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Endocrine-Related Cancer (2008) 15 1099–1114
Protein western array analysis in humanpituitary tumours: insights and limitations
Antonio Ribeiro-Oliveira Jr1,2, Giulia Franchi1,3, Blerina Kola1,Paolo Dalino1,4, Sergio Veloso Brant Pinheiro 2, Nabila Salahuddin1,Madalina Musat1, Miklos I Goth5, Sandor Czirjak 6, Zoltan Hanzely 6,Deivid Augusto da Silva 2, Eduardo Paulino Jr 2, Ashley B Grossman1
and Marta Korbonits1
1Department of Endocrinology, Barts and the London School of Medicine and Dentistry, London EC1M 6BQ, UK2Department of Internal Medicine, Pediatrics and Pathology of Federal University of Minas Gerais, 30330-120 Belo Horizonte/Minas
Gerais, Brazil3Endocrinology Unit, ‘Vita-Salute’ San Raffaele University Hospital, via Olgettina 48, 20132 Milan, Italy4Endocrinology Unit, Department of Endocrinology, Niguarda Ca’ Granda Hospital, Piazza Ospedale Maggiore, 3, 20162 Milan, Italy5Endocrinology Unit, Internal Medicine, National Medical Center, 1135 Budapest, Hungary6Endocrinology Unit, National Institute of Neurosurgery, 1143 Budapest, Hungary
(Correspondence should be addressed to A Ribeiro-Oliveira Jr who is now at Laboratorio de Pesquisas em Endocrinologia, Federal
University of Minas Gerais-Brasil, Avenida Alfredo Balena, 190, sala 151, Belo Horizonte, Brazil; Email: [email protected])
Abstract
The molecular analysis of pituitary tumours has received a great deal of attention, although themajority of studies have concentrated on the genome and the transcriptome. We aimed to study theproteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was usedto study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas(NFPAs). Individual protein expression levels in the tumours were compared with the expressionprofile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples,116 proteins had not previously been described inhumanpituitary tissue.Four prominent differentiallyexpressed proteins with potential importance to tumorigenesis were chosen for validation byimmunohistochemistry and western blotting. In the protein array analysis heat shock protein 110(HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potentialregulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, whileC-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. Theimmunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptorand underexpression of CSK and annexin II in pituitary adenoma cells when compared with theircorresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data:HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptorwas significantly overexpressed in prolactinomas, annexin II was significantly underexpressedin somatotrophinomas, while CSK did not show significant underexpression in any tumour. Proteinexpression analysis of pituitary samples disclosed both novel proteins and putative proteincandidates for pituitary tumorigenesis, though validation using conventional techniques arenecessary to confirm the protein array data.
Endocrine-Related Cancer (2008) 15 1099–1114
Introduction
Pituitary tumours are very common neoplasms, with a
reported prevalence of 17% in the general population,
and they constitute 10% of intracranial neoplasms (Asa
&Ezzat 2002, Ezzat et al. 2004).Although these tumours
Endocrine-Related Cancer (2008) 15 1099–1114
1351–0088/08/015–001099 q 2008 Society for Endocrinology Printed in Gr
cause considerable morbidity and mortality due to their
hormonal and local space-occupying effects, they only
very rarely transform into true metastatic cancers
(Kaltsas et al. 2005). Pituitary adenomas therefore offer
an important model to study how cell proliferation is
eat Britain
DOI: 10.1677/ERC-08-0003
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A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
regulated, and how dysregulation may be compatible
with adenoma transformation.
Studies over recent years have established that
sporadic pituitary tumours rarely show somatic
mutational changes in genes implicated in genetic
pituitary tumour syndromes: menin (in multiple
endocrine neoplasia-1 (MEN 1) syndrome (Chandra-
sekharappa et al. 1997)), CDKN1B (causing extremely
rarely the MEN1 syndrome (Pellegata et al. 2006,
Georgitsi et al. 2007)), protein kinase A regulatory
subunit-1A (PRKAR1A, in a proportion of Carney
complex cases (Kirschner et al. 2000)) and AIP (aryl
hydocarbon receptor-interacting protein, causing a
proportion of familial isolated pituitary adenoma
cases (Vierimaa et al. 2006, Daly et al. 2007, Leontiou
et al. 2008)). In terms of mRNA expression, MEN1,
CDKN1B and PRKAR1A mRNA expression is not
changed in sporadic pituitary adenomas (Dahia et al.
1998, Satta et al. 1999, Kaltsas et al. 2002) while AIP
mRNA is up-regulated rather than down-regulated in
sporadic pituitary adenomas (Leontiou et al. 2008). In
terms of protein expression, MEN1 and PRKAR1A
show no change while CDKN1B protein is down-
regulated in pituitary adenomas, especially in cortico-
trophinomas (Lidhar et al. 1999, Lloyd et al. 2002)
and, again contrary to expectation, AIP protein is
up-regulated in sporadic pituitary adenomas (Leontiou
et al. 2008). Transgenic animal models suggest that
changes in the regulation of the cell cycle are likely to
be important in pituitary oncogenesis (Fero et al.
1996), although it is probable that many of these
changes are secondary to more proximal abnormalities
in cell signalling pathways. Therefore, it has been of
benefit to explore the phenotypic and genotypic
changes seen in pituitary adenomas as compared with
normal human pituitary tissue in order to identify the
dysregulated pathway(s).
Several dysregulated genes and proteins are
expected to occur in different pituitary tumours, and
we and others have recently published analyses of
pituitary tumour mRNA using microarray technology
(Zhan et al. 2003, Moreno et al. 2005, Morris et al.
2005). However, it is well known that a number of cell
cycle proteins in particular are regulated at the protein
level, and it is probable that mRNA analysis would not
detect such changes. We have chosen to investigate the
level of protein expression in pituitary tumours with a
protein array using pituitary adenomas, and compared
them with normal pituitary protein expression patterns,
in order to identify novel expressed proteins in human
pituitary tissue and to search for putative proteins
involved in pituitary tumorigenesis. Furthermore, we
used both immunohistochemistry and immunoblotting
1100
to quantify protein expression individually on a subset
of these proteins in order to attempt validation of the
findings. Our results confirm that protein array may be
a useful approach to identify novel candidate proteins,
but the results obtained must be confirmed by more
established techniques. This in turn suggests that the
results of proteomic analysis must always be
considered in the light of the other data.
Subjects and methods
Tissue specimens
Samples from pituitary adenomas were removed at
transsphenoidal surgery and frozen immediately.
Autopsy normal pituitary samples were collected
within 24 h of death from patients with no evidence
of any endocrine abnormality. For the BD PowerBlot
Western array (BD Biosciences Pharmingen, La Jolla,
CA, USA), a total of 20 human pituitary adenomas
were selected, representative of each of the main
adenoma subtypes (growth hormone-(GH), prolactin-
(PRL) and corticotrophin (ACTH)-secreting tumours
and non-functioning pituitary adenomas (NFPAs)),
whilst 5 normal pituitaries were used as controls. For
the immunohistochemistry, three GH-, three PRL-,
three ACTH-secreting tumours, three NFPA and four
normal pituitary samples were studied. For the western
blotting, 10 GH-, 7 PRL-, 7 ACTH-secreting tumours,
10 NFPAs and 12 normal pituitary samples were
studied. The tumour type was determined on the basis
of clinical and biochemical findings before surgery,
and by morphological and immunohistochemical
analysis of the removed tissue samples, as described
previously (Lidhar et al. 1999). Informed consent was
obtained from all patients, and the protocol was
approved by the institutional Research Ethics
Committee.
Protein extraction and quantification
For the BD PowerBlot Western array, five samples for
each tumour subtype and five samples of normal
pituitary were combined in order to obtain a single
sample of 150 mg tissue for each diagnosis. Samples
were lysed and the protein concentration measured as
previously described (Musat et al. 2005).
BD powerblot western array
The BD PowerBlot was performed by BD Biosciences
Pharmingen utilising a high-resolution two-dimen-
sional electrophoresis as previously described (Phelan
& Nock 2003). Protein concentrations were quantified
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Endocrine-Related Cancer (2008) 15 1099–1114
and samples were run on six high-resolution 4–15%
gradient SDS–polyacrylamide gels in triplicate. Each
of the four subtypes of pituitary adenomas and the
normal pituitary tissue was studied using a single array.
After electrophoresis, the gels were transferred to
PVDF membranes. Each membrane was divided into
41 lanes by applying a chamber-forming grid and
probed with 1005 high-quality monoclonal antibodies
per run. After 1 h of incubation at room temperature,
the chambers were rinsed and incubated with single
fluorescent secondary antibody under the same
conditions (Alexa 680-labelled goat anti-mouse IgGs;
Molecular Probes, Eugene, OR, USA). Image data
were captured using the Odyssey Infrared Imaging
System (LI-COR). Molecular masses were determined
using molecular weight standards (BD Biosciences).
Samples were run in triplicate and analysed using a
3!3 matrix comparison method. For example, run 1 of
the control was compared with runs 1, 2 and 3 of the
experimental groups. Run 2 of the control was compared
with runs 1, 2 and 3 of the experimental groups. Run 3 of
the control was compared with runs 1, 2 and 3 of the
experimental groups – altogether 9 comparisons.
Comparative analysis (i.e. fold change in expression
level in treated versus control) followed, and protein
expression level changes were determined. Changes
were divided into 10 categories based on reproducibility
and signal intensity, with ‘level 10’ (the highest)
representing changes greater than twofold in all 9
comparisons that were from good quality signals and
passed a visual inspection. Only the proteins which were
expressed in pituitary samples and reached at least ‘level
1’ of confidence are reported (316 altogether from the
1005 tested antibodies).
We then filtered the data in order to identify the most
overexpressed or underexpressed proteins, correspond-
ing to thosewith good replicates (coefficient of variation
!10%) and a confidence level of 10 for each pituitary
adenoma subtype. We then selected four proteins with
prominent changes seen in all pituitary adenoma
subtypes, and with potential relevance to human
tumorigenesis, as putative protein candidates for further
study and confirmation using immunohistochemistry
and western blotting.
