MicroRNAs in Cancer: Small Molecules With a Huge Impact

Abstract

Every cellular process is likely to be regulated by microRNAs, and an aberrant microRNA expression signature is a hallmark of several diseases, including cancer. MicroRNA expression profiling has indeed provided evidence of the association of these tiny molecules with tumor development and progression. An increasing number of studies have then demonstrated that microRNAs can function as potential oncogenes or oncosuppressor genes, depending on the cellular context and on the target genes they regulate. Here we review our current knowledge about the involvement of microRNAs in cancer and their potential as diagnostic, prognostic, and therapeutic tools.

Full text

MicroRNAs in Cancer: Small Molecules With a Huge Impact
Marilena V. Iorio and Carlo M. Croce
From the Molecular Biology Unit,
Department of Experimental Oncology,
Fondazione Istituto di Ricovero e Cura a
Carattere Scientifico, Istituto Nazionale
Tumori, Milano, Italy; and Department
of Molecular Virology, Immunology and
Medical Genetics and Comprehensive
Cancer Center, Ohio State University,
Columbus, OH.
Submitted May 8, 2009; accepted
September 11, 2009; published online
ahead of print at www.jco.org on
November 2, 2009.
Supported by grants from the National
Cancer Institute (C.M.C.), and a My
First AIRC Grant from Associazione
Italiana per la Ricerca sul Cancro
(M.V.I.).
Authors’ disclosures of potential con-
flicts of interest and author contribu-
tions are found at the end of this
article.
Corresponding author: Carlo M. Croce,
MD, Department of Molecular Virology,
Immunology and Medical Genetics and
Comprehensive Cancer Center, Ohio
State University, Biological Research
Tower Room No. 1082, 460 12th Ave,
Columbus, OH 43210; e-mail: carlo
.croce@osumc.edu.
© 2009 by American Society of Clinical
Oncology
0732-183X/09/2734-5848/$20.00
DOI: 10.1200/JCO.2009.24.0317
ABSTRACT
Every cellular process is likely to be regulated by microRNAs, and an aberrant microRNA
expression signature is a hallmark of several diseases, including cancer. MicroRNA expression
profiling has indeed provided evidence of the association of these tiny molecules with tumor
development and progression. An increasing number of studies have then demonstrated that
microRNAs can function as potential oncogenes or oncosuppressor genes, depending on the
cellular context and on the target genes they regulate. Here we review our current knowledge
about the involvement of microRNAs in cancer and their potential as diagnostic, prognostic, and
therapeutic tools.
J Clin Oncol 27:5848-5856. © 2009 by American Society of Clinical Oncology
INTRODUCTION
After the initial discovery in 1993, when a small RNA
encoded by the lin-4 locus was associated to the
developmental timing of the nematode Caenorhab-
ditis elegans by modulating the protein lin-14,
1
mi-
croRNAs have undergone a long period of silence. It
took indeed several more years to realize that these
small (19 to 22 nucleotides [nts]) RNA molecules
are actually expressed in several organisms, in-
cluding Homo sapiens, are highly conserved across
different species, are highly specific for tissue and
developmental stage, and play crucial functions in
the regulation of important processes, such as devel-
opment, proliferation, differentiation, apoptosis,
and stress response. In the last few years, microRNAs
have indeed took their place in the complex circuitry
of cell biology, revealing a key role as regulators of
gene expression.
MicroRNA genes represent approximately 1%
of the genome of different species, and each of them
has hundreds of different conserved or noncon-
served targets: it has been estimated that approxi-
mately 30% of the genes are regulated by at least
one microRNA.
2
MicroRNAs are transcribed for the most
part by RNA polymerase II as long primary
transcripts characterized by hairpin structures
(pri-microRNAs) and processed into the nucleus by
RNAse III Drosha into 70- to 100-nts long premi-
croRNAs. These precursor molecules are exported
by an Exportin 5-mediated mechanism to the cyto-
plasm, where an additional step mediated by the
RNAse III Dicer generates a dsRNA of approxi-
mately 22 nts, named miR/miR*. The mature single-
stranded microRNA product is then incorporated
in the complex known as microRNA-containing
ribonucleoprotein complex (miRNP), miRgonaute,
or microRNA-containing RNA-induced silencing
complex (miRISC), whereas the other strand is
likely subjected to degradation. In this complex, the
mature microRNA is able to regulate gene expres-
sion at the post-transcriptional level, binding
through partial complementarity for the most part
to the 3⬘UTR of target mRNAs, and leading at the
same time to some degree of mRNA degradation
and translation inhibition (Fig 1).
Our laboratory was involved in an attempt to
identify tumor suppressors at chromosome 13q14,
involved in the pathogenesis of chronic lymphocytic
leukemia (CLL), the most common human leuke-
mia in the Western world. Deletions of chromo-
some 13 at band q14 are detected by cytogenetic
studies in approximatively 50% of CLLs, whereas
loss of heterozygosity studies indicate deletions at
13q14 in approximatively 70% of CLLs. By taking
advantage of chromosome translocations and small
deletions, we found, however, that the critical region
of 13q14 does not contain a protein-coding tumor
suppressor gene, but rather two microRNA genes,
miR-15a and miR-16-1, that are expressed in the
same polycistronic RNA. This result indicated that
the deletion of chromosome 13q14 caused the loss of
these two microRNAs, representing the first evi-
dence that microRNAs could be involved in the
pathogenesis of human cancer.
3
Study of a large
collection of CLLs showed knockdown or knockout
of miR-15a and miR-16-1 in approximatively 69%
JOURNAL OF CLINICAL ONCOLOGY BIOLOGY OF NEOPLASIA
VOLUME 27 䡠NUMBER 34 䡠DECEMBER 1 2009
5848 © 2009 by American Society of Clinical Oncology

of CLLs. Because such alteration is present in most indolent CLLs, we
speculated that loss of miR-15a and miR-16-1 could be the initiating
event or a very early event in the pathogenesis of the indolent form of
this disease.
3
Immediately after these initial observations, we mapped all the
known microRNA genes and found that many of them are located in
regions of the genome involved in chromosomal alterations, such as
deletion or amplification, in many different human tumors, in which
the presumed tumor suppressor genes or oncogenes, respectively,
failed to be discovered after many years of investigation.
4
Here we will show that alterations in microRNA expression are
not isolated, but the rule, in human cancer. After these early studies
indicating the role of microRNA genes in the pathogenesis of human
cancer, we and others have developed platforms to assess the global
expression of microRNA genes in normal and diseased tissues and
have carried out profiling studies to assess microRNA dysregulation in
human cancer. This was an attempt to establish whether mi-
croRNA profiling could be used for tumor classification, diag-
nosis, and prognosis.
MICRORNAs PROFILING IN CANCER DIAGNOSIS
AND PROGNOSIS
Profiling of different cell types and tissues indicated that the pattern of
expression of microRNAs is cell type and tissue specific, suggesting that
the program of expression of microRNAs is exquisitely cell-type depen-
dent and tightly associated with cell differentiation and development.
MicroRNAs aberrantly expressed in tumors are listed in Table 1.
Leukemia/Lymphoma
CLL. As mentioned, the first evidence of alterations of mi-
croRNA genes in human cancer came from studies of CLL. In a large
study of indolent versus aggressive CLL, Calin et al
6
discovered a
signature of 13 microRNAs capable of distinguishing between indo-
lent and aggressive CLL. Interestingly, it was found that miR-155,
overexpressed in different lymphomas, including the activated B-cell–
like type of diffuse large B-cell lymphoma, is also upregulated in
aggressive CLLs, whereas members of the miR-29 family and miR-181
were found to be underexpressed and later demonstrated to directly
regulate the TCL1 oncogene, which is overexpressed in the aggressive
form of CLL.
5
Because of the “wait and watch” approach to the
treatment of CLL, a signature able to distinguish between CLL with
good and bad prognosis was also found. Sequencing of many mi-
croRNAs, including those in the signature, allowed the identifica-
tion of germline and somatic mutations of microRNA genes,
including miR-15,miR-16-1, and miR-29 family members. Inter-
estingly, mutations in the miR-15/16 precursor were also identi-
fied, affecting the processing of the pri-miR into the pre-miR. In
two cases, the mutant was in homozygosity in the leukemic cells,
whereas normal cells of the two patients were heterozygous for this
abnormality, indicating a loss of the normal miR-15/16 allele in the
leukemic cell.
6
Thus miR-15a and miR-16-1 behave like typical
tumor suppressors in CLL. Interestingly, Raveche et al
36
have
mapped a gene responsible for an indolent form of CLL in the New
Zealand Black mouse strain on chromosome 14, in a region ho-
mologous to 13q14 in humans. Sequence analysis of this region
showed a mutation in the precursor of miR-15/16 in the New
Zealand Black mouse strain 6 nts 3⬘to miR-16-1 (in the human
cases, the mutation was 7 nts 3⬘to miR-16-1), that also affected the
processing of the miR-15/16 precursor. Thus germline mutation of
miR-15/16 can cause the indolent form of CLL both in human and
mouse. By using different algorithms to identify targets of miR-15a
and miR-16-1, it was found that BCL2, an oncogene protecting
cells from apoptosis, was a putative target of both miR-15a and
miR-16-1. Knockdown experiments showed this to be the case.
7
Thus loss of miR-15a and miR-16-1 leads to high constitutive level
of the oncogene BCL2, contributing to the development of an
indolent B-cell leukemia. In follicular lymphoma, another com-
mon indolent B-cell malignancy, the BCL2 gene becomes dysregu-
lated as result of a t(14;18) chromosome translocation, because of
its juxtaposition to immunoglobulin enhancers, indicating that
constitutive overexpression of BCL2 causes an indolent B-cell tu-
mor. More recently, it was also found that loss of miR-15a and
miR-16-1 causes, although indirectly, overexpression of MCL1,
Nucleus
Exportin 5
Translational repression mRNA cleavage
miR: miR*
7mGpppG 7mGpppG
AAA...AnAAA...An
✂
DGCR8
Drosha
Pol II
Pri-miR 5' 3'
Pre-miR
5'
3'
*
5'
3' 3' 5'
*
Pre-miR
5'
3'
*
Dicer
Helicase
RISC
RISC
Fig 1. Biogenesis, processing, and maturation of microRNAs (miRs). miRs are
transcribed mainly by RNA polymerase II as long primary transcripts character-
ized by hairpin structures (pri-miRs) and processed in the nucleus by RNAse III
Drosha in a 70-nucleotide-long pre-miR. This precursor molecule is exported by
the Exportin 5 to the cytoplasm, where RNAse III Dicer generates a dsRNA of
approximately 22 nucleotides, named miR/miR*. The mature miR product is then
incorporated in the complex known as miRISC, whereas the other strand is likely
subjected to degradation. As part of this complex, the mature miR is able to
regulate gene expression binding through partial homology the 3⬘UTR of target
mRNAs and leading to mRNA degradation in case of perfect matching or
translation inhibition. RISC, RNA-induced silencing complex.
MicroRNAs in Cancer
www.jco.org © 2009 by American Society of Clinical Oncology 5849

another oncogene of the BCL2 family of inhibitors of apoptosis.
37
Interestingly, a recent clinical trial of patients with CLL with
ABT737, an inhibitor of BCL2 developed by Abbott Laboratories
(Abbott Park, IL), showed partial resistance of the leukemic cells to
the drug, because ABT737 is specific for BCL2 but not for MCL1.
Thus treatment with either miR-15a or miR-16-1 may abrogate
resistance to the drug and improve responsiveness. Additional ex-
periments in vitro and in vivo also showed that miR-15a or miR-16-1
can be exploited to cause death of leukemic cells, suggesting the pos-
sibility of a microRNA-based therapeutic intervention.
37
Acute myelocytic leukemia. Acute myelocytic leukemia (AML) is
a heterogeneous disease that includes several entities with different
genetic abnormalities and clinical features. Garzon et al
38
have re-
ported unique microRNA profiles in the main molecular and cytoge-
netic subgroups of AML. In addition, a subset of these microRNAs was
associated with overall and disease-free survival. The members of the
miR-29 family are located in two clusters on two human chromo-
somes: miR-29b1/29a are located on chromosome 7q32, whereas
miR-29b2/c are located on chromosome 1q23. Importantly, chromo-
some 7q is the region frequently deleted in myelodysplastic syndrome
and therapy-related AML.
39
Members of the miR-29 family have been
shown to be downregulated in aggressive CLL,
6
invasive breast can-
cer,
18
lung cancer,
40
and cholangiocarcinoma.
8
Transfection of miR-
29b induces apoptosis in cholangiocarcinoma cell lines and reduces
the tumorigenicity of lung cancer cells in nude mice. Very recently, it
was shown that rhabdomyosarcoma loses miR-29 expression because
of an elevation of NFkB and YY1 levels, and introduction of miR-29s
into the tumor delays rhabdomyosarcoma progression in mice.
41
MiR-29s were also found to directly target MCL1,
8
an oncogene over-
expressed in AMLs, and the de novo DNA methyltransferases
DNMT-3A and -3B, although indirectly, through regulation of the
transactivator Sp1, the maintenance DNA methyl transferase
DNMT1.
40,42
Thus loss of miR-29 family members results in the
constitutive overexpression of MCL1 and of DNMT, causing
epigenetic changes characteristic of AML. These recent results
suggest that loss of miR-29s may be important, perhaps critical,
for the pathogenesis of a major group of myelodysplastic syn-
dromes and AMLs (Fig 2).
Lymphoma. Early studies have shown that miR-155 is upregu-
lated in a subgroup of Burkitt’s lymphoma, diffuse large B-cell lym-
phoma, primary mediastinal B-cell lymphoma, and Hodgkin’s
lymphoma.
9,10
This microRNA is encoded by the terminal portion of
the BIC (B-cell integration cluster) gene, which was originally identi-
fied as a common retroviral integration site in avian leukosis virus–
induced B-cell lymphomas.
43
Our group demonstrated that mice
overexpressing miR-155 in B lymphocytes develop polyclonal
preleukemic pre–B-cell proliferation followed by full-blown
B-cell malignancy.
11
More recently, two knockout mice models
have demonstrated a critical role of miR-155 in immunity by
showing that BIC/miR-155⫺/⫺have defective dendritic cell func-
tions, impaired cytokine secretion, and T
H
cells intrinsically biased
toward T
H
2 differentiation.
44,45
Moreover, miR-155 could repre-
sent the connection between inflammation, immunity, and cancer,
because its expression can be induced by mediators of inflamma-
tion and is involved in response to endotoxic shock.
46
He et al
12
reported that miR-17-92 polycistron was upregulated
in 65% of patients with B-cell lymphoma and demonstrated in a
mouse model that this microRNA cluster cooperates with the onco-
gene MYC in accelerating tumor development. More recently, a dif-
ferent group observed that the overexpression of miR-17-92 in
lymphocytes caused a lymphoproliferative disease, autoimmunity,
and premature death.
