MicroRNAs as therapeutic targets in cancer

S. PATRICK NANA-SINKAM, and CARLO M. CROCE

COLUMBUS, OHIO

Cancer remains a worldwide epidemic. An improved understanding of the underlying molecular mechanisms and development of effective targeted therapies are still required for many deadly cancers. The discovery of microRNAs (miRNAs or miRs) nearly 20 years ago introduced a new layer of complexity to gene regulation, but it also afforded us the opportunity to further our understanding of the molecular pathogenesis of cancers. Dysregulation of miRNAs is fundamental to the pathogenesis of many cancers based on their involvement in basic cellular functions. In addition, these previously underrecognized, noncoding RNAs have the capacity to target tens to hundreds of genes simultaneously. Thus, they are attractive candidates as prognostic biomarkers and therapeutic targets in cancer. However, several challenges remain in translating our current understanding of miRNAs to clinical therapies. Herein, we provide a review of the current knowledge of miRNAs in both solid and hematological malignancies with a focus on their potential application as therapeutic targets in cancer. (Translational Research 2011;157:216225)
Abbreviations: AMO Anti-miRNA oligonucleotides; CLL chronic lymphocytic leukemia; DLEU deleted in leukemia; EMT epithelial mesenchymal transition; HCC hepatocellular carcinoma; IGF-1 insulin growth factor 1; IRS-1 insulin receptor substrate 1; LNA locked nucleic acid; MBL monoclonal B-cell lymphocytosis; MDR minimal deleted region; miRNA microRNA; RISC RNA-induced silencing complex; UTR untranslated region

any solid and hematological malignancies carry poor survival rates with few therapeutic options. We are in an era in which there is an increased recognition of the gap that often exists between genotype and clinical phenotype in cancer presentation and response to treatment. This recognition has fueled intense investigation into understanding molecular phenotypes and into the development of novel

From the Division of Pulmonary, Allergy, Critical Care and Sleep; Department of Molecular Virology, Immunology and Medical Genetics; James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio. Submitted for publication December 2, 2010; revision submitted January 19, 2011; accepted for publication January 19, 2011. Reprint requests: Carlo M. Croce, MD, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, 410 West 10th Avenue, Columbus, OH 43210; e-mail: Carlo. Croce@osumc.edu. 1931-5244/$ - see front matter 2011 Published by Mosby, Inc. doi:10.1016/j.trsl.2011.01.013

targeted therapeutics. The integration of multiple platforms targeted to understanding disease at the gene, protein, and metabolic level is successfully being applied to clarify heterogeneity and potentially guide therapeutic decisions. Approximately 20 years ago, investigators rst determined that other components of the genome that traditionally had been considered nonfunctional had, in fact, gene regulatory capacity.1,2 Later termed, microRNAs (miRNAs or miRs), these 1825 nucleotide molecules represent one of a family of noncoding RNAs that have emerged as key regulators of fundamental biological processes.2 It is estimated that more than 30% of the human genome is targeted by miRNA.3 By targeting multiple genes simultaneously, single or groups of miRNAs can regulate or redirect biological pathways that are essential to cancer cell behavior. miRNA can alter cellular differentiation, angiogenesis, survival, and growth.3 In addition, miRNAs may function as tumor suppressors, oncogenes, or in some cases, both. Importantly, in many cases, these functions are disease or tissue specic. Some of the earliest observations implicated global dysregulation of miRNAs in both solid and

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hematological malignancies.3 miRNA dysregulation and chromosomal abnormalities, coupled with frequent miRNA location at amplied, deleted, or translocated chromosomal regions (fragile sites), further supports their role in cancer development.3,4 To date, many studies have been conducted that focus on either functional single miRNA/target gene relationships, global miRNA expression proling as a means for biomarker development, or in vivo targeted miRNA delivery. miRNAs are clearly integral to the biology of cancer development. However, obstacles such as reproducibility and tissue-specic delivery must be addressed in order for miRNA biology to move into clinical application in cancer. First, reproducibility of high-throughput array studies is essential to the identication of specic miRNAs that may eventually guide therapeutic decisions. Second, in vivo organ-specic delivery systems with minimal off-target effects are only beginning to be realized in cases of liver and lung disease and have yet to be applied to other organs.

IMPAIRED miRNA PROCESSING AND CANCER

miRNA processing and targeting is complex and involves multiple enzymes and proteins. Within the nucleus, long primary (pri)-miRNA transcripts are transcribed by RNA polymerase II.3,5 The pri-miRNAs are then processed to a smaller stem-loop approximately 70 nucleotide (nt) precursor miRNA (pre-miRNA) molecule by a universal RNase III endonuclease, RNASEN, better known as Drosha in invertebrates.5 The premiRNA is then transported to the cytoplasm by the double-stranded (ds)RNA binding protein, Exportin 5. In the cytoplasm, the pre-miRNA is further cleaved to double-stranded 22-nt miRNA molecules by Dicer. The miRNA is separated into 2 single-stranded molecules; the mature strand is incorporated in the RNAinduced silencing complex (RISC), whereas the sense strand undergoes degradation. Several proteins important to the degradation and/or silencing of mRNA targets are located in the RISC complex, including argonautes, helicases, deadenylases, mRNA decapping proteins, and methyltransferases.6 Base-pairing between the 30 untranslated region (UTR) of the mRNA and the seed sequence located in the 50 end of the miRNA (28 positions) is critical to determining the effect on either degradation of mRNA or inhibition of translation. The complexity of miRNA/mRNA targeting, however, goes well beyond targeting of the 30 UTR. miRNAs may directly target the 5 region, promoters, ribonucleoproteins, and other miRNA and indirectly regulate gene expression through targeting of transcriptional factors.7

Global repression of miRNA expression seems to be a common characteristic of cancers. The causes for this repression or downregulation are multifactorial including functional defects in key components of miRNA maturation (Dicer, Drosha, and Ago), epigenetic silencing, and regulation by tumor suppressors such as p53 and oncogenes such as MYC.8-10 Karube et al demonstrated in a cohort of 67 lung cancer patients reduced lung tumor expression of Dicer.11 In addition, Dicer predicted a poor post-resection prognosis independent of disease stage. Chiosea et al described a reduction in Dicer expression with more advanced invasive adenocarcinoma.12 In vitro targeting of Drosha, DGCR8, and Dicer promoted lung cancer cell colony formation and growth in soft agar.13 A more recently described mechanism by which miRNAs may be globally repressed is through defective export of pre-miRNAs. Melo et al demonstrated the presence of a mutated form of the Exportin 5 gene (XPO5) within a subset of cancer cell lines harboring microsatellite instability.14 The mutated form of XPO5 localized to the nucleus interfered with both pre-miRNA processing and subsequent select target inhibition. Re-introduction of a wild-type form in these cells resulted in an upregulation of several miRNAs with known tumor-suppressive functions. Another study determined that a haplotype in RNASEN (Drosha), which occurred with a frequency of 2%, was associated with survival in early stage lung cancer patients.15 These studies further support the importance of targeted investigation of components of the miRNA processing machinery as potential therapies.
miRNA AND THE HALLMARKS OF CANCER

Over time, cancers have developed sophisticated networks of biological activities that contribute to their ability to develop and, in some cases, evade treatment. This complex program relies on the communication between multiple cell types, including both the primary tumor as well as the stromal cells.16 Hanahan and Weinberg have reviewed and described 6 essential characteristics of cancer progression: (1) selfsufciency in growth signals, (2) insensitivity to anti-growth signals, (3) evading apoptosis, (4) limitless replicative potential, (5) sustained angiogenesis, and (6) tissue invasion and metastasis.17 Dysregulated miRNAs may function as either tumor suppressors or oncogenes in cancer by targeting each of these characteristics (Fig 1). Self-sufciency in growth signals/insensitivity to anti-growth signals: By targeting regulators of cell cycle, miRNAs have been described as independently promoting or inhibiting proliferation/growth in cancer. Let-7 is perhaps the best studied miRNA in cancer. Let-7 targets the RAS

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Fig 1. miRNA targeting the hallmarks of cancer. (Color version of gure available online.)

30 UTR and reduces tumor growth.18,19 Transgenic animal models of lung cancer demonstrated that let-7 overexpression inhibited tumor development and growth.20 miRNAs have also been linked to regulators of cell cycle to promote cellular proliferation, including miR-1792 (PTEN), miR-221/222 (p27 (Kip1)), and miR-21 (PTEN), to name a few. Evading apoptosis: miRNAs have the capacity to regulate components of the apoptotic signaling pathway and thus render cancer cells resistant or susceptible to extrinsic factors. miR-15a and 16-1 represent classic examples of miRNAs that alter cell survival. In 2002, Calin et al determined that miR-15a/16-1, which are located in 13q14.3, were downregulated in 68% of patients with chronic lymphocytic leukemia (CLL).21 CLL is characterized by a proliferative, prosurvival, anti-apoptotic cellular phenotype characterized by overexpression of the prosurvival molecule Bcl2. miR-15/16 is inversely related to Bcl2 in CLL and gain of function of miR-15a/16-1 reduced Bcl2 leading to apoptosis.22 We recently determined that specic members of the miR-34 family (miR-34a) that are targets of p53 were in part responsible for PRIMA-1 (p-53-dependent reactivation and induction of massive apoptosis) induced apoptosis.23 Sustained angiogenesis: Tumor angiogenesis is regulated directly by several factors, including hypoxia, angiogenic factors, and vascular endothelial growth factor (VEGF) and indirectly by both tumor suppressors and oncogenes. Ghosh et al recently identied miR-424 as a hypoxia-responsive miRNA that promotes HIF-1a and HIF-2a target gene production and thus angiogenesis.24 In contrast, in colon cancer cells, p53 mediated

transcriptional regulation of miR-107 reduces hypoxic signaling cascades by targeting HIF-beta.25 Tissue invasion and metastases: Metastatic potential of tumors is dependent on several complex steps, including proliferation, epithelial mesenchymal transition (EMT) that leads to cell motility, invasion, and hematogenous spread. Global miRNA expression has been used to distinguish primary tumors from metastases as well as to identify subsets of tumors with high metastatic potential and poor outcome among patients.26 The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, miR429) has been extensively studied in breast, bladder, and lung cancer. Gregory et al determined that miRNAs within this family were reduced in response to TGFbinduced EMT, and overexpression of these miRNAs prevented TGFb-induced EMT.27 In a murine model of multistage pancreatic tumorigenesis, members of miR-200 were reduced in metastatic tumors.28 The reciprocal regulation between transcriptional repressors of E-Cadherin, such as ZEB1 and ZEB2, and miR-200 members supports their critical role in EMT.29-31 High miR-103/107 expression has been associated with high metastatic probability and poor outcomes in breast cancer.32 Allied in vitro and in vivo studies revealed that miR-103/107 increased migration, invasion, and metastases through functional targeting of Dicer. Knockdown of miR-103/107 reduced metastases. Lastly, miR-103/107 controlled EMT through the regulation of miR-200.32
miRNA IN SELECT MALIGNANCIES

An increasing number of studies suggest that tumor development, progression, and metastases rely on gene regulation by multiple miRNAs (Table I). Therefore, the role for miRNAs in cancer should be considered as either redirecting or reprogramming molecular pathways or networks in cancer rather than the reductionist approach of single-miRNAsingletarget relationships. Several common miRNAs are central to these key pathways and to certain malignancies. For example, among 17 groups of human normal tissues and 1107 total samples, clusters of miRNAs (miR-29, miR-30, miR-15/16) were linked to cell survival.33 Unlike normal tissues, cancers had distinct networks of miRNAs. miR-30c and miR-16 were the most connected hubs in cancers compared with normal tissues. In addition, clusters of miRNA seemed to be more or less prominent according to tumor type. Lung cancer. Lung cancer is the number 1 cause of cancer-related deaths among men and women and still carries a poor survival particularly for late-stage disease. However, novel targeted therapies based on molecular characteristics are in various stages of

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Table I. miRNAs implicated in select human malignancies

Condition Overexpressed Downregulated

Lung cancer Breast cancer Hepatocellular cancer Colorectal cancer Pancreatic cancer Prostate cancer CLL AML

miR-155, miR-21, miR-17-92, miR-221/222 miR-155, miR-21, miR-182, miR-17-92, miR-200, miR-9 miR-21, miR-221/222 miR-155, miR-21, miR-17-92 mir-21, miR-155 miR-21 miR-155 miR-10a/b, miR-29, miR-155

let-7, miR-1, miR-29, miR-126 let-7, miR-143/145, miR-10b, miR-125b. miR-126. miR-9 miR-1, miR-26a, let-7, miR-34 let-7, miR-15a/16-1, miR-34 let-7, miR-15a/16-l, miR-221 miR-15a/16-1, miR-29b, miR-181b, miR-34 miR-181, miR-204

development. Initial, early studies on miRNAs in lung cancer have focused on global proling of miRNA in lung tumor tissues compared with adjacent uninvolved lung. Two separate proling studies identied an association between dysregulation of specic miRNA and patient outcome. In 1 study, increased miR-155 and low let7a-2 expression correlated with poor survival in adenocarcinoma of the lung.34 In a second study, investigators identied miR-137, miR-182*, and miR-372 as high-risk miRNAs.35 Recent examination of miRNA expression in 165 adenocarcinomas and 125 squamous cell carcinomas revealed a signature of 34 miRNAs that could distinguish between histological types in male smokers.36 Five miRNAs (miR-25, miR-34c-5p, miR-191, let-7e, and miR-34a) predicted survival in a group of 107 male smokers with early stage squamous cell carcinoma.36 Among a large cohort of 639 patients treated with surgical resection and adjuvant chemotherapy, miRNA expression correlated with several clinical variables including lymphoid inltration, histology, and lymphatic invasion.37 However, none of the miRNAs among a preselected panel (miR-21, miR-29b, miRa/ b/c, miR-155, and let-7a) carried a prognostic value. These ndings highlight the inherent variability in high-throughput miRNA studies in lung cancer. Individual miRNAs have been used in vitro and in vivo to target lung cancer cell behavior. These miRNAs include let-7, miR-29, miR-221/222, miR-21, and miR-17-92. Let-7 has been extensively studied in human lung cancer and functions as a tumor suppressor partially through functional targeting of RAS and HMAG2.19 miR-21 is overexpressed in several solid malignancies and targets tumor suppressors, including PDCD4. The potential for miR21 as a therapeutic target in lung cancer is evidenced by a recent series of elegant studies demonstrating that in vivo miR-21 overexpression increased K-RAS driven lung cancer and that targeted deletion of miR21 could reduce lung tumorigenesis.38

Breast cancer. Like lung cancer, breast cancers also exhibit global miRNA dysregulation. miR- 155, miR21, miR-145, miR-125b, and miR-10b may all be potential targets. miR-155 is dysregulated in both solid and hematological malignancies and numerous nonmalignant diseases. In addition, its expression correlated with chemoresistance, tumor progression, and survival. Systemic targeting of miRNAs has been applied in breast cancer. For example, antagomiR-10b reduced metastases in a murine model of breast cancer.39 Another study showed that miR-145 reduced breast cancer cell proliferation and induced apoptosis in breast cancer cell lines.40 The effects of miR-145 were partially mediated through TP53 pathway activation and targeting of the estrogen receptor a (ER-a). Circulating miRNAs are also being explored as noninvasive biomarkers for disease diagnosis and surveillance in several malignancies. In 1 study of 20 women with early stage breast cancer and 20 healthy matched controls, the authors were able to identify 26 plasma miRNAs that distinguished the 2 groups.41 Most studies on circulating miRNAs have been conducted in small cohorts and thus require larger validation. The prospect that miRNAs may be used as a noninvasive biomarker is of clinical relevance. Hematological malignancies. In 2002, Calin et al determined that miR-15a/16-1, which are located in 13q14.3, were either deleted or downregulated in 68% of patients with CLL.21 13q14 deletion is associated with mutated forms of IgVH and low expression of ZAP-70 and with a more indolent form of disease. Overexpression of miR-15a/16-1 reduced the prosurvival molecule Bcl2 to induce apoptosis.22 Both miR15a/16-1 and deleted in leukemia (DLEU) genes are located in the minimal deleted region (MDR) of 13q14. Transgenic animals that either lacked the MDR or the miR-15a/16-1 cluster developed B-cell abnormalities, including CLL/SLL, NHL, and a CD51 monoclonal B-cell lymphocytosis (MBL) (42% MDR and 26% miR-15a/16-1 -/-).42

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MiRNA proling has also been used to distinguish clinical subtypes of CLL. In 94 CLL, 9 miRNAs (181b, 155, 146, 24-2, 23b, 23a, 222, 221, and 29c) distinguished patients with a short interval to therapy from patients with a longer interval to therapy.43 In another study, the authors detected miRNAs that distinguished aggressive from indolent disease in 17p deletion cases.44 miR-29c and miR-223 downregulation were associated with poor treatment-free survival and overall survival in a cohort of 110 CLL patients with CD191CD51CD231 phenotype.45 The (TCL1) gene located at 14q32.1 is overexpressed in 25% to 35% of CLL cases.46 TCL1 overexpression is increased in CLL patients with unmutated IgVH and elevated ZAP-70. TCL1A transgenic animals represent the best in vivo model for the development of CLL. This model may be leveraged for targeted therapies in CLL. Both miR-29 and miR-181 were predicted and eventually validated as targeting TCL1.47 miR-29ab transgenic animals developed primarily an indolent form of CLL, whereas a small percentage (20%) developed an aggressive form of disease.48,49 Colorectal cancer. Colon cancer is a major cause of morbidity in the United States. Expression patterns of miRNAs have also been linked to clinical outcomes in colon cancer. miRNA expression in 2 independent cohorts demonstrated 37 miRNAs differentially expressed in colon cancer tumors.50 Of note, miR-21 was the highest expressed miRNA. Multivariate analysis demonstrated a correlation between elevated miR-21 and poor survival. This association between miR-21 and survival in colon cancer was later validated in an independent cohort.51 Both miR-143 and miR-145 represent some of the rst identied dysregulated miRNAs in colon cancer. miR143 and miR-145 are decreased in colon cancer.52 Important proteins such as ERK5, NFKB, and BCL-2 are functional targets for miR-143. This targeting lead to an inhibition of cellular growth. miR-145 targets insulin receptor substrate 1 (IRS-1) and insulin growth factor 1 (IGF-1) to reduce proliferation in colon cancer cells.53 High levels of miR-155 increased microsatellite instability by direct targeting of the key mismatch repair genes: MLH1, MSH2, and MSH6.54 Other miRNA implicated in colon cancer include miR-31.55 Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) represents the fth most common malignancy worldwide and has poor survival rates. The development of HCC depends on a series of steps including an initial hepatitis or cirrhosis that may be induced by any number of factors including alcohol or Hepatitis viral infection.56 miRNAs have been implicated in molecular pathways critical to liver tumor progression

including survival pathways (miR-122, miR-29, miR221) migration/invasion (miR-21, miR-221, miR-181b, miR-1), and proliferation (miR-122, miR-221, miR-1).56 The liver-enriched miR-122 represents the most thoroughly examined miRNA in HCC. Overexpression of miR-122 reduced cell proliferation, metastases, and cell survival through targeting of key proteins such as cyclin G1, disintegrin, metalloproteinase members ADAM 10 and ADAM 17, and Bcl-W.57 In addition, decreased miR-122 correlated with poor patient survival. The observed loss of miR-122 is primarily driven by the liver transcription factors HNF1A, HNF3A, and HNF3B.56 However, polymorphisms in 30 UTR binding sites of miR-122 target genes may also interfere with miR-122 targeting. Gao et al identied a functional insertion/ deletion polymorphism in the 30 UTR for the miR-122 target IL-1 alpha that was associated with HCC susceptibility.58 In a large study of 3 independent cohorts consisting of a total of 455 patients with HCC, investigators determined that low levels of miR-26 correlated with poor patient survival but an improved response to interferon alpha.59 The biological relevance of miR-26 was corroborated by in vitro ndings that miR-26 overexpression reduced HCC cell proliferation.60 Pancreatic cancer. miRNA proling can be used for both diagnostic and prognostic purposes in pancreatic cancer. Initial proling studies identied several upregulated miRNAs in pancreatic cancer including miR21, miR-221, miR-25, miR-181b-1, and members of the miR-17-92 family.61 A second independent study identied the upregulation of several mature miRNAs including miR-21, miR-221, and miR-181a.62 In addition, miRNAs distinguished pancreatic cancer from both chronic pancreatitis and normal pancreas.63 Also of note, high tumor expression of miR-196a-2 was associated with poor survival. A follow-up study by the same group of investigators conrmed that miR-21 was overexpressed in pancreatic cancer and correlated with outcome in node-negative disease.64 As a result of these studies, miR-21 has now emerged as a target of interest for the identication and modulation of chemoresistance in pancreatic cancer. In vitro studies have conrmed that miR-21 functions as an oncomiR with overexpression in pancreatic cell lines inducing proliferation, increased invasion, and chemoresistance. Ovarian cancer. Ovarian cancer represents one of the deadliest gynecological malignancies. Epigenetic silencing and alterations in DNA copy number have been implicated as mechanisms for miRNA dysregulation in ovarian cancer.65 In one of the earliest studies,

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Zhang et al identied 35 miRNAs that were differentially expressed between epithelial ovarian cancer cell lines and primary cultured human ovarian surface epithelial cells.65 In addition, 44 miRNAs distinguished early from late stage primary ovarian tumors. A second study focusing on primary ovarian cancer tissues identied several dysregulated miRNAs including upregulation of miR-200 members and downregulation of miR-125b1 and miR-145.66 An increase in miR-200 in epithelial ovarian cancers may contribute to the transition from an initial mesenchymal phenotype in normal ovarian epithelial cells to a more epithelial phenotype during transformation to malignancy.29,67 miRNAS are also associated with both chemotherapeutic response and prognosis in ovarian cancer. miR-200 members were associated with survival in advanced ovarian cancer.68 Decreased let-7i was associated with chemoresistance and a poor survival.69 Ratner et al identied an association between a variant in the KRAS 30 UTR that interferes with let-7 binding and the risk of developing ovarian cancer.70
APPROACHES TO miRNA TARGETING

gle miRNA targeting with antisense oligonucleotides.72 In addition, development of stable sponges may assist in recapitulating the effects of downregulation of aberrantly expressed miRNAs. In an orthotopic model of breast cancer, targeting of the antimetastatic miR-31 using a sponge approach resulted in a signicant induction of lung metastases.73 Conversely, inhibition of the MYC-driven miR-9 by a miRNA sponge reduced lung metastases in a murine breast cancer model.10 MiRNA sponges have also been applied to models of cardiac hypertrophy and multiple sclerosis.74 Nanoparticles: Nanoparticle formulations have been used primarily for in vitro delivery of siRNAs. Few studies to data have used this technology for miRNA delivery. Chen et al recently demonstrated that by using liposomepolycation-hyaluronic acid (LPH) particles as a carrier for miRNA modied with a tumor targeting monoclonal antibody (GC4 single-chain variable fragment), they could target lung metastases in a murine model of metastatic melanoma.75
IN VIVO APPROACHES TO miR MANIPULATION: GETTING FROM BENCH TO BEDSIDE

Targeting of miRNAs is based on either selective inhibition of miRNA expression or binding or selective overexpression. Most studies to date have been in vitro, but in vivo studies based on systemic or localized target delivery of miRNA are increasing in number. Enthusiasm for targeting of multiple genes and pathways must be tempered by the recognition of the potential disadvantages that include off-target effects and lack of tissue specicity. There are several approaches to miRNA targeting, each of which harbor their own advantages and disadvantages. These approaches are as follows. Anti-miRNA oligonucleotides (AMOs): AMOs are single-stranded molecules that form direct complementarity and thus inhibit specic miRNA.71 AntagomiRs: AntagomiRs are single-stranded molecules that form complementarity to miRNAs but may also be modied with a cholesterol conjugated 20 -O-methyl in order to maintain stability while minimizing degradation.71 Locked nucleic acids (LNAs): Locked nucleic acids have a methylene bridge that functionally locks ribose conformation. This change results in increased binding afnity and stability.71 miRNA sponges: MiRNA sponges represent a newly identied approach to miRNA. Sponges function by using multiple complementary 3UTR mRNA sites for a specic miRNA. Sponges competitively bind to miRNA, thus interfering with normal targeting of miRNA.72 There are several advantages to miRNA sponges, including the ability to target and inhibit a family of miRNAs as opposed to sin-

Currently, the primary approaches to in vivo miRNA manipulation include transgenic murine models, systemic delivery via intravenous or intraperitoneal route, or direct lung delivery (Fig 2). Transgenic murine models are the most widely used models for in vivo miRNA investigation. For example, transgenic overexpression of Let-7 reduced tumor burden in a KRAS model of nonsmall-cell-lung cancer.20 In vivo delivery of let-7 by intratracheal instillation reduced lung tumor development. In a separate study, Huynh et al demonstrated that intraperitoneal injection of 20 modied phosphorothioated miR-182 anti-sense oligonucleotides could reduce liver micrometastases of melanoma cells.76 Furthermore, the lack of alterations of other miRNAs within tumor cells and lack of signicant hepatotoxicity supports this approach to miRNA delivery. In a separate study, systemic delivery of miR-26 could successfully reduce tumor burden while minimizing toxicity.60 Systemic delivery of a DNALNA miR-122 anti-miRNA (SPC3649) in the only nonhuman primate model of chronic HCV infection resulted in a potent reduction (300 fold) in HCV burden.77 To date, this remains the only miRNA for systemic delivery that has reached clinical trial. Chemotherapeutics and miRNAs. Chemotherapy remains the mainstay of treatment for most solid and hematological malignancies. However, some cancers harbor either acquired or inherent mechanisms for resistance.78 Because miRNAs regulate multiple biological functions, it seems plausible that miRNA expression may guide drug response. Several such studies exist,

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Fig 2. Current approaches to in vivo miRNA targeting: (1) Systemic delivery (intravenous and intraperitoneal), (2) transgenic/knockout, (3) intranasal/intratracheal delivery of AMOs or antagomiRs, and (4) nanoparticle delivery. (Color version of gure available online.)

but most studies have been conducted in vitro. For example, miR-1 is often reduced in solid malignancies. Overexpression of miR-1 increased lung cancer cell sensitivity to doxorubicin.79 miR-155 is overexpressed in both solid and hematological malignancies as well as inammatory states. miR-155 knockdown rendered breast cancer cells sensitive to chemotherapy through regulation of FOXO3a.80 miR-21 has been implicated in gemcitabine resistance in pancreatic cancer.81 The role for miRNAs in smallcell-lung cancer remains relatively unexplored. In a recent study, miR-92-a-2*, miR-147, and miR-5745p were associated with chemoresistance in smallcell-lung cancer.82 In CLL, Ferracin et al identied 37 miRNAs that distinguished CLL patients who either responded or did not respond to udarabine.83 These dysregulated miRNAs were functionally linked to the p53 pathway. A similar study was conducted in a cohort of 19 test patients and a 21-patient validation cohort receiving udarabine therapy.84 Fludarabineresistant patients were characterized by increased miR-181a and decreased miR-29a. Two large studies have attempted to correlate patterns of miRNA expression miRNAs (Let-7i, miR-16, miR-21) in A549 lung cancer cells.85 In an early study, Blower et al studied 3 specic miRNAs, and the authors correlated each miRNA with drug sensitivity in a panel of preselected chemotherapeutics. In addition, examination for global expression of 279 miRNAs and

response to 3089 compounds in the NCI-60 cell panel led to the identication of 31 miRNAs that signicantly correlated with drug potency.86 Liu et al conducted a study in which they integrated miRNAs and mRNAs expression in the NCI-60 panel with drug sensitivity.87 As expected, miRNAs clustered according to both malignancy of origin in certain malignancies (leukemia, colon, renal, melanoma) and by drug response. miRNAs as circulating biomarkers. Noninvasive measurement of miRNA expression is another area of great interest to both investigators and clinicians. Detection of circulating miRNAs has been described in several malignancies including colorectal, lung, breast, and ovarian cancer.88-90 Mitchell et al identied circulating miRNA in a stable form in the serum.91 The point of origin for circulating miRNAs remains unclear. However, studies suggest that miRNAs may circulate in a stable form within exosomes and microvesicles with potential for cellcell communication.92 Although the concept of circulating miRNAs is exciting, it is unclear whether they have a functional role in the circulation. From a technical perspective, the optimal approach to circulating miRNA detection has to be determined.93 Lastly, most studies to date have involved relatively small cohorts that require independent validation. Studies are ongoing to determine whether circulating miRNAs may be used as surrogate markers for disease activity or response to therapy.

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CONCLUSION: WHAT IS THE CURRENT ROLE FOR miRNAs AS CANCER THERAPEUTICS?

The global dysregulation of miRNAs has now been described in several malignancies including lung cancer, colon cancer, pancreatic cancer, breast cancer, and leukemia. Furthermore, it is clear that miRNAs can alter biological processes fundamental to tumor initiation and progression. The question is how might this information be used to either guide current therapies or in the development of new therapies? High-throughput proling studies have proven to be of value in teasing out phenotypic heterogeneity and in identifying candidate miRNAs, but reproducibility of these studies is critical. From the multitude of studies conducted so far, miRNAs such as miR-155, miR-122, miR 21, let-7, miR-29, miR-30, and miR-221/222 seem as candidates to focus in on the development of therapeutics. Altering miRNAs as either direct therapy or as a means for enhancing chemosensitivity to traditional therapies may be viable therapeutic strategies. In addition, improved in silico programming will assist in identication of miRNA/target relationships. Linking our understanding of exactly how miRNAs may program cancer-specic networks to the pathways targeted by current treatments is also required. Such an understanding may allow miRNAs to be used to predict which subgroups of patients may respond to a given chemotherapeutic agent. The translation from in vitro to in vivo delivery systems remains a work in progress. Establishing ideal delivery systems with organ specicity while minimizing toxicity and off-target effects will be essential to moving the eld forward.
The authors would like to thank Dr. Tim Eubank for his assistance in the preparation of both the gures.

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