J. Biomedical Science and Engineering, 2010, 3, 476-483
doi: 10.4236/jbise.2010.35066 Published Online May 2010 (http://www.SciRP.org/journal/jbise/ JBiSE
Published Online May 2010 in SciRes. http://www.scirp.org/journal/jbise
Inhibition of mammalian target of rapamycin induces
phenotypic reversion in three-dimensional cultures of
malignant breast epithelial cells
Ross Booth, Soonjo Kwon*, Eric Monson
Department of Biological Engineering, Utah State University, Logan, USA.
Email: soonjo.kwon@usu.edu
Received 27 February 2010; revised 11 March 2010; accepted 17 March 2010.
Inhibition of mammalian target of rapamycin (m-
TOR) is a potential method for cancer treatment.
Effects of rapamycin (RAP) on the reversion of ma-
lignant breast epithelial cells were investigated on
three-dimensional (3D) basement membrane extract
(BME) cultures. Through continuous exposure to 20
nM of RAP, cell colony size was sig nificantly reduced
in 3D BME cultures of malignant breast epithelial
cells, while normal cell colony size appeared unaf-
fected. In unfixed 3D BME cultures of normal and
RAP-treated malignant breast epithelial cells, the
presence of luminal cell death was confirmed by
ethidium bromide and propidium iodide labeling.
Increased structural organization was observed by
im- munofluorescence staining of F-actin and β-cat-
enin in RAP-treated malignant breast epithelial cells.
In monolayer cultures of normal and malignant
breast epithelial cells, continuous exposure to 20 nM
of RAP increased caspase 3/7 activity and decreased
proliferation. Reverse transcriptase polymerase ch-
ain reaction (RT-PCR) array analysis indicated a fold
increase in the expression of a number of proteins
related to polarity, cell-cell adhesion, and cell-matrix
adhesion in the presence of RAP. Our data showed
that phenotypic reversion of malignancy can be ach-
ieved through RAP exposure on 3D BME cultures.
This 3D BME culture system will provide correct
microenvironments for observing the effects of other
mTOR inhibitors on phenotypic reversion of malig-
nant breast epithelial cells.
Keywords: Rapamycin; Three-Dimensional Culture;
Breast Cancer Reversion; Basement Membrane Extract;
mTOR Inhibitors
The use of traditional two-dimensional (2D) cultures for
breast cancer studies provides abundant fundamental
information on the regulation of breast cancer, but its
results often fail to properly translate into clinical appli-
cations [1]. The need to effectively address this problem
has led to a demand for culture systems which more ac-
curately represent the tissue environment found in vivo.
In recent years, three-dimensional (3D) culture systems
have been developed which use extra-cellular matrix
(ECM) proteins [2,3]. 3D cultures provide epithelial
cells with spatial freedom to allow the formation of 3D
structures. Furthermore, the presence of reconstituted
basement membrane extract (BME) promotes polariza-
tion of breast epithelial cells into acinar structures with
luminal cell death through communication with mem-
brane-bound integrins [3-7]. BME products are derived
from the Engelbreth-Holm Swarm (EHS) tumor and are
rich in laminin-1, with other ECM components including
procollagen IV, heparin sulfate proteoglycan, and entac-
tin [8]. However, most breast cancer cell lines fail to
exhibit a normal phenotype in BME cultures unless ac-
companied with one or more signal molecules [9]. Sev-
eral signal molecules have previously been shown to
affect phenotypic reversion through limited cell prolif-
eration, increased multi-cellular organization, basolateral
polarization, and programmed luminal cell death. These
reversion factors include inhibitors of β1-integrin, epi-
dermal growth factor receptor (EGFR), and the MAPK
& PI3K kinases [10-12]. Such reversion studies have
provided valuable insight for potential methods of can-
cer treatment.
