Potential Use of RNA Interference in Cancer Therapy...Potential use of RNA interference in cancer...

Potential use of RNA interferencein cancer therapy

Connor Phalon1, Donald D. Rao1 and John Nemunaitis1,2,3,4,*

RNA interference (RNAi) is an evolutionary conserved mechanism for specificgene silencing. This mechanism has great potential for use in targeted cancertherapy. Understanding the RNAi mechanism has led to the development ofseveral novel RNAi-based therapeutic approaches currently in the earlyphases of clinical trials. It remains difficult to effectively deliver the nucleicacids required in vivo to initiate RNAi, and intense effort is under wayin developing effective and targeted systemic delivery systems for RNAi.Description of in vivo delivery systems is not the focus of this review. In thisreview, we cover the rationale for pursuing personalised cancer therapy withRNAi, briefly review the mechanism of each major RNAi therapeutic technique,summarise and sample recent results with animal models applying RNAifor cancer, and provide an update on current clinical trials with RNAi-basedtherapeutic agents for cancer therapy. RNAi-based cancer therapy is still inits infancy, and there are numerous obstacles and issues that need to beresolved before its application in personalised therapy focusing on patient-cancer-specific targets can become standard cancer treatment, either aloneor in combination with other treatments.

Recent advances in understanding tumoursurvival signalling pathways have supported amigration of cancer treatment from untargetedcytotoxic therapy to selectively targetedtherapeutics. Targeted therapeutics directedagainst specific molecules in malignant cellshave been approved by the US Food and DrugAdministration (FDA) and enable initial

attempts at personalised cancer treatment. Byanalysing genetic abnormalities of a patient’stumour, unusual gene and protein expressioncan be determined and used to guide selectionfor specific targets.

RNA interference (RNAi) is a sequence-specific,naturally occurring mechanism for gene silencinginduced by double-stranded (ds) RNA that was

1Gradalis, Inc., Dallas, TX, USA.2Mary Crowley Cancer Research Centers, Dallas, TX, USA.3Texas Oncology PA, Dallas, TX, USA.4Baylor Sammons Cancer Center, Dallas, TX, USA.

*Correspondingauthor: JohnNemunaitis, 1700PacificAve, Suite 1100,Dallas, TX75201,USA.E-mail:[email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1Accession information: doi:10.1017/S1462399410001584; Vol. 12; e26; August 2010

©Cambridge University Press 2010

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first discovered in the nematode Caenorhabditiselegans – work for which Andrew Fire and CraigMello were awarded the 2006 Nobel prize forphysiology or medicine (Ref. 1). The pathway ofRNAi has been found in cells of nearly everymulticellular organism and might have evolvedas a defence against viral dsRNA (Ref. 2).Target-specific RNAi can knockdown a genewith high specificity and selectivity, therebyproviding an important tool for personalisedcancer therapy, as demonstrated in numerousanimal xenograft studies (Table 1). However,many issues need to be resolved for RNAitreatments to translate from the laboratory tothe clinic, including an effective targeteddelivery system and a more comprehensiveunderstanding of possible off-target effects(Ref. 3). RNAi-based approaches are reviewedhere as part of a new division being establishedfor cancer management.

Personalised cancer gene therapyHuman tumours show profiles of gene expressionthat are different not only from normal tissue, butalso from each other. This can explain the differingresponses to treatment (Ref. 4). Part of thedifferential gene expression involves alteredlevels of microRNA (miRNA) in malignant cells(Ref. 5). miRNAs are endogenous, noncodingRNA molecules that play a key role inregulating the gene expression pattern of avariety of developmental and physiologicalprocesses (Ref. 6). Classifying tumours by theirmiRNA levels provides further information onthe aberrant gene expression pattern ofindividual tumours (Ref. 5). Recent studies havedemonstrated that the pattern of geneexpression can be classified into categories forclinical outcome prediction and for treatmentresponse (Ref. 7).In our evaluation, targets for therapy should be

based on the degree of connectivity of dynamicgenomic–proteomic nodes in a patient-data-based network model, rather than targeting themore highly active metabolic pathways inrapidly proliferating cancer cells in accord withtraditional chemotherapy principles (Ref. 8).Highly connected targets are more vulnerable toattack because of the impact on the entirenetwork (Ref. 9). The disordered cancer circuitryin malignant tissue can cause the cells tobecome highly dependent on a specific rewiredpathway. The disruption of pathways that

produce robustness to certain attacks often alsocauses increased fragility to other perturbations(Ref. 10). Therefore, in theory, knockdown of arewired oncogenic tumour-specific pathwayshould lead to cell death in malignant tissueswithout significantly disturbing normal cellfunctionality (Ref. 3).

