Download Systemic Delivery of a miR34a Mimic as a Potential Therapeutic for

Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0209
Molecular
Cancer
Therapeutics
Large Molecule 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
The development of lipid nanoparticle technologies
that provide efficient delivery of RNA oligonucleotides
to hepatocytes and liver tumors (1) has created an opportunity for RNAi-based therapies. Among the most interesting RNAi therapeutic candidates for cancer are mimics
of tumor-suppressor miRNAs. Like multikinase inhibitors that have increased the survival rates of patients with
cancer (2), tumor-suppressor miRNAs inhibit cancer by
co-regulating many different oncogenes and oncogenic
pathways. One of the best characterized tumor-suppressor miRNAs, miR34a, functions within the p53 pathway to
regulate a diverse set of oncogenes (reviewed in ref. 3) and
reduced levels of the tumor-suppressor miRNA have
been observed in a variety of cancer types, including liver
cancer (4, 5). When introduced into cancer cells, miR34a
induces cell-cycle arrest (6), senescence (7), and apoptosis
(7). The miRNA also inhibits cancer stem cell development (8–10).
We have developed a miR34a mimic that has proved to
be effective in inhibiting the growth of tumors in mouse
models of lymphoma (11), lung cancer (12), and prostate
cancer (8). To address whether miR34a can inhibit liver
tumor growth, we encapsulated our miR34a mimic with
an amphoteric liposomal formulation and evaluated the
resulting nanoparticles for therapeutic activity using mice
Mirna Therapeutics, Inc., Austin, Texas.
Corresponding Author: David Brown, Mirna Therapeutics, Inc, 2150
Woodward, Austin, TX 78744. Phone: 512-681-5260; Fax: 512-6815200; E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-14-0209
2014 American Association for Cancer Research.
2352
with orthotopic Hep3B and HuH7 liver cancer xenografts.
Systemic delivery of the encapsulated miR34a mimic
reduced the expression levels of several miR34a target
mRNAs and caused significant growth inhibition and
even regression of the liver tumors. Toxicity studies
revealed no detrimental side effects or immune stimulation at therapeutic doses.
Materials and Methods
Cell culture
Hep3B cancer cells were purchased from ATCC in
January 2013. HuH7 cells were purchased from the
National Institute of Biomedical Innovation in Japan in
January 2013. Hep3B cells were cultured as recommended
by the manufacturer (ATCC). HuH7 cells were cultured in
DMEM (ATCC) supplemented with 10% FBS (HyClone).
All cells were grown at 37 C in 5% CO2–95% O2.
miRNAs and amphoteric liposomal formulation
The miR34a mimic was synthesized and purified by
Avecia Biotechnology using standard procedures for
RNA oligonucleotide synthesis and high-performance
liquid chromatography purification. Synthesis of a-(30 O-cholesteryloxycarbonyl)-d-(N-ethylmorpholine)-succinamide (MoChol) and production of amphoteric
liposomes were performed as described (13).
Orthotopic liver cancer models
Female NOD/SCID mice between 7 and 8 weeks of age
were purchased from Jackson Laboratory. All animals
were housed in disposable Micro-Isolator cages (Innovive)
and animal husbandry and procedures were carried out in
accordance with the institutional guidelines for proper
animal care and maintenance. Animals were maintained
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Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0209
miR34-Based Cancer Therapy Candidate
in aseptic conditions for all aspects of experimentation.
Mice were anesthetized under 3% isoflurane/O2 mixture
throughout surgery. Once sufficiently anesthetized, the
abdominal hair was removed with electric hair clippers
and the incision site sterilized with betadine and 70%
EtOH. A 1 cm incision was made along the midline of the
abdomen with the uppermost portion being just inferior
to the sternum. Separate incisions were required to safely
expose the peritoneum and the abdominal organs. The left
lateral lobe of the liver was exteriorized with cotton swabs
and a bolus of 2 106 Hep3B or 1 106 HuH7 cells in a 25
mL volume of PBS was injected using a 27-gauge needle
(BD Biosciences) attached to a 100 mL glass syringe
(Hamilton). Any bleeding was stopped by applying slight
pressure to the injection site with cotton swabs. The liver
was placed back in the abdomen, the peritoneum was
closed with 6.0 silk sutures, and the skin was closed with
wound clips. All wound clips were removed 7 days after
surgery.
