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Large Molecule Therapeutics

Systemic Delivery of a miR34a Mimic as a PotentialTherapeutic for Liver Cancer

Christopher L. Daige, Jason F. Wiggins, Leslie Priddy, Terri Nelligan-Davis, Jane Zhao, and David Brown

AbstractmiR34a is a tumor-suppressor miRNA that functions within the p53 pathway to regulate cell-cycle

progression and apoptosis. With apparent roles in metastasis and cancer stem cell development, miR34a

provides an interesting opportunity for therapeutic development. Amimic of miR34a was complexed with an

amphoteric liposomal formulation and tested in two different orthotopic models of liver cancer. Systemic

dosing of the formulated miR34a mimic increased the levels of miR34a in tumors by approximately 1,000-fold

and caused statistically significant decreases in themRNA levels of severalmiR34a targets. The administration

of the formulatedmiR34amimic caused significant tumor growth inhibition in bothmodels of liver cancer, and

tumor regression was observed in more than one third of the animals. The antitumor activity was observed in

the absence of any immunostimulatory effects or dose-limiting toxicities. Accumulation of the formulated

miR34a mimic was also noted in the spleen, lung, and kidney, suggesting the potential for therapeutic use in

other cancers. Mol Cancer Ther; 13(10); 2352–60. �2014 AACR.

IntroductionThe development of lipid nanoparticle technologies

that provide efficient delivery of RNA oligonucleotidesto hepatocytes and liver tumors (1) has created an oppor-tunity for RNAi-based therapies. Among the most inter-esting RNAi therapeutic candidates for cancer aremimicsof tumor-suppressor miRNAs. Like multikinase inhibi-tors that have increased the survival rates of patients withcancer (2), tumor-suppressor miRNAs inhibit cancer byco-regulating many different oncogenes and oncogenicpathways. One of the best characterized tumor-suppres-sormiRNAs,miR34a, functionswithin thep53pathway toregulate a diverse set of oncogenes (reviewed in ref. 3) andreduced levels of the tumor-suppressor miRNA havebeen observed in a variety of cancer types, including livercancer (4, 5). When introduced into cancer cells, miR34ainduces cell-cycle arrest (6), senescence (7), and apoptosis(7). The miRNA also inhibits cancer stem cell develop-ment (8–10).

We have developed a miR34a mimic that has proved tobe effective in inhibiting the growth of tumors in mousemodels of lymphoma (11), lung cancer (12), and prostatecancer (8). To address whether miR34a can inhibit livertumor growth, we encapsulated our miR34a mimic withan amphoteric liposomal formulation and evaluated theresulting nanoparticles for therapeutic activity usingmice

with orthotopicHep3B andHuH7 liver cancer xenografts.Systemic delivery of the encapsulated miR34a mimicreduced the expression levels of several miR34a targetmRNAs and caused significant growth inhibition andeven regression of the liver tumors. Toxicity studiesrevealed no detrimental side effects or immune stimula-tion at therapeutic doses.

Materials and MethodsCell culture

Hep3B cancer cells were purchased from ATCC inJanuary 2013. HuH7 cells were purchased from theNational Institute of Biomedical Innovation in Japan inJanuary 2013.Hep3B cellswere cultured as recommendedby themanufacturer (ATCC).HuH7 cellswere cultured inDMEM (ATCC) supplemented with 10% FBS (HyClone).All cells were grown at 37�C in 5% CO2–95% O2.

miRNAs and amphoteric liposomal formulationThe miR34a mimic was synthesized and purified by

Avecia Biotechnology using standard procedures forRNA oligonucleotide synthesis and high-performanceliquid chromatography purification. Synthesis of a-(30-O-cholesteryloxycarbonyl)-d-(N-ethylmorpholine)-succi-namide (MoChol) and production of amphotericliposomes were performed as described (13).

