A Knock-In Mouse Model of the Gcm2 Variant p.Y392S Develops Normal Parathyroid Glands.

Context: The glial cells missing 2 (GCM2) gene functions as a transcription factor that is essential for parathyroid gland development, and variants in this gene have been associated with 2 parathyroid diseases: isolated hypoparathyroidism in patients with homozygous germline inactivating variants and primary hyperparathyroidism in patients with heterozygous germline activating variants. A recurrent germline activating missense variant of GCM2, p.Y394S, has been reported in patients with familial primary hyperparathyroidism. Objective: To determine whether the GCM2 p.Y394S missense variant causes overactive and enlarged parathyroid glands in a mouse model. Methods: CRISPR/Cas9 gene editing technology was used to generate a mouse model with the germline heterozygous Gcm2 variant p.Y392S that corresponds to the human GCM2 p.Y394S variant. Wild-type (Gcm2+/+) and germline heterozygous (Gcm2+/Y392S) mice were evaluated for serum biochemistry and parathyroid gland morphology. Results: Gcm2 +/Y392S mice did not show any change compared to Gcm2+/+ mice in serum calcium and parathyroid hormone levels, parathyroid gland histology, cell proliferation, or parathyroid gland size. Conclusion: The mouse model of the p.Y392S variant of Gcm2 shows that this variant is tolerated in mice, as it does not increase parathyroid gland cell proliferation and circulating calcium or PTH levels. Further investigation of Gcm2+/Y392S mice to study the effect of this variant of Gcm2 on early events in parathyroid gland development will be of interest.

Metastatic Grade 3 Neuroendocrine Tumor in Multiple Endocrine Neoplasia Type 1 Expressing Somatostatin Receptors.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) may occur in 30% to 90% of patients with multiple endocrine neoplasia type 1 (MEN1). However, only 1% of GEP-NETs are grade 3 (G3). Given the rarity of these aggressive tumors, treatment of advanced G3 GEP-NETs in MEN1 is based on the treatment guidelines for sporadic GEP-NETs. We report a 43-year-old male with germline MEN1 followed at our institution, with clinical features including hyperparathyroidism, a nonfunctional pancreatic NET, and Zollinger-Ellison syndrome. On routine surveillance imaging at age 40, computed tomography/positron emission tomography imaging showed 2 arterially enhancing intraluminal masses on the medial aspect of the gastric wall. Anatomical imaging confirmed 2 enhancing masses within the pancreas and a rounded mass-like thickening along the lesser curvature of the stomach. The gastric mass was resected, and pathology reported a well-differentiated G3 NET with a Ki-67 >20%. The patient continued active surveillance. Eighteen months later cross-sectional imaging studies showed findings consistent with metastatic disease within the right hepatic lobe and bland embolization was done. On follow-up scans, including 68Ga-DOTATATE (68Ga-DOTA(0)-Tyr(3)-octreotate) imaging, interval increase in number and avidity of metastatic lesions were compatible with disease progression. Given a paucity of treatment recommendations for G3 tumors in MEN1, the patient was counseled based on standard NET treatment guidelines and recommended 177Lu-DOTATATE treatment. PRRT (peptide receptor radionuclide therapy) with 177Lu-DOTATATE (177Lu-tetraazacyclododecanetetraacetic acid-octreotide) is an important therapeutic modality for patients with somatostatin receptor-positive NETs. However, prospective studies are needed to understand the role of PRRT in G3 NETs.

Mice With RIP-Cre-mediated Deletion of the Long Noncoding RNA Meg3 Show Normal Pancreatic Islets and Enlarged Pituitary.

Context: Maternally expressed gene 3 (MEG3) is a long noncoding RNA (lncRNA) that has been implicated as a tumor suppressor. Objective: The expression of MEG3 RNA is downregulated in various human tumors, including pituitary adenoma and pancreatic islet tumors due to MEG3 gene deletion or DNA hypermethylation. Mouse models with conventional germline deletion of Meg3 have shown that Meg3 is essential for perinatal or postnatal development and survival. However, a direct role of Meg3 loss in tumorigenesis has not been shown. Methods: To observe a causal relationship between Meg3 loss and tumorigenesis, we have generated a mouse model with conditional deletion of Meg3 mediated by the RIP-Cre transgene that initiated Meg3 deletion in pancreatic islet ß cells and anterior pituitary. Results: Meg3 loss did not lead to the development of islet tumors. Interestingly, RIP-Cre-mediated Meg3 loss led to the development of an enlarged pituitary. The genes in the Meg3 region are transcribed together as a 210 kb RNA that is processed into Meg3 and other transcripts. Whether these tandem transcripts play a functional role in the growth of pancreatic endocrine cells and pituitary cells remains to be determined. Conclusion: Our mouse model shows that Meg3 loss leads to hyperplasia in the pituitary and not in pancreatic islets, thus serving as a valuable model to study pathways associated with pituitary cell proliferation and function. Future mouse models with specific inactivation of Meg3 alone or other transcripts in the Meg3 polycistron are warranted to study tissue-specific effects on initiating neoplasia and tumor development.

