© 2009 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
MicroRNAs (miRNAs) play essential roles in many physi-
ological and pathological processes, including tumor development,
by regulating the expression of a plethora of mRNAs. Although the
importance of miRNAs in tumorigenesis is well established, only
recently have reports elucidated miRNAs as promoters or suppres-
sors of metastasis. The miR-200 family has been shown to inhibit
the initiating step of metastasis, epithelial-mesenchymal transi-
tion (EMT), by maintaining the epithelial phenotype through
direct targeting of transcriptional repressors of E-cadherin, ZEB1
and ZEB2. These findings shed light into a miRNA-mediated
regulatory pathway that influences EMT in a developmentally and
pathologically relevant setting.
Since the discovery of microRNAs (miRNAs) in mammals in
2001,1-3 there has been a tremendous surge in effort to catalogue
and attribute biological functions to these small (~21–23 nt), non-
coding RNA molecules. These efforts have reaped great rewards as
our understanding of the role of miRNAs in normal physiology
and pathology has increased significantly. Aside from participating
in normal physiological processes such as muscle differentiation,4
miRNAs also function in the onset and/or progression of several
pathologies such as Alzheimer’s disease,5,6 dilated cardiomyopathy,7
Huntington’s disease8 and cancer.9,10
MiRNAs are initially synthesized by RNA polymerase II as long
primary transcripts, which are subsequently capped and polyade-
nylated.11 These transcripts are processed into ~70 nt stem-loop
pre-miRNAs by Drosha RNase III endonuclease12 and are trans-
ported out of the nucleus by Ran-GTP/exportin 5.13 Pre-miRNAs
are further processed by Dicer in the cytoplasm to yield a ~21–23 nt
duplex.14 One strand of the duplex is incorporated into the
RNA-induced silencing complex (RISC) and is used to regulate
the expression of target genes. Binding of miRNAs to the 3' UTR
[RNA Biology 5:3, 115-119; July/August/September 2008]; ©2008 Landes Bioscience
of mRNAs with perfect or near-perfect complementary sequences
induces mRNA degradation, whereas imperfect complementarity
often induces translational repression. The seed sequence of miRNAs,
representing 7–8 nt in the 5' end, is critical for efficient targeting and
miRNAs harboring similar seed sequences can theoretically regulate
the expression of a similar subset of genes.
Role of miRNAs in Tumor Development
In addition to deregulated expression of protein-coding genes,
alterations in the expression of non-coding genes, such as miRNA
genes, have also been documented as contributors to cancer
pathology.9,15 More than 50% of human miRNAs are located in
fragile chromosomal regions that are prone to mutations during
tumor progression.16 In addition, recent expression profiling analyses
have uncovered miRNA signatures that have the power to classify
certain human cancers.17-19 More importantly, functional charac-
terization has revealed a role for miRNAs as oncogenes (miR-21,
miR-155, miR-17-92 cluster) or tumor-suppressor genes (miR-34a,
let-7, miR-15a, miR-16) through the silencing of target tumor-
suppressor or oncogenic protein-coding genes, respectively (Fig. 1).
Interfering with miRNA processing also has been shown to enhance
experimental tumorigenesis, further implicating the role of miRNAs
in cancer.20 Although there is a clear role of miRNAs in oncogenesis,
the contribution of miRNAs to malignant progression of human
tumors has only recently been investigated and characterized.
