New insights into the regulatory role of microRNA in tumor angiogenesis and clinical implications

Inducing angiogenesis is one of the hallmarks of cancer [1]. Tumor-associated neovasculature is important for delivering nutrients and oxygen to growing tumors, and also playing key roles in multi-aspect of tumor biology, including tumor dissemination/metastasis [2], metabolic deregulation [3] and cancer stem cell maintenance [4, 5]. For its fundamental role in tumor growth and progression, angiogenesis has become an appealing target in cancer treatment.

Angiogenesis is a complex process by which new blood vessels are formed from pre-existing ones by sprouting, remodeling and expansion of primary vascular networks [6]. In normal conditions, following this morphogenesis, the vasculature becomes largely quiescent. However, within tumors, an “angiogenic switch” is always activated, causing continuous generation of new vessels [7]. The “angiogenic switch” is governed by pro-angiogenic and anti-angiogenic signals elicited by tumor cells or tumor microenvironment [8, 9]. When pro-angiogenic signals are activated or anti-angiogenic signals are inhibited, the “angiogenic switch” is turned on. Understanding the regulation of tumor angiogenesis is critical to developing therapeutic strategies against cancer.

MicroRNAs (miRNAs) are small non-coding RNAs [10], that negatively regulate gene expression at post-transcriptional level. Through sequence-specific interaction with the 3′-untranslated region (UTR) of target mRNA, miRNAs suppress gene expression via transcript degradation or inhibition of protein translation. To date, miRNAs have been reported to play key roles in diverse biological processes of cancer, including tumor growth, metastasis, angiogenesis and drug resistance [11‐14]. Extensive studies have shown that miRNAs could modulate the function of endothelial cells (ECs) via non-cell-autonomous (genetic and epigenetic alterations in a cell affect the phenotypes of other cells through changing microenvironment or intercellular interaction) and cell-autonomous (genetic and epigenetic alterations in a cell affect the cell’s own phenotypes) manner during tumor angiogenesis. MiRNAs in tumor cells regulate the expression of pro- or anti-angiogenic factors, thereby modulating the proliferation and migration of ECs in a paracrine manner. Endothelial miRNAs regulate the response of ECs to multiple angiogenic stimuli mainly by targeting growth factor receptors and signaling molecules autonomously. Recently, extracellular vesicles (EVs) are emerging as mediators of intercellular communication [15]. Cancer cell-derived miRNA-containing EVs could be transferred to ECs where they induce pro-angiogenic effects. Importantly, several miRNAs have been shown to serve as predictive biomarkers for anti-angiogenic therapy response, especially as non-invasive biomarkers in the circulation. Furthermore, with the development of RNA delivery technology, miRNA-based interventions may act as novel therapeutic means to target tumor angiogenesis.

In our review, we will summarize the non-cell-autonomous and cell-autonomous roles of miRNAs, as well as EV-transferred miRNAs in the regulation of tumor angiogenesis. Moreover, we highlight the clinical implications of miRNAs as predictive biomarkers for anti-angiogenic therapy response in both tissue and circulation, and several miRNA delivery approaches that have been applied to suppress angiogenesis in vivo.

Angiogenesis is under the control of pro-angiogenic and anti-angiogenic factors [7]. Tumor cells could produce and secrete such factors into surrounding environment to promote vessel growth. Endogenous activators and inhibitors are frequently targeted in tumor cells by miRNAs, which thereby regulate angiogenesis in a non-cell-autonomous manner (Table 1).

The importance of miRNAs in ECs was demonstrated by the silencing of Dicer, an enzyme responsible for miRNA maturation. Knockdown of Dicer in ECs inhibited cell proliferation, migration and cord formation [79], indicating that miRNAs are important in the function of ECs. Many studies have shown that miRNAs in ECs regulate the cellular response to angiogenic factors by targeting surface receptors and signaling molecules (Table 2).