13
The enhanced proliferation of the transgenic
lymphocytes was mediated by direct regulation of pro-apoptotic
phosphatase and tensin homolog gene (PTEN) and Bim. O’Donnell et
Table 1. MicroRNAs Aberrantly Expressed in Tumors
Tumor Type Upregulated MicroRNA Downregulated MicroRNA Target
CLL
5-7
miR-29, miR-181 TCL1
miR-155
miR-15a, miR-16-1 BCL2
AML
8
miR-29 MCL1
DNMT
Lymphoma
9-15
miR-155
miR-17-92 PTEN, BIM,E2F1
miR-106b-25 E2F1
MM
16,17
miR-21
miR-19a, miR-19b SOCS1
Breast cancer
18-27
miR-21 PTEN, PDCD4, TPM1
miR-125b HER2, HER3
miR-205 HER3
miR-10b (associated with metastasis) HOXD10
miR-373
miR-200 ZEB
Lung cancer
28-32
let-7 RAS, HMGA2, C-MYC
miR-155
HCC
28,34,35
miR-122a Cyclin G1
miR-221 p27
miR-34a MET
Abbreviations: CLL, chronic lymphocytic leukemia; AML, acute myelocytic leukemia; MM, multiple myeloma; HCC, hepatocellular carcinoma.
Iorio and Croce
5850 © 2009 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

al
14
investigated the regulation of miR-17-92 in lymphoma, demon-
strating that the expression of this cluster is directly activated by the
oncogene c-Myc. Moreover, miR-17-92 cluster, as well as its paralog,
miR-106b-25,
15
establish with the transcription factor E2F1, a down-
stream target of c-Myc, a negative feedback loop: E2F1 indeed repre-
sents a direct target of the two microRNA clusters, but it also induces
their expression. Thus MYC simultaneously activates E2F1 transcrip-
tion and limits its expression, allowing a tightly controlled prolifera-
tive signal.
Multiple myeloma. Few recent evidences have linked microRNAs
to this plasma cell malignancy, as the aberrant expression of miR-
335, miR-342-3p, and miR-561 in comparison with normal plasma
cells
47
or the Stat3-mediated activation of the oncogenic miR-21 in
response to interleukin-6.
16
More recently, Pichiorri et al
17
de-
scribed a microRNA signature characteristic of this neoplasia.
They evaluated by both microarray analysis and real-time poly-
merase chain reaction the expression of microRNAs in multiple
myeloma (MM) – derived cell lines, CD138
⫹
bone marrow
peripheral cells from subjects with MM or monoclonal gammopa-
thy of undetermined significance, and normal donors, identifying
the oncogenic miR-21 and miR-181 among the microRNAs aber-
rantly expressed. Two miRNAs, miR-19a and 19b, part of the
miR-17-92 cluster, were also shown to downregulate expression of
SOCS-1, a gene frequently silenced in MM that plays a critical role
in inhibiting interleukin-6 growth signaling. Moreover, xenograft
studies using human MM cell lines treated with miR-19a and b precursors
or miR-181a and b antagonists resulted in significant suppression of
tumor growth in nude mice, confirming the involvement of these mi-
croRNAs in the development of MM and supporting the idea of a possible
use of these molecules as a therapeutic tool.
MicroRNAs in Solid Malignancies
Breast cancer. One of the first solid tumors to be profiled for
microRNA expression was, in 2005, breast cancer. Iorio et al
18
de-
scribed the first microRNA signature characteristic of breast carci-
noma, identifying 13 microRNAs able to discriminate tumors and
normal tissues with an accuracy of 100%. Among the most significant
microRNAs differentially expressed, some were extensively studied
since their initial discovery and revealed an important role on the
biology of breast cancer: miR-21, overexpressed in breast carcinoma,
has been demonstrated to mediate cell survival and proliferation di-
rectly targeting the oncosuppressor genes PTEN,PDCD4, and TPM1,
and it has been associated with advanced clinical stage, lymph node
metastasis, and poor patient prognosis.
19,20
This microRNA, one of
the first cancer microRNAs described, has been found overexpressed
in a variety of other malignancies: glioblastoma,
48,49
ovary,
50
lung,
28,51
and more.
52
In colorectal cancer and pancreas endocrine and exocrine
tumor, miR-21 overexpression is also associated with poor survival
and poor therapeutic outcome.
53-55
Conversely, downregulated microRNAs, as miR-125b and miR-
205, regulate oncogenes as tyrosine kinase receptors HER-2 and
HER-3, respectively.
21,22
Ectopic expression of miR-205 in a breast
cancer cell line decreases proliferation and improves the responsive-
ness to tyrosine kinase inhibitors as gefitinib or lapatinib.
22
MicroRNA
expression is also related to some histopathologic features of breast
carcinoma, such as estrogen receptor (ER) and progesterone receptor
expression, grade and stage, and presence of invasion.
18
Further stud-
ies have investigated the correlation between microRNA expression
and the classification in different subtypes of breast cancer.
56
Very
recently, few groups have reported experimental evidence support-
ing the correlation between microRNAs and ER status: miR-206
directly targets ER
␣
, and miR-221 and -222 confer tamoxifen resis-
tance regulating p27
57
and ER
␣
.
58
Unpublished data from our labo-
ratories
58a
describe a regulatory loop between ER
␣
and miR-221 and
miR-222: the two microRNAs are able to directly target ER
␣
receptor,
which in turn negatively regulates their expression, binding estrogen-
responsive elements on their promoter region.
Lung cancer. Let-7, tumor suppressor microRNA initially dis-
covered in C. elegans, where it induces cell cycle exit and terminal
differentiation, has been described as a new regulator of self-renewal
and tumorigenicity of breast cancer cells,
59
targeting molecules
originally described in lung cancer: RAS
29
as well as the oncogene
HMGA2,
29,30
and even MYC itself.
31
Overexpression of let-7 mi-
croRNA family can suppress tumor development in mouse models of
Apoptosis
Epigenetic changes
Proliferation
miR-15a
miR-16-1
miR-29
miR-181
ncRNA
TCL1
PCG
DNMT
ncRNA
MCL1
BCL2
PCG
AML
Lung cancer
mutation
deletion
Indolent CLL
Aggressive CLL deletion
Fig 2. Molecular alterations in chronic
lymphocytic leukemia (CLL) and acute
myelocytic leukemia (AML). Deletion or
downregulation of microRNA (miR)-15a/miR-
16-1 cluster, located at chromosome 13q14.3
and directly involved in the regulation of BCL2
and MCL1 expression, represent an early
event in the pathogenesis of CLL. During the
evolution of malignant clones, other micro
RNAs (miRs) can be deleted (such as miR-29)
or overexpressed (such as miR-155), contrib-
uting to the aggressiveness of B-cell CLL.
Such abnormalities can influence the expres-
sion of other protein-coding genes (PCGs), as
TCL1 oncogene, directly regulated by miR-29
and miR-181, or affect other noncoding RNAs
(ncRNAs). The consequences of this steady
accumulation of abnormalities are repre-
sented by the reduction of apoptosis and the
induction of survival and proliferation of ma-
lignant B cells, leading to the evolution of
more aggressive clones. Members of the
miR-29 family, lost in AML and in other tumor
types as lung cancer, have also been shown
to directly target MCL1 and DNMT3A and B.
MicroRNAs in Cancer
www.jco.org © 2009 by American Society of Clinical Oncology 5851

breast and lung cancer.
59,60
In the two most common forms of non–
small-cell lung cancers (NSCLCs; adenocarcinomas and squamous
cell carcinomas), high expression of miR-155 and low expression of
oncosuppressor let-7 are correlated with poor prognosis.
