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

Molecular Cancer Large Molecule Therapeutics Therapeutics

Systemic Delivery of a miR34a Mimic as a Potential Therapeutic for Liver Cancer

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

Abstract miR34a 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. A mimic 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 the mRNA levels of several miR34a targets. The administration of the formulated miR34a mimic caused signiﬁcant tumor growth inhibition in both models 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.

Introduction with orthotopic Hep3B and HuH7 liver cancer xenografts. The development of lipid nanoparticle technologies Systemic delivery of the encapsulated miR34a mimic that provide efficient delivery of RNA oligonucleotides reduced the expression levels of several miR34a target to hepatocytes and liver tumors (1) has created an oppor- mRNAs and caused significant growth inhibition and tunity for RNAi-based therapies. Among the most inter- even regression of the liver tumors. Toxicity studies esting RNAi therapeutic candidates for cancer are mimics revealed no detrimental side effects or immune stimula- of tumor-suppressor miRNAs. Like multikinase inhibition at therapeutic doses. tors that have increased the survival rates of patients with Materials and Methods cancer (2), tumor-suppressor miRNAs inhibit cancer by co-regulating many different oncogenes and oncogenic Cell culture pathways. One of the best characterized tumor-suppres- Hep3B cancer cells were purchased from ATCC in sor miRNAs, miR34a, functions within the p53 pathway to January 2013. HuH7 cells were purchased from the regulate a diverse set of oncogenes (reviewed in ref. 3) and National Institute of Biomedical Innovation in Japan in reduced levels of the tumor-suppressor miRNA have January 2013. Hep3B cells were cultured as recommended been observed in a variety of cancer types, including liver by the manufacturer (ATCC). HuH7 cells were cultured in cancer (4, 5). When introduced into cancer cells, miR34a DMEM (ATCC) supplemented with 10% FBS (HyClone). induces cell-cycle arrest (6), senescence (7), and apoptosis All cells were grown at 37 Cin5%CO2–95% O2. (7). The miRNA also inhibits cancer stem cell development (8–10). miRNAs and amphoteric liposomal formulation We have developed a miR34a mimic that has proved to The miR34a mimic was synthesized and purified by be effective in inhibiting the growth of tumors in mouse Avecia Biotechnology using standard procedures for models of lymphoma (11), lung cancer (12), and prostate RNA oligonucleotide synthesis and high-performance 0 cancer (8). To address whether miR34a can inhibit liver liquid chromatography purification. Synthesis of a-(3 - tumor growth, we encapsulated our miR34a mimic with O-cholesteryloxycarbonyl)-d-(N-ethylmorpholine)-succi- an amphoteric liposomal formulation and evaluated the namide (MoChol) and production of amphoteric resulting nanoparticles for therapeutic activity using mice liposomes were performed as described (13).

Orthotopic liver cancer models Mirna Therapeutics, Inc., Austin, Texas. Female NOD/SCID mice between 7 and 8 weeks of age Corresponding Author: David Brown, Mirna Therapeutics, Inc, 2150 were purchased from Jackson Laboratory. All animals Woodward, Austin, TX 78744. Phone: 512-681-5260; Fax: 512-681- were housed in disposable Micro-Isolator cages (Innovive) 5200; E-mail: [email protected] and animal husbandry and procedures were carried out in doi: 10.1158/1535-7163.MCT-14-0209 accordance with the institutional guidelines for proper 2014 American Association for Cancer Research. animal care and maintenance. Animals were maintained