Orthotopic liver cancer modelsFemale NOD/SCID mice between 7 and 8 weeks of age

were purchased from Jackson Laboratory. All animalswere housed in disposableMicro-Isolator cages (Innovive)and animal husbandry and procedureswere carried out inaccordance with the institutional guidelines for properanimal care and maintenance. Animals were maintained

Mirna Therapeutics, Inc., Austin, Texas.

Corresponding Author: David Brown, Mirna Therapeutics, Inc, 2150Woodward, Austin, TX 78744. Phone: 512-681-5260; Fax: 512-681-5200; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0209

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(10) October 20142352

on January 19, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0209

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in aseptic conditions for all aspects of experimentation.Mice were anesthetized under 3% isoflurane/O2 mixturethroughout surgery. Once sufficiently anesthetized, theabdominal hair was removed with electric hair clippersand the incision site sterilized with betadine and 70%EtOH. A 1 cm incision was made along the midline of theabdomen with the uppermost portion being just inferiorto the sternum. Separate incisions were required to safelyexpose the peritoneumand the abdominal organs. The leftlateral lobe of the liverwas exteriorizedwith cotton swabsand a bolus of 2� 106 Hep3B or 1� 106 HuH7 cells in a 25mL volume of PBS was injected using a 27-gauge needle(BD Biosciences) attached to a 100 mL glass syringe(Hamilton). Any bleedingwas stopped by applying slightpressure to the injection site with cotton swabs. The liverwas placed back in the abdomen, the peritoneum wasclosed with 6.0 silk sutures, and the skin was closed withwound clips. All wound clips were removed 7 days aftersurgery.

Animal studiesTarget gene identification. NOD/SCID mice with

Hep3B or HuH7 orthotopic liver cancer xenografts weresubjected to a single tail vein injection with liposomedilution buffer, 1.0 mg/kg of formulated miR34a mimic(MRX34), or liposome formulation alone with lipid con-centrations equivalent to 1.0 mg/kg dose of formulatedmiR34a mimic. Twenty-four hours after injection, tumorswere recovered from sacrificed mice and RNA was iso-lated and subjected to mRNA array analysis using anIllumina Bead (Hep3B) or Affymetrix (HuH7) microAr-ray. Microarray analyses were performed by Asuragen.Array data are available at Gene Expression Omnibus(GEO) using accession numbers GSE58893 (Hep3B) andGSE58800 (HuH7).Efficacy studies. NOD/SCID mice bearing Hep3B or

HuH7 orthotopic liver cancer xenografts were subjectedto multiple tail vein injections with liposome dilutionbuffer: 0.3, 1.0, or 3.0 mg/kg MRX34 or liposome formu-lation alone with lipid concentrations equivalent to0.3, 1.0, or 3.0mg/kgMRX34. Tumor growthwas assessedby periodic blood collections and subsequent assaying ofa-fetoprotein (AFP) from sera. Threedoseswere givenperweek (Monday/Wednesday/Friday) for twoweeks and aseventh dose was given on Monday. Twenty-four hoursafter the final injections, animals were subjected to nec-ropsy and tumors were resected and weighed. As bothHep3B and HuH7 tumors typically grow as single bul-bous tumor masses, they were easily dissected fromsurrounding host liver tissue.Toxicology assessments. Seven- to 8-week-old female

Balb/C mice (Charles River) were housed appropriatelyand animal husbandry and procedures were carried outin accordance with institutional guidelines for properanimal care and maintenance. Animals were injectedintravenously with MRX34 or dilution buffer. Blood wascollected from mice via retro orbital sinus draws. Bloodsamples were added to serum collection tubes for subse-

quent serum separation by centrifugation. Serum cyto-kine levels were determined using a mouse FluorokineMultianalyte Profiling Kit (R&D Systems) and the Lumi-nex 100 IS instrument. For blood chemistry analyses,serum from animals dosed was sent to the ComparativePathology Laboratory at University of California, Davis(Davis, CA).

