review article therapeutic use of micrornas in lung...

Review ArticleTherapeutic Use of MicroRNAs in Lung Cancer

Orazio Fortunato Mattia Boeri Carla Verri Massimo Moro and Gabriella Sozzi

Tumor Genomics Unit Department of Experimental Oncology and Molecular MedicineFondazione IRCCS Istituto Nazionale dei Tumori Via Venezian 1 20133 Milan Italy

Correspondence should be addressed to Gabriella Sozzi gabriellasozziistitutotumorimiit

Received 5 June 2014 Accepted 8 August 2014 Published 16 September 2014

Academic Editor Paolo Gandellini

Copyright copy 2014 Orazio Fortunato et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Lung cancer is a leading cause of cancer deaths worldwide Although the molecular pathways of lung cancer have been partlyknown the high mortality rate is not markedly changed MicroRNAs (miRNAs) are small noncoding RNAs that actively modulatecell physiological processes as apoptosis cell-cycle control cell proliferation DNA repair and metabolism Several studiesdemonstrated that miRNAs are involved in the pathogenesis of lung diseases including lung cancer and they negatively regulategene and protein expression by acting as oncogenes or tumor suppressors In this review we summarize the current knowledgeon the role of miRNAs and their target genes in lung tumorigenesis and evaluate their potential use as therapeutic agents in lungcancer In particular we describe methodological approaches such as inhibition of oncogenic miRNAs or replacement of tumorsuppressor miRNAs both in in vitro and in vivo assays Furthermore we discuss new strategies to achieve in vivo tissue specificdelivery potential off-target effects and safety of miRNAs systemic delivery

1 Introduction

Lung cancer is the most frequent cause of cancer mortalityworldwide accounting for more than 14 million deaths peryear [1]The twomajor histotypes of lung cancer are nonsmallcell lung cancer (NSCLC) (about 80 of all lung cancers) andsmall cell lung cancer (about 20) [2]

Despite improvements in early diagnosis and new thera-peutic strategies the overall 5-year survival remains only of10ndash20 [1] The poor prognosis is due to diagnosis at advan-ced stage tumor heterogeneity and relatively limited under-standing of lung cancer biology Surgical resection is so farthe most common treatment for early stages tumors in com-bination with chemotherapeutic agents for advanced lungcancer patients or chemotherapy treatment alone for meta-static disease The platinum-based treatment is commonlyused in the clinical practice with small benefit in survivalof lung cancer patientsThe identification of driver mutationsand genetic rearrangements in approximately 50ndash60 ofNSCLC has led to a change in the treatment of lung cancerpatients [3ndash5] by identifying subgroups of patients charac-terized by different molecular profile K-RAS mutations havebeen found in approximately 17 of all NSCLC especially

in adenocarcinomas (27ndash34) whereas the discovery ofactivating mutations in the EGFR gene (23) and rearrange-ments of anaplastic lymphoma (ALK) (5) were found andhad also relevant impact in the treatment of lung cancerpatient through their responsiveness to tyrosine kinase inhi-bitor (TKI) agents such as erlotinib crizotinib and gefitinib[6 7]

Although new targeted therapies entering clinical trialshave shown positive results a large number of patients stillremain without any potential known target for therapyTherefore the identification of novel treatment strategies iscritical and essential for lung cancer management

MicroRNAs (miRNAs) are small noncoding 22 nt-longRNAs that are able to bind complementary sequences oftarget mRNAs and to induce either their degradation ortranslational repression [8] In mammals miRNAs controlthe activity of more than 50 of all protein-coding genes [9]MiRNAs are expressed in a tissue or developmental specificmanner thereby greatly contributing to cell-type-specificprofiles of protein expression MiRNAs potentially targethundreds of different mRNAs and thus coordinate or fine-tune the expression levels of many proteins thus regulating awide variety of cellular processes [10] To datemore than 1000

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 756975 8 pageshttpdxdoiorg1011552014756975

2 BioMed Research International

Table 1 Principal miRNAs involved in lung carcinogenesis

miRNAs Expression inlung cancer Cellular processes affected and targets Reference

Let-7 family Decreased(i) Cell proliferation (KRAS MYC and HMGA2) [28ndash30](ii) miRNA maturation Dicer mediated [31](iii) Cell-cycle regulation (CDC25A CDK6 and cyclin D2) [32]

mir-34 family Decreased (i) Transcriptionally activated by p53 [40](ii) TRAIL-induced cell death and cell proliferation (BCL-2 METand PDGFR-120572120573) [41ndash43]

mir-21 Increased(i) Apoptosis cellular proliferation and migration (TPM1 PDCD4and PTEN) [50ndash53]

(ii) TKI-treatment resistance [54]

mir-1792a cluster Increased (i) Transcriptionally regulated by c-MYC [60](ii) Cellular proliferation and cancer development (PTEN HIF-1aCL2L11 CDKNA and TSP-1) [57 61ndash63]

mir-15a16 cluster Decreased Cell cycle regulation (cyclin D1 D2 and E1) [66 67]

mir-200 family Decreased Promotion of EMT and metastasization(ZEB transcription factorsCDH-1 and vimentin) [68ndash73]

miRNA-29 family Decreased Epigenetic regulation of gene expression(DNMT-3A and DNMT-3B) [74]

mir-221mir-222 Increased (i) TRAIL resistance and cellular migration (PTEN and TIMP3) [75 76](ii) Transcriptionally regulated by EGFR and MET and gefitinibresistance (BIM and APAF1) [77]

mir-548 Decreased Tumor cell growth(CCND ERBB2 DMNT3A and DNMT3B) [78]

human miRNAs were found in the genome and a consider-able number of them were found deregulated in cancer cellscompared to normal cells [11ndash14]

The rationale of miRNA therapy in lung cancer is basedon two criteria one is that miRNAs play an essential role inlung development [15] and their expression levels are deregu-lated in lung cancer compared to normal tissues [16] and alsoin the blood of patients [17] and the second one is that severalstudies demonstrated that modulation of miRNA expressionboth in vitro and in vivo can modify the cancer phenotype[18ndash22]

Different strategies of miRNAs therapeutics can be envis-aged according to the expression status of miRNAs in thetumor (i) miRNAs inhibition when it is overexpressed usingantagomir which are synthetic miRNAs complementary tothe miRNAs of interest or miRNAs sponges that competewith the targets of the miRNA and (ii) miRNAs replacementusing oligonucleotide mimics or viral vectors when themiRNAs is downmodulated [23]

In this review the role of those miRNAs which regulatefundamental proteins and pathways involved in lung tumori-genesis will be discussed Furthermore we will describe gen-eral therapeutic strategies involving inhibition of oncogenicmiRNAs and replacement of tumor suppressor miRNAsincluding recent experimental methods developed to bypassblood clearance and poor intracellular uptake

2 miRNA Role in Lung Carcinogenesis

Human cancers exhibit an altered expression of miRNAswith oncogenic or tumor-suppressive activity providing an

explanation of their role in tumor development and pro-gression Recent studies reported an aberrant expression ofmiRNAs in lung tumor tissues when compared to the corre-sponding normal lung tissues supporting miRNAs involve-ment in lung carcinogenesis (Table 1) Lethal-7 (let-7) isone of the earliest identified tumor suppressormiRNA in lungcancer and its downmodulation is associated with poorprognosis [16 24ndash27] Furthermore let-7 can regulate severaloncogenic pathways for example this miRNA negativelymodulates multiple cell-cycle oncogenes as KRAS mutationof which are commonly implicated in lung adenocarcinomas[28] MYC and HMGA2 [29 30] Furthermore an involmentof let-7 in the regulation ofmiRNAmaturation process-Dicermediated has also been described [31] Let-7 overexpressionin lung adenocarcinoma A549 cell line inhibits in vitro assayscell growth and reduces cell-cycle progression by targetingCDC25A CDK6 and cyclin D2 [32] Accordingly in vivoexperiments show that let-7b inhibits lung cancer cell xeno-grafts in immunodeficient mice [33] In addition the thera-peutic potential of let-7 in lung cancer treatment is supportedalso by experimental evidence that altered expression of let-7family members associated with radiation resistance [34]

Mir-34 is a tumor suppressor miRNA largely investigatedin cancers and its deregulation has been reported in varioustumor types including lung cancer [35ndash39]Mir-34members(a b and c) are regulated by p53 and mir-34 expression isfrequently reduced in p53 mutant tumors [40] mir-34 familymembers are involved in cell-cycle control apoptosis andcellular senescence through specific targeting of BCL-2MYC andMETgenes [41] In additionGarofalo et al showed

BioMed Research International 3

that mir-34a and mir-34c are downregulated in NSCLC celllines and described an inverse correlation between PDGFR-120572120573 and miR-34ac expression in lung tumor samples [42]The inhibition of PDGFR-120572 and PDGFR-120573 by miR-34acreplacement antagonizes tumorigenicity and increases sensi-tivity to TRAIL-induced cell death suggesting an importanttherapeutic application for lung cancer The potential ther-apeutic use of mir-34 is further supported by Kasinski andSlack who investigated the effects of miR-34 replacement ontumor formation and progression in a mouse model [43]

Unlike let-7 and mir-34 mir-21 has been described asa oncogenic miRNA and it was found overexpressed inlung cancer [44] The expression levels of mir-21 weresignificantly higher in tissues from NSCLC patients withlymph node metastasis than in patients without lymph nodeinvasion [45] and the diagnostic and prognostic values asbiomarkers were confirmed in several works [45ndash49] Thesedata suggest that the overexpression of mir-21 plays a criticalrole in lung tumorigenesis In vitro inhibition of mir-21expression in NSCLC cell lines showed a deregulation ofcellular mechanisms as programmed cell death cellular pro-liferation and migration Validated target for mir-21 includeTPM1 PDCD4 and PTEN [50ndash53] and recently mir-21overexpression was indeed associated with the acquiredresistance of EGFR-TKI in NSCLC due to the activation ofPI3KAKT pathway through PTEN and PDCD4 inhibitionbymir-21 [54]The role ofmir-21 as an oncogenicmiRNAandits therapeutic value were also demonstrated in an in vivoassay using K-rasLA2murinemodel of multifocal lung cancerwhere the downmodulation ofmir-21 expression resulted in areduction of tumor number incidence and size [55]

Oncogenic activity was also reported for mir-1792a clus-ter (mir-17 mir-18a mir-19a mir-20a mir-19b and mir-92)that is upregulated in several cancer types both hematopoi-etic and solid tumors [56ndash59] miRNAs from this cluster areregulated directly by c-MYC and have a central role in thecontrol of cellular proliferation [60] Reporter assays revealedtarget sequences for mir-19a and mir-19b-1 in the 31015840UTR ofPTEN [61] and the introduction of mir-19a and mir-19b-1 orthe full cluster was sufficient to induce c-MYC-driven B-celllymphoma development in mouse model by restoring PTENexpression levels [57 62] In lung cancer this cluster is fre-quently amplified and in particularmir-17-5p andmir-20a areoverexpressed These miRNAs are fundamental for lungcancer development by targeting HIF-1a PTEN BCL2L11CDKNA and TSP-1 [63]

