miRNA signaling networks in cancer stem cells

Abstract

Cancer stem cells (CSCs) are a small cell subpopulation in many cancer types and are involved in various processes of tumor progression, such as initiation, metastasis and recurrence. The distinguished features of CSCs include a variety of biological properties, including self-renewal, multidifferentiation, stemness marker expression, and resistance to chemotherapy and radiotherapy. Despite their great potential of clinical importance, the CSC signaling pathways are not well understood at the molecular level. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs that play an important role in the regulation of several cellular, physiological, and developmental processes. Aberrant miRNA expression is associated with many human diseases, including cancer. miRNAs have been implicated in the regulation of CSC properties; therefore, a better understanding of miRNA-induced modulation of CSC gene expression could aid in the identification of promising biomarkers and therapeutic targets. In the present review, we summarize the major findings of the impacts of miRNAs on CSC signaling networks; we then discuss the recent advances that have improved our understanding of CSC regulation by miRNA-mediated signaling networks and that may lead to the development of miRNA therapeutics specifically targeting CSCs.

1. Introduction

The cancer stem cell (CSC) theory was initially proposed more than 150 years ago [1,2], and the basis of this theory is that cancer cells are derived from stem cell populations in tissues. Although there are conflicting data about cancer stem cells, tumor-initiating cells, and the origin of cancer cells, accumulating evidence suggests that so-called CSCs and normal stem cells have many similarities with regard to biological properties, e.g., self-renewal, differentiation capacity and specific marker expression [3]. The differences between CSCs and normal tissue stem cells are tumorigenicity and the chemoresistance ability. Although some exceptions have been reported [4], the CSC theory is generally accepted for various types of cancers in both the basic research and cancer therapeutic fields.

Common signaling pathways and networks were found in CSCs and normal stem cells. These pathways and networks, including Wnt/β-catenin, JAK/STAT, PI3/AKT, and NF-kB, are known to regulate stem cell self-renewal and differentiation. Abnormalities in the Wnt/β-catenin pathway were shown to enhance self-renewal in leukemia stem cells [5]. In the case of myxoid liposarcoma, the JAK-STAT pathway regulates cancer stem cell properties such as chemoresistance [6].

MicroRNAs (miRNAs) are noncoding RNAs that have versatile functions in physiology and pathophysiology. It is well known that miRNAs play a pivotal role in cancer initiation and progression. On the other hand, several miRNAs are also known to regulate signaling pathways [7]. In this review, we discuss the recent findings of miRNA-related signaling pathways in cancer biology, particularly focusing on those involved in CSCs. A better understanding of the characteristics of CSCs in the biological properties of miRNA-related signaling pathways is important for basic science as well as clinical applications.

1.1. Biogenesis and functions of miRNAs

miRNAs are noncoding RNAs of 21–24 nucleotides that generally negatively regulate gene expression at the posttranscriptional level. Mechanistically, miRNAs directly bind to partially complementary sequences in the 3′-untranslated regions (3′UTRs) of target genes and lead to the degradation of target mRNAs or repression of mRNA translation [8]. The majority of miRNA biogenesis occurs by transcribing miRNAs by RNA polymerase II as pri-miRNAs, which are long primary transcripts, and miRNAs are processed in the nucleus by the RNase III Drosha in combination with cofactors such as DGCR8 into precursor miRNAs (pre-miRNAs) 70–100 nucleotides long. DGCR8 is an evolutionarily conserved protein that interacts with proline-rich peptides through its WW domain, and heterozygous deletion results in the most common human genetic deletion syndrome, known as DiGeorge syndrome [9]. The pre-miRNA is exported from the nucleus to the cytoplasm by exportin-5, a family member of RanGTP-binding transport receptors [10], and is finally cleaved in a complex composed of the RNase III Dicer into mature miRNAs. The mature miRNA (also called the guide strand of mature miRNA) is incorporated into an RNA-induced silencing complex (RISC). The RISC consists of Argonaute (Ago) proteins and GW182. Although miRNAs basically act as negative regulators of target mRNAs by binding to the 3′UTRs of target mRNAs, it has also been reported that miRNAs can also bind to the 5′UTR or the open reading frame and regulate the translation of target mRNAs [11,12].

