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Therapeutic Delivery

miRNA therapy targeting cancer stem cells: a new paradigm for cancer treatment and prevention of tumor recurrence

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that retain the properties of self-renewal and tumorigenicity in vivo. Although CSCs have been reported in multiple cancers, the regulation of CSCs has not been described at the molecular level. miRNAs are endogenous small noncoding RNAs that posttranscriptionally regulate the expression of their target genes via RNA interference and are involved in almost all cellular processes. Since aberrant miRNA expression occurs in CSCs, such dysregulated miRNAs may be promising therapeutic targets. In this review, we summarize the current knowledge regarding miRNAs that regulate CSC properties and discuss an in vivo delivery system for synthetic miRNA mimics and miRNA inhibitors for the development of innovative miRNA therapy against CSCs.

Advances in preclinical and clinical cancer research have led to new diagnostic and treatment options for cancer patients and resulted in remarkable progress in cancer cure and prevention  [1] . Nevertheless, cancer remains one of the leading causes of death worldwide. Cancer development is a multistep process in which genomic instability and genetic diversity are considered hallmarks of cancer that contribute to treatment failure and disease progression [2] . These cellular alterations include unlimited replication potential, evasion of growth suppressors, self-sufficiency for growth signals, evasion of apoptosis, sustained angiogenesis, and tissue invasion and metastasis to other organs. Cancer was previously considered a homogeneous mass of rapidly proliferating cells, and therapies were designed to eliminate highly proliferative cells. However, recent studies have suggested that cancer cells are heterogeneous with respect to proliferation and differentiation [3–5] . In several types of malignancies, the capacity to initiate and maintain cancer growth resides in a small population of cells called cancer stem cells (CSCs)  [6–8] . Although the concept of the CSC, which may arise from a rare population of cells with stem cell properties, was proposed approximately 150 years ago [9,10] ,

10.4155/TDE.14.122 © 2015 Future Science Ltd

recent new technologies such as development of flow cytometry and cell sorters and establishment of immunodeficient animal models have provided evidence supporting the existence of CSCs. Like normal pluripotent stem cells, CSCs have the ability to self-renew and produce the variety of proliferating and differentiated cells that comprise the bulk of a tumor, suggesting that CSCs themselves contribute to tumor development (tumor initiation) and progression. Moreover, CSCs are reported to be resistant to conventional chemotherapy and radiotherapy compared with their differentiated progeny [11,12] . These data may explain why tumor recurrence and relapse occur. CSCs are likely present and concealed in the tumor microenvironment after chemotherapy and radiotherapy. Therefore, CSCs are strongly suggested to play a critical role in cancer progression, which includes chemotherapy and radiotherapy resistance, metastasis and recurrence. Thus, elimination of CSCs would likely lead to a complete cure in cancer patients. So far, small molecule inhibitors to downregulate signaling that impacts chemo- and radioresistance to CSCs and oncolytic virus in order to target CSCs were used for developing CSC-targeting therapies. To

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Mitsuhiko Osaki*,1,2, Futoshi Okada1,2 & Takahiro Ochiya3 1 Division of Pathological Biochemistry, Department of Biomedical Sciences, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori 683–8503, Japan 2 Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori 683–8503, Japan 3 Division of Molecular & Cellular Medicine, National Cancer Center Research Institute, 5–1–1 Tsukiji, Chuo-ku, Tokyo 104–0045, Japan *Author for correspondence: Tel.: +81 859 38 6242 Fax: +81 859 38 6240 [email protected]

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ISSN 2041-5990

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Key terms Cancer stem cells: Cells within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. miRNA: Small noncoding RNA molecule (containing approximately 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression. Tumorigenicity: Ability to make a tumor in vivo. Chemoresistance: Acquiring resistance against anticancer drug(s) by activating drug efflux pumps and so on. Delivery system: Systems to transport a pharmaceutical molecule in the body as needed to safely achieve its desired therapeutic effect. Self-renewal: Ability to make the same stem cell, when one stem cell divides into two cells.

allow the CSCs-specific eradication, many investigators have identified CSC-specific markers expressed on the cell surface in many types of cancer (Table 1)  [13– 30] . Further studies have reported that abnormalities in noncoding RNAs, especially miRNAs, are present in various types of cancers. Moreover, the dysregulated expression of miRNAs may contribute to CSC properties such as tumorigenicity, asymmetric cell division and chemoresistance. In this review, we describe the general and tissue-type-specific properties of CSCs in several human cancers and correlate these properties with