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in aseptic conditions for all aspects of experimentation. quent serum separation by centrifugation. Serum cyto- Mice were anesthetized under 3% isoflurane/O2 mixture kine levels were determined using a mouse Fluorokine throughout surgery. Once sufficiently anesthetized, the Multianalyte Profiling Kit (R&D Systems) and the Lumi- abdominal hair was removed with electric hair clippers nex 100 IS instrument. For blood chemistry analyses, and the incision site sterilized with betadine and 70% serum from animals dosed was sent to the Comparative EtOH. A 1 cm incision was made along the midline of the Pathology Laboratory at University of California, Davis abdomen with the uppermost portion being just inferior (Davis, CA). to the sternum. Separate incisions were required to safely expose the peritoneum and the abdominal organs. The left AFP analysis lateral lobe of the liver was exteriorized with cotton swabs Sera were prepared from mouse blood samples by and a bolus of 2 106 Hep3B or 1 106 HuH7 cells in a 25 centrifugation and AFP was measured by ELISA (DRG mL volume of PBS was injected using a 27-gauge needle EIA-4331). A standard curve was produced using purified (BD Biosciences) attached to a 100 mL glass syringe AFP so that ELISA readings could be converted to ng of (Hamilton). Any bleeding was stopped by applying slight AFP/mL of serum. pressure to the injection site with cotton swabs. The liver was placed back in the abdomen, the peritoneum was Human whole blood immunostimulation assay closed with 6.0 silk sutures, and the skin was closed with To determine whether the formulated miR34a mimic wound clips. All wound clips were removed 7 days after induces a human cytokine response, formulated miRNAs surgery. were incubated for 24 hours in whole blood collected from three healthy male volunteers. Heparin was added to Animal studies the whole blood to prevent coagulation and the formu- Target gene identification. NOD/SCID mice with lated miRNA was present at a final concentration of 600 Hep3B or HuH7 orthotopic liver cancer xenografts were nmol/L. After the incubation, plasma was separated by subjected to a single tail vein injection with liposome centrifugation and IL6, IL8, and TNFa were analyzed by dilution buffer, 1.0 mg/kg of formulated miR34a mimic ELISA (R&D Systems). (MRX34), or liposome formulation alone with lipid concentrations equivalent to 1.0 mg/kg dose of formulated Quantitative RT-PCR miR34a mimic. Twenty-four hours after injection, tumors Total RNA was isolated from tissues and blood using were recovered from sacrificed mice and RNA was iso- mirVana PARIS reagents (Ambion). All tissues were lated and subjected to mRNA array analysis using an homogenized in PARIS lysis buffer and total RNA was Illumina Bead (Hep3B) or Affymetrix (HuH7) microAr- isolated according to the manufacturer’s specifications. ray. Microarray analyses were performed by Asuragen. For mRNA or miRNA analysis, cDNA was generated Array data are available at Gene Expression Omnibus from 10 ng total RNA, respectively, with MMLV reverse (GEO) using accession numbers GSE58893 (Hep3B) and transcriptase (Invitrogen). Quantitative PCR analysis GSE58800 (HuH7). was performed using the 7900 Real-time PCR System. Efficacy studies. NOD/SCID mice bearing Hep3B or For analysis of miR34 target mRNAs, TaqMan primer/ HuH7 orthotopic liver cancer xenografts were subjected probe sets (Life Technologies) specific for ERC1, RRAS, to multiple tail vein injections with liposome dilution PHF19, WTAP, CTNNB1, SIPA1, DNAJB1, MYCN, and buffer: 0.3, 1.0, or 3.0 mg/kg MRX34 or liposome formu- TRA2A were used along with ubiquitously expressed lation alone with lipid concentrations equivalent to GAPDH and Cyclophilin-A to generate raw mRNA 0.3, 1.0, or 3.0 mg/kg MRX34. Tumor growth was assessed expression data used for relative standard curve PCR by periodic blood collections and subsequent assaying of analysis (RSC). For RSC, standard curves were gener- a-fetoprotein (AFP) from sera. Three doses were given per ated from diluted negative control samples and raw week (Monday/Wednesday/Friday) for two weeks and a data from each primer/probe set were expressed in seventh dose was given on Monday. Twenty-four hours terms of these curves to account for differences in after the final injections, animals were subjected to nec- individual target reverse transcription and PCR efficien- ropsy and tumors were resected and weighed. As both cies. The normalized data were compared for each test Hep3B and HuH7 tumors typically grow as single bul- primer/probe set against geometric means of RSC nor- bous tumor masses, they were easily dissected from malized GAPDH and Cyclophilin-A mRNA levels. surrounding host liver tissue. TaqMan primer/probe sets (Life Technologies) were C Toxicology assessments. Seven- to 8-week-old female used to measure miRNA levels and t values were con- Balb/C mice (Charles River) were housed appropriately verted to copies of miRNA using a standard curve gen- and animal husbandry and procedures were carried out erated with the miR34a mimic that was used for injections. in accordance with institutional guidelines for proper animal care and maintenance. Animals were injected Statistical analysis intravenously with MRX34 or dilution buffer. Blood was Results are reported as the mean SD. The statistical collected from mice via retro orbital sinus draws. Blood significance of the differences in tumor weights and AFP samples were added to serum collection tubes for subse- levels in the miR34a efficacy experiments was assessed by

