Differential expression of menin in sporadic pituitary adenomas

Differential expression of menin insporadic pituitary adenomas

M Theodoropoulou1, I Cavallari 2, L Barzon 3, D M D’Agostino 2, T Ferro 2,T Arzberger 4, Y Grubler 1, L Schaaf 1, M Losa 5, F Fallo 3, V Ciminale 2,G K Stalla1 and U Pagotto 6

1Max Planck Institute of Psychiatry, Neuroendocrinology Group, 80804 Munich, Germany2Department of Oncological and Surgical Sciences, University of Padova, Padova, Italy3Department of Medical and Surgical Sciences, Division of Endocrinology, University of Padova, Padova, Italy4Institute of Pathology, Division of Neuropathology, University of Wurzburg, Wurzburg, Germany5Neurosurgical Department, San Raffaele Hospital, Milan, Italy6Endocrine Unit, Department of Internal Medicine and Gastroenterology and Center for Applied Biomedical

Research (C.R.B.A.), S. Orsola-Malpighi General Hospital, Via Massarenti 9, 40125 Bologna, Italy

(Requests for offprints should be addressed to M Theodoropoulou; Email: [email protected])

Abstract

Pituitary adenomas represent one of the key features of multiple endocrine neoplasia type 1. The geneinvolved in this syndrome (MEN1) is a putative tumor suppressor, that codes for a 610-amino acidnuclear protein termed ‘menin’. Analyses of sporadic pituitary adenomas have so far failed to revealMEN1 mutations or defects in MEN1 transcription in these tumors. In the present study we detectedmenin protein expression in a panel of normal and tumoral pituitary tissues, using a monoclonalantibody against the carboxy-terminus of menin. In the normal human pituitary gland, strong nuclearstaining for menin was detectable in the majority of the endocrine cells of the anterior lobe, without aclear association with a particular hormone-producing type. In sporadic pituitary adenomas, meninexpression was variable, with a high percentage of cases demonstrating a significant decrease inmenin immunoreactivity when compared with the normal pituitary. Interestingly, metastatic tissuesderived from one pituitary carcinoma had no detectable menin levels. Altogether, our data provide thefirst information regarding the status of menin expression in human normal and neoplastic pituitary asdetermined by immunohistochemistry (IHC).

Endocrine-Related Cancer (2004) 11 333–344

Introduction

Pituitary adenomas are common neoplasms representing

approximately 10% of intracranial tumors, which can

arise sporadically or as part of the hereditary syndrome

multiple endocrine neoplasia type 1 (MEN1). This

autosomal dominant disorder is characterized by neuro-

endocrine tumors of the pituitary gland, parathyroid,

pancreas and duodenum, and, less frequently, by tumors

of the adrenal and thyroid glands and by angiofibromas,

leiomas and lipomas (Marx et al. 1999, Pannett &

Thakker 1999). The gene mutated in MEN1 patients

was mapped to chromosome 11q13 (Larsson et al. 1988)

and was identified by positional cloning (Chandrasek-

harappa et al. 1997, Lemmens et al. 1997). It encodes a

610-amino acid protein termed ‘menin’, which has no

homology to any known protein nor any recognized

functioning motifs. Menin is widely expressed in most

adult tissues (Chandrasekharappa et al. 1997) and is

predominantly located in the nucleus (Guru et al. 1998).

Menin is known to interact with the JunD transcrip-

tion factor (Agarwal et al. 1999), Smad3, a key effector in

the TGF-b signal transduction pathway (Kaji et al. 2001),

the putative tumor metastasis suppressor nm23 (Ohkura

et al. 2001), NF-kappaB (Heppner et al. 2001), glial

fibrillary acidic protein and vimentin (Lopez-Egido et al.

2002), and replication protein A (Sukhodolets et al. 2003).

Functional studies indicated that menin uncouples JunD,

c-Jun, and Elk1 phosphorylation from MAPK, thereby

inhibiting active Fos/Jun accumulation (Gallo et al. 2002).

Menin suppresses the RAS-mediated tumor phenotype

(Kim et al. 1999), and its expression might be subjected to

cell-cycle regulation (Kaji et al. 1999). In addition, menin


Endocrine-Related Cancer (2004) 11 333–344 Online version via http://www.endocrinology.org

1351-0088/04/011–333 # 2004 Society for Endocrinology Printed in Great Britain

was found to inhibit DNA synthesis under DNA-

damaging conditions (Ikeo et al. 2000).