alterations in miRNA expression. In addition, we also discuss an in vivo delivery system for synthetic miRNAs and miRNA inhibitors for the development of miRNA therapy against CSCs. CSCs A CSC is defined as a cell within a tumor that possesses the capacity to self-renew and that produces heterogeneous lineages of cancer cells that comprise the tumor. The term ‘CSC’ was adopted at the American Association for Cancer Research (AACR) workshop in 2006 [31] . Identification of the CSC was first demonstrated by Dick et al. who showed that tumorigenic properties can be attributed only to a minority population of leukemia cells [13] . Subsequent clinical and laboratory studies provided additional evidence supporting the role of CSCs in drug resistance and cancer metastasis, thereby contributing to the poor outcomes experienced by patients with pancreatic, brain, breast, colon, ovarian and prostate tumors [14–15,23,25,32–34] . CSCs can be identified by expression of certain surface markers, which distinguish them from nontumorigenic cells (non-CSCs) [35] . In other words, CSCs represent a distinct cell population that can be identified and prospectively isolated from a wide variety of tumor tissues using CSC-specific markers (Table 1) . CSCs also express specific stem cell genes [36] . However, CSCs differ from normal stem cells in their resistance to anticancer drugs, tumor-initiating activity and metastatic ability [36,37] . To attain such features,

Table 1. Representative cell surface markers for human cancer stem cells. Tumor type

CSC marker

 Ref.

Acute myeloid leukemia

CD34+/CD38-

Brain tumor

CD133+

 

CD15

Breast cancer

CD44+/CD24-/low

[17]

 

ALDH

[18]

Colorectal cancer

CD133+

 

CD44+/EpCAM+/CD166+

[21]

Ovarian cancer

CD44+

[22]

 

CD133+

[23]

 

CD117

[24]

Prostate cancer

CD44+/α2β1+/CD133+

[25]

Lung cancer

Sca1+/CD45−/CD31-/CD34+

[26]

 

CD133+

[27]

Gastric cancer

CD44+

[28]

Liver cancer

EpCAM/AFP

[29]

Pancreatic cancer

ESA+/CD44+/CD24+

[30]

[13] [14,15] [16]

[19,20]

AFP: α-fetoprotein; ALDH: Aldehyde dehydrogenase; EpCAM: Epithelial cell adhesion molecule; ESA: Epithelial specific antigen.

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miRNA therapy targeting cancer stem cells 

genetic and epigenetic alterations result in changes in multiple signaling pathways (e.g., Notch signaling pathway, Wnt/β-catenin pathway, Sonic Hedgehog pathway, etc.) that confer cell adaptation to microenvironmental stress including inflammation, low pH, hypoxia, starvation, anticancer drugs and irradiation [38–41] . Dysregulation of the Wnt/β-catenin pathway promotes self-renewal activity during leukemia stem cell propagation, as reported by Jamieson et al. in 2004 [42] . Korkaya et al. also reported that alteration of the Wnt/β-catenin pathway is involved in the regulation of breast CSCs [43] . The Notch signaling pathway and the Wnt/β-catenin pathway are activated in breast CSCs [44] , and similar dysregulation of the Notch signaling pathway was reported in glioblastoma and colon CSCs [44,45] . Also, alterations in Hedgehog signaling have been reported in colon, breast and glioblastoma CSCs [46–48] . miRNAs regulate the properties of CSCs by acting on various signaling pathways. The progression from a stem cell to a terminally differentiated cell depends on a temporal balance between proliferation and differentiation programs. This balance is altered in tumors, partly as a consequence of miRNA deregulation, leading to the maintenance of proliferation and self-renewal of CSCs [49] . Additionally, a connection between CSCs and epithelial-mesenchymal transition (EMT) may exist. EMT is an evolutionarily conserved biological process in which epithelial cells acquire mesenchymal characteristics. EMT is involved in apoptosis resistance, tumor motility and invasion. Molecules associated with this process include transcription factors, such as the zinc-finger E-box-binding homeobox proteins (ZEB1 and 2), Snail, Slug and Twist1, which repress E-cadherin and induce vimentin and fibronectin expression  [50] . The EMT process is also strongly regulated by miRNAs. miRNA: biogenesis & role in stem cells miRNAs are endogenous noncoding RNAs approximately 22 bp in length that directly bind to target RNAs in a sequence-complimentary manner to inhibit translation and facilitate degradation of their target transcripts. miRNAs play important roles in a wide range of physiological and pathological processes [51,52] . miRNAs are encoded in the genome and are generally transcribed by RNA polymerase II as part of introns of mRNA genes or from intergenic regions. The miRNA primary precursor is then processed in the nucleus by the Microprocessor complex [53,54] , which is comprised of the cleavage enzymes Drosha, DiGeorge Critical Region Gene 8 (DGCR8) and other factors, releasing a stem-loop precursor (pre-miRNA). The pre-miRNA is then exported to the cytoplasm and cleaved by the

