Exploring the roles and molecular mechanisms of RNA binding proteins in the sorting of noncoding RNAs into exosomes during tumor progression

Graphical abstract

Highlights

• •RNA binding proteins (RBPs) impact tumor development and play a role in sorting noncoding RNAs (ncRNAs) into exosomes.
• •The RBP-ncRNA-exosome mechanism is crucial in tumor regulation and provides insights for innovative treatment strategies.
• •This mechanism influences tumor metastasis, multidrug resistance, angiogenesis, the immunosuppressive microenvironment, and tumor progression.
• •Potential therapeutic strategies include targeted drug discovery and genetic engineering of therapeutic exosomes.

Abstract

Background

RNA binding proteins (RBPs) play a role in sorting non-coding RNAs (ncRNAs) into exosomes. These ncRNAs, carried by exosomes, are involved in regulating various aspects of tumor progression, including metastasis, angiogenesis, control of the tumor microenvironment, and drug resistance. Recent studies have emphasized the importance of the RBP-ncRNA-exosome mechanism in tumor regulation.

Aim of review

This comprehensive review aims to explore the RBP-ncRNA-exosome mechanism and its influence on tumor development. By understanding this intricate mechanism provides novel insights into tumor regulation and may lead to innovative treatment strategies in the future.

Key scientific concepts of review

The review discusses the formation of exosomes and the complex relationships among RBPs, ncRNAs, and exosomes. The RBP-ncRNA-exosome mechanism is shown to affect various aspects of tumor biology, including metastasis, multidrug resistance, angiogenesis, the immunosuppressive microenvironment, and tumor progression. Tumor development relies on the transmission of information between cells, with RBPs selectively mediating sorting of ncRNAs into exosomes through various mechanisms, which in turn carry ncRNAs to regulate RBPs. The review also provides an overview of potential therapeutic strategies, such as targeted drug discovery and genetic engineering for modifying therapeutic exosomes, which hold great promise for improving cancer treatment.

Introduction

In 1967, Wolf first observed extracellular vesicles (EVs) in plasma, termed platelet dust [1], and later referred to the vesicles secreted from cells collectively as EVs [2]. EVs are mainly divided into two categories according to vesicle size: microvesicles and exosomes [3]. This article focuses on exosomes, which are a “small” vesicle subgroup of EVs [4],with a diameter of approximately 40–160 nm (average 100 nm) [5]. At the initial stage of discovery, exosomes were considered to be the waste disposal system of cells [6], maintaining the normal physiological function of cells by removing waste from the body [7]. With the deepening of the study, exosomes are currently considered a new way of communication between cells, which are internalized through the interaction between the plasma membrane and cell surface [8] to transmit and exchange information between cells [9]. Exosomes mainly transmit various cargos in vesicles, such as nucleic acids (including DNAs, mRNAs, ncRNAs), proteins, lipids, etc. [10], [11], [12], to maintain the homeostasis of recipient cells. Exosomes play a key role in the development of tumors and can regulate tumor proliferation, metastasis, angiogenesis, immune escape, multidrug resistance and so on [13], [14]. Meanwhile, exosomes can be a potential drug delivery system due to their unique characteristics such as strong biocompatibility, good targeting ability, stability and long circulation time, and transport capacity.

RBPs are a kind of proteins that can directly bind RNAs [15] and regulate the transcription, translation and posttranslational modification of RNAs (which mainly include mRNAs and ncRNAs) [16], [17]. Some studies found that RBPs can selectively mediate the assembly of ncRNAs into exosomes [18], and different exo-ncRNAs (ncRNAs in exosomes) affect different fate outcomes of tumors [19], [20]. However, few studies elucidated the mechanism by which RBPs specifically sort ncRNAs into exosomes to transmit cancer information and promote the development of tumors, and such studies are of great significance. Therefore, this review takes RBPs, ncRNAs and exosomes as the research object, summarizes the relationships among them through existing research, and further describes the whole process of information transmission between tumor cells and other cells based on the mechanisms of RBP-ncRNA-exosome. At the same time, anti-tumor treatment strategies aimed at RBP-ncRNA-exosome mechanisms are proposed.

Exosome biogenesis

Exosomes are derived by cellular endocytosis [21], and the process of exosome formation comprises the following four steps: i) the cytoplasmic membrane invaginates to form an early endosome, ii) multiple mechanisms are initiated to promote the formation of intraluminal vesicles (ILVs), and vesicles with multiple ILVs are termed multivesicular body (MVB) biogenesis, iii) endosome maturation by acidification, iv) late endosomes release ILVs as exosomes by fusion with the plasma membrane [22], [23], [24]. In the process of exosome formation, the cytoplasmic membrane first invaginates to form a vesicle called the early endosome. Subsequently, the dependent endosomal sorting complex required for transport (ESCRT) pathway and independent ESCRT pathway are initiated to promote sprouts inward from the membrane and ILVs formation, which is the key to exosome formation and cargo sorting and is also the most important link in the whole process [25], [26] (Fig. 1).

Fig. 1.

Exosome biogenesis and the pathway of MVBs formation. The process of exosome formation comprises the following four steps: i) the cytoplasmic membrane invaginates to form an early endosome, ii) Then ILVs were formed in the MVBs, iii) endosome maturation, iv) and late endosomes release ILVs as exosomes by fusion with the plasma membrane. The ILVs were formed through the following pathways in the MVBs: the ESCRT-dependent pathway, the Alix-dependent pathway, the HD-PTP-dependent pathway, and the ESCRT-independent pathway.

The ESCRT pathway includes the classical ESCRT-dependent pathway and the noncanonical ESCRT-dependent pathway [3], [27]. The classical ESCRT-dependent pathway consists of ESCRT-0, -I, -II, -III subcomplexes and the ATPase VPS4, which cooperate to mediate the formation of ILVs [28]. ESCRT-0 is a heterotetramer composed of two HRs subunits and two STAM subunits [29] with ten ubiquitin-binding sites [30],which facilitates the capture of ubiquitinated cargo [31]. The FYVE domain of HRs can bind to phosphatidylinositol 3-phosphate (PI3P) on the endosomal membrane [32]. At the same time, HRs can bind to Clathrin, a protein best known for its involvement in endocytosis [33], which promotes ESCRT-0 accumulation in the electron-dense microdomain of the endosomal membrane [34]. Subsequently, ESCRT-0 recruits ESCRT-I and hands over the ubiquitylated cargo, and ESCRT-I interacts with ESCRT-II to jointly promote endomembrane sprouting [35]. ESCRT-II consists of EAP45, EAP30 and EAP20. ESCRT-III is composed of CHMP6, CHMP4, CHMP3 and CHMP2 [36], and the EAP20 subunit of ESCRT-II recruits CHMP6 in ESCRT-III, which is a key component driving membrane contraction. Then, CHMP4, CHMP2, and CHMP3 form filaments [37], and the synergistic action of ATPase VPS4 with filaments mediates the constriction and scission of the ILV neck and ultimately forms ILVs [38], [39]. Notably, deubiquitinating enzymes are also recruited during this process, which remove ubiquitin from cargo proteins in ILVs, but not all cargos need to be deubiquitinated [40]. Meanwhile, ordered regions of the MVB membrane, such as lipid rafts, are involved in sorting exosomal cargo [41], [42].

The noncanonical ESCRT-dependent pathway includes the Alix‑dependent pathway and HD-PTP‑dependent pathway. The Syndecan-Syntenin-Alix pathway is the most studied Alix-dependent pathway [43]. Syndecan is a transmembrane protein that connects with syntenin, which then binds to Alix via LYPX(n)L motifs to recruit nucleated ESCRT-III and VPS4 to support the biogenesis of ILVs and exosomes [44]. The HD-PTP pathway is mainly dependent on the HD-PTP protein, whose Ub binding site can replace the cargo sorting function of ESCRT-0. HD-PTP can also directly bind to ESCRT-0 and ESCRT-1 [45] and recruit CHMP4 of ESCRT-3 [46]. The V structure of HD-PTP can combine with the MIT domain of Vps4 to stimulate the activity of ATPase VPS4 and jointly assist in the biogenesis of ILVs [47].

