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Epigenetic Mechanisms Modulated by Glucocorticoids With a Focus on Cushing Syndrome

Posted on April 19, 2024 by MaryO

Abstract

In Cushing syndrome (CS), prolonged exposure to high cortisol levels results in a wide range of devastating effects causing multisystem morbidity. Despite the efficacy of treatment leading to disease remission and clinical improvement, hypercortisolism-induced complications may persist. Since glucocorticoids use the epigenetic machinery as a mechanism of action to modulate gene expression, the persistence of some comorbidities may be mediated by hypercortisolism-induced long-lasting epigenetic changes. Additionally, glucocorticoids influence microRNA expression, which is an important epigenetic regulator as it modulates gene expression without changing the DNA sequence. Evidence suggests that chronically elevated glucocorticoid levels may induce aberrant microRNA expression which may impact several cellular processes resulting in cardiometabolic disorders.

The present article reviews the evidence on epigenetic changes induced by (long-term) glucocorticoid exposure. Key aspects of some glucocorticoid-target genes and their implications in the context of CS are described. Lastly, the effects of epigenetic drugs influencing glucocorticoid effects are discussed for their ability to be potentially used as adjunctive therapy in CS.

epigenetic, glucocorticoids, Cushing syndrome

Issue Section:

Mini-review

In Cushing syndrome (CS), adrenocorticotropic hormone (ACTH) hypersecretion by a pituitary adenoma or an ectopic source, or autonomous cortisol hypersecretion by an adrenal tumor, induces chronic endogenous hypercortisolism with loss of the cortisol circadian rhythm (1). CS is more prevalent in women than men and frequently occurs in the fourth to sixth decades of life (2).

Glucocorticoids (GC) have extensive physiological actions and regulate up to 20% of the expressed genome, mainly related to the immune system, metabolic homeostasis, and cognition. Therefore, the prolonged exposure to high cortisol levels results in a wide range of devastating effects, including major changes in body composition (obesity, muscle atrophy, osteoporosis), neuropsychiatric disturbances (impaired cognition, depression, sleep disturbances), the metabolic syndrome (obesity, hypertension, insulin resistance, and dyslipidemia), hypercoagulability, and immune suppression (3, 4). The consequences of hypercortisolism lead to compromised quality of life and increased mortality rate (5). The mortality rate in patients with CS is 4 times higher than the healthy control population (6). Risk factors such as obesity, diabetes, and hypertension contribute to the increased risk of myocardial infarction, stroke, and cardiac insufficiency. As a result, cardiovascular disease is the leading cause of the premature death in CS (5). Infectious disease is also an important cause of death in CS (5). Therefore, prompt treatment to control hypercortisolism is imperative to prevent complications and an increased mortality rate.

Despite the efficacy of treatment leading to disease remission, the clinical burden of CS improves, but does not completely revert, in every patient (7). Indeed, obesity, neuropsychiatric disturbances, hypertension, diabetes, and osteoporosis persist in a substantial number of biochemically cured patients. For instance, in a study involving 118 CS patients in remission for about 7.8 years (median), resolution of comorbidities such as diabetes occurred in only 36% of cases, hypertension in 23% of cases, and depression in 52% of the cases (8). It has been proposed that epigenetic changes as a consequence of hypercortisolism is a mechanism of the persistence of some comorbidities (9-12).

Epigenetics is a reversible process that modifies gene expression without any alterations in DNA sequence; frequently it is mediated by histone modification and DNA methylation together with microRNAs (13-15). GCs use the epigenetic machinery as a mechanism of action to regulate gene expression in physiological circumstances, such as metabolic actions and stress response. Its networks involve DNA and histone modifying enzymes, such as DNA methyltransferases (DNMTs), histone acetyltransferases (HATs), and histone deacetylases (HDACs) (16). (Fig. 1) The DNA methylation process catalyzed by DNMTs is usually associated with downregulation of gene expression (17). Histone modifications catalyzed by HAT enzymes induce gene transcription, while those by HDAC enzymes induce transcriptional repression (17). Drugs interfering with these enzymes (so-called epigenetic drugs) may affect the GC genomic actions confirming the interaction between GC and the epigenetic system (18, 19). Furthermore, GC can modulate HDAC and DNMT expression and activity (16, 19, 20). Based on these data it might be speculated that in CS, epigenetic modifications induced by long-term GC exposure plays a role in the development of the disease-specific morbidity (9, 10).

Figure 1.

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Glucocorticoid (GC) and its epigenetic machinery. GC through its receptor interacts with DNA and histone modifying enzymes, such as DNA methyltransferases (DNMTs), histone acetyl transferases (HATs), and histone deacetylases (HDAC) to modulate gene expression.

In this review we provide an overview of epigenetic aspects of GC action in physiological conditions and in the context of CS. We start with a detailed characterization of how GC, using the epigenetic system, can change chromatin structure in order to activate or silence gene expression. (Fig. 2) Subsequently, we describe the role of epigenetic mechanisms in the regulation of expression of several GC-target genes related to CS. Finally, we present the current evidence of epigenetic changes caused by the long-term of GC exposure and the potential use of epidrugs influencing GC actions.

Figure 2.

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Epigenetic mechanisms of the glucocorticoid action to regulate gene expression. The GR is located in cytoplasm in a multi-protein complex; after GC binding, GR dissociates from the multi-protein complex, crosses the nuclear membrane, dimerizes, and binds to the GRE of the target gene. One of the mechanisms of action of GC is through the recruitment of co-regulators together with epigenetic enzymes, such as HAT, to change the chromatin structure, resulting in activation of gene transcription. Also, GR decreases gene expression by tethering other transcriptional factors and recruiting HDAC2, causing histone deacetylation, which leads to a repressed chromatin. GC can cause hypomethylation through downregulation in the expression of DNMT1. Abbreviations: Ac, acetylation; DNMT1, DNA methyltransferase 1; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid responsive elements; HAT, histone acetyltransferase; HDAC, histone deacetylases; Me: methylation.

Search Strategy

A search of the PubMed database was conducted using the advanced search builder tool for articles in the English language on the following terms “glucocorticoids,” “glucocorticoid receptor,” “Cushing,” “hypercortisolism,” “epigenetic,” “DNA methylation,” “histone deacetylase,” “histone acetyltransferase,” “microRNA” “fkbp5,” “clock genes,” and “POMC.” Moreover, references were identified directly from the articles included in this manuscript. The articles were selected by the authors after being carefully analyzed regarding their importance and impact.

Epigenetic Aspects of Genomic Action of Glucocorticoids

GCs regulate gene expression positively or negatively. GC-responsive genes include genes encoding for proteins associated with inflammation, metabolic processes, blood pressure and fluid homeostasis, apoptosis, cell cycle progression, circadian rhythm, and intracellular signaling (21).

The GC actions are cell type–specific (22). For instance, in an in vitro study, the comparison of GC-expressed genes between 2 cell lines, corticotroph (AtT20) and mammary (3134) cell lines, showed a different set of GC-regulated genes, revealing the cell type–specific nature of GC effects (23). GC function depends on the accessibility of glucocorticoid receptor (GR)-binding sites in the DNA of the target tissue, which in turn is mostly established during cell differentiation. Therefore, different chromatin organization explains the distinct GR-binding sites among different tissues (22, 24, 25). The chromatin accessibility is determined by histone modifications such as acetylation, methylation, phosphorylation, and/or DNA methylation, processes that are both dynamic and reversible (26).

Furthermore, gene expression is regulated in a GC-concentration-dependent manner which is tissue-specific. Only a few genes can be upregulated or downregulated at low concentrations of GC. For example, a dose of dexamethasone (Dex) as low as 0.5 nM selectively activated PER1 (period 1, transcription factor related to circadian rhythm) expression in lung cancer (A549) cells (21, 27). Additionally, continuous GC exposure or pulsed GC (cortisol fluctuation during circadian rhythm) may cause different responses with respect to gene expression (26, 28). For example, constant treatment with corticosterone induced higher levels of PER1 clock gene mRNA expression compared with pulsatile treatment, as demonstrated in an in vitro study using 3134 cell line (28).

The time course for gene expression in response to Dex is fast, with repression occurring slightly slower compared to activation. Half of activated and repressed genes are detected within, respectively, about 40 minutes and 53 minutes following Dex exposure (21).

In short, the transcriptional output in response to GC depends on cell type, as well as on the duration and intensity of GC exposure (21, 24, 26, 27). GCs act as a transcriptional regulatory factor resulting in activating or repressing the expression of genes. The GC exerts its function through binding to corticosteroid receptors, specifically, the mineralocorticoid receptor and the GR, members of the nuclear receptor superfamily (29, 30).

Glucocorticoid Receptor

The GR is located in the cytoplasm in a chaperone complex which includes heat-shock proteins (70 and 90) and immunophilins (such as FK506 binding protein [FKBP5]). Cortisol diffuses across the cell membrane and binds with high affinity to the GR. The activated GR bound to GC dissociates of the multi-protein complex and is transferred to the nucleus, where it ultimately regulates gene expression (26, 31).

GR is a transcription factor encoded by nuclear receptor subfamily 3, group C member 1 (NR3C1) gene, located in chromosome 5, and consisting of 9 exons. It is composed of 3 major functional domains, namely a DNA binding domain (DBD), the C-terminal ligand-binding domain (LBD) and the N-terminal domain (NTB). The LBD recognizes and joins the GC. NTB contains an activation function-1 (AF1) which connects with co-regulators and the members of the general transcription machinery to activate target genes. The DBD comprises 2 zinc fingers motifs that are able to identify and bind to glucocorticoid responsive elements (GREs) (32, 33).

GRα is the most expressed and functionally active GR. GRβ is another isoform which is the result of an alternative splicing in exon 9 of the GR transcript. The difference between the 2 isoforms is the distinct ligand-binding domain in GRβ. This variance prevents the GRβ from binding to GC. In fact, the GRβ counteracts GRα function by interfering with its binding to a GRE in the target gene, and GRβ expression is associated with GC resistance (32). In addition, GRβ has its own transcriptional activity which is independent and distinct from GRα (34).

