Biochemical and Biophysical Research Communications xxx (2015) 1e6

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Biochemical and Biophysical Research Communications

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MTA1 regulation of ERb pathway in salivary gland carcinoma cells

Kazufumi Ohshiro*, Rakesh Kumar
Department of Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, George Washington University, 2300 Eye Street, Washington,
DC 20037, USA

a r t i c l e i n f o a b s t r a c t s

Article history: Although Metastatic-tumor antigen 1 (MTA1) is differentially expressed in metastatic cancer and cor-
Received 23 June 2015 egulates the status and activity of nuclear receptors, its role upon estrogen receptor b (ERb) e a potent
Accepted 8 July 2015 tumor suppressor, remains poorly understood. Here we investigated whether MTA1 regulates the
Available online xxx
expression and functions of ERb, an ER isoform predominantly expressed in salivary gland cancer cells.
We found that the depletion of the endogenous MTA1 in the HSG and HSY salivary duct carcinoma cell
Keywords:
lines enhances the expression of ERb while MTA1 overexpression augmented the expression of ERb in
Coregulators
salivary duct carcinoma cells. Furthermore, MTA1 knockdown inhibited the proliferations and invasion of
Estrogen receptor b
Growth regulation
HSG and HSY cells. The noted ERb downregulation by MTA1 overexpression involves the process of
Salivary gland cancer proteasomal degradation, as a proteasome inhibitor could block it. In addition, both MTA1 knockdown
Cell migration and ERb overexpression attenuated the cell migration and inhibited the ERK1/2 signaling in the both cell
lines. These findings imply that MTA1 dysregulation in a subset of salivary gland cancer might promote
aggressive phenotypes by compromising the tumor suppressor activity of ERb, and hence, MTA1-ERb axis
might serve a new therapeutic target for the salivary gland cancer.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
Metastatic-tumor antigen 1 (MTA1), an integral coregulatoy
component of the nucleosome remodeling and histone deacetylase
(NuRD), is differentially expressed in metastatic cancer cells and
regulates diverse cellular processes during tumorigenesis [1e3].
Fundamental to coregulatory activity of MTA1 is its ability to
regulate the expression of target gene products at the levels of
transcription and/or post-transcription [4e10]. Initially, MTA1 was
identified as a corepressor of the estrogen response element (ERE)-
driven transcription through its direct binding to histone deacety-
lase (HDAC) and estrogen receptor-alpha (ERa) [8]. The MTA1-
NuRD complex also utilizes the ERE motifs in the BRCA1 pro-
moter to repress the transcription of BRCA1, contributing to
abnormal centrosome number and chromosomal instability [9]. In
addition, MTA1-HDAC complex also binds to the Gai2 regulatory
element to represses its transcription, leading to activation of the
Ras signaling pathway [10]. In addition, the stability of MTA1 is
regulated by the E3 ubiquitin ligase COP1-mediated proteasomal
* Corresponding author.
E-mail address: bcmkxo@gwu.edu (K. Ohshiro).
degradation and hence, MTA1 is needed for optimal repair of the
double-strand DNA break following ionizing radiation [4].
A subtype of estrogen receptor, the ERb is a nuclear receptor
activated by the sex hormone estrogens. The ERb is composed of
eight exons and its gene products have five isoforms (ERb1-ERb5)
resulting from alternative splicing of the C-terminal region [11].
ERb forms a homodimer with another molecule of ERb and a het-
erodimer with ERa in the cells [12]. Upon activation, ERb trans-
locates to the nucleus, binds to estrogen response DNA elements in
the target genes to modulate the transcription of the target genes as
a part of its genomic action [13]. In addition, activated ERb also
stimulates non-genomic signaling pathways such as mitogen-
activated protein kinase (MAPK/ERK) and participates in the
cellular functions in a manner that is independent of its DNA-
binding activity [14].
ERb is expressed in a wide range of tumors including breast,
ovarian and prostate [15e18] and thought to be also a predominant
estrogen receptor subtype in human oral epithelium and salivary
glands [19]. Although ERb is considered to act as a potent tumor
suppressor, the precise role of ERb remains contradictory in nature
[15e18]. One of our previous studies found a correlation between
the levels of coactivator proline-rich, glutamic acid-rich, and
leucine-rich protein-1 (PELP1) and ERb in the salivary duct carci-
nomas (SDC) [20]. We found a strong ERb levels in 52 out of the 70

http://dx.doi.org/10.1016/j.bbrc.2015.07.043
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Please cite this article in press as: K. Ohshiro, R. Kumar, MTA1 regulation of ERb pathway in salivary gland carcinoma cells, Biochemical and
Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.043
2 K. Ohshiro, R. Kumar / Biochemical and Biophysical Research Communications xxx (2015) 1e6
SDC tumor specimens analyzed [20]. The exclusive expression of
ERb in most of the SDC specimens suggested a potential role of this
ER isotype in the progression and the development of SDCs. Sub-
sequently, ERb was found to be also involved in the rapid estrogen
signaling and in the regulation of cytoskeletal remodeling and
migration of salivary gland adenocarcinoma cells [14]. Further-
more, the absence of ERb in SDC specimens correlate well with an
increased local and regional cancer recurrence, suggesting that the
down-regulation of the ERb expression may be closely associated
with an unfavorable clinical outcomes in patients with SDCs [21].
Recent studies from the salivary gland cancers such as pleomorphic
adenomas and adenoid cystic carcinoma suggest that ERb may
contribute in the progression and development of salivary gland
carcinomas [22,23].
In the context of coregulators, MTA1 expression negatively
correlates with the ERb expression in ovarian cancer tissues [24].
Although MTA1 overexpression in head and neck cell cancers, to
which SDC broadly belong, correlates well with the cell migration
and invasion [25e28], there is no study on the role of MTA1 and its
putative target in the salivary gland tumors. In the current study,
we investigated whether MTA1 signaling regulates the expression
and functions of ERb in salivary gland cancer cells. We also dis-
cussed the implication of MTA1 regulation of ERb tumor suppressor
as a potential new therapeutic target for the salivary gland
carcinomas.
2. Materials and methods
2.1. Cell cultures and reagents
Salivary duct carcinoma cell lines HSG and HSY cells were
maintained in DMEM/F12 (1:1) supplemented with 10% fetal
bovine serum (FBS) and 1  antibiotics/antimycotics, and
A253 cells were maintained with RPMI 1640 supplemented with
10% FBS and 1  antibiotics/antimycotics. MG132 was purchased
from Calbiochem. We used the following antibodies: ERb (Ab-3,
Calbiochem); MTA1 (Bethyl Laboratories); Myc (Neomarkers); GFP
(Clontech); Vinculin (Sigma-Aldrich); phospho-p42/p44 ERK (Cell
Signaling); ERK1, ERK2, paxillin (Santa Cruz Biotechnology). GFP-
ERb expression vector was kindly provided by Dr. Ratna Vadlumdi
(University of Texas Health Science Center).
2.2. Cell extracts and immunoblotting
Cells were washed once with PBS and then lysed in radio-
immunoprecipitation assay buffer [50 mmol/L TriseHCl (pH 7.5),
150 mmol/L NaCl, 0.5% NP40, 0.1% SDS, 0.1% sodium deoxycholate,
1  protease inhibitor cocktail (Roche Life Science), and phospha-
tase inhibitors (SigmaeAldrich)] for 10 min on ice. The lysates were
spun in a centrifuge at 4  C for 10 min. The cell lysates containing an
equal amount of protein (50 mg) were then resolved on an SDS-
polyacrylamide gel (8%), transferred to a polyvinylidene difluoride
membrane, probed with the appropriate antibodies, and developed
using the enhanced chemiluminescence method.
2.3. Cell proliferation, migration and invasion assays
For cell proliferation assays, three equal numbers of cells
(2  104) per sample were plated on 6-well plates, and then the
numbers of the proliferated cells were measured by counting them
with a Coulter counter (Beckman Coulter). For the migration assay,
cells were loaded onto the upper well of an uncoated Boyden
chamber (BD Biosciences) at a concentration of 1  105 cells per
well in triplicate. The lower side of the separating filter was filled
with culture medium. We stained, swabbed inside the upper well
Please cite this article in press as: K. Ohshiro, R. Kumar, MTA1 regulation of ERb pathway in salivary gland carcinoma cells, Biochemical and
Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.043
and then counted the cells that successfully migrated through the
filter. The same protocol was followed for measuring the invasion
potential with principal differences being the use of Matrigel-
coated Boyden chamber (BD Biosciences).
2.4. Immunofluorescent labeling and confocal microscopy
The cellular localization of proteins was determined by indirect
immunofluorescence. HSG or HSY cells were grown on sterilized
glass coverslips or 8 well chamber slides, fixed in 4% para-
formaldehyde, permeabilized in 0.1% Triton X-100, and blocked in
10% normal goat serum-PBS. The cells were incubated with primary
antibodies for 1 h, washed with PBS three times, and then incubated
with goat anti-mouse or goat anti-rabbit secondary antibody con-
jugated with Alexa 488 (green) or Alexa 555 (red) from Molecular
Probes. For actin cytoskeletal staining, phalloidin conjugated with
Alexa 488 was used (Molecular Probes). The DNA dye DAPI (Mo-
lecular Probes) was used as nuclear stain (blue). Microscopic ana-
lyses were done using an Olympus FV1000 laser scanning confocal
microscope in accordance with established methods, using
sequential laser excitation to minimize fluorescence emission bleed-
through. Each image was obtained at the same magnification.
2.5. siRNA and plasmid transfection
MTA1 specific and control nonspecific small interfering RNAs
(siRNAs) were purchased from GE Dharmacon. siRNAs were
transfected into the cells using pooled siRNA duplexes and mainly
Lipofectamine RNAiMAX (Life Technologies) with Opti-MEM (Life
Technologies) according to the manufacturer's protocol in 60 mm
dishes or 6 well plates. Plasmid vectors were transiently transfected
into the cells using FuGENE 6 or Lipofectamine LTX (Life Technol-
ogies) with Opti-MEM in accordance with the manufacturer's in-
structions in 60 mm dishes or 6 well plates.
3. Results
3.1. MTA1 regulates ERb protein in the salivary gland cancer cells
We previously validated the expression of ERb as a substantial
nuclear receptor in the salivary ductal carcinoma cell lines HSG and
HSY [19]. To examine whether MTA1 affects the expression of ERb in
the salivary gland cancer cells, we explored the effect of MTA1
silencing by specific siRNA on the levels of ERb. Western blotting
analysis showed that MTA1 knockdown leads to a distinctive in-
crease in the levels of ERb in the HSG, HSY and A253 cells (Fig. 1A).
Similarly, confocal microscopic analysis corroborated with the
finding that MTA1 depletion augments the staining intensity of ERb
in the HSG and HSY cells (Fig. 1B). To independently validate the
noted relationship between the MTA1 and ERb, we next determined
whether MTA1 overexpression could inversely influence ERb’s
expression. The overexpression of Myc-MTA1 attenuated the
expression of ERb in all three salivary gland cancer cell lines (Fig. 1C).
To investigate the potential mechanism by which MTA1 affects
ERb expression, ERb mRNA expression was detected by real-time
RT-PCR after MTA1 knockdown. The levels of ERb mRNA hardly
changed or decreased a little (Fig. 1D). Next, we examined the effect
of a proteasome inhibitor MG132 in the observed MTA1 regulation
of ERb protein. The MTA1 overexpression decreased the ERb protein
expression levels, while the treatment with MG132 restored
blocked the decrease of ERb expression upon MTA1 overexpression
in the HSG and HSY cells (Fig. 1E), suggesting that MTA1 regulates
the level of ERb protein through a proteasomal-degradation
pathway. These observations suggest that MTA1 regulates the
 K. Ohshiro, R. Kumar / Biochemical and Biophysical Research Communications xxx (2015) 1e6 3

A
HSG HSY A253 B
MTA1 HSG ERβ MTA1 DAPI Merge DIC

MTA1 Control
ERβ

siRNA
Vinculin
+ - + - + - Control
- + - + - + MTA1
C siRNA
HSG HSY A253 HSY
Myc-

MTA1 Control
MTA1

siRNA
ERβ

Actin Vinculin
Vector MTA1 Vector MTA1 Vector MTA1
D 1.6 E
HSG HSY HSG HSY
1.4
mRNA expression

1.2 ERβ
1 Myc-
ERβ MTA1
0.8 MTA1
0.6 Vinculin
0.4
Vector MTA1Vector MTA1 Vector MTA1Vector MTA1
0.2
Control MG132 Control MG132
0
siRNA

Fig. 1. MTA1 knockdown increase ERb protein expression and MTA1 overexpression decreases ERb protein expression in salivary gland carcinoma cells. A) After transfection of
control or MTA1 siRNAs into HAG, HSY and A253 cells, cell lysates were subjected to western blot analysis with MTA1, ERb and Vinculin antibodies. B) Effect of MTA1 knockdown to
ERb expression was determined by confocal microscopy. DIC: differential interference contrast. C) HSG, HSY and A253 cells were transfected with Myc or Myc-MTA1 plasmids and
cell lysates were subjected to western blotting with Myc, ERb and Vinculin antibodies. D) MTA1 knockdown did not change ERb mRNA expression. After transfection of control or
MTA1 siRNAs into HSG and HSY cells, ERb mRNAs were detected by real-time RT-PCR. E) Downregulation of ERb was blocked by proteasome inhibitor MG132. After transfection of
Myc or Myc-MTA1 plasmids, HSG and HSY cells were untreated or treated with MG132 (10 mM) for 3 h and cell lysates were subjected to western blotting.

expression of ERb at a post-translational level and thus, consistent
with the notion of no effect of MTA1 on the levels of ERb mRNA.
3.2. MTA1 influences cancerous phenotypes in the salivary gland
cancer cells via ERb
The preceding results suggested a role of MTA1 in the noted
regulation of ERb in the salivary ductal carcinoma cell lines. Next
we examined the biological consequences of MTA1 regulation of
ERb expression in the HSG and HSY cells. MTA1 knockdown
remarkably inhibited the proliferation of the salivary gland cancer
cells (Fig. 2A). However, there was no effect of ERb overexpression
on the growth rate of the HSG and HSY cells (Fig. 2B).
Because cell invasiveness is profoundly reflected in the cyto-
skeleton remodeling and motility, we next determined the impact
of MTA1 knockdown on the cytoskeletal reorganization in the HSG
and HSY cells. The confocal microscope observation illustrated a
notable disappearance of F-actin fibers in the cytoplasm and its
accumulation in the cell peripheral of the HSG (Fig. 3A) and HSY
(Fig. 3B) cells upon MTA1 knockdown. Furthermore, the focal
adhesion assemblies visualized by paxillin staining were also
increased in the cytoplasm of the HSG and HSY cells (Fig. 3C). In
contrast to the cells with MTA1-knockdown, the cytoskeleton of the
control cells remained well spread (Fig. 3A,B,C). These results
suggested that salivary gland cancer cells undergo remodeling of its
cytoskeleton in favor of low motility, in part, due to the depletion of
the endogenous MTA1.
In order to determine whether the motility of the HSG and HSY
cells is indeed influenced by MTA1 silencing, we next performed
the cell migration assays using the Boyden chambers. MTA1
knockdown markedly prevented the ability of the salivary cancer
cells to migrate through the chamber (Fig. 4A). In addition, over-
expression of ERb also effectively blocked the cell motility of the
salivary gland cancer cells (Fig. 4A). Furthermore, the invasiveness
of the HSG and HSY cells was also sufficiently compromised by
MTA1 knockdown (Fig. 4B).
One of the signaling pathways known to regulate cell motility
and invasion is the activation of p-ERK1/2 [29]. Next we investi-
gated whether the ERK phosphorylation is inactivated by MTA1
silencing as well as by ERb overexpression. We found that indeed,
MTA1 knockdown-mediated increased expression of ERb leads to
inhibition of ERK1/2 phosphorylation in the HSG and HSY cells,
while the net expression levels of total ERK1/2 remains unchanged
(Fig. 4C). Similarly, direct overexpression of ERb (as was the case
with MTA1 knockdown) also inhibited the activation of ERK1/2
phosphorylation in the both cell lines (Fig. 4D). To determine
whether the motility promoted by MTA1 overexpression is indeed
inhibited by the inactivation of p-ERK1/2, the migration assay was
performed with MEK inhibitor U0126. MTA1 overexpression-
increased motility of HSG was effectively inhibited by U0126
(Fig. 4E). Taken together, these findings suggest that the MTA1 may
regulate the cell biological functions of salivary ductal carcinoma
cell lines, in part, due to its regulation of ERb expression.
4. Discussion
In the current study, we found that MTA1 upregulation may
potentially contribute to ERb downregulation in salivary gland

Please cite this article in press as: K. Ohshiro, R. Kumar, MTA1 regulation of ERb pathway in salivary gland carcinoma cells, Biochemical and
Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.043
4 K. Ohshiro, R. Kumar / Biochemical and Biophysical Research Communications xxx (2015) 1e6

A HSG HSY
100000
100 60000
60
MTA1 Ctl siRNA MTA1 Ctl siRNA
80000
80 50000
50

Cell Number (x103)

Cell Number (x103) Vinculin
40
40000
Vinculin
60
60000 Ctl MTA1 siRNA Ctl MTA1 siRNA
30000
30
40
40000
20
20000
20
20000 MTA1 siRNA 10
10000 MTA1 siRNA

00 00
Day 3 Day 5 Day 7 Day 3 Day 5 Day 7
B
HSG HSY
70
70000 45000
100
60
60000 40000
80 GFP-ERβ

Cell Number (x103)

Cell Number (x103)

35000
50
50000
30000
40
40000 60
25000
Vector
30
30000 Vector 20000
40
20 15000
20000
10000
20
10
10000
GFP-ERβ 5000
00 00
Day 3 Day 5 Day 7 Day 3 Day 5 Day 7

Fig. 2. MTA knockdown inhibits salivary grand adenocarcinoma cell proliferations. A) After transfection of control or MTA1 siRNAs into HAG and HSY cells, cell proliferations were
investigated by cell counting over 7 days. Inlet panels of western blotting data indicate efficient MTA1 knockdown in HSG and HSY cells. B) After transfection of control or GFP-ERb
plasmids into HAG and HSY cells, cell proliferations were investigated by cell counting over 7 days.

A C
HSG F-actin MTA1 DAPI Merge DIC HSG Paxillin MTA1 DAPI Merge DIC
MTA1 Control

MTA1 Control
siRNA

siRNA

B HSY
HSY
MTA1 Control
MTA1 Control

siRNA
siRNA

Fig. 3. MTA1 knockdown changes cytoskeletal dynamics and focal contact assembly of salivary gland adenocarcinoma. A, B) Effect of MTA1 knockdown to cytoskeletal remodeling
of HSG and HSY cells was determined through observation of stained F-actin by confocal microscopy. HSG and HSY cells after MTA1 silencing were fixed, double-stained with Alexa-
568 or -488 conjugated phalloidin and MTA1 antibody, and then stained with Alexa-488 or -555 conjugated secondary antibody and DAPI. C) Effect of MTA1 knockdown to focal
contact assembly of HSG and HSY cells was determined by confocal microscopy. HSG and HSY cells after MTA1 silencing were fixed, double-stained with paxillin and MTA1 an-
tibodies, and then stained with Alexa-488 and -555 conjugated secondary antibodies and DAPI. DIC: differential interference contrast.

carcinoma cells. This is in-line with a widely recognized role of co-
repressive activity of MTA1 on a wide range of target molecules
including ERa, BRCA1, Gai2 and E-cadherin in breast cancer cells
[6,8e10]. Recently, the MTA1 expression has been shown to nega-
tively correlate with the expression of ERb in ovarian cancer tissues
and MTA1 repression of the ERb pathway facilitates anchorage-
independent growth of ovarian cancer cells [24]. In ovarian can-
cer, the reduction in the levels of ERb by MTA1 was associated with
a decreased ERb mRNA level [24]. However, the underlying mech-
anism of the ERb downregulation by MTA1 in the present study
appears to be mediated via a proteasomal degradation pathway as a
proteasome inhibitor MG132 could effectively block it (Fig. 1E). In
this context, ERa is known to be degraded by a 26S proteasome
pathway in estrogen-dependent manner [30,31]. The ubiquitin-
proteasome pathway is required for the regulation of the hor-
monal response of ERa through the receptor degradation in breast
cancer cells. As for ERb, a recent study found that the phosphory-
lation within activation function 1 domain (AF1) of ERb regulates
the recruitment of the ubiquitin ligase E6-associated protein (E6AP)
to ERb and its turnover in the absence of hormone [32].

Please cite this article in press as: K. Ohshiro, R. Kumar, MTA1 regulation of ERb pathway in salivary gland carcinoma cells, Biochemical and
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 K. Ohshiro, R. Kumar / Biochemical and Biophysical Research Communications xxx (2015) 1e6 5

A B

Migration (cell #/site)

300 500 150 400 140 60

Invasion (cell #/site)

HSG HSY HSG HSY 120 HSG HSY
250 400 300 100
200 100 40
300 80
150 200
200 60
100 50 40 20
100 100
50 20
0 0 0 0 0 0
MTA1 ERβ MTA1
Vinculin Vinculin Vinculin
siRNA siRNA

C D E
HSG HSY HSG
HSG HSY 240
p-ERK
MTA1

Migration (Cell #/site)

Total-
GFP-ERβ 200 ERK
Vinculin
ERβ 160
p-ERK1/2 Cont U0126

p-ERK1/2 120
ERK1/2
80
ERK1/2 Vinculin
40
Vinculin
0

Myc Control

Myc U0126

Myc-MTA1 Control

Myc-MTA1 U0126
siRNA

Myc Myc-MTA1

Fig. 4. MTA knockdown inhibits cell migration and invasion of salivary gland adenocarcinoma. A) HSG and HSY cells were transfected with control or MTA1 siRNAs (left) and
transfected with control or GFP-ERb plasmids (right) and their cell migrations were investigated. Upper panels indicate representative pictures of migrated cells. Cells migrated
through membrane filter were stained, taken pictures under inverted microscope and counted. B) After transfection of control or MTA1 siRNAs into HAG and HSY cells, cell invasions
were investigated. Cells invaded through Matrigel-coated membrane filter were stained, taken pictures under inverted microscope and counted. Upper panels indicate repre-
sentative pictures of invaded cells. HSG and HSY cells were silenced with MTA1 siRNA C) and overexpressed with GFP-ERb plasmid D). AeD, Cell lysates were subjected to western
blot analysis using p-ERK1/2 and ERK1/2 antibodies. E) HSG cells were transfected with transfected with Myc or Myc-MTA1 plasmids and treated with U0126 (30 mM) and the cell
migration was investigated.

Interestingly, subsequent studies demonstrated that ERb degrada-
tion is stimulated by the activation of ErbB2/ErbB3 receptors by
growth factor heregulin-b1 in breast cancer cells [33]. However, our
earlier study using breast cancer model systems discovered that the
activation of ErbB2/HER2 with heregulin-b1 suppresses ERE-driven
transcription through stimulation of MTA1 expression in breast
cancer cells [8]. In addition, MTA1 also destabilize E3 ubiquitin
ligase COP1 that in-turn, controls the stability of MTA1 itself [4].
Taken together, our present findings demonstrate a role of MTA1 in
the ubiquitin-proteasome mediated stabilization of ERb status in
salivary gland cancer cells. The expression level of ERb was higher
in MTA1-transfected than vector-transfected cells in the presence
of MG132. However, the reason of the higher expression of ERb
remains unclear.
Since the discovery of ERb, the expression and function of ERb
are studied in wide types of cancer tissues and cell lines including
in breast cancer [17]. The ERb protein expression in breast cancer
generally diminishes during breast cancer tumorigenesis [17].
However, the conflicting data exists for the status of the full-length
form of ERb1 and the spliced isoform ERb2 in the context of clini-
copathological parameters and clinical outcomes [17]. These
different conclusions may be, in part, could be attributed to
different protocols, different antibodies used and different scoring
systems.
There are a limited number of studies on the expression and role
of ERb in oral cancers. An earlier study from our laboratory found a
high level of the ERb expression (74%) in SDCs, suggesting a po-
tential role of this isotype in the progression and development of
SDCs [20]. Results from another laboratory suggest that ERb is
expressed in the ductal epithelium of normal salivary gland tissue
and in 73% of SDCs studied and that the patients with ER- and
androgen receptor (AR)-negative phenotypes have a lower survival
than the patients with the tumors expressing ERb and both ERb and
AR [21]. In these studies, the negative expression status of ERb was
identified to correlate well with various clinicopathological vari-
ables for a high risk for local and regional recurrence in patients
with SDCs [21]. In this context, our present data shows ERb upre-
gulation upon experimental silencing of MTA1 influences physio-
logical functions including, the proliferation, the motilities, and the
invasiveness of salivary gland carcinoma cells. Accordingly, these
observations helped us to further validate a tumor suppressor role
of ERb in physiological functions in SDCs.
MTA1 also regulates epithelialemesenchymal transition (EMT)
though its repression of E-cadherin in cancer cells [6,31,34]. In this
context, ERb expression positively correlates with E-cadherin level
and that ERb represses EMT through destabilizing EGFR in breast
cancer [35]. Furthermore, ERb also represses mesenchymal char-
acteristic in aggressive prostate cancer as revealed by the

Please cite this article in press as: K. Ohshiro, R. Kumar, MTA1 regulation of ERb pathway in salivary gland carcinoma cells, Biochemical and
Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.043
6 K. Ohshiro, R. Kumar / Biochemical and Biophysical Research Communications xxx (2015) 1e6
modulation of E-cadherin expression [36]. Taken together, findings
presented here suggest that a sub-set of salivary gland cancers
might acquire aggressive phenotypes due to compromised func-
tions of ERb as a tumor suppressor due to MTA1 dysregulation. Our
findings also raise the possibility that MTA1 status might serve a
potential biomarker of the aggressive phenotype of salivary gland
cancer, and might be useful for developing for new-targeted ther-
apies against the cancer.
Acknowledgments
This work is supported in part by the National Institutes of
Health grants CA098823 and CA090970 to RK.
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