Suppression of Breast Tumor Growth and Metastasis by an Engineered Transcription Factor

Abstract

Maspin is a tumor and metastasis suppressor playing an essential role as gatekeeper of tumor progression. It is highly expressed in epithelial cells but is silenced in the onset of metastatic disease by epigenetic mechanisms. Reprogramming of Maspin epigenetic silencing offers a therapeutic potential to lock metastatic progression. Herein we have investigated the ability of the Artificial Transcription Factor 126 (ATF-126) designed to upregulate the Maspin promoter to inhibit tumor progression in pre-established breast tumors in immunodeficient mice. ATF-126 was transduced in the aggressive, mesenchymal-like and triple negative breast cancer line, MDA-MB-231. Induction of ATF expression in vivo by Doxycycline resulted in 50% reduction in tumor growth and totally abolished tumor cell colonization. Genome-wide transcriptional profiles of ATF-induced cells revealed a gene signature that was found over-represented in estrogen receptor positive (ER+) "Normal-like" intrinsic subtype of breast cancer and in poorly aggressive, ER+ luminal A breast cancer cell lines. The comparison transcriptional profiles of ATF-126 and Maspin cDNA defined an overlapping 19-gene signature, comprising novel targets downstream the Maspin signaling cascade. Our data suggest that Maspin up-regulates downstream tumor and metastasis suppressor genes that are silenced in breast cancers, and are normally expressed in the neural system, including CARNS1, SLC8A2 and DACT3. In addition, ATF-126 and Maspin cDNA induction led to the re-activation of tumor suppressive miRNAs also expressed in neural cells, such as miR-1 and miR-34, and to the down-regulation of potential oncogenic miRNAs, such as miR-10b, miR-124, and miR-363. As expected from its over-representation in ER+ tumors, the ATF-126-gene signature predicted favorable prognosis for breast cancer patients. Our results describe for the first time an ATF able to reduce tumor growth and metastatic colonization by epigenetic reactivation of a dormant, normal-like, and more differentiated gene program.

Full text

Suppression of Breast Tumor Growth and Metastasis by
an Engineered Transcription Factor
Adriana S. Beltran
1.
, Angela Russo
3.
, Haydee Lara
1,4
, Cheng Fan
2
, Paul M. Lizardi
5
, Pilar Blancafort
1,2
*
1Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2Lineberger Comprehensive Cancer
Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3Department of Pharmacology, University of Illinois at Chicago,
Chicago, Illinois, United States of America, 4Department of Marine Biotechnology, CICESE Research Institute, Ensenada, Mexico, 5Department of Pathology, Yale
University School of Medicine, New Haven, Connecticut, United States of America
Abstract
Maspin is a tumor and metastasis suppressor playing an essential role as gatekeeper of tumor progression. It is highly
expressed in epithelial cells but is silenced in the onset of metastatic disease by epigenetic mechanisms. Reprogramming of
Maspin epigenetic silencing offers a therapeutic potential to lock metastatic progression. Herein we have investigated the
ability of the Artificial Transcription Factor 126 (ATF-126) designed to upregulate the Maspin promoter to inhibit tumor
progression in pre-established breast tumors in immunodeficient mice. ATF-126 was transduced in the aggressive,
mesenchymal-like and triple negative breast cancer line, MDA-MB-231. Induction of ATF expression in vivo by Doxycycline
resulted in 50% reduction in tumor growth and totally abolished tumor cell colonization. Genome-wide transcriptional
profiles of ATF-induced cells revealed a gene signature that was found over-represented in estrogen receptor positive (ER+)
‘‘Normal-like’’ intrinsic subtype of breast cancer and in poorly aggressive, ER+luminal A breast cancer cell lines. The
comparison transcriptional profiles of ATF-126 and Maspin cDNA defined an overlapping 19-gene signature, comprising
novel targets downstream the Maspin signaling cascade. Our data suggest that Maspin up-regulates downstream tumor and
metastasis suppressor genes that are silenced in breast cancers, and are normally expressed in the neural system, including
CARNS1,SLC8A2 and DACT3. In addition, ATF-126 and Maspin cDNA induction led to the re-activation of tumor suppressive
miRNAs also expressed in neural cells, such as miR-1 and miR-34, and to the down-regulation of potential oncogenic
miRNAs, such as miR-10b, miR-124, and miR-363. As expected from its over-representation in ER+tumors, the ATF-126-gene
signature predicted favorable prognosis for breast cancer patients. Our results describe for the first time an ATF able to
reduce tumor growth and metastatic colonization by epigenetic reactivation of a dormant, normal-like, and more
differentiated gene program.
Citation: Beltran AS, Russo A, Lara H, Fan C, Lizardi PM, et al. (2011) Suppression of Breast Tumor Growth and Metastasis by an Engineered Transcription
Factor. PLoS ONE 6(9): e24595. doi:10.1371/journal.pone.0024595
Editor: Austin John Cooney, Baylor College of Medicine, United States of America
Received April 15, 2011; Accepted August 15, 2011; Published September 13, 2011
Copyright: ß2011 Beltran et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported from National Cancer Institute/National Institutes of Health grants 1R01CA125273, 3R01CA125273-03S1 and Department of
Defense (DoD) W81XWH-10-1-0265 (PB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: pilar_blancafort@med.unc.edu
.These authors contributed equally to this work.
Introduction
Mammary Serine Protease Inhibitor (Maspin,SERPINB5)isa
multifunctional protein possessing tumor and metastasis suppres-
sive functions [1,2]. Additionally, Maspin over-expression inhibits
in vivo angiogenesis [3]. The multifaceted nature of Maspin affecting
many molecular mechanisms during neoplastic disease progression
makes it a very attractive target in cancer biology. Importantly,
clinical data shows that high Maspin levels are associated with
better prognosis in breast, lung and prostate carcinomas [4,5,6].
As a class II tumor suppressor gene, Maspin is not mutated,
rearranged or deleted in tumor cells. Instead, its expression is
regulated by means of transcription factors [7] and epigenetic
modifiers [8,9]. While Maspin is expressed at high levels by
epithelial cells, it is down-regulated in mesenchymal cells, such as
stromal fibroblasts. In breast cancer cell lines and cancer
specimens, silencing of Maspin correlates with acquisition of
invasive and metastatic behavior. Epigenetic mechanisms control-
ling Maspin silencing include both, DNA [9] and H3K9 histone
methylation [10]. Hence epigenetic mechanisms are reversible yet
inherited during cell division, blockade of Maspin promoter
silencing offers a potent strategy to reactivate tumor suppressor
function. To this end, we have previously described the
construction of Artificial Transcription Factors (ATFs) made of
sequence-specific six Zinc Finger (ZF) domains[11] designed to
bind unique 18-base pair recognition sites in the Maspin proximal
promoter [12]. The ZFs were linked to a VP64 transactivator
domain, which mediates a strong promoter up-regulation by
recruitment of the polII transcriptional complex. In cell systems,
both in lung and breast cancer cell lines, retroviral transduction of
one of the ATFs, ATF-126, led to a potent induction of apoptosis
and inhibition of cell invasion [12,13]. Furthermore, these ATFs
were able to directionally demethylate the Maspin promoter and
this effect depended on upon the orientation of the ATF along the
DNA [13]. Consistently, we found that ATFs synergized with both
methyltransferase and histone deacetylase inhibitors to reactivate
silenced Maspin [13,14,15].