Immunohistochemistry
In order to study the distribution of the four selected
proteins (heat shock protein (HSP110), B2 bradykinin
receptor, CSK (C-terminal Src kinase) and annexin II),
and also to compare their presence in neoplastic cells with
their respective non-neoplastic cells, we utilised confocal
immunofluorescent analysis of the GH-, PRL- and
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ACTH-secreting adenomas and normal pituitary tissue,
or the streptavidin–biotin method (DakoCytomation Inc.,
Carpinteria, CA, USA) for the NFPAs. The clinical-
pathological diagnosis of all pituitary adenomas had been
confirmed using immunohistochemical determination of
the expressed pituitary hormones: for the adenoma
samples, all slides utilised showed more than 90% of
the specific tumour, as confirmed by haematoxylin and
eosin. Normal pituitary architecture was evaluated by
reticulin staining. For confocal immunofluorescent
analysis of GH-, PRL- and ACTH-secreting adenomas,
after deparaffinisation and hydrating procedures, the
sections were incubated in blocking solution (1% BSA
and 0.1%Tween 20) at room temperature for 1 h and then
incubated overnight at 4 8C with one of the primary
antibodies (purified mouse anti-HSP110 antibody
(1:200), anti-mouse B2 bradykinin receptor antibody
(1:200), goat polyclonal to CSK antibody (1:200) and
goat polyclonal to annexin II antibody (1:400)). In order
to colocalise the selected proteins in different non-
neoplastic cells, rabbit anti-GH (1:200), rabbit anti-
prolactin (1:200) or rabbit anti-ACTH (1:200), supplied
by Dako (Oxford, UK), were also added to the incubation
buffer used in normal pituitary samples. After an
overnight incubation, slides were rinsed five times in
PBS and then incubated for 1h in the dark at room
temperature with anti-mouse IgG-conjugated with FITC
(1:400, Jackson Immunoresearch,WestGrove,PA,USA)
or anti-goat conjugated with Cy5 (1:400, Jackson),
according to the primary antibody previously used. An
anti-rabbit conjugatedwithCy3 (1:400, Jackson)was also
added to the incubation buffer used on normal pituitary
samples. Following washes with PBS, sections were
mounted in 90%glycerol/10%TRIS1Mand imageswere
captured through confocal microscope (Zeiss LSM 510),
40! objective, and 400! original magnification. All
confocal settings were determined at the beginning of
imaging session and remained unchanged. For quan-
titative analysis, images were captured at eight bits and
analysed in grey scale. Three to four images were
captured from each sample and three measurements
were obtained for each image. ImageJ (NIH, Bethesda,
USA) software was used to quantify fluorescence
intensity and area. Background fluorescence was then
subtracted from the region of interest. The intensity of
fluorescence corresponded to the unit ‘grey level’,
varying from zero (black) to 255 (white), as an average
of the area (sum of grey value of all pixels divided by the
number of pixels/area).
For the non-functional pituitary adenomas, while it is
accepted that the majority of them are of gonadotrophic
origin, many do not express gonadotrophins uniformly
or may express only one subunit (follicle-stimulating
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A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
hormone, FSH, luteinizing hormone and/or human
chorionic gonadotrophin, HCG) rendering the double-
labelling technique difficult. The NFPA tumours were
therefore selected on the basis of their immunohisto-
chemical expression of FSH using monoclonal anti-
FSH (Novocastra, Newcastle-upon-Tyne, UK).
Conventional immunohistochemistry using consecu-
tive cuts was performed, as described previously
(Lidhar et al. 1999), and FSH staining positive areas
were compared between NFPA and normals. Sections
were assessed by a single observer blinded to the
diagnosis. In each section, at least 200 cells were
analysed. The percentage of positive cells was then
expressed as a ratio of positive cells to the total number
of cells counted. Photographs were taken with a Canon
PowerShot A530 colour video camera and processed in
Corel photo-Paint, version 12.0.0.458, 2003, Corel
Corporation.
Western blotting
Human pituitary adenoma tissue and normal pituitary
tissuewere studied for the assessment of the four selected
proteins: HSP110, B2 bradykinin receptor, CSK and
annexin II. In each case, 10 mg protein samples were
subjected to 10% SDS-PAGE separation, with protein
transfer to a PVDF membrane. The membrane was
blocked with 5% non-fat milk for 90 min and incubated
overnight at 4 8C with the primary antibody. The
membrane was washed six times with PBS containing
0.01–0.05% Tween and subsequently incubated for
90min at room temperature. A chemiluminescent
peroxidase substrate, ECL (Amersham-Pharmacia),
was applied according to themanufacturer’s instructions,
and the membranes were exposed briefly to X-ray film.
The PVDF membrane was then stripped with 0.985 g
Tris–Hydrochloride, 2 g SDS, 781 ml 2-mercaptoethanol
(pH 4), and reprobed. The antibodies and the respective
dilutions thatwere used are the following: purifiedmouse
anti-HSP110 antibody at 1:1000 (BD Biosciences
Pharmingen), mouse anti-B2 Bradykinin Receptor
antibody at 1:1000 (Fitzgerald, Concord, MA, USA),
polyclonal goat anti-CSK (1:500) and anti-annexin II
(1:200) antibody (Abcam,Cambridge,UK) and for equal
loading rabbit polyclonal anti-GAPDH antibody at
1:10 000 (Santa Cruz Biotechnology, Santa Cruz, CA,
USA). An anti-goat antibody at 1:5000, an anti-rabbit
antibody at 1:2000, and an anti-mouse antibody at 1:2000
Dakowere the secondary antibodies used. Densitometric
readings of the resulting bands were evaluated using the
ImageJ program, and the ratio toGAPDHwas calculated
and compared with controls.
1102
Statistical analysis
The data were tested for statistical significance with the
non-parametric Mann–Whitney test or multivariate
analysis using the Kruskal–Wallis test followed by
Conover–Inman analysis (StatsDirect, Ian Buchan,
Cambridge, UK). Significance was taken as P!0.05.
All values given are meansGS.E.M).
Results
The 1005 antibodies utilised in the BD PowerBlot
Western array detected a total of 316 proteins from the
human pituitary tissues. These proteins were cate-
gorised into different functional groups, according to
their areas of activity, and they are summarised in Fig. 1
and listed with their functional categories in Supple-
mentary Table 1, which can be viewed online at http://
erc.endocrinology-journals.org/supplemental/. Out of
these 316 proteins, we found through a literature survey
and interrogation of a human pituitary database (Zhan&
Desiderio 2003, Zhao et al. 2005a) that 116 proteins had
not previously been described as being expressed in
human pituitary tissue, and 81 of these have also not
been studied in the pituitary of any other species.
Expression profile of adenomas
The expression profiles for GH-, PRL-, ACTH-secreting
and non-functioning adenomas and NFPAs, and the
expression profile comparisons to normal autopsy
pituitary tissues, using the BD PowerBlot Western array,
were analysed. The main criteria used to select potential
candidate proteins were ‘level 10’ of confidence for all
pituitary adenoma subtypes. We also considered in this
selection process the consistency within the replicates in
the array, our previous microarray data (Morris et al.