AFP analysisSera were prepared from mouse blood samples by

centrifugation and AFP was measured by ELISA (DRGEIA-4331). A standard curvewasproducedusingpurifiedAFP so that ELISA readings could be converted to ng ofAFP/mL of serum.

Human whole blood immunostimulation assayTo determine whether the formulated miR34a mimic

induces a human cytokine response, formulatedmiRNAswere incubated for 24 hours inwhole blood collected fromthree healthy male volunteers. Heparin was added tothe whole blood to prevent coagulation and the formu-lated miRNA was present at a final concentration of 600nmol/L. After the incubation, plasma was separated bycentrifugation and IL6, IL8, and TNFa were analyzed byELISA (R&D Systems).

Quantitative RT-PCRTotal RNA was isolated from tissues and blood using

mirVana PARIS reagents (Ambion). All tissues werehomogenized in PARIS lysis buffer and total RNA wasisolated according to the manufacturer’s specifications.For mRNA or miRNA analysis, cDNA was generatedfrom 10 ng total RNA, respectively, with MMLV reversetranscriptase (Invitrogen). Quantitative PCR analysiswas performed using the 7900 Real-time PCR System.For analysis of miR34 target mRNAs, TaqMan primer/probe sets (Life Technologies) specific for ERC1, RRAS,PHF19, WTAP, CTNNB1, SIPA1, DNAJB1, MYCN, andTRA2A were used along with ubiquitously expressedGAPDH and Cyclophilin-A to generate raw mRNAexpression data used for relative standard curve PCRanalysis (RSC). For RSC, standard curves were gener-ated from diluted negative control samples and rawdata from each primer/probe set were expressed interms of these curves to account for differences inindividual target reverse transcription and PCR efficien-cies. The normalized data were compared for each testprimer/probe set against geometric means of RSC nor-malized GAPDH and Cyclophilin-A mRNA levels.

TaqMan primer/probe sets (Life Technologies) wereused to measure miRNA levels and Ct values were con-verted to copies of miRNA using a standard curve gen-eratedwith themiR34amimic thatwasused for injections.

Statistical analysisResults are reported as the mean � SD. The statistical

significance of the differences in tumor weights and AFPlevels in themiR34a efficacy experiments was assessed by

miR34-Based Cancer Therapy Candidate

www.aacrjournals.org Mol Cancer Ther; 13(10) October 2014 2353




one-way ANOVA and the Dunnett posttest. All statisticswere generated using GraphPad Prism 5.04 software forWindows, GraphPad Software (www.graphpad.com).The level of significance was set at P < 0.05.

ResultsSystemic delivery and activity of a miR34a-basedtherapeutic candidate

Amimic ofmiR34awas encapsulated using a liposomalformulation featuring amphoteric lipids that are cationicduring liposome formation to ensure efficient encapsula-tion of the miRNAmimic and anionic during storage anddelivery to maximize shelf-life, circulation time, and tis-sue uptake (14). We call the liposome-formulated miR34amimic MRX34. To characterize the delivery pattern of theamphoteric liposomal formulation, we injected MRX34into the tail veins of NOD/SCID mice bearing orthotopicHep3B liver tumor xenografts and measured the levels ofmiR34a infive tissues of animals 24hours after injection.Asingle intravenous injection of MRX34 introduced more

than 60 million copies of the miRNA per ng of total RNAfrom liver andmore than 9million copies per ng of tumorRNA (Fig. 1A). The single injection of MRX34 alsoincreased the levels of miR34a by 100,000 to 4,000,000copies per ng of total RNA from lung, spleen, and kidney(Fig. 1A). The endogenous levels of miR34a in the fivetissues ranged from 10,000 to 100,000 copies per ng of totalRNA. The endogenous levels ofmiR34a and the deliveredcopies ofmiR34awere similar for a secondorthotopic livercancer model that featured xenografts of the HuH7human liver cancer cell line (data not shown).