Each patient has a genetically different tumour,with a distinct rewiring of protein pathways. Inorder to find the optimal targets, malignant andnonmalignant tissue can be analysed in thecontext of global protein-interaction networks,providing information to create a prioritised list ofpotential gene and protein targets for each patient(Ref. 11). For example, we recently identified twoscaffold proteins [RACK1 (GNB2L1) and stathmin1 (STMN1)] as uniquely elevated withinmalignant tissue in comparison with matchedsame-organ nonmalignant tissue, and highlyconnected with several procancer gene signals(Ref. 11). We constructed and verified activity of aunique bifunctional short hairpin RNA (bi-shRNA) targeting stathmin 1 (Ref. 12) and arecurrently processing the Investigational New Drug(IND) application to treat patients demonstratingelevated stathmin 1 in their malignant tissue.RNAi therapy of patients demonstrating targetsignal upregulation is also under way in severalclinical trials around the world (Table 2).

Mechanism of RNAiSingle-stranded antisense RNA was initiallyshown to cause interference as a result ofhybridisation of the antisense and the mRNAtarget site (Ref. 13). However, it was alsoobserved that treatment with the sense strandcaused a similar reduction of gene function(Ref. 14). It was then demonstrated that dsRNAcaused much more interference than each strandindividually. Only a few molecules of dsRNAwere required to achieve this effect, whichindicated hybridisation was not the onlymechanism of gene knockdown (Ref. 1).

RNAi can be modelled by both cleavage-dependent and cleavage-independentmechanisms (Fig. 1). It can be induced by smallinterfering RNA (siRNA; dsRNA of 21/22nucleotides with a two-nucleotide overhang onthe 3′ end), microRNA (miRNA; endogenouslyproduced noncoding RNA of approximately 22nucleotides in length), or short hairpin RNA(shRNA; a DNA plasmid encoding short RNAwith a stem–loop structure).




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siRNA mechanism: cleavage-dependentRNAiThe mechanism of RNAi begins with the cleavageof long dsRNA molecules into siRNAs by theRNAse III enzyme Dicer (DICER1) in the

cytoplasm. Dicer protein contains an N-terminalhelicase domain, an RNA-binding domain calledPiwi/Argonaute/Zwille (PAZ), two RNAse IIIdomains, and a dsRNA-binding domain(Ref. 15). The PAZ domain of Dicer specifically

RNA interference (RNAi) pathwaysExpert Reviews in Molecular Medicine © Cambridge University Press 2010

LiposomeCell surface

Nucleus

Cytoplasm

Plasmids encodingshRNAs

TranscriptionProcessing

Loading onto RISCUnwinding and departureof passenger strand

Imperfect

complementarity

Perfect

complementarityRibosome

AAA AAA

pre-shRNA

Exportin 5

Guide strand

RISC

Target mRNATarget mRNA

Cleavage

Cleavage-independentmechanism:

translational block

Cleavage-dependentmechanism

Dicer

Figure 1. RNA interference (RNAi) pathways. (See next page for legend.)




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recognises andbinds to the 5′ phosphate of dsRNAwith a two-nucleotide 3′ overhang (Ref. 16). Thetwo RNAse III domains each cut a single strandof the dsRNA and their alignment results in thetwo-nucleotide overhang. The 21/22-nucleotidesiRNA product length is due to the distancebetween the PAZ binding domain and the activesites of the RNAse III domains (Ref. 17). Dicerinteracts with the TAR RNA-binding protein(TRBP/TARBP2P) or protein kinase R (PKR)-activating protein (PACT) to mediate siRNAproduction. TRBP and PACT are structurallyrelated but provide opposing regulation ofRNA-dependent protein kinase (PKR) (Ref. 18).The complex of ds siRNA, Dicer and TRBP/

PACT is then loaded into the RNA-inducedsilencing complex (RISC) (Ref. 19). Perfectlyprocessed siRNA (most exogenously appliedsiRNA) can be loaded onto RISC without theTRBP/PACT–Dicer complex, but every otherRNAi source must be processed for loading(Ref. 3). The siRNA–TRBP/PACT–Dicer complexrecruits Argonaute 2 (Ago2/EIF2C2) throughTRBP/PACT to form the RISC loading complex(RLC) (Ref. 20). The RLC initiates siRNAunwinding and determines which strand isassembled into the RISC by preferentiallybinding the strand with lower thermodynamicstability at the 5′ end, which becomes the guidestrand (Ref. 21). The passenger strand is cleavedby Ago2 and departs the complex, facilitatingassembly of RISC (Ref. 22). RISC mediatessequence-specific binding of the guide strandRNA to the corresponding target mRNA mainlywith the 5′ seed region (nucleotides 2–8) of thesiRNA (Ref. 23). The mRNA is then cleaved byAgo2 at the centre of the oligonucleotide

duplexed with it, concluding the cleavage-dependent RNAi mechanism (Refs 24, 25). RISCremains bound to the single-stranded siRNAand can execute several rounds of cleavage(Ref. 26). The protein Tudor-SN (SND1) is also apart of RISC and degrades RNA and DNAnonspecifically, suggesting it degrades themRNA after cleavage (Ref. 27).

