Crosstalk Between TGF-b Signaling

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    Crosstalk between TGF-b signaling andthe microRNA machinery

    Henriett Butz1, Karoly Racz1, LaszloHunyady2 and Attila Patocs3,412nd

    Department

    of

    Medicine,

    Faculty

    of

    Medicine,

    Semmelweis

    University,

    Budapest,

    Hungary2Department

    of

    Physiology,

    Semmelweis

    University,

    Budapest,

    Hungary3Molecular

    Medicine

    Research

    Group,

    Hungarian

    Academy

    of

    Sciences,

    Budapest,

    Hungary4Department

    of

    Laboratory

    Medicine,

    Semmelweis

    University,

    Budapest,

    Hungary

    The activin/transforming growth factor-b (TGF-b) path-

    way

    plays

    an

    important

    role

    in

    tumorigenesis

    either

    by

    its tumor suppressor or tumor promoting effect. Loss of

    members of the TGF-b signaling by somatic mutations

    or

    epigenetic

    events,

    such

    as

    DNA

    methylation

    or

    regu-

    lation by microRNA (miRNA), may affect the signaling

    process. Most members of the TGF-b pathway are

    known to be targeted by one or more miRNAs. In addi-

    tion, the biogenesis of miRNAs is also regulated by TGF-

    b both directly and through SMADs. Based on these

    interactions, it appears that autoregulatory feedback

    loops between TGF-b and miRNAs influence the fate

    of tumor cells. Our aim is to review the crosstalk be-

    tween TGF-b signaling and the miRNA machinery to

    highlight

    potential

    novel

    therapeutic

    targets.

    Members and functions of the TGF-b signaling pathway

    The activin/TGF-b family

    consists

    of

    evolutionarily

    con-

    served

    polypeptides,

    which

    play

    prominent

    roles

    in the

    regulation

    of

    embryonic

    development,

    reproduction

    andtumor

    formation

    [1]. TGF-b signaling

    is

    often

    considered

    a pathogenic factor in tumorigenesis due to its tumor

    suppressor

    or

    tumor

    promoting

    effects

    (see

    below).

    Altered

    TGF-b signaling has been frequently shown in different

    tumor

    types,

    including

    breast,

    prostate,

    endometrial,

    colo-

    rectal,

    thyroid,

    parathyroid

    and

    pancreas

    neoplasms

    [2].

    The TGF-b family comprises more than 35 members,

    including

    TGF-bs, bone

    morphogenetic

    proteins

    (BMPs),

    growth

    differentiation

    factors

    (GDFs),

    activins,

    inhibins,

    Mullerian inhibiting substance (MIS), Nodal and leftys [1].

    They

    are

    secreted

    in

    an

    inactive

    form,

    and

    TGF-bs

    become

    active

    after

    cleavage

    of

    the

    N-terminal

    pro-region,

    referred

    to

    as

    the

    latency-associated

    peptide

    (LAP).

    Until

    thiscleavage occurs, the LAP-associated TGF-bs (L-TGF-bs)

    cannot interact

    with

    their

    receptors,

    and

    hence

    they

    are

    biologically

    inactive

    [1]. The

    active

    TGF-b

    molecule

    is

    a

    dimer composed of two TGF-b molecules linked by a disul-

    fide

    bridge

    between

    the

    ninth

    cysteine

    of

    each

    monomer

    [1].

    TGF-b

    receptors

    are

    serine/threonine

    kinase

    receptors

    and are divided into three groups: type I, type II and type

    III. In

    mammals

    there

    are

    seven

    different

    members

    of

    type

    I (ALK1ALK7)

    and

    five

    members

    of

    type

    II receptors

    (ACVR2, ACVR2B, AMHR2, BMPR2, TGF-bR2). Upon

    ligand

    binding,

    an

    active

    ligand-type

    I/type

    II

    receptor

    complex is formed, type II receptors activate type I recep-

    tors

    by

    phosphorylation,

    and

    type

    I

    receptors

    subsequently

    phosphorylate

    downstream

    SMAD

    proteins

    which

    trans-

    mit the signal to the nucleus (Figure 1). Type III receptors

    (betaglycan

    and

    endoglin)

    have

    a

    higher

    molecular

    weight

    than

    type

    I

    and

    type

    II receptors.

    Betaglycan

    is

    a

    mem-

    brane-anchored proteoglycan that can bind TGF-bs and

    facilitates

    interaction

    of

    TGF-b-type

    II

    receptor

    with

    TGF-

    b [1]. It can also promote binding of inhibin to type II

    receptor,

    thereby

    antagonizing

    activin

    signaling.

    Endoglin

    is a

    membrane

    glycoprotein

    with

    a

    large

    extracellular

    domain containing an integrin recognition motif and a

    short

    cytoplasmic

    tail

    with

    serine

    and

    threonine

    residues,

    which can be phosphorylated by the TGF-b receptors.

    Endoglin

    phosphorylation

    seems

    to

    play

    a

    regulatory

    role

    for

    ALK1-dependent

    endothelial

    cell

    growth

    and

    adhesion,

    which is confirmed by the findings that endoglin was found

    to be

    overexpressed

    in several

    tumor

    types

    [3,4].

    Tumor suppressor and tumor promoting effects of theTGF-b pathway

    TGF-b

    may

    inhibit

    cell

    proliferation

    at

    multiple

    levels.

    Well-known

    tumor

    suppressors,

    such

    as

    p15Ink4b and

    p21Waf1 are

    induced

    by

    TGF-b

    signaling,

    whereas

    oncogen-

    ic

    factors

    such

    as

    c-Myc,

    a

    transcription

    factor

    that

    pro-

    motes cell proliferation, and Id proteins, nuclear factors

    that

    inhibit

    cell

    differentiation,

    are

    repressed

    via

    TGF-b

    signaling

    [2].

    TGF-b

    can

    also

    activate

    apoptosis

    [5].

    This

    function is mediated by its downstream targets, such as

    death-associated

    protein

    kinase

    (DAPK),

    growth

    arrest

    and DNA-damage

    inducible

    b

    (GADD45b),

    and

    Bcl2-like

    11 (BCL2L11 or BIM). TGF-b signaling also inhibits tumor

    growth

    by

    repressing

    hepatocyte

    growth

    factor

    (HGF),macrophage-stimulating protein (MSP) and TGF-a [2].

    In

    addition

    to

    its

    tumor

    suppressor

    effects,

    TGF-b

    sig-

    naling has tumor promoting downstream targets. For

    example,

    through

    induction

    of

    deleted

    in

    esophageal

    can-

    cer

    1

    (DEC1),

    platelet-derived

    growth

    factor

    beta

    polypep-

    tide (PDGF-B), protein snail homolog 1 (SNAIL) and high

    mobility

    group

    AT-hook

    2

    (HMGA2),

    TGF-b

    signaling

    may

    mediate

    antiapoptotic

    effects,

    growth

    stimulation

    and

    epi-

    thelialmesenchymal transition (EMT). EMT is a biologi-

    cal

    process

    through

    which

    a

    polarized

    cell

    that

    normally

    interacts with a basement membrane (epithelial pheno-

    type) switches to a mesenchymal phenotype that is char-

    acterized by invasiveness and increased cell mobility [6].

