Indispensable role of the Runx1-Cbfbeta transcription complex for in vivo-suppressive function of FoxP3+ regulatory T cells.

Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here, we showed that Treg cell-specific deficiency of Cbfbeta, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyperproduction of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfbeta heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3.

PHLDA3 is a novel tumor suppressor of pancreatic neuroendocrine tumors.

The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.

Enhancement of catecholamine release from PC12 cells by the traditional Japanese medicine, rikkunshito.

BACKGROUND: Rikkunshito is a traditional Japanese herbal medicine that is used to treat appetite loss associated with cancer and other disorders. The formulation contains various constituents that influence cell signaling, and rikkunshito may accordingly affect human homeostasis through multiple regulatory pathways, including those governed by the endocrine system. We investigated the actions of rikkunshito on catecholamine release from PC12 cells, an adrenal chromaffin cell line. METHODS: The actions of rikkunshito on PC12 cells were evaluated by measuring intracellular cAMP levels, tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP) mRNA expression levels, and catecholamine levels in the culture medium. The transcriptional activation of VIP gene by rikkunshito was assessed by using a VIP promoter-driven reporter gene assay. RESULTS: Rikkunshito dose-dependently enhanced forskolin-induced elevations in cAMP in PC12 cells, and also increased the gene expression of TH and VIP. The transcriptional activation of VIP gene by rikkunshito was confirmed. Norepinephrine and dopamine secretion into the culture medium of PC12 cells were also dose-dependently augmented by rikkunshito and/or forskolin, but experiments with a protein kinase C (PKC) activator and a phosphodiesterase inhibitor revealed that the effects of rikkunshito were not simply due to the modulation of PKC or phosphodiesterase activity. CONCLUSIONS: These findings suggest that rikkunshito enhances the release of catecholamines by a novel mechanism involving cAMP.

Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1.

Naturally arising CD25+CD4+ regulatory T cells (T(R) cells) are engaged in the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses, such as autoimmune disease and allergy. T(R) cells specifically express the transcription factor Foxp3, a key regulator of T(R)-cell development and function. Ectopic expression of Foxp3 in conventional T cells is indeed sufficient to confer suppressive activity, repress the production of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and upregulate T(R)-cell-associated molecules such as CD25, cytotoxic T-lymphocyte-associated antigen-4, and glucocorticoid-induced TNF-receptor-family-related protein. However, the method by which Foxp3 controls these molecular events has yet to be explained. Here we show that the transcription factor AML1 (acute myeloid leukaemia 1)/Runx1 (Runt-related transcription factor 1), which is crucially required for normal haematopoiesis including thymic T-cell development, activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells through binding to their respective promoters. In natural T(R) cells, Foxp3 interacts physically with AML1. Several lines of evidence support a model in which the interaction suppresses IL-2 and IFN-gamma production, upregulates T(R)-cell-associated molecules, and exerts suppressive activity. This transcriptional control of T(R)-cell function by an interaction between Foxp3 and AML1 can be exploited to control physiological and pathological T-cell-mediated immune responses.

Analysis of genotype-phenotype correlations and survival outcomes in patients with primary hyperparathyroidism caused by multiple endocrine neoplasia type 1: the experience at a single institution.

PURPOSE: To examine the clinical characteristics and survival outcomes of patients with primary hyperparathyroidism (PHPT) in multiple endocrine neoplasia type 1 (MEN1) in relation to the MEN1 gene mutation. METHODS: The study population included the patients, positive for the MEN1 gene mutation, who underwent parathyroidectomy between 1983 and 2009 at a single tertiary referral center. Manifestations of the syndrome, other tumors and causes of death were retrospectively correlated with the specific types and locations of MEN1 gene mutations. RESULTS: Thirty-two patients from 19 families were diagnosed as having MEN1 on genetic examinations. Mutations were most common in exons 2, 7 and 10. A phenotypic analysis of the main MEN1 tumor types among the 32 patients revealed that PHPT was the most common (100 %), followed in order by pancreatic neuroendocrine tumors (PNETs) (53 %) and pituitary tumors (38 %). Death due to MEN1-related disease occurred in five patients (16 %), including malignant PNET in three cases (exons 2, 3), pituitary crisis in one case (exon 2) and thymic cancer in one case (large deletion). CONCLUSIONS: Premature deaths related to MEN1 are due to the development of malignant PNET, pituitary crisis or thymic tumors associated with mutations in exons 2, 3 and a large deletion.