Another splice variant of human GR, GRγ, is associated with GC resistance in lung cell carcinoma and childhood acute lymphoblastic leukemia (33, 35). There is an additional amino acid (arginine) in the DBD of the GRγ that reduces, by about half, the capacity to activate or suppress the transcription of the target gene, as compared with GRα (32). One study identified GRγ in a small series of corticotroph adenomas (36).

Glucocorticoid Mechanism of Action

The GR-GC complex induces or represses gene expression directly by binding to DNA, indirectly by tethering other transcription factors or yet in a composite manner that consists in binding DNA in association with binding to other co-regulators (35, 37).

The GR has the ability to reorganize the chromatin structure to become more or less accessible to the transcriptional machinery. In the classical mechanism of direct induction of gene expression, the GR dimerizes and binds to a GRE in DNA. The receptor recruits co-regulators, such as CREB binding protein, which has intrinsic histone acetyltransferase (HAT) activity that modifies the chromatin structure from an inactive to an active state. This model, called transactivation, upregulates the expression of some genes related to glucose, protein, and fat metabolism. Gene repression, on the other hand, is accomplished by GR binding to a negative GRE (nGRE) leading to the formation of a chromatin remodeling complex composed by co-repressor factors, such as NCOR1 and SMRT, and histone deacetylases (HDACs), that ultimately turn chromatin less accessible and suppress gene transcription. The gene repression through direct binding events occurs less frequently when compared to gene induction (25, 35, 38).

Another mechanism of GC action is through binding to other transcription factors (tethering). In case of switching off inflammatory genes, GR binds to transcriptional co-activator molecules, such as CREB binding protein with intrinsic HAT activity, and subsequently recruits HDAC2 to reverse histone acetylation, thus resulting in a suppression of the activated inflammatory gene (39). In the same model, GC interacts with other cofactors, such as the STAT family, to induce chromatin modifications resulting in increased gene expression (26).

Furthermore, the transcriptional dynamics of some genes follow a composite manner. In this model, GR, in conjunction with binding to GRE, also interacts with cofactors in order to enhance or reduce gene expression (35).

GCs can also modulate gene expression by influencing the transcription of epigenetic modifiers. An experimental study demonstrated that GC mediated the upregulation of HDAC2 in rats exposed to chronic stress, which in turn decreased the transcription of histone methyltransferase (Ehmt2) that ultimately upregulated the expression of Nedd4. Nedd4 is a ubiquitin ligase, expression of which has been related to cognitive impairment (40). Additionally, GC was found to interact with another epigenetic eraser, namely JMJD3, a histone demethylase, suppressing its transcription in endothelial cells treated with TNFα that led to decreased expression of other genes related to the blood-brain barrier (41).

GCs have the ability to induce (de)methylation changes in DNA, ultimately affecting gene expression. The DNA methylation process triggered by GC involves the family of DNA methyltransferases (DNMT) and ten-eleven translocation (TET) protein (20, 42-44). The DNMT, DNMT1, DNMT3A, and DNMT3B are able to transfer a methyl group to a cytosine residue in DNA, forming 5-methylcytosine (5mC), which negatively impacts gene expression. In contrast, TET protein chemically modifies the 5mC to form 5-hydroxymethylcytosine (5hmC), which ultimately leads to unmethylated cytosine, positively influencing gene expression (45).

Glucocorticoids mainly induce loss of methylation events rather than gain of methylation across the genome (11, 46). The DNA demethylation process can be either active or passive. The active mechanism is linked to the upregulation of TET enzyme expression that follows GC treatment, which was described in retinal and osteocyte cell line model studies (42, 43). The passive demethylation event involves the downregulation (Fig. 2) or dysfunction of DNMT1. DNMT1 is responsible for maintaining the methylation process in dividing cells (45). In case of GC exposure, GC can cause hypomethylation through downregulation in the expression of DNMT1, a process described in the AtT20 corticotroph tumor cell model, or through GC hindering DNMT activity, particularly DNMT1, as demonstrated in the retinal cell (RPE) line (20, 42, 44).

Glucocorticoid-Induced Epigenetic Changes

There are several molecular mechanisms connecting GR activation and epigenetic modifications ultimately affecting gene expression (Fig. 2). As described above, GC uses epigenetic machinery, such as DNA and histone modifying enzymes, to restructure the chromatin in order to induce or silence gene transcription (16, 47).

In an in vitro study using murine AtT20 corticotroph tumor and neuronal cell lines, after chronic GC exposure followed by a recovery period in the absence of GC, the cells retained an “epigenetic memory” with persistence of loss of methylation content in FKBP5 gene but with no increased gene expression at baseline. The functionality of this “epigenetic memory” only became evident in a second exposure to GC, when the cells responded sharply with a more robust expression of FKBP5 gene compared to the cells without previous exposure to GC (44). Another in vitro study, using a human fetal hippocampal cell line, confirmed long-lasting DNA methylation changes induced by GC. The cells were treated for 10 days with dexamethasone, during the proliferative and cell differentiation phases of the cell line, followed by 20 days without any treatment. The second exposure to GC resulted in an enhanced gene expression of a subset of GC-target genes (48). Additionally, using an animal model subjected to chronic stress, a distinct gene expression profile was demonstrated in response to acute GC challenge compared to those without chronic stress history. The proposed mechanism was that chronic stress resulted in GC-induced enduring epigenetic changes in target genes, altering the responsiveness to a subsequent GC exposure (49).

In general, it seems that the majority of differential methylation regions (DMRs) induced by GC are loss of methylation rather than gain of methylation. In an experimental study, an association between hypomethylation and GC exposure was demonstrated in mice previously exposed to high levels of GC. Further analysis demonstrated that the genes linked with DMR were mostly related to metabolism, the immune system, and neurodevelopment (11).

Human studies have also shown that excess of cortisol can induce modifications in DNA methylation. DNA methylation data obtained from whole blood samples from patients with chronic obstructive pulmonary disease (COPD) treated with GC revealed DMR at specific CpG dinucleotides across the genome. These DMR were confirmed by pyrosequencing and annotated to genes, such as SCNN1A, encoding the α subunit of the epithelial sodium channel, GPR97, encoding G protein coupled receptor 97, and LRP3, encoding low-density lipoprotein receptor-related protein 3 (50). Furthermore, it has been proposed that the negative impact of chronic GC exposure on the immune system, which increases the risk of opportunistically infections, may be epigenetically mediated (51). In a clinical study, using whole blood samples, an analysis of genome-wide DNA methylation was performed on patients before and after exposure to GC (51). Long-term GC exposure disrupts, through a persistent modification of the cytosine methylation pattern, the mTORC1 pathway which affects CD4+ T cell biology (51).

Taken together, these data clearly show the interplay between GC signaling and methylation and histone modifications processes suggesting that GC interferes in the epigenetic landscape modulating gene expression. It is possible that most of these GC-induced epigenetic events are dynamic and temporary, while others may persist leading to long-lasting disorders. Further research to provide insight into what makes some events reversible is warranted.

Epigenetic Changes as a Consequence of Long-Term Glucocorticoid Exposure in Cushing Syndrome

The comorbidities associated with CS are associated with increased mortality mainly due to cardiovascular events (52). GC-induced comorbidities in CS may be at least in part epigenetically mediated. Previous study using whole blood methylation profile demonstrated that specific hypomethylated CpG sites induced by GC were associated with Cushing comorbidities, such as hypertension and osteoporosis (46). The study identified a methylator predictor of GC excess which could be used as a biomarker to monitor GC status (46).

The long-term exposure to high cortisol levels may be crucial for the persistence of some morbidities in CS through epigenetic changes. Hypercortisolism-induced persistent changes in visceral adipose tissue gene expression through epigenetic modifications was investigated in a translational study (12). This study combined data from patients with active CS and data from an animal model of CS in active and remitted phase. Interestingly, the study demonstrated long-lasting changes in the transcriptome of adipose tissue that were associated with histone modifications induced by GC. Therefore, these epigenetic fingerprints observed even after the resolution of hypercortisolism may elucidate the mechanism of persistent modifications in gene expression in the visceral adipose tissue (12).

With regard to the persistence of GC-induced DMR, a genome-wide DNA methylation analysis showed a lower average of DNA methylation in patients in remission of CS compared to controls. Interestingly, the most common biologically relevant affected genes were retinoic acid receptors, thyroid hormone receptors, or hormone/nuclear receptors, important genes related to intracellular pathways and regulators of gene expression (9).

In summary, this large body of evidence supports the concept that prolonged GC exposure modulates the epigenetic landscape across the genome by inducing DMR and histone modifications. Some epigenetic modifications are persistent, and this may partially explain the incomplete reversibility of some of CS features following clinical remission.

Glucocorticoid-Target Genes in Cushing Syndrome

A detailed identification and characterization of GC-target genes may shed light in the understanding of the pathophysiology and treatment response in patients with CS. For instance, the GC regulation of pro-opiomelanocortin (POMC) expression as part of the physiologic GC negative feedback may be impaired in Cushing disease (CD), which is an important mechanism for the maintenance of high GC levels (53). Another example is the interaction between GC and clock genes, which may interfere in the loss of the GC circadian rhythm and may contribute to metabolic disorders in CS (54). Furthermore, the suppressive action of GC on drug targets, such as the somatostatin receptor (subtype 2), may influence the efficacy of first-generation somatostatin receptor ligands in normalizing cortisol levels in CD (55). Here we describe how GCs using epigenetic machinery influence the expression of important target genes and their implications in CS.

FKBP5

FK506 binding protein (FKBP5) plays an important role in the regulation of hypothalamic-pituitary-adrenal (HPA) system (56). As part of the GC negative feedback loop, GC binds to hypothalamic and pituitary GR. In the cytoplasm, GR is bound to a multi-protein complex including FKBP5. FKBP5 modulates GR action by decreasing GR binding affinity to GC and by preventing GR translocation from cytoplasm to nucleus (57, 58). In other words, an increase of FKBP5 expression is inversely correlated with GR activity and results in GC resistance leading to an impaired negative feedback regulation in the HPA axis (59).