2005), and thepossible relevanceof theproteins for human
tumorigenesis. Themost significant and consistently over-
and underexpressed ‘level 10’ proteins occurring in all
adenoma types studied are shown in Table 1. ‘Level 10’
proteins that were specific to only one or two adenoma
subtypes are shown in Table 2. Interestingly, there were
some ‘level 10’ proteins that were present only in certain
tumours and not present in normal pituitary tissue, while
others were present in normal pituitary tissue but not
present in certain tumours (Table 3).
In all pituitary adenomas subtypes, the number of
‘level 10’ overexpressed proteins was higher than
the number of ‘level 10’ underexpressed ones
(Table 4). However, the fold-change of underexpression
was higher for the underexpressed proteins when
compared with the fold change for the overexpressed
ones (Table 4).
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Figure 1 Functional categories of 316 characterised proteins covered by the protein microarray. Some proteins were categorised tomore than one functional group. The proteins with areas of research reaching less than 2% in the array were grouped to others(Calcium Signalling, Stats, Immunology, Nitric Oxide, Protein Phosphatase, Protein Sorting and Modification). See Supplementalmaterial Table 1, which can be viewed online at http://erc.endocrinology.org/supplemental/, for each individual protein.
Endocrine-Related Cancer (2008) 15 1099–1114
Identification of potential candidate proteins
We selected four proteins which showed significant
changes in all four adenoma subtypes, two being
overexpressed (HSP110 and B2 bradykinin receptor)
and two underexpressed (CSK and annexin II) for
further study . HSP 110 and C-terminal Src kinase had
not specifically been described before as part of the
human pituitary proteome database (Zhan & Desiderio
2003, Zhao et al. 2005a).
HSP110 showed an average of 17-fold overexpres-
sion in all adenoma subtypes as compared with normal
pituitary tissues (33-, 9-, 10- and 14-fold overexpres-
sion for GH-, PRL-, ACTH-secreting adenomas and
NFPAs respectively) in the protein array analysis. In
our previous pituitary microarray study HSP110 gene
expression showed underexpression although this was
not significant (Morris et al. 2005).
The B2 bradykinin receptor showed an average of
11-fold overexpression in all adenomas subtypes as
compared with normal pituitary tissues (16-, 20-, 5- and
4-fold overexpression for GH-, PRL-, ACTH-secreting
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adenomas and NFPAs respectively). No data from our
previousmicroarraywere available for the B2 bradykinin
receptor. Other studies have suggested a role for this
receptor in anteriorpituitary regulation (Sharif et al. 1988,
Kuan et al. 1990), and the importance of its up-regulation
in other human cancers has been emphasised (Ikeda et al.
2004, Dlamini & Bhoola 2005, Zhao et al. 2005b).
The CSK protein showed an average of 117-fold
underexpression in all adenoma subtypes as compared
with normal pituitary tissues (25-, 168-, 134- and 140-
fold underexpression for GH-, PRL-, ACTH-secreting
adenomas and NFPA respectively). Our previous
microarray data also showed reduced mRNA
expression in pituitary adenomas (Morris et al. 2005).
Finally, annexin II showed an average of 27-fold
underexpression in all adenoma subtypes as compared
with normal pituitary tissues (25-, 20-, 32- and 31-fold
underexpression for GH-, PRL-, ACTH-secreting ade-
nomas and NFPAs respectively). A significant level of
underexpression had also been shown in our previous
microarray data, where an average fourfold under-
expression was found (Morris et al. 2005).
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Table 1 Most consistently changing proteins in all adenoma subtypes
Average fold change for each adenoma
subtype
Protein Swiss-Prot ID GH PRL ACTH NFPA
Direction
of change
ARF3 (ADP-ribosylation factor 3) P16587 14 26 N/A 28 CAnnexin I P46193 12 13 31 24 K
Annexin II P07355 25 20 32 31 K
B2 bradykinin receptor P30411 16 20 05 04 C
CSK tyrosine-protein kinase CSK (C-Src Kinase) P32577 25 168 134 140 KHeat shock-110 kDa Q60446 33 09 10 14 C
MEF 2D (myocyte-specific
enhancer factor 2D)
Q63943 20 15 06 21 C
P54nrb (54 kDa nuclear RNA-
and DNA-binding protein)
Q15233 39 50 29 68 C
Rab3 (Rab-protein 3, member
RAS oncogene)
04 N/A 14 11 C
RBP (retinol-binding protein I, cellular) P09455 06 177 N/A 26 K
SCAMP1 (secretory carrier membrane protein 1) O15126 12 10 N/A 06 C
Average fold change for each adenoma subtype of the most consistent over- (C) or underexpressed (K) ‘level 10’ proteins from thearray in alphabetical order. N/A, not available.
A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
Immunohistochemistry for HSP110, B2
bradykinin receptor, CSK and annexin II
In order to compare the expression levels of an individual
protein between specific cell types in the normal pituitary
and the specific cell type-derived tumours, we performed
confocal immunofluorescent analysis for GH-, PRL- and
ACTH-secreting adenomas. For theNFPA, conventional
immunohistochemistry with consecutive cuts was
performed and FSH-positive tumour areas were
compared with FSH-positive areas from the normal
pituitary. To this end, 12 pituitary tumours and 4 normal
pituitaries were selected.