Rapamycin (RAP), the primary inhibitor for mam-
malian target of rapamycin (mTOR), has been used for
its immunosuppressive properties under the drug name
Sirolimus [13]. However, the mTOR signaling pathway
has been shown to be a component of several signaling
pathways related to breast cancer, including Akt [14],
PTEN [15], and PI3K [16]. The web of signaling path-
ways related to mTOR is complex, and its malignant
effects include increased cell growth, proliferation, and
R. Booth et al. / J. Biomedical Science and Engineering 3 (2010) 476-483 477
Copyright © 2010 SciRes. JBiSE
resistance to apoptosis [17]. Presently, RAP and its ana-
logues are under investigation as potential cancer treat-
ments [15]. In breast epithelial cells transformed to ex-
hibit hyperactivity of the Akt kinase, RAP exposure in-
hibits morphological disruptions caused by Akt overac-
tivity [18]. However, the effects of mTOR inhibition on
breast cancer cell lines cultured in 3D BME are not well
To prov ide further insight into the value of mTOR in-
hibition as a potential cancer treatment, we ob served and
analyzed the phenotypic reversion of malignant breast
epithelial cells in 3D BME following exposure to RAP.
Two malignant breast epithelial cells (BT-483 and
MDA-MB-468) were exposed to RAP and compared to
normal breast epithelial cells (184-B5). Changes in mul-
ti-cellular morphology, cell proliferation, polarity, and
gene expression were investigated in the presence of
RAP on 3D BME cultures.
2.1. Cell Culture
Malignant human breast epithelial cells (BT-483 and
MDA-MB-468) an d normal human breast epithelial cells
(184-B5) were purchased from ATCC (VA). For mo-
nolayer cultures: BT-483 was cultured using RPMI-
1640 (Sigma-Aldrich, MO) with 20% of fetal bovine
serum (FBS) (Thermo Fisher Scientific, UT) and 0.01
mg/mL of bovine insulin, MDA-MB-468 was cultured
using Leibovitz’s L-15 (Sigma-Aldrich) with 10% of
FBS in air with no added CO2, and 184- B5 was cultured
using MEGM (Lonza, Switzerland) with 1 ng/mL of
cholera toxin. To achieve luminal morphology, cells
were cultured using 3D on-top method as previously
described [2,3]. 3D culture matrix BME (phenol red
free), with reduced growth factors, was obtained from
Trevigen (MD). The 3D culture media were modified to
contain 2-10% of BME, with 2% of FBS for malignant
cells with or without EGF (epidermal growth factor) su-
pplements. All media were supplemented with 1% of
Penicillin/Streptomycin (Invitrogen, CA) and 0.1% of
Fungizone (Hyclone, UT). Cells were seeded at 0.25 ×
105 (184-B5), 0.22 × 105 (BT-483), and 0.18 × 105
(MDA-MB-468) cells/cm2. Media were changed every
two days subsequent to seeding. RAP (Sigma-Aldrich,
MO) was reconstituted in DMSO and stored at -20°C at
a stock concentration of 1 μM.
2.2. Morphological Observation
Morphology was observed using a TE2000-S Eclipse
Microscope (Nikon, Japan) or a TS100 Eclipse Micro-
scope (Nikon, Japan). All images were taken with a
Digitial Sight DSQi1Mc Camera (Nikon, Japan). Flu-
orescence images were detected with an Intensilight
C-HGFI lamp (Nikon, Japan). For the detection of
non-viable (apoptotic) cells, non-fixed cultures were
incubated for 15-30 minutes with 1 µM of ethidium
bromide or 500 nM of propidium iodide and observed
for fluorescence. Cell colonies were extracted using
PBS-EDTA solution, fixed using 4% of paraformalde-
hyde, and blocked with 10% of goat serum and 1% of
goat F (ab’) IgG (Sigma-Aldrich, MO). Monoclonal
anti-actin (1A4) and monoclonal anti-β-catenin (15B8)
were obtained from Sigma-Aldrich (St. Louis, MO) for
use as primary antibodies. Secondary antibody for
FITC-conjugated goat anti-mouse was obtained from
Santa Cruz Biotechnology (CA). Image analysis was
conducted using NIS-Elements v3.0 (Nikon). Areas of
cell co lonies (in the x -y plane) in all images were ana-
lyzed for the calculation of mean areas under different
2.3. Caspase 3/7 Assay
Monolayers of BT-483 and 184-B5 cells were prepared
in a 96-well plate to analyze the effects of RAP on
caspase activity. Caspase activity was measured at 24
and 48 hours following exposure to RAP at 0, 5, and 20
nM. One hundred µL of Caspase-Glo 3/7 Reagent
(Promega, WI) was added to each sample, including
two blank wells containing 100 µL of growth media,
and incubated for 2 hours. Luminescence was then
measured using a Synergy 4 Plate Reader (Biotek, VT).