Animal studies
Target gene identification. NOD/SCID mice with
Hep3B or HuH7 orthotopic liver cancer xenografts were
subjected to a single tail vein injection with liposome
dilution buffer, 1.0 mg/kg of formulated miR34a mimic
(MRX34), or liposome formulation alone with lipid concentrations equivalent to 1.0 mg/kg dose of formulated
miR34a mimic. Twenty-four hours after injection, tumors
were recovered from sacrificed mice and RNA was isolated and subjected to mRNA array analysis using an
Illumina Bead (Hep3B) or Affymetrix (HuH7) microArray. Microarray analyses were performed by Asuragen.
Array data are available at Gene Expression Omnibus
(GEO) using accession numbers GSE58893 (Hep3B) and
GSE58800 (HuH7).
Efficacy studies. NOD/SCID mice bearing Hep3B or
HuH7 orthotopic liver cancer xenografts were subjected
to multiple tail vein injections with liposome dilution
buffer: 0.3, 1.0, or 3.0 mg/kg MRX34 or liposome formulation alone with lipid concentrations equivalent to
0.3, 1.0, or 3.0 mg/kg MRX34. Tumor growth was assessed
by periodic blood collections and subsequent assaying of
a-fetoprotein (AFP) from sera. Three doses were given per
week (Monday/Wednesday/Friday) for two weeks and a
seventh dose was given on Monday. Twenty-four hours
after the final injections, animals were subjected to necropsy and tumors were resected and weighed. As both
Hep3B and HuH7 tumors typically grow as single bulbous tumor masses, they were easily dissected from
surrounding host liver tissue.
Toxicology assessments. Seven- to 8-week-old female
Balb/C mice (Charles River) were housed appropriately
and animal husbandry and procedures were carried out
in accordance with institutional guidelines for proper
animal care and maintenance. Animals were injected
intravenously with MRX34 or dilution buffer. Blood was
collected from mice via retro orbital sinus draws. Blood
samples were added to serum collection tubes for subse-
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quent serum separation by centrifugation. Serum cytokine levels were determined using a mouse Fluorokine
Multianalyte Profiling Kit (R&D Systems) and the Luminex 100 IS instrument. For blood chemistry analyses,
serum from animals dosed was sent to the Comparative
Pathology Laboratory at University of California, Davis
(Davis, CA).
AFP analysis
Sera were prepared from mouse blood samples by
centrifugation and AFP was measured by ELISA (DRG
EIA-4331). A standard curve was produced using purified
AFP so that ELISA readings could be converted to ng of
AFP/mL of serum.
Human whole blood immunostimulation assay
To determine whether the formulated miR34a mimic
induces a human cytokine response, formulated miRNAs
were incubated for 24 hours in whole blood collected from
three healthy male volunteers. Heparin was added to
the whole blood to prevent coagulation and the formulated miRNA was present at a final concentration of 600
nmol/L. After the incubation, plasma was separated by
centrifugation and IL6, IL8, and TNFa were analyzed by
ELISA (R&D Systems).
Quantitative RT-PCR
Total RNA was isolated from tissues and blood using
mirVana PARIS reagents (Ambion). All tissues were
homogenized in PARIS lysis buffer and total RNA was
isolated according to the manufacturer’s specifications.
For mRNA or miRNA analysis, cDNA was generated
from 10 ng total RNA, respectively, with MMLV reverse
transcriptase (Invitrogen). Quantitative PCR analysis
was 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, and
TRA2A were used along with ubiquitously expressed
GAPDH and Cyclophilin-A to generate raw mRNA
expression data used for relative standard curve PCR
analysis (RSC). For RSC, standard curves were generated from diluted negative control samples and raw
data from each primer/probe set were expressed in
terms of these curves to account for differences in
individual target reverse transcription and PCR efficiencies. The normalized data were compared for each test
primer/probe set against geometric means of RSC normalized GAPDH and Cyclophilin-A mRNA levels.
TaqMan primer/probe sets (Life Technologies) were
used to measure miRNA levels and Ct values were converted to copies of miRNA using a standard curve generated with the miR34a mimic that was used for injections.
