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1
The pathophysiology of pituitary adenomas
Dorota Dworakowska, MD, PhD,Doctor a,b, Ashley B. Grossman, MD, PhD,Professor of Neuroendocrinology a,*
a Centre for Endocrinology, Barts and the London School of Medicine, EC1M 6BQ, UKb Department of Endocrinology and Internal Medicine, Medical University of Gdansk, 80-211, 7 Debinki Str., Poland
Keywords:
pathophysiology
pituitary adenoma
molecular alteration
The pathogenesis of tumour formation in the anterior pituitary has
been intensively studied, but the causative mechanisms involved
in pituitary cell transformation and tumourigenesis remain elusive.
Most pituitary tumours are sporadic, but some arise as a compo-
nent of genetic syndromes such as the McCuneAlbright
syndrome, multiple endocrine neoplasia type 1, Carney complex
and, the most recently described, a MEN1-like phenotype (MEN4)
and pituitary adenoma predisposition syndromes. Some specificgenes have been identified that predispose to pituitary neoplasia
(GNAS, MEN1, PRKAR1A, CDKN1B and AIP), but these are rarely
involved in the pathogenesis of sporadic tumours. Mutations of
tumour suppressor genes or oncogenes, as seen in more common
cancers, do not seem to play an important role in the great
majority of pituitary adenomas. The pituitary tumour transforming
gene (PTTG; securin) was the first transforming gene found to be
highly expressed in pituitary tumour cells, and seems to play an
important role in the process of oncogenesis. Many tumour
suppressor genes, especially those involved in the regulation of the
cell cycle, are under-expressed, most often by epigenetic modu-
lation usually promoter hypermethylation but the regulator ofthese co-ordinated series of methylations is also unclear. Cell sig-
nalling abnormalities have been identified in pituitary tumours,
but their genetic basis is unknown. Both Raf/MEK/ERK and PI3K/
Akt/mTOR pathways are over-expressed and/or over-activated in
pituitary tumours: these pathways share a common root, including
initial activation related to the tyrosine kinase receptor, and we
* Corresponding author. Centre for Endocrinology, St Bartholomews Hospital, West Smithfield, London EC1A 7BE, UK. Tel.:
44 20 76018343; Fax: 44 20 76018505.
E-mail address: [email protected](A.B. Grossman).
Contents lists available atScienceDirect
Best Practice & Research Clinical
Endocrinology & Metabolismj o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / lo c a t e / b e e m
1521-690X/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.beem.2009.05.004
Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 525541
mailto:[email protected]://www.sciencedirect.com/science/journal/1521690Xhttp://www.elsevier.com/locate/beemhttp://www.elsevier.com/locate/beemhttp://www.sciencedirect.com/science/journal/1521690Xmailto:[email protected] -
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speculate that a change to these receptors or their relationship to
membrane matrix-related proteins may be an early event in
pituitary tumourigenesis.
2009 Elsevier Ltd. All rights reserved.
Pituitary adenomas are the third most common intracranial tumours in surgical practice,
accounting for approximately 1025% of intracranial neoplasms.1 Pituitary tumours are frequently seen
in autopsy specimens (14%).2 Radiological series suggest that pituitary incidentalomas may be present
in one in six people. Furthermore, recent epidemiological data suggest that clinically apparent pituitary
adenomas have a prevalence of approximately one in 1000 in the general population.3
Although only very rarely malignant, pituitary tumours may cause significant morbidity in affected
patients. First, given the critical location of the gland, large tumours may lead to mass effects, and,
second, proliferation of hormone-secreting pituitary cells leads to endocrine syndromes. Approxi-
mately 50% of newly diagnosed pituitary adenomas are prolactinomas (PRL-omas). Endocrinologically
inactive adenomas non-functioning pituitary adenomas (NFPAs) represent about 30%, somatotrophadenomas (GH-omas) 1520%, corticotroph adenomas (ACTH-omas) 510% and thyrotroph adenomas
(TSH-omas) less than 1%.4,5 True gonadotroph-secreting pituitary adenomas resulting in clinical
syndromes are extremely rare, but it seems likely that the great majority, if not all, NFPAs are in fact
very-low-grade FSH/LH-omas. Pituitary carcinomas comprise around 0.2% of pituitary adenomas1,
distinct from thew1% of pituitary mass lesions, which are metastatic tumours from non-pituitary sites.
The initiating molecularaberrations in the great majority of sporadicpituitarytumours remain elusive.6
The majority of pituitary adenomas are sporadic, although some arise as a component of familial
syndromes. Due to clonal analysis (which allows one to make the important distinction between a poly-
clonal proliferation in response to a stimulatory factor versus a monoclonal expansion of a genetically
aberrant cell), it has been demonstrated that almost all hormone-secreting as well as non-functioning
pituitary adenomas are unicellular in origin, arising from a monoclonal expansion of a genetically mutatedcell.68 Pituitary adenomas have attracted considerable attention because they can act as a model system
for more aggressive cancers. The aim of this review is to summarise recent advances in pituitary tumour
pathophysiology rather than to provide exhaustive information about all molecular abnormalities seen in
these tumours, to which several recent reviews have been dedicated.5,914
Genetic/familial syndromes
Pituitary adenomas may be a manifestation of an underlying germ line syndrome; such disorders
include the McCuneAlbright syndrome (MAS), multiple endocrine neoplasia type 1 (MEN1), Carney
complex (CNC) and, most recently, the MEN1-like phenotype and pituitary adenoma predisposition
(PAP) syndromes.11,13
The genes for GNAS (at 20q13), menin (at 11q13) and protein kinase A regulatorysubunit-1-a (PRKAR1A, at 17q24) have been associated with MAS, MEN1 and CNC, respectively. The
cyclin-dependent kinase inhibitor 1B (CDKN1B, which codes for p27, at 12p13) and aryl hydrocarbon
receptor (AHR)-interacting protein (AIP, also at 11q13, but distinct from menin)15,16 are associated with
an MEN1-like phenotype and PAP, respectively. CDKN1B was identified from a rat phenotype referred
to as MENX, but still only accounts for a fraction of human MEN1-like syndromes, which are MEN1-
gene negative.17 Indeed, the syndrome, now designated MEN4 by OMIM, seems less often than
originally thought to include pituitary adenomas. AIP is mainly mutated in the subset of families with
familial isolated pituitary adenomas, which have familial acromegaly and prolactinomas, isolated
familial somatotropinomas (IFSs)18, but even in this syndrome the majority of patients do not show
a mutation in AIP, and other genes are almost certainly involved.19
The McCuneAlbright syndrome (MAS) is caused by mosaicism for a mutation in the GNASoncogene. The GNAS1 mutation is the only mutation that has been identified in a significant proportion
of sporadic pituitary tumours, occurring in approximately 3040% of GH-omas. However, in spite of
extensive work, there is little direct evidence that the gsp mutation plays an essential primary onco-
genic role in such tumours, or that it alters tumour growth or recurrence rate.20,21
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Multiple endocrine neoplasia type 1 (MEN1) is familial disorder with a mutation in the MEN1gene
located on chromosome 11q13, inherited as an autosomal-dominant trait.22 Even though allelic
deletions on 11q13 are often observed in sporadic tumours23, somatic MEN1 mutations or loss of
expression24 are very rare in sporadic pituitary tumours25, and the evidence to date suggests that
mutation or loss of this gene plays only a limited role in sporadic tumours.