28
The asso-
ciation of let-7a with survival was also confirmed by an independent
study performed by Yu et al,
32
who identified an microRNA signature
as an independent predictor of cancer relapse and survival of patients
with NSCLC.
As in other tumor types, also in lung cancer microRNAs can
represent accurate diagnostic markers: very recently, it has been de-
scribed that squamous and nonsquamous NSCLCs can be distin-
guished according to the expression of miR-205.
61
Hepatocellular carcinoma. In hepatocellular carcinoma (HCC),
Murakami et al
62
reported that miR-222, miR-106a, and miR-17-92
clusters are associated with the degree of tumor differentiation,
whereas high levels of the oncosuppressor miR-125b are correlated
with good survival.
63
MiR-125b has also been shown to induce
growth inhibition in vitro in a model of human thyroid anaplastic
carcinoma.
64
Other studies focused on the identification of molecules
targeted by microRNAs deregulated in HCC: miR-122a, downmodu-
lated in HCC, directly regulates Cyclin G1,
33
and miR-221, upregu-
lated in this neoplasia, directly targets p27,
34
as also shown in thyroid
cancer,
64
glioblastoma,
65
prostate cancer,
66
and melanoma.
67
One of
the first evidences proving miR alteration in human melanoma is a
genomic study performed by Zhang et al,
68
who reported DNA copy
abnormalities in microRNA genes also in two other epithelial tumors,
breast and ovary. Interestingly, the results obtained by this genomic
analysis were largely overlapping with the expression profiles on the
same tumor types.
18,50
MICRORNAs IN INVASION, ANGIOGENESIS, AND METASTASIS
MicroRNAs have been implicated not only in the development of
primary tumors, but also in affecting progression and the metastatic
phase of the disease. Indeed, several evidences show how microRNAs
are involved in the regulation of biologic processes leading to the
acquisition of metastatic potential, as adhesion, migration and inva-
sion, and angiogenesis.
One of the first studies reporting a prometastatic role for a mi-
croRNA was published by Ma et al.
24
They observed that miR-10b was
downmodulated in all the breast carcinomas from metastasis-free
patients, as previously reported,
18
but surprisingly, 50% of metastasis-
positive patients had elevated miR-10b levels in their primary tumors.
Induced by transcription factor Twist, miR-10b inhibits the transla-
tion of mRNA encoding homeobox D10 (HOXD10), releasing the
expression of the prometastatic gene RHOC and thus leading to tumor
cell invasion and metastasis.
Through a functional screen aimed to discover microRNAs pro-
moting cell migration in vitro, Huang et al
25
identified miR-373 and
validated its metastatic potential in tumor transplantation experi-
ments using breast cancer cells.
MiR-34a, lost in several tumor types and involved in the network
mediated by the well-known “genome guardian” p53,
35
inhibits mi-
gration and invasion by downregulation of MET expression in human
HCC cells.
69
Being the epithelial-mesenchymal transition (EMT) thought to
promote malignant tumor progression, several groups have recently
investigated whether microRNAs are involved in this process, and a
number of evidences support this hypothesis. Indeed, members of the
miR-200 family of microRNAs and miR-205 have been shown to
reduce cell migration and invasiveness targeting ZEB transcription
factors, known inducers of EMT,
26,27
and PKC␧, as recently demon-
strated in prostate cancer.
70
The oncogenic miR-21 stimulates inva-
sion, extravasation, and metastasis in different tumor types, included
colorectal cancer
71
and breast cancer,
72
whereas oncosuppressor miR-
205 has opposite effects, reducing invasion in vitro and suppressing
lung metastasis in vivo.
23
With the same aim of searching for regula-
tors of breast cancer metastasis, Tavazoie et al
73
identified miR-126
and miR-335 as metastasis suppressors: reduced levels of the two
microRNAs are associated with poor metastasis-free survival of pa-
tients with breast cancer, whereas their re-expression inhibits metas-
tasis in a cell transplantation model.
Interestingly, it has been recently observed that primary tumors
and metastasis from the same tissue show a similar pattern of mi-
croRNA expression.
74
Being a more accurate classifier than mRNA
expression studies, microRNA profiling has thus revealed the poten-
tial to solve one of the most demanding issues in cancer diagnostics:
the origin of metastasis of unknown primary tumors.
In the metastatic process, neoangiogenesis is the crucial step
allowing cells to reach and disseminate through the systemic circula-
tion. microRNAs can also control tumor progression at this level,
either promoting or inhibiting the proliferation of endothelial cells.
miR-221 and miR-222 repress proliferative and angiogenic properties
of c-Kit in endothelial cells,
75
whereas hypoxic reduction of miR-16,
miR-15b, miR-20a, and miR-20b, directly targeting vascular endothe-
lial growth factor, supports the angiogenic process.
76
On the other
hand, vascular endothelial growth factor levels can be indirectly in-
creased by miR-27b, through reduction of the zinc finger protein
ZBTB10 and the consequent activation of Sp transcription factor,
77
and by miR-126, through repression of Sprouty-related protein
Spred-1 and phosphoinositol 3-kinase regulatory subunit 2.
78
Angio-
genesis can be also promoted by miR-210, activated by hypoxia and
directly repressing endothelial ligand ephrin A3,
79
and by miR-17-92
cluster, which sustains MYC angiogenic properties through repres-
sion of connective tissue growth factor and the antiangiogenic
adhesive glycoprotein thrombospondin 1,
80
also targeted by miR-
27b and let-7f.
81
MICRORNA AND EPIGENETICS
MiRNA expression can be altered by several mechanisms in human
cancer (Fig 3): chromosomal abnormalities, as suggested by the evi-
dence that microRNAs are frequently located in regions of the genome
involved in alterations in cancer,
4
and recently confirmed by a genetic
study in ovarian carcinoma, breast cancer, and melanoma
68
; muta-
tions, as the inherited mutations in the primary transcripts of miR-15a
and miR-16-1 responsible for reduced expression of the two microRNAs
in vitro and in vivo in CLL
36
; and polymorphisms (SNPs), as
described in lung cancer.
82
The deregulated microRNA expression
in cancer can also be due to defects in the microRNA biogenesis
machinery, as supported by the changes in microRNA levels con-
sequent to altered Drosha or Dicer activity in different tumor
types,
83-86
and epigenetic changes, as altered DNA methylation. An
extensive analysis of genomic sequences of miRNA genes have
Iorio and Croce
5852 © 2009 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

shown that approximately half of them are associated with CpG
islands, suggesting that they could be subjected to this mechanism
of regulation.
87
Several evidences have indeed proved that an
altered methylation status can be responsible for the deregu-
lated expression of microRNAs in cancer, as the silencing of
putative tumor suppressor microRNAs: treating T24 bladder
cancer cells and human fibroblasts with DNMT inhibitor 5-Aza-
2⬘-deoxycytidine, Saito et al
88
observed a strong upregulation of
miR-127, microRNA characterized by a CpG island promoter, able
to target the proto-oncogene BCL-6, and silenced in several cancer
cells. With the same approach of unmasking epigenetically silenced
microRNAs inducing chromatin-remodeling by drug treatment, it
has been demonstrated that miR-9-1 is hypermethylated and con-
sequently downmodulated in breast cancer,
89
as well as the clus-
tered miR-34b and miR-34c in colon cancer.
90
Conversely, the upmodulation of putative oncogenic mi-
croRNAs in cancer can be due to DNA hypomethylation, as shown
in lung adenocarcinoma for let-7a-3
91
or in epithelial ovarian
cancer for miR-21.