1.2. Relationship between cancer stem cells and miRNAs

Similar to protein-coding genes, abnormal expression of miRNAs has been reported in various types of cancers [13]. The functional investigation of miRNAs has clearly demonstrated that oncogenic miRNAs and tumor suppressor miRNAs play pivotal roles in cancer initiation and progression. Additionally, some miRNAs are known to be related to the regulation of CSC properties, such as asymmetric cell division, tumorigenicity, and drug resistance.

One of the most famous oncogenic miRNAs is the miR-17–92 cluster. The miR-17-92 cluster is polycistronically expressed from the chromosome 13q31 locus, which is known to be amplified in lung cancer [14]. The expression levels of miR-17–92 are higher in tumor tissues than in normal tissues, and increased miR-17–92 expression significantly promotes tumorigenesis in lymphoma [15]. Based on this evidence, it is considered an oncogenic miRNA. A bioinformatics analysis identified the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome ten) as a direct target gene of the miR-17–92 cluster [16]. Because PTEN induces apoptotic cell death via the P13K-Akt-PKB pathway, miR-17–92 indirectly regulates this signaling pathway.

Conversely, the expression levels of some miRNAs are decreased in tumorigenesis. Generally, this kind of miRNA is considered to work as a tumor suppressor miRNA. Tumor suppressor miRNAs negatively regulate oncogenes or oncogenic pathways and inhibit tumor development. The most classic and famous tumor suppressor miRNAs are the let-7 family [17]. Let-7 was originally discovered in Caenorhabditis elegans, and the let-7 sequences are conserved among species. It has been reported that let-7c suppresses the self-renewal of stem cells and inhibits the estrogen-induced activation of Wnt signaling in breast cancer stem cells [18]. Another well-known tumor suppressor miRNA, miR-34, which is a direct target of p53, was shown to inhibit Notch signaling pathways [19]. As such, in this review, we focus on the roles of miRNAs in the signaling pathways involved in CSC properties and summarize the therapeutic potential of miRNAs targeting CSCs.

2. Involvement of miRNAs in maintaining CSCs

CSCs, which are characterized by the capacity for self-renewal and differentiation, were first identified in acute myeloid leukemia in 1997 [20,21]. CSCs usually share specific stemness-related markers, such as CD44 and CD133, but they can be different depending on the cancer type [[22], [23], [24], [25]]. Moreover, several stemness-related markers, such as NANOG, OCT4, SOX2, and KLF4, can be targeted by a single miRNA, namely, miR-145, in embryonic stem cells and CSCs [26,27]. Furthermore, various other miRNAs are involved in stemness by regulating several signaling pathways, including the Wnt/β-catenin, PI3K/Akt, and NF-kB pathways, and the detailed functions of the miRNAs in each signaling pathway are described below (Fig. 1).

Fig. 1.

Overview of miRNAs in CSCs. miRNAs which regulate signaling pathways and miRNAs which are regulated by CSC-related signal pathways are shown. As CSC-related pathways based on the literatures, Wnt/β-catenin signaling, JAK/STAT signaling, NF-κB signaling, PI3K/AKT signaling, NOTCH signaling and Hippo signaling were illustrated to interacted with miRNAs.

