Publications by authors named "Cynthia L Andoniadou"

35 Publications

Loss of SDHB Promotes Dysregulated Iron Homeostasis, Oxidative Stress, and Sensitivity to Ascorbate.

Cancer Res 2021 Jul 14;81(13):3480-3494. Epub 2021 Jun 14.

PARCC, INSERM UMR970, Equipe Labellisée par la Ligue Contre le Cancer, Paris, France.

Succinate dehydrogenase is a key enzyme in the tricarboxylic acid cycle and the electron transport chain. All four subunits of succinate dehydrogenase are tumor suppressor genes predisposing to paraganglioma, but only mutations in the SDHB subunit are associated with increased risk of metastasis. Here we generated an knockout chromaffin cell line and compared it with deficient cells. Both cell types exhibited similar SDH loss of function, metabolic adaptation, and succinate accumulation. In contrast, cells showed hallmarks of mesenchymal transition associated with increased DNA hypermethylation and a stronger pseudo-hypoxic phenotype compared with cells. Loss of SDHB specifically led to increased oxidative stress associated with dysregulated iron and copper homeostasis in the absence of NRF2 activation. High-dose ascorbate exacerbated the increase in mitochondrial reactive oxygen species, leading to cell death in cells. These data establish a mechanism linking oxidative stress to iron homeostasis that specifically occurs in -deficient cells and may promote metastasis. They also highlight high-dose ascorbate as a promising therapeutic strategy for SDHB-related cancers. SIGNIFICANCE: Loss of different succinate dehydrogenase subunits can lead to different cell and tumor phenotypes, linking stronger 2-OG-dependent dioxygenases inhibition, iron overload, and ROS accumulation following SDHB mutation.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2936DOI Listing
July 2021

Dynamic Expression of Imprinted Genes in the Developing and Postnatal Pituitary Gland.

Genes (Basel) 2021 Mar 30;12(4). Epub 2021 Mar 30.

Department of Medical and Molecular Genetics, Faculty of Life Sciences and Medicine, King's College London, London SE19RT, UK.

In mammals, imprinted genes regulate many critical endocrine processes such as growth, the onset of puberty and maternal reproductive behaviour. Human imprinting disorders (IDs) are caused by genetic and epigenetic mechanisms that alter the expression dosage of imprinted genes. Due to improvements in diagnosis, increasing numbers of patients with IDs are now identified and monitored across their lifetimes. Seminal work has revealed that IDs have a strong endocrine component, yet the contribution of imprinted gene products in the development and function of the hypothalamo-pituitary axis are not well defined. Postnatal endocrine processes are dependent upon the production of hormones from the pituitary gland. While the actions of a few imprinted genes in pituitary development and function have been described, to date there has been no attempt to link the expression of these genes as a class to the formation and function of this essential organ. This is important because IDs show considerable overlap, and imprinted genes are known to define a transcriptional network related to organ growth. This knowledge deficit is partly due to technical difficulties in obtaining useful transcriptomic data from the pituitary gland, namely, its small size during development and cellular complexity in maturity. Here we utilise high-sensitivity RNA sequencing at the embryonic stages, and single-cell RNA sequencing data to describe the imprinted transcriptome of the pituitary gland. In concert, we provide a comprehensive literature review of the current knowledge of the role of imprinted genes in pituitary hormonal pathways and how these relate to IDs. We present new data that implicate imprinted gene networks in the development of the gland and in the stem cell compartment. Furthermore, we suggest novel roles for individual imprinted genes in the aetiology of IDs. Finally, we describe the dynamic regulation of imprinted genes in the pituitary gland of the pregnant mother, with implications for the regulation of maternal metabolic adaptations to pregnancy.
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http://dx.doi.org/10.3390/genes12040509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066104PMC
March 2021

Single-cell molecular profiling of all three components of the HPA axis reveals adrenal ABCB1 as a regulator of stress adaptation.

Sci Adv 2021 Jan 27;7(5). Epub 2021 Jan 27.

Department of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry, Munich, Bavaria 80804, Germany.

