Downregulation of the tumor-suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development.

Abstract

Experimental and clinical evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA expression in mammary tumorigenesis.
We performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16.
We found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells.
In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin- and growth factor-induced growth in breast cancer.

Full text

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Downregulation of the tumor-suppressor miR-16
via progestin-mediated oncogenic signaling
contributes to breast cancer development
Rivas et al.
Rivas et al. Breast Cancer Research 2012, 14:R77
http://breast-cancer-research.com/content/14/3/R77 (14 May 2012)

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RESEARCH ARTICLE Open Access
Downregulation of the tumor-suppressor miR-16
via progestin-mediated oncogenic signaling
contributes to breast cancer development
Martin A Rivas1,4, Leandro Venturutti1, Yi-Wen Huang2, Roxana Schillaci1, Tim Hui-Ming Huang3and
Patricia V Elizalde1*
Abstract
Introduction: Experimental and clinical evidence points to a critical role of progesterone and the nuclear
progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of
progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short
ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in
progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA
expression in mammary tumorigenesis.
Methods: We performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in
breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer
and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected
with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target
genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E.
Reporter gene assays were performed to confirm that cyclin E 3’UTR is a direct target of miR-16.
Results: We found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary
tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as
one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-
16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription
factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E,
contains conserved putative miR-16 target sites in its mRNA 3’ UTR region. We found that, similar to the molecular
mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of
cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce
cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16
also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role
for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG)
downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast
tumor cells.
Conclusions: In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel
role for miR-16 as a tumor suppressor in progestin- and growth factor-induced growth in breast cancer.
* Correspondence: patriciaelizalde@ibyme.conicet.gov.ar
1Laboratory of Molecular Mechanisms of Carcinogenesis, Instituto de Biología
y Medicina Experimental (IBYME), CONICET, Vuelta de Obligado 2490,
C1428ADN Buenos Aires, Argentina
Full list of author information is available at the end of the article
Rivas et al. Breast Cancer Research 2012, 14:R77
http://breast-cancer-research.com/content/14/3/R77
© 2012 Rivas et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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Introduction
Progestins have arisen as important players in breast can-
cer etiology. Compelling experimental and clinical evi-
dence points to a critical role for progesterone and the
nuclear progesterone receptor (PR) in controlling mam-
mary gland tumorigenesis [1-8]. However, the molecular
mechanisms through which progesterone controls breast
cancer growth are not yet fully understood. Multiple
findings have shown that progestins either support sus-
tained in vitro growth of breast cancer cells [2-4,8-11] or
induce cells to progress through one or multiple rounds
of cell division, followed by growth arrest at the G1/S
phase [12]. Consistent with the proliferative role of PR, a
series of G1/S cell cycle phase proteins are induced upon
progestin stimulation of breast cancer cells including
cyclins E and D1, c-Fos, and c-Myc [13,14]. Moreover,
animal models strongly implicate PR in the genesis of
breast cancer. Studies in genetically modified mice
revealed that: 1) a PR knockout mouse shows dramati-
cally reduced susceptibility to carcinogenesis [15], 2) pro-
gesterone increases genomic instability in p53 null
mouse models of breast cancer [16], and 3) treatment of
Brca-1-deficient mice with the anti-progestin mifepris-
tone (RU486) prevented mammary tumorigenesis [17]. In
addition, progestins exert a sustained proliferative
response in vivo in the ER- and PR-positive C4HD model
of mammary carcinogenesis induced by the synthetic
progestin medroxyprogesterone acetate (MPA) in female
BALB/c mice [9,11,18]. Moreover, this effect is fully abro-
gated by antiprogestins [19]. Notably, progesterone was
recently shown to activate adult mammary stem cells
within the mammary stem cell niche during the repro-
ductive cycle, where mammary stem cells are putative
targets for cell transformation events leading to breast
cancer [20]. Finally, clinical observations as well as the
recent extensive, randomized, and controlled Women’s
Health Initiative trial revealed that postmenopausal
women who undergo a combined estrogen and progestin
hormone replacement therapy (HRT) suffer a higher inci-
dence of breast cancer than women who take estrogen
alone [21-23]. Interestingly, the decline in breast cancer
incidence seen during the last years in developed coun-
tries appears to be linked to drops in HRT use [24].
Upon progestin binding, PR translocates to the nucleus
and binds to progesterone response elements (PREs) in
the promoter of target genes. In addition to its direct
transcriptional effects, PR activates signal transduction
pathways through a rapid or nongenomic mechanism
[8,25,26]. Consequently, progestins are broad genome
regulators, acting either directly or indirectly on different
sets of genes. Moreover, PR participates in extensive
crosstalk with many cellular proteins and transcription
factors. Our own findings demonstrated that progestins
induce the transcriptional activation of signal transducer
and activator of transcription 3 (Stat3), which is an abso-
lute requirement for progestin-mediated breast cancer
growth [18].
Micro RNAs (miRNAs) are a recently discovered class
of noncoding endogenous RNAs with regulatory func-
tions. Only in the past few years has the role of miRNAs
in cancer and metastasis been identified [27-29]. Many
papers have shown relationships between miRNA dereg-
ulation and different malignancies since then. In particu-
lar, it has been shown that miRNAs are aberrantly
expressed in breast cancer and that the overall level of
miRNA expression could clearly separate normal versus
cancerous tissues [30]. Most recently, miRNAs were
shown to be involved in different stages of breast cancer
progression and metastasis [31-35].
miR-16 belongs to the miR-15/miR-16 cluster that is
located on the noncoding gene deleted in leukemia 2
(DLEU2) [36]. Validated targets of miR-16 include many
genes related to the control of cell-cycle progression, such
as cyclin D1 [37] and cyclin E [38], among others [39-41].
At present, few works have assessed the relationship
between steroid hormones and miRNAs in breast cancer
[42,43]. miRNA signatures predict ER, PR and human epi-
dermal growth factor receptor 2 (ErbB-2/neu) status in
breast tumors, suggesting that differences in miRNAs are
related to hormone-receptor expression [44]. It has also
been shown that the overexpression of ErbB-2/neu, a
member of the ErbB family of membrane receptor tyrosine
kinases with a major role in breast cancer, causes an
increase in the oncogenic miRNA miR-21, which confers
an aggressive breast cancer phenotype via the downregula-
tion of the metastasis suppressor protein PDCD-4 [32]. In
a recent paper, estradiol was shown to regulate miRNAs,
which control the estradiol response in breast cancer cells
by targeting the oncogene c-Myc and the transcription
factor E2F2 [45]. These authors showed that estradiol
increased the expression of Dicer, the RNase responsible
for releasing mature miRNA, implying that steroid hor-
mones have a profound effect on miRNA regulation in
breast cancer cells [45]. Furthermore, miR-22 has been
shown to inhibit estrogen signaling by directly targeting
estrogen receptor-a mRNA [46]. In addition, a causal rela-
tionship between miR-221 and miR-222 expression and
resistance to the anti-estrogen drug tamoxifen has been
identified in breast cancer [47,48]. As a whole, these find-
ings suggest that steroid-modulated miRNAs are potent
regulators of protein expression and cell fate that act on
multiple levels in breast cancer growth, invasion, metasta-
sis and hormone-therapy resistance.
