Download Breast Cancer Amplified Sequence 2, a Novel Negative Regulator of

Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023
Cell, Tumor, and Stem Cell Biology
Breast Cancer Amplified Sequence 2, a Novel Negative Regulator
of the p53 Tumor Suppressor
Ping-Chang Kuo,1 Yeou-Ping Tsao,2 Hung-Wei Chang,1 Po-Han Chen,1 Chu-Wei Huang,1
Shinn-Tsuen Lin,1 Yu-Tzu Weng,1 Tzung-Chieh Tsai,4 Sheau-Yann Shieh,3 and Show-Li Chen1
1
Graduate Institute of Microbiology, College of Medicine, National Taiwan University; 2Department of Ophthalmology, Mackay Memorial
Hospital; 3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan and 4Department of Microbiology & Immunology,
National Chiayi University, Chiayi, Taiwan
Abstract
Breast cancer amplified sequence 2 (BCAS2) was reported
previously as a transcriptional coactivator of estrogen receptor. Here, we report that BCAS2 directly interacts with p53 to
reduce p53 transcriptional activity by mildly but consistently
decreasing p53 protein in the absence of DNA damage. However, in the presence of DNA damage, BCAS2 prominently
reduces p53 protein and provides protection against chemotherapeutic agent such as doxorubicin. Deprivation of BCAS2
induces apoptosis in p53 wild-type cells but causes G2-M arrest in p53-null or p53 mutant cells. There are at least two
apoptosis mechanisms induced by silencing BCAS2 in wildtype p53-containing cells. Firstly, it increases p53 retention
in nucleus that triggers the expression of apoptosis-related
genes. Secondly, it increases p53 transcriptional activity by
raising p53 phosphorylation at Ser46 and decreases p53 protein degradation by reducing p53 phosphorylation at Ser315.
We show for the first time that BCAS2, a small nuclear protein
(26 kDa), is a novel negative regulator of p53 and hence a
potential molecular target for cancer therapy. [Cancer Res
2009;69(23):8877–85]
Materials and Methods
Introduction
The sequence of breast cancer amplified sequence 2 (BCAS2) is
the same as DAM1 (Mus musculus DNA amplified in mammary carcinoma mRNA; GenBank accession no. AB020623) deposited at National Center for Biotechnology Information in July 2001 (1–3). The
BCAS2 gene maps to chromosome 1p13.3-21 region and contains
678 bp encoding 225 amino acids, with a predicted molecular mass
of 26 kDa (2). BCAS2 was recently characterized as a transcriptional cofactor that enhances estrogen receptor–mediated gene expression (4). In this article, we found that BCAS2 associates with
the tumor suppressor p53 protein; we have also probed the cellular
and molecular effects of this interaction.
Tumor suppressor protein p53 is a multifunctional protein that
regulates cell growth and death by responding to various genotoxic
and oncogenic stresses (5). Tight regulation of p53 activity is essential for maintaining normal cell growth and for preventing tumorigenesis. Mutations in p53 are a hallmark of at least 50% of all
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
P-C. Kuo and Y-P. Tsao contributed equally to this article.
Requests for reprints: Show-Li Chen, Graduate Institute of Microbiology, College
of Medicine, National Taiwan University, 7F, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan.
Phone: 886-2-2312-3456, ext. 88289; Fax: 886-2-2391-5293; E-mail: [email protected].
©2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-09-2023
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human cancers (6, 7). The p53 protein has been shown to interact
with several cellular and viral proteins including SV40 large T, human papillomavirus E6 protein, adenovirus E1b, MDM2, E2F, p300/
CBP, NUMB, and ASPP proteins (8, 9). These proteins are a mixture
of general and apoptosis-specific regulators of p53 and regulate
how p53 protein determines the fate of cells (10).
In this study, BCAS2 interaction partners were identified and
characterized using glutathione S-transferase (GST) pull-down
assays combined with mass spectrometry. One such protein
shown to bind BCAS2 was p53; its interaction was confirmed
by further GST pull-down in vitro and through reciprocal immunoprecipitation in vivo. Because BCAS2 is a nuclear protein, we
further examined whether BCAS2 could regulate p53 transactivation and apoptosis. Here, we show for the first time that BCAS2
is a novel negative regulator of p53 that directly interacts with
p53 protein, decreasing p53 transcription activity. Deprivation of
BCAS2 induces cell apoptosis in p53 wild-type cells but causes
G2-M arrest in p53-null or p53 mutant cells. It seems that, beyond p53, other cellular factors exist that interact with BCAS2
and are involved in the inhibition of cell growth.
In vitro GST pull-down analysis. The GST and GST fusion protein
plasmids were transformed into Escherichia coli BL21 (DE3) cells.
These fusion proteins were purified using glutathione-Sepharose 4B
beads (GE Life Sciences) according to the manufacturer’s procedures.
His-tagged p53 was synthesized by transformed pRSET-p53 plasmid
into E. coli BL21 (DE3) cells and then purified by Ni-NTA agarose
beads (Qiagen). The purified His-tagged p53 protein was incubated with
the immobilized GST or GST-fused proteins beads and these bound
proteins were subjected to Western blotting with anti-His or anti-GST
antibodies.
Preparation of cytoplasmic and nuclear extracts. Nuclear and cytoplasmic fractions were prepared from MCF-7 cells treated with the appropriate anti-BCAS2 short hairpin RNAs (shRNA) for 48 h as described
previously (11).
Fluorescence microscopy assay. MCF-7 cells were fixed and then
doubly stained with anti-p53 and anti-BCAS2 antibodies followed by incubation with FITC-conjugated anti-rabbit (Abcam) and Alexa Fluor–conjugated
anti-mouse antibodies (Invitrogen). Nuclei were stained with 4′,6-diamidino2-phenylindole. Fluorescent images were monitored using a Carl Zeiss
confocal microscope (LSM 510 META).
Coimmunoprecipitation and Western blot analysis. To determine
BCAS2-p53 binding, MCF-7 cells were transfected with full-length or
deletion p53 and BCAS2 plasmids. Forty-eight hours after transfection, cell
lysates were harvested and immunoprecipitated with the antibodies indicated in procedures described previously (11, 12).
Transient transfection and luciferase assays. Transient transfections
were done by plasmid electroporation into MCF-7 or H1299 cells. Forty-eight
hours after transfection, the cells were assayed using a Dual-Glo Luciferase
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Assay System (Promega) according to the manufacturer’s instructions and
detected using a Luminoskan Ascent luminometer (Thermo). The intensity
of the firefly luciferase signals generated was normalized against an internal
control (Renilla luciferase pRL-CMV).
Cell cycle analysis. Cells transfected with the indicated plasmids were
collected and fixed and then stained with propidium iodide and analyzed
by flow cytometry (BD FACSCalibur).
Cell proliferation assay. MCF-7, H1299, C33A, and LNCaP cells were
transfected by electroporation. Twenty-four hours after transfection,
MCF-7, C33A, and H1299 cells were replated at 1 × 105 per well in 12-well
culture plates. LNCaP cells were reseeded at 2 × 104 per well in 24-well
culture plates. The total cell numbers were counted at 24, 48, 72, and 96
h time points by trypan blue staining.
Real-time quantitative PCR. Total cellular RNA was extracted using
Trizol reagents (Invitrogen) according to the manufacturer’s instructions.
Real-time PCR was done with a Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol.
Chromatin immunoprecipitation-quantitative PCR assay. Chromatin immunoprecipitation assays were done as described previously (11).
For chromatin immunoprecipitation-quantitative PCR (qPCR), DNA was
purified with a QIAquick PCR purification kit (Qiagen) and subjected
to analysis by real-time qPCR. One percent of the preclear supernatant
from the reaction lacking primary antibody was saved as total input of
chromatin.
Statistical analysis. Statistical procedures were carried out using SPSS
16.0 software. ANOVA analysis was done. P values < 0.05 were considered
significant.
Results
Protein-protein Interaction between BCAS2 and p53 tumor
suppressor. To characterize BCAS2 function, we first examined
which proteins could interact with BCAS2 by examining the nuclear extracts of MCF-7 cells by GST pull-down assays followed by
mass spectrometry (Fig. 1A, top). Tumor suppressor protein p53
was just one of numerous potential BCAS2-interacting proteins
identified (data not shown); immunoblotting confirmed that p53
was bound to GST-BCAS2 fusion protein (Fig. 1A, bottom).
We then asked whether BCAS2 could directly interact with p53.
MDM2 protein is a well-studied p53 binding protein, and residues
1 to 110 are required for p53 binding (13). GST-MDM2 (120) covering MDM2 amino acid sequence 1 to 120 was constructed and
regarded as a positive control for p53 binding. The His-p53 protein
synthesized by E. coli was incubated with the various purified GSTfusion proteins in GST pull-down assay. The results showed that
the GST-MDM2 (120) and GST-BCAS2 proteins could be pulled
down with the purified His-p53 protein (Fig. 1B, top, lanes 2 and
4). These data indicate that BCAS2 directly interacted with p53.