Statistical analysis
Results are reported as the mean SD. The statistical
significance of the differences in tumor weights and AFP
levels in the miR34a efficacy experiments was assessed by
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Daige et al.
one-way ANOVA and the Dunnett posttest. All statistics
were generated using GraphPad Prism 5.04 software for
Windows, GraphPad Software (www.graphpad.com).
The level of significance was set at P < 0.05.
Results
Systemic delivery and activity of a miR34a-based
therapeutic candidate
A mimic of miR34a was encapsulated using a liposomal
formulation featuring amphoteric lipids that are cationic
during liposome formation to ensure efficient encapsulation of the miRNA mimic and anionic during storage and
delivery to maximize shelf-life, circulation time, and tissue uptake (14). We call the liposome-formulated miR34a
mimic MRX34. To characterize the delivery pattern of the
amphoteric liposomal formulation, we injected MRX34
into the tail veins of NOD/SCID mice bearing orthotopic
Hep3B liver tumor xenografts and measured the levels of
miR34a in five tissues of animals 24 hours after injection. A
single intravenous injection of MRX34 introduced more
than 60 million copies of the miRNA per ng of total RNA
from liver and more than 9 million copies per ng of tumor
RNA (Fig. 1A). The single injection of MRX34 also
increased the levels of miR34a by 100,000 to 4,000,000
copies per ng of total RNA from lung, spleen, and kidney
(Fig. 1A). The endogenous levels of miR34a in the five
tissues ranged from 10,000 to 100,000 copies per ng of total
RNA. The endogenous levels of miR34a and the delivered
copies of miR34a were similar for a second orthotopic liver
cancer model that featured xenografts of the HuH7
human liver cancer cell line (data not shown).
NOD/SCID mice with orthotopic Hep3B or HuH7 liver
tumor xenografts were subjected to a single dose of
MRX34, empty liposome, or dilution buffer and sacrificed
24 hours later. RNA samples from the tumors resected
from the mice were subjected to microarray analyses
and the resulting data were analyzed by ranking genes
based upon their level of differential expression in mice
dosed with MRX34 compared to mice dosed with dilution
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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Molecular Cancer Therapeutics
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miR34-Based Cancer Therapy Candidate
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)
CDKN1A (þ)
BID (þ)
CASP3 (þ)
CCNG2 (þ)
FRAT1 ()
CSNK2A1P ()
CTNNB1 ()
FBXWW11 ()
NFATC3 ()
PDGF ()
RRAS ()
PRKX ()
NFkb2 ()
ELK4 ()
THBS1 ()
RAB23 ()
PRKX ()
FBXW11 ()
VEGFB ()
MAPKAPK2 ()
NFATC3 ()
MAPK1 ()
cMET ()
MAPK3 ()
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
dosed with MRX34. Thirty-three of the downregulated
mRNAs were identified via TargetScan (15) as having
potential binding sites for miR34a. The levels of 59
mRNAs were reduced by at least 30% in the HuH7 liver
tumors of mice dosed with MRX34. Twenty of the downregulated mRNAs were identified via TargetScan as having potential binding sites for miR34a. Comparing the
downregulated mRNAs with miR34a-binding sites from
the two liver cancer models revealed that nine mRNAs are
common between the two datasets. Quantitative reverse
transcription-PCR (qRT-PCR) analysis confirmed that
the mRNA levels of all nine genes were significantly lower
in the liver tumors of mice dosed with MRX34 than the
mice dosed with dilution buffer or empty liposomes in
the Hep3B and HuH7 tumor RNA samples (Fig. 1B and C).
The complete list of genes that were differentially expressed (up or down) in the liver tumors of the MRX34-dosed
mice by 30% or greater includes 25 genes that are associated with pathways that are commonly altered in primary
liver tumors—p53, WnT/b-catenin, MapK, Hedgehog,
VEGF, and c-MET (refs. 16–18; Table 1).