Despite the undeniable role of progestins in breast
cancer progression, progestin-mediated regulation of
miRNAs has only recently been explored [49]. Bearing
in mind the importance of progestins in breast cancer
growth, we hypothesized that progestins may also
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govern a set of miRNAs that regulate the expression of
genes relevant to breast cancer growth. Our findings
indicate that progestins, acting through the classical PR
and via Stat3 and c-Myc, downregulate miR-16, which is
a potent tumor suppressor in breast cancer. Moreover,
we found that miR-16 is a suppressor not only of pro-
gestin-induced breast cancer growth but also of heregu-
lin (HRG)-induced breast cancer cell proliferation.
Materials and methods
Animals and tumors
Experiments were carried out using female BALB/c mice
raised at the Instituto de Biología y Medicina Experi-
mental (IBYME). Animal studies were conducted as pre-
viously described [18] in accordance with the highest
standards of animal care, as outlined in the US National
Institutes of Health Guide for the Care and Use of
Laboratory Animals [50] and these procedures were
approved by the IBYME Animal Research Committee.
The C4HD tumor line displays high levels of estrogen
receptor (ER) and PR, overexpresses ErbB-2 and ErbB-3,
exhibits low ErbB-4 levels and lacks epidermal growth
factor receptor (EGF-R) expression [2,3,18,51]. This
tumor line does not express glucocorticoid or androgen
receptors. Progestins exert a sustained proliferative
response in vitro and in vivo in the C4HD tumor model
[52].
Reagents
MPA, RU486 and Dulbecco’s modified Eagle’s medium:
Ham’s F12 1:1 (DMEM) were purchased from Sigma-
Aldrich (Saint Louis, MO, USA). Heregulin-b (HRG)
was from R&D Systems (Minneapolis, MN, USA), and
FCS was from Gibco Life Technologies (Carlsbad, CA,
USA).
Antibodies
The following antibodies were used for Western blots:
anti-Stat3 (C-20) and anti-cyclin E (M-20) from Santa
Cruz Biotechnology (Santa Cruz, CA, USA); anti-c-Myc
(D84C12) from Cell Signaling (Beverly, MA, USA); anti-
PR (clone hPRa7) and anti-b-actin (clone ACTN05)
from Neomarkers (Freemont, CA, USA); anti-b-tubulin
from Sigma-Aldrich and HRP-conjugated secondary
antibodies from Vector Laboratories (Burlingame, CA,
USA).
Cell cultures, treatments and proliferation assays
Human breast cancer cell lines T47D and BT-474 were
supplied by the American Type Culture Collection and
maintained in DMEM + 10% FCS and in Roswell Park
Memorial Institute medium (RPMI) + 10% FCS, respec-
tively. Proliferation assays and treatments were
performed in DMEM supplemented with 5% FCS that
had been stripped of steroids by treatment with active
charcoal (ChFCS). T47D-Y cells were a generous gift
from K. Horwitz (University of Colorado Health Sciences
Center, Denver, CO, USA). The tumorigenic BT-474.m1
cell line was kindly provided by D. Yu (The University of
Texas MD Anderson Cancer Center, Houston, TX, USA)
and was maintained in DMEM + 10% FCS.
Primary cultures of epithelial cells from C4HD tumors
were performed as described [18,51]. For the proliferation
assays, 1 × 104C4HD cells/well were plated in 96-well
plates and allowed to attach overnight. Cells were starved
in DMEM supplemented with 0.1% ChFCS. Treatments
were performed in 0.1% ChFCS with 10 nM MPA or the
control vehicle (ethanol 1:1000) for 48 hours. Cell prolif-
eration was evaluated by the incorporation of 1 μCi [3H]-
thymidine during the last 16 hours of incubation (New
England Nuclear, DuPont, Boston, MA, USA; specific
activity 20 Ci/mmol) as previously described [53]. T47D
cell proliferation was assessed after 24 hours of culture.
Assays were performed in octuplicate. The proliferation of
the C4HD cells was also assessed by counting the cells in
the presence of Trypan Blue dye at 48 and 120 hours in
the presence of 10 nM MPA or ethanol.
Western blots
Lysates were prepared from cells subjected to the different
treatments and proteins were resolved by SDS-PAGE as
described previously [51,54,55]. Membranes were immu-
noblotted with the antibodies described in each experi-
ment and filters were reprobed, after stripping, with
antibodies against total b-actin or b-tubulin protein as a
loading control.
siRNA transfections
siRNAs targeting PR, Stat3 and c-Myc were synthesized
by Dharmacon, Inc. (Lafayatte,
CO, USA). The following constructs were used: PR
siRNA #1, 5’-AUAGGCGAGACUACAGACGUU-3’; PR
siRNA #2, 5’-AAGUUCCGGAAACCUGGCAGA-3’; Stat3
siRNA #1, 5’-GGUCAAAUUUCCUGAGUUGUU-3’;
Stat3 siRNA #3, 5’-CCACGUUGGUGUUUCAUAAUU-3’;
c-Myc siRNA #5, 5’-GAAACGACGAGAACAGUUG-3’
and c-Myc siRNA #6, 5’-CCACUCACCAGCACAACUA-
3’. A nonsilencing siRNA oligonucleotide from Dharma-
con that does not target any known mammalian gene was
used as a negative control. Transfection of siRNA duplexes
was performed using the DharmaFECT 1 transfection
reagent according to the manufacturer’s instructions. In
every case, experiments were performed with the two dif-
ferent siRNA sequences for each protein [see Additional
file 1], but the results obtained with only one of them are
presented here.
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miRNA profiling
RNA was extracted from four independent primary cul-
tures of epithelial cell of C4HD tumors, which were treated
with 10 nM MPA or with the control vehicle (ethanol)
using an miRVANA PARIS miRNA isolation kit from
Ambion (Applied Biosystems, Austin, TX, USA) according
to the manufacturer’s instructions. For miRNA profiling,
490 ng total RNA was run on Applied Biosystems Mouse
Low Density qPCR miRNA Array A and B cards. A total of
585 miRNA were surveyed. The cards were read on a
7900HT Fast Real-Time PCR System at the core lab facil-
ities of the Comprehensive Cancer Center of Ohio State
University.
miR-16 and U6 snRNA qPCR
Levels of miR-16 were analyzed by real time quantitative
RT-PCR (RT-qPCR) using a TaqMan®MicroRNA assay
specific for miR-16 of human or mouse origin (Assay ID
00391, miR-16 sequence: 5’-UAGCAGCACGUAAAUAU
UGGCG-3’) according to the manufacturer’s instructions.
Briefly, total RNA was extracted using miRVANA PARIS,
and 100 ng of RNA was retrotranscribed to cDNA using
the aforementioned assay. cDNA was amplified in an
ABI7500 Real Time PCR instrument (Applied Biosystems)
using specific TaqMan primers and TaqMan®Universal
PCR Master Mix, No AmpErase®UNG (both from
Applied Biosystems) to detect miR-16 and U6 snRNA
(Assay ID 01973, U6 snRNA sequence: 5’-GTGCTCGCT
TCGGCAGCACATATACTAAAATTGGAACGATACA-
GAGAAGATTAGCATGGCCCCTGCGCAAGGATGA-
CACGCAAATTCGTGAAGCGTTCCATATTTT-3’). The
latter was used as an endogenous control to correct varia-
tions in the experiment. Only mature miR-16 and U6 are
detected using these assays. ΔΔCt values were used to
assign a fold value, which was calculated as 2-ΔΔCt. All
experiments were done in triplicate.