These properties, together with the fact that MEN1

mutations are of the loss-of-function type and that the

wild-type allele is lost in tumors derived from MEN1-

affected patients, identify menin as a tumor suppressor.

Indeed, MEN1þ/� mice develop parathyroid and pitui-

tary tumors and insulinomas (Crabtree et al. 2001).

Despite extensive studies, the mechanisms underlying

the pathogenesis of pituitary adenomas are not yet fully

understood. Few defects have been demonstrated in

oncogenes and tumor-suppressor genes that are known

to be involved in the development of other types of cancer

(Asa & Ezzat 1998, Farrell & Clayton 2000). Oncogenic

mutations have been found in the gsp gene in a subset of

acromegalic-associated tumors (Vallar et al. 1987), while a

small number of tumor suppressor genes, such as p16

(Simpson et al. 1999), p27/Kip1 (Bamberger et al. 1999,

Lidhar et al. 1999) and ZAC (Pagotto et al. 2000), were

found to be downregulated in other types of pituitary

adenomas.

In this study, we investigated whether menin protein

expression is altered in pituitary tumors, using a new

monoclonal antibody raised against the C-terminus of

menin (Cavallari et al. 2003). The menin expression

pattern was determined in the human normal pituitary

gland and in a panel of sporadic benign and metastatic

pituitary adenomas.

Materials and methods

Patients and tissues

Seven normal human pituitary glands from autopsy cases

of sudden death without any evidence of endocrine

diseases taken 8–12 h after demise, 68 sporadic benign

pituitary adenomas (21 acromegaly-associated pituitary

adenomas (ACRO), eight corticotrophinomas (CUSH),

six prolactinomas (PROL) and 33 nonfunctioning pitui-

tary adenomas (NFPA)) and one prolactin (PRL)-

producing pituitary carcinoma were included in this

study. The pituitary adenomas were characterized on the

basis of their clinical, radiologic, surgical and immuno-

histologic diagnosis and graded according to a modified

Hardy’s classification (Bates et al. 1997). All patients with

sporadic pituitary adenomas had normal calcium and

glucose levels, had no known family history of MEN1 and

did not present any of the two other major MEN1 lesions

(hyperparathyroidism and enteropancreatic tumor) (Pan-

nett & Thakker 2001, Verges et al. 2002). In the case of the

PRL-producing pituitary carcinoma, three sequential

biopsies were obtained; the first after transphenoidal

adenomectomy, the second 6 months later after transcra-

nial surgery, and the third 3 years later upon autopsy,

with samples taken from a parasellar invasion and

metastases localized in the orbital, medulla oblongata

and femural bone (Winkelmann et al. 2002). All tissues

were snap-frozen at �808C. Informed consent was

obtained from all living patients or from their relatives.

Genetic analysis

Peripheral blood for genetic analysis was available from

12 out of 68 patients with sporadic pituitary adenoma.

DNA was isolated from leukocytes and frozen pituitary

adenoma tissues by proteinase K digestion and the

phenol/chloroform method. To screen for mutations in

the MEN1 gene, exons 2–10 and the neighboring splice

junctions of the MEN1 gene were amplified by PCR as 15

partially overlapping fragments (Debelenko et al. 1997).

PCR was carried out in 25 ml reaction volumes containing

1.5mM MgCl2, 200 mM each dNTP, 500 mM each primer,

template DNA and 0.5U Taq DNA polymerase. An

initial denaturation step at 94 8C for 5min was followed

by 35 cycles at 94 8C for 1min, 62 8C for 1min, 72 8C for

1min, and a 7-min extension at 72 8C. PCR products were

separated through nondenaturing polyacrylamide gel, and

individual bands were purified and sequenced on both

strands with an ABI PRISM 310 DNA sequencer and the

ABI PRISM BigDye Terminator Cycle Sequencing Kit

with AmpliTaq DNA Polymerase FS (Perkin Elmer,

Foster City, CA, USA). Sequencing data were analyzed

with the Sequencing Analysis 3.0 computer program

(Perkin Elmer) and compared with the MEN1 sequence

reported in the GenBank database (accession number

U93237).

DNA from leukocytes and tumor tissue was screened

for LOH with the intragenic microsatellite marker

D11S4946 and two flanking 11q13 markers, PYGM and

D11S4933. Primer sequences were obtained from the

Genome Database. PCR was performed as described

above, and PCR products were resolved on 6% denatur-

ing polyacrylamide gels and visualized with silver staining.

Complete or nearly complete (90% decrease in intensity)

absence of an allele was interpreted as LOH. The common

C<T (D418D) polymorphism in exon 9 of the MEN1

gene was also utilized to assess LOH.

Immunohistochemistry (IHC)

A recently described monoclonal antibody (mAb C126)

generated against the 126-amino acid C-terminal portion

of menin (Cavallari et al. 2003) was used to assess

expression of menin in normal pituitary glands, 68

sporadic pituitary adenomas and metastases derived

from the carcinoma. The specificity of the antibody was

confirmed by Western blot and IHC on cells transfected

with menin-expressing plasmid, as we showed in our

Theodoropoulou et al: Expression of menin in sporadic pituitary adenomas

334 www.endocrinology.org

previous work (Cavallari et al. 2003). A further indirect

proof of the mAb C126 specificity was obtained when it

failed to detect any immunoreactivity in a familiar MEN1

gastrinoma which carried a germline 11bp deletion in

exon 9 of the MEN1 gene and had lost the second MEN1

allele, as demonstrated previously by our recent report

(Cavallari et al. 2003).

Frozen tissues were cut into 8 mm sections, fixed in

phosphate-buffered 4% paraformaldehyde and dehy-

drated. They were either analyzed immediately or stored

in 96% ethanol for no longer than 24 h; in fact, as

previously reported (Cavallari et al. 2003), prolonged

storage after paraformaldehyde treatment resulted in a

substantial decrease in menin immunoreactivity. IHC was

performed as follows: sections were incubated in a 1 : 10

dilution of normal goat serum for 30min at room

temperature and then treated with the Vector Avidin/

Biotin Blocking Kit (Vector Lab, Burlingame, CA, USA)

according to the manufacturer’s instructions. The sections

were then incubated overnight at 4 8C with the 1 : 100

dilution of mAb C126, followed by sequential 30-min

incubations at room temperature with biotinylated goat

antimouse immunoglobulin G (1 : 300, Vector Lab) and

avidin–biotin–peroxidase complex (Vectastain Elite Kit,

Vector Lab); sections were washed three times in TBS for

5min between all steps. Reactivity was detected using

diaminobenzidine (DAB, 1mg/ml) as chromogen and

0.01% H2O2 as substrate. Sections were then counter-

stained with toluidine blue, dehydrated and mounted with

Entellan (Merck, Darmstadt, Germany).

The integrity of the tissues was tested by IHC using

antibodies against the nuclear protein Pit-1 (rabbit antirat

Pit-1, 1 : 100; Santa Cruz, Heidelberg, Germany) in the

case of ACRO and PROL, steroidogenic factor 1 (rabbit

antimouse SF-1, 1 : 300; Upstate Biotechnology, Lake

Placid, NY, USA) for NFPA and ZAC for CUSH, since

we have shown in a previous report that this transcription

factor is highly expressed in corticotrophinomas (Pagotto

et al. 2000). The hormone content was assessed in normal

pituitaries and pituitary tumors, using the following

mouse monoclonal antibodies: antihuman b-follicle-stimulating-hormone (FSH) (1 : 800), antihuman b-lutei-nizing hormone (LH) (1 : 800), antihuman b thyrotropin

stimulating hormone (TSH) (1 : 800), antihuman PRL

(1 : 400), antihuman a-subunit (a-sub) (1 : 500) (all pur-

chased from Immunotech, Karlsruhe, Germany), antihu-

man adrenocorticotropin hormone (ACTH) (1 : 100),

(Dako Diagnostika, Hamburg, Germany) and antihuman

growth hormone (GH) (1 : 800; a gift from Dr CJ

Strasburger). Double IHC was performed as follows:

after completing the IHC for menin, sections were

extensively washed and incubated with pituitary hormone

antibodies. The following day, sections were incubated

with antimouse immunoglobulin G (1 : 100; Sigma,

Deisenhofen, Germany) and mouse alkaline phosphatase

(AP)–anti–AP complex (1 : 50; Sigma) for 1 h each.

Immunoreactivity was detected with Vector Red for 40

min in the presence of 10 mmol/l levamisole (Sigma).

