Role of exosomal microRNAs in lung cancer biology and clinical applications

Abstract Exosomes, small extracellular vesicles ranging from 30 to 150 nm, are secreted by various cell types, including tumour cells. Recently, microRNAs (miRNAs) were identified to be encapsulated and hence protected from degradation within exosomes. These exosomal miRNAs can be horizontally transferred to target cells, in which they subsequently modulate biological processes. Increasing evidence indicates that exosomal miRNAs play a critical role in modifying the microenvironment of lung cancers, possibly facilitating progression, invasion, angiogenesis, metastasis and drug resistance. In this review, we summarize the novel findings on exosomal miRNA functions during lung cancer initiation and progression. In addition, we highlight their potential role and challenges as biomarkers in lung cancer diagnosis, prognosis and drug resistance and as therapeutic agents.

with limited treatment options and 5-year survival rates of only 4%. 5 Therefore, the identification of reliable diagnostic biomarkers and effective therapeutic strategies is an unmet medical need in lung cancer.
Exosomes, small extracellular vesicles ranging between 30 and 150 nm in size, are secreted by various cell types, including tumour cells. 6 It has been shown that they deliver their cargo (proteins or nucleic acids) to specific cell types, which subsequently act as important messengers in cancer. 7 It was also reported that cancer cells produce 10-fold more exosomes than normal cells and that exosomes derived from cancer cells can facilitate cellular communication via delivery of growth factors, chemokines, miRNAs, etc. 8,9 Interestingly, researchers have shown that exosomes derived from different cells possess unique mRNA and miRNA expression profiles that may differ from their donor cells. 10 Furthermore, accumulating evidence reveals that cancer-derived exosomal miRNAs play important roles in the recruitment and reprogramming of constituents of the tumour environment. 11 Therefore, exosomal miRNAs are regarded as potentially ideal non-invasive tools for early diagnosis as well as therapeutic targets, since they contain key information on signalling pathways related to tumour biological responses. In this review, we summarize the recent findings on exosomal miRNA involvement in cancer initiation and progression, mainly focusing on lung cancer. Furthermore, we highlight the potential future use of exosomal miRNAs as biomarkers and therapeutic targets or agents in lung cancer.

| PROP OS ED ROLE OF E XOSOMAL MIRNA S IN LUNG C AN CER
Exosomes are membrane-encapsulated vesicles present in many biological fluids. They are important mediators of cell-to-cell communication and regulators of biological processes. Exosomes contain multifaceted cargoes, including proteins, lipids, DNAs, mRNAs and miRNAs. 12 Among these molecules, miRNAs, which are short and endogenous non-coding RNAs, are the most intriguing and extensively studied due to their powerful regulatory role in gene expression at the post-transcriptional and translational levels 12,13 (Figure 1). In the tumour microenvironment, including that of lung cancer, transfer of exosomal miRNA between cancer and stromal cells has been demonstrated to be linked with cancer initiation and progression. [14][15][16][17][18] In patients with lung cancer, the concentrations of both circulating exosomes and exosomal miRNA are elevated compared with the respective concentrations in controls. 19 Moreover, exosomal miRNA levels are elevated in both plasma and bronchoalveolar lavage (BAL) samples from patients with non-small-cell lung cancer (NSCLC) compared with those in non-tumour patients. 20 In addition, recent functional studies identified relationships between exosomal miRNAs and lung cancer hallmark pathways ranging from metabolism to intercellular communication ( Figure 2). To this end, exosomal miRNAs have been implicated in a series of biological processes in lung cancer, including proliferation, angiogenesis and metastasis (Table 1). Moreover, exosomal miRNAs have been found to affect the lung tumour microenvironment and to signal to the immune system. As detailed below, the exosomal miRNA effects in lung cancer are largely similar to those of miR-NAs of non-exosomal origin.