The ESCRT-independent pathway includes the neutral sphingomyelinase 2 (nSMase2)-ceramide pathway [48], which has been reported to induce vesicle curvature and inward budding from the MVB, ultimately inducing ILVs formation [49]. Caveolin‑1 [50], flotillins [51], cholesterol [52] and tetraspanins [53], [54] can also sort exosomal cargo and promote exosome production, but the specific mechanism is still unclear.

RBPs and their regulation of ncRNAs sorting in exosomes

RBPs play a crucial role in the regulation of various cellular processes, including post-transcriptional gene regulation. RBPs are roughly divided into the heterogeneous nuclear ribonucleoprotein (hnRNP) family, the IGF2BP family, the PCBP family, the tristetraprolin (TIS11/TTP) family, and the ELAV family [55]. These RBPs can interact with ncRNAs [56],ncRNAs are divided into different categories based on their length and shape according to their length and shape, namely microRNAs (miRNAs), long non-coding ncRNAs (lncRNAs) and circRNAs [57]. Such interactions enable RBPs to regulate their translation [58], localization, and the stability [59]. And several RBPs have been identified to contribute to the selective packaging of ncRNAs into exosomes.

One well-studied RBP involved in exosomal sorting is the hnRNP family. For example, hnRNPA2B1 has been shown to interact with specific motifs present in lncRNAs and promote their packaging into exosomes [60]. Another RBP known to regulate ncRNAs sorting into exosomes is the RBP Musashi (MSI). MSI protein was involved in stem cell maintenance and found to interact with specific motifs in mRNAs and ncRNAs to control their localization and stability. Studies shown that MSI protein can regulate the sorting of various ncRNAs, including miRNAs and circRNAs, into exosomes [61], [62]. In addition to hnRNPs and MSI proteins, other RBPs such as AGO2 (Argonaute 2) and YBX1 have also been implicated in the selective sorting of specific ncRNAs into exosomes [63], [64]. These RBPs interact with different ncRNA species and mediate their packaging into exosomes through various mechanisms, including recognition of RNA secondary structure and sequence-specific binding.

One important mechanism involves post-transcriptional modifications of ncRNAs, such as methylation or alternative splicing, which can regulate their binding affinity for RBPs. Other regulatory factors include RNA secondary structures, RNA-protein interactions, and specific motifs within the RNA sequence recognized by RBPs. Additionally, RBPs themselves can be regulated at the transcriptional or post-translational level, influencing their availability and affinity for binding to ncRNAs. Changes in cellular conditions, such as stress or disease states, can alter RBP expression or activity, thereby influencing the sorting of specific ncRNAs into exosomes.

Overall, the sorting of ncRNAs into exosomes is a complex and regulated process that involves the interaction of various RBPs with specific RNA motifs or structures. Further research is needed to fully understand the mechanisms underlying RBP-mediated sorting of ncRNAs into exosomes and its implications in intercellular communication.

The roles of ncRNAs, RBPs, and exosomes in cellular function and pathophysiology

NcRNAs regulate the function of cells, such as chromatin remodeling, cell cycle regulation, splicing, translation, mRNA degradation, and posttranscriptional modification [65], [66]. Moreover, exo-ncRNAs have been found to affect the pathophysiological processes of cells. For example, exo-lncRNA PTENP1 inhibited the growth of bladder cancer [67]. Exo-lncRNA H19 was released by tumor-associated fibroblasts which induced chemoresistance of colorectal cancer [68]. Exo-circTRPS1, secreted by bladder cancer cells, promoted CD8⁺ T-cell exhaustion in the tumor microenvironment [69]. And M2 macrophage-derived exosomes delivered miR-942 to promote lung adenocarcinoma cell invasion and angiogenesis [70]. These ncRNAs were selectively incorporated into exosomes, although the underlying mechanisms remain unclear. It is worth noting to discusses the relationship among RBPs, ncRNAs and exosomes (Fig. 2).

Fig. 2.

The role of ncRNAs, RBPs, and exosomes in cellular function and pathophysiology. (A) RBPs and ncRNAs are captured by exosomes, with the caveolin-1, IC3 proteins, ESCRT complex and MVBs closely involved in the formation of exosomes, facilitating the capture of RBPs and ncRNAs into exosomes. (B) RBPs mediate the sorting of ncRNAs into exosomes. An array of RNA-binding proteins, including hnRNPQ, hnRNPA1, SRSF1, hnRNPA2B1, and hnRNPC1, bound to ncRNAs via specific motifs to allow for the sorting of ncRNAs into exosomes. (C) Exo-ncRNAs regulate RBPs in target cells. For example, internalization of Exo-LncAY927529 by bone marrow stromal cells led to upregulation of the expression of the RBP CXCL14.

RBPs and ncRNAs are captured by exosomes

There is a class of RBPs and ncRNAs that are captured by exosomes into vesicles and through the transport function of exosomes to regulate the function of cells [71]. Caveolin‑1 is a hairpin-like membrane protein located on the MVB membrane that plays a crucial role in the formation of exosomes and the sorting of cargo. Caveolin-1 facilitated the localization of hnRNPK (heterogeneous nuclear ribonucleoprotein K) to MVBs through a membrane raft-dependent mechanism. Subsequently, hnRNPK recruited miRNAs containing the AsUGnA motif, enabled the loading of these miRNAs into EVs [72]. The LC3-conjugation machinery was reported to sort RBPs into exosomes, which mediated IC3 protein binding to hnRNPK. Additionally, the factor associated with nSMase2 activity (FAN) transiently bound to the IC3 protein via the LIR motif on the MVB limitation membrane, which worked together to successfully capture hnRNPK into exosomes and hnRNPK further regulated ncRNAs in exosomes [73]. YBX1 is a kind of RNA binding protein ubiquitous in the cells, which was identified as the directly-bound target protein for ESCRT complex [74]. And a 'cold shock' domain (CSD) of YBX1 specifically bound with miR223 through a 5′ proximal sequence motif UCAGU to mediated miR-223 into exosomes [75], [76]. YBX1 was also confirmed to specifically bind to and is required for the sorting of miR-133 into exosomes [77]. HuR protein (also called ELAVL1) belongs to the ELAV family, which bound to miR-122 under starvation conditions, MVBs induced HuR ubiquitination and reduced the ability of HuR to bind to miRNA, subsequently causing miR-122 to unbind from HuR and load into exosomes [78]. The HuR protein can also assist in packaging miR-1246 into exosomes and releasing it into the serum of gastric cancer patients. The involvement of the HuR protein made exo-miR-1246 a potential biomarker for diagnosing gastric cancer [79]. In summary, exosomes can capture RBPs and ncRNAs into vesicles through proteins on the MVB limitation membrane, and RBPs also regulate ncRNAs in exosomes.

RBPs mediate the sorting of ncRNAs into exosomes

In addition to the active capture of RBPs and ncRNAs by exosomes, RBPs also mediate the selective sorting of ncRNAs into exosomes. Some mechanisms of exo-ncRNAs sorting by RBPs have been reported. SYNCRIP (also named hnRNPQ) contains an amino-terminal domain called NURR (N-terminal unit for RNA recognition), which could directly bind to certain miRNAs with a conserved minimal GGCU motif (known as the hEXO motif). Moreover, three RRM domains on SYNCRIP significantly increased miRNA binding affinity through αββ motif coupling to a NURR domain and finally mediated the exosomal partitioning of a set of miRNAs with the hEXO motif [80], [81]. Similarly, the RBP SRSF1 was found to bind specifically to a 6-bp motif and selectively sort miRNAs containing the commonly shared motif to the exosomes of pancreatic cancer cells [18]. HnRNPA2B1 is a ubiquitously expressed RBP [82], and sumoylated hnRNPA2B1 could specifically bind miRNAs through the recognition of a short nucleotide GGAG sequence (named EXO motifs GGAG) and control their loading into exosomes [83]. Unexpectedly, hnRNPA2B1 could negatively regulate the exosomal sorting of ncRNAs and prevented the exosomal export of miR–503 in endothelial cells due to its high affinity for miR-503 [84].

