RNA interference against polo-like kinase-1 in advanced non-small cell lung cancers.

0.52 MB·English

by  Kawata, Eri

Kawataetal.JournalofClinicalBioinformatics2011,1:6 JOURNAL OF
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CLINICAL BIOINFORMATICS
REVIEW Open Access
RNA interference against polo-like kinase-1 in
advanced non-small cell lung cancers
Eri Kawata1,2, Eishi Ashihara1,3*, Taira Maekawa1
Abstract
Worldwide, approximately one and a half million new cases of lung cancer are diagnosed each year, and about
85% of lung cancer are non-small cell lung cancer (NSCLC). As the molecular pathogenesis underlying NSCLC is
understood, new molecular targeting agents can be developed. However, current therapies are not sufficient to
cure or manage the patients with distant metastasis, and novel strategies are necessary to be developed to cure
the patients with advanced NSCLC.
RNA interference (RNAi) is a phenomenon of sequence-specific gene silencing in mammalian cells and its
discovery has lead to its wide application as a powerful tool in post-genomic research. Recently, short interfering
RNA (siRNA), which induces RNAi, has been experimentally introduced as a cancer therapy and is expected to be
developed as a nucleic acid-based medicine. Recently, several clinical trials of RNAi therapies against cancers are
ongoing. In this article, we discuss the most recent findings concerning the administration of siRNA against polo-
like kinase-1 (PLK-1) to liver metastatic NSCLC. PLK-1 regulates the mitotic process in mammalian cells. These
promising results demonstrate that PLK-1 is a suitable target for advanced NSCLC therapy.
Introduction of new TKIs, new mutations in EGFR exon 20, develop-
Worldwide, approximately one and a half million new ing resistance to EGFR TKIs, have emerged in the trea-
cases of lung cancer are diagnosed each year [1]. About ted NSCLC [6,7], and current therapies are not
85% of lung cancer are non-small cell lung cancer sufficient to cure or manage the patients with distant
(NSCLC), including adenocarcinoma, squamous cell, metastasis [8,9]. Therefore, novel strategies are necessary
and large cell carcinoma [2], and NSCLC is the leading to be developed so that the patients with NSCLC can be
cause of cancer-related deaths. Surgery is generally cured.
regarded as the best strategy for lung cancers. However, RNA interference (RNAi) is a process of sequence
only 30% of patients are suitable for receiving potentially specific post-transcriptional gene silencing induced by
curative resection [3], and it is necessary for other double-strand RNA (dsRNA) and this phenomenon was
patients to be treated with chemotherapy. As we gain a discovered in Caenorhabditis elegans (C. elegans) [10].
better understanding of the molecular pathogenesis RNAi has been shown to function in higher organisms
underlying NSCLC, new molecular targeting agents can including mammals, and methods that exploit RNAi
be developed. Tyrosine kinase inhibitors (TKIs) targeting mechanisms have been developing. RNAi has now been
the epidermal growth factor receptor (EGFR), such as well-established as a method for experimental analyses
gefitinib and erlotinib, have shown remarkable activity of gene function in vitro as well as in high-throughput
in the patients with NSCLC, and particularly these TKIs screening, and recently, RNAi has been experimentally
are more effective to NSCLC with EGFR mutations in introduced into cancer therapy. To apply the RNAi phe-
19 exon (in-frame deletions) and exon 21 (L858R point nomenon to therapeutics, it is important to select suita-
mutation), which are found to be more prevalent in ble targets for the inhibition of cancer progression and
Asian patients [4,5]. However, despite the development also to develop effective drug delivery systems (DDSs).
Recently a lot of useful non-viral DDSs for small inter-
*Correspondence:[email protected] fering RNAs (siRNAs) have been developed [11-17].
1DepartmentofTransfusionMedicineandCellTherapy,KyotoUniversity Besides selecting suitable targets, an important consid-
Hospital,Kyoto,Japan
eration for siRNA-mediated treatment is to predict and
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Kawataetal.JournalofClinicalBioinformatics2011,1:6 Page2of6
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avoid off-target effects, which are the silencing of an strand is also degraded [36,37]. The targeted RNA dis-
unintended target gene, and potential immunostimula- sociates from the siRNA after the cleavage, and the
tory responses. To avoid those effects, the most specific RISC cleaves additional targets, resulting in decrease of
and effective siRNA sequence must be validated. Modifi- expression of the target gene (Figure 1) [38].
