Download Three-dimensional (3D) culture and immunostaining

A novel effect of β-adrenergic receptor on mammary branching
morphogenesis and its possible implications in breast cancer
Lucía Gargiulo1, María May1, Ezequiel M Rivero1, Sabrina Copsel1,2, Caroline
Lamb1, John Lydon3, Carlos Davio2, Claudia Lanari1, Isabel A Lüthy1, Ariana
Bruzzone4
1
Instituto de Biología y Medicina Experimental-CONICET, Vuelta de Obligado 2490,
C1428ADN CABA, Argentina
2
Laboratorio de Farmacología de Receptores, Departamento de Farmacología, Facultad de
Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956, 1113 CABA, Argentina
3
Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston,
Texas, 77030, USA
4
Instituto de Investigaciones Bioquímicas de Bahía Blanca, CONICET-Universidad
Nacional del Sur, Camino La Carrindanga km 7, 8000 Bahía Blanca, Argentina
Short running title: β-AR regulates normal and tumor breast morphogenesis.
Corresponding author: Dr. Ariana Bruzzone, Instituto de Investigaciones Bioquímicas de
Bahía Blanca (INIBIBB), Camino la Carrindanga km 7, 8000 Bahía Blanca, Buenos Aires,
Argentina.
Phone:
++54-291
4861201,
[email protected]
1
fax:
++54-291
4861200,
e-mail:
ABSTRACT
Understanding the mechanisms that govern normal mammary gland development is crucial
to the comprehension of breast cancer etiology. β-adrenergic receptors (β-AR) are targets of
endogenous catecholamines such as epinephrine that have gained importance in the context
of cancer biology. Differences in β2-AR expression levels may be responsible for the
effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing
higher levels of β2-AR.
To study regulation of the breast cell phenotype by β2-AR, we over-expressed β2-AR in
MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A
breast cells. In MCF-10A cells having knocked-down β2-AR, epinephrine increased cell
proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7
cells overexpressing the β2-AR, epinephrine decreased cell proliferation and migration and
increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus
underscoring that β2-AR expression level is a key player in cell behavior.
β-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing
in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo.
Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice,
demonstrating that the effect of β-adrenergic stimulation on branching is dependent on the
estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture
was nonetheless found. Isoproterenol significantly increased the expression of ERα,
Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased
tumor growth and induced tumor differentiation.
This study highlights that catecholamines, through β-AR activation, seem to be involved in
mammary gland development, inducing mature duct formation. Additionally, this
differentiating effect could be resourceful in a breast tumor context.
2
KEYWORDS
Mammary gland development
Beta-adrenergic receptor
Breast cancer
Non-tumorigenic breast epithelial cells
Normal mammary gland
Estrogen receptor
3
INTRODUCTION
At birth the mammary gland of most species is a rudimentary ductal system that grows
isometrically before the onset of puberty [1]. Under ovarian hormone stimulation, the
mammary glands of mice begin a proliferative phase of development, growing rapidly via
terminal end buds (TEB) that are bulb-like structures consisting of relatively
undifferentiated epithelial cells located at the tip of each growing duct. These structures
invade and communicate with the fat pad stroma leaving differentiated ducts behind as they
grow. Under cyclical ovarian stimulation short tertiary branches, called alveolar buds,
sprout laterally from the primary ducts. In humans, the site of active proliferation is a TEBlike structure, which presents morphological differences with mouse TEB. At puberty, the
ductal tree elongates and undergoes sympodial branching. Terminal duct may give rise to
an average of 11 surrounding alveolar buds, leading to the formation of a terminal duct
lobular unit (TDLU) type 1. These structures subsequently develop into TDLU types 2 and
3 with recurrent menstrual cycles [1].
Estrogens are the main hormones driving ductal elongation during puberty through
activation of estrogen receptor alpha (ER𝛼) that is expressed in ~ 50% of the mammary
epithelial cells [2]. The epithelium and stroma communicate through numerous factors and
mechanisms downstream of ERα, which are ultimately responsible for driving ductal
development. Among these factors, some FGF family members emerge as key mediators of
mammary branching [3, 4].
Breast cancer is by far the most frequent cancer among women (25% of all cancers),
ranking second overall when considering both sexes [5]. Although breast cancer is
normally detected later in life, cancer initiation occurs many years before clinical detection,
where some early carcinogenic events may be established during mammary gland
development. Previous research has concluded that early-life stress could cause
morphologic and molecular changes in the mammary glands of adult mice affecting the risk
of developing mammary tumors [6, 7]. Therefore, understanding the factors that contribute
to normal breast development is crucial to comprehending the etiology of breast cancer and
improving its prevention.
β-adrenergic receptors (β-AR) are targets of endogenous catecholamines such as
epinephrine (EPI) and serve to mediate the stress response. These receptors were originally
4
associated mainly with cardiovascular and central nervous system functions. However, in
the last years β-AR have gained importance in the context of cancer treatment [8]. In
addition, the β-adrenergic system has been associated with almost every hallmark of cancer
[9]. The β-AR are classic G protein-coupled receptors (GPCR) that activate adenylyl
cyclase (AC) through Gαs, increasing second messenger cyclic AMP (cAMP) levels. These
receptors, especially the β2 subtype, have been found in normal mammary gland of
different species, in non-tumorigenic breast cells, as well as in breast cancer tissue samples
and cell lines [10-16].
The evidence relating β-AR to breast cancer has been controversial. While some authors
have demonstrated that activation of β-AR increased cell proliferation and tumor growth
[17-19], others claimed that β-AR stimulation is correlated with an inhibition of these
parameters [10-12, 20]. Previous research has also shown that both the activation of β2-AR
and its expression levels regulate breast tumor cell phenotype by modifying proliferation,
adhesion and cell migration [21]. It has also been reported that EPI behaves differently in
non-tumorigenic and breast tumor cell lines. In non-tumorigenic breast cells, EPI tended to
maintain a benign phenotype while in tumor cells it induced a malignant phenotype. A
suggestion has been that β2-AR expression levels are, in part, responsible for the
differential behavior observed between these lines, where the non-tumorigenic cells have
higher levels of β-AR than tumor cells [21]. Thus, low expression levels of this receptor
could be an important indicator of cell malignancy and, consequently, of tumor progression
[21].
To gain a greater insight on the implication for β-AR in breast cancer, we evaluated β-AR
activation in normal and tumor breast models, including in vitro and in vivo. The aim of
this study was to understand the role of β-AR, as stress mediators, in normal breast
development and cancer.
5
MATERIALS AND METHODS
Drugs, reagents and antibodies
Epidermal growth factor (EGF), hydrocortisone, adrenergic drugs, bovine serum albumin
(BSA), carmine and aluminum potassium sulfate were from Sigma-Aldrich (Saint Louis,
MO, USA). Culture medium, fetal bovine serum (FBS), Lipofectamine 2000, siRNAs
(ADRB2 Stealth Select RNAiTM, HSS100258, HSS100259 and HSS100260) and other
products for cell culture were from Invitrogen (Waltham, MA, USA). FuGENE® was
purchased from Promega (Madison, WI, USA) and Transwell inserts were from BD
Biosciences (San Jose, CA, USA).
Ephrin-B1 (sc-1011), EphA2 (sc-10746), FGF10 (sc-7917), FGF2 (sc-79), FGFR2 (sc-122)
and ERK1 (sc-94) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).
Monoclonal rabbit anti-human ERα (IR084) was from DAKO (Glostrup, Denmark). β-actin
antibody (A5441) was from Sigma. Fulvestrant (FULV) was a kind gift from LKM
Laboratorios SA (Buenos Aires, Argentina). Insulin was donated by Denver FarmaLaboratorios Beta (Buenos Aires, Argentina). Tamoxifen citrate (TAM) was a gift from
Laboratorios Gador and telapristone acetate (TELA) was from Repros Therapeutics.
Mifepristone (RU) was from Sigma-Aldrich.
Cell lines, culture and transfection
The MCF-7 cell line was originally obtained from a pleural effusion of a human breast
cancer [22] and represents a model for luminal breast cancer model that accounts for more
than 70% of breast cancers [23]. The MCF-10A cell line arose from a breast cyst of a
premenopausal woman [24] and has been considered triple negative [25]. Whereas MCF-7
is a well-accepted luminal breast cancer in vitro model, MCF-10A is a well-accepted nontumorigenic breast model.
MCF-10A and MCF-7 cells were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA) and were cultured as already described [26] with HEPESbuffered DMEM:HamF12 culture medium (basal medium) supplemented with antibiotics
(100 µg/mL streptomycin, 100 IU/mL penicillin), 10% FBS and 2 µg/mL human insulin
(complete medium). For MCF-10A cells, the medium was also supplemented with 20
6
ng/mL EGF and 0.1 µM hydrocortisone. Cells were sub-cultured by trypsinization (0.25%
trypsin–0.025% EDTA) and the medium was changed three times a week.
For transfection, 5x105 cells/well were seeded into 24 well-plates in complete medium. The
receptors were knocked down with small interfering RNA (siRNA) by using a mixture of
three different sequences of siRNA targeting β2-RA, or 10 nM non-targeting scrambled
siRNA (sc) as a control [27, 21]. β2-AR was overexpressed by transfecting cells with 1.5 µg
per well of a plasmid as described elsewhere [28]. Control cells (mock) were transfected
with the empty vector pcDNA3.1. Plasmids and siRNAs were prepared as previously
described [21]. Proliferation, adhesion and migration assays were performed between 48
and 72 h post-transfection. Receptor silencing and over-expression were checked by
radioligand binding assay of whole cells at 4 °C, as described [27]. Briefly, 7x104 cells/well
were seeded into 48 well-plates. The number of binding sites was evaluated using the
antagonist [3H]-GCP12177 in the presence or absence of 100 µM isoproterenol (ISO).
Incubation was stopped by dilution with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.4. After
three washes with 3 ml of ice-cold buffer, the bound fraction was collected in 200 μl of
ethanol. Radioactivity in the eluted fractions was measured using LLC Optiphase Hisafe III
(Perkin Elmer) in a Wallac 1410 liquid scintillation counter.
Proliferation, cell adhesion and migration assays
For the proliferation assays, transfected cells were treated daily with 1 μM epinephrine in
medium supplemented with 2% charcoal-stripped FBS. The proliferation response in
transfected cells was evaluated by cell-counting using Neubauer chamber. Cell adhesion
was measured as previously described [16]. Briefly, the medium was removed and cells
were treated with or without 1 μM EPI in basal medium for 15 min. The medium was
subsequently removed and cells were incubated in Mg2+/Ca2+-free PBS containing 0.5 mM
EDTA and 0.25% trypsin with constant agitation at room temperature (15 min for MCF10A cells or 5 min for MCF-7) [21]. Cells that resisted the treatment and remained adhered
to the plastic were harvested following additional 30 min incubation in Mg2+/Ca2+-free PBS
containing 2.5 mM EDTA and 1.25% trypsin and were counted as the number of attached
7
cells. The percentage of adherent cells was calculated as follows: attached cells x 100/
(attached cells + detached cells).
Cell migration was analyzed using Transwell inserts with 8 μm pores as previously
described [21]. Cells (2x104) were seeded onto the porous membrane and were allowed to
adhere for 4 h. The medium was then removed and changed to basal medium with or
without EPI. After 16 h, the medium was removed and the cells were fixed and stained with
0.05% crystal violet in methanol for 10 min. Non-migrated cells were removed from the top
of the membrane using a cotton swab. Cells fixed to the underside of the membrane were
counted on an inverted microscope. The results were expressed as fold over the control
cultures incubated in the absence of EPI.
Three-dimensional (3D) culture and immunostaining
Cells were cultured for 15 days using the overlay method [29]. Cells (5x104cells/chamber
in eight-well chambers (Nunc, Rochester, NY, USA) were seeded on a 1–2 mm thick
solidified layer of Growth Factor-Reduced Matrigel (BD Biosciences). Cells were refed
with 400 μl medium containing 2% Matrigel every 4 days. Treatments were replaced daily.
After 15 days, the medium was aspirated and the structures formed were immediately fixed
with freshly-prepared 4% paraformaldehyde in PBS (pH 7.4) and 1% glutaraldehyde for 15
min at 4°C, then permeabilized with PBS containing 0.5% Triton X-100 for 20 min. Cells
were incubated with 1:50 phalloidin-Alexa Fluor 488 (Invitrogen) for 30 min. To
counterstain nuclei, cells were incubated with PBS containing 0.1 mg/mL propidium iodide
for 10 min, then were mounted with Vectashield H-1000 (Molecular Probes, Eugene, OR,
USA) and allowed to dry. Stained structures were analyzed on a Nikon laser confocal
microscope.
Animals and treatment
Animal care and manipulation were in agreement with institutional guidelines and the
Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal
Resources Commission on Life Sciences and National Research Council, 1996). The
animal protocol was accepted by the Ethics Committee of the Instituto de Biología y
Medicina Experimental, Buenos Aires, Argentina. Female virgin BALB/c mice of 3 weeks
8
old (weaned) or six-eight weeks old (pubertal), and mice carrying a null mutation of the
progesterone receptor (PRKO) derived from C57BL/6 strain were used [30]. PRKO mice
were checked for homozygosity by PCR. Animals were housed in ventilated racks, were
fed ad libitum, and were kept in air-conditioned rooms at 20±2 °C with a 12 h light–dark
period. For ovariectomy, animals were anesthetized with ketamine (100mg/Kg) and
xylazine (10 mg/kg) intraperitoneally. Pellets of telapristone acetate (6mg) were prepared
as described previously [31] and placed SC under anesthesia. Animals received daily
injections of ISO (1 mg/Kg, SC) for 15 days while control mice received physiological
saline solution. FULV was administered once a week at a dose of 5 mg/kg. RU (10 mg/kg,
SC) and TAM (5mg/kg, SC) were administered daily. At necropsy, the right 4th inguinal
mammary gland was removed and placed on a microscope slide for whole mount, whereas
the left glands were fixed in formalin, paraffin-embedded and cut in thin-sections. Standard
hematoxylin and eosin (H&E) staining was performed for histological evaluation.
Mammary gland whole mounts
The 4th right mammary gland was spread on a glass slide and fixed in Carnoy's fixative
solution (60% ethanol, 30% chloroform, 10% glacial acetic acid) for 2 to 4 h at room
temperature. After fixation, the tissue was washed in 70% ethanol for 15 min, and gradually
hydrated to distilled water and incubated overnight with carmine alum aqueous solution for
staining (0.2% carmine 0.5 % aluminum potassium sulfate). The tissue was dehydrated
with graded alcohols (70%, 96%, and 100%), cleared in xylene and mounted in permanent
mounting media.
Immunohistochemistry and scoring of slide sections
Immunohistochemical procedures were performed as previously described [32]. Briefly,
3µm tissue sections were deparaffinized and endogenous peroxidase inhibited with H2O2 in
water. Antigen retrieval was performed with 10 mM citrate buffer-0.001% Tween (pH 6) in
a water bath at 95° for 50 min. The tissue sections were washed with 2.5% BSA-PBS and
then sequentially incubated with the primary antibody (1:100) in 2.5% BSA overnight.
Slides were washed and incubated with the appropriate secondary antibody and the ABC
kit (Vector Lab, Burlingame, USA). After washing with PBS, samples were exposed to
9
3,3′-diaminobenzidine chromogen solution (DAKO) according to the manufacturer's
protocol, then stopped with tap water and counterstained with hematoxylin for 10 seconds
at room temperature. Samples were dehydrated, cleared and mounted to be visualized using
bright field microscopy.
Membranous, cytoplasmic, and nuclear staining were differentially scored and expressed as
percentage of stained cells (0-100%) and intensity (0-3), giving rise to a scale ranging from
0 to 300. In the case where nuclear staining was found, each stained cell was also counted
and normalized as the number of positive cells to total cell number. Negative controls were
prepared by excluding the primary antibody.
Western Blot
Cells were seeded into 6-well plates (2.5 x106 cells/well) and cultured for 24 h in complete
medium followed by 24 h in basal medium. Cells were subsequently incubated with ISO in
basal medium for 24 h and were then rapidly washed with ice-cold PBS and lysed in RIPA
buffer (10 mM Tris, pH=7.5, 150 mM NaCl, 2 mM Na ortho-vanadate, 0.1% SDS, 1%
Igepal, 1% Na deoxycholate). Equal amounts of proteins were separated on SDS-PAGE
and transferred to nitrocellulose (Millipore, Billerica, MA, USA). Anti-FGF2 and -FGF10
antibodies were used at a 1:100 concentration, while anti-FGFR2 was used at 1:200.
Immunocomplexes were revealed by chemiluminescence using horseradish peroxidase–
conjugated secondary antibody (Amersham Biosciences GE Healthcare, Little Chalfont,
England). Immunostaining for β-actin or ERK1 (1:5000) was used for protein loading
normalization. Chemiluminescence detection was performed using an enhanced detection
solution (1.25 mM luminol, 0.2 mM p-coumaric acid, 0.06% (v/v) hydrogen peroxide, 100
mM Tris-HCl pH 8.8). Immunoblots were exposed to autoradiographic film (Thermo
Scientific, Waltham, MA) and quantified by densitometry with ImageJ (US National
Institute of Health).
Statistical analysis
Experiments were repeated at least three times with similar results. The analyses performed
were Student´s t test or ANOVA followed by Bonferroni or Dunnet´s tests. Differences
were considered significant when p<0.05.
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RESULTS
EPI has different effects on non-tumorigenic and tumor breast cell lines that could be due to
differences in β2-AR levels, where non-tumorigenic cells have higher levels of β-AR
expression than tumor cells [21]. To elucidate the role of β2-AR in regulating the breast cell
phenotype, we used MCF-7 and MCF-10A cell lines that are well-established models to
study tumorigenic and non-transformed breast epithelial cells, respectively.
We successfully over-expressed β2-AR in MCF-7 cells and knocked-down the receptor in
MCF-10A cells as previously described [21]. Both cell lines were subsequently stimulated
with 1 μM EPI, a concentration that mimics tissue catecholamine levels found during acute
stress [8]. In MCF-10A cells transfected with the scrambled siRNA (sc), EPI decreased cell
proliferation and migration, and stimulated cell adhesion (Fig 1a, c and e). On the contrary,
in MCF-7 tumor cells transfected with empty vector (mock), EPI induced cell proliferation
and cell migration (Fig 1b, d and f). Of note, the transfected cells, either with scrambled
sequence or with empty vector, behaved as wild type cells [21]. Interestingly, when we
knocked down β2-AR in MCF-10A cells, EPI increased cell proliferation and cell migration
(Fig 1a, c and e), thus resembling the response of MCF7 cells to EPI. Furthermore, MCF-7
cells with overexpressed β2-AR decreased cell proliferation and migration in response to
EPI, and increased cell adhesion (Fig 1b, d and f), thus mimicking the response of MCF10A cells to EPI. These results underscore that β2-AR expression level is a key player in
cell behavior.
To gain further insight to the role of β2-AR in mammary cells, we studied the effect of βAR stimulation using several normal and tumor models in 3D cell culture. When MCF-10A
cells were cultured for 15 days on Matrigel, gland-like organoids were formed (Fig 2a).
Under control conditions, tubular structures resembling mammary ducts were observed
with secondary branches and a few tertiary ones, similar to the formations found in TDLU
type 1 [33]. No sign of lumen formation was observed by confocal microscopy (Fig 2b).
There were approximately 12 alveolar buds per secondary structure (Fig 2c). Cells treated
with 1 μM of the β-AR agonist ISO formed complex structures, showing a massive increase
in alveolar buds, corresponding to TDLU type 2 (ISO: 45±3 vs control: 12±3, p<0.001, Fig
2a, c). In addition, under ISO treatment it was possible to identify the presence of a lumen,
both in acinar and in tubular structures, which is characteristic of more differentiated
11
structures (Fig 2b). Similar to the response to ISO, 1 μM EPI and 10 μM forskolin (FK, an
AC activator which increases cAMP levels) induced the formation of differentiated
structures. The effect of ISO or EPI on MCF-10A cells cultured on Matrigel was reverted
by 10 μM ICI-118551 (ICI, a β2-AR selective antagonist), while ICI alone had no effect per
se as compared to the control (Fig 2a).
In order to study the effect of β-AR activation on the normal mammary gland, ISO was
administered to 3-weeks old (weaned) and/or 6-8 weeks old (pubertal) mice. Mammary
gland morphology was subsequently analyzed from H&E stained sections and mammary
gland whole mounts. ISO induced a significant increase in mammary branching both in
weaned and pubertal mice (Fig 2d). In weaned mice, the number of secondary and tertiary
branches after ISO treatment was increased (ISO: 150 ± 32 vs control: 57 ± 21
branches/field, p<0.05). Mammary glands of weaned control mice were composed of 90%
adipose tissue and 10% epithelial ductal structures. The majority of these ducts were
composed of stratified epithelium with rough and tiny additional lumens. Moreover, the
mammary stroma was primarily composed of an immature multi-vacuolated adipose tissue
and new capillaries. This is in line with the description of brown adipose tissue observed in
postnatal mammary gland of mice [34]. In addition, it was described that brown adipose
tissue negatively regulates the differentiation of mammary epithelial cells in a systemic
manner during prepubertal ductal outgrowth [34]. In contrast, in ISO-treated mice, ducts
were composed mainly of only one layer of epithelial cells, with evidence of recruitment of
fibroblasts and deposition of dense matrix in near almost all ductal structures (Fig 2d). In
the mammary glands of pubertal mice, the ductal system displayed a regular number of
mature ducts composed of one layer of epithelial cells resting on a visible myoepithelial
layer. Again, ISO treatment statistically increased the number of secondary and tertiary
branches: 196 ± 16 vs 94 ± 14 branches/field, p<0.01). In addition, in histological sections
of ISO treated mice, normal ductal structures with slightly enlarged lumens were observed
(Fig 2d).
Using a microarray profiling strategy, Kouros-Mehr and Werb identified novel genes that
may regulate mammary branching [35] . Among them, Ephrine-B1 and Eph-A2 were found
to be differentially expressed in the mature mammary gland. We therefore examined their
expression and response to ISO by immunohistochemistry. In adult mice, ISO significantly
12
increased Ephrine-B1 expression in both epithelial and stroma compartments (p<0.05, Fig
2e), although these differences were not seen for Eph-A2 expression. Given our results
showing that β-adrenergic stimulation induces differentiation of breast cells growing in 3D
culture as well as induction of mammary branching in vivo, we also analyzed if the ability
of the mammary gland to feed pups was affected by ISO treatment. Pup weight was
measured as an indicator of successful lactation. ISO administered 15 days before
pregnancy (BP) and/or during lactation (DL) caused no differences in offspring growth,
(Fig 2f). No further differences were observed in other reproductive output measures, such
as gestation length or the number of pups born. These findings confirm that the lactation
capacity of the gland does not seem to be affected by ISO treatment.
As mammary gland development occurs mainly during postnatal life under control of
female reproductive hormones, we evaluated if ISO action was mediated through the
estrogen receptor (ER) and/or progesterone receptors (PR), which both mediate mammary
branching. In order to analyze the role of PR, we tested the effect of exogenous ISO on
mice treated with or without TELA (a selective PR modulator) or RU (a non-selective
antiprogestin), and on PRKO mice. The doses and methods of administration used were
effective in achieving other physiological effects as previously described [36, 31]. In these
three different approaches, ISO reproduced exactly the same effect as that on untreated
BALB/c mice (control), which was a significant increase in the number of ductal structures
(Fig 3a). We subsequently analyzed the role of ER by depleting it via FULV (a selective
ER degrader), by modulating it via TAM (a selective modulator of ER) and by depleting
ovarian estrogens by ovariectomy (OVX). It is well known that when estrogen signaling is
downregulated, the mammary gland becomes atrophic leading to scant and tiny ducts, with
a constricted lumen [37]. Under these conditions, the effect of ISO on ductal branching was
almost totally abolished, suggesting that ER plays a central role in the β-adrenergic effect
on mammary branching (Fig 3b). Nevertheless, H&E sections showed that ISO caused a
clear effect on the architecture of ducts, restoring the number of cells per duct and also the
lumen area in both OVX and FULV treated mice (p<0.05, Fig 3c-d). Furthermore, ISO
treatment did not modify the presence of protein content in the lumens under any condition.
This finding is consistent with the fact that this process is mainly modified later, under the
influence of pregnancy/lactation hormones (Fig 3d).
13
On account of the fact that ER seems essential for the in vivo effect of ISO, and because
MCF-10A cells in 2D culture have been described as ER negative [38], we assessed
whether incubation with ISO as well as 3D culture conditions modify the expression of this
receptor. Western blotting revealed that not only 3D culture conditions but also 24h
incubation with ISO increased ERα expression in MCF-10A and MCF-7 cells (p<0.05 and
p<0.01, Fig 4a). In addition, FULV treatment of MCF-10A cells in 3D culture partially
reversed the formation of complex structures induced by ISO (Fig 4b). Under FULV
treatment, ISO induced the development of smaller but well-developed lumens. ISO
treatment also significantly increased ERα expression in murine mammary glands, both in
control mice (p<0.001) and in OVX- and FULV-treated mice (p<0.05) (Fig 4c).
In the mammary gland, ERα transforms endocrine signals into local signals, where FGFR2,
FGF2 and FGF10 have key roles in different aspects of mammary gland development [4].
Glands of ISO-treated mice displayed a significant increase in the nuclear expression of
FGFR2 in the epithelial compartment. This increase was also observed in FULV-treated
and OVX animals, thus suggesting that this increase in FGFR2 appears to be independent
of ER (p<0.001, Fig 5a). ISO also significantly increased FGF10 and FGF2 expression in
control and FULV-treated animals (Fig 5 b and c). However, ISO had no effect on FGF10
and FGF2 expression in OVX animals. In addition, in MCF-10A cells, ISO increased
FGFR2, FGF10 and FGF2 expression (Fig 5). Overall, these findings suggest that ISO
exerts its effect, at least in part, through the FGF family of growth factors.
The role of β-AR in breast cancer is still controversial. Previous research has reported that
β-AR activation is related to a decrease in breast cancer cell proliferation and tumor growth
in vivo [10, 21, 11, 12]. When MCF-7 cells growing in 3D were treated with ISO or FK, the
structures acquired a polarized phenotype with well-established lumens (Fig 6a-b). EPI
induced larger spheroids without hollow lumens when compared with control conditions,
thus suggesting more proliferation and less differentiation. The β-AR blocker ICI was
observed to abolish the effect of ISO but not EPI (Fig 6a). This finding is consistent with
our initial and previous results (Fig 1), which show that EPI induces a more aggressive
behavior in breast tumor cells [21]. Furthermore, in an in vivo model using a poorly
differentiated PR+ and ER+ murine ductal carcinoma [36], ISO decreased tumor growth
(p<0.05, Fig 6c). Moreover, after ISO treatment, histological imaging showed that tumor
14
cells arrange forming new incipient ductal structures making the tumor a less aggressive
entity (Fig 6d). Supporting this morphological finding, an increase in nuclear GATA3
expression was observed after ISO treatment (p<0.001, Fig 6d). GATA3 is an important
transcription factor required for mammary luminal epithelial cell differentiation whose
expression is progressively lost during luminal breast cancer progression (as reviewed in
[39]). Taking into account these results from the in vitro and in vivo tumor models, it seems
that β-adrenergic stimulation also play a differentiating role in the tumoral context.
15
DISCUSSION
Our results highlight the importance of β2-AR as modulator of benign breast cell behavior.
Tumorigenic and non-tumorigenic breast cell lines express α2 and β-AR [26, 40, 16] and
EPI binds them with equal affinity. Breast tumor cells respond to EPI with increased cell
proliferation and migration, whereas in non-tumorigenic cells, EPI induces a decrease in
cell proliferation and migration and an increase in cell adhesion [21]. We previously
suggested that the β2-AR expression level was in part responsible for this differential
behavior between these cell lines, given the higher levels of β-AR in non-tumorigenic cells
[21]. In this context, we found that the effects of EPI were reversed towards a
predominantly α2-adrenergic response in β2-AR knocked-down MCF-10A cells. It could be
assumed that the effects of EPI on these cells were at least partly mediated by a α2-AR
since the same was observed when MCF-10A cells were stimulated with an α2-adrenergic
agonist [21]. In contrast, in β2-overexpressing MCF-7 cells, EPI completely reversed tumor
cell behavior. It has in fact been reported that whereas β-AR expression in wild type MCF7 cells is modest [21], α2-AR expression is high [26].
β-AR have attracted attention in breast cancer treatment as a result of the studies carried out
in patients treated with β-blockers. After initial data confirming the protective role of
propranolol [41, 42], one meta-analysis of clinical trials revealed a non-significant trend
towards a reduced risk for cancer development [43]. In contrast, another meta-analysis
showed a significant improvement in breast cancer-specific survival, although no
association with overall survival was found [44]. In some cases, β-agonists and β-blockers
exerted the same action on tumor growth in vivo [10]. In our experiments, ICI was used
instead of propranolol which was discarded because of its partial agonist action in certain
parameters in vitro, as previosly described [45]. In addition, ICI has a greater affinity for
β2-AR than for β1 and β3-AR [46], and β2 is the main β subtype expressed in both tumor
and non-tumor breast cells [47, 48, 16]. In our study, ICI blocked the effect of ISO,
demonstrating the involvement of β2-AR in ISO-induced differentiation in 3D cultures of
MCF-10A and MCF-7.
Three dimensional culture is an important tool to study normal and tumor mammary gland
[49], where mammary epithelial cells growing in 3D recapitulate features of the breast
16
epithelium in vivo, including formation of acini [50]. In our experiments, these cells not
only formed acini but also branches resembling the terminal ductal lobular units in the
breast, which was enhanced by β-AR stimulation. After ISO and EPI treatment, more
differentiated structures could be observed. Increased cAMP levels after ISO treatment
regulate lumen formation in mammary cells in 3D [51]. On the other hand, MCF-7 cells in
3D culture only showed a few signs of cell differentiation after β-adrenergic stimulation.
Previous studies have shown that differentiation of the mammary gland is related to a
reduction of breast cancer risk [52, 53].
The effect of β-AR stimulation on the mammary glands of mice was also studied. ISOtreated mammary glands were functional because these animals demonstrated the ability to
achieve a successful lactation. In agreement with our results, several reports have shown
that psychosocial stressors produce morphologic and molecular changes in the mammary
glands of adult mice [54, 7]. Maternal separation (stress) of newborn mice accelerates
mammary gland development, inducing the maturation of TEBs and increasing branching
[7]. Compared with untreated mice, adult mice that suffered prolonged maternal separation
showed a 200% increase in ERα levels in the mammary gland [7]. In line with this, social
isolation of mice decreased the number of pre-TEB and decreased the risk for developing
mammary tumors [6]. Previous research showed that implants of cholera toxin pellets
which locally elevate cAMP levels stimulate normal ductal morphogenesis in C57 OVX
mice [37]. Although authors of those findings suggested that cAMP operates through a
normal morphogenetic pathway, they suggested that cAMP could replace the normal
hormonal requirements for this type of development [37]. That proposal is contrary to our
findings where β-AR stimulation, probably via their classical cAMP second messenger,
depends on the functional ER to exert their effect on mammary branching.
Under the influence of ISO, apart from the remarkable effect observed on branching, there
was an increase in the differentiated state of structures in the epithelial compartment as well
as in the stroma. Overall, taking all the effects observed under ISO influence together, we
can propose that β-AR stimulation increases development and maturation of mammary
epithelial structures.
Epithelial ER𝛼 signaling is required for ductal elongation [55]. On the other hand, PR
signaling is required for side branching and alveologenesis during pregnancy, ultimately
17
giving rise to a lactation-competent gland [56]. The fact that ISO induced branching in
TELA- and MIFE-treated mice, in PRKO mice as well as in weaned mice, led us to
disregard PR as a mediator of the effects of ISO. In line with our results, the outgrowth of
ducts in PRKO mice during puberty was delayed but not abolished [56], thus indicating that
PR is not totally essential for the branching of the mammary gland in this early stage.
In contrast, we demonstrated that the presence of ER is essential for the effect of ISO on
mammary branching. However, we also found that ISO affects the lumen architecture
regardless of ER presence. Given the developmental functions of Ephrine-B1 and its
expression pattern in mature ducts, particularly in sites of lateral budding, it was suggested
that it has a regulatory role [35]. In agreement with this, ISO increased Ephrin-B1
expression in the mammary gland.
Another interesting observation worthy of note from our study is the ability of ISO to
directly modulate important factors involved in mammary branching development such as
FGFR2, FGF2 and FGF10. In MC3T3-E1 cells, FK increased FGF2, FGFR2 and FGFR1
transcripts and produced protein translocation to the nucleus [57]. The FGF2 promoter
contains a known consensus AP-1–like site. In rat osteosarcoma cells, cAMP production
induces CREB binding to an AP-1 consensus sequence [58]. All the above could indicate
that ISO exerts the reported effect on lumen architecture via Ephrine-B1 and/or through
FGF family of growth factors.
To our knowledge, ours is the first study showing that β-adrenergic stimulation induces
ERα expression in MCF-7 and MCF-10A cells. The MCF-10A cell line in monolayer has
been reported to express ERβ but not to express ERα [38, 59]. Interestingly, 3D culture
conditions of MCF-10A cells stimulated the expression of ERα, an effect which has not
been reported elsewhere. It has been demonstrated that gene expression in mammary
epithelial cells cultured in monolayer are significantly different from those in 3D cultures
[60]. MCF-10A cells produce milk proteins under certain 3D incubation conditions [61].
Therefore, the expression of ERα in MCF-10A growing in 3D culture is not an unexpected
phenomenon. Further studies to determine how β-AR increases ERα expression in vitro and
in vivo are needed to better understand how this GPCR affects mammary development.
Another highlight of our study is that β-adrenergic effect on normal mammary
differentiation can be translated to tumors models. Apart from the differentiating effect on
18
tumor cells in 3D, ISO caused a growth inhibition in a murine mammary tumor. The
diminution of tumor growth can be partially explained by the pronounced ISO-induced
differentiation. Although several authors have shown that β-adrenergic stimulation
promotes breast cell proliferation in vitro [19, 17, 18], several others have reported a direct
effect on breast tumor consisting in tumor growth diminution [12, 10, 20].
Even though it is well-known that estrogens are the master regulators of mammary gland
development, our study highlights that catecholamines could also be involved in this
process. Because the mammary gland is innervated by the sympathetic nervous system, and
because changes in the sympathetic-adrenal tone have been reported during lactation (36),
it seems tempting to speculate that the β2-AR have a physiological role, which still remains
largely unknown. Sternlicht et al. suggested that a GPCR may influence the mammary
branching mechanism governed by ERα [62]. It is thus possible that β-adrenergic signaling
during important developmental periods may cause transient molecular changes of the
mammary gland that ultimately have implications on tumor development. To decipher how
β-AR impinge as stress mediators on the basic framework of the mammary gland
development appears to be promisingly exciting and could have relevant clinical
implications in cancer research.
Acknowledgments
In the memory of our friend Hervé Paris. We gratefully thank Federico Carrizo from
Laboratorios Beta and Denver Farma Argentina for kindly providing insulin and Michel
Bouvier from Département de Biochimie, Université de Montréal for kindly providing β2AR plasmid. Thanks are also due to KLM for fulvestrant and Bruno Luna and Anita
Sahores for their technical help. Authors declare there are no conflicts of interest. This
work was supported by a grant from PICT N° 103 (ANPCyT) and PIP N° 539 from
CONICET, Fundación René Barón and Fundación Roemmers.