The discovery of extracellular vesicles (EVs) represents an exciting area of research that offers novel mechanisms for intercellular communication. EVs, generally including exosomes (typically 30–100 nm) and microparticles or microvesicles (typically 100 nm-1 μm), are small secretory vesicles that contain proteins, DNA, and coding and non-coding RNAs [90]. EVs can be released from various cell types under physiological and pathological conditions and transfer their cargo to proximal or distal cells [91]. In many published articles, the collective term “microvesicles” was used for all the EVs between 30 and 1000 nm in- diameter.

Recent evidence has demonstrated that cancer cell-derived EVs can be transferred to ECs where they induce pro-angiogenic effects. Glioblastoma cells released exosomes containing mRNA, miRNAs and angiogenic proteins. These exosomes were taken up by brain microvascular endothelial cells and modulated angiogenesis [92]. A set of pro-angiogenic mRNAs and miRNAs were enriched in the microvesicles of CD105-positive renal cancer stem cells, therefore conferring an activated angiogenic phenotype to ECs [93].

MiRNAs are the most abundant non-coding RNAs enriched in EVs, and studies have also showed that EVs contain components of the RNA-induced silencing complex (RISC), suggesting potent regulatory role of EV-miRNA in recipient cells [94]. Several studies have reported the regulation of signaling pathways in ECs by miRNAs delivered via tumor-derived EVs (Table 3). MiR-494 containing EVs secreted by tumor cells were taken up by ECs and promoted the migration of EC via targeting of PTEN and subsequent activation of Akt/eNOS pathway [95]. EV-encapsulated miR-9 transferred from tumor cells to ECs reduced SOCS5 level, leading to JAK-STAT pathway activation to promote endothelial cell migration and sprout [96]. Colorectal cancer cells secreted miR-1246 via EVs, which activated Smad1/5/8 signaling through targeting promyelocytic leukemia (PML) mRNA in recipient ECs, promoting angiogenic activities [97].

Hypoxia is a potent trigger of angiogenesis. Some studies have focused on clarifying how hypoxia affects tumor angiogenesis through tumor cell-derived EVs. Hsu et al. found that more exosomes were produced under hypoxic conditions than normoxic conditions by lung cancer cells. Tumor-derived exosomal miR-23a directly suppressed the expression of prolyl- hydroxylase 1 and 2 (PHD1 and 2), leading to the accumulation of HIF-1α in ECs and increased angiogenesis [98]. Tadokoro et al. reported that exosomes derived from leukemia cells under hypoxia are potent inducers of angiogenesis through transfer of miR-210 to ECs, downregulating the expression of receptor tyrosine kinase ligand Ephrin-A3 in HUVECs [99]. Neutral Sphingomyelinase 2 (nSMase2) was shown to regulate exosomal miR-210 secretion by metastatic cancer cells [100].

Tumor-induced vascular vessels commonly have abnormal structure with increased permeability and incomplete cellular junction [101]. Zhou et al. showed that in endothelial monolayers, exosomal miR-105 derived from breast cancer cells efficiently destroyed tight junctions and the integrity of natural vascular endothelial barriers by targeting ZO-1 in ECs, thus promoting breast cancer metastasis [102]. Similarly, lung cancer cells-secreted exosomal miR-23a also inhibited tight junction protein ZO-1, thereby increasing vascular permeability and cancer trans-endothelial migration [98]. Collectively, these observations suggest that tumor-secreted EV miRNAs participate in intercellular communication and function as a novel pro-angiogenic mechanism.

As angiogenesis is essential for tumor growth and progression, anti-angiogenic therapies have been developed to improve the outcomes of cancer patients. Several agents, including bevacizumab, aflibercept and most recently ramucirumab that target the VEGF/VEGFR signaling pathway, have been approved across some cancer types. In addition, multi-targeted tyrosine kinase inhibitors (TKIs), such as sorafenib and sunitinib, also block angiogenesis signaling. However, the overall response rate of these anti-angiogenic therapies is unsatisfactory in the clinic. Therefore, selecting out the patients, who would benefit from anti-angiogenic treatment, will help to promote therapy efficacy and avoid unnecessary toxic adverse effect. Due to the high tissue specificity and stability of miRNAs and their altered expression in tumor angiogenesis, miRNAs have been suggested as potent predictive biomarkers for therapeutic response (Table 4).