Chronic activation and dysregulation of the neuroendocrine stress response have severe physiological and psychological consequences, including the development of metabolic and stress-related psychiatric disorders. We provide the first unbiased, cell type-specific, molecular characterization of all three components of the hypothalamic-pituitary-adrenal axis, under baseline and chronic stress conditions. Among others, we identified a previously unreported subpopulation of cells involved in stress adaptation in the adrenal gland. We validated our findings in a mouse stress model, adrenal tissues from patients with Cushing's syndrome, adrenocortical cell lines, and peripheral cortisol and genotyping data from depressed patients. This extensive dataset provides a valuable resource for researchers and clinicians interested in the organism's nervous and endocrine responses to stress and the interplay between these tissues. Our findings raise the possibility that modulating ABCB1 function may be important in the development of treatment strategies for patients suffering from metabolic and stress-related psychiatric disorders.
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http://dx.doi.org/10.1126/sciadv.abe4497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840126PMC
January 2021

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.

Elife 2021 Jan 5;10. Epub 2021 Jan 5.

Centre for Craniofacial and Regenerative Biology, King's College London, London, United Kingdom.

In response to physiological demand, the pituitary gland generates new hormone-secreting cells from committed progenitor cells throughout life. It remains unclear to what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to pituitary growth and renewal. Moreover, neither the signals that drive proliferation nor their sources have been elucidated. We have used genetic approaches in the mouse, showing that the WNT pathway is essential for proliferation of all lineages in the gland. We reveal that SOX2 stem cells are a key source of WNT ligands. By blocking secretion of WNTs from SOX2 PSCs in vivo, we demonstrate that proliferation of neighbouring committed progenitor cells declines, demonstrating that progenitor multiplication depends on the paracrine WNT secretion from SOX2 PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion and homeostasis, acting as paracrine signalling centres to coordinate the proliferation of neighbouring cells.
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http://dx.doi.org/10.7554/eLife.59142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803373PMC
January 2021

Insulin and obesity transform hypothalamic-pituitary-adrenal axis stemness and function in a hyperactive state.

Mol Metab 2021 01 4;43:101112. Epub 2020 Nov 4.

Department of Internal Medicine III, University Hospital Carl Gustav Carus, Dresden University of Technology, Fetscherstraße 74, Dresden, 01307, Germany. Electronic address:

Objective: Metabolic diseases are an increasing problem in society with the brain-metabolic axis as a master regulator of the human body for sustaining homeostasis under metabolic stress. However, metabolic inflammation and disease will trigger sustained activation of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the role of metabolic stress on progenitor cells in the hypothalamic-pituitary-adrenal axis.

Methods: In vitro, we applied insulin and leptin to murine progenitor cells isolated from the pituitary and adrenal cortex and examined the role of these hormones on proliferation and differentiation. In vivo, we investigated two different mouse models of metabolic disease, obesity in leptin-deficient ob/ob mice and obesity achieved via feeding with a high-fat diet.

Results: Insulin was shown to lead to enhanced proliferation and differentiation of both pituitary and adrenocortical progenitors. No alterations in the progenitors were noted in our chronic metabolic stress models. However, hyperactivation of the hypothalamic-pituitary-adrenal axis was observed and the expression of the appetite-regulating genes Npy and Agrp changed in both the hypothalamus and adrenal.

Conclusions: It is well-known that chronic stress and stress hormones such as glucocorticoids can induce metabolic changes including obesity and diabetes. In this article, we show for the first time that this might be based on an early sensitization of stem cells of the hypothalamic-pituitary-adrenal axis. Thus, pituitary and adrenal progenitor cells exposed to high levels of insulin are metabolically primed to a hyper-functional state leading to enhanced hormone production. Likewise, obese animals exhibit a hyperactive hypothalamic-pituitary-adrenal axis leading to adrenal hyperplasia. This might explain how stress in early life can increase the risk for developing metabolic syndrome in adulthood.
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http://dx.doi.org/10.1016/j.molmet.2020.101112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691554PMC
January 2021

Requirement of FAT and DCHS protocadherins during hypothalamic-pituitary development.

JCI Insight 2020 10 27;5(23). Epub 2020 Oct 27.

Pituitary developmental defects lead to partial or complete hormone deficiency and significant health problems. The majority of cases are sporadic and of unknown cause. We screened 28 patients with pituitary stalk interruption syndrome (PSIS) for mutations in the FAT/DCHS family of protocadherins that have high functional redundancy. We identified seven variants, four of which putatively damaging, in FAT2 and DCHS2 in six patients with pituitary developmental defects recruited through a cohort of patients with mostly ectopic posterior pituitary gland and/or pituitary stalk interruption. All patients had growth hormone deficiency and two presented with multiple hormone deficiencies and small glands. FAT2 and DCHS2 were strongly expressed in the mesenchyme surrounding the normal developing human pituitary. We analyzed Dchs2-/- mouse mutants and identified anterior pituitary hypoplasia and partially penetrant infundibular defects. Overlapping infundibular abnormalities and distinct anterior pituitary morphogenesis defects were observed in Fat4-/- and Dchs1-/- mouse mutants but all animal models displayed normal commitment to the anterior pituitary cell type. Together our data implicate FAT/DCHS protocadherins in normal hypothalamic-pituitary development and identify FAT2 and DCHS2 as candidates underlying pituitary gland developmental defects such as ectopic pituitary gland and/or pituitary stalk interruption.
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http://dx.doi.org/10.1172/jci.insight.134310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714405PMC
October 2020

