Role of the Inhibin/Activin System and Luteinizing Hormone in Adrenocortical Tumorigenesis
F. Beuschlein 1 B. D. Looyenga 2 M. Reincke 1 G. D. Hammer 2
Abstract
Adrenal masses are one of the most common endocrine tumors diagnosed. Although most adrenal tumors are inactive adeno- mas, a considerable proportion is associated with hormonal hy- perfunction and/or malignancy. The adrenocortical carcinoma (ACC) is a rare but highly malignant tumor. Most ACCs in adults are diagnosed in an advanced tumor stage limiting therapeutic options. Accordingly, despite some progress in diagnostic and therapeutic approaches, the overall survival rate of patients with ACC remains poor. However, the prerequisite for the devel- opment of new diagnostic tools and therapeutic options in the management of patients with ACC is the elucidation of the molecular pathogenesis of adrenal tumorigenesis. Although our understanding of adrenal tumor biology has increased substan- tially over the last decades, the regulation of many molecular pathways involved in adrenocortical growth and differentiation
awaits further elucidation. Luteinizing hormone (LH) and activin have only recently emerged as hormones likely to play opposite roles in adrenocortical hormone secretion and cellular prolifera- tion. Recent evidence from studies on human surgical tumor sample expression and detailed characterization of murine adre- nal tumor models suggests stimulatory effects of LH on adreno- cortical growth and function. On the contrary, activin, which plays a critical role as a paracrine and autocrine factor regulating cellular growth and differentiation, has been demonstrated to in- duce apoptosis and suppress proliferation in the human and murine adrenal cortex. In this review, we will summarize molec- ular and functional aspects of adrenal tumorigenesis and high- light some prospects for future clinical applications.
Key words
Inhibin . Tumorigenesis . Adrenal gland · Mouse
Tumors of the Adrenal Cortex
The most common adrenal disorder encountered by clinicians today is the incidentally discovered adrenal mass termed “adre- nal incidentaloma.” The rate of detection of clinically silent adre- nal masses has increased dramatically through the widespread use of modern abdominal imaging techniques over the last two decades. As a consequence, the management of adrenal inciden- talomas has become a common clinical problem. In the absence
of a known malignancy of non-adrenal origin, the vast majority of adrenal incidentalomas are benign. Nonfunctioning cortical adenomas are the most common lesions, while hormonally ac- tive tumors of the adrenal cortex are much less common [1]. However, the proportion of malignant adrenocortical carcinoma in adrenal incidentalomas on which surgery was performed is as high as 12% [2].
Affiliation
1 Division of Endocrinology and Metabolism, Department of Internal Medicine II, Klinikum der Albert-Ludwigs-Universität, Freiburg, Germany
2 Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
Correspondence
F. Beuschlein, M. D. . Division of Endocrinology and Metabolism · Department of Internal Medicine II . Klinikum der Albert-Ludwigs-Universität Freiburg · Hugstetter Str. 55 . 79106 Freiburg ·
Phone: +49 (761) 270-7327 · Fax: +49 (761) 270-3372 · E-Mail: beuschlein@medizin.ukl.uni-freiburg.de
Received 18 December 2003 . Accepted without revision 27 February 2004
Bibliography
Horm Metab Res 2004; 36: 392-396 @ Georg Thieme Verlag KG Stuttgart . New York . DOI 10.1055/s-2004-814584 . ISSN 0018-5043
Adrenocortical carcinomas (ACCs) form a rare but highly malig- nant endocrine tumor entity with a worldwide incidence of ap- proximately two new cases per million persons per year [3]. Based on clinical and chromosomal characteristics, adenomas and carcinomas of the adrenal cortex are thought to be distinct entities [4]. However, discerning malignancy in small adrenal masses can be challenging, even on a histological basis. Unfortu- nately, existing therapies for ACC patients are generally ineffec- tive. Retrospective studies on combined surgical and medical therapy indicate an overall 5-year survival of 15-35% [5]. The therapeutic results are largely dependent on tumor stage; the most severe prognostic factor is the presence of metastases. Ac- cording to a recent meta-analysis, 62% of ACCs are hormonally active, which may eventually lead to early diagnosis [6]. In the remaining cases, ACC tumors are usually discovered incidentally at an advanced stage by radiological investigations or alterna- tively in the setting of the symptoms of bulky disease [7]. Ac- cordingly, a large proportion of adult patients have distant me- tastasis at the time of presentation [8]. In children, adrenocorti- cal neoplasms can occur in the context of several clinical syn- dromes such as Li-Fraumeni syndrome [9] or Beckwith-Wiede- mann syndrome [10]. However, most ACCs in children and al- most all adult cases occur sporadically with no defined underly- ing genetic predisposition. Besides epidemiological evidence suggesting an increased risk of ACC in association with cigarette smoking in men and oral contraceptives in women, no other risk factors have been defined [11].