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RNase III enzyme Dicer, which is also involved in the maturation of short interfering RNAs (siRNAs) [55,56] . The mature miRNA is finally incorporated into the RNA-induced silencing complex as a single strand [57] . At this stage, miRNAs can downregulate target gene expression by two mechanisms: inhibition of translation to protein or direct digestion of target mRNA. The choice is made based on the degree of complementarity between the miRNA and the target gene in combination with an Argonaute family protein [58] . miRNAs were first identified in Caenorhabditis elegans as RNA molecules that are complementary to the 3′ untranslated regions of target transcripts, such as the lin-4 and let-7 genes [59,60] . Based on miRBase release 21, about 2000 human miRNAs have been registered [61] , with a large number that are evolutionarily conserved  [62,63] . miRNAs are expressed in all animal cells and play fundamental roles in cellular activities such as development, cellular differentiation, proliferation, cell cycle control, apoptosis, metabolism and cancer  [63] . In addition, the potential for self-renewal and differentiation in normal stem cells is regulated by many miRNAs. The absence of Dicer1, an enzyme that is essential for biogenesis of miRNAs, results in depletion of stem cells in mouse embryos [64] , and restoration of Dicer1 in mouse embryos restores the stem cell phenotype [65] . DGCR8-deficient mouse embryonic stem cells show altered expression of stemness markers such as NANOG, SOX2 and OCT4 as well as differentiation markers; dysregulation of the cell cycle and differentiation are also seen [66] . The expression of such pluripotency genes is regulated in part by the miR-34 family, a direct target of P53 [67] , suggesting that wild-type P53 may repress pluripotency by upregulating members of the miR-34 family. The let-7 family also attenuates the pluripotency property in embryonic stem cells by inhibiting Lin28, a marker of undifferentiated embryonic stem cells that may induce pluripotent stem cells [68] . Thus, these miRNAs may be tumor-suppressive miRNAs. In other words, downregulation of stemness-attenuating miRNAs may result in tumor initiation and development. Dysregulation of miRNAs in several types of CSCs The aberrant expression of miRNAs has also been observed in CSCs, and the aberrant expression profile of miRNAs may be the cause of initiation, development or/and maintenance of the tumor. miRNAs also regulate CSC features by influencing signaling pathways and CSC signature genes. Particular miRNAs that are differentially expressed in the CSC population in various cancers are potential CSC markers as described below and in Table 2.

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Review  Osaki, Okada & Ochiya Brain tumors

Although primary brain tumors constitute only 2% of all adult cancers [97] , they result in a disproportionate share of cancer morbidity and mortality. Gliomas are the most frequent primary brain tumors in adults, and prognosis depends on histological tumor type, grade and tumor genetics [98] . Despite multimodal treatment with surgery, radiotherapy and chemotherapy, gliomas remain largely incurable [98,99] . Brain CSCs are characterized by the expression of cell surface markers such as CD133 [14,15] and CD15 [16] . The pentaspan membrane glycoprotein CD133, also known as Prominin-1, was first identified as a marker of hematopoietic stem cells and progenitor cells, and was subsequently used to identify malignancies [100,101] . In solid cancers, CD133 was first used to identify CSCs in different types of human brain tumors, and tumor cells from patients have been separated into CSC-including populations and bulk tumor cells based on the expression of CD133 [14,102] . The CD133+ cell population is highly tumorigenic in vivo, whereas CD133- cells do not form tumors, even in high numbers. Higher expression of miR199–5p reduces the CD133+ population and downregulates stem cell genes such as Nanog, Oct4 and Klf5 in brain tumor cells [73] . The study also revealed that HES1, a transcription factor involved in Notch signaling is a regulator of stemness of the cells. miR-199–5p blocks Notch signaling, inhibiting the self-renewal ability of these cells by reducing the CD133+ population in vitro, and miR-199–5p may also inhibit tumor growth in vivo. In addition, lower expression of miR199–5p is correlated with poor prognosis in medulloblastoma patients. Recently, miR-34a was shown to regulate Notch signaling by targeting Notch-1 and Notch-2 in medulloblastoma cells [103] and to reduce the CSC number by inducing cell differentiation of glioma cells [69] . Therefore, miR-199b-5p and miR-34a are important for the self-renewal potential of glioma CSCs by diminishing their stemness. miR-128 is one of the most highly expressed miRNAs in brain and is preferentially expressed in neurons. miR128 is downregulated in gliomas, especially in patients with high-grade disease [72] . Moreover, the stem cell self-renewal factor BMI-1 is a target of miR-128. miR124 is another highly expressed miRNA in the adult brain and is downregulated in gliomas [70,71] and medulloblastomas  [104] . miR-124 specifically inhibits the selfrenewal capacity of glioma CSCs by directly targeting SNAI2 in CD133+ cells in which stem cell genes such as Nanog, Nestin and Bmi-1 are downregulated; inhibition of neurosphere formation also occurs [105] . Another study reported that miR-451 inhibits neurosphere formation and reduces cell viability in the glioblastoma

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cell line A172. Furthermore, miR-451 overexpression also enhances the efficacy of imatinib mesylate, a drug used to inhibit neurosphere formation; however, the target of miR-451 for this function has not been identified  [74] . Because BMI-1 and SNAI2 contribute to the maintenance of stemness, these miRNAs may abolish stem-like characteristics of glioma cells. Breast cancer

