Pheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile

doi:10.1016/j.neuroscience.2007.05.008

Neuroscience

Volume 147, Issue 4, 29 July 2007, Pages 928-937

https://doi.org/10.1016/j.neuroscience.2007.05.008 Get rights and content

Abstract

Pheochromocytomas are adrenal medullary tumors that typically occur in adult patients, with increased frequency in multiple endocrine neoplasia type 2, von Hippel-Lindau disease, familial paraganglioma syndromes and neurofibromatosis type 1 (NF1). Pheochromocytomas arise in adult mice with a heterozygous knockout mutation of exon 31 of the murine Nf1 gene, providing a mouse model for pheochromocytoma development in NF1. We performed a microarray-based gene expression profiling study comparing mouse pheochromocytoma tissue to normal adult mouse adrenal medulla to develop a basis for studying the pathobiology of these tumors. The findings demonstrate that pheochromocytomas from adult neurofibromatosis knockout mice express multiple developmentally regulated genes involved in early development of both the CNS and peripheral nervous system. One of the most highly overexpressed genes is receptor tyrosine kinase Ret, which is known to be transiently expressed in the developing adrenal gland, down-regulated in adult adrenals and often overexpressed in human pheochromocytomas. Real-time polymerase chain reaction validated the microarray results and immunoblots confirmed the overexpression of Ret protein. Other highly expressed validated genes include Sox9, which is a neural crest determinant, and Hey 1, which helps to maintain the progenitor status of neural precursors. The findings are consistent with the recently proposed concept that persistent neural progenitors might give rise to pheochromocytomas in adult mouse adrenals and suggest that events predisposing to tumor development might occur before formation of the adrenal medulla or migration of cells from the neural crest. However, the competing possibility that developmentally regulated neural genes arise secondarily to neoplastic transformation cannot be ruled out. In either case, the unique profile of gene expression opens the mouse pheochromocytoma model to new applications pertinent to neural stem cells and suggests potential new targets for treatment of pheochromocytomas or eradication of their precursors.

Section snippets

Experimental procedures

A microarray-based gene expression profiling study was performed to compare MPC tumor tissue to normal adult mouse adrenal medulla. Nine tumors from separate animals were compared with three sets of pooled normal adult mouse adrenal medullas, each representing approximately 50 adrenals, that were harvested and reverse transcribed completely independently on separate occasions. The overexpression of representative developmentally regulated genes considered to be important in early neural

Results

Principal component analysis based on all probe sets showed tight clustering of the three separate batches of pooled adrenals with each other and distinction from any of the tumors (Fig. 1A). Component 1 represented a “hybrid” probe set (or a group of probe sets with similar expression patterns) that distinguished the normal (Points J–L) and tumor samples (Points A–I), Component 2 represented a “hybrid” probe set (or a group of probe sets with similar expression patterns) that separated the 12

Discussion

Our findings show that pheochromocytomas arising in heterozygous Nf1 knockout mice express a novel profile of developmentally regulated genes involved in formation of the CNS and peripheral nervous system. Many of the overexpressed genes are not entirely specific to developing neurons. However, chromaffin cells are of neural crest origin and the overexpression of large numbers of genes related to neural development is unlikely to be coincidental.

Genes associated with an immature phenotype are

Conclusion

In summary, multiple up-regulated and down-regulated genes distinguish MPC tissue from normal mouse adrenal medulla. Many of the up-regulated genes are associated with neural stem cells or early committed neural progenitors in the CNS and peripheral nervous system, consistent with a hypothesized model of defective developmental culling in the pathogenesis of pheochromocytomas. However, the competing possibility that developmentally regulated neural genes are re-expressed during neoplastic

Acknowledgments

These studies were supported by NIH grants NS 36785 (A.S.T.) and GM 46588 (M.J.E.) and by Philip Morris USA and Philip Morris International (M.J.E.).

