Aberrant expression of G1/S regulators is a frequent event in sporadic pituitary adenomas
David J. Simpson,
Simon J. Frost,
John E. Bicknell,
John C. Broome1,,
Anne Marie McNicol2,,
Richard N. Clayton and
William E. Farrell3,
Centre for Cell and Molecular Medicine, School of Postgraduate Medicine, Keele University, North Staffordshire Hospital, Stoke on Trent ST4 7QB,
1 Department of Neuropathology, Walton Centre for Neurology and Neurosurgery, NHS Trust, Lower Lane, Fazakerley, Liverpool L9 7LJ and
2 University Department of Pathology, Glasgow Royal Infirmary, Glasgow G4 0SF, UK
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Abstract
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Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution we have simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.
Abbreviations: CDK, cyclin dependant kinase; IHC, immunohistochemistry; pRb, retinoblastoma protein; TSGs, tumour suppressor genes.
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Introduction
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Human pituitary tumours account for ~1015% of all intracranial tumours (1), however, the precise molecular mechanisms underlying their tumourigenesis has yet to be elucidated. Studies by several groups have described aberrations in both oncogenes and tumour suppressor genes (TSGs) in pituitary tumours (reviewed in refs 2,3). Until recently the only oncogene significantly associated with pituitary tumours was gsp, which encodes a constitutively active
-subunit of the Gs protein (reviewed in 4). However, Zhang et al. (5) have recently described a novel oncogene termed pituitary tumour transforming gene (PTTG) that is overexpressed in the majority of pituitary tumours investigated. Furthermore, Hibberts et al. (6) have shown overexpression of cyclin D1 in sporadic pituitary tumours that was not associated with CCND1 gene amplification. With regard to TSGs, a recent study (7) has shown methylation of the CpG island within the p16 gene, which is associated with loss of p16 protein in non-functioning tumours but not somatotrophinomas. Conversely, loss of retinoblastoma protein (pRb) expression is more frequently associated with somatotrophinomas (8) and is associated with either CpG island methylation or deletion within the protein-binding pocket domain (9).
The ability of a cell to maintain control of its proliferative status is essential for normal growth and homeostasis, with deregulation of genes that are involved in the progression from G1 to S phase of the cell cycle being a frequent event in numerous tumour types, including lung cancer (10), melanoma (11) and oesophageal carcinoma (12). The transition from G1 to S phase is regulated by the interplay between proteins that mediate phosphorylation of pRb. The cyclin D1cyclin dependant kinase (CDK) 4/6 complex phosphorylates pRb, whilst p16, a cyclin-dependent kinase inhibitor, through its interaction with CDK4, inhibits this process. Phosphorylation of pRb results in the release of transcription factors, such as members of the E2F family, which are involved in the activation of genes necessary for entry into S phase. In addition to loss of pRb itself, unchecked phosphorylation of pRb due to loss of p16, overexpression of cyclin D1 or CDK4 may lead to uncontrolled cellular proliferation, a hallmark of tumourigenesis (reviewed in refs 1315).
Whilst some reports would suggest that aberrations of this cell cycle checkpoint are mutually exclusive (14; reviewed in ref. 16), other studies show that more than one component may be targeted in a particular tumour type (10,17). Recent reports have described deregulated expression of pRb (9), p16 (7,18) and cyclin D1 (6) associated with particular pituitary tumour sub-types, however, these studies examined individual components of this transitional pathway in distinct tumour cohorts. In this study of pituitary tumours comprising non-functioning tumours and somatotrophinomas we have determined the coordinate immunohistochemical expression status of pRb, p16 and cyclin D1 and examined the CDK4 gene for a well-characterized mutation. Statistical analysis was then performed to determine any associations between aberrations in these cell cycle regulatory molecules.