Menin immunoreactivity was observed by two inde-

pendent investigators. The intensity pattern was classified

into four categories: absent (0), weak (þ), moderate (þþ)

and strong (þþþ). The specificity of the IHC pattern

generated by the mAb C126 was verified by a competition

assay with recombinant GST-C126men, the immunogen

used to generate mAb C126; in brief, replica sections were

incubated for 30min at room temperature with the mAb

C126 in the presence of either 25 mg of purified GST-

C126men or of 25mg of purified unfused GST. Negative

controls were carried out with mouse nonimmune ascites

(in the case of menin staining), or omitting the primary

antibody.

Immunofluorescence/laser scanningmicroscopy (IF/LSM)

Menin expression was also studied by IF/LSM in 16 out

of 68 sporadic pituitary adenoma cases (six ACRO, two

CUSH and eight NFPA). IF/LSM was performed by

incubating the tissues with normal goat serum (1 : 70) for

30 min at room temperature and then overnight at 4 8Cwith a 1 : 80 dilution of C126 mAb, followed by a 1 : 500

dilution of Alexa 488-conjugated antimouse secondary

antibody (Molecular Probes, Eugene, OR, USA). Dual

labeling experiments were carried out by staining with

0.1mg/ml propidium iodide (PI). LSM was carried out

with a Zeiss LSM 510 microscope, using argon (488 nm)

and helium–neon (543 nm) laser sources. Images were

obtained using 10�, 20� or 63� objectives with the

digital zoom set at either 1� ð512� 512 pixels) or

2� ð1024� 1024 pixels). Laser intensity, pinhole aper-

ture, and photomultiplier parameters were standardized

to enable comparison of signals obtained in different

samples. Fluorescence signals were analyzed using a 505–

530 nm band-pass filter for Alexa 488 and a long-pass

560 nm filter for the PI.

Results

Analysis of MEN1 gene mutations

Paired tumor and leukocyte DNA samples were available

from 12 patients with sporadic pituitary adenomas. All

the patients were informative for at least one locus

analyzed at 11q13. Loss of heterozygosity was found in

four cases (nos 6, 9, 43 and 44; Table 1), one of which

exhibited chromosomal instability (no. 6 in Table 1),

determined by examining three different loci (data not


www.endocrinology.org 335

Table 1 List of the 68 tumors used for the menin study plus the tissues derived from pituitary carcinoma (no. 69: 1st intervent; no.

70: 2nd intervent: no. 71 parasellar invasion; no. 72 orbital invasion: no. 73 metastasis to medulla oblongata; no. 74 metastasis to

femural bone. The samples nos. 71–74 were taken at autopsy). Information is given about age, sex and clinical diagnosis of each

patient. Tumor grade is given in column (G). In the column (LOH) are listed the data concerning allelic status as determined using

four informative markers. In the column (IHC) are listed the findings of the immunohistochemical examination for the five hormones

and a-subunit in each case. Menin immunoreactivity (menin ir) was determined by two independent investigators and categorized in

four classes: (0): no menin ir; (þ) weak ir; (þþ) moderate ir; (þþþ) strong ir. Menin immunofluorescence (menin IF) was also

determined in a small number of tumors and categorized in the same way as menir ir

Age/sex Diagnosis IHC GLOH

Menin ir Menin IF

PYGM D11S4946 D11S4933 D418D

1. 28/M ACRO GH III – – – – ++ n.d.

2. 43/M ACRO GH/PRL III ROH NI NI NI + n.d.

3. 44/F ACRO GH II – – – – +++ n.d.

4. 35/F ACRO GH III – – – NI ++ n.d.

5. 36/M ACRO GH/PRL II – – – – + n.d.

6. 33/M ACRO GH III LOH LOH NI LOH + n.d.

7. 31/F ACRO GH III NI NI NI – ++ n.d.

8. 64/F ACRO GH/a-sub/FSH III – – – – + ++

9. 65/F ACRO GH III LOH – – LOH + n.d.

10. 50/M ACRO GH/PRL III – – – – + ++

11. 64/M ACRO GH/PRL III ROH NI NI – +++ n.d.

12. 36/F ACRO GH/PRL III – – – – ++ n.d.

13. 64/M ACRO GH/PRL II – – – – +++ n.d.

14. 80/M ACRO GH/PRL II – – – – + n.d.

15. 42/F ACRO GH/PRL/a-sub III – – – – +++ n.d.

16. 32/M ACRO GH/PRL II – – – – ++ n.d.

17. 58/F ACRO GH/PRL II – – – – +++ +++

18. 52/F ACRO GH/FSH/LH II – – – – + n.d.

19. 38/F ACRO GH/PRL/TSH/a-sub II – – – – ++ ++

20. 75/F ACRO GH/PRL II – – – – + +

21. 18/M ACRO GH/PRL/a-sub III – – – – +++ +++

22. 30/M CUSH ACTH I – – – – ++ n.d.

23. 16/M CUSH ACTH I – – – – +++ n.d.

24. 24/F CUSH ACTH II – – – – ++ n.d.

25. 62/F CUSH ACTH II – – – – + n.d.

26. 34/M CUSH ACTH I – – – – +++ n.d.

27. 24/F CUSH ACTH II – – – – + n.d.

28. 64/F CUSH ACTH III – – – – ++ ++

29. 31/F CUSH ACTH I – – – – +++ +++

30. 44/F PROL PRL II – – – – ++ n.d.

31. 63/M PROL PRL III – – – – ++ n.d.

32. 42/F PROL PRL III – – – – + n.d.

33. 48/F PROL PRL III – – – – + n.d.

34. 37/F PROL PRL III – – – – + n.d.

35. 43/F PROL PRL III NI ROH – NI ++ n.d.

36. 42/M NFPA ACTH III – – – – ++ n.d.

37. 69/M NFPA ACTH II – – – – + n.d.

38. 51/F NFPA a-sub II – – – – ++ n.d.

39. 63/M NFPA LH/a-sub II – – – – ++ n.d.

40. 51/F NFPA FSH/LH II – – – – ++ n.d.

41. 72/M NFPA FSH/LH II – – – – +++ n.d.

42. 61/M NFPA FSH II – – – – +++ n.d.

43. 46/M NFPA FSH III – – – LOH + +

44. 28/M NFPA FSH III LOH LOH NI – + +

45. 50/M NFPA FSH III – – – – ++ n.d.

46. 35/M NFPA FSH III – – – – + n.d.

47. 76/M NFPA a-sub/FSH/LH II – – – – +++ n.d.

48. 31/F NFPA FSH II ROH ROH NI NI + n.d.

49. 37/F NFPA a-sub/FSH/LH III – – – – ++ n.d.



shown). Sequencing of the second allele in these cases

revealed no abnormalities in the MEN1 gene. The

remaining eight patients showed no abnormalities in the

11q13 locus.

Menin expression in normal human pituitary

Immunohistochemical examination of seven normal

human pituitary glands revealed strong menin immuno-

reactivity in almost all the endocrine cells of the anterior

lobe (Fig. 1A) and in some pituicytes of the posterior lobe

(data not shown). The signal was nuclear, in agreement

with previous observations in transfected cells (Guru et al.

1998) and in pancreatic tissues (Cavallari et al. 2003). The

specificity of the signal was determined by using mouse

preimmune ascites instead of mAb C126 (Fig. 1B) and by

preabsorbing the mAb C126 antibody with the immuno-

gen GST-menin (Fig. 1C).

To determine whether menin is differentially

expressed in pituitary cells, we carried out double IHC;

results revealed menin immunoreactivity in all types of

hormone-producing cells (ACTH in Fig. 1E and PRL in

Fig. 1F; for the other hormones, data not shown),

indicating that its expression is not restricted in one

particular cell population. The nuclei of the fibroblasts

and endothelial cells were devoid of menin immuno-

reactivity (Fig. 1A and D).

Menin expression in pituitary adenomas

IHC analysis of 68 sporadic pituitary adenomas revealed

the same nuclear distribution as in normal pituitary tissue.

Although all the endocrine cells in each tumor expressed

menin, our analysis revealed ample fluctuations in the

intensity of the immunoreactivity, which varied from

levels comparable to those detected in normal pituitary

(considered as strong, þþþ; NP2 and 3 in Fig. 2A and B)

to an almost undetectable signal (considered as weak, þ;

Table 1). Fifteen out of 68 sporadic pituitary adenomas

examined had a strong menin signal (scored as þþþ, as in

Fig. 2C), 22 displayed an intermediate level of expression

(þþ, as in Fig. 2D), and 31 had weak menin immuno-

reactivity (þ, as in Fig. 2E and F). The levels of menin

expression correlated neither with the histologic and

clinical features of the tumor nor with the grade and

invasiveness (Table 1). In analogy to what was observed

Table 1 continued

Age/sex Diagnosis IHC GLOH

Menin ir Menin IF

PYGM D11S4946 D11S4933 D418D

50. 65/M NFPA FSH III – – – – + n.d.