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TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation
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The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.
Domain landscapes of somatic NF1 mutations in pheochromocytoma and paraganglioma
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Pheochromocytoma and paraganglioma (PPGL), are rare neuroendocrine tumors arising from the adrenal medulla and extra-adrenal paraganglia, respectively. Up to about 60% are explained by germline or somatic mutations in one of the major known susceptibility genes e.g., in NF1, RET, VHL, SDHx, MAX and HRAS. Targeted Next Generation Sequencing was performed in 14 sporadic tumors using a panel including 26 susceptibility genes to characterize the mutation profile. A total of 6 germline and 8 somatic variants were identified. The most frequent somatic mutations were found in NF1 (36%), four have not been reported earlier in PCC or PGL. Gene expression profile analysis showed that NF1 mutated tumors are classified into RTK3 subtype, cluster 2, with a high expression of genes associated with chromaffin cell differentiation, and into a RTK2 subtype, cluster 2, as well with overexpression of genes associated with cortisol biosynthesis. On the other hand, by analyzing the entire probe set on the array and TCGA data, ALDOC was found as the most significantly down regulated gene in NF1-mutated tumors compared to NF1-wild-type. Differential gene expression analysis showed a significant difference between Nt - and Ct-NF1 domains in mutated tumors probably engaging different cellular pathways. Notably, we had a metastatic PCC with a Ct-NF1 frameshift mutation and when performing protein docking analysis, Ct-NF1 showed an interaction with Nt-FAK suggesting their involvement in cell adhesion and cell growth. These results show that depending on the location of the NF1-mutation different pathways are activated in PPGLs. Further studies are required to clarify their clinical significance.
Toxic Responses of the Adrenal Medulla
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The adrenal medulla is a modified sympathetic ganglion composed principally of neuroendocrine cells, known as chromaffin cells, derived from the neural crest. Chromaffin cells secrete the catecholamines epinephrine and norepinephrine, together with regulatory peptides, granin proteins, and other constituents of neuroendocrine secretory granules, in response to stress. Glucocorticoids from the adrenal cortex induce the expression of catecholamine biosynthetic enzymes, thereby maintaining high levels of catecholamine stores. To a lesser extent, released catecholamines reciprocally influence adrenal cortical steroidogenesis. Secretion is stimulated principally by preganglionic sympathetic nerve endings that originate in the splanchnic nerve and synapse on chromaffin cells. The process of stimulus–secretion coupling involves activation of and cross talk between multiple signaling pathways that also contribute to replenishment of secretory products, determine the composition of secretions, and, under some circumstances, stimulate chromaffin cell proliferation.
Proliferative lesions, classified according to somewhat arbitrary criteria as adrenal medullary hyperplasia or pheochromocytoma, are the most common pathological findings in the adrenal medulla in toxicological studies. These lesions can occur spontaneously in the course of aging or can be induced by xenobiotic agents. Both spontaneous and induced medullary proliferative lesions tend to be much more frequent in rats than in mice and, in rats, most studies report a higher incidence in males than in females. The frequency of these lesions in rats is also affected by dietary modifications. Most of the agents that induce adrenal medullary proliferative lesions are nongenotoxic, suggesting that mechanisms involved in normal chromaffin cell function contribute to the pathogenesis of the lesions. Experimental evidence supporting this hypothesis is available for a few agents.
Adrenal cortical and chromaffin stem cells: Is there a common progeny related to stress adaptation?
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Citation Excerpt :
It has been suggested that a subpopulation of proliferation competent progenitor cells continues to exist in the adult adrenal medulla (Bornstein et al., 2012; Ehrhart-Bornstein et al., 2010). In addition to the adaptation to physiological needs, these progenitor cells may be involved in the development of stress-related hyper activation and hyperplasia of the adrenal and potentially in tumor development (Molatore et al., 2010; Powers et al., 2007). Within recent years we embarked on isolating chromaffin progenitor cells from the adult adrenal medulla.
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.
Neurofibromin protein loss in desmoplastic melanoma subtypes: Implicating NF1 allelic loss as a distinct genetic driver?
2016, Human Pathology
