micrornas in cancer - from bench to bedside (cortez et. al. 2010)

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microRNAs in Cancer:

From Bench to BedsideMaria Angelica Cortez,* Cristina Ivan,* Peng Zhou,{

Xue Wu,z Mircea Ivan,z and George Adrian Calin*

*Department of Experimental Therapeutics and The RNA

Interference and non-codingRNA Center, The University of

Texas MD Anderson Cancer Center, Houston, Texas, USA{

Department of Biological Science, Purdue University Calumet,

Hammond, Indiana, USAz

Department of Medicine, Microbiology and Immunology, Indiana University

Melvin and Bren Simon Cancer Center, Indianapolis, Indiana, USA

I. IntroductionA. What Are miRNAs?B. miRNA Biogenesis and Mechanism of Action

II. Alterations of miRNA Expression in CancerIII. Causes of miRNA Expression Variations

A. Cancer Associated with Genomic RegionsB. Mutations and Single-Nucleotide Polymorphisms

C. Epigenetic Regulation of miRNA ExpressionD. Roles of Hypoxia-Inducible Factor and Hypoxia in miRNA ExpressionE. Regulation of miRNA Expression by Transcription FactorsF. Regulation of miRNA Expression by Estrogens

G. Posttranscriptional Regulation of miRNA ExpressionIV. Pathways Involving miRNA Alterations

A. Self-Sufficiency in Growth SignalsB. Insensitivity to Antigrowth SignalsC. Evasion of ApoptosisD. Limitless Replicative PotentialE. Angiogenesis

F. Invasion and MetastasisV. Clinical Applications

A. miRNAs Biomarkers for Cancer Diagnosis and PrognosisB. Potential Use of Circulating miRNAs in Cancer DiagnosisC. Therapy with miRNAs

VI. Concluding RemarksReferences

microRNAs (miRNAs) are master regulators of gene expression. By degrading orblocking translation of messenger RNA targets, these noncoding RNAs can regulate the

expression of more than half of all protein-coding genes in mammalian genomes.Aberrant miRNA expression is well characterized in cancer progression and has prog-nostic implications for cancer in general. Over the past several years, accumulatingevidence has demonstrated that genomic alterations in miRNA genes are correlated

Advances in CANCER RESEARCH 0065-230X/10 $35.00Copyright 2010, Elsevier Inc. All rights reserved. DOI: 10.1016/S0065-230X(10)08001-2

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with all aspects of cancer biology. In this review, we describe the effects of miRNAderegulation in the cellular pathways that lead to the progressive conversionof normal cells into cancer cells as well as in cancer diagnosis and therapy in humans.# 2010 Elsevier Inc.

I. INTRODUCTION

A. What Are miRNAs?

microRNAs (miRNAs) are short (1924 nt) noncoding RNAs (ncRNAs,RNAs that do not encode proteins) that play important roles in posttran-scriptional gene silencing of target messenger RNAs (mRNAs) (Bartel,

2004). miRNAs are involved in virtually all biological processes, such ascell proliferation and apoptosis, development, differentiation, metabolism,immunity, neuronal patterning, stress response, aging, and cell-cycle control(Ambros and Lee, 2004; Bartel, 2004; He and Hannon, 2004; Kato andSlack, 2008; Plasterk, 2006). miRNAs are strongly conserved among inver-tebrates, vertebrates, and plants (Ambros, 2003), and researchers haveidentified more than 700 miRNAs in humans (Griffiths-Jones et al., 2008).More than 70% of miRNAs are transcribed from individual miRNA genes,introns or exons of protein-coding genes, or polycistronic transcripts that

encode related miRNAs (Lee et al., 2004). Investigators have estimated thatmore than 50% of all protein-coding genes are regulated by miRNAs inmammalian genomes (Friedman and Jones, 2009; Lewis et al., 2005).

B. miRNA Biogenesis and Mechanism of Action

An miRNA is transcribed in the nucleus as a long, capped, polyadenylated

precursor primary precursor (pri-miRNA) by RNA polymerase II or III (Leeet al., 2002; Zeng et al., 2003). The resulting pri-miRNA is processed by theribonuclease (RNase) III Drosha and the double-stranded DNA-bindingprotein DGCR8/Pasha (Ambros and Lee, 2004) to form a precursor miRNA(pre-miRNA) (Lee et al., 2003). The nuclear export receptor exportin 5/RanGTP (Lund et al., 2004; Yi et al., 2003) actively transports pre-miRNAs to thecytoplasm, where they are processed by the RNase III endonuclease Diceralong with the double-stranded transactivation-responsive RNA-binding pro-tein (TRBP), resulting in a small double-stranded RNA structure (22 nt).

This miRNA duplex is unwound into mature single-stranded form andincorporated into the RNA-induced silencing complex (RISC), which guidesthe complex into the complementary 30-untranslated region (UTR) of thetarget mRNA (Gregory et al., 2006). However, authors recently reported

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that miRNAs can also target the 50-UTRs of target mRNAs and open readingframes as well as promoter regions (Kloosterman, etal., 2004; Lee etal., 2009;Lytle et al., 2007; Place et al., 2008). Negative regulation of gene expression

occurs via either mRNA cleavage, when it is perfectly complementary to the 30

-UTR of the target mRNA, or translational repression in cases of partialcomplementarity (Fig. 1; Bohnsack et al., 2004; Gregory et al., 2006; He andHannon, 2004). In mammals, regulation mediated by miRNAs is accom-plished by imperfect base pairing along with protein translational repressionof the target gene (Mathonnet et al., 2007; Petersen et al., 2006). In addition,studies demonstrated that miRNAs can upregulate the expression of theirtargets; for example, miR-369-3p upregulates tumor necrosis factor-a (TNF-a) expression (Vasudevan et al., 2007).

Many miRNAs exhibit diverse temporal and spatial expression patterns.Additionally, the relative level of expression of a particular miRNA can varyby several orders of magnitude depending on the cell type. Researchers havedeveloped combined experimental and computational methods to determinewhen, where, and in what quantity a specific miRNA exists and identify itsbiological function. This miRNA analysis is performed in two steps. First,the level of miRNA expression is measured using one of the several availablehigh-throughput technologies (e.g., microarray, real-time polymerase chainreaction, microbeads analysis). Second, the miRNA expression is clustered

to distinguish biologically meaningful information that can be used toclassify and identify specific molecular pathways for a given disease. Becausea single miRNA can target hundreds of mRNAs, aberrant miRNA expres-sion is capable of disrupting the expression of several mRNAs and proteins(Chin and Slack, 2008). Therefore, alterations in miRNA expression areinvolved in the initiation of many diseases, including cancer.

II. ALTERATIONS OF miRNA EXPRESSION IN CANCER

Initially identified in cases of B cell chronic lymphocytic leukemia (CLL)(Calin et al., 2002), investigators have since detected miRNA alterations inmany types of human tumors. Researchers have broadly applied genome-wide miRNA expression profiling using high-throughput technologies suchas microarrays in the study of several cancer types. Based on the results ofthese studies, authors have reported disease-specific expression profiles withimportant diagnostic and prognostic implications in many human cancers,

including B cell CLL (Calin et al., 2004), breast carcinoma (Iorio et al.,2005), primary glioblastoma (Ciafre et al., 2005), hepatocellular carcinoma(Murakami et al., 2006), papillary thyroid carcinoma (He et al., 2005),lung cancer (Yanaihara et al ., 2006), gastric and colon carcinomas

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(Michael et al., 2003), and endocrine pancreatic tumors (Volinia et al.,2006). In addition, miRNA expression profiles have displayed signatures

related to tumor classification, diagnosis, and disease progression and haveproven useful in determining the primary site for cancers of unknown origin(Calin and Croce, 2006; Lu et al., 2005; Rosenfeld et al., 2008; Yanaiharaet al., 2006).

Cleavage

Pri-miRNA

Transcription(RNA pol II ou III)

Drosha

DGCR8

Nucleus

Pre-miRNA

Ran-GTPExportin 5

Cytoplasm

Pre-miRNA

Ribosoms

Repression of translationmRNA degradation

Dicer

miRNA: miRNA*

miRNA gene

RISCAAAAm7Gppp

m7Gppp AAAA

OH 5P

Export

miRNA*

Fig. 1 miRNA biogenesis and mechanism of action. miRNAs are first transcribed by RNApolymerase II or III in the nucleus as primary transcripts (pri-miRNAs) and then processed bythe RNase III Drosha and the double-stranded DNA-binding protein DGCR8 to produce pre-miRNAs. The pre-miRNAs (hairpins) are actively transported to the cytoplasm by exportin

5/Ran-GTP. In the cytoplasm, pre-miRNAs are processed by the RNase III endonuclease Diceralong with the TRBP, yielding a small double-stranded RNA (miRNA: *miRNA). The maturesingle-stranded miRNA (*miRNA) is incorporated in the RISC, which is guided to the comple-mentary 30-UTR of the target mRNA. miRNA-negative regulation occurs via either mRNAcleavage or translational repression.



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miRNAs reportedly function as either oncogenes or tumor suppressors(Esquela-Kerscher and Slack, 2006). For instance, miR-10b, which is highlyexpressed in metastatic breast cancer cells, is a known oncogenic miRNA

that suppresses HOXD10, which increases the expression ofRHOC, a geneassociated with tumor cell proliferation and metastasis (Ma et al., 2007).Also, researchers identified miR-21 as a potentially oncogenic miRNAwhose expression is upregulated in various solid tumors as well as hemato-logical malignancies (Krichevsky and Gabriely, 2009). miR-21 regulatesimportant suppressor genes, such as PTEN (Meng et al., 2007) andPDCD4 (Asangani et al., 2008). In addition, not only single transcribedmiRNAs but also clusters of miRNAs, such as miR-1792 (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1), enhance tumorigenicity.

Interestingly, miR-17

92 is located on 13q31.3, a chromosomal region ampli-fied in diffuse large B cell lymphomas (DLBCLs), follicular lymphomas, Bur-kitt lymphoma, and lung carcinomas (Ota et al., 2004). Furthermore,miR-1792 has a pleiotropic function, as it is able to promote proliferation,increase angiogenesis, and sustain cell survival via posttranscriptional repres-sion of a number of target mRNAs (Olive et al., 2010). On the other hand,studies have identified several miRNAs that act as tumor suppressors. Amongthe most well-characterized tumor suppressor miRNAs are the miR-34 familymembers, which are important effectors of TP53 activation (Bommer et al.,

2007; Chang et al., 2007). Ectopic expression ofmiR-34 genes has promotedcell-cycle arrest, induced cellular senescence, and inhibited proliferation(Hermeking, 2010). Also, expression of members of the let-7 family oftumor suppressor miRNAs is downregulated in many malignancies and inhi-bits cancer growth by targeting various oncogenes, such as RAS, and inhibitingkey regulators of several mitogenic pathways, such as HMGA2 (Johnson etal.,2005; Peter, 2009). However, the initial categorization of miRNAs as onco-genes or tumor suppressor genes based on their levels of expression in tumorsversus normal tissues has proven to be inaccurate, as experiments have shown

that many of them have dual natures as both oncogenes and tumor suppressorgenes according to cancer type. Nonetheless, further studies should elucidatethe nature of deregulation of miRNA expression as well as its role intumorigenesis.

III. CAUSES OF MIRNA EXPRESSION VARIATIONS

Over the past few years, investigators have made much progress withrespect to understanding the regulatory mechanisms of specific miRNAs.Currently, we can assume that the expression of virtually every miRNA isregulated and finely tuned by a variety of transcription factors in a fashion



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similar to the effect of regulatory mechanisms on the expression of conven-tional genes. In this section, we concentrate on regulatory mechanismsrelevant to malignant cells, particularly specific transcription factors directly

involved in tumorigenesis.

A. Cancer Associated with Genomic Regions

Genomic variation in miRNA genes can affect their processing and, con-sequently, their ability to properly regulate the expression of target genes.miRNAs are frequently located in cancer-associated genomic regions(CAGRs) that are often subject to rearrangements, breakpoint regions, loss

of heterozygosity sites, deletions, and amplifications in cancer cells and areaberrantly expressed in a variety of malignancies (Calin et al., 2004). Thefirst evidence of the involvement of miRNAs in cancer came in a study ofmiR-15a and miR-16a, located on chromosomal region 13q14, which isdeleted in more than half of all B cell CLL cases (Calin et al., 2002). miR-15aand miR-16a induce apoptosis by targeting the mRNA of the antiapoptoticBCL2 gene (Cimmino et al., 2005). Also, copy-number changes for somemiRNA genes are common to several tumor types, such as ovarian cancer,breast cancer, and melanoma, whereas other such copy-number changes are

unique to specific tumor types (Zhang et al., 2006). A study demonstratedfrequent, marked overexpression, with occasional gene amplification, of themiR-1792 cluster in intron 3 of C13orf 25 gene on 13q31.3 in lung cancercases(Hayashita etal.,2005). Moreover, specific miRNAexpression signatureshave proven to be associated with specific translocations in hematopoieticmalignancy and solid tumor (Dixon-McIver et al., 2008; Garzon et al., 2008;Varambally et al., 2008). For example, the fusion gene AML1/ETO, which isproduced by the t(8;21) translocation, promotes heterochromatic silencing of

pre-miR-223 in patients with leukemia (Fazi et al., 2007).

B. Mutations and Single-Nucleotide Polymorphisms

Although single-nucleotide polymorphisms (SNPs) are rare in miRNAgenes, they can affect miRNA function in pri-miRNA transcription, pri-miRNA and pre-miRNA processing, and miRNA and mRNA binding sites(Saunders et al., 2007; Wu et al., 2009). In addition, several studies indicatedthat some SNPs in both miRNA genes and miRNA target genes increase the

risk of certain cancers. Initially, investigators discovered a mutation in themiR-189 binding site of SLIT and SLITRK1, which is associated withTourette syndrome (Abelson et al., 2005). Afterward, other studies demon-strated an association between the presence of SNPs in miRNA genes and



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cancer risk. Recent studies demonstrated that the presence of SNPs in pri-miRNAs is related to processing and the level of expression of maturemiRNAs, such as that occurs in pri-miRNA regions of let-7e and miR-16

(Nicoloso et al., 2010; Ryan et al., 2010). Also, the presence of the SNPrs531564 in pri-miR-124-1 was associated with increased bladder andesophageal cancer risk (Yang et al., 2008; Ye et al., 2008). Researchersalso showed an association of the pre-miR-196a-2 SNP rs11614913 withbreast cancer risk (Hoffman et al., 2009). In contrast, the pre-miR-27a SNPrs895819 decreases the risk of breast cancer (Kontorovich et al., 2010).Furthermore, miRNA: mRNA base pairing is crucial in driving miRNAstoward target genes. Increasing evidence shows that SNPs can abolish orcreate new binding sites. For example, researchers found an SNP in binding

sites in the complementary sites of let-7 in the 30

-UTR of KRAS gene,increasing the risk of lung cancer in moderate smokers (Chin et al., 2008).Importantly, the SNP rs2910164, which is located in the 3p strand of miR-146a, is an example of a functional SNP in miRNAs. It promotes mispairingin the hairpin of the precursor, altering the expression of miR-146a andleading to an increased risk of papillary thyroid carcinoma (Jazdzewskiet al., 2008). Alterations in miRNA expression caused by sequence varia-tions such as SNPs may be another important factor contributing to cancerpredisposition. Moreover, because specific miRNAs have numerous targets,

inherited SNPs in miRNA genes may have important consequences on theexpression of various target oncogenes and tumor suppressor genes involvedin cancer pathogenesis. Nevertheless, examination of the impact of miRNAgene SNPs on cancer risk is only just a beginning, and new findingsshould elucidate the potential of these variations in affecting human cancerprognosis and progression.

C. Epigenetic Regulation of miRNA Expression

DNA hypermethylation of tumor suppressor genes, global genomic hypo-methylation, and aberrant histone modifications are the most common hall-marks of epigenetic alterations associated with cancer (Herman and Baylin,2003). Emerging evidence indicates that epigenetic mechanisms contributeto the aberrant expression of miRNAs in cancer cells, especially the trans-criptional inhibition of tumor suppressor miRNAs. Silencing of miRNAswith tumor-suppressive roles by epigenetic mechanisms includes promoter-associated CpG island methylation and repressive histone modifications

(Agirre et al., 2009). Chim et al. (2010) found that the miR-34a promoteris methylated in 75% of lymphoma and 37% of melanoma cell lines com-pared with its unmethylated status in normal controls. Expression ofmiR-124a is reduced in acute lymphoblastic leukemia (ALL) by hypermethylation



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of promoter and histone modifications (Agirre et al., 2009). Epigeneticsilencing of specific miRNAs induces overexpression of their targeted onco-genes. For example, epigenetic silencing ofmiR-124a in acute lymphoblastic

leukemia cells increases CDK6 expression, which contributes to abnormalproliferation of the cells via phosphorylation of retinoblastoma 1 (RB1)(Agirre et al., 2009). Another study showed that increased SOX4 expressionin endometrial cancer cells is caused by aberrant methylation of the miR-129-2 promoter and that restoration of this miRNA expression is associatedwith decreased SOX4 expression and reduced proliferation of cancer cells(Huang et al., 2009b).

Like the widely discussed miRNA signatures in cancer, a similar conceptregarding epigenetic miRNA signatures in cancer may contribute to its

diagnosis and prognosis. Lujambio and colleagues proposed that themiRNA hypermethylation profile may be used to characterize tumor meta-stasis and found that the hypermethylation of miR-148a, miR-34b/c, andmiR-9 is significantly associated with metastasis (Lujambio et al., 2008).

Similar to the transcript factor-miRNA regulatory feedback loop, recentstudies showed that some important parts of epigenetic machinery, includingDNA methyltransferases, histone deacetylases, and histone methyltrans-ferases, are direct targets of miRNAs. For example, authors reported thatmiR-29b induced global hypomethylation in acute myeloid leukemia (AML)

by directly targeting DNMT3A and DNMT3B and indirectly targetingDNMT1 (Garzon et al., 2009). Also, DNMT3 is a direct target ofmiR-143, which is frequently downregulated in colorectal cancer cells.Restoration of miR-143 expression in colorectal cancer cells reduced theirgrowth and colony formation in a soft agar assay (Ng et al., 2009b). Thediscovery that methylation is implicated in miRNA expression opens up thepossibility of future use of epigenetic drugs as DNA-demethylating agents incancer therapy.

D. Roles of Hypoxia-Inducible Factor and Hypoxia

in miRNA Expression

Hypoxia is a central feature of the cancer microenvironment (Harris, 2002)and well-documented contributor to the development of resistance to antineo-plastic therapy (Giaccia et al., 2004; Semenza, 2004). The hypoxia-induciblefactor (HIF) family of transcriptional regulators is widely acknowledged to

coordinate molecular mechanisms of response to oxygen deprivation bydirectly regulating the expression of hundreds of genes.Recent data suggest that the wide spectrum of hypoxia- and HIF-triggered

responses extend beyond protein-encoding genes. Increasingly, groups have



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reported hypoxia-regulated miRNAs, including miR-210, miR-373, miR-103, miR-24-1, miR-181c, miR-26b, and miR-26a-2 (Camps et al., 2008;Crosby et al., 2009; Fasanaro et al., 2008; Kulshreshtha et al., 2007).

Although at least one study has shown that more than 20 miRNAsrespond to hypoxia (Kulshreshtha et al., 2007), miR-210 stands out as thecommon denominator in all the reported studies. Indeed, hypoxic inductionofmiR-210 is not limited to transformed cells, as this miRNA is also a keyplayer in the response of endothelial cells to low levels of oxygen tension(Fasanaro et al., 2008), which affects angiogenesis. Apart from being theprototypical miRNA modulated by oxygen, miR-210 is very likely to signif-icantly impact clinical outcomes of a variety of cancer types. Virtuallyuniversally overexpressed in tumor cells, especially in breast, pancreatic,

and head and neck cancers, miR-210 expression is strongly correlated withthe hypoxia metagene expression in vivo and negatively affects clinical out-comes (Camps et al., 2008). Studies have indicated that HIF1A is the leadingcandidate regulator of hypoxia-responsive miRNAs, particularly miR-210and miR-373 (Camps et al., 2008; Crosby et al., 2009; Fasanaro et al., 2008;Huang et al., 2009a; Kulshreshtha et al., 2007). The researchers in thesestudies employed multiple strategies, including transduction of active formsof HIFs and, conversely, inactivation by using short hairpin RNA lenti-viruses or small interfering RNA duplexes. Additionally, chromatin immu-

noprecipitation analysis indicated recruitment of endogenous HIF1to specific hypoxia response element (HRE) sequences in the miR-210 pro-moter, and luciferase-based reporters driven by fragments of select HREpromoters. Similar findings were reported for miR-373, another miRNAwidely overexpressed in cancer cells (Crosby et al., 2009). Consistent withHIF role in the expression of miRNAs, miR-210 is particularly overexpressedin clear cell renal cell carcinoma cases (Juan et al., 2010). These tumors areknown to have abnormally high levels of HIF expression because of geneticinactivation of the tumor suppressor VHL (Ivan and Kaelin, 2001).

What are the biological and biochemical implications of upregulation ofmiR-210 expression induced by hypoxia? Although relevant data are justnow emerging, several groups have reported that miR-210 links hypoxiawith reactive oxygen species generation, decreased Krebs cycle activity, andelectron transport in mitochondria via downregulation of iron-sulfur clusterscaffold homolog (ISCU) expression (Chen et al., 2010; Fasanaro et al.,2009; Favaro et al., 2010). ISCU is critical for the assembly of FeS clustersat least at the level of mitochondrial complex 1 and aconitase enzymeactivity; therefore, downregulation of ISCU expression in response to miR-

210 overexpression results in decreased mitochondrial energy metabolismand increased reliance of glycolysis. The importance of this pathway is sup-ported by clinical data showing that a variety of cancer types with low ISCUand high miR-210 expression exhibit worse prognoses (Favaro et al., 2010).



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Other metabolic players are emerging, such as the phosphate dehydrogenaseGPD1L (Fasanaro etal., 2009). However, their roles in the response to hypoxiaremain elusive. Thus, these studies were the first to show that miR-210 is a

nodal point linking the microenvironment, metabolism, and the clinical coursein cancer cases.

E. Regulation of miRNA Expression by

Transcription Factors

Not surprisingly, some of the most important positive regulators of pro-

oncogenic miRNAs expression are transcription factors encoded by proto-oncogenes. One of the best documented cases of this regulation involvesMYC and the miR-1792 cluster. MYC upregulates the expression of thiscluster, and this mechanism contributes to robust angiogenesis and growthin tumors. Although the picture is far from complete, miR-1792 seems toexert these effects largely by targeting antiangiogenic thrombospondin 1 andrelated proteins (Dews et al., 2006). Another target ofmiR-1792 relevantto cancer is the transcription factor E2F1. MYC activation of miR-1792leads to downregulation of E2F1 expression, providing a regulatory loop

potentially aimed at limiting MYC-triggered proliferation (ODonnellet al., 2005).E2F1 has been at the center stage of research on miRNA expression

regulation. Recently, multiple studies reported the existence of negativeregulatory loops between all E2F family members and several miRNAs asa safety mechanism for prevention of excessive proliferation (Coller et al.,2007; ODonnell et al., 2005; Sylvestre et al., 2007; Woods et al., 2007).One study of different subtypes of AML showed a mutual negative feedbackloop between E2F1 and miR-223 involved in granulopoiesis (Pulikkan et al.,

2010). E2F1 inhibits miR-223 transcription, whereas repression of E2F1mediated by miR-223 prevents myeloid cell-cycle progression (Pulikkanet al., 2010). Authors reported on another feedback loop in gastric cancercases in which the E2F1-induced miR-106b25 oncogenic cluster inhibitsE2F1 expression (Petrocca et al., 2008). Regulation of miRNAs by tran-scription factors in cancer cells occurs in a cancer- and tissue-specific fash-ion, one example being induction of miR-449a/b by E2F1 in testes, lungs,and trachea but rarely in other cancer cells (Lize et al., 2010).

Because it is the most frequently mutated transcription factor in cancer

cells, the fact that TP53s impact on miRNA expression has been a focus ofintensive investigation is hardly surprising. The first documented TP53-induced miRNAs were the members of the miR-34 family, which haveevolutionarily conserved TP53 binding upstream of the coding sequences



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(Corney et al., 2007; He et al., 2007; Tarasov et al., 2007). miR-34 familymembers are increasingly viewed as more than bystanders during TP53activation, with involvement in reprogramming of critical gene expression

and regulation of apoptosis and the cell cycle. Thus, these miRNAs mayaccount in part for well-established biological effects of TP53, potentially toan extent similar to classic targets of this tumor suppressor. In fact, Guessouset al. (2010) reported evidence that miR-34a acts as a bona fide suppressorby downregulating the expression of oncogenes such as METand NOTCH11 and 2 and inhibiting glioma xenograft growth.

Following the discovery of an miR-34based response to TP53 activation,studies identified additional miRNAs that behave in a similar fashion. Forexample, expression of the homologous miRNAs miR-192 and miR-215 is

upregulated in a TP53-dependent manner after exposure to genotoxic stressand lower in colon tumors than in normal colon tissue (Braun et al., 2008),potentially reflecting loss of wild-type TP53. miR-192 and miR-215 inducecell-cycle arrest by coordinately targeting several transcription factorsinvolved in mediation of G1-S and G2-M checkpoints, which is consistentwith their status as biologically relevant targets of TP53. Also, investigatorsshowed that these TP53-induced miRNAs were involved in TP53 regulationof hypoxia signaling (Boominathan, 2010; Yamakuchi et al., 2010). Forexample, miR-107 is an miRNA with TP53-induced expression in colon

cancer cells that potentially suppresses hypoxia signaling, tumor angiogene-sis, and growth by targeting hypoxia-inducible factor HIF1B. Consistently,in human colon tumor specimens, expression ofmiR-107has been inverselyassociated with expression of HIF1B.

Although activation of miRNAs by TP53 has been extensively studied, therepression of particular miRNAs can be relevant to the function of TP53.Reports demonstrated that TP53 inhibits the level of miR-1792 clustertranscripts under hypoxic conditions, and overexpression of these transcriptssignificantly suppresses hypoxia-induced apoptosis. Yan and colleagues iden-

tified relevant TP53 and TATA-binding protein binding sites in miR-17

92,observing that transcriptional repression results from competition for bindingsites between the two factors (Yan et al., 2009).

In addition to specific transcriptional regulation of miRNA expression,recent data indicated that TP53 broadly affects miRNA expression levels(Suzuki et al., 2009). Specifically, TP53 interacts with the Drosha processingcomplex by associating with the RNA helicase p68 and facilitates the proces-sing of pri-miRNAs to pre-miRNAs with growth-suppressive functions, in-cluding miR-16-1, miR-143, and miR-145. Transcriptionally inactive TP53

mutants interfere with functional assembly of the Drosha complex with RNAhelicase p68, leading to attenuation of miRNA processing activity and thuspotentially contributing to a reported general decrease in miRNA expressionin cancer cells. Also, preliminary evidence indicates that TP53 as well as the



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related tumor protein p73 and tumor protein p63 interact with and conse-quently regulate the major components of miRNA processing, includingDrosha-DGCR8, Dicer-TRBP2, and Argonaute proteins. Additionally, pro-

moters of Dicer and retinoblastoma-binding protein 6 contain candidateTP53-response elements (Boominathan, 2010). Such miRNAs, and potential-ly other noncoding transcripts, continue to expand the already complex TP53network, and their role will likely become more apparent in the near future.Which, if any, of the TP53-responsive miRNAs are essential for the functionof TP53 as a tumor suppressor and guardian of the genome remains to beestablished.

F. Regulation of miRNA Expression by Estrogens

Estrogens are widely accepted as major contributors to breast cancerdevelopment. Ligand-activated estrogen receptor (ER) a and b regulatetranscription by directly binding to estrogen response elements locatedupstream of the target genes or indirectly by tethering to nuclear proteinssuch as JUN and Sp1 transcription factor (Kushner et al., 2000). SeveralmiRNA microarray analyses have revealed specific, although somewhat

discrepant, miRNA expression patterns after estrogen-based treatment ofERa-positive breast cancer cell lines (Bhat-Nakshatri et al., 2009; Castellanoet al., 2009; Kovalchuk et al., 2007; Maillot et al., 2009). Some estrogen-regulated miRNAs are associated with estrogen response elements, whereasseveral others are located in the intergenic regions of estrogen-regulatedgenes. A few miRNAs are regulated by secondary estrogen responses viaestrogen-regulated transcript factors and are likely associated with epigenet-ic alteration (Bhat-Nakshatri et al., 2009).

In studies of chronic (612 weeks) exposure to estradiol (E2), in a mam-

mary carcinogenesis model in female rats (Kovalchuk et al., 2007), expres-sion of a group of miRNAs (including miR-22, miR-99a, miR-127,miR-29c, and miR-499) was downregulated, whereas expression ofmiR-20a/b, miR-21, miR-17-5p, and miR-106a/b was upregulated. Interest-ingly, following even longer exposure to E2, the spectrum of miRNAexpression changed significantly, as expression of only miR-139 was down-regulated, whereas expression ofmiR-21, miR-103, miR-107, miR-129-3p,and miR-148a was upregulated.

In a study on human breast cancer cells, Maillot et al. (2009) noted that 23

of 125 miRNAs tested were repressed in an E2

-dependent manner in MCF-7cells after treatment with E2. Of note, several E2-repressed miRNAs, espe-cially miR-26a and miR-181a, also suppressed E2-dependent cellproliferation.



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Expression of one of the best documented cancer-associated miRNAs,miR-21, is significantly upregulated in ERa-positive breast cancer cells(Iorio et al., 2005). However, the direct impact of E2 on its expression is

still controversial, as different groups have reported contrasting effects ofthis exposure (Bhat-Nakshatri et al., 2009). A variety of additional factorsseem to be involved in the effects of estrogens on miRNA expression, such asthe AKT (Bhat-Nakshatri et al., 2009). Additionally, upregulation of pri-miR-1792 under E2 stimulation is thought to be mediated by direct inter-action between MYC and its promoter in an E2-dependent manner(Castellano et al., 2009).

G. Posttranscriptional Regulation of miRNA ExpressionIn addition to specific transcription factors, the multistep miRNA matu-

ration process can be targeted by regulatory mechanisms. Frequentlyobserved lack of correlation among expression of pri-miRNAs, pre-miRNAs, and mature miRNAs indicates the existence of an extensive post-transcriptional regulation mechanism (Thomson et al., 2006).

Early evidence of such mechanisms emerged with a study showing thatDicer processing of pre-miR-138-2 was blocked by an unknown inhibitory

factor in the cytoplasm (Obernosterer et al., 2006). In another study, Dicerprocessing was blocked by nuclear sequestration of pre-miRNA in miR-31,miR-105, and miR-128a in several cancer cell lines (Lee et al., 2008).Drosha processing of primary let-7 is selectively inhibited in embryoniccells by the RNA-binding protein Lin-28, which interacts with let-7s con-served loop region (Newman et al., 2008; Viswanathan et al., 2008). Wide-spread downregulation of miRNA expression caused by blockade duringDrosha processing has occurred in mice during their development and in awide range of primary tumors (Thomson et al., 2006). Furthermore, Argo-

naute, a well-known RISC slicer with RNase activity, is reported to beinvolved in miRNA posttranscriptional regulation via its enhancement ofthe production or stability of mature miRNAs (Diederichs and Haber,2007).

The global efficiency of miRNA biogenesis can be affected by well-knownphysiological or pathological factors, and large-scale alterations in posttran-scriptional regulation of miRNA expression may contribute to cancer devel-opment. A high cell density can globally activate miRNA biogenesis in bothnontransformed and cancer cells. This broad enhancement of miRNA

expression is associated with elevated processing of pri-miRNAs by Droshaand increasingly efficient incorporation of mature miRNAs into RISC(Hwang et al., 2009). Kumar et al. (2007) reported that global repressionof miRNA maturation by infection with short hairpin RNAs targeting



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components of miRNA processing machinery (Drosha, DGCR8, andDicer1) promoted cellular transformation and tumorigenesis in human can-cer cell lines and animal models, which was consistent with observations

that cancer cells exhibit generally reduced expression of miRNAs.

IV. PATHWAYS INVOLVING miRNA ALTERATIONS

Recent reports suggested that multiple miRNAs work in concert to regu-late related targets in common pathways. Indeed, genes with diverse func-tions in multiple pathways can be simultaneously regulated by miRNAs.miRNA expression is globally lower in cancer cells than in normal tissuecells; thus, aberrantly expressed miRNAs act in cross-talk pathways topromote tumorigenesis. Tumorigenesis is a multistep process during whichcancer cells acquire characteristics such as self-sufficiency in growth, insen-sitivity to growth-inhibitory signals, evasion of apoptosis, limitless replica-tive potential, sustained angiogenesis, invasion, and metastasis (Hanahanand Weinberg, 2000). Herein, we review the participation of miRNAs inthese processes in cancer cells.

A. Self-Sufficiency in Growth Signals

Cancer cells constitutively activate different pathways that sustain cellproliferation and survival, making them independent from extracellulargrowth factor signals. Interestingly, modulation of cancer cell interactionswith their microenvironments is necessary for cancer self-sufficiency ingrowth signals (Guo et al., 2006). Deregulation of miRNA expression incancer cells can result in aberrant regulation of growth factor and receptor

expression during growth signaling. One of the most well-established path-ways by which cancer cells avoid growth factor dependency is activation ofRAS signaling. RAS is a key molecule in cellular growth-regulatory path-ways and is mutated in several types of malignancies. Importantly, RAS isregulated by let-7, one of the first miRNAs identified, whose expression isdownregulated in many cancers (Johnson et al., 2005). Interestingly, let-7targets the oncogene HMGA2 (Lee and Dutta, 2007), which contributes tothe growth of cancer cells in an anchorage-independent manner. Researchersshowed that underexpression of let-7 is an indicator of poor prognosis for

lung cancer (Esquela-Kerscher and Slack, 2006) and head and neck squa-mous cell carcinoma (Childs et al., 2009). Also, downregulation of RAS bytreatment with all-trans retinoic acid relies on transcriptional induction oflet-7expression by NFKBIA gene enhancer in AML (Garzon et al., 2007).



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Moreover, let-7has induced tumor regression in in vivo lung cancer models(Esquela-Kerscher et al., 2008). These studies suggest clinical relevance forlet-7 because of antiproliferative effects and potential in cancer therapy

development. In addition to let-7, miR-21 is related to RAS oncogenicsignaling. miR-21 is induced by the transcription factor complex JUN,which participates in RAS downstream signaling and is negatively con-trolled by the miR-21 target PDCD4 (Talotta et al., 2009). Therefore,induction of miR-21 expression by JUN represents a positive feedbackloop that sustains JUN activity in response to RAS signaling (Talotta et al.,2009). miR-143 targets the RAS family member KRAS, suppressing cellproliferation (Chen et al., 2009). KRAS is mutated in various malignancies,and studies showed that its expression was inversely correlated with miR-

143 expression (Chen et al., 2009). Interestingly, by inhibiting KRAS expres-sion, miR-143 also inhibits constitutive phosphorylation of ERK1/2 (Chenet al., 2009), which is located in an important pathway of cellular growthsignal transduction. Authors reported that by repressing the oncogeneERBB2/3, miR-125a, and miR-125b also negatively regulate ERK1/2 andAKT phosphorylation (Scott et al., 2007). Also, miR-143, along with miR-145, targets ERK5, another member of the ERK family. ERK5 is known topromote cell growth and proliferation in response to growth factors andtyrosine kinase activation. In addition, the activity of several transcription

factors, such as MYEF2, FOS, FOSL1, PSAP, MYC, and NFKB1, are regu-lated by ERK5 (Terasawa et al., 2003). In addition to acting on ERK5expression, miR-145 suppresses the insulin receptor substrate IRS1, a dock-ing protein for IGF1R that plays a critical role in transformation events byfunctioning as an antiapoptotic agent to enhance cell survival (Shi et al.,2007).

Growth signaling involves the interaction of growth factors and/or cyto-kines with transmembrane receptors. Cancer cells overexpress surface recep-tors and consequently develop hypersensitivity to growth factors at low

concentrations. As demonstrated with growth factors, miRNA expressionderegulation can result in aberrant of cell surface receptors. Investigatorsrecently demonstrated that miR-205 targets ERBB3, a member of the tyro-sine kinase receptor (TKR) family, and inhibits activation of the downstreammediator AKT (Iorio et al., 2009). Interestingly, researchers showed thatmiR-7 also suppresses AKT activation, which plays a critical role in EGFsignaling (Webster et al., 2009). Indeed, miR-7targeted this receptor, whichfrequently is mutated or exhibits upregulated expression in cancer cells.ERBB2 is another important receptor that is overexpressed in several can-

cers whose expression is regulated by miR-331. Consequently, by regulatingthe expression of this receptor, miR-331 also blocks the downstream PI3Kand AKT signaling pathways (Epis et al., 2009). Furthermore, members ofthe miR-34 family and miR-199 suppress MET receptor expression, which is



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related to this oncogenic pathway in papillary renal carcinoma cells(Migliore and Giordano, 2008). Finally, miR-140 targets the expression ofPDGF growth factor receptor, which is a known oncogenic factor, especially

in ovarian cancer (Eberhart et al., 2008).

B. Insensitivity to Antigrowth Signals

The ability of cancer cells to become insensitive to antigrowth signals isassociated with alterations in the mechanisms that regulate the cells transitthrough the G1 phase of the cell cycle. One of the most important regulatorsof antigrowth signals is TGFB1. Besides controlling several pathways,

TGFB1 prevents phosphorylation and inactivation of the tumor suppressorRB1 (Hannon and Beach, 1994). Dephosphorylation of proteins in the RBfamily promotes growth arrest via sequestration of E2F and inhibition of cellcycle progression (Iaquinta and Lees, 2007). Studies demonstrated thatexpression of TGFB1 and RB1 is regulated by miR-20a and miR-106a,respectively (Volinia et al., 2006). Interestingly, E2Fs transcription factoractivity is also controlled at the posttranscriptional level by miR-20a alongwith miR-17-5p, miR-92, and miR-106b (ODonnell et al., 2005; Petroccaet al., 2008; Sylvestre et al., 2007). miR-20a, miR-106a, and miR-106b are

members of the highly homologous clusters miR-17

92, miR-106a

92,and miR-106b25, respectively (Tanzer and Stadler, 2004). Reciprocally,E2F-activating transcription factors can regulate the expression of theseclustered miRNAs, which target apoptotic and growth-inhibitory proteinssuch as BCL2L11 (apoptosis facilitator) and CDKN1A (p21) (Petroccaet al., 2008; Sylvestre et al., 2007; Woods et al., 2007). Furthermore, miR-1792 is important for integration of signals during the G1 phase of the cellcycle, protecting cells against MYC-induced apoptotic E2F responses andleading to uncontrolled cellular proliferation (Coller et al., 2007). Research-

ers recently demonstrated that let-7a induces cell-cycle arrest at the G1

/Sphase by suppressing E2F2 and cyclin D2 expression in prostate cancer cells(Dong et al., 2010).

Other clusters, such as miR-106b93 and miR-221-222, are also involvedin the insensitivity of cancer cells to external inhibitory signals by repressingimportant antigrowth signals such as CDK. Expression of these miRNAs isupregulated in several types of cancer (Fornari et al., 2008; Kim et al., 2009;le Sage et al., 2007), and researchers showed that these miRNAs directlyrepress all members of the Cip/Kip family of CDK inhibitors (p57Kip2,

p21Cip1, and p27Kip1) (Kim et al., 2009). Also, other miRNAs regulatethe expression CDK proteins. For example, miR-34a expression is inducedby TP53 activation and mediates cell-cycle arrest at the G1 phase by sup-pressing multiple targets, including CDK4, CDK6, cyclins D1 and E2, and



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MET (He et al., 2007). miR-15a and miR-16 target G1 cyclins such ascyclins D1, D2, and E1, inducing cell-cycle arrest at the G1G0 phase(Bandi et al., 2009).

C. Evasion of Apoptosis

The ability to evade apoptosis is one of the main characteristics of tumori-genesis. Several studies demonstrated that miRNAs play significant roles inapoptosis regulation in different types of cancer cells. miRNAs can act inboth proapoptotic and antiapoptotic regulatory pathways according to thecell type and specific proapoptotic and antiapoptotic target genes. Accord-

ingly, expression of the majority of proapoptotic miRNAs is downregulatedin cancer cells (Subramanian and Steer, 2010). Among the proapoptoticmiRNAs are miR-101 and miR-1, which target the BCL2 homologousprotein MCL1 (Su et al., 2009) and heat shock proteins HSPD1 andHSPA4 (Xu et al., 2007), respectively.

Interestingly, the most important examples of proapoptotic miRNAs areassociated to TP53 regulation. TP53 is the most extensively studied tumorsuppressor and is mutated in almost 50% of all human cancers. TP53 isknown to be the guardian of the genome, with a critical role in both cell

cycle and apoptosis regulation. DNA damage or genotoxic stress can acti-vate TP53, which modulates the transcription of several target genes andexpression of more than 30 miRNAs (Subramanian and Steer, 2010). Forexample, TP53 activates miR-34a (He et al., 2007), which targets importantgenes involved in apoptosis and cell proliferation, such as CDK4, MYCN,SIRT1, E2F3, and E2F5 (Wei et al., 2008; Welch et al., 2007; Yamakuchiet al., 2008). Reciprocally, miR-34 family members are essential for theproper execution of TP53-dependent cellular responses (He et al., 2007).Other major proapoptotic miRNAs whose expression is induced by TP53

activation include the miR-15a/miR-16-1 cluster, which represses the anti-apoptotic BCL2 protein expression and activates the intrinsic apoptoticpathway APAF-1/CASPASE-9/PARP (Calin et al., 2008). Interestingly,investigators showed that members of the miR-29 family activate TP53 byrepressing PIK3R2 and CDC42 (Park et al., 2009b). In addition, overex-pression ofmiR-29b downregulates the expression of MCL1 and sensitizescancer cells to TRAIL (Mott et al., 2007), and promotes the expression ofproapoptotic genes silenced by methylation by targeting the DNA-methylatinggenes DNMT3A and DNMT3B (Fabbri et al., 2007).

Conversely, the fact that MYC, the major regulator of cell proliferationand apoptosis, is associated with antiapoptotic miRNA regulation is not asurprise. MYC can transactivate the miR-1792 cluster, which targetsproapoptotic genes such as E2F1 (ODonnell et al., 2005), p21, and



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BCL2L11 (Inomata et al., 2009). In addition to the miR-1792 cluster,other miRNAs exhibit antiapoptotic functions by targeting several tumorsuppressor genes. One of the most well-known antiapoptotic miRNAs is

miR-21. Expression of this miRNA is upregulated in many cancer types andit represses the expression of apoptosis-related genes such as PTEN (Menget al., 2007), PDCD4 (Asangani et al., 2008), and TPM1 (Zhu et al., 2007).Also, miR-221 and miR-222 repress genes that promote apoptosis, such asKIT (Felli et al., 2005), p27 (le Sage et al., 2007) and CDKN1C (p57)(Fornari et al., 2008). In addition, miRNAs regulate genes in the apoptosissignaling pathway such as miR-133, which represses caspase-9 expression(Xu et al., 2007), and miR-155, which is responsible for silencing of TP53functions by directly repressing TP53INP1 (Gironella et al., 2007), an

important mediator of TP53 antioxidant and proapoptotic activities (Canoet al., 2009). A schematic of the main miRNAs involved in apoptosis isshown in Fig. 2.

D. Limitless Replicative Potential

Cancer cells have unlimited replicative potential. In contrast, normal cellsbecame senescent when they complete the limited doubling in response to a

multitude of different stimuli, such as DNA-damage signaling, oxidativestress, telomere attrition, and oncogene activation (Kuilman et al., 2008;Pascal et al., 2005). Several mechanisms regulate cellular senescence and theresponses to these stimuli, including miRNA regulation. Regarding this,researchers found that loss of miR-138 expression may contribute to gainof human telomerase reverse transcriptase (hTERT) protein expression inthyroid carcinoma cells, inducing consequent telomerase deregulation(Mitomo et al., 2008). In an miRNA-screening library study, researchersfound that miR-373 and miR-372 repressed the expression ofLATS2, which

interacts with a negative regulator of TP53 and may function in a positivefeedback loop with TP53 that responds to cytoskeleton damage. Therefore,miR-373 and miR-372 are capable of facilitating transformation of primarycells harboring oncogenic RAS and wild-type TP53 expression via neutrali-zation of TP53-mediated CDK inhibition and thus preventing prematuresenescence induced by oncogene activation (Voorhoeve et al., 2006).Because TP53 is a key regulator of senescence, the miRNAs that are acti-vated by TP53 are also important in this process. For example, miR-34family members participate in senescence via the E2F signaling pathway

(Kumamoto et al., 2008; Tazawa et al., 2007). Recently, authors reportedstrong induction ofmiR-34a and miR-146a expression during senescence inprimary human TIG3 fibroblasts after constitutive activation of the smallnuclear ribonucleoprotein SNRPE (Christoffersen et al., 2010). Moreover,



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Proapoptotic

Apoptosis

Antiapoptotic

DNA damage or genotoxic stress

TP53INP1

TP53

miR-155

miR-34amiR-15a/16-1 cluster

miR-17-92 cluster

Mitochondria

miR-29 family

miR-21

miR-133

APAF-1Pro-caspase-9

BCL2

p85 CDC42

Pro-caspase-9

Caspase-9

Caspase-3

miR-221- and miR-222

CDK4 MYCN

SIRT1 E2F3

E2F5

E2F1PTEN PDCD4

TPMI CDKNIC

p27 c-Kit

CDKN1A BIM

MYC

Fig. 2 miRNAs activity in proapoptotic and antiapoptotic pathways. DNA damage orgenotoxic stress activates TP53, the main regulator of the proapoptotic pathway. TP53 thenactivates the transcription of several miRNAs (e.g., miR-34a) that target important genesinvolved in apoptosis such as MYCN, E2F3, E2F5, and the miR-15/miR-16-1 cluster, whichrepresses BCL2 and activates the intrinsic apoptotic pathway APAF-1/CASPASE-9/PARP. miR-

29 family members activate TP53 by repressing PIK3R2 (p85) and CDC42 expression. In theantiapoptotic pathway, MYC transactivates the miR-1792 cluster, which targets proapoptoticgenes such as E2F1, CDKN1A, and BIM. miR-21 suppresses the expression ofPTEN, PDCD4,and TPM1, and miR-221/miR-222 represses the expression of proapoptotic proteins such asc-Kit, p27, and CDKNIC, inhibiting apoptosis. Expression of the mediator of TP53 function,TP53INP1, is downregulated by miR-155, which suppresses cell-cycle arrest and apoptosis.Finally, miR-133 represses the expression of caspase-9, impairing apoptosis.



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they demonstrated that during oncogene-induced senescence miR-34a isregulated independently of TP53 and targets the important proto-oncogeneMYC, coordinately controlling a set of cell-cycle regulators. Furthermore,

investigators observed downregulated expression of 15 miRNAs in senes-cent cells and breast tumors harboring wild-type TP53 suggesting thatexpression of these miRNAs is repressed by TP53 in an E2F1-mediatedmanner (Brosh et al., 2008).

E. Angiogenesis

During tumor progression, normal endothelium quiescence is lost, and

proliferation is activated by proangiogenic factors, resulting in promotion ofneoangiogenesis (Suarez and Sessa, 2009). Neoangiogenesis is the process bywhich new blood vessels form through the growth of existing blood vessels(Carmeliet, 2005). Angiogenesis is driven in part by hypoxia, which stimu-lates tumor-cell production of angiogenic factors such as bFGF, PGF, andVEGF (Kerbel, 2008). VEGF is one of the most important angiogenicfactors, as it is highly expressed in most tumors and promotes angiogenesisby enhancing the survival, migration, and invasion of endothelial cells(Suarez and Sessa, 2009). In addition, several reports demonstrated that

miRNAs are important modulators of tumor-induced neoangiogenesis(Fig. 3). For example, investigators identified a group of miRNAs includingmiR-16, miR-15b, miR-20a, and miR-20b as potential modulators of VEGFunder hypoxic conditions (Hua et al., 2006). Interestingly, miR-126 had theopposite biological effect on VEGF regulation according to the cellularcontext. In a study, miR-126 directly repressed VEGF expression in vitroand in vivo and induced cell-cycle arrest at the G1 phase in lung cancer cells(Liu et al., 2009). Contrarily, researchers found that miR-126 expressionwas upregulated during angiogenesis and repressed negative regulators of

the VEGF pathway in endothelial cells (Fish et al., 2008). Nonetheless, miR-126 targets the components of MAPK and PI3K signaling pathways,SPRED1 and PIK3R2, making it a key positive regulator of angiogenicsignaling in endothelial cells (Fish et al., 2008).

Other miRNAs also promote angiogenesis in cultured endothelial cells.For example, endothelial cells exposed to serum overexpress miR-130a,which targets the antiangiogenic homeobox genes HOXA5 and GAX(Chen and Gorski, 2008). Interestingly, miR-210 directly modulates theTKR ligand EFNA3, a repressor of VEGF-dependent endothelial cell migra-

tion and tubulogenesis (Fasanaro et al., 2008). Cocultured endothelial cellsoverexpress miR-296 in response to VEGF stimulation, which promotesangiogenic signaling by degrading VEGF receptor and PDGF receptor viaHGF substrate repression (Wurdinger et al., 2008). In addition, miR-27a



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represses the expression of the zinc finger gene ZBTB10 and consequentlyinduces its targets, such as the specificity proteins Sp1, Sp3, and Sp4, whichpromote the transcription of both survival and angiogenic genes (i.e., survi-vin, VEGF, VEGFR) (Mertens-Talcott et al., 2007). Other miRNAs thatpromote angiogenesis are miR-378, which targets two tumor suppressorgenes (SuFu and Fus-1) and enhances cell survival and angiogenesis (Lee

et al., 2007), and the miR-17

92 cluster, which targets antiangiogenicproteins including the secreted factors TSP1 and CTGF (Dews et al., 2006).Recently, a study demonstrated that miR-107can mediate TP53 regulationof hypoxic signaling and tumor angiogenesis (Yamakuchi et al., 2010).

miR-17-92 cluster

Tumor cell

Endothelialcell

Growth factors

Nucleus

Genes for angiogenesis

Hypoxia

VEGF

miR-378miR-210

cKit eNos Ephrin-A3

AKT

PI3KRAS

HGS

TSP1

CTGF

RAF

PIK3R2 SPRED-1

P53HIF-1

miR-107

miR-216 miR-296

SuFu Fus-1HOXA5 GAX

miR-221/222miR-130a

Fig. 3 miRNA regulation of angiogenesis. miRNAs play roles in proangiogenic and antian-

giogenic pathways. The hypoxia-inducible miR-210 acts as a proangiogenic factor by regulatingthe expression ofEFNA3 (Ephrin A3), which is a repressor of VEGF-dependent endothelial cellmigration. miR-126 positively regulates angiogenesis by regulating SPRED1 and PIK3R2,which are components of MAPK/PI3K signaling pathway. Because of growth factor exposition,endothelial cells overexpress miR-130a, which represses expression of the antiangiogenic ho-meobox genes HOXA5 and GAX, thus promoting angiogenesis. VEGF stimulation inducesoverexpression ofmiR-296, which represses and thus promotes angiogenic signaling. miR-378also participates in the proangiogenic pathway by repressing the tumor suppressors SuFu andFus-1. Also, the miR-1792 cluster promotes neoangiogenesis by targeting secreted antiangio-genic factors such as thrombospondin 1 and CTGF. Additionally, miR-221/miR-222 suppressesthe expression of c-Kit and eNOS, impairing angiogenesis.



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In addition, the authors showed that miR-107is a potential regulator of one ofthe subunits of HIF1. In contrast, studies have demonstrated that miRNAsinhibit angiogenesis. For example, miR-221 and miR-222 target c-Kit and

eNOS, which are important regulators of proangiogenic endothelial cell func-tion (Poliseno et al., 2006). Recently, researchers demonstrated that miR-519c isa pivotal regulator of tumor angiogenesis and plays an important role in HIF1A-mediated angiogenesis. miR-519c suppresses HIF1A, leading to reduced tumorangiogenesis. Studies in mice demonstrated that miR-519c-overexpressing cellsexhibited dramatically reduced HIF1A levels, which was followed by suppressedtumor angiogenesis, growth, and metastasis in the mice (Cha et al., 2010).

F. Invasion and MetastasisInvasion and metastasis play important roles in the spread of cancer to

distant sites. Cell invasion consists of migration and penetration of cancercells into surrounding tissue, whereas metastasis results from cancer cellsreaching the bloodstream and colonizing in distant organs. Several proteins,transcription factors, and miRNAs have roles in these processes. Interesting-ly, some miRNAs that affect processes in tumorigenesis, such as neoangio-genesis and apoptosis, also play roles in invasion and metastasis. For

example, miR-21 is one of the most upregulated miRNAs in cancer cellsand is a key regulator of invasion and metastasis. Besides controlling cellsurvival and proliferation, this miRNA promotes cell motility and invasionby targeting PTEN, a known tumor suppressor that inhibits cell invasion byblocking the expression of several matrix metalloproteinases (MMPs), suchas MMP2 and MMP9 (Meng et al., 2007). In addition, miR-21 promotesinvasion, intravasation, and metastasis by directly modeling the cell cyto-skeleton via TPM1 suppression and indirectly regulating the expression ofthe prometastatic receptor UPAR (Zhu et al., 2007). Recent studies demon-

strated that miR-21 targets the protein kinase C substrate MARCKs which isinvolved in cell adhesion and motility via regulation of the actin cytoskele-ton (Li et al., 2009), TIMP3 and RECK (Gabriely et al., 2008). Also, besidesrepressing the expression genes involved in cell-cycle arrest at the G1 phase,apoptosis, and senescence, the pleiotropic putative tumor suppressor miR-34a regulates tumor cell scattering, migration, and invasion by downregu-lating MET and its downstream signaling cascades (Li et al., 2009).

Other miRNAs are also implicated in invasion and metastasis. For exam-ple, Ma and colleagues observed that miR-10b targets the homeobox tran-

scription factor HOXD10 and consequently upregulates expression of theG-protein RHOC, which is involved in metastasis and is repressed byHOXD10 (Ma et al., 2007). These researchers demonstrated that miR-10bis modulated by the metastasis-promoting transcription factor TWIST.



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TWIST is a key molecule in metastasis that induces epithelial-to-mesenchy-mal transition (EMT) (Yang et al., 2004), which is the conversion of polar-ized immotile epithelial cells into motile mesenchymal cells. This process

primarily occurs during embryonic development and is implicated in thepromotion of tumor invasion and metastasis (Thiery, 2002). Recently,researchers identified several miRNAs as regulators of EMT. Specifically,expression of members of the miR-200 family (miR-200a, miR-200b,miR-200c, miR-141, and miR-429) as well as miR-205 was significantlydownregulated in EMT (Gregory et al., 2008). In addition, members of themiR-200 family may be downstream factors in the TGFB1 pathway duringEMT. Interestingly, in comparison, miR-155 contributes to RHOA suppres-sion and therefore is related to cell migration and invasiveness (Kong et al.,

2008). Studies demonstrated that miR-200c and miR-200b target the maininductors of EMT, ZEB1 and ZEB2, respectively (Christoffersen et al.,2007; Hurteau et al., 2007). Interestingly, other studies demonstrated thatZEB1 regulates transcription of miR-200 family members, a characteristicof a reciprocal negative feedback loop (Bracken et al., 2008; Burk et al.,2008). Importantly, members of the let-7family also target important genes,such as RAS, MYC, and HMGA2, which use the RAS/MAPK kinase path-way to promote EMT (Watanabe et al., 2009).

In addition to the miR-200 family, other miRNAs act as metastasis sup-

pressors. For example, low expression of miR-335 or miR-126 in humanprimary tumors is significantly associated with poor metastasis-free survival.Experimentally, knockdown ofSOX4 and TNCdecreased invasion in vitroand metastasis in vivo, indicating that these proteins are critical effectors ofmetastasis activated by loss ofmiR-335 (Tavazoie et al., 2008). Also, miR-7has an implicated role in tumor invasion and metastasis in that it inhibits thetumor invasion promoter PAK1 (Reddy et al., 2008). Moreover, miRNAsthat target important genes involved in cell adhesion and motility signalpathways are implicated to be metastasis suppressors. Examples include

miR-126, which targets CRK, a member of the adaptor protein family(Crawford et al., 2008), and miR-183, which suppresses the expression ofezrin, a member of the ERM family of cell migration and metastasis-mediatingproteins (Wang et al., 2008). In addition, miR-122 represses ADAM17,RHOA, and RAC1 (Coulouarn et al., 2009; Tsai et al., 2009). Conversely,several miRNAs are known to promote metastasis.

Of note is that most miRNAs that promote invasion and metastasis arelocated in the regulatory pathways of suppressor genes. miR-373 and miR-520c, which belong to the same family, have proinvasive and promigratory

effects in that they suppress CD44 expression. CD44 is a cell surface recep-tor and acts as a metastatic suppressor. Also, ectopic expression of miR-182stimulates migration of melanoma cells in vitro and in vivo by directlyrepressing the transcription factor FOXO3, which functions as a trigger



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for apoptosis by inducing expression of genes necessary for cell death(Segura et al., 2009). Recently, studies showed that miR-9 directly targetsE-cadherin and activates the b-catenin signaling which contributes to upre-

gulation ofVEGF expression and thus increases the rate of tumor angioge-nesis (Ma et al., 2010b). Overexpression ofmiR-9 in nonmetastatic breastcancer cells enables them to form pulmonary micrometastases in mice.Moreover, miR-9 expression correlates with MYCN amplification and, inturn, is activated by MYC and MYCN. Taken together, these remarkablefindings are important for understanding malignant transformation and mayhave implications for treatment of advanced cancer (Fig. 4).

V. CLINICAL APPLICATIONS

A. miRNAs Biomarkers for Cancer Diagnosis

and Prognosis

miRNAs are active players in human tumorigenesis that can be potentiallyused as novel tools for cancer treatment and risk stratification. A growingnumber of studies identified miRNAs as diagnosis and prognosis markers in

cancer. One of the most important causes of death in patients with cancer ismetastasis. For example, miR-10b is highly expressed in cultured metastaticcancer cells as well as human metastatic breast tumors, making it an inte-resting target of therapy for metastasis (Ma et al., 2007). Recently, investi-gators demonstrated that the miR-10b antagomir apparently is a promisingantimetastasis agent that does not act in a cytotoxic fashion against primarytumor cells but instead blocks their ability to form metastases (Ma et al.,2010a,b). Importantly, downregulation of the miR-1792 cluster in T cellspromotes decreased persistence of tumor-specific T cells and tumor control.

Thus, genetically engineered T cells expressing miR-17

92 miRNAs may bea promising approach to cancer immunotherapy (Sasaki et al., 2010).In addition, several studies demonstrated that miR-21 expression is upre-

gulated in many types of cancer, including breast (Yan et al., 2009) andgastric (Motoyama et al., 2010) cancer, making this miRNA an interestingtarget in cancer treatment. Recently, Hwang et al. (2010) demonstrated thatlow miR-21 expression was associated with increased survival followingadjuvant treatment in a study of two independent cohorts of pancreatictumor samples. In addition, they found that anti-miR-21 treatment

increased anticancer drug activity in vitro, suggesting that miR-21 is usefulas an adjuvant in personalized cancer therapy. Furthermore, by regulatingthe expression of B7-H3 protein, the miR-29 family is implicated in theescape of solid tumors of the immune system (Xu et al., 2009). Therefore,



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Tumorcell

Invasion

Metastasis

Metastasis

Metastasis

EMT

miR-21

miR-200 family

miR-10b

miR-126 miR-183

miR-520c miR-182miR-373

MYC

RAS HMGA2

Let-7 family

PTEN

CRK Ezrin

CD44

Twist

HOXD10

RHOC

ZEB2

ZEB1

FOXO3

MMP2

MMP9TPM1

RECK

TIMP3

EMT

Invasion

Bloodvessel

Tumorcell

Fig. 4 Modulation of invasion and metastasis signaling by miRNAs. Invasion and metastasisare complex cellular processes involving EMT and the participation of miRNA regulation.Several miRNAs are implicated to promote invasion and metastasis. For example, miR-21 is a

key regulator of invasion and metastasis that controls cell survival, proliferation, motility, andinvasion by targeting PTEN, MMP2, MMP9, TPM1, TIMP3, and RECK. miR-10b promotesmetastasis and RHOCoverexpression by regulating TWIST and HOXD10. miR-373 and miR-520c, which belong to the same family, have proinvasive and promigratory effects by targeting



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this family is an interesting group of immunoinhibitory molecules withpotential utility in cell-mediated immunotherapy and antibody-based tar-geted therapeutic strategies (Xu et al., 2009).

Perdesen et al. (2009) showed that treatment with anti-TNF drugs such aseternacept and infliximab was sufficient to reduce both miR-155 expression andproliferation of DLBCL cells. In addition, they observed a substantial decreasein the tumor burden in DLBCL xenografts in response to treatment witheternacept. However, the use of downregulated miRNAs may be an effectiveapproach to cancer therapy based on these small miRNAs. For instance, becausemiR-15 and miR-16-1 are natural BCL2 regulators, they may be used in therapyfor BCL2-overexpressing cancers (Calin and Croce, 2006). Interestingly, down-regulation of miR-34a expression is an independent prognostic marker for

relapse of non-small-cell lung cancer (NSCLC) (Gallardo et al., 2009) and haspredicted response of CLL associated with TP53 inactivation (Zenz et al.,2009). These studies identified miR-34 as a novel prognostic marker withpotential applications in therapy for NSCLC and CLL. Using a chemicallymodified synthetic miR-143, Akao et al. (2010) achieved a significant suppres-sive effect on colorectal tumor xenografts, suggesting that miR-143 is an inter-esting candidate agent for the treatment of colorectal tumors. Finally, studiesshowed that let-7administration was effective against lung (Kumar et al., 2008)and pancreatic (Torrisani et al., 2009) cancers in mouse models, suggesting that

this miRNA is useful in miRNA-based replacement therapy. These findingssupport the use of miRNA-based cancer therapeutics and suggest that miRNAsare candidate biomarkers for cancer diagnosis (Table I).

B. Potential Use of Circulating miRNAs in Cancer

Diagnosis

In addition to studies of miRNA expression profiling of primary tumor

samples, the usefulness of circulating miRNAs as diagnostic markers wasalso indicated by several studies. With the exception of leukemia cases, inwhich malignant cells are easily obtainable, solid tumor samples are obtainedfor profiling via either biopsy or surgery. Therefore, studies demonstrating the

the metastatic suppressor CD44. Also, miR-182 stimulates cell migration by directly repressingthe transcription factor FOXO3, which functions as an apoptotic promoter. Conversely, variousmiRNAs suppress invasion and metastasis. Members of the miR-200 family target ZEB1 andZEB2, which are the main inducers of EMT. ZEB1 regulates transcription ofmiR-200 family

members, characterizing a reciprocal negative feedback loop. Let-7 miRNA family memberstarget important genes such as RAS, MYC, and HMGA2, which promote EMT via RAS/MAPKkinase pathway. miR-126 targets a member of the adaptor protein family CRK and miR-183suppresses the expression of ezrin, a member of metastasis-mediating proteins family.



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Table I Deregulated miRNAs with Potential Prognostic and Therapeutic Implication

miRNAs deregulated in

tumor/chromosomal region Location/organization Target genes and tumor type P

UpregulatedmiR-10b/2q31.1 Intergenic/single HOXD10/breast cancer

(Ma et al., 2007)System

breamiR-1792

cluster/13q31.3Intronic/cluster E2F1/B cell lymphomas

(He et al., 2005)HIF1A/lung cancers

(Hayashita et al., 2005)

Downdimtum(Sas

miR-21/17q23.1 Intergenic/single PTEN/human hepatocellular cancer(Meng et al., 2007)

PDCD4/colorectal cancer(Asangani et al., 2008)

RECK/gastric cancer (Gabrielyet al., 2008)

MARCKS/prostate cancer(Li et al., 2009)

miR-2

cellset al

Low mviva(Hw

miR-29a/7q32.3 Intergenic/cluster DNMT3A and DNMT3B/non-small cell lung cancer(Fabbri et al., 2007)

The abexprtum(Xu

miR-29b-1/7q32.3 Intergenic/clustermiR-29c/1q32.2

miR-155/21q21.3 Intergenic/single TP53INP1/pancreatic cancer

(Gironella et al., 2007)

Anti-T

exprwithsuggDLB


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Table I (continued)

miRNAs deregulated in

tumor/chromosomal region Location/organization Target genes and tumor type P

DownregulatedmiR-15a/13q14.2 Intergenic/cluster BCL2/CLL (Calin and Croce, 2006) miR-1

interBCL

miR-16-1/13q14.2

miR-34a/1p36.22 Intergenic/single E2F3/neuroblastoma(Welch et al., 2007)

BCL2/NSCLC cancer(Bommer et al., 2007)

Low lehighbe u

Low mTP5resp

miR-34b/11q21.1miR-34c/11q21.1

miR-143/5q32 Intergenic/cluster ERK5/CLL (Akao et al., 2007)KRAS/colorectal cancer(Chen et al., 2009)

miR-1hummodof co

miR-145/5q32

let-7 family Intergenic/cluster RAS/lung cancer(Johnson et al., 2005)

HMGA2/ovarian cancer(Shell et al., 2007)

An SNsignsuggcept

Restoristroprol

(Tor


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diagnostic and prognostic usefulness of circulating miRNAs are of greatinterest. To date, the majority of the published studies using circulating miR-NAs have used serum or plasma samples. Initially, the serum levels ofmiR-21

were associated with relapse-free survival in patients with DLBCL (Lawrieet al., 2008). Because miR-21 expression is upregulated in several cancers, it isa potential diagnostic biomarker for them. Another study found that measure-ment of serum levels of miR-141 made distinguishing patients with prostatecancer from healthy subjects possible (Mitchell et al., 2008). The same studyindicated the presence of specific circulating tumor-derived miRNAs in thebloodstream in a murine prostate cancer xenograft model. Chen et al. (2008)demonstrated that by analyzing serum directly or RNA extracted from serum,they could identify unique miRNA expression profiles in patients with lung

cancer, colorectal cancer, or diabetes and distinguish them from profiles inhealthy subjects. In that study, the expression profiles showed 28 miRNAsexclusive to the healthy subjects and 63 miRNAs exclusive to the patients.In another study, circulating miRNAs such as miR-21, miR-92, miR-93, miR-126, and miR-29a were significantly overexpressed in patients with ovariancancer but not in healthy subjects (Resnick et al., 2009).

Wong et al. (2009) reported that plasma levels of miR-184 were signifi-cantly higher in patients with squamous cell carcinoma of the tongue than inhealthy individuals. Moreover, they observed that plasma miR-184 levels

were significantly reduced in these patients after primary tumor resection. Inanother study, expression ofmiR-17-3p and miR-92 was significantly upre-gulated in plasma samples obtained from patients with colorectal cancer butsignificantly reduced after surgery (Ng et al., 2009a). In the same study,miR-92 expression distinguished colorectal cancer from gastric cancer, in-flammatory bowel disease, and normal tissue. In comparison, another studyfound that the levels of circulating mRNAs were predictive of malignancyand survival in patients with renal cell carcinoma (Feng et al., 2008).Recently, a study demonstrated that plasma miR-17-5p, miR-21,

miR-106a, and miR-106b concentrations were significantly higher inpatients with gastric cancer than in normal subjects, indicating the potentialof these miRNAs as complementary cancer markers (Tsujiura et al., 2010).

In addition to serum and plasma, a few studies have assessed miRNAs inother body fluids as diagnostic markers for cancer. In one study, miR-126 andmiR-152 indicated the presence of bladder cancer at a specificity of 82% anda sensitivity of 72% (Hanke et al., 2009). Another study demonstrated thatmiR-125a and miR-200a expression was present in saliva at significantlylower levels in patients with oral squamous cell carcinoma than in normal

subjects (Park et al., 2009a). Several other studies have provided furtherevidence that miRNAs in body fluids are useful as biomarkers for cancerdiagnosis. Nonetheless, investigators must assess studies of large populationsand certain aspects of the experimental reliability before using miRNA in



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serum or plasma as a biomarker. Likewise, given that most of the currentapproaches to cancer screening are invasive and cannot detect early-stagedisease, determining when tumor-related circulating miRNAs can be

detected in the bloodstream during cancer progression.

C. Therapy with miRNAs

Although the use of miRNAs as cancer drugs is still at the preclinical stage,several studies have demonstrated the potential use of miRNAs or com-pounds that interact with them as new therapeutic agents for cancer. Becausea single miRNA targets genes involved in the same pathway, use of RNA

inhibition techniques may be more advantageous than other techniques(Spizzo et al., 2009). Several studies of antisense-mediated inhibition ofoncogenic miRNAs and replacement of miRNAs with mimics in demon-strated the potential use of these molecules in cancer therapy. Specifically,Anti-miRNA oligonucleotides (AMOs) are antisense oligonucleotides di-rected against miRNAs. Researchers showed that use of AMOs especiallythose with 2-O-methyl modifications, is a powerful technique for miRNAtargeting (Weiler et al., 2006). For example, modified AMOs decreased cellgrowth by inhibiting miR-21 expression in an in vivo model of breast cancer

and in glioblastoma cases (Si et al., 2007). Also, AMOs conjugated withcholesterol give rise to antagomirs (Krutzfeldt et al., 2007). In a neuroblas-toma model, researchers treated tumors subcutaneously induced in micewith antagomir-17-5p for 2 weeks, which resulted in tumor-growth inhibi-tion and complete tumor regression in 30% of the cases (Fontana et al.,2008). Alternatively, in vivo studies have shown that lock nucleic acid(LNA)-based oligonucleotides are very promising in therapy for cancer(Vester and Wengel, 2004). One study showed that miR-21 could be silencedin vitro using LNA-modified antisense oligonucleotides, leading to signifi-

cantly reduced cell viability accompanied by elevated intracellular caspaselevels (Chan et al., 2006). Also, studies of African green monkeys demon-strated that administration of three doses of 10 mg/kg LNA-antimiR effi-ciently silenced miR-122, leading to a long-lasting, reversible decrease intotal plasma cholesterol level without any evidence of associated toxiceffects or histopathological changes in the liver (Elmen et al., 2008).

Recently, Ma et al. (2010a,b) demonstrated that systemic treatment ofbreast cancer with miR-10b antagomirs suppressed metastasis in mice. Also,silencing of miR-10b with antagomirs in vitro and in vivo significantly

increased the levels of the functionally important miR-10b targetHOXD10. Although administration of miR-10b antagomirs in mice didnot reduce primary mammary tumor growth, it markedly suppressed theformation of lung metastases in a sequence-specific manner. Ebert et al.



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(2007) developed an alternative strategy to the use of single anti-miRs withnew molecules called miRNA sponges. These molecules are syntheticmRNAs that contain multiple binding sites for an endogenous miRNA,

preventing interaction with their endogenous targets. Preliminary in vitrostudies showed that miRNA sponges derepressed miRNA targets with effi-ciency comparable with modified AMOs.

Researchers have also investigated small molecule inhibitors that specificallytarget miRNAs. For example, treatment with the compound azobenzenereduced miR-21 biogenesis in HeLa cells and did not produce any cytotoxiceffects at a concentration of 10 mmoL (Gumireddy et al., 2008). Nonetheless,in vivo studies are necessary to confirm the use of this compound in miRNA-based cancer therapy. Because expression of most miRNAs is downregulated in

cancer cells in general, clinical restoration of the expression of specific miRNAsabnormally expressed represents an interesting approach to treat cancer. Studiesshowed that treatment with miRNA mimics induced cell death and significantlyreduced tumorigenic potential for miR-15a/miR-16 cluster in a leukemia cellmodel (Calin etal., 2008) and for members of themiR-29 family in a lung cancermodel (Fabbri et al., 2007). Furthermore, a study demonstrated that in anestablished murine orthotopic lung cancer model, intranasal let-7 administra-tion reduced tumor formation in vivo in animals with expression of a KRASoncogene mutation (Esquela-Kerscher et al., 2008).

The use of viral vectors is another potential approach to cancer therapy.Kota et al. (2009) demonstrated that miR-26a expression in hepatocellularcarcinoma cells induced cell-cycle arrest associated with direct targeting ofcyclins D2 and E2. Systemic administration of this miRNA in mice using anadeno-associated virus inhibited cancer cell proliferation, induced apopto-sis, and dramatically protected against disease progression without havingany toxic effects.

These studies established the basis for use of miRNAs as therapeuticmolecules in clinical trials of cancer and that the use of miRNAs as adjuvants

in cancer therapy appears to have great potential. Nonetheless, furtherstudies are necessary to assess the impact of specific miRNA-mediatedtherapies for prevention of off-target effects and improvement of deliveryefficiency while preventing inflammatory responses.

VI. CONCLUDING REMARKS

miRNAs represent a layer of complexity in gene expression regulation.Since the discovery of these tiny molecules, authors have reported a tremen-dous amount of published data. Several studies have shed light on miRNAbiogenesis, function, and genomic variations. One of the most important



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insights was the implication of miRNA deregulation in many diseases,including cancer. Importantly, studies demonstrated that miRNAs partici-pate in the primary phenotypic changes in cancer. In addition, the potential

clinical applications of miRNAs in cancer were assessed by several studies,suggesting that they are potential candidate biomarkers in cancer diagnosisand therapy. Further studies will improve our understanding of the nature ofmiRNAs and of their potential use in converting untreatable tumors intotreatable ones and increasing cancer cure rates.

ACKNOWLEDGMENTS

G.A.C. is supported as a fellow at The University of Texas M. D. Anderson Research Trust, as afellow of The University of Texas System Regents Research Scholar, and by the LadjevardianRegents ResearchScholarFund. Work in Dr Calins laboratory is supported in part by an NIH RO1grant, by a DOD Breast Cancer Idea Award, by a Breast Cancer SPORE Developmental ResearchAward, by an Ovarian Cancer SPORE Developmental Research Award, by a CTT/3I-TD grant,and by 2009Seena MagowitzPancreatic Cancer Action NetworkAACR Pilot Grant. M.I. is anAmerican Cancer Society Research Scholar.

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