Molecular Mechanisms of Glucocorticoid Resistance in Corticotropinomas: New Developments and Drug Targets

Cushing's disease is characterized by excessive adrenocorticotropin hormone (ACTH) secretion caused by a corticotroph tumor of the pituitary gland, leading to hypercortisolism and increased morbidity and mortality. The molecular causes of the disease are not completely understood, therefore more research is needed to discover novel molecular targets and more effective treatments. To date, the SSTR-analog pasireotide is the only approved drug for Cushing's Disease treatment that is directly targeting the source of the disease. Targeting directly the activity of glucocorticoid receptor or the factors modulating it might be a new valid option for the medical management of Cushing's disease. Here, we briefly review the molecular mechanisms involved in the glucocorticoid negative feedback and glucocorticoid resistance and examine novel targets and therapies that might effectively restore glucocorticoid sensitivity

Novel molecular mechanisms involved in the pathogenesis of GH-secreting pituitary adenomas

Pituitary tumors usually show a particular benign nature, with a slow growth potential. Despite these characteristics, they can cause severe complications due to their excessive hormone secretion. Still, further investigations are needed to understand the molecular mechanisms implicated in their development in detail. AHRR (Aryl Hydrocarbon Receptor (AHR) Repressor) and HSP90 (Heat Shock Protein 90) are good candidates for playing a role in pituitary tumorigenesis due to their tumorigenic role reported in different types of tumors and their action in the metabolism of endocrine disruptors (EDs), in which AIP (Aryl Hydrocarbon Receptor Interacting Protein) is also implicated. AHR Repressor (AHRR) is the main regulator of AHR activity through a negative feedback mechanism, which impairment leads to tumor-promoting events. Accordingly, AHRR downregulation was reported in several malignancies including colon, breast and lung cancer and was correlated with a more malignant phenotype (e.g. resistance to apoptosis, increased cellular motility and angiogenic potential) in different tumor cell lines. Although several studies investigated the potential role of AHR signaling in pituitary tumorigenesis, so far no data has been available on the role of AHRR in pituitary tumorigenesis. Therefore, the primary aim of the present study was to evaluate the possible role of AHRR in influencing the tumor phenotype in the somato-lactotroph cell line GH3 in which AHRR was stably silenced or overexpressed. AHRR-silenced cells showed a less aggressive phenotype due to a decreased cell viability and to a more pronounced resistance to apoptosis-inducing stimulus in comparison to the control. Correspondingly, opposite results were obtained for the stable overexpression of AHRR. In addition, AHRR silencing increased senescence-associated beta galactosidase (SA-β Gal) staining in accordance with a slow-growing phenotype observed at proliferation level, without having effect on cell cycle progression. No correlation was observed between AHRR mRNA expression in human pituitary adenomas and tumor aggressiveness, although GH-secreting tumors displayed a higher expression of AHRR in comparison to normal pituitaries. The second part of this thesis was aimed at the role of HSP90 in the pathogenesis of GH-secreting tumors. In addition to the stabilization of AHR, this protein plays a pivotal role in maturation and stabilization of other proteins implicated in oncogenic signaling and cancer progression, and treatment with HSP90 inhibitors has recently shown promising results in pituitary corticotroph adenomas. Therefore, the function of HSP90 was investigated in GH-secreting pituitary adenomas. An intense HSP90 immunoreactivity was reported in 8 out of 25 GH-secreting pituitary tumors. In order to study the therapeutic potential of HSP90 inhibition in these tumors, the cell line GH3 was treated with different HSP90 inhibitors. The C-terminal HSP90 inhibitors novobiocin and KU174 dose-dependently decreased GH promoter activity, whereas only KU174 decreased GH secretory levels. Conclusively, the data presented herein provide novel insights in the pathogenesis of GH-secreting pituitary adenomas. AHRR might have an oncogenic role by influencing tumor aggressiveness, as shown in the cellular model. Moreover, the results obtained in the second part suggest that HSP90 might have a role in the tumorigenesis of GH-secreting pituitary tumors and a potential for HSP90 C-terminal inhibitors in managing the excess of GH secretion

Recent advances in understanding corticotroph pituitary tumor initiation and progression [version 1; referees: 2 approved]