| Exosomal miRNAs and cellular proliferation in lung cancer
Proliferation, characterized by altered expression and/or activity of cell cycle-related proteins, plays a critical role in cancer development and progression. 14 Exosomes transfer genetic information between cells in the tumour environment via exosomal miRNAs, thereby promoting lung cancer cell proliferation. For example, Harel et al found that exosomal miR-512 halted lung tumour cell proliferation by targeting TEA domain family member 4 (TEAD4), indicating that miR-512 possesses tumour-suppressive effects. 16 Moreover, miR-208a packaged in exosomes from A549 NSCLC cells was demonstrated to act as a transfer messenger and to target p21 with corresponding activation of the AKT/mechanistic target of rapamycin (mTOR) pathway, thereby inhibiting NSCLC cell proliferation. 21 In addition, miR-96-containing exosomes from H1299 cells could promote cell proliferation by directly targeting and inhibiting LIM-domain only protein 7 (LMO7) expression, demonstrating their tumour-promoting role. 22

| Exosomal miRNAs and angiogenesis in lung cancer
Tumour angiogenesis, essential for tumour growth and metastasis, is modulated by exosomes released by different cell types that act as cell-to-cell mediators. 23 To this end, one study indicated that exosomal miR-9 stimulates angiogenesis by activating the JAK/STAT signalling pathway. 24 Tissue inhibitor of metalloproteinases-1 (TIMP-1) upregulated exosomal miR-210 derived from lung adenocarcinoma (LUAD) samples and ultimately stimulated angiogenesis in stromal cells. 25 In addition, Liu et al found that exosomal miR-21 led to activation of signal transducer and activator of transcription (STAT) 3, increasing VEGF expression and inducing angiogenesis and malignant transformation of human bronchial epithelial cells (HBECs). 17

| Exosomal miRNAs in EMT and metastasis in lung cancer
Metastasis is a complex process that requires cancer cells to invade blood or lymph vessels, disseminate to a new location and establish colonies at the new site. 27 Epithelial-to-mesenchymal transition (EMT), characterized by epithelial cells losing cell-to-cell adhesion and cellular polarity and acquiring a mesenchymal migratory and invasive phenotype, 28 is essential for tumour progression and metastasis. 29,30 Several lines of evidence have confirmed that exosomes and their cargo (especially miRNAs) play critical roles in different steps of the metastatic process. For example, it was demonstrated that exosomal miR-23a was significantly increased after induction of EMT with transforming growth factor (TGF)-β1 in A549 cells. 31 Moreover, Tang et al reported changes in the exosomal miRNA profile upon EMT in the human NSCLC cell lines A549 and H1299 and that some miRNAs specifically contained in exosomes derived from mesenchymal phenotype cells were associated with EMT and metastasis. 32 However, the mechanisms by which miRNAs transferred by exosomes affect tumour metastasis remain poorly understood. 33 There appear to be three mechanisms of exosomal miRNA transfer during lung cancer metastasis. 34 First, exosomes from invasive cells transfer miRNAs to less invasive cells, thereby altering the status of recipient cells and ultimately prompting metastasis.

F I G U R E 1
Biology of exosomal miRNAs. In animals, microRNA (miRNA) genes are transcribed into primary miRNAs (pri-miRNAs) by RNA polymerase II (Pol-II) and then processed by the Drosha complex to form precursor miRNAs (pre-miRNAs), which are exported into the cytoplasm by the exportin5 complex. The pre-miRNAs are digested by the Dicer complex to become double-stranded miRNAs. With the help of a helicase, they are then turned into single-stranded mature miRNAs. Mature miRNAs are sorted into multivesicular bodies (MVBs). The MVBs are then transported along microtubules to the plasma membrane and released as exosomes. Exosomes with special miRNAs from the parent cell can interact with the recipient cell through different ways, such as by fusion via clathrin-dependent endocytosis, clathrin-independent endocytosis (micropinocytosis or phagocytosis), caveolae-mediated endocytosis or lipid raft-dependent endocytosis. Once exosomes enter recipient cells, exosomal miRNAs may act in target repression F I G U R E 2 Exosomal miRNAs in lung cancer. Lung cancer cells export exosomal miRNAs to parent cells to affect their proliferation, angiogenesis, EMT and metastasis. Lung cancer cells are also able to export exosomal miRNAs to immune cells and influence the function of immune cells  [39] (Continues) For example, exosomes derived from adenocarcinoma cells were able to target non-transformed cells (lymph node stromal cells and lung fibroblasts) and to modulate pre-metastatic organ cells predominantly through transferred miR-494 and miR-542-3p. 35 Another example is LUAD cell-derived exosomal miR-21, which was shown to be transferred to osteoclast progenitor cells and to target programmed cell death 4 (PDCD4) to facilitate osteoclastogenesis. 18