    Review

    Corresponding author: Patocs, A. ([email protected]).

    Keywords: TGF-b signaling pathway; miRNA; endocrine neoplasm.

    382 0165-6147/$ see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2012.04.003 Trends in Pharmacological Sciences, July 2012, Vol. 33, No. 7

    mailto:[email protected]://dx.doi.org/10.1016/j.tips.2012.04.003http://dx.doi.org/10.1016/j.tips.2012.04.003mailto:[email protected]
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    During this process, basement membrane degradation

    occurs,

    cells

    lose

    E-cadherin

    and

    produce

    vimentin,

    a

    mesenchymal

    cell-specific

    intermediate

    filament.

    EMT

    has key roles in embryogenesis and wound healing, and

    has

    also

    been

    described

    as

    a

    crucial

    mechanism

    for

    the

    acquisition

    of

    malignant

    phenotypes

    of

    epithelial

    cells

    [7].

    Various factors are reported to mediate TGF-b-induced

    EMT,

    including

    SNAIL1,

    SNAIL2

    (Slug),

    ZEB1,

    ZEB2

    (SIP1).

    These

    are

    transcriptional

    repressors

    that

    bind

    to

    E-box motifs of the DNA and repress transcription of

    various

    genes,

    including

    E-cadherin

    [2].

    It

    is

    generally accepted that TGF-b signaling

    exerts

    tumor suppressor

    effects

    during

    the early

    phase

    of

    tumori-

    genesis and, in certain situations, it switches from a

    tumor suppressor

    to

    a

    tumor promoter

    [2].

    For the tumor

    promoting effect, additional secondary molecular mecha-

    nisms,

    includingmutation

    of

    theTP53

    gene

    encodingtumor

    suppressor

    p53

    [8] or

    Ras

    activation [9],

    are also involved.

    Ras enhances induction of SNAIL by TGF-b; however, this

    process

    is

    also regulated by

    glycogen

    synthase kinase-3b

    (GSK-3b).

    GSK-3b is

    apparently

    one of

    themajor

    mediators

    of environment-dependent TGF-b-induced responses, as a

    downstream

    collector

    of

    multiple signaling

    pathways,

    in-

    cluding mitogen-activated

    protein

    kinase

    (MAPK),

    PI3K/

    Akt and Wnt [2].

    In

    addition

    to

    the effects of genes encoding members

    of the TGF-b signaling, regulation

    of

    TGF-b expression

    by epigenetic mechanisms

    via DNA

    methylation or

    miR-

    NAs has also been demonstrated in different tumors

    [10,11].

    TGF-/activin

    TGFBR II TGFBR I

    SARA

    Smad

    2/3

    Smad2/3

    Smad2/3

    Smad2/3

    P

    P

    Cytoplasm

    Nucleus

    P

    Smad4

    Smad4

    Smad4

    Smad4

    Smurf1/2

    Smad3

    P

    Smad4Smad2/3

    TFP

    SBE TGF-specific miRNA genesTGF-specific genesSBE

    TFSmad2/3

    Smad4

    P

    miRNA genes

    pri-miRNA

    pre-miRNA

    miRNAs

    p68

    p68

    p72

    miRNA processing

    p72

    AAAA

    DGCR8

    DGCR8

    Drosha

    Drosha

    m7G

    TRENDS in Pharmacological Sciences

    Figure 1.

    Outlineof thecrosstalkbetweenmembers of thecanonical TGF-b signalingand themiRNAmachinery. TheTGF-b/activinbindsto its

    receptor. Through ligandbinding

    the type II receptors phosphorylate (hence activate) type I receptors. This complexthen intracellularly activates SMADmolecules (R-SMADs) byphosphorylation. Bindingof R-

    SMADs to thetype I receptoris mediated by theprotein namedSARA(SMADanchor for

    receptor activation). SMAD2andSMAD3are

    TGF-b-specific, whereas SMAD1, SMAD5

    andSMAD8areBMP-,AMH- andGDF-specific. After activation

    byTGF-b,

    SMAD2/3 interactswithSMAD4and this complex translocates to thenucleus.Here

    SMADcomplex can

    interact withother cofactors and transcription factors, and binds to specific DNAsequences, referred to as SBE (SMAD-binding element), in promoters of TGF-b targetgenes.

    Among SMAD family members, SMAD6 and SMAD7

    have inhibitory roles

    (I-Smad). SMAD6 preferentially inhibits phosphorylation of SMAD1/5/8 via BMP type I receptor,

    whereas SMAD7 in a complex withSmurf1/2 (E3ubiquitinligases) translocates fromnucleus andassociates with TGF-b/activin type I receptor causing its degradation.SMAD7

    can inhibit both the TGF-b

    andBMPsignaling pathways [1,2,10].

    TGF-b target

    genes includegenes encoding both proteins andmiRNAs (left

    lower part of the figure). Red and

    green arrows indicate the connections between TGF-b signaling and biogenesis of miRNAs. In the nucleus, SMAD3 can interact with

    Drosha, a member of miRNA

    microprocessing complex, andcan enhance processing of boththe T/BmiRNAs(see details in thetext) andmiRNAs transcribed fromnon-T/BmiRNAgenes (right lower part of

    the figure). After the pre-miRNAmature miRNA process, mature miRNA can target

    severalmembers of TGF-b

    signaling (highlightedas red

    combs).

    Review Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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    Canonical miRNA biogenesis

    miRNA

    miRNAnmiRNA2

    Transcription

    Transcription

    Non-canonical steps

    RNA Pol II

    miRNA1

    Individual miRNA gene

    miRNA genes in cluster

    pri-miRNA

    pri-miRNA

    pre-miRNA

    pre-miRNA

    pre-miRNA

    ac-pre-miRNA

    miRISC

    3UTR

    Ribosome5

    AGO2

    AAAA

    A

    Mature miRNAmiRNA:miRNA* duplexmiRNA*

    Host gene mRNACytoplasm

    Nucleus

    Splicing

    Exon1

    Exon1 Exon2

    Exon2Exon1

    miRNA

    miRtron

    Exon2

    Exportin 5

    Ran GTP

    DGCR8

    Drosha

    Splicosome

    Microprocessor

    complex

    Processing

    RNA Pol II

    RNA Pol II

    Cleavage

    Incorporationto miRISC

    Cleavage

    Passenger strand degradation

    Translational repression,mRNA degradation by cleavage or deadenylation

    Targeting mRNA

    AGO2

    m7G

    m7G

    m7G

    AAAA

    AAAA

    AAAA

    TRBP

    Dicer

    TRENDS in Pharmacological Sciences

    Figure 2.