Several papers showed that mir-15a16 are implicated incell-cycle control and likely contribute to NSCLC tumorige-nesis [64ndash67] mir-15a16 are frequently deleted or downreg-ulated in squamous cell carcinomas and adenocarcinomas ofthe lung and their expression is inversely correlated with theexpression of cyclin D1 In NSCLC cell lines physiologic con-centrations of mir-15a16 regulated cyclin D2 and cyclin E1expression Using luciferase reporter constructs of the dif-ferent cyclin genes miRNAs were able to downregulateluciferase activity and this effect was reverted by cotrans-fection with anti-mir-15a16 In addition mir-15a16-inducedcell-cycle arrest can be partially restored by overexpression ofCCND1 or CCNE1

Recent works investigated mir-200 family (mir-200bmir-200a mir-429 and mir-200c) and its role in the promo-tion of EMT inNSCLC through regulation of ZEB1 transcrip-tion factors and regulation of CDH-1 and vimentin expres-sion [68ndash70] In addition in a mouse model that developsmetastatic lung adenocarcinoma overexpression ofmir-200blocus in highly metastatic cells eliminated their ability toundergo EMT andmetastasize and also this miRNA could beused to distinguish lung tumors cell lines based on their site oforigin with higher levels in cells from a primary tumor thanin cell lines derived frommetastatic sites Recently Tejero andcolleagues demonstrated that high levels of miR-141 andmiR-200c are associated with shorter overall survival in a cohortof NSCLC patients with adenocarcinoma [71ndash73]

Among the reported miRNAs that are downregulated inlung cancer themiRNA-29 family (29a 29b and 29c) expres-sion in lung cancer tissue is associated with DNA methyl-transferase DNMT-3A and DNMT-3B levels two importantenzymes for DNA methylation that are frequently upregu-lated and associated with poor prognosis [74]The expressionof mir-29 family members in lung cancer cell lines restorednormal patterns of DNA methylation inducing reexpressionof tumor suppressor genes such as FHIT and WWOX thatwere previously silenced by methylation thereby inhibitingtumorigenesis in vitro and in vivo These findings support therole of mir-29s in epigenetic normalization of nonsmall celllung cancer (NSCLC) providing a rationale for the devel-opment of miRNA-based therapeutic interventions for thetreatment of lung cancer

Finally mir-221 and mir-222 are reported to be involvedin lung cancer by targeting PTEN and TIMP3 tumor sup-pressors inducing TRAIL resistance and enhancing cellularmigration [75 76] Mir-221222 are modulated by bothepidermal growth factor (EGF) and MET receptors and werefound to be involved in gefitinib resistance in cooperationwith mir-30b and mir-30c by inhibiting APAF1 and BIM celldeath genes SinceMET amplification plays an important rolein the resistance to anti-EGFR agents a modulation of thesemiRNAs could have therapeutic applications to sensibilizelung tumors to TKI therapy [77]

Recently the work ofHu et al identified a newmember ofthemiR-548 family in the intron of human FHIT gene whichis a tumor suppressor gene altered in human cancer as lunghead and neck esophageal stomach pancreatic and cervicalcancer This miRNA inhibited human tumor cell growth invitro and in vivo xenograft mouse model targeting CCND1ERBB2 DNMT3A and DNMT3B [78]

3 miRNA as Therapeutic Agents

Delivery of miRNAs as synthetic miRNA mimics or anta-gomirs has emerged as a promising approach to treat cancer(Table 2) To date MRX34 is the only phase I clinical trialfor miRNA replacement in patients with primary liver canceror with liver metastasis from other cancers (Mirna Ther-apeutics) MRX34 is based on the formulation of miR-34mimic and the liposomal delivery technology SMARTICLES(Marina Biotech) Interestingly mir-34 as mentioned above


Table 2 Strategies for in vivomiRNA delivery

Type of particles Advantages Disadvantages Reference

Synthetic Mimicinhibitor

Safe and simpleEasy to produceLow immunogenicity

Poor cellular uptakeRapid degradation and clearance [94]

Neutral lipoplexesliposomesMaxSuppressorSLNsSMARTICLESsiPORT

Low immunogenicityLung accumulationEasy to produceAvailable commercially

Nonspecific uptakeLow transfection efficiencyCytotoxicity

[79 80 84 86]

Cationic lipoplexesliposomes PU-PEIDOTMA

Low immunogenicityEasy to produceHigh cellular uptake

CytotoxicityTend to form aggregatesModerate transfection efficiencyNonbiodegradability

[81 83]

Viral vectors AdenovirusLentivirus

High transfectionefficiencyStable expression

Difficult to produceHigh immunogenicityHigh toxicity

[33 43]

is one of the most deregulated tumor suppressor miRNA inlung cancer suggesting also a potential use of this methodol-ogy for the treatments of lung cancer patients

The first evidence of miRNAs replacement in lung cancerwas reported in 2008 The authors showed that restoringlet-7 expression affected tumor growth in xenograft modelsderived from lung cancer cells H460 or A549 (NSCLC celllines carrying mutations in K-RAS gene) injected subcuta-neously in immunodeficient mice In this work cells weretransiently transfected with 30 nM of let-7 miRNA mimicprior to subcutaneous injection into NODSCID mice and adelay in tumor growth was observed They also useda conditionally inducible K-RAS mutated mouse model(KrasLSL-G12D+) developing orthotopic lung adenocarcinomato demonstrate that let-7 expression interferes with lungtumorigenesis They treated mice intranasally with aden-ovirus expressing cre recombinase (Ad-cre) (5 times 108 pfu) toinduce lung cancer growth and treatment with a second ade-novirus expressing let-7 resulted in significant tumor growthinhibition [33]