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Materials and methods
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Patient material
Twenty-nine clinically non-functioning adenomas (13 non-invasive and 16 invasive) and 16 somatotrophinomas (nine non-invasive and seven invasive) with matched peripheral leukocyte samples were obtained from patients who had undergone hypophysectomy. Tumours were collected retrospectively after standard histological assessment and graded as previously described (19). Routine immunohistochemical assessment showed that the somatotrophinomas stained for growth hormone. The non-functioning adenomas failed to stain for any mature hormone, although in some cases they were positive for the
-subunit. In addition, post-mortem histologically normal pituitaries and matched spleen samples were obtained simultaneously, within 12 h of death, and stored at 20°C.
Tissue and DNA preparation
Sections (5 µM) were taken from pituitary tumours and histologically normal post-mortem pituitaries that had been formalin fixed and paraffin embedded. A single section was subjected to haematoxylin and eosin staining to allow tumour identification and subsequent microdissection of tissue from the remaining unstained sections. DNA was then extracted using proteinase K extraction as previously described (19). Leukocyte DNA was extracted from matched blood samples using commercially available reagents (Nucleon 1; Scotlab, Strathclyde, UK).
Mutational analysis of codon 24 within the CDK4 gene
Wolfel et al. (20) reported a presumed activating mutation resulting from an Arg
Cys amino acid substitution at codon 24 (R24C) within the CDK4 gene, resulting from a single base pair mutation at nt 204 (numbering according to GDB Z48970). Tumour and matched blood DNA was subjected to PCR amplification of codon 24 and the surrounding region using gene-specific oligonucleotide primers (sense, 5'-GCTACCTCTCGATATGAGC-3'; antisense, 5'-ACTCTTGAGGGCCACAAAG-3'). PCR was carried out in 25 µl volumes with 1.5 mM MgCl2, 200 µM each dATP, dCTP, dGTP and dTTP, 2 pmol each primer and 1 U Taq DNA polymerase. Amplification occurred using annealing temperatures of 60°C for 3 cycles, 58°C for 12 cycles, 56°C for 12 cycles and 54°C for 13 cycles, with extension of PCR products and denaturation of template DNA carried out at 72°C and 94°C, respectively.
The single nucleotide substitution within codon 24 creates a recognition site for the restriction endonuclease StuI. The resulting PCR product (10 µl) was digested with 2 U StuI for 3 h at 37°C. The digested products (CC genotype, 105 bp; CT genotype, 105, 64 and 41 bp; TT genotype, 64 and 41bp) were electrophoresed on 8% non-denaturing polyacrylamide gels, fixed in 10% methylated spirit, 0.5% acetic acid for 6 min and then incubated in 0.1% aqueous silver nitrate for 15 min. Following two brief washes in distilled water, the products were visualized by development in 1.5% sodium hydroxide, 0.1% formaldehyde.
Direct sequencing of CDK4 gene
In addition to the PCR/enzyme digestion method we also analysed 10 randomly chosen sporadic pituitary adenomas for mutations within codon 24 and the surrounding area using a direct sequencing approach. PCR amplification using gene-specific oligonucleotides was carried out as described above, PCR amplicons were then subjected to cycle sequencing reactions according to the manufacturer's instructions (Big Dye Terminator cycle sequencing kit; PE Applied Biosystems, Warrington, UK). Cycle sequencing products were subjected to capillary electrophoresis using an automatic 310 Genetic Analyser (PE Applied Biosystems) and analysed using Sequencing Analysis v.3.0 software (PE Applied Biosystems).
Immunohistochemistry (IHC)
The expression status of pRb and cyclin D1 were analysed in a pituitary tumour cohort (n = 45), previously examined for p16 expression (7) and p16 immunoreactivity was assessed according to the criteria of Geradts et al. (21). In accordance with previous publications, low level cytoplasmic staining, in some cases accompanied by focal nuclear staining, was interpreted as negative (7,21). In addition, a proportion of the tumours had previously been examined for the expression status of pRb (8) and cyclin D1 (6), however, in order to fully investigate coordinated aberrations within the pRb/p16/cyclin D1 pathway tumours were subjected to IHC using the appropriate antibody as described below.
