The Role of the ACTH Receptor in Adrenal Tumors: Identification of a Novel Microsatellite Marker
O. Zwermann 1 F. Beuschlein 1 A. Klink 1 M. Stahl1 M. Reincke2
Abstract
In vitro, the growth inhibiting effect of ACTH on adrenocortical cells is well documented, even though there are reports of oppo- site effects under defined cell culture conditions. In vivo, activa- tion of the ACTH receptor (ACTHR) has a trophic effect on the adrenal cortex, while the effects on proliferation are still under discussion, especially since other POMC derived peptides have been characterized. However, ACTH is thought to act as a differ- entiation factor with inhibiting effects on tumor growth. In un- differentiated adrenocortical carcinomas, ACTHR expression is frequently lost, which is associated with extensive tumor growth. We describe a new microsatellite marker within the in- tron of the ACTHR gene termed ACTHRint1. In a series of 114 pa- tients with various adrenal and non-adrenal tumors, the rate of
heterozygosity was 100%. Only one out of 57 patients with adre- nocortical adenoma showed LOH at the ACTHR locus, whereas 4 of 10 carcinomas had loss of one allele. Patients suffering from tumors with LOH showed a more aggressive disease course and had earlier recurrences with poor prognosis. These data confirm earlier findings that adrenocortical carcinomas frequently show loss of ACTHR expression, which is associated with a more ag- gressive tumor growth. However, whether the ACTHR is directly involved in tumor growth or acts a marker of differentiation that is lost in more advanced tumor stages is still not clear.
Key words
ACTH . Corticotropin . ACTH receptor . Corticotropin receptor . MC2R . Adrenal tumor . Adrenal carcinoma . ACTHRint1 . LOH
Introduction
The ACTH receptor (ACTHR) is a G-protein coupled, seven-trans- membrane receptor that transmits the ACTH signal to induce glucocorticoid and androgen production in adrenocortical cells. Ligand binding is followed by Gs protein and Protein kinase A (PKA) activation and increased intracellular cAMP levels [1]. Be- sides activation of the PKA pathway, activation of the MAPK pathway [2], phospholipase C and calcium channel opening have been described [3]. Interestingly, activation of the ACTHR upregulates its own transcription, forming an unusual positive feedback loop [4] that might increase adrenal responsiveness to ACTH in the context of physiological stress. Inactivating muta- tions of the ACTHR are the cause of isolated familial glucocorti- coid deficiency, which is characterized by ACTH insensitivity
and adrenal hypoplasia [5]. Furthermore, hypophysectomy re- sults in decreased adrenal volume [6], whereas treatment with ACTH leads to hypertrophy of the adrenal cortex [7]. Therefore, effects of the ACTHR on adrenal cell proliferation and tumor growth were suspected in addition to its role in steroid biosyn- thesis regulation. However, in spite of numerous studies being performed in vitro and in vivo, the effects of ACTH and activation of the ACTHR on cell proliferation are still subject to discussion.
Effects of ACTHR Activation on Cell Growth In Vitro
Initial experiments concerning the effects of ACTH on cell growth in vitro were performed more than 30 years ago. Masui et al. first reported the growth-inhibiting effect of ACTH on mouse adreno-
Affiliation
1 Division of Endocrinology, Department Internal Medicine 2, Clinic of the Albert-Ludwigs-University Freiburg, Freiburg, Germany
2 Medizinische Klinik - Innenstadt, Ziemssenstr. 1, 80336 München, Germany
Correspondence
Prof. M. Reincke · Medizinische Klinik - Innenstadt · Ziemssenstr. 1 . 80336 München . Germany .
Phone: + 49 (89) 51 60 21 00 · Fax: + 49 (89) 51 60 44 28 · E-Mail: martin.reincke@med.uni-muenchen.de
Received 9 January 2004 . Accepted after revision 3 March 2004
Bibliography
Horm Metab Res 2004; 36: 406-410 @ Georg Thieme Verlag KG Stuttgart . New York .