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one-way ANOVA and the Dunnett posttest. All statistics than 60 million copies of the miRNA per ng of total RNA were generated using GraphPad Prism 5.04 software for from liver and more than 9 million copies per ng of tumor Windows, GraphPad Software (www.graphpad.com). RNA (Fig. 1A). The single injection of MRX34 also The level of significance was set at P < 0.05. increased the levels of miR34a by 100,000 to 4,000,000 copies per ng of total RNA from lung, spleen, and kidney Results (Fig. 1A). The endogenous levels of miR34a in the five Systemic delivery and activity of a miR34a-based tissues ranged from 10,000 to 100,000 copies per ng of total therapeutic candidate RNA. The endogenous levels of miR34a and the delivered A mimic of miR34a was encapsulated using a liposomal copies of miR34a were similar for a second orthotopic liver formulation featuring amphoteric lipids that are cationic cancer model that featured xenografts of the HuH7 during liposome formation to ensure efficient encapsula- human liver cancer cell line (data not shown). tion of the miRNA mimic and anionic during storage and NOD/SCID mice with orthotopic Hep3B or HuH7 liver delivery to maximize shelf-life, circulation time, and tis- tumor xenografts were subjected to a single dose of sue uptake (14). We call the liposome-formulated miR34a MRX34, empty liposome, or dilution buffer and sacrificed mimic MRX34. To characterize the delivery pattern of the 24 hours later. RNA samples from the tumors resected amphoteric liposomal formulation, we injected MRX34 from the mice were subjected to microarray analyses into the tail veins of NOD/SCID mice bearing orthotopic and the resulting data were analyzed by ranking genes Hep3B liver tumor xenografts and measured the levels of based upon their level of differential expression in mice miR34a in five tissues of animals 24 hours after injection. A dosed with MRX34 compared to mice dosed with dilution single intravenous injection of MRX34 introduced more buffer or empty liposomes. The levels of 624 mRNAs were