| Exosomal miRNAs in drug resistance in lung cancer
The development of drug resistance is one of the main factors of poor prognosis in lung cancer. Lung cancer patients easily develop resistance to not only conventional chemotherapy drugs, but also molecular targeted drugs, such as epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Therefore, exploring the potential mechanisms that impair drug efficacy will help to improve cancer treatments. with mTOR as its potential target gene, and thereby modulate cell sensitivity to DDP. 39 Wei et al also revealed that gemcitabine-resistant A549 (A549-GR) cells could effectively assemble miR-222-3p into A549-GR-secreted exosomes, which could then be transported into parental sensitive cells and promote migration, invasion, anoikis resistance and gemcitabine resistance by directly targeting the promoter of SOCS3. 40 Furthermore, the expression of miR-96 was significantly higher in lung cancer than in normal lung tissue. The same tendency was observed in exosomes isolated from lung cancer patients and controls. MiR-96 could alter the chemotherapeutic sensitivity of lung cancer cells by downregulating the drug resistance-related gene LMO7. 22 Additionally, the levels of exosomal miR-146a-5p TA B L E 1 (Continued) were also found to be significantly lower in advanced NSCLC patients with higher recurrence rates than in those with lower recurrence rates. Exosomal miR-146a-5p was demonstrated to be linked with DDP responses by targeting autophagy-related protein 2 (Atg2) to inhibit autophagy. 41 Finally, exosome-derived miR-512 and miR-373 were also shown to be associated with increased sensitivity to DDP and suppression of tumour progression. 16 In addition, exosomal miRNAs were also involved in resistance to EGFR-TKIs. For example, Jing et al 42

| Exosomal miRNAs and immunity in lung cancer
Immune checkpoint molecules play a critical role in regulating the immune system to maintain self-tolerance and prevent autoimmunity. 44 Targeting immune checkpoint molecules principally represented by programmed cell death protein 1 (PD-1) and its ligand PD-L1 has resulted in improved survival for advanced NSCLC patients. 45 A network of miRNAs has been demonstrated to control immune checkpoint-related processes. For example, miR-34 was shown to be controlled by p53 and directly binds to the PD-L1 3'-UTR and represses its expression in NSCLC models. 46 MiR-200 was also revealed to control PD-L1 expression. 47 Increasing evidence indicates that exosomes participate in tumour progression by delivering immunosuppressive molecules and factors. 48 Exosomal miRNAs are important carriers that can influence the function of immune cells, including dendritic cells (DCs) and T-lymphocytes, in cancer. 14

| Exosomal miRNA from non-tumour cells in lung cancer
Although numerous studies have focused on the role of only exosomal miRNAs derived from cancer cells, an increasing number of studies have focused on those exosomal miRNAs derived from non-cancer cells within the tumour microenvironment. After being transferred from non-cancer cells to recipient cells, exosomal miRNAs can effectively influence the recipient cell phenotype (including epithelial and stromal cells) and then play a crucial role in the growth and progression of lung malignancies by modulating a wide range of pathways.
For instance, by secreting cigarette smoking-induced exosomes containing miR-21, human bronchial epithelial cells (HBECs) have been shown to enhance VEGF levels through STAT3 deregulation, thereby promoting angiogenesis and tumour growth. 17 Similarly, Fujita and colleagues also reported that the transfer of miR-210 in cigarette smoke extract (CSE)-induced HBEC-derived exosomes promoted the myofibroblast differentiation and autophagy of lung fibroblasts (LFs). 50 Moreover, exosome transfer of miR-223 from platelets to lung cancer cells has been reported to modulate invasion through the suppression of EPB41L3. 51 In addition, exosomal miRNAs from MSCs are also reported to be involved in tumour growth. As stated above, exosomes derived from hypoxic BMSCs were demonstrated to transfer miR-193a-3p, miR-210-3p and miR-5100 to lung cancer cells, thus activating STAT3 signalling-induced EMT and promoting metastasis. 15

