Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy?

Abstract

MicroRNAs (miRNAs) were discovered more than a decade ago as noncoding, single-stranded small RNAs (approximately 22 nucleotides) that control the timed gene expression pattern in Caenorhabditis elegans life cycle. A number of these evolutionarily conserved, endogenous miRNAs have been shown to regulate mammalian cell growth, differentiation and apoptosis. miRNAs are multispecific by nature. The individual miRNA is capable of modulating the expression of a network of mRNAs that it binds by imperfect sequence complementarity. Human cancers commonly exhibit an altered expression profile of miRNAs with oncogenic (miR-21, miR-106a and miR-155) or tumor-suppressive (let-7, miR-15a/16, miR-34a and miR-143/145) activity. As consistent with the natural function of miRNAs in specifying cellular phenotype, miRNA-based cancer gene therapy offers the theoretical appeal of targeting multiple gene networks that are controlled by a single, aberrantly expressed miRNA. Reconstitution of tumor-suppressive miRNA, or sequence-specific knockdown of oncogenic miRNAs by 'antagomirs,' has produced favorable antitumor outcomes in experimental models. We discuss pending issues that need to be resolved prior to the consideration of miRNA-based experimental cancer gene therapy. These include the need for definitive mRNA target validation, our incomplete understanding of rate-limiting cellular components that impact the efficiency of this posttranscriptional gene-silencing phenomenon, the possibility for nonspecific immune activation and the lack of a defined, optimal mode of delivery.

Full text

REVIEW
Modulation of miRNA activity in human cancer: a new paradigm
for cancer gene therapy?
AW Tong
1,2,3
and J Nemunaitis
1
1
Mary Crowley Cancer Research Centers, Dallas, TX, USA;
2
Gradalis, Inc., Dallas, TX, USA and
3
Cancer
Immunology Research Laboratory, Baylor Charles A Sammons Cancer Center, Dallas, TX, USA
MicroRNAs (miRNAs) were discovered more than a decade ago as noncoding, single-stranded small RNAs (B22 nucleotides) that
control the timed gene expression pattern in Caenorhabditis elegans life cycle. A number of these evolutionarily conserved,
endogenous miRNAs have been shown to regulate mammalian cell growth, differentiation and apoptosis. miRNAs are multispecific
by nature. The individual miRNA is capable of modulating the expression of a network of mRNAs that it binds by imperfect
sequence complementarity. Human cancers commonly exhibit an altered expression profile of miRNAs with oncogenic (miR-21,
miR-106a and miR-155) or tumor-suppressive (let-7, miR-15a/16, miR-34a and miR-143/145) activity. As consistent with the
natural function of miRNAs in specifying cellular phenotype, miRNA-based cancer gene therapy offers the theoretical appeal of
targeting multiple gene networks that are controlled by a single, aberrantly expressed miRNA. Reconstitution of tumor-suppressive
miRNA, or sequence-specific knockdown of oncogenic miRNAs by ‘antagomirs,’ has produced favorable antitumor outcomes in
experimental models. We discuss pending issues that need to be resolved prior to the consideration of miRNA-based experimental
cancer gene therapy. These include the need for definitive mRNA target validation, our incomplete understanding of rate-limiting
cellular components that impact the efficiency of this posttranscriptional gene-silencing phenomenon, the possibility for
nonspecific immune activation and the lack of a defined, optimal mode of delivery.
Cancer Gene Therapy (2008) 15, 341–355; doi:10.1038/cgt.2008.8; published online 28 March 2008
Keywords: micro-RNA; oncogene knockdown; antagomirs
Introduction
MicroRNAs (miRNAs) are a class of endogenous, small
(B22 nucleotides), noncoding RNA molecules that
regulate a wide array of developmental and physiologic
processes. The first miRNA, lin-4, was discovered in
Caenorhabditis elegans by Victor Ambrose,
1
and initially
termed an stRNA (short temporal RNA) in view of its
chronologically restricted expression, and its role in
orchestrating the transition from larval to adult stage
gene expression in C. elegans. The second miRNA, let-7,
was subsequently identified in a large number of species,
including vertebrates. Thousands of miRNAs have now
been cloned in higher eukaryotes.
2
These miRNAs are
highly conserved across species, and exhibit the common
function of negatively regulating gene expression at the
posttranscriptional level.
Approximately 450 miRNAs have been identified in
mammalian cells, although sequence analysis predicts the
presence of up to 1000 different miRNAs. However, only
a limited number has been defined functionally through
overexpression, misexpression and in vitro knockdown.
3–5
miRNAs modulate gene expression by utilizing much of
the cellular apparatus involved in the phenomenon of
RNA interference and perform endogenous functions that
were previously assigned exclusively to small interfering
RNAs (siRNAs).
6
miRNAs are commonly encoded as
polycistronic clusters. Processing by the RNase III
enzyme Drosha and its cofactor, Pasha (also known as
DGCR8/diGeorge syndrome critical region gene-8) con-
verts the pri-miRNA transcripts into stem-loop (hairpin)-
configured, 60–110-nucleotide pre-miRNAs in the nucleus
(Figure 1). The pre-miRNA is exported into the
cytoplasm by exportin 5 and involves Ran, a GTP-binding
cofactor.
7
Another RNase III enzyme, Dicer, cleaves the
pre-miRNA into a transient, B22-nucleotide miRNA/
miRNA* duplex intermediate prior to its integration into
the miRNA-associated multiprotein RNA-induced silen-
cing complex (miRISC).
8
The miRISC assembly comprises of the Argonaute
proteins (AGO2 and AGO1), as well as other proteins
(TRBP and PACT) that interact with the Dicer-linked
miRNA/miRNA* duplex. Unwinding of the duplex likely
involves an ATP-dependent RNA helicase.
9
The antisense
strand of the miRNA, retained by the complex by virtue
Received 14 August 2007; revised 22 December 2007; accepted 19
January 2008; published online 28 March 2008
Correspondence: Dr AW Tong, Cancer Immunology Research
Laboratory, Baylor Charles A Sammons Cancer Center, 3500
Gaston Avenue, Dallas, TX 75246, USA.
E-mail: atong@gradalisbio.com
Cancer Gene Therapy (2008) 15, 341–355
r
2008 Nature Publishing Group All rights reserved 0929-1903/08 $30.00
www.nature.com
/
cgt

of its pharmacodynamic features,
10
is guided by the
miRISC effector complex to complement with the target
messenger RNA (mRNA) sequence, forming an A-form
double-stranded helix.
11
mRNA target recognition com-
monly requires a perfect 6–7-nucleotide match at the
5
0
end of the miRNA, known as the ‘seed’ sequence, and
extensive base pairing to the remaining sequence of the
miRNA.
12
AGO2, an endonucleolytic component of the
miRISC, appears responsible for the cleavage and
displacement of the passenger strand (miRNA*), as well
as cleavage of the targeted mRNA, which complements
perfectly with the miRISC-loaded miRNA.