FKBP5 is a GC-responsive gene; its upregulation by GC is part of an intracellular negative short-feedback loop (60). The mechanism by which GC regulates FKBP5 expression was shown to include inhibition of DNA methylation (44). In a model for CS, mice treated with corticosterone for 4 weeks had a reduced level of DNA methylation of FKBP5 in DNA extracted from whole blood, which was strongly correlated in a negative manner with GC concentration. Interestingly, a negative correlation was also observed between the degree of FKBP5 gene methylation measured at 4 weeks of GC exposure and the percentage of mice visceral fat (61). Accordingly, previous studies have provided compelling evidence of decreased methylation in the FKBP5 gene in patients with active CS compared to healthy control (10, 46). Even in patients with CS in remission, previous data have suggested a small decrease in FKBP5 methylation levels compared to healthy controls (9, 10). In an in vitro study, it was demonstrated that, by decreasing DNMT1 expression, GC is able to reduce FKBP5 methylation levels and, therefore, increase its expression (44).

Likewise, FKBP5 mRNA is also sensitive to GC exposure. A time-dependent increase in blood FKBP5 mRNA after single-dose prednisone administration has been demonstrated in healthy humans (62). Accordingly, patients with ACTH-dependent CS had higher blood FKBP5 mRNA levels compared with healthy controls, and after a successful surgery, FKBP5 mRNA returned to baseline levels (63). Furthermore, in another study, blood FKBP5 mRNA was inversely correlated with FKBP5 promoter methylation and positively correlated with 24-hour urine free cortisol (UFC) levels in patients with CS (46). Taken together, this fine-tuning of FKBP5 DNA methylation and mRNA according to the level of GC suggests that FKBP5 can be used as a biomarker to infer the magnitude of GC exposure.

POMC and Corticotropin-Releasing Hormone

The partial resistance of the corticotroph adenoma to GC negative feedback is a hallmark of CD. Indeed, the lack of this inhibitory effect constitutes a method to diagnose CD, that is, with the dexamethasone suppression test. One of the mechanisms related to the insensitivity to GC can be attributed to GR mutations which are, however, rarely found in corticotrophinomas (64). Another mechanism that was uncovered in corticotroph adenomas is an overexpression of the HSP90 chaperone resulting in reduced affinity of GR to its ligand and consequently GR resistance (53, 65).

In addition, the loss of protein expression of either Brg1, ATPase component of the SWI/SNF chromatin remodeling complex, or HDAC2 has been linked to GC resistance in about 50% of some adenomas (66). The trans-repression process on POMC transcription achieved by GC involves both the histone deacetylation enzyme and Brg1. One mechanism of corticotropin-releasing hormone (CRH)-induced POMC expression is through an orphan nuclear receptor (NR) related to NGFI-B (Nur77). NGFI-B binds to the NurRE sequence in the promoter region of POMC gene and recruits a co-activator to mediate its transcription. In a tethering mechanism, the GR directly interacts with NGFI-B to form a trans-repression complex, which contains the GR itself, Brg1, the nuclear receptor, and HDAC2; the latter being essential to block the gene expression through chromatin remodeling process (53, 66).

In CD, hypercortisolism exerts a negative feedback at CRH secretion from the hypothalamus (67). The mechanism involved in GR-induced suppression of CRH expression is through direct binding to a nGRE in the promoter region of CRH gene and subsequent recruitment of repressor complexes. In a rat hypothalamic cell line, it was demonstrated that Dex-induced CRH repression occurs through coordinated actions of corepressors involving Methyl-CpG-binding protein 2 (MeCP2), HDAC1, and DNA methyltransferase 3B (DNMT3B). Possibly, GR bound to nGRE recruits DNMT3B to the promoter in order to methylate a specific region, subsequently binding MeCP2 on these methylated sites followed by the recruitment of chromatin modify corepressor HDAC1, ultimately resulting in CRH suppression. Another possibility is that 2 independent complexes, one consisting of GR with DNMT3 for the methylation and the other the MeCP2, bound to methylated region, interact with HDAC1 to induce repression (68).

Clock Genes

The clock system and the HPA axis are interconnected regulatory systems. Cortisol circadian rhythm is modulated by the interaction between a central pacemaker, located in the hypothalamic suprachiasmatic nuclei, and the HPA axis (69). At the molecular level, mediators of the clock system and cortisol also communicate with each other, both acting as transcription factors of many genes to influence cellular functions.

In CS, the impact of chronic GC exposure on clock genes expression was recently evaluated using peripheral blood samples from patients with active disease compared with healthy subjects. The circadian rhythm of peripheral clock gene expression (CLOCK, BMAL, PER1-3, and CRY1) was abolished as a result of hypercortisolism, and that may contribute to metabolic disorders observed in Cushing patients (70). Another study, which investigated persistent changes induced by hypercortisolism in visceral adipose tissue, found that the expression of clock genes, such as PER1, remained altered in association with persistent epigenetic changes in both H3K4me3 and H3K27ac induced by hypercortisolism even after the resolution of hypercortisolism (12). This suggests that chronic exposure to GC may induce sustained epigenetic changes that can influence clock genes expression. Nevertheless, further studies are warranted to better elucidate how long-term exposure to GC impacts clock genes expression using the epigenetic machinery.

Glucocorticoid Effects on MicroRNAs

Along with histone modification and DNA methylation, microRNAs (miRNAs) have emerged as an epigenetic mechanism capable of impacting gene expression without changing DNA sequence (15). Interestingly, miRNA expression itself is also under the influence of epigenetic modifications through promoter methylation like any other protein-encoding genes (71).

MicroRNAs are small (about 20-25 nucleotides in length) non-coding RNAs that are important in transcriptional silencing of messenger RNA (mRNA). By partially pairing with mRNA, miRNAs can either induce mRNA degradation or inhibit mRNA translation to protein. MiRNAs regulate the translation of about 50% of the transcriptome, allowing them to play an important role in a wide range of biological functions, such as cell differentiation, proliferation, metabolism, and apoptosis under normal physiological and pathological situations. Some miRNAs can be classified as oncogenes or tumor suppressing genes, and aberrant expression of miRNAs may be implicated in tumor pathogenesis (71-73).

Insight into the regulation of miRNA expression is, therefore, crucial for a better understanding of tumor development and other human diseases, including cardiac, metabolic, and neurological disorders (73, 74). There are different regulatory mechanisms involved in miRNA expression, including transcriptional factors such as GR-GC. GC may modulate miRNA expression through direct binding to GRE in the promoter region of the host gene, as observed in hemopoietic tumor cells (75). In addition to transcriptional activation, in vascular smooth muscle cells, Dex treatment induces downregulation of DNMT1 and DNMT3a protein levels and reduces the methylation of miRNA-29c promoter, resulting in an increased expression of miRNA-29c (76). Interestingly, it was demonstrated that the increased expression of miRNA-29 family (miRNA-29a, -29b, and -29c) associates with metabolic dysfunction, such as obesity and insulin resistance, which pertains to CS (77, 78). With regard to metabolic dysfunction, miRNA-379 expression was shown to be upregulated by GC and its overexpression in the liver resulted in elevated levels of serum triglycerides associated with very low-density lipoprotein (VLDL) fraction in mice (79). In obese patients, the level of hepatic miRNA-379 expression was higher compared to nonobese patients and positively correlated with serum cortisol and triglycerides (79). Hence, GC-responsive miRNA may be, at least in part, a mediator to GC-driven metabolic conditions in CS.

In pathological conditions, such as seen in CS, prolonged exposure to an elevated cortisol level results in a wide range of comorbidities. It can be hypothesized that the chronic and excessive glucocorticoid levels may induce an aberrant miRNA expression that might impact several cellular processes related to bone and cardiometabolic disorders. A recent study addressed the impact of hypercortisolism on bone miRNA of patients with active CD compared to patients with nonfunctional pituitary adenomas. Significant changes in bone miRNA expression levels were observed, suggesting that the disruption of miRNA may be partially responsible for reduced bone formation and osteoblastogenesis (80). Similarly, altered expression levels of selected miRNAs related to endothelial biology in patients with CS may point to a contribution to a high incidence of cardiovascular disorders in Cushing patients (81). Therefore, dysregulated miRNAs as a consequence of high cortisol levels may underpin the development and progression of comorbidities related to CS. To the best of our knowledge, it is currently not clear whether miRNA dysregulation persists after resolution of hypercortisolism, thus contributing to the persistence of some comorbidities. This hypothesis needs to be further investigated.

MicroRNA can also be used as a diagnostic tool in CS. A study was performed to identify circulating miRNA as a biomarker to differentiate patients with CS from patients with suspected CS who had failed diagnostic tests (the control group) (82). It was observed that miRNA182-5p was differentially expressed in the CS cohort compared to the control group; therefore, it may be used as a biomarker (82). However, a large cohort is necessary to validate this finding (82). In corticotroph tumors, downregulation of miRNA 16-1 expression was observed relative to normal pituitary tissue (83). In contrast, the plasma level of miRNA16-5p was found to be significantly higher in CD compared to ectopic Cushing (EAS) and healthy controls (84). This finding suggests that miRNA16-5p may be a biomarker capable to differentiate the 2 forms of ACTH-dependent Cushing (84).

Epidrugs and Glucocorticoid Action in Cushing’s Syndrome

The interest in understanding the epigenetic mechanism of GC action in the context of CS is based on reversibility of epi-marks, such as DNA methylation and histone modifications, using epidrugs (85, 86). The biological characteristics of epigenetic drugs and their target have been extensively explored. Their effectiveness as antitumor drugs have been tested on corticotroph tumors using in vitro studies (87-89). However, a limited number of studies have explored the role of epidrugs as a therapeutic tool in reversing the genomic action of GC in CS, particularly in comorbidities induced by hypercortisolism (90, 91).