Trang and coworkers investigated the therapeutic role oflet-7 in established lung tumors [79] They injected intratu-morally 625 120583g of let-7 mimics mixed with siPORT-amine(siPORT) a lipid-based reagent in xenograft mouse modelsof H460 lung cancer cell lineMice were treated on days 11 1417 and 20 after xenograft implantation and tumor size mea-sure on day 21 resulted in a robust tumor growth inhibitionThis group also obtained a reduction in tumor growth withintranasal injections of lentiviral (106 pfu) or adenoviral (5 times108 pfu) vectors expressing let-7 in KrasLSL-G12D+mice Local

delivery by intratumorally or intranasal injection of miRNAdemonstrated a significant tumor reduction but is probablyinadequate for the clinical setting of miRNA therapy

In 2010 Wiggins et al reported a tumor growth reduc-tion by miR-34 treatment in H460 and A549 NSCLCxenograftmodels in immunodeficientmice [80] NODSCIDmice were treated with different concentration of mir-34mimic (5 and 1mgkg) formulated with MaxSuppressor invivo RNALancerII a neutral lipid emulsion (NLE) deliveryreagent (BIOO Scientific Inc) injected intratumorally or

intravenously on days 12 15 and 18 after xenograft implan-tation Interestingly they performed a blood chemistry anal-ysis to test possible toxicity in liver kidney and heart andobserved that miRNA treatment was well tolerated Usinga different mouse model they proved that miRNA treatmentdid not induce nonspecific immune responses in mice bymeasuring serum cytokines In summary this study providedevidence for the safe and effective therapeutic delivery of amiRNA mimics However further studies have to demon-strate thatmiRNAs replacement has no effect on normal cells

One year later the same authors also showed that usingNLE rather than the standard cationic liposomes it was pos-sible to transfect miRNAs systemically by intravenous injec-tions in mice and these particles with miRNA were taken upespecially by lung tissue thus showing that NLE could vehicletherapeutic miRNAs to lung tumors Indeed using NLEKrasLSL-G12D+mice were treated with 1mgkg of let-7 or mir-34 for 8 consecutive days and showed a significant reductionof tumor burden [81] The authors suggest that NLE maybypass some of the shortcomings that can be attributed tocharge of cationic lipids These particles form less aggregatesin biofluids are not filtered by the liver and do not adhere tothe endothelium or are taken up by macrophages [82]

More recently it was created a double transgenic mousemodel KrasLSL-G12D+ Trp53LSL-R172H+ where KRAS muta-tion was induced by Ad-cre in the cells with a p53 nullbackground to recapitulate human lung carcinogenesis [43]In this study mir-34 was able to reduce progression of pre-formed tumors and also totally prevented tumor formationFor these experimentsmice were intubated and 3times106 TUs oflentiviral particles expressing mir-34 were administrateddirectly in the thoracic region

New methodologies for miRNA delivery were settled upin order to reduce cytotoxicity to avoid liver capture andto better target lung cells Among these the biodegradablecationic polyurethane short branch polyethylenimine (PU-PEI) system was conjugated with mir-145 and showed veryhigh transfection efficiency when injected intratumoral inlung adenocarcinoma cancer stem cells (LAC-CSCs) xeno-graft models suppressing the stem-like properties and


sensitizing them to chemo- or radiotherapy [83] These non-viral gene vectors could represent a genetically and immuno-genically safe delivery method for miRNAs-based therapiesbut since intratumoral administration is not a feasible clinicalroute PU-PEI should be subjected to further characterizationfor systemic delivery

In an effort to find an appropriate delivery method ofmiRNAs for lung cancer treatment Shi et al used solid lipidnanoparticles (SLNs) containing dimethyldioctadecylammo-nium bromide (DDAB) that condense miRNAs and enhancecellular uptake They demonstrated that intravenously injec-tion of 05mg of SLNs loaded with Cy3-miR-34 was taken upby lung tissue in just 1-2 hours resulting in a cytosolic deliveryof the miRNA [84] Their results indicate that miSLNsenhance miR-34a uptake into the lung due to the ability ofthe delivery vehicle to avoid liver capture and to be physicallyentrapped in the capillary of the lung compared to lipofec-tamine particles which were captured by liver Recently Wuet al developed a cationic lipoplexes-based carrier systemcontaining miR-29b (LP-miR-29b) to treat mice by intra-venous injection and reducing tumorigenicity of the A549xenograft models [85] LPs formulation contained DOTMAa cationic lipid that confers a positive surface charge for cellu-lar uptake cholesterol to improve transfection efficiency andprotect from degradation and a short polyethyleneglycolmolecule linked to vitamin E to increase the stability andcirculation time of LPs in vivo Furthermore Cortez et aldeveloped a systemic delivery method using miR-200c-loaded liposomes (NOV340miR-200c SMARTICLES) toenhance radiosensitivity in a mouse model of lung cancerThey described mir-200c accumulation in tumor liver brainand lung after subcutaneous injection in mice InterestinglyNOV340miR-200c is a liposomal nanoparticle that containsa synthetic double-stranded mimic of the tumor suppressormiRNA miR-200c which is composed of amphoteric lipidsthat change their net charge depending on the pH of theenvironment such that it becomes cationic at low pH andanionic at higher pH to prevent unwanted interactions and toadhere only at tumor site [86]