Surgery is the treatment of choice for patients with resectable primary and secondary tumors or recurrent disease [12]. Because most ACCs in adults are commonly diagnosed in advanced stages, however, effective medical treatment has been sought. Mitotane (o,p’-DDD) has potent adrenolytic effects and may re- tard the growth of individual ACCs [13]. Although on a functional level, treatment with mitotane can induce hypercortisolism re- mission in the majority of patients, objective tumor response re- mains uncertain. Several cytotoxic agents have been used as monotherapy or in combination to treat advanced disease, yet, the response rates have generally been low [14,15]. In light of this unsatisfactory clinical situation facing the management of ACC patients, the search for better medical treatment protocols is a continuing challenge. Development of effective ACC thera- pies, however, is dependent on the detailed knowledge of the causative molecular events leading to adrenocortical tumorigen- esis.
Inhibin and Activin in Adrenal Physiology and Tumorigenesis
Inhibin and activin, members of the TGF family of ligands, are dimeric glycoproteins formed by two of three different subunits. While inhibin is mainly expressed in the gonads, adrenal cortex and pituitary, activin has a more widespread tissue distribution. Initially identified as opposing regulators of pituitary FSH secre- tion [16], both inhibin and activin have since been shown to play critical roles as paracrine and autocrine factors that regulate growth and differentiation in a number of organs including the gonads and the adrenal gland [17]. In humans, inhibin is specifi- cally localized to the fetal zone of the developing adrenal cortex, raising speculation that the growth dynamics of the fetal zone
are regulated, at least in part, by this hormone [18]. As for adre- nal tumors, marked immunohistochemical staining for inhibin in archival surgical material was demonstrated only in virilizing tu- mors presumed to have arisen from the inner reticular zone con- trasted to the weak or no staining in tumors of the fasciculata and glomerulosa origin [19].
Binding of radio-labeled activin and inhibin to the adrenal cortex [20] and expression studies on members of the activin/inhibin signaling pathway [21] have demonstrated the presence of acti- vin receptors and inhibin binding proteins in the adult and fetal adrenal and cultured adrenocortical cells. Thus, cells of the adre- nal cortex not only serve as a source of inhibin and activin, but are also a potential target of activin and inhibin action. Interest- ingly, ACTH regulates adrenal inhibin and activin expression, suggesting that inhibin and activin might modulate the effects of ACTH in a paracrine or autocrine fashion [21]. In addition, an increasing body of evidence suggests a significant impact of acti- vin on the regulation of adrenal growth and function. Specifical- ly, activin has been shown to inhibit proliferation, induce apop- tosis and modulate ACTH-induced cortisol secretion in the adre- nal fetal zone [17,22,23]. Furthermore, in vitro activin treatment inhibits steroidogenesis in adrenocortical tumor cells in a time and dose-dependent manner [21].
The activin signaling pathway requires the action of two integral membrane receptor serine/threonine kinases as well as the in- tracellular Smad-family proteins in order to transduce a signal from membrane to nucleus [24]. Two different cell-membrane activin receptors have been identified. Both type I and type II re- ceptors further sub-classified into IA, IB, IIA and IIB are required on the cell surface to mediate activin signaling. Recently, inhibin- binding protein (InhBP) and betaglycan have been identified as two candidates that bind inhibin with high affinity and could re- semble the inhibin receptor [25,26]. InhBP can dissociate activin receptor complexes in the presence of inhibin and antagonize the action of activin by rendering the receptor complex physically unable to associate. Similarly, by binding betaglycan and ActRIA, inhibin blocks activin binding to the type II receptor and subse- quent recruitment of the signaling type I receptor [25]. However, in addition to this activin counteracting function of inhibin, acti- vin-independent signaling pathways might be in place to trans- duce specific action of inhibin in target cells [27].
Luteinizing Hormone and Adrenal Physiology
An increasing body of evidence suggests that hormone produc- tion by a substantial number of adrenal tumors and hyperplasias is controlled by aberrant hormone receptors. Although the regu- lation of cortisol secretion by LH in adrenal Cushing’s syndrome was only recently demonstrated in vivo [28,29], there have been several reports indicating that the regulation of steroidogenesis in some pure androgen-secreting tumors is stimulated by hCG or GnRH [30-35]. The LH/hCG receptor is one of the largest sev- en-transmembrane receptors with an unusually long extracellu- lar ligand-binding domain. The four copies of hLH receptor genes are localized on chromosome 2p16-21 loci. Interestingly, expres- sion of the LH/hCG receptor has been reported in the human adrenal zona reticularis [36], and hCG can stimulate DHEAS se-
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cretion in human fetal adrenal cells [37]. Taken together, these findings indicate that LH can induce steroidogenesis in the adre- nal cortex and in a subset of (LH receptor-expressing) adrenal tu- mors and hyperplastic tissues. However, whether LH also exerts growth promoting effects on adrenocortical cells awaits further elucidation. In general, it has been hypothesized that ectopic ex- pression of any G-protein coupled receptor could induce the stimulation of adrenal cells by trophic factors lacking regulatory negative feedback by cortisol. This stimulus may lead to in- creased function and confer a proliferative advantage [38]. Thus, hormone stimulated LH receptor expression could act as an adre- nocortical tumor promoter when ectopically expressed in the adrenal cortex. However, whether LH receptor expression is suf- ficient to induce adrenocortical tumorigenesis in the absence of other oncogenic events is uncertain.