The use of histone deacetylase inhibitors (HDACi) may reduce the genomic action of GC (90-92). It has been demonstrated that the use of the HDAC inhibitor valproic acid increases the acetylation level of GR, consequently attenuating the genomic action of GC. In an experimental Cushing model in rats, the use of valproic acid decreased expression of genes related to lipogenesis, gluconeogenesis, and ion regulators in the kidney that ultimately reduces hepatic steatosis, hyperglycemia, and hypertension in ACTH-infused rats (90, 91).

More studies evaluating the effects of epidrugs influencing the GC actions are warranted to further elucidate the underlying mechanisms and to explore potential treatment modalities to reverse long-lasting consequences of chronic corticoid exposure.

Conclusions

In physiologic conditions, GC are secreted in pulses following a circadian rhythm pattern, as opposed to a constant, chronic, and high GC exposure in CS. This pathological pattern may account for numerous devastating effects observed in CS (7). Yet, the expressed genome in response to chronic GC exposure may potentially be abnormal, leading to dysregulation in clock genes, among other effects.

GC levels may return to a normal circadian pattern in response to a successful treatment, but with incomplete reversibility of some CS features, which may in part be explained by epigenetic changes. The epigenetic machinery is used by GC to induce dynamic changes in chromatin to modulate gene expression. (Fig. 2) It seems that most of chromatin modifications are reversible, but some may persist resulting in long-term epigenetic changes. (Table 1)

Table 1.

Evidence of interaction between glucocorticoid and epigenetic machinery

Epigenetic changes/epigenetic enzymes	Action
Histone acetylation (HAT)	Glucocorticoid receptors (GR) recruit co-regulators, such as CREB binding protein (CBP), which has intrinsic histone acetyltransferase (HAT) activity that modifies the chromatin structure from an inactive to an active state (25, 33, 35).
Histone deacetylation (HDAC)	GR recruit histone deacetylases (HDACs) to turn chromatin less accessible and suppress gene transcription (25, 35). The trans-repression process on POMC transcription achieved by glucocorticoids (GC) involves the histone deacetylation enzyme (HDAC2). GC mediates the upregulation of HDAC2 in rats exposed to chronic stress (40).
Histone demethylase (JMJD3)	GC suppress transcription of JMJD3 in endothelial cells treated with TNFα (41).
Histone modifications	Using ChIP-seq, a study in mice treated for 5 weeks with corticosterone showed higher levels of histone modifications (H3K4me3, H3K27ac) compared to control mice. In mice after a 10-week washout period, persistence of this epigenetic fingerprint was observed, which was associated with long-lasting changes in gene expression (12).
DNA methylation (DNMT3B) and histone deacetylation (HDAC1)	GC mediates CRH downregulation through DNMT3B to the promoter in order to methylate a specific region and recruitment of chromatin modify corepressor HDAC (68).
DNA hypomethylation	GC induces downregulation of DNMT1 in AtT20 (mouse corticotroph adenoma cell line) (20). GC induces upregulation of TET enzyme expression which was described in retinal and osteocyte cell line model (42, 43). An experimental study in mice previously exposed to high levels of GC showed differentially methylated regions (DMR) induced by GC treatment, of which the majority was loss of the methylation (11). Reduced DNA methylation in FKBP5 gene was found in patients in active disease and also in remission state of Cushing syndrome (CS) as compared to a healthy control group (10). A genome-wide DNA methylation analysis showed a lower average of DNA methylation in patients in remission of CS compared to controls (9). A study using whole blood methylation profile demonstrated an association between cortisol excess and DNA hypomethylation in patients with CS (46).

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Further studies are needed to elucidate how chronic exposure to GC leads to incomplete reversibility of CS morbidities via sustained modulation of the epigenetic machinery and possibly other mechanisms. Subsequent identification of therapeutic targets may offer new perspective for treatments, for example, with epidrugs, aiming to reverse hypercortisolism-related comorbidities.

Funding

The authors received no financial support for this manuscript.

Disclosures

T.P., R.A.F., and L.J.H. have nothing to declare.

Data Availability

Data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study.

From https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgae151/7633538?searchresult=1

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The Genomic Landscape of Corticotroph Tumors: From Silent Adenomas to ACTH-Secreting Carcinomas

Posted on May 21, 2022 by MaryO

Abstract

Corticotroph cells give rise to aggressive and rare pituitary neoplasms comprising ACTH-producing adenomas resulting in Cushing disease (CD), clinically silent ACTH adenomas (SCA), Crooke cell adenomas (CCA) and ACTH-producing carcinomas (CA). The molecular pathogenesis of these tumors is still poorly understood. To better understand the genomic landscape of all the lesions of the corticotroph lineage, we sequenced the whole exome of three SCA, one CCA, four ACTH-secreting PA causing CD, one corticotrophinoma occurring in a CD patient who developed Nelson syndrome after adrenalectomy and one patient with an ACTH-producing CA. The ACTH-producing CA was the lesion with the highest number of single nucleotide variants (SNV) in genes such as USP8, TP53, AURKA, EGFR, HSD3B1 and CDKN1A. The USP8 variant was found only in the ACTH-CA and in the corticotrophinoma occurring in a patient with Nelson syndrome. In CCA, SNV in TP53, EGFR, HSD3B1 and CDKN1A SNV were present. HSD3B1 and CDKN1A SNVs were present in all three SCA, whereas in two of these tumors SNV in TP53, AURKA and EGFR were found. None of the analyzed tumors showed SNV in USP48, BRAF, BRG1 or CABLES1. The amplification of 17q12 was found in all tumors, except for the ACTH-producing carcinoma. The four clinically functioning ACTH adenomas and the ACTH-CA shared the amplification of 10q11.22 and showed more copy-number variation (CNV) gains and single-nucleotide variations than the nonfunctioning tumors.

Keywords: corticotroph; Cushing disease; ACTH-secreting carcinoma; single nucleotide variation; copy number variation; exome

1. Introduction

The pathological spectrum of the corticotroph includes ACTH (adrenocorticotropic hormone)-secreting pituitary adenomas (PA), causing Cushing disease (CD), silent corticotroph adenomas (SCA), Crooke cell adenomas (CCA) and the rare ACTH-secreting carcinoma (ACTH-CA). Pituitary carcinomas account for 0.1 to 0.2% of all pituitary tumors and are defined by the presence of craniospinal or distant metastasis [1,2,3]. Most pituitary carcinomas are of corticotroph or lactotrope differentiation [3]. Although a few cases present initially as CA, the majority develop over the course of several months or years from apparently benign lesions [3,4]. CCA are characterized by the presence of hyaline material in more than 50% of the cells of the lesion, and most of them arise from silent corticotroph adenomas (SCA) or CD-provoking ACTH-secreting adenomas [5]. SCA are pituitary tumors with positive immunostaining for ACTH but are not associated with clinical or biochemical evidence of cortisol excess; they are frequently invasive lesions and represent up to 19% of clinically non-functioning pituitary adenomas (NFPA) [6]. ACTH-secreting PA represents up to 6% of all pituitary tumors and causes eloquent Cushing disease (CD), which is characterized by symptoms and signs of cortisol hypersecretion, including a two- to fivefold increase in mortality [7,8]. The 2017 World Health Organization (WHO) classification of PA considers not only the hormones these tumors synthesize but also the transcription factors that determine their cell lineage [9]. TBX19 is the transcription factor responsible for the terminal differentiation of corticotrophs [9]. All tumor lesions of corticotroph differentiation are positive for both ACTH and TBX19.

ACTH-secreting PA causing CD are among the best genetically characterized pituitary tumors, with USP8 somatic variants occurring in up to 25–35% of sporadic cases [9]. Yet, information regarding the molecular pathogenesis of the lesions conforming to the whole pathological spectrum of the corticotroph is scarce. The aim of the present study is to characterize the genomic landscape of pituitary tumors of corticotroph lineage. For this purpose, we performed whole exome sequencing to uncover the mutational burden (single-nucleotide variants, SNV) and copy-number variations (CNVs) of these lesions.

2. Results

2.1. Clinical and Demographic Characteristics of the Patients

A total of 10 tumor samples from 10 patients were evaluated: 4 ACTH-secreting adenomas causing clinically evident CD, three non-functioning adenomas that proved to be SCA upon immunohistochemistry (IHC), one ACTH-secreting CA with a prepontine metastasis, one rapidly growing ACTH-secreting adenoma after bilateral adrenalectomy (Nelson syndrome) in a patient with CD and one non-functioning, ACTH-producing CCA (Table 1). All except one patient were female; the mean age was 38.8 ± 16.5 years (range 17–61) (Table 1). They all harbored macroadenomas with a mean maximum diameter of 31.9 ± 13 mm (range 18–51). Cavernous sinus invasion was evident on MRI in all but one of the patients (Table 1). Homonymous hemianopia was present in seven patients, whereas right optic nerve atrophy and amaurosis were evident in patient with the ACTH-CA, and in patient with CD and pituitary apoplexy (Table 1). Detailed clinical data are included in Supplementary Table S1. Death was documented in only the patient with pituitary apoplexy, and one patient was lost during follow-up, as of October 2018.

Table 1. Clinical features of the tumors analyzed and SNV present in each tumor.

2.2. General Genomic Characteristics of Neoplasms of Corticotrophic Lineage

Overall, approximately 18,000 variants were found, including missense, nonsense and splice-site variants as well as frameshift insertions and deletions. Of these alterations, the majority corresponded to single-nucleotide variants, followed by insertions and deletions. The three most common base changes were transitions C > T, T > C and C > G; most of the genetic changes were base transitions rather than transversions (Figure 1). There were several genes across the whole genome affected in more than one way, meaning that the same gene presented missense and nonsense variants, insertions, deletions and splice-site variants (Figure 2). Many of these variants are of unknown pathogenicity and require further investigation. Gains in genetic material were found in 44 cytogenetic regions, whereas 72 cytogenetic regions showed loss of genetic material in all corticotroph tumors.

Figure 1. Panel (A) shows the gadolinium-enhanced magnetic resonance imaging of the patient with ACTH-CA, highlighting in red the metastatic lesion in the prepontine area. Panel (B) shows the hematoxylin and eosin staining displaying the hyaline structures in the perinuclear areas denoting a Crooke cell adenoma. Panel (C,D) depict a representative corticotroph tumor with positive ACTH and TBX19 immunohistochemistry, respectively. Panel (E) shows four graphics: variant classification, variant type, SNV class and transition (ti) or transversion (tv) describing the general results of exome sequencing of the corticotroph tumors.

