ReviewArticle - Cancer Pharmacology LabPancreatic ductal adenocarcinoma (PDAC) is an extremely...

Review ArticleMolecular Mechanisms Underlying the Role of MicroRNAs inthe Chemoresistance of Pancreatic Cancer

Ingrid Garajovaacute12 Tessa Y Le Large1 Adam E Frampton3

Christian Rolfo4 Johannes Voortman1 and Elisa Giovannetti15

1 Department of Medical Oncology VU University Medical Center Cancer Center Amsterdam De Boelelaan 11171081 HV Amsterdam The Netherlands

2Department of Experimental Diagnostic and Speciality Medicine University of Bologna SantrsquoOrsola-Malpighi HospitalVia Massarenti 9 40138 Bologna Italy

3 HPB Surgical Unit Department of Surgery amp Cancer Imperial College Hammersmith Hospital Campus White CityLondon W12 0NN UK

4Phase I-Early Clinical Trials Unit Department of Medical Oncology Antwerp University Hospital Wilrijkstraat 102650 Edegem Belgium

5 Start-Up Unit University of Pisa Lungarno Pacinotti 43 56126 Pisa Italy

Correspondence should be addressed to Elisa Giovannetti elisagiovannettigmailcom

Received 2 July 2014 Accepted 28 July 2014 Published 28 August 2014

Academic Editor Paolo Gandellini

Copyright copy 2014 Ingrid Garajova et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pancreatic ductal adenocarcinoma (PDAC) is an extremely severe disease where the mortality and incidence rates arealmost identical This is mainly due to late diagnosis and limited response to current treatments The tumor macroenviron-mentmicroenvironment have been frequently reported as the major contributors to chemoresistance in PDAC preventing thedrugs from reaching their intended site of action (ie the malignant duct cells) However the recent discovery of microRNAs(miRNAs) has provided new directions for research on mechanisms underlying response to chemotherapy Due to theirtissue-disease-specific expression and high stability in tissues and biofluids miRNAs represent new promising diagnostic andprognosticpredictive biomarkers and therapeutic targets Furthermore several studies have documented that selected miRNAssuch as miR-21 and miR-34a may influence response to chemotherapy in several tumor types including PDAC In this reviewwe summarize the current knowledge on the role of miRNAs in PDAC and recent advances in understanding their role inchemoresistance through multiple molecular mechanisms

1 Introduction

A surprising revelation from the human genome project wasthat 75 of the genome is transcribed into RNA [1ndash3] but lessthan 2 is composed of protein-coding genes [4] NoncodingRNAs (ncRNAs) represent an extremely interesting class ofRNAs that can be divided into three types according to thesize Short ncRNAs are lt50 nucleotides (nt) those between50 nt and 200 nt are referred to asmidsize ncRNAswhile longncRNAs (lncRNAs) are gt200 nt [5ndash8] miRNAs are a classof short ncRNAs containing approximately 19ndash24 nt Theyhave a key regulatory role in development differentiation

and apoptosis of normal cells as well as in the determinationof the final phenotype of cancer cells affecting carcino-genesis and metastatic potential [9] Remarkably miRNAsexhibit tissue-specific and disease-specific expression thatcould provide the basis for their development as noveldiagnostic prognostic andor predictive biomarkers as wellas therapeutic targets [8] Furthermore several studies havedocumented that selectedmiRNAsmay influence response tochemotherapy [10]

Cancer chemoresistance can occur by multiple mecha-nisms It can arise fromphysiological barriers to drug absorp-tion or penetration into target tissues or from biological

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 678401 17 pageshttpdxdoiorg1011552014678401

2 BioMed Research International

mechanisms within individual tumor cells which reduce theeffectiveness at their intended site of action such as increasedexpression of enzymes involved in drug catabolism or anti-apoptotic proteins [10]

The dense stromal reaction which characterizes mostPDACs has been frequently reported as the main causeof chemoresistance preventing the drugs from reachingtheir intended site of action [11] However detailed geneticanalyses have unraveled the pivotal mechanisms controllingpancreatic carcinogenesis and cluster analysis of recurrentlymutated genes has defined twelve different core pathwaysthat lead to aberrant signaling in PDAC cells [12] Suchstudies suggest that the best hope for the development ofagents targeting critical points in the altered pathways liesin the study of mechanisms involved in gene expressionregulation Therefore in the present review we summarizethe role of miRNAs in PDAC and focus on the miRNA-basedmechanisms of PDAC chemoresistance

2 Discovery of miRNAs andTheir Role in Cancer

The first miRNA molecule lin-4 was identified in 1993 byLee and collaborators [13] In 2000 Reinhart et al identifiedlethal-7 (let-7) another miRNA and discovered its role inthe posttranscriptional regulation of gene expression [14]Currently it has been reported that there are around 2600unique mature human miRNAs (miRBase version 20) [15]miRNAs regulate more than one-third of all human geneswhich suggest their remarkable influence on human biology[16] It is known that more than 50 of miRNA genes arelocalized within genomic regions that are either frequentlyamplified or deleted in different tumor types resulting inmiRNAs deregulation and aberrant expression [17 18] ThealteredmiRNAsmay have different effects on the tumors [19]Some of these miRNAs have been characterized as potentoncogenes (oncomiRs) while others have been identified astumor suppressors (tsmiRs) based on the consequences oftheir expression on the phenotype of several experimentalmodels [4] OncomiRs such as miR-21 are commonlyupregulated in cancer [20] while tsmiRs such as let-7 aredownregulated [21] resulting in unique combinations ofmiRNAs (ie overexpressed oncomiRs and underexpressedtsmiRs) characterizing different tumors [22]

The multiple roles of these miRNAs can be explained bystarting from the analysis of their biological synthesis andfunctions Biosynthesis of miRNAs is a multistep processinvolving both nuclear and cytoplasmic components [23]Initially they are transcribed in the nucleus by RNA poly-merase II into large RNA precursors called pri-miRNAs [24ndash26] which can be several hundreds to several thousands of ntin lengthThe first slicing step performed by the ribonuclease(RNase) III Drosha-DGCR8 (DiGeorge syndrome criticalregion 8) enzyme leads to the formation of 70-base longpre-miRNAs [27ndash29] Pre-miRNAs are actively transportedfrom the nucleus to the cytoplasm by Exportin-5 [30] wherethey are subjected to further cleavage by the RNase Dicerto achieve the final size and each molecule is combined

with proteins of the Argonaute (AGO) family to obtain itsfunctional form [31ndash33] thus forming the miRNA-inducedsilencing complexes (miRISCs) Typically these complexesbind to the 31015840-untranslated region (31015840 UTR) of target mRNAwith perfect or near-perfect complementarity When miR-NAs form perfect base pairs with their target mRNA theyresult in its degradation Nevertheless most of the humanmiRNAs bind to their target 31015840-UTRs with imperfect com-plementarity and therefore induce translational repression[34] A result of all these interactions is that the targetsequence is not translated or there is a variation of translationand subsequently the encoded protein is modified or notproduced at all According to the role of this protein thisleads to structural or functional alterations to the involvedcells thus having a direct effect on their phenotype [9]Remarkably each miRNA can regulate the expression ofnumerous target genes and also the same target gene can beregulated by several types of miRNAs which create a complexnetwork of interactions [35ndash37] However the regulatory roleof miRNA in mRNA stability and translation into protein isa complex biological process which is not restricted throughthe binding of miRNA only in the 31015840-UTR of the mRNA [19]miRNAs can also interact with the 51015840 UTR of protein-codinggenes and cause translational repression [38] or activationof the targeted proteins [39] Similarly miRNAs can alsotarget the coding sequence and repress the translation oftargeted genes [40] Moreover some miRNAs can interactwith regulatory protein complexes such as AGO2 and fragileX mental retardation-related protein 1 (FXR1) and indirectlyupregulate the translation of a target gene [41]

The involvement of miRNAs in cancer was first discov-ered in a quest to identify tumor suppressors in the frequentlydeleted 13q14 region in chronic lymphocytic leukaemia(CLL) and the miRNA cluster miR-15a-miR-16-1 was foundto be deleted or downregulated via epigenetic silencing in69 of the patients [42] One of the most striking themes inthe study of miRNAs and cancer is indeed the large alterationof miRNA expression in malignant cells compared to theirnormal counterparts Most cancers have a specific miRNAsignature or ldquomiRNomerdquo that characterizes the malignantstate and defines some of the clinicopathological featuresof the tumors (eg grade stage aggressiveness vascularinvasion andor proliferation index) [43] miRNAs have avariety of roles in cancer development and progression [8]acting not only as tumor suppressors or oncogenes [44] butalso as key activators or suppressors of tumor metastasis[45] Variations in miRNA genes and their precursors as wellas the target sites and genes encoding components of themiRNA processing machinery can affect the cell phenotypeand disease susceptibility [46 47] Finally a subclass ofmiRNAs known as epi-miRNAs can directly control theepigenetic variations [48] andmiRNA expression can also bedownregulated via promotor hypermethylation [49] addinganother piece to the puzzle of regulatory gene expressionnetworks

Research in various cancers has found that miRNAs alsohave great potential as biomarkers for early diagnosis andprognosis [17 50 51] In particular circulating miRNAs havehigh translational potential as noninvasive biomarkers [52]

BioMed Research International 3

Indeed miRNA expression levels can be detected in a varietyof human specimens including both fresh and formalin-fixed paraffin-embedded tissues fine-needle aspirates andin almost all human body fluids including serum plasmasaliva urine and amniotic fluid [53 54] The impressivestability of miRNAs in tissues and biofluids is a key advantageover proteins and mRNAs [55] Circulating miRNAs mayhave cellular or extracellular origin and are presumably notnaked miRNA which would be degradated within secondsdue to the high levels of nucleases in blood Several reportshave demonstrated that stability results from the formation ofcomplexes between circulatingmiRNAs and specific proteins[56] Other studies have foundmiRNAs contained within cir-culating exosomes or other microvesicles and it is also pos-sible that cell lysis or an increase in the number of exosomesshed from the diseased cells can contribute to increased levelsof certain circulatingmiRNAs [56]miRNAs have been foundwithin circulating exosomes or other microvescicles whichcan be taken up by acceptor cells playing a role in cell-to-cellcommunication Although the mechanism of secretion andincorporation of miRNAs has not been elucidated secretorymiRNAs may play a pivotal role as signaling moleculesin physiological and pathological events In general thereare three mechanisms of shedding which lead to release ofvesicles into the extracellular space that is via exocytosisbudding of microvesicles directly from a plasma membraneor through the membranous microvesicles shed from cellsduring apoptosis [56] However before applying large-scaleefforts to miRNA biomarker discovery baseline parameterssuch as intraindividual and interindividual variability ofmiRNAs must be explored very carefully Currently there areno validated guidelines for the collection and extraction ofsamples for miRNA analysis Differences in specimen types(tissue type or plasmaserum) can have a profound effect onmiRNA levels For example miRNA content in both plasmaand serum can be influenced by cell remnant contaminationfrom erythrocytes leukocytes or platelets Standardization ofmany analysitical parameters is essential for the evaluation ofmiRNA as ideal biomarkers

Further research is also necessary to understand whethermiRNAs have clinical potential as prognostic factors and aspredictive biomarkers for chemotherapy resistance in specifictumor types The present review summarizes the currentknowledge on the role of miRNAs in PDAC reportingthe most recent studies on miRNA-based mechanisms ofchemoresistance

3 miRNA and PDAC

PDAC is a highly aggressive malignancy and fourth leadingcause of cancer-related death in developed countries [57]Themedian survival after diagnosis is 2ndash8 months and approxi-mately only 3ndash6 of all patients with PDAC survive 5 yearsafter diagnosis [58] For resectable or borderline resectablepatients (ie patients with stages T1 T2 or T3 tumors)surgical resection remains the cornerstone of managementof PDAC However the average survival of resected patientsis between 12 and 20 months with a high probability ofrelapse [9] Owing to vague symptoms in early stages 80

of PDACs are diagnosed when already advanced and nocurative therapy is currently available [59ndash61]

Tumors of the pancreas are divided into those arisingfrom the exocrine pancreas and those arising from theendocrine cells PDACs represent 75 of exocrine malignan-cies [61] It has been established that PDAC does not arisede novo but is preceded by histologically distinct noninvasiveprecursor lesions within the pancreatic ducts The mostcommon precursors are pancreatic intraepithelial neoplasia(PanIN) which show a defined histological progression fromthe low-grade PanIN-1 through to the intermediate-gradePanIN-2 and culminating in the high-grade PanIN-3 (carci-noma in situ) [62] Key shared genetic alterations associatedwith PDAC progression include earliest genetic events suchas mutation of K-RAS and overexpression of HER-2neuAt later stages inactivation of the p16 tumor suppressorgene occurs followed by the loss of TP53 SMAD4 andBRCA2 signaling pathways and the genomic-transcriptomicalterations that facilitate cell cycle deregulation cell survivalinvasion and metastases [4] Importantly several miRNAsfunctionally interact with these genetic lesions as describedin the following paragraphs (see also Figure 1)