    Biogenesis and function of miRNAs. Genes encoding miRNAs can be located in the genome individually or in clusters (upper part of the figure) of noncoding

    sequences, or in introns of protein-coding genes (called miRtrons) [74]. miRNA clusters are transcribed together. Both individual and clustered miRNA genes are

    transcribed by RNA polymerase II. They have a 7-methyl guanosine cap and are polyadenylated similar to mRNA molecules [117119]. The primary transcript (primary

    miRNA, pri-miRNA) is processedby an RNase III (Drosha) containing complex [120]. Pri-miRNAs areprocessed by an RNase III (Drosha)containing complex.Droshacleaves

    both strands into a 6070 nucleotide precursor-miRNA (pre-miRNA), which has hairpin secondary structure. The microprocessor complex contains an RNA-binding

    protein (DGCR8or Pasha) and other components including DEAD boxhelicases p68and p72 [120].

    Thepre-miRNA molecule is transported to the cytoplasmby Exportin-5

    [121] and processed by another RNase III enzyme (Dicer) complexed with transactivation-responsive RNA binding protein (TRBP). Dicer cleaves pre-miRNA into 21 nt

    miRNA:miRNA* duplexes[122]. One strand of thisRNAduplex (guide strand or maturedmiRNA) is incorporated intomiRNA-induced silencing complex (miRISC),whereas

    the other strand (passenger strand ormiRNA*) is usuallydegraded[123].

    However,recentdatasuggestthat miRNA* could alsobe loaded intomiRISC,which has functional

    Review Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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    Biogenesis and function of miRNAs

    miRNAs are approximately 1925 nucleotides

    long

    (with

    an

    average of

    22 nucleotides),

    noncoding RNA

    molecules

    that post-transcriptionally regulate gene expression via

    RNA

    interference

    by binding either

    to

    the 30UTR or

    50UTR or the coding sequence of

    protein-encoding

    mRNAs [1216] (Figure 2). Approximately 3050% of

    all protein-coding genes

    might be controlled by miRNAs[17,18]. One miRNA has the potential to affect the ex-

    pression

    of

    several

    proteins,

    and one protein

    is influenced

    by several

    miRNAs. As the expression

    pattern of the

    miRome is highly tissue-specific, miRNAs provide fine-

    tuning of

    protein

    expression

    level

    that

    renders

    them

    cell-

    and context-specific regulators of the adaptation process

    [19]. As

    miRNAs may influence many different

    mRNAs,

    they

    can participate in the regulation

    of

    several

    physio-

    logical and pathological cellular processes. Their roles

    have already been

    considered

    in development,

    cell pro-

    liferation, differentiation,

    apoptosis

    and

    tumorigenesis

    [20,21].

    Experimentally

    validated

    interactions

    between

    TGF-b

    signaling and miRNAs suggest that miRNAs influence theTGF-b

    pathway

    at

    multiple

    levels.

    In

    addition,

    TGF-b

    signaling itself enhances the maturation of miRNAs

    [22], resulting

    in

    a

    bidirectional

    functional

    link.

    Regulation of the TGF-b signaling by miRNAs via direct

    interaction with downstream members of canonical

    signaling pathways

    Most, if not all, members of the canonical TGF-b signaling

    pathway

    may

    be

    influenced

    by

    miRNAs.

    Using in silico

    miRNAmRNA

    target

    predictions,

    several

    possible

    inter-

    actions can be obtained. However, these predictions always

    need

    to

    be

    confirmed

    experimentally.

    Owing

    to

    the

    lack

    of

    high-throughput

    screening

    (HTS)

    methods

    for

    monitoringmiRNAmRNA

    interactions,

    few

    such

    interactions

    have

    been demonstrated to date (Table 1). Starting from TGF-b

    receptors, it

    has

    been

    shown

    that

    TGF-b

    type

    1

    receptor

    (TGF-bR1)

    and

    SMAD2

    were

    upregulated

    in

    most

    primary

    anaplastic thyroid carcinoma-derived cells, whereas miR-

    NAs

    (miR-30

    and/or

    miR-200

    families)

    potentially

    target-

    ing

    these

    molecules

    were

    downregulated

    [23]. Inhibition

    of

    the TGF-bR1 in these cells induced EMT and a concomi-

    tant

    increase

    of

    the

    miR-200

    family,

    suggesting

    their

    role

    in TGF-b-mediated EMT.

    In acute promyelocytic leukemia (APL) cells, miR-146a

    was downregulated by all-trans-retinoid acid treatment

    during

    APL

    differentiation.

    This

    miRNA

    may

    possibly

    influence

    cell

    proliferation

    in this

    cell

    line

    via

    SMAD4

    [24].

    In addition to tumorigenesis, TGF-b signaling also plays

    an

    important

    role

    in

    the

    development

    of

    several

    organs.

    Regulation

    of

    the

    TGF-bR1 by

    let-7

    may

    modulate

    and

    control

    TGF-b

    signaling

    activity

    to

    the

    necessary

    level

    at

    each

    developmental

    stage

    [25]. The

    miR-23b

    cluster

    (in-

    cluding miR-23b, miR-27b and miR-24-1) was found to

    repress

    bile

    duct

    gene

    expression

    in

    fetal

    hepatocytes

    through

    downregulation

    of

    SMADs

    (SMAD3,

    SMAD4

    and SMAD5), but low levels of the miR-23b cluster was

    required

    in

    cholangiocytes

    to

    allow

    TGF-b

    signalingnecessary for bile duct formation [26].

    TGF-b

    has

    also

    been

    implicated

    in

    regulation

    of

    fibro-

    genesis.

    In

    the

    heart,

    downregulation

    of

    miR-133

    and

    miR-

    590 via TGF-b1 and TGF-bR2 contribute to the enhance-

    ment

    of TGF-b

    signaling

    [27], whereas

    in

    the

    liver

    and

    lung, miR-21 targeting the negative regulator SMAD7 can

    also

    enhance

    TGF-b

    signaling

    [28,29]. Structural

    remodel-

    ing in

    vascular

    smooth

    muscle

    cell

    (VSMC)

    can

    lead

    to

    atherosclerosis or abdominal aortic aneurysm. In this

    process,

    miR-26a

    was

    found

    to

    be

    a

    potential

    pathogenic

    factorby

    altering

    TGF-b

    signaling

    through

    direct

    targeting

    of SMAD1 [30]. miR-141 and miR-200a directly inhibit

    TGF-b2

    in

    rat

    proximal

    tubular

    epithelial

    cells

    (NRK52E),

    and their downregulation may be responsible for the de-velopment

    and

    progression

    of

    TGF-b-dependent

    EMT

    and

    fibrosis [31].

    SMAD3

    has

    also

    been

    found

    to

    be

    a

    potential

    miRNA

    target

    in

    stem

    cells

    and

    in

    the

    pituitary.

    Interestingly,

    five

    of seven miRNAs that negatively correlated with tumor

    size

    in

    pituitary

    adenomas

    have

    been

    potentially

    predicted

    to

    target

    SMAD3,

    and

    among

    them

    miR-140

    was

    already

    validated experimentally [11,32].