Role of miRNAs in Metastasis
Metastasis is a multi-step process by which cells from the primary
tumor invade adjacent stroma, enter the systemic circulation, trans-
locate and arrest at distal capillaries, extravasate and finally proliferate
to form distant secondary tumors.21 Several recent reports have
elucidated the role of certain miRNAs as promoters22-25 or suppres-
sors26 of metastasis using a variety of approaches (Fig. 1). Tavazoie
et al.26 performed array-based miRNA profiling of the MDA-
MB-231 human breast cancer cell parental population as well as its
highly bone- or lung-metastatic derivatives to uncover the role of
miR-335 and miR-126 as metastasis suppressors in human breast
cancer. In contrast, Ma et al.24 identified miR-10b as a pro-metastatic
miRNA of breast cancer by functionally testing miRNAs deregulated
Point of View
The emerging role of miR-200 family of microRNAs in
epithelial-mesenchymal transition and cancer metastasis
Manav Korpal and Yibin Kang*
Department of Molecular Biology; Princeton University; Princeton, New Jersey USA
Abbreviations: EMT, epithelial-mesenchymal transition; MET, mesenchymal-epithelial transition; MiRNA, microRNA; TF, transcription
factor; RISC, RNA-induced silencing complex
Key words: microRNA, E-cadherin, ZEB, epithelial-mesenchymal transition, metastasis
*Correspondence to: Yibin Kang; Department of Molecular Biology; Washington
Road; Lewis Thomas Laboratory 255; Princeton University; Princeton, New Jersey
08544 USA; Tel.: 609.258.8834; Email: email@example.com
Submitted: 05/03/08; Accepted: 07/07/08
Previously published online as an RNA Biology E-publication:
www.landesbioscience.com RNA Biology 115
© 2009 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
E-cadherin regulation, such as epigenetic silencing and transcriptional
The role of miR-200 family in EMT and metastasis
116RNA Biology 2008; Vol. 5 Issue 3
in breast cancer previously reported by Iorio et al.27 In a recent study,
Huang et al.23 identified miR-373 and miR-520c as pro-metastasis
miRNAs by applying a forward genetic screen to search for miRNAs
that can induce migration and invasion in the non-migratory and
non-metastatic MCF-7 cell line. It is clear from these studies and
others that miRNAs may influence multiple steps of metastatic
cascade, such as tumor cell migration,23,24,26 invasion22-26 and intra-
vasation.22,24 However, the initiating, and perhaps the most critical
step in malignant conversion of tumor cells is thought to be the
activation of a cellular program in tumor cells that drives epithelial-
mesenchymal transition (EMT). EMT is a process whereby epithelial
tumor cells are stimulated by extracellular cytokines, such as TGFβ,
or intracellular cues, such as oncogenic Ras, to lose their epithelial
polarity and gain mesenchymal phenotypes with increased migra-
tory and invasive capabilities.28,29 One of the molecular hallmarks
driving this transition is the functional loss of E-cadherin (CDH1),
a cell adhesion protein and a major constituent of adherens junc-
tions thought to be a suppressor of migration/invasion during
carcinoma progression.30 The characterization of different modes of
repression, during tumor progression has enhanced our understanding
of the mechanisms that drive malignancy. In light of the importance
of E-cadherin as a suppressor of metastatic progression, elucidating
how miRNAs regulate epithelial cell plasticity and ultimately EMT
during tumor progression may hold tremendous therapeutic poten-
tial in reducing the incidence of metastasis. However, since the
aforementioned studies primarily used mesenchymal-like cell lines—
which may have already undergone the EMT process—for their
initial miRNA screens, it is unlikely that the identified miRNAs are
important regulators of EMT.
