Impact of Etoposide on BRCA1 Expression in Various Breast Cancer Cell Lines
Drugs R D
Impact of Etoposide on BRCA1 Expression in Various Breast Cancer Cell Lines
Xi Zhang 0 1
Simone Hofmann 0 1
Nadia Harbeck 0 1
Udo Jeschke 0 1
Sophie Sixou 0 1
Key Points 0 1
0 Faculty of Pharmacy, University Paul Sabatier Toulouse III , 31062 Toulouse Cedex 09 , France
1 Department of Obstetrics and Gynecology, Breast Center, Ludwig-Maximilians University of Munich , Maistrasse 11, 80337 Munich , Germany
Breast cancer 1 (BRCA1), as a tumor suppressor, exerts an effective influence on protecting DNA integrity to suppress the development of breast cancer (BC). BRCA1 expression is induced in response to DNA-damaging agents such as etoposide. Germline BRCA1 gene mutations are associated with development of hereditary BC. However, besides BRCA-mutated BCs, some sporadic cancers may also exhibit a BRCA-like phenotype, displaying so-called 'BRCAness'. This common phenotype may respond to similar therapeutic approaches as BRCA-mutated tumors and may thus have important implications for the clinical management of these cancers. In order to determine whether and how etoposide regulates the protein levels of BRCA1 in BC cells, we exposed a panel of five selected cell lines to etoposide, compared the results to untreated control cells, and then stained the cells with the specific, reliable, and reproducible MS110 antibody directed against phosphorylated Ser1423 BRCA1. By evaluating cytoplasmic BRCA1 protein levels, we were able to distinguish three aggressive BC subtypes with BRCAness characteristics. In addition, determination of early and late apoptosis helped to complete the analysis of BRCA1 functions in the DNA damage pathway of aggressive BC. In conclusion, our study suggested that high cytoplasmic BRCA1 protein levels could be considered as a potential predictive marker for response to chemotherapy in both sporadic and hereditary BC. Tumors with either BRCAness phenotype or germline BRCA1 mutation are both aggressive BCs associated with poor prognosis and could both be subjected to targeted therapies against BRCA1-mutated BC in future clinical management strategies.
The function of BRCA1 in the DNA damage
pathway of aggressive BC cells may link to
Cytoplasmic BRCA1 expression has potential to be a
predictive biomarker in response to chemotherapy in
Breast cancer (BC) is the leading cause of death among
women diagnosed with cancer worldwide [
]. In 2012, it
alone comprised 25% of all cancer cases and 15% of all
cancer deaths among females [
], making it the most
common female cancer. However, BC is a complex and
extremely heterogeneous disease [
]. Thus, a deep
understanding of its biology and of certain prognostic factors is of
great significance in predicting disease outcome and
developing new target therapeutic strategies. Breast cancer 1
(BRCA1) is a susceptibility gene responsible for hereditary
predisposition to BC. Since it was first found to encode a
DNA repair enzyme involved in BC susceptibility in 1990
], and subsequently was successfully cloned in 1994 [
BRCA1 has received a great deal of attention in BC. It has
been mapped to chromosome 17q21 containing 24 exons,
encoding a pleiotropic full-length protein of 1863 amino
acids in humans [
]. BRCA1 full-length form is the
bestdefined BRCA1 gene product that contains multiple
functional domains, including a highly conserved N-terminal
RING domain, two nuclear localization signals located in
the exon 11, a serine-glutamine (SQ) cluster between amino
acids 1280–1524 [
], and tandem C-terminal BRCA1
(BRCT) domains [
]. BRCA1 is a serine phosphoprotein
that is regulated in a cell cycle-specific manner [
hyper-phosphorylated in response to DNA damage [
As a tumor suppressor, BRCA1 mediates many different
molecular processes including repair of double-strand DNA
breaks, transcriptional activation, apoptosis, cell-cycle
checkpoint control, and chromosomal remodeling, binds
different functional proteins (c-myc, E2F, p53, RAD50,
cyclins, CDKs, RNA polymerase, etc.), and suppresses
development of BC and ovarian cancers [
Therefore, genomic sequencing of BRCA1 (and
BRCA2) in women with a familial history of one or more
incidences of early-onset BC or ovarian cancer provides a
powerful tool to detect disease predisposition. However,
the genomic test is expensive and not suitable for detection
of sporadic cancers associated with somatic events.
Overall, about 9.3% of female BC patients carry predisposing
]. Germline mutations of BRCA1 and BRCA2
are responsible for about 50% of hereditary BC [
nevertheless, these mutations account for only 3–8% of all
BCs. Most BCs are sporadic and occur in absence of
BRCA1 mutations [
]. In sporadic breast tumors,
many researchers have postulated that loss of
heterozygosity (LOH) reduces BRCA1 messenger RNA (mRNA)
and protein levels, induces incorrect subcellular
], and impairs methylation of the BRCA1
promoter region [
]. These events lead to noticeable loss
of BRCA1 function and provide evidence for a BRCA1
tumor suppressor function in sporadic forms . Besides
BRCA-mutated BC, sporadic cancers may exhibit a
socalled ‘BRCAness’ feature, as they display a BRCA1
mutation phenotype without any mutation [
Nonetheless, BRCAness is generally associated with
mutations of other genes of the same signaling pathway. In
addition to its involvement in the tumor-suppressing
process, BRCA1 is also considered a key player in
establishing chemotherapy sensitivity and could thus be
considered a predictive factor for patient management . In
preclinical and clinical studies, the role of BRCA1 in
response to DNA-damaging agents and other types of
chemotherapy agents has only partly been elucidated
]. To the best of our knowledge, numerous studies
have investigated the clinic pathological value of the
BRCA1 protein level or of its subcellular localization in
clearly defined breast carcinomas, including sporadic and
BRCA1-mutated tumors. Nonetheless, in spite of the
findings concerning BRCA1 expression, the clinical value of
its subcellular localization is still controversial, mostly due
to limited techniques and approaches [
To address this issue, we evaluated BRCA1 nuclear and
cytoplasmic expression using immunofluorescence in a
panel of cultured breast cell lines with specific properties.
In addition, we used etoposide, as a DNA-damaging
reagent, to validate its effect on BRCA1 protein regulation,
and shed light on BRCA1 expression patterns in
representative cell line models of the different BC types with or
without etoposide treatment.
2.1 Cell Culture and Etoposide Treatment
The human adenocarcinoma cell lines MCF-7 and
MDAMB-231, both with the BRCA1 wild-type gene, were
obtained from the European Collection of Authenticated
Cell Cultures (ECACC, Salisbury, UK). The human breast
epithelial cell line MCF10A and ductal carcinoma cell line
HCC1937 (the latter with BRCA1 mutation 5382insC
]) were obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA). Human breast
ductal carcinoma cell line HCC3153 with BRCA1 mutation
(943ins10)  was kindly provided by Adi F. Gazdar
(Hamon Center for Therapeutic Oncology Research and
Department of Pathology, University of Texas
Southwestern Medical Center at Dallas, Dallas, TX, USA).
Cryopreservation of cell cultures ranged from passages 1 to 10.
Cells were used during up to 20 passages. To minimize the
heterogeneity that arises from different cultured conditions,
and in agreement with our own and literature data [
all cell lines were incubated routinely in Dulbecco’s
modified Eagle’s medium (DMEM) (Biochrom, Berlin,
Germany), supplemented with 10% FCS (Fetal calf serum)
(PAA, Pasching, Austria), in a humidified atmosphere of
95% air and 5% CO2 at 37 C. A 50 mM etoposide
(Sigma-Aldrich, Saint Louis, MO, USA) solution was
prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich,
Saint Louis, MO, USA) as a stock solution for treatment. In
preliminary experiments (data not shown), we used
different dilutions (25, 50, 75, and 100 lM) and incubation
times (6, 12, 24, and 48 h). As a result of this optimization
procedure, we used 100 lM of etoposide for 48 h as
unique treatment for the five cell lines. Hence, cells were
treated using a 1:500 dilution of the stock solution
(etoposide 100 lM) and vehicle (DMSO 100 lM) was used as
control in all experiments. For immunofluorescence and
apoptosis assays, 5 9 105 cells were grown on slides
(ThermoFisher Scientific, Braunschweig, Germany)
overnight to 70–80% confluency, and then treated in 10% FCS
with etoposide solution 100 lM for 48 h.
2.2 Fluorescence Labeling of Breast Cancer 1 (BRCA1) or Phosphorylated BRCA1 with Parallel 40-6-Diamidino-2-Phenylindole (DAPI) Analysis
After 48 h of treatment, culture slides were washed in PBS
(phosphate-buffered saline) (Fischer, Saarbru¨cken,
Germany), then immediately fixed in 3.7% neutral buffered
formalin (Fischer, Saarbru¨cken, Germany) in PBS for
15 min at room temperature and permeabilized in cold
(- 20 C) methanol (Sigma-Aldrich, Steinheim, Germany)
for 2 min. After washing in PBS, Ultra V Blocking
medium (ThermoFisher Scientific, Fremont, CA, USA) was
used for 15 min. This blocking step and all the following
steps were performed in a humidified chamber at room
temperature. Both antibodies were diluted in Dako
Antibody Diluent with Background Reducing Components
(Dako, Carpinteria, CA, USA). Cells slides were incubated
for 1 h with either a monoclonal mouse anti-human
BRCA1 antibody (1:200 dilution) (MS110, ab16780,
Abcam, Cambridge, UK) or a polyclonal rabbit anti-human
phosphorylated BRCA1 (1:200 dilution) (phospho S1423,
ab47325, Abcam, Cambridge, UK), washed in PBS,
incubated for 30 min with a secondary either goat anti-mouse
or anti-rabbit IgG labeled with DyLight488 (Jackson
ImmunoResearch, West Grove, PA, USA), and washed in
PBS. After drying (30 min, at room temperature), the slides
could be mounted with Vectashield Mounting Medium
with 40-6-diamidino-2-phenylindole (DAPI) (Vector
Laboratories, Burlingame, CA, USA) before manual analysis
with a computerized fluorescence microscope Axioskop
(Carl Zeiss Micro Imaging GmbH, Go¨ttingen, Germany)
for phase and fluorescence, with 409 magnification. An
AxioCam MR camera and AxioVision software were used
to capture, analyze, and save high-resolution images for
two fluorescence channels, considered independently or in
combination (Carl Zeiss Microscopy, Go¨ttingen,
Germany). Definite threshold values of exposure time for
BRCA1 were determined. The percentage of cells
expressing no (-), low (?), average (??), or high (???)
levels of BRCA1 in cytoplasm (BRCA1) or nuclei
(phosphorylated BRCA1) were calculated by analyzing 1500
cells in each slide. Three independent experiments were
systematically performed to calculate the mean values and
standard error (SE).
2.3 WST-1 Cell Viability Assay
After 48 h of treatment, cell viability was evaluated using
the WST-1 reagent (Roche, Mannheim, Germany), based
on the enzymatic cleavage of the tetrazolium salt WST-1 to
formazan by cellular mitochondria dehydrogenases present
in viable cells. Cells (1 9 104/well) were plated in 96-well
plates in DMEM medium containing 10% FCS. 24 h later,
cells were treated or not in 10% FCS with 100 lmol of
etoposide. After 48 h, WST-1 reagent was added to the
medium according to the manufacturer’s instructions. After
30 min, the absorbance of the samples was measured using
the microplate reader (MRX, DYNEX Technologies,
Denkendorf, Germany) at 450 nm wavelength. The relative
cell viability percentage in each cell line was calculated by
comparison to that of the control group. Each condition
was performed three times in each experiment and for each
cell line, and three independent experiments were then
performed to calculate the mean values and SE.
2.4 In Situ Nick-Translation (ISNT) Apoptosis
After 48 h treatment, the in situ nick-translation (ISNT)
technique was used to stain DNA fragmentation and
apoptotic bodies in the cells for late apoptosis detection [
Slides were washed in PBS, then immediately fixed in
acetone (Sigma-Aldrich, Steinheim, Germany) for 10 min.
After rinsing with distilled water, the endogenous
peroxidase was quenched with 0.3% hydrogen peroxide for
10 min. After being rinsed in distilled water again, the slides
were equilibrated in nick buffer (0.1 M Tris, 0.1 M MgCl2,
0.75% b-mercaptoethanol, 2 mg/mL bovine serum albumin
[BSA]) at room temperature for 10 min. ISNT was then
carried out by incubating the slides with deoxynucleotides
(dNTPs) (1:50 dilution) (ThermoFisher Scientific, Fremont,
CA, USA) and biotinylated 14-deoxyadenosine triphosphate
(dATP) (1:20 dilution) (ThermoFisher Scientific, Fremont,
CA, USA) diluted in nick buffer for 50 min at 37 C.
Terminating buffer (0.3 M sodium chloride and 0.03 M sodium
citrate) was used to rinse the chamber slides at room
temperature for 15 min. After washing in PBS and 1% FCS PBS
for 10 min each, slides were incubated with extravidin–
peroxidase (Sigma, Steinheim, Germany) at room
temperature for 30 min. AEC-substrate (Dako, Glostrup, Denmark)
was used for color development. Afterwards, the slides were
counterstained with Mayer’s hemalum (Merck, Darmstadt,
Germany), then immediately mounted with Aquatex
(Merck, Darmstadt, Germany) before manual analysis with a
Diaplan light microscope (Leitz, Wetzlar, Germany), with
109 and 409 magnifications. The late apoptosis ISNT was
calculated by analyzing 1500 cells in each slide. Three
independent experiments were systematically performed to
calculate the mean values and SE.
2.5 M30 Cyto Death Apoptosis Assay
The M30 cyto Death assay was developed to detect
caspase-cleaved Cytokeratin 18, which is one of the earliest
apoptosis markers in epithelial cells [
treatment, cells were immediately fixed in pure methanol at
- 20 C for 30 min, washed in washing buffer (0.1%
PBSTween) and blocked. Afterwards, cells were incubated with
a mouse monoclonal antibody (1:25 dilution) (clone M30,
Roche, Mannheim, Germany) overnight at 4 C in a
humidified chamber and then with a secondary goat
antimouse IgG labeled with DyLight488. After drying (30 min
at room temperature), the slides were mounted with
Vectashield Mounting Medium with DAPI (Vector
Laboratories, Burlingame, CA, USA) before manual analysis with a
computerized fluorescence microscope Axioskop (Carl
Zeiss Micro Imaging GmbH, Go¨ttingen, Germany) with
409 magnification. The early apoptosis by M30 cyto Death
staining was calculated by analyzing 1500 cells in each
slide. Three independent experiments were systematically
performed to calculate the mean values and SE.
2.6 Statistical Analysis
IBM SPSS Statistics for Windows, Version 24.0 (IBM,
Ehningen, Germany) was used for collection, processing,
and statistical data analysis. The student’s t test was
performed for comparison between control and treated group
in each cell line. p values B0.05 were considered
3.1 High Cytoplasmic BRCA1 Protein Levels in Aggressive Breast Cancer (BC) Cell Lines
To gain insights into the importance of BRCA1 expression,
we characterized and compared five representative breast
cell lines with or without etoposide treatment. BRCA1
protein levels were investigated by immunofluorescence in
the human breast normal cell line MCF10A and in four
human BC cell lines: MCF-7 (wild-type BRCA1),
MDAMB-231 (wild-type BRCA1, but ‘BRCAness’ phenotype),
HCC1937, and HCC3153 (both BRCA1 mutated). BRCA1
mutations in the HCC1937 and HCC3153 cells were in
exons 20 and 11, respectively, and the mutated BRCA1
still includes the epitope of the MS110 antibody, with
truncation sites far away from the N-terminal end [
Staining results are presented in Fig. 1a. The original 409
magnification shows that in control cells, BRCA1 was
expressed in the nucleus as well as in the cytoplasm. The
enlarged pictures show higher BRCA1 protein levels in the
cytoplasm compared with the nuclei of each cell line. For
etoposide-treated cells, original magnifications and
enlargements demonstrate higher nuclear and cytoplasmic
BRCA1 protein levels than in controls, with a more
dramatic effect in cytoplasm. Because of this obvious visual
difference, we concentrated on solely analyzing BRCA1
cytoplasmic staining to better clarify and quantify the
etoposide effect. We counted 1500 cells in each cell slide
and evaluated the intensity of BRCA1 cytoplasmic protein
levels (no [-], low [?], average [??], and high [???])
among all cell lines with or without etoposide treatment
(Electronic Supplementary Material Table 1 for all data;
Fig. 1b for cytoplasmic high expressions). It is noteworthy
that within each cell line, cells did not exhibit the same
intensity of BRCA1 cytoplasmic staining. Moreover, very
few cells exhibit no fluorescence intensity at all (3.3% in
untreated MCF-10A and 7% in untreated MCF-7). In the
control groups, all five cell lines were found with
predominantly low or average protein levels: 71.4 and 80.0%
of cells expressing low BRCA1 cytoplasmic staining in
MCF-10A and MCF-7 cells; 81, 92.4, and 84.9% of cells
expressing low or average staining in MDA-MB-231,
HCC1937 and HCC3153 cells, respectively. In the
untreated cells, a certain percentage of the population
expressed only high levels of cytoplasmic BRCA1 in the
MDA-MB-231, HCC1937, and HCC3153 cells (19.1, 7.6,
and 15.1%, respectively).
After etoposide treatment, all cell lines showed stronger
BRCA1 cytoplasmic staining; in particular, the same
MDA-MB-231, HCC1937, and HCC3153 cells expressed
high of cytoplasmic BRCA1 levels with 80.4%
(p = 0.005), 70.6% (p = 0.002), and 80.7% (p = 0.01),
respectively, thus demonstrating a significant rise in the
highest protein levels in the entire population (only 1.4% of
the HCC1937 still expressed a low cytoplasmic expression,
but no cells in the MDA-MB-231 and HCC3153). Besides,
only 2.3% (p = 0.02) and 11% (p = 0.003) of the
MCF10A and MDA-MB-231 cells reached such high
cytoplasmic expression, but 50.8% (p = 0.05) of the MCF-10A
cells and 67.9% (p = 0.009) of the MCF-7 cells now
expressed intermediate intensities, demonstrating the same
action of etoposide—still significant, but to a lower extent
than in the three other cell lines. In summary, high
cytoplasmic BRCA1 expression characterizes only a minority
Fig. 1 BRCA1 expression in control and etoposide-treated breast
cancer cell lines. Breast cancer cell lines were treated (ETOPOSIDE)
or not (CONTROL) with 100 lM of etoposide for 48 h, then
immunostained with BRCA1 antibody. a Immunofluorescence
labelling of BRCA1 (green) was performed together with DAPI nuclear
staining (blue). White arrows indicate enlargement parts. Original
magnification before enlargement, 940. Scale bar 50 lm. b The
percentage of cells exhibiting high BRCA1 cytoplasmic staining after
analysis of 1500 cells for each experiment (mean value and standard
error, n = 3). The correlation is statistically significant for *p B 0.05,
**p B 0.01, or ***p B 0.001. BC breast cancer, BRCA1 breast cancer
1, DAPI 40-6-diamidino-2-phenylindole
of cells in the three more aggressive untreated cell lines
(MDA-MB-231, HCC1937, and HCC3153) and etoposide
treatment induces a dramatic increase of these cytoplasmic
protein levels in all cell lines. For the less aggressive,
hormone-dependent model of BC (MCF-7 cells) and for the
normal breast cells (MCF-10A model), this specific high
cytoplasmic BRCA1 expression only appears in a minority
of the etoposide-treated cells.
3.2 High Nuclear Phosphorylated BRCA1 Protein
Levels in Aggressive Etoposide-Treated BC Cell
Phosphorylation of BRCA1 is regulated during the cell
cycle and in response to DNA damage. We then studied
phosphorylated BRCA1 expression, for the five cell lines
and in the conditions described in Sect. 3.1 (Fig. 2a). We
clearly observed that, in contrast to BRCA1 expression, the
phosphorylated BRCA1 staining was all nuclear, with basal
protein levels in all cells of the five untreated cell lines. We
then semi-quantified the nuclear protein levels of
phosphorylated BRCA1, according to the various intensities
(again low [?], average [??], or high [???]), as
presented in Electronic Supplementary Material Table 2 for
all data and in Fig. 2b for nuclear high expressions.
Untreated cells expressed predominantly low/average
levels of phosphorylated BRCA1: 100% of the MCF10A,
94.6% of the MCF-7, 91.7% of the MDA-MB-231, 98.5%
of the HCC1937, and 88.1% of the HCC3153. Although
very rare in any untreated cell line, the high protein levels
of nuclear phosphorylated BRCA1, were nonetheless
slightly increased in all cell lines after etoposide treatment
to 3.2% (p = 0.04) of the MCF-10A, 8.4% (p = 0.12) of
the MCF-7, most notably and significantly in 71.5%
(p = 0.007) of the MDA-MB-231, 70.8% (p = 0.001) of
the HCC1937, and 70.4% (p = 0.003) of the HCC3153.
MCF-10A and MCF-7 cells still exhibited significant low
nuclear phosphorylated BRCA1 staining (61 and 46.3%,
respectively). In summary, high nuclear protein levels of
phosphorylated BRCA1 predominantly characterize the
three more aggressive cell lines (MDA-MB-231,
HCC1937, and HCC3153) after etoposide treatment.
3.3 Effect of Etoposide on Cell Viability of Breast
Cancer Cell Lines
To further investigate the effect of etoposide, cell viability
was determined by WST-1 assay. As demonstrated in
Fig. 3, etoposide inhibited the viability of all five cell lines
at a concentration of 100 lM. Nonetheless, a significant
minor effect was observed on the normal breast cell model
MCF-10A (87.4% viability; p = 0.05) compared to
dramatic effects on all the BC cell lines: 35.9% (p = 0.004)
MCF-7, 22.6% (p = 0.0001) MDA-MB-231, 33.2%
(p = 0.005) HCC1937, and 30.4% (p = 0.03) HCC3153.
3.4 Effect of Etoposide on Late and Early Apoptosis
We then wanted to correlate the viability results to
apoptosis and performed in parallel assays for late apoptosis
analysis by ISNT and for early apoptosis by M30 staining
using conditions already described (Fig. 4a, b,
respectively). The rate of late apoptosis (Fig. 4c) detected in the
untreated and etoposide-treated MCF10A cells had a
similar mean value of 0.5 and 0.6% (p = 0.6), respectively,
demonstrating that etoposide did not significantly stimulate
apoptosis of the normal breast cell model MCF-10A. The
normal rate of apoptosis in the untreated MCF-7,
MDAMB-231, HCC1937, and HCC3153 had minimal means of
1, 0.9, 1, and 1.1%, respectively, while exposure to
etoposide significantly increased apoptosis in MCF-7, and to a
higher extent in MDA-MB-231, HCC1937, and HCC3153
to 2.4% (p = 0.009), 4.3% (p = 0.005), 3.3% (p = 0.01),
and 3.1% (p = 0.006), respectively.
The rates of early apoptosis were found to be very
similar to those of late apoptosis (Fig. 4d). The normal
breast model, MCF10A cells, control or treated, again had
a similar mean value of 0.8 and 0.9% (p = 0.74),
respectively. Besides, the normal rates of apoptosis in the four
untreated BC cell lines were confirmed to be very low,
inferior to 2%, whereas they were significantly elevated to
2.7% (p = 0.0005), 6.5% (p = 0.004), 6.4% (p = 0.008),
and 7.0% (p = 0.001) after etoposide treatment (MCF-7,
MDA-MB-231, HCC1937, and HCC3153, respectively).
Since the 1990s, the importance of BRCA1 expression and
of its subcellular localization as a marker in sporadic BC
has been under debate. Chen et al. [
] first reported that
BRCA1 was found in the nuclei of epithelial cells, and
detected mainly in the cytoplasm of malignant mammary
cells. In contrast, Scully et al. [
] showed that BRCA1
was located predominantly in the nuclei of both normal and
malignant cells, whereas Jensen et al. [
] contradicted this
by stating that BRCA1 was observed in cytoplasm and cell
membrane. Following this, there has been a slow stepwise
progression in the understanding of the subcellular
distribution of BRCA1, often hampered by technical problems
attributable to cross-reactivity and low specificity of certain
BRCA1 antibodies. In recent years, advanced technologies
and approaches enabled to detect more phosphorylated
than non-phosphorylated forms of BRCA1 in nuclear and
mitochondrial genomes than in cytoplasm [
demonstrated that BRCA1, as a shuttle protein, shuttles
Fig. 2 Phosphorylated BRCA1 expression in control and
etoposidetreated breast cancer cell lines. Breast cancer cell lines were treated
(ETOPOSIDE) or not (CONTROL) with 100 lM etoposide for 48 h,
then immunostained with phosphorylated BRCA1 antibody. a
Immunofluorescence labelling of phosphorylated BRCA1 (green) was
performed together with DAPI nuclear staining (blue). White arrows
indicate enlargement parts. Original magnification before
enlargement, 940. Scale bar 50 lm. b The percentage of cells
exhibiting high BRCA1 nuclear staining after the analysis of 1500
cells for each experiment (mean value and standard error, n = 3). The
correlation is statistically significant for *p B 0.05, **p B 0.01, or
***p B 0.001. BC breast cancer, BRCA1 breast cancer 1, DAPI
Fig. 3 Viability of
etoposidetreated breast cancer cell lines.
Breast cancer cell lines were
treated (ETOPOSIDE) or not
(CONTROL) with 100 lM
etoposide for 48 h, then cell
viability was analyzed by
WST1. The quantitative assessment
of viability is presented as the
mean value and standard error
(n = 3). The correlation is
statistically significant for
*p B 0.05, **p B 0.01, or
***p B 0.001
between specific sites within the nucleus and cytoplasm,
including DNA repair foci, centrosomes, and mitochondria,
and uses its different transport sequences to form distinct
protein complexes with various protective roles [
However, little is known about how BRCA1 shuttling
between the nucleus and cytoplasm is controlled . The
specificity of the antibodies selected for BRCA1 detection
is also a key point to explore. Wilson et al. [
] first tried to
comprehensively characterize 19 anti-BRCA1 antibodies,
suggesting that the monoclonal antibody MS110 (Ab-1),
targeting the 304 first amino acids from the N-terminal end
of BRCA1, is highly specific and allows evaluation of
BRCA1 localization and relative protein levels in normal
and malignant human breast and ovarian tissues.
PerezValles et al. [
] demonstrated that this MS110 antibody
gives the most accurate, reliable, and reproducible results
in familial and sporadic non-BRCA1 associated breast
carcinomas among a four-antibody panel. Using the same
MS110 antibody, Milner et al. [
] proposed the
measurement of nuclear BRCA1 expression by
immunohistochemistry (IHC) on breast and ovarian tumor tissue
sections, as patient selection biomarker by focusing
exclusively on cells in the S/G2 phase where BRCA1
protein staining is expected. Wei et al. [
] aimed to
investigate the associations of BRCA1 nuclear expression
and clinic pathological characteristics in young Chinese
BC patients, and Mylona et al. [
] applied IHC on
sporadic BC patients to explore a different prognostic
significance of BRCA1 protein, according to its subcellular
distribution. In this study, we further investigated BRCA1
protein levels, by selecting five representative mammary
cell lines: MCF-10A, a human normal breast epithelial cell
line, which is a widely used in vitro model for studying
normal breast cell function and transformation, in spite of
some controversies [
], MCF-7 and MDA-MB-231,
sporadic BC models, and HCC1937 and HCC3153,
BRCA1mutated BC cell models. Of note, the MCF-7 cell line is a
model of non-aggressive hormone-dependent cancer cells
(luminal A), whereas MDA-MB-231, HCC1937, and
HCC3153 belong to aggressive triple-negative BC (TNBC)
]. Regarding the MDA-MB-231 cell line, it shares
many features with BRCA1-mutated tumors [
] and is
associated to the BRCAness phenotype, defined as a
phenocopy of BRCA1 or BRCA2 mutations, initially different
from BRCA1 mutations [
]. We selected the widely used
antibody MS110 [
24, 70–72, 78, 79
] and demonstrated
BRCA1 protein levels in both the nucleus and cytoplasm of
the five normal and cancerous subtypes, which is consistent
with other reports [
47, 68, 80–83
]. In this article, we
wanted to detect whether BRCA1 protein
expression—irrespective of BRCA1 gene mutation—could differentiate
BC subtypes: normal/sporadic/BRCA1-mutated or
aggressive/non-aggressive. Some sporadic BC cell lines have no
mutation of the BRCA1 gene, such as MDA-MB-231, but
nonetheless exhibit BRCAness. Consequently, we aimed to
define the relationship between BRCA1 expression and
different types BC cell lines. As all cell lines were
observed to express predominantly null, low, or average
protein levels of BRCA1, with heterogeneous expressions
within each cell line, it made it difficult to differentiate BC
subtypes using either nuclear or cytoplasmic BRCA1
protein levels. Nonetheless, it is noteworthy that 7–19% of
cells expressed high levels of cytoplasmic BRCA1 only in
the three more aggressive TNBC cell lines.
Etoposide, as topoisomerase II poison, induces
doubleand single-strand breaks in DNA [
]. This plant alkaloid
is an oral drug used eventually in anthracycline and taxane
pre-treated metastatic BC [
] or may be useful in
combination with new targeted therapy such as
anti-vascular endothelial growth factor (VEGF), histone
deacetylase, and DNA damage response (DDR) inhibition
]. In HeLa cervix carcinoma cells and
SK-OV-3 ovarian cancer cells, BRCA1 mRNA levels were
increased by etoposide treatment [
], while BRCA1
expression displayed only a minimal increase in MCF-7
nuclei . Using the conditions we optimized (100 lM
concentration and 48 h duration), our data demonstrate that
etoposide treatment induced higher cytoplasmic BRCA1
Fig. 4 Late and early apoptosis in etoposide-treated breast cancer
cell lines. Breast cancer cell lines were treated (ETOPOSIDE) or not
(CONTROL) with 100 lM etoposide for 48 h, then apoptosis was
detected by in situ nick translation (ISNT) assay for late apoptosis
(a) and M30 cyto Death assay for early apoptosis (b). Apoptotic cells
were stained brown in (a) (black arrows) and green in (b) (white
arrows). The related percentages of apoptotic cells are presented after
the analysis of 1500 cells for each experiment in (c) and (d),
respectively (mean value and standard error, n = 3). The correlation
is statistically significant for **p B 0.01 or ***p B 0.001
levels in the five breast models, with more than 70% of
cells expressing high cytoplasmic levels of BRCA1 in the
three aggressive BRCA1-deficient or -mutated cell lines,
MDA-MB-231, HCC1937, and HCC3153. In comparison,
only 2 and 11% of the MCF10A and MCF-7 cells
expressed these high cytoplasmic levels of BRCA1:
BRCA1 cytoplasmic protein levels increased essentially
from low to average intensities in most cells of these
nontumorigenic MCF-10A and luminal A type MCF-7 models.
Thereby, we could distinguish even better the three
aggressive TNBC BRCA1-deficient or -mutated cell lines
from the normal and luminal subtypes according to
BRCA1 cytoplasmic protein levels after using etoposide.
Cytoplasmic expression of BRCA1 could be explained by
two probable mechanisms: cytoplasmic retention and
nuclear export. BRCA1 is trapped in the cytoplasm
following overexpression of the anti-apoptotic factor Bcl-2,
which redirects BRCA1 to mitochondria and endoplasmic
]. In addition, it is notable that HCC1937 has
a phosphatase and tensin homolog on chromosome 10
(PTEN) deletion, and the PTEN inactivation causes an
increase in cellular PIP3 levels subsequently activating
PI3 K/AKT signaling. This causes an increased expression
of several genes for cell growth, cell survival, and cell
migration, including BRCA1. AKT1 kinase was also
reported to suppress homologous recombination
(HR)mediated DNA repair through the cytoplasmic retention of
BRCA1 and Rad51 [
]. Meanwhile, the nuclear export
of BRCA1 was directly linked to p53-independent
proapoptotic activity [
]. BRCA1 and p53 are both tumor
suppressors, which are involved in many cellular processes.
BRCA1 has been reported to bind directly to p53, thereby
enhancing p53-mediated transcriptional activation
]. Nuclear run-on experiments and luciferase
reporter assays demonstrate that the changes in BRCA1
expression are mainly due to transcriptional repression
induced by p53 [
]. Nuclear export of BRCA1 occurred
in response to ionizing radiation DNA damage in cells with
functional p53 but in cells lacking wild-type p53 BRCA1
was retained in the nucleus [
]. Compared to p53
wildtype MCF-7 and MCF10A, both HCC1937 and
MDA-MB231 are p53 mutants, while, to our knowledge, the p53
status of HCC3153 is unknown, although its protein level is
]. In our study, MCF-7 and MCF10A
demonstrated an increase of cytoplasmic BRCA1
expression after treatment, which is consistent with the former
study. But due to an abnormal BRCA1 and p53 status, the
other three cell lines showed much stronger cytoplasmic
expressions before treatment. Fedier et al. [
that BRCA1 deficiency in p53-null cells was associated
with increased sensitivity to the topoisomerase II poisons
etoposide, which could be a mechanism to explain our
observations. A study claimed to observe a correlation
between cytoplasmic localized BRCA1 and activation of
the intrinsic caspase cleavage pathway, in particular after
DNA damage [
]. As mentioned earlier, p53, PTEN
status, and other tumor suppressors that are also crucial for
therapy outcome might have functional interplay with
BRCA1 and thus lead to BRCA1 expression alteration and
cellular shuttling. To date, the actual mechanism by which
cytoplasmic-localized BRCA1 elicits cell death is not fully
understood but may be a reason for the increased rate of
apoptosis shown in the following apoptosis assay.
As BRCA1 is a serine phosphoprotein regulated in
response to DNA damage [
], it has been reported that
DNA damage induces both nuclear redistribution of
BRCA1, which may also explain increased cytoplasmic
staining and an increased phosphorylation of the protein
through DNA damage-activated kinases [
14, 107, 108
Several phosphorylation sites have been identified under
these conditions, including Ser-1423 [
]. We used
phospho-Ser-specific antibodies recognizing the Serine in
position 1423 of BRCA1 to further explore the regulation
of BRCA1 phosphorylation in non-treated and
etoposidetreated cells. Our study demonstrated that phosphorylated
BRCA1 was mainly located in the nuclei, before and after
treatment. BRCA1 being a serine phosphoprotein regulated
in a cell cycle-specific manner, its phosphorylation starts
when cells enter S-phase. Phosphorylated BRCA1 then
accumulates in the nucleus where it functions in the
cellular response to DNA damage and regulates specific
processes including cell cycle checkpoint activation, DNA
repair, and chromatin remodeling. Coene et al. [
support a universal role for BRCA1 in the maintenance of
genome integrity in nucleus. In addition, DNA damage also
induces an increased phosphorylation of the protein
through DNA damage-activated kinases. Our results
reasonably demonstrate the same trend as a low or medium
basal nuclear expression of phosphorylated BRCA1
characterized all non-treated cell lines, with no cell line
exhibiting high levels of phosphorylated BRCA1. As
expected, etoposide treatment moderately increased the
percentage of normal and luminal A cells expressing high
nuclear levels of phosphorylated BRCA1 (reaching 3.2 and
8.4%, respectively). In contrast, more than 70% of the
TNBC, BRCA1-deficient or -mutated, cells expressed high
nuclear phosphorylated BRCA1. This extremely elevated
expression may be the result of the inefficiency of the
mutated or deficient BRCA1 in these cell lines. These
results obtained by immunofluorescence for BRCA1
protein levels and phosphorylation status in five different cell
lines confirm preliminary data we generated using
immunocytochemistry colorimetric, non-fluorescent
staining (data not shown). However, samples are pre-treated
differently according to the protein analysis technique and
this may profoundly influence the ability of a given
antibody to bind specifically to its target [
]. So in the
future, the results and conclusions of our study will have to
be extended using alternate protein analysis technique as
western-blot. Moreover, manipulation of BRCA1
expression using RNA interference may demonstrate the
importance of BRCA1 for prediction of response to
Our data suggest that etoposide could induce apoptosis, as
we observed an obvious reduction, 60–80%, in the four BC
cell populations compared to control cells, whereas the
normal breast cells exhibited only a slight decrease. We
confirmed that etoposide did induce early and late apoptosis
among the four BC cell lines, around a two-fold increase for
the MCF-7 and three- to five-fold increases in the three
aggressive TNBC cell lines. This higher apoptosis induction
rate in the BRCA1-deficient/-mutated cells may relate to the
higher expression of cytoplasmic BRCA1 and of nuclear
phosphorylated BRCA1. All the results we generated
strongly suggest that these three aggressive TNBC cell lines
might share some identical pathways related to BRCA1
during DNA damage repair. The elevated expression of
(phosphorylated) BRCA1 in cytoplasm or nucleus, before or
after treatment, may be associated with the prognosis and
further studies are needed to develop this approach as
diagnostic assay in BC. In the near future, (phosphorylated)
BRCA1 could be first analyzed in the tumors of a large
cohort of patients with different BRCA1 status. Unlike the
two BRCA1-mutated HCC cell lines, MDA-MB-231 is a
model of sporadic BC without BRCA1 mutation. But as a
member of basal-like BCs (BLBCs), MDA-MB-231 shares
many features with BRCA1-mutated tumors [
]. In the
meantime, three-quarters of BRCA1-associated tumors are
]. Dysfunctions of the BRCA1 pathway
detected in BLBCs mainly regards the impairment of
doublestrand break (DSB) repair through HR, leading to genomic
instability. The hallmark of BLBCs is the ‘BRCAness’ [
previously, the concept of BRCAness referred to the fact that
sporadic tumors characterized by reduced or absent BRCA1
expression share the same phenotype of familial BRCA
]. Over 20 years, a reassessment of the concept of
BRCAness was required and nowadays it describes the
situation in which an HR repair (HRR) defect exists in a tumor
in the absence of a germline BRCA1 or BRCA2 mutation
]. BRCAness is then a common characteristic for
MDAMB-231, HCC1937, and HCC3153. Since the role of
BRCA1 in DNA repair is mainly related to the HR, the new
proposed biomarker (cytoplasmic BRCA1) should be
compared to the classical (Rad-51 foci in cyclin-A positive cells)
or even novel HR assays [
There is limited information on BRCA2 mutations in the
discussed cell lines. Distribution of histologic types
of BRCA1-associated BCs differs from sporadic BCs in
various aspects: having distinct morphology, being more
often medullary-like, being triple negative, and showing a
‘basal’ phenotype; but BRCA2-associated BCs do not
appear to exhibit a specific pathologic phenotype [
In BRCA1-mutant tumors, the capability of DNA damage
repair is decreased, which makes tumor cells more
sensitive to DNA-damaging drugs than normal BC cell lines
. Consistent with the HRR defect, tumors with
BRCAness might also share therapeutic vulnerabilities
with germline BRCA1 or BRCA2 mutation tumors, such as
sensitivity to platinum-based drugs and then Poly
(ADPribose) polymerase inhibitor (PARPi) [
]. It was recently
suggested that inhibition of the DDR (cell cycle arrest and
DNA repair) could increase the efficacy of conventional
DNA-damaging agents. In particular, like PARPi, which
targets the DDR in specific tumor cells, it can selectively
kill tumor cells carrying BRCA mutations but not normal
To date, BRCA1 protein measurement evaluated as a
potential diagnostic and prognostic biomarker for BC has
never reached a consensus. In our study, with etoposide
induction, we can better distinguish BRCA1-associated BC
cell line representative subtypes by evaluating cytoplasmic
BRCA1 protein level. Meanwhile, our results also show
that the increased sensitivity of BRCA1-deficient cells to
etoposide may be due to the specific DSB created by
topoisomerase II. However, a larger set of BC cell lines
with specific sensitivity to various DNA damage agents and
different levels of cytoplasmic BRCA1 should be
characterized to confirm our hypothesis using other accurate and
reliable technologies. Therefore, we suggest that
cytoplasmic BRCA1 protein levels level could be considered
and further explored as a potential predictive marker for
response chemotherapy in both sporadic and hereditary
BC. Although this evaluation could not specifically help in
guiding treatment, we intend to analyze tumor samples
through further collaboration with clinicians in the future.
Our results also raise several issues concerning the
functions of BRCA1 in the DNA damage pathway and
biochemical details of signaling conferred by nuclear
phosphorylated BRCA1. BRCAness phenotype and
germline BRCA1 or BRCA2 mutation tumors are both aggressive
BCs with a poor prognosis which could share common
clinical management strategies. Many targeted therapies
have been developed against BRCA1-mutated BC, of which
PARPi are most promising drugs.
Acknowledgements We would like to thank C. Kuhn for technical
advice and the China Scholarship Council (CSC) for a scholarship to
Authors Contributions SS and UJ conceived and designed the
project. XZ wrote the paper and performed most experiments. SH
assisted with cell culture. SS contributed to manuscript writing and
editing. NH and UJ conceived the topic and contributed to manuscript
editing. SS supervised the research. All authors read and approved the
Compliance with Ethical Standards
Conflict of Interest Xi Zhang, Simone Hofmann, Nadia Harbeck,
Udo Jeschke, and Sophie Sixou declare that they have no conflict of
Funding Sophie Sixou’s salary was supported by the University Paul
Sabatier in Toulouse (France).
Ethics approval and consent to participate This article does not
contain any studies with human participants or animals performed by
any of the authors.
Open Access This article is distributed under the terms of the
Creative Commons Attribution-NonCommercial 4.0 International
License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits any noncommercial use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made.
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