So far, extracellular vesicles (EVs) have played an important role in the progression of multiple cancers by regulating the Wnt pathway. The migration of cancer cells in breast cancer is closely related to stromal mobilization, and studies have reported that this can be achieved by Wnt/planar cell polarity (Wnt/PCP)PCP signaling regulation by EVs ^[1][66]. In cervical cancer-derived EVs, Wnt7b mRNA is expressed at high levels. It could be transferred to human umbilical vein endothelial cells to promote Wnt7b synthesis in recipient cells, affecting the proliferation and angiogenesis of recipient cells by regulating the β-catenin signaling, ultimately promoting tumor progression ^[2][67]. However, the regulation of Wnt signaling by EVs in hepatocellular carcinoma (HCC) is beginning to be researched. Current studies have found that Evs can transfer contents between different HCC cells and activate Wnt signaling, thereby regulating the biological behavior of recipient cells and promoting the progression of malignant phenotypes. In addition, stromal cell-derived Evs can also deliver contents to HCC cells and other stromal cell types. These, on the one hand, lead to HCC resistance and, on the other hand, lead to immune cells expressing more exhaustion markers and fewer effector molecules. The following contentsis review summarizes the current status and mechanisms underlying EV regulation of Wnt signaling in HCC to help us gain a better understanding of this phenomenon and provide ideas for deriving new therapeutic strategies for HCC.

The regulation of Wnt signaling by Evs in HCC is primarily achieved by modulating the classical pathway. Several studies have found that Evs regulate Wnt signaling via transport between different types of HCC cells (Table 1). Solid tumor cells are often surrounded by a hypoxic microenvironment ^[3][68], a condition favoring metastatic tumor progression ^[4][69]. Yu et al. discovered that this hypoxic environment contributes to exosome production in HCC. Furthermore, exosomes secreted by hypoxic tumor cells were found to promote malignant phenotypes of normoxic cells, including their proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT). A study of the underlying mechanisms found that miR-1273f levels increased significantly in exosomes secreted by Huh7 cells under a hypoxic environment, and recipient HCC cells showed enhanced malignancy that was closely related to this miRNA in vitro. The search for downstream targets revealed that miR-1273f functions by targeting a tumor suppressor gene (LHX6) ^[5][70]. Several previous studies have demonstrated that LHX6 can inhibit the progression of multiple tumor types, including glioma, lung cancer, and breast cancer. LHX6 regulates Wnt/β-catenin signaling by inhibiting β-catenin expression, thereby inhibiting tumor progression. Furthermore, this process can be reversed by the downregulation of LHX6 by miR-1273f ^[6][7][8][9][71,72,73,74].

Accumulating evidence suggests that via exosome-mediated material transfer, high-metastatic HCC cells can enhance the migration and invasion abilities of low-metastatic HCC cells ^[10][11][12][75,76,77]. An in vitro study that isolated exosomes from highly metastatic HCC cells and used them to treat low-metastatic HCC cells observed reduced apoptosis and enhanced proliferative capacity in the latter. The abilities of the recipient cells to invade and migrate were also simultaneously enhanced. Animal experiments concluded that the tumorigenic rate of mice injected with exosome-treated tumor cells increased significantly, and multiple metastases were found in the liver and lungs, suggesting enhanced tumor metastasis ability. Interestingly, high levels of miR-25 were expressed in both highly metastatic HCC cell-derived exosomes and recipient cells. Mechanistically, miR-25 activates Wnt/β-catenin signaling by reducing the expression level of serine/threonine-protein kinase 1 (SIK1) ^[13][78].

Yu et al. analyzed paired tumor tissues and adjacent non-tumor tissues of HCC patients, including 35 paraffin-embedded and 40 frozen specimens. They found that DEAD-box helicase 55 (DDX55) was overexpressed in HCC tissues, mainly located in the nucleus and cytoplasm. They studied its function and mechanism and found that DDX55 interacted with Bromodomain-containing protein 4 (BRD4) to form a transcriptional regulatory complex that positively regulated the transcription of PIK3CA, a core gene regulating Akt and Wnt pathways. Subsequently, the PI3K/Akt/GSK-3β pathway was activated, stabilizing β-catenin and regulating the downstream gene expression, ultimately promoting cell cycle progression and EMT. On further investigation, it was noted that DDX55 could be transferred from HCC cells with relatively high DDX55 expression to those with relatively low DDX55 expression via exosomes. They also observed that these DDX55-rich exosomes could also be transferred into endothelial cells, inducing increased angiogenesis, and the underlying mechanism might be related to the activation of the β-catenin signal ^[14][79].

In one study, serum samples were collected from randomized healthy donors, patients with chronic HBV infection, and patients with liver disease (cirrhosis, early HCC, and late HCC) without any treatment. Then exosomes were extracted from the serum. Interestingly, the researchers found that patients with late HCC had the greatest number of serum exosomes. Then they co-cultured these exosomes with Huh7 cell lines and were surprised to find that exosomes from HCC patients increased the growth and motility of Huh7 cells. In addition, exosomes derived from patients with late HCC were superior to those from cirrhotic patients and even more superior than those from patients with early HCC. This finding was linked to the promotion of malignant biological behavior of HCC cell lines by exosomes in vitro or the promotion of tumor formation and metastasis in orthotopic liver transplantation models in vivo. The research focused on the underlying mechanism and found that the polymeric immunoglobulin receptor (pIgR) was abundant in exosomes derived from advanced HCC patients. These exosomes were primarily secreted by tumor cells. When recipient HCC cells took up these exosomes, β-catenin was transferred from the cytoplasm to the nucleus, which was closely related to the activation of PDK1/Akt/GSK-3β signaling. These exosomes ultimately enabled the recipient cells to acquire stem cell properties and enhance their invasion and metastasis abilities ^[15][80].