EMT and ZEB Factors
During metazoan embryogenesis, EMT plays a key role in the
formation of various tissues and organs such as the neural crest, heart,
musculoskeletal and peripheral nervous systems. However, during
adult life, only a certain subset of cells retain the ability to undergo
EMT-keratinocytes, for example, during wound healing. Epithelial
tumor cells often activate latent embryonic programs such as EMT
to acquire malignant properties, including enhanced motility and
invasiveness during tumor progression (Fig. 1). Several mouse models
Figure 1. Schematic of miRNAs involved in tumor progression. MiRNAs that play a role in tumor promotion (miR-21, miR-155, miR-17-92 cluster) or suppres-
sion (miR-let-7, miR-15a, miR-16, miR-34a) have been identified in various recent studies. Several miRNAs have also characterized as metastasis promoters
(miR-10b, miR-21, miR-373, miR-520c) or suppressors (miR-126, miR-335). The miR-200 family has recently been identified as suppressors of EMT and it is
speculated that they may inhibit metastasis. Epithelial cells (left, green outline) express low levels of Snail (yellow) and ZEB transcriptional factors (brown oval)
and high levels of the miR-200 family as two separate clusters. MiR-200 family miRNAs reduce the production of ZEB factors. Conversely, ZEB1 can repress
miR-200c/141 expression. High levels of miR-200 family and hence low levels of ZEB factors result in high expression of E-cadherin (blue oval), which is
essential for maintaining the epithelial state. Under the stimulation of certain cytokines, such as TGFβ, the expression of Snail and ZEB factors is increased.
ZEB factors inhibit the expression of miR-200c/141 and directly suppress E-cadherin expression to stimulate a morphological change to the mesenchymal
phenotype (right, purple outline). Thus, the balance of the levels and activities of miR-200 family and ZEB factors determine the bistable states of epithelial
and mesenchymal phenotypes of tumor cells. Solid lines show confirmed regulations and dashed lines indicate putative regulations.
© 2009 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
miR-200a/141, AACACU, by only one nucleotide. Although target
The role of miR-200 family in EMT and metastasis
of breast cancer progression have implicated EMT as a promoter for
metastasis through enhancing motility and invasive properties.31-33
The EMT process requires a complex genetic program coordinated
in a large part by the nuclear translocation of several transcriptional
repressors of CDH1. The characterization of the zinc-finger factor
snail (SNAI1) as a transcriptional repressor of CDH1 and an inducer
of EMT34,35 partially uncovered the molecular mechanisms that
govern tumor invasion.36 Since then, many other transcriptional
repressors of CDH1 have been discovered and because of their poten-
tial importance in tumor malignancies, there is a tremendous effort
in characterizing their molecular function.29
ZEB1 (ZFHX1A/TCF8/δEF1/Nil-2α) and ZEB2 (ZFHX1B/
SIP1), members of the ZEB family, are such CDH1 repressors that
have been implicated in EMT, tumorigenesis and metastasis.37-41
ZEB1 and ZEB2 can interact with DNA binding sites composed of
bipartite E-boxes (CACCT and CACCTG).42 The CDH1 promoter
contains such E-box sequences and it has been shown that ectopic
expression of ZEB factors in mammary gland epithelial cells43 or
MDCK cells,44 is sufficient to induce dissociation of adherens junc-
tions43,44 and enhance invasiveness/motility respectively.44 Both
ZEB factors appear to have common tissue expression patterns
during development and seemingly have redundant roles,45 although
some differences in tissue expression45 and functionality have
The ZEB factors can regulate the expression of various EMT- and
tumor-related genes. For example, they have been shown to repress
the expression of genes encoding proteins critical to maintaining the
epithelial phenotype such as E-cadherin, plakophilin 2 and ZO3.47
Conversely, the ZEB factors can also activate the expression of genes
promoting migratory/invasive phenotypes, such as the pro-invasion
Although a great deal is known of the signaling pathways that
regulate the expression of the EMT-promoting snail superfamily
members SNAI1 and SNAI2 (also know as slug), little informa-
tion is available regarding the specific regulation of the ZEB factors
during tumor progression.36 It has been elucidated that SNAI1 is
an activator of ZEB1,49 and that SNAI1 can indirectly regulate the
expression of ZEB2 by inducing the expression of a natural anti-
sense transcript of ZEB2 that promotes the translation of its own
mRNA during SNAI1-induced EMT50 (Fig. 1). In addition to Snail
proteins, miRNAs may also serve to regulate the expression of the