Table 2 Most consistently changing proteins for each adenoma su
Protein Swiss-
Acetylcholine receptor b P25109
ASS (argininosuccinate synthase) P00966
Apoptosis regulator BAX Q07812
Dematin Q08495
EBP 50 (Ezrin–radixin–moesin-binding
phosphoprotein 50)
O14745
ECA39 (branched-chain-amino-acid aminotransferase) P54687
Epithelial-cadherin precursor P12830
HILP/XIAP(inhibitor of apoptosis protein 3/X-linked IAP) P98170
Lamin A/C Q969I8
L-Caldesmon Q05682
MEPHX (microsomal epoxide hydrolase) P07099
The 11 most consistent ‘level 10’ proteins for pituitary adenoma sudirection of change (C/K).
1104
The confocal immunofluorescent analysis showed
that HSP110was expressed in the normal pituitary, with
mild coexpression in normal GH, PRL and ACTH cells.
It also showed the overexpression of HSP110 in GH-,
PRL- and ACTH-secreting tumour cells when
compared with respective normal cells from normal
pituitary (P!0.01 for all comparisons). HSP110 was
also overexpressed in FSH-staining tumour when
compared with FSH cells from normal pituitary
(P!0.01). These data confirm the overexpression of
this protein in tumorous cells when compared with the
corresponding normal pituitary cells (Fig. 2).
btype
Prot ID Adenoma subtype
Average fold
change
Direction
of change
ACTH 14 CPRL 21 C
NFPA 15 C
ACTH and GH 10 and 10 C
PRL and NFPA 19 and 15 C
NFPA 14 C
PRL 39 C
NFPA and ACTH 18 and 11 CACTH 14 K
ACTH 18 C
GH, PRL and NFPA 14, 17 and 10 K
btypes in alphabetical order, including average fold change and
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Table 3 Proteins that were not detected in normal pituitary or in one adenoma type
Protein Swiss Prot ID Adenoma subtype Comment
14-3-3e-23KD 14-3-3 protein epsilon
(mitochondrial import stimulation factor
L subunit)
P42655 Underexpression in GH secreting
adenomas and NFPA
Cannot be detected in GH
secreting tumours and
NFPA
CPG16/CaM kinase VI
(calcium/calmodulin-dependent protein
kinase type I-like)
O08875 Overexpression in ACTH-secreting
adenomas
Cannot be detected in normal
pituitary
GRIM-19 (cell death-regulatory
protein 19)
Underexpression in ACTH-secreting
adenomas
Cannot be detected in
ACTH-secreting tumours
HRF (histamine-releasing factor) P13693 Underexpression in ACTH-secreting
adenomas
Cannot be detected in
ACTH-secreting tumours
L22-18-19 KD (60s ribosomal
protein L22)
P35268 Overexpression in PRL-secreting
adenomas
Cannot be detected in
normal pituitary
MEK5-53-57KD (dual specificity
mitogen-activated protein kinase
kinase 5; MAP kinase kinase 5)
Q13163 Overexpression in PRL-secreting
adenomas
Cannot be detected in
normal pituitary
PP2A catalytic a-34KD (protein
phosphatase 2, catalytic subunit, alpha)
Overexpression in PRL-secreting
adenomas
Cannot be detected in
normal pituitary
Reps1(RalBP1-associated Eps
domain-containing protein 1;
RalBP1-interacting protein 1)
O54916 Overexpression in GH-secreting
adenomas
Cannot be detected in
normal pituitary
RONa (macrophage-stimulating protein
receptor precursor; MSP receptor;
p185-Ron; CD136 antigen; CDw 136)
Q04912 Overexpression in GH-secreting
adenomas
Cannot be detected in
normal pituitary
Consistent level 10 proteins for specific adenoma subtypes that showed gross over or underexpression compared with normalpituitary. NFPA, non-functional pituitary adenoma.
Endocrine-Related Cancer (2008) 15 1099–1114
The confocal immunofluorescent analysis showed
that B2 bradykinin receptor was expressed in the
normal pituitary, with mild coexpression in normal
GH, PRL and ACTH cells. It also showed over-
expression of this protein in GH-, (P!0.05), PRL-,
(P!0.01) and ACTH-secreting tumour cells
(P!0.01) when compared with their respective
pituitary cells (P!0.01). B2 bradykinin receptor was
also overexpressed in FSH-staining tumour when
compared with FSH cells from normal pituitary
(P!0.01). These data confirm the overexpression of
B2 bradykinin receptor in tumorous cells when
Table 4 The number of the most consistently changing proteins in
‘Level 10’ proteins GH
Number of ‘level 10’ overexpressed proteins 29
Number of ‘level 10’ underexpressed proteins 12
MeansGS.E.M. of fold changes in ‘level 10’
overexpressed proteins
8.9G0.8*
MeansGS.E.M. of fold changes in ‘level 10’
underexpressed proteins
10.6G1.0
Number of ‘level 10’ proteins and profile of their over and underexp*P!0.05 for comparisons between fold changes in ‘level 10’ over anfold changes in ‘level 10’ over and underexpressed proteins.
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compared with the corresponding normal pituitary
cells (Fig. 3).
For the CSK protein, the confocal immunofluor-
escent analysis showed expression of this protein in
normal pituitary, with coexpression in GH, PRL and
ACTH cells. It also showed underexpression in GH-,
PRL- and ACTH-secreting tumour cells when
compared with their respective normal pituitary cells
(P!0.01). CSK was also underexpressed in FSH-
staining tumour when compared with FSH cells from
normal pituitary (P!0.01). These data confirm the
underexpression of CSK in all adenoma tumour cells
each adenoma subtype
PRL ACTH NFPA
29 36 36
11 22 20
10.9G1.1† 6.7G0.5* 9.0G1.1†
25.7G7.0 13.8G2.0 16.4G2.4
ressions in each adenoma subtype compared with controls.d underexpressed proteins. †P!0.01 for comparisons between
1105
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Figure 2 Double immunofluorescent staining using HSP110 (blue staining) and polyclonal GH, PRL and ACTH (yellow staining)antibodies in normal pituitary, and HSP110 (blue staining) in GH-, PRL- and ACTH-secreting adenomas (3A, 3B and 3Crespectively). Colocalisation is shown by green colour (scale bar 50 mm): In figure 2D, conventional immunohistochemistry stainingusing HSP110 (brown) in consecutive cuts of normal pituitary tissue and non-functional pituitary adenomas (FSH positive); scale bar:25 mm. All data are shown as meansGS.E.M.; AU, arbitrary units; *P!0.05, **P!0.01.
