Oncotarget, Vol. 7, No. 2

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Epigenetic regulation of ZEB1-RAB25/ESRP1 axis plays a criticalrole in phenylbutyrate treatment-resistant breast cancer
Mariko Kikuchi1, Keishi Yamashita1,2, Mina Waraya1, Naoko Minatani1, Hideki
Ushiku1, Ken Kojo1, Akira Ema1, Yoshimasa Kosaka1, Hiroshi Katoh1, NorihikoSengoku1, Takumo Enomoto1, Hirokazu Tanino1, Masakazu Sawanobori2 andMasahiko Watanabe1
1Departments of Surgery, Kitasato University School of Medicine, Kanagawa, Japan

2

Epigenetic treatment research group, Japan

Correspondence to: Masahiko Watanabe, email: gekaw@med.kitasato-u.ac.jp
Keywords: phenylbutyrate, breast cancer, epigenetic, histone deacetylase (HDAC), ZEB1
Received: July 19, 2015

Accepted: November 15, 2015

Published: December 05, 2015

Abstract
Phenylbutyrate (PB) is a histone deacetylase antagonist that also exhibits
antitumor activity. In this study, we used 7 breast cancer cell lines to identify
biomarker candidates that predict PB sensitivity in breast cancer.
Comprehensive gene expression profiles were compared using microarrays,
and the importance of the identified genes to PB sensitivity was confirmed in genetransfection experiments. CRL and MDAMB453 cells were identified as PB-sensitive,
while MDAMB231 cells were PB-resistant. RAB25 and ESRP1 were identified as key
regulators of PB sensitivity, while ANKD1, ETS1, PTRF, IFI16 and KIAA1199 acted as
PB resistance-related genes. Expression of these genes was dramatically altered by
DNA demethylation treatments. RAB25 expression inhibited IFI16 and PTRF, while
ESRP1 expression suppressed ANKRD1, ETS1, and KIAA1199. Both RAB25 and ESRP1
were suppressed by ZEB1, which was in turn regulated via epigenetic mechanisms.
Thus, PB sensitivity is influenced by epigenetic expression alteration of ZEB1. The
genes associated with PB sensitivity are downstream targets of ZEB1. Epigenetic
regulation of ZEB1 may prove valuable as a critical biomarker for predicting resistanceto breast cancer therapies.

Introduction

Breast cancer is the most prevalent cancer amongfemales in Europe and the United States [11]. Breast
cancer is also the most prevalent carcinoma in Japanesewomen, ranking as the fifth leading cause of death amongfemales, despite the nation-wide spread of surveillancesystems and the emergence of novel anticancer drugs[12]. Progress in hormone therapy, chemotherapy, andmolecular therapies has dramatically improved clinicaloutcomes for breast cancer. However, resistance to these
therapies is a major obstacle to breast cancer treatment[13-15], and the molecular mechanisms underlying
resistance remain largely unknown.
In this study, we clarified the effects of PB in breast
cancer and identified PB-sensitive breast cancer cell lines.
We also investigated gene expression profiles to identify
biomarkers predictive of PB sensitivity.

Phenylbutyrate (PB) has been used to treat children
with hyperammonemia resulting from an inherited
urea cycle abnormality [1]. PB causes the excretion of
glutamine in urine, which decreases blood ammonia levels[2]. In addition, PB is a histone deacetylase (HDAC)
antagonist that also exhibits anticancer effects in vitro [3].
A preclinical study showed that PB has cytotoxic effects at
concentrations greater than 0.5 mM [4]. A phase I clinical
trial recommended a dosage of 27 g/day for patients withsolid tumors, as the blood PB concentration reached 0.5-3mM under that regimen [4] Significant clinical anticancer
effects were reported for leukemia [5-8], colorectal cancer[9] and prostatic cancer [10]. However, no reports have
yet described the clinical efficacy of PB for treating breast
cancer.
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Results