Cushing’s disease is the most frequent form of hypercortisolism and is caused by hypophyseal corticotroph adenomas secreting excessive amounts of adrenocorticotropic hormone. Most of the tumors develop sporadically and only a limited number of corticotroph adenomas have been found to be associated with different neuroendocrine syndromes or with familial isolated pituitary adenomas. The pathogenic mechanisms of corticotroph adenomas are largely unknown, but the discovered aberrant chaperoning activity of heat shock protein 90 on the one hand and the presence of ubiquitin-specific protease 8 mutations on the other hand partially explained the causes of their development. Corticotroph tumors arise initially as benign microadenomas but with time form invasively growing aggressive macroadenomas which can switch to corticotroph carcinomas in extremely rare cases. The mechanisms through which corticotroph tumors escape from glucocorticoid negative feedback are still poorly understood, as are the processes that trigger the progression of benign corticotroph adenomas toward aggressive and malignant phenotypes. This review summarizes recent findings regarding initiation and progression of corticotroph pituitary tumors

Translational research in pituitary tumours

Although effective treatment regimens (surgical resection, drug treatment with dopamine agonists or somatostatin analogues, radiotherapy) have been established for the therapy of most pituitary tumours, a considerable proportion of affected patients cannot completely cured due to incomplete resection or drug resistance. Moreover, even if hormone levels have been normalized, patients with hormone-secreting tumours still show persistent pathophysiological alterations in metabolic, cardiovascular or neuropsychiatric parameters and have an impaired quality of life. In this review reasons for the discrepancy between biochemical cure and incomplete recovery from tumour-associated comorbidities are discussed and the clinical management is delineated exemplarily for patients with acromegaly and Cushing's disease. In view of the development of additional treatment concepts for the treatment of pituitary adenomas we speculate about the relevance of RSUME as a potential target for the development of an anti-angiogenic therapy. Moreover, the role of BMP-4 which stimulates prolactinoma development through the Smad signalling cascade is described and its role as putative drug target for the treatment of prolactinomas is discussed. Regarding the well-known resistance of a part of somatotropinomas to somatostatin analogue treatment, recently identified mechanisms responsible for the drug resistance are summarized and ways to overcome them in future treatment concepts are presented. Concerning novel therapeutic options for patients with Cushing's disease the impact of retinoic acid, which is currently tested in clinical studies, is shown, and the action and putative therapeutic impact of silibinin to resolve glucocorticoid resistance in these patients is critically discussed.Fil: Stalla, Günter K. No especifíca;Fil: Dimopoulou, Christina. Technische Universitat München; AlemaniaFil: Jung Sievers, Caroline. No especifíca;Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Paez Pereda, Marcelo. Max Planck Institut Fur Psychiatrie; AlemaniaFil: Theodoropoulou, Marily. Technische Universitat München; AlemaniaFil: Ciato, Denis. Technische Universitat München; AlemaniaFil: Renner, Ulrich. Max Planck Institut Fur Psychiatrie; Alemani

A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAF(V600E) may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Novel molecular mechanisms involved in the pathogenesis of GH-secreting pituitary adenomas