NOD/SCIDmicewith orthotopic Hep3B orHuH7 livertumor xenografts were subjected to a single dose ofMRX34, empty liposome, or dilution buffer and sacrificed24 hours later. RNA samples from the tumors resectedfrom the mice were subjected to microarray analysesand the resulting data were analyzed by ranking genesbased upon their level of differential expression in micedosedwithMRX34 compared tomice dosedwith dilutionbuffer or empty liposomes. The levels of 624mRNAswere

Figure 1. Systemic delivery and activity of a miR34a mimic in mice with liver tumors. NOD/SCID bearing orthotopic Hep3B or HuH7 liver cancer xenograftswere injected with 1 mg/kg MRX34, dilution buffer, or empty liposomes (3 mice/dose group) and sacrificed 24 hours after injection. Tissue sampleswere recovered and assessed by qRT-PCR analysis. A, copies of miR34a above endogenous levels in liver, Hep3B liver tumor, lung, spleen, and kidneyof mice dosed with 1.0 mg/kg MRX34. B, relative Hep3B tumor mRNA expression of nine putative miR34a target genes in liver tumors from micedosed with dilution buffer, empty liposomes, or MRX34 (3 mice per dose group). C, relative HuH7 tumor mRNA expression of nine putative miR34a targetgenes in liver tumors from mice dosed with MRX34, dilution buffer, or empty liposomes (4 mice/dose group).

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reduced by at least 30% in the Hep3B liver tumors of micedosed with MRX34. Thirty-three of the downregulatedmRNAs were identified via TargetScan (15) as havingpotential binding sites for miR34a. The levels of 59mRNAs were reduced by at least 30% in the HuH7 livertumors of mice dosed with MRX34. Twenty of the down-regulated mRNAs were identified via TargetScan as hav-ing potential binding sites for miR34a. Comparing thedownregulated mRNAs with miR34a-binding sites fromthe two liver cancermodels revealed that ninemRNAs arecommon between the two datasets. Quantitative reversetranscription-PCR (qRT-PCR) analysis confirmed thatthemRNA levels of all nine geneswere significantly lowerin the liver tumors of mice dosed with MRX34 than themice dosed with dilution buffer or empty liposomes intheHep3BandHuH7 tumorRNAsamples (Fig. 1BandC).The complete list of genes thatwere differentially express-ed (up or down) in the liver tumors of the MRX34-dosedmice by 30% or greater includes 25 genes that are associat-ed with pathways that are commonly altered in primaryliver tumors—p53, WnT/b-catenin, MapK, Hedgehog,VEGF, and c-MET (refs. 16–18; Table 1).

miR34a anticancer effects in Hep3B liver orthotopicmiceWe conducted a study to evaluate the therapeutic

activity of MRX34 usingmice with orthotopic liver cancerxenografts. A total of 2 � 106 Hep3B cells were surgicallyimplanted into the left lateral lobes of the livers of femaleNOD/SCIDmice and tumorswere allowed to develop for3weeks.We used a serum biomarker of liver cancer, AFP,tomonitor the growth of the xenografts. SerumAFP levelscorrespond with tumor mass in orthotopically grownHep3B xenografts (data not shown) and provide an effec-tive way to assess tumor size without the need for imag-ing. When AFP reached mean levels of 1,600 ng/mL ofserum,micewere separated into test andcontrol groupsof6 animals per group (Fig. 2A). Animals received singleintravenous administrations of MRX34 at 0.3 or 3.0 mg ofmiRNA/kg of body weight (mg/kg), 3.0 mg/kg equiva-lent (eq) of empty liposomes, or dilution buffer three timesperweek (Monday/Wednesday/Friday) for 2weekswith

a final injection on the following Monday for a total of 7doses. To monitor tumor growth in the animals, serumAFPwas measured every 3 to 4 days after the initiation ofdosing. At every time point following the initiation oftreatment, AFP levels in the twoMRX34dose groupsweresignificantly lower than the levels in the two controlgroups (Fig. 2B). Mean serum AFP levels of animals inthe empty liposome and dilution buffer groups weresimilar in magnitude reaching nearly 1,000,000 ng/mLby the end of 2 weeks of dosing (Fig. 2B). In contrast, theserum AFP levels of the mice on the MRX34 dose groupsranged from less than 2-fold higher than the starting levelsto barely above background for the AFP ELIAS (Fig. 2Band C).