2.1. Wnt/β-catenin signaling

Wnt signaling is an evolutionarily conserved pathway that plays various important roles, including serving as a regulator of stem cell proliferation and self-renewal [28]. Wnt signaling through its β-catenin receptors is called the canonical signaling pathway, and the noncanonical signaling pathways are also functional [28]. Inhibition of Wnt/β-catenin signaling leads to the upregulation of pri-let-7a and pri-miR-200c [29]. The expression of let-7 is high in differentiated cancers, and let-7 decreases the expression of OCT4 and SOX2 by targeting the 3′-UTR of ARID3B and HMGA2 [30,31]. Conversely, LIN28B suppresses the expression of let-7 and induces stem-like genes [32]. Thus, the expression of the let-7 family is associated with favorable prognosis in patients with lung cancer [33]. Moreover, the miR-200 family can directly target SOX2, and in contrast, lncRNA Sox2ot promotes stemness by sponging the miR-200 family [34]. Furthermore, miR-200c suppresses clonogenicity by targeting BMI1 [35]. Therefore, let-7 and the miR-200 family suppress stemness properties.

On the other hand, circ_001680 exerts its oncogenic function, including stemness, by sponging miR-340, which also targets BMI1 [36]. Moreover, miR-22 represses the expression of miR-200 and induces stemness by directly targeting the TET family [37]. Conversely, lncRNA MIR22HG suppresses Wnt/β-catenin signaling by modulating both mature miR-22-3p and miR-22-5p [38]. In addition, miR-600, miR-128-3p, miR-302c, and miR-320 also weaken stemness by targeting the mediators of Wnt/β-catenin signaling SCD1, NEK2, CARF, and CTNNB1, respectively [[39], [40], [41], [42]]. Hypoxia is also a key promoter of CSCs, and miR-1275 is regulated by HIF-1α and maintains stemness by targeting multiple genes in the Wnt signaling pathway, such as DKK3, SFRP1, GSK3β, and RUNX3 [43]. Therefore, various miRNAs and competing endogenous RNAs (ceRNAs) are involved in the regulation of Wnt/β-catenin signaling.

2.2. JAK/STAT signaling

JAK/STAT signaling has crucial roles in inflammation, proliferation, and survival. Several cytokines and growth factors, such as IL-6, IGF, and TGF-β1, can activate the pathway through their receptors, and nuclear STAT mediates transcriptional regulation of various target genes [44].

First, regarding stemness, nuclear CD44/acetylated STAT3 signaling is required for tumorigenicity and can be associated with metastasis [45]. In addition, IGF-1 is one of the key stimulators of signaling, and downregulation of miR-28-5p promotes CSC self-renewal by targeting IGF-1 [46]. As a downstream target of IGF/STAT3 signaling, NANOG is activated, and moreover, miR-135a enhances the expression of NANOG by modulating methylation of the promoter region [47,48]. Then, STAT3 signaling further increases the expression of miR-21 and miR-92a, and they induce stemness by modulating Wnt signaling [[49], [50], [51]]. However, IL-6/STAT3 signaling suppresses miR-34a, resulting in cancer progression [52].

Furthermore, radiation promotes the release of extracellular vesicles containing miR-603, and miR-603 then confers stemness properties and radioresistance by targeting both IGF and IGF1R [53]. On the other hand, the miR-1181 directory suppresses SOX2 and STAT3, and miR-7 suppresses STAT3 by targeting SETDB1 [54,55].

Therefore, JAK/STAT3 signaling is also important for stemness and is regulated by several oncogenic and tumor suppressor miRNAs. Moreover, STAT3 is also involved in the regulation of NF-κB signaling, which is discussed in the next section.

2.3. NF-κB signaling

The NF-κB family consists of the following five transcribed genes: p65 (RelA), RelB, c-Rel, p105/p50, and p100/p52, which are critical regulators of inflammation and immune responses [56]. In addition, NF-κB is activated downstream of oncogenic pathways, such as the RAS, BCR-ABL, p53, and PTEN pathways [56].