31 K-RAS Mutations Over 90 of PDACs harbor an acti-vating K-RAS gene mutation The vast majority of thesemutations are at codon 12 and occur very early in pancreaticcarcinogenesis [63] K-RAS is a 21 kDa intracellular mem-brane bound protein that belongs to the GTPase superfamily[64 65] In physiological conditions the GAP proteins andspecifically the RAS GTPases do promote GTP hydroly-sis and reversal of the RAS activation step [66] Duringoncogenic transformation the mutated RAS is constitutivelyactivated and cannot be deactivated by theGAPproteins [65]RAS signaling involves multiple branches (B-RAF PI3K andPLC pathways) Together these branches cover most aspectsof cellular life including regulation of the cell cycle differ-entiation proliferation and apoptosis [62] Several recentstudies have identified specific miRNAs that regulate the K-RAS signaling pathway in pancreatic oncogenesis and viceversa Preclinical studies have shown that K-RAS regulatesmiR-21 expression levels in precancerous lesions and thepeak of miR-21 expression correlates with the degree ofprogression to more aggressive forms [67] K-RAS is alsoa direct target of miR-217 thus upregulation of miR-217decreases K-RAS protein levels and reduces the constitutivephosphorylation of downstream AKT [68] Another studyidentified K-RAS as a direct target of miR-96 [69] Indeedoverexpression of miR-96 decreased cancer cell invasionmigration and slowed tumor growth and was associated withK-RAS downregulation [69] Recent studies have shown thatmiR-126 and let-7d can also regulate K-RAS levels in PDACIn particular miR-126 can directly target K-RAS thus miR-126 downregulation can allow overexpression of K-RAS [70]

32 HER2neu Overexpression Up to 29 of PDACs haveHER2 overexpression [71ndash73] There is direct correlationbetween the expression levels of the Her2neu and theshorter survival in patients with PDAC suggesting that


Ang

ioge

nesis

EGFR

path

way

NO

TCH

path

way

TGF-120573

path

way

Wnt

pat

hway

Hed

geho

gpa

thw

ay

VEGF EGFR Her2neu NOTCH PTCA1-SMO TGF120573RIII Frizzled

CD

CSL

GLI

p16

BRCA2Rad51

CDK46CyclinD1 Rb E2F Cell cycle

Failed repair of genes

MiR-21MiR-200let-7d

MiR-222MiR-132MiR-212MiR-148

MiR-494

SMAD4 120573-catenin

SMAD3 SMAD2 GSK-3 120573PI3K

AKT

SOS JAK

KRAS STAT

p53

BCL2

BRAF

MEK

ERK

MYC

MiR-21MiR-217MiR-96MiR-126let-7d


p27

MD

M2

mTO

R NF-120581

B

FOX

Figure 1 MicroRNA and their involvement in oncologenic signaling cascades in pancreatic cancer EGFR pathway Activation of the EGFreceptor results in autophosphorylation of key tyrosine residues which subsequent activation of downstream signalling cascades includingthe RASextracellular signal regulated kinase (ERK) pathway the phosphatidylinositol 3-kinase (PI3) pathway and the Janus kinaseSignaltransducer and activator of transcription (JAKSTAT) pathway All of them result in cell survival promotion Notch pathway A ligand onone cell induces a series of proteolytic cleavage events in a Notch receptor on a contacting cell These cleavage events release the Notchintracellular domain (NICD) which translocates to the nucleus to activate the transcription of Notch target genes together with CSL(CBF1Suppressor of HairlessLAG-1)The notch signaling pathway is important for cell-cell communication which involves gene regulationmechanisms that control multiple cell differentiation processes Hedgehog pathway Hedgehog is a secreted ligand that binds to its receptorPatched (PTCA1) When PTCA1 is activated it leads to inhibition of the Smoothened (Smo) receptor Smo is then able to inhibit thephosphorylation and cleavage of Gli which prevents the formation of repressive Gli (GliR) and promotes the formation of activated Gli(GliA) GliA then translocates into the nucleus and initiates transcription of target genes which play a role in stem cell regulation TGF-120573pathway TGF receptors are activated after binding with their ligand which leads to further phosphorylation of receptor-regulated SMADs(mainly SMAD2 and SMAD3) Phosphorylated SMAD23 form heteromeric complexes with SMAD4 which accumulate in the nucleus andactivate transcription of different genes including those responsible for cell cycle arrest Wnt pathway In the absence of signal action ofthe destruction complex (CKI120572 GSK3120573 APC and Axin) creates a hyperphosphorylated 120573-catenin which is a target for ubiqitination anddegradation by the proteosome Binding of Wnt ligand to a FrizzledLRP-56 receptor complex leads to stabilization of hypophosphorylated120573-catenin which interacts with TCFLEF proteins in the nucleus to activate transcription

the HER2neu signaling pathway is a central regulator ofpancreatic oncogenesis [74]

The HER2neu pathway has been primarily studied inbreast cancer cell lines where miR-21 expression levels cor-relate with the HER2neu upregulation [65] More recentlydysregulation of miR-125a-5p125b and HER2 emerged as anearly event in the gastric (intestinal-type) and esophageal(Barretts) oncogenesis [75] In these oncogenic lesionsmiR-125 expression correlates inversely with HER2 statusTherefore miR-125a-5p125b can be considered among thetherapeutic targets in HER2-positive esophageal and gastricadenocarcinoma Similarly the role of newer anti-HER2agents agents interacting with regulating miRNA in HER2-positive PDAC remains to be explored [74 76]

33 p16CDKN2A Inactivation CDKN2A is a tumor sup-pressor gene which is somatically inactivated in approxi-mately 95 of PDACs [77] Most of these inactivating muta-tions lead to loss of function of the protein p16 the product ofthe CDKN2A gene The p16 protein binds cyclin-dependentkinases 4 and 6 (CDK4 and CDK6) and specifically inhibitstheir pRb phosphorylating activity which is required for G1Stransition [62] Inherited mutations in the p16CDKN2Agene cause the familial atypical multiple mole melanomasyndrome with increased risk for developing PDAC andmelanoma [78] Several miRNAs that participate in thederegulation of the cell cycle genes are essential during PDACdevelopment and progression For example miR-222 targetsp27 and p57 which are both pivotal cell cycle inhibitors [79]


Other studies have shown that downregulation of miR-132and miR-212 causes G2M cell cycle arrest and results inreduced cell proliferation [80] while miR-148 directly targetsAMPactivated protein kinase (AMPK)which plays a key roleas a master regulator of cellular energy homeostasis and caninduce cell cycle arrest and apoptosis [81]

34 TP53 Mutations The TP53 gene is inactivated in 75 to85 of PDACs [63] Genetic inactivation of TP53 abrogatesimportant cell functions such as regulation of cellular pro-liferation and apoptosis in response to DNA damage Whencellular stress and DNA damage are detected degradationof TP53 is inhibited by different mechanisms leading toaccumulation of its active form [82] Preclinical studies haveshown that TP53 directly regulates miR-34 which furtherdownstream targets Notch and therefore plays a role inthe maintenance and survival of PDAC initiating cells [82]Moreover TP53-induced nuclear protein 1 gene has beendescribed to be downregulated by miR-155 acceleratingpancreatic tumor development [83] MiR-222 and miR-203are also able to target p53 and affect its function as a crucialregulator of the cell cycle [84]

35 SMAD4 Inactivation The SMAD4 gene is inactivated inapproximately 60of PDACs [63]Theprotein product of theSMAD4 gene is involved in the transmission of intracellularsignals from transforming growth factor beta (TGFb) recep-tors within the cell membrane to the nucleus [85] In normalcells TGF-A receptors are activated after binding with theirligand which leads to further phosphorylation of receptor-regulated SMADs (mainly SMAD2 and SMAD3) Phospho-rylated SMAD2 and SMAD3 form heteromeric complexeswith SMAD4 which accumulate in the nucleus and activatetranscription of different genes including those responsiblefor cell cycle arrest This pathway is of key importance forpancreatic cells [62] PDACs with loss of SMAD4 expressionhave higher rates of distantmetastases and a poorer prognosis[86 87] A recent study showed that loss of SMAD4 inPDAC cells leads to increased levels of FOXM1 nuclearlocalization of 120573-catenin and reduced levels of miR-494 [88]Transgenic expression of miR-494 in PDAC cells producedthe same effects as reducing expression of FOXM1 or blockingnuclear translocation of 120573-catenin reducing cell prolifera-tion migration and invasion and increasing their sensitivityto gemcitabine Reduced expression of miR-494 correlatedwith PDACmetastasis and reduced survival times of patientsThis study suggested that miR-494 might be developed as aprognostic marker or a therapeutic target for patients withPDAC Other studies have shown that in human PDACspecimens the expression levels of both miR-421 and miR-483-3p are inversely correlated to SMAD4 expression andectopic expression of these miRNAs significantly repressesSMAD4protein levels in PDACcell lines suggesting that theyare potent regulators of SMAD4 in PDAC [89 90]

36 BRCA2 and PALB2 Mutations The BRCA2 gene isinactivated in fewer than 10 of PDACs [91] Impor-tantly germline mutations in BRCA2 are associated with

an increased risk of PDAC [92] Similarly germline trun-cating mutations in the PALB2 gene which encodes for aBRCA2 binding protein [93] have been identified in sim3of individuals with familial pancreatic cancer [94 95] Ofnote a recent study for the prediction of BRCA12 mutation-associated hereditary breast cancer identified a 35-miRNAclassifier for the prediction of BRCA12 mutation status witha reported 95 and 92 accuracy in the training and the testset respectively [96] These miRNA signatures might be ofinterest also in PDAC in order to complement current patientselection criteria for gene testing by identifying individualswith high likelihood of being BRCA12 mutation carriers

4 MicroRNA-Based Mechanisms of AnticancerDrug Resistance in PDAC

Chemotherapy remains the primary treatment formetastaticnonresecable PDAC However the best currently availabletreatments prolong life by only a few months [97 98] andPDAC chemoresistance renders most drugs ineffective

Drug resistance can be divided into two groups intrinsicor acquired Intrinsic resistance is caused by a preexistingphenotype whereas acquired resistance develops due torepeated use of the same drug The most common reason forthe acquisition of resistance to a broad range of anticancerdrugs is the overexpression of one ormore energy-dependenttransporters that detect and eject anticancer drugs fromcells resulting in multidrug resistance (MDR) [10 99] How-ever drug resistance can occur for many causes includingincreased drug efflux alterations in drug target DNA repaircell cycle regulation and evasion of apoptosis [100]

Up- andor downregulation of miRNAs can influence theexpression of multiple target mRNAs and therefore multipleproteins leading to variations in the chemosensitivity ofcancer cells via various cellular processes In particularseveral miRNAs have been demonstrated to alter cellularresponse to anticancer agents via modulation of drug effluxand targets cell cycle survival pathways andor apoptoticresponse as reported in the following paragraphs and inFigure 2

41 Upregulation of Drug Efflux Transporters Resistance tovarious anticancer agents has been associated with increasedexpression of drug efflux pumps [99] keeping the intracel-lular drug concentration below a cell-killing threshold [100]miRNAs have also been shown to be involved in chemother-apy resistance through the regulation of ATP-binding cassette(ABC) membrane transporters [100] They transport drugsfrom the cytosol to the extracellular space Activation of theMDR1 gene results in overexpression of P-glycoprotein (P-gp) which is a multidrug efflux pump and confers cancercell resistance to a broad spectrum of drugs [10 101] P-glycoprotein is localized at the apical level in cells membranesof different cellular compartments such as liver intestinekidney and placenta This strategic localization gives P-gp a crucial role as responsible for drugs absorption andaccumulation [101] It has been shown thatmiR-27a andmiR-451 are activators of drug resistant process by modulationof MDR1P-gp expression in human ovarian and cervical


Drug effluxtransporters

Alterations indrug target

Alteration in DNA repair

Aberrant regulation of the cell cycle

Evasion of apoptosis

miR-27miR-451

miR-181-5pmiR-218-5pmiR-130a-3pmiR-424-3p

miR-192miR-215miR-211

let-7

miR-21


miR-21miR-148amiR-17-5p

ATPATP ATP

lowast

Figure 2 MicroRNA and their involvement in anticancer drug resistance Drug resistance can occur at many levels including drug effluxalterations in drug target DNA repair cell cycle regulation and evasion of apoptosis Some selected miRNAs which have been demonstratedto alter these mechanisms are shown in this figure

cancer cells [10 102] A recent study evaluated the role ofmiRNAs in MDR in PDAC monitoring the modulation ofsome specificmiRNAs by the treatment of a wild type cell lineand in the corresponding cell line with P-gp overexpressionand unsensitive to several antineoplastic treatments [103]This study showed the different modulation of 4 miRNAs(miR-181a-5p miR-218-5p miR-130a-3p and miR-424-3p)using a specific P-gp substrate and suggested new molecularmechanisms potentially involved in chemoresistance suchas the modulation by miR-424 of the protein cullin 2 ascaffolding protein displaying a pivotal role in the assemblyof the ubiquitin ligase system thereby stabilizing HIF-1120572

42 Alterations in Drug Targets and DNA Repair Chemore-sistance can be caused by either quantitative (ie modulationof expression levels) or qualitative (ie mutation) alterationsof the drug targets [100] Examples of quantitative alterationshave been reported for several antimetabolites which influ-ence various steps the metabolism of nucleic acids throughinhibition of key enzymes such as thymidylate synthaseand ribonucleotide reductase MiR-192 and miR-215 targetthymidylate synthase (TS) which is the main drug targetof the fluoropyrimidine-based therapy in colorectal cancer

which is also used in PDAC patients [104] However down-regulation of TS bymiR-192215 did not lead to an increase in5-FU sensitivity suggesting that the activity of miR-192215was not mediated by TS In contrast overexpression of bothmiRNAs resulted in a reduction of cell proliferation andtherefore diminished the effectiveness of S-phase specificdrugs like 5-FU suggesting thatmiR-192 andmiR-215 can stillplay a role in 5-FU resistance

Two recent studies suggested the key role ofmiR-211 in themodulation of ribonucleotide reductase subunit 2 (RRM2)which is an important cellular target of gemcitabine ThismiRNA had significantly higher expression in long- versusshort-OS PDAC patients evaluating high-resolution miRNAprofiles with Torayrsquos 3D-Gene-miRNA-chip detecting morethan 1200 human miRNAs [105] The preclinical analysesdemonstrated that induction of the miR-211 expression inPDAC cells increased the sensitivity to gemcitabine throughreduced expression of its target RRM2 [106] Similarly it hasbeen demonstrated that let-7 negatively regulates RRM2 andlet-7 expression is inversely correlated with RRM2 expressionin gemcitabine-resistant PDAC cells Additionally silencingRRM2 or overexpression of let-7 was shown to sensitizePDAC cells to gemcitabine [107]


miRNAs can also alter cellular response to several anti-cancer drugs via interfering with DNA repair In particularthe inhibition of ribonucleotide reductase by gemcitabineresults in deoxyadenosine triphosphate depletion causingDNA replication errors Moreover gemcitabine is incor-porated into DNA and arrests DNA replication Both themispaired bases and the gemcitabine-modified DNA basescan be the substrates for postreplicative DNA mismatchrepair (MMR) machinery [108] which influences cancer cellsensitivity

Similarly defects in MMR proteins have been associ-ated with reduced or absent benefit from 5-FU adjuvantchemotherapy [109] MMR alterations reduce the incorpora-tion intoDNAof the 5-FUmetabolites that causeG2Marrestand induce apoptosis after 5-FU treatment Colorectal can-cer cells with miR-21 overexpression exhibited significantlyreduced 5-FU-induced G2M damage arrest and apoptosissuggesting that miR-21-dependent downregulation of coreMMR component (hMSH2ndashhMSH6) might be responsiblefor both primary and acquired resistance to 5-FU [110 111]Of note miR-21 is included in the miRNA metasignaturefor recognising PDAC [112 113] Furthermore high miR-21highmiR-31 and lowmiR-375 tumoral expressions have beenvalidated as independent prognostic biomarkers for pooroverall survival in PDAC

43 Aberrant Regulation of the Cell Cycle The cell cycle is anordered set of events culminating in cell growth and divisioninto two daughter cells Uncontrolled cellular proliferationis one of the hallmarks of cancer and these alterations arecommonly caused by genetic damages to regulator genessuch p16 and cyclin D1 that govern phosphorylation of theretinoblastoma protein (RB) and control exit from the G1phase of the cell cycle or the tumor suppressor TP53 whichcan arrest growth by holding the cell cycle at the G1Sregulation point on DNA damage recognition [114] Recentstudies showed that the members of the miR-34 family aredirect TP53 targets and their upregulation induced apoptosisand cell cycle arrest [115] ThemiR-34 family comprises threemiRNAs encoded by two different genesmiR-34a is encodedby its own transcript whereasmiR-34bmiR-34c share a com-mon primary transcript Moreover the promoter region ofmiR-34a miR -34b and miR -34c contains CpG islands Anaberrant CpGmethylation reduces miR-34 family expressioninmultiple types of cancer including PDAC [116]Therefore arecent study investigated the functional significance of miR-34a in PDAC progression through its epigenetic restorationwith chromatin modulators demethylating agent 5-Aza-21015840-deoxycytidine and HDAC inhibitor Vorinostat [117] Therestoration of miR-34a in human PDAC and pancreaticcancer stem cells (CSCs) strongly inhibited cell prolifera-tion cell cycle progression self-renewal epithelial to mes-enchymal transition and invasion while inducing apoptosisThese results provided not only mechanistic insight but alsopromising therapeutic approaches whichmight also improveesponse to existing chemotherapies in PDAC

Another example of protein of interaction between pro-teins regulating the cell cycle and miRNA is representedby Cyclin-dependent kinase inhibitor 1B (CDKN1B p27 or

p27Kip1) which is a cell cycle inhibitor and tumor suppressorThis enzyme has been identified as a direct target of miR-221and miR-222 [53] The expression of miR-221 is significantlyupregulated in PDAC cell lines and tumor tissues comparedto normal pancreatic duct epithelial cells and normal pan-creas tissues and has been proposed as candidate plasmabiomarkers in PDAC [118] However transfection of miR-221 inhibitor suppressed the proliferative capacity of PDACcells with concomitant upregulation of CDKN1B as well asof PTEN and PUMA which are other tumor suppressorsamong the predicted targets of miR-221 [119]The same studyshowed that the expression of miR-221 was modulated by thetreatment with isoflavone mixture (G2535) formulated 331015840-diindolylmethane (BR-DIM) or synthetic curcumin ana-logue (CDF) leading to the inhibition of cell proliferation andmigration and supporting further studies on these potentialnontoxic agents in novel targeted therapeutic strategy that arecapable of downregulation of miR-221

44 Evasion of Apoptosis Apoptotic evasion is considered tobe one of the main causes of chemotherapeutic and radio-therapeutic resistance that characterizes the most aggressivetumor [120] Cancer cells can resist apoptosis if they havean overexpression of antiapoptotic proteins involved in thetwo main apoptosis pathways extrinsic and intrinsic Theextrinsic pathway is regulated mainly by ldquodeath receptorsrdquoof the TNF-receptor family while the intrinsic pathway isregulated by Bcl-2 proteins Various anticancer drugs such asantimetabolites DNA cross-linking and intercalating agentsalkylating agents topoisomerase III inhibitors and TKIshave been reported to induce intrinsic or extrinsic apoptoticresponse in tumor cells resulting in caspases activation [121]Although the extrinsic and the intrinsic apoptosis pathwaysare activated by different stimuli both these pathways canbe regulated by specific miRNAs For example upregulationof Bcl-2 directly induced by miR-21 is associated withapoptosis chemoresistance to gemcitabine and proliferationof MIA PaCa-2 cells [110] Using western blot and luciferaseactivity assay Bcl-2 was identified also as a target ofmiR-148aand the expression of Bcl-2 lacking in 31015840UTR could abrogatethe proapoptotic function of miR-148a in PANC-1 and AsPC-1 cells [122] Similarly exogenous expression of miR-204 andmiR-320 reduced the protein level of their targets Bcl-2 andMcl-1 respectivelyMcl-1 is an antiapoptoticmember of Bcl-2family and induction of miR-320 activity leads to apoptosisthrough Mcl-1 suppression sensitizing cholangiocarcinomacells to 5-FU [123] However miR-204 was also reported to besignificantly downregulated in gemcitabine-resistant PDAC[124] and Li et al identified the role of the entire miR-200family of miRNAs in gemcitabine-resistant PDAC cells [125]

Conversely miR-17-5p downregulates the proapoptoticmember of the Bcl-2 protein family Bim and PDAC cellstransfected with miR-17-5p inhibitor showed growth inhibi-tion spontaneous apoptosis higher caspase-3 activation andincreased chemosensitivity to gemcitabine [126] Pathwaysdelivering an antiapoptotic signal such as PI3KAkt playalso a pivotal role in the balance between proapoptoticand survival signals which determines the fate of cancercells An increased miR-21 expression has been associated


with the activation of PI3KAKTmTOR pathway whilecombination of anti-miR-21 strategies with drugs target-ing PI3KAKTmTOR pathway reduced pAKT levels andenhanced apoptosis when used in combination with gem-citabine [127] Importantly the antiapoptotic role of miR-21 is possibly tumor specific with inhibition of miR-21increasing sensitivity and apoptosis induction by gemcitabinein PDAC and cholangiocarcinoma but not in colon cancercells [127] This suggests that its oncogenic properties couldbe cell and tissue dependent and that its potential role inchemoresistance should be contextualized with respect to thetumor type and the treatment [128]

5 miRNA in PDAC Resistance to ConventionalTherapy and Target Therapy

Pancreatic cancer is a genetically heterogenous disease witha very limited response to most treatments [129] includingboth conventional (also known as standard-dose chemother-apy which includes chemotherapeutic agents and regimensthat have been in use from the past 15 to 40 years) and targetedtherapies (a newer type of cancer treatment that uses drugs orother substances tomore precisely identify specificmoleculesinvolved in cell growth and survival and attack cancer cells)as described in the following paragraphs

51 Conventional Chemotherapy Conventional chemother-apy also known as standard-dose chemotherapy includeschemotherapeutic agents and regimens that have been in usefrom the past 15 to 40 years The three different therapeuticoptions for PDAC in the metastatic setting include gemc-itabine as monotherapy or in combinations the combinationof 5-FU leucovorin irinotecan and oxaliplatin (FOLFIRI-NOX) and the most recent combination of gemcitabine withnab-paclitaxel Although only 20 of patients present withlocalized disease amenable to potentially curative resectionon the basis of a few randomized trials [130ndash132] the currentaccepted standard of care is adjuvant gemcitabine or 5-FU chemotherapy while there have been no conclusionsregarding the role or timing of adjuvant chemoradiation[133]

52 Gemcitabine Monotherapy and Gemcitabine-Based Com-binations Since 1997 gemcitabine is being used inmetastaticPDAC Patients receiving gemcitabine have amedian survivalof 62 months and a 1-year survival rate of 20 [134]Meta-analysis of randomized trials with a combination ofgemcitabine and platinum analogues or of gemcitabine andcapecitabine suggested a survival benefit for these combi-nations for patients with a good performance status [135ndash137] In contrast an Italian phase III trial examining gemc-itabine and cisplatin did not confirm a survival benefit forthis combination [138] In a retrospective study on laser-microdissected PDAC specimens patients with high miR-21expression had a significantly shorter overall survival both inthe metastatic and in the adjuvant setting Multivariate anal-ysis confirmed the prognostic significance of miR-21 [127]The reduced expression ofmiR-21 was associated with benefitfrom gemcitabine treatment in two independent cohorts of

PDAC patients [139 140] as well as in a cohort of intraductalpapillarymucinous neoplasms (IPMNs) of the pancreas [141]These results might be explained by the effects of miR-21expression on certain phenotypic characteristics in PDACcell lines [139 142] Overexpression of miR-21 promotes cellproliferation increases the metastatic ability through expres-sion of matrix metalloproteinase-2 and metalloproteinase-9 as well as VEGF and decreases gemcitabine sensitivitywhereas miR-21 repression delivers the opposite results [143]Furthermore as reported in the previous chapters Hwanget al [139] and Dong et al [144] provided experimentalevidence for a role of miR-21 in chemoresistance thoroughmodulation of apoptosis by directly regulating Bcl-2 andPTEN expression More recently Frampton et al identifiedthree miRNAs (miR-21 miR-23a and miR-27a) that actedas cooperative repressors of a network of tumor suppressorgenes that included PDCD4 BTG2 andNEDD4L [145] In 91PDAC samples from PDAC radically resected patients highlevels of a combination of thesemiRNAswere associated withshorter survival times Thus high expressors of this triplemiRNA combination (miR-2123a27a) may be identifiedas having a much worse prognosis and may benefit fromanti-miRNA therapy although the best way to deliver sucha treatment and potential off-target effects are unknownAnother recent study demonstrated that miR-10b might be anovel diagnostic and predictive biomarker for PDAC [146]MiR-10b is indeed overexpressed in PDAC patients andreduced expression of miR-10b was associated with improvedresponse tomultimodality neoadjuvant therapy likelihood ofsurgical resection delayed time to metastasis and increasedsurvival [146] Finally several studies reported miR-155among the miRNA which are commonly overexpressed inPDACs and their precursor lesions [147] and although onlyone study reported that its elevated expression correlatedwith shorter survival [84] Xia et al [148] demonstrated thatgemcitabine treatment induced the expression of miR-155in PDAC cells suggesting its role in acquired chemoresis-tance Other miRNAs that have been linked to gemcitabinechemoresistance in PDAC are reported in Table 1

Gemcitabine plus nanoparticle albumin-bound nab-paclitaxel represents a novel acceptable alternative toFOLFIRINOX This combined therapy was associated withsignificantly higher objective response rate (23) and signifi-cantly longer median overall (85 months) and progression-free survival (55 months) in comparison to gemcitabinealone [149] Combination treatment with gemcitabine andnab-paclitaxel increases intratumoral gemcitabine levelsattributable to a marked decrease in the primary gemcitabinemetabolizing enzyme cytidine deaminase Correspondinglypaclitaxel reduced the levels of cytidine deaminase proteinin cultured cells through reactive oxygen species-mediateddegradation resulting in the increased stabilization of gem-citabine [150] Nab-paclitaxel alone or in combination withgemcitabine has been demostrated to reduce the desmo-plastic stroma [151] Moreover it is hypothesized that thealbumin-bound nab-paclitaxel may selectively accumulate inthe pancreatic stroma via its binding to secreted protein acidicand rich in cysteine (SPARC) matricellular glycoproteinwhich binds albumin and is overexpressed in tumor stroma