13,14
By
comparison, mRNAs that binds with imperfect comple-
mentarity to the miRNA are subject to translational
blockade, likely due to the formation of a bulge sequence
in the middle of the A-form helix that is not suitable for
RNA cleavage (reviewed in Rana
15
). Translational
repression is considered to be the predominant mechan-
ism in mammalian cells,
16–19
and requires the general
translation repressor protein RCK (also known as p54
20
),
which has a putative role of oligomerizing with the target
mRNAs and shuttling them to P-bodies, cytoplasmic foci
that serve as depot for storage and/or sequence-specific
miRISC processing.
16,21
Deadenylation and subsequent
mRNA degradation constitutes yet another mechanism
for the downregulation of mRNA target from imperfectly
matched miRNA binding.
22,23
Effector function through imperfect complementation
implies that an individual miRNA can potentially
regulate many mRNA targets.
19
Indeed, sequence analysis
Figure 1 Biogenesis of mammalian microRNA (miRNA). miRNAs, often encoded as polycistrons, are transcribed into primary miRNA
transcripts, then processed by the RNase III enzyme Drosha and its cofactor, Pasha into 60–110-nucleotide pre-miRNA hairpins in the nucleus.
The pre-miRNA is exported into the cytoplasm by exportin 5 and its cofactor Ran, where it is cleaved by Dicer into a transient, B22-nucleotide
miRNA/miRNA* duplex intermediate. The antisense strand of the miRNA, retained by the complex by virtue of its pharmacodynamic features, is
guided by the multiprotein RNA-induced silencing complex (miRISC) effector complex to complement with the target messenger RNA (mRNA)
sequence, forming an A-form double-stranded helix. AGO2, an endonucleolytic component of the miRISC, appears responsible for the cleavage
and displacement of the passenger strand (miRNA*), as well as endonucleolytic cleavage of the targeted mRNA that binds with the loaded
miRNA by perfect sequence complementation. By comparison, mRNAs that bind with imperfect complementarity to the miRNA are subject to
translational blockade, likely due to the formation of a bulge sequence in the middle of the A-form helix that hinders the cleavage process.
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has identified multiple, eligible binding sites in the 3
0
-
UTRs of different mRNAs, hence giving rise to divergent
(a single miRNA modulating multiple mRNA targets)
and convergent (multiple miRNAs modulating a single
mRNA target) paradigms of gene regulation.
10,12,24
For
example, the 3
0
-UTR of the pivotal angiogenic factor
VEGF (vascular endothelial growth factor) contains eligible
binding domains for 13 miRNAs, including let-7b,
miR-16, miR-17-5p, miR-20a, miR-20b, miR-106a and
miR-106b in the nasopharyngeal carcinoma line CNE. To
examine the interactive outcome of these miRNAs, Hua
et al.
25
have carried out cotransfection studies with the
predicted miRNA-binding regions cloned into the 3
0
-
UTR of a luciferase reporter gene. miRNAs with distinct
binding sites (miR-20a and miR-361) exhibited coordinate,
additive suppressive activity, whereas the suppressive
activity of one miRNA was usurped by another with an
overlapping binding domain (miR-125a and miR-378). As
consistent with the divergent paradigm, coregulatory
activities were also observed, whereby VEGF-suppressive
miRNAs also downregulated other angiogenesis-related
genes, including uPAR, COX2 and c-MET.
25
Thus, a
relatively small number of miRNA networks can effec-
tively modulate multiple factors involved in angiogenesis.
It is estimated that up to one-third of protein-coding
human mRNAs are susceptible to this complex miRNA-
regulatory network.
26
Genes that are key regulators or
essential for cell function may have high rates of
transcription and low rates of translation.
27
This ‘genetic
buffering’ effect can be achieved by negative translational
modulation by miRNAs, allowing the cell to make many
mRNA copies but to have a low and carefully controlled
amount of protein.
28
Thus, appropriate actions by an
miRNA can achieve rapid changes in protein synthesis
without altering transcriptional activity or mRNA pro-
cessing. A subset of intragenic, intronic miRNAs has been
identified recently in mammalian cells to share the same
promoter with their encoded target genes. The expression
of a gene results not only in the gain of function of that
gene but also the coordinate loss of function of other
downstream genes with complementarity to the mature
intronic miRNA.
29
This mechanism allows for a precise,
immediate and rapidly reversible way of controlling
expression of proteins.
30
Altered miRNA expression in human cancers
Multiple mechanisms lead to altered miRNA expression
in human cancers, although a majority of studies point to
chromosomal aberrations as the primary contributor.
Aberrant miRNA expression in human cancers was first
described in B-CLL (B-cell chronic lymphocytic leukemia),
where hemizygous and/or homozygous chromosomal
deletion at the 13q14 locus results in the loss of
miR-15 and miR-16 expression.
31,32
The preponderance
of this phenomenon in indolent CLLs and a large
proportion of mantle cell lymphomas and multiple
myelomas
33
implicate a pathophysiologic role for the loss
of miR-15/16 function in these hematopoietic malignan-
cies.
34–36
Of the 186 miRNA sequences tested by Calin
et al.,
32
approximately 50% were localized to cancer-
associated genomic regions of instability, including fragile
sites (FRAs) and human papilloma virus (HPV) integra-
tion sites, homeobox (HOX) genes, minimal regions of
loss of heterozygosity (LOH) and regions of amplifica-
tion. The high frequency of genomic alterations in
miRNA loci in human cancers was recently confirmed
by high-resolution array comparative genomic hybridiza-
tion analyses.
37
Notably, miR-125b, a homolog of lin-4,is
located in a fragile site on chromosome 11q24 and now
known to be deleted in a subset of patients with breast,
lung, ovarian and cervical cancer (Table 1). Chromosomal
deletion at 5q32 was seen in colon
71
and breast
51
cancers,
accounting for the downregulated expression of the
miR-143/145 clusters. Gene copy number changes were
believed to be responsible for the altered expression of 41
miRNAs (26 with gains and 15 with losses) in at least
15% of breast and ovarian cancers, and melanoma.
37
Recent findings indicate that epigenetic aberrations,
83–87
and miRNA processing and degradation defects also
affect miRNA expression.
88–91
About half of the miRNA
genes are associated with CpG islands and thus represent
candidate targets for the DNA methylation machinery.
87
Methylation status has been correlated with altered levels
of miRNAs in a number of recent studies, such as
decreased miR-124a expression that was attributed to
DNA hypermethylation in colon, breast and lung
carcinomas.
85
Conversely, DNA hypomethylation likely
led to increased miR-21, -203 and -205 in ovarian cancer
cells,
52
and let-7-3a in lung adenocarcinomas.
86
As a
whole, miRNA promoters contain a lower percentage of
CpG islands as compared with coding gene promoters,
suggesting that CpG island modification may play a
comparatively less important role in the transcriptional
regulation of miRNA.
In spite of normal expression levels of pri-miRNA, a
number of human primary cancers displayed reduced
levels of mature miRNAs.