51. 46/F NFPA LH II – – – – + n.d.

52. 53/M NFPA FSH/LH/a-sub II – – – – ++ n.d.

53. 77/F NFPA FSH/LH/a-sub II – – – – +++ +++

54. 63/M NFPA FSH/a-sub/LH II – – – – + +

55. 29/F NFPA FSH/a-sub III – – – – + +

56. 49/F NFPA LH III – – – – + +

57. 42/F NFPA None II – – – – ++ ++

58. 48/M NFPA None III – – – – ++ n.d.

59. 77/M NFPA None II – – – – +++ n.d.

60. 70/M NFPA None III – – – – + n.d.

61. 63/F NFPA None II – – – – + n.d.

62. 69/F NFPA None II – – – – + n.d.

63. 52/F NFPA None III – – – NI + n.d.

64. 49/M NFPA None III – – – – + n.d.

65. 59/F NFPA None III – – – – + +++

66. 60/M NFPA None II NI – – – ++ n.d.

67. 51/F NFPA None III – – – – + n.d.

68. 73/F NFPA None II – – – – +++ n.d.

69. 50/M 1st intervent PRL IV – – – – + n.d.

70. 50/M 2nd intervent PRL IV – – – – + n.d.

71. 53/M Parasellar PRL IV – – – – 0 n.d.

72. 53/M Orbit PRL IV – – – – 0 n.d.

73. 53/M Medulla PRL IV – – – – 0 n.d.

74. 53/M Femur PRL IV – – – – 0 n.d.

IHC, immunohistochemistry; G, grade; LOH, loss of heterozygosity; ROH, retention of heterozygosity; NI, not informative; n.d., not

determined; M, male; F, female.



in the normal pituitary, the nuclei of endothelial cells in all

pituitary adenomas were menin immunonegative.

In the case of the PRL-producing pituitary carci-

noma, the samples derived from the first and the second

resections, obtained 6 months apart, displayed weak

menin immunoreactivity (nos 69 and 70 Table 1; Fig.

2G), while the autopsy tissues obtained 3 years later from

a parasellar and orbital invasion and from metastases

derived from medulla oblongata, spinal canal and left

femur were all immunonegative (nos 71–74, Table 1; for

example, metastasis to medulla oblongata in Fig. 2H).

Double IHC with PRL on the metastatic tissues

confirmed the prolactinoma origin of the sections (inset

in Fig. 2H).

The integrity and preservation of the samples studied

were confirmed after performing IHC for Pit1, in the case

of ACRO and PROL (insets in Fig. 2C, F, G and H), and

SF1, for NFPA (insets in Fig. 2D and E). All the 68

tumors, the carcinoma and the metastatic tissues, as well

as all the normal human pituitaries (insets in Fig. 2A and

B), were included in the present study after their integrity

was confirmed by IHC for Pit1 and SF1.

Preimmune

GST-Menin GST

C D

E F

Figure 1 Menin expression in a human adenohypophysis (NP1). (A) Menin is present in the nuclei of all endocrine cells, but not in

the nuclei of endothelial cells. The specificity of the mAb C126 is demonstrated by depletion of the signal after incubation with the

preimmune serum (B) or after preabsorption of the antibody with the GST-menin immunogen (C). (D) Preabsorption with the GST

alone does not alter the antibody activity. (E) Menin colocalization with PRL. (F) Menin colocalization with ACTH.



#8

Pit1

#17 #66

#46

Pit1

SF-1

SF-1

Pit1Pit1

Carcinoma/ 1st intervent Medulla PRL

E F

NP 3NP 2

SF-1Pit1A B

C D

G H

Figure 2 Menin immunoreactivity in two human anterior pituitary glands (NP2 and 3; A and B). Menin immunoreactivity in pituitary

adenomas. In the figure are depicted examples of sporadic pituitary adenomas with strong (C), moderate (D), and weak (E and F)

menin immunoreactivity. IHC on tissues derived from different stages of a PRL-producing carcinoma progression reveal that menin

immunoreactivity is weak in tumor obtained at the second intervent (G) and totally absent in the autoptic tissue derived from the

metastasis to the medulla oblongata (H). Immunostaining for PRL in a parallel section proves the pituitary origin of the tissue (H

inset). The insets in panels A to H show immunostaining for the transcription factors Pit1 or SF-1.