cation of two nucleosides of the sense strand also com-
pletely co-inhibited the immunological activities of the Polo-like kinase-1
antisense strand, while the silencing activity of the To develop RNAi therapy against cancers, it is essential
siRNA was maintained [18]. that suitable gene targets are selected. Such targets
Polo-like kinase-1 (PLK-1) belongs to the family of include antiapoptotic proteins, cell cycle regulators,
serine/threonine kinases and regulates cell division in transcription factors, signal transduction proteins, and
the mitotic phase [19,20]. PLK-1 is overexpressed in factors associated with malignant biological behaviors of
many types of malignancies and its overexpression is cancer cells. All of these genes are associated with the
associated with poor prognosis of cancer patients poor prognosis of cancer patients. PLKs belong to the
[21,22]. In this review, we discuss possible RNAi strate- family of serine/threonine kinases and are highly con-
gies against PLK-1 in advanced lung cancers. served among eukaryotes. PLK family has identified
PLK-1, PLK-2 (SNK), PLK-3 (FNK), and PLK-4 (SAK)
Mechanisms of RNAi in mammalians so far and PLKs function as regulators
The precise mechanisms of RNAi are discussed in sev- of both cell cycle progression and cellular response to
eral reviews [23-25]. In the initiation phase of RNAi DNA damage [19,39-41]. PLK-1 has an N-terminal ser-
processes, following introduction of dsRNA into a target ine/threonine protein kinase domain and two polo box
cell, dsRNA is processed into shorter lengths of 21-23 domains at the C-terminal region. Polo box domains
nucleotides (nts) dsRNAs, termed siRNAs, by the ribo- regulate the kinase activity of PLK-1 [21,42]. PLK-1 reg-
nuclease activity of a dsDNA-specific RNAse III family ulates cell division at several points in the mitotic phase:
ribonuclase Dicer. Dicer consists of an N-terminal heli- mitotic entry through CDK1 activation, bipolar spindle
case domain, an RNA-binding Piwi/Argonaute/Zwille formation, chromosome alignment, segregation of chro-
(PAZ) domain, two tandem RNAse III domains, and a mosomes, and cytokinesis [19,43]. PLK-1 gene expres-
dsRNA-binding domain [26,27]. Mammals and nema- sion is regulated during cell cycle progression, with a
todes have only a single Dicer, which acts to produce peak level occurring at M phase. Similar to its gene
both siRNAs and miRNAs [28-30], while other organ- expression, PLK-1 protein expression and its activity are
isms have multiple Dicers which perform separate, low in G0, G1, and S phases, and begin to increase in
specialized functions. Drosophila has two Dicers: Droso- G2 phase with peak in M phase [44-47].
phila Dicer-1 is required for generating miRNAs, Whereas PLK-1 is scarcely detectable in most adult
whereas Drosophila Dicer-2 produces siRNAs [25,31]. tissues [45,48,49], PLK-1 is overexpressed in cancerous
dsRNA precursors are sequentially processed by the two tissues. Its expression levels were tightly correlated with
RNAse III domains of Dicer, and cleaved into smaller
dsRNAs with 3’ dinucleotide overhangs [26,32].