50
A different approach to identifying epigenetically regulated
microRNAs was represented by the microRNA profiling of
DNMT1- and DNMT3b-deficient colorectal cancer cells: among
the 18 microRNAs upmodulated in comparison with wild-type
cells, the only one resulting unmethylated in normal tissue but
hypermethylated, and thus silenced, in tumor was miR-124a, em-
bedded in a large CpG island and able to target cyclin D kinase 6,
which mediates the phosphorylation of the RB tumor suppres-
sor gene.
92
Methylation is not the only epigenetic mechanism that can affect
microRNA expression: Scott et al
93
showed that in SKBR3 breast
carcinoma cells, histone deacetylase inhibition is followed by the ex-
tensive and rapid alteration of microRNA levels.
The existence of epigenetic drugs, such as DNA demethyl-
ating agents and histone deacetylase inhibitors, able to reverse
Proliferation
Inhibition of Apoptosis
↑ Onco-miRs
(miR-21, miR-155, miR-17-92...)
↓ Oncosuppressor miRs
(miR-15/16, let-7, miR-125, miR-205...)
Migration and Metastasis
Anti-miRs
miRs
Fig 4. MicroRNAs (miRs) as therapeutic
tools. The reintroduction by transfection of
synthetic miRs lost during cancer develop-
ment or progression or the inhibition of
oncogenic miRs by using anti-miR oligonu-
cleotides could contribute to counteract tu-
mor proliferation, extended survival, and the
acquisition of a metastatic potential, thus
representing potential therapeutic tools.
Drosha
Pol II
5' 3'
*
Exportin 5
Pre-miR
5'
3'
*
5'
3'
*
Pre-miR
Genomic changes:
deletions, amplifications,
translocations
Transcription
factors
Epigenetic
changes
SNPs,
mutations
Altered expression or
function
Mature miR
Pri-miR
promoter miR gene
Dicer
Fig 3. Mechanisms of microRNA (miR)
regulation. The deregulated microRNA ex-
pression observed in cancer can be caused
by chromosomal abnormalities, mutations,
polymorphisms (SNPs), transcriptional de-
regulation, defects in the microRNA biogen-
esis machinery, and epigenetic changes.
MicroRNAs in Cancer
www.jco.org © 2009 by American Society of Clinical Oncology 5853

an aberrant methylation or acetylation status, raises the intrigu-
ing possibility of regulating microRNA levels, for example, to
restore the expression of tumor suppressor microRNAs, thus
reverting a tumoral phenotype.
To complicate the scenario connecting microRNAs and epige-
netics, microRNAs themselves can regulate the expression of compo-
nents of the epigenetic machinery, creating a highly controlled
feedback mechanism: the miR-29 family directly targets the de novo
DNA methyltransferases DNMT-3A and -3B, although indirectly,
through regulation of the transactivator Sp1, the maintenance DNA
methyl transferase DNMT1. Interestingly, introduction of miR-29s
into lung cancers and AMLs results in reactivation of silenced tumor
suppressors and inhibition of tumorigenesis.
40,42
Loss of miR-290
cluster in Dicer-deficient mouse embryonic stem cells leads to the
downregulation of DNMT3a, DNMT3b, and DNMT1 through up-
modulation of their repressor, RBL-2, proven target of miR-290
94,95
;
miR-1, involved in myogenesis and related diseases, directly tar-
gets HDAC4.
96
MICRORNA/ANTI-MICRORNAs IN CANCER TREATMENT
The evidences collected to date demonstrate how microRNAs could
represent valid diagnostic, prognostic, and predictive markers in can-
cer. Indeed, the aberrant microRNA expression is correlated with
specific biopathologic features, disease outcome, and response to spe-
cific therapies in different tumor types.
Considering the importance of microRNAs in development,
progression, and treatment of cancer, the potential usefulness of a
microRNA-based therapy in cancer is now being exploited, with
the attempt to modulate their expression, reintroducing micro
RNAs lost in cancer, or inhibiting oncogenic microRNAs by using
anti-microRNA oligonucleotides (Fig 4). For example, transfec-
tion of miR-15a/16-1 induces apoptosis in leukemic MEG01 cells
and inhibits tumor growth in vivo in a xenograft model,
37
whereas the
inhibition of miR-21 with antisense oligonucleotides generates a pro-
apoptotic and antiproliferative response in vitro in different cellular
models and reduces tumor development and metastatic potential
in vivo.
97
Moreover, microRNAs involved in specific networks, as the ap-
optotic, proliferation, or receptor-driven pathways, could likely influ-
ence the response to targeted therapies or to chemotherapy: inhibition
of miR-21 and miR-200b enhances sensitivity to gemcitabine in
cholangiocytes, probably by modulation of CLOCK,PTEN, and
PTPN12,
98
whereas reintroduction of miR-205 in breast cancer cells
can improve the responsiveness to tyrosine kinase inhibitors through
HER-3 silencing.
22
Beside targeted therapies and chemotherapy, microRNAs could
also alter the sensitivity to radiotherapy, as recently reported by Slack
et al
99
: in lung cancer cells, the let-7 family of microRNAs can suppress
the resistance to anticancer radiation therapy, probably through
RAS regulation.
Evidence described to date represents the experimental bases
for the use of microRNAs as both targets and tools in anticancer
therapy, but there are at least two primary issues to address to
translate these fundamental research advances into medical practice:
the development of engineered animal models to study cancer-
associated microRNAs and the improvement of the efficiency of
miRNAs/anti-microRNAs delivery in vivo. To this aim, modified
microRNA molecules with longer half-lives and efficiency have been
developed, such as anti-microRNA oligonucleotides,
100
locked nu-
cleic acid–modified oligonucleotides,
101
and cholesterol-conjugated
antagomirs.
102
Interestingly, Ebert et al
103
have recently described a
new approach to inhibit microRNAs function: synthetic mRNAs
containing multiple binding site for a specific microRNA, called
microRNA sponges, are able to bind up the microRNA, preventing its
association with endogenous targets.
To improve the in vivo delivery of either microRNAs or anti-
microRNAs, the methods that have been tested in preclinical studies
over the last decades for short-interfering RNAs (siRNA) or short
heteroduplex RNA (shRNA)
104
could be applied also to micro
RNAs. Moreover, the advantage of microRNAs over siRNA/
shRNA is their ability to affect multiple targets with a single hit,
thus regulating a whole network of interacting molecules.
In conclusion, 15 years ago, when microRNAs seemed just a peculiar
discovery in C elegans, the scientific world probably did not even
imagine that those small noncoding molecules would have a large
impact on our understanding of cellular biology and gene regulation.
MicroRNAs contribute to maintaining the balance among genes
regulating cells’ fate, and their deregulation, a frequent hallmark in
different human malignancies, can destabilize this equilibrium, thus
contributing to cancer development and/or progression, from initia-
tion to metastatic disease. However, despite the increasing and en-
couraging evidences linking microRNAs to cancer biology, many
important questions remain to be addressed; in fact, although the
identification and validation of microRNA targets greatly improved in
the last few years, we still know very little about the cellular and
molecular circuits where they are involved. The scenario is surely
complicated by the ability of microRNAs to target multiple molecules,
sometimes belonging to related pathways, and at the same time by the
redundancy existing among microRNAs. This gives rise to a complex
regulatory network in which biologic effects and properties of a par-
ticular microRNA do not always allow a linear explanation.
Improvement of computation programs of microRNA target
prediction and experimental methods of validation will certainly con-
tribute to elucidating their mechanisms of action, and genetically
modified murine models will likely help in determining the oncogenic
and tumor suppressor potential of individual microRNAs.
