Recent advances in understanding corticotroph pituitary tumor initiation and progression

Introduction

Corticotroph adenomas (CAs) derive from normal corticotroph cells of the anterior pituitary and account for about 4% to 8% of all clinically hormone-active anterior pituitary tumor types¹. Including clinically inactive silent corticotroph tumors, CAs encompass up to 15% of all pituitary tumors². The prevalence for hormone-active CAs is about 40 cases per million and the incidence ranges from 1.2 to 2.4 per million per year. In adults, CAs are diagnosed mainly in the fourth to sixth decade of life and are about three times more prevalent in women¹. No gender preference has been observed in CAs in children, in whom these tumors are rare (<10% of all CAs)³.

CAs have rarely been described with hereditary background, and in the vast majority of sporadic CAs, the genetic background is still largely unknown⁴. Whereas silent CAs are immunopositive for adrenocorticotropic hormone (ACTH), but do not manifest biochemical or clinical hypercortisolism⁵, the majority of clinically active CAs secrete excessive amounts of ACTH, causing chronic hypercortisolism due to lack of hypothalamus–pituitary–adrenal (HPA) axis feedback regulation at the level of the CA, leading to manifestations of the clinical features of Cushing’s disease¹. Normally, there is a delay of several years between the onset of the disease and its diagnosis because early symptoms of Cushing’s disease are rather unspecific. At the time of diagnosis, about 90% of the patients present with corticotroph microadenomas less than 1 cm in diameter; in some cases, these tumors are hardly detectable with magnetic resonance imaging¹. Transsphenoidal surgery is generally the initial treatment of choice but is not always successful. In the case of incomplete resection (for example, if the tumor is critically located in proximity to the optical nerves), the tumor might expand and become more aggressive over time, requiring repeated surgery, radiotherapy, pharmaceutical therapy, or adrenalectomy¹. In rare cases, CAs finally transform to metastasizing corticotroph carcinomas for which temozolomide treatment has recently been established with some success in single cases^6,7.

Genetics of hereditary corticotroph adenomas

Less than 5% of all CAs are familial adenomas representing rare tumor manifestations in various hereditary endocrine syndromes—multiple endocrine neoplasia 1 (MEN1), MEN4, Carney complex (CNC), and DICER1 syndrome—or in familial isolated pituitary adenomas (FIPAs)^4,8. Patients with MEN1, which is caused by a mutation of the MEN1 gene encoding the tumor suppressor menin, mainly manifest prolactinomas and somatotropinomas and only about 5% of their totality is represented by corticotroph tumors^8,9. So far, a single case of CA has been described in MEN4, in which the cell cycle regulator p27^Kip1 is impaired by inactivating mutations of the CDKN1B gene¹⁰. In CNC, caused by inactivating germline mutations of the PRKAR1A gene which encodes the type 1α regulatory subunit of protein kinase A, one case of CA has been reported¹¹. In DICER1 syndrome, which is associated with the development of different types of blastomas during embryogenesis, 2% of the affected patients develop corticotroph blastomas as the only pituitary blastoma manifestation^12,13. Patients with DICER1 syndrome bear an autosomal dominant mutation of the DICER1 gene which encodes the DICER protein, a microRNA (miR) processing endoribonuclease. This suggests that a disturbed production of miRs and thus an altered regulation of their target mRNAs may be responsible for the development of the corticotroph blastomas. However, it is not yet known which miRs are affected and how they induce corticotroph tumor development or why only a small subset of affected patients develops corticotroph blastomas^12,13.

Hereditary disorders, in which the patients manifest only pituitary tumors, are designated FIPAs. About 20% of all patients with FIPA bear mutations of the aryl hydrocarbon receptor-interacting protein (AIP), whereas in the majority of FIPAs, the underlying mutations are not known. CAs account for about 5% of all pituitary tumors in FIPAs¹⁴.

In several studies with big cohorts of patients with sporadic pituitary adenoma, only three cases of pathogenetic AIP mutations and three cases of menin mutations have been found in sporadic corticotropinomas, suggesting that mutations of genes causing hereditary corticotroph tumors play only a minor role in sporadic CA development^4,15,16.

Genetic and epigenetic modifications in sporadic corticotroph adenomas

Most of the CAs (>95%) are sporadic tumors of monoclonal origin^17,18. Loss of heterozygosity in combination with different mutations leading to loss of tumor suppressor genes or activation of oncogenes (or both) may play a role in the initiation of sporadic CAs¹⁹.

Despite considerable progress during the last decade, sufficient knowledge about the genetic background of CAs is still missing. Important progress has been made with the recent detection of recurrent gain-of-function mutations in the gene encoding a protein called ubiquitin-specific protease 8 (USP8) in about 20% to 60% of CAs in adult patients^20–22. As the mutation was not found in any other type of pituitary tumors, it is evident that the mutation is specific for CAs²³. USP8 is involved in the ubiquitination/deubiquitination process, which is a modality of post-translational protein modification^24,25. In this complex process, specific proteins are ubiquitinated and then degraded by the lysosome or are rescued from degradation by deubiquitination. USP8 is an enzyme that mediates the deubiquitination of target proteins, and described mutations in CAs lead to an increased activity of the protein. CAs with mutated USP8 exhibit more stable or higher levels of proteins whose activities are regulated by ubiquitination/deubiquitination^20,21.

Multiple proteins are known to be regulated at least in part by ubiquitination/deubiquitination, and in principal all USP8 regulated proteins, if expressed in CAs, could be affected by USP8 mutations^24,25. In this context, two candidate proteins that might play a pathogenic role and be a pharmacological target in CAs with mutated USP8 have already been identified: the epidermal growth factor receptor (EGFR)^20,21 and the somatostatin receptor type 5 (SSTR5)²⁶. The tyrosine kinase receptor EGFR is expressed in corticotroph cells and mediates the ACTH-stimulating effect of the epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-α)^27–29. In CAs with USP8 mutations and thus enhanced deubiquitination, EGFR is stabilized and its expression is increased. Therefore, the ACTH-stimulating activity of EGF/TGF-α is elevated and may contribute to the excessive ACTH production. Indeed, comparative studies between CAs with and without mutated USP8 have already shown that the expression and activity of the EGFR are enhanced in CAs with USP8 mutations,^20,21 although this could not be confirmed in another study²⁶. In the case of SSTR5, USP8 mutations would lead to an increased expression and activity of this receptor and suggest that CAs with mutated USP8 may respond better to the treatment with pasireotide, a somatostatin analogue that suppresses CA growth and ACTH secretion through SSTR5^26,30. In addition, a Cushing’s disease–relevant protein, whose expression or function or both are regulated at least in part by ubiquitination/deubiquitination (and thus may be affected by USP8 mutations), is the glucocorticoid receptor (GR)³¹. The latter regulates the responsiveness to glucocorticoids, resistance to which is a typical feature of Cushing’s disease. Whether a reduced sensitivity to glucocorticoids by CAs is related to the mutational status of USP8 still needs to be clarified. Interestingly, a recent article has shown evidence that in normal rat corticotroph cells the proopiomelanocortin (POMC) peptide is directly degraded by ubiquitination and therefore that the treatment of rat corticotropes with the polyubiquitination inhibitor K48R enhanced ACTH production³². Whether this is also relevant in human corticotroph tumor cells has yet to be proven. Comparative investigations have demonstrated that CAs with mutated USP8 are smaller and that the prevalence for USP8 mutations is higher in women^20,21,33. In a recent study, a lower incidence of USP8 mutations was found in children with Cushing’s disease compared with adults^33,34. Whereas the prevalence of USP8 mutations was reduced in Crooke’s cell adenomas²⁶, the proportion of mutant USP8 in so-called Nelson’s tumors (see the “Corticotroph pituitary tumor types” section) was identical to that in corticotropinomas in general³⁵. Patients with USP8 mutations were diagnosed significantly earlier and had higher preoperative 24-hour urinary-free cortisol levels³⁶. After surgery, recurrences were more abundant and appeared significantly earlier in patients with USP8 mutant corticotropinomas³⁶. In contrast to ACTH-secreting CAs, no USP8 mutations have been detected in a small number (n = 13) of silent CAs studied³³ but this has to be confirmed in a bigger cohort of this tumor type. Interestingly, no mutant USP8 has been found in tumors from a big cohort of dogs with Cushing’s disease, suggesting that the mutation is primate-specific³⁷.

So far, apart from mutated USP8, no other recurrent mutations have been found in CAs^4,23,38. Thus, more than half of the CA cases may have a variable genetic background and may be associated with mutations of different single genes or may be caused by epigenetic mechanisms (see below). So far, several mutations of different genes have been described in CAs, which mainly affected cell cycle–regulating proteins, signal transduction proteins, oncogenes, and tumor suppressors^4,23,38,39. Very recently, loss-of-function mutations of the Cdk5 and ABL enzyme substrate 1 (CABLES1) gene were found in four patients from a cohort of 146 pediatric and 35 adult patients with Cushing’s disease: two in children and two in young adults⁴⁰. The mutated CABLES1 protein lost its inhibitory action on corticotropinoma cell growth, which may explain why all affected patients had corticotroph macroadenomas⁴⁰.

Given that mutations play a tumorigenic role in only a part of the corticotropinomas, it is thought that epigenetic changes such as DNA methylation, histone methylation/acetylation, and miR are of considerable impact for corticotropinoma formation^41,42. Most studies on this subject have been performed in unselected cohorts of different types of pituitary adenomas; among them, corticotropinomas and epigenetic changes of different factors have been detected in subsets of the adenomas. Corticotropinoma-specific studies on histone deacetylases have demonstrated that a loss of HDAC2 might play a role in inducing glucocorticoid resistance⁴³, and treatment with histone deacetylase inhibitors reduced survival and ACTH secretion in corticotroph tumor cells⁴⁴. Many of the epigenetic changes caused by DNA methylation or histone methylation/acetylation have direct or indirect effects on the expression of the tumor suppressors p53 and retinoblastoma (Rb) protein, suggesting that these factors are prominent targets of epigenetic changes^41,42. A case report of loss of Rb expression in a corticotroph carcinoma⁴⁵ supports the concept that this tumor suppressor plays a role in corticotropinoma development but apparently it is involved in tumor progression rather than in adenoma initiation.

In regard to miRs, which bind to specific messenger RNAs and block their translation into corresponding proteins, several entities have been reported to be either over- or under-expressed in pituitary adenomas in general^46–48 or specifically in CAs^49–51. For instance, invasive characteristics of pituitary tumors were found to be associated with the over-expression of the miR-106b~25 cluster consisting of miR-25, miR-93, and miR-106b⁵⁰. Moreover, aggressive corticotroph Crooke’s cell adenomas had a higher expression of miR-106b~25 than other corticotropinomas, suggesting that this miR cluster is also associated with corticotroph tumor aggressiveness⁵⁰. In another study about miR expression specifically in corticotropinomas, patients presenting reduced miR-141 levels showed a better chance of remission, whereas the reduced expression of other miRs (miR-15a, miR-16, miR-21, miR-141, miR-143, miR-145, miR-150, and let-7a) had no association with corticotropinoma phenotype or clinical parameters of the affected patients⁴⁹. The targets and pathological consequences of miRs in CAs have been identified in only a few cases^48,51, and much more work has to be done to identify the pathological mechanisms of aberrant miR expression.

Glucocorticoid resistance in corticotroph adenomas

In normal corticotroph cells, ACTH production and release are under negative feedback control of glucocorticoids and are well balanced. If needed, the ACTH and thus glucocorticoid production can be transiently adapted to specific physiological or pathophysiological conditions (stress, infections, inflammation, and so on)⁵². Corticotroph tumor cells produce excessive amounts of ACTH despite strongly elevated glucocorticoid serum levels, indicating that the negative glucocorticoid feedback is impaired. It is still not clear why corticotroph tumor cells are resistant to glucocorticoids. Inactivating mutations of the GR encoding nuclear receptor subfamily 3 group C member 1 (NR3C1) gene are rare, and as corticotroph tumor cells show mostly enhanced GR levels, the missing response to glucocorticoids cannot be explained by GR downregulation^23,39,53,54. Thus, an alternate impairment of the function of GR must be responsible for the glucocorticoid resistance, and two candidates—the testicular orphan nuclear receptor 4 (TR4) and heat shock protein 90 (HSP90)—are discussed to play important roles in GR dysfunction.