Real time quantitative RT-PCR (RT-qPCR)
cDNA was amplified by RT-qPCR performed with an ABI
Prism 7500 sequence detector using SYBR green PCR
master mix (Applied Biosystems, Foster City, CA, USA).
Primers with the following sequences were used to amplify
a transcribed region of the cyclin E1 gene: 5’-CACCACT-
GAGTGCTCCAGAA-3’
and
CAGTGGAGA-3’. The full list of primers used to amplify
miR-16 candidate target genes is shown in Additional file
2. qPCR was performed with 15 seconds of denaturing at
95°C followed by 40 amplification cycles of annealing and
extension at 60°C for one minute.
5’-CTGTTGGCTGA-
In silico analysis
A heat map of miRNA expression was built using the
unsupervised clustering function in MultiExperiment
Viewer version 4.4.1 [56,57]. miR-16 targets were searched
using the search engine miRecords [58,59]. To narrow the
list of predicted targets, a filter was applied to show
miRNA target interactions predicted by at least five target-
prediction programs. PREs were located on the DLEU2
promoter gene using MatInspector [60].
Pre-miR transfection
Pre-miR precursors were obtained from Applied Biosys-
tems and were used in accordance with the manufac-
turer’s instructions. Briefly, 6 nM pre-miR-16 or a pre-
miR-control (pre-miR-CTRL) that does not form any
known mammalian miRNA, were transfected using the
transfection reagent siPORT NeoFx (Ambion). After 48
hours, levels of miR-16 were augmented 2,500-fold com-
pared with pre-miR-control-transfected C4HD cells.
Transient transfections and luciferase reporter assay
In several experiments, T47D-Y cells were transiently
transfected using the FuGENE HD transfection reagent
(Roche Diagnostics Corporation, Indianapolis, IN, USA)
with 1 μg of an expression plasmid previously generated
and characterized by Horwitz and co-workers [61]. This
plasmid encodes a PR-B engineered to contain a point
mutation in a conserved cysteine in the first zinc finger
of the DNA-binding domain (C587A-PR) and lacks the
ability to bind to DNA. T47D-Y cells were also trans-
fected with a mutant PR-B (PR-BmPro) engineered to
contain alanines instead of three key prolines (P422A,
P423A, P426A), thus abolishing PR binding to all SH3
domains and inhibiting the activation of c-Src family tyr-
osine kinases [26].
Luciferase constructs were bought from SwitchGear
Genomics (Menlo Park, CA, USA) and contain the wild-
type CCNE1 3’ UTR downstream of the Promega desta-
bilized luciferase reporter gene in the pSGG_3’UTR vec-
tor (pSGG-luc-CCNE1-3’-UTR), or an EMPTY multiple
cloning site (pSGG-luc-EMPTY). Additional constructs
carrying the wild-type CCNE1 3’ UTR, or a minimal
region from the CCNE1 3’-UTR which has a response
site for miR-16 (luc-3’ 1×TS), or the mutated response
site for miR-16 (luc-3’ mTS) were kindly provided by
Dr. Vassella from the Institute for Pathology, University
of Bern, Switzerland [62]. Cells were co-transfected in
12-well plates using FuGENE HD Transfection Reagent
with 250 ng of pSGG-luc-CCNE1-3’-UTR or pSGG-luc-
EMPTY firefly luciferase reporter vector + 10 ng of
pRL-CMV, which encodes Renilla luciferase. After 48
hours, cells were re-transfected with either pre-miR-16
or pre-miR-CTRL following the protocol described
above. Luciferase activity was measured 24 hours later
(Dual Luciferase Reporter, Promega, Madison, WI, USA)
using Renilla luciferase for normalization.
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Chromatin immunoprecipitation (ChIP) assays
ChIP was performed as described elsewhere previously
[55,63], with minor modifications. Briefly, chromatin was
sonicated to an average of about 500 bp. Sonicated chro-
matin was then immunoprecipitated by using 4 μg of the
following antibodies: Anti-trimethyl-Histone H3 (Lys9)
and anti-Acetyl H4 (both from Millipore, Billerica, MA,
USA), anti-c-Myc (N-262, Santa Cruz Biotechnology) or
an irrelevant IgG as control. The immunoprecipitate was
collected by using protein A beads (Millipore, Temecula,
CA, USA), which were washed repeatedly to remove non-
specific DNA binding. Chromatin was eluted from beads,
and crosslinks were removed overnight at 65°C. DNA was
then purified using the QIAquick PCR Purification Kit
(QIAgen, Valencia, CA, USA) and quantified by real-time
PCR. The following pair of primers was used: 5’-ACGG-
CAAAAGCTCTACAAGC-3’ and 5’-GGGTCCTGCTTA
GGAGAAAA-3’ that amplify a genomic region encom-
passing the E-box located just upstream of Dleu2 exon 1A.
In vivo tumor growth
C4HD cells (2 × 106cells per mouse) were transiently
transfected with the precursor of miR-16 (pre-miR-16) or
with a control precursor (pre-miR-CTRL) and were then
injected s.c. into animals treated with a 40 mg MPA depot
in the opposite flank to the cell inoculum. Tumor volume,
growth rate and growth delay were determined as
described previously [18]. At specific times, tumor
volumes in the different groups were compared using ana-
lysis of variance (ANOVA) followed by Tukey’s test. A lin-
ear regression analysis was performed on tumor growth
curves and slopes were compared using ANOVA followed
by a parallelism test to assess the statistical significance of
the observed differences.
BT-474.m1 cells (20 × 106cells per mouse) were
injected s.c. in NIH(S)-nude mice obtained from La Plata
University (Argentina) concomitantly with a contralateral
0.72 mg estradiol pellet. After seven days tumor bearing
mice (n = 12) were administered or not a MPA depot
[64,65]. A week later, the tumors were excised and total
RNA and proteins were prepared for subsequent analysis.
Immunohistochemistry
Formalin-fixed paraffin-embedded C4HD tumors were cut
with a microtome in 10 μm sections. Antigen retrieval was
performed in 10 mM sodium citrate buffer pH 6 for 20
minutes at 96° to 98°C. Slides were incubated with primary
antibodies anti cyclin E (M-20) (Santa Cruz Biotechnology,
dilution 1:100 overnight at 4°C) or were incubated with
control rabbit immunoglobulin G (IgG). Sections were
subsequently incubated with the polydetector HRP system
(Bio SB, Santa Barbara, CA, USA) and developed in 3-3’-
diaminobenzidine tetrahydrochloride. Immunostainings
were run with known positive and negative tumor controls
and were blindly evaluated by a pathologist who ignored
sample identity. Cyclin E expression was quantitated
through the H-index, which was calculated as (% of tumor
cells weakly stained) + (% of tumor cells moderately
stained × 2) + (% of tumor cells strongly stained × 3) [66].
Statistical analysis
For the proliferation assays and cell counts, differences
between control and experimental groups were analyzed
by ANOVA followed by Tukey’s test.
Results
Progestins modulate miRNAs in breast cancer cells
To identify progestin-regulated miRNAs in breast cancer,
we performed miRNA array profiling of breast cancer cells
that were treated with the synthetic progestin MPA or left
untreated. We used primary cultures of C4HD epithelial
cells from the MPA-induced model of mammary carcino-
genesis in female BALB/c mice. In these cells, progestins
induce a potent and sustained mitogenic response [52].
