The crosstalk between circular RNAs and the tumor microenvironment in cancer metastasis

The “seed-soil” description proposed by Stephen Paget in 1889 [1] is the classical theory of cancer metastasis. Cancer cells move from the primary neoplasm to a target distant location. The host environment, described as the “soil”, is remodeled to facilitate tumor metastasis, and the “seed” represents the cancer cells colonizing a new site suitable for tumor growth and progression. The cancer invasion-metastasis cascade consists of a series of discrete biological processes. Initially, cancer cells invade the surrounding tissue and enter the circulation, followed by their extravasation into a new site where they evade immune surveillance and anoikis to develop into metastatic tumors [2]. Various studies have considered the influence of the tumor microenvironment (TME) on cancer metastasis. The TME is composed of stromal cells, immune cells, vasculature, and extracellular matrix (ECM) [3]. Pervasive stromal reprogramming and ECM remodeling in the TME play vital roles in cancer metastasis [4].

Circular RNAs (circRNAs) are a novel type of non-coding RNAs that form covalently closed loop structures without 5′-caps or 3′-polyadenylated tails. They are ubiquitous and play critical roles in carcinogenesis. Cumulative evidence has shown that circRNAs are closely associated with cancer metastasis [5‐7]. The interaction between circRNAs and the TME has significant clinical relevance to cancer therapy [8, 9]. Therefore, in the current review, we focus on the crosstalk between circRNAs and the TME in cancer metastasis.

CircRNAs are produced by back splicing, which is different from the classical splicing of linear RNAs. Exon skipping and intron pairing narrow the distance between splice sites and facilitate the back-splicing of pre-mRNA [10]. This results in circRNAs lacking 3′ and 5′ ends and being more stable. CircRNAs are mainly classified into four types: exon circRNA (ecRNA), circular intron RNA (ciRNA), exon-intron circRNA (ElciRNA) and tRNA intronic circRNA (tricRNA) [11]. RNA-binding proteins (RBPs) and the spliceosome can regulate the circRNA biogenesis, whereas some of them are regulated by the TME [12, 13]. For example, the expression of circARSP91 in human hepatocellular carcinoma (HCC) is suppressed via upregulation of ADAR1 p110 caused by the androgen receptor (AR) [12]. HNRNPC is another RBP involved in circRNA biogenesis during hypoxic stress in cervical, breast, and lung cancer cell lines [13]. The function of circRNAs depends on their cellular localization; in the cytoplasm, they can be translated into peptides and sponge miRNAs, while in the nucleus, they may bind RBPs to influence transcription and modulate targeted pathways [14].

The TME is composed of stromal cells, ECM components, immune cells, vasculature, other cell types, and various signaling entities, including exosomes [15]. The crosstalk between cancer cells and other TME components drives tumor progression and metastasis [16]. Cell types within the TME with specific roles in tumor metastasis include fibroblasts, endothelial cells, immune cells, adipocytes, and neuroendocrine (NE) cells. Apart from the ECM, other acellular components, such as extracellular vesicles (EVs) and cytokines released by tumor and non-tumor cells have also been identified [17‐19]. Tumor cells communicate with stromal cells to promote tumor metastasis as part of the invasion-metastasis cascade. Cancer-associated fibroblasts (CAFs) represent the largest proportion of stromal cells in the TME; interestingly, their origin remains unclear. CAFs are activated by growth factors and cytokine including transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF), which are released by tumor, stromal, and immune cells. Upon activation, CAFs secrete growth factors to support tumor progression, such as vascular endothelial growth factor (VEGF) that induces vascular permeability and angiogenesis. The YAP transcription factor is required for the CAF-mediated remodeling of the ECM to promote tumor progression [16]. In addition, CAFs can drive and direct cancer cell migration via fibronectin alignment [20]. Tumor-associated macrophages (TAMs), another important cell type within the TME, can be recruited by factors released from tumor cells and contribute to tumor progression during the metastatic cascade [21]. Moreover, they receive tumor-cell derived signals in the form of exosomes to promote metastasis [22]. Endothelial cells in the TME release adhesion molecules and chemokines to promote tumor progression, and can be stimulated by multiple factors secreted by tumor cells to promote angiogenesis, such as the basic fibroblast growth factor (bFGF) and VEGF [23, 24]. With regard to the acellular TME components, the ECM builds a three-dimensional network composed of collagen, fibronectin, elastin, proteoglycans, laminins, and other glycoproteins [18]. These components bind to cell surface receptors and transmit signals to cells from the ECM, thereby inducing tumor migration, differentiation, metabolic alterations, and other changes in cellular behavior [25]. A change in cell adhesion to the ECM is an important step in metastasis. Notably, the matrix metalloproteinase (MMP) family members are the main proteolytic enzymes to degrade the ECM component and promote tumor cell migration via the basement membrane (BM). MMPs can be produced by stromal and tumor cells. Specifically, MMP13 expression is mainly observed in CAFs, but this enzyme may also be synthesized by other cells to promote invasiveness and angiogenesis [26]. MMP10 supports lung cancer development and metastasis [27]. Recent studies have shown that primary tumor sites may send signals to distant homing locations via primary tumor-derived exosomes. The delivery of such signals may contribute to organ tropism, immune evasion, metastasis, and can be used to predict patient outcomes [16].