The above RBPs mainly regulate the sorting of exo-ncRNAs by binding to specific motifs on ncRNAs. However, some RBPs were also reported to be involved in sorting exo-ncRNAs, but the specific mechanism is still unclear. For example, hnRNPC1 was involved in the sorting of miR-30d in exosomes, and silencing hnRNPC1 significantly reduced the level of miR-30d in endometrial exosomes [85]. HnRNPA1 bound miR-4835p to mediate exosomal sorting and transport cellular miR-483-5p out of tubular epithelial cells into the urine. MiR-483-5p restrained renal interstitial fibrosis, and a high glucose environment improved the binding efficiency between hnRNPA1 and miR-4835p to accelerate the entry of miR-4835p into urine and promoted the progression of renal interstitial fibrosis [86]. Although the mechanism by which RBPs mediate ncRNAs sorting into exosomes as described above is unclear, RBPs have the ability to selectively sort exo-ncRNAs and occupy an important position in the assembly of exosomal cargoes.

Exo-ncRNAs regulates RBPs in target cells

Interestingly, it has been found that exosomes selectively load ncRNAs into vesicles for internalization into other cells through the transport function of exosomes, and such exo-ncRNAs regulated RBPs within target cells to activate downstream signaling pathways. For example, LncAY927529 was highly expressed in prostate cancer cells and was specifically assembled into exosomes. Exo-LncAY927529 was internalized by bone marrow stromal cells, which upregulated the level of the RBP CXCL14 to activate the ERK signaling pathway in the bone microenvironment and stimulated autophagy of bone marrow stromal cells, thus regulated the bone microenvironment and promoted the occurrence and development of prostate cancer [87]. In summary, RBPs, ncRNAs and exosomes regulate each other and work together to mediate cell-to-cell communication.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in tumors

With the intensive exploration of tumor research, it now has been discovered that tumor cells use a variety of strategies to regulate exosome production and assemble cargo in exosomes, which deliver effective messages to determine the fate of tumors. RBPs and ncRNAs are currently involved in the sorting of exosomal cargo and information transmission. However, how RBPs, exosomes and ncRNAs deliver tumor signaling remains largely unknown. Elucidation of the RBP-ncRNA-exosome mechanisms may help to discover new opportunities for cancer treatments. Next, we focus on the interamctions among RBPs, ncRNAs, and exosomes, which together regulate tumor development (see Table 1).

Table 1.

Tumor-promoting and tumor-suppressing roles and mechanisms of RBP-ncRNA exosomes in cancer.

RBPs	ncRNAs	Host cells/ Recipient cells	Expression in cancers	Targets/Mechanisms	Biological roles	Ref.
hnRNPA1	miR-27b-3p	EMT-CRC cells / HUVECs	miR‐27b‐3p was highly enriched in the EMT‐HCT116 exosomes	decrease VE-cadherin and p120 in HUVEC	enhances blood vessel permeability to mediate CRC metastasis	[92]
hnRNPA1	lncRNA BCYRN1	BCa cells/ HLECs	BCYRN1 is overexpressed in BCa cell‐secreted exosomes	activated the VEGF-C/VEGFR3 signaling pathway	promote lymphatic metastasis	[121]
hnRNPA2B1	lncRNA LNMAT2	BCa cells/ HLECs	LNMAT2 expression was increased in BCa tissues and enriched in BCa cell–secreted exosomes	increase PROX1 in HLECs	promote lymphatic metastasis	[124]
hnRNPA2B1	miR-122-5p	NSCLC cells/liver cells	miR-122-5p highly enriched in EVs from NSCLC Cells	promote migration of normal liver cells	establishes a pre-metastatic microenvironment for hepatic metastasis of lung cancer	[93]
IGF2BP3	circFOXK2 and miR-370	highly metastatic BC cells/BC cells	circFOXK2 upregulated in highly metastatic BC cells	circFOXK2 could act with IGF2BP3 and miR-370	promote BC metastasis	[94]
hnRNPA2B1	lncRNA H19	gefitinib-resistant NSCLC cells/ NSCLC cells	H19 was increased in gefitinib-resistant NSCLC cells	/	promotes gefitinib resistance in NSCLC	[97]
PUM2	miR-130a	CAFs/ NSCLC cells	miRNA-130a was highly expressed in CAFs	/	contributing to the development of cisplatin resistance in NSCLC cells	[98]
hnRNPA1	miR-196a	CAFs/ HNC cells	miR-196a was increased in CAF-derived exosomes	decrease CDKN1B and ING5 in HNC cells	promoting cisplatin resistance in HNC cells	[99]
hnRNPA1	lncFERO	GC cells/ GCSCs	lncFERO was significantly increased in GC	exo-lncFERO /hnRNPA1/SCD1 axis	suppressing ferroptosis and acquired chemo-resistance in GC	[125]
hnRNPA1	miR-522	CAFs/ GC cells	miR-522 were both up-regulated in exosomes and GC cells	targete USP7/ hnRNPA1/exo-miR-522/ALOX15 axis to inhibit ferroptosis	promotes acquired chemo-resistance in gastric cancer	[103]
hnRNPA1	miR-1246	glioma cells/ monocytes	miR-1246 was increased inglioma cell-derived exosomes	exo-miR1246 activated the DUSP3/ERK signaling pathway	drives the differentiation and activation of MDSCs to contribute to the immunosuppressive microenvironment of glioma	[108]
EWSR1 and hnRNPA2B1	circNEIL3	glioma cells/ TAMs	CircNEIL3 was significantly upregulated in glioma cells	EWSR1/ circNEIL3/YAP/ LOX/CCL2 signaling pathway to drive macrophage, and hnRNPA2B1/ exo-circNEIL3/ IGF2BP3 signaling pathway to promote macrophage polarization	Mediates macrophage immunosuppressive polarization and contribute to an immunosuppressive microenvironment of glioma	[109]
PABPC1	miR-21-5p	ESCC cells/ HUVECs	PABPC1 is highly expressed in ESCC	targets PABPC1/IFI27/ exo-miR-21-5p/ CXCL10 signaling pathway	promotes turom angiogenesis	[114]
HUR	CircSHKBP1	GC cells/ GC cells	circSHKBP1 was upregulated in GC cells	exo-CircSHKBP1 targets miR-582-3p/HUR/VEGF signaling pathway	promotes VEGF secretion and induced angiogenesis	[115]
hnRNPA	miR-320	leukemia cell/ BMMSCs	miR-320 was highly enriched in leukemic exosomes	Exo-miR-320 inhibits the β-catenin signaling pathway	inhibited osteogenesis to remodel the normal niche into a malignant niche, leading to leukemia progression	[117]
hnRNPA2B1	miR-92a-2-5p and miR-373-3p	Myeloma/ monocytes or MSCs	hnRNPA2B1 was significantly upregulated in plasma cells of myeloma	exo-miR-92a-2-5p increased the expression of NFATc1 in monocytes and exo-miR-373-3p inhibits the expression of RUNX2 in MSCs	induce osteoclastogenesis and inhibit osteoblast formation to promote the progression of myeloma	[118]

Roles and regulatory mechanisms of RBP-ncRNA-exosome in tumor metastasis

Metastasis is an important cause of poor prognosis and death in cancer patients [88]. In the process of metastasis, cancer cells interact with the surrounding extracellular matrix environment (including crossing the basement membrane, modulating the stromal microenvironment and migrating across the barrier), break through the defenses into the blood vessels or lymphatic vessels, and spread the tumor from the primary site to distant tissues and organs [89], [90]. One of the most important metastatic strategies is the release of exo-ncRNAs by cancer cells to promote tumor metastasis [91], and RBPs now have been found to regulate exo-ncRNAs to help tumor metastasis (Fig. 3).