Data available to date clearly support the involvement of mi-
croRNA in cancer etiology and strongly suggest a possible use of
these molecules as markers of diagnosis and prognosis and, even-
tually, as new targets or tools for a specific therapy. Stepping from
the bench to clinical applications would be the next great challenge
in cancer research.
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
The author(s) indicated no potential conflicts of interest.
AUTHOR CONTRIBUTIONS
Conception and design: Carlo M. Croce
Manuscript writing: Marilena V. Iorio, Carlo M. Croce
Final approval of manuscript: Carlo M. Croce
Iorio and Croce
5854 © 2009 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

REFERENCES
1. Lee RC, Feinbaum RL, Ambros V: The C.
elegans heterochronic gene lin-4 encodes small
RNAs with antisense complementarity to lin-14. Cell
75:843-854, 1993
2. Bartel DP: MicroRNAs: Genomics, biogene-
sis, mechanism, and function. Cell 116:281-297,
2004
3. Calin GA, Dumitru CD, Shimizu M, et al:
Frequent deletions and down-regulation of micro-
RNA genes miR15 and miR16 at 13q14 in chronic
lymphocytic leukemia. Proc Natl Acad SciUSA
99:15524-15529, 2002
4. Calin GA, Sevignani C, Dumitru CD, et al:
Human microRNA genes are frequently located at
fragile sites and genomic regions involved in can-
cers. Proc Natl Acad SciUSA101:2999-3004, 2004
5. Pekarsky Y, Santanam U, Cimmino A, et al:
Tcl1 expression in chronic lymphocytic leukemia is
regulated by miR-29 and miR-181. Cancer Res 66:
11590-11593, 2006
6. Calin GA, Ferracin M, Cimmino A, et al: A
MicroRNA signature associated with prognosis and
progression in chronic lymphocytic leukemia. N Engl
J Med 353:1793-1801, 2005
7. Cimmino A, Calin GA, Fabbri M, et al: MiR-15
and miR-16 induce apoptosis by targeting BCL2.
Proc Natl Acad SciUSA102:13944-13949, 2005
8. Mott JL, Kobayashi S, Bronk SF, et al: Mir-29
regulates Mcl-1 protein expression and apoptosis.
Oncogene 26:6133-6140, 2007
9. Metzler M, Wilda M, Busch K, et al: High
expression of precursor microRNA-155/BIC RNA in
children with Burkitt lymphoma. Genes Chromo-
somes Cancer 39:167-169, 2004
10. Kluiver J, Poppema S, de JD, et al: BIC and
miR-155 are highly expressed in Hodgkin, primary
mediastinal and diffuse large B cell lymphomas.
J Pathol 207:243-249, 2005
11. Costinean S, Zanesi N, Pekarsky Y, et al:
Pre-B cell proliferation and lymphoblastic leukemia/
high-grade lymphoma in E(mu)-miR155 transgenic
mice. Proc Natl Acad SciUSA103:7024-7029, 2006
12. He L, Thomson JM, Hemann MT, et al: A
microRNA polycistron as a potential human onco-
gene. Nature 435:828-833, 2005
13. Xiao C, Srinivasan L, Calado DP, et al: Lym-
phoproliferative disease and autoimmunity in mice
with increased miR-17-92 expression in lympho-
cytes. Nat Immunol 9:405-414, 2008
14. O’Donnell KA, Wentzel EA, Zeller KI, et al:
C-Myc-regulated microRNAs modulate E2F1 ex-
pression. Nature 435:839-843, 2005
15. Petrocca F, Visone R, Onelli MR, et al: E2F1-
regulated microRNAs impair TGFbeta-dependent
cell-cycle arrest and apoptosis in gastric cancer.
Cancer Cell 13:272-286, 2008
16. Lo¨ ffler D, Brocke-Heidrich K, Pfeifer G, et al:
Interleukin-6 dependent survival of multiple my-
eloma cells involves the Stat3-mediated induction of
microRNA-21 through a highly conserved enhancer.
Blood 110:1330-1333, 2007
17. Pichiorri F, Suh SS, Ladetto M, et al: MicroRNAs
regulate critical genes associated with multiple my-
eloma pathogenesis. Proc Natl Acad SciUSA
105:12885-12890, 2008
18. Iorio MV, Ferracin M, Liu CG, et al: Mi-
croRNA gene expression deregulation in human
breast cancer. Cancer Res 65:7065-7070, 2005
19. Yan LX, Huang XF, Shao Q, et al: MicroRNA
miR-21 overexpression in human breast cancer is
associated with advanced clinical stage, lymph node
metastasis and patient poor prognosis. RNA 14:
2348-2360, 2008
20. Qian B, Katsaros D, Lu L, et al: High miR-21
expression in breast cancer associated with poor
disease-free survival in early stage disease and high
TGF-beta1. Breast Cancer Res Treat 117:131-140,
2009
21. Scott GK, Goga A, Bhaumik D, et al: Coordi-
nate suppression of ERBB2 and ERBB3 by enforced
expression of micro-RNA miR-125a or miR-125b.
J Biol Chem 282:1479-1486, 2007
22. Iorio MV, Casalini P, Piovan C, et al:
MicroRNA-205 regulates HER3 in human breast
cancer. Cancer Res 69:2195-2200, 2009
23. Wu H, Zhu S, Mo YY: Suppression of cell
growth and invasion by miR-205 in breast cancer.
Cell Res 19:439-448, 2009
24. Ma L, Teruya-Feldstein J, Weinberg RA:.
Tumour invasion and metastasis initiated by
microRNA-10b in breast cancer. Nature 449:682-
688, 2007
25. Huang Q, Gumireddy K, Schrier M, et al: The
microRNAs miR-373 and miR-520c promote tumour
invasion and metastasis. Nat Cell Biol 10:202-210,
2008
26. Gregory PA, Bracken CP, Bert AG, et al:
MicroRNAs as regulators of epithelial-mesenchymal
transition. Cell Cycle 7:3112-3118, 2008
27. Park SM, Gaur AB, Lengyel E, et al: The
miR-200 family determines the epithelial phenotype
of cancer cells by targeting the E-cadherin repres-
sors ZEB1 and ZEB2. Genes Dev 22:894-907, 2008
28. Yanaihara N, Caplen N, Bowman E, et al:
Unique microRNA molecular profiles in lung cancer
diagnosis and prognosis. Cancer Cell 9:189-198,
2006
29. Johnson SM, Grosshans H, Shingara J, et al:
RAS is regulated by the let-7 microRNA family. Cell
120:635-647, 2005
30. Mayr C, Hemann MT, Bartel DP: Disrupting
the pairing between let-7 and Hmga2 enhances
oncogenic transformation. Science 315:1576-1579,
2007
31. Sampson VB, Rong NH, Han J, et al: Mi-
croRNA let-7a down-regulates MYC and reverts
MYC-induced growth in Burkitt lymphoma cells.
Cancer Res 67:9762-9770, 2007
32. Yu SL, Chen HY, Chang GC, et al: MicroRNA
signature predicts survival and relapse in lung can-
cer. Cancer Cell 13:48-57, 2008
33. Gramantieri L, Ferracin M, Fornari F, et al:
Cyclin G1 is a target of miR-122a, a microRNA
frequently down-regulated in human hepatocellular
carcinoma. Cancer Res 67:6092-6099, 2007
34. Fornari F, Gramantieri L, Ferracin M, et al:
MiR-221 controls CDKN1C/p57 and CDKN1B/p27
expression in human hepatocellular carcinoma. On-
cogene 27:5651-5661, 2008
35. He L, He X, Lim LP, et al: A microRNA
component of the p53 tumour suppressor network.
