Targeted silencing of the oncogenic transcription
factor SOX2 in breast cancer
Sabine Stolzenburg1,2, Marianne G. Rots1, Adriana S. Beltran2, Ashley G. Rivenbark2,3,
Xinni Yuan2, Haili Qian4, Brian D. Strahl3,5and Pilar Blancafort2,5,*
1Epigenetic Editing, Department of Pathology and Medical Biology, University Medical Center Groningen,
University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands,2Department of Pharmacology,
3Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA,
4State Key Laboratory of Molecular Oncology, Cancer Hospital/Institute, Chinese Academy of Medical
Sciences, Pan Jia Yuan Nan Li 17, Chaoyang District, Beijing 100021, P.R. China and5UNC Lineberger
Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
Received January 17, 2012; Revised March 22, 2012; Accepted April 11, 2012
The transcription factor (TF) SOX2 is essential for
the maintenance of pluripotency and self-renewal
in embryonic stem cells. In addition to its normal
stem cell function, SOX2 over-expression is asso-
ciated with cancer development. The ability to se-
lectively target this and other oncogenic TFs in cells,
however, remains a significant challenge due to the
‘undruggable’ characteristics of these molecules.
Here, we employ a zinc finger (ZF)-based artificial
TF (ATF) approach to selectively suppress SOX2
gene expression in cancer cells. We engineered
four different proteins each composed of 6ZF
arrays designed to bind 18bp sites in the SOX2
promoter and enhancer region, which controls
SOX2 methylation. The 6ZF domains were linked
to the Kruppel Associated Box (SKD) repressor
domain. Three engineered proteins were able to
bind their endogenous target sites and effectively
suppress SOX2 expression (up to 95% repression
down-regulation of SOX2 expression resulted in
decreased tumor cell proliferation and colony for-
mation in these cells. Furthermore, induced expres-
sion of an ATF in a mouse model inhibited breast
cancer cell growth. Collectively, these findings
potential of engineered ATFs to mediate potent
and long-lasting down-regulation of oncogenic TF
expression in cancer cells.
Transcription factors (TFs) are crucial molecules orches-
trating gene programs involved in self-renewal, differenti-
ation and organism’s
Maintaining the proper threshold of expression of TFs is
critical for the normal homeostatic function of cells and
tissues. Aberrant regulation of TF expression is frequently
found in human malignancies and associated with specific
tumor subtypes (1). Over-expression of oncogenic TFs is
well documented in the mammary gland, particularly in
poorly differentiated,triple negative breast
(TNBCs) (2). TNBCs are characterized by the lack of ex-
pression of Estrogen Receptor (ER?), Progesterone
Receptor (PR?) and Epidermal Growth Factor Receptor
2 (Her2?). Recent progress revealed that some TNBCs
belonging to the basal-like and claudin-low intrinsic
subtypes of breast cancers are highly aggressive and resist-
ant to treatment (3–5). It has been proposed that these
breast cancers are enriched in stem cells, which might be
critical for tumor initiation, progression and resistance to
chemotherapy and radiation (6–11). Albeit their funda-
mental role in tumor etiology and progression, TFs are
approaches due to their lack of small molecule binding
pockets. Thus, novel strategies are required to efficiently
silence the aberrant expression of oncogenic TFs in cancer
cells. Ideally these novel approaches should restore and
stably maintain the expression pattern of these TFs, like
it is observed in normal epithelial cells.
The SOX2 gene encodes a TF belonging to the
high-mobility group (HMG) family (12). SOX2 expression
is critical for the maintenance of self-renewal in embryonic
stem cells (ESCs) and neural progenitor cells (13–15).
*To whom correspondence should be addressed. Tel: +1 919 966 1615; Fax: +1 919 966 5640; Email: firstname.lastname@example.org
Published online 4 May 2012 Nucleic Acids Research, 2012, Vol. 40, No. 146725–6740
? The Author(s) 2012. 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.
While SOX2 is highly transcribed in self-renewal condi-
tions, its promoter undergoes epigenetic silencing during
the onset of differentiation of stem cells (16,17). In neural
stem cells epigenetic modifications in two SOX2 enhancer
elements, SRR1 and SRR2, control the onset of differen-
tiation gene programs (18). Thus, in the majority of
differentiated cells, including mammary epithelial cells,
the SOX2 promoter is silenced (19). However, SOX2 has
been detected in normal gastric mucosae and promoter
silencing by DNA methylation has been reported in
some human gastric carcinomas (20,21). In contrast to
gastric cancers, SOX2 has been found over-expressed in
multiple malignancies. The SOX2 gene was found
amplified in a subset of squamous cell lung and esophageal
cancers in which the amplification/upregulation of SOX2
was associated with improved clinical outcome (22).
Several publications report over-expression of SOX2 in
glioblastomas (23), non-small cell lung cancer (24,25),
prostate cancer (26), hepatocellular carcinomas (27) and
breast carcinomas (28), supporting a role of SOX2 as an
oncogene in these tissues. SOX2 was found over-expressed
in 28% of all invasive breast carcinomas and in 43% of
basal-like TNBCs (29). These reports suggest that SOX2
could activate important gene cascades involved in tumor
initiation and progression and in the maintenance of a
poorly differentiated state.
Previous studies targeting SOX2 in breast cancer cell
lines have shown that shRNA-mediated knock-down of
SOX2 resulted in cell cycle arrest by down-regulation of
Cyclin D1 (30). This arrest in the cell cycle was
accompanied by an inhibition of tumor cell proliferation
in xenograft models (30). Although shRNA or siRNA
approaches are widely used to silence gene expression,
there are potential limitations associated with inhibitory
RNA (RNAi). First, oncogenes are expressed at very high
levels in the mammary tissue, and thereby these targets are
difficult to knock-down completely by RNAi. Second,
siRNAs have a transient effect in tumor cells due to the
short half-life of the small RNAs, which limits the
long-term effect of RNAi in tumor cells. We reasoned
that molecules able to directly silence the promoter and
DNA regulatory regions necessary for oncogenic tran-
scription would result in potent transcriptional down-
regulation of the targeted gene.