Among the many characteristics of cancers, abnormal sialylation has often been found to be important for the progression of tumors ^[16][81]. Wang et al. discovered that when α2,6-sialyltransferase I (ST6Gal-I) was silenced, the expression level of CD63, a regulator of exosome production and secretion, decreased. The level of α2,6-sialylated glycoconjugates on the surface of HCC-derived exosomes also decreased, reducing the internalization of exosomes by recipient cells. Furthermore, silencing of this molecule could attenuate the highly metastatic attributes of HCC-derived exosomes on the malignant biological behavior of low metastatic HCC cells, related to the suppressed function of highly metastatic HCC-derived exosomes, resulting in the failure of Akt/GSK-3β/β-catenin signaling and JNK1/2 signaling ^[17][82].

[86,87]. Liu et al. found that miR-92a-2-5p is highly expressed in M2 macrophages and can be secreted into exosomes. When the exosomes were used to treat HCC cells, they found that the invasive ability of the recipient cells was enhanced. Then they explored the mechanism and observed reduced androgen receptor (AR) expression in recipient cells caused by the inhibition of AR mRNA translation by miR-92a-2-5p. The PHLPP/p-AKT/β-catenin signaling was altered by miR-92a-2-5p/AR axis ^[23][88]. Numerous studies have confirmed the association of tumor immune escape with CD8+ T cell dysfunction and severe depletion ^[24][25][26][89,90,91]. Pu et al. found increased expression of miR-21-5p in M2 macrophage-derived exosomes. After CD8+ T cells were treated with these exosomes, the levels of CD8+ T cell surface exhaustion markers, such as PD-1 and T cell immunoglobulin and mucin domain-containing protein 3, were significantly increased, and that of tumor suppressor factors, such as interferon-γ, interleukin-2, and tumor necrosis factor-α, were reduced. In animal experiments, it was found that exosomes can promote HCC tumorigenesis and malignant behavior and reduce the infiltration of CD8+ T cells in HCC tissues. Mechanistically, miR-21-5p acted mainly by binding to YOD1 mRNA, reducing YOD1 level, and thereby activating YAP/β-catenin signaling ^[27][92]. Previous studies had found that the activity of YOD1 was essential for antigen presentation, which was also the key associated with the effective functioning of antigen-specific CD8+ T cells ^[28][93]. In other tumor types, YAP has been found to induce the expression and secretion of multiple chemokines, which recruits myeloid-derived suppressor cells (MDSCs) and inhibits the function of cytotoxic T lymphocytes (CTLs), thereby promoting the formation of immunosuppressive TME ^[29][30][94,95].

In addition to transferring cargos between tumor cells, EVs also act as a bridge between tumor cells and stromal cells and even between different types of stromal cells (Table 2). CAFs are important components of TME and are important for cancer progression and resisting treatment. Qin et al. discovered that Gremlin 1 was highly expressed in CAF-derived exosomes, which could be transferred into HCC cells to promote invasion, migration, and EMT of the recipient cells by activating Wnt/β-catenin signaling. As EMT is closely related to sorafenib resistance, they also investigated the effect of exosome-mediated Gremlin 1 transfer on the sensitivity of HCC cells to sorafenib. Their results indicated that HCC cells were less sensitive to sorafenib after exosome treatment through a similar mechanism, as described above ^[18][83]. Additionally, another study found that the expression of miR-320a notably decreased in HCC tissues and liver cancer cell lines. Lu et al. found that miR-320a overexpression in the HepG2 cells exhibited a marked inhibitory effect on cell proliferation. Mechanically, miR-320a exerted its tumor-suppressive function via downregulating the Wnt/β-catenin signaling pathway. However, in this study, for the first time, miR-320a was reported as a negative regulator of β-catenin ^[19][84]. Additionally, Zhang et al. found a significant reduction in the miR-320a expression in CAF-derived exosomes. CAF-mediated HCC tumor progression is partially related to the loss of miR-320a in the exosomes of CAFs, and transferring stromal cell-derived miR-320a might be a potential treatment option against HCC progression ^[20][85].

Macrophages are an important part of immune cells and mainly include two subtypes, namely: M1 type and M2 type. Among them, the M1 type plays a tumor suppressor role, whereas the M2 type has tumor-promoting properties, TAMs, with the M2 phenotype, in many tumors associated with malignant tumor progression ^[21][22]

Previous studies showed that the cellular localization of β-catenin was closely related to its function. When accumulated in the nucleus, it promoted EMT by mediating the expression of target genes. However, when localized on the plasma membrane (PM), it interacted with E-cadherin to increase cell-to-cell adhesion ^[31][32][96,97]. Several studies have found that EVs alter the cellular localization of β-catenin, which modulates β-catenin signaling in HCC (Table 3). Han et al. found that Vps4A overexpression could mediate β-catenin relocalization, promoting β-catenin expression on PM and secreting it out of the cell through exosomes, thereby reducing nuclear β-catenin level, which downregulated β-catenin signaling and suppressed EMT ^[33][98]. Then the research group found a similar phenomenon on another molecule. They found that exosomes derived from p62-overexpressing HCC cells can be taken up by p62-low-expressing HCC cells, following which p62 increased intracellular GSK3β levels, finally promoting β-catenin degradation. Unexpectedly, they observed that high levels of p62 promoted the malignant phenotype of HCC, including cell proliferation, migration, and invasion. This may be related to the p62-mediated extracellular secretion of exosome-coated β-catenin ^[34][99].