Sixteen out of the 68 sporadic pituitary adenomas

included in the study were also screened for menin using

IF/LSM. As shown in Table 1 and Fig. 3, in most cases

IF/LSM analysis of pituitary adenomas confirmed the

IHC results, demonstrating variable levels of menin

expression, which was classified as high (Fig. 3A–D),

average (Fig. 3E–H) or low (Fig. 3I–M) menin expression.

However, in three cases (nos 8, 10 and 65), the intensity of

the menin signal appeared to be higher in IF/LSM than

IHC, most likely due to the higher sensitivity of IF/LSM.

Discussion

The fact that one of the most common manifestations of

the polyendocrine syndrome MEN1 is the development of

pituitary adenomas (Verges et al. 2002), together with the

increasing evidence that MEN1 is a tumor-suppressor

gene, has drawn attention to a possible role for menin in

the pathogenesis of sporadic pituitary adenomas. Using a

monoclonal antibody against the menin C-terminus, we

studied the expression pattern of menin in normal and

adenomatous pituitary. Other commercial antibodies

against menin are available, but, to our knowledge,

none of them have been validated until now for IHC in

primary human tissues. We decided to perform IHC, since

normal and tumoral pituitaries are heterogeneous tissues

composed of a mixture of endocrine and nonendocrine

cells. This is also why IHC, which is only a semiquanti-

tative method, was preferred to Western blotting. IHC of

human normal pituitary gland revealed that most endo-

crine cells of the adenohypophysis display a high level of

menin expression, which is not restricted to a particular

hormonal type. On the other hand, analysis of a large

number of pituitary adenomas showed that, although

most cells composing the mass of the adenoma retain a

nuclear staining pattern for menin, the majority of the

samples displayed a weaker signal when compared with

the normal adenohypophysis. These data are in line with

#20

#19

E G H

I L M

#19 #19 #19

#20 #20 #20

Pit

Pit

A C D

#21 #21 #21 #21

PitB

F

K

Figure 3 IF/LSM on pituitary adenomas — comparison with IHC. (A, E and I) PI staining nuclei red. Sporadic pituitary adenomas

present with strong (B), moderate (F) and weak (K) signal for menin (green). (C, G and L) Overlap of menin and PI signals produced

a yellow signal. (D, H and M) IHC on adjacent sections of the same pituitary adenomas. The insets show immunostainings for Pit1.



our previous work on normal and tumoral pancreatic

tissues, which showed different levels of menin expression

in normal exocrine and endocrine cell populations and in

tumors (Cavallari et al. 2003).

Our results are in partial agreement with the only

previous study showing menin expression in pituitary

adenomas (Wrocklage et al. 2002). Using commercially

available menin antibody to examine 11 pituitary tumors

and four normal pituitaries by Western blotting, Wrock-

lage et al. demonstrated a variable expression of menin in

the tumors examined. However, the lowest expression was

detected in the normal pituitaries. Our experience from

using the mAb C126 on pancreatic (Cavallari et al. 2003)

and pituitary tissues suggests that the protein is suscep-

tible over time to degradation. Therefore, it is possible

that the low menin level found in four normal pituitaries

by Wrocklage et al. may be due to postmortem degrada-

tion. In our study, seven postmortem pituitaries were

collected only 8–12 h after demise. IHC for nuclear

transcription factors as SF-1, Pit-1 and ZAC (Pagotto et

al. 2000) confirmed the preservation of the tissues.

The present study also included an analysis of menin

expression carried out using the IF/LSM technique. The

restricted quantities of tissues available forced us to

confine this analysis to a limited number of cases.

Nevertheless, IF analysis revealed variable menin expres-

sion among the different pituitary adenoma cases, thus

confirming the results obtained by IHC.

Since menin protein is susceptible over time to

degradation after tissue fixation, it is important that the

tissue is properly frozen, stored at �80 8C and fixed only

for each individual IHC or IF experiment. However, if

these requirements are fulfilled, mAb C126 is suitable for

the study of menin expression in a wide range of tissues,

not only by conventional IHC but also by IF/LSM.