Direct alteration of endogenous gene expression at
DNA level requires a sequence-specific DNA-recognition
module and an effector domain, which modulates tran-
scriptional activity. Zinc-finger (ZF)-based artificial tran-
scription factors (ATFs) are currently the state-of-the art
molecules able to bind genomic sequences with potentially
single locus specificity (31,32). Because ZFs bind endogen-
ous DNA sequences with high selectivity, they provide an
opportunity to modify, edit, and sculpt the epigenetic and
transcriptional state of endogenous promoters. In the
past, several genes have been targeted with ZF-based
ATFs for transcriptional up- and down-regulation of
targeted promoters (33–36). Recently, our laboratory has
reported ATFs able to reactivate the expression of the
tumor-suppressor gene MASPIN, which is silenced by epi-
genetic mechanisms in metastatic tumor cells. Expression
of our ATFs in breast cancer cells decreased tumor growth
and metastasis in vivo (37,38). Likewise, ATFs have been
designed to repress potential oncogenes, such as Epithelial
Cell Adhesion Molecule (EpCAM), human Telomerase
(34,39–41). In this article, we investigated the capability
of ATFs to down-regulate the oncogenic TF SOX2 in
breast cancer cell lines. Retroviral delivery of three
out of four designed ATFs led to a potent (?95%)
down-regulation of endogenous SOX2 mRNA and
protein expression in two breast cancer cell lines. This
strong suppression of the endogenous SOX2 promoter
activity was accompanied by a long-term inhibition of
growth. Furthermore, one of our ATFs was able to effi-
ciently inhibit tumor growth in a xenograft model of
breast cancer. Importantly, repression of SOX2 was
still maintained in the tumors in vivo even 48 days
post-injection of the tumor cells. Overall, our data
outline the therapeutic potential of ATFs to effectively
repress oncogenic TFs that are highly expressed in
MATERIALS AND METHODS
Cell lines and cell culture
The packaging cell line 293T-GagPol cells and the human
breast cancer cell lines MDA-MB-231, MDA-MB-435s
were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% Fetal Bovine Serum
(FBS, BenchMark, Gemini Bio Products) and 1%
Penicillin streptomycin (Pen/Strep, Invitrogen, Carlsbad,
CA). Culture media of MDA-MB-435s cells contained
MCF7 breast cancer cells were cultured in Minimum
sodium bicarbonate, 0.1mM non-essential amino acids
(NEAAs), 1mM sodium pyruvate, 0.01mg/ml Bovine
Insulin, 10% FBS and 1% Pen/Strep. MCF7 and MCF-
12A cells were cultured in DMEM containing 20ng/ml
Epithelial Growth Factor (EGF), 100ng/ml cholera
hydrocortisone, 5% Horse serum and 1% Pen/Strep.
MDA-MB-468 breast cancer cells were cultured in L15
media supplemented with 10% FBS and 1% Pen/Strep.
ZR-75-1 and BT549 cells were cultured in RPMI 1640
media supplemented with 10% FBS and 1% Pen/Strep.
SUM102 and SUM149 cells were cultured in human
mammary epithelial cell (HuMEC) media containing
HuMEC supplemental bullet kit (Gibco/Invitrogen),
bovine pituitary extract (Gibco/Invitrogen) and 1%
Pen/Strep. For SUM149 cells media contained addition-
ally 5% FBS. SUM159 breast cancer cells were cultured in
Ham’s F12 media containing 5mg/ml Bovine Insulin, 1mg/
ml hydrocortisone, 10mM Hepes, 5% FBS and 1%
Pen/Strep. SK-Br-3 cells were cultured in McCoy’s 5a
Pen-Strep. MDA-MB-453s breast cancer cells were
cultured in Leibovitz’s L15 Medium, supplemented with
10% FBS and 1% Pen-Strep. All cell lines were purchased
10% FBS and 1%
6726 Nucleic Acids Research, 2012,Vol.40, No. 14
Manassas, VA, USA) maintained at 37?C and 5% CO2.
ATCC (AmericanType Culture Collection,
The ZF target sites within the SOX2 promoter were se-
lected using the website www.zincfingertools.org (42). The
selection of three 18-bp target sites was based on the close
proximity to the transcriptional start site and the high
content of GNN-triplets in the target sequence. One
ATF was designed to target an 18-bp sequence in the
SOX2 enhancer region 1, ?4-kb upstream of the TSS
(Figure 1B). Specific primers were designed coding for
the amino acids in the recognition helix of the ZFs respon-
sible for the binding to the target sequence (Figure 1C).
The ZF proteins were generated by overlapping PCR
as described (43), SfiI-digested fragments were subcloned
into the retroviralvector
generating pMX-ZF552SKD, pMX-ZF598SKD, pMX-
ZF619SKD and pMX-ZF4203SKD. Each ATF contains
an internal SV40 nuclear localization signal (NLS) and
a terminal hemagglutinin (HA) decapeptide tag. The
correct ZF-sequence of the obtained product was con-
firmed by plasmid sequencing.
Retrovirus infection of MDA-MB-435s
The pMX retroviral vectors containing the SOX2-ATFs
were first co-transfected with the plasmid (pMDG.1) ex-
pressing the vesicular stomatitis virus envelope protein
into 293TGagPol cells to produce retroviral particles.
(Invitrogen, Carlsbad, CA) as recommended by the manu-
facturer. For cell proliferation and soft agar assays, cells
were harvested 24h after the last infection. For flow
cytometry analysis, protein, and mRNA extraction,
transduced cells were harvested 48h post-infection.
MDA-MB-435s breast cancer cells were transfected with
D-011778-01-04), an irrelevant (non-specific) siRNA
2n o i geR y r o t a l ugeR1no i geR y r o t a l ugeR
SOX2 Core Promoter Region
ZF1 ZF2 ZF3ZF4 ZF5ZF6
ZF6 ZF5ZF4 ZF3 ZF2 ZF1
Figure 1. Design of ATFs to down-regulate SOX2 expression. (A) Schematic representation of a 6 ZF ATF bound to DNA with the orientation of
the domains depicted. (B) Schematic illustration of the SOX2 promoter outlining the ZF-552SKD, ZF-598SKD, ZF-619SKD and ZF-4203SKD
targeted sequences and their location relative to the transcription start site (TSS). Highlighted are the core promoter (red), regulatory region 1
(green), and regulatory region 2 (blue). Arrows show the orientation of the 18-bp binding site in the promoter (from 50to 30). (C) Alpha-helical ZF
amino acid sequences chosen to construct the ATFs. Residues at position –1, +3 and +6 making specific contacts with the recognition triplets are
indicated in color (red refers to position –1, blue to position+3 and green to+6 of the ZF recognition helix). (D) Quantification of SOX2 expression
in 12 breast cancer cell lines by western blot.
Nucleic Acids Research, 2012,Vol.40, No. 14 6727
pool targeting the TF PATZ1 (siGENOME M-013539-00)
or human a positive control for transfection, a cytotoxic
siRNA pool UBB (siGENOME MU-013382-01-0002).
(Dharmacon, Lafayette, CA) according to manufacturer’s
protocol. Cells were collected 72h after transduction for
RNA or protein preparations.
Generation of MCF7 stable cell lines
The coding region of the ATFs ZF-552SKD and ZF-
598SKD was subcloned using the BamHI/EcoRI restric-
tion sites into the expression vector pRetroX-Tight-Pur
ticles from pRetroX-Tight-ZF-552SKD (ZF-552SKD),
Tight-empty vector (empty vector), and pRetroX-Tet-
Lipofectamin transfection (Invitrogen, Carlsbad, CA) of
293T-Gagpol cells according to manufacturer’s recom-
mendation. Virus-containing supernantant was harvested
48h post-transfection, filtered, and concentrated using
Billerica, MA). MCF7 cells were co-transduced with
ATF-expressing retroviral particles, and the second con-
taining the transactivator pTet-On particles (CloneTech,
Mountain View, CA) in a ratio of 1:1. Double stable
MCF7 cells were selected with 2mg/ml geneticin (Gibco/
Invitrogen) and 5mg/ml puromycin (InvivoGen, San
Diego, CA) for 10 days. ATF expression was induced
using Doxycycline (Dox, 100mg/ml) for 72h.
Lentiviral transduction of SOX2 cDNA
HEK 293T cells were transfected with a lentiviral vector
encoding the SOX2 cDNA
Cambridge, MA), together with the accessory plasmids
Gagpol, VSVG and RSV-REV as described (19). As
control, parallel transfections were
empty plasmid. Viral supernatants from either SOX2
cDNA or control transfections were used to infect the
MCF7 cell lines stably transduced with either empty
vector, ZF-552SKD, or ZF-598SKD, with a density of
5?105cells in 10cm plates. These cells were cultured
for 48h and cells were un-induced or induced with Dox
for 72h and then analyzed with a cell culture microscope.
Treatment with 5-aza-20-deoxycytidine (5-Aza)
MCF7 cells stably transduced with empty vector or
ZF-552SKD were plated in a density of 5?105cells in
10cm plates. Cells were un-induced or induced with Dox
(+Dox) and simultaneously treated with either vehicle or
5mM 5-Aza (+5-Aza, Sigma Aldrich, Milwaukee, WI).
Cells were cultured for 48h and then processed for quan-
titative real-time PCR (qRT-PCR).
Total RNA was extracted using RNeasy Kit (Qiagen;
Valencia, CA) and 3mg of RNA was converted into
cDNA using the High Capacity cDNA Archive Kit
(Applied Biosystems, Foster City, CA). qRT-PCR was
carried out as described (38). SOX2 mRNA expression
levels were detected using Taqman primer and probes
(Hs01053049s1) and GAPDH (FAM/MGB #4333764F).
Detection of Cyclin D1 mRNA was carried out using
Absolute Blue QPCR SYBR Green Low ROX Mix
(Thermo Scientific, Rockford, IL) with the following
Cyclin D1 detection primers (Applied Biosystems, Foster
City, CA): forward 50-GCTCCTGGTGAACAAGCT
CAA-30and reverse 50-TTGGAGAGGAAGTGTTCAA
TGAAA-30. For detection of human GAPDH as endogen-
ous control the primers forward 50-CCATGTTCGTCAT
GGGTGTGA-30and reverse 50-CATGGACTGTGGTC
ATGAGT-30were used. Data were analyzed using the
comparative ?Ct method (ABPrism software, Applied
Biosystems, Foster City, CA) using GAPDH as an
internal normalization control. Data represented an
average of at least three independent experiments and stat-
istical analysis was performed using Student’s t-test.
Nuclear extract preparation and western blotting
transduction and MCF7 cells were harvested 72h after
Dox-induction. Nuclear protein was extracted using
NE-PER Nuclear and Cytoplasmic Extraction Reagents
(Pierce, Thermo Scientific, Rockford, IL) according to
manufacture’s instruction. For western blot 25mg of
nuclear protein per lane was loaded and resolved on
10% pre-cast NuPAGE Bis-Tris Mini Gels (Invitrogen,
Carlsbad, CA). Proteins were transferred from the gel
on a Sequi-Blot PVDF membrane (BioRad, Hercules,
CA). Membranes were blocked with 5% non-fat dry
milk/TBST for at least 1h, and then probed with the fol-
lowing antibodies: rabbit anti-SOX2 polyclonal antibody
(Cell Signalling Technology, Danvers, MA) diluted
(Covance, Princeton, NJ) 1:2500 or anti-H3 (Active
Motif, Carlsbad, CA) diluted 1:10,000. The horseradish
peroxidase-conjugated secondary mouse anti-rabbit and
rabbit anti-mouse antibodies were used for detection
(Jackson Immunoresearch, West Grove, PA) diluted
1:10000 and visualized using ECL plus kit (Amersham,
Immunofluorescence and Immunohistochemistry
MCF7 cells were plated in 24-well plates coated with
fibronectin (Sigma-Aldrich, St Louis, MO). Immuno-
antibody (AB 5603, Millipore, Billerica, MA) diluted
1:200 and an anti-HA antibody 1:500 (Covance, Prince-
ton, NJ). For staining of tumor sections we used the fol-
lowing antibodies: anti-SOX2 (AB 5603, Millipore,
Billerica, MA) 1:500, an anti-Ki67 antibody (ab833,
antibody (Covance, Princeton, NJ) 1:1000. SOX2 was
(Invitrogen, Carlsbad, CA) 1:1000 dilution in MCF7
cells and 1:750 on tumor sections. Detection of the HA
epitope tag was performed with an Alexa-Fluor488
6728 Nucleic Acids Research, 2012,Vol.40, No. 14
dilution in MCF7 cells and 1:500 on tumor sections.
Images were taken using a confocal Leica microscope at
Chromatin immunoprecipitation (ChIP) assay
IgG (Invitrogen, Carlsbad,CA) 1:1000
Doxycycline-induced and un-induced MCF7 cells were
fixed, sonicated, and incubated with either an anti-HA
antibody, respectively (1mg/reaction). DNA complexes
were immunoprecipitated using Protein A Sepharose 4
Fast Flow beads (GE Healthcare, Pittsburgh, PA). DNA
was amplified by PCR using the SOX2-specific primers: 50-
ATCCTTCTTCATAA-30, with the following conditions:
cycle 1, 5min at 95?C; cycle 2, 1min at 95?C; cycle 3,
1.30min at 53?C; cycle 4, 1min at 72?C; repeat cycle 2 to
4, 35 times followed by a final step of 10min at 72?C.
PCR-products were visualized on a 1.8% agarose gel.
Cell proliferation assays
Six replicates of MDA-MB-435s/MCF7 cells were plated
in 96-well flat bottom plates in a density of 1000 cells per
well. Cell proliferation was assessed every 24h using a
CellTiter Glo assay (Promega; Madison, WI) according
to the manufacturer’s instructions. Emitted luminescence
was detected in a PHERAstar plate reader (BMG
readings obtained immediately after seeding of the cells
(day=0). Statistical analyses were performed by 2-way
analysis of variance (ANOVA).
Anchorage independent colony formation assays
For colony formation assays, 1.8% Agarose/PBS was
diluted with cell culture media to a final concentration
of 0.6%, and 2ml/well media/agar solution was plated
in the bottom layer of a 6 well plate. For the top layer
5000 cells were re-suspended in 0.3% media/agar solution
and plated in a volume of 2ml/well on the solidified
bottom layer. The soft agar was covered with 0.5ml
culture media and cultured in 5% CO2humidified incuba-
tor at 37?C for 50 days. Experiments were performed in
three replicates. Plates were counted visually for the
presence of colonies that were greater than 2mm in
diameter. Statistical analysis was performed with a
Student’s t-test with level of significance P<0.05.
Female NUDE mice (age 4 weeks) were purchased
from Taconic Farms and housed under pathogen-free
conditions. The Institutional Animal Care and Use
Carolina at Chapel Hill
17b-Estradiol (Sigma-Aldrich Corp. St. Louis, MO) and
8mg Cellulose (Sigma-Aldrich Corp. St. Louis, MO), were
subcutaneously implanted in the animals 7 days prior of
approved all experiments
University of North
the injection of the cells. MCF7 cells (2?106) were col-
lected and re-suspended with matrigel (BD Bioscience,
San Diego, CA) 1:1 volume ratio in a total volume of
100ml. 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 approxi-
mately 50–100mm3, Doxycycline (+Dox) was adminis-
tered to the mice in the form of green food pellets
(200mg/kg of mice chow) for a period of 28 days.
During the entire experiment the mice weight was moni-
tored to ensure absence of toxicity. Animals were
euthanized 28 days after Dox induction. Statistical differ-
ences between control and ATF animals were assessed by
Wilcoxon Ranks Sum Test analysis.
Delivery of SOX2-specific ATFs in breast cancer
cells suppresses SOX2 expression
In order to down-regulate SOX2 expression in tumor cells,
we designed ATFs consisting of arrays of 6ZF domains
linked to a potent repressor domain, the Kruppel
Associated Box (SKD) domain. Each ZF domain recog-
nizes 3bp of genomic DNA, and arrays of 6ZF domains
will read an 18-bp stretch of endogenous DNA (Figure
1A). Using the helix grafting or modular approach, ini-
tially developed by the Barbas group (44), we engineered
four distinct ATFs. Three ATFs were designed to bind the
proximal SOX2 promoter (ZF-552SKD, ZF-598SKD,
and ZF-619SKD) and one ATF (ZF-4203SKD) was
directed against the SOX2 regulatory region I (SRR1),
which controls SOX2 silencing in stem cells (Figure 1B)
(18). ZF-552SKD was engineered to recognize a sequence
that was perfectly conserved between the murine and
the human promoters. The ZF proteins were constructed
by PCR using the helix grafting approach as we have pre-
viously described (43). The specific a-helical sequences
used for the assembly of the proteins are shown in
We first investigated SOX2 expression levels in a panel
of 12 breast cancer cell lines by western blot (Figure 1D).
We found that SOX2 was over-expressed in several breast
cancer lines relative to non-transformed breast epithelial
cells, such as MCF-12 A. The highest expression of SOX2
was detected in the ER+luminal MCF7 cell line, followed
by the claudin-low triple negative MDA-MB-435s cell
line. SOX2 was also found up-regulated in the ER+
luminal line ZR-75-1 (Figure 1D). Gene expression micro-
arrays have recently questioned whether the cell of origin
of the MDA-MB-435 line is melanoma or basal breast
cancer (45,46). More recently, with the discovery of the
new mesenchymal intrinsic subtype of breast cancer (47),
MDA-MB-435s cells have been clustered within the
claudin-low subtype of breast cancer (5). To examine if
the ATFs were able to silence the endogenous SOX2
promoter, we chose the highest SOX2 expressing lines
MCF7 and MDA-MB-435s as model cell lines.
For transduction of MDA-MB-435s cells, the retroviral
vector pMX-IRES-GFP was used. These cells were
transduced with up to 80–90% efficiency, as measured
Nucleic Acids Research, 2012,Vol.40, No. 146729
by flow cytometry (data not shown). Quantitative changes
in SOX2 mRNA expression upon transduction of
MDA-MB-435s cells were assessed by real-time expression
analyses (qRT-PCR; Figure 2A). As shown in Figure 2A,
a significant down-regulation of SOX2 mRNA expression
was achieved with ZF-552SKD, ZF-598SKD but not with
Furthermore, the targeting of the regulatory region I of
ZF-552SKD ZF-552SKD ZF-598SKD
DOX - + - + - +
Dox -+ -+ -+
Figure 2. ATFs down-regulate SOX2 expression in MDA-MB-435s and MCF7 breast cancer cells. (A) Quantification of SOX2 mRNA expression by
qRT-PCR in MDA-MB-435s cells. PMX-IRES-GFP (empty vector), ZF proteins –552SKD, –598SKD, –619SKD, –4203SKD, or a pool of 107ZF
domains [Library-SKD (48)] were retrovirally delivered in the cells and total mRNA was extracted. Mock-treated cells (Mock) are also indicated as
control. Real-time quantification of gene expression was normalized to empty vector control samples. As positive controls for knock-down, cells were
transfected with an anti-SOX2 siRNA. A non-specific siRNA targeting another TF (PATZ1) was used as a negative control for siRNA transfection.
Error bars represent the standard deviation of three independent experiments. Statistical significance was analyzed using t-test (***P<0.001, *P<0.05)
(B) Detection of SOX2 protein levels by western blot in MDA-MB-435s cells transduced with the same constructs as in (A). An anti-Histone H3
antibody was used as a loading control. ATFs were designed with a C-terminal Hemagglutinin (HA) tag, used for ATF detection. (C) Quantification of
SOX2 mRNA by qRT-PCR in MCF7 breast cancer cells. MCF7 cells were stably transfected with either empty vector control, ZF-552SKD, or
ZF-598SKD. The ATF expression was induced by Doxycyclin as indicated in the x-axis (?/+ Dox). Error bars show SD of three independent
experiments and statistical significance was analyzed using Student’s t-test (***P<0.001, **P<0.01). (D) Detection of SOX2 protein by western
blot in MCF7 cells. An anti-Histone H3 antibody was used as a loading control. Samples are the same as in (C). (E) Immunofluorescence analysis of
MCF7 cells transduced with ZF-552SKD and ZF-598SKD. Detection of ZF-552SKD and ZF-598SKD is indicated in green (a HA-tag) and nuclear
staining in blue (Hoechst). The left panel shows un-induced (–Dox) and the right panel induced (+Dox) MCF7 cells. Images are taken at 40?.
6730Nucleic Acids Research, 2012,Vol.40, No. 14
SOX2 by ZF-4203SKD led to a potent down-regulation of
SOX2 mRNA (Figure 2A). When siRNA was used to
knock-down the SOX2 mRNA, only 50% SOX2 mRNA
down-regulation was achieved by the SOX2-specific
siRNA relative to control cells transduced with a non-
specific siRNA. Importantly, ZF-552SKD, ZF-598SKD
down-regulation of SOX2 mRNA levels relative to
empty vector control. Consistent with the results in
Figure 2A, reduction of SOX2 mRNA expression
resulted in strong suppression of SOX2 protein expression
by ZF-552SKD, ZF-598SKD and ZF-4203SKD, but not
with ZF-619SKD (Figure 2B; Supplementary Figure S1).
The ZF-619SKD construct was not properly expressed in
the tumor cells, as assessed by western blotting using an
anti-HA antibody to detect the terminal HA-tag in the ZF
protein (Figure 2B). Improper translation of designed
proteins could be due to instability of the protein or inef-
fective codon usage. Thus, this construct had no signifi-
cant effect on SOX2 mRNA expression. As an unspecific
SKD control we used a diversity library of 6ZF domains
comprising more than 107different ZFs capable of target-
ing any 50-(GNN)6-30sequence in the genome (48). These
6ZF-library members were linked to the SKD repressor
domain (library-SKD). Some down-regulation of SOX2
(38%) was observed upon transduction of this library in
the cells, which was expected based on the potential of
multiple library constituents to regulate not only the
SOX2 cis-regulatory regions but also other regulatory se-
quences, which could indirectly affect SOX2 expression.
The effect of library members on gene expression has been
well documented (48–52). However, the repressive effect
of the 6ZF library was significantly lower than the effect of
the proteins –552, –598 and –4203, demonstrating the
Similarly, the retroviral delivery of the SOX2-specific
6ZFs in absence of effector domain had no impact in
SOX2 transcriptional regulation. In addition, when the
same 6ZFs were linked to the transcriptional activator
VP64, a significant up-regulation of SOX2 mRNA expres-
sion was achieved with ZF-598VP64 and ZF-4203VP64 in
MDA-MB-435s cells (Figure 3). These data indicated that
the regulatory effect of the engineered proteins required
both, a sequence specific DNA binding domain and a
functional effector domain. The down-regulation of
SOX2 by the ZF proteins was validated at protein level
by western blot (Figure 2B and D) and is quantitated in
Supplementary Figure S1.
We next focused on the two most potent proximal
proteins, ZF-552SKD and ZF-598SKD, to assess their
capability to suppress SOX2 expression in a second cell
line, MCF7. Since MCF7 cells have lower transduction
efficiencies than MDA-MB-435s, we generated stable cell
lines using the pRetroX-tight retroviral vector system, by
which the expression of the ZF protein is controlled by
Doxycyclin (Dox). The induction of ZF-552SKD and
ZF-598SKD in MCF7 cells (+Dox) resulted in potent
down-regulation of both SOX2 mRNA and protein ex-
pression, compared with un-induced control (–Dox). In
contrast, no change of SOX2 expression levels were
detected in +Dox cells transduced with empty vector
(Figure 2C and D; Supplementary Figure S1). The expres-
sion of the ZF proteins in MDA-MB-435s and MCF7 cells
was validated by western blotting and immunofluores-
cence (IF), respectively, using an anti-HA antibody to
(Figure 2B and E). Collectively these results demonstrated
that the ZF silencers resulted in strong suppression of
SOX2 expression in MDA-MB-435s and MCF7 cells.
The engineered ATFs ZF-552SKD and ZF-598SKD
bound their targeted DNA in vivo
To verify the binding of our engineered proteins to their
target sites in the SOX2 promoter in vivo, ChIP assays
were performed. MCF7 cells stably transduced with
either ZF-552SKD or ZF-598SKD were induced with
Dox (+Dox) or maintained in –Dox media. Cells were
fixed, cross-linked, and chromatin was extracted. First,
ZF–DNA complexes were immunoprecipitated with an
anti-HA antibody, which detects the C-terminal tag of
the engineered constructs. The ChIP products were next
amplified by PCR using specific primers flanking the 18-bp
ZF binding sites (Figure 4A). As shown in Figure 4B in-
duction of ZF-552SKD and ZF-598SKD by Dox led to a
products, indicating that the ZF constructs were binding
to their target sites in the context of the endogenous SOX2
promoter. In addition, when the ChIP experiments were
performed with an anti-RNA Polymerase II (RNA Pol II)
antibody, a decrease of RNA Pol II-immunoprecipitated
products was detected in +Dox cells relative to the
un-induced cells (Figure 4B). These experiments indicate
that the engineered ZF proteins were physically associated
with the SOX2 promoter and directed potent transcrip-
tional repression. This silencing of SOX2 expression was
relative SOX2 mRNA expression
Figure 3. 6ZF domains linked to transcriptional activators enhance
SOX2 mRNA expressionin MDA-MB-435s
retrovirally transduced with either ZF-552, ZF-598 or ZF-4203 (retro-
viral constructs expressing the specific DNA-binding domains but
lacking the SKD effector domain or with the same ZFs linked to the
ZF-4203VP64). Library-VP64 sample refers to a pool of ?106different
6ZF domains (48). Quantification of SOX2 mRNA cells was analyzed
by qRT-PCR and normalized to empty vector control. (*P<0.05).
Nucleic Acids Research, 2012,Vol.40, No. 14 6731
Figure 4. ATFs bind their targeted site in the endogenous SOX2 promoter. (A) Schematic illustration of the chromatin Immunoprecipitation (ChIP)
assay. (B) ZF-598SKD (upper panel) and ZF-552SKD (lower panel) are binding their target sites, as assessed by ChIP using an anti-HA antibody.
Genomic DNA bound by the corresponding ATF was amplified using SOX2-specific primers. An anti RNA-polymerase II (RNA-Pol II) antibody
and no antibody (No AB) samples were used in the same assay, as positive and negative controls, respectively. A quantification of the ChIP assay by
densitometry analyses of the bands from the same gels is outlined below. (C) A schematic illustration of the proposed repressive mechanism induced
by ZF silencers in the SOX2 promoter. Upon recruitment of the co-repressor KAP1 (KRAB-associated protein 1) and NuRD (nucleosome
remodeling and deacetylase) by SKD in the targeted site, a repressive complex including HDACs (histone deacetylases), SETDB1 (histone
methyltransferase), and HP1 (heterochromatin protein 1) is assembled. This repressive complex catalyzes the formation of condensed chromatin
by de-acetylation of histones, demethylation of H3K4me3, and incorporation of H3K9me3.
6732Nucleic Acids Research, 2012,Vol.40, No. 14
not induced by DNA methylation, as subsequent treat-
ment of the ATF-transduced cells with the methyl-
transferase inhibitor 5-Aza-20deoxycytidine (5-Aza) did
not result in a reactivation of SOX2 (Supplementary
These findings are in agreement with the molecular
mechanism of SKD-mediated repression (Figure 4C).
In this model, SKD interacts with the co-repressor
KAP1 (53). Subsequent recruitment of the nucleosome
remodeling and histone deacetylase (NuRD) complex,
histone deacetylases (HDACs), histone methyltrasferase
(SETDB1) and heterochromatin protein 1 (HP1) catalyzes
the formation of condensed chromatin, which is inaccess-
ible for the binding of RNA Pol II.
ATF-mediated down-regulation of SOX2 expression
decreased cell proliferation and anchorage-independent
growth of MDA-MB-435s and MCF7 cells
Ectopic expression of the SOX2 cDNA has been associa-
ted with an induction of oncogenic properties in different
cancer cell types, including breast cancer. Reciprocally,
shRNA-mediated knock-down of SOX2 in breast (30,54)
and lung (55,56) cancer cell lines resulted in decreased
regulation of SOX2 expression mediated by our SOX2-
specific ATFs would also entail a decreased tumorigenic
phenotype of breast cancer cells. To this end, MDA-
ZF-552SKD or ZF-598SKD were first subjected to cell
vector, ZF-552SKD or ZF-598SKD over time for a total
period of 96h (Figure 5A). We found that cells expressing
ZF-552SKD and ZF-598SKD exhibited a significant re-
duction in tumor cell growth relative to un-transduced
mock cells or empty vector-transduced cells (both ATFs
P<0.001). In MCF7 cells stably transduced with the same
constructs, Dox treatment of the ZF-transduced cells
controls, even at 120h after seeding of the cells
To further validate that the down-regulation of SOX2
expression by the ZF silencers resulted in a decreased
tumorigenic phenotype, we performed colony formation
assays, which monitor anchorage-independent growth
(Figure 5C and D). MDA-MB-435s un-transduced mock
cells, empty vector, ZF-552SKD- and ZF-598SKD-
transduced cells were seeded in soft agar and the
number of colonies was quantified. While mock treated
and empty vector transduced cells formed abundant
foci in soft-agar, down-regulation of SOX2 by either
ZF-552SKD or ZF-598SKD abolished colony formation
(Figure 5C). These results were also validated in the
MCF7 cell line stably transduced with ZF-552SKD and
ZF-598SKD, where induction of the ATFs by Dox effect-
ively suppressed colony formation (Figure 5D).
The oncogenic properties of SOX2 have been associated
with activation of Cyclin D1 promoter, by direct binding
and trans-activation of the SOX2 TF. Reciprocally,
down-regulation of SOX2 was shown to arrest the prolif-
eration of the breast cancer cells by down-regulation of
Cyclin D1 (30). We therefore analyzed Cyclin D1 mRNA
levels in MCF7 cells stably transduced with either
ZF-552SKD, ZF-598SKD or controls. As shown in
Supplementary Figure S3, induction of the ZF silencers
resulted in a significant down-regulation of Cyclin D1
mRNA relative to control cells. Overall, these data dem-
down-regulation of tumor cell proliferation and anchor-
age independent growth.
To confirm that the phenotype of the ZF silencers in
inhibiting tumor cell proliferation was dependent on the
SOX2 target, rescue experiments with the SOX2 cDNA
were performed (Supplementary Figure S4). MCF7 cells
stably transduced with either ZF-552SKD or ZF-598SKD
were challenged with either a SOX2 cDNA-expressing
lentiviral vector (pSinSOX2) or with an empty vector
control. 48h after adding the lentiviral supernatants, the
cells were either maintained in a –Dox medium or
switched to a Dox-containing medium to activate the ex-
pression of the ZF proteins. As shown in Supplementary
Figure S4, the delivery of SOX2 cDNA in –Dox cells
resulted in enhanced cell proliferation relative to control,
consistent with the oncogenic function of SOX2 cDNA in
breast cancer. The delivery of the SOX2 cDNA in+Dox
cells rescued the cell proliferation phenotype of the ZF
proteins. These functional assays demonstrate that the
observed phenotype can be directly attributed to SOX2
Down-regulation of SOX2 using ZF-598SKD inhibited
tumor growth in a breast cancer xenogaft model in
To analyze the effect of the SOX2 ZF silencers in vivo, we
focused on ZF-598SKD since this protein mediated potent
repression of breast tumor proliferation in vitro. We took
advantage of the Tet-ON inducible ZF-598SKD and
empty vector control MCF7 cell lines to analyze whether
Dox induction of the ZF repressor resulted in long-term
repression of SOX2 and decreased tumor cell growth in a
mouse model. Unlike constitutive viral vectors, inducible
systems have the unique capability to interrogate the role
of the therapeutic agent when tumors are already estab-
lished (37). A total of 2?106
transduced with either ZF-598SKD or empty vector
control were implanted into the flank of nude mice.
Tumor growth was monitored every other day using a
digital caliper. Tumor volume was determined by meas-
urement of length (L) and the width (W) as described (37).
post-injection) half of the animals for each group
(N=6) were switched to a Dox-containing diet (+Dox),
whereas the other half (N=6) was maintained in
ZF-598SKD animals induced with Dox underwent a sig-
nificant inhibition of tumor growth relative to the dox-free
diet (–Dox) animals. In contrast, control tumors main-
tained an exponential growth during the entire experi-
ment. Moreover, the ZF-mediated inhibition of tumor
MCF7 cells stably
Nucleic Acids Research, 2012,Vol.40, No. 146733
growth was evident in most of the animals even 27 days
post-induction (Figure 6B). A significant (P=0.015) re-
duction of tumor growth was observed in ZF-598SKD
induced animals relative to the ZF-598SKD –Dox
animals. In contrast, empty vector animals did not
exhibit significant reduction on tumor volume upon induc-
tion with Dox (P=0.269) (Figure 6C). Examination of
the tumors by qRT-PCR demonstrated that repression of
SOX2 was maintained in ZF-598SKD induced animals
relative to un-induced ZF-598SKD and controls (Figure
6D). Pathological analysis of ZF-598SKD –Dox tumors
by hematoxylin-eosin staining revealed an amorphous
tissue with higher density of closely packed tumor cells
(Figure 6E, left panel). The same morphology was found
empty vector -Dox
empty vector +Dox
Fold increase in ATP
release relative to t=0
Fold increase in ATP
release relative to t=0
number of soft
Dox - + -+ -+
number of soft
ZF-552SKD empty vectorZF-598SKD
Figure 5. Repression of SOX2 decreases cell viability and anchorage-independent growth. (A) Cell viability analysis of MDA-MB-435s cells
transduced with either empty vector, ZF-598SKD or ZF-552SKD. Mock-transfected cells (Mock) were used to assess background. Cell viability
over time was monitored over a period of 96h after the initial seeding of the infected cells. Cell viability was monitored using a CellTiter Glo Assay
(19). (B) Cell viability assays in MCF7 cell cells. Empty vector, ZF-552SKD- or ZF-598SKD-transduced cells were induced with Doxycyclin every
48h. Un-induced (–Dox) cells were used as controls. The y-axis indicates fold increase in ATP release relative to time point 0 measured by
luminescence. Statistical significance was analyzed using two-way ANOVA. The P-values for both, ZF-552SKD and ZF-598SKD +Dox samples
versus the same samples in –Dox conditions were P<0.001 at the last time point (120h). (C and D) Anchorage-independent growth of
MDA-MB-435s and MCF7 cells. (C) Quantification of the number of soft-agar colonies from un-transduced MDA-MB435s cells (Mock),
MDA-MB435s cells transduced with empty vector, ZF-552SKD and ZF-598SKD. (D) Quantification of the number of soft-agar colonies from
MCF7 cells transduced with either empty vector, ZF-552SKD or ZF-598SKD. –Dox and+Dox indicate un-induced and induced cells, respectively.
Left panels show the quantification of colony numbers. Right panels show representative pictures of the soft-agar plates. Error bars represent SDs of
three independent experiments. Statistical significance was analyzed by Student’s t-test (**P<0.01, *P<0.05).
6734 Nucleic Acids Research, 2012,Vol.40, No. 14
in tumors derived from empty vector control (data not
shown). In contrast, the ZF-598SKD +Dox tumors ex-
hibited a more organized tissue with increased amount
of intervening stroma separating small islands of tumor
cells (Figure 6E, right panel). In addition, immunofluor-
escence analyses of the tumor sections demonstrated that
the ZF proteins were expressed in the nucleus of the
majority of tumor cells in ZF-598SKD +Dox animals,
but not in un-induced animals (Figure 7A) or controls
(data not shown). This induction of ZF expression was
accompanied by a decreased nuclear SOX2 staining
(Figure 7A), and by a decreased proliferation of the
tumor cells, as indicated by a Ki67 staining of the
tumor sections (Figure 7B). In summary, our in vivo
analyses indicated that the tumor suppressive functions
long-term inoculation of the tumor cells, resulting in
the maintenance of the SOX2 down-regulation and
decreased tumor cell proliferation in animal models of
were maintained after
tumor volume mm3
Dox - + - +
tumor volume mm3
empty vector +
Figure 6. A SOX2-specific ATF inhibits the growth of pre-existing s.c xenografts of MCF7 cells. (A) Time course plot of tumor volume monitored
by caliper measurements. Animals (N=6) were either maintained in a Dox-free diet (-Dox) or induced with Dox diet (arrow) at day 21
post-injection. (B) Picture of representative tumors collected at day 28 post-induction from induced empty vector, un-induced ZF-598SKD, and
induced ZF-598SKD animals. (C) Tumor volume measurements at day 21 post-induction from empty vector and ZF-598SKD groups (N=6 animals
per group). Differences between groups were assessed by a Wilconxon rank sum test. (D) Quantification of SOX2 mRNA expression by qRT-PCR in
tumor samples from a representative tumor xenograft. Bar graphs represent the mean and SD of three tumor samples. Differences in gene expression
were calculated with a Student’s t-test, *P=0.01 (E) Hematoxylin-Eosin staining of representative ZF-598SKD –Dox and +Dox tumor sections.
Un-induced (–Dox) animals revealed highly compact tumors. Induced (+Dox) ZF-598SKD sections comprised discrete islands of tumor cells,
separated by intervening stroma. Pictures were taken at 10? and a detail of a 40? magnification is shown.
Nucleic Acids Research, 2012,Vol.40, No. 146735
In this study, we investigated the capability of ATFs to
promote sequence-specific silencing of the oncogenic tran-
scription factor (TF) SOX2. SOX2 is a self-renewal TF
crucial to maintain pluripotency in embryonic stem cells
self-renewal gene promoters undergo several layers of epi-
genetic silencing by means of DNA, H3K4, H3K9 and
H3K27 methylation (57–59). Although the function of
SOX2 in the normal mammary gland hierarchy has not
been well explored, our lab has found that the gene is
silenced in human mammary epithelial cells (HUMECs)
derived from mammoplastic reductions (19). In contrast,
over-expression of SOX2 is frequently associated with the
development of many malignancies, including breast
cancer (30,54). Over-expression of SOX2 in breast carcin-
omas has been associated with disease progression and
poor clinical outcome (28). It has been proposed that
SOX2 is expressed in a subpopulation of cells within the
tumor with tumor-initiating characteristics (2). This
subpopulation of cells shares remarkable similarities in
their overall gene expression profiles with stem cells and
exhibit important phenotypic characteristics, such as sus-
tained proliferation and resistance to apoptotic insults
(60). Therefore, being able to target SOX2 and other
TFs involved in tumor initiation and maintenance
would provide a unique opportunity for anti-cancer inter-
vention. However, because of their lack of small molecule
binding pockets, TFs are currently an example of
‘undruggable targets’. Thus, novel strategies to effectively
down-regulate these targets are required; these agents are
anticipated to block specific gene programs involved in the
maintenance of proliferation of the bulk of the tumor,
stably abolishing tumor growth.
Previously, knock-down experiments using shRNAs
targeting SOX2 demonstrated that down-regulation of
SOX2 in cancer cells resulted in decreased tumor cell pro-
liferation by down-regulation of Cyclin D1 and induction
of cell cycle arrest (30,61). Although RNAi is widely used
to induce specific gene silencing, one potential limitation
of interference approaches has been the achievement of
complete knock-down of highly expressed gene tran-
scripts, such as oncogenic TFs. In contrast with post-
transcriptional approaches, transcriptional and epigenetic
silencing of targeted genes provides additional advantage
since only two genomic copies of the target promoter need
to be silenced. Such genome-based approaches would
prevent gene expression by silencing the promoter with
no opportunity for ‘residual’ oncogenic transcriptional
activity. In addition, genomic approaches have the
unique property to impact the epigenetic state of the
targeted promoter, which have the potential to enhance
the longevity of the silencing and the therapeutic effect
in vivo. Indeed, we have recently demonstrated that 6ZF
proteins can target DNMT3a into specific promoter sites
in vivo, resulting in stable, phenotypic reprogramming of
the tumor cell (62).
In order to down-regulate SOX2 expression directly at
DNA level, we generated four sequence-specific ZF
DNA-binding domains (31). Three ATFs were designed
Figure 7. Induction of ZF-598 SKD reduces SOX2 expression and tumor cell proliferation in vivo. (A) SOX2 (red) and ZF-598SKD (a-HA, green)
detection by immunofluorescence (IF) analyses of representative sections from ZF-598SKD un-induced (–Dox) and induced (+Dox) animals.
(B) Ki67 expression (red) analyzed by IF in the same samples. Nuclei were labelled with Hoechst (blue). Images were taken at 40?.
6736Nucleic Acids Research, 2012,Vol.40, No. 14
to bind the core promoter of SOX2 in close proximity to
the transcriptional start site (TSS) and one ATF was
designed to bind in the regulatory region of SOX2
(4200bp upstream the TSS). These multi-modular ZF
genomic ‘readers’ were linked to the transcriptional re-
pressor domain Kruppel-Associated box (SKD domain)
(63). SKD recruits the co-repressor KRAB-associated
protein 1 (KAP1). By assembling a complex with hetero-
chromatin protein 1 (HP1), the histone methyltransferase
SETDB1, nucleosome-remodeling (NuRD) and histone
deacetylases (HDAC), KAP1 facilitates heterochromatin
formation through methylation of H3K9 (53). In this
manuscript the SKD domain was recruited to the SOX2
promoter via the 6ZF proteins to promote gene silencing
and chromatin condensation in breast cancer cells lines
expressing high levels of SOX2. Our ChIP analyses
demonstrated that retroviral delivery of our ZF proteins
results in decreased RNA-Pol II recruitment to the SOX2
promoter. These results support the notion that the ATFs
were able to impact the epigenetic state of SOX2 by pre-
venting the binding of the transcription complex. The
ATF-induced condensation of active chromatin is most
likely not based on DNA-methylation, since co-treatment
of ATF-transduced cells with the DNA methyltransferase
inhibitor 5-aza-20-deoxycytidine (5-Aza) failed to rescue
SOX2 expression (Supplementary Figure S2). Instead,
theSKD domain could
deacetylation and/or histone methylation resulting in
potent SOX2 silencing and chromatin condensation.
The importance of the epigenetic modifications in the
regulation of SOX2 and in the phenotype of tumor cells
has been well documented in other reports. The SOX2
promoter has been found hypomethylated in glioblastoma
tumor specimens as compared with normal cell lines or
normal adjacent tissue (64,65). Treatment of SOX2-
re-activation of the endogenous gene thereby supporting
the role of DNA methylation as a critical regulator of
SOX2 silencing in glioblastoma (64). In addition to
DNA methylation maps, genome-wide high-throughput
profiling of histone modifications of embryonic, pluripo-
tent and lineage-committed cells demonstrated that
H3K27me3, could play a role in determining the transcrip-
tional state of SOX2. In embryonic stem cells, the SOX2
locus presented a high abundance of H3K4me3 marks,
together with an enrichment of H3K36me3 in the 30of
the gene. Moreover, SOX2 was found flanked by two
bivalent CpG islands, which could poise the gene for re-
pression (66). In this regard, more analyses need to be
performed in the breast cancer cells to uncover the role
of specific histone combinations in the transcriptional
status of SOX2 and the resulting phenotypic outcomes.
Three out of four ATFs mediated strong silencing
of SOX2 mRNA expression, even with higher potency
than siRNA. The ATFs ZF-552SKD and ZF-598SKD,
designed to bind the core promoter region, down-
regulated SOX2 mRNA expression by 74 and 94%,
respectively, and thus, nearly abolished expression of
SOX2 in MDA-MB-435s cells. ZF-4203SKD, which was
designed to bind the enhancer regulatory region I (18),
resulted in 88% repression of SOX2 expression. This
finding demonstrated that ATFs targeting regulatory
regions in chromatin promote potent down-regulation of
endogenous promoter activity. In our hands, the modular
approach for engineering of ZF proteins yielded 75%
success rate; hence three out of four ZF proteins were
able to silence a highly expressed oncogene in breast
cancer cells. When ZF-552SKD and ZF-598SKD were ex-
pressed in MCF7 cells by means of inducible retroviral
vectors, an arrest in tumor cell proliferation was
observed. Our xenograft experiments demonstrated that
ZF-598SKD inhibited tumor growth of breast cancer
cells in vivo, and this inhibitory phenotype was maintained
long-term, even 48 days post-injection. Pathological exam-
ination of the tumors revealed that ZF-598SKD induced
animals exhibited decreased proliferation, as demons-
trated by Ki67 staining, relative to un-induced or
control tumors. In addition, expression of the ZF
proteins and stable down-regulation of SOX2 in the
tumors was validated by qRT-PCR and immunofluores-
cence. Interestingly, the hematoxylin-eosin staining of the
ZF-598SKD induced tumors revealed small structured
islands of tumor cells separated by large areas of
intervening stroma, free of tumor cells. This phenotype
was in contrast with the highly dense and compact
growth of the tumor cells in un-induced and control
tumors. The significance of this distinct phenotype
induced by the ZF proteins is not known. However, it is
reminiscent with the notion that transcriptional and/or
epigenetic silencing of SOX2 could induce cell arrest re-
sulting in a more structured or normal-like growth of the
tumor cells in vivo.
To date, multimodular proteins composed of 6ZF
domains represent the state of the art molecules for the
engineering of designer transcription factors since they are
potentially capable of regulating single genes (32). The
specificity of our 6ZF silencers for SOX2 was further
evaluated by SOX2 cDNA rescue experiments, which sug-
gested that the cell proliferation defect mediated by the
ATFs was dependent on the down-regulation of SOX2.
Nevertheless, we arecurrently
genome-wide mapping of 6ZF binding sites by ChIP-seq
in our MCF7 cell lines stably expressing the 6ZFs. These
experiments will provide important insights regarding the
endogenous specificity of our proteins in the breast cancer
Previously our group has reported the ATF-mediated
re-activation of the tumor suppressor gene Mammary
demonstrated that MASPIN
cancer cells resulted in tumor and metastasis suppression
in breast cancer and non-small cell lung carcinoma cell
lines (37,38). The reactivation of MASPIN using the
VP64 activator domain was mediated at least partially
by DNA demethylation (38). Herein we have reported
the capability of the SKD domain to down-regulate
highly expressed oncogenic TFs in breast cancer cells.
Overall these results indicate that ATFs can modify the
transcriptional landscape of tumor cells to direct cell fate.
Thereby, this work opens the door to design an ‘alphabet’
of chromatin ‘editors’, with the ultimate goal to stabilize
Nucleic Acids Research, 2012,Vol.40, No. 146737
the longevity of the epigenetic, transcriptional, and pheno-
typic state. Ideally, such ‘ZF editors’ will be able to repro-
gram the tumor cells epigenetic landscape like it is
observed in normal epithelial cells.
Metastatic resistance and disease recurrence, which ul-
timately affect multiple pathways, including activation of
‘undruggable’ oncogenic TFs, are presently the main
causes of death of cancer patients. Novel treatments able
to suppress disease recurrence pathways will provide great
hope for targeting this disease, potentially in combination
with small molecules. Moreover, the delivery in vivo of
ATFs into the tumor cells has historically been a major
challenge and limitation for clinical applications. To this
aim, we are developing targeted nanoparticles encapsulat-
RNA-based delivery of nanoparticles circumvents several
problems associated with plasmid-based DNA delivery.
RNA has a negligible chance of integration in the chromo-
some, it is less toxic, and less immunogenic than
DNA. The in vitro synthesis of RNA incorporating
ribonucleotide analogues enhances the stability of the
RNA and the half-life inside the cells (Wang et al.
nanoparticles encapsulating an ATF-mRNA designed to
up-regulate the MASPIN promoter in ovarian cancer cell
lines demonstrated potent regulation of the endogenous
gene and robust therapeutic effect in vivo (Lara et al.,
submitted for publication). These data confirm that
ATFs can be delivered into the tumors in vivo and
achieve targeted and potent anti-tumor effects. In the
future, encapsulation of multiple agents, for example
small molecule inhibitors in combination with chemically
modified RNA, which has been successfully performed
with siRNA and doxorubicin in prostate cancer cells
(67), could improve therapeutic outcome. Delivery of
multiple agents together with ZF-encoded mRNA or
even protein (68), is particularly interesting given the
inherent plasticity of ZF domains to be designed for onco-
genes and tumor suppressor genes, the availability of epi-
genetic editors, which could stabilize the longevity of the
therapeutic effect in vivo, and the reported synergisms of
ATFs with chromatin remodeling drugs (69). In summary,
our data suggest that the targeted down-regulation of
highly expressed oncogenes using ATF-based technologies
can be used as a powerful tool for the long-term targeting
of oncogenic TFs with potential application in cancer
biology and other human diseases.
Supplementary Data are available at NAR Online:
Supplementary Figures 1–4.
(Department of Pathology and Laboratory Medicine,
UNC, Chapel Hill) for pathological evaluation of xeno-
graft tumor samples.
[1R01CA125273, 3R01CA125273-03S1]; DoD awards
to P.B. and B.D.S.]. Funding for open access charge:
Conflict of interest statement. None declared.
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