A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
when compared with the corresponding normal
pituitary cells (Fig. 4).
The confocal immunofluorescent analysis showed
expression of annexin II in normal pituitary, with
coexpression in GH, PRL and ACTH cells. It also
showed underexpression of this protein in GH-, PRL-,
and ACTH-secreting tumour cells when compared
with their respective normal pituitary cells (P!0.01
for all comparisons). Annexin II was also under-
expressed in FSH-staining tumour when compared
with FSH cells from normal pituitary (P!0.01). These
data confirm the underexpression of annexin II in all
adenoma tumour cells when compared with the
correspondent normal pituitary cells (Fig. 5).
1106
Western blotting for HSP110, B2 bradykinin
receptor, CSK and annexin II
In order to validate the expression patterns of HSP110,
B2 bradykinin receptor, CSK and annexin II as shown
by protein array, conventional western blotting was
also performed on the proteins from 34 pituitary
tumours and 12 normal pituitaries. Certain samples
were the same as the ones included in the protein array,
to these we added additional tumours and normal
pituitary samples for this validation process.
HSP110 immunoblotting confirmed overexpression
in NFPAs (P!0.01 compared with normal controls)
and prolactin-secreting adenomas (P!0.05), but no
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Figure 3 Double immunofluorescent staining using B2 bradykinin receptor (blue staining) and polyclonal GH, PRL and ACTH(yellow staining) antibodies in normal pituitary, and B2 bradykinin receptor (blue staining) in GH-, PRL- and ACTH-secreting adenomas(4A, 4B and 4C respectively). Colocalisation is shown by green colour (scale bar 50 mm). In figure 3D, conventionalimmunohistochemistry staining using B2 bradykinin receptor (brown) in consecutive cuts of normal pituitary tissue and non-functionalpituitary adenomas (FSH positive); scale bar 25 mm. All data are shown as meansGS.E.M.; AU, arbitrary units; *P!0.05, **P!0.01.
Endocrine-Related Cancer (2008) 15 1099–1114
significant overexpression was detected in GH- or
ACTH-secreting adenomas (Fig. 6A). These data
therefore confirmed in part, the protein overexpression
shown in the protein array to all adenomas subtypes.
Immunoblotting for the B2 bradykinin receptor
confirmed overexpression in prolactin-secreting ade-
nomas (P!0.05), correlating well with the degree of
overexpression shown for this adenoma subtype in the
protein array; ACTH-secreting adenomas also showed
a trend towards overexpression (PZ0.1), but neither
the NFPAs nor the GH-secreting adenomas showed
significant changes (Fig. 6B). These data thus
confirmed the overexpression of B2 bradykinin
receptor shown in the protein array for the prolactin-
secreting adenomas alone.
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For the underexpressedCSKprotein, the results on the
conventional western blotting were not confirmed; none
of the adenoma subtypes showed significantly reduced
expression by immunoblotting (Fig. 6C). Annexin II,
another prominent underexpressed protein in the protein
array, was underexpressed in GH-secreting adenomas
(PZ0.01) and there was also a trend for underexpression
in the NFPAs (PZ0.09). However, this was not seen in
either ACTH- or PRL-secreting adenomas (Fig. 6D).
Discussion
We have utilised a proteomics technique to identify a
series of proteins that are differentially expressed in
pituitary adenomas when compared with the normal
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Figure 4 Double immunofluorescent staining using CSK (red staining) and polyclonal GH, PRL and ACTH (green staining)antibodies in normal pituitary, and CSK (red staining) GH-, PRL- and ACTH-secreting adenomas. Colocalisation is shown by orangecolour (scale bar 50 mm). In figure 4D, conventional immunohistochemistry staining using CSK (brown) in consecutive cuts of normalpituitary tissue and non-functional pituitary adenomas (FSH positive); scale bar 25 mm. All data are shown as meansGS.E.M.; AU,arbitrary units; *P!0.05, **P!0.01.
A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
pituitary. Due to the magnitude of the amount of
protein required for protein array, and the relatively
small amount of material available from each tumour,
we had to amalgamate tissue from five separate
tumours for each tumour subtype sample. Following
the analysis of protein array data, we explored the
expression of four grossly over- and underexpressed
proteins seen in all tumour subtypes by immunohis-
tochemistry and conventional western blotting. The
proteomic array data were fully reproduced by the
immunohistochemistry but only partially reproduced
by western blotting.
High-resolution two-dimensional gel electro-
phoresis (2-DE) has been the standard tool for
expression proteomics since its introduction more
than 30 years ago. The protein array that we describe
1108
here covered 1005 proteins with monoclonal
antibodies, out of which 316 were expressed in
pituitary tissue. Excellent previous studies have set
up a database of proteins expressed in pituitary
tumours (Zhan & Desiderio 2003, Zhao et al. 2005a)
and now our data add a large number of novel proteins
into this database. These proteins are shown in
Supplementary Table 1 according to their functional
categories and direction of changes observed in
adenomas. Despite various improvements of the
2-DE technique, one of the main inherent limitations
of this method is the fact that many proteins that are
expressed at low levels may escape detection. On the
other hand, the findings shown in Table 4 of many-fold
changes in underexpression suggest a considerable
sensitivity for this array.