88
Thomson and co-workers
88
subsequently localized this discrepancy to a processing
defect from the loss of the RNase III Drosha. Conversely,
enhanced Drosha activity, through its fusion with the
translocated, oncogenic All1 gene, appeared to be
responsible for miR-191 upregulation in human leukemias,
89
which in turn contributed to a poor prognostic outcome
for acute myeloid leukemias. In a proportion of
non-small cell lung cancers (NSCLCs), however, de-
creased Dicer endonuclease activity, but not Drosha,
correlated with reduced let-7 expression, an unfavorable
postoperative survival and poor tumor differentiation
status.
90
The loss of Dicer likely represents a somatic
alteration, as Dicer-deficient mice failed to thrive beyond
gastrulation due to the lack of multipotent stem cell
development.
91
Another caveat is that these enzymes may
perform functions in addition to miRNA processing.
92
Hence, common oncogenetic and epigenetic defects likely
impact both coding and noncoding RNA transcription,
contributing independently or in concert to the altered
expression of miRNAs.
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Table 1 Commonly altered miRNA expression in human cancer
miRNAs Cancer cell type Putative targets Reference Notes
Br Co Ce Gl Hp Ov Pn LL Ln Pr Te Thy
let-7 kk kk c-MYC, RAS, HMGA2
38–45
Level inversely correlated with survival in NSCLCs; modulated by p53
miR-10b m HOXD10
46
Regulates metastatic/invasive function in breast cancer; modulated by p53
miR-15a, miR-16 kkkkBcl-2
2,31,32,43,45,47,48
miR-17-92 mmmtsp-1, CTGF
45,48–50
miR-18, miR-19 bind to antiangiogenic proteins in myc-overexpressing
tumors, modulated by p53
miR-20a k E2F1, TGFbR2
45
Modulated by p53, has antiapoptotic role
miR-21 m m mmm m m PTEN, TPM1, PDCD4
44,48,51–58
miR-26a k PLAG1
59
miR-30c, Mir-d kk
49,59
miR-34a, -34b, -34bc m,kk k k E2F3, NOTCH1, DLL1
45,51,53,60–65
p53-Dependent induction by DNA damage and oncogenic stress;
expression leads to apoptosis and cellular senescence
miR-106a mmmmRB-1
66,67
Regulated by c-myc
miR-122a k Cyclin G1
44
Hepato-specific expression
miR-125b kk k kk kErbB2/3, EIF4EBP1
37,51,59,68
Targets TNFa production in macrophages
miR-127 kkk kkk Bcl-6
69
Epigenetically downregulated in the majority of human cancer lines
miR-128 k
70
miR-143, miR-145 kkk kk k k ERK5, Raf1, G-protein 7
41,44,51,56,71–73
miR-155 mmmmAT1R, TP53INP 1
48,51,74
miR-200a, -200b, -200c mk TCF8 for miR-200c
75
miR-221/222 mm k2m mP27Kip1, c-KIT
59,70,76–81
Directly correlated with NSCLC survival, miR-221 induced by
MYCN in neuroblastoma.
miR-372, miR-373 m LATS2
82
Abbreviations: AT1R, angiotensin II type I receptor; Bcl-2, B-cell lymphoma 2; Br, breast; Ce, cervical; Co, colon; CTGF, connective tissue growth factor; E2F1, E2F
transcription factor 1; EIF4EBP1, eukaryotic elongation initiation factor 4E binding protein 1; ERK5, extracellular signal-regulated kinase-5; G-protein, guanine nucleotide-binding
protein; Gl, glioblastoma; Hp, hepatocellular; c-kit, tyrosine kinase; LATS2, large tumor suppressor homolog 2; LL, leukemia and lymphoma; Ln, lung; miRNA, microRNA; c-myc,
myelocytomatosis viral oncogene; NSCLC, nonsmall cell lung cancer; Ov, ovarian; PDCD4, programmed cell death 4 tumor suppressor protein; PLAG1, pleomorphic adenoma
gene 1; Pn, pancreatic, Pr, prostate; PTEN, phosphatase and tensin homolog; Raf1, serine/threonine protein kinase; Ras, rat sarcoma viral oncogene homolog; RB-1,
retinoblastoma 1; TCF8 (ZEB1, deltaEF1), transcription factor 8; Te, testes; TGFb2R, transforming growth factor beta-2 receptor; Thy: thyroid carcinomas; TP53INP 1, tumor
protein p53-induced nuclear protein 1; TPM1, tropomyosin 1; tsp-1, thrombospondin-1.
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miRNA profiling in human cancers
Concurrent with studies that define the pivotal role of
miRNAs in regulating normal tissue differentiation, high-
throughput analyses have been used in determining the
miRNA phenotype of human cancers. Lu et al.
38
showed
that primary and metastatic lung adenocarcinomas
exhibit a unique miRNA expression profile from normal
lung, based on liquid-phase array hybridization with 131
miRNA probes. Volinia
66
identified a solid cancer
miRNA signature having common features for lung,
breast, stomach, prostate, colon and pancreatic tumors,
and comprised 43 significantly altered miRNAs that
embodied the cancer-associated miRNAs miR-17-5p,
miR-20a, miR-21, miR-92, miR106a and miR-155.As
consistent with the tissue-specific pattern of miRNA
expression, miRNA profiling successfully identified the
tissue origin of 15 distinct tumor types with 80%
accuracy.
93
Recent studies indicated that miRNA expression
profiles correlated with cancer pathophysiological features.
Bandres
49
showed that colorectal cancer development was
associated with significantly downregulated miR-30c,
-124a, -133a and miR-145, and increased expression
of 98 miRNAs including the miR-17-92 cluster.
Similarly, miRNA expression profiling of 76 primary
breast cancer samples have identified an array of
dysregulated miRNAs (decreased miR-145 and miR-
125b, and increased miR-21, -34 and -155) that reflected
biopathologic features, including estrogen/progesterone
receptor expression, tumor stage, vascular invasion or
proliferation index.
51
miR-10b was overexpressed in
metastatic breast cancer cells and likely constituted an
inducing component for distant metastases.
46
While
differential expression of coding mRNA between normal
and malignant tissues may differ by orders of magnitude,
relatively modest differences (one- to twofold or less)
were commonly observed with respect to individual
miRNAs,
15,28,30
which may account for some conflicting
findings regarding the absolute expression level of
individual miRNAs. Of the two widely reported high-
throughput techniques, the solid-phase, array-based plat-
form is generally considered as semiquantitative, often
requiring transcript amplification/labeling and carries an
inherent limitation of cross hybridization among miRNAs
of the same family.
88,94,95
Flow-based, liquid-phase
profiling has the theoretical edge of increased specificity
in discriminating the expression of closely related
miRNAs,
38,49
but is technically demanding with respect
to quality consistency in the production of miRNA
probes and the intricacy of the flow analytic processing
for reactant subset discrimination. This technique report-
edly is more sensitive in detecting modest decreases in
downregulated miRNAs.