Fig. 3.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in tumor metastasis. (A) STAT3 bound to the promoter to upregulate hnRNPA1 protein, which then mediated the transfer of miR-27b-3p into exosomes. Exo-miR-27b-3p targeted VE-cadherin and p120, to enhance blood vessel permeability and mediating CRC metastasis. (B) HnRNPA1 bound to lncRNA BCYRN1 to activate the WNT/β-catenin pathway, led to the secretion of VEGF-C. The hnRNPA1-BCYRN1 complex was internalized by HLECs via exosomes to stabilize VEGFR3 mRNA, thereby activating the VEGF-C/VEGFR3 signaling pathway to promote lymphatic metastasis in bladder cancer. (C) HnRNPA2B1 mediated LNMAT2 into exosomes, subsequently, exo-LNMAT2 and hnRNPA2B1 formed a complex to bind to the PROX1 promoter to upregulate PROX1 expression in HLECs to promote lymphatic metastasis in BCa. (D) HnRNPA2B1 specifically bound to the EXO motif on miR-122-5p, mediated its sorting into exosomes of non-small cell lung cancer (NSCLC) cells. Exo-miR-122-5p promoted the migration of hepatocytes and hepatic metastasis in lung cancer.

HnRNPA1 was reported to promote tumor metastasis through the regulation of exo-ncRNAs, STAT3 binding to the hnRNPA1 promoter was observed in epithelial-mesenchymal transition (EMT) colorectal cancer (CRC) cells, leading to an upregulation of hnRNPA1 protein, hnRNPA1 facilitated the packaging of miR-27b-3p into exosomes, and exo-miR-27b-3p translocated into human umbilical vein endothelial cells (HUVECs). Once inside HUVECs, exo-miR-27b-3p enhanced blood vessel permeability by targeting VE-cadherin and p120, thereby facilitating the migration of CRC cells into the vasculature. This process ultimately contributed to the generation of circulating tumor cells (CTCs) and promoted CRC metastasis [92]. HnRNPA2B1 mediated hepatic metastasis of lung cancer, which specifically bound to EXO motif on miR-122-5p and mediated its sorting into exosomes of non-small cell lung cancer (NSCLC) cells. These exo-miR-122-5p then promoted migration of hepatocytes and established a premetastatic microenvironment for hepatic metastasis of lung cancer [93]. RBP IGF2BP3 and circFOXK2 together promoted miR-370 expression in exosomes of breast cancer to mediate breast cancer metastasis [94].

Previously, exo-ncRNAs were thought to play an important role in tumor metastasis. However, the current study unveiled the essential role of RBPs in this process. RBPs selectively mediate the packaging and release of exo-ncRNAs and activate downstream signaling pathways by taking advantage of the transport function of exosomes to achieve tumor metastasis by increasing vascular permeability or promoting lymphatic vessel generation. These findings highlight a more comprehensive understanding of the mechanisms underlying tumor metastasis, extending beyond the scope of exo-ncRNAs alone, and offer new insights for further research in this field.

Impact of RBP-ncRNA-exosome on multidrug resistance in tumors

Multidrug resistance is a critical obstacle to overcome in cancer treatment, and almost all cancer patients will ultimately be resistant to antineoplastic drugs [95]. The mechanisms underlying multidrug resistance are highly complicated [96], and exploring the molecular mechanisms of multidrug resistance can help to overcome this barrier. Recent research suggests that the RBP-ncRNA-exosome mechanism plays a crucial role in mediating chemoresistance in tumors (Fig. 4). For instance, the hnRNPA2B1 protein specifically mediated the assembly of lncRNA H19 into the exosomes of resistant NSCLC cells and was then released and transferred to nonresistant cells, thus promoted gefitinib resistance in NSCLC [97]. Similarly, PUM2, a RNA-binding protein, specifically mediated the sorting of miRNA-130a into the exosomes of tumor-associated fibroblasts (CAFs), and exo-miRNA-130a was internalized by NSCLC cells, contributing to the development of cisplatin resistance in NSCLC cells [98]. Other RBPs were also revealed to be involved in the assembly of ncRNAs in the exosomes of CAFs. For example, hnRNPA1 specifically bound miR-196a through a specific sequence (UAGGUA) on the 5′ end of miR-196a. This interaction facilitates packaging of miR-196a into CAF-derived exosomes, which are then internalized by tumor cells. Once inside the tumor cells, miR-196a bound to its target genes CDKN1B and ING5, resulting in downregulation of their mRNA transcription and protein expression. This process promoted resistance to cisplatin in head and neck cancer (HNC) cells [99].

Fig. 4.

Impact of the RBP-ncRNA-exosome on multidrug resistance in tumors. (A) HnRNPA2B1 mediated lncRNA H19 into exosomes, which were transferred to nonresistant NSCLC cells to promote gefitinib resistance. (B) Cisplatin and paclitaxel activated the USP7/hnRNPA1 axis in CAFs. HnRNPA1 mediated miR-522 into exosomes, which suppressed the expression of ALOX15, resulting in the inhibition of ferroptosis and chemotherapy resistance in GC cells. (C) PUM2 mediated the sorting of miRNA-130a into CAF-secreted exosomes. Exo-miRNA-130a was internalized by NSCLC cells and contributed to cisplatin resistance. Meanwhile, hnRNPA1-mediated miR-196a was sorted into CAF-secreted exosomes, and exo-miR-196a was internalized by HNC cells to target CDKN1B and ING5, ultimately promoting cisplatin resistance. (D) Cisplatin and paclitaxel upregulate USP7 expression in GC cells, which further increased hnRNPA1 expression. HnRNPA1 promoted lncFERO into exosomes, which were internalized by GCSCs to upregulate SCD1. SCD1 inhibited the production of lipid ROS, ultimately preventing ferroptosis in GCSCs to promote chemoresistance.

RBP-ncRNA-exosome mechanisms are also involved in mediating drug resistance in gastric cancer (GC). Additionally, Ferroptosis is tightly associated with multidrug resistance in tumors [100]. Ferroptosis is a novel mode of nonapoptotic cell death caused by iron-dependent accumulation of toxic lipid peroxides (lipid-ROS) [101], [102], and induction of ferroptosis reverses drug resistance. Two important regulators of lipid metabolism and ferroptosis in cancer are Stearoyl-CoA-desaturase (SCD1) and arachidonic acid lipoxygenase 15 (ALOX15). Research shown that cisplatin and paclitaxel increased the expression of ubiquitin-specific protease 7 (USP7), which then stabilized hnRNPA1 mRNA in GC cells through deubiquitination. This, in turn, leading to increased expression of hnRNPA1. HnRNPA1 was directly involved in the assembly of miR-522 into exosomes derived from cancer-associated fibroblasts (CAFs). The exo-miR-522 suppressed the expression of ALOX15 in GC cells, reducing the accumulation of lipid ROS and inhibiting ferroptosis in GC cells. Consequently, this led to decreased sensitivity to chemotherapy [103].

The above studies reveal the function of RBP-ncRNA-exosome mechanisms in multidrug resistance for cancer. Through these mechanisms, the process of cell-to-cell transmission of information is comprehensively described, which includes the transcription and expression from upstream exo-ncRNAs to downstream target genes and finally regulates tumor chemoresistance, which helps researchers explore new molecular drugs and open up new research fields to discover the complex mechanisms underlying drug resistance.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in the tumor immune microenvironment

The tumor immune microenvironment (TIME) plays a crucial role in determining the fate of tumors. Initially, the immune system can work as an anti-tumor immune system to eliminate tumors, but in later stages, cancer gradually shape the TIME into an immunosuppressive state to counteract antitumor immunity [104]. Cancer have evolved various mechanisms to evade immune surveillance by shaping the immunosuppressive microenvironment [105].