In stemness-enriched cancer cells, miR-205 upregulated by NF-κB signaling suppresses COMMD1, and then, downregulation of COMMD1 promotes intrinsic and TNF-α-induced inflammatory responses and RelA expression to maintain signaling activation [57]. RelA promotes CD44 expression but is targeted by the tumor suppressor miR-7-5p [58]. Previously, we identified that CD44 induces miR-629-3p expression and confers cisplatin resistance [59]. Moreover, NF-κB suppresses the expression of miR-194-5p by directly binding to its promoter region, and downregulation of miR-194-5p contributes to tumor progression by targeting IGF1R and PPFIBP1 [60]. Therefore, NF-κB is also important for cancer development, including stemness.

2.4. PI3K/AKT signaling

PI3K/AKT signaling regulates key metabolic processes, including glucose, lipid, protein, and nucleotide synthesis [61]. In addition to the direct metabolic functions of AKT, it also activates key downstream effectors, such as the mTORC1, GSK3 and FOXO transcription factors [61].

The AKT/mTOR pathway is activated by miR-135b and increases the expression of stemness markers [62]. However, PI3K signaling is attenuated by PTEN, and thus, miR-10b promotes CSC self-renewal by targeting PTEN [61,63]. Moreover, miR-106b-5p increases the expression of p-Akt by targeting PTEN and p21, resulting in resistance to irradiation [64]. Furthermore, SOX2 directly binds to the promoter region of miR-486-5p, and increased expression of miR-486-5p also suppresses expression of PTEN and FOXO1 [65]. Therefore, these miRNAs promote stemness by targeting negative regulators of PI3K signaling.

In addition, PD-L1 expression is positively correlated with stemness marker expression, and miR-873 inactivates PI3K/Akt and ERK1/2 signaling by directly targeting PD-L1 [66]. Moreover, miR-1976 suppresses CSC properties by targeting PIK3CG, and higher expression of miR-1976 is associated with favorable prognosis in patients with triple-negative breast cancer [67].

Therefore, aberrant PI3K/AKT signaling, which is frequently observed in various cancers, is also important for the maintenance of stemness.

2.5. Notch signaling

Notch signaling, which usually requires cell–cell contact, is essential for crosstalk between cancer cells and other components of the tumor microenvironment [68]. Notch signaling is one of the regulators of the CSC division mode, symmetrically or asymmetrically, and symmetric division is associated with high self-renewal ability and exponential tumor growth [69,70]. Mechanistically, miR-200b-3p targets NOTCH1 and inhibits symmetric division of CSCs by modulating the Notch/Numb ratio [69]. Moreover, lncRNA TUSC-7 increases the expression of NUMB by sponging miR-146 and inactivates Notch signaling [70]. Therefore, NOTCH1 promotes symmetric division by producing two daughter cells with high NOTCH1 expression, while Numb promotes asymmetric division.

Notch signaling also promotes multidrug resistance in various cancers, and miR-34, which is suppressed by EZH2-mediated H3 lysine 27 trimethylation (H3K27me3), targets Notch1 in CSCs [71,72]. Moreover, the tumor suppressors miR-21-5p and miR-26a-5p are also silenced by H3K27me3, and thus, EZH2 can be one of the targets to inactivate NOTCH signaling and weaken self-renewal [71,73]. Similarly, the expression of miR-7-5p is regulated by DNA methylation in the promoter region, and its downregulation promotes tumor progression by targeting Hes1, a key effector of Notch signaling [74]. Moreover, NOTCH2/3 is targeted by miR-195-5p, miR-181b, and miR-136 [[75], [76], [77]].

In addition, according to a double-negative feedback loop of the miR-200 family and ZEB1 in epithelial–mesenchymal transition, ZEB1 can indirectly activate Notch signaling because the miR-200 family has several other target genes involved in Notch signaling, such as Jag1, Maml2, and Maml3 [78]. Interestingly, exosomes can also activate Notch signaling. Exosomes containing miR-142-3p are secreted by bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) in the tumor microenvironment, and miR-142-3p promotes Notch signaling by downregulating Numb in CSCs [79]. Therefore, Notch signaling does not necessarily require cell–cell contact and is deeply involved in stemness.

2.6. Hippo signaling

Hippo signaling is involved in organ development, tissue regeneration, and tumorigenesis, including stemness [80]. YAP and TAZ are two main downstream targets of signaling, and YAP is highly expressed in the self-renewal of embryonic stem cells [80].

In lung squamous cell carcinoma, high expression of miR-214-3p is associated with favorable prognosis and suppresses tumorigenesis by targeting YAP1 [81]. On the other hand, miR-30b enhances CSC-like properties by targeting MYPT1, an upstream gene of the Hippo signaling pathway [82]. Importantly, YAP1 regulates miRNA biogenesis depending on the cell density, and at a low cell density, YAP1 activation results in global miRNA suppression [83]. Therefore, the Hippo pathway can be a strong regulator of the miRNA-regulated stemness described in this review. These miRNAs were listed in Table 1.

Table 1.

Stemness associated miRNAs and their target genes.

miRNA	target gene	cell type	Reference
let-7	ARID3B and HMGA2	Oral SCC	[11]
let-7 family	HMGA2	Ovarian cancer	[12]
miR-106b	PTEN and p21	Colorectal cancer	[45]
miR-10b	PTEN	Breast cancer	[44]
miR-1181	SOX2 and STAT3	Pancreatic cancer	[36]
miR-1275	DKK3, SFRP1, GSK3β, RUNX3, and NUMB	Lung adenocarcinoma	[24]
miR-128-3p	NEK2	Breast cancer	[20]
miR-135a	DNMT1	Liver cancer	[29]
miR-135b	JADE1	Pancreatic cancer	[43]
miR-136	NOTCH3	Ovarian cancer	[57]
miR-142-3p	NUMB	Colon cancer	[60]
miR-145	OCT4, SOX2, and KLF4	Human embryonic stem cells	[7]
miR-145	NANOG, OCT4, and SOX2	Uterine cervical SCC	[8]
miR-146	NUMB	Lung adenocarcinoma	[51]
miR-181b	NOTCH2 and RBPJ	NSCLC	[58]
miR-194-5p	IGF1R and PPFIBP	Ovarian cancer	[41]
miR-195-5p	NOTCH2 and RBPJ	Colorectal cancer	[56]
miR-1976	PIK3CG	Breast cancer	[48]
miR-200 family	SOX2	Pancreatic ductal adenocarcinoma	[15]
miR-200 family	JAG1, MAML2, and MAML3	Several cell lines	[59]
miR-200b-3p	NOTCH1, TRIM2, PROX1, and NUMB	Pancreatic cancer	[50]
miR-200c	BMI1	Breast cancer	[16]
miR-205	COMMD1	Head and Neck SCC	[38]
miR-21	TGFβR2	Colon cancer	[30]
miR-214-3p	YAP1	Lung SCC	[62]
miR-21-5p	JAG1	Hepatocellular carcinoma	[54]
miR-22	TET family	Breast cancer	[18]
miR-22-3p	SFRP2	Glioblastoma	[19]
miR-22-5p	PXDH15	Glioblastoma	[19]
miR-26a-5p	JAG1	Hepatocellular carcinoma	[54]
miR-28-5p	IGF-1	Liver cancer	[27]
miR-302c	CARF	Colon cancer	[21]
miR-30b	MYPT1	Ovarian cancer	[63]
miR-320	CTNNB1	Prostate cancer	[23]
miR-340	BMI1	Colorectal cancer	[17]
miR-34a	IL-6R	Colorectal cancer	[33]
miR-34a	NOTCH1, NOTCH2, and JAG1	Cholangiocarcinoma	[52]
miR-34a	NOTCH1	Breast cancer	[53]
miR-486-5p	PTEN and FOXO1	Glioblastoma	[46]
miR-600	SCD1	Breast cancer	[22]
miR-603	MGMT	Glioblastoma	[34]
miR-7	SETDB1	Breast cancer	[35]
miR-7	RELA, Slug, and LncRNA XIST	Breast cancer	[39]
miR-7-5p	SMO and HES1	Gastric cancer	[55]
miR-873	PD-L1	Breast cancer	[47]
miR-92a	DDK1	Ovarian cancer	[32]

SCC, squamous cell carcinoma; NSCLC, non-small cell lung cancer.

