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Is Down-Regulated in Human Breast Cancer Cells1
Equipe Facteurs de Croissance, Laboratoire de Biologie du Développement, UPRES-EA 1033, Villeneuve d’Ascq, France [A-S. V-E., X. L. B., B. B., H. H.]; Laboratoire de Chimie Biologique, UMR 8576 Centre National de la Recherche Scientifique, Université des Sciences et Technologies de Lille, 59650 Villeneuve d’Ascq Cedex, France [J. L.]; Laboratoire d’Oncologie Moléculaire Humaine, Centre de Lutte Contre le Cancer, de la région Nord-Pas de Calais (Centre Oscar Lambret), BP307, 59020 Lille, France [H. L., F. R., J-P. P.]; and Department of Anatomical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia [V. N.]
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ABSTRACT |
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form of 14-3-3 has been
shown to interact with cyclin-dependent kinases and to control
the rate of entry into mitosis. To test for a direct role for 14-3-3 in
breast epithelial cell neoplasia, we have quantitated 14-3-3 protein
levels using a proteomic approach based on two-dimensional
electrophoresis and matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-TOF). We show here that 14-3-3 protein is
strongly down-regulated in the prototypic breast cancer cell lines
MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with
normal breast epithelial cells. In contrast, levels of the 
, ß,
, or 
isoforms of 14-3-3 were the same in both normal and
transformed cells. The data support the idea that 14-3-3 is involved
in the neoplastic transition of breast epithelial cells by virtue of
its role as a tumor suppressor; as such, it may constitute a robust
marker with clinical efficacy for this pathology. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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form of 14-3-3 seems
to play a particular role in the control of such kinetics because its
overexpression causes cell cycle arrest (5)
. Most
importantly, it has recently been shown that gene expression of
14-3-3 is silenced in breast cancer cells (6)
and that
transfection of breast cancer cells with 14-3-3 inhibits
cyclin-dependent kinase activity, and thus cell cycle progression
(7)
, which suggests involvement of 14-3-3 in breast
tumorigenesis. However, there is as yet no clear data that shows that
14-3-3 protein levels correlate directly with breast cell
transformation. Here, we have used a proteomic approach to rigorously
test the hypothesis that levels of 14-3-3 protein correlate with breast
epithelial cell tumorigenesis and may, thus, constitute a robust new
marker for its prognosis. Our results demonstrate that 14-3-3
protein level may indeed constitute such a marker and should be tested
for clinical efficacy. |
Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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2-DE.
2-DE were performed as previously described (9)
with the
Investigator 2-D Electrophoresis System from Millipore (Paris,
France); all of the reagents were purchased from Oxford
Glycosystems (Abingdon, England). Ten to 30 µl of sample
(containing 106
cpm for labeled proteins, or 50
µg proteins for nonlabeled samples) were separated by IEF in rod gels
containing 2% pH 4–8 and 2% pH 3–10 carrier ampholytes. The
separation was performed at 20,000 Vh (
1143 V over 17.5 h).
After IEF, the strips were equilibrated twice for 2 min in 0.3
M Tris base [0.075 M Tris-HCl (pH 9.0), 3%
SDS, 50 mM DTT, and 0.01% bromophenol blue]. The second
dimensional separation was done using 10% SDS-PAGE for 5 h at
20,000 mW. After 2-DE, the proteins were detected by silver-staining,
and the gels dried and autoradiographed (X-Omat AR Kodak; Sigma). For
preparative 2-DE, 0.5–1 mg of protein were separated by IEF as
described above. The gels were then equilibrated twice for 20 min in
the equilibration buffer. After the second dimension, the gels were
stained in 0.2% (w/v) Coomassie Blue in 50% methanol and 10% acetic
acid.
Computer Analysis.
Both gels and autoradiographs were scanned (scanner SM3, Pharmacia)
using the Diversity One program (Pharmacia) and were analyzed using the
MELANIE II program (Bio-Rad) on a SUN-SPARC station. Molecular masses
and pI were determined after comparison with reference gels in
the SWISS-2DPAGE (Expasy)
database.5
Protein Identification by MALDI-TOF.
Protein identification was performed as described previously (10
, 11)
with modification. Spots were cut out from the gel and
washed three times with 400 µl of a 125-mM ammonium
carbonate/ACN 1:1 (v/v) solution for 20 min with shaking. The wash
solution was discarded, and the pieces were dried at room temperature
for 2 h. Enzymatic cleavage was initiated by reswelling the gel in
ammonium carbonate solution (125 mM), whereupon 50
mM acetic acid was added and the digestion finally
initiated by adding 50 mM acetic acid containing 7–7.5
units of trypsin (Promega, Lyon, France). After absorption of
the protease solution, aliquots (5 µl) of pure water were added
sequentially. The gel slices were placed in an Eppendorf tube and a
minimum volume of water was added to totally immerse the gel pieces.
The digestion was carried for 12–16 h at 37°C. The liquid was
collected and the resulting peptides recovered after two extractions
with a solution containing 45% ACN/10% formic acid. To recover very
hydrophobic peptides, a third extraction with 95% ACN/5% formic acid
was performed. The extract was finally dried using a Speed
Vac-concentrator (Savant).
MALDI-TOF analysis of trypsin digests was performed on a Vision 2000 (Finnigan, Bremen, Germany) reflector instrument in positive ion mode at an accelerating voltage of 6 kV. Peptides were resuspended in CH3CN/H2O (1:1) containing 0.5% formic acid of which 0.5 µl was mixed directly onto the target with 1 µl of 2,5 dihydroxybenzoic acid matrix solution (10 mg/ml in CH3OH/H2O, 7:3). Between 30 and 50 laser shots were accumulated to obtain a final spectrum. Mass measurements were then made after peak smoothing and internal calibration using the average mass of the two autolysis trypsin fragment ions at m/z 843.014 and 2212.425, resulting in mass accuracy better than 0.3 Da. Protein sequence database searching was performed using MS-Fit6 using the average molecular weight of [M+H]+ peptide ions.
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Results |
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illustrates the 2-DE separation of 35S-proteins
from primary culture of NBECs. More than 1000 polypeptides were
detected on autoradiograms and localized in pI 4–8 and molecular mass
range 20–200 kDa. Computer analysis of the autoradiograms
allowed determination of molecular masses and isoelectric point after
comparison with standard 2-DE gels of the Swiss-2DPAGE database. In
total, we were able to identify 50 spots (corresponding to 35 different
proteins) in 2-DE gels using MALDI-TOF analysis; the positions of some
of the identified proteins are shown in Fig. 1
. All of the identified
spots can be considered as abundant proteins, because they are
detectable with Coomassie Blue. For each identified protein, >50% of
the trypsin-generated fragments matched with theoretical masses.
For example, in case of annexin III, 14 fragments were generated by
trypsin digestion, and the mass of 8 of them matched with the
theoretical masses obtained for annexin III tryptic fragments in
sequence database. Some of these proteins, such as the cytoskeletal
elements actin, -actinin, ß-tubulin, vimentin, and tropomyosins,
have already been described in breast cancer cells. Some signal-related
proteins (annexins, numatrin, rho GDI) were also detected.
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and allow the characterization of /ß, /, and forms. The
masses of the tryptic fragments of 14-3-3 that were used for the
identification are reported in Table 1
. After database searching, matching masses represent more than 60% of
the fragment masses obtained, with a difference between measured and
calculated masses below 0.2 Da. The isoform /ß represents
different levels of phosphorylation of the same protein, as does the
isoform / (1)
; this presumably explains the
elongated form observed for the spots corresponding to these isoforms
in 2-DE gels.
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, and their quantitation is shown in Fig. 4
. The isoforms /ß and / of 14-3-3 showed no significant
variation in their expression, whereas the isoform, detected as a
major spot in normal breast cells, was clearly down-regulated in MCF-7
and MDA-MB-231 cells as well as in breast carcinomas. Careful
examination of the spot reveals two forms of 14-3-3 that migrate
differently in IEF, which suggests that it is subject to different
levels of phosphorylation as has been described for the /ß and
/ forms (1)
. The fact that several proteins shown in
Fig. 1
are missing in Fig. 3
is attributable to differences in
the method of protein visualization. Fig. 1
represents an autoradiogram
(35S-proteins), whereas Fig. 3
depicts silver staining
of the gel; differences in protein sensitivity to these two methods of
display are usually observed. Fig. 5
shows the quantification of the 14-3-3 protein in 35 primary breast
carcinomas compared with NBECs. The level of 14-3-3 reported for
NBECs represents the average value (±SE) obtained from eight 2-DE gels
produced from eight different samples. The level of 14-3-3
was found to be at an average of 10-fold lower in breast carcinomas
than in NBECs. In all of the breast carcinomas studied, the quantity of
14-3-3 was down-regulated compared with normal breast epithelium and
was not even detected in 5 of 35 samples.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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form of the 14-3-3 protein is down-regulated in breast cancer cells
as compared with NBECs. Because these proteins are modulators of
transduction signals related to cellular growth, our results suggest
that this down-regulation contributes to the deregulation of growth
observed in breast cancer. The hypothesis that 14-3-3 may constitute
a marker for the discrimination of noncancerous from cancerous
breast epithelial cells is thus supported by the study.
The widely expressed and highly conserved protein forms of 14-3-3 (,
ß, , , ) help regulate those signaling pathways that control
cell proliferation, differentiation, and survival (1)
.
They are known to associate directly or indirectly with such signaling
proteins as Raf, MEK kinase, PI3-kinase, and cdc-25, although the
precise molecular mechanism by which 14-3-3 proteins regulate these
elements remains unclear (1)
. Recently, Tzivion et
al. (2)
and Roy et al. (3)
have shown that Raf activation requires formation of a complex
containing Raf and 14-3-3 forms, and that the cellular levels of 14-3-3
have a significant impact on the control of Raf-1 activity. Varying
levels of Raf-1, MEK kinase, PI3-kinase, and cdc-25 activation may
account for the ability of 14-3-3 to induce either cell cycle arrest or
cell proliferation (1
, 4 , 5
, 12)
. Together these data
strongly suggest that certain levels of 14-3-3 expression are critical
for the control of cell growth, and that the down-regulation of
14-3-3 observed in breast cancer cells is involved in the neoplastic
transformation.
Interestingly, we have shown here that the amounts of the , ß,
, and forms of 14-3-3 do not significantly vary between normal
and cancer cells, which suggests that it is only the regulation of the
form of 14-3-3 that is related to neoplastic transformation. In
keratinocytes and in bladder, the expression of 14-3-3 is lower in
transformed cells than in normal cells (13
, 14)
, although
other forms of 14-3-3 were not monitored. The form of 14-3-3 is a
p53-regulated inhibitor of G2-M progression, and
its overexpression can cause cell cycle arrest (5)
. In
addition, it is able to both up-regulate cdc2 phosphorylation via Wee1
(15)
and down-regulate cdc25c (4)
, thus
controlling the entry of cells into mitosis by maintaining the
G2 checkpoint (16)
. Recently it was
also demonstrated that 14-3-3 directly associates with
cyclin-dependent kinases to negatively regulate cell cycle
progression (7)
. Gene expression of 14-3-3 was found to
be 7- to 10-fold lower in breast cancer cells than in normal breast
cells (6
, 17)
because of the high frequency of
hypermethylation of the 14-3-3 locus (6)
. We found here
that the 14-3-3 protein is present in breast cancer biopsies at a
level that is at an average of 10-fold lower than in NBECs.
Interestingly, Fergusson et al. (6)
have
reported that mRNA for 14-3-3 was undetectable by Northern blot
analysis in 45 of 48 primary breast carcinomas studied; in contrast, we
have detected the 14-3-3 protein in 30 of 35 primary tumor samples,
which indicates the high sensitivity provided by proteomic analysis.
The higher level of 14-3-3 in NBECs may, therefore, contribute to
the prevention of the high cellular proliferation characteristic of the
transformed phenotype.
Like breast cancer cells, NBECs express specific tyrosine kinase
receptors for peptides such as NGF (18)
and FGF-2
(19)
. Both NGF and FGF-2 activate the
Ras/Raf/mitogen-activated protein kinase pathway in breast cancer
cells, which results in stimulation of the cell proliferation
(18
, 20)
. However, neither NGF nor FGF-2 has a mitogenic
effect on NBECs (18
, 21)
. Paradoxically, therefore, NBECs
express both NGF and FGF receptors but do not proliferate in response
to either cognate factor. The reason for the lack of sensitivity to
these mitogens is not understood, but the growing body of evidence
implicating 14-3-3 involvement in cell cycle progression suggests
that the high level that we have found in normal cells may block the
mitogenic effect of such growth factors. These data support the idea
that the expression of 14-3-3 in NBECs participates in the control
of cellular growth by preventing proliferation, whereas in breast
cancer cells the down-regulation in 14-3-3 allows growth factor
stimulation and cell cycle progression.
This study also confirms that proteomic analysis is a powerful tool for the discovery of such molecular markers. Complementing the burgeoning field of genomics, proteomic analysis allows the characterization of picoquantities of proteins with mass spectrometry and, thus, changes in the levels inherent to the pathophysiology of any cell type, tissue, or whole organism (22) . It has always been hoped that it would allow the identification of markers to discriminate cancerous from normal cells, as well as the different stages of this pathology. As demonstrated here, proteome analysis may well become central to the discovery of new indicators for the diagnosis and prognosis of cancer progression.
In conclusion, our data show a correlation between breast epithelial
cell malignancy and down-regulation of 14-3-3, which suggests the
latter’s involvement in breast epithelial cell transformation. This
protein form, therefore, qualifies as a marker for the noncancerous
state of breast epithelial cells.
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FOOTNOTES |
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1 This work was supported by the Fondation pour la
Recherche Médicale, the Association pour la Recherche contre le
Cancer, Contrat 5360, the Ligue Nationale Contre le Cancer,
Comité du Nord, the French Ministry of Education, and the
National Health and Medical Research Council of Australia. 
2 A-S. V-E. and J. L. contributed equally to
this work.
3 To whom requests for reprints should be
addressed, at UPRES-EA 1033 de Biologie du Développement,
bâtiment SN3, Université de Lille1, 59650 Villeneuve
d’Ascq Cedex, France. Phone: 33-3-20-43-40-97; Fax: 33-3-20-43-40-38;
E-mail: Hubert.Hondermarck{at}univ-lille1.fr
4 The abbreviations used are: NBEC, normal
breast epithelial cell; 2-DE, two-dimensional electrophoresis; ACN,
acetonitrile; IEF, isoelectric focusing; MALDI-TOF, matrix-assisted
laser desorption/ionization-time of flight; NGF, nerve growth factor;
FGF, fibroblast growth factor.
5 Internet address:
http://expasy.hcuge.ch/ch2d/ch2d-top.html.
6 Internet address:
http://prospector.ucsf.edu/htmlucsf3.0/msfit.htm.
Received 5/25/00. Accepted 11/13/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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is a p53-regulated inhibitor of G2/M progression. Mol. Cell, 1: 3-11, 1997.[Medline]
locus leads to gene silencing in breast cancer. Proc. Natl. Acad. Sci. USA, 97: 6049-6054, 2000. negatively regulates cell cycle progression. J. Biol. Chem., 275: 23106-23112, 2000. is required to prevent mitotic catastrophe after DNA damage. Nature (Lond.), 401: 616-620, 1999.[Medline]
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