38,96
Regardless of the high-
throughput platform, independent validation by a second
technique such as northern blotting or quantitative, real-
time PCR, is de rigueur for confirming miRNA expression
profile. Further, the identification of histotype-specific
mRNA target and/or affected cellular pathways is critical,
to establish the pathophysiologic relevance of the
individual, aberrantly expressed miRNAs as related to
cancer initiation and progression.
let-7, multiple mRNA targets and impact on patient
survival
The term ‘oncomir’ was coined by Hammond and
co-workers
76
to denote miRNAs that are integral to the
molecular architecture of oncogene and tumor suppressor
networks, and best exemplified by the let-7 miRNA.
let-7 is one of the two stRNAs initially discovered in
C. elegans. Owing in part to its highly conserved nature
even in humans, let-7 was the first miRNA identified in
mammalian cells.
97
In C. elegans, let-7 suppresses the
expression of let-60,
98
an ortholog of the human RAS
family of oncogenes. Indeed, a variety of studies have
demonstrated the tumor suppressor role of let-7 in
mammalian lung tissues.
39
let-7 expression was consis-
tently reduced by 50% in lung tumors, as compared with
adjacent normal tissues.
98
Decreased let-7 expression was
an independent prognosticator for poor survival
40
in a
retrospective study on 143 cases of surgically resected
NSCLCs. Patients with low let-7 expression had a greater
risk of earlier death and a hazard ratio of 2.2 as compared
with patients with high let-7 expression. Lower expression
of let-7 was correlated with a markedly upregulated levels
of RAS oncoprotein, another biomarker for poor survival
for NSCLC.
99
In vitro studies showed that exogenously
introduced let-7a and let-7f, two of the dominant isoforms
of the let-7 family, markedly reduced in vitro clonogeni-
city of the human lung cancer cell line A549,
40
whereas
inhibition of let-7 function led to increased cell division in
A549 and the hepatoma line HepG2, likely due to a
blockade of G
1
/S transition.
39
The loss of let-7 also led to
radioresistance in vitro, suggesting that altered
let-7 expression likely impacts the response of a cancer
cell to treatment.
100
Recently, Karube
101
showed that NSCLCs also dis-
played decreased Dicer expression. Poorly differentiated
tumors exhibited a higher incidence of reduced Dicer
expression, and lower Dicer expression was an indepen-
dent prognostic indicator for surgically treated NSCLC.
Hence, one could speculate that cellular dysfunction
stemming from an altered let-7 genotype, its 3
0
-UTR
miRNA-binding sites, or pathways that process or
regulate let-7 expression may contribute critically to lung
cancer oncogenesis and disease progression.
47
The cancer growth-suppressive activity of let-7 has
since been validated in other human cancers, including
colon,
41,102
hepatocellular,
103
gastric
104
and ovarian
cancers.
42
In addition to members of the RAS family,
Johnson showed that the cell-cycle regulators CDK6
(cyclin D kinase 6) and CDC25A are also likely, direct
targets for let-7, based on differential, high-throughput
expression array analysis, in silico target prediction and
validation by reporter gene expression analysis in let-7-
transfected lung and liver cancer lines.
39
The tumor-
suppressive role of let-7 may also come from its binding to
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the high-mobility group A2 (hmga2) oncogene.
105
The
loss of the let-7-binding/repression elements in the 3
0
-
UTR of hmga2 mRNA was associated with the develop-
ment of abdominal lipomatosis, lymphomas, pituitary
adenomas and lung adenomas. The targeting of hmga2
mRNA by let-7 was recently confirmed by expression
analysis with the NCI60 panel of human tumor cell lines,
where loss of let-7 expression was a marker for less
differentiated cancers.
42
For MYC-dysregulated hemato-
poietic cancers such as Burkitt lymphoma, let-7 expres-
sion was inversely correlated with that of the c-myc
oncogene mRNA.
43,106
Transfection with pre-let-7a
downregulated myc mRNA and its target genes, and
reduced proliferation. These findings illustrate the context
dependence of let-7 targets in cancers of different
histological types. The tumor-suppressive role of let-7
appears to be directly related to its capacity to modulate
multiple oncogenes, including RAS, MYC and HMGA2.
miRNAs target oncogenes and tumor suppressor genes
Evidence is accumulating with regards to the capacity of
other aberrantly expressed miRNAs to modulate onco-
genic and tumor suppressor activity, hence providing
explanations for their pathophysiologic role in human
oncogenesis and tumor progression (Table 1 and Figure 2).
The target gene for miR-15a and miR-16-1 has been
identified to be bcl-2, the archetypical antiapoptotic gene
whose activation is considered to be critical for the
oncogenic process of human lymphomas, leukemias and
lung cancer. The loss of these miRNAs correlated with
BCL-2 overexpression, while mir-15/16 reconstitution
increased tumor apoptotic activity through their inter-
action with bcl-2 mRNA.
107
The proto-oncogene c-myc encodes a transcription
factor that regulates cell proliferation, growth and
apoptosis, whose activity is in turn modulated by
miRNAs. The miR-17-92 polycistron, commonly elevated
in C-MYC
þ
human B-cell lymphomas, was found to act
in concert with this oncogene to accelerate tumor
development in a mouse lymphoma model.
76
For primary
neuroblastomas, miR-17-92 cluster (including miR-106a)
expression correlated positively with N-MYC amplification.
77
Increased neovascularization in MYC-overexpressing
tumors was mediated at least in part through miR-17-92
cluster upregulation,
108
resulting in the downregulation
of antiangiogenic proteins with thrombospondin motifs
including tsp-1 (thromospondin-1)bymiR-19 and the
CTGF (connective tissue growth factor)bymiR-18.
Conversely, miR-17-5 and miR-20a of the miR-17-92
cluster also exhibited tumor suppressor activity through
its downregulation of E2F1 or other C-MYC-dependent
transcriptional factors. E2F1 is a downstream target gene
of c-myc, best known for driving cell-cycle progression in
the G1 stage. Other C-MYC target genes (RPS6KA5,
BCL-11B, PTEN (phosphatase and tensin homolog) and
HCFC2) are also predicted templates for translational
regulation by the miR-17-92 cluster.
109
These findings,
when contrasted with the oncogenic activity of the miR-
17-92 cluster in diffuse large cell lymphoma and B-CLL,
indicate that the regulatory roles of miR-17-92 are highly
contextural, exerting their influence on cell proliferation
and tumorigenesis according to the milieu of target
mRNAs that are expressed.
71
A number of solid cancers displayed reduced steady-
state expression of the miR-143 and miR-145 cluster,
including adenomatous and cancer stage of colorectal
neoplasia, breast, prostate, cervical, hepatocellular carci-
nomas, as well various B-cell malignancies.
71,72,44
The
tumor-suppressive activity of miR-143 and miR-145 has
been demonstrated in vitro,
41
with ERK5 (extracellular
signal-regulated kinase-5) identified as the target gene for
miR143 in colon cancer DLD-1
41
and Burkitt lymphoma
RAJI
72
lines. ERK5, a recently discovered member of the
MAPK (mitogen-activated protein kinase) family and
downstream mediator of multiple extracellular signals
and oncogenes, is a critical component for mitotic entry
and for the survival of proliferating cells.
110
Other eligible
targets for miR-143/145
71
include MTHFR (methylene-
tetrahydrofolate reductase), whose variant expression was
associated with aberrant DNA methylation patterns in
colorectal tumors,
111
and the sodium- and potassium-
dependent ATPase A subunit (ATP1A1) shown to be
reduced in premalignant mucosa and in colorectal
tumors.
112
Hence, the tumor suppressor activity of
miR-143 and miR-145 may manifest in a wide array of
Figure 2 The Yin and Yang of microRNA (miRNA) activity in
cancer pathophysiology. While all miRNAs act as negative,
posttranslational silencing regulators, their aberrant activity may
lead to opposing pathophysiologic outcome in accordance to their
targeted messenger RNAs (mRNAs). Representative miRNAs with
tumor-suppressive and/or oncogenic miRNAs are shown. The miR-
17-92 cluster, an example of context-dependent miRNAs having
oncogenic as well as tumor-suppressive features, is shown in both
hemispheres.
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Cancer Gene Therapy

pathophysiological functions. It is noteworthy that
the hairpin precursor transcripts of miR-143 and
miR-145 were not downregulated in colon tumor
tissues,
71
suggesting that a disruption in processing may
account for the decreased expression of the mature
miR-143/145.
The miR-221/222 cluster has been shown to be
upregulated in various malignancies of neural crest
tissues, including glioblastomas, neuroblastoma with N-
myc amplification and thyroid papillary carcinoma.
78,70,79
Earlier studies suggest that miR-221/222 targeted the
proto-oncogene/tyrosine kinase receptor c-kit in papillary
thyroid cancers,
76,70,79
although this finding was not
experimentally validated. Three independent studies
recently identified experimentally that the multifunctional
cyclin-dependent kinase inhibitor and tumor suppressor
p27 (kip1) was a direct target of miR-221 in glioblastoma,
78
papillary carcinoma
59
and prostate cancer.
80
The findings
of miR-221/222 dependence for tumor survival have
prompted the investigation of miR-221/222 inhibitors as
a venue for experimental therapy of these tumor cell
types.
79
The human proto-oncogene bcl-6 encodes a BTB/POZ-
zinc-finger transcriptional repressor that is necessary for
germinal center formation, and is implicated in the
pathogenesis of B-cell lymphoma.
113,114
BCL-6 suppresses
p53 expression and modulates DNA damage-induced
apoptotic responses in germinal center B cells. bcl-6 is a
predicted target of miR-127, an miRNA that is part of the
miR-136, -431 and -433 cluster.
69
This miRNA cluster is
downregulated or completely silenced in the majority of
human tumor cell lines. However, DNA demethylation
and histone deacetylase inhibition can activate the
expression of the tumor-suppressive miR-127, corres-
pondingly downregulating bcl-6.
Intermodulation of miRNAs and tumor suppressor genes
A number of miRNAs have now been shown to serve as
effectors for the tumor suppressors p53 and rb. The tumor
suppressor gene p53, best known as a key regulator of
cell-cycle control and apoptosis,
115
may impact miRNA
activity by at least two mechanisms, by functioning as an
RNA-binding protein and recruiting miRNA to the
actively translated mRNA complex, or as a transcription
factor to upregulate miRNAs and downstream cellular
mRNAs.
60
Conditional expression of the p53 transgene,
or activation of endogenous, wt p53, both led to
pronounced upregulation of miR-34a in human cancer
cells, indicating that miR-34a is encoded by a p53-
responsive gene.
45
Studies by Raver-Shapira,
61
Chang
62
and others
45
showed that miR-34a is a direct transcrip-
tional target of p53 that can mediate the proapoptotic
effects of p53.
61
Hence, altered miR-34a expression may
contribute to tumorigenesis by attenuating p53-dependent
apoptosis.
In the p53
null
HCT-116 colon carcinoma line, ectopic
wt-p53 expression led to increased expression of 11
miRNAs, including miR-181 and miR-132 with cell
proliferation regulatory activities, and miR-21 with
antiapoptotic function.
30
miR-21 was also upregulated
in glioblastoma cells,
53
and recently found to target the
tumor suppressor gene, TPM1 (tropomyosin 1) that
modulated human breast cancer cell growth.
54
wt-p53
expression decreased the expression of 43 miRNAs,
including miR-15b, miR-125a and miR-191, whereas the
loss of wt-p53 expression increased expression of SPIB,a
regulator of transcription from Pol II promoter, and
translation initiation factor 4A and 5A, which are
susceptible to miR-15b and miR-125a modulation. For
the H1299 lung cancer line, conditional expression of the
p53 wild-type transgene led to increased expression of 34
miRNAs, whose putative silencing targets include E2F3/
NOTCH1/DLL1 (miR-34a), E2F1 (miR-20a, miR-17-5a),
BCL-2 (miR-15a and miR-16) SDF-1 and BRN-3b (miR-
23a).
45
Hence, the loss of tumor suppressor activity from
p53 aberrancy may manifest by downstream alterations
of multiple miRNAs serving as intermediate effectors
for increased cell cycling and decreased apoptotic
consequences.
Like p53, the loss of RB1 function impacts cellular
differentiation, survival and programed cell death. For
colon cancer cells, the lack of RB1 protein expression
appears to be a translation defect, in view of normal rb1
transcriptional activity. In gastric, prostate and lung
tumor samples, downregulated RB1 has been inversely
correlated with the overexpression of miR-106a.
66
miR-
106a has now been shown to bind to the UTR of rb1
mRNA,
66
reflecting its capacity to regulate RB1 activity
posttranscriptionally. Conversely, breast cancers with
modestly increased RB1 levels exhibited lower miR-
106a.
66
The preliminary findings of Lujambio indicated
that epigenetic loss of miR-124a was linked with RB
phosphorylation and activation of oncogenic CDK6 in
human colon cancer cell lines.
67
Thus, miRNAs may act
both as effectors or modulators of oncogenes and tumor
suppressor genes in controlling cell differentiation and
apoptosis.
Theoretical and practical considerations of miRNA-based
cancer gene therapy
The presence of functional redundancy in a thriving
biologic system, such as cancer, likely buffers the
populational impact by any single gene/target modifica-
tion on the malignant process, with rare exception (for
example, chronic myeloid leukemia).
116
However, highly
connected targets do allow for ‘attack vulnerability.’ In
other words, the disordered circuitry characteristic of
malignancy results in a change, such that the otherwise
robust oncogenic process can become, almost paradoxi-
cally, more highly dependent on a specific rewired
pathway (‘pathway addiction’).