4 Challenges for miRNA Therapy

The principal advantage of using miRNA as therapeuticsagents is that they could target several genes of redundantpathways involved in cancer development [87] For thisreason a small number ofmiRNAs could be used to achieve abroad silencing of protumoral pathways and they becomemore attractive than mixture of small interfering RNAsalready used for therapy In addition miRNA mutations areextremely rare due to the small size of the sequence andprobably a potential resistance to miRNA therapeutics wouldrequire multiple mutations in several genes Furthermoreseveral studies demonstrated that relatively small changes inmiRNAs and their corresponding targets expression couldinduce phenotypic alterations reinforcing the idea that cor-rection of a limited number of miRNA could revert themalignant phenotype [88] The challenges for the use ofmicroRNA in therapy that have to be addressed are tissuespecific delivery potential off-target effects and safety

In this review we described several strategies of miRNAsreplacementinhibition such as intratumoral injections andregulation of miRNAs expression by viral vectors whichrepresent delivery routes unlikely to be applied in a clinicalcontext Intratumoral injections could be used only for asmall number of easily accessible tumors Similarly modu-lation of miRNAs expression with viral vectors could poten-tially show the sameweaknesses encountered in gene therapysuch as limited infectivity and problems in the transcriptionof the gene product In addition cancer cells frequentlyshow deficiencies in the maturation of miRNA precursors soexpression with a viral vector is a less preferable approach

Thus the most promising approach for miRNAs ther-apeutics is the systemic delivery of miRNA mimics orinhibitors The main obstacle for the systemic delivery ofmiRNAs in lung cancer therapy is to determine their uptakein lung cancer cells this delivery has to overcome miRNAsdegradation by nucleases renal clearance failure to crossthe capillary endothelium ineffective endocytosis by targetcells and activation of the host immune responses [89ndash92]A major aspect to be considered is how to deliver the ther-apeutic molecules into the target cells without inducingunwanted responses in cells other than the intended onesFurthermore other potential side effect ofmiRNAs therapy isthat the same miRNA can affect hundreds of targets genes indifferent cell types and regulates different mechanismsTherefore miRNA modulation might have beneficial effectsin one cell type but harmful effects in another Furthercomplication is the finding thatmiRNAs generated from eachstrand of the same hairpin RNA structure termed 5p and 3pmay bind to differentmRNAs and display different behaviourFor example it has been reported that miR-125a-5p andmiR-125a-3p which are downregulated in NSCLC exhibitdistinct effects in vitro on the migration and invasion of lungcancer cells [93]This information has direct implications forthe design of miRNA gene therapy in fact when using aprecursor miRNA inserted into a viral vector and bothstrands are produced the function of each strand in the cellsmust be identified

5 Conclusion

The challenges for the future development of miRNA therapyas the improvement of stability delivery and control of off-target effects of miRNAs need to be addressed

We hope that some of the miRNAs strategies describedabove will be further developed to improve specificity andefficacy and finally will be used alone or in combination withchemotherapy for the treatment of lung cancer patients

Conflict of Interests

Gabriella Sozzi and Mattia Boeri are coinventors for twopatent applications regarding miRNA signature for lungcancer detection

Authorsrsquo Contribution

Orazio Fortunato Mattia Boeri and Carla Verri contributedequally to this work


Acknowledgments

This work was supported by grants from the Italian Associ-ation for Cancer Research (AIRC) IG research Grant nos10096 1227 11991 and 10068 and Special Program ldquoInnova-tive Tools for Cancer Risk Assessment and early Diagnosisrdquo5x1000 no 12162 and Italian Ministry of Health (RF-2010)Research funding was received from Gensignia Inc

References

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[39] A G Bader ldquoMiR-34 a microRNA replacement therapy isheaded to the clinicrdquo Frontiers in Genetics vol 3 article 1202012

[40] L He X He L P Lim et al ldquoA microRNA component of thep53 tumour suppressor networkrdquo Nature vol 447 no 7148 pp1130ndash1134 2007

[41] G T Bommer I Gerin Y Feng et al ldquop53-mediated activationof mirna34 candidate tumor-suppressor genesrdquo Current Biol-ogy vol 17 no 15 pp 1298ndash1307 2007

[42] M Garofalo Y-J Jeon G J Nuovo et al ldquoMiR-34ac-dependent PDGFR-120572120573 downregulation inhibits tumorigenesisand enhances TRAIL-induced apoptosis in lung cancerrdquo PLoSONE vol 8 no 6 Article ID e67581 2013

[43] A L Kasinski and F J Slack ldquoMiRNA-34 prevents cancer initi-ation and progression in a therapeutically resistant K-ras andp53-induced mouse model of lung adenocarcinomardquo CancerResearch vol 72 no 21 pp 5576ndash5587 2012

[44] S Volinia G A Calin C-G Liu et al ldquoAmicroRNA expressionsignature of human solid tumors defines cancer gene targetsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 103 no 7 pp 2257ndash2261 2006

[45] H Stenvold T Donnem S Andersen et al ldquoHigh tumor cellexpression of microRNA-21 in node positive non-small celllung cancer predicts a favorable clinical outcomerdquoBMCClinicalPathology vol 14 no 1 p 9 2014

[46] W Gao H Shen L Liu J Xu J Xu and Y Shu ldquoMiR-21 over-expression in human primary squamous cell lung carcinomais associated with poor patient prognosisrdquo Journal of CancerResearch and Clinical Oncology vol 137 no 4 pp 557ndash566 2011

[47] A Capodanno L Boldrini A Proietti et al ldquoLet-7g andmiR-21 expression in non-small cell lung cancer correlationwith clinicopathological and molecular featuresrdquo InternationalJournal of Oncology vol 43 no 3 pp 765ndash774 2013

