Xiuli Jiang

Activating Hotspot L205R Mutation in PRKACA and Adrenal Cushing's Syndrome

Adrenal Cushing’s syndrome is caused by excess production of glucocorticoid from adrenocortical tumors and hyperplasias, which leads to metabolic disorders. We performed whole-exome sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing adrenocortical adenomas (ACAs), adrenocorticotropic hormone–independent macronodular adrenocortical hyperplasias (AIMAHs), and adrenocortical oncocytomas (ADOs). We identified a hotspot in the PRKACA gene with a L205R mutation in 69.2% (27 out of 39) of ACAs and validated in 65.5% of a total of 87 ACAs. Our data revealed that the activating L205R mutation, which locates in the P+1 loop of the protein kinase A (PKA) catalytic subunit, promoted PKA substrate phosphorylation and target gene expression. Moreover, we discovered the recurrently mutated gene DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight potentially functional mutated genes in adrenal Cushing’s syndrome. Adrenal Cushing’s syndrome involves recurrent mutations in a key signal transduction pathway [Also see Perspective by Kirschner] Candidate Cushings culprit identified Cushings syndrome is a rare condition resulting from the excess production of cortisol. About 15% of Cushings syndrome cases are associated with an adrenocortical tumor. However, the genetic etiology of these adrenocortical tumors is ill defined (see the Perspective by Kirschner). Cao et al. and Sato et al. both performed whole-exome sequencing of tumors from individuals with adrenal Cushings syndrome and compared it with the patients own matched non-tumor DNA and identified recurrent mutations in the protein kinase A catalytic subunit alpha (PRKACA) gene, as well as less frequent mutations in other putative pathological genes. The most common recurrent mutation activated the kinase, which may suggest a potential therapeutic target. Science, this issue p. 913, p. 917; see also p. 804

Nuclear-Cytoplasmic Shuttling of Menin Regulates Nuclear Translocation of β-Catenin

ABSTRACT Menin, which is encoded by the multiple endocrine neoplasia type 1 (MEN1) gene, is a tumor suppressor and transcriptional regulator. Menin controls proliferation and apoptosis of cells, especially pancreatic β cells. We have found that menin contains two functional nuclear export signals and that there is nuclear accumulation of β-catenin in Men1-null mouse embryonic fibroblasts and insulinoma tissues from β-cell-specific Men1 knockout mice. It is reported that the deregulation of Wnt/β-catenin signaling caused by inactivation of tumor suppressors results in abnormal development or tumorigenesis. We further revealed that overexpression of menin reduces β-catenin nuclear accumulation and its transcriptional activity. Menin is able to directly interact with β-catenin and carry β-catenin out of the nucleus via nuclear-cytoplasmic shuttling in a CRM1-dependent manner. These results imply that menin may control cell proliferation through suppression of Wnt/β-catenin signaling.

Whole exome sequencing of insulinoma reveals recurrent T372R mutations in YY1

Functional pancreatic neuroendocrine tumours (PNETs) are mainly represented by insulinoma, which secrete insulin independent of glucose and cause hypoglycaemia. The major genetic alterations in sporadic insulinomas are still unknown. Here we identify recurrent somatic T372R mutations in YY1 by whole exome sequencing of 10 sporadic insulinomas. Further screening in 103 additional insulinomas reveals this hotspot mutation in 30% (34/113) of all tumours. T372R mutation alters the expression of YY1 target genes in insulinomas. Clinically, the T372R mutation is associated with the later onset of tumours. Genotyping of YY1, a target of mTOR inhibitors, may contribute to medical treatment of insulinomas. Our findings highlight the importance of YY1 in pancreatic β-cells and may provide therapeutic targets for PNETs.

Hepatic menin recruits SIRT1 to control liver steatosis through histone deacetylation

BACKGROUND & AIMS The development and progression of non-alcoholic fatty liver disease are associated with aging, obesity, and type 2 diabetes. Understanding the precise regulatory networks of this process will contribute to novel therapeutic strategies. METHODS Hepatocyte-specific Men1 knockout mice were generated using Cre/loxP technology. Lipid and glucose metabolic phenotypes and mechanisms were investigated in aging and high-fat diet fed mice. RESULTS The expression of menin, encoded by multiple endocrine neoplasia 1 (Men1) gene, is reduced in the liver of aging mice. Hepatocyte-specific deletion of Men1 induces liver steatosis in aging mice. Menin deficiency promotes high-fat diet-induced liver steatosis in mice. Menin recruits SIRT1 to control hepatic CD36 expression and triglyceride accumulation through histone deacetylation. CONCLUSIONS Our work reveals that the adaptor protein menin is critical for the progression of hepatic steatosis during aging and metabolic imbalance.