Animals were sacrificed from each group 24 hours afterthe final dose was administered and large tumors weredetected in the livers of all mice from the two controlgroups, while much smaller and paler tumors, in general,were detected in bothMRX34 dose groups. In fact, tumorswere visibly absent in 1/7 and 3/7 animals dosed at 0.3and3.0mg/kgMRX34, respectively (Fig. 2CandD). Theseresults have been repeated multiple times in the Hep3Bliver orthotopic mouse model and these data are repre-sentative of those results.

MRX34 inhibits growth of established HuH7orthotopic liver tumors

We used a second orthotopic liver cancer model fea-turing HuH7 cells that express mutant p53 (19) to deter-mine whether the therapeutic activity of MRX34 extendsbeyond Hep3B xenografts. HuH7 and Hep3B cells havedistinct genetic backgrounds and cancermodels featuringthese two cell lines are often used to evaluate howbroadlyapplicable a therapeutic candidate for liver cancer mightbe. We surgically implanted HuH7 human liver cancercells into the left lateral lobes of NOD/SCID mice andallowed tumors to develop for 2 weeks.When serumAFPlevels in 24mice averaged 1,200 ng/mL, we separated themice into three dosing groups (Fig. 3A). The difference inmean serum AFP levels between the HuH7 and Hep3Befficacy studies (1,200ng/mLvs. 1,600ng/mL)wasdue toslightly different growth rates of the tumors in the two

Table 1. Genes in tumor-suppressor and oncogenic pathways that are commonly altered in liver tumorsthat were differentially expressed in Hep3B and/or HuH7 xenografts of mice dosed with MRX34

p53 WnT/b-catenin MapK Hedgehog VEGF c-MET

CHEK2 (þ) FRAT1 (�) PDGF (�) RAB23 (�) VEGFB (�) c�MET (�)CDKN1A (þ) CSNK2A1P (�) RRAS (�) PRKX (�) MAPKAPK2 (�) MAPK3 (�)BID (þ) CTNNB1 (�) PRKX (�) FBXW11 (�) NFATC3 (�)CASP3 (þ) FBXWW11 (�) NFkb2 (�) MAPK1 (�)CCNG2 (þ) NFATC3 (�) ELK4 (�)

THBS1 (�)

NOTE: The symbols (þ/�) in parentheses indicate whether the expression of the gene was elevated (þ) or reduced (�) in the tumors ofmice dosed with MRX34 compared with tumors from mice dosed with dilution buffer and empty liposomes.






liver cancer models. Tumor-bearing mice received threetail vein injections ofdilution buffer, 1.0mg/kgMRX34, or1.0 mg/kg equivalent of empty liposomes per week for 2weeks and all mice received a seventh dose 15 days afterthe initiation of dosing. Unlike the Hep3B study, wherewe had enough mice with liver tumors to accommodatetwo MRX34 dose groups, we only had enough mice withHuH7 tumors to accommodate a single MRX34 dosegroup. For the HuH7 study, we used an MRX34 doselevel (1.0 mg/kg) that was between the 0.3 and 3.0mg/kgdoses thatwereused for theHep3B study.Consistentwiththe study using the Hep3B liver cancer model, tail veininjections of MRX34 significantly reduced the accumula-

tion of human AFP in the sera of mice compared with thedilution buffer and empty liposome dosing groups (Fig.3B). The serum AFP levels in the mice from both controlgroups increased on average by more than 115-foldbetween the first and last day of dosing. In contrast, theaverage serum AFP levels for the animals in the MRX34dose group increased by less than 2-fold and 2 of the 8mice had substantially lower AFP levels at the end of thedosing period than at the beginning (Fig. 3C). We sacri-ficed animals 24 hours after the final dose and tumorswere detected in all of the mice from the two controlgroups, whereas only 5 of the 8 animals in the MRX34group had detectable tumors (Fig. 3D). The average liver