DOI 10.1055/s-2004-814566 . ISSN 0018-5043
cortical Y1 cells [8]. Weidman et al. and Morera et al. confirmed these results in Y1 cells, the former group showing a growth ar- rest in G1 phase [9,10]. Ramachandran et al. demonstrated DNA synthesis inhibition by thymidine incorporation assay accompa- nied by an increased corticosterone production in primary rat adrenocortical cell cultures [11]. The same effects could be shown in primary bovine and human adrenal cell cultures [12-14]. However, there were also contradictory results - Armato et al. ob- served an increase in DNA synthesis in human adrenocortical pri- mary cultures [15] and an increased shift of these cells into the S and M phases of the cell cycle, resulting in enhanced proliferation [16]. Because all experiments at that time had been performed in the presence of fetal calf serum, Menapace et al. investigated the effects of ACTH 1-24 on rabbit adrenocortical primary cells in a synthetic medium and found a stimulatory effect on DNA syn- thesis and cell proliferation [17]. In this study, the effects were not transduced by cAMP alone, and a role of phospholipase C in ACTHR signaling on cell growth was proposed. In 1993, Arola et al. suggested that ACTH might have a biphasic effect on primary rat adrenocortical cell growth [18]. They observed an initial de- cline in proliferation after 24 h with a 2.5-fold increase in prolif- eration after 3 days. Lotfi et al. proposed an inverse biphasic ef- fect on Y1 cells using synthetic ACTH 1-39 and native porcine ACTH [2]. They demonstrated a weak stimulatory effect on DNA synthesis within 2 h of stimulation after serum starvation for two days. Following the two-hour interval, growth inhibition was observed for up to 24 h. An overview of the publications cited is given in Table 1. One reason for the contradictory results in these studies might be the ACTH preparation used, which dif- fered from native porcine extracts to synthetic ACTH 1-24 and ACTH 1-39. On the other hand, for example, ACTH 1-24 showed stimulatory [17,19] and inhibiting [10] effects as did other pre- parations. Therefore, the ACTH preparation does not explain the discrepancies. The earlier reports showing an inhibitory effect used high amounts of fetal calf serum. Menapace attributed their finding of a stimulatory effect to the absence of FCS, but again there are reports of growth stimulatory effects with serum [18] and reports of growth inhibition with serum concentrations as low as 0.2% FCS [12]. In addition, timing and concentration of ACTH seems crucial for the results as seems to be the observation period. In conclusion, although a growth inhibitory effect of ACTH on adrenocortical cell growth is widely accepted and repro- duced frequently until today, one has to remember that these ef- fects seem to be closely related to certain experimental condi- tions with limited predictive value on physiological conditions.
Effects of the ACTHR on Adrenal Growth In Vivo
ACTH is necessary for normal development and maintenance in the adrenal cortex. Preliminary experiments on hypophysecto- mized rats suggested a growth-promoting effect [6], while ACTH treatment in guinea pigs induced DNA synthesis and thy- midine kinase in the adrenal cortex [20,21]. These changes might be restricted to the zona glomerulosa; Payet et al. injected vary- ing ACTH preparations into rats and found a 16-fold increase in mitosis rate in the zona glomerulosa, while there was no effect on proliferation in the zona fasciculata, which developed a marked hypertrophy [22]. The size of the whole gland was in- creased, which was mostly due to hypertrophy of the fasciculata
and hyperemia. ACTH 1-24 slow-release formula (Synacthen De- pot®) was the most potent ACTH preparation in this study.