miR34a anticancer effects in Hep3B liver orthotopic
mice
We conducted a study to evaluate the therapeutic
activity of MRX34 using mice with orthotopic liver cancer
xenografts. A total of 2 106 Hep3B cells were surgically
implanted into the left lateral lobes of the livers of female
NOD/SCID mice and tumors were allowed to develop for
3 weeks. We used a serum biomarker of liver cancer, AFP,
to monitor the growth of the xenografts. Serum AFP levels
correspond with tumor mass in orthotopically grown
Hep3B xenografts (data not shown) and provide an effective way to assess tumor size without the need for imaging. When AFP reached mean levels of 1,600 ng/mL of
serum, mice were separated into test and control groups of
6 animals per group (Fig. 2A). Animals received single
intravenous administrations of MRX34 at 0.3 or 3.0 mg of
miRNA/kg of body weight (mg/kg), 3.0 mg/kg equivalent (eq) of empty liposomes, or dilution buffer three times
per week (Monday/Wednesday/Friday) for 2 weeks with
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a final injection on the following Monday for a total of 7
doses. To monitor tumor growth in the animals, serum
AFP was measured every 3 to 4 days after the initiation of
dosing. At every time point following the initiation of
treatment, AFP levels in the two MRX34 dose groups were
significantly lower than the levels in the two control
groups (Fig. 2B). Mean serum AFP levels of animals in
the empty liposome and dilution buffer groups were
similar in magnitude reaching nearly 1,000,000 ng/mL
by the end of 2 weeks of dosing (Fig. 2B). In contrast, the
serum AFP levels of the mice on the MRX34 dose groups
ranged from less than 2-fold higher than the starting levels
to barely above background for the AFP ELIAS (Fig. 2B
and C).
Animals were sacrificed from each group 24 hours after
the final dose was administered and large tumors were
detected in the livers of all mice from the two control
groups, while much smaller and paler tumors, in general,
were detected in both MRX34 dose groups. In fact, tumors
were visibly absent in 1/7 and 3/7 animals dosed at 0.3
and 3.0 mg/kg MRX34, respectively (Fig. 2C and D). These
results have been repeated multiple times in the Hep3B
liver orthotopic mouse model and these data are representative of those results.
MRX34 inhibits growth of established HuH7
orthotopic liver tumors
We used a second orthotopic liver cancer model featuring HuH7 cells that express mutant p53 (19) to determine whether the therapeutic activity of MRX34 extends
beyond Hep3B xenografts. HuH7 and Hep3B cells have
distinct genetic backgrounds and cancer models featuring
these two cell lines are often used to evaluate how broadly
applicable a therapeutic candidate for liver cancer might
be. We surgically implanted HuH7 human liver cancer
cells into the left lateral lobes of NOD/SCID mice and
allowed tumors to develop for 2 weeks. When serum AFP
levels in 24 mice averaged 1,200 ng/mL, we separated the
mice into three dosing groups (Fig. 3A). The difference in
mean serum AFP levels between the HuH7 and Hep3B
efficacy studies (1,200 ng/mL vs. 1,600 ng/mL) was due to
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
tail vein injections of dilution buffer, 1.0 mg/kg MRX34, or
1.0 mg/kg equivalent of empty liposomes per week for 2
weeks and all mice received a seventh dose 15 days after
the initiation of dosing. Unlike the Hep3B study, where
we had enough mice with liver tumors to accommodate
two MRX34 dose groups, we only had enough mice with
HuH7 tumors to accommodate a single MRX34 dose
group. For the HuH7 study, we used an MRX34 dose
level (1.0 mg/kg) that was between the 0.3 and 3.0 mg/kg
doses that were used for the Hep3B study. Consistent with
the study using the Hep3B liver cancer model, tail vein
injections of MRX34 significantly reduced the accumula-
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tion of human AFP in the sera of mice compared with the
dilution buffer and empty liposome dosing groups (Fig.
3B). The serum AFP levels in the mice from both control
groups increased on average by more than 115-fold
between the first and last day of dosing. In contrast, the
average serum AFP levels for the animals in the MRX34
dose group increased by less than 2-fold and 2 of the 8
mice had substantially lower AFP levels at the end of the
dosing period than at the beginning (Fig. 3C). We sacrificed animals 24 hours after the final dose and tumors
were detected in all of the mice from the two control
groups, whereas only 5 of the 8 animals in the MRX34
group had detectable tumors (Fig. 3D). The average liver
Molecular Cancer Therapeutics
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miR34-Based Cancer Therapy Candidate
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
buffer and empty liposome groups were 155 and 159 mg,
respectively. The average tumor mass recovered from the
five MRX34-dosed mice that had liver tumors was 8 mg.
qRT-PCR analysis of the recovered tumors revealed that
animals dosed with MRX34 had approximately 107 more
copies of miR34a per ng of total tumor RNA than the
animals in the control groups (Fig. 3E). These results have
been individually reproduced in a separate efficacy study
with MRX34 in the HuH7 liver orthotopic mouse model
and these data are representative of those results.