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide, accounting for 23% (1.38 million) of all new cancer cases and 14% (458,400) of all cancer deaths in 2008 [1] . Al-Haji et al. reported the first solid tumor CSCs and described that the CD44+/CD24-/low cell population has a markedly high tumor-initiating capacity in breast cancer [17] . Thereafter, several common CSC markers, including CD44, CD133, ALDH, c-kit, ESA and ATP-binding cassette including ABCG2, have been identified in primary breast cancer tissues [17,106–107] . Yu  et al. demonstrated that downregulation of let-7 expression promotes the self-renewal property and inhibits differentiation by regulating HRAS and HMGA2 [77] . HRAS is responsible for the self-renewal property, and HMGA2 affects the differentiation capacity of the cells. Huang et al. showed that miR-888 is upregulated in the side population of the human breast cancer cell line, MCF-7, which has CSC properties [76] . Interestingly, overexpression of miR-888 significantly reduces tumor formation when miR-888-expressing side population cells are inoculated into mice, suggesting that miR-888 maintains CSC properties. On the contrary, Zhang et al. showed that miR-7 is downregulated in CSCs from the human breast cancer cell lines, MCF-7 and MDA-MB-231 [78] . miR-7 inhibits cell invasion and metastasis, decreases the breast CSC population and partially reverses EMT in MDA-MB-231 cells by directly targeting the oncogene, SETDB1, which induces STAT3 expression by binding to the STAT3 promoter. Suppression of STAT3 by miR-7 leads to the downregulation of c-myc and twist, which maintain the self-renewal property and EMT phenotype. STAT3 activation by SETDB1, a target gene of miR7, may be an essential pathway for maintaining the stem-like properties of breast CSCs. Another study showed that miR-7 suppresses brain metastasis by downregulating the transcription factor KLF4, which is an essential gene for induced pluripotent stem cells, and an miR-7 target gene [108] . The metastatic property is attributed to downregulated miR-7 in CSCs because CSCs are considered the first cells to occur during metastasis.

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miRNA therapy targeting cancer stem cells 

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Table 2. miRNAs regulating cancer stem cells. Tumour type/target miRNAs

Up/Down†

Target gene

Function

Ref.

miR-34

Down

NOTCH-1, -2

Inhibition of self-renewal

[69]

miR-124

Down

SNAI2

Inhibition of self-renewal

[70,71]

miR-128

Down

BMI-1

Inhibition of self-renewal

[72]

miR-199b-5p

Down

HES1

Reduction of CD133+ cells

[73]

miR-451

Down

(not identified)

Inhibition of neurosphere formation

[74]

miR-155

Up

C/EBPbeta

Inhibition of invasion and metastasis

[75]

miR-888

Up

E-cadherin

Promotion of EMT

[76]

let-7

Down

HRAS, HMGA2

Inhibition of self-renewal

[77]

miR-7

Down

SETDB1

Inhibition of invasion and metastasis

[78]

miR-200c

Down

BMI-1, SUZ12

Inhibition of self-renewal

[79]

miR-21

Up

TGFBR

Modulation of Wnt signaling

[80]

miR-140

Up

HDAC4, CXCL2

Chemosensitivity

[81,82]

miR-215

Up

DTL

Chemosensitivity

[83]

miR-34

Down

NOTCH1

Suppression of asymmetric cell division

[84]

miR-93

Down

HDAC8, TLE4

Inhibition of proliferation

[85]

miR-328

Down

ABCG2

Inhibition of chemoresistance

[86]

miR-451

Down

MIF, COX-2

Inhibition of self-renewal and tumorigenicity

[87]

miR-451

Down

ABCB1

Inhibition of chemoresistance

[87]

miR-214

Up

P53/NANOG

Promotion of self-renewal

[88]

miR-199a

Down

CD44

Inhibition of proliferation and invasion

[89]

miR-199a

Down

mTOR

Induction of chemoresistance of CD133+ cells

[90]

miR-200a

Down

ZEB2

Inhibition of migration and invasion

[91]

miR-34

Down

CD44

Inhibition of proliferation and metastasis

[92]

miR-34

Down

NOTCH

Inhibition of NOTCH and androgen signaling

[93]

miR-101

Down

EZH2

Inhibition of self-renewal

[94]

miR-128

Down

BMI-1

Inhibition of self-renewal

[95]

miR-320

Down

β-catenin

Inhibition of Wnt signaling

[96]

Brain tumor

Breast cancer

Colorectal cancer

Ovarian cancer

Prostate cancer

Expression status in cancer stem cells. Down: Downregulated; Up: Upregulated.

