletion increases intracellular accumulation of endosomal vesicles and decreases exosome secretion. 14 Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are trans-membrane proteins and SNARE complexes mediate membrane fusion and regulate the release of exosomes.

Ternary SNARE complexes consist of a SNARE on vesicle membrane
(v-SNARE) and two SNAREs on target membrane (t-SNARE). 15,16 Synaptosomal-associated protein (SNAP) such as SNAP23 is t-SNAREs and vesicle-associated membrane protein (VAMP) such as VAMP3 and VAMP8 are v-SNAREs. [17][18][19] The phosphorylation of SNAP23 enhanced the stability of the SNARE complex and promoted the secretion of exosomes. 20,21 LncRNA-APC1 regulates the production of exosomes by interacting with Rab5b mRNA. 22 The interplay of lncRNA-APC and Rab5b mRNA reduces the stability of Rab5b mRNA and inhibits Rab5b expression, leading to a reduction in exosomes. On the contrary, HOTAIR enhances the release of exosomes by modulation of several processes. 23 It regulates the docking process by modulating Rab35 expression and localization. In addition, HOTAIR facilitates the fusion process by controlling the colocalization of VAMP3 and SNAP23. HOTAIR also enhances the release of exosomes via phosphorylation of SNAP23.
Exosomes contain multiple bioactive molecules, including lipids, proteins, RNA and DNA. [24][25][26][27] The components of plasma membranes such as cholesterol, sphingomyelin, hexosylceramides, phosphatidylserine and saturated fatty acids are also present in the exosomes. 28 Rab GTPases and annexins, the proteins associated with membrane transport and fusion, are found abundantly in the exosomes. ESCRT components, ALIX and TSG101 are consistently detected in exosomes. Moreover, exosomes are enriched in heat-shock proteins, HSP70 and HSP90; tetraspanins, including CD9, CD63, CD81 and CD82; MHC class II proteins; members of the human epidermal receptor family; and epithelial cell adhesion molecules. [29][30][31][32][33] LncRNAs lncARSR and LNMAT2 have been reported in the exosomes derived from cancer cells. 34,35 Various investigations have revealed the presence of miRNAs, such as miR-21 and miR-221 in the exosomes. 36,37 Single-stranded DNA and double-stranded DNA are also found in the exosomes. 38,39 Earlier, exosomes were regarded as cellular garbage bags with non-functional cellular molecules or excess constituents. Emerging studies demonstrated the various functional roles of exosomes in physiological and pathological processes. 40,41 As exosomes released from donor cells are accepted by recipient cells, one of the most important roles of exosomes is intercellular communication. Exosomes contain multiple biological active molecules and the message included in cargoes was transferred from one cell to another. Exosomal components, such as proteins, miRNAs and lncRNAs regulate the biological processes of recipient cells.
The cargoes inside the exosomes reflect the pathophysiological state of the donor cells. Exosomal constitution may vary depending on the donor cells. 25,27,42,43 Moreover, exosomes are detected in multiple physiological fluids, including plasma or serum, saliva, amniotic fluid, breast milk, urine, nasal secretion, cerebrospinal fluid, semen LncRNAs also play important roles in disease occurrence and tumour progression. They are found to regulate cell proliferation, migration and invasion of cancer cells, and modulate cell cycle, apoptosis, lymphatic metastasis and drug sensitivity. 58-61 VEGF-C is a critical factor in lymphatic metastasis of human cancers. 62,63 It is overexpressed in many types of cancers and associated with regional lymph node metastasis and poor survival. LncRNA BLACAT2 has been reported to promote lymphatic metastasis of bladder cancer via regulating VEGF-C expression. 64 WDR5 is a core subunit of H3K4 methyltransferase complexes and a binding protein of BLACAT2.
BLACAT2 interacts with WDR5 and enhances the H3K4me3 levels of VEGF-C promoter thereby up-regulating VEGF-C expression.
Another lncRNA in bladder cancer, LNMAT1, promotes lymphatic metastasis through modulation of tumour microenvironment. 65 LNMAT1 overexpression is seen in bladder cancer with lymph node metastasis and associated with poor survival. In addition, LNMAT1 can recruit hnRNPL to CCL2 promoter and up-regulate the levels

| E XOSOMAL LN CRNA S IN C AN CER-B IOLOG IC AL FUN C TIONS
LncRNAs play important roles in cancer progression through multiple mechanisms. Exosomal lncRNAs are transferred to recipient cells and exert oncogenic roles or tumour suppressive roles in the recipient cells (Table 1). 67-71  73,74 In multiple myeloma, exosomal LINC00461 has been reported to enhance cell proliferation via binding to miR-15a/16 and promoting the expression of BCL-2. 75 Similarly, in oesophageal squamous cell carcinoma (ESCC) cells, exosomal lncRNA PCAT1 promotes cell proliferation through binding to miR-326. 76 Exosomal ZFAS1 also promotes the cell proliferation of ESCC cells by regulating the miR-124/STAT3 axis. 77

| Metastasis
Metastasis is one of the major malignant characteristics of cancer, and patients with metastatic cancer have a poor prognosis.
Lymphangiogenesis and epithelial-mesenchymal transition (EMT) facilitate the process of cancer metastasis.