Compensatory adrenal growth following unilateral adrenalec- tomy is an established model for adrenal growth studies. In early studies, ACTH was presumed to be the factor leading to hypertro- phy and hyperplasia following unilateral adrenalectomy. How- ever, Weidman et al. used an antiserum raised against ACTH to block ACTH induced hypertrophy of the remaining adrenal. As expected, steroidogenic capacity was impaired, but the antise- rum had no influence on DNA content or cell number. They con- cluded that ACTH is responsible for steroidogenesis induction, but they proposed a different factor as responsible for the in- creased proliferation of adrenal cells after unilateral adrenalec- tomy [9]. Using the same model, Dallman et al. showed that ACTH inhibited hyperplasia and hypertrophy of the remaining gland [23]. In spite of ACTH, they proposed neural regulators with involvement of the ventromedial hypothalamus as the most important factors in compensatory adrenal growth [24]. At the same time, experiments on N-terminal POMC peptides showed a mitogenic activity on the adrenal cortex [25-27]. Cloning a serine protease expressed in the rat adrenal cortex (adrenal specific protease, AsP) that cleaves N-terminal POMC shed new light on these findings [28]. It was presumed that ACTH is not the mitogen for the adrenal gland, but N-terminal POMC peptides that are produced by the pituitary in equimolar amounts to ACTH. The growth promoting effects of synthetic POMC 1-28 with correct disulfide bridges in vitro has been shown recently by Fassnacht et al. [29]. However, ACTH alone (in pharmacological doses) was able to induce adrenal weight gain and normalization of adrenocortical structure in POMC-/- knock out mice showing adrenal atrophy [30]. In conclusion, ACTH in physiological doses does not seem to act as a mitogen on the adrenal cortex, although this issue is still an area of active research.
LOH of the ACTHR Locus in Adrenocortical Tumors
Since the role of the ACTHR on adrenocortical cell growth was not clear, the question was raised whether the ACTHR might be involved in adrenocortical tumor growth. For this reason, we identified a PstI polymorphism 3 kb upstream the ACTHR coding region. One out of sixteen adrenocortical adenomas and two out of four carcinomas showed LOH at this locus. Investigation of flanking markers confirmed the loss of one allele of the ACTHR coding sequence. Northern blot analysis confirmed decreased ACTHR mRNA expression in the tumors affected [31].
Since only 53% of patients were heterozygous and also informa- tive for the described polymorphism, we aimed to screen for a microsatellite marker at the ACTHR locus. For this reason, 6 cos- mids from the RZPD human cosmid library (library no. 111) were isolated using an ACTHR coding region PCR product as a probe. Primer pairs for ACTHR coding region were 5’GCT GTG TTC AAG AAT AAG AAT C3’; and 5’GAT CTT CCT GGT GTG GGA TC3’. Hybridization was performed in Quik-Hyb solution (Stratagene, Heidelberg, Germany) at 50℃ for 2h. One of the cosmids, ACTHRcos1, was digested with BamHI, Dral, MspI, Sacl and Xbal and hybridized to a 30-bp (CA)n oligonucleotide. The (CA)n oligo-
D18S1086
ACTH-R promoter
ACTH-R ORF
D18S1073
18p tel
cen
12 kb
9.5 kb
0.5 kb
3.1 kb
0.9 kb
SalI
BamHI SalI PstI PstI
BamHI
PstI
location of ACTHRint1
ATG
TAG
exon 1
intron (~18kb)
exon 2
nucleotide was labeled with y-32P-dATP using 10 units of T4 poly- nucleotide kinase (New England Biolabs, Schwalbach/Taunus, Germany). Two or three bands were obtained for each digest (data not shown). A 2.0 kb Sacl band was subcloned in pBlue- script KS and 1.1 kb of the sequence determined, which revealed a potential microsatellite marker with 17 CA repeat units.
To locate the novel microsatellite marker with respect to the ACTHR gene, ACTHRcos1 was digested with several restriction enzymes, Southern-blotted and hybridized to the coding region PCR product, an ACTHR promoter PCR product and to the 2.0 kb Sacl, subfragment of ACTHRcos1 containing the CA repeat. Pri- mer pairs for amplification of ACTHR promoter were 5’CTG CAG GGC ATG TTG CGG3’ and 5’GAA GCA GGA ACT TTC TGG G3’. A common 9.5 kb BamHI band was detected by the coding region probe and the 2.0 kb Sacl fragment. A common 12 kb Sall frag- ment hybridized to the promoter probe and the 2.0 kb Sacl frag- ment (data not shown). In each hybridization, these bands were the only bands obtained with the respective enzyme. As the pro- moter and the coding region lie 18 kb apart [32], this allows to locate the 2.0 kb Sacl fragment in a 3.5 kb interval lying 9.5 kb to 6 kb upstream of the ACTH-R coding exon, within the intron of the ACTH-R gene (Fig. 1). The novel microsatellite marker was thus designated ACTHRint1. The nucleotide sequence data have been submitted to GenBank and have been assigned the acces- sion number AF166123.