116
This thesis forms the
theoretic basis of attempting to correct the tumor
phenotype through the targeting of the dominant,
offending oncogenic pathway.
miRNA-based cancer gene therapy
AW Tong and J Nemunaitis
347
Cancer Gene Therapy

miRNA-based cancer gene therapy offers the theore-
tical alternative of targeting multiple gene networks that
are controlled by an individual miRNA, as consistent
with the natural function of this class of molecules in
specifying cellular phenotype through posttranscriptional
modulation.
92
As illustrated by Lim et al.,
117
the
physiological phenotype of a cell may be balanced on
the activity of a limited number of miRNAs. Ectopic
expression of miR-124 in the cervical cancer line HeLa
shifted gene expression profile toward that of the brain,
whereas delivery of miR-1 shifted its gene expression
toward that of skeletal muscles. Correspondingly, high
endogenous miR-124 and miR-1 expression was found in
brain and skeletal muscles, respectively. With respect to
hematopoietic differentiation, unilineage erythroid differ-
entiation requires the downregulation of miR-221 and
miR-222, two miRNAs that are highly expressed in
human cord blood-derived CD34 þ progenitor cells,
118
whereas ectopic expression of miR-181a or miR-
223 þ miR-142 was adequate in directing lymphoid
progenitor differentiation toward B- or T-cell lineage,
respectively.
119
Thus, miRNA-based cancer therapeutics
holds the appeal of efficacy and widespread phenotypic
ramifications.
Efficacy
MicroRNAs offer the venue of modulating gene expres-
sion programs in an immediate and reversible manner,
based on their natural function of fine-tuning gene
expression at the posttranscriptional level.
30
Reconstitu-
tion of downregulated miRNAs offers the theoretical edge
of correcting the malignant defect with endogenously
occurring molecules of arguably preoptimized binding
avidity through natural selection. As discussed in a
subsequent section, lessons from the study of ectopically
introduced miRNA indicate that relatively small changes
in miRNA gene dosage and corresponding gene modifier
effects can achieve substantive phenotypic alterations,
92
reinforcing the notion that correction of a limited small
number of miRNA-networked transcripts can conceivably
reshape the malignant phenotype.
The potential clinical benefits of reconstituting miRNAs
with tumor suppressor function can be gleaned from
parallel findings by siRNAs in animal models. Typically,
small RNA molecules or transgenes have repeatedly
proven to be more robust in terms of consistency of
transcript knockdowns as compared with antisense
oligonucleotides or ribozymes in achieving loss of
function of the targeted offending gene/s (reviewed in
Tong et al.
120
), and at threshold concentrations that are
several orders of magnitude below typically used antisense
oligonucleotides.
121,122
The efficacy of localized siRNA-
based therapeutics is currently being explored in a number
of Phase I/II clinical trials. These studies examine local
delivery of siRNAs against VEGF and its receptor,
VEGF-R1, for exudative, age-related macular degenera-
tion, and the intranasal delivery of siRNA (ALN-RSV01)
to specifically inhibit respiratory syncytial virus infec-
tion.
123
Other agents are under preclinical development in
the arenas of infectious disease (HIV- and HBV-siRNAs),
inflammation (IL-4R) and cancer (VEGF).
Emerging evidence indicate that perturbation of
miRNA-mediated regulatory pathways can achieve
similar in vivo knockdown effects as siRNAs. Introduc-
tion of Pol II-mediated, artificial intronic miRNA system
against b-catenin or noggin into chicken embryos resulted
in long-term (41 month) silencing of the specific gene
and a correspondingly altered phenotype.
29
With the use
of a plasmid-driven artificial miRNA based on the the
murine miR-155 sequence, Li and co-workers
124
demon-
strated the in vivo silencing of PRL-3, a protein tyrosine
phosphatase, resulting in growth suppression of perito-
neal metastases and improved survival of nude mice
bearing gastric carcinoma xenografts. Conversely,
miRNAs can be inhibited in vitro with specific
2
0
-O-methyl oligonucleotide antagonists (AMOs),
124–126
or ‘antagomirs’ when delivered in the form of liposomal
complex into adult mice,
56
and conceivably can be used to
negate the translational-regulatory effects of ‘oncogenic’
miRNAs.
Functionality
The multispecific nature of miRNA-based gene regulation
is both a strength and a weakness, and dictates that
certain minimal criteria should be fulfilled by the miRNA
(or antagomir) candidate prior to clinical consideration.
First, the putative gene target/s should be defined and
independently verified by multiple laboratories, prefer-
ably with multiple cell lines and primary tumor tissues of
the same histologic type. Since miRNA expression and
function are highly dependent on the cellular differentia-
tion stage, the application of miRNA-based treatment
should initially be restricted to narrowly defined histo-
pathological stages for each malignancy. Consistent with
the development of all novel therapeutics, in vivo safety
and efficacy should be well-defined in preclinical animal
models.
While no miRNA or antagomir currently fulfills these
criteria, differential expression of a number of miRNAs in
human tumors has been documented extensively, with
some having experimentally verified primary targets
(Table 1). For example, miR-34a, which was down-
regulated in approximately 36% of 25 primary colon
carcinomas, displayed an inverse relationship of expres-
sion with the E2F family protein in the HCT 116 and
RKO colon cancer lines. The tumor suppressor role of
miR-34 has been confirmed in vivo with these tumor
xenografts, following p53 activation by low-dose adria-
mycin.
63
Thus far, E2F-3 remains to be a predicted target
for miR-34a. Further studies are required to experimen-
tally validate that miR-34a acts through the E2F signaling
pathway.
Conversely, the knockdown of miR-21 represents a
potentially promising experimental therapeutic venue.
miR-21 expression was upregulated in large numbers of
glioblastomas,
53
breast,
55
pancreatic,
127
head and neck,
128
cervical,
56
colorectal,
129,130
ovarian,
52
and hepatocellular
57
primary tumors and cell lines tested. In vitro knockdown
of miR-21 led to increased apoptosis and suppressed cell
miRNA-based cancer gene therapy
AW Tong and J Nemunaitis
348
Cancer Gene Therapy

growth in glioblastoma,
53
breast
55
and hepatocellular
carcinomas.
57
The proapoptotic, antitumor activity of
miR-21 knockdown was confirmed in vivo by Corsten,
58
where the use of locked nucleic acid-anti-miR-21
oligonucleotides reduced U87 glioblastoma xenograft
growth by 470%. This antiglioma effect was further
augmented by combined treatment of bystander neural
precursor cells with the proapoptotic protein S-TRAIL
(secretable variant of tumor-necrosis factor-related
apoptosis-inducing ligand), resulting in tumor xenografts
eradication.
Similar findings of in vivo efficacy against breast cancer
cells have been reported. Treatment with anti-miR-21
oligonucleotides reduced MCF-7 xenograft growth by
approximately 50% for up to 2 weeks.