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Figure 5 Double immunofluorescent staining using annexin II (red staining) and polyclonal GH, PRL and ACTH (green staining)antibodies in normal pituitary, and annexin II (red staining) in GH-, PRL- and ACTH-secreting adenomas. Colocalisation is shown byorange colour (scale bar 50 mm). In figure 5D, conventional immunohistochemistry staining using annexin II (brown) in consecutivecuts of normal pituitary tissue and non-functional pituitary adenomas (FSH positive); scale bar 25 mm. All data are shown asmeansGS.E.M.; AU, arbitrary units; *P!0.05, **P!0.01.
Endocrine-Related Cancer (2008) 15 1099–1114
The molecular analysis of pituitary tumours has
been extensively studied, and a number of factors
involved in pituitary tumorigenesis have been
described. The genome of a given cell or organism is
relatively static, subject to epigenetic modulation,
whereas the transcriptome and proteome are more
dynamic, and change with time and conditions
reflecting the different state of the disease. Indeed,
the proteome is much more complex than the
transcriptome due to post-translational modifications,
translocation, protein–protein interactions and
regulation, but otherwise may provide more direct
information when compared with the transcriptome in
relation to oncogenesis. An important aim of proteo-
mics has been to understand the cellular function at the
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protein level by the dynamic proteome, clarifying the
functions of proteins and disclosing new biomarkers
for disease states.
The four proteins chosen from the protein array for
validation by immunoblotting showed consistent pro-
minent over- or underexpression in all adenoma
subtypes in the original array. Furthermore, two of
these proteins (HSP110 and CSK) had not previously
been described as expressed in normal human pituitary
tissue. The data obtained from the immunohistochemi-
cal analysis confirmed these changes for all four
proteins. Immunoblotting showed only partial paralle-
lism with the array data; it confirmed the results
shown in the protein array of overexpression of
HSP110 in NFPAs and prolactin-secreting adenomas,
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Figure 6 Expression of (A) HSP110, (B) B2 bradykinin receptor, (C) CSK and (D) annexin II in each adenoma subtype comparedwith normal pituitary tissue by western blotting. Data are shown as meansGS.E.M., *P!0.05, **P!0.01.
A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
and also overexpression of the B2 bradykinin
receptor in prolactin-secreting adenomas. Furthermore,
it confirmed the underexpression of annexin II in
GH-secreting adenomas, although it completely failed
tomirror the results for CSK.These data implicate a role
for HSP110, the B2 bradykinin receptor, CSK and
annexin II in the process of pituitary tumorigenesis in
these tumour subtypes, although whether these changes
are causal or consequential remains unclear.
HSP110 is expressed in most tissues, and like other
stress proteins, HSP110 is a chaperone protein
associated with protein folding. It can be induced by
heat shock and its induction is associated with
thermotolerance. HSP’s overexpression has been
recently described as important in a series of human
cancers (Feng et al. 2005, Melle et al. 2006). Our
previous microarray data showed a trend for HSP110 in
the opposite direction (Morris et al. 2005), suggesting
that some form of post-transcriptional modification
accounts for these results. The novel data regarding the
overexpression of molecular chaperone HSP110 in
NFPAs and prolactin-secreting tumours demonstrates
the added value of proteomic analysis subsequent to
mRNA analysis. Recent proteomic studies on breast
cancer cells have shown that HSPs, including HSP110,
showedprotein expression patterns thatmight be altered
by 17b-oestradiol treatment (Lee et al. 2006). Further-
more, APG-2 protein, a member of the HSP110 family,
has shown developmental changes in its expression at
the protein level (Okui et al. 2000). These data
demonstrate the utility of an analysis at the protein
level. As recent publications have characterised
HSP110 and other HSPs as potential anti-tumour agents
(Wang et al. 2001, Casey et al. 2003), it is possible that
HSP110 overexpression in these adenomas may be a
1110
protective mechanism to reduce adenomatous
proliferation, and may relate to the benign nature of
the majority of such tumours. Other heat shock-induced
proteins have also been demonstrated to be differen-
tially induced in tumorous tissue (Feng et al. 2005,
Melle et al. 2006).
The B2 bradykinin receptor is a member of the
kallikrein system, a subgroup of the serine protease
family of enzymes, which also includes the enzyme
known as prostate-specific antigen, an important
clinical marker in human prostate cancers (Diamandis
& Yousef 2002). The B2 bradykinin receptor was
overexpressed in prolactin-secreting tumours; previous
work has shown that bradykinin increases prolactin,
but not GH release, and that this stimulation could be
antagonised by a B2-type bradykinin receptor antagon-
ist and inhibited by dopamine (Kuan et al. 1990). Other
studies have suggested a role for this receptor in
anterior pituitary regulation (Sharif et al. 1988). The
B2 bradykinin receptor belongs to the kallikrein-kinin
system, and several of its components have been
reported to be tumour markers for many malignancies
(Diamandis & Yousef 2002), especially those of
endocrine-related organs (Yousef & Diamandis
2003). The importance of its up-regulation in other
human cancers has also been described (Ikeda et al.
2004, Dlamini & Bhoola 2005, Zhao et al. 2005b).
Furthermore, it has been suggested that some
kallikreins may be part of a novel enzymatic cascade
pathway that is activated in some cancers, with a
particular potential role for kallikreins in central
nervous system (Yousef & Diamandis 2003, Yousef
et al. 2003). While its function in pituitary oncogenesis
remains obscure, it may offer a novel therapeutic target
(Stewart et al. 1997).
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Endocrine-Related Cancer (2008) 15 1099–1114
CSK is a non-receptor protein tyrosine kinase, its
major function being to specifically phosphorylate a
conserved C-terminal tyrosine of Src family kinases.