TR4 is a nuclear receptor which can both activate and repress transcription in different types of organs. TR4 is overexpressed in CAs and corticotroph tumor cell lines and is able to activate the ACTH encoding POMC gene by binding to its promoter^55,56. The activation is further enhanced if TR4 is phosphorylated through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. In nude mice harboring corticotroph tumors, TR4 overexpression stimulated ACTH secretion and tumor cell growth⁵⁵. It was shown that TR4 interacts with the GR and could overcome the negative regulation of GR on POMC transcription and ACTH secretion, suggesting that TR4 might promote resistance to negative glucocorticoid feedback^55,56. Suppression of ERK-mediated TR4 phosphorylation by the ERK inhibitor MEK-162 in a murine model of corticotroph tumors reduced ACTH production and inhibited tumor growth, indicating that targeting TR4 action or suppression (or both) could be a novel therapy for Cushing’s disease⁵⁷.

HSP90 is a chaperone protein that stabilizes and regulates different proteins by inducing conformational changes. HSP90 interferes with the GR to facilitate ligand binding and receptor translocation to the nucleus and therefore plays an important role in the function of the GR⁵⁸. However, under certain conditions, HSP90 can impair GR function^59,60. HSP90 expression is strongly enhanced in the anterior pituitary in patients with CA in comparison with the normal anterior pituitary, and it was shown that HSP90 overexpression restrains the release of mature GR from the chaperone, leading to partial glucocorticoid resistance⁶¹. C-terminal inhibitors of HSP90 such as novobiocin and the herbal compound silibinin could induce the release of mature GR in corticotroph tumor cells in vitro, and in vivo silibinin was able to revert glucocorticoid resistance in a corticotroph tumor allograft mouse model, leading to a partial restoration of symptoms of the disease⁶¹. This suggests that silibinin, a safe drug already used in humans for the treatment of liver disease and amanitin intoxication⁶², may represent a novel therapeutic option for the treatment of Cushing’s disease and corresponding clinical studies are currently in preparation.

Progression of corticotroph adenomas

Conclusions

CAs are mostly sporadic tumors; only a small proportion of CAs have a hereditary background and are associated with neuroendocrine syndromes or are FIPAs. In sporadic CAs, a recurrent activating mutation of the USP8 gene, which is present in about 40% of ACTH-secreting CAs, has recently been found. The USP8 mutation participates in CA pathophysiology by stimulating EGFR expression and thus enhances EGF-induced ACTH production. As mutated USP8 also increases SSTR5 expression, CAs with USP8 mutation respond better to treatment with the somatostatin analogue pasireotide which acts through SSTR5. No other recurrent mutations have been identified in CAs, suggesting that single different mutations or epigenetic changes (or both) may play a role in CA initiation. The ACTH secretion by corticotroph tumor cells, in contrast to normal corticotroph cells, is resistant to inhibitory glucocorticoid feedback control, and HSP90 overexpression has recently been shown to be involved in Cushing’s disease. Thus, HSP90 inhibitors like silibinin may be promising drugs to restore glucocorticoid responsiveness of CAs and to reverse the pathophysiological changes of Cushing’s disease. During the progression of CAs from microadenomas to aggressive macroadenomas and finally rare cases of corticotroph carcinomas, multiple factors (genes, miRs, proteins, and peptides) are up- or down-regulated and thus may provide putative targets for new pharmacological treatment strategies. However, despite remarkable progress in understanding corticotroph tumor initiation and progression, a lot of work is still needed to explore the underlying mechanisms, and the translation of new findings into clinical applications for diagnosis and therapy of corticotropinomas is still unsatisfactory.

Abbreviations

ACTH, adrenocorticotropic hormone; AIP, aryl hydrocarbon receptor-interacting protein; CA, corticotroph adenoma; CABLES1, Cdk5 and ABL enzyme substrate 1; CNC, Carney complex; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FIPA, familial isolated pituitary adenomas; GR, glucocorticoid receptor; HIF-1, hypoxia-inducible factor 1; HSP90, heat shock protein 90; MAPK, mitogen-activated protein kinase; MEN, multiple endocrine neoplasia; MGMT, O⁶-methylguanine DNA methyltransferase; miR, microRNA; MMP, matrix metalloproteinase; NFPA, non-functioning pituitary adenoma; POMC, proopiomelanocortin; Rb, retinoblastoma; RSUME, RWD-domain-containing sumoylation enhancer; SSTR5, somatostatin receptor type 5; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TR4, testicular orphan nuclear receptor 4; USP8, ubiquitin-specific protease 8; VEGF-A, vascular endothelial growth factor-A; WHO, World Health Organization.