Two distinct classes of thymic tumors in patients with MEN1 show LOH at the MEN1 locus.

Patients with the multiple endocrine neoplasia type 1 (MEN1) syndrome carry germline heterozygous loss-of-function mutations in the MEN1 gene which predisposes them to develop various endocrine and non-endocrine tumors. Over 90% of the tumors show loss of heterozygosity (LOH) at chromosome 11q13, the MEN1 locus, due to somatic loss of the wild-type MEN1 allele. Thymic neuroendocrine tumors (NETs) or thymic carcinoids are uncommon in MEN1 patients but are a major cause of mortality. LOH at the MEN1 locus has not been demonstrated in thymic tumors. The goal of this study was to investigate the molecular aspects of MEN1-associated thymic tumors including LOH at the MEN1 locus and RNA-sequencing (RNA-Seq) to identify genes associated with tumor development and potential targeted therapy. A retrospective chart review of 294 patients with MEN1 germline mutations identified 14 patients (4.8%) with thymic tumors (12 thymic NETs and 2 thymomas). LOH at the MEN1 locus was identified in 10 tumors including the 2 thymomas, demonstrating that somatic LOH at the MEN1 locus is also the mechanism for thymic tumor development. Unsupervised principal component analysis and hierarchical clustering of RNA-Seq data showed that thymic NETs formed a homogenous transcriptomic group separate from thymoma and normal thymus. KSR2 (kinase suppressor of Ras 2), that promotes Ras-mediated signaling, was abundantly expressed in thymic NETs, a potential therapeutic target. The molecular insights gained from our study about thymic tumors combined with similar data from other MEN1-associated tumors may lead to better surveillance and treatment of these rare tumors.

Functional Defects From Endocrine Disease-Associated Mutations in HLXB9 and Its Interacting Partner, NONO.

The insulin-secreting pancreatic neuroendocrine tumors, insulinomas, characterized by increased pancreatic islet ß-cell proliferation, express the phosphorylated isoform of the ß-cell differentiation factor HLXB9 that interacts with NONO/p54NRB, a survival factor. Interestingly, two different homozygous germline mutations in HLXB9, p.F248L and p.F272L, were reported in neonatal diabetes, a condition with functional ß-cell deficiency. Also, two somatic heterozygous NONO mutations were found in endocrine-related tumors, p.H146R (parathyroid) and p.R293H (small intestine neuroendocrine tumor). However, the biological consequence of the mutations, and the role of HLXB9-NONO interaction in normal or abnormal ß cells, is not known. Expression, localization, and functional analysis of the clinically relevant HLXB9 and NONO mutants showed that HLXB9/p.F248L mutant localized in the nucleus but lacked phosphorylation, and NONO/p.R293H mutant was structurally impaired. The HLXB9 and NONO mutants retained the ability to interact, and overexpression of wild-type or mutant HXLB9 in MIN6 cells suppressed cell proliferation. To further understand the biological consequence of the HLXB9-NONO interaction, we mapped the NONO-interacting region in HLXB9. An 80-amino acid conserved region of HLXB9 could compete with full-length HLXB9 to interact with NONO; however, in functional assays, nuclear expression of this HLXB9-conserved region in MIN6 cells did not interfere with cell proliferation. Overall, our results highlight the importance of HLXB9 in conditions of ß-cell excess (insulinomas) and in conditions of ß-cell loss or dysfunction (diabetes). Our studies implicate therapeutic strategies for either reducing ß-cell proliferation in insulinomas or alleviating normal ß-cell deficiency in diabetes through the modulation of HLXB9 phosphorylation.

Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b).

Pancreatic islet ß-cells that lack the MEN1-encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the ß-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting ß-cell tumors (insulinomas). In this study we investigated the binding partners and target genes of phospho-HLXB9 in mouse insulinoma MIN6 ß-cells. Co-immunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54-kDa nuclear RNA-binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phospho-HLXB9 as a pro-oncogenic factor by interacting with a survival factor and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing ß-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in ß-cell expansion strategies to alleviate ß-cell loss in diabetes.

Consequence of Menin Deficiency in Mouse Adipocytes Derived by In Vitro Differentiation.

Lipoma in patients with the multiple endocrine neoplasia type 1 (MEN1) syndrome is a type of benign fat-cell tumor that has biallelic inactivation of MEN1 that encodes menin and could serve as a model to investigate normal and pathologic fat-cell (adipocyte) proliferation and function. The role of menin and its target genes in adipocytes is not known. We used in vitro differentiation to derive matched normal and menin-deficient adipocytes from wild type (WT) and menin-null (Men1-KO) mouse embryonic stem cells (mESCs), respectively, or 3T3-L1 cells without or with menin knockdown to investigate cell size, lipid content, and gene expression changes. Adipocytes derived from Men1-KO mESCs or after menin knockdown in 3T3-L1 cells showed a 1.5-1.7-fold increase in fat-cell size. Global gene expression analysis of mESC-derived adipocytes showed that lack of menin downregulated the expression of many differentially methylated genes including the tumor suppressor long noncoding RNA Meg3 but upregulated gene expression from the prolactin gene family locus. Our results show that menin deficiency leads to fat-cell hypertrophy and provide model systems that could be used to study the regulation of fat-cell size.

Epigenetic regulation of the lncRNA MEG3 and its target c-MET in pancreatic neuroendocrine tumors.

Biallelic inactivation of MEN1 encoding menin in pancreatic neuroendocrine tumors (PNETs) associated with the multiple endocrine neoplasia type 1 (MEN1) syndrome is well established, but how menin loss/inactivation initiates tumorigenesis is not well understood. We show that menin activates the long noncoding RNA maternally expressed gene 3 (Meg3) by histone-H3 lysine-4 trimethylation and CpG hypomethylation at the Meg3 promoter CRE site, to allow binding of the transcription factor cAMP response element-binding protein. We found that Meg3 has tumor-suppressor activity in PNET cells because the overexpression of Meg3 in MIN6 cells (insulin-secreting mouse PNET cell line) blocked cell proliferation and delayed cell cycle progression. Gene expression microarray analysis showed that Meg3 overexpression in MIN6 mouse insulinoma cells down-regulated the expression of the protooncogene c-Met (hepatocyte growth factor receptor), and these cells showed significantly reduced cell migration/invasion. Compared with normal islets, mouse or human MEN1-associated PNETs expressed less MEG3 and more c-MET. Therefore, a tumor-suppressor long noncoding RNA (MEG3) and suppressed protooncogene (c-MET) combination could elicit menin's tumor-suppressor activity. Interestingly, MEG3 and c-MET expression was also altered in human sporadic insulinomas (insulin secreting PNETs) with hypermethylation at the MEG3 promoter CRE-site coinciding with reduced MEG3 expression. These data provide insights into the ß-cell proliferation mechanisms that could retain their functional status. Furthermore, in MIN6 mouse insulinoma cells, DNA-demethylating drugs blocked cell proliferation and activated Meg3 expression. Our data suggest that the epigenetic activation of lncRNA MEG3 and/or inactivation of c-MET could be therapeutic for treating PNETs and insulinomas.

GSK-3ß protein phosphorylates and stabilizes HLXB9 protein in insulinoma cells to form a targetable mechanism of controlling insulinoma cell proliferation.

Insulinomas (pancreatic islet ß cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic ß cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3ß inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3ß are elevated in ß cells with menin knockdown, in MEN1-associated ß cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3ß blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G2/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3ß and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3ß and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with ß cell proliferation.

Germline and somatic mutations in cyclin-dependent kinase inhibitor genes CDKN1A, CDKN2B, and CDKN2C in sporadic parathyroid adenomas.