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The parallel pathogenesis of these diseases underscores a potential interaction for neurofibromin in RET signaling. Pheochromocytoma cell lines derived from Nf1 knockout mice overexpress RET and respond to the GDNF activating ligand [42]. Given the higher incidence of neurofibromin loss in PDM and this precedent for RET overexpression in neurofibromin-deficient tumors, RET-GDNF activation coupled with neurofibromin loss is an intriguing model for PDM.
Loss of the NF1 allele, coding for the protein neurofibromin, and polymorphism in the proto-oncogene RET (RETp) are purportedly common in desmoplastic melanoma (DM). DM is categorized into pure (PDM) and mixed (MDM) subtypes, which differ in prognosis. Most NF1 mutations result in a truncated/absent protein, making immunohistochemical screening for neurofibromin an ideal surrogate for NF1 allelic loss. Using antineurofibromin, our aims were to ascertain the incidence of neurofibromin loss in DM subtypes and to evaluate the relationship with RET, perineural invasion (PNI) and established histopathologic prognosticators. A total of 78 archival samples of DM met criteria for inclusion (54 cases of non-DM serving as controls). Immunohistochemistry was performed for neurofibromin, whereas direct DNA sequencing was used for RETp and BRAF mutation status. Statistical analyses included χ² test as well as Fisher exact test. Neurofibromin loss was more common in DM than non-DM (69% versus 54%; P = .02). In DM, significant differences in neurofibromin loss were noted in the following: non–head and neck versus head and neck biopsy site (88% versus 55%) and PDM versus MDM variants (80% versus 56%). No significant associations were noted with sex, presence of a junctional component, Breslow depth, ulceration, mitoses, host response, RETp, BRAF status, or PNI. RETp was marginally associated with PNI-positive DM versus PNI-negative DM (36 versus 18%; P = .08). Our findings, the largest to date investigating neurofibromin in DM, validate the incidence of NF1 mutations/allelic loss in DM and suggest that the DM subtypes have distinct genetic drivers.
Adrenomedullary progenitor cells: Isolation and characterization of a multi-potent progenitor cell population
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Citation Excerpt :
Increasing evidence indicates that these tumors, at least in part, may derive from persistent sympathoadrenal progenitor cells within the adrenal medulla. This hypothesis is based on the fact that pheochromocytomas overexpress multiple genes that are involved in early neural development (Molatore et al., 2010; Powers et al., 2007; Qin et al., 2014) and that the expression of some of these genes also characterize sympathoadrenal progenitor cells from the adult adrenal medulla (Chung et al., 2009; Ehrhart-Bornstein et al., 2010; Santana et al., 2012). In addition to forming the adrenal medulla, neural crest derived sympathoadrenal progenitors also play an important role in the development of the sympathetic nervous system.
The adrenal is a highly plastic organ with the ability to adjust to physiological needs by adapting hormone production but also by generating and regenerating both adrenocortical and adrenomedullary tissue. It is now apparent that many adult tissues maintain stem and progenitor cells that contribute to their maintenance and adaptation. Research from the last years has proven the existence of stem and progenitor cells also in the adult adrenal medulla throughout life. These cells maintain some neural crest properties and have the potential to differentiate to the endocrine and neural lineages. In this article, we discuss the evidence for the existence of adrenomedullary multi potent progenitor cells, their isolation and characterization, their differentiation potential as well as their clinical potential in transplantation therapies but also in pathophysiology.

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Cellular neurosciencePheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile

Abstract

Section snippets

Experimental procedures

Results

Discussion

Conclusion

Acknowledgments

Mech Dev

Cell

Brain Res Bull

Gene Expr Patterns

Dev Biol

Cancer Cell

Neuron

Toxicol Appl Pharmacol

Neuropharmacology

Exp Neurol

Neuroscience

Mol Cell Neurosci

Dev Biol

J Biol Chem

Brain Res Dev Brain Res

Trends Cell Biol

Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems

Proc Natl Acad Sci U S A

A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas

PLoS Genet

Cancer: stem cells and brain tumours

Nature

Malignant pheochromocytoma: current status and initiatives for future progress

Endocr Relat Cancer

The epigenetic progenitor origin of human cancer

Nat Rev Genet

Identification and characterization of the Hesr1/Hey1 as a candidate trans-acting factor on gene expression through the 3′ non-coding polymorphic region of the human dopamine transporter (DAT1) gene

J Biochem (Tokyo)

Cellular neuroscience
Pheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile