MEN I gene mutations in sporadic adrenal adenomas

Loss of heterozygosity (LOH) on chromosome 11q13 occurs in about 20% of sporadic adrenal neoplasms. Adrenal lesions, mostly benign, occur in up to 40% of patients from MEN I kindreds. The MEN I gene, positioned on 11q13, has been considered a primary candidate gene in these lesions. We studied a group of 15 patients with sporadic adrenal adenoma, and 1 patient with multinodular hyperplasia. Of the 16 patients, 4 had incidentally discovered masses, 5 had Conn's syndrome, 6 suffered from Cushing's syndrome, and 9 had high sex hormone production. Studies with the markers D11S480, PYGM, D11S449, and D11S987 in 13 patients (12 of whom were from our group of 16) revealed 4 losses of heterozygosity on D11 S480 on 11q13, but the deletion did not affect the MEN I gene in any case. We present complete direct DNA sequencing data of the menin gene in 14 sporadic adrenal adenomas and one with adrenal hyperplasia. We identified one heterozygous missense mutation, T552S, in a hormonally inactive adrenal adenoma. One base exchange was identified close to the intron-exon boundary in intron 9 of a nodular adrenal hyperplasia. mRNA expression studies found that MEN I was transcribed in all 13 samples analyzed. In summary, our study identified the second patient with sporadic benign adrenal tumor presenting a menin gene mutation. Our complete direct sequencing approach adds evidence that menin gene mutations may account only for a minority of benign adrenal tumors if at all. Another tumor-suppressor gene inactivated in sporadic adrenal neoplasms may be located on chromosome 11q13.     

Menin induces apoptosis in murine embryonic fibroblasts

Multiple endocrine neoplasia type I (MEN1) is a hereditary tumor syndrome characterized by multiple endocrine and occasionally non-endocrine tumors. The tumor suppressor gene Men1, which is frequently mutated in MEN1 patients, encodes the nuclear protein menin. Although many tumor suppressor genes are involved in the regulation of apoptosis, it is unclear whether menin facilitates apoptosis. Here we show that ectopic overexpression of menin via adenoviruses induces apoptosis in murine embryonic fibroblasts. The induction of apoptosis depends on Bax and Bak, two proapoptotic proteins. Moreover, loss of menin expression compromises apoptosis induced by UV irradiation and tumor necrosis factor-alpha (TNF-alpha), whereas complementation of menin-null cells with menin restores sensitivity to UV- and TNF-alpha-induced apoptosis. Interestingly, loss of menin reduces the expression of procaspase 8, a critical protease that is essential for apoptosis induced by death-related receptors, whereas complementation of the menin-null cells up-regulates the expression of procaspase 8. Furthermore, complementation of menin-null cells with menin increases the activation of caspase 8 in response to TNF-alpha treatment. These results suggest a proapoptotic function for menin that may be important in suppressing the development of MEN1.     

The negative feedback-loop between the oncomir Mir-24-1 and menin modulates the Men1 tumorigenesis by mimicking the "Knudson's second hit"

Multiple endocrine neoplasia type 1 (MEN1) syndrome is a rare hereditary cancer disorder characterized by tumors of the parathyroids, of the neuroendocrine cells, of the gastro-entero-pancreatic tract, of the anterior pituitary, and by non-endocrine neoplasms and lesions. MEN1 gene, a tumor suppressor gene, encodes menin protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors, in agreement with the Knudson's two-hit hypothesis. In silico analysis with Target Scan, Miranda and Pictar-Vert softwares for the prediction of miRNA targets indicated miR-24-1 as capable to bind to the 3'UTR of MEN1 mRNA. We investigated this possibility by analysis of miR-24-1 expression profiles in parathyroid adenomatous tissues from MEN1 gene mutation carriers, in their sporadic non-MEN1 counterparts, and in normal parathyroid tissue. Interestingly, the MEN1 tumorigenesis seems to be under the control of a "negative feedback loop" between miR-24-1 and menin protein, that mimics the second hit of Knudson's hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Our data show an alternative way to MEN1 tumorigenesis and, probably, to the "two-hit dogma". The functional significance of this regulatory mechanism in MEN1 tumorigenesis is also the basis for opening future developments of RNA antagomir(s)-based strategies in the in vivo control of tumorigenesis in MEN1 carriers.     

Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1

We recently identified a novel metastasis suppressor gene, BRMS1, in breast cancer. Since the BRMS1 gene maps to chromosome 11q13.1-q13.2 and since chromosome 11q defects have been described in various stages of human melanoma progression, we hypothesized that BRMS1 may function as a tumor or metastasis suppressor in melanomas as well. Quantitative real-time RT-PCR revealed that BRMS1 mRNA expression was high in melanocytes, considerably reduced in early melanoma-derived cell lines, and barely detectable in advanced/metastatic cell lines. Stable transfectants of BRMS1 in the human melanoma cell lines MelJuSo and C8161.9 did not alter the tumorigenicity of either cell line, but significantly suppressed metastasis compared to vector-only transfectants. Orthotopic tumors continued to express BRMS1, but expression was lost in lung metastases. In vitro morphology, growth rate, and histology of BRMS1 transfectants were similar to controls. BRMS1 transfectants were less invasive in a collagen sandwich assay and had restored homotypic gap junctional intercellular communication (GJIC). Thus, BRMS1 functions as a metastasis suppressor in more than one tumor type (i.e., breast carcinoma and cutaneous melanoma) by modifying several metastasis-associated phenotypes.     

Menin and JunD regulate gastrin gene expression through proximal DNA elements

Mutations in the MEN1 gene correlate with multiple endocrine neoplasia I (MEN1). Gastrinomas are the most malignant of the neuroendocrine tumors associated with MEN1. Because menin and JunD proteins interact, we examined whether JunD binds to and regulates the gastrin gene promoter. Both menin and JunD are ubiquitous nuclear proteins that we showed colocalize in the gastrin-expressing G cells of the mouse antrum. Transfection with a JunD expression vector alone induced endogenous gastrin mRNA in AGS human gastric cells, and the induction was blocked by menin overexpression. We mapped repression by menin to both a nonconsensus AP-1 site and proximal GC-rich elements within the human gastrin promoter. Chromatin immunoprecipitation assays, EMSAs, and DNA affinity precipitation assays documented that JunD and Sp1 proteins bind these two elements and are both targets for menin regulation. Consistent with menin forming a complex with histone deacetylases, we found that repression of gastrin gene expression by menin was reversed by trichostatin A. In conclusion, proximal DNA elements within the human gastrin gene promoter mediate interactions between JunD, which induces gastrin gene expression and menin, which suppresses JunD-mediated activation.     

Sequence analysis of the MEN I gene in two patients with multiple cutaneous lipomas and endocrine tumors.

The discovery of mutations of the menin gene in a few multiple endocrine neoplasma type 1 (MEN I)-associated lipomas and loss of heterozygosity (LOH) on chromosome 11q13 in some sporadic lipomas has stimulated the hypothesis that lipomas may belong to the group of sporadic tumors caused by defects of the gene responsible for MEN I. Since it is unclear if the above hypothesis applies to all patients with lipoma or just to specific subsets, we searched to enlarge the database on this topic. For this purpose, we identified two patients with multiple cutaneous lipomas. One had an additional pituitary adenoma and familial presentation of multiple lipomas, the other had recurrent goiter in the setting of a family history of adenomatous goiter. Deoxyribonucleic acid (DNA) was analyzed by complete direct DNA sequencing of all coding exons and splice junctions of the MEN I gene. No mutation was identified in the coding exons of the menin gene. In contrast to former data on sporadic lipomas, these data are the first to render evidence that mutations of the MEN I gene may not be responsible for the formation of multiple lipomas, even if they appear in the context of other endocrine tumors.     

Somatostatin stimulates menin gene expression by inhibiting protein kinase A

Somatostatin is a potent inhibitor of gastrin secretion and gene expression. Menin is a 67-kDa protein product of the multiple endocrine neoplasia type 1 (MEN1) gene that when mutated leads to duodenal gastrinomas, a tumor that overproduces the hormone gastrin. These observations suggest that menin might normally inhibit gastrin gene expression in its role as a tumor suppressor. Since somatostatin and ostensibly menin are both inhibitors of gastrin, we hypothesized that somatostatin signaling directly induces menin. Menin protein expression was significantly lower in somatostatin-null mice, which are hypergastrinemic. We found by immunohistochemistry that somatostatin receptor-positive cells (SSTR2A) express menin. Mice were treated with the somatostatin analog octreotide to determine whether activation of somatostatin signaling induced menin. We found that octreotide increased the number of menin-expressing cells, menin mRNA, and menin protein expression. Moreover, the induction by octreotide was greater in the duodenum than in the antrum. The increase in menin observed in vivo was recapitulated by treating AGS and STC cell lines with octreotide, demonstrating that the regulation was direct. The induction required suppression of protein kinase A (PKA) since forskolin treatment suppressed menin protein levels and octreotide inhibited PKA enzyme activity. Small-interfering RNA-mediated suppression of PKA levels raised basal levels of menin protein and prevented further induction by octreotide. Using AGS cells, we also showed for the first time that menin directly inhibits endogenous gastrin gene expression. In conclusion, somatostatin receptor activation induces menin expression by suppressing PKA activation.     

Molecular and genetic mechanisms of tumorigenesis in multiple endocrine neoplasia type-1

Multiple endocrine neoplasia type 1 (MEN1) is a rare but informative syndrome for endocrine tumorigenesis. Since its isolation, several groups have begun to determine the role of menin, the protein product of MEN1, in sporadic endocrine tumors as well as tumors of the MEN1 syndrome. Mutations of menin have been reported in more than 400 families and tumors, most of which are truncating mutations, thus supporting the function of menin as a tumor suppressor. The exact function of menin is unknown, but overexpression of menin inhibits proliferation of Ras-transformed NIH3T3 cells. Since menin interacts with proteins from both the TGF beta and AP-1 signaling pathways, perhaps its tumor suppressor function is related to these key cell growth pathways. In this review we will discuss the various clinical manifestations of MEN1 syndrome, potential mechanisms of MEN1 tumorigenesis, and mutations associated with MEN and sporadic endocrine tumors.     

Genome-wide characterization of menin-dependent H3K4me3 reveals a specific role for menin in the regulation of genes implicated in MEN1-like tumors

Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies.     

Menin, a gene product responsible for multiple endocrine neoplasia type 1, interacts with the putative tumor metastasis suppressor nm23

Although the gene responsible for multiple endocrine neoplasia type 1 (MEN1) has been identified, the function of its gene product, menin, is unknown. To examine the biological role of the MEN1 gene, we searched for associated proteins with a yeast two-hybrid system using the MEN1 cDNA fragment as bait. On screening a rat fetal brain embryonic day 17 library, in which a high level of MEN1 expression was detected, we identified a putative tumor metastasis suppressor nm23/nucleoside diphosphate (NDP) kinase as an associated protein. This finding was confirmed by in vitro interaction assays based on glutathione S-transferase pull down experiments. The association required almost the entire menin protein, and several missense MEN1 mutations reported in MEN1 patients caused a loss of the binding activity for nm23. This result suggests that this interaction may play important roles in the biological functions of the menin protein, including tumor suppressor activity.     

Heterozygous Men1 mutant mice develop a range of endocrine tumors mimicking multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome characterized by the occurrence of multiple endocrine tumors of the parathyroid, pancreas, and anterior pituitary in patients. To study tumorigenesis related to the MEN1 syndrome, we have generated Men1 knockout mice using the gene targeting approach. Heterozygous Men1 mutant mice developed the same range of major endocrine tumors as is seen in MEN1 patients, affecting the parathyroid, pancreatic islets, pituitary and adrenal glands, as well as the thyroid, and exhibiting multistage tumor progression with metastatic potential. In particular, extrapancreatic gastrinoma, pancreatic glucagonoma, and mixed hormone-producing tumors in islets were observed. In addition, there was a high incidence of gonadal tumors of endocrine origin, i.e. Leydig cell tumors, and ovary sex-cord stromal cell tumors in heterozygous Men1 mutant mice. Hormonal disturbance, such as abnormal PTH and insulin levels, was also observed in these mice. These tumors were associated with loss of heterozygosity of the wild-type Men1 allele, suggesting that menin is involved in suppressing the development of these endocrine tumors. All of these features are reminiscent of MEN1 symptoms in humans and establish heterozygous Men1 mutant mice as a suitable model for this disease.     

Complete sequencing and mRNA expression analysis of the MEN-I gene in adrenal myelolipoma.

The molecular pathogenesis of adrenal myelolipoma is unclear. Endocrine activity of these tumors and association with other endocrine tumors have stimulated the hypothesis that it may belong to the group of sporadic tumors caused by defects of the gene responsible for multiple endocrine neoplasia type I (MEN-I). DNA of blood and tumoral sections from two patients with adrenal myelolipoma were analyzed by examination of variable number of tandem repeats (VNTR) loci PYGM, D11S987, D11S480, and D11S449 on chromosome 11q13 and by complete direct DNA sequencing of all coding exons and splice junctions of the MEN-I gene. Menin expression was examined by RT-PCR. RT-PCR did not detect menin expression in one adrenal myelolipoma. No loss of heterozygozity on chromosome 11q13 was identified. Intragenic heterozygozity was retained in codon 418 of the menin gene in both patients. No mutation was identified in the coding exons of the menin gene. Complete DNA sequencing yielded no hint that defects of the MEN-I gene are responsible for the formation of adrenal myelolipomas. Adrenal myelolipomas do not share the loss of heterozygozity on chromosome 11q13 observed in some benign adenomatous and many malignant adrenocortical tumors.