RNA in situ hybridization for human papillomavirus testing in oropharyngeal squamous cell carcinoma on a routine clinical diagnostic platform.

J Oral Pathol Med 2021 Jan 1;50(1):68-75. Epub 2020 Sep 1.

Head and Neck Pathology, Guy's and St Thomas' NHS Foundation Trust, London, UK.

Background: The current diagnostic standard for detection of high-risk human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma is via a two-stage algorithm, namely p16 immunohistochemistry followed by HPV DNA in situ hybridization in p16 positive cases. This study evaluated the feasibility of automated RNA in situ hybridization on a clinical platform as a single-step alternative to the two-stage algorithm within a routine diagnostic histopathology setting.

Methods: Thirty-eight cases positive for both p16 and DNA in situ hybridization, 42 p16 negative cases and 20 cases positive for p16 but negative for DNA in situ hybridization were randomly selected. High-risk HPV RNA in situ hybridization was undertaken on all cases on an automated clinical platform. Manufacturer-recommended and on-slide additional p16/HPV positive and negative controls were used. Test quality assurance and diagnostic RNA in situ hybridization were independently assessed by two observers. A consensus diagnosis was reached in the presence of a third observer on discordant cases. All RNA in situ hybridization results were then correlated against p16 and DNA ISH status.

Results: Inter-slide RNA in situ hybridization staining variation was observed in control sections. RNA in situ hybridization demonstrated a high inter-observer agreement rate (κ = .897, P < .001). Following consensus review, there was full concordance between RNA in situ hybridization and the current standard.

Conclusion: Human papillomavirus testing by standalone automated RNA in situ hybridization on a clinical diagnostic platform currently available in routine diagnostic histopathology laboratories is a feasible alternative to the two-step algorithm of p16 and DNA in situ hybridization. Control tissue staining procedures need to be adapted to achieve the most accurate results.
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http://dx.doi.org/10.1111/jop.13103DOI Listing
January 2021

Cancer Stem Cells in Pheochromocytoma and Paraganglioma.

Front Endocrinol (Lausanne) 2020 25;11:79. Epub 2020 Feb 25.

Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Pheochromocytoma (PCC) and paraganglioma (PGL) are rare neuroendocrine tumors associated with high cardiovascular morbidity and variable risk of malignancy. The current therapy of choice is surgical resection. Nevertheless, PCCs/PGLs are associated with a lifelong risk of tumor persistence or recurrence. A high rate of germline or somatic mutations in numerous genes has been found in these tumors. For some, the tumorigenic processes are initiated during embryogenesis. Such tumors carry gene mutations leading to pseudohypoxic phenotypes and show more immature characteristics than other chromaffin cell tumors; they are also often multifocal or metastatic and occur at an early age, often during childhood. Cancer stem cells (CSCs) are cells with an inherent ability of self-renewal, de-differentiation, and capacity to initiate and maintain malignant tumor growth. Targeting CSCs to inhibit cancer progression has become an attractive anti-cancer therapeutic strategy. Despite progress for this strategy for solid tumors such as neuroblastoma, brain, breast, and colon cancers, no substantial advance has been made employing similar strategies in PCCs/PGLs. In the current review, we discuss findings related to the identification of normal chromaffin stem cells and CSCs, pathways involved in regulating the development of CSCs, and the importance of the stem cell niche in development and maintenance of CSCs in PCCs/PGLs. Additionally, we examine the development and feasibility of novel CSC-targeted therapeutic strategies aimed at eradicating especially recurrent and metastatic tumors.
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http://dx.doi.org/10.3389/fendo.2020.00079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051940PMC
March 2021

Novel medications inducing adrenal insufficiency.

Nat Rev Endocrinol 2019 10;15(10):561-562

Department of Medicine, University Hospital Carl Gustav Carus, Dresden, Germany.

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http://dx.doi.org/10.1038/s41574-019-0248-9DOI Listing
October 2019

Editorial.

Semin Cell Dev Biol 2019 07 20;91. Epub 2018 Dec 20.

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http://dx.doi.org/10.1016/j.semcdb.2018.12.004DOI Listing
July 2019

Isolation and characterization of adrenocortical progenitors involved in the adaptation to stress.

Proc Natl Acad Sci U S A 2018 12 4;115(51):12997-13002. Epub 2018 Dec 4.

Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany;

The adrenal gland is a master regulator of the human body during response to stress. This organ shows constant replacement of senescent cells by newly differentiated cells. A high degree of plasticity is critical to sustain homeostasis under different physiological demands. This is achieved in part through proliferation and differentiation of adult adrenal progenitors. Here, we report the isolation and characterization of a Nestin population of adrenocortical progenitors located under the adrenal capsule and scattered throughout the cortex. These cells are interconnected with progenitors in the medulla. In vivo lineage tracing revealed that, under basal conditions, this population is noncommitted and slowly migrates centripetally. Under stress, this migration is greatly enhanced, and the cells differentiate into steroidogenic cells. Nestin cells cultured in vitro also show multipotency, as they differentiate into mineralocorticoid and glucocorticoid-producing cells, which can be further influenced by the exposure to Angiotensin II, adrenocorticotropic hormone, and the agonist of luteinizing hormone-releasing hormone, triptorelin. Taken together, Nestin cells in the adult adrenal cortex exhibit the features of adrenocortical progenitor cells. Our study provides evidence for a role of Nestin cells in organ homeostasis and emphasizes their role under stress. This cell population might be a potential source of cell replacement for the treatment of adrenal insufficiency.
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http://dx.doi.org/10.1073/pnas.1814072115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304967PMC
December 2018

Non-secreting pituitary tumours characterised by enhanced expression of YAP/TAZ.

Endocr Relat Cancer 2019 01;26(1):215-225

Centre for Craniofacial and Regenerative Biology, King's College London, London, UK.

Tumours of the anterior pituitary can manifest from all endocrine cell types but the mechanisms for determining their specification are not known. The Hippo kinase cascade is a crucial signalling pathway regulating growth and cell fate in numerous organs. There is mounting evidence implicating this in tumour formation, where it is emerging as an anti-cancer target. We previously demonstrated activity of the Hippo kinase cascade in the mouse pituitary and nuclear association of its effectors YAP/TAZ with SOX2-expressing pituitary stem cells. Here, we sought to investigate whether these components are expressed in the human pituitary and if they are deregulated in human pituitary tumours. Analysis of pathway components by immunofluorescence reveals pathway activity during normal human pituitary development and in the adult gland. Poorly differentiated pituitary tumours (null-cell adenomas, adamantinomatous craniopharyngiomas (ACPs) and papillary craniopharyngiomas (PCPs)), displayed enhanced expression of pathway effectors YAP/TAZ. In contrast, differentiated adenomas displayed lower or absent levels. Knockdown of the kinase-encoding Lats1 in GH3 rat mammosomatotropinoma cells suppressed Prl and Gh promoter activity following an increase in YAP/TAZ levels. In conclusion, we have demonstrated activity of the Hippo kinase cascade in the human pituitary and association of high YAP/TAZ with repression of the differentiated state both in vitro and in vivo. Characterisation of this pathway in pituitary tumours is of potential prognostic value, opening up putative avenues for treatments.
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http://dx.doi.org/10.1530/ERC-18-0330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215911PMC
January 2019

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

Acta Neuropathol 2018 05 14;135(5):757-777. Epub 2018 Mar 14.

MRC London Institute of Medical Sciences, Imperial College London, London, UK.

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.
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http://dx.doi.org/10.1007/s00401-018-1830-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904225PMC
May 2018

Basic Research Advances on Pituitary Stem Cell Function and Regulation.

Neuroendocrinology 2018 14;107(2):196-203. Epub 2018 Mar 14.

