Differential expression of menin in sporadic pituitary adenomas

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1 Endocrine-Related Cancer (2004) Differential expression of menin in sporadic pituitary adenomas M Theodoropoulou 1, I Cavallari 2, L Barzon 3, D M D Agostino 2, T Ferro 2, T Arzberger 4, Y Grübler 1, L Schaaf 1, M Losa 5, F Fallo 3, V Ciminale 2, G K Stalla 1 and U Pagotto 6 1 Max Planck Institute of Psychiatry, Neuroendocrinology Group, Munich, Germany 2 Department of Oncological and Surgical Sciences, University of Padova, Padova, Italy 3 Department of Medical and Surgical Sciences, Division of Endocrinology, University of Padova, Padova, Italy 4 Institute of Pathology, Division of Neuropathology, University of Wurzburg, Wurzburg, Germany 5 Neurosurgical Department, San Raffaele Hospital, Milan, Italy 6 Endocrine 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, Bologna, Italy (Requests for offprints should be addressed to M Theodoropoulou; marily@mpipsykl.mpg.de) Abstract Pituitary adenomas represent one of the key features of multiple endocrine neoplasia type 1. The gene involved in this syndrome (MEN1) is a putative tumor suppressor, that codes for a 610-amino acid nuclear protein termed menin. Analyses of sporadic pituitary adenomas have so far failed to reveal MEN1 mutations or defects in MEN1 transcription in these tumors. In the present study we detected menin protein expression in a panel of normal and tumoral pituitary tissues, using a monoclonal antibody against the carboxy-terminus of menin. In the normal human pituitary gland, strong nuclear staining for menin was detectable in the majority of the endocrine cells of the anterior lobe, without a clear association with a particular hormone-producing type. In sporadic pituitary adenomas, menin expression was variable, with a high percentage of cases demonstrating a significant decrease in menin immunoreactivity when compared with the normal pituitary. Interestingly, metastatic tissues derived from one pituitary carcinoma had no detectable menin levels. Altogether, our data provide the first information regarding the status of menin expression in human normal and neoplastic pituitary as determined by immunohistochemistry (IHC). Endocrine-Related Cancer (2004) 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 neuroendocrine 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 (Chandrasekharappa 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 transcription 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) /04/ # 2004 Society for Endocrinology Printed in Great Britain Online version via

2 Theodoropoulou et al: Expression of menin in sporadic pituitary adenomas was found to inhibit DNA synthesis under DNAdamaging 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 pituitary 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 pituitary 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 immunohistologic 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) (Pannett & 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 transcranial 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 1.5 mm MgCl 2, 200 mm each dntp, 500 mm each primer, template DNA and 0.5 U Taq DNA polymerase. An initial denaturation step at 94 8C for 5 min was followed by 35 cycles at 94 8C for 1min, 62 8C for 1 min, 72 8C for 1 min, 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% denaturing 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 334

3 Endocrine-Related Cancer (2004) 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 dehydrated. 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 30 min 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 5 min between all steps. Reactivity was detected using diaminobenzidine (DAB, 1 mg/ml) as chromogen and 0.01% H 2 O 2 as substrate. Sections were then counterstained 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-folliclestimulating-hormone (FSH) (1 : 800), antihuman b-luteinizing hormone (LH) (1 : 800), antihuman b thyrotropin stimulating hormone (TSH) (1 : 800), antihuman PRL (1 : 400), antihuman a-subunit (a-sub) (1 : 500) (all purchased from Immunotech, Karlsruhe, Germany), antihuman 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 independent 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 30 min at room temperature with the mab C126 in the presence of either 25 mg of purified GST- C126men or of 25 mg 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 scanning microscopy (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 8C with 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 0.1 mg/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 ð pixels) or 2 ð pixels). Laser intensity, pinhole aperture, and photomultiplier parameters were standardized to enable comparison of signals obtained in different samples. Fluorescence signals were analyzed using a 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 335