Figure 2. Hep3B tumor regression with MRX34 treatment. Serum AFP levels before MRX34 administration (A) and during 2 weeks of administration of MRX34(B). C, percent change in serumAFP between initiation on day 0 and upon termination 24 hours following the final administration on day 13 for MRX34 groupsonly. D, resected tumor weights display the effects of MRX34 dosing on tumor size.

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tumor masses recovered from the mice in the dilutionbuffer and empty liposome groups were 155 and 159 mg,respectively. The average tumor mass recovered from thefive MRX34-dosed mice that had liver tumors was 8 mg.qRT-PCR analysis of the recovered tumors revealed thatanimals dosed with MRX34 had approximately 107 morecopies of miR34a per ng of total tumor RNA than theanimals in the control groups (Fig. 3E). These results havebeen individually reproduced in a separate efficacy studywith MRX34 in the HuH7 liver orthotopic mouse modeland these data are representative of those results.

MRX34 evaluation in immunocompetent systemsThe safety profile of MRX34 was evaluated using

immunocompetent BALB/c mice. Three to 4 animals per

group were dosed intravenously with 1.5 mg/kg MRX34every other day (q.o.d.) for 2 weeks. Animal health wasmonitored daily and liver, spleen, and whole blood werecollected 24 hours after the last intravenous administra-tion. Whole organ weights were collected and comparedbetween groups and serum clinical chemistries wereanalyzed. No body weight changes were observed overthe 2-week period between animals in the test (MRX34)and control (dilution buffer) groups (Fig. 4A). Spleenweights were normalized to body weight and no changesin spleen size were observed in mice dosed with MRX34compared with animals dosed with dilution buffer (Fig.4B). In a separate study, we measured levels of cytokinesindicative of immune stimulation (IL1b, IL6, andTNFa) inBALB/c mouse sera following single administration of

Figure 3. HuH7 liver tumor growth inhibition and regression in mice treated with MRX34. A, eighteen days after surgically implanting HuH7 cells into livers,NOD/SCIDmicewere bled for serumpreparation. Tenmicroliters of serumwas assayed using a human-specificELISA for AFP (DRG International) to estimatetumor size. Serum AFP was approximately 1,200 ng/mL and groups of similar AFP mean were formed (n ¼ 8). B, mice in the Empty Liposome andMRX34 groupswere dosed (1.0mg/kg) for 14 days and tumor growthwasmonitored bymeasuring serumAFP levels every 3 to 4 days. C, the percent changein serum AFP levels between the final day of the dosing period (day 14) and the first day of dosing (day 0) was calculated for the mice dosed withMRX34. Two animals had lower AFP at the end of 2 weeks of treatment than their initial AFP levels. D, all animals were sacrificed on day 14, and 3 ofthe 8 animals in the MRX34 group had no visible tumors, while all of the animals in the Empty Liposomes and Dilution Buffer groups had tumors. E, miR34aaccumulation in liver tumors. Levels of miR34a are represented as copies per ng of total tumor RNA.






MRX34 at 1 mg/kg and found no significant elevationscompared with normal levels measured in animals dosedwith PBS (Fig. 4C). An ex vivomodel was used to confirmthat MRX34 is not immunostimulatory in human blood(20). MRX34 (600 nmol/L), along with 600 nmol/L for-mulated NC2 and unformulated PBS, were incubated inwhole blood from threemale donors for 24 hours and thenplasmawas prepared and assayed for TNFa, IL6, and IL8.Consistent with the mouse studies, MRX34 failed to stim-ulate a cytokine response (Fig. 4D).