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

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Table 1. Genes in tumor-suppressor and oncogenic pathways that are commonly altered in liver tumors that 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 ()cMET () 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 of mice dosed with MRX34 compared with tumors from mice dosed with dilution buffer and empty liposomes. reduced by at least 30% in the Hep3B liver tumors of mice a final injection on the following Monday for a total of 7 dosed with MRX34. Thirty-three of the downregulated doses. To monitor tumor growth in the animals, serum mRNAs were identified via TargetScan (15) as having AFP was measured every 3 to 4 days after the initiation of potential binding sites for miR34a. The levels of 59 dosing. At every time point following the initiation of mRNAs were reduced by at least 30% in the HuH7 liver treatment, AFP levels in the two MRX34 dose groups were tumors of mice dosed with MRX34. Twenty of the down- significantly lower than the levels in the two control regulated mRNAs were identified via TargetScan as hav- groups (Fig. 2B). Mean serum AFP levels of animals in ing potential binding sites for miR34a. Comparing the the empty liposome and dilution buffer groups were downregulated mRNAs with miR34a-binding sites from similar in magnitude reaching nearly 1,000,000 ng/mL the two liver cancer models revealed that nine mRNAs are by the end of 2 weeks of dosing (Fig. 2B). In contrast, the common between the two datasets. Quantitative reverse serum AFP levels of the mice on the MRX34 dose groups transcription-PCR (qRT-PCR) analysis confirmed that ranged from less than 2-fold higher than the starting levels the mRNA levels of all nine genes were significantly lower to barely above background for the AFP ELIAS (Fig. 2B in the liver tumors of mice dosed with MRX34 than the and C). mice dosed with dilution buffer or empty liposomes in Animals were sacrificed from each group 24 hours after the Hep3B and HuH7 tumor RNA samples (Fig. 1B and C). the final dose was administered and large tumors were The complete list of genes that were differentially express- detected in the livers of all mice from the two control ed (up or down) in the liver tumors of the MRX34-dosed groups, while much smaller and paler tumors, in general, mice by 30% or greater includes 25 genes that are associat- were detected in both MRX34 dose groups. In fact, tumors ed with pathways that are commonly altered in primary were visibly absent in 1/7 and 3/7 animals dosed at 0.3 liver tumors—p53, WnT/b-catenin, MapK, Hedgehog, and 3.0 mg/kg MRX34, respectively (Fig. 2C and D). These VEGF, and c-MET (refs. 16–18; Table 1). results have been repeated multiple times in the Hep3B liver orthotopic mouse model and these data are repre- miR34a anticancer effects in Hep3B liver orthotopic sentative of those results. mice We conducted a study to evaluate the therapeutic MRX34 inhibits growth of established HuH7 activity of MRX34 using mice with orthotopic liver cancer orthotopic liver tumors xenografts. A total of 2 106 Hep3B cells were surgically We used a second orthotopic liver cancer model fea- implanted into the left lateral lobes of the livers of female turing HuH7 cells that express mutant p53 (19) to deter- NOD/SCID mice and tumors were allowed to develop for mine whether the therapeutic activity of MRX34 extends 3 weeks. We used a serum biomarker of liver cancer, AFP, beyond Hep3B xenografts. HuH7 and Hep3B cells have to monitor the growth of the xenografts. Serum AFP levels distinct genetic backgrounds and cancer models featuring correspond with tumor mass in orthotopically grown these two cell lines are often used to evaluate how broadly Hep3B xenografts (data not shown) and provide an effec- applicable a therapeutic candidate for liver cancer might tive way to assess tumor size without the need for imag- be. We surgically implanted HuH7 human liver cancer ing. When AFP reached mean levels of 1,600 ng/mL of cells into the left lateral lobes of NOD/SCID mice and serum, mice were separated into test and control groups of allowed tumors to develop for 2 weeks. When serum AFP 6 animals per group (Fig. 2A). Animals received single levels in 24 mice averaged 1,200 ng/mL, we separated the intravenous administrations of MRX34 at 0.3 or 3.0 mg of mice into three dosing groups (Fig. 3A). The difference in miRNA/kg of body weight (mg/kg), 3.0 mg/kg equiva- mean serum AFP levels between the HuH7 and Hep3B lent (eq) of empty liposomes, or dilution buffer three times efficacy studies (1,200 ng/mL vs. 1,600 ng/mL) was due to per week (Monday/Wednesday/Friday) for 2 weeks with slightly different growth rates of the tumors in the two

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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 serum AFP between initiation on day 0 and upon termination 24 hours following the ﬁnal administration on day 13 for MRX34 groups only. D, resected tumor weights display the effects of MRX34 dosing on tumor size.

liver cancer models. Tumor-bearing mice received three tion of human AFP in the sera of mice compared with the tail vein injections of dilution buffer, 1.0 mg/kg MRX34, or dilution buffer and empty liposome dosing groups (Fig. 1.0 mg/kg equivalent of empty liposomes per week for 2 3B). The serum AFP levels in the mice from both control weeks and all mice received a seventh dose 15 days after groups increased on average by more than 115-fold the initiation of dosing. Unlike the Hep3B study, where between the first and last day of dosing. In contrast, the we had enough mice with liver tumors to accommodate average serum AFP levels for the animals in the MRX34 two MRX34 dose groups, we only had enough mice with dose group increased by less than 2-fold and 2 of the 8 HuH7 tumors to accommodate a single MRX34 dose mice had substantially lower AFP levels at the end of the group. For the HuH7 study, we used an MRX34 dose dosing period than at the beginning (Fig. 3C). We sacri- level (1.0 mg/kg) that was between the 0.3 and 3.0 mg/kg ficed animals 24 hours after the final dose and tumors doses that were used for the Hep3B study. Consistent with were detected in all of the mice from the two control the study using the Hep3B liver cancer model, tail vein groups, whereas only 5 of the 8 animals in the MRX34 injections of MRX34 significantly reduced the accumula- group had detectable tumors (Fig. 3D). The average liver

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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/SCID mice were bled for serum preparation. Ten microliters of serum was assayed using a human-specific ELISA for AFP (DRG International) to estimate tumor 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 and MRX34 groups were dosed (1.0 mg/kg) for 14 days and tumor growth was monitored by measuring serum AFP levels every 3 to 4 days. C, the percent change in 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 with MRX34. 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 of the 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, miR34a accumulation in liver tumors. Levels of miR34a are represented as copies per ng of total tumor RNA. tumor masses recovered from the mice in the dilution group were dosed intravenously with 1.5 mg/kg MRX34 buffer and empty liposome groups were 155 and 159 mg, every other day (q.o.d.) for 2 weeks. Animal health was respectively. The average tumor mass recovered from the monitored daily and liver, spleen, and whole blood were five MRX34-dosed mice that had liver tumors was 8 mg. collected 24 hours after the last intravenous administra- qRT-PCR analysis of the recovered tumors revealed that tion. Whole organ weights were collected and compared animals dosed with MRX34 had approximately 107 more between groups and serum clinical chemistries were copies of miR34a per ng of total tumor RNA than the analyzed. No body weight changes were observed over animals in the control groups (Fig. 3E). These results have the 2-week period between animals in the test (MRX34) been individually reproduced in a separate efficacy study and control (dilution buffer) groups (Fig. 4A). Spleen with MRX34 in the HuH7 liver orthotopic mouse model weights were normalized to body weight and no changes and these data are representative of those results. in spleen size were observed in mice dosed with MRX34 compared with animals dosed with dilution buffer (Fig. MRX34 evaluation in immunocompetent systems 4B). In a separate study, we measured levels of cytokines The safety profile of MRX34 was evaluated using indicative of immune stimulation (IL1b, IL6, and TNFa)in immunocompetent BALB/c mice. Three to 4 animals per BALB/c mouse sera following single administration of

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Figure 4. Evaluation of toxicologic endpoints in Balb/c mice and human whole blood. Percent body weight changes in mice following 2 weeks of treatment with test articles (A) and percent Spleen:Body weight following 2 weeks of MRX34 administration (B). C, serum IL1b, IL6, and TNFa levels following MRX34 dosing in Balb/C mice. D, human whole blood cytokine release ex vivo evaluation. Formulated NC2 and MRX34 at a ﬁnal concentration of 600 nmol/L as well as LPS and PBS were incubated overnight in whole blood collected from three healthy human males. Plasma was prepared from each sample and cumulative cytokine release (IL6, IL8, and TNFa) was measured using the Fluorokine Multianalyte Proﬁling Kit (R&D Systems) and the Luminex 100 IS instrument.

MRX34 at 1 mg/kg and found no significant elevations The profound efficacy exhibited by the systemic deliv- compared with normal levels measured in animals dosed ery of the miR34a mimic likely derives from the ability of with PBS (Fig. 4C). An ex vivo model was used to confirm the small RNA to regulate multiple genes and pathways that MRX34 is not immunostimulatory in human blood that are important for hepatocellular carcinoma develop- (20). MRX34 (600 nmol/L), along with 600 nmol/L for- ment. A single administration of the miRNA inhibited mulated NC2 and unformulated PBS, were incubated in multiple genes within oncogenic pathways of WnT/b- whole blood from three male donors for 24 hours and then Catenin, MapK, c-Met, Hedgehog, and VEGF and plasma was prepared and assayed for TNFa, IL6, and IL8. stimulated multiple genes within the p53 pathway. Consistent with the mouse studies, MRX34 failed to stim- Hyperactivation of any of the five oncogenic pathways ulate a cytokine response (Fig. 4D). or suppression of the p53 pathway have proved to stimulate the development of hepatocellular carcinoma (HCC) Discussion in mice (16–19, 21) The systemic delivery of a formulated mimic of miR34a p53 is the most commonly mutated gene in patients caused tumor regression in two different orthotopic mod- with HCC (19, 22) and reduced p53 activity appears to be a els of liver cancer. Repeated dosing of the formulated key early event in the development of liver tumors (23, 24). tumor-suppressor miRNA did not impact normal animal Not surprisingly, the p53-regulated miR34a has reduced behavior nor did it produce any histologic evidence of expression levels in liver tumors (4). Consistent with a toxicity at any dose tested. Furthermore, there was no recent publication that describes the ability of miR34a to evidence of immune stimulation in mice or in human stimulate the p53 pathway in cancer cells lacking p53 (25), whole blood. The combination of antitumor activity and we observed increased expression levels of the p53-relat- favorable safety profile suggests that the formulated ed genes CHEK2, CDKN1A, BID, CASP3, and CCNG2 miR34a mimic might be effective as a targeted therapy (21, 26) in the liver tumors of mice dosed a single time for cancer. with MRX34. This result suggests that the potency of

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MRX34 might derive at least partially from its capacity to kinase, RAS, and STAT3 pathways, all of which have been stimulate the p53 pathway in cancer cells that lack p53 implicated in HCC development (16). In more advanced activity as is the case with the Hep3B cell line. tumors, c-Met activation leads to HCC proliferation (27) The hyperactivation of the WnT/B-catenin pathways is and additionally increases incidences of intrahepatic an early signature of hepatocellular carcinogenesis (27). tumor metastases through HGF (42). Reduced expression Abnormal WnT/b-catenin signaling impacts cellular pro- of c-Met and MAPK3 were observed in the liver tumors of liferation, metabolic function, and stem cell behavior (28) mice dosed with MRX34. Targeting the c-Met pathway and stimulates HCC formation (29). Following a single alone might be sufficient to inhibit growth of liver tumors administration of MRX34, we observed the inhibition of but the collective activation of the p53 pathway and FRAT1, CSNK2AP, CTNNB1, FBXW11, and NFATC3, attenuation of the Wnt/b-catenin, MapK, Hedgehog genes that are involved in propogating WnT/b-catenin VEGF, and c-MET pathways likely mitigates the ability signaling (30, 31). The ability to suppress WnT/b-catenin of liver tumors to proliferate by compensatory mechan- signaling with MRX34 likely contributes to the efficacy isms and aids in explaining the pronounced efficacy observed in the two xenograft models of HCC that we observed in our studies. used in our studies. The in vivo data represented here, coupled with the lack The MAPK pathway has been shown to be elevated in of adverse general health and immunostimulatory effects 91% of human samples of HCC (32). In these tumor types, in human cells, give MRX34 a clean and straightforward dysregulation of JNK1 and p38 is facilitated by MAPK path to evaluation in human clinical trials as an efficacious signaling and a cascade effect ultimately results in abnor- and safe means for treating HCC. In addition, many of the mal cellular proliferation, survival, and differentiation miR34a-regulated HCC oncogenes are also associated (33). With a single injection of MRX34 there were notable with the development of cancers that metastasize to the reductions in the expression of five genes that are com- liver, including colon, lung, pancreatic, and breast can- monly associated with the MAPK pathway, PDGF, RRAS, cers. This provides potential clinical applications for PRKX, NFkB, and ELK4. patients with advanced cancer with liver involvement The Hedgehog pathway plays a significant role in HCC (43, 44). proliferation, invasion, and metastasis by upregulating the protein expression of MMP9 via the ERK pathway (34, Disclosure of Potential Conflicts of Interest 35). In fact, formulated siRNA targeting Hedgehog is C.L. Daige, J.F. Wiggins, L. Priddy, T. Nelligan-Davis, and J. Zhao received commercial research grants from Cancer Prevention and sufficient to eliminate Hedgehog expression and marked- Research Institute of Texas (CPRIT). D. Brown received a commercial ly reduce metastasis in orthotopic mouse models of HCC research grant from CPRIT and has ownership interest (including patents) (36). A single injection of MRX34 reduced the tumor levels in Mirna Therapeutics. No potential conflicts of interest were disclosed by the other authors. of RAB23, PRKX, and FBXW11 mRNAs. All three genes fall within the Hedgehog pathway (37). Activation of the Authors' Contributions Hedgehog pathway has been detected in 67% of human Conception and design: C.L. Daige, J.F. Wiggins, D. Brown liver tumors (35). Development of methodology: C.L. Daige, J.F. Wiggins, L. Priddy, The VEGF pathway regulates angiogenesis and T. Nelligan-Davis, D. Brown Acquisition of data (provided animals, acquired and managed patients, enhances HCC tumor development when hyperactivated provided facilities, etc.): C.L. Daige, L. Priddy (38). Interestingly, VEGF expression has been reported to Analysis and interpretation of data (e.g., statistical analysis, biostatis- be highest in cirrhotic regions of the liver which surround tics, computational analysis): C.L. Daige, J.F. Wiggins, L. Priddy, T. Nelligan-Davis, J. Zhao, D. Brown HCC tumors in human patients (39), and agents are Writing, review, and/or revision of the manuscript: C.L. Daige, D. Brown currently being designed to target both tumor and sur- Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): C.L. Daige, J.F. Wiggins, L. Priddy, rounding liver tissue as both appear critical in tumor T. Nelligan-Davis, J. Zhao formation and propagation (40, 38). Administering a Study supervision: C.L. Daige, J.F. Wiggins, D. Brown single injection of MRX34 inhibited mRNA expression of MAPKAPK2, NFATC3, and MAPK1, which all function Grant Support within the VEGF pathway (41). The ability to diminish This work was supported by a commercialization award from the CPRIT that was awarded to all of the authors. angiogenesis via the VEGF pathway is a likely contributor The costs of publication of this article were defrayed in part by the to the efficacy observed in our mouse models of HCC. payment of page charges. This article must therefore be hereby marked c-MET plays a role in early HCC development and cell advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. proliferation (4). In fact, the activation of c-MET is sufficient to initiate hepatocellular carcinogenesis in mice (27), Received March 12, 2014; revised July 1, 2014; accepted July 18, 2014; presumably via the activation of the phosphoinositide 3- published OnlineFirst July 22, 2014.

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2360 Mol Cancer Ther; 13(10) October 2014 Molecular Cancer Therapeutics

Systemic Delivery of a miR34a Mimic as a Potential Therapeutic for Liver Cancer

Christopher L. Daige, Jason F. Wiggins, Leslie Priddy, et al.

Mol Cancer Ther 2014;13:2352-2360. Published OnlineFirst July 22, 2014.

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