    Regulation of TGF-b signaling by miRNAs that interact

    directly with TGF-b target genes

    TGF-b

    target

    genes

    can

    also

    be

    regulated

    by miRNAs

    (Figure 3a). The

    miR-106b/25

    cluster

    (miR-106b,

    miR-93and

    miR-25)

    was

    found

    to

    be

    upregulated

    and

    correlated

    with the loss of tumor suppressor activity of TGF-b signal-

    ing.

    These

    miRNAs

    directly

    target

    the

    cell

    cycle

    inhibitor

    p21Waf1/Cip1 and

    the

    pro-apoptotic

    protein

    BCL2L11

    (BIM)

    in gastric cancer by interfering with TGF-b-induced cell

    cycle

    arrest

    and

    TGF-b-mediated

    apoptosis

    [5].

    The

    miR-

    106b/25

    cluster

    accumulates

    prostate

    and

    pancreatic

    can-

    cers, neuroendocrine tumors, neuroblastoma and multiple

    myeloma.

    In

    B

    cells,

    BIM

    expression

    is

    also

    affected

    by

    a

    well-characterized oncogenic miRNA cluster, miR-17/92

    [33]. This cluster impairs TGF-b effects not only by target-

    ing individual TGF-b responsive genes as p21Waf1/Cip1 and

    BIM

    but

    also

    by

    targeting

    canonical

    TGF-b

    signaling

    molecules

    (TGF-bR2,

    SMAD2,

    SMAD4)

    [34,35].

    As discussed above, miRNAs regulate EMT by targeting

    ZEB1,

    ZEB2

    (see

    also

    below),

    SNAIL1,

    SNAIL2

    and

    consequences in certain cases [124,125]. This mechanism is called canonical miRNA processing. There are two noncanonical steps in themiRNAmaturization process.

    Some pre-miRNAs are transcribed from very short introns (called miRtrons) as a result of splicing anddebranching [126].

    In the cytoplasmsome of these pre-miRNAs are

    cleaved by AGO2, an argonaute protein into AGO2-cleaved precursor miRNA (ac-pre-miRNA). The single-stranded matured miRNA directly associates with argonaute

    proteins(in mammals AGO14), which are core components of miRISC. HeremiR interacts with 30UTR of its targetmRNA by base-pairingand represses expression of the

    targets. In this process theseed regionofmiRNA is essential, although there is evidence that the central loop regionof miRNA is also involvedin the target determination

    [127]. The seed region is defined as the consecutive stretch of approximately seven nucleotides starting from either the first or the second nucleotide at the 50end of the

    miRNA molecule. The mechanistic details of miRNA-mediated translational repression are not fully understood. Of the numerous factors that influence pairing, each

    predicted miRNAmRNA target pair needs to be experimentally verified because a simple, high-throughput method for biological validation of miRNA targets does not

    exist. Although several studies revealed spatial or temporal avoidance of miRNA coexpression with target genes, these may support the negative correlation between

    miRNA and its potential target mRNA expression strengthening target pairing [8,22].

    However, the negative correlation between mRNA and miRNA expression is not

    exclusive because experimentally valid targets can be found even without changes in mRNA expression [32].

    The repression of the target is realized by three major

    processes: mRNA cleavage by AGO2, mRNA degradation by deadenylation and inhibition of different steps of the translation process [128132].

    Review Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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    HMGA2. TGF-bR2 and SNAIL2 are direct targets of miR-

    204. The expression of this miRNA was significantly lower

    in

    the

    NCI60

    tumor

    cell

    line

    panel

    than

    in

    normal

    tissues

    [36]. In

    addition,

    SNAIL2

    is

    an

    essential

    mediator

    of

    EMT,

    which underlines the pathogenic role of miR-204 in pro-

    moting

    tumor

    dissemination

    [37]. The

    HMGAs

    are

    low

    molecular

    weight,

    nonhistone

    chromosomal

    proteins

    that

    interact with the minor groove of manyAT-rich promoters

    and

    enhancers,

    and

    mutations

    of

    the HMGA gene associ-

    ates

    with

    many

    common

    diseases,

    including

    benign

    and

    malignant tumors [38,39]. HMGA2 was found to cooperate

    with

    the

    TGF-b

    pathway

    in

    regulating

    the

    expression

    of

    SNAIL1

    and

    SNAIL2

    [40,41]. There

    is

    a

    physical

    interac-

    tion

    between

    HMGA2

    and

    SMAD

    molecules,

    leading

    to

    an

    increased binding of SMADs to the SNAIL1 promoter [41].

    Because

    SNAIL1

    is

    a

    master

    downstream

    effector

    of

    HMGA2 during induction of EMT, miRNAs may also

    influence EMT by regulating HMGA2. Indeed, the fre-

    quently

    downregulated

    miRNA

    let-7

    targets

    HMGA2

    resulting

    in

    its

    overexpression,

    which

    has

    already

    been

    demonstrated in numerous tumors, including ovarian car-

    cinoma,

    retinoblastoma,

    uterine

    leiomyosarcoma,

    neuro-

    endocrine

    tumors

    and

    pituitary

    adenomas

    [4246].

    In

    these tumors, expression of HMGA proteins is associated

    with

    malignant

    phenotypes

    and

    poor

    prognosis.

    TGF-b alters miRNA expression directly and by

    regulating the maturation process of miRNAs

    As

    described

    above,

    miRNAsdirectly regulate the expres-

    sion

    of

    members

    of the canonical TGF-b signaling and

    the

    expression of TGF-b target genes. However, TGF-b itself

    can

    alter

    the expression

    of numerous miRNAs

    through

    Table

    1.

    Regulation

    of

    members

    of

    canonical

    TGF-b

    signaling

    by

    miRNAs

    Name of miRNA miRNA target Tissue/cell where this miRNAmRNA regulation was demonstrated Species Refs

    miR-133 TGF-b1 Atrial fibroblast cells Dog [27]

    miR-744 TGF-b1 Proximal tubular epithelial cells (HK-2) Human [133]

    miR-200a TGF-b2 NRK52E kidney proximal tubular epithelial cells Rat [31]

    miR-141 TGF-b2 NRK52E kidney proximal tubular epithelial cells Rat [31]

    miR-210 AcvR1B (ALK4) ST2 bone marrow-derived stromal cells Human [60]

    miR-21 TGF-b components

    (TGF-bR2, TGF-bR3;DAXX, BMPR2)

    U251and U87glioblastoma cells Human [58]

    let-7 TGF-bR1 Adult and 912 week human embryonic liver tissues Human [25]

    miR-200 family,

    miR-141

    TGF-bR1 Anaplastic thyroid carcinoma tissues and ATC-derived cells,

    proximal tubular epithelial cells (NRK52E)