55
With the use of a
transfected luciferase reporter gene system, Frankel
131
recently demonstrated the direct targeting of the tumor
suppressor PDCD4 (programmed cell death 4), also in the
MCF-7 breast cancer line. PDCD4 binding has been
confirmed experimentally in colorectal cell lines,
130
where
anti-miR-21 treatment resulted in reduced invasion/
metastatic activity. Other validated targets include the
tumor suppressor genes TPM1 in MCF-7 cells
54
and
PTEN in hepatocellular carcinomas.
57
It is conceivable
that miR-21 targets different negative regulators of the
PI3K/AKT/mTOR survival pathway. PDCD4 expression
is controlled by the mTOR pathway, in which PTEN acts
as the PI3K antagonist. TPM1 belongs to the family
of TPM proteins that regulate both microfilament
organization and anchorage-independent growth,
critical components of the cell transformational
process.
132
Thus, a likely explanation is that miR-21
modulates apoptotic function via PDCD4, and anchorage
dependence via TPM1 within its gene regulatory network
in MCF-7 cells.
Other candidates for miRNA-based cancer therapy
include members of the let-7 family,
66
miR-17-5p
108
and
miR-16,
107
through their capacity to downregulate
RAS þ HMGA2, E2F-1 and BCL-2 expression, respec-
tively. A member of the let-7 family, let-7a-3, was recently
found to be overexpressed in a minor proportion of
human NSCLC cases, and promoted anchorage-indepen-
dent colony formation in the A549 lung cancer line. These
findings contrast the notion that all members of the let-7
gene family exhibit similar, tumor-suppressive activities.
86
Moreover, the functional impact of these miRNAs has
not been characterized in vivo, particularly in considera-
tion of the ‘context-dependent’ miRNAs that are best
exemplified by the miR-17-92 cluster. The miR-17-92
cluster displayed oncogenic, antiapoptotic activity in
hematopoietic stem cell-derived tumors. However,
members of the miR-17-92 cluster exhibited feedback,
cancer-suppressive activity in MYC-overexpressing B
lymphomas and repressed the protein levels of the
transcription factor E2F1 in HeLa cells,
109
reflecting its
antiproliferating role in cancers with amplified C-MYC.
Yet, in other solid cancers such as small cell lung
carcinoma, miR-17-92 cluster is overexpressed and may
exerted oncogenic activity by the reduction of PTEN and
RB2 tumor suppressor activity.
50
As new technologies continue to develop for identifica-
tion, expression profiling, target gene validation and
manipulation of miRNA expression in vivo,
133
additional
miRNA with a defined contribution to the malignant
process may be revealed as candidates for novel, cancer
therapeutic agents. Alternatively, eligible miRNA candi-
dates of undefined target activity may be uncovered as
clinically relevant through differential expression analysis
of distinct patient subsets. In a recently initiated miRNA
profiling analysis using a multiplex bead-based liquid
hybridization technique with 114 miRNA oligonucleo-
tide-probes (mirMASA; microRNA multianalyte suspen-
sion array), we have identified a differential miRNA
expression in prostatectomy specimens from early
chemical relapse prostate cancer patients, as compared
with those from patients without clinical relapse for a
period of over 10 years.
134
While malignant tissues from
both clinical subsets displayed significantly reduced
expression of 5 miRNAs (mir-23b, -100, -145, -221 and
-222) according to mirMASA and quantitative, real-time
PCR analyses, patients with chemical relapse (postsurgery
elevation of prostate-specific antigen) displayed a distinct
expression profile of 16 miRNAs, including a lower level
of miR-122a. These findings, when coupled with in silico
prediction of miRNA targets and functional validation,
can be used to identify miRNA candidates whose altered
function contributes to prostate cancer progression.
Other epidemiology correlations of miRNA expression
profiles with specific biopathologic features have been
described. Colon cancers that were characterized by
microsatellite stability displayed a distinct miRNA gene
expression profile from the histopathologically and
clinically distinct subset with high microsatellite instabi-
lity, particularly in reference to members of the oncogenic
miR-17-92 family.
135
The expression of miR-92, miR-20,
and miR-18 was also inversely correlated with the
differentiative stage of hepatocellular carcinomas.
136
For
breast cancers, miR-30 family overexpression was asso-
ciated with estrogen/progesterone receptor expression.
51
miR-10b expression was intricately linked to potent pro-
metastatic features in breast cancer cells, according to the
recent elegant findings of Ma et al.
46
Ectopic miR-10b
expression can drive tumor invasion and metastasis in
otherwise nonmetastatic breast tumors in vitro and in vivo.
miR-10b was experimentally shown to downregulate the
translation of homeobox D10 (HOXD10), which in turn
led to robust expression of the downstream, previously
repressed genes involved in cell migration and extra-
cellular matrix remodeling such as RHOC. These and
other findings suggest that an elevated miR-10b may be a
unique feature of the pro-metastatic breast cancer
subset,
137
although it remains to be established whether
silencing of miR-10b can reverse the metastatic phenotype
of highly malignant breast cancer cells in vivo.
Delivery
As in all other forms of cancer gene therapy platforms,
the manner by which the reconstituting miRNA or
antagomir is delivered is likely to critically impact success
of the experimental approach (Table 2). For cancer
miRNA-based cancer gene therapy
AW Tong and J Nemunaitis
349
Cancer Gene Therapy

patients with advanced disease, multiorgan metastatic foci
are largely responsible for the morbidity and mortality.
Hence, the effective and global systemic coverage of target
cells represents a critical factor for optimizing therapeutic
outcome. Experimental strategy remains to be defined for
the stable transfection miRNAs and for achieving
prolonged knockdown. On the basis of animal studies
on siRNA delivery and the parallel biologic features of
siRNA and miRNAs, the two potentially applicable
systemic delivery modes for small RNA transgenes are
viral vectors
138,139
and nonviral, lipid-based (cationic
liposomes, liposome-protamine/DNA) particles
140–142
(Table 2).
Late-generation adenoviral constructs are ‘reengi-
neered’ to replicate selectively in cancer cells with defined
cytogenetic defects (p53 and pRb) through modification of
viral promoter/enhancer elements. The oncolytic adeno-
virus is activated only by nuclear transcriptional factor/s
that are unique or upregulated in the cancer cell, and has
produced significant clinical gains as a stand-alone agent
or in combination with conventional chemotherapy.
143
We recently demonstrated that incorporation of a
K-ras
v12
-specific siRNA hairpin construct into the deleted
E3b region of the late-generation, oncolytic adenovirus
ONYX-411 led to a 10-fold increased antitumor potency
in human lung cancer cells expressing the relevant
K-ras
v12
mutation. This construct was markedly
more effective in reducing the growth of subcutaneous
H79 pancreatic cancer xenografts than parental ONYX-
411,
144
indicating that ras oncogene suppression and
viral oncolysis can generate a two-pronged attack on
tumor cell growth. Alternatively, cell-specific immuno-
liposomes, which have been used successfully to deliver
chemotherapy drug by target-specific cell surface recep-
tors, may also be a viable small RNA delivery plat-
form.
