Targeting microRNAs in Pancreatic Cancer: Microplayers in the Big Game

Review
Sheema Khan1, Ansarullah2, Deepak Kumar3, Meena Jaggi1, and Subhash C. Chauhan1
Abstract
The prognosis of patients with pancreatic cancer is extremely poor, and current systemic therapies result in only marginal survival rates for patients. The era of targeted therapies has offered a new avenue to search for more effective therapeutic strategies. Recently, microRNAs (miRNA) that are small noncoding RNAs (18–24nucleotides)havebeenassociatedwithanumberofdiseases,includingcancer.DisruptionofmiRNAs may have important implications in cancer etiology, diagnosis, and treatment. So far, focus has been on the mechanisms that are involved in translational silencing of their targets to ?ne tune gene expression. This review summarizes the approach for rational validation of selected candidates that might be involved in pancreatic tumorigenesis, cancer progression, and disease management. Herein, we also focus on the major issues hindering the identi?cation of miRNAs, their linked pathways and recent advances in understanding their role as diagnostic/prognostic biomarkers, and therapeutic tools in dealing with this disease. miRNAs are expected to be robust clinical analytes, valuable for clinical research and biomarker discovery. Cancer Res; 73(22); 6541–7.
2013 AACR.
Pancreatic Cancer and miRNA Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States (1). The recent sequencing of the pancreatic cancer genome has underscored the considerable heterogeneity of somatic DNA alterations and dysregulation of genetic pathways between individual tumors and hence challenges for molecularly targeted therapies. The progression model of PDAC is distinguished by three PDAC precursor lesions: pancreatic intraepithelial neoplasia (PanIN I, II, III), a less common precursor lesion mucinous cystic neoplasm (MCN), and intraductal papillary mucinous neoplasm (IPMN; ref. 1). PanIN, which is found in the smaller-caliber pancreatic ducts, is the most common and extensively studied lesion due to the suggested biologic relationship and common genetic alterations documented for initial increased incidence of PanINs in patientswithPDAC.Key sharedgenetic alterationsassociated withPDAC progression include earliest genetic events such as mutation of K-RAS and overexpression of HER-2/neu. At later stages, inactivation of the p16 tumor suppressor gene occurs,
followed by the loss of p53, SMAD4, and BRCA2 signaling pathways and the genomic-transcriptomic alterations that facilitate cell-cycle deregulation, cell survival, invasion, and metastases (1). Therefore, detecting molecular alterations at precancerous conditions together with targeting PDAC at an early stage represent an assault on the Achilles heel of cancer. Recently, miRNA therapy has emerged as a promising therapeutic strategy for various diseases. miRNAs are small noncoding RNAs consisting of 18–24 nucleotides that regulate their target mRNA stability and translation by binding imperfectly with their 30UTR region. Apart from the role of miRNAs in cancer progression and invasion, their differential expression has been associated with patient survival and regulation of the disease response (resistance or sensitization) towards chemotherapeutic drugs. The clinical potential of miRNAs is based on the fact that a single miRNAcanregulate multipleoncogenicpathways commonly deregulated in cancer. A uniform pattern of miRNA dysregulation in PDAC is still lacking. This is due to the highly heterogeneous nature of pancreatic tissue that contains not only pancreatic ducts and acinar cells from which ductal type tumors may arise, but also the predominance of dense desmoplastic non-neoplastic stromal and in?ltrating in?ammatory cells. miRNAs can regulate several molecules, including mucins that harbor the complex tumor–stromal microenvironment and contribute to tumor progression and chemoresistance. Thus, the rapid and coordinated manipulation of protein levels across multiple pathways endows these regulatory RNAs with the ability to instantly switch between cellular programs.Also,theidenti?cationofmiRNAsduringtransition from PanIN I, PanIN II, and PanIN III to adenocarcinoma and the speci?c roles they exert in such a process is warranted to properly design strategies to prevent and/or attenuate the
Authors’ Af?liations: 1Department of Pharmaceutical Sciences and Center for Cancer Research, University of Tennessee Health Science Center, Memphis, Tennessee; 2The Sanford Project, Children Health Research Center, Sanford Research/USD, Sioux Falls, South Dakota; and 3Cancer Research Laboratory, Department of Biological and EnvironmentalSciences, UniversityoftheDistrictofColumbia, Washington,Districtof Columbia Corresponding Author: Subhash C. Chauhan, Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 SouthManassas,CancerResearchBuilding,Memphis,TN38163.Phone: 901-448-2175; Fax: 901-448-2099; E-mail: schauha1@uthsc.edu doi: 10.1158/0008-5472.CAN-13-1288
2013 American Association for Cancer Research.
Cancer Research
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malignant phenotype. Thus far, very few studies have investigatedmiRNAchangesassociatedwithPDACprogression.This review summarizes the strategies to tackle pancreatic cancer at genetic level through the miRNAs.