The involvement of RBP-ncRNA-exosome mechanisms are also implicated in the establishment of an immunosuppressive microenvironment in glioma (Fig. 5). Hypoxia is a recognized characteristic of the glioma microenvironment [106]. Hypoxia activated HIF-1α, which upregulated the expression of POU5F and hnRNPA1. POU5F was a transcription factor of miR-1246, which bound to the miR-1246 promoter to increase its expression. HnRNPA1 then bound to miR-1246 via UAGGUA motif and mediated the selective sorting of miR-1246 into the exosomes of glioma cells. Exo-miR-1246 was internalized by monocytes to activate the DUSP3/ERK signaling pathway to promote the polarization of monocytes to myeloid-derived suppressor cells (MDSCs). MDSCs are immunosuppressive cells [107] that suppress the proliferation of CD8⁺ T cells and contribute to the establishment of an immunosuppressive microenvironment [108]. In addition, EWS RNA-binding protein 1 (EWSR1) and hnRNPA2B1 were also involved in regulating the immune microenvironment of glioma cells. EWSR1 stimulated cyclization of circNEIL3 and upregulated its expression. circNEIL3 activated YAP1 signaling, which promoted the secretion of lysyl oxidase (LOX) and C–C motif chemokine ligand 2 (CCL2), which drived macrophage infiltration into the tumor microenvironment. HnRNPA2B1 further mediated the packaging of circNEIL3 into glioma cell-derived exosomes and translocation into tumor-associated macrophages (TAMs). Exo-circNEIL3 blocked ubiquitination of IGF2BP3 protein to stabilize its expression, promoted macrophage polarization toward an immunosuppressive phenotype, and ultimately suppressed the antitumor immune response to contribute to an immunosuppressive microenvironment [109].

Fig. 5.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in the tumor immune microenvironment. In the glioma tumor immune microenvironment, HIF-1α upregulated POU5F to increase the expression of miR-1246 and hnRNPA1. HnRNPA1 mediated miR-1246 into the exosomes of glioma cells. Exo-miR1246 was internalized by monocytes, to activate the DUSP3/ERK signaling pathway and promote the polarization of monocytes to MDSCs. Meanwhile, EWSR1 upregulated the expression of circNEIL3. CircNEIL3 activated YAP1 signaling, resulting in the secretion of LOX and CCL2, which drove macrophage infiltration into the tumor microenvironment. HnRNPA2B1 mediated circNEIL3 into exosomes, and exo-circNEIL3 stabilized the expression of IGF2BP3, to promote macrophage polarization toward an immunosuppressive phenotype. These mechanisms ultimately contribute to the formation of an immunosuppressive microenvironment.

Previously, we only knew that tumor cells can regulate immune cells in the tumor microenvironment to establish an immunosuppressive microenvironment for their own growth, but there is still a large gap in the study of the mechanism of how tumor cells regulate immune cells. The above study provides us with new perspectives on RBP-ncRNA-exosome mechanisms as a focus to fully unveil the immune microenvironment regulated by tumors.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in tumor angiogenesis

Tumors are characterized by high proliferation [110], and therefore, they need to rapidly establish a neovascular network to obtain the necessary nutrients for their growth. Nevertheless, abnormal development of this neovascular network [111] can exacerbate the risk of tumor hypoxia and metastatic spread [112], [113]. The RBP-ncRNA-exosome mechanisms have been demonstrated to play a role in regulating angiogenesis (Fig. 6). The RBP PABPC1 was involved in angiogenesis in esophageal squamous cell carcinoma (ESCC). The binding of Sp1 with p300 led to enrichment of H3K27ac at the PABPC1 promoter, which activated the transcription of PABPC1. Subsequently, PABPC1 interacted with eIF4G to prevent the degradation of IFI27 mRNA and increase its expression. PABPC1 and IFI27 collaborated to regulate the expression of miR-21-5p in ESCC cells, with PABPC1 further promoting the packaging and released of exo-miR-21-5p. Upon uptake by HUVECs, exo-miR-21-5p inhibited the expression of CXCL10, thereby promoting angiogenesis [114].

Fig. 6.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in tumor angiogenesis. In esophageal squamous cell carcinomas (ESCCs), Sp1 and p300 increased the expression of PABPC1. Furthermore, PABPC1 and IFI27 co-upregulated the expression of miR-21-5p, and PABPC1 promoted the packaging and release of exo-miR-21-5p. Once taken up by human umbilical vein endothelial cells (HUVECs), exo-miR-21-5p inhibited CXCL10 to promote angiogenesis. Meanwhile, in gastric cancers (GCs), exosomes secreted circ-SHKBP1, which sponged miR-582-3p to increase the expression of HUR. HUR directly bound to VEGF mRNA to enhance its stability and promote angiogenesis.

Interestingly, exo-ncRNAs also play a role in regulating the expression of RBPs. In GC, exo-circSHKBP1 was found to be highly expressed in exosomes secreted by GC cells. Exo-circSHKBP1 acted as a sponge for miR-582-3p, leading to an increase in the expression of the RBP HUR. HUR, in turn, directly bound to VEGF mRNA and enhanced its stability, thereby promoting angiogenesis [115]. Angiogenesis is a hallmark feature of tumors, and hypoxia is the main factor that induces angiogenesis [116]. Combined with the abovementioned studies, it has been found that tumors can secrete specific exo-ncRNAs to control angiogenesis. These findings uncover additional molecular mechanisms involved in angiogenesis and providing a new research direction for this field.

Roles and regulatory mechanisms of RBP-ncRNA-exosome in tumor progression

The RBP-ncRNA-exosome mechanisms not only function in regulating tumor chemoresistance, metastasis, establishment of an immunosuppressive microenvironment, and angiogenesis but also directly contribute to tumor progression. For example, hnRNPA specifically bound to miR-320 via the AGAGGG motif and facilitated its packaging into exosomes derived from leukemia cells. These exosomes carrying miR-320 were then taken up by bone marrow mesenchymal stromal cells (BMMSCs), leading to the inhibition of the β-catenin signaling pathway and suppression of osteogenesis in BMMSCs. Consequently, the malignant ecological niche was remodeled, promoting the acceleration of leukemia progression [117]. HnRNPA2B1 also inhibited osteoblast formation by recruiting the miRNA microprocessor complex protein (DGCR8) and forming a complex. This complex, in turn, specifically engaged primary miR-92a-2-5p and primary miR-373-3p, causing an upregulation in their expression. Subsequently, hnRNPA2B1 facilitated their packaging into myeloma-derived exosomes, which were transported to monocytes or mesenchymal stem cells (MSCs). Exo-miR-92a-2-5p suppressed the expression of interferon regulatory Factor 8 (IRF8) and increased the expression of NFATc1, thereby promoting osteoclastogenesis. Exo-miR-373-3p downregulated the expression of RUNX2, to inhibit osteoblast formation and accelerate bone destruction, then promoted the progression of myeloma [118].

The complex-RBPs and ncRNAs in exosomes regulate tumor progression

Most of the studies mentioned above demonstrated that RBPs sort ncRNAs into exosomes and then transport them into recipient cells, thereby activating or inhibiting signaling pathways to regulate tumor progression. Interestingly, some studies have also found that RBPs to ncRNAs to form complexes, which then transported together by exosomes to affect cancer development. Activation of the VEGF-C/VEGFR3 signaling pathway induced lymphangiogenesis in tumors to facilitate lymph node metastasis [119], [120]. HnRNPA1 directly bound to lncRNA BCYRN1, leading to the upregulation of WNT5A expression and activation of the WNT/β-catenin pathway in bladder cancer (BCa) cells. The hnRNPA1-BCYRN1 complex was subsequently internalized by human lymphatic endothelial cells (HLECs) via exosomes, thereby stabilizing VEGFR3 mRNA in HLECs and collectively activating the VEGF-C/VEGFR3 signaling pathway, promoting lymphatic metastasis of BCa [121] (Fig. 3). HnRNPA2B1 interacted with LNMAT2 to form a complex that was co-packaged into exosomes. Subsequently, Exo-LNMAT2-hnRNPAB1 was internalized by HLECs, and this complex promoted H3K4 trimethylation of the PROX1 promoter. This ultimately induced proliferation of HLECs by upregulating PROX1 expression [122], [123] and facilitated lymphatic metastasis of BCa [124] (Fig. 3). Cisplatin and paclitaxel activated the USP7/hnRNPA1 axis to upregulation of RNPA1 protein expression in GC cells. HnRNPA1 then promoted the packaging of lncFERO into GC cell exosomes, which were internalized by gastric cancer stem cells (GCSCs). Upon bound to hnRNPA1 in GCSCs, exo-lncFERO-hnRNPA1 formed a complex and subsequently bound to the 5′UTR of SCD1 mRNA. This complex promoted SCD1 translation, further inhibiting lipid ROS production, which inhibited GCSC ferroptosis and mediated chemoresistance [125] (Fig. 4). These studies firstly clarified the sorting effect of RBPs on ncRNAs, secondly confirmed the binding ability between them and finally verified the tumor regulatory effects of the bound complexes. These complexes may appear new spatial structures, and changes in spatial structure may expose new binding sites to increase or decrease affinity with downstream RNAs, then affecting the post-translational regulation of RNAs. Thus, RBPs not only have the ability to sort ncRNAs, but the complexes formed by their binding to ncRNAs may have more sophisticated regulation of RNAs, ultimately affecting tumor progression.