ZEB factors to influence the epithelial phenotype (Fig. 1). In fact, a
compilation of data from a few studies suggests a negative correlation
between the expression of miR-200s with that of the ZEB factors,
suggesting a miR-200 mediated targeting of ZEB factors in embry-
MiR-200 Family, the Epithelial Phenotype and EMT
The miR-200 family consists of five members organized as two
clusters, miRs-200b/a/429 and miRs-200c/141, on chromosomes
1 and 12 in humans and 4 and 6 in mice. Analysis of the miR-200
expression levels in various cell lines in our laboratory suggests that
miRNAs from each cluster are co-expressed whereas the expression of
miRNAs from the two clusters do not appear to be highly correlated
(Korpal M et al., unpublished result).53,54 The five miR-200 family
miRNAs contain very similar seed sequences, with the seed sequence
of miR-200b/c/429, AAUACU, differing from the seed sequence of
prediction algorithms predict a significant difference in the spectrum
of genes targeted by miR-200b/c/429 and miR-200a/141, microarray
analyses of cells transfected with members of these subfamilies indi-
cate a much higher degree of overlap in target genes, suggesting that
multiple members of the miR-200 family may target a large common
subset of genes to enhance the efficiency of genetic regulation.54
Several recent developmental studies show an enrichment of the
miR-200 family in differentiated epithelial tissues. Choi et al.55
showed that the miR-200 family is required for the differentiation
of olfactory progenitor cells in the zebrafish and mouse. Similarly,
an independent report demonstrated that this family is highly
expressed in skin epidermal cells during mouse skin morphogenesis.56
In support, other reports have found that miR-200a and miR-
200b are expressed in the ectoderm and the endoderm but largely
excluded from the mesoderm during chick embryo development.57
In zebrafish embryos, miR-200a, miR-200b and miR-141 are
expressed in the epidermis, proctodeum, the lateral line organs, and
the sensory epithelial structures that can sense chemicals such as nose
epithelium and taste buds, as assessed by in situ hybridization.58
These data strongly imply an underlying correlation between the
miR-200 family and the epithelial phenotype, as well as a negative
correlation with ZEB factors during embryonic development.
Collectively, these studies imply that the miR-200 family might regu-
late CDH1 expression through targeting of the ZEB factors (Fig. 1).
Two reports in 2007 functionally linked miR-200b and miR-200c
with the epithelial phenotype through targeting of ZEB1/ZEB2 in a
developmental setting59 and in cancer cell lines.59,60 Christoffersen
et al.59 showed that miR-200b and ZEB2 show overlapping expres-
sion patterns in the mouse brain and in vitro assays revealed that
miR-200b targets ZEB2 through binding to multiple sites in the 3'
UTR. In addition, Hurteau et al.60 reported the targeting of ZEB1
by miR-200c in cancer cell lines and showed that ectopic expression
of miR-200c enhanced CDH1 expression and promoted an epithelial-
like morphology. Although these two reports were the first to show
targeting of ZEB factors by the miR-200 family, they only partially
uncovered the full potential of this family of miRNAs to regulate the
epithelial phenotype and the underlying clinical implications.
Three recent reports overwhelmingly linked the miR-200 family
with the epithelial phenotype, EMT, and its inverse process, mesen-
chymal-epithelial transition (MET).53,54,61 Inhibition of endogenous
miR-200 expression levels was sufficient to induce EMT54,61 and
ectopic expression induced MET in normal and cancer cell lines
through direct targeting of ZEB1/253,54,61 and reduced motility and
aggressiveness of breast carcinoma cells.53,54 Interestingly, ectopic
expression of even single miR-200 members was sufficient to target
ZEB1/ZEB2 and induce CDH1 expression, albeit with different
efficacies.53,54 Park et al.54 reported a significant correlation between
the expression of miR-200 and the E-cadherin/Vimentin ratio across
all NCI60 cells, suggesting that the expression levels of the miR-200
family is a powerful, universal regulator of the epithelial phenotype
of cancer cells. In clinical patient samples, there is a significant
correlation between E-cadherin and miR-200c expression in primary
serous papillary ovarian tumors54 and the expression of miR-200
family is significantly lower in sarcomatoid metaplastic breast tumors
as compared to the more epithelial ductal breast carcinomas.61 These
data collectively suggest that the miR-200 family may serve as a poten-
tial therapeutic target to reduce the rates of invasive carcinomas.