The molecular pathogenesis of sporadic parathyroid adenomas is incompletely understood. The possible role of cyclin-dependent kinase inhibitor (CDKI) genes was raised by recognition of cyclin D1 as a parathyroid oncogene, identification of rare germline mutations in CDKI genes in patients with multiple endocrine neoplasia type 1; that in rodents, mutation in Cdkn1b caused parathyroid tumors; and subsequently through identification of rare predisposing germline sequence variants and somatic mutation of CDKN1B, encoding p27(kip1), in sporadic human parathyroid adenoma. We therefore sought to determine whether mutations/variants in the other six CDKI genes CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and CDKN2D, encoding p21, p57, p14(ARF)/p16, p15, p18, and p19, respectively, contribute to the development of typical parathyroid adenomas. In a series of 85 sporadic parathyroid adenomas, direct DNA sequencing identified alterations in five adenomas (6 %): Two contained distinct heterozygous changes in CDKN1A, one germline and one of undetermined germline status; one had a CDKN2B germline alteration, accompanied by loss of the normal allele in the tumor (LOH); two had variants of CDKN2C, one somatic and one germline with LOH. Abnormalities of three of the mutant proteins were readily demonstrable in vitro. Thus, germline mutations/rare variants in CDKN1A, CDKN2B, and CDKN2C likely contribute to the development of a significant subgroup of common sporadic parathyroid adenomas, and somatic mutation in CDKN2C further suggests a direct role for CDKI alteration in conferring a selective growth advantage to parathyroid cells, providing novel support for the concept that multiple CDKIs can play primary roles in human neoplasia.

The embryonic transcription factor Hlxb9 is a menin interacting partner that controls pancreatic ß-cell proliferation and the expression of insulin regulators.

The multiple endocrine neoplasia type 1 (MEN1) syndrome is caused by germline mutations in the MEN1 gene encoding menin, with tissue-specific tumors of the parathyroids, anterior pituitary, and enteropancreatic endocrine tissues. Also, 30-40% of sporadic pancreatic endocrine tumors show somatic MEN1 gene inactivation. Although menin is expressed in all cell types of the pancreas, mouse models with loss of menin in either pancreatic α-cells, or ß-cells, or total pancreas develop ß-cell-specific endocrine tumors (insulinomas). Loss of widely expressed tumor suppressor genes may produce tissue-specific tumors by reactivating one or more embryonic-specific differentiation factors. Therefore, we determined the effect of menin overexpression or knockdown on the expression of ß-cell differentiation factors in a mouse ß-cell line (MIN6). We show that the ß-cell differentiation factor Hlxb9 is posttranscriptionally upregulated upon menin knockdown, and it interacts with menin. Hlxb9 reduces cell proliferation and causes apoptosis in the presence of menin, and it regulates genes that modulate insulin level. Thus, upon menin loss or from other causes, dysregulation of Hlxb9 predicts a possible combined mechanism for ß-cell proliferation and insulin production in insulinomas. These observations help to understand how a ubiquitously expressed protein such as menin might control tissue-specific tumorigenesis. Also, our findings identify Hlxb9 as an important factor for ß-cell proliferation and insulin regulation.

Tocopherol succinate: modulation of antioxidant enzymes and oncogene expression, and hematopoietic recovery.

PURPOSE: A class of naturally occurring isoforms of tocopherol (tocols) was shown to have varying degrees of protection when administered before radiation exposure. We recently demonstrated that α-tocopherol succinate (TS) is a potential radiation prophylactic agent. Our objective in this study was to further investigate the mechanism of action of TS in mice exposed to (60)Co γ-radiation. METHODS AND MATERIALS: We evaluated the effects of TS on expression of antioxidant enzymes and oncogenes by quantitative RT-PCR in bone marrow cells of (60)Co γ-irradiated mice. Further, we tested the ability of TS to rescue and repopulate hematopoietic stem cells by analyzing bone marrow cellularity and spleen colony forming unit in spleen of TS-injected and irradiated mice. RESULTS: Our results demonstrate that TS modulated the expression of antioxidant enzymes and inhibited expression of oncogenes in irradiated mice at different time points. TS also increased colony forming unit-spleen numbers and bone marrow cellularity in irradiated mice. CONCLUSIONS: Results provide additional support for the observed radioprotective efficacy of TS and insight into mechanisms.

Effects of genistein administration on cytokine induction in whole-body gamma irradiated mice.