In the second effector phase, smaller dsRNAs enter (cid:86)(cid:86)(cid:86)(cid:86)(cid:76)(cid:76)(cid:76)(cid:76)(cid:53)(cid:53)(cid:53)(cid:53)(cid:49)(cid:49)(cid:49)(cid:49)(cid:36)(cid:36)(cid:36)(cid:36) (cid:71)(cid:71)(cid:71)(cid:71)(cid:86)(cid:86)(cid:86)(cid:86)(cid:53)(cid:53)(cid:53)(cid:53)(cid:49)(cid:49)(cid:49)(cid:49)(cid:36)(cid:36)(cid:36)(cid:36)
into an RNA-induced silencing complex (RISC) assem- (cid:55)(cid:55)(cid:55)(cid:55)(cid:85)(cid:85)(cid:85)(cid:85)(cid:68)(cid:68)(cid:68)(cid:68)(cid:81)(cid:81)(cid:81)(cid:81)(cid:86)(cid:86)(cid:86)(cid:86)(cid:73)(cid:73)(cid:73)(cid:73)(cid:72)(cid:72)(cid:72)(cid:72)(cid:70)(cid:70)(cid:70)(cid:70)(cid:87)(cid:87)(cid:87)(cid:87)(cid:76)(cid:76)(cid:76)(cid:76)(cid:82)(cid:82)(cid:82)(cid:82)(cid:81)(cid:81)(cid:81)(cid:81)(cid:3)(cid:3)(cid:3)(cid:3)
bly pathway [33]. RISC contains Argonaute (Ago) pro- (cid:70)(cid:70)(cid:70)(cid:70)(cid:92)(cid:92)(cid:92)(cid:92)(cid:87)(cid:87)(cid:87)(cid:87)(cid:82)(cid:82)(cid:82)(cid:82)(cid:83)(cid:83)(cid:83)(cid:83)(cid:79)(cid:79)(cid:79)(cid:79)(cid:68)(cid:68)(cid:68)(cid:68)(cid:86)(cid:86)(cid:86)(cid:86)(cid:80)(cid:80)(cid:80)(cid:80)
teins, a family of proteins characterized by the presence (cid:39)(cid:39)(cid:39)(cid:39)(cid:76)(cid:76)(cid:76)(cid:76)(cid:70)(cid:70)(cid:70)(cid:70)(cid:72)(cid:72)(cid:72)(cid:72)(cid:85)(cid:85)(cid:85)(cid:85)
of a PAZ domain and a PIWI domain [34]. The PAZ
domain recognizes the 3’ terminus of RNA, and the
(cid:53)(cid:53)(cid:53)(cid:53)(cid:44)(cid:44)(cid:44)(cid:44)(cid:54)(cid:54)(cid:54)(cid:54)(cid:38)(cid:38)(cid:38)(cid:38)
PIWI domain adopts an RNAse H-like structure that (cid:11)(cid:11)(cid:11)(cid:11)(cid:53)(cid:53)(cid:53)(cid:53)(cid:49)(cid:49)(cid:49)(cid:49)(cid:36)(cid:36)(cid:36)(cid:36)(cid:3)(cid:3)(cid:3)(cid:3)(cid:76)(cid:76)(cid:76)(cid:76)(cid:81)(cid:81)(cid:81)(cid:81)(cid:71)(cid:71)(cid:71)(cid:71)(cid:88)(cid:88)(cid:88)(cid:88)(cid:70)(cid:70)(cid:70)(cid:70)(cid:72)(cid:72)(cid:72)(cid:72)(cid:71)(cid:71)(cid:71)(cid:71)(cid:3)(cid:3)(cid:3)(cid:3)(cid:86)(cid:86)(cid:86)(cid:86)(cid:76)(cid:76)(cid:76)(cid:76)(cid:79)(cid:79)(cid:79)(cid:79)(cid:72)(cid:72)(cid:72)(cid:72)(cid:81)(cid:81)(cid:81)(cid:81)(cid:70)(cid:70)(cid:70)(cid:70)(cid:76)(cid:76)(cid:76)(cid:76)(cid:81)(cid:81)(cid:81)(cid:81)(cid:74)(cid:74)(cid:74)(cid:74)(cid:3)(cid:3)(cid:3)(cid:3)(cid:70)(cid:70)(cid:70)(cid:70)(cid:82)(cid:82)(cid:82)(cid:82)(cid:80)(cid:80)(cid:80)(cid:80)(cid:83)(cid:83)(cid:83)(cid:83)(cid:79)(cid:79)(cid:79)(cid:79)(cid:72)(cid:72)(cid:72)(cid:72)(cid:91)(cid:91)(cid:91)(cid:91)(cid:12)(cid:12)(cid:12)(cid:12)
can catalyze the cleavage of the guide strand. Most spe- (cid:55)(cid:55)(cid:55)(cid:55)(cid:68)(cid:68)(cid:68)(cid:68)(cid:85)(cid:85)(cid:85)(cid:85)(cid:74)(cid:74)(cid:74)(cid:74)(cid:72)(cid:72)(cid:72)(cid:72)(cid:87)(cid:87)(cid:87)(cid:87)(cid:3)(cid:3)(cid:3)(cid:3)(cid:80)(cid:80)(cid:80)(cid:80)(cid:53)(cid:53)(cid:53)(cid:53)(cid:49)(cid:49)(cid:49)(cid:49)(cid:36)(cid:36)(cid:36)(cid:36)
cies have multiple Ago proteins, but only Ago2 can
(cid:39)(cid:39)(cid:39)(cid:39)(cid:72)(cid:72)(cid:72)(cid:72)(cid:74)(cid:74)(cid:74)(cid:74)(cid:85)(cid:85)(cid:85)(cid:85)(cid:68)(cid:68)(cid:68)(cid:68)(cid:71)(cid:71)(cid:71)(cid:71)(cid:68)(cid:68)(cid:68)(cid:68)(cid:87)(cid:87)(cid:87)(cid:87)(cid:76)(cid:76)(cid:76)(cid:76)(cid:82)(cid:82)(cid:82)(cid:82)(cid:81)(cid:81)(cid:81)(cid:81)(cid:3)(cid:3)(cid:3)(cid:3)(cid:82)(cid:82)(cid:82)(cid:82)(cid:73)(cid:73)(cid:73)(cid:73)(cid:3)(cid:3)(cid:3)(cid:3)(cid:55)(cid:55)(cid:55)(cid:55)(cid:68)(cid:68)(cid:68)(cid:68)(cid:85)(cid:85)(cid:85)(cid:85)(cid:74)(cid:74)(cid:74)(cid:74)(cid:72)(cid:72)(cid:72)(cid:72)(cid:87)(cid:87)(cid:87)(cid:87)(cid:3)(cid:3)(cid:3)(cid:3)