†

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327

Review  Osaki, Okada & Ochiya The miR-200 family consists of five members and can be divided into two clusters: miR-200a/miR200b/miR-429 and miR-200c/miR-141, which map to chromosome 1 and 12, respectively. A molecular link between EMT and the miR-200 family is provided by ZEB1/ZEB2, which are transcriptional repressors of E-cadherin [109,110] . miR-200 family members are downregulated in normal human and mouse mammary stem cells and breast CSCs [79] . miR-200c inhibits the formation of mammary ducts from mammary stem cells and tumor formation from breast CSCs [79] . Downregulation of miR-200c in breast CSCs suppresses the expression of BMI-1 and SUZ12, which regulate stem cell self-renewal. miR-200c inhibits the clonal expansion of breast CSCs in vitro  [79] . In addition to the miR-200 family, several other miRNAs are involved in EMT in breast cancer. miR-155 plays an important role in transforming growth factor-beta-induced EMT, cell migration, and invasion by targeting Rho A in a mammary epithelial cell model [111] and CCAAT/enhancer binding protein-beta in breast cancer cells [75] . miR-9 promotes EMT, and thus metastasis, in breast cancer cells by suppressing E-cadherin [112] . Moreover, high expression of miR-9 is correlated with poor prognosis in human breast cancer patients [113] . Colorectal cancer

Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females, with over 1.2 million new cancer cases and 608,700 deaths estimated to have occurred in 2008 [1] . CD133 [19,20] , CD44  [21] , CD166 [21] , EpCAM [21] and ALDH [114] are used to identify colon CSCs. miR-93, miR-328 and miR-451 are downregulated in colon CSCs. Yu et al. showed that miR-93 inhibits cell renewal and proliferation of SW116 CSCs, possibly by targeting HDAC8 and TLE4 [85] . miR328 increases the sensitivity of the cells to hydroxycamptothecin and 5-fluorouracil by targeting ABCG2 in CSCs in colorectal cancer cell lines (SW116 and SW480) and cancer tissues [86] . The sphere formation assay demonstrated that downregulation of miR-451 is required for the self-renewal property via upregulation of macrophage migration inhibitory factor and cyclooxygenase-2, which are involved in the activation of the Wnt pathway that is essential for the maintenance of colon CSCs. Moreover, downregulation of miR-451 leads to acquisition of chemoresistance to irinotecan via upregulation of ABCB1, a target of miR-451 [87] . Another study reported that miR-140, miR-215 and miR-21 are significantly upregulated in colon CSCs. On the one hand, miR-140 is responsible for chemoresistance to 5-fluorouracil and methotrexate

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by suppressing HDAC-4 and CXCL2, target genes of miR-140 [81,82] . On the other hand, miR-215 mediates chemoresistance to methotrexate and Tomudex by suppressing DTL, a target gene of miR-215 [83] . Upregulation of miR-21 suppresses TGFBR2 expression, followed by activation of the Wnt pathway [80] . Bu et al. showed that miR-34a determines whether colon CSCs undergo symmetric or asymmetric cell division and that low expression of miR-34a is maintained in CSCs [84] . Because Notch signaling, which induces the self-renewal activity of intestinal stem cells, is inhibited by miR-34a by targeting NOTCH receptors  [103] , activation of Notch signaling in conditions in which miR-34a expression is low may maintain CSCs, whereas upregulation of miR-34a abrogates Notch signaling, followed by generation of non-CSCs. Ovarian cancer

Ovarian cancer is the seventh major cause of cancer mortality in women [1] . More than 220,000 new cases of ovarian cancer are reported worldwide each year, and more than 60% of patients die within 5 years [1] . The high mortality rate in ovarian cancer patients results from diagnosis at a late stage when the cancer has spread into the peritoneal cavity and metastasized to vital organs [115] . The cell surface markers expressed by ovarian CSCs include CD44 [22] , CD133 [23] and CD117 [24] . Downregulation of miR-200a is observed more in ovarian CD133+ CSCs than in CD133- CSCs, and gain of function of miR-200a in CD133+ cells inhibits the migration and invasion of CD133+ ovarian CSCs. ZEB2 is a target of miR-200a [91] . Xu et al. examined the role of miR-214 in initiation of the ovarian CSC phenotype and found that forced expression of miR-214 targets the P53/Nanog axis and contributes to ovarian CSC confluence and self-renewal [88] . In addition, miR214 expression increases the chemoresistance of the cells to cisplatin and doxorubicin. CD44 is a marker of ovarian CSCs. miR-199a represses CD44 expression and inhibits the proliferation, migration and invasion of CD44+CD117+ ovarian CSCs. The inhibition of CD44 by miR-199a reduces expression of the multidrug resistance gene ABCG2 and thereby increases the chemosensitivity of ovarian CSCs  [89] . In addition, miR-199a is also implicated in cisplatin resistance because inhibition of miR-199a increases mammalian target of rapamycin expression and decreases cisplatin-induced apoptosis in vitro [90] . Prostate cancer

Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in

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miRNA therapy targeting cancer stem cells 

males, accounting for 14% (903,500) of the total new cancer cases and 6% (258,400) of the total cancer deaths in males in 2008 [1] . As most prostate cancer patients are treatable, the survival rate has increased significantly over the years, although fatalities still occur, especially with aggressive tumors that are resistant to chemotherapy. Considerable effort has resulted in identification and characterization of prostate CSC populations, allowing targeting of prostate CSCs to treat this condition. Prostate CSCs are characterized by expression of CD44, CD133, stem cell antigen 1 (Sca-1), alpha2beta1 integrin and ABCG2 [25,116] . Liu  et al. reported that miR-34a expression is reduced in CD44+ prostate CSCs [92] . Moreover, miR-34a overexpression in CD44- CSCs downregulates the expression of CD44, followed by inhibition of tumor formation and metastasis in a xenograft model. Another study reported that miR-34a attenuates prostate cancer aggressiveness by abrogating NOTCH and androgen receptor signaling in prostate cancer cells [93] , suggesting that miR-34a inhibits the maintenance of stem-like properties in prostate CSCs. Low expression of miR-128 has been shown in prostate CSCs, and cells that express low levels of miR-128 possess higher clonal, clonogenic and tumorigenic activities [95] . BMI1, a stem cell regulator, was identified as a direct and functionally relevant target of miR-128, suggesting that miR-128 as well as miR-34a described previously inhibit maintenance of stem-like properties in prostate CSCs. In addition, Li et al. reported that EZH2, along with BMI1, is a polycomb family transcriptional repressor, is highly expressed in prostate CSCs compared with non-CSCs and that the expression of EZH2 is attenuated by miR-101 expression [94] . miR-320 also regulates CSC properties by directly downregulating beta-catenin in prostate CSCs [96] . Interestingly, knockdown of miR-320 significantly increases cancer stem-like properties, such as tumorsphere formation, chemoresistance and tumorigenic abilities by suppressing the Wnt/β-catenin signaling pathway in a xenograft model [96] . miRNA therapy against CSCs The development of therapies targeting CSCs involves efficient strategies that target various cancers, and both bulk cancer cells and CSCs must be eliminated. Because CSCs are molecularly distinct from bulk tumor cells, they can be targeted by exploiting their molecular differences as described above (Figure 1) . However, no drugs have been identified so far that can effectively target CSCs. Important features in CSCs that are maintained by dysregulated miRNAs include activation of self-renewal and tumor-initiating ability, and resistance to anti-cancer drugs by activating

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drug efflux pumps. Thus, one of the most promising approaches to eliminating CSCs is the cell-based delivery of miRNAs or miRNA inhibitors to abolish CSCs by modulating their function and/or expression of important proteins to at least that of non-CSCs. Downregulation of miR-128 in brain tumors [72] , let-7 in breast cancer [77] , and miR-93 and miR-451 in colorectal cancer [85,87] results in gain of the self-renewal property. Thus, introduction of synthesized miRNAs into CSCs may induce ablation of their ‘stemness,’ suggesting that promoting terminal differentiation of CSCs may make them sensitize to chemotherapy and radiotherapy. Chemoresistance is also regulated by drug efflux pumps such as ABC transporters, which are potently activated in CSCs. The family of ABC transporters is composed of 49 transmembrane proteins, three of which, MDR1 (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2), are correlated with multidrug resistance  [117] . Selective expression of ABC transporters has been reported in several types of CSCs [118,119] , and their expression contributes to multiple drug resistance in various human cancers [117] . Colon cancer spheres exhibit decreased expression of miR-451, a negative regulator of ABCB1, compared with parental colorectal cancer cells [120] . Transfection of miR-451 confers a decrease in tumorigenicity and self-renewal to colon spheres via downregulation of ABCB1. Xu et al. reported that ABCG2 is a target of miR-328 in a colorectal CSC-like cell population, and overexpression of miR-328 increases drug sensitivity and inhibits cell invasion by CSCs [119] . These studies indicate that ABC transporters are key players not only as drug efflux pumps but also concurrent regulators of proteins related to stemness and tumorigenicity for the maintenance of CSCs. miRNA expression is controlled by treatment with natural dietary compounds, but not synthesized miRNAs or miRNA inhibitors. One such compound is resveratrol, which is a polyphenolic antioxidant compound contained in grapes, berries, peanuts and red wine [121] . Hagiwara et al. revealed that treatment with resveratrol upregulates Ago2 expression, followed by enhanced function of tumor suppressive miRNAs (e.g., miR-143, miR-200c, etc.), which leads to the suppression of CSC properties [122] . Another study reported that berbamine, which is contained in the berberis amurensis plant, and its analog induce apoptosis in human glioblastoma stem-like cells by upregulating miR-4284 [123] . Bao et al. found that CDF, a novel synthetic analog of curcumin, inhibited the sphere-forming ability, known as one of CSCs’ properties, via downregulation of miR-21 in pancreatic cancer cells  [124] . These results strongly support the idea that