Table 1 Selected miRNA candidates which are correlated to gemcitabine chemoresistance in pancreatic cancer

miRNA Expression Targets ReferencemiR-21 Upregulated EGFR HER2neu PDCD4 BCL2 PTEN TIMP2 and TIMP3 [139 142]miR-222 and miR-221 Upregulated p27 PUMA PTEN and Bim [84 185]miR-10a and miR-10b Upregulated HOXB8 HOXA1 [186 187]miR-214 Upregulated PTEN ING4 [188 189]mir-320c Upregulated SMARCC1 [190]miR-155 Upregulated PI3K SMG-1 [148]miR-34∘ Downregulated BCL-2 [43]Let-7 Downregulated E2F2 c-Myc KRAS and MAPK [125]miR-142-5p Downregulated Unknown [124]miR-204 Downregulated MIC-1 [124]miR-200a miR-200b and miR-200c Downregulated EP300 [125 191]

[57] High SPARC expression has been correlated to poorsurvival outcome and has been suggested as a possible pre-dictive biomarker for nab-paclitaxel in the phase-II trial [151]However no data on SPARC are available from the phase IIItrial andNeesse et al showed that the effects of nab-paclitaxelwere largely dose-dependent and that SPARC expression inthe tumor stroma did not influence drug accumulation in aPDACmouse model Further studies are therefore warrantedto evaluate tissue and plasma SPARC expression as a potentialpredictive biomarker for nab-paclitaxel [11]

No data are available on miRNA affecting nab-paclitaxelbut several miRNAs have been associated to resistance topaclitaxel Regarding miRNA potentially affecting the drugtarget TUBB3 has been unraveled as a target for miR-200c in ovarian and endometrial cancer cells and theectopic expression of this miRNA downregulated TUBB3and enhanced sensitivity to microtubule-targeting agentsincluding paclitaxel [152]

As example ofmiRNAaffecting survival pathwaymiR-17-5p has been identified as one of most significantly downregu-lated miRNAs in paclitaxel-resistant lung cancer cells whichmight cause upregulation of beclin 1 gene one of the mostimportant autophagy modulators [153] Moreover miRNAmiR-17-5p which is a member of the miR-17-92 cluster isupregulated in pancreatic cancer and some present findingssuggest that miR-17-5p plays important roles in pancreaticcarcinogenesis and cancer progression and is associated witha poor prognosis in pancreatic cancer [154]

53 FOLFIRINOX (5-FU Leucovorin Irinotecan and Oxali-platin) A phase III trial using FOLFIRINOX regimen inPDAC patients has shown a response rate of 316 a mediansurvival of 111 months [155] Therefore FOLFIRINOX pro-tocol confers a significant improvement in the overall sur-vival in stage IV PDAC and can be considered as a noveltherapeutic option for patients with a good performancestatus [136] No predictive biomarkers are actually used inclinical practice but a few studies suggested the role ofcandidate miRNAs to predict the sensitivityresistance to 5-FU and the other drugs in this regimen 5-FU activity mightindeed depend on the expression of its target TS or by the

modulation of cell cycle and apoptosis induction by severalmiRNAs as reported above

Interestingly a pharmacogenetic study evaluated 18 poly-morphisms both in miRNA-containing genomic regions(primary and precursor miRNA) and in genes related tomiRNA biogenesis with outcome in metastatic colorectalcancer patients treated with 5-FU and irinotecan [156] Asignificant association with tumor response and time toprogression was observed for the SNP rs7372209 in pri-miR26a-1 The genotypes CC and CT were favorable whencompared with the TT variant genotype Similarly the SNPrs1834306 located in the pri-miR-100 gene significantlycorrelated with a longer time to progression

54 Targeted Therapy From its introduction cancerchemotherapy has been encumbered by its poor selectivitybecause most antineoplastic drugs are toxic also to fast-replicating cells of the blood compartment skin cellsand gastrointestinal tract lining cells This unsatisfactorysituation and the development of technology leadingto the sequencing of the genome have driven intensiveresearches and development over the last few decadestowards more specific and less toxic anticancer drugsthat block the growth and spread of cancer by interferingwith specific molecules involved in tumor growth andprogression and are therefore called ldquotargeted therapiesrdquoSome of these therapeutic regimens especially designedto intercept deregulated dominant oncogenes have provento be effective treatment in ldquooncogene addictedrdquo tumors[157] In particular the epidermal growth factor receptor(EGFR) has been successfully targeted either by mAbs orsmall molecules inhibiting the tyrosine kinase domainThe mAb cetuximab blocks the extracellular domain ofEGFR thereby competing with the ligands and resultingin the inhibition of the receptor This mAb is approvedfor the treatment of advanced colorectal cancer while theEGFR-TKIs gefitinib and erlotinib have been approvedas upfront therapy replacing chemotherapy in late-stageNSCLC patients harboring activating-EGFR mutations

55 Anti-EGFR Therapy in PDAC The SWOG group con-ducted a randomized Phase III clinical trial randomizing


patients with stages III-IV PDAC to receive either gemc-itabine alone or in combination with cetuximab which didnot improve the clinical outcome Negative results for thiscombination were also observed in the adjuvant setting [158]Similarly other EGFR and HER2 targeted therapies includ-ing trastuzumab and lapatinib have not shown a survivalbenefit in PDAC patients [136] In contrast a combinationof gemcitabine and erlotinib has been approved for use bythe United States Food and Drug Administration (FDA)and European Medicines Agency (EMEA) as a treatment forPDAC patients on the basis of a randomized trial showing aoverall gain inmedian survival of 2 weeks [159] Examinationof K-RAS mutational status and EGFR gene copy numberin 26 of patients from this trial failed to identify eitherchange as molecular predictors of response [160] Howeveraccumulating evidence suggests that dysregulation of specificmiRNAs may be involved in the acquisition of cancer cellresistance to EGFR-targeted agents In particular miR-7emerged as a critical modulator of a regulatory networkfor EGFR signaling in lung cancer cells with the abilityof coordinately downregulating the expression of severalmembers of the EGFR signaling cascade [161] The bindingof c-Myc to the miR-7 promoter enhanced its activitywhile ectopic miR-7 promoted cell growth and orthotopictumor formation in nude mice In these models quantitativeproteomic analysis revealed that miR-7 decreased levels ofthe Ets2 transcriptional repression factor ERF which is adirect target of miR-7 Accordingly the inhibition of miR-7expression suppressed EGFR mRNA and protein expressionin different lung cancer cell lines as well as the growth ofthe A549 lung adenocarcinoma cells [162] Of note miR-7 ispreferentially expressed in endocrine cells of the developingand adult human pancreas [163] However its role in theregulation of the insulin growth factor-1 receptor expressionmight affect the development of diabetes-associated PDAC[164]

Other studies in lung cancer cell lines showed thatdecreased miR-424 levels were indicative of increased resis-tance to erlotinib while the gefitinib resistant cell line-HCC827GR had a significant upregulation of miR-214 [165]The inhibition of miR-214 has been also correlated withdecreased apoptosis and miR-214 and PTEN were indeedinversely expressed while knockdown of miR-214 altered theexpression of PTEN and p-AKT resensitizingHCC827GR togefitinib MiR-214 has been identified as aberrantly expressedin PDAC and in vitro experiments showed that overexpres-sion of miR-214 decreased the sensitivity of the BxCP-3 cellsto gemcitabine [166]

The sensitivity to erlotinib was also predicted by a 13-gene miRNA signature identified in sensitive towards resis-tant lung cancer cell lines Ontological annotation of thesemiRNA (miR-140-3p miR-628-5p miR-518f miR-636 miR-301a miR-34c miR-224 miR-197 miR-205 miR135b miR-200b miR-200c and miR-141) and their potential targetsrevealed enrichment in the components of EMT includingWnt pathway which may explain the ability of this sig-nature to separate primary from metastatic tumor samplesas well as why the treatment with TGF1205731 modulated boththe expression of these miRNA and cell migration [167]

Interestingly EMT has been inversely correlated with theresponse of cancers to EGFR-targeted therapy and the TGF120573-mitogen-inducible gene 6-miR200 network orchestrates theEMT-associated kinase switch hat induces resistance to EGFRinhibitors in primary tumor xenografts of patient-derivedlung and pancreatic cancers carrying wild type EGFR [168]These data support the low ratio of Mig6 to miR200 as apromising predictive biomarker of the response of PDAC toEGFR-TKIs

6 miRNA Affecting PDACChemoresistance through Modulation ofIts Microenvironment

PDAC is characterized by a dense fibrotic stromalmatrix [11]composed of activated fibroblastsstellate cells inflammatorycells and other cell types such as endothelial cells PDAC isone of themost stroma-richmalignancies [169] Such desmo-plasia facilitates a mechanopathology known as growth-induced solid stress resulting in collapsed or compressedintratumoral blood vessels or lymphatics which respectivelylead to increased hypoxia and interstitial fluid pressure bothattenuating chemosensitivity [170]

Hypoxia is an essential component of the PDACmicroen-vironment as demonstrated by the characteristic avascularappearance on computed tomography and low oxygen ten-sion measurements of these tumors [171 172] Several studiesshowed that hypoxia plays a pivotal role in cancer progressionthrough induction of the hypoxia-inducible factor (HIF)which leads to increased expression of VEGF [173] Howeverhypoxic conditions in solid malignancies may also conferresistance to conventional radiation and chemotherapy [174]A functional link between hypoxia and miRNA expressionwas shown in colon and breast cancer cell lines [175] andin several other cancers including PDAC [176] MiR-210in particular is induced by hypoxia and the levels of thismiRNA are significantly higher elevated in PDAC patientsand may potentially serve as a useful biomarker for PDACdiagnosis [177] Furthermore miR-210 regulates the inter-action between PDAC cells and stellate cells promotingthe progression and chemoresistance of tumor cells [178]However the same study showed that stellate cells-inducedmiR-210 upregulation was inhibited by inhibitors of ERK andPI3KAkt pathways suggesting novel therapeutic combina-tions to counteract the interaction between stellate cells andPDAC which is at least in part responsible for the innateresistance to chemotherapy in pancreatic tumors by creatingbarriers against circulating therapeutic compounds

Hypoxia induces also the overexpression of miR-21 [179]while the treatment with the novel curcumin-derived ana-logue CDF downregulated the expression ofmiR-21 andmiR-210 as well as Nanog Oct4 and EZH2 mRNAs and theproduction of VEGF and IL-6 CDF also led to decreased cellmigrationinvasion angiogenesis and formation of pancre-atospheres under hypoxia supporting further studies on itsrole to overcome microenvironment-mediated chemoresis-tance of PDAC [180]

Other important factors playing a key role in PDACmicroenvironment and chemoresistance include cells of


the immune response and CSCs Recent data indicated thattumor-associated macrophages (TAMs) which are abundantin the microenvironment of PDAC secrete protumorigenicfactors that contribute not only to cancer progression anddissemination but also to chemoresistance by reducinggemcitabine-induced apoptosis In particular TAMs induceupregulation of cytidine deaminase the enzyme that metab-olizes gemcitabine following its transport into the cell [181]Moreover immune cells within the tumormicroenvironmentcan also activate pancreatic stellate cells which orchestratethe strong desmoplasia that characterizes PDAC and theresulting hypoxia [182] Importantly severalmiRNAs includ-ing miR-155 which is commonly overexpressed in PDACare involved in the control of macrophage production andactivation suggesting that reprogramming miRNA activityin TAMs andor their precursors might be effective forcontrolling tumor progressionchemosensitivity [183]

The existence of CSCs has been widely accepted tobe responsible for tumor aggressiveness in PDAC becauseCSCs have the capacity for increased cell growth cellmigrationinvasionmetastasis and also treatment resistanceHowever a recent study detected deregulated expressionof over 400 miRNAs including let-7 miR-30 miR-125band miR-335 in PDAC CD44+CD133+EpCAM+ (triple-marker-positive) CSCs [184] In the same study as a proof ofconcept knockdown of miR-125b resulted in the inhibitionof tumor aggressiveness consistent with the downregulationof CD44 EpCAM EZH2 and snail These results clearlysuggest the importance of miRNAs in the regulation of CSCscharacteristics and their potential role as novel targets toimprove therapeutic efficacy

7 Conclusions and Future Perspectives

PDAC is a common cause of cancer-death and has theworst prognosis of any major malignancy with less than5 of patients alive 5 years after diagnosis miRNAs havebeen documented to be involved in PDAC tumorigenesisprogression and recent evidence support their utility aspromising biomarkers in cancer diagnosis and prognosis Inthe present review we evaluated studies on the associationbetween candidate miRNAs and drug responseresistanceImportantly miRNAs remain intact in routinely collectedformalin-fixed paraffin-embedded tumor tissues and bioflu-ids and hopefully in the near future the expression profilesof specific miRNAs could provide new information aboutresistance of individual tumors to different treatments beforestarting therapy while modulation of the expression of othermiRNAs during treatment might offer a new tool for theprediction of acquired resistance

However as with previous studies on gene profilingmost emergingmiRNA signatures of chemoresistance are notoverlapping and no conclusive evidence has been obtainedon their clinical utility The controversial results might beexplained by different specimens (frozen versus paraffin-embedded micro- versus nonmicrodissected) experimentalplatforms used (quantitative PCR versus different miRNAarray or in situ hybridization systems) stage and regimens

as well as small sample size ethnic differences and lack ofappropriate statistical analyses