As a central regulator of major physiological processes, the pituitary gland is a highly dynamic organ, capable of responding to hormonal demand and hypothalamic influence, through adapting secretion as well as remodelling cell numbers among its seven populations of differentiated cells. Stem cells of the pituitary have been shown to actively generate new cells during postnatal development but remain mostly quiescent during adulthood, where they persist as a long-lived population. Despite a significant body of research characterising attributes of anterior pituitary stem cells, the regulation of this population is poorly understood. A better grasp on the signalling mechanisms influencing stem proliferation and cell fate decisions can impact on our future treatments of pituitary gland disorders such as organ failure and pituitary tumours, which can disrupt endocrine homeostasis with life-long consequences. This minireview addresses the current methodologies aiming to understand better the attributes of pituitary stem cells and the normal regulation of this population in the organ, and discusses putative future avenues to manipulate pituitary stem cells during disease states or regenerative medicine approaches.
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http://dx.doi.org/10.1159/000488393DOI Listing
January 2019

Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma.

Nat Commun 2017 11 28;8(1):1819. Epub 2017 Nov 28.

Developmental Biology and Cancer Programme, Birth Defects Research Centre, UCL Institute of Child Health, London, WC1N 1EH, UK.

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2- cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.
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http://dx.doi.org/10.1038/s41467-017-01992-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703905PMC
November 2017

Two missense mutations in KCNQ1 cause pituitary hormone deficiency and maternally inherited gingival fibromatosis.

Nat Commun 2017 11 3;8(1):1289. Epub 2017 Nov 3.

IGF, CNRS, INSERM, Univ. Montpellier, F-34094, Montpellier, France.

Familial growth hormone deficiency provides an opportunity to identify new genetic causes of short stature. Here we combine linkage analysis with whole-genome resequencing in patients with growth hormone deficiency and maternally inherited gingival fibromatosis. We report that patients from three unrelated families harbor either of two missense mutations, c.347G>T p.(Arg116Leu) or c.1106C>T p.(Pro369Leu), in KCNQ1, a gene previously implicated in the long QT interval syndrome. Kcnq1 is expressed in hypothalamic GHRH neurons and pituitary somatotropes. Co-expressing KCNQ1 with the KCNE2 β-subunit shows that both KCNQ1 mutants increase current levels in patch clamp analyses and are associated with reduced pituitary hormone secretion from AtT-20 cells. In conclusion, our results reveal a role for the KCNQ1 potassium channel in the regulation of human growth, and show that growth hormone deficiency associated with maternally inherited gingival fibromatosis is an allelic disorder with cardiac arrhythmia syndromes caused by KCNQ1 mutations.
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http://dx.doi.org/10.1038/s41467-017-01429-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668380PMC
November 2017

Hypothalamic sonic hedgehog is required for cell specification and proliferation of LHX3/LHX4 pituitary embryonic precursors.

Development 2017 09 14;144(18):3289-3302. Epub 2017 Aug 14.

Developmental Biology and Cancer Programme, Birth Defects Research Centre, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK

Sonic hedgehog (SHH) is an essential morphogenetic signal that dictates cell fate decisions in several developing organs in mammals. data suggest that SHH is required to specify LHX3/LHX4 Rathke's pouch (RP) progenitor identity. However, studies have failed to reveal such a function, supporting instead a crucial role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3/LHX4 RP identity in mouse embryos. First, we show that conditional deletion of in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of expression in RP epithelium at 9.0 days post coitum (dpc) and total loss of pituitary tissue by 12.5 dpc. Conversely, overactivation of the SHH pathway by conditional deletion of in RP progenitors leads to severe hyperplasia and enlargement of the Sox2 stem cell compartment by the end of gestation.
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http://dx.doi.org/10.1242/dev.153387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612255PMC
September 2017

MAPK pathway control of stem cell proliferation and differentiation in the embryonic pituitary provides insights into the pathogenesis of papillary craniopharyngioma.

Development 2017 06 15;144(12):2141-2152. Epub 2017 May 15.

Developmental Biology and Cancer Programme, Birth Defects Research Centre, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK

Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here, we show that the over-activation of the MAPK pathway, through conditional expression of the gain-of-function alleles and in the developing mouse pituitary, results in severe hyperplasia and abnormal morphogenesis of the gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted, leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2 stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring also contains Sox2 cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a crucial function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2 cells and suggest that persistent proliferative capacity of Sox2 cells may underlie the pathogenesis of PCP.
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http://dx.doi.org/10.1242/dev.150490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482995PMC
June 2017

Adrenal cortical and chromaffin stem cells: Is there a common progeny related to stress adaptation?

Mol Cell Endocrinol 2017 02 13;441:156-163. Epub 2016 Sep 13.

Department of Internal Medicine III, Technische Universität Dresden, Dresden, Germany; Department of Endocrinology and Diabetes, King's College London, London, UK.

