Differential Expression of Menin in Various Adrenal Tumors
The Role of Menin in Adrenal Tumors
Mohammad M. R. Bhuiyan, M.B.B.S., Ph.D.1 Makoto Sato, M.D., Ph.D.1 Koji Murao, M.D., Ph.D. Hitomi Imachi, M.D., Ph.D.1 Hiroyoshi Namihira, M.D.1 Toshihiko Ishida, M.D., Ph.D.1 Jiro Takahara, M.D., Ph.D.1 Akira Miyauchi, M.D., Ph.D.2
1 First Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan.
2 Kuma Hospital, Kobe, Japan.
The authors thank Settara C. Chandrasekharappa (National Human Genome Research Institute of the National Institutes of Health, Bethesda, Maryland 20892-4442) for providing the generous gift of plasmid A11 DNA containing a full-length menin CDNA.
Address for reprints: Makoto Sato, M.D., Ph.D., First Department of Internal Medicine, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan; Fax: (011) +81-87- 891-2147; E-mail: makoto@kms.ac.jp
Received May 28, 2001; accepted June 19, 2001.
BACKGROUND. Adrenocortical tumors occur as sporadic tumors, as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome, or as part of other hered- itary disorders. MEN1 is a tumor suppressor gene located on chromosome 11q13 that encodes a 610-amino acid protein called menin, and plays an important role in the development of MEN1 syndrome. Recent reports indicate that heterozygous germline mutations of this gene are responsible for the disease onset of MEN1.
METHODS. To investigate the role of menin in sporadic adrenocortical tumors, the authors examined a series of adrenocortical adenoma cases and a single case of carcinoma and adrenomedulary tumors with the corresponding adjacent tumor tissues using reverse transcriptase-polymerase chain reaction (RT-PCR) for menin mRNA and Western blot analysis for menin protein. Both RNA and protein from these tumors were applied to RT-PCR and Western blot analysis, respectively, although they are not truly quantitative. Primers for RT-PCR were designed to amplify the sequence between exons 2 and 3 of the MEN1 gene. A specific antibody against menin was generated in guinea pigs immunized with the recombinant peptide from the amino acid residues 443-535 of menin made by using glutathi- one-S-transferase gene fusion.
RESULTS. Based on the results of RT-PCR and Western blot analysis, both MEN1 mRNA and menin protein appeared to be highly expressed in Cushing syndrome resulting from adrenocortical adenomas and carcinoma. However, their expression was found to be greatly decreased in primary aldosteronism compared with their expression in Cushing syndrome. Although weak expression of MEN1 mRNA also was detected in pheochromocytoma on RT-PCR, menin expression was not de- tected in any case of pheochromocytoma by Western blot analysis, possibly due to the lower sensitivity of this assay compared with RT-PCR. Neither MEN1 mRNA nor menin protein was detected in any of the corresponding adjacent tumor tissues examined.
CONCLUSIONS. The findings of the current study indicate that menin expression appears to be up-regulated in Cushing syndrome, suggesting that adrenocortical proliferation might be one of the primary lesions in the MEN1 syndrome in which menin might play a significant role in some specific cellular functions. Cancer 2001;92:1393-401. @ 2001 American Cancer Society.
KEYWORDS: MEN1, MEN1 gene, menin, Cushing syndrome, tumor suppressor, mutation, adrenal tumors.
M ultiple endocrine neoplasia type-1 (MEN1) is an autosomal dominant familial tumor syndrome mainly characterized by the combined occurrence of tumors in the parathyroid gland, enteropan- creatic endocrine tissue, and the anterior pituitary gland.1-3 Parathy- roid tumor is the most common manifestation of this disorder.3 In
| Patients | Age (yrs) | Gender | Diagnosis | Tumor location | Tumor size | Clinical symptoms |
|---|---|---|---|---|---|---|
| 1 | 57 | F | Cushing adenoma | Lt adrenal cortex | 2.6 × 2.2 cm | Thirsty, hyperglycemia |
| 2 | 47 | F | Cushing adenoma | Rt adrenal cortex | 3.4 × 2.0 cm | General fatigue |
| 3 | 56 | M | Cushing adenoma | Lt adrenal cortex | 2.9 × 2.8 cm | Moon face, hypertension |
| 4 | 57 | F | Cushing adenoma | Lt adrenal cortex | 3.2 × 2.2 cm | Moon face, edema |
| 5 | 64 | M | Cushing adenoma | Rt adrenal cortex | 1.8 × 1.8 cm | Lumbago due to osteoporosis |
| 6 | 38 | M | Adrenocortical carcinoma | Lt adrenal cortex | 11.1 × 5.9 cm | Hypertension |
| 7 | 54 | F | Primary aldosteronism | Rt adrenal cortex | 1.6 × 0.8 cm | Hypertension |
| 8 | 33 | M | Primary aldosteronism | Rt adrenal cortex | 1.8 × 1.5 cm | Hypertension, general fatigue |
| 9 | 28 | F | Pheochromocytoma | Lt adrenal medulla | 1.8 × 1.3 cm | No symptoms |
| 10 | 25 | F | Pheochromocytoma | Lt adrenal medulla | 9.7 × 8.3 cm | Headache |
| 11 | 67 | M | Pheochromocytoma | Lt adrenal medulla | 7.0 × 6.1 cm | Hypertension |
| 12 | 31 | M | Pheochromocytoma | Rt adrenal medulla | 2.7 × 3.0 cm | Back pain |
| 13 | 26 | F | Pheochromocytoma (MEN2A) | Rt adrenal medulla | 3.5 × 3.0 cm | No symptoms |
| 14 | 16 | M | Pheochromocytoma (MEN2B) | Rt adrenal medulla | 0.9 × 0.7 cm | No symptoms |
F: female; Lt: left; RT: right; M: male.
addition, adrenocortical tumors, foregut carcinoids, and lipomas also have been observed in association with MEN1.1 The genetic basis for MEN1 is the ho- mozygous inactivation of the putative tumor suppres- sor gene on chromosome 11q13.4 Sporadic adrenocor- tical tumors are relatively common neoplasms, having been reported in 2-10% of the general population.5,6 The frequency of small benign adrenocortical tumors gradually increases with age and has been reported in up to 10%5-8 of individuals age > 50 years (Table 1). The majority of these tumors are discovered inciden- tally (incidentalomas) by abdominal computed to- mography or magnetic resonance imaging. Patients with MEN1 syndrome develop adrenocortical lesions, with a frequency of up to 40%.9,10 The majority of these lesions are hormonally silent and exhibit a be- nign clinical phenotype.9,11 In contrast, adrenocortical carcinomas are rare and highly malignant tumors, with an approximate prevalence of 1:1.7 million per year.8
Larsson et al.12 were instrumental in initially iden- tifying chromosome 11q13 as the location of the gene responsible for MEN1; such patients frequently are found to have somatically lost the wild-type allele of markers in the vicinity of the gene.12,13 Moreover, al- lelic deletions on chromosome 11q13 have been re- ported in 63-100% of MEN1-associated parathyroid tumors and in 25-35% of sporadic parathyroid tumors, suggesting the MEN1 gene plays an important role in the pathogenesis of such tumors.14-16
The MEN1 gene contains 10 exons and expresses a 2.8-kilobase transcript that encodes a 610-amino acid protein called menin. Although analysis of the pre- dicted menin amino acid sequence was not found to
exhibit any apparent similarities to any known pro- teins, nuclear localization signals were identified in the C-terminals of menin.17,18 It has been reported that the wild-type allele consistently is lost in MEN1 tumors, and both alleles of the MEN1 gene often are inactivated in sporadic tumors, indicating that tumor- igenesis is very likely due to loss of function of the protein product as a tumor suppressor.15,16,19 Thus the MEN1 gene appears to be an excellent example of a classic tumor suppressor. The loss of one allele in adrenocortical tumors has been observed in several chromosomal loci including 11q20,21 and 13q.22 Be- cause 11q13 harbors the MEN1 gene13 and 11q loss of heterozygosity (LOH), this suggests that MEN1 gene mutation might contribute to the pathogenesis of MEN1-associated adrenal tumors20,21 and not spo- radic adrenal tumors.
The objective of the current study was to investi- gate the role of menin in a series of well documented adrenal tumors obtained from patients with Cushing syndrome, primary aldosteronism, and pheochromo- cytoma.
MATERIALS AND METHODS
Freshly frozen tumor samples from surgical speci- mens of adrenal tumors from different patients with Cushing syndrome, pheochromocytoma, primary al- dosteronism, and carcinoma with all corresponding adjacent tumor tissues were used in this study. Tissues collected after surgical removal were frozen immedi- ately into liquid nitrogen and then stored at -80 ℃. The diagnosis was confirmed based on histopatho- logic findings and clinical outcome. Informed consent was obtained from all patients for the use of the tissue
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samples. Total RNA from each tumor and correspond- ing adjacent tumor tissue was extracted using a Qia- gen RNA/DNA mini kit (Qiagen, Hilden, Germany). Total proteins extracted from the corresponding sam- ples with 1% Nonidet P-40 were used for Western blot analysis.
Reverse Transcriptase-Polymerase Chain Reaction
Total RNA (2 µg) was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Life Sci- ence Co., Petersburg, FL) and random primer (Takara Shuzo Co., Osaka, Japan) and the synthesized cDNA was amplified by polymerase chain reaction (PCR) as described previously.21 The primers used for reverse transcriptase (RT)-PCR were 5’-GAG CTG TCC CTC TAT CCT CG-3’ (sense) and 5’-TGA CCT CAG CTG TCT GCT CC-3’ (antisense) designed to amplify the sequence between exons 2 and 3 of the MEN1 gene (Fig. 1). The primers for amplification of ß-actin se- quence in the corresponding tissues were designed as described previously.23 The initial denaturation was performed for 7 minutes at 94 ℃ followed by 30 cycles of PCR for both the MEN1 gene and ß-actin using a thermal cycler (Sanko Junyaku, Tokyo, Japan) accord- ing to a step program of 94 ℃ for 80 seconds, 55 ℃ for 80 seconds, and 72 ℃ for 80 seconds, followed by a 15-minute extension at 72 ℃. Eight uL of each PCR product was separated electrophoretically on a 1.5% agarose gel containing 0.5 µg/mL ethidium bromide and photographed. As an internal control, ß-actin was amplified and analyzed under identical conditions us- ing an appropriate set of primers.
Generation of Menin Antibody Using Glutathione-S- transferase Gene Fusion System
A fragment of menin cDNA corresponding to the amino acid residues 443-535 of menin from parathy- roid tissue containing restriction sites for HindIII and XbaI was amplified by PCR (Sigma Chemical Co., St. Louis, MO) as described previously24 using an appro- priate set of primers matching the published sequence of the MEN1 gene.18 The purified PCR product then was digested with HindIII and XbaI and ligated to the HindIII and Xba1 restriction sites of the pGEX2T plas- mid vector (Pharmacia Biotech, Inc., Buckingham- shire, UK) by T4DNA Ligase (Takara Shuzo Co. Ltd., Osaka, Japan) forming a recombinant plasmid DNA. Competent Escherichia coli then were transformed with the recombinant plasmid DNA and cultured for glutathione-S-transferase (GST) fusion protein expres- sion. The protein expression was induced by 0.1 mM isopropyl B-D thiogalactoside. Stable transformation of the cells were maintained by their resistance to ampicillin. Cells were harvested and lysed by mild sonication. The GST fusion protein was isolated with glutathione sepharose 4B beads (Pharmacia Biotech Inc.) from the bacterial lysate and used to generate a specific antibody in guinea pigs directed against the amino acid residues 443-535 of the reported sequence of the menin containing 610-amino acid residues.18 The immunoglobulin (Ig) G fraction was purified us- ing an ImmunoPure Plus immobilized Protein A IgG Purification Kit (Pierce, Rockford, IL) and used for Western blot analysis to detect menin in various tu-
mor tissues in which a horseradish peroxidase-conju- gated goat antiguinea pig IgG (Sigma Chemical Com- pany, St. Louis, MO) was used as the second antibody.