The comprehensive mechanism through which tumor cells regulated the growth of other cells, utilizing RBP-ncRNA-exosome mechanisms, was thoroughly elucidated. The whole process by which tumor cells regulate other cells to help their own growth through RBP-ncRNA-exosome mechanisms was elucidated in detail. Here, we realized that the escape of the tumor is attributed not only to itself but also to other cells that assist it in escaping, which play a great auxiliary role. Therefore, it is essential to focus not only on tumor cells but also on their downstream target cells in oncology research, which can help us gain insight into the underlying mechanisms of tumors and find novel therapeutic approaches.

RBP-ncRNA-exosome mechanisms as novel targets for cancer biology and treatment

In this paper, RBP-ncRNA-exosome mechanisms are shown to regulate tumor multidrug resistance, metastasis, the immunosuppressive microenvironment, angiogenesis, and tumor progression. Among them, hnRNPA2B1, hnRNPA1, hnRNPK, HuR, PABPC1 and other RBPs are the main target proteins that mediate the sorting of ncRNAs into exosomes. These RBPs mainly bind to specific motifs on ncRNAs to mediate the loading of ncRNAs into exosomes and subsequently convey tumor information through exosomal transport. Simultaneously, these RBPs are also potential markers for predicting tumors. For example, the abnormal expression of the hnRNP family, observed in gastric, colorectal, esophageal, liver, and pancreatic cancers, could contribute to early diagnosis [126]. The high expression of hnRNPA1 promoted invasive metastasis of gastric cancer cells [127], and the overexpression of hnRNPA2B1 drived colorectal cancer metastasis [128] and esophageal cancer progression [129]. PABPC1 was aberrantly expressed in tumor tissues of lung, stomach, breast, liver and esophageal cancers and can be used as a prospective biomarker for tumor therapy [130]. HuR stabilized ncRNAs to accelerate the progression of gallbladder cancer [131]. Consequently, targeting these RBPs, destroying the binding motif between RBPs and ncRNAs or inhibiting exosome formation is conducive to blocking the RBP-ncRNA-exosome mechanism for anti-tumor purposes and is a potential clinical therapeutic target.

RBP as targets for cancer treatment

The RBP-ncRNA-exosome mechanism, mainly involving hnRNPA2B1, hnRNPA1, hnRNPK, HuR, and PABPC1, has been found to be engaged in promoting tumor development. Accordingly, identifying drugs that can effectively suppress these RBPs presents a promising therapeutic strategy. Cotyledon orbiculate, a small shrub with fleshy leaves belonging to the Crassulaceae family, was found to alter the ability of hnRNPA2B1 to bind mRNA and induced apoptosis in colorectal cancer cells by splicing hnRNPA2B1 from the B1 isoform to the A2 isoform [132]. Esculetin inhibited the export of the hnRNPA1/mRNA complex from the nucleus to the cytoplasm by downregulating hnRNPA1 protein expression. This, in turn, downregulated the transcription and translation of BCLXL and XIAP mRNA, effectively suppressing endometrial cancer proliferation [133]. Quercetin, a natural polyphenol compound, bound to the C-terminal region of hnRNPA1 protein and impaired the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm. This led to the accumulation of hnRNPA1 in the cytoplasm, resulting in downregulation of cIAP1 protein levels and inhibition of prostate cancer proliferation [134]. Quercetin was shown to reduce hnRNPA1 expression, overcoming enzalutamide resistance in prostate cancer cells and enhancing the anti-tumor effect of BET inhibitors [135], [136].

At the same time, quercetin also inhibited the activity of the HuR protein to prevent the migration and progression of triple-negative breast cancer cells [137]. Muscone derivative ZM-32 prevented HuR RRM1/2-VEGF-A mRNA complex formation by competitively binding to the RRM1/2 structural domain on HuR to exert anti-breast cancer angiogenesis effects [138]. Another drug, suramin, also competed with the HuR protein to break the binding ability of HuR to mRNA to suppress progressive oral cancer [139].

Nujiangexathone A (NJXA), a novel compound from Garcinia nujiangensis, inhibited cervical cancer growth by downregulating hnRNPK protein expression [140]. Withanone and withaferin A, the active ingredients extracted from Ashwagandha (an edible herb), were combined to depress hnRNPK transcription. The group also found that withanone in combination with cucurbitacin B decreased hnRNPK protein levels, preventing tumor metastasis and angiogenesis [141], [142]. The above small molecule compounds have been proven to block the binding of RBPs to mRNA by inhibiting the expression and activity of RBPs, weakened the ability of RBPs to shuttle between the nucleus and cytoplasm, competitively bound to RBPs, or directly disrupted the binding ability of RBPs to mRNA. Ultimately, these compounds inhibited the downstream pro-cancer signaling pathways to achieve antitumor effects. Hence, targeting RBPs may be a potential approach for antitumor therapy.

The binding motifs of ncRNAs as potential novel therapeutic targets

We have found that RBPs bind to specific motifs on ncRNAs to mediate ncRNAs assembly into exosomes. A specific class of motifs, called RAFT motifs, has also been identified. These motifs bound to the lipid raft region of the exosome membrane [143], and ncRNA possessing such motifs have been found to be more readily sorted into exosomes, and disruption of these motifs can inhibit ncRNA entry into exosomes [144], [145].

NcRNAs not only affect disease progression but can also be used in clinical therapy. Some ncRNAs are currently being used for the treatment of certain diseases. For example, the RNAi drug patisiran, a siRNA that specifically silenced the TTR gene to treat hereditary transthyretin amyloidosis (hATTR) combined with polyneuropathy [146]. Exosomes as carrier tools [147] have been reported to have extraordinary potential in drug delivery [148] and have also been recognized to hold great promise in oncology therapy [149].

Consequently, we believe that these motifs can be used to modify ncRNAs by inserting specific motifs into therapeutic ncRNAs. This modification aims to increase the affinity between therapeutic ncRNAs and exosomes so that they can be effectively sorted into exosomes and then targeted to recipient cells through the transport function of exosomes for anti-tumor purposes. Nevertheless, there is still a huge gap in the research on modifying the binding motifs of ncRNAs. Research and development in this area may be a novel technological tool for antitumor therapy.

Targeted exosome formation and synthetic manipulation

The main function of exosomes is to serve as carriers for transport, and their ability to regulate target cells is mainly derived from the various cargoes in exosomes. Here, we mainly discuss exo-ncRNAs. As mentioned earlier, ncRNAs can play dual roles in disease progression as well as disease treatment. Therefore, it is vital to consider exo-ncRNAs from two perspectives: i) exo-ncRNAs that impede tumor progression can be leveraged by enhancing their secretion or through artificial synthesis, and ii) exo-ncRNAs that facilitate tumor progression can be counteracted by inhibiting their production.

Some exo-ncRNAs have been declared to have cancer-suppressive effects. For example, exo-LncPTENP1, secreted by normal cells, blocked the progression of BCa [67]. Similarly, exo-miR-139, released by CAFs, inhibited the advancement of GC [150]. On the other hand, promoting the secretion of anti-cancer exo-ncRNAs has proved to be an effective anti-tumor strategy. Hyperthermia was reported to enhance exosome secretion in adriamycin-resistant MCF-7/ADR cells by modulating Rab7b, consequently improving drug sensitivity [151]. Calendula officinalis (SC), a natural compound isolated from Calendula officinalis, was reported to inhibit the growth of a variety of tumors [152], [153]. Interestingly, it now was found to act as a promoter of exosome production [154]. Since there are only a few naturally occurring drugs that stimulate exosome release, further exploration of additional drugs or genetic technologies is necessary to address the current limitations in this field.