miRNA mechanismThemiRNA family of RNA is transcribed by RNApolymerase II (pol II) into a capped andpolyadenylated primary transcript (pri-miRNA). The pri-miRNA transcript is processedinto a stem–loop configuration of 60–110nucleotides, known as pre-miRNA, in thenucleus by a ‘microprocessor’ complex,containing the nuclear RNase III enzyme Drosha(RNASEN) and its cofactor DGCR8 (a ds-RNA-binding domain protein) (Ref. 28). Drosha alsogenerates a two-nucleotide 3′ overhang that canbe recognised by Dicer for further processing inthe cytoplasm (Ref. 25). This pre-miRNA isexported to the cytoplasm by exportin 5 (XPO5)(Ref. 28). Dicer cleaves the pre-miRNA to formmature miRNA and integrates the miRNAduplex with argonaute-containing RISC, muchas it does for siRNA (Ref. 29). In addition toAgo2, the argonaute proteins Ago1, Ago3 andAgo4, which do not have endonuclease activity,can also be assembled into RISC for cleavage-independent interference (Ref. 30).

mRNA target recognition by miRNA usuallyrequires a perfect nucleotide match in the seedsequence of the miRNA (Ref. 31). If the miRNAis perfectly complementary to the mRNA, Ago2cleavage of the mRNA will occur just as it does

Figure 1. RNA interference (RNAi) pathways. (See previous page for figure.) Cleavage-dependent and-independent RNAi pathways are illustrated here with reference to bifunctional shRNA. The shRNAexpression vector is shown entering the cell cytoplasm via a liposome delivery system, and enters thenucleus for expression. After nuclear entry, shRNA expression vectors are transcribed into primarytranscripts by host RNA polymerases, and the transcripts are then processed by a microprocessor complexin the nucleus into a stem–loop structure and transported into the cytoplasm. In the cytoplasm, thestem–loop structure is further processed into mature shRNAs by Dicer in the RISC-loading complex,resulting ultimately in a single guide strand loaded onto the RISC for activity. In the bifunctional shRNAapproach, two stem–loop RNA molecules are encoded, with the aim of increasing the efficiency of geneknockdown through efficient loading onto multiple types of RISC loading complexes: one has perfectcomplementarity for the target sequence, which results in cleavage of the target mRNA; the other hasimperfect complementarity, which results in knockdown via translational repression. By contrast, siRNA andconventional shRNA rely on cleavage-dependent RISC for activity, whereas cleavage-independent RNAi isthe predominant mechanism undertaken by miRNA. Note that RNAi via shRNA and vector based miRNA-mimics requires expression from engineered constructs in the nucleus, whereas siRNAs or oligonucleotide-based miRNA mimics or antagomirs remain in the cytoplasm seeking entry into the RISC.




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for siRNA (Ref. 32). However,mRNA that binds tomiRNA with imperfect complementarityexperiences translational blockage due to theformation of a bulge sequence in the middle ofthe A-form helix, making it unsuitable forcleavage. This cleavage-independent mechanismof RNAi is the predominant mechanismundertaken by miRNA (Ref. 33). As a result ofthe ability to affect translation of mRNAs withimperfect complementarity, a single miRNA canact on several mRNA targets and mRNAs canbe the target of multiple miRNAs (Ref. 31). Upto a third of protein-coding mRNAs aresusceptible to regulation by miRNAs (Ref. 34).

shRNA mechanismshRNAs are synthesised in the nucleus of cells,further processed and transported to thecytoplasm, and incorporated with the RISC forsilencing activity (Ref. 35) The processing ofshRNA is presumed to be very similar to thematuration pathway of miRNA, so studies onmiRNA have provided the foundation of ourunderstanding of shRNA synthesis (Ref. 36).shRNA is transcribed by either RNA pol II or IIIthrough promoters on the expression cassette.RNA pol II creates a transcript with a hairpin-like stem–loop structure that is processed in thenucleus by the microprocessor complex(Ref. 37). This creates pre-shRNAs containing atwo-nucleotide 3′ overhang, which aretransported to the cytoplasm by exportin 5,much like pre-miRNA. The loop of the hairpinis removed by the complex containing Dicer andTRBP/PACT to form ds siRNA with a two-nucleotide 3′ overhang, which is loaded ontoRISC as described earlier (Ref. 38) Pre-shRNAhas been found to be a part of the RLC, so it ispossible that pre-shRNA directly associates withthe RLC by a different Dicer–TRBP/PACTcomplex (Ref. 39). After loading onto RLC, andpassenger-strand departure, shRNA shouldbehave the same as siRNA or miRNA, causingcleavage of mRNA or translational blockage.