Up to 1025% of patients with MEN1 have normal MEN1 genes26,27, implicating other predispositiongenes in this phenotype. A germ line nonsense mutation in the human cyclin-dependent kinase
inhibitor 1B (CDKN1B, also known as p27 and KIP1) gene was recently identified in a family with an
MEN1-like condition15; however, the occurrence of this mutation seems to be very rare in such
families.17 No mutations in the p27 gene have been found in sporadic pituitary tumours.28 As discussed
below, p27 negatively regulates cell cycle progression by inhibiting cyclin and cyclin-dependent kinase
complexes, with p27 protein showing under-expression in most human pituitary tumours. 2931 More
recent studies indicate that p27 is an important down-stream signal in the menin signalling pathway
and a possible target of oncogenic RET in endocrine cells.32 It is also a direct transcriptional target of
AHR, the partner of AIP, which is known to affect cell proliferation.33
Carney complex (CNC) is an inherited neoplasia syndrome characterised by spotty skin pigmen-
tation, myxomas, endocrine tumours and Schwannomas. Some 40% of CNCs are associated withinactivating mutations in the gene encoding for the protein kinase A (PKA) type 1A regulatory (R1a)
subunit (PRKAR1A).34 PRKAR1A, like menin, acts as a tumour suppressor gene in affected tissues; loss
of its normal allele at chromosomal region 17q2224 is present in pituitary tumours associated with
CNC35, but it is normally expressed and unmutated in almost all sporadic somatotroph tumours. 36,37
Familial isolated pituitary adenoma (FIPA) is a rare syndrome that embraces IFS as well as the PAP
syndrome. The most frequently described forms of FIPA are familial somatotropinomas or prolacti-
nomas; however, pituitary adenomas of all types can occur in a familial setting. FIPA differs from MEN1
in terms of a lower proportion of prolactinomas and more frequent somatotropinomas. Patients with
FIPA are significantly younger at diagnosis38 and have significantly larger pituitary adenomas than
matched sporadic pituitary adenoma counterparts.12,39 PAP constitutes the group of patients with FIPA,
especially those with IFS, who show germ line mutations in AIP. A linkage in patients with IFS tochromosome 11q13.111q13.3 was first established by Gadelha et al.40, and it is now known that many
such patients show a mutation in AIP.
In a recent series from our group, about 66% of patients with AIP mutations showed pituitary
adenomas.41 Mutations to the AIP gene also resulted in unchanged cell proliferation when inserted into
GH3 pituitary cell lines, HEK293 cell lines and TIG3 cell lines (compared with markedly reduced cell
proliferation in cell lines engineered to over-express AIP).41 AIP thus appears to be a tumour suppressor
gene, and mutations in this gene are involved at least in some patients with IFS. On the other hand,
approximately 85% of the FIPA cohort and 50% of those with familial somatotropinomas were negative
for AIP mutations.39 Furthermore, AIP can be ruled out as a candidate gene for involvement in the great
majority of sporadic pituitary adenomas, as it is extremely rarely mutated and, if anything, is over-
expressed (possibly as a compensatory phenomenon) in all tumour types.41
Others tumour suppressor genes, oncogenes, growth factors and cell cycle regulators
Molecular studies on pituitary tumours have shown that alterations in the best-known tumour
suppressor genes in other neoplasms (e.g.,P53 andRB) or common oncogenes (e.g., theRas-family) are
only rarely involved in the development of these tumours.4245 Although point mutations of the Ras
Practice points and research agenda
Current studies have greatly increased our knowledge of the genetic basis of MAS, MEN1, CNC
and familial pituitary adenoma syndromes, but unfortunately they have done little to elucidate
the causes of sporadic pituitary tumours.
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oncogene43, loss of heterozygosity (LOH) near the RB locus on chromosome 1346,47 and LOH on
chromosome 1144,45 have all been implicated in some pituitary tumours, these are clearly in the
minority. For instance, a mutation of the H-Ras gene (codon 12, Gly to Val) was found in recurrent,
highly invasive prolactinomas.43 A recent study showed the presence of P53 gene mutations in
pituitary carcinomas but confirmed their absence in pituitary adenomas (in carcinomas, a P53gene
mutation was additionally related to p53 protein over-expression in tumour cells, which may bediagnostically helpful).48
Several other genetic alterations have been implicated in pituitary tumourigenesis: increased
expression of pituitary tumour transforming gene (PTTG), alternative transcription initiation of pitu-
itary-tumour-derived fibroblast growth factor receptor 4 (Pdt-FGFR4) or promoter methylation of
tumour suppressor genes (e.g., RB). Growth arrest and DNA damage-inducible protein gamma
(GADD45G), maternally expressed protein 3A (MEG3A), the zinc-finger protein pleiomorphic adenoma
gene-like 1 (ZAC), death-associated protein kinase (DAP kinase), pituitary tumour apoptosis gene
(PTAG) and p27 (Kip 1) gene have all been found to be abnormally expressed in pituitary adenomas and
thus may play a possible role in pituitary tumourigenesis.14,4952
The novel PTTG was identified and cloned in rat GH4 pituitary tumour cells.53 PTTG over-expression
occurs in a wide variety of endocrine and non-endocrine tumours, including those of the pituitary,thyroid, ovary, breast, prostate, lung, esophagus, colon and the central nervous system.54 By acting as
a securin protein and inhibiting premature sister chromatid separation, PTTG plays an important role in
mitosis. Cohesin binds sister chromatids during metaphase, and is subsequently degraded by separin to
allow sister chromatid separation at anaphase (Fig. 1).55 It has been shown that PTTG blocks sister
chromatid separation during metaphase by binding to separin and preventing cohesin degradation.56
PTTG induces cell transformation when over-expressed in vitro in NIH 3T3 fibroblasts, and tumour
formation in nude mice. It stimulates basic fibroblast growth factor (bFGF), which is a potent mitogenic
and angiogenic factor,in vitro49, and interacts with theP53tumour suppressor gene andc-mycproto-
oncogene.54 In addition, phosphoinositol-3-kinase (PI3K) and MAPK both physically interact with
Fig. 1. Molecular mechanism of chromosome segregation. At the metaphaseanaphase transition, APC/CCdc20 ubiquitinates securin
(PTTG). Degradation of securin activates separase. Separase then cleaves the Scc1 subunit of cohesin, allowing chromosome
segregation. In response to sister-chromatids not being properly attached to the mitotic spindle, the spindle checkpoint promotes
the assembly of checkpoint protein complexes that inhibit the activity of APC/C, leading to the stabilisation of securin, preservation
of sister-chromatid cohesion, and a delay in the onset of anaphase. 63
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PTTG, thus suggesting that PTTG phosphorylation occurs through these two cascades.57,58 Interaction
of PTTG with Sp1, an important transcription factor involved in the regulation of several genes
associated with cellular growth and differentiation, is well documented.54 PTTG and Sp1 were found to
cooperatively mediate G1/S-phase transition59, and recently it was shown that PTTG binds to the p21
promoter region through Sp1, thereby suppressing p21activity.60 Conversely, knock-out of PTTG causes
decreased cellular proliferation and premature cellular senescence.61 Thus, PTTG acts at the centre ofa network of processes that control cell proliferation and division, and both up- and down-regulation
are inimical to regulated cell growth. However, specific mutation of PTTG has not been shown in
sporadic pituitary adenomas.
Changes in PTTG expression may relate to pituitary tumour angiogenesis as well as to tumour
invasiveness and aggressiveness.49,54,62 PTTG expression was found to be correlated with Ki-67
expression in pituitary adenomas.62 In addition, in pituitary adenomas, a significant positive correla-
tion was found between VEGF and PTTG mRNA expression, as well as between PTTG and KDR (kinase
insert domain receptor, one of the family of VEGF receptors) mRNA 63,64:in situhybridisation revealed
PTTG expression in NFPAs and in GH-secreting adenomas but not in normal pituitary tissue.49
Immunohistochemistry showed lack of PTTG expression in normal pituitary tissue and a very high rate
of PTTG positivity in different types of pituitary tumours (approximately 90% of tumours showed PTTGexpression, but with considerable variability).62 More sensitive techniques based on reverse
transcriptase polymer chain reaction (RT-PCR) showed that PTTG expression was present in normal
pituitaries, but at a much lower level than in pituitary adenomas. PTTG was mostly over-expressed in
adenomas with a >50% increase in NFPA, GH-, PRL- and ACTH-omas. In some tumours, more than
a 10-fold increase in PTTG expression was observed, suggesting that PTTG is indeed involved in some
manner in pituitary tumourigenesis.49 It has not yet been clarified whether PTTG functions as
a cytoplasmic or a nuclear protein. In pituitaryadenomas, some studies showed that PTTG might be
predominantly expressed in the cytoplasm64,65, but another showed PTTG to be expressed predomi-
nantly in the nucleus.62 PTTG was found to be a secretory protein in human pituitary adenomas and in
mouse pituitary tumour cell lines, and may exert autocrine and/or paracrine functions.66 In hormone-
secreting tumours, over-expression of PTTG correlated with tumour invasiveness, since higher PTTGexpression was observed in tumours that had invaded the sphenoid bone (stages III and IV; 95%
confidence interval (CI): 3.1189.715) compared with tumours that were confined to the pituitary fossa
(stages I and II; 95% CI: 1.6813.051).49
Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) are known to be
important for a variety of biological processes, including mitogenesis, differentiation, development,
angiogenesis and tumourigenesis.67 The FGFs mediate their biological effects by binding to high-
affinity tyrosine kinase receptors, the FGFRs.68 FGF-2 (also known as basic or bFGF) is over-expressed
by pituitary tumour cells with higher levels in more aggressive tumours.69 The role of the pituitary
tumour-derived FGFR-4 isoform (ptd-FGFR4), a constitutively phosphorylated protein with trans-
forming properties in vitro and in vivo70, has been recently investigated in sporadic pituitary adenomas.
Cytoplasmic expression of ptd-FGFR4 was found in around 60% of cases (containing GH-, ACTH-, FSH/LH- and NFPAs) but was relatively rare in PRL-omas and absent in normal adenohypophysial tissue.
Expression of ptd-FGFR4 was stronger in macroadenomas in comparison to microadenomas, and
correlated with cell proliferation assessed by Ki-67.71 Up-stream regulators, particularly transcription
factors of theicarusgroup, controlling this aberrant splice variant are under study, and, if confirmed in
other laboratories, could signify a novel pathway regulating proliferation.