Pituitary tumors usually show a particular benign nature, with a slow growth potential. Despite these characteristics, they can cause severe complications due to their excessive hormone secretion. Still, further investigations are needed to understand the molecular mechanisms implicated in their development in detail. AHRR (Aryl Hydrocarbon Receptor (AHR) Repressor) and HSP90 (Heat Shock Protein 90) are good candidates for playing a role in pituitary tumorigenesis due to their tumorigenic role reported in different types of tumors and their action in the metabolism of endocrine disruptors (EDs), in which AIP (Aryl Hydrocarbon Receptor Interacting Protein) is also implicated. AHR Repressor (AHRR) is the main regulator of AHR activity through a negative feedback mechanism, which impairment leads to tumor-promoting events. Accordingly, AHRR downregulation was reported in several malignancies including colon, breast and lung cancer and was correlated with a more malignant phenotype (e.g. resistance to apoptosis, increased cellular motility and angiogenic potential) in different tumor cell lines. Although several studies investigated the potential role of AHR signaling in pituitary tumorigenesis, so far no data has been available on the role of AHRR in pituitary tumorigenesis. Therefore, the primary aim of the present study was to evaluate the possible role of AHRR in influencing the tumor phenotype in the somato-lactotroph cell line GH3 in which AHRR was stably silenced or overexpressed. AHRR-silenced cells showed a less aggressive phenotype due to a decreased cell viability and to a more pronounced resistance to apoptosis-inducing stimulus in comparison to the control. Correspondingly, opposite results were obtained for the stable overexpression of AHRR. In addition, AHRR silencing increased senescence-associated beta galactosidase (SA-β Gal) staining in accordance with a slow-growing phenotype observed at proliferation level, without having effect on cell cycle progression. No correlation was observed between AHRR mRNA expression in human pituitary adenomas and tumor aggressiveness, although GH-secreting tumors displayed a higher expression of AHRR in comparison to normal pituitaries. The second part of this thesis was aimed at the role of HSP90 in the pathogenesis of GH-secreting tumors. In addition to the stabilization of AHR, this protein plays a pivotal role in maturation and stabilization of other proteins implicated in oncogenic signaling and cancer progression, and treatment with HSP90 inhibitors has recently shown promising results in pituitary corticotroph adenomas. Therefore, the function of HSP90 was investigated in GH-secreting pituitary adenomas. An intense HSP90 immunoreactivity was reported in 8 out of 25 GH-secreting pituitary tumors. In order to study the therapeutic potential of HSP90 inhibition in these tumors, the cell line GH3 was treated with different HSP90 inhibitors. The C-terminal HSP90 inhibitors novobiocin and KU174 dose-dependently decreased GH promoter activity, whereas only KU174 decreased GH secretory levels. Conclusively, the data presented herein provide novel insights in the pathogenesis of GH-secreting pituitary adenomas. AHRR might have an oncogenic role by influencing tumor aggressiveness, as shown in the cellular model. Moreover, the results obtained in the second part suggest that HSP90 might have a role in the tumorigenesis of GH-secreting pituitary tumors and a potential for HSP90 C-terminal inhibitors in managing the excess of GH secretion.I tumori ipofisari manifestano normalmente un fenotipo benigno, caratterizzato da un potenziale di crescita ridotto. Anche in assenza di effetti patogenetici dovuti all´espansione della massa tumorale nelle strutture adiacenti, possono causare severe complicazioni a causa della loro eccessiva secrezione ormonale. Studi approfonditi sono ancora necessari per riconoscere nuovi meccanismi molecolari implicati nel loro sviluppo. AHRR (Aryl Hydrocarbon Receptor (AHR) Repressor) e HSP90 (Heat Shock Protein 90), oltre ad avere un ruolo nella tumorigenesi in tessuti di differente origine, potrebbero potenzialmente essere coinvolti nello sviluppo dei tumori ipofisari per via della loro attività nel metabolismo di Endocrine Disruptors (EDs), in cui anche la proteina AIP (Aryl Hydrocarbon Receptor Interacting Protein) è implicata. AHRR é il principale regolatore dell’attività di AHR, e lo sbilanciamento di questo sistema di regolazione può avere effetti tumorigenici. Nella fattispecie, l´espressione ridotta di AHRR è stata descritta in tumori di differente origine anatomica tra i quali colon, seno e polmone; e sembra essere associata ad una maggiore aggressività del fenotipo tumorale (in termini di resistenza all´apoptosi, aumento della proliferazione e potenziale angiogenico) in differenti modelli cellulari. Nonostante diversi studi abbiano investigato il potenziale ruolo della via di segnale di AHR nei tumori ipofisari, non vi sono evidenze riguardo al ruolo specifico di AHRR. Scopo principale di questo lavoro di tesi è stato perciò valutare il possibile ruolo di AHRR nella patogenesi dei tumori ipofisari. La linea cellulare somato-lattotropa GH3 è stata utilizzata come modello per generare cloni stabili in cui AHRR è silenziato o overespresso. Il silenziamento di AHRR ha portato alla manifestazione di un fenotipo meno aggressivo dovuto alla diminuzione della capacità proliferativa e alla maggiore resistenza a stimoli apoptotici rispetto al controllo, mentre risultati opposti sono stati ottenuti overesprimendo stabilmente AHRR. È stato inoltre evidenziato come il silenziamento di AHRR abbia indotto la manifestazione di un fenotipo più senescente rispetto al controllo, in correlazione con la riduzione della proliferazione cellulare. Non sembra però che ciò coinvolga un cambiamento sulla distribuzione del ciclo cellulare. Inoltre, forse a causa del numero ridotto di campioni analizzati, la valutazione quantitativa dell’espressione a livello dell’mRNA di AHRR nei tumori ipofisari non sembra essere in relazione con l’aggressività tumorale, anche se nel sottogruppo adenomi GH-secernenti vi sia una maggiore espressione rispetto a campioni di ipofisi normali. La seconda parte di questo lavoro di tesi ha avuto come scopo la valutazione del ruolo di HSP90 nella patogenesi dei tumori ipofisari. Oltre alla stabilizzazione di AHR, questa proteina svolge un ruolo fondamentale nella maturazione e nella stabilizzazione di altre proteine implicate nella trasformazione e nella progressione tumorale, e la sua inibizione specifica ha recentemente dimostrato risultati promettenti negli adenomi ACTH-secernenti. Sulla base di queste premesse, la valutazione del ruolo di HSP90 è stata estesa ai tumori GH-secernenti. L´analisi immunoistochimica effettuata su adenomi GH secernenti ha evidenziato una intensa immunoreattivitá di HSP90 in 8/25 casi. Visto il potenziale coinvolgimento di HSP90 in questa tipologia di adenomi ipofisari, la linea cellulare GH3 è stata utilizzata come modello per investigare il potenziale terapeutico di differenti inibitori di HSP90. Nella fattispecie, il trattamento con gli inibitori C-terminali di HSP90 novobiocina e KU174 ha provocato una diminuzione dose-dipendente dell´attivitá promotoriale del GH, mentre solo KU174 ha significativamente diminuito la secrezione di GH

Constructs used in transfection experiments.

<p>(A) Either the wild type, the c.-456_-453delCCTT or the c.-469C>T containing 5′UTRs were cloned upstream the firefly luciferase gene. (B) The wild type/c.-456_-453delCCTT 5′UTR were subcloned upstream the <i>CDKN1B</i> gene. (C) The introduction of the c.-428A>T substitution in the two former plasmids reported in panel A restores uORF length and intercistonic distance. (D) Constructs reported in panel B were mutated introducing a c.-74insC leading to the translation of a chimeric protein in the double mutant.</p

Comparison of the <i>CDKN1B</i> gene among species.

<p>(A) Schematic representation of the <i>CDKN1B</i> gene in different species. The uORF has been reported as grey box while <i>CDKN1B</i> as a yellow one. Similarities at nucleotide level with human sequences have been reported within boxes. Intercistronic length is also shown. * In chicken <i>CDKN1B</i> 5′UTR a second uORF has been described. (B) uORF amino acid sequences in seven species. In all but one, conservation of the uORF encoded peptide is even higher than p27^KIP1. The considered nucleotide and protein sequences are the following: Human, NM_004064.3, NP_04055.1; Mouse, NM_009875.4, NP_034005.2; Dog, HE804769, CCH35981; Opossum, HE804772, CCH35984; Chicken, NM_204256, NP_989587; Sloth, HE804770, CCH35982; Megabat, HE804771, CCH35983. Nucleotide and protein sequences alignments among species have been performed by LALIGN and PRALINE, respectively.</p

The c.-456_-453delCCTT affects protein translation by reducing reinitiation.

<p>Relative luciferase activity for the wild type and the mutants (c.-456_-453delCCTT, c.-469C>T) 5′UTRs was measured in GH3 (A) and HeLa (B) cells. The effects of the 4 bp deletion on luciferase mRNA in HeLa and GH3 cells (C) and on p27^KIP1 level in HEK293 cells (D) are shown. Panel E shows the presence of the chimeric product in HEK293 cells transfected with the double mutant construct (c.-456_-459delCCTT+c.-74insC), followed by substantial reduction of p27^KIP1 expression, demonstrated by western blot. P-values were calculated using a two-tailed t-test. *p<0,01; EV, empty vector; wt, wild type; error bars, standard deviation.</p