Bifunctional shRNA mechanismA bifunctional shRNA is designed to induce boththe cleavage-dependent and cleavage-independent mechanisms of RNAi (Ref. 12). Itconsists of two stem–loop shRNA structures:one of fully matched passenger and guidestrand to incorporate with the cleavage-dependent RISC, and one composed of

mismatched strands for incorporation with thecleavage-independent RISC. This design shouldincrease efficacy as a result of the uniqueloading into the RISC complex. A higher level ofefficacy (greater knockdown at equivalentdosing) and altered kinetics in a head to headcomparison with siRNA to the same target hasin fact been observed (Refs 3, 12).

Delivery methodsOne of the biggest obstacles for RNAi use in vivo isachieving efficient delivery of the dsRNA requiredto induce RNAi. Delivering shRNA has more incommon with traditional gene therapy thanwith the delivery of siRNA or antisenseoligonucleotides because the nucleic acid mustbe delivered to the nucleus (Ref. 40) Therefore,previous research on delivering plasmid DNAcan be adapted for the delivery of shRNA, andantisense delivery technology can be modifiedfor the delivery of siRNA (Ref. 41).

Numerous methods of delivery are beingused; natural and synthetic polymers, lipidcomplexes, and viral vectors are the mostcommon (Ref. 42). In addition, effort is beingmade to create targeting systems so the nucleicacid is delivered only to tumour cells. Deliveryvehicles can be complexed with monoclonalanitbodies, peptides, small-molecule ligands, oraptamers to recognise markers on the cellsurface (Ref. 43).

In vivo use of RNAiRNAi has become a standard procedure for geneknockdown in cells, which can be used todetermine gene function by inhibiting a specificgene and analysing the phenotypic change. Thisknowledge of gene function is being used tocharacterise and identify signalling pathways fortumourigenesis and to evaluate possible targetsfor drugs or future RNAi therapy (Ref. 44).There have been numerous in vivo studies forcancer treatment using RNAi targeting aplethora of genes, and some of them aresummarised here (Table 1). Moreover, systemicadministration of cancer-relevant miRNAs orantagomirs (antagonists of miRNA) mayprovide opportunity for further novel cancertherapy investigation.

p21-activated kinase 6Malignant prostate tissue overexpresses p21-activated kinase 6 (encoded by PAK6). The effect




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Table1.

Exa

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Intratum

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Cytok

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poptotic

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aSMMC77

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opha

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Pretrea

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pede

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47

VEGFC

Colorec

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pretreated

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grow

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Osteo

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BGC-823

cell

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shRNA

Stableex

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Sup

pres

sedtumou

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49

MYC

Colon

HT-29

cell

xeno

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Pretrea

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Table1.

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ntinue

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Target

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tinProstate

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miR-221

and

miR-222

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Red

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STA

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miR-26a

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of PAK6 inhibition with siRNAwas investigated invivo in combination with the chemotherapy drugdocetaxel. The siRNA was deliveredintratumourally to nude mice with prostatetumour xenografts. After six weeks, the controlgroup’s average tumour size was 475 mm3; micetreated with docetaxel alone had an averagetumour size of 210 mm3; the group treated withsiRNA alone averaged 68 mm3; and thecombination treatment led to an average tumoursize of 47 mm3. The PAK6-siRNA inhibited cellgrowth of prostate cancer cells by causing cellcycle arrest at the G2–M phase. Tumour growthwas inhibited by both PAK6-siRNA anddocetaxel alone, yet groups treated with bothPAK6-siRNA and docetaxel showed a largerdecrease in tumour volume than eithertreatment alone (Ref. 45).

Cytokine-induced antiapoptotic moleculeHepatocellular carcinoma (HCC) overexpressescytokine-induced antiapoptotic molecule(CIAPIN1). CIAPIN1 knockdown in HCC cells invivo by adenovirus-mediated siRNA(AdsiCIAPIN1) delivery inhibited cell growth byblocking entry into the S phase of the cell cycle.Nude mice with HCC xenografts treated withAdsiCIAPIN1 injected intratumourally hadtumours that were more than sixfold smallerthan the control group after eight weeks. Thetreatment was also shown to induce apoptoticcell death in the tumours (Ref. 46).

β-Cateninβ-Catenin (CTNNB1) is overexpressed in manyforms of cancer, especially oesophageal cancer.A plasmid with U6-promoter-driven expressionof shRNA was used to induce RNAi to silenceβ-catenin expression in oesophageal squamouscell carcinoma (ESCC) cells. Nude micexenografted with ESCC cells showed an averagetumour size of 909.3 mm3 when treated with theβ-catenin-silencing plasmid, compared with2684.4 mm3 in mice treated with a differentplasmid, and 2722.6 mm3 in untreated mice. β-Catenin silencing showed growth inhibition byG0–G1 cell cycle arrest but did not induceapoptosis (Ref. 47).