[48] M Yang H Shen C Qiu et al ldquoHigh expression of miR-21 andmiR-155 predicts recurrence and unfavourable survival in non-small cell lung cancerrdquo European Journal of Cancer vol 49 no3 pp 604ndash615 2013

[49] Z-X Wang H-B Bian J-R Wang Z-X Cheng K-MWang andW De ldquoPrognostic significance of serummiRNA-21expression in human non-small cell lung cancerrdquo Journal ofSurgical Oncology vol 104 no 7 pp 847ndash851 2011

[50] I Bhatti A Lee V James et al ldquoKnockdown of microRNA-21 inhibits proliferation and increases cell death by targetingprogrammed cell death 4 (PDCD4) in pancreatic ductal ade-nocarcinomardquo Journal of Gastrointestinal Surgery vol 15 no 1pp 199ndash208 2011

[51] I A Asangani S A K Rasheed D A Nikolova et al ldquoMicro-RNA-21 (miR-21) post-transcriptionally downregulates tumorsuppressor Pdcd4 and stimulates invasion intravasation andmetastasis in colorectal cancerrdquo Oncogene vol 27 no 15 pp2128ndash2136 2008

[52] J-G Zhang J-J Wang F Zhao Q Liu K Jiang and G-HYang ldquoMicroRNA-21 (miR-21) represses tumor suppressorPTENand promotes growth and invasion in non-small cell lungcancer (NSCLC)rdquo Clinica Chimica Acta vol 411 no 11-12 pp846ndash852 2010

[53] S Zhu H Wu F Wu D Nie S Sheng and Y-Y MoldquoMicroRNA-21 targets tumor suppressor genes in invasion andmetastasisrdquo Cell Research vol 18 no 3 pp 350ndash359 2008

[54] B Li S Ren X Li et al ldquoMiR-21 overexpression is associatedwith acquired resistance of EGFR-TKI in non-small cell lungcancerrdquo Lung Cancer vol 83 no 2 pp 146ndash153 2014

[55] M E Hatley D M Patrick M R Garcia et al ldquoModulation ofK-Ras-dependent lung tumorigenesis by MicroRNA-21rdquo Can-cer Cell vol 18 no 3 pp 282ndash293 2010

[56] Y Hayashita H Osada Y Tatematsu et al ldquoA polycistronicMicroRNA cluster miR-17-92 is overexpressed in human lungcancers and enhances cell proliferationrdquo Cancer Research vol65 no 21 pp 9628ndash9632 2005

[57] H Y Jin H Oda M Lai et al ldquoMicroRNA-17sim92 plays a cau-sative role in lymphomagenesis by coordinating multiple onco-genic pathwaysrdquo EMBO Journal vol 32 no 17 pp 2377ndash23912013

[58] Q Wu Z Yang F Wang et al ldquoMiR-19b20a92a regulatesthe self-renewal and proliferation of gastric cancer stem cellsrdquoJournal of Cell Science vol 126 part 18 pp 4220ndash4229 2013

[59] B L Murphy S Obad L Bihannic et al ldquoSilencing of the miR-17sim92 cluster family inhibits medulloblastoma progressionrdquoCancer Research vol 73 no 23 pp 7068ndash7078 2013

[60] K A OrsquoDonnell E AWentzel K I Zeller C V Dang and J TMendell ldquoc-Myc-regulated microRNAs modulate E2F1 expres-sionrdquo Nature vol 435 no 7043 pp 839ndash843 2005

[61] C Xiao L Srinivasan D P Calado et al ldquoLymphoproliferativedisease and autoimmunity in mice with increased miR-17-92expression in lymphocytesrdquo Nature Immunology vol 9 no 4pp 405ndash414 2008

[62] A Ventura A G Young M M Winslow et al ldquoTargeteddeletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusterrdquo Cell vol 132 no 5 pp875ndash886 2008

[63] H Matsubara T Takeuchi E Nishikawa et al ldquoApoptosisinduction by antisense oligonucleotides against miR-17-5p andmiR-20a in lung cancers overexpressing miR-17-92rdquo Oncogenevol 26 no 41 pp 6099ndash6105 2007

[64] F Chen S K HouH J Fan and Y F Liu ldquoMiR-15a-16 repressesCripto and inhibits NSCLC cell progressionrdquo Molecular andCellular Biochemistry vol 39 no 1-2 pp 11ndash19 2014

[65] Y Ke W Zhao J Xiong and R Cao ldquoDownregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cellsrdquo FEBS Letters vol 587 no 18 pp3153ndash3157 2013


[66] N Bandi S ZbindenM Gugger et al ldquomiR-15a andmiR-16 areimplicated in cell cycle regulation in a Rb-dependent mannerand are frequently deleted or down-regulated in non-small celllung cancerrdquo Cancer Research vol 69 no 13 pp 5553ndash55592009

[67] N Bandi and E Vassella ldquoMiR-34a and miR-15a16 are co-regulated in non-small cell lung cancer and control cell cycleprogression in a synergistic and Rb-dependent mannerrdquoMolec-ular Cancer vol 10 article 55 2011

[68] P Ceppi G Mudduluru R Kumarswamy et al ldquoLoss of miR-200c expression induces an aggressive invasive and chemore-sistant phenotype in non-small cell lung cancerrdquo MolecularCancer Research vol 8 no 9 pp 1207ndash1216 2010

[69] Y Yang Y H Ahn Y Chen et al ldquoZEB1 sensitizes lung ade-nocarcinoma to metastasis suppression by PI3K antagonismrdquoJournal of Clinical Investigation vol 124 no 6 pp 2696ndash27082014