| CLINI C AL IMPLI C ATI ON S OF E XOSOMAL MIRNA S IN LUNG C AN CER
A number of studies have shown that exosomal cargoes can potentially be used as diagnostic, prognostic and predictive biomarkers for lung cancer. To date, the most widely studied exosomal cargo related to lung cancer is miRNA ( Table 2). and 66%-80%, respectively. 54 Additionally, Wu et al revealed that exosomal miR-96 had the potential to be of diagnostic value in lung cancer patients. 22 Another study reported that exosomal miR-126 correlated with NSCLC and held substantial promise as a diagnostic biomarker for the disease. 55 Exosomal miR-23a and miR-30b/30c have also been identified as possible biomarkers for the diagnosis of lung cancer. 26,56 Recently, a noteworthy study focused on early-stage NSCLC and histologic heterogeneity, and found that exosomal miRNAs (miR-let-7b-5p, miR-let-7e-5p, miR-23a-3p and miR-486-5p) were promising diagnos-

| Exosomal miRNAs as predictive biomarkers in lung cancer
Acquired resistance to chemotherapy, radiotherapy and targeted therapies presents a major clinical challenge in the treatment of lung cancer. 63 Exosomes can serve as vehicles for miRNAs that impact drug resistance and hence as biomarkers that predict therapeutic responses. To this end, exosomal miR-221-3p and miR-222-3p derived from plasma were associated with the response to osimertinib in EGFR-mutated NSCLC. 64 In addition, exosomal miR-29a-3p and miR-150-5p were identified as circulating biomarkers of delivered radiotherapy dose, which could possibly be exploited to predict response or toxicity. 65 Exosomal miR-208a in sera might influence the radiosensitivity of lung tumour cells by targeting p21. 21 Moreover, Yuwen et al showed that serum exosomal miR-146a-5p might serve as a new biomarker predicting the efficacy of cisplatin in NSCLC and might be useful for real-time monitoring of drug resistance. 41 Exosomal serum miR-222-3p levels were recently proposed as a potential prognostic biomarker of gemcitabine sensitivity in NSCLC. 40 Although clinical studies predicting therapy efficacy using exosomal miRNA are sparse, fundamental research studies are abundant. For example, exosomal miR-4443 and miR-100-5p secreted by A549 cells responded to cisplatin treatment, 38 and exosomal miR-100-5p was able to transfer cisplatin resistance to recipient cells by targeting rapamycin (mTOR) in vitro and in vivo. 39 Moreover, exosomal miR-96 derived from H1299 cells enhanced cisplatin resistance, 22 while exosomal miR-521 and miR-373 were associated with cisplatin sensitivity in lung cancer cells. 16 In summary, since exosomal miRNAs can provide information on donor cells and change the cellular state of target cells, they are positioned to regulate tumour resistance and could be used to monitor therapy response/relapse in personalized treatment. TA B L E 2 (Continued) of these two miRNAs for recurrence was also validated in another large cohort, indicating that elevated plasma exosomal miR-21 and miR-4257 levels were linked with shorter disease-free survival.

| Exosomal miRNAs as biomarkers of prognosis in lung cancer
Additionally, another study reported that the presence of serum exosomal miR-146a-5p correlated with higher recurrence rates in advanced NSCLC. 41 Liu et al 70  we cannot ignore the problems of miRNA itself. One study reported that imported miRNA could lead to cellular toxicity. 14 In addition, the mechanisms of miRNA packaging into exosomes are still unclear.
Thus, applying exosomal miRNAs as delivery systems is challenging and needs further exploration.

| CON CLUS I ON S AND PER S PEC TIVE S
In conclusion, the discovery of exosomes and their multiple func-

ACK N OWLED G EM ENTS
The authors would like to thank PhD Ilona E. Kammerl and Dr Mircea-Gabriel Stoleriu for their support and critical reading.

CO N FLI C T S O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
CH and JC wrote the manuscript; SM and GT reviewed and edited the manuscript before submission; and CL prepared the figures.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.