Western Blot Analysis of Menin
The plasmid cytomegalovirus (pCMV)-sport menin clone (A11) containing a full-length menin cDNA17 from the National Human Genome Research Institute of the National Institutes of Health was used for trans- fection of HEK-293T cells. Forty-eight hours after transfection, cells were harvested by washing with phosphate-buffered saline (PBS) and mechanical scraping from the flask, and total cellular proteins were extracted with 1% Nonidet P-40 for Western blot analysis. Cellular proteins from tissue samples that were stored at -80 ℃ were prepared,25 resuspended under reducing conditions, 30 µg was fractionated by size on a 10% sodium dodecyl sulfate (SDS)-polyacryl- amide gel for menin and 10 µg was fractionated on a 15% gel for cyclophilin A as an internal control, and transferred to polyvinylidene difluoride (PVDF) mem- brane (Millipore, Bedford, MA) using Trans-Blot (Bio- Rad Laboratories, Richmond, CA) for immunoblot- ting.26 The membranes were blocked overnight at 4 ℃ with PBS containing 0.1% Tween 20 (PBS-T) and 7.5% skimmed milk followed by incubation for 2 hours at room temperature with the antimenin antibody (1: 2000 dilution) or the anticyclophilin A antibody (Bi- omol Research, Plymouth Meeting, PA) (1:1000 dilu- tion). The membranes were washed with PBS-T, incubated with the secondary antibody conjugated to peroxidase for 1 hour at room temperature, and then washed with PBS-T. The staining signal was detected using the enhanced chemiluminescence detection system (ECL; Amersham Corporation, Arlington Heights, IL) followed by exposure to Kodak X-ray film (Eastman-Kodak, Rochester, NY).
RESULTS
RT-PCR Analysis of MEM Gene
Menin mRNAs were amplified successfully to an ex- pected size (286 base pair [bp]) by RT-PCR using exon specific primers in various tumor tissues. As an inter- nal control, ß-actin was amplified to an expected size of 297 bp. Representative data regarding menin and B-actin mRNAs from each tissue are shown in Figure 1. Menin mRNA expression appeared to be up-regu- lated and in approximately equivalent amounts in Cushing adenoma and carcinoma as indicated by Lanes 2, 4, 6, and 8; no such expression was found in any of the corresponding adjacent tumor tissues as indicated by Lanes 1, 3, 5, and 7 in Figure 1. Expres- sion of menin mRNA appeared to be greatly down- regulated in both primary aldosteronism and pheo-
chromocytoma as well as in some other adjacent tumor tissues (data not shown) on RT-PCR, which is a more sensitive assay than Western blot analysis. The expression level of ß-actin mRNA as an internal con- trol was nearly constant in all the samples examined. In addition, we examined the expression of menin mRNA in several other adrenal tumor tissues, includ- ing primary aldosteronism and pheochromocytoma.
Western Blot Analysis of Menin
Menin protein was detected with an estimated molec- ular size of approximately 67 kilodaltons (kD) in HEK- 293T cells transfected with the plasmid A11 DNA con- taining a full-length menin cDNA (gift from SC Chandrasekharappa, SETTARA, Department of Health and Human Services, National Institutes of Health). 17 However, in the untransfected cells menin expression was much weaker although it still was detectable, sug- gesting that these cells expressed endogenous menin (data not shown). Our antibody was able to detect endogenous menin of approximately 67 kD, predom- inantly in Cushing adenoma and carcinoma. Menin protein was expressed strongly and in nearly equiva- lent amounts on Western blot analysis with a molec- ular size of approximately 67 kD in all cases of Cushing adenoma (Fig. 2), which was consistent with the re- sults of RT-PCR. In contrast, expression of menin pro- tein was much weaker in primary aldosteronism cases, as indicated in Lanes 8 and 10 compared with Cushing adenoma and carcinoma, in which menin expression appeared to be up-regulated as shown in Figure 3. Menin expression was not detected in any of the cases of pheochromocytoma as indicated by Lanes 4, 6, 8, and 10 as well as in their corresponding adjacent tumor tissues, which were examined as shown in Fig- ure 4. However, a relatively stronger expression of menin protein was detected in Cushing adenoma (Fig. 4). Figure 5 shows menin expression in Cushing ade- noma and in pheochromocytoma associated with MEN2A and MEN2B. However, in pheochromocyto- mas associated with MEN2A and MEN2B and their corresponding adjacent tumor tissues, expression of menin was nearly undetectable (Fig. 5). Western blot analysis of cyclophilin A as an internal control re- vealed an estimate size of approximately 18 kD pro- tein, which was expressed nearly equally in all tissues examined.
DISCUSSION
Involvement of the adrenal gland has been reported in approximately 40% of MEN1 patients and has been found to represent bilateral hyperplasia, adenoma, and, in a few cases, carcinoma.9,10,27 In a previous study,28 our screening involved parathyroid tumors
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resulting from primary hyperparathyroidism and sec- ondary hyperparathyroidism, and menin was found to be greatly up-regulated. Therefore, as the next step, the current study focused on the detection and anal- ysis of menin in Cushing syndrome due to adrenocor- tical adenoma and carcinoma and in tumors from cases of primary aldosteronism and pheochromocy- toma. Although the approaches used in the current study were not truly quantitative, the results have shown that the expression of menin at both mRNA and protein levels in adrenocortical tumors in patients with Cushing syndrome appears to be up-regulated compared with that in primary aldosteronism and pheochromocytoma, indicating that Cushing ade- noma may harbor an abundance of menin, which is important for the development and progression of this
tumor form. Alternatively, one might hypothesize that high levels of menin expression simply reflect an in- creased mitotic index rather than a cause of neoplasia. In the current study, menin protein expression ap- peared to be greatly down-regulated in primary aldo- steronism whereas it was nearly undetected in pheo- chromocytoma and in all corresponding adjacent tumor tissues examined. The near absence of menin expression in the adjacent tumor tissues on Western blot analysis possibly was due to atrophy of such tis- sues by the tumors. However, on RT-PCR, menin mRNA expression still was detectable in both primary aldosteronism and pheochromocytoma and also in some other adjacent tumor tissues (data not shown), possibly due to the higher sensitivity of this assay compared with that of Western blot analysis. Due to
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such an abundance of menin in both parathyroid28 and adrenocortical tumors, one might speculate that menin might play an important role as a nuclear pro- tein17,29 in specific cellular functions in some specific tissues, including parathyroid and adrenocortical tu- mors.
Many observations have indicated that germline MEN1 gene mutations most likely result in loss of function of its protein product, a finding that is con- sistent with a tumor suppressor mechanism.12,18,30 A wide range of studies established that 11q13 LOH was common not only in MEN1 tumors19,31 but also in many types of sporadic tumors, especially sporadic endocrine tumors of the types observed in MEN1.2
However, MEN1 gene mutation in adrenocortical tu- mors has been reported to be less common.32 Al- though we did not check menin for mutation, previous studies have shown no mutation of menin in sporadic adrenal tumors. Adrenocortical tumors in MEN1 pa- tients always are observed in conjunction with tumors in the endocrine pancreas.9,27 A potential role of the MEN1 gene in adrenocortical tumor formation was suggested by previous studies regarding 11q13 LOH in MEN1-associated adrenocortical tumors as observed frequently in carcinoma but rarely in adenoma and hyperplasia.9,11,33 Allelic deletions at 11q13 have been reported in sporadic adrenocortical tumors,21 al- though mutation of the MEN1 gene rarely is involved
in this disease process.33 Because 11q13 harbors the MEN1 gene13 11q LOH thus suggests that MEN1 gene mutation might contribute to the pathogenesis of MEN1-associated adrenal tumors20,21 and not of the sporadic tumors. Therefore, the involvement of the MEN1 gene in the pathogenesis of adrenocortical tu- mors cannot be ruled out completely. Clearly, one allele of the MEN1 gene frequently is lost, and in the current study we speculated that there might be some reasons why MEN1 gene mutations were not found in adrenocortical lesions with loss of one copy of the MEN1 gene. Possible reasons in this particular study may include mechanisms other than mutations within the coding region of the gene, such as splice mutations or hypermethylations in the promoter region of MEN1 gene or mutations within the noncoding region that might inhibit the transcription of the second copy of the MEN1 gene in the tumor tissues, thus leading to RNA instability.34,35 Furthermore, other tumor sup- pressor genes on 11q13 also may be involved in the development of these tumors.33
In the current study, although RT-PCR and West- ern blot analysis are not truly quantitative, they ap- peared to be similar with regard to the results. The combined results of these two assays demonstrated that the expression of both MEN1 mRNA and menin protein appears to be higher in Cushing syndrome and much weaker in primary aldosteronism. Weak expres- sion of MEN1 mRNA also was detected in pheochro- mocytoma on RT-PCR. However, menin protein ex- pression was not revealed in any case of pheochromocytoma by Western blot analysis, possibly due to its lower sensitivity compared with that of RT-PCR. These observations suggest that the MEN1 gene might play an important role in some other spe- cific cellular functions in adrenocortical tumors in addition to the development of MEN1 syndrome, al- though potential pathogenic mechanisms remain to be determined.
Recent reports have shed light on the cellular function of menin. Some authors have reported that menin principally is a nuclear protein,17,36 in which it possibly plays a role in transcriptional regulation, DNA replication, or cell cycle regulation. It also has been indicated that menin protein interacts directly with the AP1 transcription factor JunD and represses transcriptional activation mediated by JunD.29,36 In addition, Agarwal et al.29 also have reported that nat- urally occurring and clustered MEN1 missense muta- tions disrupt menin interaction with JunD. Many other observations have indicated that many of the mutations detected to date in the MEN1 gene most likely result in loss of function of its protein product, a finding that is consistent with a tumor suppressor
mechanism.12,18,30,37 Further studies aimed at the nor- mal function of menin and discovery of its specific metabolic pathway(s) should contribute to cell biology and could lead to new strategies in tumor therapy. In the current study, we were concerned especially with the importance of the MEN1 gene that appears to be overexpressed both at the mRNA and protein levels in Cushing syndrome, which is the most obvious finding of the current study, thus suggesting that a factor(s) other than tumorigenesis participates in the control of MEN1 gene expression in this tumor. A prospective study of this finding will allow us to assert strictly the prognostic value of this marker.
The AP1 transcription factors are a group of in- ducible proteins comprised of fos-related antigens (fra) and jun proteins that recognize and bind to spe- cific AP-1 DNA sequences in the promotor regions of genes.38 It has been reported that the rat adrenal gland contains a high basal level of AP-1 DNA binding ac- tivity in which the AP-1 protein complex is comprised of fra and c-jun proteins.39 It also has been indicated that mRNA for c-jun is several times higher in the cortex than in the medulla.4º Although it has been reported that menin protein interacts directly with the AP1 transcription factor JunD and represses transcrip- tional activation mediated by JunD,29,36 menin does not appear to inhibit transcription mediated by c-jun; rather, it appears to augment transcription activated by c-jun many times over.29 Steroidogenic acute reg- ulatory protein (StAR), specific to the adrenal cortex, enhances the mitochondrial conversion of cholesterol into pregnenolone by the cholesterol side-chain cleav- age enzyme cytochrome P450. Recently, StAR mRNA was expressed at high levels in normal human adrenal tumors and adrenocortical tumors and was up-regu- lated in parallel with cytochrome P450.41,42 Another interesting protein of potential importance is steroi- dogenic factor 1 (SF-1), a recently identified orphan nuclear hormone receptor that is a key regulator of steroid hydroxylases in adrenocortical cells.43 There- fore, we speculate that menin expression that appears to be up-regulated, particularly in Cushing syndrome, may play an important role in some specific cellular functions in the regulation of StAR, SF-1, and cyto- chrome P450 expression.
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