Here, C4HD cells were treated for 24 hours with 10 nM
MPA or with the control vehicle (ethanol) and total RNA
was extracted. Out of the 585 mouse miRNA assayed, 350
were expressed in at least one of the two conditions [see
Additional file 3]. The comparison between control- and
MPA-treated cells revealed that 16 miRNAs were signifi-
cantly modulated by more than two-fold (P < 0.05, Figure
1A), nine miRNAs were upregulated (miR-191*, miR-17*,
miR- 470*, miR-451, miR-702, miR-434-3p, miR-493,
miR-23a* and miR-485*) and seven were downregulated
(miR-378*, miR-376a, miR-224, miR-190b, miR-16, miR-
410 and miR-197) (Figure 1B). Among the differentially
expressed miRNAs, we were particularly interested in
miR-16, a previously reported tumor suppressor in leuke-
mia [67,68], which was downregulated by treatment with
MPA.
Progestins downregulate miR-16 via the classical PR and
a hierarchical interplay between Stat3 and c-Myc
To characterize the modulation of miR-16 by MPA, we
performed a time-course experiment. RNA from C4HD
cells treated for 0 to 24 hours was reverse transcribed and
analyzed by RT-qPCR to detect the presence of miR-16.
We observed that miR-16 levels were decreased compared
with control levels (Figure 2A) as early as six hours after
MPA treatment and they remained low until at least 24
hours. A similar time course was observed for the human
T47D breast cancer cell line (Figure 2A). Treatment of
C4HD cells with the anti-progestin RU486 or silencing of
PR expression using siRNAs overcame the MPA-induced
miR-16 downregulation, indicating that this effect is
mediated through the classical PR (Figure 2B). No modu-
lation of miR-16 levels was observed following the sole
addition of RU486 or after knockdown of PR expression in
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unstimulated cells (Figure 2B). These results suggest that
miR-16 is regulated as part of the ligand-induced PR
effects observed in breast cancer, but would not be
involved in PR modulation of breast cancer growth in the
absence of the ligand.
In addition, MPA was unable to modulate miR-16 in
the T47D-Y cell line, a variant of the parental T47D cell
line that lacks PR expression (Figure 2C). Reconstitution
of PR-B levels in T47D-Y cells [55] restored MPA capa-
city to downregulate miR-16 (Figure 2C). In order to
explore whether rapid signaling through PR and/or geno-
mic effects participate in the MPA-downregulation of
miR-16, we transfected T47D-Y cells with a PR mutant,
PR-BmPro, in which three prolines (P422A, P423A,
P427A) were converted to alanines (T47D-Y-PR-BmPro
cells). Previous studies have defined the proline-rich
domain of human PR as an absolute requirement for the
interaction between progestins and c-Src [25,26] and the
consequent rapid activation of signaling cascades [8,26].
Our present findings demonstrated that at least from six
hours (Figure 2C) to 24 hours (data not shown) later,
miR-16 levels were not regulated in response to MPA in
T47D-Y-PR-BmPro cells. In addition, we restored the
expression in T47D-Y cells of a PR-B mutant engineered
to contain a point mutation in a conserved cysteine in
the first zinc finger of the DNA binding domain (C587A),
causing it to be transcriptionally crippled. Consistent
with pioneering works [61], our own findings demon-
strated that the C587A-PR mutant is also unable to parti-
cipate in nonclassical PR tethering transcriptional
mechanisms [55]. As shown in Figure 2C, MPA had no
effect on miR-16 levels in T47D-Y-C587A-PR cells.
These findings demonstrate the participation of both
rapid (nongenomic) and transcriptional PR effects in pro-
gestin-induced miR-16 downregulation. Moreover, we
found an inverse relationship between the levels of miR-
16 and the proliferative state of C4HD and T47D cells.
As shown in Figure 2D, MPA induces a strong prolifera-
tive response in both cell lines, which correlates with its
ability to induce the downregulation of miR-16 levels
Figure 1 Progestins modulate miRNAs expression in breast cancer cells. A, Heat map depicting the expression profile of miRNA genes with
changes ≥2 fold after 24 hours of MPA treatment. Total RNA was extracted from primary cultures of C4HD cells treated with 10 nM MPA or left
untreated for 24 hours and used for miRNA profiling with the Applied Biosystems Mouse Low Density qPCR miRNA Array (n = 4). The small
nuclear RNA U6 (U6 snRNA) was used as an endogenous control to normalize the results. B, Average fold changes of miRNAs significantly
modulated by MPA (P < 0.05, n = 4) in primary cultures of C4HD cells. Each graph depicts the fold change of a specific miRNA in C4HD cells
treated with MPA or left untreated; all values were normalized to U6 snRNA. The data shown represent the means of three independent
experiments ± SEM (P < 0.01 for b versus a). MPA, medroxyprogesterone acetate; SEM, standard error of the mean.
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Figure 2 Progestins induce miR-16 downregulation via the classical PR. A, C4HD and T47D cells were treated with 10 nM MPA for the
times shown. B, C4HD cells were either pretreated with 10 nM RU486 (left panel) or transfected with 100 nM PR and control (CTRL) siRNAs, and
were then stimulated with MPA for 24 hours or left untreated (middle panel). The western blot (WB) in the right panel shows the effect of
siRNAs on PR expression in C4HD cells. The experiment shown was performed with PR siRNA #1, but the same results were obtained with PR
siRNA #2. C, T47D and T47D-Y cells were treated with MPA for the indicated time or T47D-Y cells were transiently transfected with the PR-B
isoform (T47D-Y-PR-B), PR-BmPro mutant (T47D-Y-PR-BmPro cells) or the C587A-PR mutant (T47D-YC587A-PR cells) before MPA stimulation. In A
to C, miR-16 expression levels were determined by RT-qPCR. The fold change of miR-16 expression levels upon MPA treatment was calculated
by normalizing the absolute levels of miR-16 to those of U6 snRNA, which was used as internal control, and setting the value of untreated cells
to 1. D, C4HD cells were treated with 10 nM MPA for 48 hours (left panel) or T47D cells were treated with 10 nM MPA for 24 hours (right panel)
and the incorporation of [3H]-thymidine was used as a measure of DNA synthesis. The middle panel shows cell counts for C4HD cells that were
treated with 10 nM MPA for 48 hours and then stained with Trypan blue dye. Experiments shown in A to D were repeated in triplicate with
similar results. The data shown represent the means of three independent experiments ± SEM (P < 0.001 for b versus a and c versus b). MPA,
medroxyprogesterone acetate; PR, progesterone receptor; SEM, standard error of the mean.
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(Figure 2A). These findings constitute the first piece of
evidence to suggest a role for miR-16 as a tumor suppres-
sor in progestin-induced breast cancer growth.