Rationale for Developing miRNA-Based Therapy Considerable research shows that restoring the expression of tumor suppressor miRNAs seeks to reinstate those cellular programs that are active in normal cells and interfere with oncogenic programs necessary for a malignant phenotype. However, antagonizing the function of overexpressed oncogenic miRNAs inhibits multiple oncogenic pathways. It has been successfully shown that intravenous injection of antagomirs, 20-O- methyl-antisense oligonucleotides conjugated with a cholesterol moiety at the 30-end, leads to long-lasting inhibition of speci?c miRNAs in mice (2). In contrast, restoration of expression of downmodulated tumor-suppressive miRNAsisusuallyachievedusingadeno-andlentiviralvectors, non-viral lipid-based strategies that have recently been developed for systemic miRNA delivery and applied successfully to lung and prostate cancer xenograft models (3). Miravirsen (LNA oligomer targeting miR-122), after showing positive effects against HCV infection in chimpanzees (4), showed prolonged dose-dependent reductions in HCV RNA levels without evidence of viral resistance in patients with chronic HCVgenotype1infection(5).Standardcomputationalanalysis is used in a combined genome-wide association study, miRNA pro?ling for the identi?cation of response predictors in patients with metastatic breast cancer (NCT01598285, ClinicalTrial.gov). The success of the preclinical trial of MRX34 (proprietary tumor suppressor miRNA, miR-34) in an orthotopic mouse model of hepatocellular carcinoma through the SMARTICLES oligonucleotide delivery technology has prompted the start of its phase I clinical trials (Mirna Therapeutics, Inc) recently, in May 2013 (http://www.clinicaltrials.gov/ct2/show/NCT01829971?term¼MRX34&rank¼1). miR-34-basedtherapyandtheMRX34isthe?rstmiRNAmimic to advance into a human clinical trial. Similar approaches can beusedtotargetthehighly ?broticpancreatictumors.Overall, these strategies, besides being powerful approaches for functional validation of miRNAs relevant for a speci?c disease, constitute a prerequisite for the development of potential miRNA-based therapies. Herein, we discuss several important miRNAs, which have consistentlybeenshowntobeinvolvedinPDACprogressionby many research groups. Also, we discuss strategies of miRNA modulator delivery in cancer models, a subject that remains the key challenge to the establishment of miRNA therapeutics.
miRNAs in Pancreatic Cancers. Where Are We Now? ThefunctionalactivityofonlyahandfulofmiRNAshasbeen experimentally modeled in the context of pancreatic cancer. Some of these miRNAs have been characterized as potent oncogenes (oncomiR) or tumor suppressors (tsmiR), based on the consequences of their expression on the phenotype in experimental models.
Oncogenic microRNAs miRNA pro?ling and functional studies reveal several deregulatedmiRNAsinPDACprogression,butveryfewstudies have shown their role in the key shared genetic alterations associated with their precursor lesions. The most speci?cally overexpressed miRNA in PanIN III lesions (carcinoma in situ) identi?ed is miRNA-196b (6). Aberrant expression of several miRNAs has been identi?ed early in PanIN lesions as well as throughout the multistep progression of PDAC. They include miR-21, miR-155, and miR-221 (7), which have been linked to ?brogenesis through TGF-b that promotes their expression. Theyplayanimportantroleinfunctioningasproto-oncogenes and have been associated with decreased survival in patients with PDAC (8). miR-155 targets a key tumor suppressor, TP53INP1 (9), which is expressed in normal tissues in early stagesofpancreaticcancerdevelopment.miR-21decreasesthe expression of SMAD7, a negative regulator of TGF-b signaling (10). On the other hand, several studies have reported significant overexpression of miR-21 in PDAC associated with chemoresistance (11). Thus, inhibition of miR-21 in PDAC would be potentially useful to inhibit the desmoplastic reactions via repression of TGF-b signaling in PDAC (12) to overcome drug resistance and inhibit tumor surveillance. Immunosuppressive tumor microenvironments can restrain antitumor immunity, particularly in PDAC. CD40 agonists alter tumor stroma and show ef?cacy against pancreatic carcinoma in mice and humans. miR-224 and miR-486 are upregulated and target CD40, thus downregulating its protein expression at cell surfaces in highly invasive and metastatic PDAC(13). The oncogenic role ofselected miRNAs has mainly been associated with their ability to enhance the proliferative potentialofcancercellsthroughthesuppressionofcell-cycle– related protein pRb (14), as well as members of the E2F family oftranscriptionfactors.Forinstance,miR-132andmiR-212are upregulated in PDAC tissues and these miRNAs promote tumorigenesis by reducing pRB, thus increasing expression of E2F gene products (14). A critical tumor suppressor, DPC4/Smad4, is involved in the progression of PDAC and is targeted by miR-27a (15). Attenuation of miR-27a in PDAC cells reduced growth, colony formation, migration, and also downregulated sprouty2 (spry2), a negative feedback regulator of multiple receptor tyrosine kinases exerting an oncogenic effect. MicroRNA-10 a/b are overexpressed in PDAC, promote its invasiveness, and correlate with a poor prognosis (16–18). HOX genes have been identi?edas theirpotentialtargets inmanycancers, including PDAC (19). An association between aberrant miRNA expression and malignancy in PDAC was recently shown with an oncogenic positive feedback loop. miR-301a inhibits proapoptotic Bim expression (20) and suppresses NF-kB repressing factor (NKRF) in PDAC, leading to NF-kB activation, which in turn activates the transcription of miR-301a. Inhibition of miR-301 or overexpression of NKRF reverses the process and slows down tumor growth, suggesting that miR-301a contributes to oncogenesis by activating NF-kB (21). In addition, increased expressionofmiR-155,miR-21,miR-203,miR-210,andmiR-222 has reportedly been associated with poor survival in patients
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with cancer (8). Moreover, high expression of miR-196a-2 is associated with patients’ poor outcome (22), which suggests it may be a prognostic marker for PDAC. In addition, applying silencing strategies to these miRNAs may likely alter the outcome of conventional therapeutics and overall survival of patients with pancreatic cancer.
Tumor suppressor microRNAs Recently, the miR-200 a/b/c family has been observed to suppress stemness of cancer cells by suppressing stem cell factors such as Sox2 and inhibiting SIP 1 and ZEB1 (negative regulator of E-cadherin), thereby inhibiting EMT and invasion in pancreatic cancer cells (23). But the miR-200 family (overexpressed in PanINs/miR-200c only in low grade PanINs), including Let-7, miR-194, and miR429, are found to be significantly overexpressed with epigenetic alterations in highly invasiveandmetastaticPDACtargetingthetumorandmetastasis suppressor, EP300 mRNA, and protein (24). Thus, the epigenetic modi?cation responsible for abnormal expression of miRNAs in pancreatic tumors may be a crucial mechanism contributing to cancer invasion and metastasis. Differential expression of certain miRNAs detected in PanINs correlates with their expression in invasive PDAC. A well-documented tumor suppressor in PDAC is miR-375, which targets 14-3-3 zeta (15, 22) and induces caspase-depen
dent apoptosis. Its overexpression downregulates the PDK1/ Aktsurvivalpathwayandleadstodownregulationofpotential biomarkers such as IGFBP5 and CAV-1 (25). Very recently, diverse tumor-suppressive functions have been associated with miR-15a, which modulates the survival pathways by targeting WNT3A (Wnt/b-catenin pathway) and FGF7 (26). Ectopic expression of miR-96 increases cell apoptosis reduces migration, proliferation, and invasion of PDAC cell lines by inhibition of the KRAS/Akt (27). In addition, the downregulationofmiR-34aisseenalongwiththelossofp53function(28), implicating its role in PDAC.
Therapeutic Implications of miRNAs, a Way toward the Clinic The therapeutic application of miRNAs involves two strategies. One strategy is directed against a gain-of-function and aims to inhibit oncogenic miRNAs by using miRNA antagonists. The second strategy against a loss-of-function, miRNA replacement, involves the reintroduction of a tumor suppressor miRNA to restore a loss of function.
miRNA Gain-of-Function It is our understanding that miR-21, miR-155, miR-196a, miR-210,andmiR-221(Table1)areoverexpressedinPDACand
Table 1. Selected miRNA candidates that might be clinically relevant in the management of pancreatic cancer
miRNA Status Role Targets Signi?cance Ref. miR-21 ” O EGFR, HER2/neu, PDCD4, Bcl2, PTEN, TIMP2, TIMP3 Postresection short DFS/worse OS (11, 30, 31, 45) Gem chemoresistance Biomarker (Dtc: plasma, serum, feces) miR-155 ” O TP53INP1 Postresection poor OS and prognosis (8, 9) miR-196a-2; miR-196b ” O HOXB8, ANXA1, HMGA2 Postresection (22, 46–48) Biomarker (Dtc: plasma) miR-222/221 ” O CDKN1B (p27), PUMA, PTEN, Bim Postresection poor OS and prognosis (8, 22, 45) Gem chemoresistance Biomarker (Dtc: plasma) miR-210 ” O HOXA1, FGFRL1, HOXA9, COX10, E2F3, RAD52, ACVR1B, MNT Postresection poor OS (8, 22, 45) Biomarker (Dtc: plasma, feces) miR 10a/b ” O HOXB8, HOXA1 Poor OS; Gem chemo-resistance (16–19) miR-375 ” O PDK1, 14-3-3zeta Biomarker (Dtc: plasma, feces) (22, 25, 49) miR-301a ” O Bim, NKRF Proliferation and metastasis (20, 21) miR-200a/b/c # TS EP300 Postresection high expression improved OS (22, 24, 40, 50) Gem chemosensitivity miR-34a # TS Bcl-2 Improved OS after treatment, marker for Gem chemosensitivity (38) Let-7 # TS E2F2, c-Myc, KRAS, MAPK Enhances chemosensitivity (40) miR-34b # TS Smad3 Reduces invasion/metastasis (51) miR-96 # TS KRAS, AKT Inhibits tumor growth and invasion (27) Abbreviations: DFS, disease-free survival; Dtc, detection; Gem, gemcitabine; OS, overall survival.
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currently represent the most promising candidates as oncogenic miRNAs.
Antisense oligonucleotides It has been shown that antisense oligonucleotides (ASO) can inhibit upregulated oncogenic miRNAs in tumors in a speci ?c, ef?cient, and long-lasting manner (29). ASOs for miR-21 and miR-221 increase the levels of their targets (PTEN, RECK, and CDKN1B), reduce proliferation, and increase apoptosis of pancreatic cancer cells (30, 31). ASOs also sensitize PDAC cells to gemcitabine and generate synergistic antitumor effects. Preclinical studies have shown potent activity of AEG35156 (targets X-linked inhibitor of apoptosis mRNA) in combination with gemcitabine in PDAC (32). These results imply that targeting miRNAs with ASOs could be a potential new therapeutic strategy for PDAC.
AntagomiRs The miRNA antagonists carry chemical modi?cation that degrades and traps the endogenous miRNA in a con?guration that is unable to be processed by RISC. A locked nucleic acid (LNA) is an ASO modi?ed by LNA technology [bicyclic high-af?nity RNA mimicking N-type (C30-endo) conformation by the introduction of a 20-O,4-C methylene bridge]. An anti-miRNAoligonucleotide(AMO)isasingle-strandedASO, chemically modi?ed (usually with a 20-O-methyl group) 17 to 22 nucleotide long nucleic acid molecule. A study showed that an antagomiragainstmiR-21sensitized culturedcells to 5-?uorouracil (5-FU) treatment, suggesting this miRNA therapy is a promising treatment for tumors that are resistant to 5-FU (11). Also, MTg-AMO (multiple-target AMO) is an innovative approach in which multiple antisense units are engineered into a single unit for simultaneous silencing of multiple-target miRNAs (33). Studies suggest that the MTgAMO is an improved approach for miRNA target ?nding and miRNA function validation (34). The combined targeted effect against the oncomirs will validate the physiologic relevance of their ability to inhibit invasion or metastasis and the development of miRNA-based therapeutic modality for PDAC.
Agents targeting epigenetic alterations Studies have shown that subsets of miRNAs are also modulated by aberrations in epigenetic regulation. Studies show garcinol (a potent inhibitor of histone acetyltransferases) synergizes with gemcitabine to modulate speci?c miRNA biomarkers (miR-21, miR-196a, miR-495, miR-605, miR-638, and miR-453) that sensitize PDAC cells to gemcitabine treatment, thus attenuating the drug-resistance phenotype (35). Another study shows that trichostatin A, a histone deacetylase inhibitor, induces downregulation of oncogenic miR-21 and upregulation of tumor suppressor miR-200c in pancreatic cancer cells, resulting in cell-cycle arrest and increased apoptosis (36). Therefore, epigenetic regulation of miRNAs with a histone deacetylase inhibitor may be an effective therapeutic option for pancreatic cancer.
miRNA Loss-of-Function In our opinion, the miRNAs, Let-7, miR-34a, miR-96, miR375, miR-200 family (Table 1) currently represent the most promising candidates as tumor-suppressive miRNAs.
miRNA replacement therapy Ef?cient reconstitution of miRs through in vivo delivery of miRNA precursors is a crucial factor for the development of successful miRNA-based treatment modalities, a practical application of which is still pending. Restoration of lost Let-7 expression in gemcitabine-resistant PDAC cells inhibits proliferation,restoresepithelialphenotype,andrendersthetumor cell sensitive to gemcitabine. Also, ectopic expression of members of the Let-7 family of miRNAs in cancer cells increased their in vitro radiation response (37). on the basis of this evidence, rational design of combined therapeutic approaches comprising anticanceragents andspeci?cmiRNA modulatorscanbeenvisagedtoimprovetherapeuticresponse. Functional studies carried out in PDAC models attribute an oncosuppressive function to miR-34, because it is a key regulator of cancer cell growth and invasion. Restoration of miR34canhavesigni?cantpromiseasanovelmoleculartherapyas it reduces tumor-initiating cancer stem cells mediated by inhibition of its downstream targets BCL2 and NOTCH (38) and sensitizes to chemo/radiation therapies (87%). Therefore, miR-34 is prospectively a potential target to be restored in tumors for modulating many oncogenic signaling pathways. Themostcommonlyusedstrategiesagainstloss-of-functionto probe miRNA functions in vivo are synthetic miRNA mimics (miRNAoverexpression),vector-mediatedmiRNAoverexpression, and small-molecule therapy.
Small-molecule drugs targeting speci?c miRNAs Fromtheperspectiveoftheirsecondarystructures,miRNAs appear to be “druggable” targets. The formation of stem loops found in pre-miRNAs and the bulges in miRNAs facilitate small-molecule targeting, thereby, making the development of small-molecule drugs (SMIR) a promising prospect. SMIRs interactandbindtothegroovesandpocketsonthesurfacesof miRNAs directly, thereby interfering with the biologic functions of targeted miRNAs and inhibiting the processing of miRNAs(39).Anumberofagents,includingiso?avoneand3,3diindolylmethane, has been shown to alter expression of miR200b, miR-200c, Let-7b, Let-7c, Let-7d, and Let-7e in gemcitabine-resistant cancer cells (40). A naturally occurring ?avonoid, curcumin (diferuloylmethane) from the rhizome of Curcumalonga,hasbeenshowntohavetremendouseffectsonthe expression pro?les of miRNAs in pancreatic cancer models. It causes overexpression of miR-22 and downregulation of miR199a (41), regulating Sp1 transcription factor (SP1) and estrogen receptor 1 (ESR1), which are involved in cell growth, metastasis, and apoptosis.
Nanovector therapy Non-viral lipid-based complexes, liposomes, and nanoparticleshavebeenpromisingavenuesusedforsystemicmiRNA delivery. Studies show that systemic nanovector therapy
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modulates direct and indirect miRNA targets in a pancreatic cancerxenograftmousemodel(42).Inouropinion,thisisan effective delivery system, displaying carrier-de?ned speci?city to deliver miRNA or miRNA-modulating agents to relevant tissues or organs. The SMARTICLES oligonucleotide delivery technology is currently being used to deliver MRX01 (proprietary tumor suppressor miRNA, miR-34) in a hepatocellular carcinoma orthotopic mouse model. Preclinical trials are projected to begin this year (Mirna Therapeutics, Inc).
Correlation of miRNAs in Clinicopathologic Parameters miRNAs have potential as diagnostic and prognostic biomarkers and as therapeutic targets in cancer. Poor survival of patients with pancreatic cancer, especially with PDAC, is attributed to diagnosis at an advanced stage. Therefore, the developmentofminimallyinvasiveearlydetectionbiomarkers for effective clinical management is urgently needed. A company named Asuragen has developed a molecular diagnostic test to distinguish between benign and PDAC that can be performed on ?ne-needle aspirate (FNA) biopsies of lesions. This is used in conjunction with indeterminate and benign FNAcytopathologyandisbasedontheexpressionlevelsofthe seven miRNAs and their scores. The detection of biomarkers in bodily ?uids (blood, sera, plasma, and feces) offers the possibility of detecting disease at an early stage (43, 44). However, the diagnostic and prognostic value of circulating miRNAs in PDAC has not yetbeenabundantlyproven.ThediscoveryofserummiRNAs aspotentialbiomarkersovercomestheproblemofcollecting tissue samples by an invasive process. In our opinion, the combination of multiple serum miRNAs can serve as a more comprehensive indicator for tumor detection than the conventional single protein–based or single carbohydrate molecule–based biomarkers, such as CA19-9 and CEA. A comparison of the miRNA expression patterns in serum and tissue/cells may provide additional evidence supporting the use of circulating miRNAs as reliable diagnostic biomarkers (43). However, past studies have not provided direct evidence that serum miRNAs are actively secreted or passively leaked from tumor cells. Therefore, more research in this area will provide a proof-of-principle approach for the use of miRNAs as a diagnostic tool for early pancreatic cancer detection.
Future Perspectives The contribution of miRNAs to the biology of PDAC is still at an early stage even though evidence of deregulated expression suggests miRNAs possess crucial roles in disease development/progression. Collectively, given the myriad of diagnostic, prognostic, and therapeutic opportunities arising from miRNA modulation, the translation of miRNAtargeted therapeutics into the clinic will probably witness rapid progress. However, it remains unclear whether the identi?cation of miRNA associated with the transition of an
early PanIN and other premalignant lesions into PDAC prevents and/or modulates the malignant phenotype. Focus is required on the critical shifts of these miRNAs in the behavior of these lesions and how they relate to underlying molecular alterations, checkpoint responses, genomic complexity, and the activation of key signaling cascades. Pathobiologic and clinical information, along with the development of uniform standards for miRNA measurement to facilitate comparison across studies, could potentially explain the whole story. Moreover, the role of miRNAs in various genetic networks and regulatory pathways, including the mesenchyme and stroma, needs to be analyzed in larger cohort neoplastic and normal tissues. This will be important for the identi?cation and validation of speci?c miRNAs as diagnostic/prognostic markers. In addition, studies regarding the mechanisms of widespread miRNA dysregulation are clearly needed to understand the origin of the highly metastatic properties of PDAC and provide insight for the improvement of the prognosis of this fatal disease. From a therapeutic standpoint, more details regarding the role of miRNAs in the maintenance of normal cellular homeostasis as well as development of transformed phenotypes would stimulate rational therapeutic bene?ts. For this purpose, the development of proper in vivo experimental models is important to recapitulate in vivo miRNA functions. In addition, getting a small RNA to interfere with the speci?c mRNA in the target tissue at a safe and therapeutic level by systemic administration remains a great challenge. Hence, adequate assessment of the functional effects after miRNA inhibition and the long-term miRNA antagonism in in vivo are of key importance for anti-miR–based functions. Owing to the severity and limited treatment paradigms in cancer the higher tolerance provided by regulatory authorities for novel anticancer agents would overcome the time frame barrier for miRNA therapeutics to reach clinic.
Disclosure of Potential Con?icts of Interest No potential con?icts of interest were disclosed.
Authors’ Contributions Conception and design: S. Khan, M. Jaggi, S.C. Chauhan Development of methodology: S. Khan, M. Jaggi Analysisandinterpretationofdata(e.g.,statisticalanalysis,biostatistics, computational analysis): S. Khan, D. Kumar, M. Jaggi Writing, review, and/or revision of the manuscript: S. Khan, Ansarullah, D. Kumar, M. Jaggi, S.C. Chauhan Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Khan, M. Jaggi Study supervision: M. Jaggi, S.C. Chauhan
Acknowledgments The authors thank Cathy Christopherson for editorial assistance.
Grant Support ThisworkwaspartiallysupportedbygrantstoS.C.ChauhanfromGovernor’s Cancer2010,theNIHResearch Project GrantProgram (RO1)(CA142736),(UO1) (CA162106A),P20GM103548,andtheCentersofBiomedicalResearchExcellence (P20 RR024219).
Received May 2, 2013; revised August 15, 2013; accepted August 16, 2013; published OnlineFirst November 7, 2013.
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