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The role of miR-200 family in EMT and metastasis
118 RNA Biology2008; Vol. 5 Issue 3
Strategies for Manipulating miR-200 Family Levels
to Influence Metastasis
Since many diseased states have been correlated with deregulated
expression of various miRNAs, developing strategies to manipulate
miRNA expression levels in vivo may hold considerable potential in
the treatment of previously intractable diseases. Methods of silencing
miRNA expression in vivo via antagomirs62 and miRNA sponges63
or overexpressing miRNAs using viral/non-viral vectors, in vivo have
been tested but need to be optimized.
Other strategies for influencing miR-200 expression in tumors in
vivo are to manipulate the activity of transcription factors (TFs) that
regulate the expression of these miRNA genes. Previous studies have
shown that miRNA promoters can express protein coding reporter
genes and it has been shown that a translocation event forced MYC
to be expressed from a miRNA promoter in an aggressive B-cell
leukemia.64 Taken together, these data suggest that regulation of
miRNA expression may involve many of the same TFs that regulate
the expression of protein-encoding genes. A recent report by Ma et
al.24 showed that Twist, another CDH1 suppressor, could induce
the expression of miR-10b by binding directly to its promoter.
Interestingly, Burk et al.65 recently showed that ZEB1, a target of the
miR200 family, could bind directly to the promoter and suppress the
expression of miR-141 and miR-200c, triggering a feed forward loop
that stabilizes EMT and promotes invasion of cancer cells. In addi-
tion to binding sites for ZEB1, the promoter element also contains
Snail binding sites and overexpression of SNAI1 reduces expression
of miR-141 and miR-200c, although to a lesser extent65 (Fig. 1).
This suggests that multiple EMT factors may coordinately reduce
miR-200 family expression and disrupt the epithelial phenotype,
thereby driving the EMT process.
Human tumors upregulate the normally cytostatic cytokine
TGFβ late in tumor progression, enhancing the expression of EMT
inducers such as the ZEB factors, to promote invasiveness. Since
the miR-200 family members are repressed during TGFβ-induced
EMT in NMuMG53 and MDCK cells,61 it is likely that enhanced
TGFβ production by primary tumors may reduce expression of the
miR-200 family by enhancing the expression of ZEB factors, thus
promoting EMT and invasiveness (Fig. 1). Indeed, TGFβ mRNA
is highly expressed in NCI60 cell lines that express high levels of
ZEB1/2 and vimentin.54 In light of this, the simplest therapeutic
strategy may be to disrupt TGFβ-signaling activity in advanced
stage tumors to reduce the likelihood of EMT occurring through the
action of the miR-200 family in tumor cells in vivo.
The recently described role of the miR-200 family in regulating
EMT enhances our understanding of the regulatory pathways influ-
encing this key developmental and pathological process, opening up
new avenues for therapeutic intervention in patients with localized
primary lesions. Therapeutic targeting of the miR-200 family may
be achieved at many levels, the simplest strategy of which being
disrupting TGFβ-signaling activity using various inhibitors currently
in preclinical and/or clinical trials.
This work is supported by the US Army Medical Research
and Material Command (W81XWH-06-1-0481) with additional
support from the American Cancer Society (RSG MGO-110765),
the Susan Kormen for the Cure (BCTR0503765). MK is supported
by a pre-doctoral fellowships from the Natural Sciences and
Engineering Research Council of Canada and the Department of
Defense. We thank Dr. Yong Wei, Nilay Sethi and Mario A. Blanco
for their helpful suggestions and discussion.
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