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Downregulation of ER60 Protease Inhibits Cellular Proliferation by Inducing G1S Arrest in Breast Cancer Cells In Vitro.

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THE ANATOMICAL RECORD 295:410–416 (2012)
Downregulation of ER60 Protease
Inhibits Cellular Proliferation by
Inducing G1/S Arrest in Breast Cancer
Cells In Vitro
Department of Anatomy, National University of Singapore, Singapore
Department of Biological Sciences, National University of Singapore, Singapore
ER60 protease, a 58-kDa molecular chaperone in the endoplasmic
reticulum, is involved in glycoprotein synthesis. ER60 protease has been
reported to be differentially expressed in various cancers including breast
carcinoma. This study explored the relationship of ER60 protease with
cell proliferation in breast cancer in vitro. ER60 protease expression was
first determined in a panel of breast cell lines by real-time RT-PCR and
Western blot analysis and found to be most abundantly expressed in
T47D breast cancer cells. The ER60 protease gene was then successfully
knocked down in T47D breast cancer cells using two different sequences
of small-interfering RNA. The silencing efficiencies of siER-1 and siER-2
at 48-hr post-transfection were found to be >80% at the mRNA level with
concomitant downregulation of the ER60 protease protein by >60% when
compared with control T47D breast cancer cells. Downregulation of ER60
protease was also associated with inhibition of cell proliferation when
assessed by the AlamarBlue assay. Cell cycle analysis performed on the
siER-1- and siER-2-transfected cells, revealed an increase in G1 phase
population and a decrease in the S and G2/M phase populations compared
with control cells, implicating G1/S cell cycle arrest. It would appear that
ER60 protease is involved in breast tumorigenesis and could therefore be
a prospective target for cancer therapeutics. Anat Rec, 295:410–416,
C 2012 Wiley Periodicals, Inc.
2012. V
Key words: ER60; endoplasmic reticulum; breast cancer; gene
Breast cancer, the most prevalent female cancer in
many parts of the world (Hery et al., 2008; Matsuda
et al., 2010; Vuong et al., 2010), is also the leading cause
of cancer death among women in both developed and
developing countries (Jemal et al., 2011). The incidence
rate of breast cancer is considered low in Asia [with an
age-standardized rate (ASR) 24.0–32.5 per 100,000
depending on which part of Asia] when compared with
Europe, Australia/New Zealand, and North America
(ASR 76.7–89.9 per 100,000) (Jemal et al., 2011). However, the incidence of breast cancer in Singapore is much
higher than that reported in the other parts of Asia.
According to the Singapore Cancer Registry interim
report, the ASR for breast cancer in Singapore is 58.5
per 100,000 accounting for 17.1% of all female cancer
deaths between 2004 and 2008 (National Registry of Diseases Office of Singapore). As a heterogeneous disease
Grant sponsor: Singapore National Medical Research Council;
Grant number: NMRC/1081/2006.
*Correspondence to: Boon-Huat Bay, MBBS, PhD, Department
of Anatomy, Yong Loo Lin School of Medicine, National
University of Singapore, 4 Medical Drive, MD10, Singapore 117
597, Singapore. E-mail: [email protected]
Received 6 June 2011; Accepted 24 December 2011.
DOI 10.1002/ar.22413
Published online 20 January 2012 in Wiley Online Library
with distinct molecular profiles (Hsiao et al., 2010),
breast cancer presents a great challenge to healthcare
professionals in terms of diagnosis, management, and
prognostication. Currently, clinical classification of
breast cancer subtypes for deciding treatment regimes
are based on the expression profile of common biomarkers including estrogen receptor, progesterone
receptor, human epidermal growth factor receptor 1/2,
and cytokeratin 5/6.
ER60 protease, a protein with high sequence similarity to phosphoinositide-specific phospholipase C (PI-PLC)
(Urade and Kito, 1992; Otsu et al., 1995; Urade et al.,
2000), was first isolated from guinea pig uterus and was
mistakenly identified as an isoenzyme of PI-PLC (Bennett et al., 1988). It was later purified as a novel
cysteine protease from the endoplasmic reticulum of rat
liver (Urade and Kito, 1992). ER60 protease is also
known by several names including: ER60, ERp60,
ERp57, GRp58, or 1,25D3-MARRS (Membrane Associated, Rapid Response Steroid binding) receptor (Khanal
and Nemere, 2007a,b). ER60 has been studied extensively as an endoplasmic reticulum luminal chaperone
protein involved in the quality control of newly synthesized glycoproteins (Khanal and Nemere, 2007b) as well
as in the assembly of major histocompatibility complex
class-I (MHC-I) molecules (Vigneron et al., 2009; Chapman and Williams, 2010). Studies involving various
types of tissues have shown differential expression of
ER60 protease in normal tissues as compared with cancerous tissues such as breast, lung, gastric, and ovarian
carcinomas (Celli and Jaiswal, 2003; Leys et al., 2007;
Cicchillitti et al., 2009). However, much remains to be
elucidated to fully understand the role of ER60 protease
in cancer.
In this study, we elucidated the relationship of ER60
protease with cell proliferation (a hall mark of cancer) in
breast cancer cells in vitro.
Cell Culture
Three human breast cancer cell lines, viz., MCF-7,
T47D, and MDA-MB-231 breast cancer cells, as well as
immortalized normal breast epithelial MCF-12A cells,
were purchased from American Type Culture Collection
(ATCC). Breast cancer cell lines were cultured in either
Dulbecco’s-Modified Eagle Medium (DMEM) or Roswell
Park Memorial Institute (RPMI 1640) medium supplemented with 10% fetal bovine serum. The normal breast
epithelial MCF-12A cell line was cultured in DMEM-F12
(Invitrogen, Carlsbad, CA) medium with additional 20
ng/mL epidermal growth factor (Sigma-Aldrich, St.
Louis, MO), 0.01 mg/mL insulin (Sigma-Aldrich), 100 ng/
mL cholera toxin (Sigma-Aldrich), 500 ng/mL hydrocortisone (Sigma-Aldrich), and 5% fetal bovine serum
(Hyclone, Thermo Fisher Scientific, Rockford, IL). All
the breast cell lines were cultured in a 5% CO2 incubator at 37 C.
following the manufacturer’s protocol. The quantitative
real-time polymerase chain reaction was performed
using hot-start QuantiTect SYBR Green (Qiagen) and
LightCycler 2.0 System (Roche Applied Science, Penzberg, Germany). The cycling conditions used were one
cycle of 95 C for 15 min, 45 cycles of 94 C for 15 sec,
60 C for 25 sec, and 72 C for 12 sec. The primer sequences were 30 -AAG-CTC-AGC-AAA-GAC-CCA-AA-50 and 50 CAC-TTA-ATT-CAC-GGC-CAC-CT-30 for ER60 protease,
latter was the housekeeping gene used as the internal
control for normalizing the expression level of ER60.
Relative fold change was based on the formula 2DDCT
(Livak and Schmittgen, 2001) with the MCF-12A breast
epithelial cell line as the calibrator.
Western Blot
Total protein was extracted from cells using M-PERV
Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) with added HaltTM protease inhibitor cocktail
(Pierce). The extract was quantified by the Bradford
method (Bradford, 1976), and 20 lg was fractionated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene
difluoride membrane using the Semi-Dry Transfer Cell
unit (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked overnight at 4 C with 5% nonfat milk
dissolved in tris-buffered saline containing 0.1% Tween.
For detection of targeted proteins, the membrane was
incubated with the following antibodies for 1 hr at room
temperature: rabbit anti-ER60 (1:1,000; Abcam, Cambridge, UK) and mouse anti-b-actin (1:6,000; SigmaAldrich), horseradish peroxidase (HRP)-conjugated goat
anti-rabbit antibody (1:6,000; Pierce ECLTM, GE Healthcare, Piscataway, NJ), and HRP-conjugated donkey antimouse antibody (1:6,000; Pierce ECLTM). The protein
bands were visualized by incubation with chemiluminescence reagent (Pierce) and subsequent exposure of the
X-ray film (Kodak, Rochester, NY). For quantification,
the film was scanned and densitometric analysis performed to calculate the optical density (OD) ratio.
Immunocytochemical Staining
Cells were fixed with 4% paraformaldehyde for 30 min
at room temperature followed by quenching of endogenous peroxidase activity by incubation with 0.5%
hydrogen peroxide dissolved in methanol for 30 min.
The cells were then blocked with 5% goat serum for 1 hr
at room temperature before incubation with rabbit polyclonal anti-ER60 primary antibody at 1:200 dilution for
1 hr at room temperature. The VECTASTAIN ABC kit
(Vector Laboratories, Burlingame, CA) was used following the manufacturer’s protocol and immunostaining
was demonstrated using 3,-30 -diaminobenzidine (DAB)
as the substrate before visualization of the slides under
a light microscope.
Quantitative Real-Time RT-PCR
Total RNA was extracted using RNeasy Mini kit (Qiagen, Venlo, Netherlands). One microgram of total RNA
was converted to cDNA using random hexamers with
Superscript III 1st Strand Synthesis System (Invitrogen)
Immunofluorescence Staining
Cells were fixed as described above and blocked with
1% BSA for 1 hr at room temperature. Cells were then
incubated with rabbit anti-ER60 primary antibody at
Fig. 1. ER60 mRNA expression in breast cell lines. A: ER60 mRNA
expression was measured using real-time RT-PCR. Relative fold
change was calculated using 2DDCT using GAPDH as internal control
and MCF-12A normal breast epithelial cell line as the calibrator cell
line. Error bar ¼ SEM. **P < 0.01. B: Single bands with the correct
product size (160 bp for ER60 and 177 bp for GAPDH) as verified by
1.2% agarose gel electrophoresis. Lanes 1 and 5 ¼ MCF-12A, lanes
2 and 6 ¼ MCF-7, lanes 3 and 7 ¼ MDA-MB-231, and lanes 4 and 8
¼ T47D. For verification of the specificity of the PCR products, a
standardized 45 PCR cycle was performed and the band intensities
do not reflect the expression level. C: Representative melting curves
of primers for ER60 and GAPDH. Single peak for each primer further
validates the absence of primer dimers and nonspecific
1:200 dilution for 1 hr at room temperature followed by
Cy3-conjugated goat anti-rabbit secondary antibody
(1:200; Sigma-Aldrich). The nuclei of the breast cancer
cells were stained with DAPI (40 -6-diamidino-2-phenylindole) dye. The slides were subsequently visualized by
confocal microscopy.
mg/mL RNase A (Roche Applied Science), and 20 lg/mL
propidium iodide (Sigma-Aldrich) dissolved in phosphate-buffered saline before analysis by the Dako
Cytomation Cyan LX flow cytometer (Dako, Glostrup,
Silencing of ER60 Protease Gene by
Small-Interfering RNA
Two ER60 specific small-interfering RNAs (siRNAs;
labeled siER60-1 and siER60-2) and one sequence of
nontargeting siRNA (siNeg) were purchased from
Ambion (Applied Biosystems, Austin, TX). DharmaFECT
1 transfection reagent was purchased from Dharmacon
(Chicago, IL). Briefly, T47D cells were seeded into 6-well
plates at 2.5 105 cells per well and allowed to stabilize
for 24 hr before transfection with 2 mL of RPMI medium
containing 10% FBS, 5 nM siRNA, and 4 lL transfection
reagent per well.
Statistical Analysis
Comparisons between groups were analyzed with the
one-way ANOVA and post-hoc Tukey’s test using the
PASW Statistics 18 (SPSS, Chicago, IL). P-value < 0.05
was considered to be statistically significant.
ER60 Protease Expression in Breast Cancer
Cell Lines
Cell proliferation was assessed using the AlamarBlue
assay (Invitrogen) by measuring the fluorescence reading at 570-nm excitation wavelength and 585-nm
emission wavelength using a fluorescence spectrometer.
After 24-hr post-transfection, cells were synchronized by
serum starvation for 24 hr. The medium was then
replaced with fresh medium containing 10% FBS and
cells were allowed to grow for 1 day before subjecting
them to cell viability assay. Cells were incubated in 2
mL of complete culture medium containing 10% AlamarBlue solution for 90 min before transferring 120-lL
solution from each well into a 96-well plate for measurement of fluorescence reading.
ER60 protease was expressed in all the four breast
cell lines examined (MCF-7, MDA-MB-231, T47D, and
MCF-12A cells) with T47D cells exhibiting the highest
expression. As shown in Fig. 1A, ER60 protease mRNA
transcript in T47D cells was almost 10 folds higher compared with the other three cell lines. The specificity of
the primers was validated by DNA gel electrophoresis
(Fig. 1B) and melting curve analysis (Fig. 1C). Western
blotting also revealed that T47D cells had the highest
ER60 protease protein expression (Fig. 2A), which was
verified by quantification of the intensity of the protein
bands using optical densitometry (Fig. 2B).
The ER60 protease protein was observed to be localized primarily in the cytoplasm of the cells particularly
at the perinuclear region by immunocytochemistry (Fig.
3A) and confirmed by immunofluorescence staining (Fig.
3B), corroborating the notion that ER60 is an endoplasmic reticulum luminal protein.
Cell Cycle Analysis
Silencing the ER60 Protease Gene
siRNA-transfected cells were synchronized as
described above before harvesting by trypsinization and
fixation with ice-cold 70% ethanol overnight. Cells were
stained using a cocktail containing 0.1% Triton-X, 0.2
T47D breast cancer cells were subsequently selected
for ER60 protease knockdown experiments in view of
the high expression of the protein in this breast cancer
cell line. The ER60 protease gene was successfully
Cell Proliferation Assay
knocked down using two sequences of siRNA, labeled
siER60-1 and siER60-2 with silencing efficiencies of
88% for both sequences at 48-hr post-transfection (Fig.
4A). There was concomitant downregulation of the ER60
protease protein (Fig. 4B), which was also demonstrated
qualitatively by immunocytochemistry at 72-hr posttransfection, where a markedly lower staining intensity
was observed in the siER60-1- and siER60-2-transfected
cells (Fig. 4C).
Effects on Cell Proliferation and Cell Cycle
Cell proliferation was measured by the AlamarBlue
assay 72 hr after transfection (including 24 hr of serum
starvation). Knocking down ER60 protease expression by
siRNA significantly decreased cell proliferation in T47D
breast cancer cells (Fig. 5) with the fluorescence readings decreasing by 38.5 6 2.5% for siER60-1-transfected
cells and 11.2 6 3.1% for siER60-2-transfected cells (P <
0.001 and P < 0.01, respectively).
Cell cycle analysis was carried out at 72-hr post-transfection, after 24-hr serum starvation to synchronize the
growth phase of cells. Downregulation of ER60 protease
in T47D breast cancer cells was accompanied by a significant increase in G1 population (P < 0.001), while the Sphase and G2/M populations were significantly reduced
in siER60-1- and siER60-2-transfected cells (Fig. 6), indicating that ER60 protease downregulation induced G1/S
arrest. The sub-G1 population, which represented apoptotic cells, was observed to be marginally increased.
The S þ G2M phases which denote cell proliferation
were significantly lower in the siER60-1- (26.05 6
0.58%) and siER60-2-transfected cells (22.76 6 0.56%)
when compared with the siNeg controls (P < 0.01 in
both instances).
Fig. 2. ER60 protein expression in breast cancer cell lines. A: Western blots of ER60 protease protein in breast cancer cell lines with bactin as the loading control. B: Protein bands were quantified by densitometric analysis. Relative expression was calculated by OD ratio of
ER60 protease to b-actin and normalized with the MCF-7 cell line.
Error bar ¼ SEM.
Although the ER60 protease protein has been investigated for more than two decades (Bennett et al., 1988),
the main areas of study were focused primarily on its
function as a molecular chaperone with relatively little
information available regarding its role in carcinogenesis. In fact, ER60 protease has recently attracted the
attention of researchers in the field of cancer biology. In
our study, we observed expression of ER60 protease at
both mRNA and protein levels in breast cancer cells in
vitro. MCF-12A cells, which represent normal breast
epithelium, had comparable expression with MCF-7
cells but lower than MDA-MB-231 cells and T47D cells
at the mRNA transcript level. This is in line with a
Fig. 3. Localization of the ER60 protease protein in T47D breast
cancer cells. A: Immunocytochemical staining of ER60 protease protein. Brown DAB staining in the cytoplasm indicates specific ER60
protease staining while the nuclei are stained blue by hematoxylin.
Scale bar ¼ 50 lm. B: Immunofluorescence staining of ER60
protease protein. Red Cy3 immunofluorescence represents specific
ER60 protease staining and DAPI dye stains the nucleus blue. Scale
bar ¼ 25 lm.
Fig. 4. Silencing of the ER60 protease gene in T47D breast cancer
cells. A: Silencing efficiency of siRNAs targeting the ER60 protease
gene. Relative mRNA expression of ER60 protease at 48-hr posttransfection was calculated using 2DDCT. Error bar ¼ SEM. B: Western blots of ER60 protease and b-actin (the housekeeping protein) and
bar chart showing relative ER60 protease protein expression. Error bar
¼ SEM. C: Qualitative expression of ER60 protease protein as analyzed by immunocytochemistry. ER60 protease was visualized by the
brown DAB staining. Nucleus was counterstained with hematoxylin.
Scale bar ¼ 100 lm.
previous finding reporting an upregulation of ER60 protease in some cancers including breast, uterus, lung,
and stomach cancers (Celli and Jaiswal, 2003). It has
been proposed that ER60 protease, an endoplasmic
reticulum chaperone protein (Coe and Michalak, 2010),
is involved in endoplasmic reticulum stress response
(Corazzari et al., 2007; Ni and Lee, 2007) and promotes
tumor cell survival in hypoxic microenvironment
brought about by poor vascularization (Koumenis,
However, ER60 protease expression has also been
reported to be partially downregulated or even absent in
cancers such as gastric adenocarcinoma (Leys et al.,
2007), squamous cell esophageal carcinoma (Qi et al.,
2008; Ayshamgul et al., 2011), cervical carcinoma (Mehta
et al., 2008), and renal cell carcinoma (Tanaka et al.,
2008). Some investigators have attributed this observation to ER60 protease being a key component for antigen
presentation by MHC-I molecules during immune surveillance (Garbi et al., 2007; Chapman and Williams,
2010), and therefore a lower expression would facilitate
immune evasion by cancer cells (Seliger et al., 2001;
Dunn et al., 2006; Seliger et al., 2010).
ER60 protein has also been associated with resistance
to the chemotherapeutic drug Paclitaxel in A2780 ovarian cancer cells in vitro (Cicchillitti et al., 2009).
However, overexpression of ER60 protease in Chinese
Hamster Ovary stable transfectants has been reported
to increase mitomycin C-induced DNA crosslinking leading to enhanced cytotoxicity on exposure to this
anticancer drug (Celli and Jaiswal, 2003). Hence, more
work needs to be done before one can ascertain the exact
role played by ER60 protease in carcinogenesis.
In this study, we observed that T47D cells had the
highest ER60 protease gene expression compared with
MCF-7 and MDA-MB-231 breast cancer cells. T47D and
Fig. 5. Effect of downregulating ER60 protease expression on cell
proliferation in T47D breast cancer cells. Relative fluorescence emitted
represents the relative number of cells in each sample normalized against
the control (siNeg) group. *P < 0.05, **P < 0.01, and ***P < 0.001.
cytokines and mitogens direct cells to enter the mitotic
cycle and activate a cascade of signaling pathways
including MAP kinase (MAPK) pathway (Tartaglia and
Gelb, 2010), phosphatidyl inositol-3 kinase (PI3K)/AKT
pathway (Castaneda et al., 2010) and JAK/STAT
pathway (Spano et al., 2006; Borges et al., 2008).
Dysregulation of these signaling pathways could result
in aberrant cell proliferation as in the case of cancer
(Kim and Choi, 2010). For instance, vitamin D is known
to perturb regulation of the cell cycle and impede cell
proliferation by inhibiting the MAPK–extracellular signal-regulated kinase (ERK) 1 and 2 signaling pathways
(Deeb et al., 2007). Richard et al. (2010) have also
recently demonstrated that the growth inhibition potential of vitamin Ds in MCF-7 breast cancer cells is
mediated by a novel receptor identified as 1,25D3MARRS protein. A study by Apati et al. (2003) has
shown that calcium induces cell survival and proliferation in TF-1 leukemia cell line by MAPK pathway
activation and a known function of ER60 protease is
maintenance of homeostasis of intracellular calcium.
There is also evidence that ER60 protease can modulate
STAT3 signaling (Coe et al., 2010).
In conclusion, silencing the ER60 protease gene inhibited cell proliferation and induced G1/S cell cycle arrest in
breast cancer in vitro. Several different mechanisms could
be involved stemming from the multifunctional roles of
ER60 protease. It would be worthwhile to elucidate the
mechanistic pathway(s) responsible for regulating the proliferative effect exerted by ER60 protease in breast cancer
cells. ER60 protease could therefore be a potential molecular target in breast cancer therapeutics.
The authors thank Ms. Saw Marlar from Flow Cytometry Laboratory (National University Medical Institute)
and Ms. Bay Song Lin, Multimedia Unit (Department of
Anatomy, National University of Singapore) for their
technical assistance.
Fig. 6. Effect of downregulating ER60 protease expression on regulation of the cell cycle in T47D breast cancer cells. Cell cycle was analyzed
by flow cytometry using propidium iodide staining. A representative
histogram depicting the different phases of the cell cycle in T47D breast
cancer cells is displayed above the bar chart showing the subG1, G1, S,
and G2/M cell populations. The y-axis represents the percentage of cells
in each phase of the cell cycle from a sample population of 10,000 cells.
Error bar ¼ SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
MCF-7 breast cancer cells share similar distinctive
characteristics as both cell lines exhibit the luminal epithelial ERþ/PRþ/Her2 phenotype while MDA-MB-231
breast cancer are highly invasive and ER/PR/Her2þ
(Lacroix and Leclerc, 2004). It is still unclear at this
point in time the reason for T47D breast cancer cells displaying the highest ER 60 protease expression among
the three cancer cell lines examined. We also demonstrated that siRNA-mediated silencing of the ER60
protease gene inhibited cell proliferation by inducing cell
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