The cell cycle
Cell proliferation is dependent on a highly organised and temporally linked series of kinases, which
rise and fall with each cell division. In the cell cycle the cyclins are phosphorylated (by cyclin-
dependent kinases or CDKs) and then degraded in sequence, leading to DNA synthesis, chromosomalseparation and mitosis. We have already emphasised the role of PTTG, or securin, in this process. In
addition, so important is the regulation of this process and its exquisite co-ordination, that a series of
CDK inhibitors (CDKIs) are also involved. Most importantly, the step from cyclin D/CDKs 4/6 to cyclin E/
CDK2 is precisely tuned by a series of CDKIs known as the Ink4 family (p15, p16, p18, for CDK 4 and 6)
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and the kip/cip family (p21, p27, p57 for CDK2). A cell passing through this checkpoint phosphorylates
Rb, leading to dissociation from E2F and induction of pro-proliferative capacity (Fig. 2).72 It then
becomes irreversibly committed to cell division; thus, disruption at this level is of fundamental
importance. It is of considerable interest that transgenic knockouts of many of these CDKIs will usually
greatly increase the probability of pituitary tumour formation. In rodent transformed pituitary cell
lines, p16 was shown to be under-expressed by promoter hypermethylation in the mouse AtT20 lineand p27 under-expressed by a similar process in the rat GH3 line. In the human, the vast majority of
adenomas display alterations of the RB1 pathway (90%); specifically, promoter hypermethylation of the
p15(INK4b), p16(INK4a) and RB1 genes was detected in 3236%, 5971% and 2935% of cases,
respectively.73,74 Yoshino et al. additionally demonstrated promoter hypermethylation of p14 (ARF),
p21(Waf1/Cip1) and p73 genes in 6%, 3% and 12% of adenomas, respectively, whereas the promoter of
p27(Kip1) was not hypermethylated.74 However, our group and that of Ricardo Lloyd reported under-
expression of p27 at the protein level in all types of pituitary adenomas, especially corticotroph
tumours.29,30 Thus, dysregulation of the cell cycle appears to be a hallmark of pituitary tumour
formation, occurring both by CDKI promoter methylation as well as by enhanced degradation.
Inappropriate methylation of CpG islands of other key cell cycle control and growth-regulatory
genes has been demonstrated: such genes include the growth arrest and DNA damage-inducibleprotein gamma (GADD45G)75, and death-associated protein kinase (DAP kinase).76 GADD45G nega-
tively regulates cell growth and is significantly under-expressed in GH-secreting and PRL-secreting
pituitary tumours.50 The DAP kinase gene encodes for a calmodulin-dependent serine/threonine
kinase, which positively mediates programmed cell death: loss of DAP kinase expression was
demonstrated in highly metastatic cells, whilst re-expression of the protein resulted in delayed tumour
Fig. 2. Cell cycle progression. In early G1, cyclin D activates CDK4/6 which partially phosphorylates Rb protein. After progression
beyond the restriction point, CDK2 in complex with cyclin E further phosphorylates pRb rendering it inactive and thus allowing
transcription of cyclin A. The accumulating cyclin A captures CDK2 from cyclin E and promotes progression through S phase. At the
transition between G2-M phase, cyclin B complexes with and activates CDK1 to further activate metaphase promoting complex
(MPC) required for entering M phase. CDK inhibitors oppose CDK activation: in early G1, the INK4 family bind to CDK4/6 and prevent
cyclin D activation of this kinase. At G1-S transition, WAF/KIP family members sequester CDK2 away from cyclin E/A when they are
in stoichiometric amounts with CDK2. When CDK2 exceeds p27 levels, the former phosphorylates the latter and triggers its
degradation. Resetting of pRb occurs in M-phase by dephosphorylation, probably by protein-phosphatase type 1 and this correlates
with the time of cyclin A degradation.80
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growth and a decreased incidence of metastasis. Loss of DAP kinase expression (associated with either
CpG island methylation or homozygous deletion) preferentially segregates with pituitary tumours that
show an invasive phenotype.76
A novel differentially methylated chromosome 22 CpG island-associated gene (C22orf3), also
known as pituitary tumour apoptosis gene (PTAG), has been recently identified. 52 The majority of
pituitary adenomas (80%) failed to express this gene in comparison to normal pituitary, and in thisgroup approximately 20% showed methylation of the CpG islands of the PTAG promoter.52 Where the
promoter was methylated, it was invariably associated with loss of transcript expression.52 In a model
pituitary tumour cell line, AtT20, expression of PTAG per sehad no discernible effects on proliferation,
cell cycle profile or viability. However, enforced expression was associated with a significantly
increased sensitivity to the apoptotic effects induced by the bromocriptine challenge.77
Other genes with suggested role in pituitary tumourigenesis on the basis of their promoter
methylation include maternally expressed protein 3A (MEG3A)78 and the ZAC.79 MEG3 is highly
expressed in the normal human pituitary, but is not detected in human NFPAs. Two functionally
important 50- flanking regions, immediately preceding and approximately 1.62.1 kb up-stream of the
first exon, respectively, were found to be hypermethylated in tumours without MEG3 expression.78 The
ZAC gene encodes a novel zinc-finger protein that concomitantly induces apoptosis and cell cyclearrest; it localises to chromosome 6q24q25, a well-known hot spot related to cancer. ZAC is highly
expressed in the anterior pituitary gland, and its ablation by antisense targeting promotes pituitary cell
proliferation. Strong down-regulation of ZAC mRNA and protein expression was reported in NFPAs 79,
although this was not associated with gene mutations.
We have reviewed the evidence for disruption of the cell cycle in pituitary adenomas, usually by
promoter hypermethylation of CDKIs. However, in the case of p27, this is due to a decrease in p27
protein levels,while p27 mRNA levels are normal or even increased, and somatic mutations have not
been reported.11,30 Knockout mice lacking p27 have been shown to develop multi-organ hyperplasia
and intermediate lobe pituitary tumours secreting ACTH. The expression of p27 is reduced in pituitary
adenomas (including ACTH-, PRL-, GH-, LH/FSH- and TSH-adenomas), compared with the normal cells,
and completely lost in metastatic pituitary carcinomas.30
Importantly, p27 expression is inverselyrelated to Ki-67.80 Phosphorylation of p27 is an essential step for its degradation, and this has been
shown to be higher in pituitary adenomas than in the normal pituitary, leading to the cytoplasmic
sequestration and reduced nuclear expression of p27.80 Decreased levels of p27 particularly lead to
increased levels of cyclin E, and this has been reported in corticotroph adenomas. 80,81
RNA microarrays and proteomics of pituitary adenomas
Microarray technology allows for the expression profile of many thousands of genes to be quantified
at the same time and has resulted in novel discoveries regarding the tumour biology of
pituitary adenomas. A study by Ruebel at al. underlined how major are the differences between
Practice points and research agenda
Recent studies have highlighted multiple molecular alterations in pituitary adenomas, espe-
cially involving the cell cycle. Many of the changes seem to reflect epigenetic mechanisms
associated with gene silencing, particularly of cell cycle inhibitors. PTTG is a gene that is
importantly over-expressed, which may lead to enhanced proliferation and chromosomal de-
stabilisation. The isolation of novel genes in pituitary adenomas (e.g., PTTG, Pdt-FGFR4, GADD45G,
MEG3A, ZAC, DAP kinase and PTAG) offers a significant advantage with respect to our under-
standing of tumourigenesis in pituitary tumours. The reversal of apparent gene silencing may
eventually lead to tumour cell sensitisation to chemo- and radiotherapeutic treatment strategies.
A summary of the reported changes in expression in pituitary tumours is shown in Table 1.
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Table 1
A summary of gene inactivation studies due to promoter hypermethylation in pituitary adenomas.
Gene Remarks Ref.
p15(INK4b)
p16(INK4a)RB1
91% of the adenomas displayed alterations of the RB1 pathway.
Promoter hypermethylation of the p15(INK4b), p16(INK4a), and RB1 genes wasdetected in 15 (35.7%), 30 (71.4%), and 12 (28.6%) of the adenomas, respectively.
Promoter hypermethylation of the p15(INK4b) gene coincided with a p16(INK4a)
alteration and/or RB1 methylation, whereas p16(INK4a) and RB1 methylations tended
to be mutually exclusive.
73
RB1, p14(ARF)
p15(INK4b)p16(INK4a)
p21(Waf1/Cip1)
p27(Kip1) p73
Promoter hypermethylation of the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/
Cip1), p27(Kip1), and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1
(3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the
adenomas displayed methylation of at least one of such cell cycle regulatory genes,
especially methylation of the member genes of the RB1 pathway (29 of 34; 85%).
Promoter hypermethylation of p15(INK4b) coincided with RB1 and/or p16(INK4a)
methylation, whereas RB1 and p16(INK4a) methylations tended to be mutually exclu-
sive (p 0.005).
Promoter hypermethylations of p14(ARF), p21(Waf1/Cip1), and p73 (not belonging tothe member genes of the RB1 pathway) were coincident with RB1 and/or p16(INK4a)
methylation except in one p73 methylated case.
74
RB1 Methylation of the CpG island within the RB1 promoter region was detected in 6 of 10
tumors that failed to express pRb.
18 of 20 tumors and all six histologically normal postmortem pituitaries that expressed
pRb were unmethylated.
131
GADD45G 67% of pituitary adenomas (22/33) do not express GADD45G as determined by RT-PCR
analysis.
Loss of expression was not associated with either LOH or mutations within the coding
region of this gene.
Methylation of the GADD45G genes CpG island, was found in 19 of 33 adenomas (58%)
and was significantly associated with tumours in which GADD45G transcript was notexpressed (18 of 22; 82%; P 0.002).
75
DAP kinase 34% (11/32) of tumours had undetectable DAP kinase expression, by Western blot and/
or RT-PCR analysis.
Of 11 tumours that failed to express DAP kinase, five (45%) showedde novomethylation
of the CpG island contained within the promoter region, while four (36%) had evidence
of homozygous deletion of this region.
Loss of DAP kinase expression was significantly (P
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pituitary adenomas and carcinomas, indicating 4298 genes that were differentially expressed among
the adenomas compared to the carcinoma, with 2057 genes over-expressed and 2241 genes under-
expressed in the adenomas.82 In particular, they showed that the b-galactoside binding protein
galectin-3, the human achaetescute homologue-1 ASCL1 (hASH-1) gene and ID2 (which plays an
important role in cell development and tumourigenesis), were under-expressed in adenomas
compared to the carcinomas.82
A pioneering complementary DNA microarray study indicated that 128 of 7075 genes examined
were differentially expressed in pituitary adenomas compared to normal pituitaries. Further RT-real
time quantitative PCR (RT-qPCR) examination established that at least three genes involved in
carcinogenesis in other tissues were also aberrantly regulated in the major types of pituitary
tumours, including the folate receptor gene (over-expressed in NFPAs and under-expressed in PRL-
and GH-adenomas), the ornithine decarboxylase gene (over-expressed in GH-omas adenomas and
under-expressed in ACTH-omas), and the C-mer proto-oncogene tyrosine kinase gene (over-
expressed in ACTH-omas and under-expressed in PRL-omas).83 The most recent microarray analysis
performed by the Evans group focussed on the molecular pathogenesis of human prolactinomas
(based on gene expression profiling, RT-qPCR, and proteomic analyses) and identified 726 unique
genes that were statistically significantly different between prolactinomas and normal glands,whereas proteomic analysis identified four differentially up-regulated and 19 down-regulated
proteins.84
b-Catenin is a major component of the Wingless (Wnt) signalling pathway and an important factor
in pituitary development. Its activation regulates the functioning of the somatotroph, mammotroph,
thyrotroph and gonadotroph pituitary axes, and it regulates Pit-1 expression/transcription. Recent
studies have explored the possible involvement ofb-catenin signaling in pituitary tumour develop-
ment.9 The data are sometimes discordant and contradictory, although there is indeed evidence that
there are alterations in Wnt/b-catenin signalling in these benign neoplasms, leading to aberrant
activation and transcription of specific target genes.9 In one study based on microarray analysis and
proteomics, several changes in the expression of genes involved in pituitary development were found
in NFPAs, including up-regulation of Pitx2, Pitx1, SFRP1 (secreted frizzled related protein 1) and TLE2(transducin-like enhancer of split 2, which not only represses targets ofb-catenin, but also interacts
with Hes1 during neuronal development).85 The same group has recently shown that several
components of the Notch pathway were altered in prolactinomas, and there was an increased
expression of the Pit-1 transcription factor, and the survival factor BAG1, but decreased E-cadherin and
N-cadherin expression in comparison to normal pituitaries.84 An Australian group has shown by
microarray profiling and real-time quantitative RT-PCR that the mRNA expression of a series of
inhibitors of the Wnt pathway (WIF1, SFRP2, frizzled B SFRP3 (FZDB), SFRP4) were all down-regu-
lated in pituitary tumours compared to normal pituitaries.86
A recent microarray study performed by our group showed that the expression of some genes was
altered in particular subtypes of pituitary adenomas. For instance, lysosomal-associated protein
transmembrane-4-b (LAPTM4B), a novel gene up-regulated in hepatocellular carcinoma, was signifi-cantly over-expressed in ACTH-omas and NFPAs. Bcl-2-associated athanogene (BAG1), an anti-
apoptotic protein found at high levels in a number of human cancers, was significantly over-expressed
in GH- and PRL-omas as well as in NFPAs. Furthermore, the cyclin-dependent kinase inhibitor p18,
which was shown on murine gene deletion to induce pituitary ACTH cell hyperplasia and adenomas,
was significantly under-expressed in ACTH-secreting adenomas.87 However, it should be noted that
quantitative variations in gene expression between normal pituitary and human pituitary adenoma
biopsy samples require careful interpretation, as the expression profiles of highly uniform cell pop-
ulations in human pituitary adenomas are necessarily different from the composite derived from the
complex admixture of different cell types that constitute the normal pituitary.5
There are even fewer studies on protein arrays in pituitary tumours. One study from our group
detected 316 different proteins present in pituitary tissue, of which 116 had not previously beendescribed in the human pituitary. In protein array analysis, heat-shock protein 110 (HSP110) and B2
bradykinin receptor were significantly over-expressed in all adenoma subtypes, while C-terminal src
kinase (CSK) and annexin II were significantly under-expressed in all adenoma subtypes. The immu-
nohistochemical analysis confirmed the over-expression of HSP110 and B2 bradykinin receptor and
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under-expression of CSK and annexin II in pituitary adenoma cells when compared to their corre-
sponding normal pituitary cells, but results were more variable using standard Western blotting. 88 In
NFPAs, a proteomics and transcriptomics study demonstrated secretagogin down-regulation at both
protein and mRNA levels.89
A new phosphoproteomics study showed 50 phosphorylation sites characterised in 26 proteins90,
and post-translational modifications of proteins from the human pituitary gland play an important rolein the regulation of different pituitary functions. The proteome database of the human pituitary can be
accessed at the website http://www.utmem.edu/proteomics.
Pituitary-specific signalling pathways
Since deregulation of normal feedback processes seems to occur in GH- and ACTH- secreting
tumours, abnormalities in pituitary-specific signalling pathways have been studied.
In Cushings disease, there are several possible sites of derangement of corticotroph signalling.
The ACTH receptor (ACTH-R), the second member of the melanocortin (MC-2) receptor family that
includes five seven-transmembrane G protein-coupled receptors, has been shown to be predomi-
nantly expressed in the adrenal cortex. It has been postulated that ACTH may regulate its own
secretion through ultra-short-loop feedback within the pituitary. In the study performed by ourgroup, we identified ACTH-R mRNA expression in normal pituitaries, but not in 16/22 ACTH-
adenomas. In addition, we did not find any ACTH-R gene mutation in a subgroup analysis of eight of
these tumours. Interestingly, diagnostic preoperative plasma ACTH levels were significantly lower in
the ACTH-R positive ACTH-omas, compared with those who were ACTH-R negative. In the absence of
any gene defects, we concluded that the absence of ACTH-R expression in Cushings disease is
probably a consequence of the tumoural process rather than a cause, but may aid in tumour
progression.91
The ACTH-secreting tumours are at least partially glucocorticoid resistant, but no mutations in the
glucocorticoid receptor (GR) were found in any of the tumours. A majority of the ACTH-omas tested
(16 out of 19) showed variable but increased overall GR expression, and thus these findings could
not account for the dexamethasone resistance characteristic of these tumours.92
Over-expression ofCRH-R has been shown in corticotroph tumours in comparison to normal pituitaries; interestingly, in
ACTH-secreting bronchial carcinoids, CRH-R signals were detected in only four of the six tumours.93
The CRH-R does not appear to be constitutively activated by mutation.94
On the other hand, vasopressin is an important regulator of hypothalamicpituitaryadrenal axis
activation, primarily acting through the V3 receptor (V3R). Many patients with ACTH-secreting
pituitary adenomas, but not normal individuals, respond to desmopressin, a relatively V2-specific
vasopressin agonist, with increased ACTH and cortisol levels. No mutation of the V3R gene was found in
12 ACTH-omas; the V2R gene was expressed in the majority of the samples tested, while V3R was
expressed in all of these tumours.95
Derangement has been demonstrated in ACTH-omas in the two isoenzymes 11b-hydroxysteroid
dehydrogenase (11b-HSD) 1 and 2, which convert cortisone to cortisol, and cortisol to cortisone,respectively. These enzymes may play an important pre-receptor role in regulating corticosteroid
hormone action. The 11b-HSD2 is up-regulated and 11b-HSD1 is down-regulated in some pituitary
tumours, resulting in increased cortisone and decreased cortisol production. Decreased cortisol in
the region of the GR could diminish functional feedback and allow selective secretion of
Practice points and research agenda
The techniques of modern genetics, genomics and molecular biological approaches may be
expected to reveal new mechanisms of endocrine tumourigenesis aiming at development of
better diagnostic, prognostic and therapeutic tools. However, current results have provided much
information but a less impressive increase in understanding.
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corticotrophs essentially re-setting glucocorticoid feedback control.96 However, these changes may
be a general feature of all pituitary adenomas so their functional significance in corticotroph
tumours remains unclear.97
An additional molecular basis for glucocorticoid resistance in pituitary adenomas is the observation
that approximately 50% of tumours are deficient in the expression of Brg1 (the ATPase subunit of the
Swi/Snf complex) or histone deacetylase-2 (HDAC2).98 These two proteins (Brg1 and HDAC2) areinvolved in chromatin re-modelling and may also participate in the tumourigenic process, as Brg1 is
a tumour suppressor.99 The GR-dependent repression of the pro-opiomelanocortin (POMC) gene,
which is at the centre of the hypothalamicpituitaryadrenal axis that controls glucocorticoid
synthesis, involves orphan nuclear receptors related to NGFI-B: this repression of POMC requires Brg1
to stabilise interactions between GR and NGFI-B. Brg1 also stabilises the interaction between GR and
HDAC2, which results in de-acetylation of the POMC promoter region and thus repression. Loss of
either HDAC2 or Brg1, therefore, produces glucocorticoid resistance as seen in Cushings disease.98
In acromegaly, there is less information available than for Cushings disease. However, GHRH receptor
variants were found to be common in GH-producing pituitary adenomas, but constitutively activating
mutations, as a mechanism for GH-omas, appear to be rare.100,101 The type 1 insulin-like growth factor
(IGF1)/GH negative feedback loop is one of the several factors that regulate GH secretion. It has beenpostulated that this is a result of changes at the GH-receptor (GH-R) level. However, it seems that
decreased feedback inhibition of GH because of somatic mutations of the coding region of the GH-R is
unlikely to be a common factor in the pathogenesis of GH-omas.102 Nevertheless, the decreased
expression of the GH-R and of IGF-R in 18 somatotroph tumours (both at the mRNA and protein level)
may, at least in part, help explain the continuous secretion of GH from the tumour despite the high
circulating levels of IGF-I and GH.102 Recently, a selective somatic histidine-to-leucine substitution in
codon 49 of the extracellular domain of the GH receptor has been described in a morphologic subtype of
human GH-producingpituitary tumoursthatis characterised by the presenceof cytoskeletal aggresomes.
ThisGH-R mutation significantlyimpairs glycosylation-mediatedreceptor processing,maturation, ligand
binding and signalling, and provides evidence for impaired hormone auto-feedback in the pathogenesis
of these pituitary tumours. It might explain the lack of responsiveness to somatostatin analogue therapyof this tumour type, in contrast to the exquisite sensitivity of tumours that lack aggresomes, and this has
therapeutic implications for the safety of GH antagonism as a therapeutic modality in acromegaly.103 By
contrast, no mutations of the IGF-1 receptor were found in another study.104
A relationship between somatostatin sensitivity and tumour pathogenesis has been suggested in
some GH-omas. However, no mutations affecting the sst2A protein were found in any of 15 analysed
tumours, suggesting that these mutations are unlikely to be involved in the pathogenesis of acro-
megaly.105 Molecular analysis of genomic DNA from pituitary tumour and peripheral blood obtained
from an acromegalic resistant to octreotide confirmed a lack of mutation in sst2, but showed one
polymorphism (Pro109Ser) and one germ line mutation (Arg240Trp) in sst5.106
In addition, it is well documented that cytokines of the gp130 family, such as interleukin-6, which
use gp130 as a common signalling protein, stimulate not only the proliferation but also the hormonesecretion of pituitary cells. Experiments in vivo have shown that the over-expression of the gp130
receptor resulted in abnormal pituitary growth.107 Bone morphogenetic protein-4 (BMP-4), a member
of the TGF-b family, promotes the development of prolactinomas but prevents Cushings disease
progression107; mRNA differential display showed that the BMP inhibitor noggin is down-regulated in
prolactinomas from dopamine D2-receptor-deficient mice, whereas BMP-4 is over-expressed in
prolactinomas taken from dopamine D2-receptor-deficient female mice.108 Based on Western blot
analysis, BMP-4 was found to be over-expressed in other prolactinoma models, including oestradiol-
induced rat prolactinomas and human prolactinomas, compared with normal tissue and other
pituitary adenoma types. In addition, BMP-4 stimulates, and noggin blocks, cell proliferation and the
expression of c-myc in human prolactinomas.108
On the other hand, ghrelin, originally isolated from the stomach, is an endogenous ligand of the GHsecretagogue receptor (GHS-R). Several groups, including our own, have demonstrated the presence of
both ghrelin and GHS-R mRNA in the normal human hypothalamus, as well as normal and
adenomatous human pituitary. ACTH-omas showed significantly less expression of ghrelin mRNA,
whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumours
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showed a particularly low level of expression of GHS-R mRNA. It seems that the presence of ghrelin
mRNA and peptide in the pituitary implies that the locally synthesised hormone may have an auto-
crine/paracrine modulatory effect on pituitary hormone release.109 In addition, in a rat pituitary
somatotroph cell line, ghrelin was shown to exert a proliferative effect through the mitogen-activated
protein kinase pathway.110
Abnormal cell signalling pathways
Abnormalities in cell signalling pathways are frequently seen in pituitary adenomas. Clarifying the
role of the particular component involved in regulation of these pathways may represent potential
selective targets for anti-tumour therapy, and identify the locus or loci of the initiating abnormalities.
The serinethreonine kinases, including key mediators of tumourigenesis such as Raf, mitogen-
activated protein kinase (MAPK) cascades and Akt/protein kinase B, appear to be of particular
interest.111
The PI3K/Akt/mTOR pathway is altered in many tumours, now including pituitary adenomas.
Phosphatidylinositol 3-kinase (PI3K) is activated as a result of the ligand-dependent activation of
tyrosine kinase receptors, G-protein-coupled receptors or integrins. Receptor-independent activationof PI3K can also occur in, for instance, cells expressing a constitutively active Ras protein.112,113 The
best-characterised phosphorylation target of PI3K is Akt (also known as protein kinase B), resulting in
the phosphorylation of a host of other proteins that affect cell growth, cell cycle entry and cell survival.
Akt phosphorylation activates a serinethreonine kinase mTOR (mammalian target of rapamycin),
which activates 40S ribosomal protein S6 kinase (p70S6K)114 and inactivates 4E-binding protein (4E-
BP1). It has been shown that the tuberous sclerosis complex (TSC), which mediates between PI3K/Akt
and mTOR, inhibits mTOR. Mitogenic stimuli activating Akt can directly phosphorylate TSC2 (tuberin),
causing destabilisation of TSC2 and inhibiting the formation of the TSC1/2 complex, leading to an
increase of mTOR activity through rheb (Ras homolog enriched in brain).115 4E-BP1 inhibits the initi-
ation of translation of mRNA for many factors, including c-myc and cyclin D1, through its association
with eIF-4E116,117
, and thus loss of the binding protein in response to mTOR activation will lead toenhanced proliferation. Akt is over-expressed (at both mRNA and protein levels) as well as over-
activated (through phosphorylation) in all pituitary tumours, especially NFPAs.118 This up-regulation of
Akt will increase the phosphorylation of p27, preventing its nuclear import and causing at least some of
the changes in the cell cycle discussed above. Therefore, changes in the cell cycle occurring in pituitary
adenomas may be secondary to activation of the Akt pathway.118 It is also salient that constitutive
activation of the TSC1/2 complex, as occurs in tuberous sclerosis, appears to be associated with a risk of
neuroendocrine tumourigenesis, especially insulinomas and Cushings disease.119
The Raf/MEK/ERK pathway is a hierarchical cascade originating at the cell membrane with receptors
for mitogens or growth factors, which recruit, via adapter proteins and exchange factors, the small
guanosine triphosphatase (GTPase) Ras.111 Activated Ras in turn activates Raf (MAPKKK), which is
a serinethreonine kinase. Raf activates mitogen-activated protein kinase kinase (MAPKK), also knownas MEK; MEK, in turn, phosphorylates and activates mitogen-activated protein kinase (MAPK, or ERK1
and ERK2), which translocates to the nucleus and transactivates transcription factors, changing gene
expression to promote growth and mitosis.120 This MAPK pathway activation causes phosphorylation
and activation of ribosomal S6 kinase and transcription factors such as c-myc, Elk1, c-Fos and cyclin
D1121,122, similar to the Akt/mTOR cascade and resulting in the activation of genes associated with
proliferation123 and leading to cell transformation.124 Over-activation of Akt may also lead, via changes
in the kinase GSK3b, to activation of the b-catenin pathway, which has been implicated in pituitary
tumourigenesis.9
The most sensitive Raf, B-Raf, is frequently mutated at the V600E position in melanomas and
papillary thyroid cancer leading to constitutive activity, but it is not similarly mutated in sporadic
pituitary adenomas; however, it is over-expressed in pituitary adenomas, particularly NFPAs.
125
Bcl-associated athanogene (BAG1), which is over-expressed in somatotroph adenomas and non-functioning
NFPAs, may also increase B-Raf activity.87 We have recent preliminary data indicating that MEK1/2 as
well as its down-stream regulator ERK1/2 is also over-phosphorylated, and hence over-activated in all
types of pituitaryadenomas, compared to normal pituitary.126 Over-activation of ERK, an end-product of
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the MAPK pathway, was also associated with increased cyclin D1126, as previously reported.81,127 Future
studies should be focussed on assessing the role of consecutive ERK1/2 down-stream regulators and
possible correlations between B-Raf/MEK/ERK and PI3K/Akt/mTOR pathways, especially as these two
pathways have been shown recently to play an important role not only in malignant transformation but
also in drug resistance.128 Furthermore, activation of at least one type of growth factor receptor, EGFR,
increased proliferation in a cell line model.129 This in turn raises the possibility that tyrosine kinasereceptor antagonists, such as gefitinib, may play a role in the treatment of intractable tumours.
Finally, a very recent study has shown by differential display that a sumoylation factor, now referred
to as RSUME, is over-expressed in pituitary tumours. This agent sumoylates and thereby stabilises HIF-
1a in response to hypoxia, and thereby feeds into an important angiogenic pathway implicated, in
particular, in pheochromocytomas and paragangliomas.130 Study of the interplay between this
pathway and the preceding signalling pathways will be of immense interest in unraveling pituitary
oncogenesis.
In summary, a number of genetic factors, including hormones, growth factors and cell cycle regu-
lators, are believed to act together with different epigenetic events in the process of pituitary tumourformation. Most pituitary tumours are sporadic, but some arise as a component of hereditary
syndromes. Our understanding of these genetic conditions has expanded rapidly due to the identifi-
cation of new predisposing genes, including MEN1, MEN4, PRKAR1A, CDKN1B and AIP, but there is little
information that the mutations occurring in genetic syndromes are common in sporadic tumours.
While significant progress has been made in characterising the molecular basis of pituitary
tumourigenesis, the current evidence does not point towards any of these changes being the primary
event responsible for sporadic tumours. Mutations in recognised tumour suppressor genes and
oncogenes do not seem to play an important role in the great majority of pituitary adenomas. However,
methylation-mediated or -associated gene silencing, in particular of tumour suppressor genes, has
been reported by numerous investigators. Several genes (e.g., PTTG, Pdt-FGFR4, GADD45G, MEG3A,
ZAC, DAP kinase, PTAG and p27) have been implicated to be involved in pituitary tumourigenesis. PTTG(securin) seems to be related to pituitary adenoma invasiveness and aggressiveness. Cell signalling
abnormalities have been identified in pituitary tumours, but their genetic basis is unknown. Both the
PI3K/Akt/mTOR and the Raf/MEK/ERK pathways are over-expressed and/or over-active in many pitu-
itary tumours, which results in the inhibition of cell cycle inhibitors, as may b-catenin and HIF path-
ways. These pathways may share a common root at multiple stages, including initial pathway
activation related to the tyrosine kinase receptor as well as interactions with down-stream regulators,
including c-myc or cyclin D1. For a significant proportion of pituitary tumours, the molecular defect is
not yet elucidated, and more genes for susceptibility are likely to be identified. Modern genetics,
genomics and molecular biology approaches are expected to reveal new mechanisms of endocrine
tumourigenesis aiming at the development of better diagnostic, prognostic and therapeutic tools.
Acknowledgements
Dorota Dworakowska thanks the Polish Science Foundation for a post-doctoral fellowship to Barts
and the London School of Medicine, London, England.
Practice points and research agenda
Pituitary tumours are characterised by over-activity of both the Akt pathway and the MAPK
pathways, and, while this is a finding in many tumour types, it suggests that cell cycle changes
may be secondary to cell signalling abnormalities. There are also changes in the b-catenin
pathway, which is involved in cell contact inhibition through the cadherins. We speculate that
changes at the level of the cell surface growth factor receptors and their modulation by integrins
and cadherins may hold the key to pituitary pathogenesis.
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