Vascular endothelial growth factorThe vascular endothelial growth factor (VEGF)family of growth factors is known to induceangiogenesis, and members of this family are

required for cancer progression and metastasis.A DNA plasmid expressing siRNA for vascularendothelial growth factor C (VEGFC) wascomplexed with CaCO3 nanoparticles and usedto treat colon carcinoma cells in vivo. Micexenografted with colon carcinoma cells weretreated with the CaCO3–DNA complex by directinjection into the tumour mass and showed anaverage tumour size of nearly half the untreatedor saline-treated tumours. The treatment alsosignificantly inhibited lymph-node metastasisand lymphangiogenesis (Ref. 48).

OsteopontinOsteopontin (OPN) is overexpressed in a majorityof gastric cancers and is associated withpathogenesis. Transfection of a vector expressingsiRNA for OPN into human gastric cancer cellssignificantly inhibited growth and invasivenessof those cells in vitro. It also decreasedexpression of several other proteins that areimportant in breast and melanoma cellmigration, growth and invasion. Nude micewith gastric cancer xenografts that receivedintratumoural injections of a poly(ethyleneimine) (PEI)–OPN vector complex had anaverage tumour size that was about a third ofuntreated tumours. Half of the treated micesurvived until the end of the study (60 days),whereas no untreated mice survived to day 40(Ref. 49).

MYCThe MYC proto-oncogene family is thought tocontain central regulators of cell growth, andderegulation of expression is associated withseveral types of cancer. Elevated expression ofMYC is commonly seen in colon cancer. Nudemice with colon cancer xenograft tumours thatreceived intratumoural injections of siRNAtargeting MYC showed a 40% reduction in MYCexpression and experienced reduced rates oftumour growth, exhibiting an average tumoursize that was half of that observed in untreatedmice. The treated mice had large areas ofcytonecrosis that inhibited tumour cellproliferation (Ref. 50).

Eukaryotic initiation factor 4EEukaryotic initiation factor 4E (EIF4E) plays animportant role in protein translation, which isupregulated in breast cancer. A vector using thesurvivin promoter was created to provide




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shRNA-mediated knockdown of EIF4E. Humanbreast carcinoma cells transfected with thisvector showed a reduction in EIF4E mRNA andprotein expression, as well as downregulation ofexpression of VEGF, basic fibroblast growthfactor (FGF2) and cyclin D1 (CCND1), which areall proteins associated with the progression ofcancer. Nude mice with breast cancer xenograftstreated with the vector showed significantlysmaller tumours, averaging 233.5 mm3

compared with 397.7 mm3 for mice treated witha control plasmid. Mice with treated xenograftsalso showed enhanced chemosensitivity tocisplatin, seen by decreased tumour size in micetreated with cisplatin and the shRNA plasmid,which was 134.5 mm3, compared with208.9 mm3 in mice treated with cisplatin and acontrol plasmid (Ref. 51).

Epithelial cellular adhesion moleculeEpithelial cellular adhesion molecule (EPCAM) isoverexpressed in gastric cancer, with increasedlymph-node metastasis in patients with higherlevels of EPCAM expression. Nude micereceived xenografts from two gastric cancer celllines (AGS and SGC7901) for in vivo study. Thetumour volume for both types of xenograft wasabout half the size in mice treated with EPCAM-siRNA vectors delivered by Lipofectamine 2000compared with untreated mice. EPCAM-siRNAsignificantly reduced the expression of cyclinD1, causing cell cycle arrest at the G1 phase(Ref. 52).

Urokinase-type plasminogen activatorUrokinase-type plasminogen activator receptor(u-PAR; PLAUR) is overexpressed in many typesof malignant tumours. A retroviral vectorexpressing siRNA for PLAUR was injectedintratumourally into nude mice with oralsquamous cell carcinoma xenografts; 30 daysafter treatment, the treated mice had an averagetumour size of 1382 mm3, whereas the controlgroup treated with saline had an averagetumour size of 4181 mm3. The number ofapoptotic cells increased in the treatedxenografts, with the treated group showing anaverage of 32.7 compared with 2.7 for thecontrol group. Proliferation-related Ki-67(MKI67) was inhibited by the treatment, andprotein expression levels of u-PAR, matrixmetalloproteinases MMP2 and MMP9, VEGFC,VEGFD, and the VEGF receptor VEGFR-3

(FLT4) were significantly reduced in treatedtumours (Ref. 53).

SurvivinSurvivin (BIRC5) is overexpressed in gastric cancerand its inhibition by siRNA has been assessed invivo. Gastric cancer cells were treated with aplasmid expressing siRNA targeting survivin or acontrol plasmid, and subcutaneously injected intomice. Four weeks after injection, mice that receivedtreated cancer cells showed an average tumoursize of 831 mm3; the average tumour size in micethat received cells treated with a control plasmidwas 2617 mm3; and mice that received untreatedcells had an average tumour size of 2536 mm3.Thus the treatment resulted in significant tumourgrowth inhibition. The percentage of apoptoticcells was much higher in treated cells (27.63%)than in cells with a control plasmid (2.15%) or inuntreated cells (2.31%) (Ref. 54).