The adrenal gland is a highly plastic organ with the capacity to adapt the body homeostasis to different physiological needs. The existence of stem-like cells in the adrenal cortex has been revealed in many studies. Recently, we identified and characterized in mice a pool of glia-like multipotent Nestin-expressing progenitor cells, which contributes to the plasticity of the adrenal medulla. In addition, we found that these Nestin progenitors are actively involved in the stress response by giving rise to chromaffin cells. Interestingly, we also observed a Nestin-GFP-positive cell population located under the adrenal capsule and scattered through the cortex. In this article, we discuss the possibility of a common progenitor giving rise to subpopulations of cells both in the adrenal cortex and medulla, the isolation and characterization of this progenitor as well as its clinical potential in transplantation therapies and in pathophysiology.
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http://dx.doi.org/10.1016/j.mce.2016.09.011DOI Listing
February 2017

Expression Analysis of the Hippo Cascade Indicates a Role in Pituitary Stem Cell Development.

Front Physiol 2016 31;7:114. Epub 2016 Mar 31.

Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London London, UK.

The pituitary gland is a primary endocrine organ that controls major physiological processes. Abnormal development or homeostatic disruptions can lead to human disorders such as hypopituitarism or tumors. Multiple signaling pathways, including WNT, BMP, FGF, and SHH regulate pituitary development but the role of the Hippo-YAP1/TAZ cascade is currently unknown. In multiple tissues, the Hippo kinase cascade underlies neoplasias; it influences organ size through the regulation of proliferation and apoptosis, and has roles in determining stem cell potential. We have used a sensitive mRNA in situ hybridization method (RNAscope) to determine the expression patterns of the Hippo pathway components during mouse pituitary development. We have also carried out immunolocalisation studies to determine when YAP1 and TAZ, the transcriptional effectors of the Hippo pathway, are active. We find that YAP1/TAZ are active in the stem/progenitor cell population throughout development and at postnatal stages, consistent with their role in promoting the stem cell state. Our results demonstrate for the first time the collective expression of major components of the Hippo pathway during normal embryonic and postnatal development of the pituitary gland.
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http://dx.doi.org/10.3389/fphys.2016.00114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814506PMC
April 2016

Concise Review: Paracrine Role of Stem Cells in Pituitary Tumors: A Focus on Adamantinomatous Craniopharyngioma.

Stem Cells 2016 Feb 13;34(2):268-76. Epub 2016 Jan 13.

Craniofacial Development and Stem Cell Biology, King's College London, Guy's Campus, London, United Kingdom.

The existence of tissue-specific progenitor/stem cells in the adult pituitary gland of the mouse has been demonstrated recently using genetic tracing experiments. These cells have the capacity to differentiate into all of the different cell lineages of the anterior pituitary and self-propagate in vitro and can therefore contribute to normal homeostasis of the gland. In addition, they play a critical role in tumor formation, specifically in the etiology of human adamantinomatous craniopharyngioma, a clinically relevant tumor that is associated with mutations in CTNNB1 (gene encoding β-catenin). Mouse studies have shown that only pituitary embryonic precursors or adult stem cells are able to generate tumors when targeted with oncogenic β-catenin, suggesting that the cell context is critical for mutant β-catenin to exert its oncogenic effect. Surprisingly, the bulk of the tumor cells are not derived from the mutant progenitor/stem cells, suggesting that tumors are induced in a paracrine manner. Therefore, the cell sustaining the mutation in β-catenin and the cell-of-origin of the tumors are different. In this review, we will discuss the in vitro and in vivo evidence demonstrating the presence of stem cells in the adult pituitary and analyze the evidence showing a potential role of these stem cells in pituitary tumors.
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http://dx.doi.org/10.1002/stem.2267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864894PMC
February 2016

Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

Sci Rep 2015 Sep 28;5:14479. Epub 2015 Sep 28.

Department of Craniofacial Development and Stem Cell Biology, King's College London, UK.

In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma.
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http://dx.doi.org/10.1038/srep14479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585991PMC
September 2015

Establishment of the neurogenic boundary of the mouse retina requires cooperation of SOX2 and WNT signaling.

Neural Dev 2014 Dec 9;9:27. Epub 2014 Dec 9.

UNC Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA.