Additional studies in larger homogeneous populationswith validatedmethodology are needed to clarify these issuesFurthermore new analytical techniques such as next-generation sequencing may provide useful tools to under-stand the role of miRNA as effective biomarkers also startingfrom very small amount of tissues The next step will then beto use the emerging miRNAs as markers within prospectivetrials to see if they can aid clinical decision-making

Conflict of Interests

The authors confirm that this paper content has no conflict ofinterests

Acknowledgments

Thisworkwas partially supported by grants fromNWOStim-uleringfonds Open Access AIRC Start-Up grant and ItalianMinister of Research PRIN-2009 (to Elisa Giovannetti) Theauthors would like to thankDennis Poel (Vumc Amsterdam)and Alessandro Augusti (University of Pisa) for their supportand suggestions

References

[1] E Pennisi ldquoENCODE project writes eulogy for junk DNArdquoGenomics vol 337 no 6099 pp 1159ndash1161 2012

[2] B E Bernstein E Birney I Dunham E D Green C GunterandM Snyder ldquoAn integrated encyclopedia ofDNAelements inthe human genomerdquo Nature vol 489 no 7414 pp 57ndash74 2012

[3] S Djebali C A Davis A Merkel A Dobin and T R GingerasldquoLandscape of transcription in human cellsrdquo Nature vol 489no 7414 pp 101ndash108 2012

[4] S Khan D AnsarullahM Jaggi and S C Chauhan ldquoTargetingmicroRNAs in pancreatic cancermicroplayers in the big gamerdquoCancer Research vol 73 no 22 pp 6541ndash6547 2013

[5] J Hong H Zhang Y Kawase-Koga and T Sun ldquoMicroRNAfunction is required for neurite outgrowth of mature neuronsin the mouse postnatal cerebral cortexrdquo Frontiers in CellularNeuroscience vol 3 no 7 p 151 2013

[6] S P Srivastava D Koya and K Kanasaki ldquoMicroRNAs inkidney fibrosis and diabetic nephropathy roles on EMT andEndMTrdquo BioMed Research International vol 2013 Article ID125469 10 pages 2013

[7] J Lv H Liu Z Huang et al ldquoLong non-coding RNA identifi-cation over mouse brain development by integrative modelingof chromatin and genomic featuresrdquoNucleic Acids Research vol41 no 22 pp 10044ndash10061 2013

[8] H Ling M Fabbri and G A Calin ldquoMicroRNAs and othernon-coding RNAs as targets for anticancer drug developmentrdquoNature ReviewsDrugDiscovery vol 12 no 11 pp 847ndash865 2013

[9] M Li C Marin-Muller U Bharadwaj K Chow Q Yao andC Chen ldquoMicroRNAs control and loss of control in humanphysiology and diseaserdquo World Journal of Surgery vol 33 no4 pp 667ndash684 2009

[10] E Giovannetti A Erozenci J Smit R Danesi and G J PetersldquoMolecular mechanisms underlying the role of microRNAs(miRNAs) in anticancer drug resistance and implications for


clinical practicerdquo Critical Reviews in OncologyHematology vol81 no 2 pp 103ndash122 2012

[11] A Neesse PMichl K K Frese et al ldquoStromal biology and ther-apy in pancreatic cancerrdquo Gut vol 60 no 6 pp 861ndash868 2011

[12] S Tang J Bonaroti S Unlu et al ldquoSweating the small stuffmicroRNAs and genetic changes define pancreatic cancerrdquoPancreas vol 42 no 5 pp 740ndash759 2013

[13] R C Lee R L Feinbaum and V Ambros ldquoThe C elegans het-erochronic gene lin-4 encodes small RNAs with antisensecomplementarity to lin-14rdquoCell vol 75 no 5 pp 843ndash854 1993

[14] B J Reinhart F J Slack M Basson et al ldquoThe 21-nucleotidelet-7 RNA regulates developmental timing in Caenorhabditiselegansrdquo Nature vol 403 no 6772 pp 901ndash906 2000

[15] A Kozomara and S Griffiths-Jones ldquomiRBase integratingmicroRNA annotation and deep-sequencing datardquo NucleicAcids Research vol 39 pp 152ndash157 2011

[16] A Bhardwaj S Singh and A P Singh ldquoMicroRNA-basedcancer therapeutics big hope from small RNAsrdquoMolecular andCellular Pharmacology vol 2 no 5 pp 213ndash219 2010

[17] G A Calin and C M Croce ldquoMicroRNA signatures in humancancersrdquo Nature Reviews Cancer vol 6 no 11 pp 857ndash8662006

[18] I Bhatti A Lee J Lund and M Larvin ldquoSmall RNA a largecontributor to carcinogenesisrdquo Journal of GastrointestinalSurgery vol 13 no 7 pp 1379ndash1388 2009

[19] S Sethi S Ali S Sethi and F H Sarkar ldquoMicroRNAs inpersonalized cancer therapyrdquoClinical Genetics vol 86 no 1 pp68ndash73 2014

[20] S Ali A Ahmad S Banerjee et al ldquoGemcitabine sensitivitycan be induced in pancreatic cancer cells through modulationof miR-200 and miR-21 expression by curcumin or its analogueCDFrdquo Cancer Research vol 70 no 9 pp 3606ndash3617 2010

[21] DKong EHeathWChen et al ldquoLoss the acquisition of cancerstem cell signatures that are attenuated by BR-DIMrdquo PLoS ONEvol 7 no 3 Article ID e33729 2012

[22] S Gounaris-Shannon and T Chevassut ldquoThe role of miRNA inhaematological malignancyrdquo Bone Marrow Research vol 2013Article ID 269107 12 pages 2013

[23] Y Du M Liu J Gao and Z Li ldquoAberrant micrornas expressionpatterns in pancreatic cancer and their clinical translationrdquoCancer Biotherapy and Radiopharmaceuticals vol 28 no 5 pp361ndash369 2013

[24] Y Lee M Kim J Han et al ldquoMicroRNA genes are transcribedby RNA polymerase IIrdquoThe EMBO Journal vol 23 no 20 pp4051ndash4060 2004

[25] X Cai C H Hagedorn and B R Cullen ldquoHuman microRNAsare processed from capped polyadenylated transcripts that canalso function as mRNAsrdquo RNA vol 10 no 12 pp 1957ndash19662004

[26] S-L Lin J D Miller and S-Y Ying ldquoIntronic microRNA(miRNA)rdquo Journal of Biomedicine and Biotechnology vol 2006Article ID 26818 13 pages 2006

[27] Y Lee C Ahn J Han et al ldquoThe nuclear RNase III Droshainitiates microRNA processingrdquo Nature vol 425 no 6956 pp415ndash419 2003

[28] A M Denli B B J Tops R H A Plasterk R F Kettingand G J Hannon ldquoProcessing of primary microRNAs by theMicroprocessor complexrdquo Nature vol 432 no 7014 pp 231ndash235 2004

[29] R I Gregory K Yan G Amuthan et al ldquoThe microprocessorcomplex mediates the genesis of microRNAsrdquo Nature vol 432no 7014 pp 235ndash240 2004

[30] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[31] R F Ketting S E J Fischer E Bernstein T Sijen G J Hannonand R H A Plasterk ldquoDicer functions in RNA interference andin synthesis of small RNA involved in developmental timing inC elegansrdquo Genes and Development vol 15 no 20 pp 2654ndash2659 2001

[32] S W Knight and B L Bass ldquoA role for the RNase III enzymeDCR-1 in RNA interference and germ line development inCaenorhabditis elegansrdquo Science vol 293 no 5538 pp 2269ndash2271 2001

[33] T Du and P D Zamore ldquomicroPrimer the biogenesis andfunction ofmicroRNArdquoDevelopment vol 132 no 21 pp 4645ndash4652 2005

[34] L He and G J Hannon ldquoMicroRNAs small RNAs with a bigrole in gene regulationrdquo Nature Reviews Genetics vol 5 no 7pp 522ndash531 2004

[35] R Spizzo M I Almeida A Colombatti and G A Calin ldquoLongnon-coding RNAs and cancer a new frontier of translationalresearchrdquo Oncogene vol 31 no 43 pp 4577ndash4587 2012

[36] J T Mendell and E N Olson ldquoMicroRNAs in stress signalingand human diseaserdquo Cell vol 148 no 6 pp 1172ndash1187 2012

[37] M Esteller ldquoNon-coding RNAs in human diseaserdquo NatureReviews Genetics vol 12 no 12 pp 861ndash874 2011

[38] J R Lytle T A Yario and J A Steitz ldquoTarget mRNAs arerepressed as efficiently by microRNA-binding sites in the 51015840UTR as in the 31015840 UTRrdquo Proceedings of the National Academyof Sciences of the United States of America vol 104 no 23 pp9667ndash9672 2007

[39] U A Oslashrom F C Nielsen and A H Lund ldquoMicroRNA-10abinds the 5rsquoUTR of ribosomal protein mRNAs and enhancestheir translationrdquo Molecular Cell vol 30 no 4 pp 460ndash4712008

[40] Y Tay J Zhang A M Thomson B Lim and I RigoutsosldquoMicroRNAs toNanogOct4 and Sox2 coding regionsmodulateembryonic stem cell differentiationrdquo Nature vol 455 no 7216pp 1124ndash1128 2008

[41] S Vasudevan Y Tong and J A Steitz ldquoSwitching from repres-sion to activation microRNAs can up-regulate translationrdquoScience vol 318 no 5858 pp 1931ndash1934 2007

[42] G A Calin C D Dumitru M Shimizu et al ldquoFrequent dele-tions and down-regulation of micro-RNA genes miR15 andmiR16 at 13q14 in chronic lymphocytic leukemiardquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 24 pp 15524ndash15529 2002

[43] Q Ji X Hao M Zhang et al ldquoMicroRNA miR-34 inhibitshumanpancreatic cancer tumor-initiating cellsrdquoPLoSONE vol4 no 8 Article ID e6816 2009

[44] G A Calin C Sevignani C D Dumitru et al ldquoHuman mic-roRNA genes are frequently located at fragile sites and genomicregions involved in cancersrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no9 pp 2999ndash3004 2004

[45] L Ma F Reinhardt E Pan et al ldquoTherapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor modelrdquoNature Biotechnology vol 28 no 4 pp 341ndash347 2010

[46] S E Wojcik S Rossi M Shimizu et al ldquoNon-codingRNAsequence variations in human chronic lymphocytic leukemiaand colorectal cancerrdquo Carcinogenesis vol 31 no 2 Article IDbgp209 pp 208ndash215 2010


[47] M Fabbri N Valeri and G A Calin ldquoMicroRNAs andgenomic variations from Proteus tricks to Prometheus giftrdquoCarcinogenesis vol 30 no 6 pp 912ndash917 2009

[48] 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

[49] M Fabbri ldquoMicroRNAs and cancer epigeneticsrdquo Current Opin-ion in Investigational Drugs vol 9 no 6 pp 583ndash590 2008

[50] V Ambros ldquoMicroRNA pathways in flies and worms growthdeath fat stress and timingrdquo Cell vol 113 no 6 pp 673ndash6762003

[51] C M Croce ldquoCauses and consequences of microRNA dysreg-ulation in cancerrdquo Nature Reviews Genetics vol 10 no 10 pp704ndash714 2009

[52] P S Mitchell R K Parkin E M Kroh et al ldquoCirculatingmicroRNAs as stable blood-based markers for cancer detec-tionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 30 pp 10513ndash10518 2008

[53] C Le Sage R Nagel D A Egan et al ldquoRegulation of the p27Kip1tumor suppressor bymiR-221 andmiR-222 promotes cancer cellproliferationrdquoTheEMBO Journal vol 26 no 15 pp 3699ndash37082007

[54] S Sethi D Kong S Land G Dyson W A Sakr and F HSarkar ldquoComprehensive molecular oncogenomic profiling andmiRNA analysis of prostate cancerrdquo The American Journal ofTranslational Research vol 5 no 2 pp 200ndash211 2013

[55] M Humeau J Torrisani and P Cordelier ldquomiRNA in clinicalpractice pancreatic cancerrdquo Clinical Biochemistry vol 46 no10-11 pp 933ndash936 2013

[56] M Zoller ldquoPancreatic cancer diagnosis by free and exosomalmiRNArdquoWorld Journal of Gastrointestinal Pathophysiology vol4 no 4 pp 74ndash90 2013

[57] J A McCarroll S Naim G Sharbeen et al ldquoRole of pancreaticstellate cells in chemoresistance in pancreatic cancerrdquo Frontiersin Physiology vol 5 article 141 2014

[58] S Arora A Bhardwaj S Singh et al ldquoAn undesired effectof chemotherapy Gemcitabine promotes pancreatic cancercell invasiveness through reactive oxygen species-dependentnuclear factor120581b- and hypoxia-inducible factor 1120572-mediatedup-regulation of CXCR4rdquo Journal of Biological Chemistry vol288 no 29 pp 21197ndash21207 2013

[59] K Bhat F Wang Q Ma et al ldquoAdvances in biomarker researchfor pancreatic cancerrdquo Current Pharmaceutical Design vol 18no 17 pp 2439ndash2451 2012