To explore the upstream effectors involved in the MPA-
mediated downregulation of miR-16, we first conducted
an in silico analysis. We did not find canonical or half
PREs at the proximal promoter of DLEU, the miR-16 host
gene. This paucity indicates that miR-16 downregulation
is most likely driven not by PR loading at the DLEU2 pro-
moter, but rather by nonclassical PR tethering mechan-
isms. Our literature and database searches also revealed
that the DLEU2 gene promoter contains two well-con-
served E-box sites (CACGTG elements) which function as
response elements for the oncogenic transcription factor
c-Myc [36]. Therefore, we hypothesized that c-Myc, long
known to be an immediate early gene for several prolifera-
tive signal cascades and whose induction by PR is well
acknowledged [13,69,70], may also be an upstream effector
in MPA-induced miR-16 downregulation. In accordance
with previous findings in T47D cells [13,69], we found
that MPA treatment also induced a significant increase in
c-Myc protein levels after 24 hours of treatment in C4HD
cells; this effect was abrogated by the knockdown of PR
expression (Figure 3A). To explore the direct involvement
of c-Myc in the molecular mechanism of MPA-induced
miR-16 downregulation, we silenced c-Myc expression
using siRNAs. Figure 3B (left panel) shows that knock-
down of c-Myc resulted in the inhibition of MPA-induced
effects on miR-16 expression.
Our previous studies of PR function demonstrated that
Stat3 is a key mediator of progestin effects in breast cancer
[18,71]. We found that PR induces Stat3 transcriptional
activation via a nongenomic action. In addition, Stat3 acti-
vated in response to progestins is in turn directly involved
in nonclassical PR transcriptional mechanisms [55,71].
Progestin-mediated modulation of miR-16 expression
requires an intact PR-signaling function (Figure 2C, PR-B-
mPro-transfected T47D-Y cells), the same as Stat3, and
also appears to be modulated by PR-mediated transcrip-
tional tethering mechanisms (Figure 2C, C587A-PR-trans-
fected T47D-Y cells). Because Stat3 was found to be
directly involved [55] in these mechanisms, we reasoned
that Stat3 may constitute an interesting gene whose parti-
cipation in progestin-mediated miR-16 expression was
worth studying. Our present findings showed that indeed
the knockdown of Stat3 expression resulted in a complete
abrogation of MPA-induced miR-16 downregulation
(Figure 3C). Stat3 function as an upstream activator of c-
Myc in breast cancer has already been shown [72]. Here,
we found that silencing Stat3 strongly impaired MPA-
induced c-Myc upregulation (Figure 3D) in C4HD and
T47D cells, for the first time demonstrating that Stat3
mediates the effects of progestin on c-Myc expression. We
have previously revealed that MPA induces Stat3 expres-
sion in C4HD cells [18]. Here we found that knockdown
of c-Myc expression had no effect on MPA modulation of
Stat3 protein levels (data not shown). Our findings show
that progestins downregulate miR-16 via the classical PR
and a hierarchical interplay between Stat3 and c-Myc.
In order to further elucidate the mechanism of c-Myc
induced miR-16 downregulation by MPA, we conducted
chromatin immunoprecipitation assays (ChIP) on the
DLEU2 promoter. Interestingly, the addition of MPA
induced a two-fold increase in the recruitment of c-Myc
to the E-boxes in the DLEU2 proximal promoter (Figure
3E, left panel). This result is in concordance with the ones
reported by Chang et al. [73] in which c-Myc was shown
to be recruited to E-boxes on the DLEU2 promoter. In
line with a role for c-Myc as a repressor of miR-16, the
addition of MPA caused a significant decrease of the levels
of acetylation of histone H4 (AcH4), a chromatin modifi-
cation already reported to be an activation mark for the
DLEU2 locus [74] (Figure 3E, middle panel). Furthermore,
we observed an increase in the levels of trimethylation of
the lysine 9 in histone H3 (H3K9me3), a classical chroma-
tin repressive mark (Figure 3E, right panel). The above
results support a role for MPA as a repressor of miR-16
expression via c-Myc, inducing the recruitment of proteins
with activity of chromatin remodelers which modulate
gene expression.
Pro-tumor effects of miR-16 downregulation in breast
cancer are mediated by cyclin E
A variety of targets for miR-16 has already been reported,
including the cell-cycle promoter cyclin D1 and the anti-
apoptotic protein Bcl-2 [37]. Our own recent findings
demonstrated that MPA induces the expression of cyclin
D1 in C4HD cells via the assembly of a transcriptional
complex between Stat3, ErbB-2 and PR, in which ErbB-2
acts as a Stat3 co-activator [55]. In the current study, we
found that transfection with a precursor of miR-16 (pre-
miR-16) resulted in the inhibition of MPA-induced cyclin
D1 expression in C4HD cells, indicating that cyclin D1 is
also a downstream target of miR-16 in breast tumor cells
(Figure 4A, upper panel). As shown in Figure 4A (bottom
panel), cells were efficiently transfected, reaching approxi-
mately 2,500-fold greater expression compared with
C4HD cells transfected with a pre-miR-control.
To predict novel targets for miR-16, we used miRecords,
a publicly available miRNA target prediction tool that inte-
grates the predicted targets of the most commonly used
search engines. To increase the stringency of the target
prediction protocol, we searched for mRNAs simulta-
neously predicted by five or more different target-predic-
tion programs. From a list of approximately 112 predicted
interactions with mRNAs, we chose 12 with a suspected
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role in cancer development and progression based on the
literature [See Additional file 2]. We performed RT-qPCR
to amplify those mRNAs (Figure 4B) and, interestingly, we
observed that CCNE1 mRNA, encoding the cell cycle reg-
ulator cyclin E, and RAP2C mRNA, encoding a member
of the RAS oncogene family [75], showed a profile in
response to MPA that mimicked MPA-induced prolifera-
tion; these mRNAs were also regulated inversely from
miR-16. We chose to explore the regulation of CCNE1
mRNA by miR-16 due to its acknowledged role in breast
Figure 3 Progestin induces miR-16 downregulation via c-Myc and Stat3. A, C4HD cells were treated with MPA or transfected with PR
siRNAs or CTRL siRNAs before MPA stimulation. Western blot (WB) was performed with anti-c-Myc or anti-PR antibodies and filters were
reprobed with an anti-b-actin antibody. The experiment shown was performed with PR siRNA #1, but the same results were obtained with PR
siRNA #2. B, C4HD cells were transfected with c-Myc and CTRL siRNAs and were then treated with MPA. miR-16 levels were studied by RT-qPCR,
and data analysis was performed as described in Figure 2. The WB in the right side of the figure shows the effects of siRNAs on c-Myc
expression in C4HD cells. The experiment shown was performed with c-Myc siRNA #5, but the same results were obtained with c-Myc siRNA #6.
C, C4HD cells were transfected with Stat3 siRNAs or CTRL siRNAs and then treated with MPA for 24 hours. miR-16 levels were studied by RT-
qPCR, and data analysis was performed as described in Figure 2. The experiment shown was performed with Stat3 siRNA #1, but the same
results were obtained with PR siRNA #3. D, C4HD and T47D cells were transfected with Stat3 siRNAs or CTRL siRNAs before MPA stimulation and
WBs were performed with anti-c-Myc antibodies. Filters were reprobed with an anti-b-tubulin antibody. The experiment shown was performed
with Stat3 siRNA #1, but the same results were obtained with PR siRNA #3. E, Recruitment of c-Myc, and H4 acetylation (AcH4) or trimethylation
of lysine 9 histone H3 (H3K9me3) levels at the promoter of the DLEU2 gene was studied by ChIP. Amounts of immunoprecipitated DNA were
normalized to inputs and are reported relative to the untreated control group, which was set to 1 (P < 0.001 for b versus a). Experiments shown
in A to E were repeated in triplicate with similar results. Data shown represent the means of three independent experiments ± SEM (P < 0.001
for b versus a and c versus b). ChIP, chromatin immunoprecipitation; MPA, medroxyprogesterone acetate; SEM, standard error of the mean.
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Figure 4 Cyclin E is a target of miR-16 in breast cancer cells. A, Upper panel, C4HD cells were transfected with pre-miR-16 or pre-miR-
control (CTRL) before MPA stimulation. WB was performed with an anti-cyclin D1 antibody, and filters were reprobed with an anti-b-tubulin
antibody. Bottom panel, as a control of transfection efficiency, miR-16 levels are shown in pre-miR-16-C4HD and pre-miR-CTRL-C4HD cells. B,
C4HD cells were treated with MPA or pretreated with 10 nM RU486 before MPA stimulation, and mRNA expression levels of candidate miR-16
target genes were determined by RT-qPCR. The fold change of mRNA expression levels was calculated by normalizing the absolute levels of the
gene-of-interest (GOI) mRNA to GAPDH levels, which were used as an internal control, and setting the value of untreated cells to 1. RAP2C,
member of RAS oncogene family; RAF1, v-raf-1 murine leukemia viral oncogene homolog 1; CCNT2, cyclin T2; TCFAP2D, transcription factor AP-2
delta; BCL2L2, BCL2-like 2; CCNE1, cyclin E; TRAF3, TNF receptor-associated factor 3; Akt3, v-akt murine thymoma viral oncogene homolog 3;
DMTF1, cyclin D binding myb-like transcription factor 1; WNT3A, wingless-type MMTV integration site family, member 3A; RREB1, ras responsive
element binding protein 1; BDNF, brain-derived neurotrophic factor. C, C4HD cells were transfected with PR and CTRL siRNAs and then treated
with MPA for 24 hours. Left panel, cyclin E mRNA levels were studied by RT-qPCR and data analysis was performed as described in Figure 2B.
Right panel, WB was performed with an anti-cyclin E antibody and filters were reprobed with an anti-b-actin antibody. Longer exposures
showing the expression of the low molecular weight (LMW) cyclin E isoforms are shown in the middle panel. The experiment shown was
performed with PR siRNA #1, but the same results were obtained with PR siRNA #2. D, C4HD cells were transfected with Stat3 or CTRL siRNAs
and were then treated with MPA or remained untreated. WB was performed as described in C. As a control for siRNA efficiency, the membranes
were probed with an anti-Stat3 antibody. The experiment shown was performed with Stat3 siRNA #1, but the same results were obtained with
PR siRNA #3. E, C4HD cells were transfected with c-Myc or CTRL siRNAs and then treated with MPA. WB was performed as in Figure 4C. The
experiment shown was performed with c-Myc siRNA #5, but the same results were obtained with c-Myc siRNA #6. F, C4HD cells were
transfected with pre-miR-16 or pre-miR-CTRL before MPA stimulation and WB was performed as in C. G, A scheme depicting the different
constructions used is shown in the left panel. C4HD cells were transfected with a construct carrying the CCNE1 3’ UTR cloned downstream of
the firefly luciferase reporter gene (luc-3’CCNE1), middle panel, or with a construct that carried a minimal region of CCNE1 3’UTR which
comprised only one of the miR-16 responding sites either wild type (luc-3’ 1×TS) or mutated (luc-3’ mTS), right panel. As a control, cells were
transfected with a construct that lacks the 3’ UTR cloned downstream of the luciferase gene (luc-3’EMPTY). Cells were co-transfected with pre-
miR-16 or pre-miR-CTRL (middle panel) or treated with 10 nM MPA for 24 hours (right panel). Firefly luciferase activity was measured as
described in the Methods. Renilla luciferase was used for normalization. The experiments shown in A to G were repeated in triplicate with similar
results. The data shown represent the means of three independent experiments ± SEM (P < 0.001 for b versus a and c versus b). MPA,
medroxyprogesterone acetate; PR, progesterone receptor; SEM, standard error of the mean; WB, western blot.
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cancer [76]. CCNE1 mRNA contains two highly conserved
target sites for miR-16, one at position 485-491 and the
other at position 241-247 of its 3’ UTR. Interestingly,
human and mouse CCNE1 mRNA share these target sites
which suggests the importance of their preservation in
gene regulation. As we already reported for C4HD cells,
western blot analysis revealed the presence of the full-
length, 52-kDa cyclin E isoform and a variable number of
low-molecular-weight isoforms ranging in size from 35 to
50 kDa [4]. We found that MPA treatment for 24 hours
induced a significant increase in CCNE1 mRNA and the
expression of all protein isoforms in these cells (Figure
4C). Thus, we reasoned that cyclin E might be a true tar-
get of miR-16 in breast cancer cells. Consistent with the
role of PR, Stat3 and c-Myc as upstream regulators of
miR-16, the MPA-induced cyclin E increase was blocked
by the silencing of PR (Figure 4C), Stat3 (Figure 4D) or c-
Myc using siRNAs (Figure 4E). To validate cyclin E as a
target of miR-16 action in breast cancer, we transiently
transfected primary cultures of C4HD cells with pre-miR-
16. As shown in Figure 4F, MPA did not induce cyclin E
upregulation in miR-16-overexpressing C4HD cells, indi-
cating that CCNE1 mRNA is indeed a direct target of
miR-16. For further demonstration, we transfected C4HD
cells with a construct carrying the 3’ UTR of CCNE1
downstream from the luciferase gene (luc-3’CCNE1) or
with a luciferase reporter gene which lacks the CCNE1 3’
UTR (luc-3’EMPTY). Transfection of pre-miR-16 for 24
hours greatly decreased luciferase activity in the luc-
3’CCNE1-transfected cells compared with the pre-miR-
control transfected cells (Figure 4G, middle panel). Neither
pre-miR-CTRL nor pre-miR-16 modified luciferase activity
in the luc-3’EMPTY cells. In addition, we studied miR-16
regulation of cyclin E levels in a system in which miR-16
was not being transfected but modulated endogenously by
the presence of MPA. In addition to the constructs
described above, we used a construct in which only a
minimal region of the CCNE1 3’ UTR encompassing a
miR-16 responding site was included (luc-3’ 1×TS) and
another in which the same site was mutated (luc-3’ mTS,
Figure 4G, left panel) [62]. Treatment with MPA of luc-3’
CCNE1-transfected C4HD cells resulted in a significant
increase of luciferase activity, in line with our hypothesis
that miR-16 is a negative regulator of cyclin E (Figure 4G,
right panel). In contrast, no modulation of the reporter
activity was observed when C4HD cells were transfected
with the luc-3’ mTS and treated with MPA (Figure 4G,
right panel). Noticeably, although not responsive to the
endogenous changes of miR-16 levels, a higher basal luci-
ferase activity was observed for the luc-3’ mTS construct
as compared to luc-3’ 1 xTS or CCNE1-3’UTR, adding
further evidence for a negative role of miR-16 response
sites on cyclin E expression.
miR-16 acts as a tumor suppressor in both in vivo and in
vitro progestin-induced breast cancer growth
To test the ability of miR-16 to counteract MPA-induced
proliferation, C4HD and T47D cells were transfected
with pre-miR-16 or pre-miR-CTRL, and proliferation
assays were performed by measuring [3H]-thymidine
uptake (Figure 5A) and by counting viable C4HD cells at
48 and 120 hours (Figure 5B). As shown in Figures 5A
and 5B, transfection with pre-miR-16 significantly inhib-
ited MPA-induced proliferation in C4HD and T47D
cells. The results presented here indicate for the first
time a role for miR-16 as a tumor suppressor in breast
cancer.
We next conducted a preclinical trial to test the role of
miR-16 in the MPA-induced growth of C4HD tumors in
vivo. For this purpose, C4HD cells were transfected with
pre-miR-CTRL (pre-miR-CTRL-C4HD) or pre-miR-16
(pre-miR-16-C4HD) cells and 2 × 106cells were injected
subcutaneously (s.c.) into mice treated with MPA. One
representative experiment of the two performed is
described here. Mice (n = 6) injected with pre-miR-CTRL-
C4HD cells developed tumors that became palpable 12
days after inoculation. All six mice injected with pre-miR-
16-C4HD cells also developed tumors, albeit with five days
of tumor latency, compared with the control group. The
mean volume (Figure 5C) and growth rates (Table 1) of
the tumors developed from the pre-miR-16-C4HD cells
were significantly lower than those of the tumors from the
control group. miR-16 levels in pre-miR-16-C4HD were
augmented two-fold at day 14, in comparison to pre-miR-
CTRL-C4HD tumors (Figure 5C, inset). Immunohisto-
chemistry for cyclin E revealed that pre-miR-CTRL-C4HD
tumors displayed strong, mainly cytoplasmic staining for
cyclin E (Figure 5D, central column and inset, H-index:
153 ± 33). In contrast, pre-miR-16-C4HD tumors stained
weakly for cyclin E (Figure 5D, right column and inset,
H-index: 61 ± 32), showing miR-16 efficiency in vivo nega-
tively regulated cyclin E.
To extend our results to a different experimental
model, we took advantage of the human BT-474.m1
breast cancer cell line which displays moderate levels of
ER and PR and overexpresses the receptor tyrosine
kinase ErbB-2, and which forms tumors in female nude
mice. Published findings demonstrated that MPA pro-
motes in vivo BT-474 tumor growth [64,65]. Therefore,
20 × 106BT-474.m1 cells were injected s.c. in nude
mice and after seven days tumor-bearing mice (n = 12)
were administered or not a MPA depot. As expected
[64], addition of MPA rescued the growth of BT-474
tumor (Figure 5E). After one week, tumors were excised
and studied for miR-16, c-Myc and cyclin E levels.
Interestingly, tumors growing in the presence of MPA
displayed lower levels of miR-16, which correlated with
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120h
0
0.5
1.0
1.5
48 h
0
0.3
0.6
0.9
Cell count (x 105)
--
++
pre-miR-CTRLpre-miR-16
--
++
pre-miR-CTRL pre-miR-16
0
1000
2000
3000
[3H]-Thymidine
uptake (CPM)
MPA 10 nM (48h)
--
++
pre-miR-CTRLpre-miR-16
0
1000
3000
[3H]-Thymidine
uptake (CPM)
MPA 10 nM (24h)
--
++
pre-miR-CTRLpre-miR-16
A
B
C4HD T47D
C4HD
b
a
c
b
a
c
b
a
c
b
a
c
MPA 10 nM
0
0.2
0.4
0.6
0.8
1.0
1.2
BT-474
vehicle
BT-474
+ MPA
a
b
miR-16 / U6snRNA
(Fold change)
0
0.5
1.0
1.5
2.0
Cyclin E / β-tubulin
(Fold change)
BT-474
vehicle
BT-474
+ MPA
a
b
c-Myc
β-tubulin
BT-474
vehicle
BT-474
+ MPA
F
E
010 203040
0
100
200
300
400
500
600
700
E2 + MPA
E2
Tumor volume (mm3)
Time (days)
<
***
**
****
**
0
10 2030
0
300
600
900
pre-miR-CTRL-C4HD
pre-miR-16-C4HD
Time (days)
Tumor volume (mm3)
*******
C
0
1
2
3
miR-16 / U6snRNA
(Fold change)
pre-miR-CTRL
C4HD
pre-miR-16
C4HD
Day 14
pre-miR-CTRL-C4HD pre-miR-16-C4HD
IHC: Cyclin EIsotype control
D
0
50
100
150
200
H-score
cyclin E
CTRL miR-16
pre-miR-CTRL-C4HD
Figure 5 miR-16 is a tumor suppressor in progestin-induced breast cancer growth in vitro and in vivo. A, miR-16 inhibits in vitro
progestin-induced breast cancer growth. C4HD or T47D cells were transfected with pre-miR-CTRL or pre-miR-16. After 48 hours of transfection,
cells were treated with 10 nM MPA or left untreated, and proliferation was measured by [3H]-thymidine incorporation as described in Figure 2D.
B, C4HD cells were transfected with pre-miR-CTRL or pre-miR-16. After 48 hours, cells were treated with 10 nM MPA for 48 or 120 hours or left
untreated, and proliferation was measured by cell count as described in Figure 2D. The experiments shown in A and B were repeated four times
with similar results. The data shown represent the means of the data from three independent experiments ± SEM (P < 0.001 for b versus a and
c versus b). C, miR-16 inhibits in vivo progestin-induced breast cancer growth. C4HD cells were transfected with pre-miR-CTRL or pre-miR-16 for
48 hours and then injected subcutaneously (s.c.) into BALB/c mice at 2 × 106cells/mouse. Mice were simultaneously injected with a 40 mg MPA
depot. Tumor volume was calculated as described in the Methods. Each point represents the mean volume ± SEM of six independent tumors
for both experimental groups. The experiment shown in C was repeated twice with similar results. *P < 0.01 or **P < 0.001 versus control. Inset,
levels of pre-miR-16 in pre-miR-CTRL-C4HD and pre-miR16-C4HD tumors were studied by RT-qPCR at day 14; data analysis was performed as
described in Figure 2. D, Cyclin E is an in vivo target of miR-16. Immunohistochemistry (IHC) for cyclin E in pre-miR-CTRL-C4HD and pre-miR-16-
C4HD tumors (400×). Representative images are shown. As control, IHC was performed using an irrelevant rabbit antibody. Scale bar, 50 μM.
Inset, average H-score, used to quantify the levels of cyclin E in pre-miR-CTRL-C4HD and pre-miR-16-C4HD tumors. E, BT-474.m1 cells were
injected s.c. into nude mice at 20 × 106cells/mouse. Mice were simultaneously injected with a 0.72 mg E2depot. Seven days after cell injection,
half of the mice were injected with a 40 mg MPA depot (arrow). Tumor volume was calculated as described in Methods. Each point represents
the mean volume ± SEM of six independent tumors for both experimental groups. *P < 0.01 or **P < 0.001 versus control. F, c-Myc WB was
performed in whole protein extracts from BT-474 tumors growing into mice treated or not with MPA (upper panel). WB from two representative
animals from each group is shown. miR-16 levels were measured in RNA from BT-474 tumors from mice treated or not with MPA (lower-left
panel). Quantification of cyclin E from WB performed on whole protein extracts from BT-474 tumors from mice treated or not with MPA (lower-
right panel). MPA, medroxyprogesterone acetate; SEM, standard error of the mean; WB, western blot.
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higher levels of both c-Myc and cyclin E (Figure 5F).
This result highlights the importance of miR-16 in pro-
gestin-promoted human breast cancer growth in vivo.