Figure 2. Representative rainfall plots showing the SNV alterations throughout the whole genome of corticotroph tumors (A) CCA, (B) SCA, (C) CD and (D) ACTH-CA, displaying all base changes, including transversions and transitions. No kataegis events were found. Alterations across the genome were seen in all corticotroph tumors.

2.3. ACTH-Secreting Carcinoma (Tumor 1)

SNV missense variants were found in the genes encoding TP53 (c.215G > C [rs1042522], p.Pro72Arg); AURKA (c.91T > A [rs2273535], p.Phe31Ile); EGFR (epidermal growth factor receptor, c.1562G > A [rs2227983], p.Arg521Lys); HSD3B1 (3-ß-hydroxisteroid dehydrogenase, c.1100C > A [rs1047303], p.Thr367Asn); CDKN1A (cyclin-dependent kinase inhibitor 1A or p21, c.93C > A [rs1801270], p.Ser31Arg); and USP8 (c.2159C > G [rs672601311], p.Pro720Arg). Interestingly, the previously reported USP48, BRAF, BRG1 and CABLES1 variants in pituitary CA cases were not found in this patient’s tumor (Figure 3). All SNV detected in WES experiments were validated by Sanger sequencing. The variants described were selected due to their potential pathogenic participation in other tumors and the allelic-risk association with tumorigenesis. Hereafter, all the mentioned variants in other corticotroph tumors are referred to by these aforementioned variants. Even though these same genes presented other variants, currently the significance of those variants is unknown.

Figure 3. Panel (A) shows the oncoplot from the missense variants of the selected genes and their clinical–pathological features. Panels (B–G) depict USP8, EGFR, TP53, AURKA, CDKN1A and HSD3B1 proteins, respectively, with the changes found in DNA impacting aminoacidic changes.

In general, the pituitary CA presented more CNV alterations than the benign tumors, with 27 and 32 cytogenetic regions showing gains and losses of genetic material, respectively. The cytogenetic regions showing gains were 10q11.22, 15q11.2, 16p12.3, 1p13.2 and 20p, where genes SYT15, POTEB, ARL6IP1, HIPK1 and CJD6 are coded, respectively. By contrast, 8p21.2 was the cytogenetic region showing loss of genetic material. The previously reported amplification of 1p13.2 was also detected in this tumor (Figure 4) [10].

Figure 4. Hierarchical clustering of corticotroph tumors according to their gains and losses across the whole genome (somatic chromosomes only). High contrast was used to enhance potential CNV alterations; nevertheless, there were only 44 unique cytogenetic regions that showed gains in genetic material with statistical significance, whereas only 72 unique cytogenetic regions showed loss of genetic material with statistical significance.

2.4. Crooke Cell Adenoma (Tumor 2)

The CCA showed SNV in the genes encoding TP53, EGFR, HSD3B1 and CDKN1A. However, neither the genes encoding AURKA and USP8 nor those encoding USP48, BRAF, BRG1 and CABLES were affected in this tumor. In CCA, only two and fifteen gains and losses were observed in copy-number variation, respectively. CNVs only showed gains in cytogenetic regions 17q12 and 10q11.22, harboring genes CCL3L1 and NPY4R, respectively, whereas losses were found in cytogenetic regions 18q21.1, 15q12 and 2q11.2, harboring genes KATNAL2, TUBGCP5 and ANKRD36.

2.5. Silent Corticotroph Adenomas (Tumors 3–5)

The three SCA shared SNVs in the genes encoding HSD3B1 and CDKN1A. SCA 4 and 5 showed SNV in the genes encoding EGFR, whereas SNV in the genes encoding AURKA and TP53 were present in SCA 3 and 5. None of the SCA were found to have SNV in the genes encoding USP8, USP48, BRAF, BRG1 or CABLES1.

The SCA presented only two and eighteen gains and losses (CNV), respectively. In regard to CNV, the these clinically silent tumors presented gains of genetic material in cytogenetic regions 17q22 and 10q11.22, which harbor genes encoding CCL3L1 and NPY4R. Eighteen losses were found distributed in cytogenetic regions 18q21.1, 15q12 and 2q11.2, encompassing the genes encoding KATNAL2, TUBGCP5 and ANKRD36. This CNV pattern closely resembles the one found in the CCA, which is somewhat expected if we consider that both neoplasms are clinically non-functioning

2.6. ACTH-Secreting Adenomas (Cushing Disease) (Tumors 6–9)

SNV of the genes encoding TP53 and HSD3B1 were present in tumor samples from all four CD patients, whereas none of these patients harbored adenomas with SNV in the genes encoding USP8 or CDKN1A. An SNV in the gene encoding AURKA was identified in only one of these tumors (tumor 8). EGFR SNV were found in tumors 7 and 9. None of the CD-causing ACTH-secreting adenomas showed the previously reported SNV in the genes encoding USP48, BRAF, BRG1 and CABLES1.

CNV analysis in this group of eloquent-area corticotroph tumors revealed 25 gains and 55 losses of genetic material. The gains occurred in cytogenetic regions 17q12, 2p12, 9p24 and 10q11.22, where genes CCL3L1, CTNNA2, FOXD4 and NPY4R are coded, respectively. The losses were localized in cytogenetic regions 21p12, 15q11.2, and 8p23, harboring genes USP16, KLF13 and DEF130A, respectively. We also detected the previously reported 20p13 amplification [10].

2.7. ACTH-Secreting Adenoma Causing Nelson Syndrome (Tumor 10)

This patient’s tumor showed SNV in the genes encoding USP8, TP53, HSD3B1 and CDKN1A but no alterations were found in the genes encoding EGFR and AURKA. This tumor and the ACTH-CA were the only two neoplasms that harbored a USP8 variant. No SNV were identified in the genes encoding USP48, BRAF, BRG1 and CABLES1. Interestingly, CNV analysis revealed the same gains and losses of genetic material found in tumors from other patients with CD.

2.8. Tumor Phylogenic Analysis

We performed a phylogenetic inference analysis to unravel a hypothetical sequential step transformation from an SCA to a functioning ACTH-secreting adenoma and finally to an ACTH-CA. The theoretical evolutive development of the ACTH CA, departing from the SCA, shows two main clades, with the smallest one comprising two of the three SCA and two of the five ACTH-adenomas causing CD. Since these four tumors have the same SNV profile, we can assume that they harbor the genes that must be altered to make possible the transition from a silent to a clinically eloquent adenoma; the gene encoding ATF7IP (c.1589A > G [rs3213764], p.K529R) characterizes this clade. The second and largest clade includes the CCA, the ACTH-CA, one of the three SCA and three of the five most aggressive ACTH adenomas causing CD, including the adenoma of the patient with Nelson syndrome. This clade represents the molecular alterations required to evolve from a CD-causing ACTH-adenoma to a more aggressive tumor, or even to a CA and is characterized by the gene encoding MSH3 (c.235A > G [rs1650697], p.I79V) (Figure 5).

Figure 5. Phylogenetic analysis of the corticotroph tumors. The theoretical evolutive development of the ACTH-CA, departing from the SCA shows two main clades. The first clade, characterized by ATF7IP gene, comprises 2 of the 3 SCA and 2 of the 5 ACTH-adenomas causing CD. The second clade is characterized by the gene encoding MSH3 and includes the CCA, the ACTH-CA, one of the 3 SCA and 3 of the 5 most aggressive ACTH adenomas causing CD, including the adenoma of the patient with Nelson syndrome. Red dots represent the Cushing Disease provoking adenomas, green dots represent the silent corticotroph tumors, brown dot represent the Crooke cell adenoma and the blue dot represent the corticotroph carcinoma.

2.9. Correlation between Gene Variants and Clinicopathological Features

The USP8 variant positively correlated with increased tumor mass (p = 0.019). The CDKN1A variant was significantly associated with silent tumors (p = 0.036). The rest of the genetic variants did not correlate with any of the clinicopathological features tested. The presence of the EGFR variant was not distinctly associated with any of the clinical parameters and was equally present in functional as well as non-functional tumors (p = 0.392). AURKA SNV did not correlate with any of the features, including recurrence (p = 0.524). Detailed statistical results are presented in Supplementary Table S2.

3. Discussion

Corticotrophs are highly specialized cells of the anterior pituitary that synthesize and secrete hormones that are essential for the maintenance of homeostasis. In this study, we sequenced the exome of 10 corticotroph tumors, including three SCA, four ACTH adenomas causing CD, an ACTH adenoma in a patient with Nelson syndrome, a CCA and an ACTH-CA in total, representing the broad pathological spectrum of this cell. Our results portray the genomic landscape of all the neoplasms that are known to affect the corticotroph.

The neoplasm with the highest number of genomic abnormalities, including SNV and CNV, was the ACTH-CA, followed by the CCA and the CD tissues. Of all the genes harboring SNVs, six were found to be present in at least two of our tumor samples: HSD3B1, TP53, CDKN1A, EGFR, AURKA and USP8.