After identification of the ACTHRint1 locus, flanking primers creating a 96-bp PCR product were designed: ACTHRint1f: 5’GCT ATG GAT TTT TCT ATG CC3’ and ACHTRint1r: 5’AAA TTG CAG TTC AAC GGT GC3’. PCR was carried out with 0.5 units of Taq polymerase under the following conditions: 3 min 95 ℃ fol- lowed by 35 cycles of 30 s 95 ℃, 45 s 62 ℃ and 20 s 72 ℃ followed by 3 min 72℃. MgCl2 concentration was 1.4 mM. PCR products
were resolved on 12% polyacrylamide gels. Lymphocytic DNA served as an internal control in each patient.
A hundred and fourteen patients with a variety of adrenal and non-adrenal tumors were screened for LOH. In all individuals studied two main bands were obtained by electrophoresis on a polyacrylamide gel, indicating a rate of heterozygosity of 100% in our series. In a subsequent study, 67 patients with adrenal tu- mors were investigated for LOH of the ACTHRint1 locus (Table 2). LOH was found in 4 out of 10 carcinoma patients and in one pa- tient with an oncocytic non-functional adrenal adenoma from a total of 57 adrenal adenomas (Fig. 2B). No LOH of the ACTHR gene locus was found in any of the other patients (Fig. 2A). All patients with adrenocortical carcinomas with ACTHR-LOH had recurrent disease within six months after surgery. Unfortunately, follow-up in the LOH negative patients is only available for three out of six patients. No recurrences and no deaths related to the tumor appeared in any of these three patients without LOH. This confirms and extends our previous finding that the ACTHR locus is deleted in a substantial subset of adrenal carcinoma pa- tients, and that loss of ACTHR expression might be associated with a poor clinical prognosis.
ACTHR Expression in Adrenocortical Tumors
The three tumors from our original study with LOH at the PstI polymorphism have been investigated for ACTHR mRNA expres- sion by Northern blot. An association between LOH and de- creased ACTHR mRNA expression could be demonstrated in these tumors compared to normal adrenal tissue. The two carci- nomas with LOH had advanced tumor stages compared to the unaffected tumors, and survival was drastically reduced to 0 to 3 months in the patients with LOH compared to more than 15
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| Publication | Ref.No. | Adrenocortical Cell System | ACTH preparation | ACTH concentration | ACTH dosage interval | Observation period | Fetal Calf Serum | Results |
|---|---|---|---|---|---|---|---|---|
| Masui 1971 | [8] | Y1 | ? | 3 × 10-5-0.3 U/ml | Once | 26 h | 12.5% HS 2.5% FCS | Growth inhibition |
| Ramachandran 1975 | [11] | Rat primary cells | Ovine ACTH | 1 µg/ml | Once | 4d | 10% | Growth inhibition |
| Gospodarowicz 1975 | [12] | Y1 | Porcine ACTH 1-39 | 0.75 U/ml (1.3 mg) | Once | 7 d | 0.2% | Growth inhibition |
| Weidman 1977 | [9] | Y1 | Porcine ACTH 1-39 | 0.4 U/ml | Once | 4 d | 10% | Growth inhibition |
| Hornsby 1977 | [13] | Bovine primary cells | Porcine ACTH 1-39 | 10-14 - 10-6 M | 48 h | 1-8 d | 10% | Growth inhibition |
| Armato 1978 | [16] | Human primary cells | Synthetic ACTH 1-24 | 3 × 10-6 M | 24 h | 8 d | 20% | Growth stimulation |
| Morera 1980 | [10] | Y1 | Porcine ACH 1-39 Synthetic ACTH 1-24 | 10-8-10-7 M | Once | 6 d | 0.1% | Growth inhibition |
| Simonian 1981 | [14] | Human primary cells | Porcine ACTH 1-39 Synthetic ACTH 1-24 | 10-8 M | Once | 48 h | 10% HS | Growth inhibition |
| Menapace 1987 | [17] | Rabbit primary cells | Synthetic ACTH 1 - 24 | 10-6M | 24 h | 3 d | 0% | Growth stimulation |
| Arola 1993 | [18] | Rat primary cells | ACTH 1-39 | 7 x 10-11 -7x10-7M | 24 h | 1 to 63 d | 25% | Biphasic: Inhibition for 24 h followed by stimulation |
| Lotfi 1997 | [2] | Y1 | Synthetic human ACTH 1-39 Porcine ACTH | 0.1-1000 µU/ml | 5 min to 2 h pulse, continous 14 h | 24 h | 5 min pulse or 14 h: 15% HS, 2,5% FCS | Biphasic: Growth stimulation for 2 h, followed by growth inhibition |
HS: Horse serum; FCS: Fetal calf serum.
| Diagnosis | n | ACTHR int1 LOH-positive | Not informative | % LOH |
|---|---|---|---|---|
| Endocrine inactive adenoma | 10 | 1 | 0 | 10 |
| Cortisol producing adenoma | 16 | 0 | 0 | 0 |
| Aldosterone producing adenoma | 17 | 0 | 0 | 0 |
| Adenoma with unknown activity | 14 | 0 | 0 | 0 |
| Adenoma total | 57 | 1 | 0 | 1.75 |
| Adrenocortical carcinoma | 10 | 4 | 0 | 40 |
| Total | 67 | 5 | 0 | 13.4 |
months for the patients whose tumors did not show LOH [31]. Another study dealt with ACTHR mRNA expression in 22 adreno- cortical adenomas and 6 carcinomas. While endocrine active adenomas showed unaffected ACTHR expression correlated to P450scc expression, the ACTHR mRNA expression was low in non-functioning adenomas (23 ± 11% vs. normal adrenal tissue) and carcinomas (19 ± 12%). In 4 of the 6 carcinomas studied, ACTHR mRNA expression could not be detected by Northern blot; however, expression was clearly present in two carcinomas [33]. In a study using in situ hybridization, two out of three carci- nomas were positive for ACTHR mRNA expression as were all adenomas except one [34]. In conclusion, expression of the ACTHR is impaired frequently in adrenocortical carcinomas and rarely in non-functioning adenomas. The carcinomas affected were at a more advanced tumor stage and were associated with shorter survival, so it seems reasonable to conclude that expres-
A
P1
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sion of the ACTHR is associated with a higher differentiation state and a lower proliferation rate of the tumors. Whether the ACTHR has a direct growth-inhibiting effect or whether this ef- fect is achieved via differentiation is not known. Furthermore, expression of the ACTHR might only be a marker of differentia- tion without any causative role in tumor development. Upcom- ing investigations should address this questions.
Downloaded by: University of Pittsburgh. Copyrighted material.
Conclusions
In vitro, a growth inhibiting effect of the ACTHR is well estab- lished. However, not all data published confirm this hypothesis, most probably because of different culture conditions and ex- perimental procedures.
More recent in vitro data suggest that proliferation of adrenocor- tical cells might be induced mainly by N-terminal derived POMC peptides rather than ACTH.
ACTHR expression is lost in a substantial subset of undifferenti- ated adrenocortical carcinomas, suggesting a role for the ACTHR in differentiation and proliferation of the tumors. Further studies should address whether the ACTHR is directly involved in inhibi- tion of tumor growth or acts as a differentiation marker.
Acknowledgements
This study was supported by grants from the Deutsche For- schungsgemeinschaft (DFG) and Dr. Mildred Scheel Stiftung.
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