There was no apparent correlation between menin

immunoreactivity and clinical and immunohistochemical

diagnosis of each tumor studied, a finding consistent with

the observation that pituitary adenomas in MEN1

patients are not restricted to a certain clinical or histologic

type. Furthermore, there was no correlation between

menin staining intensity and tumor grade and invasive-

ness, whereas in MEN-1 associated pituitary tumors,

prolactinomas have been shown to have a more aggressive

phenotype (Verges et al. 2002). This discrepancy is due to

the fact that in sporadic pituitary adenomas there is still

menin present, while in the MEN-1 tumors the complete

absence of wild-type menin may lead to a more aggressive

phenotype. This can be supported by the fact that in our

study the only case displaying complete loss of menin

immunoreactivity was the tumor specimen taken at

autopsy from a patient with a pituitary carcinoma.

Analysis of sequential tumor samples from this patient

revealed a weak signal detected in the earlier specimens,

but loss of menin immunoreactivity in the metastatic

tissues. This observation indicates a possible role for

menin loss in the transition to a highly malignant

phenotype. Unfortunately, we were not able to perform

a genetic analysis of the patient due to inadequate amount

of material.

Although MEN1 mutations are relatively frequently

found in sporadic pancreatic (30%) (Toliat et al. 1997,

Zhuang et al. 1997a) and parathyroid (20%) (Heppner et

al. 1997) tumors, they appear extremely rarely, if at all, in

sporadic pituitary adenomas (Zhuang et al. 1997b,

Prezant et al. 1998, Wenbin et al. 1999), excluding the

possibility of a defect in the MEN1 gene. However, there

is still the possibility that disruption of the 11q13 region

leading to loss of one allele could result in a decrease in

menin expression. Allelic loss has been described in 5–

20% of sporadic pituitary adenomas (Boggild et al. 1994),

and in our study we found allelic loss in four out of 12

cases (33%), which is a much smaller percentage than that

of the tumors that were scored with a medium or weak

menin immunoreactivity. Therefore, allelic loss may be

responsible for the lower menin levels in some, but not all,

cases with low menin immunoreactivity. However, studies

by many groups using RT-PCR (Asa et al. 1998, Farrell et

al. 1999, Satta et al. 1999) showed no variation in MEN1

mRNA levels between neoplastic and nontumoral pitui-

taries. Therefore, alterations in the transcriptional regula-

tion of the gene do not account for the reduction of menin

levels as documented by IHC and IF/LSM. Our data

indicate that translational and post-translational mechan-

isms must play an important role in the regulation of

menin expression, and suggest that defects in these

systems may be responsible for the reduced levels of

menin in sporadic pituitary adenomas.

The plausibility of this hypothesis is supported by

documented cases, since there are proteins involved in the

cell cycle and tumor formation that are regulated at the

protein level and whose fate is determined by post-

translational modifications. One paradigm is p27/Kip1, a

regulator of cell-cycle progression, which, though nor-

mally detectable at mRNA level (Jin et al. 1997, Dahia et

al. 1998), shows reduced expression at the protein level in

a significant percentage of pituitary adenomas (Jin et al.

1997, Bamberger et al. 1999, Lidhar et al. 1999). Although

the exact mechanism underlying this effect is not yet

clarified in the case of pituitary adenomas, abnormalities

in the ubiquitin-mediated degradation system have been

shown to be responsible for the downregulation of p27Kip1

in other types of tumors (Pagano et al. 1995, Loda et al.

1997). It remains to be clarified whether the same

mechanism can be applied to menin and is responsible



for the low levels of this protein in sporadic pituitary

adenomas.

In conclusion, this study provides new information

regarding the expression of menin in the normal and

adenomatous pituitary. In human pituitary, we demon-

strated that menin is highly expressed in the anterior lobe,

while tumoral transformation is associated with a

significant reduction in menin levels in a high percentage

of pituitary adenomas. Its likely role as a tumor

suppressor with an important role in cell-cycle regulation

suggests that the decrease in menin levels in sporadic

pituitary adenomas may represent a mechanism contri-

buting to pituitary oncogenesis.

Acknowledgements

This work was supported by the Deutsche Forschungs-

gemeinschaft (Pa 647/1-1 to U.P.) and by the Ministero

dell’Universita e della Ricerca Scientifica (MURST no.

990621892/007 to V.C.).

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Differential expression of menin in sporadic pituitary adenomas

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