    Human, rat [23,134]

    miR-106b,

    miR-93

    TGF-bR2 SH-SY5Y neuroblastoma cells, embryonic fibroblasts (MEF) Human, mouse [135,136]

    miR-17-5p,

    miR-20

    TGF-bR2 HCT116 and DLD1 colon carcinoma cell line Human [81]

    miR-204 TGF-bR2 hfRPE human fetal retinal pigment epithelial cells Human [36]

    miR-20a TGF-bR2 Lung cancer tissues Human [137]

    miR-21 TGF-bR2 Myometrial smooth muscle cells, leiomyoma smooth muscle cells,

    transformed LSMC and SKLM-S1 leiomyosarcoma cell line

    Human [57]

    miR-21 TGF-bR2 hADSC human adipose tissues derived stem cell Human [56]

    miR-590 TGF-bR2 Atrial fibroblast Human [27]

    miR-155 SMAD1 Burkitts lymphoma cell line (Mutu I) Human [73]

    miR-26 SMAD1 HeLa S3 Human [138]

    miR-26a SMAD1 hADSC human adipose tissues derived stem cell Human [139]

    miR-199a SMAD1 Pluripotent C3H10T1/2 stem cells Human [140]

    miR-26a SMAD1

    (SMAD2/3/4 reporter)

    Vascular smooth muscle cells Human [30]

    miR-141,

    miR-200a,c,

    miR-30d,e

    SMAD2 Anaplastic thyroid carcinoma tissues and ATC-derived cells Human [23]

    miR-155 SMAD2 THP1 monocyte cell line Human [70]

    miR-140 SMAD3 Pluripotent C3H10T1/2 stem cells Human [32]

    miR-23b cluster

    (miR-23b, -27b, -24)

    SMAD3, -4, -5 HBC-3 fetal mouse liver stem cell Mouse [26]

    miR-146a SMAD4 NB4 cell (acute promyelocytic leukemia cell) and dermal fibroblast Human [25,141]

    miR-146b-5p SMAD4 Papillary carcinoma cell lines (TPC-1 and BCPAP) Human [142]miR-18 SMAD4 HCT116 and DLD1 colon carcinoma cell line Human [63]

    miR-224 SMAD4 Preantral granulosa cells (GCs) Mouse [138]

    miR-130a SMAD4 HEK-293 kidney, A549 lung, 32Del3 myeloid precursor cell line Human [143]

    miR-124 SMAD5 HeLa S3 Human [144]

    miR-155 SMAD5 Diffuse large B cell lymphoma and Burkitts lymphoma

    cell line (Mutu I)

    Human

    [71,72]

    miR-21 SMAD7 Lungs of mice with bleomycin-induced fibrosis and

    lungs of patients with idiopathic pulmonary fibrosis

    Human,

    mouse [29]

    miR-21 SMAD7 HCV-infected human liver tissues Human [28]

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    binding of

    p68, a

    component of

    the Drosha

    microproces-

    sor

    complex.

    These issues

    are

    discussed in the following

    sections.

    Changes in miRNA expression levels after TGF-b

    treatment

    Upon

    TGF-b

    treatment,

    changes

    in

    the

    expression

    of

    nu-

    merous

    miRNAs

    have

    been

    detected

    in

    different

    cells

    (Table 2).

    The

    miR-200

    family

    (miR-200a,

    miR-200b,

    miR-200c,

    miR-141

    and

    miR-429)

    and

    miR-205

    were

    markedly

    down-regulated

    in

    response

    to

    TGF-b

    treatment

    in

    kidney

    (MDCK) and rat proximal tubular epithelial cells

    (NRK52E)

    [31]. These

    miRNAs

    regulate

    the

    expression

    of the

    E-cadherin

    transcriptional

    repressors,

    ZEB1

    and

    ZEB2, which are implicated in EMT and tumor metastasis

    [47,48].

    In

    murine

    mammary

    epithelial

    cells

    (NMuMG),

    expression

    of

    the

    miR-200

    family

    was

    lost

    after

    induction

    of

    EMT by TGF-b stimulation [47]. Enforced expression of the

    miR-200

    family

    alone

    was

    sufficient

    to

    prevent

    TGF-b-

    induced EMT, and its inhibition was sufficient to induce

    EMT in a process requiring upregulation of ZEB1 and/or

    ZEB2 [49]. However, miR-200a and miR-141 directly tar-

    get TGF-b

    and

    TGF-bR1

    in

    mesenchymal

    anaplastic

    thy-

    roid

    carcinoma-derived

    and

    NRK52E

    cells

    [23,31]. ZEB1

    directly suppresses transcription of miR-141 and miR-

    200c, and

    triggers

    a

    miRNA-mediated

    feed-forward

    loop,

    which

    stabilizes

    EMT

    and

    promotes

    invasion

    of

    cancer

    cells

    [50]. This network, with the miR-200 family in the center,

    contributes

    to

    the

    regulation

    of

    EMT

    in an

    environmental-

    ly-dependent

    manner

    [48].

    TGF-b signaling can also regulate the expression of a

    subset

    of

    miRNAs

    via

    transcription

    regulation

    by

    SMAD3.

    miRNA

    let-7d

    was

    repressed

    in tissue

    samples

    of

    patients

    with

    idiopathic

    pulmonary

    fibrosis

    (IPF)

    [51], and

    miR-24

    was also repressed in C2C12 cells leading to reduced

    expression

    of

    myogenic

    differentiation

    markers

    [52].

    An onco-miRNA,

    miR-21

    was

    found

    to

    be

    upregulated

    by

    TGF-b

    treatment

    in

    breast

    cancer

    and

    proliferating

    tubu-

    lar epithelial cells (TECs) [5355], and its direct interac-

    tion with

    TGF-bR2

    was

    also

    demonstrated

    [56,57].

    Downregulation

    of

    miR-21

    in

    glioblastoma

    cells

    caused

    growth repression, increased apoptosis and cell cycle arrest

    [58]. miR-21

    was

    also

    among

    those

    miRNAs

    (miR-21,

    miR-

    32, miR-137,

    miR-346,

    miR-136,

    miR-192,

    miR-210,

    miR-

    211), which were suggested to participate in the regulation

    of EMT

    [59].

    In addition

    to

    its

    role

    in

    tumorigenesis,

    upregulation

    ofmiR-21

    was

    also

    detected

    in

    myofibroblasts

    obtained

    both

    from lungs of mice having bleomycin-induced fibrosis and

    patients

    with

    IPF

    [29].

    Another

    miR,

    miR-210,

    was

    also

    found

    to

    be

    overex-

    pressed after BMP4 administration and was considered to

    act

    as

    a

    positive

    regulator

    of

    osteoblastic

    differentiation

    by

    inhibiting

    TGF-b

    signaling

    through

    inhibition

    of

    ACVR1B

    (ALK4) [60].

    As mentioned

    above,

    TGF-b1 is

    a

    key

    mediator

    of

    fibro-

    tic diseases. The pathomechanism of this process includes

    SMAD7, a direct target of mir-21. Upregulation of miR-21

    in primary pulmonary fibroblasts inhibits SMAD7, and

    inhibition

    of

    this

    inhibitory

    SMAD7

    results

    in

    enhanced

    TGF-b

    signaling

    [28,29]. SMAD3

    also

    regulates

    miR-192

    by binding to its promoter and hence participating in the

    regulation

    of

    renal

    fibrosis

    [61].