Application of an intracellular stability test of a novel missense menin mutant to the diagnosis of multiple endocrine neoplasia type 1.

Germline MEN1 mutation analysis is a powerful tool for an early diagnosis of multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant familial cancer syndrome characterized by the parathyroid, pituitary and gastroenteropancreatic endocrine tumors. However, the clinical significance of MEN1 gene variants, especially missense and in-frame mutations as well as some splicing mutations, is not always obvious. We have previously shown that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. We also demonstrated by a fluorescent immunocytochemical stability test that the stability of missense and in-frame deletion mutants varies widely but that unstable mutants were found only in MEN1 and related disorders and not in normal polymorphisms. In the present study, we evaluated by this stability test the pathogenicity of a novel MEN1 missense mutation, c.1118C>T, encoding a P373L mutant menin, identified in a suspected MEN1 patient. The results demonstrated that the mutant menin is highly unstable, indicating that this mutation is causative for MEN1. These findings encouraged us to proceed with presymptomatic genetic screening for this mutation among the family members, which resulted in the identification of asymptomatic mutation carriers. Thus, the information from the menin stability test was useful for genetic diagnosis and counseling of MEN1 in the case with a previously unreported MEN1 missense mutation.

A novel splice site mutation of the MEN1 gene identified in a patient with primary hyperparathyroidism.

Heterozygous germline mutation of the tumor suppressor gene MEN1 is responsible for multiple endocrine neoplasia type 1 (MEN1), a familial cancer syndrome characterized by pituitary, parathyroid and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and its incomplete forms often manifested as familial isolated hyperparathyroidism and apparently sporadic parathyroid tumor. Mutation analysis of the MEN1 gene is a powerful tool for the early diagnosis of MEN1; however, the clinical significance of the identified mutations is not always obvious. In this study, a previously unreported missense MEN1 mutation, c.824G>T was identified in a patient with primary hyperparathyroidism and evaluated for its pathogenicity. This mutation was predicted to generate a putative missense menin protein, R275M. A stability test of the menin protein demonstrated that the stability of R275M mutant was reduced only slightly as compared with wild type menin, and therefore could not preclude the possibility that it was a rare benign polymorphism. However, further analysis of leukocyte mRNA and minigene experiments indicated that the mutant c.824G>T allele gives rise to abnormally spliced menin mRNA, and thereby confirmed that c.824G>T mutation is causative for MEN1. Thus, leukocyte mRNA analysis has been demonstrated useful to identify a splicing mutation of the MEN1 gene.

Correlation of mutant menin stability with clinical expression of multiple endocrine neoplasia type 1 and its incomplete forms.

Germline mutations of the tumor suppressor gene MEN1 are found not only in typical multiple endocrine neoplasia type 1 (MEN1) but also in its incomplete forms such as familial isolated hyperparathyroidism (FIHP) and apparently sporadic parathyroid tumor (ASPT). No definitive genotype-phenotype correlation has been established between these clinical forms and MEN1 gene mutations. We previously demonstrated that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. To examine whether the intracellular stability of mutant menin is correlated with clinical phenotypes, we developed a method of evaluating menin stability and examined 20 mutants associated with typical MEN1 (17 missense, two in-frame deletion, one nonsense) and 21 mutants associated with FIHP or ASPT (19 missense, two in-frame deletion). All tested mutants associated with typical MEN1 showed reduced stability. Some missense and in-frame deletion mutants (G28A, R171W, T197I, E255K, E274A, Y353del and E366D) associated with FIHP or ASPT were almost as stable as or only slightly less stable than wild-type menin, while others were as unstable as those associated with typical MEN1. Some stable mutants exhibited substantial biological activities when tested by JunD-dependent transactivation assay. These findings suggest that certain missense and in-frame mutations are fairly stable and retain intrinsic biological activity, and might be specifically associated with incomplete clinical phenotypes. The menin stability test will provide useful information for the management of patients carrying germline MEN1 mutations especially when they have missense or in-frame variants of ambiguous clinical significance.

Caspase 8 and menin expressions are not correlated in human parathyroid tumors.

Menin is lost by the sequential inactivation of both MEN1 alleles in subsets of non-hereditary endocrine tumors as well as those associated with multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant hereditary cancer syndrome characterized by multiple tumors including parathyroid, pituitary and enteropancreatic endocrine tumors. Loss of menin has been reported to be associated with lowered caspase 8 expression and resistance to apoptosis in murine fibroblasts and in pancreatic islet tumors arising in heterozygous MEN1 gene knockout mice, the animal model of the human MEN1 syndrome. We confirmed by menin-knockdown experiments with specific siRNA that menin is crucial for caspase 8 expression in human culture cells while overexpression of menin did not increase caspase 8 protein over basal levels. We then examined expression of menin, caspase 8 and cyclin-dependent kinase inhibitors p27(Kip1) and p15(Ink4b) by Western blotting in human parathyroid tumors surgically resected from patients with MEN1 and those with non-hereditary primary hyperparathyroidism. The menin and p27(Kip1) expression levels were correlated with MEN1 mutation status that was confirmed by DNA analysis. The caspase 8 and p15(Ink4b) protein levels were variable among tumors, and were not correlated with menin protein levels. These findings suggest that human endocrine tumors lacking menin may not always exhibit lowered caspase 8 expression and hence may not be resistant to apoptosis-inducing therapy.

MEN1 gene and its mutations: basic and clinical implications.

Heterozygous germline mutations of the tumor-suppressor gene MEN1 are responsible for multiple endocrine neoplasia type 1 (MEN1), a dominantly inherited familial cancer syndrome characterized by pituitary, parathyroid, and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and related disorders. Neither mutation hot spot nor phenotype­genotype correlation has been established in MEN1 although some missense mutations may be specifically associated with a phenotype of familial isolated hyperparathyroidism. The gene product menin has been implicated in multiple roles, including gene transcription, maintenance of genomic integrity, and control of cell division and differentiation. These multiple functions are likely to be conferred by association with multiple protein factors. Occurrence of MEN1-causing missense mutations throughout menin also suggests the requirement of multiple binding factors for its full tumor-suppressive activity. The effect of menin depletion is highly tissue specific, but its underlying mechanism remains to be elucidated. A DNA test for MEN1 germline mutations is a useful tool for diagnosis of MEN1 although it needs further improvements

Parathyroid carcinoma: etiology, diagnosis, and treatment.

BACKGROUND: The goal of the present study was to make our medical practice evidence-based for patients with parathyroid carcinoma. METHODS: We posed six clinical questions relevant to the management of parathyroid cancer. A comprehensive search and critical appraisal of the literature was then carried out. RESULTS: Most of the literature retrieved was retrospective in design and differed in the definition of carcinoma. The distinction between unequivocal and equivocal carcinoma (or atypical adenoma) was not always made for the study populations. None of the studies indicated reproducibility of outcome measures. Of the histopathological features described in the literature based on the description of Schantz and Castleman, capsular/vascular invasions and trabecular growth pattern were the most specific, and fibrous bands were the most sensitive. None of the patients with "atypical adenoma" developed recurrence, whereas 25% of those with "equivocal carcinoma" did. Mutations in HRPT2, the gene responsible for hereditary hyperparathyroidism with jaw-tumor syndrome, were strongly associated with sporadic parathyroid carcinoma. Severe hypercalcemia and its related clinical symptoms, extremely high levels of parathyroid hormone, osteitis fibrosa cystica, a palpable neck mass, and a relatively large depth-width ratio on ultrasonography, are the important features of parathyroid carcinoma. Disease-specific survival rates reported in the literature were varied, reflecting the differences in the definitions of carcinoma, study populations, and interventions. CONCLUSIONS: To establish valid evidence for patient management in the future, a collaboration of endocrine specialists is essential to conduct well-designed clinical studies for this rare disease.

Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.

Menin missense mutants associated with multiple endocrine neoplasia type 1 are rapidly degraded via the ubiquitin-proteasome pathway.

MEN1 is a tumor suppressor gene that is responsible for multiple endocrine neoplasia type 1 (MEN1) and that encodes a 610-amino-acid protein, called menin. While the majority of germ line mutations identified in MEN1 patients are frameshift and nonsense mutations resulting in truncation of the menin protein, various missense mutations have been identified whose effects on menin activity are unclear. For this study, we analyzed a series of menin proteins with single amino acid alterations and found that all of the MEN1-causing missense mutations tested led to greatly diminished levels of the affected proteins in comparison with wild-type and benign polymorphic menin protein levels. We demonstrate here that the reduced levels of the mutant proteins are due to rapid degradation via the ubiquitin-proteasome pathway. Furthermore, the mutants, but not wild-type menin, interact both with the molecular chaperone Hsp70 and with the Hsp70-associated ubiquitin ligase CHIP, and the overexpression of CHIP promotes the ubiquitination of the menin mutants in vivo. These findings reveal that MEN1-causing missense mutations lead to a loss of function of menin due to enhanced proteolytic degradation, which may be a common mechanism for inactivating tumor suppressor gene products in familial cancer.

Detection of APC gene deletion by double competitive polymerase chain reaction in patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant familial cancer syndrome caused by germline mutations of the tumor suppressor adenomatous polyposis coli (APC) gene. Heterozygous apc mutations have been identified in the majority of classical FAP patients who develop more than 100 colorectal adenomas. However, classical FAP patients often fail to display germline APC mutations detectable by routine mutation analysis. These apparently mutation-negative cases may be caused by heterozygous large genomic deletions. In the present study, FAP patients who showed no APC germline mutation detectable by the protein truncation assay and direct sequencing of protein coding exons were screened for APC gene deletion by a gene dose assay based on double competitive polymerase chain reaction. Gene dosage measurements within exon 15 of the APC gene identified two patients with gene deletion and one with possible gene duplication among 41 apparently mutation-negative patients. The deleted sequences in the two patients were determined by fine gene dose mapping around the APC gene and nucleotide sequencing of the deletion breakpoints. They were approximately 435-kilobase pair (kb) and 737-kb regions including the whole APC gene and flanked by a 4-base pair repeat and LINE-1 repetitive sequences, respectively. The chimeric LINE-1 element created at the breakpoint in the latter case also contained a short sequence derived from another LINE-1 element, suggesting a complex unequal homologous recombination event. These findings indicate that this gene dose assay is a useful technique to detect large gene deletions of the APC gene and to determine their genomic breakpoints.

Menin inactivation leads to loss of transforming growth factor beta inhibition of parathyroid cell proliferation and parathyroid hormone secretion.

Primary hyperparathyroidism is a common endocrine disorder caused by parathyroid gland enlargement and excessive parathyroid hormone (PTH) secretion. However, the precise mechanisms of tumorigenesis of the parathyroids are unknown. Here we have investigated the roles of transforming growth factor (TGF)-beta and menin, the product of the multiple endocrine neoplasia type 1 (Men1) gene, in the proliferation and PTH production of parathyroid cells from either patients with secondary hyperparathyroidism or Men1. TGF-beta was expressed in the parathyroid endocrine cells. Addition of TGF-beta to parathyroid cells from patients with secondary hyperparathyroidism inhibited their proliferation and PTH secretion. These responses to TGF-beta were lost when menin was specifically inactivated by antisense oligonucleotides. Moreover, TGF-beta did not affect the proliferation and PTH production of parathyroid cells from a Men1 patient. These results indicate that menin is required for TGF-beta action in the parathyroid. We conclude that TGF-beta is an important autocrine/paracrine negative regulator of parathyroid cell proliferation and PTH secretion and that loss of TGF-beta signaling due to menin inactivation contributes to parathyroid tumorigenesis.

A family of autosomal dominant hypocalcemia with a positive correlation between serum calcium and magnesium: identification of a novel gain of function mutation (Ser(820)Phe) in the calcium-sensing receptor.

To date about 20 activating mutations in the calcium-sensing receptor (CaR) gene have been identified to cause autosomal dominant hypocalcemia (ADH) or sporadic hypoparathyroidism. We report a novel activating mutation in the CaR gene in a Japanese family with ADH. The proband, a 15-yr-old boy, and 5 other patients in 3 generations were asymptomatic, except for the proband's grandmother who had a history of seizures. They showed mild hypocalcemia (1.68-1.98 mmol/liter) with normal urinary calcium excretion and low normal serum PTH levels. Their serum magnesium concentrations were below normal in 3 adults and within the normal range in 3 teenagers. There was a significant positive correlation (r = 0.90; P < 0.05) between the serum calcium and magnesium concentrations of 6 affected members. Nucleotide sequencing revealed that the proband had a known polymorphism (Gly(990)Arg) and a novel heterozygous mutation substituting phenylalanine for serine at codon 820 (Ser(820)Phe) in the sixth transmembrane helix of the CaR. In other family members, the Ser(820)Phe mutation cosegregated with hypocalcemia. The mutation was not detected in 50 control subjects. The Gly(990)Arg polymorphism was observed in 8 of 9 family members with or without hypocalcemia and in 36 of 50 controls. The sensitivity of the Ser(820)Phe mutant CaR to calcium was assessed using transiently transfected HEK293 cells and measuring the increases in intracellular Ca(2+) concentrations in response to the changes in extracellular Ca(2+). The concentration-response curve of the mutant receptor was left-shifted, and its EC(50) (2.5 mM) was significantly (P < 0.05) lower than that of the wild-type CaR (3.3 mM). We conclude that the Ser(820)Phe mutation in the CaR caused ADH in this family. The positive correlation between serum calcium and magnesium levels observed in this family may support the concept that renal CaR acts as a magnesium sensor as well as a calcium sensor.

Gene dose mapping delineated boundaries of a large germline deletion responsible for multiple endocrine neoplasia type 1.

A deleted genomic region including the MEN1 gene was determined in a family with multiple endocrine neoplasia type 1 caused by a large germline deletion. Gene dose mapping by the modified gene dose assay technique roughly located the deletion end points, which were then precisely determined by nucleotide sequencing. The deletion was approximately 68kbp flanked by a 3-base pair repeat and included the whole MEN1, MAP4K2 and KAPPA-200 genes. This is the first case of a large germline deletion responsible for multiple endocrine neoplasia type 1, in which the size and end points were determined precisely.

Allelic loss of DNA locus of the RET proto-oncogene in small cell lung cancer.

We analyzed all 21 exons of the RET proto-oncogene of paired genomic DNA from tumors and normal tissues in 12 small cell lung cancer (SCLC) patients for the presence of genetic alteration. Polymerase chain reaction single-strand conformation polymorphism analysis and direct sequencing revealed that heterozygosity of the RET proto-oncogene was lost in the tumor tissues of six patients out of eight informative SCLC patients, although point mutation was not evident in any tumors. These results suggest that a deletion of the chromosomal region including the RET proto-oncogene is involved in the pathogenesis of SCLC.

Upregulation of ghrelin expression in cachectic nude mice bearing human melanoma cells.

Ghrelin is a gastrointestinal peptide that stimulates food intake and growth hormone (GH) secretion. We studied the biosynthesis and secretion of ghrelin in a cancer cachexia mouse model. G361, a human melanoma cell line, was inoculated into nude mice. The body weight was reduced and the plasma concentration of interleukin-1beta (IL-1beta) was markedly higher in tumor-inoculated mice compared with vehicle-treated mice. Furthermore, white adipose tissue (WAT) weight, blood sugar level, and plasma concentrations of leptin and nonesterified fatty acids (NEFA) were significantly lower in tumor-inoculated mice. The plasma concentration of ghrelin increased with the progression of cachexia. The levels of both ghrelin peptide and mRNA in the stomach were also upregulated in tumor-inoculated mice. This study demonstrates that both ghrelin biosynthesis and secretion are stimulated in the long-term negative energy balance of tumor-inoculated cachectic mice. These findings suggest the involvement of ghrelin in the regulation of energy homeostasis in cancer cachexia.