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These previous observations suggested that ATF-126 was able
to partially reprogram or revert the epigenetic state of the Maspin
promoter, resulting in a re-activation of the endogenous gene.
However, the impact of ATF-126 in inhibiting tumor progression
in preexisting tumors and/or metastases in vivo has never been
addressed. Herein, we have taken advantage of an inducible viral
vector system to control the expression of ATF-126 in pre-existing
breast tumor growths and experimental metastases in immunode-
ficient mice. Chemical induction of ATF-126 in vivo resulted in
tumor suppression as well as in inhibition of breast tumor cell
colonization. Furthermore, genome-wide DNA microarrays of
MDA-MB-231 cells induced with ATF-126 revealed that breast
tumor cells acquired a 550-gene signature that was found over-
represented in estrogen receptor positive (ER+) breast cancer cell
lines and in the normal-like intrinsic subtype of breast cancer. Our
data indicates that ATF-126 up-regulates novel Maspin-dependent
targets possessing tumor and metastasis suppressive functions,
which are found epigenetically silenced in aggressive tumors. Our
results outline a possible mechanism by which ATF-126
reprograms aggressive tumor cells towards a more ‘‘normal-like’’,
more benign, and more differentiated ‘‘epithelial-like’’ phenotype.
Results
Induction of ATF-126 by DOX results in endogenous
reactivation of Maspin in MDA-MB-231 breast cancer cells
In order to monitor the effect of ATF-126 in inhibiting tumor
progression in pre-existing tumors, we cloned the ATF-126 gene
into an inducible TetOn retroviral vector (Fig. S1A). In this
expression system the ATF expression was activated only in
presence of the chemical inducer, Doxycycline (DOX). The MDA-
MB-231-LUC cell line stably engineered with a luciferase (LUC)
gene was transduced with either a control (empty retroviral vector)
or the same vector expressing ATF-126. The LUC gene allowed
the non-invasive monitoring of tumor growth and dissemination in
a mouse model, using bioluminescence imaging, BLI (Fig. S1B).
The effects of inducing ATF-126 with DOX were first
monitored in cell culture assays. As shown in Fig. 1A, induction
of the full length ATF-126 (comprising the specific 6ZF DNA-
binding domains and the VP64 transactivator domain) resulted in
a dose-dependent ATF-126 expression, as assessed by qRT-PCR.
The induction of the ATF was accompanied by a concomitant up-
regulation of the Maspin target (Fig. 1B). For subsequent studies
we used a concentration of DOX of 100 ng/ml at which the
expression of both, ATF-126 and Maspin, reached saturation.
Efficient ATF-126 induction was also verified by immunopre-
cipitation (using an anti-HA antibody recognizing the C-terminal
HA epitope of the ATF; Fig. 1C, left panel). Maspin up-regulation
in +DOX cells was also verified by immunoprecipitation (Fig. 1C,
right panel). As shown in Fig. 1D, ATF-126 +DOX cells up-
regulated Maspin quickly after ATF-126 expression (6–12 hours
after addition of DOX), as expected from direct transcriptional
regulation. A saturation of ATF expression was reached 48 hours
after the addition of the drug, whereas that Maspin up-regulation
achieved a maximum at 72 hours post-induction (Fig. 1D).
Removal of DOX from the cell culture of ATF-126 cells also
resulted in a decay of ATF expression that was evident 24 hours
upon the retrieval of the drug. However, 72 hours after DOX
removal, substantial Maspin expression was still detected in the cells
even though ATF-126 mRNA expression was not. These results
suggested that ATF-126 was able to transiently impact the
epigenetic and transcriptional status of the Maspin promoter.
Previously we have reported that ATF-126 was able to
demethylate the Maspin proximal promoter [13]. Moreover, It is
possible that the endogenous mechanisms responsible for Maspin
silencing (including endogenous DNA-methylation processes)
would be restored 96 hours after removal of DOX in ATF-126
transduced cells.
ATF up-regulation by DOX results in Maspin-dependent
induction of apoptosis
Endogenous expression of tumor suppressors, including Maspin,
has been associated with reduction of tumor cell viability by
induction of apoptosis [12,16]. Next, we studied if ATF-126
induction resulted in a reduction of tumor cell growth. As shown in
Fig. 2A, the reactivation of ATF-126 (ATF-126 +DOX) led to
70% reduction in tumor cell viability relative to the same cells in
absence of DOX (ATF-126 2DOX). As expected, no difference in
cell viability was observed between CONTROL and ZF-126
(inactive ATF-126 lacking the activator domain) cells upon DOX
induction. Interestingly, removal of DOX in ATF-126 cells led to
a maintenance of the cell proliferation defect for 96 hrs after DOX
removal (Fig. 2B), and approximately over four cell generations.
This maintenance of tumor suppression correlates with the time
window of Maspin transcriptional activation of Fig. 1E.In
addition to cell proliferation defects, ATF-126 +DOX cells
exhibited a 40% induction of apoptosis, as assessed by Annexin-
V staining, which monitors early apoptosis (Fig. 2C). Similarly,
30–40% of ATF-126 +DOX cells were positive for the Hoechst
staining, which labels apoptotic nuclei (Fig. 2D).
To verify that the apoptotic response triggered by ATF-126 was
due to Maspin reactivation, we challenged ATF-126 cells with
either a Maspin-specific or scramble shRNA constructs. The
Maspin-shRNA in ATF-126 +DOX cells led to a 60% reduction of
Maspin mRNA expression (Fig. 2E). As expected, a scramble
shRNA construct did not significantly impact Maspin expression.
Consistently, the scramble-shRNA in ATF-126 +DOX cells
induced similar levels of cell death relative to the original ATF-
126 +DOX cell line (Fig. 2F). However, the Maspin-shRNA in
ATF-126 +DOX cells was able to rescue the phenotype, and a
significant reduction of cell death (55% of reduction) was observed
in the Maspin knock-down relative to the scramble and the ATF-
126 parental line +DOX (Fig. 2F). Although the rescue of the cell
death phenotype was not 100%, our results suggest that ATF-126
mediate reduction in tumor cell growth by primarily activation of
its designed target Maspin. The incomplete rescue of the phenotype
could be due the fact that the Maspin shRNA did not completely
knocked down the mRNA transcript levels of Maspin, as shown in
Fig. 2E. A siRNA approach further confirmed that the tumor
suppressive phenotype observed in ATF-126 +DOX cells was due
to Maspin re-activation. As shown in Figs. 2G–H,aMaspin-
specific siRNA but not a non-specific mismatch (scramble) siRNA
rescued the proliferation defect of ATF-126 +DOX cells. Overall
these results support the conclusion that the cell death phenotype
induced by ATF-126 was dependent on the Maspin target.