Nature 447:1130-1134, 2007
36. Raveche ES, Salerno E, Scaglione BJ, et al:
Abnormal microRNA-16 locus with synteny to hu-
man 13q14 linked to CLL in NZB mice. Blood
109:5079-5086, 2007
37. Calin GA, Cimmino A, Fabbri M, et al: MiR-
15a and miR-16-1 cluster functions in human leuke-
mia. Proc Natl Acad SciUSA105:5166-5171, 2008
38. Garzon R, Volinia S, Liu CG, et al: MicroRNA
signatures associated with cytogenetics and prog-
nosis in acute myeloid leukemia. Blood 111:3183-
3189, 2008
39. Le Beau MM, Albain KS, Larson RA, et al:
Clinical and cytogenetic correlations in 63 patients
with therapy-related myelodysplastic syndromes
and acute nonlymphocytic leukemia: Further evi-
dence for characteristic abnormalities of chromo-
somes no. 5 and 7. J Clin Oncol 4:325-345, 1986
40. Fabbri M, Garzon R, Cimmino A, et al:
MicroRNA-29 family reverts aberrant methylation in
lung cancer by targeting DNA methyltransferases
3A and 3B. Proc Natl Acad SciUSA104:15805-
15810, 2007
41. Wang H, Garzon R, Sun H, et al: NF-kappaB-
YY1-miR-29 regulatory circuitry in skeletal myogen-
esis and rhabdomyosarcoma. Cancer Cell 14:369-
381, 2008
42. Garzon R, Liu S, Fabbri M, et al: MicroRNA
-29b induces global DNA hypomethylation and tu-
mor suppressor gene re-expression in acute my-
eloid leukemia by targeting directly DNMT3A and 3B
and indirectly DNMT1. Blood 113:6411-6418, 2009
43. Tam W, Hughes SH, Hayward WS, et al:
Avian bic, a gene isolated from a common retroviral
site in avian leukosis virus-induced lymphomas that
encodes a noncoding RNA, cooperates with c-myc
in lymphomagenesis and erythroleukemogenesis.
J Virol 76:4275-4286, 2002
44. Thai TH, Calado DP, Casola S, et al: Regula-
tion of the germinal center response by microRNA-
155. Science 316:604-608, 2007
45. Rodriguez A, Vigorito E, Clare S, et al: Re-
quirement of bic/microRNA-155 for normal immune
function. Science 316:608-611, 2007
46. Tili E, Michaille JJ, Cimino A, et al: Modula-
tion of miR-155 and miR-125b levels following
lipopolysaccharide/TNF-alpha stimulation and their
possible roles in regulating the response to endo-
toxin shock. J Immunol 179:5082-5089, 2007
47. Ronchetti D, Lionetti M, Mosca L, et al: An
integrative genomic approach reveals coordinated
expression of intronic miR-335, miR-342, and miR-
561 with deregulated host genes in multiple my-
eloma. BMC Med Genomics 1:37, 2008
48. Chan JA, Krichevsky AM, Kosik KS:
MicroRNA-21 is an antiapoptotic factor in human
glioblastoma cells. Cancer Res 65:6029-6033,
2005
49. Ciafre` SA, Galardi S, Mangiola A, et al: Ex-
tensive modulation of a set of microRNAs in primary
glioblastoma. Biochem Biophys Res Commun 334:
1351-1358, 2005
50. Iorio MV, Visone R, Di LG, et al: MicroRNA
signatures in human ovarian cancer. Cancer Res
67:8699-8707, 2007
51. Markou A, Tsaroucha EG, Kaklamanis L, et
al: Prognostic value of mature microRNA-21 and
microRNA-205 overexpression in non-small cell lung
cancer by quantitative real-time RT-PCR. Clin Chem
54:1696-1704, 2008
52. Volinia S, Calin GA, Liu CG, et al: A microRNA
expression signature of human solid tumors defines
cancer gene targets. Proc Natl Acad SciUSA
103:2257-2261, 2006
53. Schetter AJ, Leung SY, Sohn JJ, et al:
MicroRNA expression profiles associated with prognosis
and therapeutic outcome in colon adenocarcinoma.
JAMA 299:425-436, 2008
54. Roldo C, Missiaglia E, Hagan JP, et al:
MicroRNA expression abnormalities in pancreatic
endocrine and acinar tumors are associated with
distinctive pathologic features and clinical behavior.
J Clin Oncol 24:4677-4684, 2006
55. Bloomston M, Frankel WL, Petrocca F, et al:
MicroRNA expression patterns to differentiate pan-
creatic adenocarcinoma from normal pancreas and
chronic pancreatitis. JAMA 297:1901-1908, 2007
MicroRNAs in Cancer
www.jco.org © 2009 by American Society of Clinical Oncology 5855

56. Blenkiron C, Goldstein LD, Thorne NP, et al:
MicroRNA expression profiling of human breast
cancer identifies new markers of tumor subtype.
Genome Biol 8:R214, 2007
57. Miller TE, Ghoshal K, Ramaswamy B, et al:
MicroRNA-221/222 confers tamoxifen resistance in
breast cancer by targeting p27Kip1. J Biol Chem
283:29897-29903, 2008
58. Zhao JJ, Lin J, Yang H, et al: MicroRNA-221/
222 negatively regulates estrogen receptor alpha
and is associated with tamoxifen resistance in
breast cancer. J Biol Chem 283:31079-31086, 2008
58a. Di Leva G, Gasparini P, Piovan C: A regula-
tory “miRcircuitry”involving miR-221&222 and ER
␣
determines ER
␣
status of breast cancer cells. J Natl
Cancer Inst (in press)
59. Yu F, Yao H, Zhu P, et al: Let-7 regulates self
renewal and tumorigenicity of breast cancer cells.
Cell 131:1109-1123, 2007
60. Kumar MS, Erkeland SJ, Pester RE, et al:
Suppression of non-small cell lung tumor develop-
ment by the let-7 microRNA family. Proc Natl Acad
SciUSA105:3903-3908, 2008
61. Lebanony D, Benjamin H, Gilad S, et al:
Diagnostic assay based on hsa-miR-205 expression
distinguishes squamous from nonsquamous non–
small-cell lung carcinoma. J Clin Oncol 27:2030-
2037, 2009
62. Murakami Y, Yasuda T, Saigo K, et al: Com-
prehensive analysis of microRNA expression pat-
terns in hepatocellular carcinoma and non-tumorous
tissues. Oncogene 25:2537-2545, 2006
63. Li W, Xie L, He X, et al: Diagnostic and
prognostic implications of microRNAs in human
hepatocellular carcinoma. Int J Cancer 123:1616-
1622, 2008
64. Visone R, Pallante P, Vecchione A, et al:
Specific microRNAs are downregulated in human
thyroid anaplastic carcinomas. Oncogene 26:7590-
7595, 2007
65. le Sage C, Nagel R, Egan DA, et al: Regula-
tion of the p27(Kip1) tumor suppressor by miR-221
and miR-222 promotes cancer cell proliferation.