Invasion is the initial step of metastasis. During this process, tumor cells alleviate their adherence to neighboring cells and the ECM, followed by the degradation of surrounding tissues and the acquisition of enhanced motility to navigate through tissues [2].

Intravasation refers to cancer cells disseminating into the lumen of vasculature in order to travel to distant sites for metastasis. The endothelium poses a barrier for tumor cell intravasation [68]. TAMs are the main stromal cells to localize to blood vessels and help tumor cell intravasation [69]. Two TAM subsets are involved in intravasation: migratory macrophages are responsible for guiding cancer cells toward blood vessels, while sessile perivascular macrophages assist them into vessels. Monocytes are initially recruited via CCR2 signaling. Cancer cells induce TGF-β-dependent upregulation of C-X-C motif chemokine receptor 4 (CXCR4) on monocytes. Monocytes are then attracted by perivascular fibroblasts, which express CXCL12 around blood vessels, and bring along motile cancer cells [70]. Subsequently, migratory TAMs differentiate into perivascular macrophages to promote vascular leakiness and intravasation via Tie2-mediated attachment to endothelial cells [71]. Accordingly, CXCR4-expressing macrophages are called “transporters”, which carry tumor cells toward the blood vessel and present them to perivascular macrophages. The CXCR4 expression level was significantly higher in non-small cell lung cancer (NSCLC) tissues from the patients with lymph node metastasis than in those without metastasis. Interestingly, high CXCR4 expression in tumor tissue paralleled its expression in TAMs [72]. Recently, a study on circRNAs targeting CXCR4 expression has revealed the association between tumor cells and TAM infiltration. In pancreatic adenocarcinoma (PAAD), circ-UBAP2-hsa-miR-494 potentially targets CXCR4 and ZEB1 transcripts. Expression of CXCR4 and ZEB1 regulated PAAD by modulating the infiltration and function of immune cells, including TAMs [73]. CXCR4 is the chemokine receptor in tumors and chemokine CXCL12 is the specific ligand that interacts with it. As per the study described above, the CXCR4/CXCL12 interaction is important for migration to the blood. Tumor cells expressing CXCR4 may be attracted toward blood vessels by perivascular fibroblasts that express CXCL12 via synergizing with CXCR4-expressing TAMs. Notably, circRNAs targeting CXCR4 in tumor tissue represent a new insight in TAM-associated tumor invasion. Circ_0056618 was overexpressed in GC tumor tissues and cells. Inhibition of circ_0056618 suppressed tumor cell proliferation and metastasis by preventing sponging of miR-206 and the subsequent targeting of CXCR4 [74]. Furthermore, in NSCLC, circFGFR1 (hsa_circ_0084003) expression increased in cancer tissues and promoted cell migration, invasion, and immune evasion by interacting with miR-381-3p to target CXCR4 [75]. CircRNA-modulated CXCR4 expression may represent an important regulatory mechanism for TAM-mediated intravasation (Fig. 1) (Table 1).

During invasion and intravasation, tumor cells should avoid immune recognition in order to survive before extravasating. Some reviews have elaborately summarized the association among circRNAs TME immune cells and the immune escape [8, 9]. Herein, we focus on the crosstalk between circRNAs and immune cell function in relation to invasion. Tumor immunity mainly involves in T cells and dendritic cells (DCs), and exploits immune checkpoints, which are normally critical for the prevention of autoimmunity, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA4, CD152) and programmed cell death protein 1 (PD-1) [76]. Cytotoxic CD8+ and CD4+ Th1 T cells are the major antitumor immune effector cells, whose antitumor functions are amplified by immune checkpoint blockade antibodies such as CTLA4 inhibitors and PD-1/PD-L1 inhibitors [77]. PD-1 belongs to the B7/CD28 family and is expressed on activated T cells [78]. PD-1 binds to PD-L1 and PD-L2 to decrease T cell proliferation and induce apoptosis [17, 79]. In lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC), epithelial gene markers showed a general negative correlation with the expression of immune checkpoint genes, while the opposite was seen for mesenchymal gene markers [80]. In melanoma, subsets of tumors inherently resistant to immunotherapy demonstrated innate anti-PD-1 resistance (IPRES) [81]. Immune checkpoints seem to be the main factors leading to immune suppression-associated cell-cell adhesion. As previously reported, circRNAs directly influence PD-L1 expression. In CRC, an upregulation of hsa_circ_0020397 inhibited miR-138 activity by targeting telomerase reverse transcriptase (TERT) and PD-L1, and, consequently, regulated CRC cell viability, apoptosis, and invasion [82]. CDR1-AS can regulate cell surface PD-L1 protein levels independently of microRNA to promote the malignant behavior of CC cell lines [83] (Fig. 1) (Table 1).

Angiogenesis plays an important role in the process of intravasation. It is a crucial adaptive event, allowing for tumor growth beyond 1–2 mm³ [84]. Blood supply is required for the growth of metastases by providing oxygen, growth factors, nutrients, and metabolites. VEGF is the main inducer of angiogenesis and also induces vascular permeability [2]. Some circRNAs directly target VEGF expression. VEGFA (a member of the VEGF family) and the VEGFA receptor VEGFR-1/2 play key regulatory roles in tumor endothelial tube formation [85‐87]. In bladder cancer, circRNA-MYLK binds to miR-29a to target VEGFA, which normally activates its receptor VEGFR2 and the downstream Ras/ERK signaling pathway, contributing to tube formation and cytoskeletal rearrangement [88]. Circ0001429 interacts with miR-205-5p to regulate VEGFA expression, accelerating cell propagation, migration, and invasiveness in bladder cancer [89]. In glioma, circSCAF11 (hsa_circ_0098619) overexpression can sponge miR-42 to positively regulate the expression of transcription factor SP1, which can bind to the promoter region of VEGFA to enhance its expression [90]. Furthermore, VEGFR-positive tumor cells create an autocrine loop that also affects non-angiogenic aspects of tumor cell progression [91]. CircRNAs targeting VEGFR have been reported as upstream regulators of the autocrine loop. circZNF292 was confirmed to induce tube formation through its association with genes such as VEGFR-1/2 and p-VEGFR-1/2 in glioma [92].

Isthmin (ISM) is another endogenous angiogenesis inhibitor that has the capacity to block angiogenesis by VEGF and bFGF. It mitigates VEGF-stimulated endothelial cell proliferation without affecting endothelial cell migration and binds to integrins on the endothelial surface to support adhesion [93]. In HCC, hsa_circ_0091570 was downregulated and functioned as a sponge of miR-1307 to regulate the expression of ISM1 [94]. The tumor blood supply is influenced by vasculogenic mimicry (VM) that has the features of intratumoral channels lined by tumor cells and lacking inner endothelial cell lining. Notably, erythrocytes and plasma can flow in these vessel-like structures. VMs are connected with host vessels for blood supply [4]. In HCC, hypoxia-induced circZNF292 can affect the VM through SRY (sex determining region Y)-box 9 (SOX9) nuclear translocation, a downstream transcription factor in the Wnt/β-catenin pathway, to promote tumor progression [95] (Fig. 1) (Table 1).

After intravasation, tumor cells stuggle to survive through the circulatory system and eventually undergo arrest in the vasculature, followed by extravasation to form distant metastases. It is estimated that only 0.01% of cells that intravasate into circulation can form detectable metastases [96]. Interaction with platelets and anoikis resistance both play important roles during this process.

CircRNAs derived from exosomes can influence MMP2 expression to promote distant metastasis. CircPUM1 (circ_0000043) was highly expressed in epithelial ovarian cancer (EOC) tissues and upregulated MMP2 expression by sponging miR-6753-5p. CircPUM1 was found in cancer cell-derived exosomes and could be transferred to act on the peritoneum, regulating peritoneal expression of MMP2 and, thus, promoting tumor dissemination [106].

The endothelial barrier is the main factor for intravasation and extravasation during metastasis. The endothelial-tumor cell interaction is a central determinant of where exactly metastatic tumor cells will exit circulation [16]. CircRNAs can directly influence the endothelial barrier via exosomes. Circ-IRAS packaged in exosomes was upregulated in pancreatic cancer and entered microvascular endothelial cells to promote liver metastasis, intravasation, and tumor node-metastasis stage [107]. Circ-IARS could sponge miR-122 in endothelial cells to upregulate RhoA expression. Activated RhoA increases F-actin expression and reduces the expression of tight junction protein Zonula occludens-1 (ZO-1), resulting in an enhanced inward contractile force of cells, compromised endothelial barrier function, and increased endothelial permeability [107].

Exosomal circRNAs from local tumor cells can signal to endothelial cells in distant organs in order to facilitate metastasis. Endothelial cells in different organs can express different surface proteins. A number of peptides selectively bind the endothelia of target organs [108]. In cancers, specific proteins expressed on endothelial cells in the target organ may be crucial in determining the site of tumor cell arrest. Metadherin (MTDH), a protein that is overexpressed in metastatic breast cancer, can bind to the vasculature of the lung, a common site of breast cancer metastasis [109]. CircRNAs targeting MTDH expression have been studied. In bladder cancer, hsa_circ_0137439 upregulation in urine supernatant was correlated with advanced tumor stage, high tumor grade, increased lymph node metastasis, and muscular wall invasion. Mechanistically, knocking down hsa_circ_0137439 contributed to the inhibition of cell proliferation and migration via the hsa_circ_0137439/miR-142-5p/MTDH axis [110]. Hsa_circ_0137439 may be found in cell-free urine supernatant as exosomal cargo or within other molecular vehicles. Tumor cells can use pre-existing tissue blood vessels to support themselves in the absence of angiogenic processes. This new form of tumor metastasis is called vessel co-option and has been reported in tumors of the brain, liver, lung, and lymph nodes [111]. Exosomal circRNAs targeting specific endothelial cell proteins might provide new insights into the metastatic process although the exact mechanisms remain unclear.

TAMs are the main stromal cells in the TME and may therefore contribute to tumor cell homing. CXCR4 was frequently expressed on breast cancer cells and guided tumor cell spread to the bone, lung, and regional lymph nodes expressing high levels of its ligand CXCL12 [112]. The CXCR4/CXCL12 interaction is central to tumor cell homing to distant organs. However, the underlying mechanism is not very clear. Recently, TAMs have been found to play a dominant role in mediating CXCR4/CXCL12-induced liver metastasis of CRC via exosomal miRNAs. miR-25-3p, miR-130b-3p, and miR-425-5p were upregulated in CRC cells and could be transferred to macrophages via exosomes after activation of the CXCR4/CXCL12 axis. Macrophages would uptake these miRNAs and be transformed into TAMs via PI3K signaling, thereafter promoting metastasis by enhancing the epithelial-mesenchymal transition and secreting VEGF. These miRNAs were also examined in serum exosomes and correlated with CRC progression and metastasis [22]. Thus, it can be reckoned that local tumor cell homing may be mediated by exosomes, which can facilitate the crosstalk with other cells within the TME. LUAD patients with lymph node metastasis had higher plasma levels of exo-hsa_circRNA_0056616 and CXCR4 expression than those without [113]. CircRNAs can build up a ceRNA network with miRNAs. CXCR4-related exo-circRNAs were involved in TAM-mediated metastasis toward organs with high expression of CXCL12. Elevated circ_0020710 was observed in melanoma tissues. It promoted cell proliferation, migration, and invasion in vitro and tumor growth in vivo via the miR-370-3p/CXCL12 axis [114] (Table 1). The roles of circRNAs in CXCR4/CXCL12-mediated distant metastasis are depicted in Fig. 2.

In summary, we have reviewed multiple interactions between the TME and circRNAs involved in the metastatic process. These interactions form crucial regulatory networks that can be useful for predicting tumor progression. With the discovery of increasing numbers of circRNAs, the interaction between circRNAs and the TME has become a hot topic within molecular biology and oncology. A better understanding of this reciprocal cooperation is mandatory to identify important prognostic and predictive biomarkers for the development of novel cancer therapies. For example, one study found that primary tumor cells surrounded by platelets were localized to sites in which the epithelial-mesenchymal transition occurred based on molecular and morphological changes of HER2-negative breast cancer. Moreover, primary tumor cells associated with platelets exhibited chemoresistance to common anti-cancer drugs [115]. Understanding the mechanism through which platelet-associated exo-circRNAs contribute to tumor cell survival in circulation and extravasation to distant sites may provide important new insights. As an important cell type present within the TME, TAMs are central for the promotion of metastasis. The CXCR4/CXCL12 axis may play an important role in directing distant homing. As previously described, homing-associated circRNAs have been identified; however, the specific mechanism of TAM contributiing to tumor cell migration to vessels and survival in the circulation remains unclear. Personalized single-cell sequencing has emerged as a way of identifying and characterizing the heterogeneity within cell populations, which is important for disease pathogenesis. In the future, single-cell circRNA sequencing may play a vital role in identifying different factors involved in interactions between tumor cells and stromal cells in the TME contributing to metastasis.