DiscussionThe systemic delivery of a formulatedmimic of miR34a

caused tumor regression in two different orthotopic mod-els of liver cancer. Repeated dosing of the formulatedtumor-suppressor miRNA did not impact normal animalbehavior nor did it produce any histologic evidence oftoxicity at any dose tested. Furthermore, there was noevidence of immune stimulation in mice or in humanwhole blood. The combination of antitumor activity andfavorable safety profile suggests that the formulatedmiR34a mimic might be effective as a targeted therapyfor cancer.

The profound efficacy exhibited by the systemic deliv-ery of the miR34a mimic likely derives from the ability ofthe small RNA to regulate multiple genes and pathwaysthat are important for hepatocellular carcinoma develop-ment. A single administration of the miRNA inhibitedmultiple genes within oncogenic pathways of WnT/b-Catenin, MapK, c-Met, Hedgehog, and VEGF andstimulated multiple genes within the p53 pathway.Hyperactivation of any of the five oncogenic pathwaysor suppression of the p53 pathway have proved to stim-ulate the development of hepatocellular carcinoma (HCC)in mice (16–19, 21)

p53 is the most commonly mutated gene in patientswithHCC (19, 22) and reducedp53 activity appears to be akey early event in thedevelopment of liver tumors (23, 24).Not surprisingly, the p53-regulated miR34a has reducedexpression levels in liver tumors (4). Consistent with arecent publication that describes the ability of miR34a tostimulate the p53 pathway in cancer cells lacking p53 (25),we observed increased expression levels of the p53-relat-ed genes CHEK2, CDKN1A, BID, CASP3, and CCNG2(21, 26) in the liver tumors of mice dosed a single timewith MRX34. This result suggests that the potency of

Figure 4. Evaluation of toxicologic endpoints in Balb/cmice and humanwhole blood. Percent bodyweight changes inmice following 2weeks of treatmentwithtest articles (A) and percent Spleen:Body weight following 2weeks ofMRX34 administration (B). C, serum IL1b, IL6, and TNFa levels followingMRX34 dosingin Balb/C mice. D, human whole blood cytokine release ex vivo evaluation. Formulated NC2 andMRX34 at a final concentration of 600 nmol/L as well as LPSand PBS were incubated overnight in whole blood collected from three healthy human males. Plasma was prepared from each sample and cumulativecytokine release (IL6, IL8, and TNFa) was measured using the Fluorokine Multianalyte Profiling Kit (R&D Systems) and the Luminex 100 IS instrument.

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MRX34 might derive at least partially from its capacity tostimulate the p53 pathway in cancer cells that lack p53activity as is the case with the Hep3B cell line.The hyperactivation of theWnT/B-catenin pathways is

an early signature of hepatocellular carcinogenesis (27).AbnormalWnT/b-catenin signaling impacts cellular pro-liferation, metabolic function, and stem cell behavior (28)and stimulates HCC formation (29). Following a singleadministration of MRX34, we observed the inhibition ofFRAT1, CSNK2AP, CTNNB1, FBXW11, and NFATC3,genes that are involved in propogating WnT/b-cateninsignaling (30, 31). The ability to suppress WnT/b-cateninsignaling with MRX34 likely contributes to the efficacyobserved in the two xenograft models of HCC that weused in our studies.The MAPK pathway has been shown to be elevated in

91% of human samples of HCC (32). In these tumor types,dysregulation of JNK1 and p38 is facilitated by MAPKsignaling and a cascade effect ultimately results in abnor-mal cellular proliferation, survival, and differentiation(33). With a single injection of MRX34 there were notablereductions in the expression of five genes that are com-monly associatedwith theMAPKpathway, PDGF,RRAS,PRKX, NFkB, and ELK4.The Hedgehog pathway plays a significant role in HCC

proliferation, invasion, and metastasis by upregulatingthe protein expression ofMMP9 via the ERKpathway (34,35). In fact, formulated siRNA targeting Hedgehog issufficient to eliminate Hedgehog expression andmarked-ly reduce metastasis in orthotopic mouse models of HCC(36). A single injection ofMRX34 reduced the tumor levelsof RAB23, PRKX, and FBXW11 mRNAs. All three genesfall within the Hedgehog pathway (37). Activation of theHedgehog pathway has been detected in 67% of humanliver tumors (35).The VEGF pathway regulates angiogenesis and

enhances HCC tumor development when hyperactivated(38). Interestingly, VEGF expression has been reported tobe highest in cirrhotic regions of the liver which surroundHCC tumors in human patients (39), and agents arecurrently being designed to target both tumor and sur-rounding liver tissue as both appear critical in tumorformation and propagation (40, 38). Administering asingle injection of MRX34 inhibited mRNA expression ofMAPKAPK2, NFATC3, and MAPK1, which all functionwithin the VEGF pathway (41). The ability to diminishangiogenesis via the VEGF pathway is a likely contributorto the efficacy observed in our mouse models of HCC.c-MET plays a role in early HCC development and cell

proliferation (4). In fact, the activation of c-MET is suffi-cient to initiate hepatocellular carcinogenesis inmice (27),presumably via the activation of the phosphoinositide 3-

kinase, RAS, and STAT3 pathways, all ofwhich have beenimplicated in HCC development (16). In more advancedtumors, c-Met activation leads to HCC proliferation (27)and additionally increases incidences of intrahepatictumor metastases through HGF (42). Reduced expressionof c-Met andMAPK3were observed in the liver tumors ofmice dosed with MRX34. Targeting the c-Met pathwayalone might be sufficient to inhibit growth of liver tumorsbut the collective activation of the p53 pathway andattenuation of the Wnt/b-catenin, MapK, HedgehogVEGF, and c-MET pathways likely mitigates the abilityof liver tumors to proliferate by compensatory mechan-isms and aids in explaining the pronounced efficacyobserved in our studies.

The in vivo data represented here, coupledwith the lackof adverse general health and immunostimulatory effectsin human cells, give MRX34 a clean and straightforwardpath to evaluation in human clinical trials as an efficaciousand safemeans for treatingHCC. In addition, many of themiR34a-regulated HCC oncogenes are also associatedwith the development of cancers that metastasize to theliver, including colon, lung, pancreatic, and breast can-cers. This provides potential clinical applications forpatients with advanced cancer with liver involvement(43, 44).

Disclosure of Potential Conflicts of InterestC.L. Daige, J.F. Wiggins, L. Priddy, T. Nelligan-Davis, and J. Zhao

received commercial research grants from Cancer Prevention andResearch Institute of Texas (CPRIT). D. Brown received a commercialresearch grant fromCPRIT and has ownership interest (including patents)in Mirna Therapeutics. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: C.L. Daige, J.F. Wiggins, D. BrownDevelopment of methodology: C.L. Daige, J.F. Wiggins, L. Priddy,T. Nelligan-Davis, D. BrownAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.L. Daige, L. PriddyAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): C.L. Daige, J.F. Wiggins, L. Priddy,T. Nelligan-Davis, J. Zhao, D. BrownWriting, review, and/or revision of themanuscript:C.L. Daige, D. BrownAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.L. Daige, J.F. Wiggins, L. Priddy,T. Nelligan-Davis, J. ZhaoStudy supervision: C.L. Daige, J.F. Wiggins, D. Brown

Grant SupportThis work was supported by a commercialization award from the

CPRIT that was awarded to all of the authors.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 12, 2014; revised July 1, 2014; accepted July 18, 2014;published OnlineFirst July 22, 2014.

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Daige et al.




2014;13:2352-2360. Published OnlineFirst July 22, 2014.Mol Cancer Ther Christopher L. Daige, Jason F. Wiggins, Leslie Priddy, et al. Liver CancerSystemic Delivery of a miR34a Mimic as a Potential Therapeutic for

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