The HSD3B1 gene encodes a rate-limiting enzyme required for all pathways of dihydrotestosterone synthesis and is abundantly expressed in adrenal tumors. Gain of function of this HSD3B1 variant, which has a global allelic prevalence of 0.69678 [11], results in resistance to proteasomal degradation with the consequent accumulation of the enzyme and has been associated with a poor prognosis in patients with prostate cancer [12]. Nine of the ten corticotroph tumors in our cohort harbored an SNV of the tumor suppressor gene TP53. The TP53 variant described in our cohort has been reported to be present in 80% of non-functioning pituitary adenomas and is apparently associated with a younger age at presentation and with cavernous sinus invasion [13]. Furthermore, this TP53 variant results in a reduced expression of CDKN1A and an increased expression of vascular endothelial growth factor (VEGF) as well as an increased cellular proliferation rate [13]. CDKN1A (also known as p21) is a cyclin-dependent kinase inhibitor regulating cell cycle progression. The SNV described in our study was reported to alter DNA binding ability and expression and has a global allelic frequency of 0.086945 [14]. This cyclin-dependent kinase inhibitor SNV was found to be associated with breast carcinoma [15] and lung cancer [16]. The presence of this SNV has not been previously explored in pituitary adenomas, although CDKN1A is downregulated in clinically non-functioning pituitary adenomas of gonadotrophic lineage but not in hormone-secreting tumors [17]. EGFR encodes a transmembrane tyrosine kinase receptor, activation of which leads to mitogenic signaling [18]. This gene is upregulated in several cancers and represents a target for molecular therapies [19]. The EGFR SNV described in our corticotroph tumor series was found to be associated with the response to neoadjuvant chemotherapy in patients with breast and lung cancer [18]. EGFR is normally expressed in corticotrophs, where it participates in the regulation of POMC (proopiomelanocortin) gene transcription and cellular proliferation [20]. The EGFR rs2227983 has a 0.264334 global allelic frequency [21]. AURKA is a cell-cycle regulatory serine/threonine kinase that promotes cell cycle progression by the establishment of the mitotic spindle and centrosome separation [22]. Alterations of these gene are related to centrosomal amplification, dysfunction of cytokinesis and aneuploidy [22]; it has a global allelic frequency of 0.18078 [23]. This same SNV has been associated with overall cancer risk, particularly breast, gastric, colorectal, liver and endometrial carcinomas, but it has never been formally studied in pituitary tumors [22]. Activating somatic variants of the gene encoding USP8 were recently found in 25–40% of ACTH-secreting adenomas causing CD [24,25]. Patients harboring these variants are usually younger, more frequently females and were found to have higher long-term recurrence rates in some but not all studies [26,27]. USP8 mediates the deubiquitination of EGFR by inhibiting its interaction with protein 14-3-3, which in turn prevents its proteosomal degradation. Signaling through the recycled deubiquitinated EGFR is increased, leading to increased POMC transcription and cellular proliferation. Most activating USP8 variants are located within its 14-3-3 binding motif [24,25]. Recently, USP8 and TP53 SNV were described in corticotroph tumors as drivers of aggressive lesions [28]. To our knowledge, USP8 variants have not been evaluated in patients with pituitary carcinomas, and none of the previously mentioned studies have included patients with Nelson syndrome. In our cohort, neither the CCA nor the SCA showed variants in USP8, in concordance with previously published studies [25,29], or in the genes USP48, BRAF, BRG1 and CABLES1 [9], and none of them were present in our cohort.

Genetic structural variations in the human genome can be present in many forms, from SNV to large chromosomal aberrance [30]. CNV are structurally variant regions, including unbalanced deletions, duplications and amplifications of DNA segments ranging from a dozen to several hundred base pairs, in which copy-number differences have been observed between two or more genomes [31,32]. CNV are involved in the development and progression of many tumors and occur frequently in PA [30,33]. Hormone-secreting pituitary tumors show more CNV than non-functioning tumors [34]. Accordingly, our non-functioning SCA and CCA had considerably fewer chromosomal gains and losses than the CD-causing adenomas and the ACTH-CA. Expectedly, the ACTH-CA had significantly more cytogenetic abnormalities than any other tumor in our series. Interestingly, the ACTH-adenomas causing CD, the SCA and the CCA shared the gain of genetic material in 17q12, highlighting their benign nature. The 17q12 amplification has been described in gastric neoplasms [35]. The only cytogenetic abnormality shared by all types of corticotroph tumors was the gain of genetic material in 10q11.22. Amplification of 10q11.22 was previously described in Li–Fraumeni cancer predisposition syndrome [36]. The ACTH-CA, the CCA and one SCA clustered together showing a related CNV pattern; this CNV profile could be reflective of the aggressive nature of these neoplasms, since both CCA and SCA can follow a clinically aggressive course [5,6].

Our results show that all lesions conforming to the pathological spectrum of the corticotroph share some of the SNV and CNV profiles. These genomic changes are consistent with the potential existence of a continuum, whereby silent tumors can transform into a clinically eloquent tumor and finally to carcinoma, or at least a more aggressive tumor. It can also be interpreted as the common SNV shared by aggressive tumors. It is known that silent corticotroph adenomas may switch into a hormone-secreting tumor [37] and are considered a marker for aggressiveness and a risk factor for malignancy since most of the carcinomas are derived from functioning hormone-secreting adenomas. Our phylogenetic inference analysis showed that the genes ATF7IP and MSH3 could participate in a tumor transition ending in aggressive entities or even carcinomas. ATF7IP is a multifunctional nuclear protein mediating heterochromatin formation and gene regulation in several contexts [38], while MSH3 is a mismatch-repair gene [39]. Events related to heterochromatin remodeling and maintenance have been related to aggressive pituitary adenomas and carcinomas [40]. Additionally, alterations in mismatch-repair genes are related to pituitary tumor aggressiveness and resistance to pharmacologic treatment [41,42]. The variants described in ATF7IP and MSH3 are related to prostate and colorectal cancer, respectively [43,44]. There is evidence suggesting that the ATF7IP variant could be deleterious because it leads to a negative regulation of transcription [45]. Thus, these events could be biologically relevant to corticotroph tumorigenesis, although more research is needed.

4. Conclusions

We have shown genomic evidence that within the tumoral spectrum of the corticotroph, functioning ACTH-secreting lesions harbor more SNV and CNV than non-functioning ACTH adenomas. The ACTH-secreting CA shows more genomic abnormalities than the other lesions, underscoring its more aggressive biological behavior. Phylogenetic inference analysis of our data reveals that silent corticotroph lesions may transform into functioning tumors, or at least potentially, into more aggressive lesions. Alterations in genes ATF7IP and MSH3, related to heterochromatin formation and mismatch repair, could be important in corticotroph tumorigenesis. The main drawback of our study is the limited sample size. We are currently increasing the number of samples to corroborate our findings and to be able to perform a more comprehensive complementary phylogenetic analysis of our data. Finally, further research is needed to uncover the roles of these variants in corticotroph tumorigenesis.

5. Materials and Methods

5.1. Patients and Tumor Tissue Samples

Ten pituitary tissues were collected: one ACTH-CA, one CCA, three SCA, and five ACTH-secreting PA causing CD, including the tumor of a patient who developed Nelson syndrome after bilateral adrenalectomy. All tumors included in the study were sporadic and were collected from patients diagnosed, treated and followed at the Endocrinology Service and the Neurosurgical department of Hospital de Especialidades, Centro Médico Nacional Siglo XXI of the Instituto Mexicano del Seguro Social, Hospital General de Mexico “Dr. Eduardo Liceaga” and Instituto Nacional de Neurologia y Neurocirugia “Manuel Velazquez”. All participating patients were recruited with signed informed consent and ethical approval from the Comisión Nacional de Ética e Investigación Científica of the Instituto Mexicano del Seguro Social, in accordance with the Helsinki declaration.

CD was diagnosed according to our standard protocol. Briefly, the presence of hypercortisolism was documented based on two screening tests, namely a 24 h urinary free-cortisol level above 130 µg and the lack of suppression of morning (7:00–8:00) cortisol after administration of 1 mg dexamethasone the night before (23:00) to less than 1.8 µg/dL, followed by a normal or elevated plasma ACTH to ascertain ACTH-dependence. Finally, an overnight, high-dose (8 mg) dexamethasone test, considered indicative of a pituitary source, and a cortisol suppression > 69%, provided that a pituitary adenoma was clearly present on magnetic resonance imaging (MRI) of the sellar region. In none of the 10 patients included in the study was inferior petrosal venous sampling necessary to confirm the pituitary origin of the ACTH excess. Invasiveness was defined by the presence of tumor within the cavernous sinuses (CS).

DNA was extracted from paraffin-embedded tumor tissues using the QIAamp DNA FFPE tissue kit. From frozen tumors, DNA was obtained using the Proteinase K-ammonium acetate protocol.

5.2. Construction and Sequencing of Whole Exome Libraries

Exome libraries were prepared according to the Agilent SureSelect XT HS Human All exon v7 instructions. Briefly, 200 ng of DNA was enzymatically fragmented with Agilent SureSelect Enzymatic Fragmentation Kit. Fragmented DNA was end-repaired and dA-tail was added at DNA ends; then, molecular barcode adaptors were added, followed by AMPure XP bead purification. The adaptor-ligated library was amplified by PCR and purified by AMPure XP beads. DNA libraries were hybridized with targeting exon probes and purified with streptavidin-coated magnetic beads. The retrieved libraries were amplified by PCR and purified by AMPure XP beads and pooled for sequencing in NextSeq 500 using Illumina flow cell High Output 300 cycles chemistry. All quality controls of the libraries were carried out using Screen tape assays and quantified by Qubit fluorometer. Quality parameters included a DNA integrity number above 8 and a 100X sequencing depth aimed with at least 85% of coverage.

5.3. Bioinformatics Analysis

The fastq files were subjected to quality control using FastQC v0.11.9, the adapters were removed using Cutadapt v3.4, the alignment was carried out with Burrows–Wheeler Alignment Tool v0.7.17 with the -M option to ensure compatibility with Picard and GRCh38 as a reference genome. The marking of duplicates as well as the sorting was carried out with Picard v2.26.4 with the AddOrReplaceReadGroups programs with the option SORT_ORDER = coordinate and MarkDuplicates, respectively. Variant calling was carried out using Genomic Analysis Toolkit (GATK) v4.2.2.0 following the Best Practices guide (available at https://gatk.broadinstitute.org/) [46] and with the parameters used by Genomic Data Commons (GDC), available at https://docs.gdc.cancer.gov/ [47]. The GATK tools used were CollectSequencingArtifactMetrics, GetPileupSummaries, CalculateContamination and Mutect2. Mutect2 was run with the latest filtering recommendations, including a Panel of Normal and a Germline Reference from the GATK database. Filtering was performed with the CalculateContamination, LearnReadOrientationModel and FilterMutectCalls tools with the default parameters. For the calculation of CNV GISTIC v2.0.23 was used with the parameters used by GDC. Catalog of Somatic Mutation in Cancer (COSMIC) was used to uncover pathogenic variants. For the analysis of variants and CNV, the maftool v2.10.0 and ComplexHeatmap 2.10.0 packages were used. All analyses were carried out on the GNU/Linux operating system under Ubuntu v20.01.3 or using the R v4.0.2 language in Rstudio v2021.09.0+351. A second bioinformatics pipeline was also used, SureCall software (Agilent) with the default parameters used for SNV variant calling. The variants found by both algorithms were taken as reliable SNV. Data were deposited in Sequence Read Archive hosted by National Center for Biotechnology Information under accession number PRJNA806516.