The effects of the 1-fold PB dosage on each breast
cancer cell line are shown in Table1. Sensitive cell lines
were Her2-positive (CRL and MDAMB453), while theresistant cell line was Triple Negative (TN, MDAMB231).
In the sensitive cell lines (MDAMB453 and CRL cells),
PB reduced proliferation in a dose-dependent manner evenat lower doses (0.5-fold at 0.25 mM and 0.25-fold at 0.125mM)these lower PB dosages had no effect in the resistant(MDAMB231) cells (Fig. 1B). We then compared theeffect of PB with that of Trastuzumab, which decreases
proliferation of Her2-positive cells both in vitro and invivo, in the Her2-positive, PB-sensitive cell lines [16,17]. PB reduced proliferation much more than 10µg/ml
Trastuzumab in both the MDAMB453 and CRL cell lines,
while neither PB nor Trastuzumab reduced MDAMB231
cell proliferation (Fig. 1C).

Selection of PB-sensitive and PB-resistant breast
cancer cell lines
Cell proliferation was assessed in seven breast
cancer cell lines following PB treatments. Viable cells
were counted on day 7 and compared to control cellcounts (Fig. 1A). MDAMB453 and CRL cell counts werereduced by 70-80% at the 1-fold PB dose in comparison
with the control cells. SKBR, MCF7, YMB1E, YMB1
cell counts were decreased by 30% at the 1-fold PB
dose, and decreased by 80% at the 4-fold PB dose. Only
MDAMB231 cell counts did not decrease at all at either
the 1-fold or 2-fold PB dosage, but they did decrease by
80% at the 10-fold PB dosage. These findings suggested
that, although cell proliferation could be suppressed by PB
treatment in all seven lines, differences in gene expression
confer different sensitivities to PB depending on breast
cancer cell type. Therefore, MDAMB453 and CRL cells
were designated PB-sensitive strains, while MDAMB231
cells were designated a PB-resistant strain.

Identification of genes related to PB sensitivity
and resistance using expression microarrays andsemi-quantitative RT-PCR
To explore the molecular profiles underlying PB
sensitivity, expression microarrays were performed. Heat

Figure 1: Counts of viable breast cancer cells after administration of PB and trastuzumab. A. Counts of viable cells after
7 days of PB administration compared to control cells. B. Counts of viable cells after administration of lower PB doses (0.5-fold dosage
at 0.25mM and 0.25-fold dosage at 0.125mM) in PB-sensitive cells as compared to PB-resistant cells. C. Counts of viable cells after PB
administration compared to trastuzumab administration.
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Affymetrix are shown for PB-resistant and PB-sensitive
strains (Fig. 2A). The top 29 genes highly expressed in
PB-resistant strains and minimally expressed in PBsensitive strains were designated PB resistance-related
genes (Fig. S1A). These genes included ANKLD1, AXL,
CAV1/2, LDHB, ETS1, IFI16, PTRF (cavin), KIAA1199
and VIM, which have previously been linked to drug
resistance in breast and other cancers [18-28].
The PB sensitivity-related genes (the top 27 genes
highly expressed in PB-sensitive strains and minimally
expressed in PB-resistant strains) identified in the
microarray study included CLCA2, ESRP1, FGFR4,
MUCL1, FXYD3, RAB25, SEPP1, TFAP2B, andTOX3 (Fig. S1B). Reports of reduced expression andhypermethylation of promoter regions of these genes inhuman cancer tissues [29-38] suggests that they havetumor suppressor activity.
We confirmed that mRNA expression of those PB

restistance-related genes was low in MDAMB453 andCRL cells, and high in MDAMB231 cells, using semiquantitative RT-PCR (Fig. S1C). High mRNA expressionof PB sensitivity-related genes in MDAMB453 and CRL
cells, and low expression in MDAMB231 cells, was also
confirmed using this method (Fig S1D). RT-PCR analysis
recapitulated the microarray results, indicating that
Affymetrix expression microarray results were reliable.

Demethylation treatment changes expression of
PB resistance and sensitivity-related genes
Simultaneous treatment with 5 Aza-dC and the
HDAC inhibitor TSA, a highly effective demethylation
method [39], was used to evaluate whether epigenetic
factors affect the differential expression of the genes
identified above [40, 41].
Expression of eight PB resistance-related genes
decreased in the PB-resistant MDAMB231 cells after

Figure 2: Identification of PB sensitivity-related genes and PB resistance-related genes using expression microarrays
and expression changes after demethylation treatment. A. Heat map of Affymetrix gene expression in PB-resistant breast cancer
strains compared to PB-sensitive breast cancer strains. B. Demethylation treatment decreases expression of PB resistance-related genes in
MDAMB231 cells. C. Demethylation treatment increases expression of PB sensitivity -related genes in MDAMB231 cells. D. Expressionof ZEB1 and its downstream genes in PB treated and untreated cells.

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Relationship of resistance-associated genes tosensitivity-related genes

demethylation treatment (Fig. 2B).
Conversely, expression of four PB sensitivityrelated genes increased after demethylation treatment in
MDAMB231 cells (Fig. 2C). The combination of 5-AzadC and TSA primarily reactivates target genes, so PB
sensitivity-related genes were considered sensitive toprimary upstream alterations. We focused on specificallyon MUCL1, RAB25, TFAP2B, and ESRP1 in subsequent
experiments.

Differential gene expression between PB-resistantand PB-sensitive cell lines suggested that alterations inmolecular mechanisms affected by these genes might
explain the phenotypic difference (Fig. 3A). Possible
mechanisms in cancer cells include genomic and/or
epigenetic changes. If epigenetic changes drive these
expression differences, demethylation treatment should
reactivate PB sensitivity-related genes and silence PB
resistance-delated genes. Importantly, the molecularalterations associated with PB sensitivity are likely to be
monocentric by epigenetic mechanism (Fig. 3B).

Expression of ZEB1 and its pathway genes in PB
treated and untreated cells
We confirmed expression of ZEB1 and its pathway
genes in PB (1-fold dose) treated and untreated cells. In
MDAMB453 cells, expression of PB sensitivity-related
genes (RAB25, ESRP1) did not change. Similarly, in
MDAMB231 cells, expression of PB resistance-related
genes (ZEB1, ANKRD1, IFI16, PTRF, KIAA1199, andETS1) did not change (Fig. 2D).

Transfection of PB sensitivity-related genes into
PB-resistant MDAMB231 cells
Transient transfection of a plasmid vector the
expressing
full-length ESRP1 gene into MDAMB231 cells
reduced expression of the PB resistance-related genes

Figure 3: Relationship between resistance-related and sensitivity-related genes. A. Differential gene expression in PB-

resistant and PB-sensitive cell lines. B. Molecular regulation of PB sensitivity be monocentrically dependent on epigenetic regulation
rather than multicentrically dependent on a variety of genes. C. Transfection of the ESRP1 gene into PB-resistant MDAMB231 cells. D.
Transfection of the RAB25 gene into PB-resistant MDAMB231 cells.

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ANKRD1, ETS1, and KIAA1199 (p=0.0295, p=0.0147,
and p=0.0097, respectively), suggesting that ESRP1 is an
upstream regulator of ANKRD1, ETS1, and KIAA1199(Fig. 3C). Similarly, transient transfection of a RAB25expressing vector into MDAMB231 cells reduced
expression of the PB resistance-related genes IFI16 andPTRF (p=0.0145 and p=0.0341, respectively), suggestingthat RAB25 is an upstream regulator of IFI16 and PTRF
(Fig. 3D).
Transient transfection with plasmids containing
either full-length MUCL1 or TFAP2B into MDAMB231
cells did not alter the expression of PB resistance-related
genes (data not shown). Together, these findings indicatethat ESRP1 and RAB25 may play a critical and separate
roles in drug resistance by affection the expression of
ANKRD1, ETS1, KIAA1199, IFI16, and PTRF.
In addition, administration of the 1-fold PB dosage
to MDAMB231 cells transiently transfected with the
ESRP1 gene decreased cell counts (p=0.0438) (Fig.
4A). Similarly, administration of the same PB dosage
to the MDAMB231 cells transiently transfected with
the RAB25 gene also decreased cell counts (p=0.0365)(Fig. 4B). Moreover, exposure to the 1-fold PB dosage
decreased counts in MDAMB231 cells transiently
transfected with both the ESRP1 and RAB25 genes evenfurther (p=0.0025, p=0.0161) (Fig. 4C). Thus, ESRP1 andRAB25 independently and synergistically suppressed cellproliferation following PB administration.

ESRP1 and RAB25 were not directly regulated bypromoter DNA methylation in breast cancer
Expression of ESRP1 and RAB25 was low in
MDAMB231
cells, and demethylation treatments of 5 Aza-dC andtrichostatin A robustly increased expression of these twogenes (Fig. 2C). Hypermethylation of the DNA promoter
region for RAB25 has been reported previously, andboth RAB25 and ESRP1 have tumor-suppressive effects[35]. Regulation of the expression of these two genes via
promoter methylation might also affect proliferation inhuman cancers. However, we did not find CpG islandsin the promoter DNA for RAB25, and no methylation in

Figure 4: PB administration decreases cell proliferation. A. Counts of MDAMB231 cells transiently transfected with the ESRP1

gene after PB administration. B. Counts of MDAMB231 cells transiently transfected with the RAB25 gene after PB administration.
C. Counts of MDAMB231 cells transiently transfected with both the ESRP1 and RAB25 genes after PB administration. D. Counts of
MDAMB231 cells after ZEB1 gene knockdown via transient transfection of ZEB1 siRNA and PB administration.

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the promoter region for ESRP1 was detected by direct
sequencing (data not shown). These findings suggested
that neither gene is regulated directly by promoter DNA
methylation, but their expression may be indirectly
regulated by other methylation-sensitive genes. BothRAB25 and ESRP1 are strongly downregulated by theexpression of ZEB1, the epithelial-mesenchymal transition(EMT) inducer, in a Tetracycline induced model of humancancer [42].

low in MDAMB453 and CRL cells. Interestingly, levelsof both RAB25 and ESRP1 mRNA were high when ZEB1mRNA levels were low and vice versa, which is consistent
with a previous report examining other cancers [42]. These
reciprocal relations between ZEB1 and RAB25/ESRP1
were also observed in MDAMB231 cells following
demethylation treatments (right panel, Fig. 5A). Thesefindings suggest that ZEB1 regulates RAB25/ESRP1
expression at mRNA level in human cancer.
To investigate this directly, expression of the
RAB25 and ESRP1 genes was quantified in MDAMB453
cells transfected with ZEB1 using Q-RT-PCR (Fig. 5B).
Both RAB25 and ESRP1 were suppressed in MDAMB453
cells after transient transfection of ZEB1. Conversely,
expression of both RAB25 and ESRP1 was activated in
MDAMB231 cells after ZEB1 knockdown by transient
siRNA transfection (Fig. 5C). ZEB1 knockdown in
MDAMB231 cells was synergistically suppressed by
concurrent administration of PB solution 1x as expected(Fig. 4D).

Dyregulation of ZEB1 alters RAB25/ESRP1
expression in breast cancer cell lines
In our microarray experiment, ZEB1 was expressed
only in MDAMB231 cells and not in MDAMB453 andCRL cells. The expression of ZEB1 was confirmed bymeasuring mRNA levels in MDAMB231, MDAMB453
and CRL cells using RT-PCR (left panel, Fig. 5A).
Expression of ZEB1 was high in MDAMB231 cells andFigure 5: Zeb1 expression and immunohistochemistry. A. Left panel: Expression of ZEB1 mRNA in MDAMB231, MDAMB453

and CRL cells measured using RT-PCR. Right panel: Change in expression of ZEB1 mRNA after demethylation treatment measured using
RT-PCR. B. Expression of the RAB25 and ESRP1 genes in MDAMB453 cells after transfection with the ZEB1 gene. C. Expression of
the RAB25 and ESRP1 genes in MDAMB231 cells after ZEB1 knockdown resulting from transient transfection of ZEB1 siRNA. D.
Immunohistochemistry for ZEB1 in breast cancer tissues and normal breast tissues.

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Immunohistochemistryeffective inhibitor of cell proliferation in several breast
cancer cell lines. In fact, PB suppressed HER2-positive
cell proliferation more effectively than the HER2 antibody
trastuzumab. However, proliferation of TN-positive
breast cancer cells was not affected by PB. The search fordifferences in gene expression that might contribute to PB
resistance suggested a role for the ZEB1-RAB25/ESRP1
pathway, which is involved in epithelial-mesenchymaltransition (EMT) and is known to affect chemoresistancein breast cancer.
EMT, and the associated stem cell-like phenotype, isconsidered a major cause of resistance to therapy in breast
cancer [43, 44]. The EMT activator ZEB1, in particular,
has been shown to confer stemness and resistance totreatment [45]. Levels of ZEB1 protein are reduced by
the miR-200 microRNA family, and aberrant epigenetic
silencing of miR-200 in human cancer cells and tumors
promotes EMT, tumor cell adhesion, migration, invasion
and metastasis [46]. Overexpression of ZEB1 also
downregulates miR-200 and is associated with a prosurvival and drug-resistant phenotype [46, 47].
In the present study, 8 genes (ANKLD1, AXL,
CAV1, ETS1, IFI16, LDHB, PTRF (cavin) and
KIAA1199) were identified as PB resistance-related, and

ZEB1 protein levels were rated IHC 1+ in 15 breast
cancer samples, IHC 2+ in 16 samples, and IHC 3+ in
5 samples (Fig. 5D). ZEB1 was more strongly expressedin TN breast cancer than in the other cancers (p<.0001)(Table 2). Normal breast tissue did not show any ZEB1
staining.

Expression of ZEB1 pathway genes in breast
cancer clinical materials
Significant inverse correlations were found between
ZEB1 and RAB25, ZEB1 and ESRP1, RAB25 and IFI16,
RAB25 and PTRF, ESRP1 and ANKRD1, and ESRP1 andETS1 (Fig. 6A). This was consistent with the cell line datain our current study. The correlations between ZEB1 andRAB25, ZEB1 and ESRP1, RAB25 and IFI16, and ESRP1and ANKRD1 were particularly strong (Fig. 6B).

Discussion
In this study, we demonstrated that PB is an

Figure 6: Expression of ZEB1 pathway genes in clinical breast cancer materials. A. Expression of ZEB1 pathway genes inbreast cancer samples determined using microarrays. B. Diagram of correlations between the expression ofand ESRP1,

RAB25 and IFI16, and

ZEB1 and RAB25,

ZEB1

ESRP1 and ANKRD1.

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Table1: Comparison of reduction rate by PB treatment and subtype

Table 2: Zeb1 Immunohistochemistry

the expression of most of them was controlled by RAB25
or ESRP1. Although expression of PB resistance-related
genes was downregulated by demethylation treatments,
there have been no reports of genomic amplification forthese genes, suggesting that epigenetic changes alter
their expression indirectly via upstream molecules. Thus
RAB25 and ESRP1 likely play a central role in dynamicchanges in the expression of PB resistance-related genes.
Most of the PB resistance-related genes identified
here have established effects on chemoresistance.
ANKRD1 is a novel determinant of cisplatin resistance inovarian and breast cancers [18]. AXL belongs to a tyrosine
kinase family, and its inhibition promotes apoptosis,
blocks growth and enhances chemosensitivity in humannon-small cell lung cancer [19]. IFI16 (IFN-gammainducible protein 16) epigenetically suppresses estrogen
receptor expression, and it may affect tamoxifen sensitivity
in breast cancer cells [25]. CAV1 is associated withsensitivity to cisplatin and anthracyclin [20, 21], while
ETS1 is associated with both sensitivity to gemcitabineand multidrug resistance (MDR) [23]. Polymerase I
and transcript release factor (PTRF, cavin-1), which is acomponent of caveolae along with CAV1, is associatedwith chemoresistance in glioblastoma [26]. KIAA1199
expression, which has been localized to the endoplasmic
reticulum, has not yet been linked to drug resistance, but it
does affect malignant potential in breast cancer [27]. All ofthese genes were suppressed by demethylation treatment,
and upstream epigenetic targets are likely important incontrolling their expression.
Because such upstream gene candidates must
be reactivated through demethylation, we focused on
RAB25, ESRP1, TFAP2B and MUCL1. Among these fourcandidates, only RAB25 and ESRP1 affected expressionof PB resistance-related genes. Neither TFAP2B nor
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MUCL1 altered PB resistance-related gene mRNA levels(data not shown). Interestingly, levels of each mRNA
were downregulated by ESRP1 or RAB25, but not by
the two together. ESRP1 suppressed ANKD1, ETS1 andKIAA1199, while RAB25 suppressed IFI16 and PTRF.
This suggests ESRP1 and RAB25 are independently
involved in PB drug sensitivity, and the two act
synergistically to suppress PB-resistant cell proliferation.
After transfection with RAB25, MDAMB231
cell proliferation was dramatically reduced. RAB25 is aknown tumor suppressor gene, acting through multiple
pathways to enhance apoptosis and suppress angiogenesis
and invasion by modulating VEGF-A and VEGFR-1
expression [35]. ESRP1 (epithelial splicing regulatory
protein 1), which regulates alternative splicing eventsin epithelial cells, also has tumor suppressor effects
and is down-regulated during EMT [31]. Both RAB25
and ESRP1 were robustly reactivated by demethylation
treatment, but no evidence of promoter DNA methylation
was found in either gene.
It is possible that both genes are regulated by othercancer-relevant genes that are affected by promoter DNA
methylation. ZEB1 is one candidate that may affectbreast cancer in this way. ZEB1 levels are controlled
by miR-200 family microRNAs, the expression ofwhich is also influenced by DNA methylation in humancancer. Decreased expression of miR-200 by DNA
methylation promotes ZEB1 expression [48, 49], and
ZEB1 is strongly suppressed by demethylation treatment.
Reducing ZEB1 expression increases levels of ESRP1and RAB25 in human cancer and vice versa, and in ourstudy ZEB1 directly affected transcription of both genes.
Most intriguingly, ESRP1 and RAB25 synergisticallysuppressed proliferation of PB-resistant, MDAMB231(TN) cells, and siRNA-induced knockdown of ZEB1
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Oncotargetreduced proliferation even more. This suggests ZEB1
expression may play a crucial role in PB resistance inTN breast cancers. Immunohistochemistry confirmedthat ZEB1 is strongly expressed in TN breast cancer,
as compared to other cancers. Together, these findings
support the idea of using epigenetic suppression of ZEB1
as a novel therapeutic strategy to help overcome drug
resistance in human breast cancer.
In cell lines and primary breast cancer tissue,
ZEB1 reduces the expression of Both RAB25 and
ESRP1, which have tumor suppressor functions. RAB25
induces apoptosis in TN breast cancer [50], and reduced
expression of RAB25 is involved in PB resistance. ESRP1
inhibits EMT in breast cancer by antagonizing hnRNPM[51]. Here, we provide evidence that RAB25 affects
the expression of IFI16 and PTRF, while ESRP affects
the expression of ANKD1, ETS1 and KIAA1199. Of
these, expression of IFI16 and ANKD1 were particularly
strongly associated with RAB25 and ESRP1 levels inprimary breast cancer. Expression of IFI16 plays a role
in resistance to tamoxifen treatment in breast cancer [25],and ANKD1 is involved in resistance to cisplatin resistantin ovarian and breast cancers [18]. Thus, the ZEB1
pathway affects expression of a variety of genes that play
important roles in drug resistance during cancer treatment.

maintained in DMEM Medium (Sigma Aldrich, St Louis,
MO, USA). All media contained 10% fetal bovine serum(FBS) and Penicillin-Streptomycin (GIBCO).

PB and trastuzumab treatments in breast cancer
cell lines
1×106 cells from each cell line were incubated in PB
for 24 h. One tablet of PB (1g, triButyrate®, Fyrklövern
Scandinavia AB, Sweden) was dissolved in 100 mL
distilled water, and the PB solution was filtered using
a Corning® 250 mL Vacuum Filter System (0.22µm
Pore) (Corning, Tewksbury, MA). Previous literaturedemonstrated that serum PB concentrations could reach
0.5 mM-3 mM in humans when administered at dosage of
27 or 36 g/ day [4]. We thus designated 0.5mM as 1-fold
PB dosage, and 2-fold (1 mM), 4-fold (2 mM), 10-fold (5mM), and 20-fold (10 mM) dosages of PB were also usedin this study. Cell growth was observed for 7 days, during
which the culture medium and PB solution were replaced
every 72 h. Controls were not exposed to the PB solution.
On day 7, cells were detached using Trypsin-EDTA(GIBCO) and viable cells were counted using a Countess
automated cell counter (Invitrogen Life Technologies,
Carlsbad, CA). Finally, an additional 1×106 cells from eachcell line were treated in the same manner with trastuzumab(10μg/ml) (Roche, Basel, Schweizerland) rather than PB,
and viable cells were also counted on day 7.

Conclusions
Our findings show that PB sensitivity is controlled
through epigenetic regulation of a ZEB1-RAB25/ESRP1
pathway, and several candidate genes associated with PB
resistance downstream of ZEB1 have been identified.
Epigenetic regulation of ZEB1 may serve as a criticalbiomarker for predicting resistance to breast cancer
treatments.

5-aza-2’-deoxycytidine(5
Aza-dC)andtrichostatin A (TSA) treatments in BC cell lines
Cells from the PB-resistant strain (1×106 cells/T-75
flask) were treated with 1 or 5μM of the demethylating
agent 5-aza-2′-deoxycytidine (5 Aza-dC) (Sigma-Aldrich)dissolved in 50% acetic acid (Wako pure Chemical
Industries, Osaka, Japan) once every 24h for 4 days.
Controls were mock-treatment with PBS (Phosphate
Buffered Saline, GIBCO) in the same amount of acetic
acid once every 24 h for 4 days. In addition, 300 nM of
the HDAC inhibitor trichostatin A (TSA) (Sigma-Aldrich)
was added to the medium for the final 24 h.

Materials and Methods
Cell lines
We used 7 breast cancer (BC) cell lines(MDAMB231, CRL, SKBR, YMB1, YMB1E,
MDAMB453, and MCF7). MDAMB231, CRL, SKBR,
and YMB1 cells were kindly provided by the Kyushu
University Beppu Hospital (Oita, Japan). YMB1E cells
were provided by the Cell Resource
Center of the Biomedical Research Institute ofDevelopment and Aging, Tohoku University (Sendai,
Japan). MDAMB453 and MCF7 cells were purchasedfrom RIKEN Bio Resource Centre (Ibaraki, Japan). The
CRL, SKBR, YMB1, YMB1E, and MCF7 cells weremaintained in RPMI1640 Medium (GIBCO, Carlsbad,CA), the MDAMB453 cells were maintained in L15
Medium(GIBCO), and the MDAMB231 cells werewww.impactjournals.com/oncotarget

Expression microarrays
mRNA was extracted from the PB-sensitive strains(CRL and MDAMB453 cells) and the PB-resistant strain(MDAMB231 cell), we extracted mRNA using an RNeasy
Mini Kit (QIAGEN Sciences, Maryland, USA). Gene
profiles were compared using Affymetrix 3‘ IVT Express
Kit microarrays (harboring 54,675 genes) according
to the manufacturer’s instructions. Genes expressed athigh levels in the PB-sensitive strains and at low levelsin the PB-resistant strain were categorized as sensitivity1749

Oncotargetrelated genes. Similarly, PB resistance-related genes wereexpressed at high levels in the PB-resistant strain and at
low levels in the PB-sensitive strains.

chemotherapy who underwent surgical resection of the
primary tumors at Kitasato University Hospital between
January 1, 1996 and December 31, 2000. All patientsagreed to the use of pathological specimens. Tissues
were fixed in formalin and embedded in paraffin, and4 μm thick serial sections were used. The slices wereincubated with 3 % H2O2 at room temperature for 5 min todeactivate endogenous peroxidase and then washed with
PBS. Rabbit anti-human ZEB1 polyclonal antibody (Atlas
Antibodies, Stockholm, Sweden; 1:50 dilution) was added
and the slices were incubated at 4°C overnight. Immune
complexes were amplified using a Vectastain Elite ABC
kit (Vector Laboratories, Inc, Burlingame, CA) according
to the manufacturer’s instructions. These complexes
were then detected by incubating with the chromogen
3,3′-diaminobenzidine (DAB) substrate for 5 minutes.
ZEB1 expression levels were categorized according to
the diagnostic criteria of American Society of ClinicalOncology/College of American Pathology 2007 guidelines
as follows [52]: IHC 1+: light staining of more than 10%of the specimen; IHC 2+: moderate staining of more than
10% and less than or equal to 30% of the specimen; IHC
3+: strong staining of more than 30% of the specimen.

Semi-quantitative
reversetranscription
polymerase chain reaction (RT-PCR) andquantitative RT-PCR (Q-RT-PCR)
Total RNA was extracted from all cell lines
using a RNeasy Mini Kit (QIAGEN) and reversetranscribed using a SuperScript III reverse transcriptase
kit (Invitrogen). Quantitative RT-PCR (Q-RT-PCR) was
conducted in triplicate using iQ Supermix (Bio-Rad) on
the C1000 Touch TM Thermal Cycler CFX96 Real Time
System (Bio-Rad). PCR conditions and primer sequences
are provided in Table S1.

Plasmid and transfection
Full-length cDNA sequences of the MUCL1 andRAB25 genes were isolated by PCR and sub-cloned intoa pcDNATM3.1D/V5-His-TOPO vector (Invitrogen). The
vector with self-ligation was used as a control. ESRP1,
TFAP2, ZEB1, and control plasmids were purchasedfrom OriGene Technologies (Rockville, MD, USA).
Cells were seeded in 10cm dishes overnight to reach 9095% confluence. Then they were transfected with 24μg
of plasmid vector using Lipofectamine 2000 reagent(Invitrogen) in OPTI-MEM medium (GIBCO). Six hours
later, the transfected cells were transferred to complete
medium. After 48 h, the cells were harvested and used for
RT-PCR and Q-RT-PCR.

Expression of ZEB1 pathway genes in breast
cancer clinical samples
Microarray data from the MD Anderson Cancer
Center (Institut Goustave Russy cohort, GDS4057,
N=103) were downloaded from GEO(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi acc.
GDS4057).
Expression levels of
ZEB1/RAB25, 2ZEB1/
ESRP1, 3RAB25/IFI16,
RAB25/PTRF,
ESRP1/ANKRD1, and
ESRP1/ETS1 were plotted. Note that KIAA1199 levels
were not measured in this microarray.

siRNA and transfection
Small-interfering RNA (siRNA) targeting
human ZEB1 was purchased from Sigma Aldrich (StLouis, MO, USA). The ZEB1 siRNA sequences wereasfollows:
CCAAUAAGCAAACGAUUCUGA,
antisense: AGAAUCGUUUGCUUAUUGGCA. We used
MISSION siRNA Universal Negative Control (Sigma
Aldrich) as a control. Cells were seeded in 10cm dishes
overnight to reach 30-40% confluence. Then they weretransfected with 600 pmol of siRNA using Lipofectamine
2000 reagent (Invitrogen) in OPTI-MEM medium(GIBCO). Six hours later, the transfected cells weretransferred to complete medium. After 48 h, the cells were
harvested and used for RT-PCR and Q-RT-PCR.

Conflicts of interests
No potential conflicts of interest were disclosed.

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