In addition, synthetic exo-ncRNAs show promising potential as a therapeutic treatment for cancer. Several studies have now applied synthetic exo-ncRNAs to oncology therapy and have demonstrated promising anti-tumor effects in cellular and clinical experiments. For example, Liang et al. [155] utilized electroporation to co-deliver miR-21 inhibitors and 5-FU into exosomes. The engineered exosomes, containing both 5-FU and miR-21 inhibitors, were then incubated with drug-resistant colon cancer cells at 37 °C to reverse drug resistance. Similarly, Ramazan et al. [156] employed a modified calcium chloride method to load miR-375-3p mimic into exosomes, which effectively inhibited the migration and invasion abilities of HT-29 and SW480 cells, and ultimately effectively regulated EMT in colon cancer cells. Furthermore, several therapeutic exosomes that have entered clinical trials. M.D. Anderson Cancer Center conducted a clinical trial about iExosomes in treating participants with metastatic pancreas cancer with krasG12D mutation in 2018 (NCT03608631). This phase I trial studies used mesenchymal stromal cells-derived exosomes with KrasG12D siRNA (iExosomes) to treat participants with pancreatic cancer with KrasG12D mutation that has spread to other places in the body to investigate the dosage, toxicity, and anti-tumor efficacy of iExosomes in the treatment of pancreatic cancer. And the anti-tumor effect of iExosome has been well demonstrated in mouse experiments [157]. Another ongoing clinical trial (NCT01159288) involves the development of an immunotherapy using methoxycyclophosphamide (mCTX) followed by inoculation of dendritic cell exosomes (Dex) loaded with tumor antigen. The outcome of these clinical trials holds great promise. Overall, synthetic exo-ncRNAs show considerable potential as a therapeutic approach in cancer treatment. The results from these clinical trials will provide valuable insights and contribute to the advancement of cancer therapies.

Current studies have also found that a large proportion of exo-ncRNAs have a role in promoting tumor development. To effectively target exo-ncRNAs that promote cancer, the most promising approach is to inhibit their production. The relevant pathways in the process of exo-ncRNA production have been summarized in the previous text. Thus, targeting the exo-ncRNA production process is a worthwhile therapeutic measure. A number of compounds have been developed to suppress exosome production [158], and their main mechanism of action is to target proteins important in exosome formation. For example, tipifarnib targeted RAB27a, Alix, and nSMase2 [159], GW4869 targeted nSMase2 [160], and proton pump inhibitors (PPI) targeted V-ATPases [161], [162]. Natural drugs also suppressed exosome release, such as cannabidiol (CBD), which was derived from cannabis and blocked the release of exosomes and micro-vesicles (EMVs) to exert antitumor effects [163]. Surprisingly, Amrita et al. [164] searched for a natural microbial metabolite, manumycin-A (MA), which inhibited exosome formation and tumor progression in castration-resistant PC (CRPC) cells by reducing the expression levels of Alix and HRs of ESCRT-0.

In conclusion, we believe that exosome-targeted anti-tumor therapy is a double-edged sword that can promote cancer on one hand and suppress it on the other. For researchers, it is necessary to first understand the specific functions of exo-ncRNAs before developing personalized therapeutic regimens. Exosomes from different tumor cell sources vary greatly, which means that there are great challenges in applying exosomes to a wide range of clinical treatments. However, we believe that using genetic engineering to assemble personalized therapeutic exosomes for different patients holds a better prospect.

Discussion

Over the past few decades, tumor research has evolved from solely examining tumor cells to exploring the relationship between tumors and their microenvironment. An increasing number of researchers now recognize that tumor development is not solely dependent on the tumor itself; rather, it is closely intertwined with other cells in the tumor microenvironment.

Exosomes serve as the primary transport vehicle for tumor cells to deliver messages, and studies have demonstrated that tumor cells selectively package ncRNAs into exosomes to regulate other cells. Additionally, some studies have shown that RBPs can mediate the production of exo-ncRNAs, with a few confirming that binding between specific motifs on ncRNAs forms complexes. However, it remains unclear how such complexes enter exosomes, and only a select few have postulated that this process is related to specific motifs carried by ncRNAs that increase affinity with exosomes. Despite some research on this topic, there are still many unanswered questions and a scarcity of relevant studies, leaving this mechanism largely speculative. Consequently, additional research is needed to better understand this complex process.

The comprehensive investigation of the mechanisms involving RBPs, ncRNAs, and exosomes sheds light on the specific process governing tumor regulation of other cells. However, current studies have only identified a small fraction of the RBPs involved in these mechanisms, leaving the majority of them unexplored. The search for RBPs that regulate these mechanisms presents a promising therapeutic target. Additionally, exo-ncRNAs have been observed to selectively target receptor cells, possibly through receptors on the exosome membrane. The mechanisms behind this phenomenon remain unclear, representing a significant gap in knowledge. Uncovering these specific mechanisms would improve the treatment of exosomes and address issues related to exosome targeting and off-target effects.

Recent advances in the study of RBPs, ncRNAs, and exosomes have prompted new ideas for therapeutic interventions in oncology. Abnormal expression of certain RBPs in tumors can be directly targeted with antitumor therapies, while drugs that effectively inhibit RBP function and expression can be developed.

In parallel, the development of therapeutic exo-ncRNAs remains a very promising anti-tumor strategy. As a vehicle for drug delivery, exosomes have advantages that other drug delivery systems cannot surpass, such as i) Biocompatibility: Exosomes are natural EVs that are derived from cells, making them inherently biocompatible and less likely to induce immune responses or toxicity compared to synthetic drug delivery systems [5], [165], [166], ii) Targeting Ability: Exosomes possess inherent targeting capabilities owing to the presence of specific surface proteins and ligands, which can be engineered or modified to enhance their targeting to specific cell types or tissues. This can result in improved drug accumulation in desired target sites and reduced off-target effects [167], iii) Stability and Long Circulation Time: Exosomes exhibit good stability in the circulation and extended half-life compared to free drugs or other delivery systems, as they are protected by a lipid bilayer membrane. This extended circulation time allows for increased drug availability and accumulation at target sites [168], iv) Natural Cargo Delivery: Exosomes naturally transport various biomolecules, including proteins, lipids, and nucleic acids, which makes them suitable carriers for delivering therapeutic payloads such as drugs or nucleic acids, including siRNA or miRNA. This cargo delivery capability allows for versatile applications in drug delivery and gene therapy [169], v) Intercellular Communication: Exosomes actively participate in intercellular communication by transferring their cargoes, including therapeutic agents, to recipient cells. This communication facilitates the modulation of biological processes, making exosomes suitable for targeted therapy or influencing disease progression [170]. Secondly, anti-tumor ncRNAs have the advantages of extensive involvement in gene regulation and small size, which are convenient for loading into exosomes.

Development of therapeutic exo-ncRNA modalities based on the strengths of the exosomes and ncRNAs is theoretically a promising therapeutic approach. Plenty of therapeutic exo-ncRNAs are now being discovered and synthesized and have shown promising anti-tumor effects in cellular and animal experiments. However, there is only 1 clinical trial of therapeutic exo-ncRNAs (iExosame mentioned above) in ongoing currently in the ClinicalTrials.gov (accessed on October 2023), and the results of that study are not yet available. It is still taking a long time for therapeutic exosomes to be formally applied to clinical patients, and many difficulties need to be overcome, such as low production yield, inconsistent therapeutic outcomes, and low targeting efficiency [171].