Background: Eye development in vertebrates relies on the critical regulation of SOX2 expression. Humans with mutations in SOX2 often suffer from eye defects including anophthalmia (no eye) and microphthalmia (small eye). In mice, deletion of Sox2 in optic cup progenitor cells results in loss of neural competence and cell fate conversion of the neural retina to a non-neurogenic fate, specifically the acquisition of fate associated with progenitors of the ciliary epithelium. This fate is also promoted with constitutive expression of stabilized β-Catenin in the optic cup, where the WNT pathway is up-regulated. We addressed whether SOX2 co-ordinates the neurogenic boundary of the retina through modulating the WNT/β-Catenin pathway by using a genetic approach in the mouse.

Results: Upon deletion of Sox2 in the optic cup, response to WNT signaling was expanded, correlating with loss of neural competence, cell fate conversion of the neural retina to ciliary epithelium primordium and, in addition, increased cell cycle time of optic cup progenitors. Removal of Ctnnb1 rescued the cell fate conversion; however, the loss of neural competence and the proliferation defect resulting from lack of SOX2 were not overcome. Lastly, central Sox2-deficient optic cup progenitor cells exhibited WNT-independent up-regulation of D-type Cyclins.

Conclusion: We propose two distinct roles for SOX2 in the developing retina. Our findings suggest that SOX2 antagonizes the WNT pathway to maintain a neurogenic fate and, in contrast, regulates cycling of optic cup progenitors in a WNT-independent manner. Given that WNT signaling acting upstream of SOX2 has been implicated in the tumorigenicity of embryonic stem cell-derived retinal progenitor cells, our results distinguish the endogenous role of WNT signaling in early optic cup patterning and support a WNT-independent role for SOX2 in maintaining retinal progenitor cell proliferation.
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http://dx.doi.org/10.1186/1749-8104-9-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295269PMC
December 2014

CHD7 maintains neural stem cell quiescence and prevents premature stem cell depletion in the adult hippocampus.

Stem Cells 2015 Jan;33(1):196-210

King's College London, Department of Craniofacial Development and Stem Cell Biology, Guy's Hospital Tower Wing, London, UK.

Neural stem/progenitor cells (NSCs) in the hippocampus produce new neurons throughout adult life. NSCs are maintained in a state of reversible quiescence and the failure to maintain the quiescent state can result in the premature depletion of the stem cell pool. The epigenetic mechanisms that maintain this quiescent state have not been identified. Using an inducible knockout mouse model, we show that the chromatin remodeling factor chromodomain-helicase-DNA-binding protein 7 (CHD7) is essential for maintaining NSC quiescence. CHD7 inactivation in adult NSCs results in a loss of stem cell quiescence in the hippocampus, a transient increase in cell divisions, followed by a significant decline in neurogenesis. This loss of NSC quiescence is associated with the premature loss of NSCs in middle-aged mice. We find that CHD7 represses the transcription of several positive regulators of cell cycle progression and is required for full induction of the Notch target gene Hes5 in quiescent NSCs. These findings directly link CHD7 to pathways involved in NSC quiescence and identify the first chromatin-remodeling factor with a role in NSC quiescence and maintenance. As CHD7 haplo-insufficiency is associated with a range of cognitive disabilities in CHARGE syndrome, our observations may have implications for understanding the basis of these deficits.
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http://dx.doi.org/10.1002/stem.1822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952591PMC
January 2015

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

Brain 2013 Oct 10;136(Pt 10):3096-105. Epub 2013 Sep 10.

1 Developmental Endocrinology Research Group, UCL Institute of Child Health and Department of Endocrinology, Great Ormond Street Hospital for Children, London, WC1N 1EH, UK.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.
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http://dx.doi.org/10.1093/brain/awt218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784281PMC
October 2013

SOX2 regulates the hypothalamic-pituitary axis at multiple levels.

J Clin Invest 2012 Oct 4;122(10):3635-46. Epub 2012 Sep 4.

Neural Development Unit, Institute of Child Health, and Department of Cell and Developmental Biology, University College London, London, United Kingdom.

Sex-determining region Y (SRY) box 2 (SOX2) haploinsufficiency causes a form of hypopituitarism in humans that is characterized by gonadotrophin deficiency known as hypogonadotrophic hypogonadism. Here, we conditionally deleted Sox2 in mice to investigate the pathogenesis of hypogonadotrophic hypogonadism. First, we found that absence of SOX2 in the developing Rathke pouch of conditional embryos led to severe anterior lobe hypoplasia with drastically reduced expression of the pituitary-specific transcription factor POU class 1 homeobox 1 (POU1F1) as well as severe disruption of somatotroph and thyrotroph differentiation. In contrast, corticotrophs, rostral-tip POU1F1-independent thyrotrophs, and, interestingly, lactotrophs and gonadotrophs were less affected. Second, we identified a requirement for SOX2 in normal proliferation of periluminal progenitors; in its absence, insufficient precursors were available to produce all cell lineages of the anterior pituitary. Differentiated cells derived from precursors exiting cell cycle at early stages, including corticotrophs, rostral-tip thyrotrophs, and gonadotrophs, were generated, while hormone-producing cells originating from late-born precursors, such as somatotrophs and POU1F1-dependent thyrotrophs, were severely reduced. Finally, we found that 2 previously characterized patients with SOX2 haploinsufficiency and associated hypogonadotrophic hypogonadism had a measurable response to gonadotropin-releasing hormone (GnRH) stimulation, suggesting that it is not the absence of gonadotroph differentiation, but rather the deficient hypothalamic stimulation of gonadotrophs, that underlies typical hypogonadotrophic hypogonadism.
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http://dx.doi.org/10.1172/JCI64311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461924PMC
October 2012

Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma.

Acta Neuropathol 2012 Aug 18;124(2):259-71. Epub 2012 Feb 18.

Neural Development Unit, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK.

Activating mutations in the gene encoding β-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of β-catenin (β-cat(nc)) and over-activation of the Wnt/β-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the β-cat(nc) cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these β-cat(nc) cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the β-cat(nc) cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours.
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http://dx.doi.org/10.1007/s00401-012-0957-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400760PMC
August 2012

HESX1- and TCF3-mediated repression of Wnt/β-catenin targets is required for normal development of the anterior forebrain.

Development 2011 Nov 17;138(22):4931-42. Epub 2011 Oct 17.

Neural Development Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

The Wnt/β-catenin pathway plays an essential role during regionalisation of the vertebrate neural plate and its inhibition in the most anterior neural ectoderm is required for normal forebrain development. Hesx1 is a conserved vertebrate-specific transcription factor that is required for forebrain development in Xenopus, mice and humans. Mouse embryos deficient for Hesx1 exhibit a variable degree of forebrain defects, but the molecular mechanisms underlying these defects are not fully understood. Here, we show that injection of a hesx1 morpholino into a 'sensitised' zygotic headless (tcf3) mutant background leads to severe forebrain and eye defects, suggesting an interaction between Hesx1 and the Wnt pathway during zebrafish forebrain development. Consistent with a requirement for Wnt signalling repression, we highlight a synergistic gene dosage-dependent interaction between Hesx1 and Tcf3, a transcriptional repressor of Wnt target genes, to maintain anterior forebrain identity during mouse embryogenesis. In addition, we reveal that Tcf3 is essential within the neural ectoderm to maintain anterior character and that its interaction with Hesx1 ensures the repression of Wnt targets in the developing forebrain. By employing a conditional loss-of-function approach in mouse, we demonstrate that deletion of β-catenin, and concomitant reduction of Wnt signalling in the developing anterior forebrain of Hesx1-deficient embryos, leads to a significant rescue of the forebrain defects. Finally, transcriptional profiling of anterior forebrain precursors from mouse embryos expressing eGFP from the Hesx1 locus provides molecular evidence supporting a novel function of Hesx1 in mediating repression of Wnt/β-catenin target activation in the developing forebrain.
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http://dx.doi.org/10.1242/dev.066597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201662PMC
November 2011

SOX2 haploinsufficiency is associated with slow progressing hypothalamo-pituitary tumours.

Hum Mutat 2011 Dec 11;32(12):1376-80. Epub 2011 Oct 11.

Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, London, UK.

SOX2 is an early developmental transcription factor and marker of stem cells that has recently been implicated in the development of the pituitary gland. Heterozygous SOX2 mutations have been described in patients with hypopituitarism and severe ocular abnormalities. In the majority of published cases, the pituitary gland is either small or normal in size. Here, we report two unrelated patients with SOX2 haploinsufficiency (a heterozygous gene deletion and a novel c.143TC>AA/p.F48X mutation) who developed nonprogressive pituitary tumors of early onset, suggesting a congenital etiology. The truncating mutation resulted in significant loss of function and impaired nuclear localization of the mutant protein, in addition to a failure to repress β-catenin transcriptional activity in vitro. This is the first indication that SOX2 haploinsufficiency is implicated in the generation of pituitary tumors with distinct clinical characteristics, possibly mediated via its effects on the Wnt signaling pathway.
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http://dx.doi.org/10.1002/humu.21606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487182PMC
December 2011