3. The therapeutic potential of miRNAs targeting CSCs

Because several miRNAs typically work as oncogenic or tumor suppressor miRNAs in various types of cancers, targeting miRNAs, i.e., blocking oncogenic miRNAs or enhancing tumor suppressor miRNAs in cancer, is considered to be a novel type of cancer therapy [84]. As described above in this review, some miRNAs that can regulate CSC properties could serve as therapeutics.

One of the challenges of realizing miRNA-based CSC therapeutics is the protection of miRNAs from RNase degradation in serum. To overcome this issue, there are two major strategies: one is to use chemically modified nucleic acids for higher stability, and the other is to establish an effective delivery system for miRNAs [85,86]. To increase the stability and overexpress tumor suppressor miRNAs in cancer cells, miRNA mimics are usually modified with methylation, such as 2′-O-methylation, of the passenger strand. In contrast, to block oncogenic miRNAs, inhibitors of miRNAs (also known as antimiRs) are often used by modification with locked nucleic acid (LNA) technology. Additionally, for miRNA therapeutics, tremendous efforts have been made to improve the delivery system of miRNAs using liposomes. Although the utility of liposomes in vivo has not been clinically feasible because of low uptake efficiency and high cytotoxicity, technological advancements in miRNA stability and delivery efficiency have been accomplished in the past several decades by merging biology and chemistry.

It has been reported that extracellular vesicles such as exosomes are considered to be suitable carriers for miRNA delivery in vivo. Exosomes are small extracellular vesicles with a diameter of approximately 50–150 nm. Exosomes are released into the extracellular microenvironment to transfer their components, including miRNAs, proteins and metabolites [87]. Thus, the application of exosomes as drug and gene therapy delivery systems is very promising as a natural vector system. Additionally, emerging evidence has demonstrated that surface proteins such as integrins define the organotropism of exosome delivery [88]; thus, modifying surface protein patterns on exosomes may improve targeted delivery systems of miRNAs. More recently, Usman et al. showed that antimiR against miR-125b was efficiently packaged with an electroporation method into exosomes collected from human red blood cells [89]. Local injection of antimiR in a breast cancer mouse model and systemic injection of antimiR in an AML mouse model confirmed the potential utility of antimiR-packaged exosomes for cancer therapeutics.

Considering CSC-targeted miRNA therapeutics, one of the promising targets is let-7 miRNA. The Let-7 family includes ten human isoforms, and they generally target a variety of oncogenes. Differentiated cancer cells express high levels of let-7, and loss of let-7 promotes tumor progression by suppressing RAS signaling [90]. Additionally, since let-7 targeted ARID3B and HMGA2 and induced CSC differentiation, enhanced expression of let-7 could suppress the CSC population in bulk tumor tissues [30].

4. Conclusion

Technical advances in single-cell approaches have clearly demonstrated the intratumoral heterogeneity of cancer cells and a unique subpopulation that is capable of tumor reconstitution. Several signaling pathways, such as the Wnt/β-catenin, PI3K/Akt, and NF-kB pathways, are well known to be involved in CSC maintenance and differentiation. A number of studies presented in this review have shown that miRNAs can work as regulators of these pathways and act as oncogenic or tumor suppressor miRNAs in CSCs. As such, miRNAs could be therapeutic targets for CSCs, e.g., inhibition of certain miRNAs might eradicate CSCs in tumor tissues. Collectively, a better understanding of miRNA functions associated with CSC properties could provide new insight into cancer therapeutics that possibly improve patient prognosis and treatment outcomes.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.