Phylogenetic tree inference (PTI) was run by means of the default parameters using matrices for each sample. These matrices contain an identifier for each variant, mutant read counts, counts of reference reads and the gene associated with the variant. The only PTI parameter was Allele Frequency of Mutation and was used to improve the speed of the algorithm. Briefly, PTI uses an iterative process on the variants shared between the samples. First, it builds the base of the tree using the variants shared by all the samples; second, it eliminates these variants and establishes a split node; and third, it eliminates the variants of the sample that produced the division (split). PTI iteratively performs these three steps for all division possibilities. Each tree is given a score based on an aggregated variant count, and the tree with the highest score is chosen as the optimal tree.

5.4. Sanger Sequencing forConfirmation of Exome Findings

Exome variant findings in exome sequencing were validated by Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing kit (ThermoFischer) in a 3500 Genetic Analyzer. Primers used for USP8 [48], TP53 [49], EGFR [50], AURKA [51], CDKN1A [52,53] and HSD3B1 sequencing have been previously reported.

5.5. Hormone and Transcription Factor Immunohistochemistry

Paraffin-embedded, formalin-fixed tissue blocks were stained with hematoxylin–eosin and reviewed by a pathologist. Tumors were represented with a 2-fold redundancy. Sections (3 μm) were cut and placed onto coated slides. Immunostaining was performed by means of the HiDef detection HRP polymer system (Cell Marque, CA, USA), using specific antibodies against each pituitary hormone (TSH, GH, PRL, FSH, LH and ACTH) and the lineage-specific transcription factors TBX19, POU1F1 and NR5A1, as previously described [54]. Two independent observers performed assessment of hormones and transcription factors expression at different times.

5.6. Statistical Analysis

Two-tailed Fisher exact tests and Student’s t tests were used to evaluate the relationship between the identified gene variants and clinicopathological features. A p value of <0.05 was considered statistically significant. Statistical software consisted of SPSS v28.0.1

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23094861/s1.

Author Contributions

D.M.-R., K.T.-P. and M.M. conceived, designed and coordinated the project, performed experiments, analyzed, discussed data and prepared the manuscript. S.A.-E., G.S.-R., E.P.-M., S.V.-P., R.S., L.B.-A., C.G.-T., J.G.-C. and J.T.A.-S. performed DNA purification, library preparation, sequencing experiments, bioinformatics analysis and wrote the manuscript. A.-L.E.-d.-l.-M., I.R.-S., E.G.-A., L.A.P.-O., G.G., S.M.-J., L.C.-M., B.L.-F. and A.B.-L. provided biological samples and detailed patient information. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially supported by grants 289499 from Fondos Sectoriales Consejo Nacional de Ciencia y Tecnologia, Mexico, and R-2015-785-015 from Instituto Mexicano del Seguro Social (MM).

Institutional Review Board Statement

Protocol approved by the Comisión Nacional de Ética e Investigación Científica of the Instituto Mexicano del Seguro Social, in accordance with the Helsinki declaration (R-2019-785-052).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Data were deposited in Sequence Read Archive hosted by National Center for Biotechnology Information under accession number PRJNA806516.

Acknowledgments

Sergio Andonegui-Elguera is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship 921084 from CONACYT. KTP is a recipient of Consejo Nacional de Ciencia y Tecnología (CONACyT) fellowship “Estáncias posdoctorales por Mexico 2021” program. DMR is a recipient of the National Council for Science and Technology Fellowship “Catedra CONACyT” program.

Conflicts of Interest

The authors declare no conflict of interest.

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From https://www.mdpi.com/1422-0067/23/9/4861/htm

Filed under: Cushing's, pituitary | Tagged: ACTH, adenoma, corticotroph adenomas, Cushing's Disease | Leave a comment »

Diagnostic pitfalls in Cushing’s disease impacting surgical remission rates; test thresholds and lessons learned in 105 patients

Posted on September 7, 2021 by MaryO

This article was originally published here

J Clin Endocrinol Metab. 2021 Sep 3:dgab659. doi: 10.1210/clinem/dgab659. Online ahead of print.

ABSTRACT

CONTEXT: Confirming a diagnosis of Cushing’s disease (CD) remains challenging yet is critically important before recommending transsphenoidal surgery for adenoma resection.

OBJECTIVE: To describe predictive performance of preoperative biochemical and imaging data relative to post-operative remission and clinical characteristics in patients with presumed CD.

DESIGN, SETTING, PATIENTS, INTERVENTIONS: Patients (n=105; 86% female) who underwent surgery from 2007-2020 were classified into 3 groups: Group A (n=84) pathology-proven ACTH adenoma; Group B (n=6) pathology-unproven but with postoperative hypocortisolemia consistent with CD, and Group C (n=15) pathology-unproven, without postoperative hypocortisolemia. Group A+B were combined as Confirmed CD and Group C as Unconfirmed CD.

MAIN OUTCOMES: Group A+B was compared to Group C regarding predictive performance of preoperative 24-hour urinary free cortisol (UFC), late night salivary cortisol (LNSC), 1mg dexamethasone suppression test (DST), plasma ACTH, and pituitary MRI.

RESULTS: All groups had a similar clinical phenotype. Compared to Group C, Group A+B had higher mean UFC (p<0.001), LNSC(p=0.003), DST(p=0.06), ACTH(p=0.03) and larger MRI-defined lesions (p<0.001). The highest accuracy thresholds were: UFC 72 µg/24hrs; LNSC 0.122 µg/dl, DST 2.70 µg/dl, and ACTH 39.1 pg/ml. Early (3-month) biochemical remission was achieved in 76/105 (72%) patients: 76/90(84%) and 0/15(0%) of Group A+B versus Group C, respectively, p<0.0001. In Group A+B non-remission was strongly associated with adenoma cavernous sinus invasion.

CONCLUSIONS: Use of strict biochemical thresholds may help avoid offering transsphenoidal surgery to presumed CD patients with equivocal data and improve surgical remission rates. Patients with Cushingoid phenotype but equivocal biochemical data warrant additional rigorous testing.

PMID:34478542 | DOI:10.1210/clinem/dgab659

Filed under: Cushing's, Diagnostic Testing, Treatments | Tagged: adenoma, Cushing's Disease, pituitary, remission, Transsphenoidal surgery | Leave a comment »

Cushing’s syndrome in a child

Posted on February 3, 2021 by MaryO

Abstract

Cushing’s syndrome is a rare entity in children. Adrenal tumour is the common cause of this syndrome in young children, whereas, iatrogenic causes are more common among older children. We report a 4 year old male child diagnosed with Cushing syndrome due to a right adrenal adenoma; the child presented with obesity and increase distribution of body hair. After thorough investigation and control of hypertension and dyselectrolytemia, right adrenalectomy was performed. The patient had good clinical recovery with weight loss and biochemical resolution of Cushing’s syndrome.

1. Introduction

Cushing’s syndrome (CS) is rarely encountered in children. The overall incidence of Cushing syndrome is approximately 2–5 new cases per million people per year. Only approximately 10% of the new cases each year occur in children [1]. Unlike in adults, a male-to-female predominance have been observed in infants and young toddlers [[1], [2], [3]]. Although iatrogenic causes are common in children above seven years of age, adrenal causes (adenoma, carcinoma or hyperplasia) are common in children of younger age [4]. We report a 4 year old boy diagnosed with Cushing syndrome caused by a right adrenal adenoma, who had presented with obesity and increase distribution of body hair. Right adrenalectomy was performed and clinical stabilization resulted in weight loss and biochemical resolution of Cushing’s syndrome. (see Fig. 5)

2. Case report

A 4 years old boy presented with complaints of excessive weight gain of 5 months duration and increase frequency of micturition and appearance of body hair for 4 months. There was no history of any other illness, medication or steroid intake. The child was first born at term by normal vaginal delivery and birth weight of 3 kg. Physical examination revealed a chubby boy with moon face, buffalo hump, protruding abdomen, increase body hair and appearance of coarse pubic hair (Fig. 1). His intelligent quotient (IQ) was appropriate for his age and sex. His younger sibling was in good health and other family members did not have any metabolic or similar problems.

The patient’s body length was 92cm (between -2SD to -3SD), weight 20kg (between 1 SD and 2 SD), weight for height >3SD, and BMI was 23.6 (BMI for age >3 SD). His blood pressure on right arm in lying position was 138/76 mm Hg (above 99th percentile for height and age).

Investigations: Morning 8am serum cortisol level – 27.3 μg/dl (normal: 6–23 μg/dl).

with a concurrent plasma ACTH level of < 5 pg/ml (n value < 46 pg/ml).

His serum cortisol following low dose dexamethasone suppression test (1mg dexamethasone at 11pm) at 8 am next morning was 22.1 μug/dl and his 24 hours urine catecholamine fraction was within normal limit.

HB % — 10.3 gm/dl; LDDST — 25 μg/dl; FBS — 106 mg/dl.

Serum Na⁺ – 140.6mmol/l; K⁺ – 2.83mmol/l; Ca⁺ – 8.7 mg/dl.

S. Creatinine −0.3 mg/dl.

Ultrasonography of abdomen revealed a heterogenous predominantly hypoechoic right supra renal mass. Contrast enhanced CT abdomen revealed well defined soft tissue density lesion (size −5.2 cm × 5.2 cm x 5.7cm) in right adrenal gland with calcifications and fat attenuations showing mild attenuation on post contrast study (Fig. 2).