The development of an effective pharmacological countermeasure is needed to reduce the morbidity and mortality in military and civilian populations associated with possible exposure to ionizing radiation. We previously demonstrated that a single subcutaneous (sc) administration of genistein at a non-toxic dose provided protection against acute radiation injury and that the radioprotective effects were associated with multilineage, hematopoietic progenitor cell recovery. The purpose of this study was to determine whether hematopoietic recovery was preceded by cytokine induction. In mice treated with sc genistein 24 h before irradiation (7 Gy 60Co), we quantified serum cytokine levels by multiplex Luminex and also investigated a larger number of cytokines using cytokine arrays. Genistein administration stimulated serum granulocyte-colony stimulating factor (G-CSF) 4h and 24h after sham irradiation or gamma-irradiation. Interleukin-6 (IL-6) was significantly increased in genistein-treated animals 4h after irradiation. Because G-CSF and IL-6 are important hematopoietic factors, these results support our hypothesis that the previously observed radioprotective efficacy by genistein may be a result of early recovery of hematopoietic cells due to enhanced production of G-CSF and IL-6.

Role of NF-kappaB in hematopoietic niche function of osteoblasts after radiation injury.

OBJECTIVE: Hematopoietic tissue is very sensitive to ionizing radiation (IR). In adult mammalian bone marrow, hematopoietic stem and progenitor cells (HSPC) reside next to the endosteal bone surface, which is lined primarily by osteoblastic cells. In the present study, we proposed to investigate the mechanisms by which osteoblasts in the hematopoietic niche regulate survival, proliferation, and differentiation of HSPC after radiation injury. MATERIALS AND METHODS: Human primary CD34+ HSPC were cultured with human fetal osteoblast (hFOB) cell line cells or conditioned medium (CM) from hFOB cells with or without irradiation. Survival, apoptosis, and cell cycle were analyzed using clonogenic and flow cytometric assays. Cytokine and chemokine expression were measured by cytokine array and enzyme-linked immunosorbent assay. Their regulatory activities were assessed by quantitative real-time polymerase chain reaction, small interfering (si)RNA transfection, immunoblotting, and transbinding assays. RESULTS: Survival of gamma-irradiated CD34+ HSPC was significantly enhanced by coculture with hFOB cells or by CM from hFOB cells. There were six factors in hFOB cell lysates and five factors released into hFOB CM enhanced by IR. IR induced phosphorylation of p53, c-Jun, and p38 and downstream p21 expression, as well as cell cycle arrest and apoptosis in hFOB cells. However, IR also induced phosphorylation of nuclear factor (NF)-kappaBp65 (ser536) and NF-kappaB activation in hFOB cells. Inhibition of NF-kappaB expression with siRNA upregulated p21, inhibited release of cytokines and chemokines, and induced hFOB and CD34+ cell apoptosis. CONCLUSIONS: NF-kappaB is a radiation-induced prosurvival factor in human osteoblastic cells. NF-kappaB gene knockdown abrogated the hematopoietic niche function of hFOB cells in supporting survival of CD34+ cells after IR.

5-Androstenediol promotes survival of gamma-irradiated human hematopoietic progenitors through induction of nuclear factor-kappaB activation and granulocyte colony-stimulating factor expression.

5-Androstenediol (5-AED) stimulates hematopoiesis and enhances survival in animals exposed to ionizing radiation (IR), suggesting that this steroid may act on hematopoietic progenitor cells. We used gamma-irradiated primary human CD34(+) hematopoietic progenitor cells to show that 5-AED protects hematopoietic cells from IR damage, as shown by enhanced cell survival, clonogenicity, proliferation, and differentiation. Unlike in tumor cells, IR did not induce nuclear factor-kappaB (NFkappaB) activation in primary progenitors. However, IR stimulated IkappaB(beta) release from NFkappaB/IkappaB complexes and caused NFkappaB1 (p50) degradation. 5-AED stabilized NFkappaB1 in irradiated cells and induced NFkappaB gene expression and NFkappaB activation (DNA binding). 5-AED stimulated interleukin-6 and granulocyte colony-stimulating factor (G-CSF) secretion. The survival-enhancing effects of 5-AED on clonogenic cells were abrogated by small interfering RNA inhibition of NFkappaB gene expression and by neutralization of G-CSF with antibody. The effects of 5-AED on survival and G-CSF secretion were blocked by the NFkappaB inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). 5-AED had no effect on accumulation of the proapoptotic factor p53 after IR, as determined by Western blot. The results indicate that NFkappaB1 degradation after IR may be responsible for the radiation sensitivity of CD34+ cells compared with tumor cells. 5-AED exerts survival-enhancing effects on irradiated human hematopoietic progenitor cells via induction, stabilization, and activation of NFkappaB, which results in increased secretion of hematopoietic growth factor G-CSF.