pRb IHC
Archival sections were deparaffinized, rehydrated and underwent antigen retrieval by pressure cooker/microwaving on full power in a citrate buffer (pH 6.0) in a 750 W microwave oven for 5 min. The primary antibody (Rb clone 1F8; Novocastra Laboratories, Newcastle, UK) was incubated withtissue sections for 1 h as previously described (8). The monoclonal antibody recognizes hypo- and hyperphosphorylated forms of pRb protein and was generated to recognize pRb in paraffin embedded archival material. The secondary antibody and peroxidase steps were carried out using a commercially available kit according to the manufacturer's protocol (Large volume LSAB kit; Dako, Buckinghamshire, UK). Sites of binding were visualized using diaminobenzidine as chromogen. Negative controls were substitution of mouse immunoglobulin for the primary antibody and omission of the primary antibody. A malignant melanoma was used as a positive control. Immunoreactivity was scored according to the criteria of Geradts et al. (22). Nuclear staining alone was determined as positive, with cytoplasmic staining deemed non-specific.
Cyclin D1 IHC
Archival sections were deparaffinized, rehydrated and underwent antigen retrieval by microwaving on full power in a 600 W microwave oven for 6 min. IHC was then performed with the Vectastain Universal Elite ABC kit (Vector laboratories, UK), using a mouse monoclonal cyclin D1 antibody (DCS-6; Novocastra), as previously described (6). The antibody is specific for cyclin D1 and does not show reactivity with related cyclins (D2 and D3), whilst the epitope recognized by the antibody is conformation dependent. Diaminobenzene was used as the chromogen and the resulting sections were examined blind, without any prior knowledge of pituitary sub-type or grade and classified according to the following protocol: , 010% cell stained positive; +, >10100% cells stained positive. Cyclin D1 immunopositivity was defined as those tumours showing either nuclear and/or cytoplasmic staining. A positive control (human tonsil), a negative control (pituitary tumour with the primary antibody omitted) and normal human pituitary were used for each series of staining.
Statistical analysis
The inter-relationship between the expression status of pRb, p16 and cyclin D1 and clinicopathological features was determined using the Stata v.5 statistical package (Stata Corp., TX). Significance was taken at the 5% level.
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Results
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CDK4 mutational analysis
The total pituitary tumour cohort (n = 45) was analysed for the R24C mutation within the CDK4 gene (20). PCR amplification using CDK4-specific oligonucleotides followed by digestion with the restriction endonuclease StuI showed no mutation of nt 204 in any of the tumours examined. In addition, 10 randomly selected tumours were subjected to direct sequence analysis of codon 24. No mutations within codon 24 or the surrounding region were found in any of the tumours examined.
pRb IHC
Overall, 10/45 (22%) pituitary tumours failed to express pRb. Within the pituitary tumour sub-types examined 5/29 (17%) non-functioning tumours and 5/16 (31%) somatotrophinomas failed to express detectable levels of pRb (Tables I and II
). pRb expression was observed in all the histologically normal pituitaries examined (n = 15). Figure 1A
shows a histologically normal pituitary and Figure 1B
a pituitary tumour, both exhibiting pRb immunopositivity. Figure 1C
shows a pituitary tumour that failed to express pRb; the few cells that were positive for pRb (arrows) most likely represent mesenchymal, inflammatory or normal pituitary cells.
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Table I. Summary of pRb, p16 and cyclin D1 expression status as assessed by IHC in 45 sporadic human pituitary tumours
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Table II. Summary of association of aberrations in pRb, p16 and cyclin D1 expression in the total tumour cohort, non-functioning adenomas and somatotrophinoma sub-types
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Fig. 1. Immunohistochemical analysis of pRb, p16 and cyclin D1 expression in sporadic human pituitary tumours and histologically normal post-mortem pituitaries. (A) Normal pituitary showing a heterogeneous pattern of nuclear staining for pRb. (B) Pituitary adenoma showing positive nuclear staining for pRb. (C) Pituitary adenoma in which the vast majority of nuclei are negative for pRb. The few positive cells (arrows) may represent mesenchymal cells or entrapped normal pituitary cells. (D) Normal pituitary showing a heterogeneous pattern of nuclear staining for p16. (E) Pituitary adenoma showing positive nuclear staining for p16. The intensity of staining is variable but in general less intense than normal pituitary. (F) Pituitary adenoma in which the vast majority of nuclei are negative for p16. The few positive cells (arrows) may represent mesenchymal or entrapped normal pituitary cells. (G) Absence of cyclin D1 staining in a normal pituitary. (H) Positive nuclear immunostaining for cyclin D1 is accompanied by lower level cytoplasmic staining in a pituitary tumour. (I) Cytoplasmic immunoreactivity for cyclin D1 is seen in a pituitary tumour. The expression status of p16 for the total pituitary tumour cohort was previously reported (7), whilst the expression status of 43 tumours for pRb (8) and 24 tumours for cyclin D1 (6) were also previously reported.