[60] A Jimeno and M Hidalgo ldquoMolecular biomarkers theirincreasing role in the diagnosis characterization and therapyguidance in pancreatic cancerrdquoMolecular Cancer Therapeuticsvol 5 no 4 pp 787ndash796 2006

[61] A Stathis and M J Moore ldquoAdvanced pancreatic carcinomacurrent treatment and future challengesrdquo Nature Reviews Clini-cal Oncology vol 7 no 3 pp 163ndash172 2010

[62] C Partensky ldquoToward a better understanding of pancreaticductal adenocarcinoma glimmers of hoperdquo Pancreas vol 42no 5 pp 729ndash739 2013

[63] S Jones X Zhang D W Parsons et al ldquoCore signaling path-ways in human pancreatic cancers revealed by global genomicanalysesrdquo Science vol 321 no 5897 pp 1801ndash1806 2008

[64] E Lou S Subramanian and C J Steer ldquoPancreatic cancermodulation of KRAS MicroRNAs and intercellular communi-cation in the setting of tumor heterogeneityrdquo Pancreas vol 42no 8 pp 1218ndash1226 2013

[65] A Drakaki and D Iliopoulos ldquoMicroRNA-gene signaling path-ways in pancreatic cancerrdquo Biomedical Journal vol 36 no 5 pp200ndash208 2013

[66] A Young J Lyons A L Miller V T Phan I R Alarcon and FMcCormick ldquoRas signaling and therapiesrdquo Advances in CancerResearch vol 102 pp 1ndash17 2009

[67] M C Du Rieu J Torrisani J Selves et al ldquoMicroRNA-21 isinduced early in pancreatic ductal adenocarcinoma precursorlesionsrdquo Clinical Chemistry vol 56 no 4 pp 603ndash612 2010

[68] W Zhao S Yu Z Lu Y Ma Y Gu and J Chen ldquoThe miR-217microRNA functions as a potential tumor suppressor in pancre-atic ductal adenocarcinoma by targetingKRASrdquoCarcinogenesisvol 31 no 10 pp 1726ndash1733 2010

[69] S Yu Z Lu C Liu et al ldquomiRNA-96 suppresses KRAS andfunctions as a tumor suppressor gene in pancreatic cancerrdquoCancer Research vol 70 no 14 pp 6015ndash6025 2010

[70] L R Jiao A E Frampton J Jacob et al ldquoMicroRNAs targetingoncogenes are down-regulated in pancreatic malignant trans-formation from benign tumorsrdquo PLoS ONE vol 7 no 2 ArticleID e32068 2012

[71] M Komoto B Nakata R Amano et al ldquoHER2 overexpressioncorrelates with survival after curative resection of pancreaticcancerrdquo Cancer Science vol 100 no 7 pp 1243ndash1247 2009

[72] A Chou N Waddell M J Cowley et al ldquoClinical and molec-ular characterization of HER2 amplified pancreatic cancerrdquoGenome Medicine vol 5 no 8 article 78 2013

[73] M Yan B A Parker R Schwab and R Kurzrock ldquoHER2 aber-rations in cancer implications for therapyrdquo Cancer TreatmentReviews vol 40 no 6 pp 770ndash780 2014

[74] R Talar-Wojnarowska and E Malecka-Panas ldquoMolecularpathogenesis of pancreatic adenocarcinoma potential clinicalimplicationsrdquo Medical Science Monitor vol 12 no 9 pp 186ndash193 2006

[75] M Fassan M Pizzi S Realdon et al ldquoThe HER2-miR125a5pmiR125b loop in gastric and esophageal carcinogenesisrdquoHumanPathology vol 44 no 9 pp 1804ndash1810 2013

[76] J Harder G Ihorst V Heinemann et al ldquoMulticentre phaseII trial of trastuzumab and capecitabine in patients with HER2overexpressing metastatic pancreatic cancerrdquo British Journal ofCancer vol 106 no 6 pp 1033ndash1038 2012

[77] M Schutte R H Hruban J Geradts et al ldquoAbrogation of theRbp16 tumor-suppressive pathway in virtually all pancreaticcarcinomasrdquoCancer Research vol 57 no 15 pp 3126ndash3130 1997

[78] S R Hustinx R H Hruban L M Leoni et al ldquoHomozygousdeletion of the MTAP gene in invasive adenocarcinoma of thepancreas and in periampullary cancer a potential new targetfor therapyrdquo Cancer Biology and Therapy vol 4 no 1 pp 83ndash86 2005

[79] G Andreotti and D T Silverman ldquoOccupational risk factorsand pancreatic cancer a review of recent findingsrdquo MolecularCarcinogenesis vol 51 no 1 pp 98ndash108 2012

[80] S M Gapstur P H Gann W Lowe K Liu L Colangelo andA Dyer ldquoAbnormal glucose metabolism and pancreatic cancermortalityrdquo Journal of the AmericanMedical Association vol 283no 19 pp 2552ndash2558 2000

[81] S T Chari C L Leibson K G Rabe et al ldquoPancreaticcancer-associated diabetes mellitus prevalence and temporal


association with diagnosis of cancerrdquoGastroenterology vol 134no 1 pp 95ndash101 2008

[82] D Nalls S Tang M Rodova R K Srivastava and S ShankarldquoTargeting epigenetic regulation of mir-34a for treatment ofpancreatic cancer by inhibition of pancreatic cancer stem cellsrdquoPLoS ONE vol 6 no 8 Article ID e24099 2011

[83] M Gironella M Seux M J Xie et al ldquoTumor protein 53induced nuclear protein 1 expression is repressed by miR-155 and its restoration inhibitspancreatic tumor developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 41 pp 16170ndash16175 2007

[84] T Greither L F Grochola A Udelnow C Lautenschlager PWurl and H Taubert ldquoElevated expression of microRNAs 155203 210 and 222 in pancreatic tumors is associated with poorersurvivalrdquo International Journal of Cancer vol 126 no 1 pp 73ndash80 2010

[85] R Derynck and Y E Zhang ldquoSmad-dependent and Smad-independent pathways in TGF-120573 family signallingrdquoNature vol425 no 6958 pp 577ndash584 2003

[86] C A Lacobuzio-Donahue B Fu S Yachida et al ldquoDPC4 genestatus of the primary carcinoma correlates with patterns offailure in patients with pancreatic cancerrdquo Journal of ClinicalOncology vol 27 no 11 pp 1806ndash1813 2009

[87] A Blackford O K Serrano C L Wolfgang et al ldquoSMAD4genemutations are associatedwith poor prognosis in pancreaticcancerrdquo Clinical Cancer Research vol 15 no 14 pp 4674ndash46792009

[88] L Li Z Li X Kong et al ldquoDown-regulation of MicroRNA-494via loss of SMAD4 increases FOXM1 and 120573-catenin signaling inpancreatic ductal adenocarcinoma cellsrdquo Gastroenterology vol147 no 2 pp 485e18ndash497e18 2014

[89] J Hao S Zhang Y Zhou C Liu X Hu and C ShaoldquoMicroRNA 421 suppresses DPC4Smad4 in pancreatic cancerrdquoBiochemical and Biophysical Research Communications vol406 no 4 pp 552ndash557 2011

[90] J Hao S Zhang Y Zhou X Hu and C Shao ldquoMicroRNA483-3p suppresses the expression of DPC4Smad4 in pancreaticcancerrdquo FEBS Letters vol 585 no 1 pp 207ndash213 2011

[91] M Goggins M Sehutte J Lu et al ldquoGermline BRCA2 genemutations in patients with apparently sporadic pancreaticcarcinomasrdquo Cancer Research vol 56 no 23 pp 5360ndash53641996

[92] G Lal G Liu B Schmocker et al ldquoInherited predisposition topancreatic adenocarcinoma role of family history and germ-line p16 BRCA1 and BRCA2 mutationsrdquo Cancer Research vol60 no 2 pp 409ndash416 2000

[93] B Xia Q Sheng K Nakanishi et al ldquoControl of BRCA2 cellularand clinical functions by a nuclear partner PALB2rdquo MolecularCell vol 22 no 6 pp 719ndash729 2006

[94] E P Slater P Langer E Niemczyk et al ldquoPALB2 mutations inEuropean familial pancreatic cancer familiesrdquoClinical Geneticsvol 78 no 5 pp 490ndash494 2010

[95] S Jones R H Hruban M Kamiyama et al ldquoExomic sequenc-ing identifies PALB2 as a pancreatic cancer susceptibility generdquoScience vol 324 no 5924 p 217 2009

[96] M Tanic K Yanowski G Gomez-Lopez et al ldquoMicroRNAexpression signatures for the prediction of BRCA12 mutation-associated hereditary breast cancer in paraffin-embeddedformalin-fixed breast tumorsrdquo International Journal of Cancer2014

[97] R Warsame and A Grothey ldquoTreatment options for advancedpancreatic cancer a reviewrdquo Expert Review of Anticancer Ther-apy vol 12 no 10 pp 1327ndash1336 2012

[98] C LWolfgang JMHerman D A Laheru et al ldquoRecent prog-ress in pancreatic cancerrdquo CA A Cancer Journal for Cliniciansvol 63 no 5 pp 318ndash348 2013

[99] MMGottesman T Fojo and S E Bates ldquoMultidrug resistancein cancer role of ATP-dependent transportersrdquoNature ReviewsCancer vol 2 no 1 pp 48ndash58 2002

[100] M Garofalo and C M Croce ldquoMicroRNAs as therapeutic tar-gets in chemoresistancerdquo Drug Resistance Updates vol 16 no3ndash5 pp 47ndash59 2013

[101] A Gisel M Valvano I G El Idrissi et al ldquomiRNAs for thedetection of multidrug resistance overview and perspectivesrdquoMolecules vol 19 no 5 pp 5611ndash5623 2014

[102] H Zhu H Wu X Liu et al ldquoRole of MicroRNA miR-27a andmiR-451 in the regulation of MDR1P-glycoprotein expressionin human cancer cellsrdquo Biochemical Pharmacology vol 76 no5 pp 582ndash588 2008


[104] V Boni N Bitarte I Cristobal et al ldquomiR-192miR-215influence 5-fluorouracil resistance through cell cycle-mediatedmechanisms complementary to its post-transcriptional thy-midilate synthase regulationrdquo Molecular Cancer Therapeuticsvol 9 no 8 pp 2265ndash2275 2010

[105] E Giovannetti A van der Velde N Funel et al ldquoHigh-throughputmicroRNA (miRNAs) arrays unravel the prognosticrole of MiR-211 in pancreatic cancerrdquo PLoS ONE vol 7 no 11Article ID e49145 2012

[106] M Maftouh A Avan N Funel et al ldquomiR-211 modulates gem-citabine activity through downregulation of ribonucleotidereductase and inhibits theInvasive behavior of pancreatic cancercellsrdquo Nucleosides Nucleotides and Nucleic Acids vol 33 no 4ndash6 pp 384ndash393 2014

[107] Y D Bhutia S W Hung M Krentz et al ldquoDifferential proc-essing of let-7a precursors influences RRM2 expression andchemosensitivity in pancreatic cancer role of LIN-28 and SEToncoproteinrdquo PLoS ONE vol 8 no 1 Article ID e53436 2013

[108] D Matthaios P Zarogoulidis I Balgouranidou E Chatzakiand S Kakolyris ldquoMolecular pathogenesis of pancreatic cancerand clinical perspectivesrdquo Oncology vol 81 no 3-4 pp 259ndash272 2011

[109] C M Ribic D J Sargent M J Moore et al ldquoTumor mic-rosatellite-instability status as a predictor of benefit from flu-orouracil-based adjuvant chemotherapy for colon cancerrdquo TheNew England Journal of Medicine vol 349 no 3 pp 247ndash2572003

[110] J Dong Y P Zhao L Zhou T P Zhang and G Chen ldquoBcl-2via a direct interaction is associated with apoptosisand chemo-resistancerdquo Archives of Medical Research vol 42 no 1 pp 8ndash142011

[111] N Valeri P Gasparini C Braconi et al ldquoMicroRNA-21 inducesresistance to 5-fluorouracil by down-regulating human DNAMutS homolog 2 (hMSH2)rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no49 pp 21098ndash21103 2010

[112] A E Frampton E Giovannetti N B Jamieson et al ldquoA mic-roRNA meta-signature for pancreatic ductal adenocarcinomardquoExpert Review of Molecular Diagnostics vol 14 no 3 pp 267ndash271 2014


[113] M Z Ma X Kong M Z Weng et al ldquoCandidate microRNAbiomarkers of pancreatic ductal adenocarcinoma meta-analysis experimental validationand clinical significancerdquoJournal of Experimental amp Clinical Cancer Research vol 32 no1 p 71 2013

[114] C J Sherr ldquoThe pezcoller lecture cancer cell cycles revisitedrdquoCancer Research vol 60 no 14 pp 3689ndash3695 2000

[115] 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

[116] M Vogt J Munding M Gruner et al ldquoFrequent concomitantinactivation of miR-34a and miR-34bc by CpG methylation incolorectal pancreatic mammary ovarian urothelial and renalcell carcinomas and soft tissue sarcomasrdquo Virchows Archiv vol458 no 3 pp 313ndash322 2011

[117] DNalls SN TangMRodova R K Srivastava and S ShankarldquoTargeting epigenetic regulation of miR-34a for treatment ofpancreatic cancer by inhibition of pancreatic cancerstem cellsrdquoPLoS ONE vol 6 no 8 Article ID e24099 2011

[118] T Kawaguchi S Komatsu D Ichikawa et al ldquoClinical impactof circulating miR-221 in plasma of patients with pancreaticcancerrdquo British Journal of Cancer vol 108 no 2 pp 361ndash3692013

[119] S Sarkar H Dubaybo S Ali et al ldquoDown-regulation of miR-221 inhibits proliferation of pancreatic cancer cells through up-regulation of PTEN p27kip1 p57kip2 and PUMArdquo AmericanJournal of Cancer Research vol 3 no 5 pp 465ndash477 2013

[120] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000

[121] S H Kaufmann and W C Earnshaw ldquoInduction of apoptosisby cancer chemotherapyrdquo Experimental Cell Research vol 256no 1 pp 42ndash49 2000

[122] R Zhang M Li W Zang et al ldquoMiR-148a regulates the growthand apoptosis in pancreatic cancer by targeting CCKBR andBcl-2rdquo Tumour Biology vol 35 no 1 pp 837ndash844 2014

[123] L Chen H Yan W Yang et al ldquoThe role of microRNA expres-sion pattern in human intrahepatic cholangiocarcinomardquo Jour-nal of Hepatology vol 50 no 2 pp 358ndash369 2009

[124] K Ohuchida K Mizumoto T Kayashima et al ldquoMicroRNAexpression as a predictive marker for gemcitabine responseafter surgical resection of pancreatic cancerrdquo Annals of SurgicalOncology vol 18 no 8 pp 2381ndash2387 2011

[125] Y Li T G Vandenboom II D Kong et al ldquoUp-regulation ofmiR-200 and let-7 by natural agents leads to the reversal ofepithelial-to-mesenchymal transition in gemcitabine-resistantpancreatic cancer cellsrdquo Cancer Research vol 69 no 16 pp6704ndash6712 2009

[126] H Yan W Liu W Sun et al ldquoMiR-17-5p inhibitor enhanceschemosensitivity to gemcitabine via upregulating Bim expres-sion in pancreatic cancer cellsrdquo Digestive Diseases and Sciencesvol 57 no 12 pp 3160ndash3167 2012

[127] E Giovannetti N Funel G J Peters et al ldquoMicroRNA-21in pancreatic cancer correlation with clinical outcome andpharmacologic aspects underlying its role in the modulation ofgemcitabine activityrdquo Cancer Research vol 70 no 11 pp 4528ndash4538 2010

[128] J Li H Huang L Sun et al ldquoMiR-21 indicates poor prognosisin tongue squamous cell carcinomas as an apoptosis inhibitorrdquoClinical Cancer Research vol 15 no 12 pp 3998ndash4008 2009

[129] E Costello W Greenhalf and J P Neoptolemos ldquoNewbiomarkers and targets in pancreatic cancer and their appli-cation to treatmentrdquo Nature Reviews Gastroenterology andHepatology vol 9 no 8 pp 435ndash444 2012

[130] J P Neoptolemos D D Stocken H Friess et al ldquoA randomizedtrial of chemoradiotherapy and chemotherapy after resection ofpancreatic cancerrdquo The New England Journal of Medicine vol350 no 12 pp 1200ndash1210 2004

[131] J P Neoptolemos D D Stocken C Bassi et al ldquoAdjuvantchemotherapy with fluorouracil plus folinic acid vs gemcitabinefollowing pancreatic cancer resection a randomized controlledtrialrdquo JAMAmdashJournal of the American Medical Association vol304 no 10 pp 1073ndash1081 2010

[132] H Oettle S Post P Neuhaus et al ldquoAdjuvant chemother-apy with gemcitabine vs observation in patients undergoingcurative-intent resection of pancreatic cancer a randomizedcontrolled trialrdquo Journal of the American Medical Associationvol 297 no 3 pp 267ndash277 2007

[133] M D Goodman and M W Saif ldquoAdjuvant therapy for pancre-atic cancerrdquo Journal of Periodontology Online vol 15 no 2 pp87ndash90 2014

[134] A Sultana C T Smith D Cunningham N Starling J P Neop-tolemos and P Ghaneh ldquoMeta-analyses of chemotherapy forlocally advanced and metastatic pancreatic cancerrdquo Journal ofClinical Oncology vol 25 no 18 pp 2607ndash2615 2007

[135] V Heinemann S Boeck A Hinke R Labianca and C LouvetldquoMeta-analysis of randomized trials Evaluation of benefitfrom gemcitabine-based combination chemotherapy applied inadvanced pancreatic cancerrdquo BMC Cancer vol 8 article 822008

[136] D Cunningham I Chau D D Stocken et al ldquoPhase III ran-domized comparison of gemcitabine versus gemcitabine pluscapecitabine in patients with advanced pancreatic cancerrdquoJournal of Clinical Oncology vol 27 no 33 pp 5513ndash5518 2009

[137] T Seufferlein J B Bachet E Van cutsem and P RougierldquoPancreatic adenocarcinoma ESMO-ESDO clinical practiceguidelines for diagnosis treatment and follow-uprdquo Annals ofOncology vol 23 no 7 pp vii33ndashvii40 2012

[138] G Colucci R Labianca F Di Costanzo et al ldquoRandomizedphase III trial of gemcitabine plus cisplatin compared withsingle-agent gemcitabine as first-line treatment of patients withadvanced pancreatic cancer the GIP-1 studyrdquo Journal of ClinicalOncology vol 28 no 10 pp 1645ndash1651 2010

[139] JHHwang J Voortman EGiovannetti et al ldquoIdentification ofmicroRNA-21 as a biomarker for chemoresistance and clinicaloutcome following adjuvant therapy in resectable pancreaticcancerrdquo PLoS ONE vol 5 no 5 Article ID e10630 2010

[140] N B Jamieson D C Morran J P Morton et al ldquoMicroRNAmolecular profiles associated with diagnosis clinicopathologiccriteria and overall survival in patients with resectable pancre-atic ductal adenocarcinomardquo Clinical Cancer Research vol 18no 2 pp 534ndash545 2012

[141] S Caponi N Funel A E Frampton et al ldquoThe good the badand the ugly a tale of miR-101 miR-21 and miR-155 inpancreatic intraductal papillarymucinous neoplasmsrdquoAnnals ofOncology vol 24 no 3 pp 734ndash741 2013

[142] T Moriyama K Ohuchida K Mizumoto et al ldquoMicroRNA-21 modulates biological functions of pancreatic cancer cellsincluding their proliferation invasion and chemoresistancerdquoMolecular CancerTherapeutics vol 8 no 5 pp 1067ndash1074 2009

[143] J Park E J Lee C Esau and T D Schmittgen ldquoAntisenseinhibition of microRNA-21 or -221 arrests cell cycle induces


apoptosis and sensitizes the effects of gemcitabine in pancreaticadenocarcinomardquo Pancreas vol 38 no 7 pp 190ndash199 2009

[144] J Dong Y Zhao L Zhou T Zhang and G Chen ldquoBcl-2 upregulation induced by miR-21 via a direct interaction isassociated with apoptosis and chemoresistance in MIA PaCa-2 pancreatic cancer cellsrdquo Archives of Medical Research vol 42no 1 pp 8ndash14 2011

[145] A E Frampton L Castellano T Colombo et al ldquoMicroRNAscooperatively inhibit a network of tumor suppressor genes topromote pancreatic tumor growth andprogressionrdquo Gastroen-terology vol 146 no 1 pp 268ndash277 2014

[146] M Preis T B Gardner S R Gordon et al ldquoMicroRNA-10bexpression correlates with response to neoadjuvant therapy andsurvival in pancreatic ductal adenocarcinomardquo Clinical CancerResearch vol 17 no 17 pp 5812ndash5821 2011

[147] J K Ryu HMatthaeiM DalMolin et al ldquoElevatedmicroRNAmiR-21 levels in pancreatic cyst fluid are predictive of mucinousprecursor lesions of ductaladenocarcinomardquo Pancreatology vol11 no 3 pp 343ndash350 2011

[148] Q Xia Y Ishigaki X Zhao et al ldquoHuman SMG-1 is involvedin gemcitabine-induced primary microRNA-155BIC up-regu-lation in human pancreatic cancer PANC-1 cellsrdquo Pancreas vol40 no 1 pp 55ndash60 2011

[149] A Andriulli V Festa E Botteri et al ldquoNeoadjuvant preopera-tive gemcitabine for patients with localized pancreatic cancera meta-analysis of prospective studiesrdquo Annals of SurgicalOncology vol 19 no 5 pp 1644ndash1662 2012

[150] K K Frese A Neesse N Cook et al ldquoNab-paclitaxel potenti-ates gemcitabine activity by reducing cytidine deaminase levelsin a mouse model of pancreatic cancerrdquo Cancer Discovery vol2 no 3 pp 260ndash269 2012

[151] R Alvarez M Musteanu E Garcia-Garcia et al ldquoStromaldisrupting effects of nab-paclitaxel in pancreatic cancerrdquo BritishJournal of Cancer vol 109 no 4 pp 926ndash933 2013

[152] D R Cochrane N S Spoelstra E N Howe S K Nordeenand J K Richer ldquoMicroRNA-200c mitigates invasiveness andrestores sensitivity to microtubule-targeting chemotherapeuticagentsrdquo Molecular Cancer Therapeutics vol 8 no 5 pp 1055ndash1066 2009

[153] A Chatterjee D Chattopadhyay and G Chakrabarti ldquomiR-17-5p downregulation contributes to paclitaxel resistance of lungcancer cells through altering beclin1 expressionrdquoPLoSONE vol9 no 4 Article ID 95716 2014

[154] J Yu K Ohuchida K Mizumoto H Fujita K Nakata and MTanaka ldquoMicroRNA miR-17-5p is overexpressed in pancreaticcancer associated with a poor prognosis and involved incancercell proliferation and invasionrdquo Cancer Biology amp Therapy vol10 no 8 pp 748ndash757 2010

[155] T Conroy F Desseigne M Ychou et al ldquoFOLFIRINOX versusgemcitabine for metastatic pancreatic cancerrdquoTheNew EnglandJournal of Medicine vol 364 no 19 pp 1817ndash1825 2011

[156] V Boni R Zarate J C Villa et al ldquoRole of primary miRNApolymorphic variants in metastatic colon cancer patientstreated with 5-fluorouracil and irinotecanrdquo PharmacogenomicsJournal vol 11 no 6 pp 429ndash436 2011

[157] M E Gutierrez S Kummar andG Giaccone ldquoNext generationoncology drug development opportunities and challengesrdquoNature Reviews Clinical Oncology vol 6 no 5 pp 259ndash2652009

[158] H Fensterer C Schade-Brittinger H H Muller et al ldquoMul-ticenter phase II trial to investigate safety and efficacy of

gemcitabine combined with cetuximab as adjuvant therapy inpancreatic cancer (ATIP)rdquo Annals of Oncology vol 24 no 10pp 2576ndash2581 2013

[159] M J Moore D Goldstein J Hamm et al ldquoErlotinib plusgemcitabine compared with gemcitabine alone in patients withadvanced pancreatic cancer a phase III trial of the NationalCancer Institute of Canada Clinical Trials Grouprdquo Journal ofClinical Oncology vol 25 no 15 pp 1960ndash1966 2007

[160] A Jimeno C T Aik J Coffa et al ldquoCoordinated epidermalgrowth factor receptor pathway gene overexpression predictsepidermal growth factor receptor inhibitor sensitivity in pan-creatic cancerrdquo Cancer Research vol 68 no 8 pp 2841ndash28492008

[161] Y T Chou H H Lin Y C Lien et al ldquoEGFR promotes lungtumorigenesis by activating miR-7 through a RasERKMycpathway that targets the Ets2transcriptional repressor ERFrdquoCancer Research vol 70 no 21 pp 8822ndash8831 2010

[162] R J Webster K M Giles K J Price P M Zhang J S Mattickand P J Leedman ldquoRegulation of epidermal growth factorreceptor signaling in human cancer cells by microRNA-7rdquoJournal of Biological Chemistry vol 284 no 9 pp 5731ndash57412009

[163] V Bravo-Egana S Rosero R D Molano et al ldquoQuantitativedifferential expression analysis reveals miR-7 as major isletmicroRNArdquo Biochemical and Biophysical Research Communica-tions vol 366 no 4 pp 922ndash926 2008

[164] C Chakraborty C George Priya Doss and S BandyopadhyayldquomiRNAs in insulin resistance and diabetes-associated pan-creatic cancer the rsquominute and miraclelsquo moleculemoving as amonitor in the rsquogenomic galaxylsquordquo Current Drug Targets vol 14no 10 pp 1110ndash1117 2013

[165] Y S Wang Y H Wang H P Xia S W Zhou G Schmid-Bindert andC C Zhou ldquoMicroRNA-214 regulates the acquiredresistance to gefitinib via the PTENAKT pathway in EGFR-mutant cell linesrdquo Asian Pacific Journal of Cancer Preventionvol 13 no 1 pp 255ndash260 2012

[166] X J Zhang H Ye CW Zeng B He H Zhang and Y Q ChenldquoDysregulation of miR-15a and miR-214 in human pancreaticcancerrdquo Journal of Hematology amp Oncology vol 3 article 462010