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Cancer stem cell (CSC)

Self-renewal ability

Tumor-initiating ability

miR-214 ↑ let-7 ↓ miR-34 ↓ miR-101 ↓ miR-124 ↓ miR-128 ↓ miR-200c ↓ miR-451 ↓

miR-140 ↑ miR-215 ↑ miR-199a ↓ miR-328 ↓ miR-451 ↓

miR-34 ↓ miR-93 ↓ miR-199a ↓ miR-451 ↓

Chemoresistance

Drug B

Drug A

Drug C

Drug D

Figure 1. Dysregulated miRNAs contributing to the maintenance of cancer stem cell properties. CSC: Cancer stem cell. 

treatment with natural compounds that can repress CSC properties and/or induce apoptosis of CSCs by regulating miRNA expression levels is an effective therapeutic strategy, as is treatment with synthesized miRNAs or miRNA inhibitors for cancer therapy. Recently, in a mouse model, Ono et al. revealed how human breast CSCs are maintained in a dormant state in bone marrow before cancer recurrence  [125] . This group reported that bone marrow mesenchymal stem cells secrete exosomes containing miR-23b, which could suppress cell cycling and motility by targeting myristoylated alanine-rich C kinase substrate (MARCKS). The expression of MARCKS is implicated in the pathogenesis of metastatic cancer [126] . Ono et al. also showed that overexpression of miR-23b inhibits motility and acquired chemoresistance in breast cancer cells in an in vitro study. Moreover, metastatic breast cancer cells in the bone marrow of patients show increased miR-23b and decreased MARCKS expression, suggesting that exosomal transfer of miR-23b from mesenchymal stem cells in bone marrow may confer dormancy in breast CSCs. Therefore, inhibition of miR-23b or its

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function in CSCs may prevent cancer recurrence. In addition, the interaction between CSCs and their extrinsic cells, the so-called CSC niche, is a target for eliminating CSCs, although little is known about the type of dysregulated miRNA expression that is maintained in such an environment. Drug delivery system for miRNAs Effective systemic delivery of miRNAs and antimiRNAs to target cells or tissues is strongly dependent on stability of the synthetic RNAs against RNases and appropriate delivery and distribution of these molecules to target cells, tissues or organs only. For these reasons, development of RNAi therapy has been an enormous challenge. Historically, viral and nonviral delivery systems have been tested in experimental animals. Delivery system using viral vectors

Several studies using animal model have been reported to prove the utility of viral vector delivery system. Zhu et al. reported that overexpression of miR-23b with an adenoviral vector cures

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inflammatory autoimmune diseases via intra-articular infection [127] . An adeno-associated virus has also been successful for carrying a miRNA cluster into the muscle to knock down VEGF in vivo  [128] . Furthermore, treatment with an miR-34a mimic carried by a lentiviral vector prevents lung cancer initiation and progression via transtracheal infection [129] . These studies indicated that viral-mediated gene silencing is very useful for local infection, particularly at sites that generally involve difficulty with administration. Although viral delivery has frequently shown higher efficiency than nonviral systems, preliminary clinical studies have shown that viral delivery triggers strong inflammatory reactions [130] , and these delivery vectors have caused the death of several patients in the clinic  [131,132] . Therefore, understanding the details of the inflammatory mechanism is important for developing a safer carrier system using viral vectors. Delivery system using nonviral carriers

To avoid the problems caused by viral vector administration described above, a recent focus has been on a nonviral approach based on nanoparticle systems (e.g., liposomes, polymer conjugates, micelles, dendrimers, carbon nanotubes, gold nanoparticles and atelocollagen [133]) because of the advantages of such an approach over viral vectors. These nonviral approaches are nonimmunogenic, inexpensive, and provide easy quality control. A number of reports have demonstrated a significant anticancer effect caused by systemic delivery of siRNAs with cationic liposomes [134–136] . Cationic liposomes are extensively used for siRNA delivery to protect siRNA from enzymatic degradation, facilitate tumor cell uptake and promote escape from the endosomal compartment, thus resulting in effective cytoplasmic delivery. In fact, the most challenging aspect of siRNA delivery is how to get the intact siRNA out of the endosomes, a process that requires both endosome escape and sufficient deassembling of the formulation. Similarly, a cationic polymer, polyethyleneimine, was commercialized as in vivo jetPEI™ and is supplied by Life Technologies (Illkirch, France). This polymer was used to successfully deliver siRNAs or miRNAs to cancer cells in animals  [137–139] . In addition, atelocollagen can be obtained from type I collagen from calf dermis and is also expected to be a useful carrier because of its low immunogenicity and efficiency [140–142] . We have also reported two cases of miRNA therapy in which tumor suppressive miR-16 or miR-143 mimics were successfully delivered via a systemic approach using atelocollagen. This therapy dramatically inhibits the growth of