cleave its RNA target in humans. The dsRNA is
(cid:80)(cid:80)(cid:80)(cid:80)(cid:53)(cid:53)(cid:53)(cid:53)(cid:49)(cid:49)(cid:49)(cid:49)(cid:36)(cid:36)(cid:36)(cid:36)
unwound by ATP-dependent RNA helicase activity to
form two single-strands of RNA. The strand that directs
Figure1MechanismsofRNAinterference.Aftertheintroduction
silencing is called the guide strand, and the other is
ofdsRNAintoatargetcell,thedsRNAisprocessedintosiRNA
called the passenger strand. Ago2 protein selects the
lengthof21-23nucletidesbyDicer.siRNAthenentersanRNA-
guide strand and cleaves its RNA target at the phospho- inducedsilencingcomplex(RISC)assemblypathway.ThedsRNA
diester bond positioned between nucleotides 10 and 11 unwindstoformtwosingle-strandsofRNA.Thepassengerstrand
[32,35]. The resulting products are rapidly degraded rapidlydegradesandtheguidestrandbindsandcleavesthetarget
mRNA,resultinginmRNAdegradation.
because of the unprotected ends, and the passenger
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histological grades of tumors, clinical stages, and prog- endogenousnucleaseswhenadministeredinvivo,sothat
nosis of the patients. PLK-1 mRNA levels were elevated delivery methods that protect siRNAs from such degra-
in NSCLC tissues and this transcript levels were corre- dation are essential. For these reasons, safer and more
lated with the survivals of cancer patients [50]. More- effectiveDDSsmustbedeveloped.DDSsaredividedinto
over, the immunohistoligical study showed that PLK-1 two categories: viral vector basedcarriers, and non-viral
protein was overexpressed in NSCLC tissues in patients based carriers. Viral vectors are highly efficient delivery
at progressed stages of cancer (postsurgical stage ≥II) systems and they are the most powerful tools for trans-
and in patients with poorly differentiated NSCLCs [51]. fectionsofar.However,viralvectorshaveseveralcritical
Patients with urinary bladder cancers expressing high problemsininvivoapplication.Especially,retroviraland
levels of PLK-1 have a poor prognosis compared with lentiviral vectors have major concerns of insertional
patients with its low expression. Moreover, the histologi- mutagenesis[59,60].Consequently,non-viralDDSshave
cally high-grade, deeply invasive, lymphatic-invasive, and beenstrenuouslydeveloped[11-13].
venous-invasive bladder cancers demonstrated signifi- Atelocollagen, one of powerful non-viral DDSs, is type
cantly higher PLK-1 expression [52]. As PLK-1 is over- I collagen obtained from calf dermis [61]. The molecular
expressed in other various cancers [21], PLK-1 weight of atelocollagen is approximately 300,000 and the
overexpression is a prognostic biomarker for cancer length is 300 nm. It forms a helix of 3 polypeptide
patients. chains. Amino acid sequences at the N- and C-termini
InhibitionofPLK-1activityinducesmitotic arrestand of the collagen molecules are called telopeptide, and
tumorcellapoptosis[53-55].DepletionofPLK-1mRNA they have antigenecity of collagen molecules. As the tel-
also inhibits the functions of PLK-1 protein in DNA opeptide is removed from collagen molecules by pepsin
damagesandspindleformationandcausestheinhibition treatment, atelocollagen shows low immunogenicity.
ofthe cell proliferationina time- andadose-dependent Therefore, atelocollagen has been shown to be a suitable
manner.PLK-1siRNAtreatmentinducesanarrestatthe biomaterial with an excellent safety profile and it is used
G2/MphaseinthecellcyclewiththeincreaseofCDC2/ clinically for a wide range of purposes. Atelocollagen is
Cyclin B1 [51,52,56,57]. PLK-1 siRNA-transfected cells positively charged, which enable binding to negatively
haddumbbell-like andmisalignednuclei,indicatingthat charged nucleic acid molecules, and bind to cell mem-
PLK-1 depletion induced abnormalities of cell division branes. Moreover, at low temperature atelocollagen
during M phase, and these cells were shown to yield to exists in liquid form, which facilitates easy mixing with
caspase-dependent apoptosis [51,52,56]. As mentioned nucleic acid solutions. The size of the atelocollagen-
above, the kinases of PLK family cooperatively act in nucleic acid complex can be varied by altering the ratio
mitosis.Quantitativereal-timeRT-PCRdatashowedthat of siRNA to atelocollagen. Because atelocollagen natu-
PLK-2andPLK-3transcriptswereincreasedafterPLK-1 rally forms a fiber-like structure under physiological
siRNA treatment [51]. Unlike PLK-1, PLK-2 and PLK-3 conditions, particles formed a high concentration of ate-
playinhibitoryroles.PLK-2isregulatedbyp53andPLK- locollagen persist for an extended period of time at the
3isactivatedbytheDNAdamagecheckpoint[40].These site of introduction, which is advantageous to achieve a
observationssuggestthatPLK-1depletioninducedmito- sustained release of the associated nucleic acid. Atelo-
tic catastrophe and activationof spindle checkpoint and collagen is eliminated through a process of degradation
DNA damage checkpoint, resulting in increased tran- and absorption similar to the metabolism of endogenous
scriptionofPLK-2andPLK-3. Consequently,thesePLK collagen [61]. Alternatively, particles formed under con-
family kinases cooperatively prevented G2/M transition ditions of low atelocollagen concentrations result in
and induction of apoptosis. Importantly, depletion of siRNA/atelocollagen complexes approximately 100-300
PLK-1 does not affect the proliferation of normal cells nm in size that are suitable for systemic delivery by
although PLK-1 plays an important role in cell division intravenous administration. Atelocollagen complexes
[51,53,58]. This suggests that some other kinases com- protect siRNA from degradation by nucleases and are
pensatelossof PLK-1functionduringmitosisin normal transduced efficiently into cells, resulting in long-term
cells [51,58]. Collectively, PLK-1 could be an excellent gene silencing. For instance, Takeshita et al. demon-
targetforcancertherapy. strated that the systemic siRNA delivery with atelocolla-
gen existed intact for at least 3 days in tumor tissues
Atelocollagen using a mouse model [62].
Although siRNA target molecules are overexpressed in
cancer cells, most of them are essential to maintain Preclinical application of RNAi therapy against PLK-1 in a
homeostasis of physiological functions in humans. murine advanced lung cancer model
Therefore, siRNAs must be delivered selectively into Here we introduce an application of PLK-1 siRNA
cancer cells. Moreover, naked siRNAs are degraded by against an advanced lung cancer. As described above,
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PLK-1 is overexpressed in NSCLC tumors. Liver metas-
tasis is one of the most important prognostic factors in
lung cancer patients [8,9,63,64]. However, despite the
development of new chemotherapeutic and molecular
targeting agents, current therapies are not sufficient to
inhibit liver metastasis. We investigated the effects of
PLK-1 siRNA on the liver metastasis of lung cancers
using atelocollagen as a DDS. We first established a
mouse model of liver metastasis. Spleens were exposed
to allow direct intrasplenic injections of Luciferase
(Luc)-labeled A549 NSCLC cells. Ten minutes after
injections of tumor cells, the spleens were removed.
After Luc-labeled A549 cell engraftment was confirmed
by using In Vivo Imaging System (IVIS) of biolumines-
cence imaging [65], PLK-1 siRNA/atelocollagen com-
plex, nonsense siRNA/atelocollagen complex, or PBS/ Figure 2 Application of PLK-1 RNAi therapy against liver
atelocollagen complex was administered by intravenous metastaticNSCLC(citedfrom[51]).A.PBS/atelocollagencomplex,
nonsensesiRNA/atelocollagencomplex,orPLK-1siRNA/
injection for 10 consecutive days following day 1 of
atelocollagencomplexwasadministeredbyintravenousinjection.
transplantation. On day 35, mice treated with nonsense
RepresentativemiceshowingbioluminescenceaftersiRNA
siRNA/atelocollagen complex or PBS/atelocollagen com- treatment.Thephotoncountsofeachmouseareindicatedbythe
plex showed extensive metastases in the liver when pseudocolorscales.B.GrowthcurvesofinoculatedLuc-labeled
compared to mice treated with PLK-1 siRNA/atelocolla- A549cellsmeasuredbytheIVIS(pinksquare,nonsensesiRNA/
atelocollagencomplex(25μgsiRNA)-treatedmice;bluediamond,
gen complex (Figure 2). Moreover, on day 70 after the
PBS/atelocollagencomplex-treatedmice;orangetriangle,PLK-1
inoculation of tumor cells, livers of mice treated with
siRNA/atelocollagencomplex(25μgsiRNA)-treatedmice;n=5for
nonsense siRNA/atelocollagen or PBS/atelocollagen eachgroup.Onday35afterinoculation,theluminesecenceinthe
complex had numerous large tumor nodules, whereas PLK-1siRNA/atelocollagen-treatedmicewassignificantlysuppressed
the livers of mice treated with PLK-1 siRNA/atelocolla- comparedwiththatinothergroups.*p<0.05.Mean±SD.C.
Macroscopicanalysisofmiceliversafterday70ofinoculation.
gen complex showed a much lower number of smaller
Whitenodulesaremetastaticlivertumors.TreatmentwithPLK-1
nodules. These findings indicate that PLK-1 siRNA/ate-
siRNA(25μg)remarkablyinhibitedthegrowthoflivermetastases
locollagen complex is an attractive therapeutic tool for comparedwithPBSornonsensesiRNAtreatments(25μg).
further development as a treatment against liver metas-
tasis of lung cancer [51]. Consequently, our preclinical
applications suggest that PLK-1 siRNA is a promising single specific siRNA or multiple specific siRNAs
tool for cancer therapy. [57,71-73]. In conclusion, RNAi therapy represents a
powerfulstrategyagainstadvancedlungcancersandmay
Conclusion offera novel and attractive therapeutic option. The suc-
Ourpreclinicalstudiesdemonstratedthat RNAitherapy cess of RNAi depends on the suitable selection of target
againstPLK-1usingatelocollageniseffectiveagainstliver genesandthe development of DDSs. We anticipate that
metastaticNSCLCcancers.Recently,severalclinicaltrials the continued development of effective DDSs and the
for cancer therapy are ongoing (Additional file 1: Table accumulationof evidence further proving the success of
S1, http://clinicaltrials.gov/ct2/home). Although RNAi siRNAtreatmentwilladvanceRNAiasapromisingstrat-
shows excellent specificity in gene-silencing, several egyforlungcancertherapy.
adverse effects including activation of immune reaction
[66,67] and off-target effects (induction of unintended Additional material
gene silencing) [68] are brought in in vivo application.
Safer and more efficient DDSs for systemic delivery are
Additionalfile1:TableS1ClinicaltrialsofRNAi.
warrantedtobedeveloped.Moreover,studiestoestablish
thepharmacokineticsandpharmacodynamicsofsiRNAs
ontheadministrationarenecessarystepsinthepotential
Listsofabbreviations
approvalofsiRNAasatoolforcancertherapy.Tomaxi-
Ago:Argonaute;DDSs:drugdeliverysystems;dsRNA:double-strandRNA;
mizeefficacyandtominimizeadverseeffectsofRNAi,it EGFR:epidermalgrowthfactorreceptor;IVIS:InVivoImagingSystem;Luc:
shouldbedeterminedwhethersiRNAsarebestadminis- Luciferase;NSCLC:non-smallcelllungcancer;nt:nucleotide;PAZ:Piwi/
Argonaute/Zwille;PLK-1:Polo-likekinase-1;RISC:RNA-inducedsilencing
tered alone or in combination with chemotherapeutic
complex;RNAi:RNAinterference;siRNA:smallinterferingRNA;TKI:Tyrosine
agents [69,70], and whether it is better to administer a kinaseinhibitor
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