Given the important regulatory role of miRNAs in tumor angiogenesis, targeting or delivering miRNAs to tumors holds great promise as a novel therapeutic approach. In this case, it is essential to deliver antagomiRs or miRNA mimics into tumor cells or endothelial cells. As naked miRNAs are hydrophilicity, unable to pass through cell membranes, and could be easily degraded by serum RNase [113], in vivo application of miRNA requires formulation into delivery systems. Here we summarize some miRNA delivery approaches that have been applied to suppress angiogenesis in vivo (Table 5).

Significant advances have been made in exploring the regulatory role of miRNAs in tumor angiogenesis. The rapidly increasing discoveries shall pave the way in the use of miRNAs as predictive biomarkers for anti-angiogenic treatments and as miRNA-based strategy against tumor angiogenesis in the future, though there are some challenges.

Given the low response rate of anti-angiogenic agents, a step forward would be discovering predictive biomarkers to distinguish responders from non-responders. So far, several candidate miRNA biomarkers have been identified, but they emerged from small studies and should be confirmed in large prospective trials. Besides, there has been no unified control miRNAs for normalization in the analysis of circulating miRNA expression. A proposed solution to this problem may be the introduction of exogenous control [129].

Multi-targeted anti-angiogenic approach might exert increasing antitumor efficacy. Since a single miRNA has the potential to regulate angiogenesis by targeting multiple mRNAs, miRNA holds great promise as therapeutic approach for tumor angiogenesis treatment [130]. However, targeting miRNAs systemically may affect normal angiogenesis in which these miRNAs might play regulatory roles as well. In this regard, it is important to identify and target miRNAs that could distinguish angiogenic endothelial cells in tumor vasculature from those in normal tissues [131, 132], thus achieving more specific therapeutic effect and reducing side effect.

At the same time, developing technologies to deliver miRNAs to specific cells in vivo is highly essential for their therapeutic application. Despite having many strategies reported, the successful delivery approach in vivo is still limited. Clinical translation has been hampered by dose-dependent toxicity upon systemic administration [133, 134]. Intriguingly, Kalluri’s recent study showed that exosomes derived from normal fibroblast-like mesenchymal cells exhibited a superior ability to deliver RNAi to suppress pancreatic cancer growth, and showed minimum cytotoxic effects in vivo compared to synthetic nanoparticles. The engineered exosomes (known as iExosomes) target oncogenic KRAS with enhanced efficacy probably due to that CD47 on exosomes surface contributed to the evasion from immune clearance in the circulation, and KRAS-stimulated macropinocytosis increased pancreatic cancer cells uptake of iExosomes [126]. This study offers insight into the therapeutic potential of exosomes in the delivery of nucleic acid-based drugs. However, in terms of using exosomes for specific targeting of angiogenesis-related miRNAs in tumor cells or ECs, there are several aspects to consider. New targeting moieties should be tested for selective homing of exosomes to certain tumor cells and tumor-associated ECs. New targeting moieties, such as peptides and antibody fragments, could be cloned into exosomal surface proteins, or directly attached to the surface of exosomes through chemical conjugation, therefore bypassing time-consuming cloning procedures. Furthermore, the use of miRNA sponge would allow for persistent suppression of miRNA in target tissues following exosome delivery. Additionally, it is critical to find a robust cell source that produces high quantities of exosomes for use in miRNA delivery. For instance, Myc-immortalized human ES-derived mesenchymal stem cells (MSCs) have been demonstrated as a scalable source for production of therapeutic exosomes for regenerative medicine [135].

In summary, better understanding of the regulatory network of miRNAs in tumor angiogenesis provides new insights in the biological process of tumor-associated neovasculature. Advances in profiling and delivery system offer clinical translation potential of miRNAs as predictive biomarkers for anti-angiogenic therapy response and therapeutics against tumor angiogenesis.