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p16 IHC
Of the total tumour cohort 23/45 (51%) tumours failed to express p16. Loss of expression was found more frequently in non-functioning tumours (19/29, 66%) compared with somatotrophinomas (4/16, 25%) (Tables I and II
). p16 expression was observed in all the histologically normal pituitaries examined (n = 15), with 5060% of the cells throughout the section showing immunopositivity. Figure 1D
shows a histologically normal pituitary and Figure 1E
a pituitary tumour, both exhibiting p16 immunopositivity. In agreement with other reports (7,21), the intensity of staining in the tumour was in general less intense than in normal pituitary. Figure 1F
shows a pituitary tumour which failed to express detectable levels of p16; the few positive cells (arrows) may represent mesenchymal cells or entrapped normal pituitary cells.
Cyclin D1 IHC
Overexpression of cyclin D1 was demonstrated in 22/45 (49%) tumours, with cyclin D1 immunopositivity observed more frequently in non-functioning tumours (17/29, 59%) compared with somatotrophinomas (5/16, 31%) (Tables I and II
). As previously reported (6), sub-cellular positivity was variable between the cytoplasmic and nuclear components. No cyclin D1 immunopositivity was observed in any of the histologically normal pituitaries examined (n = 6). Figure 1G
shows a histologically normal pituitary showing lack of cyclin D1 staining, together with representative examples of predominantly nuclear (Figure 1H
) and cytoplasmic (Figure 1I
) staining in pituitary tumours.
Relationship between expression status of pRb, p16 and cyclin D1
In the total pituitary tumour cohort (n = 45) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36/45 (80%) tumours, with the non-functioning tumours (27/29, 93%) demonstrating a significantly (P = 0.005) higher proportion of tumours with an aberration compared with somatotrophinomas (9/16, 56%). Seventeen tumours (five somatotrophinomas and 12 non-functioning tumours) demonstrated at least two aberrations in this pathway, whilst two non-functioning tumours showed abnormal expression of all three cell cycle regulators (Table II
). There was no significant association between tumour grade and abnormal expression of any of the cell cycle regulators examined.
Within the total tumour cohort loss of p16 and/or pRb expression was evident in 28/45 (62%) tumours. However, analysis showed these losses to be mutually exclusive, since only five of the 28 (18%) tumours showed coordinated loss of both pRb and p16 expression (Tables I and II
). Finally, since overexpression of cyclin D1 was found in a significant proportion of tumours (22/45, 49%), we analysed its expression status relative to pRb and p16. No significant association between cyclin D1 overexpression and the expression status of either pRb or p16 was observed.
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Discussion
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Transition from G1 to S phase of the cell cycle is regulated by mediators that result in pRb hyperphosphorylation, with deregulation of this pathway reported in numerous tumour types (reviewed in refs 15,23,24). Recent studies (6,7,9) have suggested that the individual components of the pRb/p16/cyclin D1 pathway are associated with different pituitary tumour sub-types. While abnormal expression of p16 and cyclin D1 was observed more frequently in non-functioning tumours, loss of pRb expression is more frequently associated with somatotrophinomas. Our current study has highlighted the importance of individual components of this transition checkpoint in pituitary tumours. Eighty per cent of tumours investigated showed abnormal expression of either pRb, p16 or cyclin D1. Furthermore, deregulation of components of this pathway was more frequent in non-functioning tumours (93%) compared with somatotrophinomas (56%).
Mutually exclusive loss of pRb or p16 has been demonstrated in a number of tumour types (14,2527) and our data for sporadic pituitary tumours is in accord with these findings. Since several studies suggest that functional pRb is necessary for cell cycle inhibition by p16, it has been postulated that there would be no growth advantage to a tumour cell losing p16 subsequent to loss of pRb (14,28). However, Gourgoulis et al. (26) and Hangaishi et al. (29) have both suggested that p16 inactivation might precede loss of pRb, with loss of pRb expression giving a further growth advantage to the tumour cell by cancelling the growth inhibitory effects of other cyclin-dependent kinase inhibitors. Since few tumours in the total cohort showed concomitant aberrations in both pRb and p16, it was not possible to show an association with a more invasive tumour phenotype.
In a number of malignancies, including breast (25) and oesophageal carcinomas (12), it has been shown that cyclin D1 overexpression is more frequently observed in those tumour cells with normal pRb immunopositivity. Indeed, our data shows that 82% of tumours which overexpress cyclin D1 also express pRb. These findings are consistent with the consensus opinion that overexpression of cyclin D1 may lead to increased phosphorylation of pRb (12,30). Therefore, concomitant deregulation of pRb and cyclin D1 would not necessarily lead to any growth advantage to the tumour cells (31). However, concomitant aberrant expression of cyclin D1 and pRb has been shown in a number of tumours, including non-small cell lung (10) and breast cancer (25). A small proportion of tumours examined in our current study of pituitary tumours showed concomitant deregulated expression of both cyclin D1 and pRb. Indeed, Shapiro et al. (32) and Marsh and Varley (33) suggested that overexpression of cyclin D1 is an early event in tumorigenesis responsible for initial clonal expansion of the tumour, whilst subsequent loss of pRb expression results in complete disruption of this G1 checkpoint, thereby giving an additional growth advantage to the tumour cell.
We did not observe a significant association between abnormal expression of p16 and cyclin D1, with an approximately equal number of tumours showing aberrations in either one or both (concomitant) components of this pathway. Similar findings have been reported in other tumour types (10,17). Lukas et al. (34) reported that deregulation of both p16 and cyclin D1 in combination provided a greater growth advantage to the tumour cell than either would singly, however, we were unable to confirm this in the present study.
Using both a PCR/restriction digestion technique and direct sequencing we did not detect any point mutations within codon 24 of the CDK4 gene. A single C
T nucleotide change results in an amino acid substitution from Arg to Cys at codon 24 and has been identified in a number of reports (20,3537). This mutation, which inhibits binding of p16 to CDK4 while allowing the formation of an active kinase complex with cyclin D1, supports the concept that CDK4 is a proto-oncogene and has the potential to disrupt the pRb regulatory pathway. In addition to mutation, other studies have reported amplification of the CDK4 gene (27,38,39), however, in common with the cyclin D1 gene, amplification is not associated with overexpression of the gene product (6,27).
Aberrations of one or more components of the pRb/p16/cyclin D1/CDK4 pathway, as demonstrated by our current study, are a frequent event (80%) in sporadic human pituitary tumours, suggesting that this regulatory pathway may present a useful target in drug or gene therapeutic approaches. A number of compounds that antagonize the cyclin-dependent kinases have been studied in detail and are undergoing clinical trials (24). Frost et al. (40) have demonstrated that transfection of full-length human p16 cDNA into a mouse pituitary tumour cell line homozygous deleted for the p16 gene results in an increased number of cells in G1 cell cycle arrest. Furthermore, other studies have shown that demethylation of silenced tumour suppressor genes, leading to re-expression of their products (41), is also possible. It will therefore be of benefit to patients for their clinical management if such targeted drug design or gene therapy can provide a viable alternative to surgical intervention.
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Notes
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3 To whom correspondence should be addressedEmail: w.e.farrell{at}keele.ac.uk 
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Received September 14, 2000;
revised April 2, 2001;
accepted April 17, 2001.