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metastatic prostate cancer (miR-16) [143] or lung metastasis of osteosarcoma (miR-143) [144] . Furthermore, chemically functionalized carbon nanotubes also show potential in novel biological applications for the delivery of caspase-3 siRNA into the brain by topical injection into the cerebral cortex. This approach reduces neurodegeneration without toxic side effects [145] . miRNAs circulate in human body fluids such as plasma, saliva and breast milk, although abundant RNase also exists in the body [146–150] . Thus, miRNAs may be protected against RNase in these body fluids. miRNAs exist in extracellular vesicles such as exosomes  [151] , which are produced by not only normal cells such as dendritic cells [152] and intestinal epithelial cells [153] but also cancer cells [154] . These exosomes that include miRNAs can be delivered to other cells and subsequently suppress the target genes in the recipient cells [155–157] . Kosaka et al. reported that proliferation of prostate cancer cells is suppressed by treatment with culture supernatant from normal epithelial prostate cells in vitro and in vivo  [158] . The study showed that the tumor-suppressive miR143 inhibits growth by downregulating KRAS and ERK5 expression in prostate cancer cells. The conjugation of a cell-specific ligand at the surface of exosomes provides increased specificity and efficiency of miRNA delivery to cancer cells. Ohno et al. generated modified exosomes derived from the human embryonic kidney cell line HEK293 expressing the GE11 peptide, which specifically binds to EGF or its receptor on the cell surface. These modified exosomes can efficiently deliver let-7a miRNA to EGF receptor-expressing xenografted breast cancer tissue in immunodeficient mice [159] . These observations suggest that exosomes can be used for miRNA replacement therapy by restoring the expression of miRNAs that are downregulated in target cells. How miRNA mimics and inhibitors are delivered to CSCs only is important for the development of CSC-targeting therapy. To solve such critical problems, conjugation of antibodies or ligands recognizing CSC-specific molecules on the surface of exosomes or nanoparticle systems could produce a more potent CSC-specific delivery system. Conclusion In this review, we summarized the current understanding of the biological targets of miRNAs that maintain the properties of self-renewal and drug resistance in CSCs from several types of cancer. The increasing knowledge about the function of miRNAs makes them interesting candidates for use in therapy against CSCs.

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Review  Osaki, Okada & Ochiya Future perspective The development of a ‘magic bullet’ that can eliminate CSCs is expected to prevent not only tumorigenesis but also the metastatic capacity, drug resistance and tumor recurrence, all of which ultimately contribute to poorer prognosis. Although more preclinical works are needed to conclusively demonstrate the true nature of CSCs, groundbreaking discoveries regarding the function of miRNAs in CSCs will shed light on strategies for the development of new cancer therapy. In addition, a challenging question remains regarding whether synthetic miRNAs and/or miRNA inhibitors can be delivered in vivo in a CSC-targeted manner. Further innovations in safer CSC-specific delivery systems will facilitate the clinical application of CSC-targeted miRNA therapy.

Acknowledgement The authors acknowledge a grant-in-aid for the Scientific Research on Applying Health Technology from the Ministry of Health, Labor and Welfare of Japan.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Cancer stem cells • A cancer stem cell (CSC) is defined as a cell within a tumor that possesses the capacity to self-renew and to produce heterogeneous lineages of the cancer cells that comprise the tumor. • CSCs represent a distinct cell population that can be identified and prospectively isolated from a wide variety of tumor tissues using CSC-specific cell surface markers. • The Notch signaling pathway, the Wnt/β-catenin pathway, the Hedgehog signaling pathway and/or epithelialmesenchymal transition are aberrantly activated in a variety of CSCs.

miRNA and its dysregulation in CSCs • miRNAs are endogenous noncoding RNAs approximately 22 bp in length that may inhibit translation and facilitate degradation of their target transcripts, thus playing important roles in a wide range of physiological and pathological processes. • Aberrant expression of miRNAs has been observed in CSCs, and they regulate CSC characteristics by influencing signaling pathways and CSC signature genes, which activate self-renewal, tumor-initiating ability, and resistance to anti-cancer drugs by activating drug efflux pumps. • Dysregulation of miRNA expression is observed in several human malignancies, including brain tumors, and cancers of the breast, colon, ovary and prostate.

miRNA delivery for CSC-targeted therapy • Correction of CSC-specific aberrant expression of miRNAs with synthesized oligonucleotides may represent the next generation of therapeutic strategies in cancer treatment. In addition, natural dietary compounds such as resveratrol may be useful for miRNA therapy against CSCs. • CSC-specific drug delivery systems have been developed by utilizing not only viral vectors but also nonviral carriers including synthesized nanoparticles and exosomes produced by cells. • Conjugation of antibodies or ligands recognizing CSC-specific molecules on the surface of exosomes or nanoparticle systems may produce more potent CSC-specific delivery systems.

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