Carcinogenesis vol.32 no.11 pp.

1724–1733, 2011
doi:10.1093/carcin/bgr196
Advance Access publication September 1, 2011

Activation of the mTOR pathway by low levels of xenoestrogens in breast epithelial cells
from high-risk women

William H.Goodson III1,2,
, Maria Gloria Luciani1, Introduction

S.Aejaz Sayeed1,3, Ian M.Jaffee2, Dan H.Moore II1,5 and
Bisphenol-A (BPA) and methylparaben (MP) are xenoestrogens (XEs),
Shanaz H.Dairkee1 i.e. non-steroidal chemicals that act like estrogens (1,2). BPA, a com-
1
California Pacific Medical Center, Research Institute, San Francisco, monly used plasticizer, is so widely dispersed in the environment that 9
CA 94107, USA, 2Department of Surgery, California Pacific Medical Center, of 10 North Americans test positive in random urine samples (3,4). BPA
San Francisco, CA 94115, USA, 3Department of Cancer Biology, Kimmel has a short physiologic half-life, but due to continuous environmental
Cancer Center, Philadelphia, PA 19107, USA, 4Department of Pathology, exposure, BPA is routinely detected in human blood (5), placenta, cord
California Pacific Medical Center, San Francisco, CA 94115, USA and (fetal) blood (6), fetal liver (7) and breast milk (8). BPA binds to estro-
5
Department of Epidemiology and Biostatistics, University of California, gen receptors (ER) a and b (9,10) and reverses antiestrogen- (11) and
San Francisco, CA 94115, USA chemotherapy-induced cytotoxicity in cancer cell lines (12). BPA in-

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To whom correspondence should be addressed. Tel: þ1 415 923 3925; duces upregulation of AKT (v-Akt murine thymoma viral oncogene
Fax: þ1 415 776 1977; homolog 1) in association with increased proliferation and decreased
Email: goodsow@cpmcri.org apoptosis of epithelial cells in breast tissue of lactationally exposed rats
Correspondence may also be addressed to Shanaz H.Dairkee. (13), as well as histological changes associated with mouse mammary
Tel: þ1 415 600 1653; þ1 415 600 1775;
carcinogenesis after in utero exposure (14). These effects are specific to
Email: dairkes@cpmcri.org
breast tissues since BPA treatment of adipocytes (15) and leukemia cells
Breast cancer is an estrogen-driven disease. Consequently, hor- (16) reduces phosphorylation of the serine/threonine protein kinase
mone replacement therapy correlates with disease incidence. AKT and promotes terminal differentiation and cell death. MP, a com-
However, increasing male breast cancer rates over the past three mon preservative in medicines, toiletries and skin care products (2), is
decades implicate additional sources of estrogenic exposure in- detected in human breast tumors (2,17) and induces estrogenic signaling
cluding wide spread estrogen-mimicking chemicals or xenoes- in the MCF7 breast cancer cell line (18,19). Because breast cancer
trogens (XEs), such as bisphenol-A (BPA). By exposing incidence is proportional to estrogen exposure (20,21), there is concern
renewable, human, high-risk donor breast epithelial cells that such estrogen mimics have contributed to increased breast cancer in
(HRBECs) to BPA at concentrations that are detectable in hu- both women and men over the last three decades (22,23).
man blood, placenta and milk, we previously identified gene To test the validity of insights acquired from animal and cancer cell
expression profile changes associated with activation of mam- line models, we developed assays based on renewable, early passage,
malian target of rapamycin (mTOR) pathway genesets likely to non-malignant, high-risk donor breast epithelial cell (HRBEC) cultures
trigger prosurvival changes in human breast cells. We now pro- derived from fresh human samples. In global gene expression analysis,
vide functional validation of mTOR activation using pairwise HRBECs exposed to a low concentration of BPA exhibited geneset alt-
comparisons of 16 independent HRBEC samples with and with- erations that predicted activation of the mammalian target of rapamycin
out BPA exposure. We demonstrate induction of key genes and (mTOR) pathway (24) thereby implicating XE-induced effects in desta-
proteins in the PI3K-mTOR pathway—AKT1, RPS6 and 4EBP1 bilizing a central function in normal cells. For example, downregulation
and a concurrent reduction in the tumor suppressor, phospha- of the mTOR pathway often occurs in a nutrient-poor microenvironment,
tase and tensin homolog gene protein. Altered regulation of thereby, limiting cell proliferation and allowing cell death through apo-
mTOR pathway proteins in BPA-treated HRBECs led to ptosis and autophagy (25). However, when activated by hormones and/or
marked resistance to rapamycin, the defining mTOR inhibitor. abundant nutrition, or when co-opted in cancer development, mTOR
Moreover, HRBECs pretreated with BPA, or the XE, methyl- signaling initiates protein synthesis, cell proliferation and evasion of
paraben (MP), surmounted antiestrogenic effects of tamoxifen apoptosis (25,26). In an independent set of HRBEC samples from similar
showing dose-dependent apoptosis evasion and induction of cell high-risk individuals, we now demonstrate activation of key mTOR
cycling. Overall, XEs, when tested in benign breast cells from pathway proteins induced by XE exposure and downstream functional
multiple human subjects, consistently initiated specific func- consequences. The use of HRBECs sidesteps issues of interspecies var-
tional changes of the kind that are attributed to malignant onset iation by testing cells from at-risk humans and bypasses issues of dose,
in breast tissue. Our observations demonstrate the feasibility of route of delivery and metabolism by examining the effects of XE con-
studying renewable human samples as surrogates and reinforce centrations found in human tissues and body fluids (27,28). Because live
the concern that BPA and MP, at low concentrations detected in HRBECs are drawn directly from the population of interest, i.e. the
humans, can have adverse health consequences. heterogeneous population of women at high risk of breast cancer occur-
rence, they serve well as surrogates for the effects of XEs on this pop-
ulation. The functional changes induced by BPA and MP closely parallel
known outcomes of mTOR pathway activation (26) and tumor behavior
(29) and reveal an underlying mechanistic basis for restricted effective-
ness of breast cancer treatment and prevention strategies.
Abbreviations: AKT, v-Akt murine thymoma viral oncogene homolog 1;
BPA, bisphenol-A; ER, estrogen receptor; HRBEC, high-risk donor breast
epithelial cell; MFI, mean fluorescence intensity; MP, methylparaben; mTOR, Materials and methods
mammalian target of rapamycin; OHT, 4-hydroxy tamoxifen; PI, propidium
iodide; PTEN, phosphatase and tensin homolog gene; QPCR, quantitative Random periareolar fine needle aspirate collection and cytopathology
polymerase chain reaction; ROS, reactive oxygen species; RPFNA, random With Institutional Review Board-approved written informed consent, non-
periareolar fine needle aspirate; XE, xenoestrogens. malignant cells were obtained by random periareolar fine needle aspiration from