First, exosome production and quality control. To be translated into the clinic, both the production and quality of exosomes need to be addressed first. At present, experimental exosomes are mainly extracted from human-derived cells, and exosomes are mainly divided into natural exosomes and artificial exosomes. For natural exosomes, Tangential flow filtration (TFF) in combination with 3D culture [172], mechanical forces [173]), acoustic stimulation [174], hypoxia [175], acidity [176] and glucose starvation [177] are reported to improve the production yield of natural exosomes, and small molecule modulators such as norepinephrine [178] and N-methyldopamine [179] also promote exosome yield. However, large batches of cultured cells are still required and exosomes are extracted by a variety of complex isolation methods. Most mammalian cells release low quantities of natural exosomes, and although various engineering techniques have helped to increase exosome production capacity, the long cycle time for culturing cells is costly and economically inefficient. Interestingly, there are studies that have been able to extract exosomes from milk and plants, and milk exosomes can survive strongly acidic conditions in the stomach and degradation in the gut [180], plants exosomes can extract from the grape, grapefruit, ginger, lemon, and broccoli, which may be new ways to overcome natural exosome production. For artificial exosomes, the main methods for manufacturing artificial exosomes, including i) extrusion of human-derived cells [181], upon extrusion, lipid bilayer fragments spontaneously assemble into spherical shapes, which are then passed through filters of different pore sizes to prepare exosomes of different diameter sizes. ii) nitrogen cavitation, cells are placed in a nitrogen cavitation vessel, filled with nitrogen to increase the pressure in the chamber, where the nitrogen can enter the cells to form small bubbles, and after a rapid depressurization, vesicles are produced when the cell membrane ruptures [182]. iii) alkaline solution, alkaline solutions can dissolve cell membranes. After neutralizing the alkaline solution, the membrane components are assembled by sonication to form vesicles [183]. Although synthetic exosomes can be produced in large quantities and rapidly, artificial exosomes are not able to control cargo sorting through a variety of mechanisms and the vesicle contents are not loaded stably and efficiently, which is therefore a disadvantage of artificial exosomes. Therefore, new technologies need to be continuously developed.

Second, exosomes are efficiently loaded with ncRNA. Currently it is acceptable to load ncRNAs into exosomes by Electroporation, incubation at 37 °C, sonication and transfection reagents, including Lipofectamine 2000, Lipofectamine 3000, Lipofectamine RNAiMAX, HiPerFect transfection reagent, Exo-fect Exosome Transfection Reagent and so on, but the import efficiency remains low. Based on the RBP-ncRNA-exosome mechanism discussed above, RBPs play a vital role in exosome cargo sorting. Therefore, exosomal membrane proteins are fused to proteins of RBPs, which include HuR, hnRNPA1, hnRNRK, hnRNPQ, SRSF1, hnRNPA2B1, YBX1, hnRNPC1, and PUM2 as mentioned above. Meanwhile, the addition of corresponding specific binding motifs to therapeutic ncRNAs, such as hEXO motif, EXO motifs GGAG, and AsUGnA motif might be the key to help therapeutic ncRNAs enter exosomes. Li et al. [184] fused CD9 (exosomal membrane protein) with HuR, HuR interacted with miR-155 with a relatively high affinity. When miR-155 was overexpressed, the fused CD9-HuR successfully enriched miR-155 into exosomes. Li et al. [185] also fused the C-terminus of Lamp2b (both localized in exosome and lysosome) with HuR, this fusion protein was able to be incorporated into the exosomes and successfully recruiting specific RNA to the lysosomes for degradation. The above studies genetically engineered exosomes for better sorting of ncRNAs. In summary, exosomal membrane protein fuse with RBPs is expected to be a promising therapeutic approach and provide new ideas for clinical translation of engineered exosomes. However, it is also important to consider whether the fused RBPs are potentially promotive to tumor cells, some of the RBPs affect tumor progression after all.

Ultimately, improving the targeting ability of therapeutic exosomes. The insufficient targeting ability of exosomes limits their clinical application. In order to enhance the targeting ability of exosomes, the current main method is to add proteins or peptides that can bind to target cells on the surface of the exosome membrane. For example, Lin et al. [186] fused the peptide HSTP1, which had better targeted ability to activate hepatic stellate cells (aHSCs), with exosome-enriched membrane protein (Lamp2b), and the modified exosome (HSTP1-Exos) could specifically target the aHSC region and enhanced the therapeutic effect on liver fibrosis. Tian et al. [187] conjugated the c(RGDyK) peptide to the exosome surface, the engineered c(RGDyK)-conjugated exosomes (cRGD-Exo) target the lesion region of the ischemic brain, meanwhile loaded curcumin onto the cRGD-Exo to inhibit the inflammatory response and cellular apoptosis in the lesion region. The overexpression of HER2 was usually associated with cell survival and tumor progression in various cancers, Wang et al. [188] packaged 293-miR-HER2 (a miRNA designed to block HER2 synthesis) into exosomes while adding a peptide to the surface of the exosomes, which adhered to HER2 on the surface of cancer cells, and then internalized into the cells to block HER2 synthesis. The above study provides a new idea to add peptides on the surface of exosomes to increase their targeting ability and load ncRNAs into exosomes to exert the therapeutic effect of exosomes, which perfectly combines the targeting and therapeutic abilities, and brings the clinical translation of exosomes one step further.

In summary, therapeutic exo-ncRNAs have now been validated in cellular experiments, and one clinical study is ongoing, the results are worth waiting for. However, clinical translation of therapeutic ncRNAs still faces some difficulties. For example, the loading efficiency of ncRNAs is not efficient, although researchers have used methods such as electroporation to package therapeutic ncRNAs with anti-tumor properties into exosomes. New loading methods are worth developing, such as inserting specific motifs on ncRNAs, and according to the specific motifs, selecting RBPs that bind to them, fusing the RBPs with exosomal membrane proteins, so that the ncRNAs can enter into the exosomes selectively through the RBPs. It is also necessary to increase the targeting ability of therapeutic ncRNAs, such as the addition of peptides to the exosome membrane, which can target the receptor cells, so that the exosomes are internalized by the receptor cells to exert an anti-tumor effect. However, these studies are currently scarce, this area warrants further research and exploration. Exosomes are efficient transport vehicles, and genetic engineering can be used to load anti-tumor drugs and gene fragments for tumor treatment. However, issues surrounding loading efficiency, stability, targeting, and mass production of exosomes persist and require technical solutions. Furthermore, some tumor cell-derived exosomes have procancer effects, and their production and secretion must be blocked. Although few natural drugs exist that can inhibit exosome formation, continued efforts are necessary to explore more drugs. The abovementioned approaches hold promise for developing new strategies and explorations in oncological treatment.

Conclusion

Exosomes play a vital role in tumorigenesis, affecting the growth and survival of tumor cells and their response to therapies mainly through ncRNAs in exosomes. However, the sorting of ncRNAs into exosomes is a complex and regulated process that is influenced by multiple factors, including RBPs and RNA modifications. Targeting these mechanisms could lead to the development of new cancer therapies. Further research is needed to understand the mechanisms underlying the sorting of ncRNAs into exosomes and how this process can be exploited for cancer treatment.

Compliance with Ethics Requirements.

This article does not contain any studies with human or animal subjects.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Ting Wang, born in 1994 in Sichuan province, China, is currently dedicated to her pursuit of a Doctoral degree at Shanghai University of Traditional Chinese Medicine in Shanghai, China. Her primary research revolves around identifying traditional Chinese medicines and their derivatives that can effectively target RNA binding proteins with anti-tumor properties. Additionally, she aims to uncover the unique mechanisms through which these traditional Chinese medicine molecules exhibit anti-tumor effects by specifically targeting RNA binding proteins, including conducting related studies on exosomes. During her master's program, Ting Wang was recognized with the esteemed national scholarship and honored as the Outstanding Graduate of Shanghai.

Hui Zhang, Ph.D., is a researcher with expertise in posttranscriptional gene regulation and biomarkers of disease. He obtained his Ph.D. in Basic Medicine from Shanghai University of Traditional Chinese Medicine, China. Dr. Zhang’s research focuses on RNA-binding proteins, non-coding RNAs, and translational control. He is particularly interested in investigating the pharmacodynamic material basis of traditional Chinese medicine and exploring antitumor mechanisms and innovative drugs derived from these remedies. Dr. Zhang is committed to advancing our understanding of molecular biology and developing new approaches for disease diagnosis and treatment.