VimentinVimentin, an intermediate filament protein, oftenhas expression patterns correlating with theadvent of metastatic cancer cells, such asincreased motility, invasive ability and poorprognosis. There is considerable sequencehomology between vimentin and miR-17-3p, amember of the miRNA cluster 17-92, suggestingthat expression of vimentin is regulated by miR-17-3p. It is thought that miR-17-3p is a tumoursuppressor because its expression is low inturmourigenic and metastatic cell lines but ishigher in less tumourigenic cell lines. The effectof an shRNA plasmid expressing miR-17-3p wastested in vivo by giving mice prostate cancerxenografts and treating half of them. After 31days, mice receiving the plasmid expressingmiR-17-3p had an average tumour size less thanhalf of that seen in mice receiving no treatment.An examination of the tumours excised from themice showed a negative correlation betweenmiR-17-3p expression and vimentin expression,as well as a negative correlation betweentumour growth and miR-17-3p expression(Ref. 55).

miR-221 and miR-222miR-221 and miR-222 are highly homologousmiRNAs and their upregulation has beendescribed in several types of cancer. Theirrole in tumourigenesis is shown by their targetmRNA, p27, a negative regulator of cell cycle




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progression. Small synthetic RNAs with perfectcomplementarity to the specific miRNAs,known as antagomirs, can be used to silence theendogenous miRNAs. Mice were given aprostate cancer xenograft on each flank; one wastreated with anti-miR-221 and anti-miR-222antagomirs and the other was treated with acontrol antagomir. At 33 days, the average sizeof treated tumours was 197.2 mm3, whereas thecontrol tumours had an average size of276.82 mm3 (Ref. 56).

Signal transducer and activator oftranscription 3Signal transducer and activator of transcription 3(STAT3) acts in a signalling pathway closelyassociated with the proliferation, differentiationand apoptosis of cells. Constant activation ofSTAT3 can promote carcinogenesis, andpersistently activated STAT3 has been observedin HCC cells and tissues. Mice given HCCxenografts were treated with shRNA targetingSTAT3, shRNA with no homology to humangene sequences, or saline (PBS) by intratumouralinjection coupled with an electric impulse(electrotransfection). The average tumour size inmice treated with PBS was 0.67 g; in micetreated with the non-targeted shRNA theaverage tumour size was 0.6 g; and mice treatedwith shRNA targeting STAT3 had an averagetumour size of 0.18 g. The protein levels ofSTAT3, phosphorylated STAT3, VEGF, survivinand MYC were downregulated in the treatedmice, yet expression of caspase 3 (CASP3) andp53 (TP53) were upregulated (Ref. 57).

miR-16miR-16 has lower levels of expression in prostatecancer and has the capacity to reduce theproliferation of prostate cancer cells (Ref. 58). Toevaluate miR-16 as a therapy for bone-metastaticprostate cancer, synthetic miRNA was created.Mice were given prostate cancer xenografts thatcan form tumours in bone and treatedintravenously with a complex of the miR-16mimic and atelocollagen, a control miRNAmimic with atelocollagen, or atelocollagen alone.Tumour development was monitored in vivo bybioluminescent imaging. At the end of theexperiment (day 28), mice in the control miRNAgroup and the atelocollagen-only group showedthe presence of tumours in several places byincreased luminescence, yet mice treated with

the miR-16 mimic showed no increase inluminescence (Ref. 59).

miR-26aPreclinical evidence supports the therapeuticpotential of miR-26a expression in HCC(Ref. 60). miR-26a directly downregulates cyclinsD2 (CCND2) and E2 (CCNE2) and induces aG1 arrest in human liver cancer cells. Expressionof miR-26a inhibits tumour cell proliferationin vitro and significantly inhibits cancerprogression in vivo. Delivery of miR-26a mayprovide therapeutic potential via targetingcyclins D2 and E2.

CD147CD147 (BSG) is a highly conserved glycoproteinthat is overexpressed on many epithelial cancercells, especially malignancies of the pancreas(Ref. 61). It is required for the function andexpression of monocarboxylate transporter 1(MCT1, SLC16A1/), MCT3(SLC16A3) and MCT4(SCL16A4), and has been linked to expression oflactate transporters. The effect of CD147 silencingwas observed in vivo by giving mice xenograftsof pancreatic cancer cells, or clones of those cellsexpressing shRNA targeting CD147. Half of eachgroup was then treated with doxycycline, creatingfour treatment groups. There was no significantdifference in tumour size between mice that didnot receive doxycycline. However, mice withunchanged pancreatic cancer xenografts treatedwith doxycycline had an average tumour size of89.7 mm3, whereas mice with a xenograftfrom cloned pancreatic cancer cells expressingshRNA targeting CD147 and treated withdoxycycline had an average tumour size of39.7 mm3 (Ref. 62).

ErythropoietinErythropoietin (EPO) is a glycoprotein hormonemade in the kidney in response to hypoxia andacts through its receptor EPOR. Ovarian cancercells showed high EPOR expression, but didnot express EPO under normal or hypoxicconditions in vitro. In addition, exogenous EPOintroduced to the ovarian cancer cells in vitrohad no biological effect. Ovarian cancer cellstransfected with shRNA targeting EPOR and anegative control vector were xenografted intomice. After seven weeks, mice with xenograftstreated with the control plasmid had an averagetumour size approximately ten times the average




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size inmicewith treated xenografts. The differencein tumour growth was predominantly due todecreased proliferation in treated cells becausethe rates of apoptosis were similar between thegroups. This suggests there is an EPO-independent, EPOR-mediated mechanism ofgrowth in some cancer cells (Ref. 63).

Clinical trialsThere are currently numerous cancer therapy trialsbasedonRNAi technology (Table 2).A clinical trialinvolving targeted delivery of siRNA by CalandoPhamaceuticals is in Phase I. The delivery systemutilises a cyclodextrin-containing polymer, whichself-assembles into a nanoparticle with nucleicacid, and this is the first targeted delivery ofsiRNA in humans. Conjugates of adamantine(AD) with polyethylene glycol (PEG) (AD-PEG)and AD-PEG-transferrin (TF) were added to thesurface to provide steric stabilisation andtargeted delivery of the nanoparticle,respectively. Transferrin is used to target cancercells because many cancer types are known tooverexpress the transferrin receptor. This systemis used to deliver siRNA against ribonucleotidereductase subunit 2 (RRM2) intravenously(Ref. 64). Anti-RRM2 siRNA exhibitsantiproliferative activity in several types ofhuman cancer cells as well as mouse, rat andmonkey cells due to the complete sequencehomology between these organisms at the targetsite (Ref. 65). A dose-escalation study inmonkeys showed no significant side effects until27 mg/kg, which is equivalent to 20–100 timeslarger than doses showing efficacy in mice. Theeffect seen was an increase in blood ureanitrogen and creatinine, indicative of renaldysfunction (Ref. 66). The trial is intended totreat adults with solid tumours who arerefractory to standard-of-care therapies and thefirst patient was treated in May 2008 (Ref. 64).Another Phase I clinical trial is being conducted

by Alnylam Pharmaceuticals on patients withadvanced liver cancers or other solid tumourswith liver involvement. The trial utilises twosiRNAs in a lipid nanoparticle that usesstable nucleic-acid–lipid particle (SNALP)technology, which was developed by TekmiraPharmaceuticals (Ref. 67). The siRNAs targetkinesin spindle protein (KSP/KIF11) andVEGF (http://www.medicalnewstoday.com/articles/145218.php). The antitumour effect ofVEGF was discussed earlier, and the effects of

KSP inhibition include cell-cycle arrest andinduction of apoptosis (Ref. 68). A study byTekmira Pharmaceuticals tested the therapeuticeffect of KSP inhibition using the SNALPdelivery method on mice with liver tumourxenografts. The median survival time of treatedmice was 28 days, compared with a mediansurvival time of 20 days in mice treated with acontrol SNALP formation (Ref. 69).

Silence Therapeutics has recently begun a PhaseI study to address the safety, tolerability andpharmacokinetics of Atu027 (http://www.silence-therapeutics.com/index.php?option=com_content&task=view&id=132). It contains an siRNAlipoplex constructed to target protein kinase N3(PKN3), and inhibition of this kinase in vitro inprimary endothelial cells impairs tube formationin the extracellular matrix and cell migration. Thecomplex has been tested in vivo by systemicinfusion in mice, rats and nonhuman primates.Mice with prostate cancer xenografts treated withAtu027 showed an average tumour volume of lessthan half that seen in mice injected with a sucrosesolution. Treated mice also showed half as manylymph-node metastases compared with thesucrose-treated control. The lymphatic vesseldensity in the tumour area was decreased intreated mice, yet the blood vessel density was notsignificantly affected. Atu027 was tested fortoxicity in Cynomolgusmonkeys at doses of 0.3, 1.0and 3.0 mg siRNA/kg every fourth day. PKN3gene expression levels were determined from lungtissue taken from the animals after the last doseand silencing was observed for all three doses at asignificant level as compared with sucrose-treatedanimals. An additional study conducted at dosesof 0.03 and 0.1 mg siRNA/kg determined that0.3 mg siRNA/kg was the lowest active dose(Ref. 70).

Concluding sectionThe ability to detect pathways that malignanttissue depends on combined with the specificgene-knockdown ability of RNAi may changecancer treatment. The capability of providingpersonalised care to cancer patients allowstherapy to be specifically tailored for each case.However, there is still work to be done for thistechnology to be readily available as a therapy.The target sequence on the mRNA of a targetedgene must be selected carefully because shiftingan siRNA by only a few nucleotides candrastically affect its silencing function (Ref. 71)




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Acarefully chosen target sequence is imperative tolimit off-target effects from harming the patientfurther. A targeted delivery system withminimal toxicity must be devised to specificallyadminister the nucleic acid treatment tomalignant cells without harming healthy cells.If these issues can be resolved, personalised

RNAi therapy focusing on patient-cancer-specifictargets should become standard cancer treatment,either alone or in combination with othertreatments. However, many limitations need tobe overcome for personalised RNAi therapy tobecome a reality. First of all, safe, robust andspecific systemic delivery of the RNAitherapeutic needs to be demonstrated early(Phase I) during clinical trial. Next, sufficientknockdown of the target protein, ideally to >75%reduction compared with baseline, needs to be

achieved (later Phase I to Phase II). Follow-upassessment of the malignant tissue would need todetermine the possibility of tumour cellsignalling adaptation; furthermore, possible off-target knockdown within nonmalignant tissueshould be investigated to demonstrate safety(Phase II). Finally, correlation of targetknockdown with tumour regression will need tobe observed at a high enough frequency duringPhase II to justify Phase III investigation.

Not addressed in this review, but of criticalimportance, is the necessity of relevant targetingdelivery vehicles that are safe and effectivelydeliver RNAi to the malignant cell sites.Moreover, confidence in the target throughappropriate bioinformatic assessment and high-throughput proteogenomic determination will berequired for expanded clinical use.

Table 2. Cancer therapy in clinical trials based on RNAi

Company RNAi agentname

Disease Target TrialPhase

Refs

Calando CALAA-01 Solid tumours RRM2 Phase I 72,73

Alnylam ALN-VSP Liver cancers andsolid tumours

KSP, VEGF Phase I 72a

SilenceTherapeutics

Atu027 Lung cancers PKN3 Phase I 72b

Benitec/City ofHope

pHIV7-shI-TAR-CCR5RZ

AIDS lymphoma HIV, TAR,CCR5

Phase I 72

University ofDuisburg-Essen

BCR-ABL siRNA Chronic myeloidleukaemia

bcr-abl Singlepatient

74

CequentPharmaceuticals

CEQ508 Familialadenomatouspolyposis

β-Catenin Phase I c

Gradalis, Inc. FANG Advanced cancer Furin Phase I 75

Hadassah MedicalOrganization

SV40/BCR-ABL Chronic myeloidleukaemia

bcr-abl Phase I d

Duke UniversityHospital

siRNAimmunotherapy

Metastaticmelanoma

Proteasome Phase I d

ahttp://www.medicalnewstoday.com/articles/145218.phpbhttp://www.silence-therapeutics.com/index.php?option=com_content&task=view&id=132chttp://www.cequentpharma.com/CequentFDA-IND12-10-09-final.pdfdhttp://clinicaltrials.gov/Abbreviations: AIDS, acquired immune deficiency syndrome; CCR5, C-Cmotif cytokine receptor 5; HIV, humanimmunodeficiency virus; KSP, kinesin spindle protein; PKN3, protein kinase N3; RRM2, M2 subunit ofribonucleotide reductase; TAR, trans-activating response region.




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Acknowledgements and fundingThe authors appreciate the input of theanonymous peer reviewers and thank them fortheir valuable comments. The authors also thankSusan Mill for her assistance in the preparationof the manuscript.

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Further reading, resources and contacts

Hannon, G.J. (2002) RNA interference. Nature 418, 244-251This article describes RNA interference and explains its significance as a means to manipulate gene expression

experimentally.

Bild, A.H. et al. (2006) Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature439, 353-357

This article discusses the importance of identifying dysregulated pathways of the cancer phenotype as the basisfor prediction of molecular intervention.

Fire, A. et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditiselegans. Nature 391, 806-811

This seminal article first described specific RNA interference in C. elegans.

Devi, G.R. (2006) siRNA-based approaches in cancer therapy. Cancer Gene Therapy 13, 819-829This paper summarises the advances in the last decade in the field of post-transcriptional gene silencing using

RNA interference approaches and provides relevant comparisons with other oligonucleotide-basedapproaches, with a specific focus on oncology applications.




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Features associated with this article

FigureFigure 1. RNA interference (RNAi) pathways.

TablesTable 1. Examples of in vivo use of RNAi.Table 2. Cancer therapy in clinical trials based on RNAi.

Citation details for this article

Connor Phalon, Donald D. Rao and John Nemunaitis (2010) Potential use of RNA interference in cancer therapy.Expert Rev. Mol. Med. Vol. 12, e26, August 2010, doi:10.1017/S1462399410001584




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