Ó The Author 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 XE-induced hallmarks of cancer

the unaffected contralateral breast of high-risk women undergoing breast surgery. Quantitation of endogenous reactive oxygen species
We use the acronym HRBEC to represent ‘high-risk donor breast epithelial cells’. Endogenous cellular reactive oxygen species (ROS) accrual was determined
HRBEC donors were recruited based on personal or family history of breast using live cell cultures loaded with the fluorogenic dye, carboxy-H2DCFDA
cancer, atypical or neoplastic histopathology on biopsy and/or high mammo- (5-(and-6)-carboxy-2#7#-dichlorodihydrofluorescein diacetate), also known as
graphic density (30). Specimen size was 0.2 ml per volunteer including extra- C-400 (Invitrogen) in phenol red-free medium for 1 h followed by 30 min
neous fat, blood and debris. Random periareolar fine needle aspirate (RPFNA) cell incubation in regular growth medium. After dye removal, cells were counter-
suspension was divided into aliquots for cytopathology and cell culture. stained with PI, and intracellular oxidation of C-400 was measured by FACS-
The cytology aliquot was collected in CytolytTM, centrifuged at 600 r.p.m. for can with the FL1 filter. Experiments were done in triplicate and 10 000 cells
10 min, transferred under vacuum to 20 mm circles on ThinPrepTM microscope were acquired for each sample. ROS activity was expressed as mean fluores-
slides using the ThinPrepTM 2000 processor (Cytec Corp., Boxborough, MA), cence intensity (MFI) of the C400 dye. MFI of PI-negative (non-necrotic) cells
fixed in 95% alcohol for 10 min and processed through an automated staining was corrected for autofluorescence of unlabeled cells.
and mounting set up (Sakura Finetek USA, Torrance, CA). The entire 20 mm
circle was evaluated by a board-certified cytopathologist (I.J.) for cytological
atypia, and the number of epithelial and stromal cell clusters was counted.
Aliquots for cell culture were transferred into DME-F12 serum-free growth
Results
medium and transported on ice to the laboratory for delivery within 2 h. Live HRBEC cultures display non-malignant breast epithelial
Propagation and chemical exposure of HRBECs attributes

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Epithelial cells within RPFNA samples were plated and propagated in MCDB In this study, HRBECs were derived from the unafflicted contralateral
170 medium supplemented with 2% fetal bovine serum as described previously breast tissue of 23 individual volunteers (mean age—58, range 40–81
(24). Twenty-three independent samples were expanded in vitro and used in the years). Sixteen women had a personal history of breast cancer, 4
assays described below. The breast cancer cell lines—T47D, MCF7 and displayed high-risk histopathology, 20 had mammographically dense
SKBR3—were used as controls and adapted to the same growth medium as breasts and 19 had family history of breast cancer (supplementary
HRBEC cultures prior to each assay. After 2–3 weeks of expansion, HRBECs
were harvested for a variety of functional response tests.
Table S2 is available at Carcinogenesis Online). Routine cytology
To determine three-dimensional growth patterns, HRBECs were plated as of the samples prior to cell culture showed multiple epithelial clusters
single cell suspensions in a semisolid growth substrate comprised 3% Matrigel (range 4 to .200). No atypia was seen (Figure 1A). RPFNA samples
(BD Biosciences, San Jose, CA) and propagated for 10 days. Colonies fixed yielded short-term epithelial cultures with variable degrees of growth
with 1:1 methanol:acetone were incubated with anti-alpha-6 integrin (BD potential. Generally, the initial cell seeding produced robust growth
Pharmingen, San Diego, CA) followed by fluorescence-tagged secondary an- within 2–3 weeks, enabling culture expansion for up to three passages
tibody and propidium iodide (PI) nuclear counterstain. Immunostained colo- and providing morphologically homogeneous populations of 106–107
nies were visualized by confocal microscopy. epithelial cells. Due to the low serum concentration of the growth
For XE exposure, cells were plated at a density of 100 000 cells per well in medium, stromal fibroblast expansion did not occur (Figure 1B).
six-well plates and exposed to continuous 7 day treatments with 17b-estradiol
(5 nM), 100 pM to 100 nM BPA or 10 nM to 1 lM MP in phenol red-free
There were no selection criteria for RPFNA sampling except that
medium supplemented with 0.2% charcoal-stripped fetal bovine serum. Where the volunteer present a clinically defined increased risk of breast
indicated, cells were exposed to 10 lM 4-hydroxy tamoxifen (OHT) or to 1, 10 cancer. HRBECs were used as they grew out in sufficient numbers
or 100 nM rapamycin for 24 h prior to functional analysis. All chemicals were for one or more assays. Identification of the samples employed is
purchased from Sigma–Aldrich (St. Louis, MO). included in the relevant data figures. Our overall cell culture experi-
ence with .130 RPFNA samples thus far yielded three spontaneously
Protein detection by western blot analysis
immortalized (IMM) HRBEC cell lines, designated as IMM-PA024,
Cells were lysed in denaturing lysis buffer in the presence of protease and IMM-PA025 and IMM-PA115, currently at passages 24–26.
phosphatase inhibitors and sonicated to disrupt cellular and nuclear mem-
branes. Equal amounts of cell lysates were loaded onto 4–15% gradient gels
Non-malignant epithelial characteristics of HRBEC cultures in-
for sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, trans- cluded:
ferred to PVDF membranes and used for immunodetection with primary anti- a. Polarized growth in Matrigel—unlike breast cancer cell lines
bodies to ERa (Santa Cruz Biotechnology, Santa Cruz, CA), ERb (Genetex, propagated in a three-dimensional matrix (which develop apolar col-
San Antonio, TX), phosphatase and tensin homolog gene (PTEN) (Cell Sig- onies with random orientation of nuclei and the basement membrane
naling Technology, Beverly, MA), PTEN phosphorylated at S380/T382/T383 protein, alpha-6 integrin), HRBEC colonies were distinctive. Polar-
(Santa Cruz Biotechnology), AKT1 (Santa Cruz Biotechnology), AKT1 phos- ized colonies, which displayed an acinar pattern of nuclear orientation
phorylated at S473 (Santa Cruz Biotechnology), RPS6 (Genetex), RPS6 phos- and characteristic basal immunolocalization of alpha-6 integrin, were
phorylated at S235/S236 (Genetex) and 4EBP1 (Santa Cruz Biotechnology). observed in all HRBEC cases tested (Figure 1C).
Actin, served as a loading control.
b. ER expression—distinct from ER-positive breast cancer cell
Real time quantitative polymerase chain reaction analysis lines, represented by T47D and MCF7, which express abundant
Total RNA was isolated using the RNeasy Kit (Qiagen, Valencia, CA) from ERa, and the ER-negative cancer cell line, SKBR3 in which ERa is
untreated control and XE-treated cells. RNA concentration was determined by undetectable, HRBEC lines (IMM-PA024, IMM-PA025 and IMM-
NanoDrop 2000 (Thermo Scientific, Lafayette, CO). Complementary DNA PA115) displayed a low to moderate range of receptor protein (Figure
was synthesized and analyzed as before (31) by an Applied Biosystems 1D). On the other hand, levels of ERb were relatively similar between
5700 Sequence Detection System (Applied Biosystems, Foster City, CA). HRBEC and cancer cell lines. Protein levels of both ER isoforms in
The Ct values of test genes were normalized to the expression of the house- six independent early passage HRBEC cultures (PA134, PA135,
keeping genes, ACTB and GAPDH, within each HRBEC sample to represent
log base 2-fold increase or decrease in test gene expression over no XE con-
PA136, PA138, PA139 and PA140) were closely similar to those of
trols. Primer sequences for test genes are listed in supplementary Table S1 spontaneously immortalized counterparts. We conclude that unlike
(available at Carcinogenesis Online). many cancer cell lines, HRBECs represent an ERa-low/moderate
status. Levels of both ERa and ERb proteins are consistent with the
Analysis of apoptotic and S-phase cell fractions characteristics of non-malignant human breast cells (32,33).
For quantitation of apoptotic cells, cultures were harvested and stained with An- Early passage HRBEC cultures (passage 2 or 3) of 23 independent
nexin V-FITC and PI (BD Biosciences) according to the manufacturer’s protocol. cases were used to generate subsets of data described below. HRBECs
Cells diluted in binding buffer were analyzed by FACScan (BD Biosciences) and from 16 cases were used for pairwise analysis in functional assays
quantified by CellQuest software (BD Biosciences). Annexin-V-positive cells
(supplementary Table S2 is available at Carcinogenesis Online). Like
were measured as ‘early’ (PI-negative) and ‘late’ (PI-positive) apoptotic cell frac-
tions. Each measurement was performed in multiple replicates. normal human epithelial cells, HRBECs display a finite life and sen-
For S-phase quantitation, cells were pulse labeled with 10 lM bromodeox- esce after three passages, providing a limited amount of starting ex-
yuridine for 1 h, stained with anti-bromodeoxyuridine (Santa Cruz Biotechnol- perimental material. For this reason, it is usually possible to pursue
ogy), FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA), only one or two whole cell-based assays from a single sample, such as
counterstained with PI and analyzed by FACScan. determination of apoptotic fraction, ROS estimation or assays based

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Fig. 1. RPFNA-derived HRBEC cultures display characteristic non-malignant epithelial phenotypes. (A) Micrographs of cell clusters present in independent
representative RPFNA samples. (B) Early passage pure epithelial cultures derived from clusters similar to those shown in A (Brightfield,  4 objective). (C) Three-
dimensional growth pattern of colonies derived from early passage HRBEC cultures in Matrigel. Basal immunolocalization of alpha-6 integrin (blue) and acinar
orientation of nuclei (red) in 10 day-old colonies. (D) Relative ERa and ERb protein levels in breast cancer cell lines (T47D, SKBR3 and MCF7), spontaneously
immortalized HRBEC lines (IMM-PA024, IMM-PA025 and IMM-PA115) and early passage HRBECs (PA134, PA135, PA136, PA138, PA139 and PA140)
quantitated by western blotting. Numerical values of each protein band representing ERa or ERb obtained by densitometric scanning were normalized to actin
levels in the lysate and plotted. Note low to moderate ERa levels in HRBECs relative to T47D and MCF7, but higher than the SKBR3 cell line, HRBECs display
a distinctive ER profile unlike conventional ER-positive or ER-negative breast cancer cell lines.

on RNA amplification, for example by quantitative polymerase chain BPA exposure modulates the signal transduction cascade of the
reaction (QPCR) or by microarrays (reported by us previously). Sim- PI3K–mTOR pathway within non-malignant breast epithelial cells
ilarly, early passage HRBEC-derived protein lysates are often insuf- By QPCR analysis of the subset of critical mTOR pathway genes,
ficient for quantitative sodium dodecyl sulfate–polyacrylamide gel early passage HRBECs derived from six individuals demonstrated
electrophoresis and western blot analysis. Therefore, we have in- identifiable shifts in expression associated with BPA exposure (Figure
cluded immortalized-HRBEC lines in protein quantitation studies. 2A). Considerable inter-subject variability in relative transcript levels

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Fig. 2. BPA exposure modulates expression of mTOR pathway components and induces functional changes in breast epithelial cells. (A) QPCR measurements of
relative transcript levels of mTOR pathway genes in early passage HRBEC cultures (PA024, PA025, PA072, PA075, PA081 and PA112) normalized to
housekeeping genes. Data represent exposure to a low dose range of BPA or to a luteal serum estradiol (E2) level. Plotted values represent fold change for each
gene in treated samples, relative to the corresponding untreated control sample. Each data point is an average of triplicate QPCRs. (B) BPA-induced alterations in
mTOR pathway proteins in early passage (PA138 and PA140), immortalized HRBECs (IMM-PA024, IMM-PA025 and IMM-115) and breast cancer cells (T47D).
Pretreatment with BPA reduces steady-state PTEN protein levels and promotes functional inactivation by increased phosphorylation (indicated by ‘P’) at sites
S380/T382/T383 compared with untreated controls. Increased expression of total and phosphorylated AKT1 (at S473), RPS6 (at S235/236) and 4EBP1 (at multiple
sites) is detectable in both non-malignant and malignant breast cells. Asterisks indicate shift in the molecular weight of phosphorylated 4EBP1. (C) Effects of BPA
pretreatment in the induction of resistance to the mTOR inhibitor, rapamycin. Data plotted to represent Annexin V-positive apoptotic populations within breast
cancer cell lines (T47D, SKBR3—left panel) and HRBEC lines (IMM-PA024, IMM-PA025, IMM-PA115—right panel). Each bar represents the mean and
standard deviation of triplicate values. Increased apoptotic ratios were observed with increasing doses of rapamycin in all cultures without BPA pretreatment.
Values demonstrating the effect of BPA in reducing rapamycin-induced apoptosis were statistically significant in all cell lines (P . 0.001).
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was associated with the cellular BPA response, due to which dose– XE exposure modulates cellular oxidative stress in HRBEC cultures
response relationships were not detectable in this small sample size. To determine whether functional changes in apoptosis evasion and
However, normalized to housekeeping genes, a consistent decline was cell survival were with a reflection of oxidative stress reduction, levels
noted in transcript levels of the suppressor genes, PTEN (P 5 0.02, of endogenous ROS were quantified in early passage HRBECs, IMM-
two-tailed t-test against theoretical ratio 5 1.0), TSC1 and TSC2 (P 5 HRBEC lines and breast cancer cell lines. The MFI of C400-stained
0.03). Conversely, downstream activators of the mTOR pathway, cells was used as an indicator of ROS. OHT treatment was used as
e1F4B and e1F4E, were upregulated in the presence of BPA (P 5 a positive control for ROS induction. Both BPA and MP induced
0.003). Expression of PIK3R1, another mTOR activator, was ele- a detectable decline in endogenously accumulated ROS in all cell
vated (P 5 0.02), transcript levels of RPS6, a downstream effector of cultures during the 7 day exposure period (Figure 4A). Representative
mTOR that is activated by phosphorylation, were unexpectedly FACS data are illustrated in Figure 4B.
lower (P 5 0.03) and there was a trend (P 5 0.07) toward increased In further experiments, neutralization of ROS was measured in re-
mTOR transcripts. sponse to increasing log concentrations of BPA and MP exclusively in
Confirmation of the observed transcriptional alterations was sub- early passage HRBECs (Figure 4C as averaged data and supplemen-
sequently pursued at the protein level comparing two early passage tary Figure S2 is available at Carcinogenesis Online for each sample).
HRBEC cultures, three immortalized HRBEC lines and an ERa-pos- The average reduction in ROS by BPA treatment was 26% (95% CI
itive breast cancer cell line, T47D (Figure 2B). As predicted by gene 24–28%; P , 0.001, two-sided t-test against theoretical ratio 5 1.0,

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transcript quantitation, BPA exposure led to the following: a signifi- Figure 4C, left panel). Similarly, the average ROS reduction by MP
cant reduction in total PTEN protein levels; an increase in the inactive treatment was 38% (95% CI 24–48%; P , 0.02, Figure 4C, right
phosphorylated form of PTEN (PTENSer380/2/3) and increased total panel). A dose–response relationship was not observed for this end
and phosphorylated (activated) forms of the mTOR activating kinase, point.
AKT1 and two major downstream targets of mTOR: RPS6 and of
4EBP1 (at multiple sites, as shown by the increased molecular weight
of the phosphorylated protein). Unlike RPS6 transcript data, pRSP6 XE-mediated cell cycle changes surmount drug-induced HRBEC
levels were representative of mTOR pathway activation. Increased growth inhibition
phosphorylation observed for upregulated proteins is strongly indic- To determine whether resistance to OHT-induced apoptosis in BPA or
ative of increased mTOR activity and consistent with the finding of MP-pretreated cells was accompanied by the maintenance of prolifera-
higher mTOR transcript levels. tive potential, we measured the efficiency of bromodeoxyuridine incor-
poration during the S-phase of the cell cycle in IMM-HRBEC lines.
Prior exposure to a dose range of either BPA or MP resulted in a dra-
XE exposure induces evasion of apoptotic cell death in HRBEC matic, concentration-dependent complete to partial evasion from the
cultures G1-phase arrest induced by 10 lM OHT and a concurrent increase in
In order to establish additional functional association with mTOR the S-phase fraction (Figure 5A and B). In contrast, the growth inhibi-
pathway regulation, we asked whether BPA pretreatment altered the tory effects of OHT were not reversed by a simulated physiological
sensitivity of IMM-HRBEC lines to the antitumor drug—rapamycin, scenario of a combination of luteal phase serum levels of 17b-estradiol
a potent mTOR inhibitor. A significant reduction in rapamycin-in- and progesterone in the absence of BPA (data not shown). As with
duced apoptotic cell death was observed after BPA exposure of im- apoptosis evasion, maintenance of S-phase in OHT-treated cells was
mortalized HRBECs and in ERa-positive as well as ERa-negative significantly correlated with increasing concentrations of BPA and
breast cancer cells (Figure 2C). This effect of BPA in circumventing MP (P , 0.001 for BPA; P , 0.001 for MP, two-sided test for mixed
cell death was most prominent at 100 nM rapamycin, a concentration effects linear regression).
at which the apoptotic ratio of drug-treated versus untreated control
was .2-fold in the absence of prior BPA exposure.
To further assess a role for XEs in the evasion of apoptotic cell Discussion
death, the antiestrogenic OHT was used to initiate cell death. Early
passage HRBECs from eight subjects were compared with IMM- We demonstrate that BPA activates the mTOR pathway in non-
HRBEC lines and with breast cancer cell lines. Percent apoptotic malignant HRBECs. Both transcript and protein quantitation analyses
cells were estimated in untreated control cells, those pretreated reflected changes in the most important representative elements of this
with 100 nM BPA or 1 lM MP prior to a 24 h OHT exposure or signaling pathway. In live cells, BPA suppresses apoptosis, enhances
those exposed to OHT alone. Although the percentage of apoptotic S-phase and decreases ROS levels, a well-known prelude to apoptosis
cells was doubled in the presence of OHT alone in all cell cultures evasion. Live cells respond in a similar manner to the XE, MP, sug-
tested, the effect of OHT was almost undetectable in XE-treated gesting that mTOR activation might be a general effect of XEs. These
cultures (Figure 3A). Representative FACS data are illustrated in functional assays serve to test and confirm the consequences of mTOR
Figure 3B. activation predicted by global expression profiling of a previous in-
In separate experiments, early passage HRBECs were evaluated dependent set of BPA-exposed HRBECs (24).
for the potential to evade apoptosis after exposure to a wide range of Chemicals tested here are so common in bodily fluids (3–8) as to
BPA and MP concentrations (Figure 3C as averaged data and sup- make unexposed control subjects functionally, if not literally, unavail-
plementary Figure S1 is available at Carcinogenesis Online for each able. We provide a pragmatic approach to overcome this problem by
sample). Maximum protection from apoptotic death was conferred using live, renewable breast epithelial cell samples from high-risk do-
by exposure to 100 nM BPA (58.39 ± 6.8%). An appreciable degree nors (HRBECs) propagated in vitro with and without the chemicals of
of apoptosis reduction was also observed at 10- to 100-fold lower interest for pairwise comparisons of known end points of mTOR acti-
BPA doses: 52.84 ± 6.8% at 10 nM, 41.24 ± 10.4% at 1 nM and 19.93 vation. HRBECs are samples from women who, based on extensive
± 7.9% at 100 pM (Figure 3C, left panel). A significant dose–re- epidemiology, are identifiable as predisposed to malignant progression,
sponse was observed when log BPA concentration was regressed on even before overt cytopathological alterations are present. The oppor-
log percent reduction in apoptosis (P 5 0.002, two-sided test for tunity to demonstrate a concordant set of multiple end points portraying
logistic regression). MP exposure of HRBECs also dramatically re- the status of the mTOR metabolic pathway—despite small sample
duced the fraction of OHT-induced apoptotic cells at all three doses size—underscores the suitability of this source of target human cells
tested: 57.82 ± 6.77% at 1 lM (Figure 4A), 55.93 ± 10.54% at 100 for recapitulating early functional changes induced by carcinogen ex-
nM and 28.14 ± 11.3% at 10 nM (Figure 3C, right panel). Similar to posure. There is, and always will be, a gap between human biology
the BPA dose response, MP-induced changes in HRBECs were also in vivo and its representation by surrogate in vitro models, but unlike the
concentration dependent (P 5 0.001). limited genotypic variation represented by common immortalized cell

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Fig. 3. XE exposure promotes apoptosis evasion in HRBEC cultures. (A) Potential for XE-induced apoptosis evasion measured as percent reduction in Annexin V-
positive cells by FACS analysis. Breast cancer cell lines (T47D and SKBR3), HRBEC cell lines (IMM-PA024, IMM-PA025 and IMM-PA115) and early passage
HRBEC (PA094, PA099, PA103, PA106, PA107 and PA130) exposed to BPA or MP were treated with OHT for 24 h prior to Annexin V staining and compared
with untreated controls. All experiments were performed in triplicate. Plots illustrate average values and the standard deviation for each culture group under
conditions of no treatment, XE exposure followed by OHT or OHT treatment alone. Values demonstrating the effect of XEs in reducing OHT-induced apoptosis are
statistically significant in all cases (P , 0.002). (B) FACS profiles of representative samples. M1 fraction—autofluorescence; M2—Annexin V-positive cells
(which increase with OHT treatment). (C) Dose–response measurements (shown as decreasing XE concentrations from left to right) in early passage HRBECs. In

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Fig. 4. XE exposure alters oxidative stress levels in breast epithelial cells. (A) Comparative analysis of intracellular ROS levels measured and quantified by FACS
analysis of C400-stained cancer cell lines (T47D and SKBR3), HRBEC lines (IMM-PA024, IMM-PA025 and IMM-PA115) and early passage HRBEC cultures
(PA094, PA099, PA103, PA106, PA107 and PA130) exposed to BPA or MP. A post-XE 24 h treatment with tamoxifen (OHT) was used to induce ROS. All
experiments were performed in triplicate. Averaged data representing the MFI of C400 are plotted, and standard deviations are shown. Values demonstrating the
effect of XEs in reducing OHT-induced ROS are statistically significant in all cases (P , 0.0001). (B) FACS profiles of representative samples. The area under
each curve reflects C400-positive cells. Note the right shift of the C400 peak in OHT-treated samples (indicating higher MFI) when compared with untreated
control populations (top two panels) and the mild reduction of MFI in cells exposed to XEs (bottom two panels). (C) ROS levels measured as C400 MFI in early
passage HRBECs exposed to various concentrations of BPA or MP. Results are expressed as percent reduction from baseline MFI of no XE controls. Each data
point represents an average of six independent HRBEC samples (shown individually in supplementary Figure S2, available at Carcinogenesis Online). Error bars
display the variation between cases.

lines, HRBECs more closely reflect the diversity of genetics, exoge- subjects displayed prosurvival changes after XE exposure does not
nous hormone use, life events such as pregnancy, etc. of women en- claim that all exposed persons will develop cancer, only that such
countered in clinical practice. Our demonstration that HRBECs from all exposure can cause changes that may facilitate malignant progression.

all cases, the protection from OHT-induced apoptosis (apoptotic evasion) was calculated as a fraction of the apoptotic response in the absence of XEs (set to 1).
Each data point represents an average of eight independent HRBEC samples (shown individually in supplementary Figure S1 is available at Carcinogenesis
Online). Error bars display the variation between cases (triplicate values for each case). Note a striking dose–response effect for both XEs, despite variability
between samples in the protection from OHT-induced apoptosis.

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Fig. 5. XE exposure compromises tamoxifen-mediated cell cycle arrest in breast epithelial cells—(A) Cell cycle data derived from bromodeoxyuridine labeling of
HRBEC lines (IMM-PA024, IMM-PA025 and IMM-PA115). Representative pie charts illustrate percent cells in different phases of the cell cycle (red—S-phase,
yellow—G2, green—G1 and blue—sub G1). Note reversal of OHT-induced G1 arrest and subsequent S-phase decline in XE-pretreated cells. IMM-PA115 is
relatively insensitive to the MP concentration shown compared with the other two HRBEC lines. (B) Graphical summary of S-phase reduction in OHT-treated cells
and maintenance of S-phase by BPA (left panel) and MP (right panel) pretreatment in response to decreasing concentrations (from left to right). Plots represent an
average of the combined bromodeoxyuridine-positive populations and standard deviations around the mean within independent HRBEC lines exposed to either
BPA or MP (IMM-PA024, IMM-PA025 and IMM-PA115), shown individually in supplementary Figure S3 (available at Carcinogenesis Online).

Human cell samples that maintain phenotypes relevant to clinical in Matrigel, pAKT localizes to peripheral cells, away from the central
targets of interest are essential for carcinogenicity testing. For exam- apoptotic region (38). Moreover, constitutive pAKT expression pre-
ple, to evaluate the role of XEs in the initiation of breast cancer, vents lactogenic differentiation (39). Expectedly therefore, pAKT is
HRBECs are particularly well suited because they consistently ex- increased during RAS-induced carcinogenesis of MCF10 xenografts
press low/moderate levels of both ERa and ERb. Earlier studies that (40) and pAKT is necessary for carcinogenic progression in this
did not distinguish ER isoforms (as continues in current clinical prac- model since blocking pAKT results in apoptotic cell death (40). Clin-
tice where primarily ERa is measured) typically reported low ER ically, a similar progression is observed whereby pAKT is signifi-
positivity in normal or benign breast tissue (32–34), suggesting that cantly higher in malignant than benign breast tissue (41) and pAKT
this phenotype is sufficient for agonistic activity of natural and syn- levels in cancer correlate directly with poor prognosis (42,43). Con-
thetic estrogens, as well as for antagonistic effects of tamoxifen in versely, in vitro PTEN inactivation by phosphorylation and reduction
reducing breast cancer incidence in randomized trials (35,36), even in in protein levels parallel Cowden’s syndrome where PTEN loss of
patients harboring ER-negative atypia (37). Thus, high ERa levels are function is associated with an increase in breast and other cancers
not a prerequisite for XEs to exert their biological effects either clin- (44,45). Downstream, mTOR acts by phosphorylating (activating)
ically or in vitro. Similarly, reversal of tamoxifen toxicity by exposure 4EBP1 and RPS6 (indirectly through p70S6K activation). Both
to the common XEs, BPA and MP, implies that the ER profile of p4EBP1 and its target eIF4E increase during experimental carcino-
HRBECs is also sufficient for the induction of cancer-associated phe- genesis (40); p4EBP1 is higher in cancer than in benign biopsies (41)
notypes by estrogenic chemicals. and associated with higher tumor grade (46); and consequently,
Against the backdrop of non-malignant ER levels simulated by p4EBP1 correlates directly with tumor recurrence in women (46).
HRBECs, BPA-induced transcriptional and protein alterations char- In vitro, pRPS6 is downregulated by exposure to rapamycin (a de-
acteristic of breast cancer were discernible. For example, activation fining mTOR inhibitor) in 12/12 cell lines (46,47). In one series,
by phosphorylation of AKT is a key regulatory step in subsequent pRPS6 did not correlate with prognosis, but it was upregulated in
mTOR activation and induction of cell growth (25). During in vitro the majority (77%) of breast cancers (46). mTOR activation by
morphogenesis and differentiation of acini from non-malignant cells BPA exposure is further defined by inhibition of rapamycin-induced

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W.H.Goodson III et al.

apoptosis (48), which is the converse of the therapeutic benefit of experiments and data analysis. D.H.M.—biostatistics. I.M.J.–cytopathology.
rapamycin analogs achieved through suppression of the mTOR path- All authors approved the final manuscript.
way (49,50). In vitro pretreatment of HRBECs with BPA essentially
replicates the continuous environmental exposure underlying positive Conflict of Interest Statement: None declared.
urine tests in the general population. The failure of rapamycin to
induce apoptosis after BPA treatment raises concern that activation
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Cancer Res., 13, 81–89. Received May 7, 2011; revised August 2, 2011; accepted August 23, 2011

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