EMBO J 26:3699-3708, 2007
66. Galardi S, Mercatelli N, Giorda E, et al: MiR-
221 and miR-222 expression affects the proliferation
potential of human prostate carcinoma cell lines by
targeting p27Kip1. J Biol Chem 282:23716-23724,
2007
67. Felicetti F, Errico MC, Bottero L, et al: The
promyelocytic leukemia zinc finger-microRNA-
221/-222 pathway controls melanoma progression
through multiple oncogenic mechanisms. Cancer
Res 68:2745-2754, 2008
68. Zhang L, Huang J, Yang N, et al: MicroRNAs
exhibit high frequency genomic alterations in human
cancer. Proc Natl Acad SciUSA103:9136-9141,
2006
69. Li N, Fu H, Tie Y, et al: MiR-34a inhibits
migration and invasion by down-regulation of c-Met
expression in human hepatocellular carcinoma cells.
Cancer Lett 275:44-53, 2009
70. Gandellini P, Folini M, Longoni N, et al:
MiR-205 Exerts tumor-suppressive functions in hu-
man prostate through down-regulation of protein
kinase Cepsilon. Cancer Res 69:2287-2295, 2009
71. Asangani IA, Rasheed SA, Nikolova DA, et al:
MicroRNA-21 (miR-21) post-transcriptionally down-
regulates tumor suppressor Pdcd4 and stimulates
invasion, intravasation and metastasis in colorectal
cancer. Oncogene 27:2128-2136, 2008
72. Zhu S, Wu H, Wu F, et al: MicroRNA-21
targets tumor suppressor genes in invasion and
metastasis. Cell Res 18:350-359, 2008
73. Tavazoie SF, Alarcon C, Oskarsson T, et al:
Endogenous human microRNAs that suppress
breast cancer metastasis. Nature 451:147-152,
2008
74. Rosenfeld N, Aharonov R, Meiri E, et al:
MicroRNAs accurately identify cancer tissue origin.
Nat Biotechnol 26:462-469, 2008
75. Poliseno L, Tuccoli A, Mariani L, et al:
MicroRNAs modulate the angiogenic properties of
HUVECs. Blood 108:3068-3071, 2006
76. Hua Z, Lv Q, Ye W, et al: MiRNA-directed
regulation of VEGF and other angiogenic factors
under hypoxia. PLoS One 1:e116, 2006
77. Mertens-Talcott SU, Chintharlapalli S, Li X, et
al: The oncogenic microRNA-27a targets genes that
regulate specificity protein transcription factors and
the G2-M checkpoint in MDA-MB-231 breast cancer
cells. Cancer Res 67:11001-11011, 2007
78. Fish JE, Santoro MM, Morton SU, et al:
MiR-126 regulates angiogenic signaling and vascular
integrity. Dev Cell 15:272-284, 2008
79. Pulkkinen K, Malm T, Turunen M, et al:
Hypoxia induces miR-210 in vitro and in vivo
ephrin-A3 and neuronal pentraxin 1 are potentially
regulated by miR-210. FEBS Lett 582:2397-2401,
2008
80. Dews M, Homayouni A, Yu D, et al: Augmen-
tation of tumor angiogenesis by a Myc-activated
microRNA cluster. Nat Genet 38:1060-1065, 2006
81. Kuehbacher A, Urbich C, Zeiher AM, et al:
Role of Dicer and Drosha for endothelial microRNA
expression and angiogenesis. Circ Res 101:59-68,
2007
82. Hu Z, Chen J, Tian T, et al: Genetic variants
of miRNA sequences and non-small cell lung cancer
survival. J Clin Invest 118:2600-2608, 2008
83. Thomson JM, Newman M, Parker JS, et al:
Extensive post-transcriptional regulation of microRNAs
and its implications for cancer. Genes Dev 20:2202-
2207, 2006
84. Nakamura T, Canaani E, Croce CM: Onco-
genic All1 fusion proteins target Drosha-mediated
microRNA processing. Proc Natl Acad SciUSA
104:10980-10985, 2007
85. Karube Y, Tanaka H, Osada H, et al: Reduced
expression of Dicer associated with poor prognosis
in lung cancer patients. Cancer Sci 96:111-115, 2005
86. Merritt WM, Lin YG, Han LY, et al: Dicer,
Drosha, and outcomes in patients with ovarian can-
cer. N Engl J Med 359:2641-2650, 2008
87. Weber B, Stresemann C, Brueckner B, et al:
Methylation of human microRNA genes in normal
and neoplastic cells. Cell Cycle 6:1001-1005, 2007
88. Saito Y, Liang G, Egger G, et al: Specific
activation of microRNA-127 with downregulation of
the proto-oncogene BCL6 by chromatin-modifying
drugs in human cancer cells. Cancer Cell 9:435-443,
2006
89. Lehmann U, Hasemeier B, Christgen M, et
al: Epigenetic inactivation of microRNA gene hsa-
mir-9-1 in human breast cancer. J Pathol 214:17-24,
2008
90. Toyota M, Suzuki H, Sasaki Y, et al: Epige-
netic silencing of microRNA-34b/c and B-cell trans-
location gene 4 is associated with CpG island
methylation in colorectal cancer. Cancer Res 68:
4123-4132, 2008
91. Brueckner B, Stresemann C, Kuner R, et al:
The human let-7a-3 locus contains an epigenetically
regulated microRNA gene with oncogenic function.
Cancer Res 67:1419-1423, 2007
92. Lujambio A, Ropero S, Ballestar E, et al:
Genetic unmasking of an epigenetically silenced
microRNA in human cancer cells. Cancer Res 67:
1424-1429, 2007
93. Scott GK, Mattie MD, Berger CE, et al: Rapid
alteration of microRNA levels by histone deacety-
lase inhibition. Cancer Res 66:1277-1281, 2006
94. Benetti R, Gonzalo S, Jaco I, et al: A mam-
malian microRNA cluster controls DNA methylation
and telomere recombination via Rbl2-dependent
regulation of DNA methyltransferases. Nat Struct
Mol Biol 15:268-279, 2008
95. Sinkkonen L, Hugenschmidt T, Berninger P,
et al: MicroRNAs control de novo DNA methylation
through regulation of transcriptional repressors in
mouse embryonic stem cells. Nat Struct Mol Biol
15:259-267, 2008
96. Chen JF, Mandel EM, Thomson JM, et al:
The role of microRNA-1 and microRNA-133 in skel-
etal muscle proliferation and differentiation. Nat
Genet 38:228-233, 2006
97. Si ML, Zhu S, Wu H, et al: MiR-21-mediated
tumor growth. Oncogene 26:2799-2803, 2007
98. Meng F, Henson R, Lang M, et al: Involve-
ment of human micro-RNA in growth and response
to chemotherapy in human cholangiocarcinoma cell
lines. Gastroenterology 130:2113-2129, 2006
99. Weidhaas JB, Babar I, Nallur SM, et al: Mi-
croRNAs as potential agents to alter resistance to
cytotoxic anticancer therapy. Cancer Res 67:11111-
11116, 2007
100. Weiler J, Hunziker J, Hall J: Anti-miRNA
oligonucleotides (AMOs): Ammunition to target
miRNAs implicated in human disease? Gene Ther
13:496-502, 2006
101. Ørom UA, Kauppinen S, Lund AH: LNA-
modified oligonucleotides mediate specific inhibi-
tion of microRNA function. Gene 372:137-141, 2006
102. Kru¨ tzfeldt J, Rajewsky N, Braich R, et al:
Silencing of microRNAs in vivo with ‘antagomirs.’
Nature 438:685-689, 2005
103. Ebert MS, Neilson JR, Sharp PA: MicroRNA
sponges: Competitive inhibitors of small RNAs in
mammalian cells. Nat Methods 4:721-726, 2007
104. Dykxhoorn DM, Palliser D, Lieberman J: The
silent treatment: SiRNAs as small molecule drugs.
Gene Ther 13:541-552, 2006
■■■
Iorio and Croce
5856 © 2009 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY