# Protein Alterations in Infiltrating Ductal Carcinomas  of the Breast as Detected by Nonequilibrium pH Gradient Electrophoresis and Mass Spectrometry

Authors: Maria Kabbage; Karim Chahed; Bechr Hamrita; Christelle Lemaitre Guillier; Mounir Trimeche; Sami Remadi; Johan Hoebeke; Lotfi Chouchane
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Improvement of breast-cancer detection through the identification of potential cancer biomarkers is
considered as a promising strategy for effective assessment of the disease. The current study has used nonequilibrium pH gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from Tunisian women. We have identified multiple protein alterations in tumor tissues that were picked, processed, and unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers. They include glycolytic enzymes, molecular chaperones, cytoskeletal-related proteins, antioxydant enzymes, and immunologic related proteins. Among these proteins, enolase 1, phosphoglycerate kinase 1, deoxyhemoglobin, Mn-superoxyde dismutase,α-B-crystallin, HSP27, Raf kinase inhibitor protein, heterogeneous nuclear ribonucleoprotein A2/B1, cofilin 1, and peptidylprolyl isomerase A were overexpressed in tumors compared with normal tissues. In contrast, the IGHG1 protein, the complement C3 component  C3c, which are two newly identified protein markers, were downregulated in IDCA tissues.


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## Body

## 1. Introduction

Breast cancer remains one of
the leading causes of death among women worldwide. The incidence rate of this
malignancy is steadily rising in developing countries. It is expected to
account for 31% of all new cancer cases among women in USA  [1]. In Tunisia, the incidence of breast
cancer is approximately 19 new cases per 100 000 women per year [2].Invasive carcinomas where the
basement membrane is totally or partially destroyed represent 70–80% of all breast
cancer and include more than 10 different types. Among these, infiltrating
ductal carcinomas (IDCA) are the most aggressive forms and have a poor
prognosis [3].Nowadays, improvement of
breast-cancer detection at its early stage through the identification of
selective diagnostic biomarkers and targets for therapeutic intervention is of
critical importance. Alterations that occur in disease versus normal tissues at
either the gene or protein level have been regarded as an appropriate way to
identify such markers of pathologies. In biomedicine, proteomics, with the
recent advances in mass spectrometry, is considered as a powerful analytical
method for deciphering protein expression alterations as a function of disease
progression [4]. Recently, proteomics-based analyses of breast tissue lysates
have resulted in the finding of a number of potential tumor biomarkers
providing, therefore, a basis for a better understanding of the intracellular
signalling pathways leading to breast-cancer development and progression [3],
and eventually serving as diagnostic and prognostic markers [5]. Probably the
most widely used proteomic technology is the identification of alterations in
protein expression between two different samples through comparative two-dimensional
gel electrophoresis (2-DE) which provides high-resolution separation of
proteins and offers a powerful method for their identification and
characterization. Actually, there is no universal 2-DE method that can be
applied for all types of samples investigated, and protocols must be optimized
and adapted according to the types of tissues and proteins of interest. It is
also crucial to minimize protein alterations and degradations that could result
in artifactual spots and to improve solubilization procedures in order to avoid loss of
proteins during extraction [6].Although 2-DE is a laborious
method, it is still considered as the principal tool in proteomics since it is
able to separate proteins according to two independent physicochemical
parameters which are isoelectric point and size, and also it provides the
highest resolution in the separation of complex
protein mixtures [6]. 2-DE remains however, poor resolutive in detecting
proteins that are smaller than 10.000 Da or are basic in charge [7]. An
alternative method for the analysis of basic proteins is found in the
application of nonequilibrium pH gradient electrophoresis (NEPHGE), which can
provide excellent resolution of basic proteins, offering thus a promising
approach for the discovery of tumor-related alterations [8].In a previous study, we have
shown using IEF and mass spectrometry that the levels of a number of proteins
including fibrinogen-E, which is a potent antiangiogenic factor, were dysregulated
in IDCAs of the breast [9]. In the current report, protein expression profiles issued
from breast IDCAs and their matched surrounding histologically normal tissues
were examined by NEPHGE two-dimensional gel electrophoresis and MALDI-TOF mass
spectrometry. Using this procedure, we established a NEPHGE-2-DE map of
proteins altered in IDCAs of the breast. By comparing 2-DE protein profiles, we
report herein the identification of several proteins of interest dysregulated
in tumor tissues. These altered proteins belong to multiple cellular pathways
and may have a pathological role in breast carcinogenesis.


## 2. Materials and Methods

### 2.1. Sample Processing

Breast
tumor samples(n=10) were selected from patients living in the middle coast of Tunisia
(median
age at diagnosis of 50 years) and classified as infiltrating ductal carcinomas.
Breast tumors were obtained with informed consent according to protocols
approved by the institutional review boards of the respective hospitals.
Carcinomas were pathologically staged according to the TNM (Tumors, Nodes,
Metastasis) classification system of UICC [10]. Histological grade was
assessed according to the modified SBR grading system [11]. All the tumors
investigated were classified as T2/N0 and the grade of the tumors was G3. Breast
tumors and surrounding histologically normal tissues were collected at the time
of surgery and analyzed by histopathology. Areas of tumor were chosen after
assessment by cryostat sectioning. The first, middle, and last sections were
stained with hematoxylin and eosin, and examined by a pathologist to ensure
that at least 70% of the tissue sections used for the proteomic study contained
tumor cells. The nontumor tissues were confirmed by cryostat section before
protein extraction. For
proteomic analyses, tissue sections verified histologically were suspended and
homogenized in  250 μL of 2-DE
analysis buffer: 7 M urea,
2 M thiourea, 4% CHAPS, 0.5% DTT, and 2% ampholytes (1 part pH 3/10, 1 part pH
5/7, 2 parts pH 6/8). The clear supernatant issued after centrifugation of the
homogenate at 12500 g for 15 minutes was transferred to a sterile microcentrifuge tube and
immediately subjected to 2-DE analysis.


### 2.2. Nonequilibrium pH Gradient Electrophoresis, Isoelectrofocalization and SDS-PAGE

Protein contents were determined according to the procedure described by Bradford and modified by Ramagli and Rodriguez [12]. Bovine serum albumin (Fraction V, Sigma) was used as a standard. Analytical 2-DE was carried out in a BioRad system (Miniprotean II). Equal amounts of proteins (100 μg) issued from tumor or nontumor breast tissues were applied to the first dimension and at least three NEPHGE gels were run for each sample. NEPHGE has been used to resolve basic proteins as described earlier [8]. IEF was performed as described previously [9]. For each experiment, extraction of proteins, solubilization, NEPHGE, and SDS-PAGE were carried under similar conditions for the tumor and nontumor tissues. Electrophoresis was performed in triplicate for each pair of samples to ensure reproducibility. The alterations reported in the current study were retained after 2-DE analysis of protein extracts issued respectively from different frozen
sections of the 10 tumor and nontumor samples investigated. The reproducibility was about 85% or more which reflected the small variances between samples or running.


### 2.3. Gel Staining

After separation, the proteins were visualized by a sensitive colloidal Coomassie G250 stain [13]. The dye solution contained 17% (w/v) ammonium sulfate, 3% (v/v) phosphoric acid, 0,1% (w/v) Coomassie G250, and 34% (v/v) methanol. The staining solution was changed once after 12 hours staining and the gel slabs subjected to a 24-hour cycle for increasing dye deposition on low-abundance proteins. The detection was then increased by placing the gel into 1% v/v acetic acid for producing a better contrast between spots and gel. Coomassie stained gels were scanned and spot detection and quantification were carried out using PDQuest 2-D software version 7.2 (Biorad). Proteins with significant and reproducible changes between tumor and nontumor tissues were considered as differentially expressed. The abundance of each protein was estimated by the percentage volume. The individual spot volumes were normalized by dividing their optical density (OD) volumes by the total OD values of all the spots present in the gel and expressed as % vol. The significance of expression between tumor and nontumor tissues was estimated by student’s t-test, P<.05.


### 2.4. Spot Picking and in-Gel Digestion

Picked
spots were washed with 100μL of 25 mM NH4HCO3 and dehydrated with 100 μL of acetonitrile (ACN). This operation was repeated twice and the pieces of
gel were dried under vacuum for 10 minutes. Reduction was achieved by 1-hour
treatment with 10 mM DTT in NH4HCO3 buffer (100 μL) at 56∘C. After discarding the DTT
solution, alkylation reaction was performed by the addition of 100 μL of 25 mM iodoacetamide in 25 mM NH4HCO3 buffer for 1 hour at  room temperature, protected from light.
Finally, the excised gel pieces were again washed 3 times for 5 minutes with 25 mM NH4HCO3 and ACN alternately. Gel pieces were completely dried under vacuum before
tryptic digestion. The dried gel volume was evaluated (about 1 to 2 μL) and 3 volumes
of trypsin (Promega, V5111), 12.5 ng/μL, in 25 mM NH4HCO3 buffer (freshly diluted), were added. The digestion was performed at room
temperature overnight. Afterwards, 5 μL of 35% H2O/60% ACN/5% HCOOH
were added and the mixture is sonicated for 30 minutes and centrifuged in order
to extract tryptic peptides.


### 2.5. Protein Identification by MALDI-TOF-MS

Mass
measurements were carried out on a BIFLEX III MALDI-TOF
(Bruker, Daltonics, Bremen, Ge) equipped with the SCOUT high-resolution optics
with X-Y multisample probe and gridless reflector. This instrument was used at
a maximum accelerating potential of 19 kV (in positive mode) and was operated
in reflector mode. A saturated solution ofα-cyano-4-hydroxycinnamic acid
(Sigma, Saint Louis, MO) in acetone was used as a matrix. A first
layer of fine matrix crystals was obtained by the spreading and fast
evaporation of 0.5 μL of matrix solution. On this fine layer of crystals, a
droplet of 0.5 μL of aqueous HCOOH (5%) solution was deposited. Afterwards, 0.5 μL tryptic digest were added and mixed to a second dried under vacuum. The
sample was washed once by applying 0.7 μL of aqueous HCOOH (5%) solution on the
target and then flushed after a few seconds. In positive mode, internal
calibration was performed with tryptic peptides coming from autodigestion of
trypsin, with respectively monoisotopic masses at m/z = 842.510 and m/z =
2211.105. Monoisotopic peptide masses were assigned and used for databases
searches. For the mass measurements, up to 1 missed tryptic cleavage and
optional methionine oxidation were considered. In most cases, the mass accuracy
was less than 50 ppm, a value which is generally considered adequate for
achieving statistically significant results for protein identification. These
files were then fed into the search engine MASCOT (Matrix Science, London, UK).
The data were searched against NCBI nonredundant protein sequence database.


### 2.6. Immunohistochemistry (IHC)

IHC analysis ofα-B crystallin was performed on formalin-fixed,
paraffin-embedded sections of IDCA breast tumors. Standard indirect
immunoperoxidase procedures were used for detection of α-B crystallin. Briefly,
five-micrometer sections were deparaffinized in toluene and subsequently
hydrated with graded ethanol, and rehydrated in water. Slides were microwaved
in 0.01 M citrate buffer at pH 6.0 for 20 minutes at 750 W. Thereafter, sections were
rinsed thoroughly with water and placed in a Tris–buffered saline
(TBS) solution (0.05 M Tris-HCl pH 7.6, 1.15 M NaCl). Endogenous peroxidase activity was blocked with hydrogen peroxide
solution for 7 minutes and sections were rinsed gently with TBS. Sections were
then incubated at 4∘C overnight with the antibody against α-B crystallin  (AB1546, Chemicon) at a dilution of 1 : 1000.
After rinsing in TBS, sections were incubated with the secondary antibody,
DakoCytomation EnVision+Dual Link System Peroxydase (DakoCytomation, Glostrup, Denmark),
for 30 minutes. Sections were washed 2 times with TBS, followed by application
of the diaminobenzidine substrate pack according to the manufacturer’s
instructions (DakoCytomation), yielding a brown-colored signal. Finally, tissue
sections were counterstained with hematoxylin and mounted. Standardization of
the incubation and development times allowed an accurate comparison of
expression levels in all cases. IHC staining was evaluated by two independent
pathologists for staining intensity. Cases were considered positive if 10% or
more of the tumor cells showed unequivocal immunoreactivity.


## 2.1. Sample Processing

Breast
tumor samples(n=10) were selected from patients living in the middle coast of Tunisia
(median
age at diagnosis of 50 years) and classified as infiltrating ductal carcinomas.
Breast tumors were obtained with informed consent according to protocols
approved by the institutional review boards of the respective hospitals.
Carcinomas were pathologically staged according to the TNM (Tumors, Nodes,
Metastasis) classification system of UICC [10]. Histological grade was
assessed according to the modified SBR grading system [11]. All the tumors
investigated were classified as T2/N0 and the grade of the tumors was G3. Breast
tumors and surrounding histologically normal tissues were collected at the time
of surgery and analyzed by histopathology. Areas of tumor were chosen after
assessment by cryostat sectioning. The first, middle, and last sections were
stained with hematoxylin and eosin, and examined by a pathologist to ensure
that at least 70% of the tissue sections used for the proteomic study contained
tumor cells. The nontumor tissues were confirmed by cryostat section before
protein extraction. For
proteomic analyses, tissue sections verified histologically were suspended and
homogenized in  250 μL of 2-DE
analysis buffer: 7 M urea,
2 M thiourea, 4% CHAPS, 0.5% DTT, and 2% ampholytes (1 part pH 3/10, 1 part pH
5/7, 2 parts pH 6/8). The clear supernatant issued after centrifugation of the
homogenate at 12500 g for 15 minutes was transferred to a sterile microcentrifuge tube and
immediately subjected to 2-DE analysis.


## 2.2. Nonequilibrium pH Gradient Electrophoresis, Isoelectrofocalization and SDS-PAGE

Protein contents were determined according to the procedure described by Bradford and modified by Ramagli and Rodriguez [12]. Bovine serum albumin (Fraction V, Sigma) was used as a standard. Analytical 2-DE was carried out in a BioRad system (Miniprotean II). Equal amounts of proteins (100 μg) issued from tumor or nontumor breast tissues were applied to the first dimension and at least three NEPHGE gels were run for each sample. NEPHGE has been used to resolve basic proteins as described earlier [8]. IEF was performed as described previously [9]. For each experiment, extraction of proteins, solubilization, NEPHGE, and SDS-PAGE were carried under similar conditions for the tumor and nontumor tissues. Electrophoresis was performed in triplicate for each pair of samples to ensure reproducibility. The alterations reported in the current study were retained after 2-DE analysis of protein extracts issued respectively from different frozen
sections of the 10 tumor and nontumor samples investigated. The reproducibility was about 85% or more which reflected the small variances between samples or running.


## 2.3. Gel Staining

After separation, the proteins were visualized by a sensitive colloidal Coomassie G250 stain [13]. The dye solution contained 17% (w/v) ammonium sulfate, 3% (v/v) phosphoric acid, 0,1% (w/v) Coomassie G250, and 34% (v/v) methanol. The staining solution was changed once after 12 hours staining and the gel slabs subjected to a 24-hour cycle for increasing dye deposition on low-abundance proteins. The detection was then increased by placing the gel into 1% v/v acetic acid for producing a better contrast between spots and gel. Coomassie stained gels were scanned and spot detection and quantification were carried out using PDQuest 2-D software version 7.2 (Biorad). Proteins with significant and reproducible changes between tumor and nontumor tissues were considered as differentially expressed. The abundance of each protein was estimated by the percentage volume. The individual spot volumes were normalized by dividing their optical density (OD) volumes by the total OD values of all the spots present in the gel and expressed as % vol. The significance of expression between tumor and nontumor tissues was estimated by student’s t-test, P<.05.


## 2.4. Spot Picking and in-Gel Digestion

Picked
spots were washed with 100μL of 25 mM NH4HCO3 and dehydrated with 100 μL of acetonitrile (ACN). This operation was repeated twice and the pieces of
gel were dried under vacuum for 10 minutes. Reduction was achieved by 1-hour
treatment with 10 mM DTT in NH4HCO3 buffer (100 μL) at 56∘C. After discarding the DTT
solution, alkylation reaction was performed by the addition of 100 μL of 25 mM iodoacetamide in 25 mM NH4HCO3 buffer for 1 hour at  room temperature, protected from light.
Finally, the excised gel pieces were again washed 3 times for 5 minutes with 25 mM NH4HCO3 and ACN alternately. Gel pieces were completely dried under vacuum before
tryptic digestion. The dried gel volume was evaluated (about 1 to 2 μL) and 3 volumes
of trypsin (Promega, V5111), 12.5 ng/μL, in 25 mM NH4HCO3 buffer (freshly diluted), were added. The digestion was performed at room
temperature overnight. Afterwards, 5 μL of 35% H2O/60% ACN/5% HCOOH
were added and the mixture is sonicated for 30 minutes and centrifuged in order
to extract tryptic peptides.


## 2.5. Protein Identification by MALDI-TOF-MS

Mass
measurements were carried out on a BIFLEX III MALDI-TOF
(Bruker, Daltonics, Bremen, Ge) equipped with the SCOUT high-resolution optics
with X-Y multisample probe and gridless reflector. This instrument was used at
a maximum accelerating potential of 19 kV (in positive mode) and was operated
in reflector mode. A saturated solution ofα-cyano-4-hydroxycinnamic acid
(Sigma, Saint Louis, MO) in acetone was used as a matrix. A first
layer of fine matrix crystals was obtained by the spreading and fast
evaporation of 0.5 μL of matrix solution. On this fine layer of crystals, a
droplet of 0.5 μL of aqueous HCOOH (5%) solution was deposited. Afterwards, 0.5 μL tryptic digest were added and mixed to a second dried under vacuum. The
sample was washed once by applying 0.7 μL of aqueous HCOOH (5%) solution on the
target and then flushed after a few seconds. In positive mode, internal
calibration was performed with tryptic peptides coming from autodigestion of
trypsin, with respectively monoisotopic masses at m/z = 842.510 and m/z =
2211.105. Monoisotopic peptide masses were assigned and used for databases
searches. For the mass measurements, up to 1 missed tryptic cleavage and
optional methionine oxidation were considered. In most cases, the mass accuracy
was less than 50 ppm, a value which is generally considered adequate for
achieving statistically significant results for protein identification. These
files were then fed into the search engine MASCOT (Matrix Science, London, UK).
The data were searched against NCBI nonredundant protein sequence database.


## 2.6. Immunohistochemistry (IHC)

IHC analysis ofα-B crystallin was performed on formalin-fixed,
paraffin-embedded sections of IDCA breast tumors. Standard indirect
immunoperoxidase procedures were used for detection of α-B crystallin. Briefly,
five-micrometer sections were deparaffinized in toluene and subsequently
hydrated with graded ethanol, and rehydrated in water. Slides were microwaved
in 0.01 M citrate buffer at pH 6.0 for 20 minutes at 750 W. Thereafter, sections were
rinsed thoroughly with water and placed in a Tris–buffered saline
(TBS) solution (0.05 M Tris-HCl pH 7.6, 1.15 M NaCl). Endogenous peroxidase activity was blocked with hydrogen peroxide
solution for 7 minutes and sections were rinsed gently with TBS. Sections were
then incubated at 4∘C overnight with the antibody against α-B crystallin  (AB1546, Chemicon) at a dilution of 1 : 1000.
After rinsing in TBS, sections were incubated with the secondary antibody,
DakoCytomation EnVision+Dual Link System Peroxydase (DakoCytomation, Glostrup, Denmark),
for 30 minutes. Sections were washed 2 times with TBS, followed by application
of the diaminobenzidine substrate pack according to the manufacturer’s
instructions (DakoCytomation), yielding a brown-colored signal. Finally, tissue
sections were counterstained with hematoxylin and mounted. Standardization of
the incubation and development times allowed an accurate comparison of
expression levels in all cases. IHC staining was evaluated by two independent
pathologists for staining intensity. Cases were considered positive if 10% or
more of the tumor cells showed unequivocal immunoreactivity.


## 3. Results

In
the current study, we have used an NEPHGE-2-DE protocol for the analyses of
basic proteins, and using this procedure established an NEPHGE-2-DE map of
proteins altered in IDCAs of the breast. Since NEPHGE is stopped before
equilibrium is attained, accurate experimental estimation of the pIs of the
dysregulated proteins cannot be made by this method. Table1 depicts the
theoretical isoelectric points (pIs) and molecular weights of altered proteins
in tumor tissues, many of which are basic. In our hands, while NEPHGE-2-DE
allowed the resolution of basic proteins, IEF-2-DE resulted as expected in the
stacking of most of basic proteins at the basic end, even if the IEF pH
gradient has been extended to higher pH values.Table 1
Results of
MALDI-TOF mass spectra and database searching for protein identification.
Identified
proteinsSpot(a) numberMr(kDa)/ pI(b)Access  N°(c)Peptides
matchedRelative(d)  volume (%)Mass
accuracy
(ppm)Sequence
coverage %Reported
functionNTComplement
component C3c
(chainD)171317/6.8278101272360.085±0.0150.02±0.0074564Immune responseIGHG1
protein245286/8.634489062390.121±0.0010.021±0.0084227Immune responseIGHG1
protein345286/8.634489062390.142±0.0080.041±0.0064329Immune responseIGHG1
protein445286/8.634489062370.12±0.0040.019±0.0095024Immune responseIGHG1
protein545286/8.634489062370.101±0.0060.014±0.0055024Immune
responseIGHG1
protein645286/8.634489062370.095±0.0070.017±0.0065024Immune responseCarbonic
anhydrase 1728909/6.594502517130.081±0.010.018±0.0097272metabolic enzymeEnolase1847481/7.014503571300.012±0.0010.131±0.0064162Glycolytic
enzymephosphoglycerate
kinase 1944973/8.3048145549280.015±0.0070.108±0.036058Glycolytic
enzymeDeoxy
hemoglobin
(ChainD)1015928/7.1427574253110.018±0.0040.123±0.0064979O2/CO2
transportDeoxy
hemoglobin
(ChainD)1115928/7.1427574253110.022±0.0020.131±0.0024980O2/CO2
transportManganese
superoxide dismutase1222290/6.86278081990.019±0.0060.075±0.0015057Anti-oxidant
enzymeAlpha
B-Crystallin1320146/6.7613937813110.022±0.0010.097±0.0145756ChaperoneRaf
Kinase
inhibitor protein1416068/8,81426193490.019±0.0080.132±0.0154554Signaltrans-
ductionPeptidyl-
prolylisomerase
A1518228/7,6813937981130.02±0.0070.109±0.0016759Immuno-
philinPeptidyl-
prolylisomerase
A1618228/7,6813937981140.011±0.0040.102±0.0056760Immuno- philinCofilin11718719/8.221514736980.018±0.0080.112±0.0123545ActinturnoverHSP271822427/7.83662841120.021±0.0010.117±0.0034851ChaperoneHSP271922427/7.83662841160.014±0.0080.097±0.0054867ChaperonehnRNPA2/B1
isoform A22036041/8.674504447210.015±0.0070.141±0.0213553Several functions(a)Index in the reference gel (Figure 1).(b)Expected
Mr and pI which were calculated on the complete sequences.(c)Accession
number in NCBI.(d)The individual spot volumes were expressed as a percentage of the total volume
in all of the spots present in the gel. N: nontumor tissue; T: tumor tissue.In
our hands, a comparison of the 2-DE protein patterns obtained at constant
voltages of 400 V for 3 hours (1200 V-Hr) or 4 hours (1600 V-Hr) showed an
optimal resolution of basic proteins at 1200 V-Hr while preventing basic proteins
from running off the basic end of the gels. By using the protein extraction
procedure listed in Section2, an average amount of 100 μg of total proteins
from each sample was used in 2-DE investigations. Figure 1 displays electrophoresis
patterns of proteins from tumor and nontumor samples subjected to NEPHGE-2-DE
electrophoresis. By using this procedure, we have found that the majority of
the proteins displayed were not different in abundance between normal and
cancer tissues. Protein alterations occurring in the tumor and normal tissues
are highlighted with arrows (Figure 1). Among the proteins identified, the
levels of 13 proteins were increased in the tumor samples, while 7 proteins
were rather observed at higher amounts in the nonneoplastic breast tissues. These
alterations were regarded as serious alterations occuring between tumor and
nontumor tissues. Altered proteins were subsequently excised and subjected to
in-gel tryptic digestion and MALDI-TOF analyses. This procedure allowed the
unambiguous identification of the proteins investigated. Figure 2 shows an
example of MALDI-TOF peptide mass fingerprints of the tryptic digests
corresponding to alpha B-crystallin. Table 1 lists all of the proteins
identified. A wealth of evidence suggests that most of these dysregulated
proteins are closely related to neoplastic transformation. These proteins could
be classified on the basis of their functions into stress-related, chaperoning
proteins, cytoskeletal-related proteins, metabolic enzymes, and other molecules
related to immunological responses. Some of these alterations depicted in
Figure 1 involve co-upregulations of glycolytic enzymes (enolase1, spot 8;
phosphoglycerate kinase 1, spot 9) in tumor tissues, as well as, proteins
involved in antioxidation pathways including peptidylprolyl isomerase A (spots
15, 16), which has been reported to protect cells against oxidative stress and
manganese superoxyde dismutase (Mn-SOD, spot 12). Two other newly identified
proteins related to immunologic responses present at lower levels in tumor
tissues include complement component C3c (spot 1) and IGHG1 (spots 2–6).Figure 1
2-DE pattern of proteins from tumor and
nontumor samples resolved by NEPHGE 2-DE and listed in Table1. Proteins
dysregulated in tumor tissues (T) as compared to nontumor tissues (N) are
highlighted with arrows. The numbers indicated correspond to those given in
Table 1. (1)–(7): proteins downregulated
in tumor tissues; (8)–(20): proteins
upregulated in tumor tissues. (1) complement component C3c; (2)–(6) IGHG1 protein
isoforms; (7) carbonic anhydrase 1; (8) enolase 1; (9) phosphoglycerate kinase
1 (PGK1); (10), (11) hemoglobin; (12) MnSOD; (13)  α-B crystallin; (14) Raf kinase inhibitor
protein; (15),    (16) peptidylprolylisomerase A (PPIA); (17) cofilin 1; (18), (19)
HSP27; (20) hnRNP A2/B1.Figure 2
MALDI-TOF peptide mass fingerprints of the tryptic
digests corresponding to alpha B-crystallin. The matched peptide sequences are
underlined.More interestingly,
the levels of Raf kinase inhibitor protein (spot 14) were found to be
overexpressed in cancer tissues indicating a possible special pathological role
for this protein in breast carcinogenesis. Other alterations more represented
with respect to the IDCA tissues include heat shock proteins (HSP27, spots 18,
19) andα-B-crystallin (spot 13). Subsequent immunohistochemical staining
against α-B-crystallin has also been performed (Figure 3). Expression of α-B-crystallin
was noted in all cases investigated and positivity has been retrieved in 80 to
90% of tumor cells. The signals detected in tumor cells were strong whereas
epithelial cells of the normal tissues express lower levels of this protein.Overexpression ofα-B crystallin in infiltrating ductal carcinoma tissues of
the breast. Formalin-fixed, paraffin-embedded sections were immunostained with
rabbit polyclonal antibody against α-B crystallin. Tissues were stained with
hematoxylin and viewed by light microscopy. A strong staining (brown) in tumor
cells (T) has been observed, whereas α-B crystallin was expressed at lower
levels in normal epithelial cells. (a) α-B crystallin expression in IDCA
tissues (200x magnification), (b)-(c) α-B crystallin expression in IDCA (400x
magnification), (d) α-B crystallin expression in nontumor tissues (400x
magnification).
(a)(b)(c)(d)


## 4. Discussion

Proteomics-based
studies are increasingly used to examine cancer-related changes in protein
expression. Such protein alterations may eventually contribute to increase our
understanding of the disease pathogenesis as well as in the development of
effective strategies for diagnosis and treatment. The present study aims to
investigate protein alterations in IDCAs of the breast using an NEPHGE-2-DE
procedure. As shown in this comparative analysis, certain specific proteins
were dysregulated in IDCAs compared with their paired normal samples. These
altered proteins were subsequently analyzed by mass spectrometry and unambiguously
assigned on the basis of MALDI-TOF spectra obtained after trypsin digestion
(Table1). The proteins identified span a wide range of functions and may have
a value as diagnostic and prognostic biomarkers for breast cancer. Among the
proteins identified, we established that there is a decreased expression of IGG1
and complement component C3c proteins in breast-tumor tissues. These
alterations related to immunologic responses constitute novel identifications
that were not previously reported as potential markers of IDCA breast tumors. As
shown in Figure 1, the IGG1 proteins were observed as multiple protein spots with different isoelectric points and similar molecular weights. As displayed
in NEPHGE-2-DE gels, these proteins appeared intact as judged by their apparent
molecular mass and were closely associated with nonmalignant tissues.Consistent
with our findings, several studies have shown that malignant diseases of
various tissue origin are associated with a shift in the IGG subclass
distribution. For example, the decrease of IGG1 serum levels reported in
colorectal cancer has been considered as an early tissue nonspecific indicator
of malignant transformation that could be useful in the primary diagnosis of
colorectal tumors [14]. Such alterations were recently reported to occur in
colorectal adenocarcinomas and normal colorectal tissues using suppression
subtractive hybridization techniques [15]. A highly reduction of the percent of
IGG1 in several gynecologic malignant diseases has also been correlated with
early carcinoma stages and was found as a reliable marker that discriminates
between cancer patients and controls with a specificity ranging between 90 and
100% [16]. The reduction in IGG1 plasmatic levels has also been reported as a
highly sensitive tumor marker for detecting primary and metastatic
squamous-cell carcinomas of the head and neck [17], and was significantly
correlated with early stages of invasive breast cancer [18]. Up to now, such
alterations in IGG1 levels were found with neoplastic diseases only and may
likely reflect changes in the host’s immune system linked to the presence of
malignant tumors. Recent data unveiled that malignant tumors of various tissues
lead to irregularity of the biosynthesis of IGG1 and IGG2 by affecting their
messenger RNA levels in B-cell lymphocytes [19]. It has been hypothesized in
this context that cancer cells may affect the synthesis of  such IGG subclasses by acting on the T-helper
1/T-helper 2 ratio in the host, and, consequently, affect the level of
specific cell-derived cytokines implicated in human IGG synthesis [20]. Our
observation of underexpression of IGG1 in IDCAs with regard to nontumor tissues
may therefore, indicate a similar tumor-associated mechanism by which cancer
cells affect IGG1 synthesis. Further investigations are needed however, to
elucidate how malignant breast tissues may interfere with the T-cell mediated
regulation of IGG suclasses. Interestingly, downregualtion of IGG1 levels and
concomitant downregulation of complement component C3c were observed in our
results. To our knowledge, no studies have yet reported the decreased
expression of this protein in IDCAs of the breast. The differential tissue
expression of this complement protein provides evidence for the involvement of
anti-inflammatory processes in IDCA tissues and may indicate a selective
adaptation by breast tumors to escape elimination by antitumor immune
responses. Until recently, several studies with normal and malignant tissues
have disclosed the presence of several immune escape strategies by which cancer
cells protect themselves from the lytic action mediated by complement. Several
evasion strategies to complement attack have been reported in malignant cells
and include complement protective proteins (CD46, CD55, CD59), membrane
proteases reported to confer complement resistance by removing cell-bound
complement components such as C3 and tumor secreted complement inhibitors [21].
Such inhibitory mechanisms, often upregulated in tumors, are thought to
increase resistance to complement in malignant tissues through inhibition of
serum complement activation upon cancer cells. A previous study using a rat
model of human breast cancer unveiled that inhibitors of complement activation
may facilitate tumor growth and enhance tumorigenicity of breast-cancer cells
by inhibiting C3 deposition [22]. The present downregulation of complement C3
component C3c in IDCAs may, therefore, indicate the involvement in breast
tumors of similar complement inhibitory mechanisms.The
Raf
kinase inhibitor protein (RKIP) known also as phosphatidylethanolamine binding
protein-1 (PEBP-1) is another basic protein that has been described as being a
component of the important feedback inhibition in G-protein coupled signal
transduction [23]. It is also involved in the regulation of the NF-kappa B
pathway and is associated with resistance to TNF-α by inhibiting activation of
the Raf 1/MEK/extracellular signal regulated kinase pathway, JNK, and
phosphatidylethanolamine (PE) externalization, which is an early symptom of
apoptotic cell death resulting in the total loss of asymmetric distribution of
aminophospholipids in the plasma membrane bilayer [24, 25]. It has also been
reported that overexpressing a new member of the PEBP family (PEBP-4) correlates
with both a decreased TNF-α-induced PE externalization and
resistance to TNF-α-induced apoptosis [26].
Interestingly, recent data revealed that silencing of PEBP-4 expression in
MCF-7 breast-cancer cells promotes TNF-α-induced apoptosis, as well as TNF-α-induced
ERK and JNK activation making this PE binding protein as a promising target for
the treatment of breast cancer [27]. Overall, the present findings in IDCAs of
the breast might reflect the previously reported results for the relation
between PE-binding proteins and TNF-related apoptosis pathway. The present
increase in RKIP may confer breast-cancer cells to an advantage for survival
and proliferation.From
our results, the high abundance of hemoglobin in breast tumors could not be
considered as a reliable parameter for distinguishing between tumor and
nontumor tissues. Indeed, the relationship between tissue hemoglobin levels and
breast tumors appears indirect and indicates rather an enhanced blood supply to
the tumor. Since tumor angiogenesis is a crucial arbiter of breast-cancer
growth/metastasis, this higher level of hemoglobin is consistent with the
higher vascular density of growing tumors compared with normal tissues.Besides these alterations, the
concomitant
upregulation of two other stress-related proteins (peptidylprolylisomerase
A, MnSOD) was observed in IDCAs. The peptidylprolylisomerase A also called
cyclophilinA (CypA) is described as being involved in protein folding and
catalyzes the cis/trans-isomerization of peptide bonds preceding a proline
residue. It is secreted by cells in response to both inflammatory stimulation and
oxidative stress [28]. Our observation on the expression of CypA is consistent
with the published results from studies on other cancers. Overexpression of
CypA has been reported in colorectal carcinomas in comparison to normal mucosa
and is involved in lung-tumor growth by affecting proliferation and apoptosis
[29]. A recent study has shown that both CypA and its receptor CD147 are upregulated
in human pancreatic adenocarcinomas compared to normal pancreatic ductal epithelium
[30]. These authors pointed that CypA is a promoter of pancreatic tumor growth
and is involved in chemotaxis and cell signalling cascades. The present finding
reveals a similar correlation, implying that breast carcinomas may share a
similar mechanism of cell proliferation mediated by CypA. The MnSOD is a
mitochondrial enzyme that has been reported to protect cells against oxidative
stress by increasing the dismutation rate of superoxide anion (O2−)
to hydrogen peroxide (H2O2) which is then converted into water by catalase and
glutathione peroxidase. The role of this antioxidant enzyme in carcinogenesis
is still however controversial and unclear. In fact although, it has been
reported to suppress apoptosis and protect cells against several insults, under
some circumstances, the MnSOD may prevent cell proliferation [31]. Although
further studies are needed, the present elevation of MnSOD may indicate that
the antioxidant defense system has been stimulated in invasive carcinomas of
the breast, highlighting the ability of tumor cells to prevent damage due to
reactive oxygen species.Interestingly, the
co-upregulation of two glycolytic enzymes, enolase 1 and phosphoglycerate
kinase 1 (PGK-1) also occurred in IDCA tissues. These enzymes are key
components of the glycolytic pathway that have shown a prior relationship to
cancer. Indeed, our observation of increased expression of enolase 1 and PGK-1
is supported in various tumor tissues and may relate to the increased requirement
of both energy and protein synthesis/degradation pathways during carcinogenesis
[32]. This increased glycolytic metabolism is also thought to regulate
hypoxia-inducible gene expression in solid tumor tissues and participates in
angiogenesis [33].The protein cofilin-1 also was
identified as differentially expressed between tumor and normal tissues. This
protein is recruited at the leading edge of cells where its activation is
essential for polarized migration. Cofilin-1 is directly implicated in severing
actin cytoskeleton filaments and regulates the polymerization/depolymerization
of actin during cell migration [34]. It has been recently demonstrated that
cofilin expression is required for tumor cell invasion since inhibition of
cofilin activity in carcinoma cells inhibits cell motility whereas increased
expression correlates with enhanced cell migration [35]. More recently, Wang et
al. [34] have demonstrated that overexpression of cofilin-1 is necessary for
the activation of the cofilin pathway resulting in cell motility, invasion and
metastasis. This protein has also been associated with melanoma cell invasion
and migration via its interaction with the αvβ3 integrin [36]. Overall, the
current findings support the recently identified role for cofilin activity and highlight
its involvement in invasive lesions of the breast as a determinant factor
required for cell migration and invasion.The molecular chaperone HSP27
and alpha B-crystallin, which is a small heat shock protein (HSP) are two other
dysregulated proteins in tumor tissues. In a previous investigation, we have
reported that three other molecular chaperones (GRP78, calreticulin, protein
disulfide isomerase) are differentially expressed in breast tumors [9]. The
concomitant upregulation of these HSPs together with alpha B-crystallin and
HSP27 is not surprising since chaperones are thought to work cooperatively to
fulfil their functions [37]. Due to the capacity of HSPs to prevent
stress-accumulated, unfolded, and nascent protein aggregation, their expression
has proven to have important pathological implications such as cell
proliferation and disease prognosis [4]. The HSP27 is a molecular chaperone
whose rate of synthesis increases many folds in response to environmental
stress and during malignant transformation [38]. As shown in
Figure 1, this
protein exists as two distinct isoforms that were confirmed by mass
spectrometry as HSP27 (Table 1). Although no evidence of posttranslational
alterations was pointed out, these isoforms as reported in renal cell
carcinomas might reflect phosphorylation or other posttranslational modifications
[39]. In the last years, the role of alpha B-crystallin in cancer pathology has
been widely discussed with regard to its potential oncogenic role. Previous
studies unveiled that this small HSP may constitute a good target for
modulating cell death pathways [40]. Its expression has been shown to inhibit
both the mitochondrial and the receptor death activation pathways of caspase 3
[41] and correlates with TRAIL resistance in a panel of cancer cell lines [42].
This protein may also be an interesting molecular target for exploring the
evolution and the origin of breast tumors since higher alpha B-crystallin
levels were reported in ductal carcinoma in situ, which is an earliest form of
detectable breast cancer [5]. The data reported herein appear to confirm this for
invasive carcinomas as well. Although further studies are needed to answer how
this oncoprotein contributes to breast cancer, the data reported herein
highlight the importance of this molecular chaperone in invasive carcinomas as
a biomarker that may play a distinctive role in the process of
carcinogenesis.


## 5. Conclusion

In summary, our data have
shown the value of using an NEPHGE 2-DE based proteomic approach in identifying
potential molecular alterations and targets for breast-cancer therapeutics and
diagnostics. We have identified a number of tumor-associated proteins including
chaperones, energy-producing enzymes, stress-related proteins, proteins
involved in apoptosis and cell motility, as well as alterations highlighting
changes in the host’s immune system linked to the presence of malignant
tissues. These protein variations indicate that multiple cellular pathways are
involved in the process of breast carcinogenesis. Such alterations may help
to better understand the pathology of this disease and may have potential
clinical applications as cancer biomarkers.



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*Source: 564127-2008-03-26.xml*