ATF-126 reduces xenograft tumor growth and
suppresses metastatic colonization of MDA-MB-231 cells
upon DOX induction in immunodeficient mice
To investigate the ability of ATF-126 to reduce tumor cell
growth in pre-existing breast tumor xenografts, we implanted
either control 1610
6
MDA-MB-231-LUC cells or ATF-126 cells
in SCID mice (N = 8 animals per group). Animals were kept in
DOX-free conditions until tumors reached 100–200 mm
3
(Fig. 3A). Eighteen days post-induction, half of the animals for
control and ATF-126 groups were maintained in DOX-free diet,
whereas the other half was switched to a +DOX diet. Tumor
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Figure 1. Induction of ATF-126 by DOX results in reactivation of the target gene
Maspin
.A.Dose-response plot monitoring ATF-126 (left
panel) and Maspin (right panel) mRNA levels upon treatment with increasing concentrations of DOX. CONTROL and ATF-126 cells were treated for
72 hours and mRNA was measured by quantitative real-time PCR (qRT-PCR). B–C. ATF-126 and Maspin mRNA expression levels by qRT-PCR (B) and
western blot (C) induced with 100 ng/ml of DOX. MDA-MB-468 is a poorly aggressive ER- breast cancer cell line expressing endogenous Maspin as a
reference control [12]. D. Time course kinetics of ATF-126 and Maspin mRNA levels by qRT-PCR upon DOX treatment. ATF-126 cells were induced with
DOX and collected at 0, 6, 12, 18, 24, 48, 72 and 96 hours. E. Time course kinetics of ATF-126 and Maspin expression levels by qRT-PCR upon DOX
treatment and removal. ATF-126 cells were induced with DOX for 48 hours, then DOX was removed from the media and cells were maintained in
DOX-free media for an additional 168 hours. Gene expression levels were normalized to the 2DOX cells. Data represents the mean 6SD of three
independent biological replicates. MDA-MB-231-LUC are un-transduced cells; CONTROL, cells transduced with an empty vector; ATF-126, a full length
ATF containing the 6 ZF DNA-binding domains and VP64 activator domain; ZF-126, a truncated or inactive ATF-126 lacking the VP64 activator
domain.
doi:10.1371/journal.pone.0024595.g001
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Figure 2. ATF-126 induced apoptosis upon DOX treatment. A. Cell viability plot of ATF-126 and ZF-126 cells treated with vehicle (2DOX) or
DOX (+DOX) for 72 hours. ATF-126 refers to a full length, active ATF and ZF-126 is a truncated, transcriptionally inactive construct lacking the VP64
activator domain. B. Cell viability plot of ATF-126 transduced cells after DOX removal. Cells were induced with DOX for 48 hours (open circles). DOX
was removed from the media and cells were kept in DOX-free media for an additional period of 96 hours. Viability of ATF-126 cells growing in
absence of DOX (2DOX, filled squares) was plotted as reference control. Cell viability was measured with an XTT assay [12]. C. ATF-126 induced
apoptosis upon DOX treatment. Either ATF-126 or ZF-126 cells were kept in vehicle-treated media (2DOX) or DOX (+DOX) and collected at 72 hours
after induction. The percentage of apoptosis was quantitatively analyzed using an Annexin-V staining [12]. D. Hoechst staining of ATF-126 2DOX and
+DOX cells at 72 hours post-induction. Arrow points to a positive apoptotic cell, showing nuclear condensation. E. Expression of Maspin by qRT-PCR
in ATF-126 cells retrovirally transduced with either a scramble or with a Maspin-specific shRNA construct. F. Percentage of cell death by trypan-blue
exclusion assay in ATF-126 cells transduced with either a scramble or a Maspin-specific shRNA. G. Maspin expression as assessed by qRT-PCR in ATF-
126 cells transfected with either a mismatch or a Maspin-specific siRNA, and treated with vehicle (2DOX) or DOX (+DOX). H. Representative pictures
of the Maspin siRNA knock-down experiments of cells treated with vehicle (2DOX) or DOX (+DOX). Bar graphs in C, E, F and G represent the average
of 3 independent experiments. Differences between samples were calculated with a student t test with level of significance *p#0.05 and ** p#0.01.
doi:10.1371/journal.pone.0024595.g002
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volume was assessed from the day before induction until tumor
collection (day 41 post-injection), and BLI imaging was performed
once a week. One of the animals belonging to the ATF-126 group
previously induced with DOX was switched back to a DOX-free
diet and sacrificed at day 53 post-injection (Fig. 3A). As shown in
Fig. 3B–C (left panels), no statistical differences in tumor growth
were observed between CONTROL +DOX and -DOX animals.
In contrast, ATF-126 animals induced with DOX exhibited a
significant reduction of tumor volume (approximately 50%
reduction) that was stably maintained until the mice were
sacrificed at day 41 (Fig. 3B–C, right panels).
Interestingly, induced ATF-126 animals that were removed
from DOX at day 41 experienced a tumor relapse (Fig. 3D). This
tumor recovery suggests that, like we have observed in vitro, long-
term absence of ATF expression results in a re-establishment of
Maspin silencing. Analysis of tumors recovered from the animals at
day 41 demonstrated that the ATF-126 +DOX animals retained
efficient ATF mRNA up-regulation (500-fold relative to un-
induced animals). This induction of ATF mRNA was accompa-
nied by a 20-fold Maspin up-regulation relative to 2DOX animals
(Fig. 3E). Overall these results demonstrate that ATF-126 was
properly up-regulated in the tumor xenograft experiment and that
this induction correlated with Maspin reactivation and with the
maintenance of tumor suppressive functions.
It is well documented for breast, prostate and lung carcinomas
that high Maspin expression correlates with a less aggressive or
metastatic behavior [17,18,19]. We next investigated the ability of
ATF-126 to inhibit breast tumor colonization or experimental
metastasis formation in immunodeficient mice. To address this,
either 1610
5
MDA-MB-231-LUC CONTROL or ATF-126 cells
were injected tail vein in SCID mice (N = 24). Twelve mice per
group were maintained in DOX-free diet and 12 in +DOX diet.
Because the need of the animals to adapt to a DOX diet, mice
were feed three days prior to the injection (Fig. 4A). Fig. 4B (top
panel) shows that CONTROL +DOX and 2DOX cells
effectively colonized the lungs and metastases were evident at
day 14 post-injection. Similarly, the majority of the animals
injected with ATF-126 maintained in DOX-free diet effectively
colonized the lungs with similar signal intensities than CON-
TROLS. In contrast, ATF-126 animals in +DOX diet completely
suppressed experimental metastatic colonization at day 21
(Fig. 4B, bottom panel). Moreover, removal of DOX from the
diet of ATF-126 animals maintained these mice free of any
detectable lung colonization even 54 days post-injection (Fig. 4B,
bottom right panel). Overall these results indicated that ATF-126
effectively suppressed metastatic colonization. Whether the ATF-
126 is equally effective in suppressing or reducing secondary tumor
growth in well-established macrometastases will require further
investigation.
ATF-126 up-regulate a gene signature over-represented
in the normal-like intrinsic subtype of breast cancer and
ER+cancer cell lines
The above results demonstrated that ATF-126 reduced tumor
growth and suppressed metastatic colonization of the MDA-MB-
231-LUC line. We next began the investigation of potential
mechanisms by which ATF-126 could mediate its suppressive
functions by performing genome-wide microarray analyses.
CONTROL and ATF-126 cells where induced with DOX for
72 hours. Genes differentially regulated between 2DOX and
+DOX groups were determined by SAM analyses, with three
independent arrays performed for each group. These analyses
generated a robust 550-gene signature, defined as group of genes
differentially up-regulated by ATF-126 (see Table S1). A Gene
Ontology (GO) analysis revealed that multiple Maspin downstream
pathways were reactivated by ATF-126, including tight junction
and cell invasion, TGF-beta, and p53 signaling (Table S2).
We next examined this signature across intrinsic breast cancer
subtypes, using the UNC337 tumor database comprising 337
breast tumor cases [20]. Our analysis shows that ATF-126 up-
regulated targets that are found over-represented in the ‘‘Normal-
like’’ intrinsic subtype of breast cancer (Fig. 5A). Both Normal-like
and Luminal A are ER+tumors associated with the best prognosis
of all breast tumor subtypes [21]. In contrast, the Basal-like and
Claudin-low carcinomas are mostly triple negative breast cancers
(ER-PR-Her2-) associated with high resistance to chemotherapy
and poor prognosis [20]. Claudin-low tumors have been recently
discovered through large-scale microarray analysis of breast
cancer specimens [20,22]. The MDA-MB-231 cell line used in
this study was originally described as Basal B and has been recently
characterized as Claudin-low [20,21]. Our finding that ATF-126-
responsive genes are enriched in ER+‘‘Normal-like’’, poorly
aggressive tumors, correlates with the fact that ATF-126 induction
in MDA-MB-231 confers a more benign, less tumorigenic
phenotype. Consistent with this, we found that the ATF-126-up-
regulated gene signature was indicative or a predictor of a better
prognosis in breast cancer patients (Fig. 5B).
The relationship between the ATF-126-gene signature and ER
status was further observed in available DNA-microarrays data of
breast cancer cell lines [21]. Fig. 5C shows that the ATF-126-
gene signature was found under-represented in Claudin-low and
Basal-like ER- lines with the lowest enrichment found in the
original MDA-MB-231 cell line. The same signature was over-
represented in luminal ER+cell lines, including the poorly
aggressive lines MCF-7 and ZR75. Overall our microarray
analysis provides support that ATF-126 initiates a transcriptional
gene program resulting in a reprogramming of the original ER-
aggressive Claudin-low MDA-MB-231 towards a less aggressive,
and more ‘‘normal-like’’ breast cancer cell line. This is also
consistent with the fact that high expression of Maspin, the primary
target gene of ATF-126, is also associated with epithelial-like
features [18].
To dissect ‘‘bona-fide’’ downstream targets of Maspin in the 550-
gene signature we cloned the Maspin cDNA into the same DOX-
inducible retroviral vector, and gene expression microarrays were
performed in the Maspin cDNA 2DOX and +DOX transduced
cell populations. 123 genes (Table S3) were found differentially
up-regulated upon Maspin cDNA induction. Among these, 19
targets were shared with ATF-126 +DOX (Fig. 6A, Table 1).
Nine of the most up-regulated Maspin-dependent candidate targets
were further validated by qRT-PCR (Fig. 6B). These genes
included potential therapeutic targets normally expressed in the
neural system, such as negative regulators of oncogenic signaling
(the epigenetic regulator of Wnt/b-catenin signaling (DACT3) and
the SRC kinase signaling inhibitor 1 (SRCIN1)), putative tumor
suppressors in cancer (DACT3,SLC8A2,CARNS1,GNG4,END2),
and the apoptosis-associated tyrosine kinase ATTK.Solute carrier
family 8 (sodium/calcium exchanger), member 2 (SLC8A2), Carnosine
synthase 1(CARNS1), and Dapper, antagonist of beta-catenin, homolog 3
(DACT3) were up-regulated by more than 10-fold in both, ATF-
126 and Maspin cDNA cells, and thus, could represent novel
‘‘bona-fide’’ Maspin-dependent targets.
We next took advantage of ATF-126 and Maspin cDNA
inducible cell lines to examine whether oncogenic and tumor
suppressive microRNAs were differentially regulated upon DOX
induction. We analyzed the expression of 90 miRNAs often dys-
regulated in cancers, using a miRNA array platform by qRT-
PCR. The expression of each miRNA was normalized to the
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MDA-MB-231 cell line. Interestingly, both, ATF-126 and Maspin
cDNA, up-regulated miRNAs with potential tumor suppressive
functions, such as miR-1 [23,24] and miR-34 [25], while down-
regulating oncogenes and metastasis promoters, including miR-
10b [26] (Fig. 6C). In addition, we found that miR-124 which is
required for somatic cells to reprogram to neural cells [27], and
miR-363 (Fig. 6C), were down-regulated in both ATF-126 and
Maspin cDNA. Thus, our target analysis suggests that ATF-126
Figure 3. ATF-126 induced tumor suppression in SCID mice. A. Time-line of the experiments involving subcutaneous tumor injections
illustrating the time of injection of tumor cells, induction and removal of DOX, and tumor collection. B. Time course plots monitoring tumor volumes
of CONTROL and ATF-126 groups, N =8 animals per group. Four animals per group were maintained in DOX-free diet (2DOX), whereas the other half
was treated with DOX (+DOX). The tumor growth was measured by caliper the day before induction (day 17) until tumor collection (day 41). C. Tumor
volume measurements at the day of tumor collection for both CONTROL (left panel) and ATF-126 animals (right panel). Differences in tumor growth
were calculated with a student t test (***p = 0.001). D. Representative bioluminescence images comparing signal intensities of luciferase photon
counts from the subcutaneous growths at day 41 for CONTROL (left) and ATF-126 animals (right), treated in absence (2DOX) and presence (+DOX) of
DOX. ‘‘DOX removal’’ indicates an ATF-126-injected animal previously induced with DOX, subsequently removed from DOX (day 41), and imaged at
day 53 (right). E. ATF-126 and Maspin expression levels by qRT-PCR in tumor samples collected at day 41 from both, CONTROL and ATF-126 animals.
doi:10.1371/journal.pone.0024595.g003
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activates downstream targets of Maspin resulting in an up-
regulation of potential tumor suppressors, and down-regulation
of oncogenic and pro-metastatic pathways.
Claudin-low carcinomas and representative cell lines are
characterized by a down-regulation of epithelial junction proteins,
such as cadherins and claudins [20]. Interestingly, multiple panels
of epithelial markers, such as E-Cadherin (CDH1), Claudin 3 and
7, Ocludins, and keratins, were re-activated upon ATF-126
expression (Fig. 7A). In addition, ATF-126 led to the generation
of a CD24 positive population (Fig. 7B). CD24 is expressed in
many ER+tumor cell lines, while its expression is absent in some
basal and Claudin-low cell lines (Fig. S2). CD44+/CD24- is
considered a cancer stem cell or tumor initiating cell signature,
and high CD44/CD24 ratios are characteristic of aggressive
Claudin-low tumors and cell lines [20]. The reactivation of CD24
by ATF-126 suggests that ATF-126 could decrease the tumori-
genic potential of MDA-MB-231 cells. In summary, the above
results suggested that ATF-126 was able to initiate a transcrip-
tional program resulting in a reprogramming of a more
mesenchymal, Claudin-low phenotype, towards a more normal-
like, epithelial-like, and less aggressive breast tumor line (Fig. 7C).
Discussion
In this paper we have investigated the ability of the ATF-126,
designed to up-regulate Maspin, to decrease tumor growth and
colonization of an aggressive MDA-MB-231 line. We found that
induction of ATF expression in vivo resulted in 50% reduction in
tumor growth. In addition, ATF expression abolished the
capability of the breast cancer to colonize the lungs.
The approach used in this work facilitated the tight control of
the ATF expression both in cell culture and in a breast cancer
xenograft model. As a direct target of the ATF, we found that the
Maspin transcript was induced with very similar time course
kinetics than the ATF. Interestingly, removal of DOX from the
cell culture media resulted in a quick decay of ATF-126
expression. However, we found that Maspin expression was still
retained for at least four cell generations even in complete absence
of ATF-126 mRNA expression. In the same time window of
Maspin transcriptional activation, the ATF-126 +DOX cells
maintained their proliferation defects, suggesting that the growth
inhibition phenotype could be propagated for several cell
generations upon DOX removal. The slow decay of Maspin
expression and the maintenance of growth inhibition in absence of
ATF expression could be the result of the modification of the
epigenetic status of the Maspin promoter upon binding of the ATF.
We have reported that two ATFs, ATF-126 and ATF-97, reduced
DNA methylation levels in the Maspin promoter [13]. In addition,
this demethylation effect was directional and depended upon de
orientation of VP64 along the promoter. In this regard, it is
possible that the slow Maspin decay in absence of ATF expression
could reflect a time delay by which the endogenous epigenetic
and/or transcriptional mechanisms restore Maspin silencing. To
address this possibility, we are presently analyzing methylation
patterns upon removal of DOX at different time points. Future
engineering of ATFs should maximize this demethylation effect,
thereby increasing the potency and therapeutic window of ATFs
targeting tumor suppressor gene promoters. This unique engi-
neering aspect of ATFs could facilitate the long-term, hereditary,
and stable transmission of tumor suppression (‘‘phenotypic
Figure 4. ATF-126 inhibits breast tumor cell colonization in the lungs. A. Time-line of the tail-vein injections experiments, indicating the
times of the DOX induction and removal, and tumor collection. B. Bioluminescence images of CONTROL and ATF-126 mice groups maintained in
DOX-free (2DOX, left panel) and DOX containing diet (+DOX, right panel). Mice were injected via tail-vein with either CONTROL or ATF-126 cells and
imaged every week to assess lung colonization. Images shown were taken at day 22 after injection of the tumor cells. DOX removal indicates ATF-126
animals previously induced by DOX and next placed in DOX-free diet.
doi:10.1371/journal.pone.0024595.g004
Suppression of Breast Tumor Progression by ATFs
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memory’’) over cell generations by targeted remodeling of silenced
chromatin.
Our results in vivo demonstrate that induction of ATF-126 in
animal models led to a 50% reduction in breast cancer cell growth.
Long-term removal of DOX from the diet of the animals resulted
in a re-establishment of the tumors in vivo. As it was observed in
our analysis in vitro, it is possible that long-term absence of ATF
expression results in re-establishment of Maspin silencing. This is in
contrast with our colonization model of breast cancer, in which
removal of DOX from the ATF-126 induced animals did not
result in a recurrence of metastasis. These results could be
explained because most circulating tumor cells or cells that
underwent an early colonization in the lungs were effectively
targeted by the ATF. Nevertheless, we do not know at present if
the ATF-126 will be also effective in suppressing well-established
metastases and this will require further investigation.
In order to investigate potential downstream genetic signatures
mediating the inhibition of tumor growth and colonization, we
performed DNA arrays. Our genome-wide analysis revealed that
ATF-126 up-regulated a 550-gene signature that was found over-
represented in the Normal-like intrinsic subtype of breast cancer,
and as well as in luminal A breast cancer cell lines. Together with
Luminal A, Normal-like breast cancers are associated with ER
expression. These tumors are small, mostly found in post-
menopausal women, tend to have normal p53 status, and are
known to be genetically more stable than other tumor subtypes
[17,18]. Hence, Normal-like and Luminal A tumors both have a
significant higher survival upon endocrine adjuvant therapy
treatments after surgery as compared to other subtypes of breast
cancers. As expected from its over-representation in Normal-like
ER+tumors, we found that the 550-gene signature predicted
favorable prognosis for breast cancer patients.
In our cell line analysis, the 550-gene signature was under-
represented in the original MDA-MB-231 line as well as in ER-
breast cancer lines, while it was highly over-represented in ER+
luminal lines. Although Normal-like cancer cell lines were absent
in the cell line database of Neve et al. [21] it is highly possible that
this signature will also be over-expressed in normal breast and
Figure 5. ATF-126 up-regulates a gene signature over-represented in Normal-Like breast cancers. A. Box-and-whisker plot for the mean
expression of the 550 up-regulated gene signature (Table S1) This signature represents the number of genes significantly up-regulated in ATF-126
cells exposed to DOX for 72 hours relative to the same cells in absence of DOX. The prevalence of this signature was evaluated across the intrinsic
molecular subtypes of breast cancers using the previously published UNC breast cancer patient database (UNC337). P values were calculated by
comparing gene expression means across all breast tumor subtypes. B. Kaplan–Meier survival estimates of relapse-free survival for the Merge 550
database (left panel), relapse-free survival and overall survival for the NKI 295 database (center and right panel, respectively). Patients were stratified
into group I (red curves) and group II (blue curves) based on 2-way hierarchy clusters. P-values were obtained from the log-rank test. C. Mean
expression analysis of the ATF-126 gene signature across breast cancer cell lines [21], showing the expression status of estrogen receptor (ER), Human
Epidermal growth factor Receptor 2 (HER2) and progesterone receptor (PR).
doi:10.1371/journal.pone.0024595.g005
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Normal-like tumor cell lines. Consistent with this idea, we found
that many junction proteins highly expressed in normal breast
epithelial preparations, such as claudins, cadherins and ocludins,
were induced upon DOX treatment. This reactivation of junction
proteins suggests that ATF-126 was able to reprogram a highly
invasive, Claudin-low, and mesenchymal breast cancer line
towards a more Normal-like or epithelial-like, and less invasive
cancer cell line. Furthermore, flow cytometric analysis of ATF-126
induced cells revealed a new CD24 positive population. Since the
CD44
+
/CD24
2
signature has been associated with tumor
initiation, the up-regulation of CD24 population suggest that
ATF-126 could decrease the tumor initiating ability of the original
line MDA-MB-231.
The above phenotypic consequences of ATF-126 reactivation,
inhibition of tumor growth and metastatic potential as well as gain
of epithelial features, are all consistent with the documented
functions of the target gene Maspin. The fact that both the Maspin
shRNA and the Maspin siRNA were able to rescue the cell growth
phenotype in ATF-126 transduced cells demonstrated that the
phenotype of ATF-transduced cells was dependent on Maspin, and
not on potential off target effects.
The molecular targets by which Maspin exerts its tumor/
metastasis suppressive functions are still under investigation.
Maspin cDNA over-expression results in re-activation of multiple
pathways involved in tumor suppression and apoptosis, motility,
and cell adhesion [18],[16]. In agreement with these functions of
Maspin, we have found that tight junction, cell adhesion, and
multiple tumor suppressive pathways were activated by ATF-126.
To dissect the downstream targets of ATF-126 that were ‘‘bona
fide’’ downstream targets of Maspin, the Maspin cDNA was cloned
into the same inducible retroviral vector, and genome-wide
transcriptional profiles were compared between the un-induced
and induced cells. An overlapping 19-target gene signature
between the ATF-126 and the Maspin cDNA expressing cells
was built, which represents ‘‘high confidence’’ hits by which
Maspin could exert its mechanism of function in Claudin-low
breast cancer cells. It is important to note that ATF-126 and
Maspin cDNA represent two different mechanisms to up-regulate
target gene expression; whereas the Maspin cDNA over-expresses
the exogenous transgene form of Maspin, the ATF reactivates the
endogenous gene, which results in up-regulation the physiologi-
cally relevant isoform, at ‘‘normal’’ cellular levels. In addition,
unlike cDNA over-expression, which is predominantly cytoplas-
mic, the ATF-126 also results in substantial activation of the
nuclear form of Maspin (manuscript in preparation), which mediates
tumor and metastasis suppressive functions [28]. Thus, it is not
surprising that transcriptional profiles of ATF-126 and Maspin
cDNA were overlapping, yet not identical. Definitive analysis of
genome-wide binding specificity of ATF-126 will require the
integration of ChIP-seq data, which is underway. Overall, our
results outlined the importance of using several methodologies to
up-regulate Maspin, which allowed the dissection of potential
therapeutic targets downstream the Maspin cascade.
Three targets belonging to this overlapping 19-gene signature
were up-regulated by 10–100 fold over un-induced cells: Solute
carrier family 8 (sodium/calcium exchanger), member 2 (SLC8A2), Carnosine
synthase 1(CARNS1), and Dapper, antagonist of beta-catenin, homolog 3
(DACT3). These genes represent novel Maspin targets with
potential tumor suppressor activity. SLC8A2 encodes a Na(+)/
Ca(2+) exchanger, participating in intracellular Ca(2+) homeosta-
sis, and its expression is restricted to the brain. Interestingly,
SLC8A2 is found silenced by methylation in human gliomas where
it has been proposed to act a tumor suppressor gene [29]. CARNS1
belongs to the ATP-grasp family of ATPases, and catalyzes the
formation of carnosine (beta-alanyl-L-histidine) and homocarno-
sine (gamma-aminobutyryl-L-histidine). Carnosine is a naturally
occurring substance discovered more than a hundred years ago,
and is present in the mammalian brain and skeletal muscle [30].
Although is function is still under investigation, Carnosine has
been proposed to have a protective effect against oxidative stress
and represents a potential therapeutic agent for treatment of aging
and Alzheimer’s disease [31]. More recently Carnosine has been
shown to inhibit proliferation of human brain cancer cells in vitro
[32]. In 2010 a report showed that Carnosine retarded tumor
growth in vivo in a NIH3T3-HER2/neu mouse model [33]. Thus,
our results suggest that CARNS1 could potentially represent a
Maspin-dependent tumor suppressor enzyme in Claudin-low
Figure 6. ATF-126 and
Maspin
cDNA co-regulated targets.
A. Venn diagram indicating the intersecting up-regulated genes
between the ATF-126 and the Maspin cDNA groups in DOX induced
cells. B. Gene expression analyses of nine differentially up-regulated
genes. The ATF-126 and Maspin cDNA stable cell lines were treated with
either vehicle (2DOX) or DOX (+DOX) for a period of 72 hrs, and
differences in expression quantified by qRT-PCR. Data was normalized
to the 2DOX cells, and represent an average of three independent
experiments. C. Common MicroRNAs differentially regulated in the ATF-
126 and the Maspin cDNA cell lines, as assessed by qRT-PCR. Data was
normalized to the 2DOX cells. Up-regulated miRNAs are indicated in
red, and down-regulated miRNAs in green.
doi:10.1371/journal.pone.0024595.g006
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carcinomas. DACT3 is expressed in the embryonic CNS, and is a
negative regulator of Wnt/b-catenin signaling [34]. DACT3 is
down-regulated in colorectal cancers by epigenetic mechanisms
including histone methylation and deacetylation [35]. Given the
importance of Wnt/b-catenin in the mammary gland tumorigen-
esis in triple negative, mesenchymal breast cancers [36], we
speculate that DACT3 represents a potential therapeutic target for
these carcinomas.
ATF-126 and in less extent Maspin cDNA +DOX cells up-
regulated the protein gamma-4 subunit/guanine nucleotide-binding protein 4
(GNG4) gene, a brain-specific protein [37] with potential tumor
suppressor activity, which, like Maspin [38], is up-regulated by
hypoxia factors [39]. Another unexplored potential target of Maspin
in the 19-gene signature is the Apoptosis-Associated Tyrosine kinase
(AATYK), a protein kinase predominantly expressed in the nervous
system. AATYK regulates apoptosis, neurite growth, and differen-
tiation of cerebellar granule cultures [40]. Lastly, ATF-126, and in
less extent Maspin cDNA, up-regulated the SRC kinase signaling
inhibitor 1 (SRCIN1), a gene encoding a novel Src-binding protein
that regulates Src activation. Gain and loss of function approaches
in breast and colon cancer cells demonstrated that SRCIN1 inhibits
EGFR and Erk1/2 signaling, blocking scatter and proliferation of
cancer cells [41]. In neurons, SRCIN1 is predominantly localized to
dendritic spines and enriched in the postsynaptic density, where it
modulates spine shape via regulation of the actin cytoskeleton [42].
In addition to potential tumor/metastasis suppressor ORFs
naturally expressed in neural cells, both ATF-126 and Maspin
cDNA up-regulated miRNAs previously associated with tumor
suppression in many types of cancers, including miR-1 [23,24] and
miR-34 [25]. Intriguingly again, miR-1 is naturally expressed in
dorsal root ganglion neurons where it has a role in modulating
neurite outgrowth [43]. Similarly, miR-34 has been shown to
target Actin in mouse neuronal cells [44]. In addition to activation
of potential tumor suppressive miRNAs, both ATF-126 and
Maspin cDNA down-regulated putative oncogenes, including
miRNA-10b.
The up-regulation of epigenetically silenced genes and miRNAs
normally expressed in the nervous system is intriguing. Once over-
expressed in tumors, these targets could be involved in differenti-
ation of tumor cells, and metastasis inhibition. As a member of the
SERPIN gene superfamily, nuclear Maspin is physically associated
with Histone Deacetylase 1 (HDAC1) and functions as an HDAC
inhibitor, remodeling chromatin and mediating gene reactivation
[28,45]. This is in agreement with our data, and could
mechanistically explain the epigenetic reactivation of multiple
tumor suppressors epigenetically silenced in poorly differentiated
tumor cells, and the overall reprogramming of a mesenchymal line
towards a more benign, more differentiated, and less aggressive cell
line. Subsequent analysis in our identified Maspin targets will reveal
whether Maspin directly binds the promoter of these genes, resulting
in tumor and metastasis suppression. We are presently addressing
the potential of ATFs to be delivered in breast tumors and
metastasis, using nanoparticle-based strategies (manuscript in prepara-
tion). Another line of investigation will ascertain if ATFs can increase
the sensitivity of ER- tumors to anti-cancer drugs used in the clinic,
such as anti-estrogens.
Materials and Methods
Cell lines
The human breast cancer cell line MDA-MB-231 (ATCC, Cat.
No. HTB-26) and all stable cell lines derived from it were cultured
in DMEM medium supplemented with 10% fetal bovine serum
and grown at 37uC in a humidified 5% CO
2
incubator.
Development of a double stable Tet-On cell lines
The ZF-126, the ATF-126, and the Maspin cDNA were cloned
into the p-RetroX-Tight (Cat. Num. 632104, CloneTech, Moun-
Table 1. Genes regulated with ATF-126 and Maspin cDNA.
GeneID Name Symbol
6543 solute carrier family 8 (sodium/calcium exchanger), member 2 SLC8A2
1043 CD52 molecule CD52
6659 SRY (sex determining region Y)-box 4 SOX4
147906 dapper, antagonist of beta-catenin, homolog 3 (Xenopus laevis) DACT3
1013 cadherin 15, type 1, M-cadherin (myotubule) CDH15
162494 rhomboid, veinlet-like 3 (Drosophila) RHBDL3
4093 SMAD family member 9 SMAD9
6615 snail homolog 1 (Drosophila) SNAI1
285489 docking protein 7 DOK7
9148 neuralized homolog (Drosophila) NEURL
3726 jun B proto-oncogene JUNB
51207 dual specificity phosphatase 13 DUSP13
80725 SRC kinase signaling inhibitor 1 SRCIN1
57571 carnosine synthase 1 CARNS1
2786 guanine nucleotide binding protein (G protein), gamma 4 GNG4
29993 protein kinase C and casein kinase substrate in neurons 1 PACSIN1
9625 apoptosis-associated tyrosine kinase AATK
126567 C2 calcium-dependent domain containing 4C C2CD4C
1907 endothelin 2 EDN2
doi:10.1371/journal.pone.0024595.t001
Suppression of Breast Tumor Progression by ATFs
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tain View, CA, US), and delivered into the MDA-MB-231-LUC
cells by retroviral transduction. After transduction the cells were
placed under selection with puromycin (1 mg/ml) and geneticin
(800 mg/ml) for ten days. The CONTROL (p-RetoX-Tight empty
vector), ZF-126 (p-RetoX-Tight-ZF-126), the ATF-126 (p-RetoX-
Tight-ATF-126), and the Maspin cDNA (p-RetoX-Tight-Maspin)
stable cell lines were expanded and used for in vitro and in vivo
experiments.
ShRNA stable cell lines
Scramble and Maspin shRNAs (Open Biosystems) were co-
transfected with pMDG.1 into 293TGag-Pol cells to produce
retroviral particles. Transfection was done using Lipofectamine
system. The viral supernatant was used to infect the MDA-MB-
231-ATF-126 stable cell line. Cells were treated with hygromycin
B (100 mg/ml) for 10 days.
siRNA Maspin gene knock-down
The p-RetoX-Tight-ATF-126 stable cell line was reverse-
transfected with either 50 nM of Maspin siRNA smart pool (4
siRNAs/pool) or mismatch siRNA, and complexed with dharma-
FECT reagent (all siRNAs and reagents from Dharmacon,
Chicago, IL, USA). Transfection conditions were optimized using
a cytotoxic siRNA targeted against human ubiquitin B (Dharma-
con). Cells were induced with DOX for 24 hours, then 2.5610
5
cells/well were transfected in 6-well plates, and DOX was added.
Cells were maintained in transfection media for 72 hours, and
subjected qRT-PCR analysis.
Gene expression, western blot, proliferation, Immunofluores-
cence and apoptosis assays were performed as described [13].
Primers and probes for gene expression assays are shown in Table
S4. Primary and secondary antibodies from the corresponding
companies were applied at the concentrations shown in Table S5.
Quantification of cell death
Cells were collected and centrifuged 5 min at 1006g, then
resuspended in 0.2% trypan blue. Cells were incubated 3 min at
room temperature and counted on a hemacytometer. The
percentage of cell death was calculated by comparing all samples
counts with the ATF-126 2DOX cells.
Subcutaneous Injections
Female SCID mice (age 4 weeks) were purchased from Taconic
Farms and housed under pathogen-free conditions. The Institu-
Figure 7. ATF-126 regulates markers associated with decreased tumorigenicity and metastasis. A. Microarray expression analysis of
selected epithelial markers in CONTROL and ATF-126 cells, in presence or absence of DOX. Cells were collected 72 hours after DOX induction. Arrays
were performed in triplicate with three different biological replicates using Agilent 44 k arrays. The Array tree was derived from an unsupervised
hierarchical clustering and the gene list is shown in Table S1. Each colored square on the upper right represents the relative mean transcript
abundance (in log2 space) with highest expression being red, average expression being black, and lowest expression being green. B. Induction of
ATF-126 by DOX modifies the tumor initiating cell signature CD44
+
CD24
2
. Representative flow cytometric analysis of CD44 and CD24 expression
levels in ATF-126 2DOX and +DOX cells collected 72 hours after treatment. The forward scatter (FCS) channel was plotted in y-axis and the
fluorescence of the cell surface antigens in the x-axis. The gate in the +DOX panel illustrates the generation of a novel CD24+population upon
induction of ATF-126. C. A model illustrating a potential mechanism by which ATF-126 could reprogram a mesenchymal, Claudin-low MDA-MB-231
cell line towards a more epithelial-like phenotype.
doi:10.1371/journal.pone.0024595.g007
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tional Animal Care and Used Committee (IACUC) at the
University of North Carolina at Chapel Hill approved all
experiments described herein. MDA-MB-231-Control-LUC or
MDA-MB-231-ATF126-LUC cells (1610
6
) were collected and re-
suspended with matrigel (BD Bioscience, San Diego, CA, US) 1:1
volume ratio in a total volume of 100 ml. The cell-matrigel mixture
was injected into the mouse flank. Tumor growth was monitored
by caliper twice a week. When the tumor reached a size of
approximately 0.5 cm, doxycycline (+DOX) was administered to
mice in form of green food pellet (200 mg/Kg of mice chow) for a
period of 25 days. For the DOX removal group, the DOX food
was replaced by normal food. During the entire experiment the
mice weight was monitored to verify whether toxicity occurred.
After DOX treatment, the tumor volume was monitored both by
caliper and Bioluminescence imaging (BLI) as described [13].
Assessment of tumor shrinkage was monitored one day previous to
DOX induction and the day of tumor collection.
Tail-vein
Three days before the injections mice were kept in normal or
DOX+diet. Animals were injected via tail-vein with either 1610
5
MDA-MB-231-Control-LUC or MDA-MB-231-ATF126-LUC
cells in 100 ml of PBS. BLI imaging was performed as described
[13]. Assessment of tumor growth was monitored in vivo once a
week for up to 8 weeks.
Flow cytometry
Cells were stained with primary antibodies anti-CD24-conju-
gated with phycoerythrin (PE) and anti-CD44 conjugated with
Fluorescein isothiocyanate (FITC) (BD Bioscience, San Diego,
CA, US) following the manufacturer’s recommendations. Analysis
was performed using a FACScalibur flow cytometer and the
CellQuestTM software.
Gene expression microarrays
A total of six cell lines were used for gene expression analyses:
CONTROL 2DOX, CONTROL +DOX, ATF-126 2DOX,
ATF-126 +DOX (all with 3 technical replicates), p-RetoX-Tight-
Maspin 2DOX, and p-RetoX-Tight-Maspin +DOX (with 2
technical replicates). For each cell line, total RNA was purified,
amplified, labeled, and hybridized [46] using Agilent Agilent
4644 K oligo microarrays (Agilent Technologies, United States).
All microarray data is available in the Gene Expression Omnibus
(GEO) Database (http://www.ncbi.nlm.nih.gov/geo/query/acc.
cgi?token = nnolvkqsosuwwlm&acc = GSE27842). The probes/
genes were filtered by requiring the lowest normalized intensity
values in both 2DOX and +DOX samples to be .10. The
normalized log2 ratios (Cy5 sample/Cy3 control) of probes
mapping to the same gene were averaged to generate independent
expression estimates. We also used available microarrays from the
breast cancer cell lines [21], the UNC337-patient [20], the
MERGE 550-patient dataset [47] and the NKI (295 patients
[48,49]). All microarray cluster analyses were displayed using Java
Treeview version 1.1.3. Average-linkage hierarchical clustering
was performed using Cluster v2.12 [50]. ANOVA tests for gene
expression data were performed using R (http://cran.r-project.
org).
ATF-126/Maspin cDNA gene signatures
In order to build an ATF-126 signature, we selected those genes
that were significantly differentially expressed between ATF-
126+DOX and ATF-126 2DOX using SAM, with ,1% FDR.
The resulting up-regulated gene list is shown in Table S1.
In order to determine the genes up-regulated with the Maspin
cDNA, two biological replicates of the non-induced cells (2DOX)
and cells induced with DOX were subjected to DNA microarray
analyses, as described above. Gene expression values from the
Maspin cDNA 2DOX (N = 2) was subtracted from the Maspin
cDNA +DOX (N = 2), and the 132 up-regulated genes are shown
in Table S5.
MicroRNA microarrays
Total RNA was purified from the ATF-126 and the Maspin
cDNA cells treated with either vehicle (2DOX) or DOX for
72 hours using Trizol. Small RNAs were enriched using the
miRNeasy Mini Kit (SA Biosciences, Frederick, MD, USA), and
cDNA was generated with the RT2 miRNA First Strand Kit
(SABiosciences, Frederick, MD, USA). Samples were subjected to
MAH-102-C Micro-RNA arrays (SABiosciences, Frederick, MD,
USA), and data was normalized to the vehicle treated cells
(2DOX). Expression of miR-10b, miR-1, miR-34a, miR363, and
miR-124 was validated in two independent assays using hydrolysis
probes (Table S4).
Prediction of relapse-free survival (RFS)
To determine if the 550-gene signature was represented in
cancer patients from whom clinical data was available, we used
two patient data sets: the NKI-295 and the MERGE-550. First, we
examined the expression of the 550 genes in the MERGE-550
database and found that 322 out of the 550 genes were detected. A
2-way hierarchy cluster stratified the 322 patients into 2 groups
(cluster I and cluster II; Fig. S3). Patients in cluster II had a
significant better relapse free survival outcome (in 7 years follow-
up) than patients in cluster I. Second, we repeated the analysis
using the NKI dataset and found 444 genes. A 2-way hierarchy
cluster stratified the 295 patients into 2 groups (cluster I and cluster
II; Fig. S4). Here again, patients in cluster II had a significant
improved relapse free survival outcome and overall survival
outcome (in 18 years follow-up) relative to patients in cluster I.
Supporting Information
Figure S1 Generation of an inducible ATF expression
system to monitor breast tumor and metastasis. A.
Schematic representation of ATF-126 comprising the six Zinc
Finger (ZF) specific DNA-binding domains and the VP64
transactivator domain. ATF-126 was targeted against a unique
18-base pair site in the Maspin promoter. B. ATF-126 induction to
monitor breast tumor progression. ATF-126 was cloned into
pRetroX-Tight inducible vector system. The pRetroX-Tight
vector is composed of a modified tetracycline response element
(TRE
Mod
) and a minimal CMV promoter (CMV
min
). The
activator protein is a tetracycline–controled transactivator (rtTA),
which binds to the TRE
Mod
sequences in presence of Doxocycline
(DOX). Viral particles were prepared and MDA-MB-231-LUC
cells (engineered with a luciferase reporter) were transduced to
generate stable cell lines. ATF-126 was induced both in vitro and in
vivo with the chemical inducer DOX.
(TIFF)
Figure S2 Expression levels of CD44 and CD24 in a
panel of breast cancer cell lines, as assessed by flow
cytometry.
(TIFF)
Figure S3 Gene clustering showing the 322 genes from
the ATF-126-550 gene signature present in the
MERGED-550 patient dataset. Two-way hierarchy cluster
Suppression of Breast Tumor Progression by ATFs
PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e24595

stratified the 550 patients into two groups (cluster I and cluster II);
patients in cluster II had a significant better relapse free survival
outcome (in 7 years follow-up) than patients in cluster I. Each
colored square on the upper right represents the relative mean
transcript abundance (in log2 space) with highest expression being
red, average expression being black, and lowest expression being
green.
(TIFF)
Figure S4 Gene clustering showing the 444 genes from
the ATF-126-550 gene signature present in the NKI-295
patient dataset. Two-way hierarchy cluster stratified the 295
patients into 2 groups (cluster I and cluster II); patients in cluster II
had a significant better relapse free survival outcome and overall
survival outcome (in 18 years follow-up) than patients in cluster I.
Each colored square on the upper right represents the relative
mean transcript abundance (in log2 space) with highest expression
being red, average expression being black, and lowest expression
being green.
(TIFF)
Table S1 Genes up-regulated in ATF-126
+
DOX versus
2
DOX.
(DOC)
Table S2 Pathway analysis of the 550-gene signature
using the David database (http://david.abcc.ncifcrf.
gov/).
(DOC)
Table S3 Antibodies used in this study.
(DOCX)
Table S4 Primers and probes used in this study.
(DOCX)
Table S5 Genes differentially regulated with the Mas-
pin cDNA.
(DOC)
Acknowledgments
The authors thank Drs. A. Prat, C.M Perou and G. Wu for their assistance
with gene expression microarray data analysis.
Author Contributions
Conceived and designed the experiments: ASB PB. Performed the
experiments: ASB AR HL. Analyzed the data: ASB CF. Contributed
reagents/materials/analysis tools: ASB AR HL PL CF. Wrote the paper:
ASB PB.
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