    Members

    of

    the

    miR-17/92

    cluster

    implicated

    in

    tumori-

    genesis (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1,

    miR-92a-1)

    were

    upregulated

    by

    TGF-b

    administration

    in

    several

    cell

    lines

    (Table

    2) [62]. However,

    miR-17/92

    di-

    rectly targets TGF-bR2 and SMAD4, and thus it partici-

    pates

    in the

    regulation

    of

    an

    autoregulatory

    feedback

    loop

    similar

    to

    that

    reported

    between

    miR-200a

    and

    TGF-b/

    TGF-bR1

    [63]. In

    this

    loop,

    a

    zinc

    finger

    protein

    512B

    which is also known as GM632 or GAM (GAM/ZFp/

    ZNF512B),

    a

    vertebrate-specific

    zinc

    finger

    factor,

    has

    a

    TGF-signaling

    (a) (b)

    Apoptosis EMT

    HMGA2PDGFB

    SNAIL

    DEC1

    BIM

    GADD45bPDCD4

    miR-106b/25miR-17/92

    miR106b/25miR-17/92

    miR-223miR-429

    miR-183 miR-204 let-7

    GAM TGFBRII

    SMAD4

    miR-17/92Drosha

    c-MYC

    TGF-signaling

    TGF-downstream targets

    ZEB1, ZEB2, p21, BIM

    DAPKc-MYC

    p15p21

    Tumor suppression

    TRENDS in Pharmacological Sciences

    Figure 3.

    Schematicrepresentation of therole of theTGF-b

    pathway in tumorigenesis. (a)Maindownstream effectors of the TGF-b

    signaling and its fine-tuningbymiRNAs

    (detailed in the text). Green arrows indicate downregulation and red arrows showupregulation. (b) Schematic regulatory feedback loop involving TGF-b

    signaling (C-MYC,

    ZEB1, ZEB2, p21, BIM), Drosha, miR-17/92 cluster and GAM.

    Review Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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    key role. miR-17/92, together with let-7, reduces GAM

    expression

    by

    directly

    targeting

    its

    30UTR,

    whereas

    GAM

    downregulates

    miR-17/92

    via

    three

    mechanisms:

    (i) impairs transcriptional activation of the miR-17/92

    cluster

    via

    C-MYC;

    (ii)

    decreases

    the

    transcriptional

    activ-

    ity of SMADs; and (iii) by interacting directly with Drosha

    is involved in pri-miRNA (primary transcript of miRNA)

    miR-17/92 processing (Figure 3b). GAM increases apopto-

    sis,

    reduces

    cell

    proliferation

    and

    modulates

    levels

    of

    E2F1

    and

    Ras.

    C-MYC

    itself

    is

    a

    TGF-b

    target

    gene

    that

    acti-

    vates the promoter of this miRNA cluster. In addition, the

    miR-17/92

    cluster

    also

    targets

    individual

    TGF-b

    respon-

    sive

    genes,

    such

    as

    p21Waf1/Cip1 and

    BIM

    [35,64].

    Expression of miR-451 was shown to be induced by

    SMAD3

    and

    SMAD4

    through

    SMAD

    target

    sites

    in

    their

    promoter

    regions

    in

    glioblastoma

    cells,

    resulting

    in

    cell

    growth inhibition [65].

    The

    miR-24

    cluster

    (miR-23a,

    miR-27a

    and

    miR-24)

    was

    also

    induced

    in

    response

    to

    TGF-b

    in

    human

    hepatocellular

    carcinoma

    cells

    (Huh-7)

    in

    a

    SMAD-dependent

    manner.

    This cluster can function as an antiapoptotic and prolifer-

    ation-promoting

    factor

    in

    liver

    cancer

    cells,

    because

    its

    expression is highly upregulated in hepatocellular carci-

    noma

    tissues

    compared

    with

    normal

    liver

    [66].

    TGF-b

    induces

    miR-216a

    and

    miR-217,

    which

    were

    shown to activate Akt. Via this mechanism, TGF-b signal-

    ing

    participates

    in

    fibrosis,

    hypertrophy

    and

    survival

    in

    glomerular mesangial cells [67]. Both miR-216a and miR-

    217 target phosphatase and tensin homolog (PTEN), an

    inhibitor ofAkt activation. Because PTEN protein acts as a

    tumor

    suppressor,

    this

    interaction

    may

    have

    a

    delicate

    role

    in carcinogenesis

    beyond

    the

    development

    of

    diabetic

    kid-

    ney disease.

    Upon TGF-b

    treatment

    of

    normal

    murine

    mammary

    gland

    (NMuMG)

    epithelial

    cells,

    miR-155

    was

    shown

    to

    be among the most significantly elevated miRNAs. This

    induction

    was

    SMAD4-dependent

    [68]. Inhibition

    of

    miR-

    155 was

    sufficient

    to

    suppress

    the

    TGF-b-induced

    EMT,

    making this miRNA a potential target in breast cancer

    treatment.

    Interestingly,

    miR-155

    was

    downregulated

    by

    TGF-b

    in

    normal

    human

    lung

    fibroblasts,

    but

    its

    ectopic

    overexpression

    increased

    cell

    migration

    [69]. In

    mouse

    models of lung fibrosis the expression level of miR-155

    was

    correlated

    with

    the

    degree

    of

    fibrosis

    [69]. TGF-b

    Table

    2.

    miRNAs

    regulated

    by

    TGF-b

    administration

    Name of miRNA Direction of miRNA

    expression changes

    Tissue/cell Species Refs

    let-7d Decreased Idiopathic fibrosis pulmonary tissue Human [51]

    miR-142-3p Elevated Limb primary mesenchymal cells Chicken [145]

    miR-145 Decreased Mesenchymal stem cells Murine [146]

    miR-145 Elevated Coronary artery smooth muscle cell Human [147]

    miR-146a Elevated Langerhans cells Human [148]

    miR-155 Elevated Normal mouse mammary gland epithelialcells (NMUMG)

    Mouse [68]

    miR-155 Decreased Lung fibroblasts Human [69]

    miR-17/92 cluster Elevated HEK-293 kidney, HepG2 liver, MCF7 breast

    cancer cell line

    Human [62]

    miR-18 Elevated HeLa (cervix epithelial adenocarcinoma) Human [149]

    miR-181b Elevated Hepatocellular carcinoma, breast cancer Human [150,151]

    miR-192 Elevated Human kidney tubular epithelial cells

    Mouse mesangial cells (MMCs)

    Rat proximal tubular epithelial cells (NRK52E)

    Human

    Mouse

    Rat

    [61,152,153]

    miR-200a/b Decreased Gastric cancer cell line Human [154]

    miR-200a/b/c Decreased Proximal tubular epithelial cells (NRK52E) Rat [31]

    miR-205;

    miR-200 family

    Decreased Mesenchymal cells Human [49]

    miR-206 Decreased C2C12 myoblasts Mouse [155]

    miR-21 Elevated Breast cancer,

    human proliferating tubular epithelial cells (TECs),

    rat proximal tubular epithelial cells (NRK52E)