4 Theodoropoulou et al: Expression of menin in sporadic pituitary adenomas 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 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 G LOH PYGM D11S4946 D11S4933 D418D Menin ir Menin IF 1. 28/M ACRO GH III ++ n.d /M ACRO GH/PRL III ROH NI NI NI + n.d /F ACRO GH II +++ n.d /F ACRO GH III NI ++ n.d /M ACRO GH/PRL II + n.d /M ACRO GH III LOH LOH NI LOH + n.d /F ACRO GH III NI NI NI ++ n.d /F ACRO GH/a-sub/FSH III /F ACRO GH III LOH LOH + n.d /M ACRO GH/PRL III /M ACRO GH/PRL III ROH NI NI +++ n.d /F ACRO GH/PRL III ++ n.d /M ACRO GH/PRL II +++ n.d /M ACRO GH/PRL II + n.d /F ACRO GH/PRL/a-sub III +++ n.d /M ACRO GH/PRL II ++ n.d /F ACRO GH/PRL II /F ACRO GH/FSH/LH II + n.d /F ACRO GH/PRL/TSH/a-sub II /F ACRO GH/PRL II /M ACRO GH/PRL/a-sub III /M CUSH ACTH I ++ n.d /M CUSH ACTH I +++ n.d /F CUSH ACTH II ++ n.d /F CUSH ACTH II + n.d /M CUSH ACTH I +++ n.d /F CUSH ACTH II + n.d /F CUSH ACTH III /F CUSH ACTH I /F PROL PRL II ++ n.d /M PROL PRL III ++ n.d /F PROL PRL III + n.d /F PROL PRL III + n.d /F PROL PRL III + n.d /F PROL PRL III NI ROH NI ++ n.d /M NFPA ACTH III ++ n.d /M NFPA ACTH II + n.d /F NFPA a-sub II ++ n.d /M NFPA LH/a-sub II ++ n.d /F NFPA FSH/LH II ++ n.d /M NFPA FSH/LH II +++ n.d /M NFPA FSH II +++ n.d /M NFPA FSH III LOH /M NFPA FSH III LOH LOH NI /M NFPA FSH III ++ n.d /M NFPA FSH III + n.d /M NFPA a-sub/fsh/lh II +++ n.d /F NFPA FSH II ROH ROH NI NI + n.d /F NFPA a-sub/fsh/lh III ++ n.d

5 Endocrine-Related Cancer (2004) Table 1 continued Age/sex Diagnosis IHC G LOH PYGM D11S4946 D11S4933 D418D Menin ir Menin IF /M NFPA FSH III + n.d /F NFPA LH II + n.d /M NFPA FSH/LH/a-sub II ++ n.d /F NFPA FSH/LH/a-sub II /M NFPA FSH/a-sub/LH II /F NFPA FSH/a-sub III /F NFPA LH III /F NFPA None II /M NFPA None III ++ n.d /M NFPA None II +++ n.d /M NFPA None III + n.d /F NFPA None II + n.d /F NFPA None II + n.d /F NFPA None III NI + n.d /M NFPA None III + n.d /F NFPA None III /M NFPA None II NI ++ n.d /F NFPA None III + n.d /F NFPA None II +++ n.d /M 1st intervent PRL IV + n.d /M 2nd intervent PRL IV + n.d /M Parasellar PRL IV 0 n.d /M Orbit PRL IV 0 n.d /M Medulla PRL IV 0 n.d /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. 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. indicating that its expression is not restricted in one particular cell population. The nuclei of the fibroblasts and endothelial cells were devoid of menin immunoreactivity (Fig. 1A and D). Menin expression in normal human pituitary Immunohistochemical examination of seven normal human pituitary glands revealed strong menin immunoreactivity 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 immunogen 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), 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,asin Fig. 2C), 22 displayed an intermediate level of expression (þþ, as in Fig. 2D), and 31 had weak menin immunoreactivity (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 337

6 Theodoropoulou et al: Expression of menin in sporadic pituitary adenomas 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. in the normal pituitary, the nuclei of endothelial cells in all pituitary adenomas were menin immunonegative. In the case of the PRL-producing pituitary carcinoma, 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 SF

7 Endocrine-Related Cancer (2004) NP 2 NP 3 A Pit1 B #17 #66 SF-1 C Pit1 D SF-1 #46 #8 E SF-1 F Pit1 Carcinoma/ 1st intervent Medulla PRL G Pit1 H Pit1 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

8 Theodoropoulou et al: Expression of menin in sporadic pituitary adenomas #21 #21 #21 #21 A B C D #19 #19 #19 #19 Pit E F G H #20 #20 #20 #20 Pit I K L M Pit 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. 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 semiquantitative method, was preferred to Western blotting. IHC of human normal pituitary gland revealed that most endocrine 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 340

9 Endocrine-Related Cancer (2004) 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, Wrocklage 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 susceptible 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 degradation. 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 expression 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 invasiveness, 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 pituitaries. Therefore, alterations in the transcriptional regulation 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 mechanisms 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 posttranslational modifications. One paradigm is p27/kip1, a regulator of cell-cycle progression, which, though normally 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 p27 Kip1 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 341

10 Theodoropoulou et al: Expression of menin in sporadic pituitary adenomas 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 demonstrated 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 contributing to pituitary oncogenesis. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Pa 647/1-1 to U.P.) and by the Ministero dell Universita` e della Ricerca Scientifica (MURST no /007 to V.C.). 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