MRX34 evaluation in immunocompetent systems
The safety profile of MRX34 was evaluated using
immunocompetent BALB/c mice. Three to 4 animals per
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group were dosed intravenously with 1.5 mg/kg MRX34
every other day (q.o.d.) for 2 weeks. Animal health was
monitored daily and liver, spleen, and whole blood were
collected 24 hours after the last intravenous administration. Whole organ weights were collected and compared
between groups and serum clinical chemistries were
analyzed. No body weight changes were observed over
the 2-week period between animals in the test (MRX34)
and control (dilution buffer) groups (Fig. 4A). Spleen
weights were normalized to body weight and no changes
in spleen size were observed in mice dosed with MRX34
compared with animals dosed with dilution buffer (Fig.
4B). In a separate study, we measured levels of cytokines
indicative of immune stimulation (IL1b, IL6, and TNFa) in
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
compared with normal levels measured in animals dosed
with PBS (Fig. 4C). An ex vivo model was used to confirm
that MRX34 is not immunostimulatory in human blood
(20). MRX34 (600 nmol/L), along with 600 nmol/L formulated NC2 and unformulated PBS, were incubated in
whole blood from three male donors for 24 hours and then
plasma was prepared and assayed for TNFa, IL6, and IL8.
Consistent with the mouse studies, MRX34 failed to stimulate a cytokine response (Fig. 4D).
Discussion
The systemic delivery of a formulated mimic of miR34a
caused tumor regression in two different orthotopic models of liver cancer. Repeated dosing of the formulated
tumor-suppressor miRNA did not impact normal animal
behavior nor did it produce any histologic evidence of
toxicity at any dose tested. Furthermore, there was no
evidence of immune stimulation in mice or in human
whole blood. The combination of antitumor activity and
favorable safety profile suggests that the formulated
miR34a mimic might be effective as a targeted therapy
for cancer.
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The profound efficacy exhibited by the systemic delivery of the miR34a mimic likely derives from the ability of
the small RNA to regulate multiple genes and pathways
that are important for hepatocellular carcinoma development. A single administration of the miRNA inhibited
multiple genes within oncogenic pathways of WnT/bCatenin, MapK, c-Met, Hedgehog, and VEGF and
stimulated multiple genes within the p53 pathway.
Hyperactivation of any of the five oncogenic pathways
or suppression of the p53 pathway have proved to stimulate the development of hepatocellular carcinoma (HCC)
in mice (16–19, 21)
p53 is the most commonly mutated gene in patients
with HCC (19, 22) and reduced p53 activity appears to be a
key early event in the development of liver tumors (23, 24).
Not surprisingly, the p53-regulated miR34a has reduced
expression levels in liver tumors (4). Consistent with a
recent publication that describes the ability of miR34a to
stimulate the p53 pathway in cancer cells lacking p53 (25),
we observed increased expression levels of the p53-related genes CHEK2, CDKN1A, BID, CASP3, and CCNG2
(21, 26) in the liver tumors of mice dosed a single time
with MRX34. This result suggests that the potency of
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miR34-Based Cancer Therapy Candidate
MRX34 might derive at least partially from its capacity to
stimulate the p53 pathway in cancer cells that lack p53
activity as is the case with the Hep3B cell line.
The hyperactivation of the WnT/B-catenin pathways is
an early signature of hepatocellular carcinogenesis (27).
Abnormal WnT/b-catenin signaling impacts cellular proliferation, metabolic function, and stem cell behavior (28)
and stimulates HCC formation (29). Following a single
administration of MRX34, we observed the inhibition of
FRAT1, CSNK2AP, CTNNB1, FBXW11, and NFATC3,
genes that are involved in propogating WnT/b-catenin
signaling (30, 31). The ability to suppress WnT/b-catenin
signaling with MRX34 likely contributes to the efficacy
observed in the two xenograft models of HCC that we
used 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 MAPK
signaling and a cascade effect ultimately results in abnormal cellular proliferation, survival, and differentiation
(33). With a single injection of MRX34 there were notable
reductions in the expression of five genes that are commonly associated with the MAPK pathway, PDGF, RRAS,
PRKX, NFkB, and ELK4.
The Hedgehog pathway plays a significant role in HCC
proliferation, invasion, and metastasis by upregulating
the protein expression of MMP9 via the ERK pathway (34,
35). In fact, formulated siRNA targeting Hedgehog is
sufficient to eliminate Hedgehog expression and markedly reduce metastasis in orthotopic mouse models of HCC
(36). A single injection of MRX34 reduced the tumor levels
of RAB23, PRKX, and FBXW11 mRNAs. All three genes
fall within the Hedgehog pathway (37). Activation of the
Hedgehog pathway has been detected in 67% of human
liver tumors (35).
The VEGF pathway regulates angiogenesis and
enhances HCC tumor development when hyperactivated
(38). Interestingly, VEGF expression has been reported to
be highest in cirrhotic regions of the liver which surround
HCC tumors in human patients (39), and agents are
currently being designed to target both tumor and surrounding liver tissue as both appear critical in tumor
formation and propagation (40, 38). Administering a
single injection of MRX34 inhibited mRNA expression of
MAPKAPK2, NFATC3, and MAPK1, which all function
within the VEGF pathway (41). The ability to diminish
angiogenesis via the VEGF pathway is a likely contributor
to 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 sufficient to initiate hepatocellular carcinogenesis in mice (27),
presumably via the activation of the phosphoinositide 3-
kinase, RAS, and STAT3 pathways, all of which have been
implicated in HCC development (16). In more advanced
tumors, c-Met activation leads to HCC proliferation (27)
and additionally increases incidences of intrahepatic
tumor metastases through HGF (42). Reduced expression
of c-Met and MAPK3 were observed in the liver tumors of
mice dosed with MRX34. Targeting the c-Met pathway
alone might be sufficient to inhibit growth of liver tumors
but the collective activation of the p53 pathway and
attenuation of the Wnt/b-catenin, MapK, Hedgehog
VEGF, and c-MET pathways likely mitigates the ability
of liver tumors to proliferate by compensatory mechanisms and aids in explaining the pronounced efficacy
observed in our studies.
The in vivo data represented here, coupled with the lack
of adverse general health and immunostimulatory effects
in human cells, give MRX34 a clean and straightforward
path to evaluation in human clinical trials as an efficacious
and safe means for treating HCC. In addition, many of the
miR34a-regulated HCC oncogenes are also associated
with the development of cancers that metastasize to the
liver, including colon, lung, pancreatic, and breast cancers. This provides potential clinical applications for
patients with advanced cancer with liver involvement
(43, 44).
Disclosure of Potential Conflicts of Interest
C.L. Daige, J.F. Wiggins, L. Priddy, T. Nelligan-Davis, and J. Zhao
received commercial research grants from Cancer Prevention and
Research Institute of Texas (CPRIT). D. Brown received a commercial
research grant from CPRIT and has ownership interest (including patents)
in Mirna Therapeutics. No potential conflicts of interest were disclosed by
the other authors.
Authors' Contributions
Conception and design: C.L. Daige, J.F. Wiggins, D. Brown
Development of methodology: C.L. Daige, J.F. Wiggins, L. Priddy,
T. Nelligan-Davis, D. Brown
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C.L. Daige, L. Priddy
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.L. Daige, J.F. Wiggins, L. Priddy,
T. Nelligan-Davis, J. Zhao, D. Brown
Writing, review, and/or revision of the manuscript: C.L. Daige, D. Brown
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.L. Daige, J.F. Wiggins, L. Priddy,
T. Nelligan-Davis, J. Zhao
Study supervision: C.L. Daige, J.F. Wiggins, D. Brown
Grant Support
This 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 marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received March 12, 2014; revised July 1, 2014; accepted July 18, 2014;
published OnlineFirst July 22, 2014.
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Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0209
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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