The role of miR-16 as a tumor suppressor in HRG-induced
breast cancer growth
To generalize our discovery of the role of miR-16 as a
tumor suppressor, we decided to explore its involvement
in the proliferation of breast cancer induced by growth
factors, which along with estradiol and progestin are the
major mitogens in breast cancer. In the first place, we con-
firmed that also in BT-474, MPA induced an increase in in
vitro cell proliferation at 24 hours of treatment (Figure 6A)
and that such increase correlated with a decrease in the
expression levels of miR-16 (Figure 6B). As demonstrated
for C4HD and T47D cells, the miR-16 decrease was pre-
ceded by the upregulation of c-Myc oncogene (Figure 6C,
left panel) and was coincident with an increase in cyclin E
expression levels (Figure 6C, right panel).
In particular, we chose to study proliferation modu-
lated by HRG, a ligand for the ErbB family of receptor
tyrosine kinases (ErbB-1, ErbB-2, ErbB-3 and ErbB-4).
HRG binds ErbB-3 and ErbB-4 and recognizes EGF-R
and ErbB-2 as co-receptors [77]. The roles of HRG and
ErbBs in breast cancer, particularly ErbB-2, are well
acknowledged [78-80]. As shown in Figure 6D, we found
that HRG treatment induced a significant downregula-
tion of miR-16 expression in BT-474 cells. This decrease
in miR-16 levels, from at least 16 to 24 hours after treat-
ment, correlated inversely with the proliferative effects of
HRG at this time point (Figure 6E, left panel). Further-
more, overexpression of miR-16 resulted in significant
inhibition of HRG-induced stimulation of BT-474 cell
growth (Figure 6E, left panel).
Our previous findings demonstrated that HRG induces a
strong Stat3 activation in breast cancer cells and, notably,
that this effect is mediated via HRG co-option of PR func-
tion as a signaling molecule [53]. We here found that
comparable to MPA, HRG also induced c-Myc upregula-
tion, in a time dependent manner (Figure 6F). To explore
whether HRG regulation of miR-16 was mediated via c-
Myc and Stat3 expression, we silenced the expression of
both proteins by the use of siRNAs and found that, com-
parable to our findings with MPA, silencing of Stat3 or of
c-Myc both impair HRG-induced miR-16 downregulation
in BT-474 cells (Figure 6G), suggesting that HRG and
MPA share some of the signaling molecules in the
mechanism of downregulation of miR-16.
Discussion
Our present results indicate that the synthetic progestin
MPA, a potent mitogen in C4HD and T47D cells, regu-
lates a subset of miRNAs in mammary tumor cells
including the tumor suppressor miR-16, which is down-
regulated. Progestins downregulate miR-16 in breast can-
cer cells via the classical PR and a hierarchical interplay
between Stat3 and c-Myc. We showed, for the first time,
that miR-16 is involved in progestin-induced tumor
growth in vitro and in vivo, having the cell-cycle promo-
ter cyclin E as a target. Remarkably, we demonstrated
that miR-16 is significantly downregulated by MPA treat-
ment in an in vivo setting. These results indicate a novel
mechanism of progestin-induced breast cancer growth
that has the potential to modulate a wide array of genes.
Interestingly, we also demonstrated the involvement of
miR-16 in HRG-induced breast cancer cell proliferation,
confirming the ability of miR-16 to act as a tumor sup-
pressor during breast cancer cell proliferation.
The capacity of steroid hormones to modulate miRNAs
has already been described in breast cancer, mainly in
estrogen-induced models [42,43]. The regulation of miR-
NAs by progesterone has been examined quite intensively
in the uterus [81,82]. In a study by Kuokkanen et al., a set
of 12 miRNAs were upregulated during the midsecretory
phase compared with late proliferative endometrium sam-
ples [81]. In accordance with the opposite proliferation
roles of progestins in the uterus and the mammary gland,
none of the miRNAs that we found to be regulated by pro-
gestins in our study were also reported to be regulated in
the uterus, revealing that the proliferative input of a given
hormone directs miRNA modulation. Another study com-
pared miRNA expression in leiomyomas, a benign tumor
of the smooth muscle cells of the uterus, with paired
normal myometrium cells. Among the downregulated
miRNAs in leiomyomas, the authors identified miR-16,
miR-197 and miR-224, three miRNAs we observed to be
downregulated in progestin-induced breast cancer [82],
highlighting the importance of miR-16 as a tumor sup-
pressor in different cellular contexts. Notably, direct effects
of progesterone on miRNA expression in normal or
Table 1 Tumor growth ratesa.
TreatmentMean tumor volume
(mm3)
Growth rate
(mm3/day)
Growth inhibition
(%)
Delay in tumor growth
(days)
pre-miR-CTRL-C4HD
pre-miR-16-C4HD
683.6 ± 192.2*
231.1 ± 107.9#
26.1*
9.6#
66.2b
5b
aGrowth rates were calculated as the slopes of growth curves. The volume, percentage of growth inhibition, and delay in tumor growth (days) in tumors from
mice injected with pre-miR16-C4HD cells relative to mice injected with pre-miR-CTRL-C4HD cells were calculated at day 28, as described in Methods. *P < 0.01,
**P < 0.001 versus pre-miR-CTRL-C4HD-treated cells.bRelative to pre-miR-CTRL-C4HD-treated cells, P < 0.001.
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Figure 6 miR-16 is a tumor suppressor in HRG-induced breast cancer growth. A, BT-474 cells were treated with 10 nM MPA for 24 hours
and proliferation was measured by [3H]-thymidine incorporation as described in Figure 2D. B, BT-474 cells were treated with 10 nM MPA for the
times shown. miR-16 expression levels were determined by RT-qPCR, and data analysis was performed as described in Figure 2A. C, BT-474 cells
were treated with MPA for the times shown and WB was performed with an anti-c-Myc antibody (left panel) or with an anti-cyclin E antibody
(right panel) and filters were reprobed with an anti-b-tubulin antibody. In the WB, cyclin E LMW isoforms are shown. D, BT-474 cells were treated
with 40 ng/ml HRG for the times shown and miR-16 levels were measured as described in Figure 2A. E, BT-474 cells were transfected with pre-
miR-16 or pre-miR-CTRL, and proliferation was evaluated by [3H]-thymidine uptake as described in Figure 2D after 24 hours of HRG treatment. In
the right panel, as a control for transfection efficiency, miR-16 levels are shown in pre-miR-16- and pre-miR-CTRL-transfected BT-474 cells. F, BT-
474 cells were treated with 40 ng/ml HRG for the times shown, and WB was performed with anti-c-Myc antibody and filters were reprobed with
an anti-b-tubulin antibody. G, BT-474 cells were transfected with Stat3, c-Myc and CTRL siRNAs and then treated with HRG for 24 hours. miR-16
levels were studied by RT-qPCR, and data analysis was performed as described in Figure 2. The experiment shown was performed with Stat3
siRNA #3 and c-Myc siRNA #5, but the same results were obtained with Stat3 siRNA #1 and c-Myc siRNA #6. Experiments shown in A to G were
repeated in triplicate with similar results. The data shown represent the means of three independent experiments ± SEM (P < 0.001 for b versus
a and c versus b). HRG, heregulin; LMW, low molecular weight; MPA, medroxyprogesterone acetate; SEM, standard error of the mean; WB,
western blot.
Rivas et al. Breast Cancer Research 2012, 14:R77
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