As a consequence of down-regulation of the Src kinase
protein expression and presumably its activity, proto-
oncogenic enzymes controlling cell growth and
proliferation would diminish. It is well known that
Src-family protein tyrosine kinases are tightly
regulated in normal cells and aberration in their
regulation can lead to constitutive activation, which
is associated with certain types of cancer (Kim et al.
2004, Chong et al. 2005, Martin 2006).
As a calcium-dependent phospholipids-binding
protein, annexin II has been suggested to play a role
in exocytosis from anterior pituitary secretory cells
(Turgeon et al. 1991, Senda et al. 1994). It has
previously been reported as being down-regulated in
some human cancers (Xin et al. 2003, Gillette et al.
2004), although overexpression of annexin II on the
cell surface has been suggested to contribute to the
development of metastases by enhancing tumour cell
detachment and tissue invasion (Choi et al. 2000, Ma
et al. 2000). Annexin II was described in a previous
human pituitary proteomics analysis (Zhan & Desi-
derio 2003); in this case our finding of underexpression
in GH-secreting adenomas is in accordance with our
previous microarray showing down-regulation of
annexin II mRNA in GH-secreting adenomas (Morris
et al. 2005). The importance of the annexin family as
potential regulatory factor in pituitary tumours has
been described, and expression of annexins I and V
have been demonstrated in pituitary tumours (Traverso
et al. 1999, Mulla et al. 2004). Specifically, annexin II
overexpression has previously been shown in a
GH-secreting carcinoma (Mulla et al. 2004). It is
therefore possible that carcinomatous transformation
per se leads to a specific annexin II increase.
While factors involved in pituitary tumorigenesis
have been subject to intensive study (Asa&Ezzat 2002,
Heaney & Melmed 2004, Kaltsas et al. 2005, Farrell
2006) there have been relatively few studies of the
specific pituitary protein changes using proteomics
(Zhan & Desiderio 2003, Zhan et al. 2003, Asa & Ezzat
2005,Moreno et al. 2005, Zhao et al. 2005a). This study
was performed using a screening commercial protein
array to track many different proteins simultaneously in
order to obtain greater insight into cellular function.
However, there are problems associatedwith the limited
sampling that is often employed, and also some
uncertainty regarding the use of autopsy controls for
comparison with fresh adenomatous tissue. In our study
all tissues were normalised for protein content, and such
tissue is the best currently available for comparison to
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tumours. Reassuringly, a considerable proportion of
proteins showed higher expression in the normal
autopsy tissue than in the surgical adenoma samples.
Despite the recent advances in human proteomics
(Garbis et al. 2005, Senzer et al. 2005), we believe
confirmation with additional technique(s) can provide
furtherusefuldata.Ourproteomicsdatawerecorrespond-
ing with the immunohistochemistry but the immuno-
blotting results were more variable. Especially, the fold
changes over- or underexpressions shown by the protein
array were only weakly correlated with the quantitative
changes observed in western blotting.
Indeed, several shortcomings have been described in
proteomic studies, and while there are few reports on
proteomics for pituitary adenomas, the findings may be
even more difficult to interpret with these small tumours
when compared with other human cancers. Furthermore,
this study was performed utilising a commercial
screening array that was not designed specifically to
focus on the pituitary. As a consequence, novel proteins
were shown to be present in pituitary while others were
not assessed. However, our findings of the expression of
the pituitary transcription factor Pit 1 in controls,
up-regulation of this protein in GH- and PRL-secreting
adenomas, and the absence of this protein in ACTH-
secreting adenomas and non-functional pituitary adeno-
mas, corroborates the hypothesis that this is indeed a
reliable protein array. Although whole tissue samples
lead to frank admixture of tumour cellswith various other
cells (stromal cells, macrophages, red blood cells, etc.) it
may be argued that these cells may also be important for
the physiology of the tumour, and that they should
therefore be included. On the other hand, it would have
been ideal that this study hadmatched for age and gender
tumour bearing patients and control pituitaries, although
the small availability of pituitary material for research
generally precludes this selection. It is also known that
the comparisons between adenomatous tissues consisting
of a monoclonal, homogeneous tumour cell population
and normal pituitaries representing various proportions
of endocrine cell types may also carry some bias. This
study, however, overcame this important obstacle,
showing by immunofluorescent and conventional immu-
nohistochemistry the expected over- or underexpression
of the four selected proteins in human pituitary tumour
cells when compared with their normal pituitary cells.
Interestingly, these results were much less obvious in the
immunoblotting study, and in some cases were absent.
The concordance of the proteomic results with the
immunohistochemistry suggests that conventional
immunoblotting in this situation is of much less
sensitivity.
1111
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A Ribeiro-Oliveira Jr et al.: Proteomics of pituitary tumours
In summary, our data show the limitations of
proteomic profiling, but do reveal novel proteins in
human pituitary tissue and some possible candidates
for pituitary oncogenesis. Our data also encourage
future studies targeting these novel protein candidates
as possible biomarkers to this diagnosis, monitoring of
disease progression, and developing of novel drug
therapeutic approaches to some specific pituitary
adenomas. However, we caution using proteomic
array data without validating its findings using
alternative more conventional techniques.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
research reported.
Funding
This work was supported by the Cancer Research Committee
of Saint Bartholomew’s Hospital and by the OTKA K68660
grant. A R O Jr was supported by CNPq (Conselho Nacional
de Desenvolvimento Cientıfico e Tecnologico).
Acknowledgements
We are grateful for the support of Cancer Research
Committee of Saint Bartholomew’s Hospital and CNPq
(Conselho Nacional de Desenvolvimento Cientıfico e
Tecnologico).
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