When investigating the interaction between endogenous BCAS2
and p53 in MCF-7 cells containing wild-type p53, p53 protein could
be detected by anti-BCAS2 antibody (Fig. 1B, bottom). The detection of BCAS2 (molecular weight, 26 kDa) by precipitated p53 antibody was hampered by the immunoglobulin light chain, which is
Figure 1. Interaction between BCAS2 and p53 in vitro and
in vivo. A, BCAS2 associates with p53 in vitro. GST-BCAS2
and GST proteins were incubated with MCF-7 nuclear
extracts and analyzed by silver staining (top).
GST-pull-down proteins were analyzed by Western blot
(WB) with p53 antibody (bottom). B, top, BCAS2 directly
interacts with p53 in vitro. The His-p53 protein incubated
with the GST, GST-BCAS2, or GST-MDM2 (120).
The bound proteins were analyzed by Western blot using
an anti-His or anti-GST antibody. Bottom, association of
endogenous BCAS2 and p53 in vivo. Cell lysates of MCF-7
were subjected to immunoprecipitation (IP) with antibody
against BCAS2 and then immunoblotted with p53 antibody
(bottom). C, nuclear colocalization of BCAS2 and p53 by
confocal laser microscopy. Bar, 10 μm.
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BCAS2, a Negative Regulator of p53
Figure 2. BCAS2-p53 complexes repress p53-mediated p21 promoter activity. MCF-7 cells were transiently transfected with p21-Luc promoter plasmid along with
increasing doses of the FLAG-BCAS2 expression construct in the absence (A) or presence (B) of 1 μmol/L doxorubicin (Dox). C, H1299 cells were cotransfected
with p53 expression plasmid in combination with increasing doses of FLAG-BCAS2 along with p21-Luc promoter plasmid. Top, all luciferase assays were done three
times as independent experiments. Mean ± SD. Bottom, p53, p21, and BCAS2 protein expression. The expression levels of p53 and p21 protein quantified by
UVP BioSpectrum-AC imaging software were normalized to β-actin. D, chromatin immunoprecipitation-qPCR assay to monitor the effect of BCAS2 on p53 binding to the
p21 promoter.
25 kDa (data not shown). Instead, we conducted the confocal
microscopy assay that showed endogenous BCAS2 and p53 colocalized inside the MCF-7 nucleus (Fig. 1C). In summary, our data
showed that BCAS2 can directly interact with p53 in vitro and
in vivo.
Overexpression of BCAS2 reduces p53 protein and transcriptional activity. The tumor suppressor p53 is a well-known
transcription factor that can activate downstream target genes
(5, 10). Having identified a direct interaction between BCAS2 and
p53, we investigated whether BCAS2 affected p53 transcriptional
activity. Transcription of the p21 gene is reportedly enhanced by
p53 binding to its promoter region, a stretch that contains two
copies of the p53 response element (14). Doxorubicin, a DNA-damaging agent, is a potent inducer of p53 protein and p53-dependent
transcriptional activity (15). Firstly, we used the p21 promoter
(5 μg; kindly provided by Dr. Bert Vogelstein) along with
p3XFLAG-BCAS2 (15 or 20 μg) and internal control pRL-CMV
(0.1 μg) as an assay model to determine p53 transcriptional activity
(16). The total amount of plasmid per dish was equalized by adding
empty vectors. The results showed that the luciferase activity of
the p21 promoter decreased (top), accompanied with increased
BCAS2 protein expression and decreased levels of p53 and p21
(bottom) in MCF-7 cells, regardless of doxorubicin treatment
(Fig. 2B) or no treatment (Fig. 2A). However, that the quantitation
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of immunoblot by increasing FLAG-BCAS2 plasmids was not detectable accordingly can be explained by the transfection efficiency
having reached its limits. Likewise, H1299 cells (lung cancer with
null p53 and weak endogenous p21) were cotransfected with a p53
expression plasmid in combination with a p21-Luc promoter and
increasing doses of FLAG-BCAS2. Similarly, the results showed the
significantly decreased p21 promoter activity (Fig. 2C, top) and the
corresponding exogenous p53 protein and endogenous p21 protein
were reduced proportionally to the increased amount of BCAS2 expression (Fig. 2C, bottom). Furthermore, we conducted chromatin
immunoprecipitation-qPCR to measure the effect of BCAS2 on p53
binding to the p21 promoter. The results of Fig. 2D showed that
BCAS2 decreased the p53 binding to its 5′ and 3′ binding site on
p21 promoter in both the absence and the presence of doxorubicin
in MCF-7 cells compared with the mock and FLAG-vector control.
Taken together, BCAS2 can reduce p53 protein expression to inhibit its transcriptional activity.
Reciprocal binding domains between BCAS2 and p53. To
verify our hypothesis that BCAS2 is an inhibitor of p53, we
mapped the domains within p53 and BCAS2 responsible for interacting with BCAS2 and p53, respectively. HA-tagged deletion
mutants of p53 were constructed and named p53-FL, p53-Δ1,
p53-Δ2, p53-Δ3, p53-Δ4, and p53-Δ5 (Fig. 3A, top). Figure 3A
showed that p53-Δ2, p53-Δ3, and p53-Δ4 complexed with BCAS2
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Figure 3. Analysis of the mutual binding domains
between BCAS2 and p53 proteins. A, schematic
representation of the various p53 deletion mutants
constructed (top). Specific domains are labeled
as follows: TA, transactivation domain; DBD,
specific DNA-binding domain; TET, tetramerization
domain; NLS, nuclear localization signal; NES,
nuclear export signal. Mapping of p53 domains
used for binding with BCAS2. MCF-7 cells were
transfected with HA-p53-FL or p53 deletion
mutants. Cell extracts were immunoprecipitated
with BCAS2 antibody. Bound proteins were
subjected to Western blot analysis (bottom).
B, map of BCAS2 domains. Two coiled-coil
domains were identified and named CC1 (residues
139-173) and CC2 (residues 192-219), respectively
(top). Mapping the BCAS2 domains required for
interacting with p53. Cells were transfected with
FLAG-BCAS2-FL or BCAS2 deletion mutants.
Cell extracts were immunoprecipitated with p53
antibody. Bound proteins were subjected to
Western blot analysis (bottom). C, effect of BCAS2
mutants on the expression of p53 and p21 protein
in MCF-7 cells. MCF-7 cells were transfected
with the indicated plasmids, and total cell lysates
were subjected to Western blot analysis (top).
The expression levels of p53 and p21 protein were
measured as described in Fig. 2.
protein (bottom), indicating that p53 residues 132 to 324 are responsible for binding BCAS2, the same region required for DNA
binding and nuclear signaling (17). Likewise, BCAS2 has two
coiled-coil domains (Fig. 3B, top) that interact with other proteins
(18, 19); we generated wild-type or single- or double-domain deletion mutants named BCAS2-FL, BCAS2-dCC1, BCAS2-dCC2, and
BCAS2-dCC1+2, respectively (Fig. 3B, top), to answer whether
coiled-coil domains of BCAS2 were involved in the interaction
with p53 protein. Figure 3B showed that those BCAS2 mutants
lacking either one or both domains were unable to bind p53 (bottom). When deletion mutants of BCAS2 were introduced into
MCF-7 cells, p53 and p21 protein expression was less disrupted
than by full-length BCAS2 (Fig. 3C). This result indicated that the
Cancer Res 2009; 69: (23). December 1, 2009
BCAS2 coiled-coil domains were responsible for binding to p53
and regulating its activity.
Depletion of BCAS2 affects p53-dependent transcription. To
evaluate the functions of BCAS2 gene by RNA interference (11, 12,
20), four BCAS2 small interfering RNA sequences were designed
and cloned into pSUPER plasmid and named according to the
BCAS2 sequence to be disrupted (Supplementary Fig. S1). As
shown in Fig. 4A, BCAS2 protein expression was significantly diminished by shBCAS2#434 and mildly diminished by shBCAS2#135
and shBCAS2#510 shRNAs in 293T cells (top). To further determine
that the shBCAS2#434 construct could knock down BCAS2 gene
expression is on-target effect, we generated the mutant plasmid
FLAG-BCAS2 (mutant), in which the sequence corresponding to
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BCAS2, a Negative Regulator of p53
shBCAS2#434-targeted sequence (position 434-452) was mutated
to 5′-AATCGAGCATGCTCAAAAA-3′. After cotransfection of wildtype or mutant BCAS2 plasmid with shBCAS2#434 into 293T cells,
Western blot analysis showed that shBCAS2#434 only inhibited
wild-type protein expression (Fig. 4A, bottom, lane 4) but not mutant type expression (lane 6). In summary, the designed sequence
of shBCAS2#434 targeted the BCAS2 gene and diminished RNA
expression (data not shown).
We have shown that BCAS2 repressed the p53 transcriptional
ability (Fig. 2). We therefore examined whether BCAS2 knockdown
could increase the p53 transcriptional activity of other p53targeted genes, such as p21, MDM2, PUMA, NOXA, and BAX
(10, 21). qPCR assays showed significantly increased gene expression of p21, PUMA, NOXA, and MDM2 but not BAX in MCF-7 cells
treated with shBCAS2#434 compared with the control (pSUPER) in
both doxorubicin treatment and no treatment (Fig. 4B). Two other
cell types, LNCaP (prostate cancer cells containing wild-type p53),
presented similar patterns of p53-induced gene expression (p21,
PUMA, MDM2, and NOXA) after BCAS2 depletion as MCF-7
(Supplementary Fig. S2A) and A549 (lung cancer cells with wildtype p53) showed the increased gene expression of PUMA,
MDM2, and NOXA but not p21 (Supplementary Fig. S2B). It can
be explained the different cell contexture influenced p53 binding
efficiency to its targeted genes. To avoid off-target effects, we further treated MCF-7 cells with the other targeting sequences,
shBCAS2#135, which could decrease BCAS2 protein expression,
too (Fig. 4A, top). Similarly, qPCR of shBCAS2#135-treated cells
showed the significantly increased gene expression of p21, PUMA,
NOXA, and MDM2 but not BAX (Fig. 4B). To directly examine how
BCAS2 depletion would affect p53-dependent transcription, RNA
interference studies showed that shBCAS2#434 could increase
p21 promoter activity in MCF-7 cells by ∼3.5-fold compared with
Figure 4. Depletion of BCAS2 expression
increases the p53 transcriptional activity. A, top,
identification of effective shRNA targets to BCAS2.
Four BCAS2 shRNA clones were analyzed for
inhibition of endogenous BCAS2 protein. Bottom,
cells were transfected with shBCAS2#434 along
with BCAS2 (wild-type) or mutant constructs and
analyzed by Western blot. B, effect of BCAS2
knockdown on p53-targeted genes expression was
analyzed by qPCR. C, silencing BCAS2 expression
increases p21 promoter reporter activity. D,
depletion of BCAS2 expression increased the
protein level of p21 and PUMA by Western blot.
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shBCAS2#331 and mock controls (Fig. 4C). Figure 4D shows that
p21 and PUMA protein levels were higher in shBCAS2#434-treated
MCF-7 cells compared with mock, pSUPER, and shBCAS2#331.
Taken together, interfering with BCAS2 expression in wild-type
p53 cancer cells induces p21 and PUMA RNA and protein expression, indicating decreasing p53 transcriptional activity.
Reducing BCAS2 expression affects p53 nuclear retention
and post-translational modifications. The expression of BCAS2
mildly reduced p53 protein expression (Fig. 2). We therefore investigated whether the depletion of BCAS2 would increase the
amount of p53 protein. As shown in Fig. 5A, the p53 protein levels
were mildly increased in MCF-7 cells treated with shBCAS2#434
compared with mock, pSUPER, and shBCAS2#331 in normal condition but in the presence of doxorubicin; the significant increase
of p53 protein was shown in shBCAS2#434 (Fig. 5A, lane 8). To further assess whether BCAS2 regulates p53 protein turnover, Fig. 5B
shows that there was more p53 protein in shBCAS2#434-treated
cells than in cells transfected with the control shRNAs. This indicates that the half-life of p53 protein in the shBCAS2#434-treated
cells was longer than in cells treated with control shRNAs.
Besides being transcription-dependent, the p53-induced apoptotic program also includes a transcription-independent pathway (5, 22), probably mediated by cytosolic p53 protein (5, 10,
22–24). In our studies, most p53 protein in MCF-7 cells is located
in the nucleus (Fig. 1C). Hence, we examined whether knockdown
of BCAS2 would affect the relative distribution of p53 between
the cytosol and the nucleus. Our results show that p53 protein
was increasingly nuclear in shBCAS2#434-treated MCF-7 cells
(Fig. 5C, lane 6) post-transfection, indicating that BCAS2 depletion increases nuclear p53 protein. Taken together, the knockdown of BCAS2 caused the induction of p53 transcriptional
activity by nuclear p53 protein.
The transactivation function of p53 is believed to be regulated
through protein accumulation and post-translational modifications such as phosphorylation (25, 26). We therefore examined
changes in post-translational modifications to p53 on BCAS2 induction and deprivation of BCAS2 (Fig. 5D). The effect on the extent of p53 phosphorylation was analyzed after normalization to
total p53 level. As shown in Fig. 5D, introduction BCAS2 in combination with doxorubicin treatment in MCF-7 cells caused a measurable decrease at Ser46 phosphorylation status and increase at
Ser315 of p53 (left). In contrast, downregulation of BCAS2 by shRNA
transfection increased the levels of Ser46 but reduced Ser315 phosphorylation (right). The protein p53 phosphorylation at Ser315 is reported to be required for its nuclear export and degradation by
Hdm2 (27), and phosphorylation at Ser46 is involved in p53-dependent transcription activity (26). It may explain BCAS2-degrading
p53 protein by increasing Ser315 phosphorylation and BCAS2-reducing p53 transcriptional activity correlated with the effect of
phosphorylation levels of Ser46.
Figure 5. Effect of BCAS2 on p53 protein
stability, nuclear retention, and post-translational
modifications. A, knockdown BCAS2 gene
expression affects p53 protein expression in cells
without and with doxorubicin treatment. B, effect of
BCAS2 on p53 stability. As described in A,
48 h after transfection, the protein synthesis
inhibitor cycloheximide (CHX) was added
(100 μg/mL) to the cells at the different time points
indicated. Band densities were quantitated using
a UVP BioSpectrum-AC Imaging System. We set
the p53 protein level in each control treatment
as 100%, so the densitometer measured changes
as a percentage of each control treatment.
C, subcellular location of p53 by shBCAS2
treatment. Blots were probed for p53, BCAS2, poly
(ADP-ribose) polymerase (PARP; for the nuclear
fraction control), and tubulin (cytosolic fraction).
D, left, BCAS2 suppresses the phosphorylation
levels of p53 at Ser46 and induces phosphorylation
at 315; right, deprivation of BCAS2 increases
p53 phosphorylation at Ser46 but reduces at Ser315.
The number presented the ratio of phosphorylated
p53 to the corresponding total p53 protein in
doxorubicin-treated experiments (control, set at 1).
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BCAS2, a Negative Regulator of p53
Figure 6. Biological significance of BCAS2 effects. Depletion of BCAS2 affects cell growth. A, knockdown of BCAS2 expression in cells containing wild-type p53
caused apoptosis in MCF-7 cells. Surviving cell numbers were counted by trypan blue staining (top). Bottom, Western blot. B, depletion of BCAS2 expression in H1299
cells (p53-null) results in growth retardation (top). Bottom, Western blot. C, silencing of BCAS2 in wild-type p53-cells induces cell apoptosis in a p53-dependent manner
by flow cytometry (top). Bottom, Western blot. D, BCAS2 rescues doxorubicin-induced growth arrest. Left, cell proliferation assay of BCAS2 rescuing growth arrest
induced by doxorubicin; top right, flow cytometry analysis; bottom, Western blot analysis of p53, p21, and BCAS2 protein expression levels from each treatment.
Reducing the expression of BCAS2 increases p53-dependent
apoptosis in cancer cells. Tumor suppressor p53 was reported
to play an important role in apoptosis and cell cycle arrest (28).
Cell proliferation assays were done to determine the biological
effects of knocking down BCAS2 gene expression on p53 activity.
MCF-7 and LNCaP cells containing wild-type p53 (29, 30) were
transfected with the pSUPER, shBCAS2#434, and shBCAS2#331
vectors separately to determine the cell growth rates. H1299
cells containing null p53 and C33A cells harboring mutant p53
were also examined. The results showed that silencing BCAS2
expression by shBCAS2#434 caused dramatic decreases in the
cell growth rates of MCF-7 and LNCaP cells compared with
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mock, pSUPER, and shBCAS2#331 control cells (Fig. 6A and
Supplementary Fig. S3A, respectively). The dead cell number significantly increased with time at 72 and 96 h in MCF-7 cells treated
with shBCAS2#434 (Supplementary Fig. S4A). The corresponding
level of BCAS2 protein decreased in shBCAS2#434-treated cells at
48 h (Fig. 6A, bottom). The results from the trypan blue exclusion
assay showed that knocking down BCAS2 expression in MCF-7
cells with shBCAS2#434 caused a dramatic increase (>40%) in
cell death after 72 h (Supplementary Fig. S4A). Similar results
were obtained by flow cytometry analysis, whereby the percentage of cells in sub-G1 phase increased at 72 h in MCF-7 cells
treated with shBCAS2#434 compared with mock and pSUPER
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vector controls (Supplementary Fig. S5; Supplementary Table S1).
Likewise, growth rates for H1299 and C33A cells treated with
shBCAS2#434 were lower compared with the control groups
(Fig. 6B and Supplementary Fig. S3B, respectively). The
corresponding level of BCAS2 protein was reduced 48 h after
treatment (Fig. 6B and Supplementary S3B, bottom, respectively).
Conversely, H1299 cells did not experience the apoptotic phenomenon seen in the MCF-7 cells following shBCAS2#434 treatment
(Supplementary Fig. S4B). The percentage of cells in sub-G 1
phase did not change significantly in H1299 cells (Supplementary Table S2) and C33A cells (Supplementary Table S3) treated
with pSUPER, shBCAS2#331, and shBCAS2#434, respectively. The
appearance of sub-G1 means that cells are undergoing apoptosis (31), induced by shBCAS2#434 in wild-type p53-containing
cells (MCF-7) but not in p53-null cells (H1299) or p53 mutant
cells (C33A). Therefore, we can infer that apoptosis caused by
silencing BCAS2 expression in MCF-7 (wild-type p53) cells can
be mediated by p53. To further establish this, shp53 plasmid
was introduced to knock down p53 gene expression in MCF-7
cells. Cell cycle analysis after the indicated treatments showed that
the sub-G1 cell populations of the shp53 and shBCAS#434-treated
cells were the same as control (Fig. 6C; Supplementary Table S4).
The immunoblot presented p53 depletion in cells treated with
shp53 (Fig. 6C, bottom, lane 2), BCAS2 depletion in cells with
shBCAS2#434 (lanes 3-5), and ablation of both p53 and BCAS2
in the combined shp53- and shBCAS2-treated cells (lane 5). In
summary, silencing BCAS2-induced apoptosis is p53-dependent.
However, shBCAS2#434 caused cell cycle arrests at the G2-M
phase in p53-null H1299 cells and p53 mutant C33A cells (Supplementary Tables S2 and S3, respectively). This suggests
that, beyond p53, BCAS2 may interact with other cellular factors
to cause p53-independent cell growth arrest (Supplementary
Fig. S6).
Ectopic overexpression of BCAS2 rescues p53-mediated
growth inhibition. Because silencing BCAS2 expression caused
either cell apoptosis (wild-type p53) or growth arrest (p53-null
and mutant), we investigated whether ectopic expression of
BCAS2 could rescue cells from p53-induced growth inhibition.
Previous reports showed that low doses (50 nmol/L) of doxorubicin caused cell growth arrest through the activation of the p53
pathway and p21 induction (15). Therefore, MCF-7 cells were
transiently transfected with FLAG-BCAS2 or FLAG-vector plasmids and then treated with 50 nmol/L doxorubicin for 48 h.
Using a cell proliferation assay, we showed that doxorubicin-induced growth arrest in MCF-7 cells could be rescued by BCAS2
overexpression as shown in Fig. 6D (left). Similar results were
also observed by flow cytometry analysis (Fig. 6D, right; Supplementary Table S5). In response to doxorubicin, the percentage of
the G1 population increased in MCF-7 control cells and in MCF-7
cells treated with FLAG-vector at the expense of the respective
S-phase populations. By comparison, in doxorubicin-treated
MCF-7 cells overexpressing BCAS2, the percentage of the G1 population decreased, whereas the S-phase population increased
(Fig. 6D, right; Supplementary Table S5). The p53, p21, and BCAS2
protein expression levels derived from each treatment were compiled with the biological results (Fig. 6D, right and bottom). Taken
together, introducing BCAS2 in MCF-7 cells treated with low
doses of doxorubicin could decrease p21 protein expression,
thereby accounting for the BCAS2-mediated protection from
doxorubicin; this would imply that BCAS2 antagonizes p53-mediated growth inhibition.
Cancer Res 2009; 69: (23). December 1, 2009
Discussion
We discovered that BCAS2 is a novel p53 binding protein.
Loss of p53 function is a characteristic of almost all human tumors (32). We show that the binding of BCAS2 reduces p53
function by BCAS2 mildly but consistently decreasing p53 protein in normal condition (Fig. 2A-C). However, in the presence
of DNA damage, BCAS2 prominently reduces p53 protein and
provides protection against chemotherapeutic agent such as
doxorubicin (Fig. 6D). How BCAS2 is involved in p53 protein
degradation will become interesting. Because BCAS2 is a smallsized protein (26 kDa) and only contains two coiled-coil domains
that are reportedly responsible for the other protein interaction,
it is less likely to function as E3 ligase activity for protein degradation. On the other hand, MDM2 induces degradation and
nuclear export of p53 by acting as a p53-specific E3 ubiquitin
ligase (6, 13, 33–35). It is interesting to examine whether BCAS2
plays as a scaffold or adaptor molecule to mediate MDM2 or
other E3 ligase-related proteins for p53 protein degradation. Various negative apoptotic regulators of p53 were recently identified
that reduce p53 protein activity through different mechanisms
(6, 8, 14, 15, 21, 36–42). For example, MDM2, PACT, aurora kinase
A, and E6 function by causing p53 protein ubiquitination and
degradation (6, 13, 33, 43). Transcriptional activation of p53 is
also repressed by p202 via binding to the PXXP motifs of p53
(39) and by LSD1 through demethylated p53 protein (31). Notably, HSP27 and PFTm function as small-sized protein inhibitors
of p53 by promoting the assembly of p53 into complexes (6, 15, 40).
Here, we describe the novel functioning of BCAS2 as a smallsized p53 binding protein, not previously known to be a p53
inhibitor.
Silencing BCAS2 for 72 h without stress treatment causes ∼40%
cells apoptotic in p53-containing cells (MCF-7; Fig. 6A; Supplementary Fig. S4A; Supplementary Table S1). There are several possible
explanations for this. Firstly, the increases of p53 Ser46 phosphorylation (Fig. 5D) prominently induce p53 activity that trigger apoptosis. Secondly, in addition to p21, PUMA is also highly stimulated
in both RNA and protein levels (Fig. 4B and D). PUMA is a key mediator of p53-dependent apoptosis and targets to p53 that can release p53 from inhibitory p53-BCL-XL complexes and then induce
mitochondrial outer membrane permeabilization (6). Thirdly, deprivation of BCAS2 only without stress in p53-null cells results in
growth arrest (Supplementary Tables S2 and S3), implying that
BCAS2 may also have direct effect in cell growth. This and our unpublished finding of accumulation of DNA damage after BCAS2
knockdown5 suggest that BCAS2 may be involved in DNA damage
repair and RNA splicing. Interestingly, BCAS2 is also known as
spf27, a protein associated with the RNA spliceosome (3). spf27
can form a pre-mRNA splicing complex with Pso4/Prp19, Cdc5L,
and Plrg1. The Pso4 complex was reported to be involved in
DNA repair and RNA splicing (44–49). The function of BCAS2 in
the cell cycle process remains unknown. It would be interesting
to identify the BCAS2-interacting proteins that induce p53-independent cell cycle arrest at G2-M.
In sum, our hypothesis model shows that the binding of BCAS2
reduces p53 function, whereby silencing the BCAS2 gene causes
p53 release to function as a transcription factor toward p21, NOXA,
MDM2, and PUMA gene expression, resulting in an amplification
8884
5
Weng and Chen, unpublished results.
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Downloaded from cancerres.aacrjournals.org on August 11, 2017. © 2009 American Association for Cancer
Research.
Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023
BCAS2, a Negative Regulator of p53
of apoptotic signals leading to cell death in p53 wild-type cells. In
p53-null or p53 mutant cells, other cellular factors exist to interact
with BCAS2 to inhibit cell growth in a p53-independent manner
(Supplementary Fig. S6).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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Acknowledgments
Received 6/3/09; revised 9/22/09; accepted 9/24/09; published OnlineFirst 11/10/09.
Grant support: National Science Council grants NSC 95-2320-B-002-051-MY3 and
NSC 96-3112-B-002-030 and National Taiwan University grant 95R0066-BM 02-05.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. June-Tai Wu and Li-Po Wang for technical assistance and Drs. ShuChun Teng and Shin-Lian Doong for critical discussion.
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Published OnlineFirst November 10, 2009; DOI: 10.1158/0008-5472.CAN-09-2023
Breast Cancer Amplified Sequence 2, a Novel Negative
Regulator of the p53 Tumor Suppressor
Ping-Chang Kuo, Yeou-Ping Tsao, Hung-Wei Chang, et al.
Cancer Res 2009;69:8877-8885. Published OnlineFirst November 10, 2009.
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