145,146
According to Chang and coworkers,
147
a
nanoscale nonviral liposome-based complex incorporat-
ing the targeting moiety of an antitransferrin receptor
single-chain antibody fragment can efficiently and
specifically deliver siRNA to both primary and metastatic
disease sites. It should be noted, however, that TLR
(Toll-like receptor)-9 activation and inflammatory
cytokine induction by lipoplexed oligonucleotides may
countermand the intended gene-specific outcomes of
systemic, small RNA delivery.
148
Nonspecific toxicity
Recently, Kay and coworkers
149
found that sustained,
high-level short hairpin (shRNA) expression produced
lethal, dose-dependent liver injury. Morbidity was asso-
ciated with the downregulation of liver-derived miRNAs,
which may reflect the displacement of endogenous
miRNA precursor processing events by the exogenously
introduced shRNAs, a likely candidate being the nuclear
transport component karyopherin exportin-5. AGO2, the
‘slicer’ enzyme in mammalian RISC that cleaves the target
mRNA, was recently found to be the main limiting factor
downstream of exportin-5.
150
The combined Ago2- and
exportin-5-transfected mice demonstrated markedly
improved RNAi activity and reduced liver toxicity than
singly transfected animals. The risk of oversaturating
endogenous small RNA pathways may be minimized by
optimizing shRNA dose and sequence. Alternatively,
siRNAs have been integrated into artificial miRNA
shuttles or embedded into the molecular context of an
miRNA, a maneuver that apparently lowered nonspecific
toxicity and improved RNAi efficiency.
151,152
These are
preliminary findings to suggest that heretofore undeter-
mined feedback mechanisms in the cellular processing,
and effector miRNA machinery may provide a measure of
safety that is unavailable for exogenous, artificial
siRNAs.
Immune activation
The global upregulation of IFN (interferon)-stimulated
genes
140,153
and inflammatory cytokines is a major
component of the ‘off-target activity’ following siRNA
administration both in vivo in mice and in vitro in human
blood.
154
In most studies, the IFN-inducing activities
were not only primarily attributable to innate immune
activation by the delivery vehicle,
10,148,155
but may also
depend on the presence or absence of immunostimulatory
motifs in the artificially designed siRNAs.
154
There is
currently limited evidence to indicate whether exogenously
introduced miRNAs (with wild-type sequence) may
similarly elicit immune-activating ‘danger signals.’ Accor-
ding to Epanchintsev,
156
introduction of the relevant
miRNA by a plasmid vector effectively knocked down
p53 expression, but neither nonsilencing control nor the
p53-specific miRNA elicited an IFN response in U20S
osteosarcoma cells. In the study by Grimm et al.,
149
where
mice died following intravenous injection of shRNA-
expressing viral vectors, inflammatory cytokines were not
Table 2 Clinical considerations for miRNA-based gene therapy
Issue Possible solutions
Decreased expression of
tumor-suppressive oncomirs
K Reconstitution with miRNA
precursor sequence
Overexpression of oncogenic
miRNA
K Introduction of antagomirs
Limitations in efficacy K Multidose regimen
K Modified oligonucleotides for
improved binding (locked nucleic
acid)
K Introduction of coding vectors
instead of oligonucleotides
K Use of miRNAs with divergent
regulatory properties
K Improved understanding of rate-
limiting cellular components for
miRNA processing
Multispecificity of miRNA
candidate
K In silico screening
K Expression and functional
validation
In vivo delivery K Conditional expression as
oncolytic viral transgene
K Immunoliposomes, nanoparticles
Cytotoxic side effects K Use of delivery agents displaying
carrier-defined specificity
Abbreviation: miRNA, microRNA.
miRNA-based cancer gene therapy
AW Tong and J Nemunaitis
350
Cancer Gene Therapy

present above normal levels, arguing against immune
activation. Potential toxicity from off-target effects,
saturation and the likelihood of immune activation
appear to all depend on siRNA or shRNA concentration,
as eloquently discussed in the recent review of Snove and
Rossi.
153
This consideration emphasizes the need to
identify the most potent miRNA candidate that can
impact tumor cell growth and to work with the lowest
concentration possible.
The multispecific nature of miRNA gene regulation
adds a unique layer of complexity with respect to the
interplay of tumor growth-regulation and immune-acti-
vating activities. For example, miR-155, encoded from the
third exon of the BIC (B-cell integration cluster)
oncogene, was overexpressed in multiple tumor cell
types
66,51,74
and has demonstrated oncogenic activity.
While the knockdown of miR-155 presents a potentially
attractive therapeutic venue, systemic loss of miR-155
activity may collaterally affect immune proficiency, as this
miRNA has been found to play important roles in
modulating TLR-orIFNb-mediated innate immune
responses,
157
and T-, B- and dendritic cell functions.
158,159
The use of tumor-specific delivery agents, such as viral
vectors or tumor-targeted nanoparticles, could conceiva-
bly obviate this concern. An improved understanding of
the universe of miRNA-regulated gene networks may
address the intriguing possibility of uncovering therapeu-
tic candidates that conceivably achieve parallel, beneficial
tumor inhibitory and immune-activating outcomes.
Conclusion
MicroRNA dysregulation is the common outcome of
multiple genetic and epigenetic events, including genomic
instability, epigenetic regulatory defects and/or intrinsic
defects in the miRNA processing and effector cellular
pathways. The loss of key, miRNA-mediated posttrans-
criptional regulatory activity appears to critically weaken
the intricate balance of oncogene and tumor suppressor
network functions, thereby contributing to oncogenesis
and cancer progression. An extension of these findings is
to consider the reconstitution of tumor-suppressive
miRNAs or the knockdown of overexpressed, oncogenic
miRNAs as a means of harnessing nature’s extraordina-
rily efficient and rapid way of regulating gene expression.
Eligible miRNA candidates in these regards have recently
emerged. Not withstanding the consideration of multi-
specificity, functional analysis indicates that individual
miRNAs only negatively regulate a small number of
dominant mRNA targets in a particular cancer cell type.
This premise needs to be validated through detailed
screening with cancer cells of the relevant histotype and/
or the use of an appropriate delivery agent that limits the
expression of the miRNA transgene to the tumor
microenvironment. Thus far, in vivo validation of
individual miRNA-based approaches has been limited.
Yet given the groundwork from preexisting, siRNA
studies, speedy progress can be anticipated in defining
the clinical applicability of miRNA-based cancer gene
therapy approaches. Through miRNA differential expres-
sion analysis on patients with distinct clinicopathological
features, additional miRNA candidates that modulate
more than one critical cancer pathway node may also be
uncovered.
Acknowledgements
We thank Patrick Chen for his assistance in generating
the graphical illustrations and Dr Anagha Phadke for the
proofreading of this manuscript. This work was sup-
ported, in part, by the Mary Crowley Cancer Research
Fund and the Jasper L and Jack Denton Wilson
Foundation.
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