Inhibin Knock-out Mouse
Studies on the inhibin null (INH-/-) mouse, which develops pri- mary gonadal tumors spontaneously and adrenocortical carcino- mas upon gonadectomy [39], demonstrate that inhibin acts as a tumor suppressor gene in both the gonad and the adrenal cortex (Fig. 1). Intriguingly, both gonadal and adrenal tumors secrete high levels of activin, indicating a balance between inhibin and activin that is disrupted by the targeted deletion of the inhibin alpha subunit. As we have demonstrated in detailed morphologi- cal and in vitro studies, the adrenal phenotype in INH-/- mice is indicative of a x-zone growth dysregulation. The x-zone of the murine adrenal cortex is thought to be the functional equivalent of the primate fetal adrenal zone, and thus the INH-/- mouse may
be considered a model of childhood adrenal cancer. Develop- ment of activin-secreting ovarian tumors in female INH-/- mice is accompanied by a decrease in adrenal weight and regression of the x-zone. The ultimate cause of this regression is the distinct x-zonal expression pattern of activin receptor subunits and the intracellular mediator Smad2, which results in a particular re- sponsiveness of the x-zone to activin. As a result, activin induces apoptosis specifically in the adrenal x-zone, thus preventing adrenal tumorigenesis in the presence of activin secreting gonadal tumors. Conversely, the removal of activin-secreting ovarian tumors by surgical gonadectomy in INH-/- is followed by unopposed x-zone growth, which ultimately results in the formation of adrenal tumors [40].
Intriguingly, cells derived from murine INH-/- adrenal tumors are unresponsive to in vitro activin treatment, which explains growth of adrenal tumors in INH-/- mice despite the fact that the adrenal tumor itself secretes high levels of activin. Cancer cells can acquire resistance to the anti-proliferative effect of TGFß and activin by a number of different mechanisms, including defects in the specific cell surface receptors and mutational inac- tivation of shared downstream effector components of the sig- naling pathway [41]. For instance, loss of expression or inactivat- ing mutations of Smad2 have been reported in a variety of differ- ent tumors including head and neck squamous cell carcinoma [42], colorectal carcinoma [43], lung carcinoma [44], and serous ovarian carcinoma [45]. In accordance with their unresponsive- ness to activin, Smad2 levels are greatly diminished in adrenal tumors of INH-/- mice, suggesting a possible escape mechanism from activin dependent growth inhibition. It is intriguing to speculate that loss of function of the activin pathway could also
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play a role in development and/or growth maintenance of References sporadic human adrenocortical carcinoma.
The mechanisms leading to adrenal tumorigenesis in INH-/- mice have been suggested to involve the lack of a gonadal factor and/or compensatory increase of gonadotropins. In order to achieve elevation of gonadotropins without the concomitant loss of a gonadal hormone, we crossed INH-/- mice with a trans- genic mouse strain that has chronically elevated LH levels [46]. Interestingly, compound mice (INH-/ -: LH+) developed larger ac- tivin-secreting ovarian tumors and died within a short period of time from activin-induced tumor cachexia, suggesting that LH acts as a growth factor for ovarian tumors (Fig. 1). However, when gonadectomized, adrenal tumors in compound INH-/ -: LH+ animals grew larger adrenal tumors then gonadectomized INH-/- mice, indicating additional growth-promoting effects of LH for adrenal tumorigenesis. As predicted for a direct effect of LH on adrenal tumor growth, LH receptor expression could be demon- strated in adrenal tumor samples from inhibin deficient mice. Taken together, these findings suggest a tumor inhibiting effect of activin (by induction of apoptosis in the pre-cancerous cells), while LH might have direct growth-promoting effects on adrenal tumorigenesis in this animal tumor model.
Clinical Impact and Future Prospects
The identification of factors and pathways involved in adreno- cortical tumour growth is the prerequisite for the development of novel therapeutic options that result in the control of hormo- nal hyperfunction and/or tumour growth. Although the last dec- ade has brought considerable progress in the understanding of cellular and molecular biology of adrenal growth and differentia- tion, many questions remain that need to be addressed in future studies. While the mouse inhibin knockout model suggests that loss of inhibin is sufficient for adrenal tumor formation, expres- sion studies in human surgical samples suggest that inhibin de- ficiency is not necessary to induce tumor growth. Thus, other in- dependent events result in tumor formation and progression. Possible candidates for oncogenic events in adrenal tumors in- clude loss of function mutations in the as yet ill-defined inhi- bin-dependent pathway, thus inactivating the proposed tumor suppressor gene function of inhibin. Moreover, adrenal tumors might escape growth inhibiting effects of activin (which itself is expressed in a wide variety of tissues) by disruption of activin dependent pathways through down-regulation or mutations in activin receptors or Smads. Lastly, ectopic expression of recep- tors such as the LH receptor might modulate growth kinetics and steroidogenic profile of adrenal tumors. From a clinical standpoint, the identification of the presence of an abnormal adrenal receptor might offer the possibility of new pharmacolog- ical approaches to control endocrine hyperfunction and/or growth by suppressing the endogenous ligands or by using specific antagonists for the abnormal receptors.
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