The child was started on oral amlodipine 2.5mg 12hourly; after 5days blood pressure became normal. For hypokalemia oral potassium was given @20 meq 8 hourly and serum potassium value became normal after 4 days. Right laparoscopic adrenalectomy was planned. but due to intra operative technical problems it was converted to an open adrenalectomy with right subcostal incision. A lobulated mass of size 9 cm × 5 cm x 4 cm with intact capsule was excised. The tumour weighed 230 gm. There was no adhesion with adjacent organs, three regional nodes were enlarged but without any tumour tissue. Inferior vena cava was spared. Histopathology report was consistent with adrenal adenoma (Fig. 3) (see Fig. 4).

Post operative management: during post operative period hypokalemia and flaxuating blood sugar level was managed with oral potassium and oral glucose supplement. patient developed mild cough and respiratory distress on post op day 2, it was managed with salbutamol nebulization and respiratory physio therapy. Patient developed minor ssi and discharged on 10 th post operative day with oral prednisolone supplementation.

Follow up: the patient was followed up 2week after discharge and then every monthly, the oral prednisolone was gradually tapered and completely withdrawn on 2nd month after surgery.The patient experienced no post-surgical complications. After 4 months of surgery he reduces 6 kgs of his body weight with BMI of 16.5 (between median and 1SD) & BP 100/74 mm hg (within normal range), the moon face, buffalo hump, central obesity disappeared, morning 8am serum cortisol level was found within normal range 14 μg/dl (n value 6–23 μg/dl).

3. Discussion

Cushing’s syndrome is caused by prolonged exposure to supraphysiological levels of circulating glucocorticoids, which may be endogenously or exogenously derived. During infancy, CS is usually associated with McCune-Albright syndrome; adrenocortical tumours most commonly occur in children under four years of age and Cushing’s disease (ACTH dependent) is the commonest cause of CS after five years of age [5]. Primary adrenocortical tumours (ACTs) account for only 0.3–0.4% of all childhood neoplasms. Almost a third of these tumours manifests as Cushing syndrome and over 70% of the unilateral tumours in young children are often malignant [2,3,6,7]. There seems to be a bimodal incidence of these tumours, with one peak at under 5 years of age and the second one in the fourth or fifth decades of life. ACTs may be associated with other syndromes, such as, Li-Fraumeni syndrome, Beckwith-wiedemann syndrome, isolated hemihypertrophy, or even a germline point mutation of P53 tumour suppressor gene as reported in a series from Brazil [8]. In comparison to adult CS, growth failure with associated weight gain is one of the most reliable indicators of hypercortisolaemia in pediatric CS. The parents often fail to notice facial changes and growth failure and hence the diagnosis is often delayed. In one study, the mean time from appearing symptoms to diagnosis in 33 children with Cushing’s disease was 2.5 years [5]. More recently the comparison of height and BMI SDS measurements provided a sensitive diagnostic discriminator in pediatric patients with CD and those with simple obesity [9]. In the present case, the parents observed noticeable changes in his face and presence of body hair, which made them to bring the child to medical attention. A review of 254 children on the International Pediatric Adrenocortical Tumour Registry identified virilization as the most common manifestation [10]. About 10% of the tumours can be non-functional at presentation, and approximately one third of pediatric patients present with hypertension. Majority of patients (192/254) in the Registry had localized disease and metastatic disease was found in less than 5% of cases. Older children with CS or mixed androgen and cortisol secreting adrenocortical tumours had a worse prognosis compared to younger children [10]. The present case had mild hypertension as well as dyselectrolytemia at presentation, which could be controlled with medication. He had a single adenoma confined to the adrenal gland and there was no evidence of malignancy. After surgical excision of the tumour and the right adrenal gland, the patient made rapid improvement in clinical condition and has been on follow up for last 7 months.

4. Conclusion

Pediatric adrenocortical tumours (ACTs) are most commonly encountered in females and in children less than four years. But our case being an 4-year-old boy forms a rare presentation of endogenous Cushing’s syndrome due to adrenal adenoma. Cushing’s syndrome in this child was controlled after right adrenalectomy.

Patient consent

Informed written consent was taken.

Funding

No funding or grant support.

Authorship

All authors attest that they meet the current ICMJE criteria for authorship.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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From https://www.sciencedirect.com/science/article/pii/S2213576620303833

Filed under: Cushing's, symptoms, Treatments | Tagged: adenoma, child, Cushing's Syndrome, hypertension, male, obesity, pediatric | Leave a comment »

Temozolomide Effective Against Cushing’s Caused by Aggressive Tumors

Posted on June 29, 2019 by MaryO

The oral chemotherapy temozolomide might be an effective treatment for Cushing’s disease caused by aggressive tumors in the pituitary gland that continue to grow after surgery and taking other medications, a case report suggests.

The study, “Successful reduction of ACTH secretion in a case of intractable Cushing’s disease with pituitary Crooke’s cell adenoma by combined modality therapy including temozolomide,” was published in the journal J-Stage.

Cushing’s disease is often caused by a tumor in the pituitary gland that secretes high levels of adrenocorticotropic hormone (ACTH), leading to high levels of cortisol and other symptoms.

Macroadenomas are aggressive, fast-growing tumors that reach sizes larger than 10 millimeters. Crooke’s cell adenoma is a type of macroadenoma that does not respond to conventional therapies, but has deficient mechanisms of DNA repair. That is why chemotherapeutic agents that damage the DNA, such as temozolomide, might be potential treatments.

Researchers in Japan reported the case of a 56-year-old woman with Cushing’s disease caused by a Crooke’s cell adenoma in the pituitary gland who responded positively to temozolomide.

The patient was diagnosed with Cushing’s disease at age 39 when she went to the hospital complaining of continuous weight gain. She also had excessive production of urine and a loss of vision in the right eye.

The lab tests showed high levels of cortisol and ACTH, and the MRI detected a tumor of 4.5 centimeters in the pituitary gland. The doctors removed a part of the tumor surgically, which initially reduced the levels of ACTH and cortisol.

However, the hormone levels and the size of the residual tumor started to increase gradually after the surgery, despite treatment with several medications.

By the time the patient was 56 years old, she went to the hospital complaining of general fatigue, leg edema (swelling from fluid), high blood pressure, and central obesity (belly fat).

Further examination showed a 5.7 cm tumor, identified as a Crooke’s cell macroadenoma. The patient underwent a second surgery to remove as much tumor as possible, but the levels of ACTH remained high. She took temozolomide for nine months, which normalized the levels of ACTH and cortisol. After the treatment, the patient no longer had high blood pressure or leg edema.

The tumor shrunk considerably in the year following temozolomide treatment. The patient started radiation therapy to control tumor growth. The levels of cortisol and ACHT remained normal, and the tumor did not grow in the seven years following temozolomide treatment.

“These clinical findings suggest that [temozolomide] treatment to patients with Crooke’s cell adenoma accompanied with elevated ACTH may be a good indication to induce lowering ACTH levels and tumor shrinkage,” researchers wrote.

Other cases of Cushing’s disease caused by aggressive macroadenomas showed positive results, such as reduction of tumor size and decrease in plasma ACTH, after temozolomide treatment. However, more studies are needed to establish the ideal course of chemotherapy to treat these tumors, the researchers noted.

From https://cushingsdiseasenews.com/2019/06/18/temozolomide-effective-cushings-disease-aggressive-tumors-case-report/

Filed under: Cushing's, Treatments | Tagged: ACTH, adenoma, Crooke’s cell adenoma, edema, high blood pressure, pituitary, Temozolomide, vision changes, weight | Leave a comment »

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Epigenetic Mechanisms Modulated by Glucocorticoids With a Focus on Cushing Syndrome

Abstract

Search Strategy

Epigenetic Aspects of Genomic Action of Glucocorticoids

Glucocorticoid Receptor

Glucocorticoid Mechanism of Action

Glucocorticoid-Induced Epigenetic Changes

Epigenetic Changes as a Consequence of Long-Term Glucocorticoid Exposure in Cushing Syndrome

Glucocorticoid-Target Genes in Cushing Syndrome

FKBP5

POMC and Corticotropin-Releasing Hormone

Clock Genes

Glucocorticoid Effects on MicroRNAs

Epidrugs and Glucocorticoid Action in Cushing’s Syndrome

Conclusions

Funding

Disclosures

Data Availability

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The Genomic Landscape of Corticotroph Tumors: From Silent Adenomas to ACTH-Secreting Carcinomas

Abstract

1. Introduction

2. Results

2.1. Clinical and Demographic Characteristics of the Patients

2.2. General Genomic Characteristics of Neoplasms of Corticotrophic Lineage

2.3. ACTH-Secreting Carcinoma (Tumor 1)

2.4. Crooke Cell Adenoma (Tumor 2)

2.5. Silent Corticotroph Adenomas (Tumors 3–5)

2.6. ACTH-Secreting Adenomas (Cushing Disease) (Tumors 6–9)

2.7. ACTH-Secreting Adenoma Causing Nelson Syndrome (Tumor 10)

2.8. Tumor Phylogenic Analysis

2.9. Correlation between Gene Variants and Clinicopathological Features

3. Discussion

4. Conclusions

5. Materials and Methods

5.1. Patients and Tumor Tissue Samples

5.2. Construction and Sequencing of Whole Exome Libraries

5.3. Bioinformatics Analysis

5.4. Sanger Sequencing forConfirmation of Exome Findings

5.5. Hormone and Transcription Factor Immunohistochemistry

5.6. Statistical Analysis

Supplementary Materials

Author Contributions

Funding

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Acknowledgments

Conflicts of Interest

References

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Diagnostic pitfalls in Cushing’s disease impacting surgical remission rates; test thresholds and lessons learned in 105 patients

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Cushing’s syndrome in a child

Abstract

1. Introduction

2. Case report

3. Discussion

4. Conclusion

Patient consent

Funding

Authorship

Declaration of competing interest

References

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Temozolomide Effective Against Cushing’s Caused by Aggressive Tumors

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