2.4. Cell Viability Assay
CellTiter-Blue Cell Viability Assay reagent was ob tained
from Promega (WI). MDA-MB-468 and 184-B5 cells
were seeded in 96-well plates, beginning on day 2, and
were treated continuously with 0 nM and 20 nM of RAP.
On day three and day seven, cell viability assays were
conducted and measured for fluorescence using a Syn-
ergy 4 Plate Reader (Biotek, VT).
2.5. Real-Time RT-PCR Arrays
Real time RT-PCR (reverse transcriptase polymerase
chain reaction) Arrays for human extracellular matrix
and adhesion molecules were obtained from SABio-
sciences (MD). MDA-MB-468 cells cultured in 3D
BME were treated with 0 nM and 20 nM of RAP for 6
days. Cell colonies were extracted using PBS-EDTA,
mRNA was extracted using RNeasy RNA isolation kit
(QIAGEN, CA), and cDNA synthesis was conducted
using the Reverse Transcriptase First Strand Kit (SABio-
sciences, MD). Real time RT-PCR arrays were run using
a Bio-Rad Opticon 2 thermocycler system (MJ Research,
Canada) and analyzed using the Opticon 2 software pro-
vided. Ct values were acquired graphically.
2.6. Statistical Analysis
With the SYSTAT 12 software, statistical analyses on all
quantitative data were carried out using ANOVA (ana-
478 R. Booth et al. / J. Biomedical Science and Engineering 3 (2010) 476-4 83
Copyright © 2010 SciRes. JBiSE
lyses of variance) to verify statistical significance (p <
3.1. Effects of EGF on Phenotype of Normal
Breast Epithelial Cells
Correct signaling from growth factors (EGF) and ECM
proteins (BME) contribute to the development and main-
tenance of a hollow luminal structure within normal hu-
man breast epithelial acini, which is schematically de-
scribed in Figure 1. Cells seeded on BME proliferated to
form acini. Several events contributing to the formation
of a luminal structure preceded the suppression of pro-
liferation within the normal breast epithelial acini. Two
populations of cells within each acinus became evident
from day 6 to 8:1) An outer layer of cells that was in
direct contact with the matrix and 2) an inner subset of
cells that underwent luminal death (apoptosis) (Figure
1(c)) and that lacked contact with the matrix. Cells in
contact with BME were apparently protected from a
global death signal. Normal (184-B5) breast epithelial
cells were initially cultured in a MEGM-based medium
containing epidermal growth factor (EGF) additive. Re-
sulting cultures contained large, morphologically dis-
rupted structures with no apparen t limitations to gro wth.
Culturing the cells in EGF-red uced media resulted in the
formation of significantly smaller structures with uni-
formity in shape (Figure 2(a)). The mean area for cell
colonies in EGF-reduced media at day 7 was signifi-
cantly lower than those in EGF-enhanced media at day 7
(Figure 2(b)).
Figure 1. Schematic of biological events during normal human
breast epithelial cells’ acinar morphogenesis. Normal human
breast epithelial cells were cultured using the 3D on-top me-
thod. Cell colonies were immunostained against. (a) actin
(green); (b) β-catenin (green); (c) ethidium bromide (red). Nu-
clei were counterstained with DAPI (blue) in (b). Experimental
procedures were adapted from [2,3].
Figure 2. (a) Effects of EGF-reduction on morphology of
normal human breast epithelial cells (184-B5) in 3D BME
cultures with and without EGF. Top: EGF-reduced media in-
duced a normal phenotype, including smaller, round morphol-
ogy with maximum size at day 5 of culture. Bottom: EGF-
enhanced media led to increased proliferation and disrupted
morphology. (Scale bars = 25 µm). (b) Effects of EGF on av-
erage cell colony size: Average cell colony areas were meas-
ured from normal human breast epithelial cells (184-B5) in 3D
BME cultures with and without EGF on day 7 of culture. Co-
lonies in EGF-reduced media showed a significantly lower
average area than the colonies in EGF-enhanced media. (Error
bars ± st. dev, * significantly lower than control, p < 0.05).
3.2. Effects of RAP Exposure on Malignant Cell
Morphologies of normal and malignant breast epithelial
cells (BT-483 and MDA-MB-468 ) were compared in 3D
BME cultures. Malignant breast epithelial cells did not
form organized structures, had no uniformity in shape,
and continued to proliferate. Malignant cell colonies
exposed to 20 nM of RAP, starting on day 2, formed
significantly smaller colonies with a maximum size
reached at 6-8 days (Figure 3(a)). The mean area for
RAP-treated malignant cell colonies was significantly
reduced than the untreated malignant cell colonies on
day 7 (Figure 3(b)).
An inner subset of cells undergoing luminal cell death
(apoptosis) was observed with ethidium bromide and
propidium iodide staining. Apoptotic colonies were
commonly found at the center of colonies in normal
R. Booth et al. / J. Biomedical Science and Engineering 3 (2010) 476-483 479
Copyright © 2010 SciRes. JBiSE
(184-B5) breast epithelial cell cultures on day 8, which
indicated luminal formation. This was also observed in
RAP-treated malignant (BT-483 & MDA-MB-468) cell
colonies, providing evidence that the treated malignant
breast epithelial cells were reverting to normal phenol-
types (Figure 4(a)).
Structural organization was also observed using an-
ti-actin and anti-β-catenin immunofluorescence staining
(Figure 4(b)). RAP-treated malignant colonies exhibited
cells arranged around the center, showing similariti es to
the structure of normal breast epithelial cells in 3D BME
Figure 3. (a) Comparison of normal, malignant, and RAP-
treated malignant human breast epithelial cells in 3D BME
culture. RAP was exposed to BT-483 malignant cells beginning
on the second day of culture at 0 and 20 nM, and compared to
184-B5 normal cells. Normal cells (bottom) were compared in
colony shape and size to RAP-treated malignant cells (middle).
Malignant cells (top) exhibited severe morphological disrup-
tion when cultured without treatment. (Scale bars = 25 µm); (b)
Effects of RAP exposure on average cell colony size: Average
cell colony areas were measured from BT-483 and MDA-MB-
468 malignant cells treated with 0 and 20 nM of RAP on day 7
of 3D BME culture. No significant difference was found
between the two cell lines, but RAP treatment significantly
reduced cell colony size of malignant cells. (Error bars ± st.
dev, * significantly lower than control, p < 0.05).
Figure 4. (a) Selective staining of non-viable cells. Top: Stain-
ing of day 8 3D cultures of normal (184-B5) human breast
epithelial cells with ethidium bromide or propidium idodie
resulted in detection of cell death at the center of cell colonies.
Bottom: Similar occurences of centrally-localized apoptosis
were detected in both malignant cell lines when treated con-
tinuously with 20 nM of RAP; (b) Immunofluorscence staining
of fixed cell colonies from 3D culture. Malignant (BT-483) cell
colonies from day 7 of culture, after continuous treatment of 20
nM RAP from day 2, exhibited multi-cellular organization
similar to day 7 cultures of normal (184-B5) human breast
epithelial cells. Top: Anti-Actin (green) counterstained with
DAPI (blue). Bottom: Anti-β-Catenin (green) counterstained
with DAPI (blue). (Scale bars = 25 µm)
cultures. Exposure to up to 20 nM of RAP had no ap-
parent effect on the morphology of either EGF- reduced
or EGF-enhanced cultures of normal (184-B5) cells
(Data not shown).
3.3. Effects of RAP Exposure on Caspase
Activity and Proliferation
Confluent monolayers of normal (184-B5) and malig-
nant (BT-483) breast epithelial cells were exposed to
RAP at 0, 5, and 20 nM. Caspase activity was meas-
ured after 24 and 48 hours of exposure (Figure 5). For
normal breast epithelial cells, the only condition which
showed significant deviation from the control was 20
nM of RAP after 48 hours of exposure (Figure 5(a)).
For malignant breast epithelial cells, 20 nM of RAP
exposure showed a significantly higher effect on cas-
pase activity than 5 nM, but no significant difference
was observed with respect to exposure time (Figure
5(b)). An increase in caspase activity indicates the
presence of programmed cell death, which is essential
for the formation of the luminal space in 3D BME
480 R. Booth et al. / J. Biomedical Science and Engineering 3 (2010) 476-4 83
Copyright © 2010 SciRes. JBiSE
Figure 5. Effect of RAP exposure on caspase activity. (a)
Normal human breast epithelial cell monolayers: Caspase
activity was measured on samples of 184-B5 exposed to
RAP at 5 and 20 nM for 0, 24, and 48 hours following con-
fluency. The only condition which yielded a significant dif-
ference from the control group was 20 nM of RAP at 48
hours of exposure. (n = 2, * significantly higher than control,
+ significantly higher than 5 nM, p < 0.05); (b) Malignant
human breast epithelial cell monolayers: Caspase activity
was measured on samples of BT-483 cells exposed to RAP at
5 and 20 nM for 0, 24, and 48 hours following confluency.
While there was no significant difference between 24 and 48
hours exposure, all treated samples were significantly higher
than the control (n = 2, * significantly higher than control, +
significantly higher than 5 nM, p < 0.05).
RAP’s effect on cell proliferation was analyzed in
monolayers of normal (184-B5) and malignant (MDA-
MB-468) breast epithelial cells (Figure 6) after 2 and
6 days of RAP exposure. Both cell lines showed sig-
nificantly decreased total cell viability when exposed
to 20 nM of RAP, with a significantly higher reduction
seen on day 7 (not significant at 5 nM of RAP; data
not shown).
3.4. Effects of RAP Exposure on Gene
Expression of Extracellular Matrix and
Adhesion Molecules
RT-PCR arrays were prepared from malignant breast
epithelial cells (MDA-MB-468) in 3D BME cultures
containing 0 and 20 nM of RAP on day 6 of culture.
Quality controls included with the arrays verified suc-
cessful RT-PCR and the absence of genomic DNA
contamination. Ct values were cut off at > 35. Genes
were grouped into six major classifications, and re-
ported with expression fold change (DDCT method) of
untreated controls compared to RAP treated cells (Ta-
ble 1). In RAP-treated samples, 25 genes related to
ECM and adhesion molecules showed a significant (at
least 5-fold) increase in expression, while no genes
showed a significant decrease in expression (Table 1).
In this study, the effects of RAP exposure on th e pheno-
typic reversion of malignant breast epithelial cells were
investigated in 3D BME culture. Microscopic observa-
tion of malignant breast epithelial cells treated with RAP
showed greater similarities in morphology to normal
cells than in untreated malignant b reast epithelial cells in
3D BME cultures. Comparison of the average horizontal
area confirmed that RAP-treated malignant cell colonies
are significantly smaller in size. Increased structural or-
ganization was observed by immunofluorescence stain-
ing of anti-Actin and anti-β-Catenin. On day 8 of 3D
BME culture, RAP-treated malignant breast epithelial
Figure 6. Effect of RAP exposure on total cell viability of nor-
mal and malignant human breast epithelial cell monolayers.
Cultures of MDA-MB-468 and 184-B5 were exposed to RAP
at 0 and 20 nM beginning on the second day of culture. Total
cell viability was measured on day 3 and day 7. Both cell lines
showed a significant decrease in proliferation at day 7 (n = 3, *
significantly lower than control, p < 0.05).
R. Booth et al. / J. Biomedical Science and Engineering 3 (2010) 476-483 481
Copyright © 2010 SciRes.
Table 1. RAP’s effects on gene expression of human extracellular matrix and adhesion molecules. Fold change was calculated using
the DDCT method, with GADPH as the normalizing gene.
Gene Category Symbol Description Fold Change
LAMA2 Laminin, alpha 2 13.77
LAMA3 Laminin, alpha 3 19.05
LAMB3 Laminin, beta 3 12.57
Basement Membrane Components
SPARC Secreted protein, acidic, cysteine-rich 13.64
CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 6.40
CTNND1 Catenin (cadherin-associated protein), delta 1 7.13
CD44 CD44 molecule (Indian bl ood group) 6.95
ITGA2 Integrin, alpha 2 9.84
ITGA6 Integrin, alpha 6 5.26
ITGAV Integrin, alpha V (vitronectin receptor) 8.99
ITGB1 Integrin, beta 1 (fibronectin receptor) 14.30
ITGB2 Integrin, beta 2 9.27
ITGB5 Integrin, beta 5 5.48
SGCE Sarcoglycan, epsilon 5.25
COL1A1 Collagen, type I, alpha 1 5.79
COL4A2 Collagen, type IV, alpha 2 9.77
Cell-Cell Adhesion
FN1 Fibronectin 1 16.51
THBS1 Thrombospondin 1 16.02
COL12A1 Collagen, type XII, alpha 1 17.50
CTNNA1 Catenin (cadherin-associated protein), alpha 1 7.82
CTNNB1 Catenin (cadherin-associated protein ), beta 1 11.40
Other ECM Molecules
LAMC1 Laminin, gamma 1 (formerly L AM B2) 6.36
TGFBI Transforming growth factor, beta-induced 10.04
Other ECM Molecules VCAN Versican 17.70
Trans-membrane Molecules ITGA4 Integrin, alpha 4 5.87
cells stained with ethidium bromide and propidium io-
dide showed similar structural organization with luminal
cell death to that observed in normal breast epithelial
cells, indicating re-polarization of the epithelial cell lay-
ers. Supporting data from monolayer cultures indicated
that levels of caspase activity were increased in the
presence of RAP, and that proliferation over time was
decreased. RT-PCR array analysis of RAP-treated ma-
lignant breast epithelial cells in 3D BME culture indi-
cated an increase in the expression of several ECM and
adhesion molecules important to polarity and structural
organization. This collection of data supports our hy-
pothesis that RAP exposure can revert malignant breast
epithelial cells to a normal phenotype.
In order to understand why breast cancer develops, as
well as to predict the outcome of pharmacological treat-
ments, we need to model the structure and function of
organs or tissues in culture so that our experimental ma-
nipulations occur under physiological contexts. 3D cul-
tures provide a model to analyze the spatial and temporal
aspects of key biological processes (e.g., proliferation
and apoptosis) and signal transduction events during
morphogenesis. Many of these studies are impractical or
impossible to perform in native breast tissue. Cultured
normal human breast epithelial cell monolayers in the
absence of BME fail to assemble organized structures,
and arrest growth when they reach confluence (Figure
7(a)). We recently found that signaling from epidermal
growth factor (EGF) and ECM proteins contributes to
the development and maintenance of a hollow luminal
Figure 7. Comparison of normal and malignant human
breast epithelial cells grown in 2D monolayer and 3D cul-
ture matrix. Normal human breast epithelial cells grown in
the 3D BME culture form a luminal structure with normal
phenotype (images not to scale). (a) Normal human breast
epithelial cells in monolayer without BME (2D); (b) Nor-
mal human breast epithelial cells in 3D BME with reduced
EGF; (c) Normal human breast epithelial cells in 3D BME
with EGF; (d) Malignant human breast epithelial cells in
3D BME with reduced EGF. Phenotypic reversion of ma-
lignancy can be achieved through RAP exposure on
EGF-reduced 3D BME cultures.
482 R. Booth et al. / J. Biomedical Science and Engineering 3 (2010) 476-4 83
Copyright © 2010 SciRes. JBiSE
structure within normal breast epithelial acini. In our 3D
culture system, non-malignant breast epithelial cells
seeded at low density developed into polarized acinar
units when EGF is withdrawn. Two populations of cells
within each acinus became evident from day 6 to 8: An
outer layer of cells that was in direct contact with the
ECM, and an inner set of cells not in contact with the
matrix that underwent apoptosis (Figures 1 and 7(b)).
This emulated the ductal structures of breast epithelial
cells found in vivo. In the presence of EGF, normal
breast epithelial cells exhibited severe morphological
deformities, including colony overgrowth, luminal fill-
ing, and resistance to apoptosis (Figure 7(c)). It was
observed that EGF disrupted the formation of luminal
structures of normal breast epithelial cells either in mo-
nolayer or 3D cultures with BME (Figures 7(a) and
7(c)). We also found evidence that rapamycin and its
derivative can revert cells’ structure from disorganized to
or ganized (Figures 3, 4, and 7).
Our criteria for phenotypic reversion are: increased
structural organization, limited colony size/proliferation,
and the presence of basolateral polarity. Immunofluo-
rescence staining of F-actin and -catenin showed a much
higher occurrence of morphologically spherical colonies,
indicating structural reorganization. This was also sup-
ported by the increase in expression of cell-cell adhesion
molecules, transmembrane molecules, and ECM prote-
ases shown by the RT-PCR array data (Table 1). Smaller
mean area of RAP-treated cell colonies provided strong
evidence of limited proliferation. A decreased number of
total cell viability over time and increased level of cas-
pase activity confirmed that RAP had a growth-limiting
effect on cancer cells. The presence of centrally located
cell death indicated the process of lumen formation, di-
rected by caspase-induced programmed apoptosis, and
was a result of polarization of the epithelial layers. Up-
modulation of caspase, in addition to increased gene
expression of basement membrane components and cell-
matrix adhesion molecules such as integrins, gave evi-
dence that RAP exposure promoted development of ba-
solateral polarity (Figure 8).
In previous studies, RAP has been seen to prevent
morphological disruption and inhibit runaway prolifera-
tion normally caused by Akt activation in breast epithe-
lial cells (MCF-10A) [18], and to significantly inhibit
growth of human lung cancer cells [14]. The value of in-
hibiting mTOR as a potential cancer treatment was fur-
ther validated by this study. However, due to its poor
water solubility and limited stability, analogues of RAP
have been developed with improved pharmaceutical
properties for parenteral delivery, including CCI-799
(tensirolimus), RAD-001 (everolimus), and AP-23573
(ARIAD) [15,19,20], which also inhibit mTOR. The
effects of these analogues on the phenotypic reversion of
malignant breast epithelial cells cultured in 3D BME
Figure 8. Summary of experimental results. With normal hu-
man breast epithelial cells, normal phenotype was achieved by
removing EGF additive from the media, leading to limited
proliferation, limited cell colony size, and luminal apoptosis.
RAP was found to limit proliferation of malignant human
breast epithelial cells and induce luminal apoptosis. RT-PCR
array data indicated an increase in expression of cell-cell adhe-
sion molecules, transmembrane molecules, and ECM prote-
should also be studied. Synergistic or additive effects of
RAP with other cancer drugs have been studied, includ-
ing paclitaxel, carboplatin and vinorelbine [21]; melle-
rian inhibiting substance [22]; doxorubicin [23]; and
gemcitabine [24]. Synergistic or additive effects of RAP
with other drugs on the phenotypic reversion of malig-
nant cells in 3D BME culture should also be investig ated.
This study can be used as an experimental template for
such future investigation.
In conclusion, by showing that RAP can revert ma-
lignant breast epithelial cells to a normal phenotype, our
study has provided supporting data for the value of
mTOR inhibition as a potential anti-tumor strategy. To
provide further insight, two further directions of research
should be taken: Further studies on the effects of RAP’s
analogues on phenotypic reversion, and studies on the
synergistic effects of mTOR inhibition with other cancer
Funding for this project was provided by NIH Grant No. 1 R21 CA
131798-01A1. Special thanks to Dr. Timothy Doyle and Dr. David
Britt for their contributions.
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