[167] J L Bryant J Britson J M Balko et al ldquoA microRNA geneexpression signature predicts response to erlotinib in epithelialcancer cell lines and targets EMTrdquo British Journal of Cancer vol106 no 1 pp 148ndash156 2012

[168] E G Izumchenko X Chang C Michailidi et al ldquoThe TGF 120573-miR200-Mig6 pathway orchestrates the EMT-associated kinaseswitch that induces resistance to EGFR inhibitorsrdquo CancerResearch 2014

[169] C Feig A Gopinathan A Neesse D S Chan N Cook and DA Tuveson ldquoThe pancreas cancer microenvironmentrdquo ClinicalCancer Research vol 18 no 16 pp 4266ndash4276 2012

[170] T Stylianopoulos J D Martin V P Chauhan et al ldquoCausesconsequences and remedies for growth-induced solid stressin murine and human tumorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no38 pp 15101ndash15108 2012

[171] T A Mace A L Collins S E Wojcik C M Croce G B Lesin-ski and M Bloomston ldquoHypoxia induces the overexpressionof microRNA-21 in pancreatic cancer cellsrdquo Journal of SurgicalResearch vol 184 no 2 pp 855ndash860 2013


[172] A C Koong V K Mehta Q T Le et al ldquoPancreatic tumorsshow high levels of hypoxiardquo International Journal of RadiationOncology Biology Physics vol 48 no 4 pp 919ndash922 2000

[173] N Ferrara H Gerber and J LeCouter ldquoThe biology of VEGFand its receptorsrdquo Nature Medicine vol 9 no 6 pp 669ndash6762003

[174] K Yokoi and I J Fidler ldquoHypoxia increases resistance of humanpancreatic cancer cells to apoptosis induced by gemcitabinerdquoClinical Cancer Research vol 10 no 7 pp 2299ndash2306 2004

[175] R Kulshreshtha M Ferracin S E Wojcik et al ldquoA microRNAsignature of hypoxiardquoMolecular andCellular Biology vol 27 no5 pp 1859ndash1867 2007

[176] Z Lei B Li Z Yang et al ldquoRegulation of HIF-1120572 and VEGF bymiR-20b tunes tumor cells to adapt to the alteration of oxygenconcentrationrdquo PLoS ONE vol 4 no 10 Article ID e7629 2009

[177] A SHoXHuangHCao et al ldquoCirculatingmiR-210 as a novelhypoxia marker in pancreatic cancerrdquo Translational Oncologyvol 3 no 2 pp 109ndash113 2010

[178] T Takikawa A Masamune S Hamada E Nakano NYoshida and T Shimosegawa ldquomiR-210 regulates the interac-tion between pancreatic cancer cells and stellate cellsrdquo Biochem-ical and Biophysical Research Communications vol 437 no 3pp 433ndash439 2013


[180] B Bao S Ali A Ahmad et al ldquoHypoxia- induced aggressive-ness of pancreatic cancer cells is due to increased expressionof VEGF IL-6 andmiR-21 which can be attenuated by CDFtreatmentrdquo PLoS ONE vol 7 no 12 Article ID e50165 2012

[181] NWeizman Y Krelin A Shabtay-Orbach et al ldquoMacrophagesmediate gemcitabine resistance of pancreatic adenocarcinomaby upregulating cytidine deaminaserdquo Oncogene vol 33 no 29pp 3812ndash3819 2013

[182] A Evans and E Costello ldquoThe role of inflammatory cells infostering pancreatic cancer cell growth and invasionrdquo Frontiersin Physiology vol 3 Article ID Article 270 p 270 2012

[183] M L Squadrito M Etzrodt M De Palma and M J PittetldquoMicroRNA-mediated control of macrophages and its implica-tions for cancerrdquo Trends in Immunology vol 34 no 7 pp 350ndash359 2013

[184] B Bao S Ali A Ahmad et al ldquoDifferentially expressed miR-NAs in cancer-stem-like cells markers for tumor cell aggres-siveness of pancreatic cancerrdquo Stem Cells and Development vol23 no 16 pp 1947ndash1958 2014

[185] I G Papaconstantinou AMantaMGazouli et al ldquoExpressionofmicrornas in patients with pancreatic cancer and its prognos-tic significancerdquo Pancreas vol 42 no 1 pp 67ndash71 2013

[186] K Ohuchida K Mizumoto C Lin et al ldquoMicroRNA-10a isoverexpressed in human pancreatic cancer and involved inits invasiveness partially via suppression of the HOXA1 generdquoAnnals of Surgical Oncology vol 19 no 7 pp 2394ndash2402 2012

[187] T Setoyama X Zhang S Natsugoe and G A CalinldquomicroRNA-10b a new marker or the marker of pancreaticductal adenocarcinomardquo Clinical Cancer Research vol 17 no17 pp 5527ndash5529 2011

[188] X J Zhang H Ye CW Zeng B He H Zhang and Y Q ChenldquoDysregulation of miR-15a and miR-214 in human pancreaticcancerrdquo Journal of Hematology and Oncology vol 3 article 462010

[189] S Volinia G A Calin C Liu et al ldquoA microRNA 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

[190] Y Iwagami H Eguchi H Nagano et al ldquoMiR-320c regulatesgemcitabine-resistance in pancreatic cancer via SMARCC1rdquoBritish Journal of Cancer vol 109 no 2 pp 502ndash511 2013

[191] J Yu K Ohuchida K Mizumoto et al ldquoMicroRNA hsa-miR-200c is an independent prognostic factor in pancreatic cancerand its upregulation inhibits pancreatic cancer invasion butincreases cell proliferationrdquoMolecular Cancer vol 9 article 1692010

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


mechanisms within individual tumor cells which reduce theeffectiveness at their intended site of action such as increasedexpression of enzymes involved in drug catabolism or anti-apoptotic proteins [10]

The dense stromal reaction which characterizes mostPDACs has been frequently reported as the main causeof chemoresistance preventing the drugs from reachingtheir intended site of action [11] However detailed geneticanalyses have unraveled the pivotal mechanisms controllingpancreatic carcinogenesis and cluster analysis of recurrentlymutated genes has defined twelve different core pathwaysthat lead to aberrant signaling in PDAC cells [12] Suchstudies suggest that the best hope for the development ofagents targeting critical points in the altered pathways liesin the study of mechanisms involved in gene expressionregulation Therefore in the present review we summarizethe role of miRNAs in PDAC and focus on the miRNA-basedmechanisms of PDAC chemoresistance

2 Discovery of miRNAs andTheir Role in Cancer

The first miRNA molecule lin-4 was identified in 1993 byLee and collaborators [13] In 2000 Reinhart et al identifiedlethal-7 (let-7) another miRNA and discovered its role inthe posttranscriptional regulation of gene expression [14]Currently it has been reported that there are around 2600unique mature human miRNAs (miRBase version 20) [15]miRNAs regulate more than one-third of all human geneswhich suggest their remarkable influence on human biology[16] It is known that more than 50 of miRNA genes arelocalized within genomic regions that are either frequentlyamplified or deleted in different tumor types resulting inmiRNAs deregulation and aberrant expression [17 18] ThealteredmiRNAsmay have different effects on the tumors [19]Some of these miRNAs have been characterized as potentoncogenes (oncomiRs) while others have been identified astumor suppressors (tsmiRs) based on the consequences oftheir expression on the phenotype of several experimentalmodels [4] OncomiRs such as miR-21 are commonlyupregulated in cancer [20] while tsmiRs such as let-7 aredownregulated [21] resulting in unique combinations ofmiRNAs (ie overexpressed oncomiRs and underexpressedtsmiRs) characterizing different tumors [22]

The multiple roles of these miRNAs can be explained bystarting from the analysis of their biological synthesis andfunctions Biosynthesis of miRNAs is a multistep processinvolving both nuclear and cytoplasmic components [23]Initially they are transcribed in the nucleus by RNA poly-merase II into large RNA precursors called pri-miRNAs [24ndash26] which can be several hundreds to several thousands of ntin lengthThe first slicing step performed by the ribonuclease(RNase) III Drosha-DGCR8 (DiGeorge syndrome criticalregion 8) enzyme leads to the formation of 70-base longpre-miRNAs [27ndash29] Pre-miRNAs are actively transportedfrom the nucleus to the cytoplasm by Exportin-5 [30] wherethey are subjected to further cleavage by the RNase Dicerto achieve the final size and each molecule is combined

with proteins of the Argonaute (AGO) family to obtain itsfunctional form [31ndash33] thus forming the miRNA-inducedsilencing complexes (miRISCs) Typically these complexesbind to the 31015840-untranslated region (31015840 UTR) of target mRNAwith perfect or near-perfect complementarity When miR-NAs form perfect base pairs with their target mRNA theyresult in its degradation Nevertheless most of the humanmiRNAs bind to their target 31015840-UTRs with imperfect com-plementarity and therefore induce translational repression[34] A result of all these interactions is that the targetsequence is not translated or there is a variation of translationand subsequently the encoded protein is modified or notproduced at all According to the role of this protein thisleads to structural or functional alterations to the involvedcells thus having a direct effect on their phenotype [9]Remarkably each miRNA can regulate the expression ofnumerous target genes and also the same target gene can beregulated by several types of miRNAs which create a complexnetwork of interactions [35ndash37] However the regulatory roleof miRNA in mRNA stability and translation into protein isa complex biological process which is not restricted throughthe binding of miRNA only in the 31015840-UTR of the mRNA [19]miRNAs can also interact with the 51015840 UTR of protein-codinggenes and cause translational repression [38] or activationof the targeted proteins [39] Similarly miRNAs can alsotarget the coding sequence and repress the translation oftargeted genes [40] Moreover some miRNAs can interactwith regulatory protein complexes such as AGO2 and fragileX mental retardation-related protein 1 (FXR1) and indirectlyupregulate the translation of a target gene [41]

The involvement of miRNAs in cancer was first discov-ered in a quest to identify tumor suppressors in the frequentlydeleted 13q14 region in chronic lymphocytic leukaemia(CLL) and the miRNA cluster miR-15a-miR-16-1 was foundto be deleted or downregulated via epigenetic silencing in69 of the patients [42] One of the most striking themes inthe study of miRNAs and cancer is indeed the large alterationof miRNA expression in malignant cells compared to theirnormal counterparts Most cancers have a specific miRNAsignature or ldquomiRNomerdquo that characterizes the malignantstate and defines some of the clinicopathological featuresof the tumors (eg grade stage aggressiveness vascularinvasion andor proliferation index) [43] miRNAs have avariety of roles in cancer development and progression [8]acting not only as tumor suppressors or oncogenes [44] butalso as key activators or suppressors of tumor metastasis[45] Variations in miRNA genes and their precursors as wellas the target sites and genes encoding components of themiRNA processing machinery can affect the cell phenotypeand disease susceptibility [46 47] Finally a subclass ofmiRNAs known as epi-miRNAs can directly control theepigenetic variations [48] andmiRNA expression can also bedownregulated via promotor hypermethylation [49] addinganother piece to the puzzle of regulatory gene expressionnetworks

Research in various cancers has found that miRNAs alsohave great potential as biomarkers for early diagnosis andprognosis [17 50 51] In particular circulating miRNAs havehigh translational potential as noninvasive biomarkers [52]




3 miRNA and PDAC







Ang

ioge

nesis

EGFR

path

way

NO

TCH

path

way

TGF-120573

path

way

Wnt

pat

hway

Hed

geho

gpa

thw

ay


CD

CSL

GLI

p16

BRCA2Rad51



MiR-21MiR-200let-7d


MiR-494



AKT

SOS JAK

KRAS STAT

p53

BCL2

BRAF

MEK

ERK

MYC



p27

MD

M2

mTO

R NF-120581

B

FOX






















miR-27miR-451



let-7

miR-21



ATPATP ATP

lowast





















































Acknowledgments


References

















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




3 miRNA and PDAC







Ang

ioge

nesis

EGFR

path

way

NO

TCH

path

way

TGF-120573

path

way

Wnt

pat

hway

Hed

geho

gpa

thw

ay


CD

CSL

GLI

p16

BRCA2Rad51



MiR-21MiR-200let-7d


MiR-494



AKT

SOS JAK

KRAS STAT

p53

BCL2

BRAF

MEK

ERK

MYC



p27

MD

M2

mTO

R NF-120581

B

FOX






















miR-27miR-451



let-7

miR-21



ATPATP ATP

lowast





















































Acknowledgments


References

















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ang

ioge

nesis

EGFR

path

way

NO

TCH

path

way

TGF-120573

path

way

Wnt

pat

hway

Hed

geho

gpa

thw

ay


CD

CSL

GLI

p16

BRCA2Rad51



MiR-21MiR-200let-7d


MiR-494



AKT

SOS JAK

KRAS STAT

p53

BCL2

BRAF

MEK

ERK

MYC



p27

MD

M2

mTO

R NF-120581

B

FOX






















miR-27miR-451



let-7

miR-21



ATPATP ATP

lowast





















































Acknowledgments


References

















































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















miR-27miR-451



let-7

miR-21



ATPATP ATP

lowast





















































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

ReviewArticle - Cancer Pharmacology LabPancreatic ductal adenocarcinoma (PDAC) is an extremely...

Documents

Transcript of ReviewArticle - Cancer Pharmacology LabPancreatic ductal adenocarcinoma (PDAC) is an extremely...