    Human,

    rat

    [5355,134,156]

    miR-216/217 Elevated Glomerular mesangial cells Human [157]

    miR-216a Elevated Mouse mesangial cell (MMCs) Mouse [152]

    miR-224 Elevated Ovarian granulosa cells Mouse [158]

    miR-23a/27a/24 cluster Elevated Huh-7, HepG2, Hep3B liver cells Human [55]

    miR-24 Decreased C2C12 myoblasts Human [52]

    miR-24 Elevated for short time,

    decreased for long time

    treatment

    HaCaT keratinocytes Human [159]

    miR-24 Elevated HeLa (cervix epithelial adenocarcinoma) Human [149]

    miR-27b Decreased Cardiomyocytes Mouse [160]

    miR-29 Decreased Primary murine hepatic stellate cells,

    immortalized murine hepatic stellate cells,C2C12 myoblasts

    Mouse [161,155]

    miR-34 Decreased HeLa (cervix epithelial adenocarcinoma) Human [149]

    miR-451 Elevated Glioblastoma stem (CD133+) cells Human [65]

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    treatment

    may

    cause

    opposite

    effects

    on

    the

    expression

    of miR-155

    in

    different

    cells,

    underlining

    again

    that

    both

    TGF-b

    and

    miRNA

    machineries

    work

    in

    a

    cell-

    and

    environment-dependent manner, and emphasizing that

    the

    exact

    mechanisms

    and

    the

    direct

    targets

    of

    miR-155

    have

    to

    be

    identified

    in

    each

    cell

    type.

    miR-155

    directly

    targets TGF-b-specific SMAD2 in THP-1 monocyte cell

    lines

    [70]

    and

    BMP-specific

    SMAD1

    and

    SMAD5

    [7173], suggesting the presence of another possible feedback

    regulatory

    loop.

    SMADS influence miRNA processing

    Daviset al. presented another mechanism that contributes

    to the modulation of miR expression [74,75]. TGF-b treat-

    ment

    resulted

    in upregulation

    of

    pre-miRNAs

    and

    matured

    miRNAs,

    but

    not

    that

    of

    pri-miRNAs.

    These

    miRs

    are

    regulated post-transcriptionally by a genome-independent

    mechanism

    through

    association

    of

    receptor-specific

    SMAD

    (R-SMAD),

    SMAD1

    and

    SMAD5,

    but

    not

    SMAD4

    proteins,

    with p68, an RNA helicase component of the Drosha mi-

    croprocessor

    complex

    [75]. This

    subset

    of

    miRNAs

    is

    called

    TGF-b/BMP-regulated miRNAs (T/B miRNAs) [75]. T/BmiRNAs

    contain

    in

    their

    primary

    transcripts

    a

    conserved

    sequence, identified as R-SBE, which is similar to SMAD-

    binding

    element

    (SBE)

    [76]. SMAD

    proteins

    through

    their

    amino

    terminus

    MH1

    domain

    directly

    associate

    with

    R-

    SBE. Davis et al. demonstrated that mutations in the R-

    SBE region

    abolished

    TGF-b/BMP-mediated

    induction

    of

    pre-miRNA

    synthesis

    and

    impaired

    pri-miRNA

    binding

    to

    Drosha and DGCR8 in vivo [76]. In the human genome, 44

    T/BmiRNAs

    have

    been

    identified

    [76]. The

    nucleocytoplas-

    mic shuttling

    of

    SMADs

    (controlled

    by

    phosphorylation

    of

    serine residues by the TGF-bR1) is crucial for SMAD-

    mediated

    miRNA

    maturation.

    MAPK

    and

    GSK-3b

    can

    also

    alter the

    subcellular

    localization

    of

    SMADs

    through

    phos-phorylation

    [77,78]. Therefore,

    it

    was

    suggested

    that

    SMAD regulation of miRNA processing could be modulated

    independently

    of

    TGF-b

    and

    BMPs

    by

    signals

    that

    alter

    the

    nuclear

    localization

    of

    SMADs

    (ERKMAPK

    and

    the

    Wnt

    pathways) [76].

    Similar

    to

    SMAD

    proteins,

    the

    RNA

    helicases

    p68

    have

    been

    shown

    to

    interact

    with

    several

    other

    transcription

    factors, including MyoD, Runx2, androgen receptor, estro-

    gen receptor

    and

    p53.

    Association

    of

    p53

    and

    p68

    facilitated

    Drosha processing of a subset of miRNAs, which were

    different from T/B miRNAs [79].

    Pharmacological

    interventions

    affecting

    TGF-b

    signaling

    The TGF-b signaling pathway is a promising target in

    cancer

    therapy.

    Indeed,

    several

    compounds

    affecting

    this

    signaling

    pathway

    are

    under

    preclinical

    development

    or

    even in clinical trial phase, as summarized in several

    recent

    reviews

    [8082].

    From

    a

    theoretical

    point

    of

    view,

    there

    are

    three

    major

    possibilities in targeting the TGF-b pathway. Ligand traps

    include

    TGF-b

    antibodies

    and

    soluble

    TGF-b

    receptors.

    In

    mice,

    anti-TGF-b

    antibodies

    suppressed

    metastasis

    forma-

    tion [83], whereas

    in

    the

    rat,

    they

    arrested

    progressive

    nephropathy [84]. TGF-b antibodies increase the immune

    response

    in

    animal

    experiments

    [85]. Anti-TGF-b1,

    -b2

    and pan-TGF-b

    antibodies

    are

    under

    preclinical/clinical

    trials, and

    have

    been

    tested

    for

    scleroderma,

    prevention

    of

    scarring,

    metastatic

    melanoma

    and

    renal

    cell

    carcinoma

    [80,84,86,87]. Soluble receptors, TGF-bR2 (sTbRII) and

    TGF-bR3

    (sTbRIII) can

    also

    inhibit

    TGF-b

    signaling.

    In

    hepatoma

    cells

    transfected

    with

    sTbRII,

    decreased

    tumor

    formation was observed in an in vivo animal model [88]. In

    a

    transgenic

    mouse

    mammary

    tumor

    model,

    increasedapoptosis in primary tumors, reduced tumor cell motility,

    reduced

    intravasation

    and

    a

    decreased

    number

    of lung

    metastases

    were

    detected

    after

    systemic

    treatment

    with

    sTbRII [89]. sTbRIII was also tested and proved to sup-

    press

    cell

    growth

    and

    metastasis

    of

    human

    breast

    cancer

    and colon carcinoma cells [90].

    Silencing

    of

    TGF-b signaling

    by

    antisense

    oligonucleo-

    tides

    is

    another

    possibility

    for

    therapy.

    DNA

    oligonucleo-

    tides can inhibit the synthesis of TGF-b1 and -b2 by

    specific

    binding

    to

    their

    mRNAs.

    It

    has

    been

    shown

    that

    administration

    of

    TGF-b

    antisense

    oligonucleotides

    can

    reactivate tumor-specific immune responses [91,92].

    TGF-b

    antisense

    nucleotides

    have

    been

    tested

    in preclini-

    cal and clinical studies for the treatment of glioma, pan-creatic,

    colorectal,

    prostate

    and

    non-small

    cell

    lung

    cancer

    [86,9399].

    A

    third

    option

    of

    TGF-b-targeted

    therapy

    is

    the

    use

    of

    intracellularly-acting

    TGF-bR1

    kinase

    inhibitors.

    Numer-

    ous compounds are under development, with promising

    results;

    they

    are

    effective

    in blocking

    TGF-b-induced

    EMT

    in

    mammary

    epithelial

    cells,

    pancreatic

    carcinoma

    cells

    and in inhibiting glioma tumor growth, as well as suppres-

    sing

    renal

    fibrosis

    in

    obstructive

    nephropathy

    [100103].

    Because

    miRNAs

    influence

    cellular

    processes

    at

    several

    points, they are both promising therapeutic agents and

    targets

    [104]. Potential

    drugs

    that

    inhibit

    overexpressed

    (oncogenic)

    miRNAs

    or

    those

    that

    substitute

    underex-pressed

    (e.g.,

    tumor

    suppressor)

    miRNAs

    would

    be

    useful

    in cancer therapy. At present, miRNA-based therapeutic

    approaches

    are

    in experimental,

    preclinical

    or

    in

    early

    clinical

    phases.

    The

    principal

    problem

    is

    the

    delivery

    of

    miRNAs to the targeted cell [105]. Treatment with miR-

    NAs

    appears

    to

    be

    difficult

    even

    when

    a

    local

    delivery

    approach

    may

    be

    suitable

    to

    ensure

    the

    cell-specific

    effect.

    Systemic delivery is more dangerous; cytotoxic effects

    related

    to

    unconjugated

    miRNAs

    or

    vectors

    used

    for

    deliv-

    ery, as well as immunogenic reactions, present major

    difficulties. For this purpose, adenoviralvectors have been

    commonly used. Their application as therapeutic agents

    has

    been

    reported

    as

    local

    or

    targeted

    treatments

    (e.g.,

    intratumoral

    delivery

    in

    hepatocellular

    carcinoma

    [106]

    and transnasal administration for lung cancer treatment

    [107]). Systemic

    administration

    of

    miR-10b

    inhibited

    the

    metastasis

    formation

    in

    a

    mouse

    mammary

    tumor

    model.

    However, cytotoxic effects related to unconjugated miR-

    NAs

    or

    vectors

    used

    for

    delivery,

    as

    well

    as

    immunogenic

    reactions,

    present

    difficulties

    and

    cause

    serious

    problems

    during systemic delivery [108,109]. For miRNA silencing,

    several

    strategies,

    including

    anti-miRNA

    oligonucleotides

    (AMOs),

    miRNA

    sponges

    and

    miRNA

    masking,

    have

    al-

    ready

    been

    tested.

    AMOs

    are

    synthetic

    antisense

    oligonu-

    cleotides that bind their target miRNAs and thereby

    competitively

    inhibit

    the

    miRNAmRNA

    interaction

    Review Trends in Pharmacological Sciences July 2012, Vol. 33, No. 7

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    [110]. In

    clinical

    trials,

    AMOs

    have

    been

    tested

    against

    hypoxia-induced

    factor

    1

    (HIF-1a),

    miR-122

    and

    protein

    kinase

    N3

    (PKN3)

    in

    solid

    tumors

    [111]

    and

    lymphomas

    [112]. Another potential area in which miRNA therapy

    may

    be

    considered

    is

    treatment

    of

    viral

    infections.

    The

    first

    experimental

    drug

    was

    an

    antagomir,

    which

    inhibits

    miR-

    122, essential for the accumulation of hepatitis C virus

    (HCV)

    in

    hepatic

    cells.

    Subcutaneous

    administration

    dra-matically reduced HCV load in the liver and blood [113].

    Effective

    treatments

    may

    require

    knockdown

    of

    multiple,

    rather

    than

    individual,

    miRNAs.

    For

    this

    specific

    aim,

    miRNA sponges have been developed. These compounds

    are

    oligonucleotide-based

    constructs

    with

    multiple

    binding

    sites against an miRNA cluster inserted invectors contain-

    ing

    a

    strong

    promoter

    [114]. Also,

    target

    protection

    or

    miRNA

    masking

    represents

    an

    interesting

    novel

    strategy

    for knockdown of the effect of miRNAs. For this purpose,

    single-stranded,

    chemically-modified

    oligoribonucleotides,

    perfectly

    complementary

    to

    the

    miRNA

    binding

    site

    of

    the

    30UTR of target mRNA, have been used in vivo in a zebra-

    fish model

    [115].

    Double-stranded RNAs, which mimic the effect of en-dogenous

    miRNAs,

    have

    been

    developed

    for

    miRNA

    sub-

    stitution [105].Application of miRNAs in combination with

    other

    therapeutic

    agents

    may

    also

    contribute

    to

    a

    more

    successful

    treatment.

    Adjuvant

    miRNA

    therapy

    may

    en-

    hance the effect of other systemic therapies through influ-

    encing

    radiosensitivity

    or

    increasing

    sensitivity

    to

    DNA-

    damaging

    drugs.

    It

    has

    been

    reported

    that

    inhibition

    of

    the

    overexpressed miR-128a (which target TGF-b-R1) leads to

    resensitization

    for

    the

    growth

    inhibitory

    effects

    ofTGF-b

    in

    letrozole-resistant

    breast

    cancer

    [116].

    Concluding remarks

    The

    TGF-b

    signaling

    pathway

    is

    a

    complex

    network

    thatcontrols

    many

    physiological

    and

    pathophysiological

    pro-

    cesses. Its regulation and interaction with miRNAs in a

    cell-

    and

    context-specific

    manner

    provides

    a

    fine-tuning,

    dynamic

    and

    adaptive

    control

    of

    protein

    expression.

    In

    the

    process of tumorigenesis, alterations of the TGF-b path-

    way

    either

    by

    genetic

    or

    epigenetic

    events

    result

    in

    a

    switch

    from

    a

    tumor

    suppressor

    to

    a

    tumor

    promoting

    effect.

    Recent knowledge on targeting members of this signaling

    cascade by

    miRNAs

    or

    other

    agents

    may

    lead

    to

    the

    devel-

    opment of novel approaches in the therapy of cancer and

    other diseases.

    Disclosure

    statement

    The

    authors

    have

    nothing

    to

    disclose.

    AcknowledgmentsThe authors acknowledge the financial support from the Hungarian

    Ministry of National Resources (ETT40/09) and TAMOP-4.2.2.B-10/B-10/

    1-2010-0013. A.P. is a recipient of the Janos Bolyai Research Fellowship.

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