[70] Y Takeyama M Sato M Horio et al ldquoKnockdown of ZEB1 amaster epithelial-to-mesenchymal transition (EMT) gene sup-presses anchorage-independent cell growth of lung cancercellsrdquo Cancer Letters vol 296 no 2 pp 216ndash224 2010

[71] D L Gibbons W Lin C J Creighton et al ldquoContextualextracellular cues promote tumor cell EMT and metastasis byregulatingmiR-200 family expressionrdquoGenes andDevelopmentvol 23 no 18 pp 2140ndash2151 2009

[72] M Pacurari J B Addison N Bondalapati et al ldquoThe micro-RNA-200 family targets multiple non-small cell lung cancerprognostic markers in H1299 cells and BEAS-2B cellsrdquo Interna-tional Journal of Oncology vol 43 no 2 pp 548ndash560 2013

[73] R Tejero A Navarro M Campayo et al ldquomiR-141 and miR-200c as markers of overall survival in early stage non-small celllung cancer adenocarcinomardquo PLoS ONE vol 9 no 7 ArticleID e101899 2014

[74] M Fabbri R Garzon A Cimmino et al ldquoMicroRNA-29 familyreverts aberrant methylation in lung cancer by targeting DNAmethyltransferases 3A and 3Brdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no40 pp 15805ndash15810 2007

[75] M Garofalo C Quintavalle G di Leva et al ldquoMicroRNAsignatures of TRAIL resistance in human non-small cell lungcancerrdquo Oncogene vol 27 no 27 pp 3845ndash3855 2008

[76] M Garofalo G Di Leva G Romano et al ldquomiR-221amp222regulate TRAIL resistance and enhance tumorigenicity throughPTEN and TIMP3 downregulationrdquo Cancer Cell vol 16 no 6pp 498ndash509 2009

[77] M Garofalo G Romano G Di Leva et al ldquoEGFR and METreceptor tyrosine kinase-altered microRNA expression inducestumorigenesis and gefitinib resistance in lung cancersrdquo NatureMedicine vol 18 no 1 pp 74ndash82 2012

[78] B Hu X Ying J Wang et al ldquoIdentification of a tumor-sup-pressive human-specific microRNA within the FHIT tumor-suppressor generdquoCancer Research vol 74 no 8 pp 2283ndash22942014

[79] P Trang P P Medina J F Wiggins et al ldquoRegression of murinelung tumors by the let-7 microRNArdquo Oncogene vol 29 no 11pp 1580ndash1587 2010

[80] J FWiggins L Ruffino K Kelnar et al ldquoDevelopment of a lungcancer therapeutic based on the tumor suppressor microRNA-34rdquo Cancer Research vol 70 no 14 pp 5923ndash5930 2010

[81] P Trang J F Wiggins C L Daige et al ldquoSystemic delivery oftumor suppressor microRNA mimics using a neutral lipid

emulsion inhibits lung tumors in micerdquoMolecularTherapy vol19 no 6 pp 1116ndash1122 2011

[82] C N Landen Jr A Chavez-Reyes C Bucana et al ldquoTherapeu-tic EphA2 gene targeting in vivo using neutral liposomal smallinterfering RNA deliveryrdquo Cancer Research vol 65 no 15 pp6910ndash6918 2005

[83] G-Y Chiou J-Y Cherng H-S Hsu et al ldquoCationic poly-urethanes-short branch PEI-mediated delivery of Mir145inhibited epithelial-mesenchymal transdifferentiation and can-cer stem-like properties and in lung adenocarcinomardquo Journalof Controlled Release vol 159 no 2 pp 240ndash250 2012

[84] Y Shi C Liu X Liu D G Tang and J Wang ldquoThe microRNAmiR-34a inhibits non-small cell lung cancer (NSCLC) growthand the CD44hi stem-like NSCLC cellsrdquo PLoS ONE vol 9 no3 Article ID e90022 2014

[85] Y Wu M Crawford Y Mao et al ldquoTherapeutic delivery ofMicroRNA-29b by cationic lipoplexes for lung cancerrdquo Molec-ular TherapymdashNucleic Acids vol 2 article e84 2013

[86] M A Cortez D Valdecanas X Zhang et al ldquoTherapeuticdelivery of miR-200c enhances radiosensitivity in lung cancerrdquoMolecular Therapy vol 22 no 8 pp 1494ndash1503 2014

[87] A Esquela-Kerscher and F J Slack ldquoOncomirs microRNAswith a role in cancerrdquo Nature Reviews Cancer vol 6 no 4 pp259ndash269 2006

[88] E van Rooij and E N Olson ldquoMicroRNAs powerful newregulators of heart disease and provocative therapeutic targetsrdquoJournal of Clinical Investigation vol 117 no 9 pp 2369ndash23762007

[89] DH Kim and J J Rossi ldquoStrategies for silencing human diseaseusing RNA interferencerdquo Nature Reviews Genetics vol 8 no 3pp 173ndash184 2007

[90] C V Pecot G A Calin R L Coleman G Lopez-Berestein andA K Sood ldquoRNA interference in the clinic challenges andfuture directionsrdquo Nature Reviews Cancer vol 11 no 1 pp 59ndash67 2011

[91] J A Broderick and P D Zamore ldquoMicroRNA therapeuticsrdquoGene Therapy vol 18 no 12 pp 1104ndash1110 2011

[92] S I Pai Y Y Lin B Macaes A Meneshian C F Hung andT C Wu ldquoProspects of RNA interference therapy for cancerrdquoGene Therapy vol 13 no 6 pp 464ndash477 2006

[93] L Jiang Q Huang S Zhang et al ldquoHsa-miR-125a-3p and hsa-miR-125a-5p are downregulated in non-small cell lung cancerand have inverse effects on invasion and migration of lungcancer cellsrdquo BMC Cancer vol 10 article 318 2010

[94] J Krutzfeldt N Rajewsky R Braich et al ldquoSilencing of microR-NAs in vivo with ldquoantagomirsrdquordquo Nature vol 438 no 7068 pp685ndash689 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Principal miRNAs involved in lung carcinogenesis

miRNAs Expression inlung cancer Cellular processes affected and targets Reference

Let-7 family Decreased(i) Cell proliferation (KRAS MYC and HMGA2) [28ndash30](ii) miRNA maturation Dicer mediated [31](iii) Cell-cycle regulation (CDC25A CDK6 and cyclin D2) [32]

mir-34 family Decreased (i) Transcriptionally activated by p53 [40](ii) TRAIL-induced cell death and cell proliferation (BCL-2 METand PDGFR-120572120573) [41ndash43]

mir-21 Increased(i) Apoptosis cellular proliferation and migration (TPM1 PDCD4and PTEN) [50ndash53]

(ii) TKI-treatment resistance [54]

mir-1792a cluster Increased (i) Transcriptionally regulated by c-MYC [60](ii) Cellular proliferation and cancer development (PTEN HIF-1aCL2L11 CDKNA and TSP-1) [57 61ndash63]

mir-15a16 cluster Decreased Cell cycle regulation (cyclin D1 D2 and E1) [66 67]

mir-200 family Decreased Promotion of EMT and metastasization(ZEB transcription factorsCDH-1 and vimentin) [68ndash73]

miRNA-29 family Decreased Epigenetic regulation of gene expression(DNMT-3A and DNMT-3B) [74]

mir-221mir-222 Increased (i) TRAIL resistance and cellular migration (PTEN and TIMP3) [75 76](ii) Transcriptionally regulated by EGFR and MET and gefitinibresistance (BIM and APAF1) [77]

mir-548 Decreased Tumor cell growth(CCND ERBB2 DMNT3A and DNMT3B) [78]

human miRNAs were found in the genome and a consider-able number of them were found deregulated in cancer cellscompared to normal cells [11ndash14]

The rationale of miRNA therapy in lung cancer is basedon two criteria one is that miRNAs play an essential role inlung development [15] and their expression levels are deregu-lated in lung cancer compared to normal tissues [16] and alsoin the blood of patients [17] and the second one is that severalstudies demonstrated that modulation of miRNA expressionboth in vitro and in vivo can modify the cancer phenotype[18ndash22]

Different strategies of miRNAs therapeutics can be envis-aged according to the expression status of miRNAs in thetumor (i) miRNAs inhibition when it is overexpressed usingantagomir which are synthetic miRNAs complementary tothe miRNAs of interest or miRNAs sponges that competewith the targets of the miRNA and (ii) miRNAs replacementusing oligonucleotide mimics or viral vectors when themiRNAs is downmodulated [23]

In this review the role of those miRNAs which regulatefundamental proteins and pathways involved in lung tumori-genesis will be discussed Furthermore we will describe gen-eral therapeutic strategies involving inhibition of oncogenicmiRNAs and replacement of tumor suppressor miRNAsincluding recent experimental methods developed to bypassblood clearance and poor intracellular uptake

2 miRNA Role in Lung Carcinogenesis

Human cancers exhibit an altered expression of miRNAswith oncogenic or tumor-suppressive activity providing an

explanation of their role in tumor development and pro-gression Recent studies reported an aberrant expression ofmiRNAs in lung tumor tissues when compared to the corre-sponding normal lung tissues supporting miRNAs involve-ment in lung carcinogenesis (Table 1) Lethal-7 (let-7) isone of the earliest identified tumor suppressormiRNA in lungcancer and its downmodulation is associated with poorprognosis [16 24ndash27] Furthermore let-7 can regulate severaloncogenic pathways for example this miRNA negativelymodulates multiple cell-cycle oncogenes as KRAS mutationof which are commonly implicated in lung adenocarcinomas[28] MYC and HMGA2 [29 30] Furthermore an involmentof let-7 in the regulation ofmiRNAmaturation process-Dicermediated has also been described [31] Let-7 overexpressionin lung adenocarcinoma A549 cell line inhibits in vitro assayscell growth and reduces cell-cycle progression by targetingCDC25A CDK6 and cyclin D2 [32] Accordingly in vivoexperiments show that let-7b inhibits lung cancer cell xeno-grafts in immunodeficient mice [33] In addition the thera-peutic potential of let-7 in lung cancer treatment is supportedalso by experimental evidence that altered expression of let-7family members associated with radiation resistance [34]

Mir-34 is a tumor suppressor miRNA largely investigatedin cancers and its deregulation has been reported in varioustumor types including lung cancer [35ndash39]Mir-34members(a b and c) are regulated by p53 and mir-34 expression isfrequently reduced in p53 mutant tumors [40] mir-34 familymembers are involved in cell-cycle control apoptosis andcellular senescence through specific targeting of BCL-2MYC andMETgenes [41] In additionGarofalo et al showed





















[79 80 84 86]




[81 83]




[33 43]

















5 Conclusion








Acknowledgments


References









































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















[79 80 84 86]




[81 83]




[33 43]

















5 Conclusion








Acknowledgments


References









































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusion








Acknowledgments


References









































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References









































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article therapeutic use of micrornas in lung...

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