Targeting β-catenin signaling for therapeutic intervention in MEN1 -deficient pancreatic neuroendocrine tumours

Inactivating MEN1 mutations are the most common genetic defects present in sporadic and inherited pancreatic neuroendocrine tumours (PNETs). The lack of interventional therapies prompts us to explore the therapeutic approach of targeting β-catenin signalling in MEN1-mutant PNETs. Here we show the MEN1-encoded scaffold protein menin regulates phosphorylation of β-catenin. β-catenin signalling is activated in MEN1-mutant human and mouse PNETs. Conditional knockout of β-catenin suppresses the tumorigenesis and growth of Men1-deficient PNETs, and significantly prolongs the survival time in mice. Suppression of β-catenin signalling by genetic ablation or a molecular antagonist inhibits the expression of proproliferative genes in menin-null PNETs and potently improves hyperinsulinemia and hypoglycemia in mice. Blockade of β-catenin has no adverse effect on physiological function of pancreatic β-cells. Our data demonstrate that β-catenin signalling is an effective therapeutic target for MEN1-mutant PNETs. Our findings may contribute to individualized and combined medication treatment for PNETs.

miR-144/451 Promote Cell Proliferation via Targeting PTEN/AKT Pathway in Insulinomas

Insulinoma is the main type of functional pancreatic neuroendocrine tumors. The functional microRNAs (miRNAs) regulating tumor growth and progression in insulinomas are still unknown. We conducted the miRNA expression profile analysis using miRNA quantitative RT-PCR array and identified 114 differentially expressed miRNAs in human insulinomas compared with normal pancreatic islets. Forty-one differentially expressed miRNAs belonged to 7 miRNA families, and 28 miRNAs in 3 of the families localized in the epigenetically regulated imprinted chromosome 14q32 region. We validated the most significant differentially expressed miRNA cluster miR-144/451 in another 8 human normal islet samples and 25 insulinomas. Our data showed that the overexpression of miR-144/451 in mouse pancreatic β-cells promoted cell proliferation by targeting the β-cell regulator phosphatase and tensin homolog deleted on chromosome ten/v-akt murine thymoma viral oncogene homolog pathway and cyclin-dependent kinase inhibitor 2D. Our findings highlight the importance of functional miRNAs in insulinomas.

Conditional Deletion of Men1 in the Pancreatic β-Cell Leads to Glucagon-Expressing Tumor Development

The tumor suppressor menin is recognized as a key regulator of β-cell proliferation. To induce tumorigenesis within the pancreatic β-cells, floxed alleles of Men1 were selectively ablated using Cre-recombinase driven by the insulin promoter. Despite the β-cell specificity of the RipCre, glucagon-expressing tumors as well as insulinomas developed in old mutant mice. These glucagon-expressing tumor cells were menin deficient and expressed the mature α-cell-specific transcription factors Brain-specific homeobox POU domain protein 4 (Brn4) and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB). Moreover, the inactivation of β-cell-specific transcription factors was observed in mutant β-cells. Our work shows that Men1 ablation in the pancreatic β-cells leads to the inactivation of specific transcription factors, resulting in glucagon-expressing tumor development, which sheds light on the mechanisms of islet tumorigenesis.

Generation and Characterization of Transgenic Mice Expressing Mouse Ins1 Promoter for Pancreatic β-Cell-Specific Gene Overexpression and Knockout.

The technologies for pancreatic β-cell-specific gene overexpression or knockout are fundamental for investigations of functional genes in vivo. Here we generated the Ins1-Cre-Dsred and Ins1-rtTA mouse models, which expressed the Cre recombinase or reverse tetracycline regulatable transactivator (rtTA) without hGH minigene under the control of mouse Ins1 promoter. Our data showed that the Cre-mediated recombination and rtTA-mediated activation could be efficiently detected at embryonic day 13.5 when these models were crossed with the reporter mice (ROSA(mT/mG) or tetO-HIST1H2BJ/GFP). The Cre and rtTA expression was restricted to β-cells without leakage in the brain and other tissues. Moreover, both the transgenic lines showed normal glucose tolerance and insulin secretion. These results suggested that the Ins1-Cre-Dsred and Ins1-rtTA mice could be used to knock out or overexpress target genes in embryos and adults to facilitate β-cell researches.

Isolation of mouse islet by collagenase perfusion through the splenic vein.

Islet transplantation has become an attractive option for the treatment of insulin-dependent diabetes mellitus. For the improvement of islet transplantation, mouse models have been widely used. The classical procedure of islet isolation is based on collagenase digestion of pancreas and separation of islets (1Y4), with the common bile duct (CBD) as the puncture site to inject the collagenase solution. Here, a novel puncture route of in situ pancreas perfusion is described, in which we cannulate into the splenic vein (SV). This protocol is feasible to be mastered and especially suitable for the conditions in which CBD method does not work. In SV protocol, the pancreas was swelled via injection of collagenase solution (0.5 mg/mL in Hank balanced salt solution) into the SV. As illustrated in Figure 1A, phrenicosplenic and gastrosplenic ligament are severed to free the spleen. Splenic pancreas is separated from descending colon and rectum by severing any ligament between them. Then the spleen and splenic pancreas are flipped over to the right to expose the spleen vein on the dorsal surface of the splenic pancreas. The distal end of the splenic vein close to the spleen is clamped with a hemostatic forceps. The diaphragm is incised and the heart is transected to terminate the pancreatic blood circulation. Under a dissecting microscope, SV is punctured with a 24G needle to a point just at the junction where it joins with the superior mesenteric vein. Then 1 to 2 mL of collagenase was injected into the splenic vein until the splenic pancreas is fully inflated. The inflated splenic pancreas is dissected free of the remaining gastric and duodenal pancreas lobes and any other attachments, and placed in the 15-mL tube containing 1 mL of collagenase solution. Then the inflated pancreas was digested and the islets were purified just like in the CBD method as previously described (2). Finally, islets were harvested by hand under microscopy. Compared to common bile duct, the SV is longer, thicker, and stronger. In addition, the anatomical structure of splenic vein is stable. In young mice (such as 2-week-old mice), the increased difficulty of puncture and perfusion often leads to failure by CBD protocol. In obese mice (such as highfat-diet-induced or ob/ob mouse model) or pregnant mice, the distal part of pancreas is exclusively not easy to be inflated by CBD protocol. By SV protocol, we isolated islets from 6-month-old ob/ob mice and 2-week-old mice with a higher success rate (Fig. 1D, E). Moreover, the SV protocol is more effective when the bile duct is blocked or damaged in some mouse models. The pancreatic duct ligation (PDL) mouse model is widely used to investigate pancreatitis and islet regeneration. We successfully isolated islets from the PDL mouse model (Fig. 1F) by the SV protocol. The quantity of islets and success rate is not limited by duct blockade in these mice. The isolated islets by the SV protocol can be cultured and used for further experiments and analysis (Fig. 1G). Different dyes are used to determine the viability of islets. Acridine orange (AO) incorporates into healthy cells and fluoresces green. Propidium iodide (PI) is a membrane-penetrated red fluorescent dye, which enters only dead or dying cells. Figure 1H and I demonstrated that SV protocol did not increase cell death significantly. For further examination of A-cell function in vivo, we performed subcapsular kidney islet transplantation in streptozotocin-induced diabetes mouse models. The transplantation of 400 islets isolated by the SV protocol could improve hyperglycemia effectively. When we isolated islets from normal mice, the CBD protocol is the first choice for us. However, if the puncture of CBD failed, the SV protocol could be performed to avoid unnecessary sacrifice of mice. In some mice (È10%), the splenic ducts joins gastric duct and opens into the duodenum with an accessory papilla, so the collagenase solution cannot inflate the splenic and gastric pancreatic lobes through the CBD method. Moreover, the common bile duct is not suitable for puncture in several models, such as obesity and PDL model. Our data showed that the SV protocol is practicable and reproducible for islet isolation in these mouse models. Gotoh et al. (5) reported rat islets could be isolated with portal vein (PV) as the collagenase injection route. However, the procedure of PV method is complicated and is not very feasible for mouse model. In conclusion, the SV is a complementary site for collagenase perfusion in the process of islet isolation. The SV protocol extends the feasibility of collagenase perfusion in multiple mouse models and reduces the number of sacrificed mice.

Whole exome sequencing of thymic neuroendocrine tumor with ectopic ACTH syndrome

OBJECTIVE Thymic neuroendocrine tumor is the second-most prevalent cause of ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS), which is a rare disease characterized by ectopic ACTH oversecretion from nonpituitary tumors. However, the genetic abnormalities of thymic neuroendocrine tumors with EAS remain largely unknown. We aim to elucidate the genetic abnormalities and identify the somatic mutations of potential tumor-related genes of thymic neuroendocrine tumors with EAS by whole exome sequencing. DESIGN AND METHODS Nine patients with thymic neuroendocrine tumors with EAS who were diagnosed at Shanghai Clinical Center for Endocrine and Metabolic Diseases in Ruijin Hospital between 2002 and 2014 were enrolled. We performed whole exome sequencing on the DNA obtained from thymic neuroendocrine tumors and matched peripheral blood using the Hiseq2000 platform. RESULTS We identified a total of 137 somatic mutations (median of 15.2 per tumor; range, 1-24) with 129 single-nucleotide mutations (SNVs). The predominant substitution in these mutations was C:G > T:A transition. Approximately 80% of detected mutations resulted in amino acid changes. However, we failed to discover any recurrent mutations in these nine patients. By functional predictions, HRAS, PAK1 and MEN1, previously reported in neuroendocrine tumors, were identified as candidate tumor-related genes associated with thymic neuroendocrine tumors. CONCLUSIONS Using whole exome sequencing, we identified genetic abnormalities in thymic neuroendocrine tumors with EAS. Thereby, this study acts as a further supplement of the genetic features of neuroendocrine tumors. Somatic mutations of three potential tumor-related genes (HRAS, PAK1 and MEN1) might contribute to the tumorigenesis of thymic neuroendocrine tumors with EAS.