19
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FIGURE LEGENDS
Figure 1. Effect of β2-AR expression level on epinephrine (EPI) response in non-tumor
MCF-10A and tumor MCF-7 human breast cell lines. Cells were transfected with
scrambled siRNA (sc), β2-AR-targeted pooled siRNA (siRNA), pcDNA3.1 (mock) or the
plasmid codifying for the β2-AR. Results are expressed as EPI response fold over control
cells (in the absence of stimulation, dotted line). Cells were stimulated or not (control) with
1 µM EPI and cell proliferation (a-b), cell adhesion (c-d) and cell migration (e-f) were
measured. Data are representative of three independent experiments. Statistical significance
was assessed using ANOVA-Bonferroni test. *p<0.05, **p<0.01, ***<p0.001 between EPI
stimulated and control cells (dotted line). ## p<0.01, ###p<0.001 between EPI response in
sc vs siRNA or mock vs β2-AR transfected cells. n.s: non-significant differences.
Figure 2. Effect of β-adrenergic stimulation on human MCF-10A cells in 3D culture
and in murine mammary gland. a) Fluorescence staining of MCF-10A growing onto
Matrigel. Cells were treated or not (control) during 15 days with 1μM isoproterenol (ISO),
1μM epinephrine (EPI) or 10 μM forskolin (FK). β2-AR selective antagonist ICI-118551
(ICI) was used at 10 μM. Cells were labeled with phalloidin (green) and nuclei were stained
with propidium iodide (red). Scale bars: 100 μm. b) Pictures show lumen presence after βadrenergic stimulation. c) Brightfield microscopy images of MCF-10A 3D culture under
control or ISO condition. d) Left: H&E-stained sections of mammary glands of weaned or
pubertal mice treated or not (control) with ISO. Insets show contra-lateral mammary whole
mounts. Right: quantification of mammary branching. e) Left: immunohistochemistry for
Ephrine B-1 and EphA2 in control and ISO-treated pubertal mice. Right: quantification of
protein staining. Scale bars: continuous: 200 μm, dotted: 50 μm. f) Growth of the offspring
of mothers who received ISO treatment before pregnancy (BP) and/or during lactation
(DL). Statistical significance was assessed using t test. *p<0.05, **p<0.01, ***p<0.001.
Data are representative of three independent experiments.
24
Figure 3. Estrogen receptor alpha (ERα) is essential for isoproterenol (ISO) exerting
its effect on mammary branching. a-b) Mammary gland whole mounts and branching
quantification of non-treated mice (control), telapristone acetate (TELA), mifepristone (also
known as RU-486, RU), PR knockout (PRKO) mice, fulvestrant (FULV), tamoxifen
(TAM) and ovarioctomized (OVX) mice, treated or not (-) with ISO for 15 days. Scale
bars: 200 μm. c) H&E-stained sections of mammary gland of WT, PRKO, FULV or OVX
mice treated or not with ISO. Scale bars: 50 μm. d) Lumen area, lumen cell number and
presence or not of lumen content in Control, FULV, OVX or PRKO mice treated or not
with ISO. Statistical significance was assessed using ANOVA-Dunnet test. *p<0.05,
**p<0.01, **p<0.001. Data are representative of three independent experiments.
Figure 4. a) Estrogen receptor α (ERα) expression in MCF-10A cell culture (2D) or
tridimensional cell culture (3D) and MCF-7 cells after Isoproterenol (ISO) treatment. Ten
μg of protein were loaded. MCF-7 and T47D were used as positive control. b) Brightfield
(left) or confocal (right) photos of MCF-10A growing onto Matrigel. Cells were treated or
not (control) for 15 days with 1μM ISO in the presence or absence of Fulvestrant (FULV).
Scale bars: 100 or 50 μm, as stated in the figure. c) ERα immunohistochemistry and
quantification in mammary gland for control, FULV or ovarioctomized (OVX) pubertal
mice treated or not (control) with ISO. Scale bars: 20 μm. Statistical significance was
assessed using Student t test (a) or ANOVA-Bonferroni test (c). *p<0.05, ***p<0.001. Data
are representative of three independent experiments.
Figure 5. Iso induces FGF family protein expression. Left: Immunohistochemistry and
quantification for a) FGFR2, b) FGF10 and c) FGF2 in mammary gland of mice treated or
not (CONTROL) with isoproterenol (ISO), in fulvestrant treated (FULV) or
ovarioctomized (OVX) pubertal mice. Scale bars: 50 μm. Right, western blot for FGFR2,
FGF10 and FGF2 of MCF-10A cells treated or not with 1μM ISO. Actin was used as
loading control. Statistical significance was assessed using ANOVA-Bonferroni test (left
panels) or Student t test (right panels). *p<0.05, **p<0.01, ***p<0.001. Data are
representative of three independent experiments.
25
Figure 6. Isoproterenol effect on a tumor context. a) Fluorescence staining of MCF-7
growing onto Matrigel. Cell were treated or not (control) for 15 days with 1μM
isoproterenol (ISO), 1μM epinephrine (EPI) or 10 μM forskolin (FK). β2-AR selective
antagonist ICI-118551 (ICI) was used at 10 μM. Cells were labeled with phalloidin (green)
and nuclei were stained with propidium iodide (red). Scale bars: 200 μm. b) Pictures show
lumen presence after β-adrenergic stimulation. c) Effect of ISO on murine mammary tumor
growth at 25 day. Statistical significance was assessed using Student t test, *p<0.05. d)
Hematoxilin-eosin staining (H&E) and immunohistochemistry for GATA3 for murine
breast tumor. Statistical significance was assessed using Student t test, ***p<0.001.
26