| Chemoresistance
Drug resistance is a big challenge in cancer treatment and understanding the underlying mechanism of drug resistance will provide therapeutic strategy for cancer patients with drug resistance. Exosomal lncRNAs were reported to take part in drug resistance ( Figure 3).
Sunitinib is an oral drug used for treating advanced renal cell carcinoma (RCC). Most of the patients receiving sunitinib therapy develop drug resistance after 6-15 months of treatment. It is reported that exosomal lncRNAs are involved in the modulation of drug resistance. LncARSR regulates the expression of AXL and c-MET by binding to miR-34/miR-449 and thereby promotes sunitinib resistance in RCC cells. 34 Exosomal lncARSR from sunitinib-resistant RCC cells was found to induce drug resistance in previously sensitive cells.
Gefitinib is a type of tyrosine kinase inhibitor that blocks epidermal growth factor receptor tyrosine kinase (EGFR-TK). In NSCLC, overexpressed lncRNA UCA1 was reported to be responsible for gefitinib resistance. 83  Cisplatin is an extensively used chemotherapeutic drug for many cancers, such as ovarian cancer, gastric cancer and cervical cancer.
In ovarian cancer, exosomal UCA1 promotes cisplatin resistance by modulating the expression of FOSL2. 89 Exosomal HOTTIP regulates the expression of HMGA1 through binding to miR-218, thereby promoting cisplatin resistance in gastric cancer. 90 Additional, HNF1A-AS1 binds to miR-34b to up-regulate TUFT1 expression and subsequently promotes cisplatin resistance in cervical cancer. 91 In general, lncRNAs serve as ceRNAs to interact with miRNAs and then protect the target genes of miRNAs, that are considered vital in drug resistance. As a result, these lncRNAs are incorporated into the exosomes which then induce drug resistance in previously sensitive cells.

| Angiogenesis
Exosomal RAMP-AS1 is up-regulated in chondrosarcoma patients and high levels of RAMP-AS1 are often associated with poor survival. RAMP-AS1 acts as a ceRNA to bind to miR-2355-5p and enhances the expression of VEGFR2, an important factor in angiogenesis. 92 Cervical cancer cells secrete exosomes containing lncRNA TUG1.

Researchers observed that when exosomal TUG1 was transferred to the human umbilical vein endothelial cells (HUVECs), it promoted
HUVECs proliferation via inhibiting caspase-3 activity and regulating apoptosis-related proteins. Thus, it was suggested that exosomal TUG1 plays a role in angiogenesis. 93 GAS5 is down-regulated in lung cancer tissues. The exosomal GAS5 binds to miR-29-3p and increases the expression of PTEN.
Additionally, GAS5 decreases the phosphorylation of AKT and PI3K.
To summarize, exosomal GAS5 represses HUVECs proliferation and enhances their apoptosis, thus, inhibiting angiogenesis in lung cancer. 94 In glioma, exosomal lncRNA CCAT2 transferred to HUVECs is shown to activate VEGFA and TGFβ. CCAT2 increased BCL-2 expression while suppressed the expression of BAx and caspase-3, which inhibited apoptosis. Overall, exosomal CCAT2 promotes angiogenesis. 95 Another report found that gliomas can induce angiogenesis via secreting exosomes enriched in LINC-POU3F3. 96

| E XOSOMAL LN CRNA S IN C AN CER-CLINI C AL APPLI C ATI ON S
The    35 With the development on the investigation of promising ln-cRNA targets, the effective delivery of oligonucleotides targeting lncRNAs to the tumour cells is a key step in cancer treatment.

| Therapeutic target
There are several oligonucleotides delivery strategies, including lipid-based carrier, polyethylenimine-based carrier, protamine delivery, antibody-mediated targeted delivery. [105][106][107] Mice treated with pre-miRNA mixed with liposomes had a higher expression of miRNA than the control ones. 108 Protamine-condensed siRNAs reduced the expression of cyclin D1 in vitro and in vivo. 109 Effective delivery of oligonucleotides will accelerate the clinical application of the exosomal lncRNAs.

ACK N OWLED G EM ENTS
We would like to apologize to those researchers whose related work we were not able to cite in this review. This work was supported by a special programme from the Chinese National Natural Science Funds (31871405 and 31571460 to FZ), Jiangsu National Science Foundation (BK20180043 and 19KJA550003 to FZ).

CO N FLI C T S O F I NTE R E S T
We have no conflicts of interest to declare.

AUTH O R CO NTR I B UTI O N S
WY conceived and drafted the manuscript. WY and ZM discussed the concepts of the manuscript. ZM drew the figures. LZ approved the version to be submitted.