Expression of adrenocorticotrophic hormone receptor mRNA in human adrenocortical neoplasms: correlation with P450scc expression
Martin Reincke, Felix Beuschlein, Anna-Claudia Latronico*, Wiebke Arlt, George P. Chrousos* and Bruno Allolio Department of Internal Medicine, University of Würzburg, FRG, and *Developmental Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, MD, USA
(Received 18 October 1996; returned for revision 5 December 1996; finally revised 6 January 1997; accepted 10 March 1997)
Summary
OBJECTIVE Adrenocorticotrophin (ACTH) is the main hormone-regulating steroid secretion from the adre- nal cortex. The ACTH receptor (ACTH-R) has recently been cloned, allowing systematic evaluation of its expression and function in adrenal tumorigenesis. We investigated ACTH-R and P450 side-chain cleav- age enzyme (P450scc) mRNA expression in a variety of neoplastic adrenocortical tissues by Northern blot and reverse-transcriptase-PCR (RT-PCR).
PATIENTS AND MEASUREMENTS We studied tissue from eight normal adrenals, six diffuse adrenocortical hyperplasias in patients with ACTH-dependent Cushing’s syndrome, 22 adrenal adenomas, six car- cinomas and two carcinoma cell lines. Poly A mRNA was electrophoresed, immobilized on a nylon mem- brane and hybridized using @32P-CTP labelled human ACTH-R and P450scc cDNAs.
RESULTS Mean ACTH-R mRNA expression showed significant differences between groups (P = 0-0001), but appeared to be independent of plasma ACTH concentrations. Compared to normal adrenal (= 100 ± 12%), expression was low in non-functional adenomas (23 ± 11%) and carcinomas (19 ± 12%), intermediate in adrenocortical hyperplasias (88 ± 6%) and cortisol-producing adenomas (81 ± 15%) and high in aldosteronomas (175 ± 29%). In adenomas, ACTH-R mRNA expression correlated closely with the expres- sion of P450sccmRNA (r = 0.8, P = 0.0001) suggesting
regulation by similar factors. However, carcinomas and cancer cell lines did not show a positive correlation between these two parameters (r = - 0-44, P = 0-3). CONCLUSIONS We have demonstrated that plasma ACTH is not the majorfactor influencing ACTH-receptor mRNA expression in neoplastic adrenal tissue. In benign tumours of the adrenal cortex there was a close positive correlation between ACTH-receptor mRNA and P450scc mRNA which was missing in adrenocortical carcinomas, probably as a result of tumour dedifferentiation.
Adrenocortical carcinoma is a highly malignant tumour with an incidence of 1:1.7 million/year (Lipsett et al., 1963). Seventy per cent of patients present at an advanced tumour stage with local invasion or distant metastases and can rarely be cured (Søreide et al., 1992). In contrast, benign adrenal lesions are very frequent. Most of these tumours are clinically silent and are detected incidentally by ultrasound or computed tomog- raphy (Reincke et al., 1992). The prevalence of these so-called ‘incidentalomas’ is estimated to be 1% in abdominal CT scans (Copeland, 1983). The majority of these tumours are non- functional adrenal adenomas not requiring specific therapy. However, the pathogenesis and clinical significance of these lesions are, respectively, unknown and controversial, although there is general agreement that the malignant potential of ‘incidentalomas’ is low (Reincke & Allolio, 1995).
Progress in the understanding of adrenal tumorigenesis has been slow. Mutations in tumour suppressor genes and oncogenes have been identified in 30% of the carcinomas but not in adenomas (Lyons et al., 1990; Reincke et al., 1993; Ilvesmäki et al., 1993a; Reincke et al., 1994; Beuschlein et al., 1994; Gicquel et al., 1994a; 1994b; 1995a; 1995b; Latronico et al., 1995; Light et al., 1995). The importance of hormonal factors and para- or autocrine growth factors in tumour development has not been studied in detail, with the exception of the recent finding of increased IGF-II expression in adrenocortical carcinomas (Ilvesmäki et al., 1993a; Gicquel et al., 1994b).
ACTH is the main hormone that regulates steroid synthesis and secretion and stimulates cell proliferation in the adrenal cortex, both via its specific receptor (Baxter & Tyrrel, 1986). After ligand binding and coupling to Gs, the stimulatory G-protein, increased adenylylcyclase activity stimulates
Correspondence: Dr Martin Reincke, Schwerpunkt Endokrinologie, Medizinische Universitätsklinik Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany. Fax: 49 931 2012283.
| Tissue | N | Age range (years) | Sex | Adrenal mass |
|---|---|---|---|---|
| Normal adrenal | 8 | 29-55 | 4 F/4 M | 4-7 g |
| Bilateral adrenal hyperplasia of ACTH-dependent Cushing's syndrome | 6 | 31-45 | 6 f | 14-25 g |
| Non-functioning adenoma | 4 | 50-70 | 4 F | 20-70 g |
| Cortisol-producing adenoma with 'preclinical Cushing's syndrome' | 4 | 43-51 | 4 F | 18-35 g |
| Cortisol-producing adenoma with Cushing's syndrome | 5 | 39-53 | 5 F | 22-35 g |
| Aldosteronoma | 9 | 33-62 | 5 F/4 M | 14-5-34 g |
| Carcinoma | 6 | 17-79 | 5 F/1 M | 119-1600 g |
| Non-functional | 1 | |||
| Functional | 5 |
F, female; M, male.
intracellular cyclic AMP which in turn activates the protein kinase A pathway. In the past, analysis of ACTH receptor function in the adrenal cortex has been hampered by technical difficulties, e.g. low biological activity of the radiolabelled ACTH ligand (Lebrethon et al., 1994). The recent cloning of the human ACTH receptor (ACTH-R) gene (Mountjoy et al., 1992) has allowed study of the expression of ACTH-R mRNA in normal and tumourous human adrenal tissue.
ACTH-R expression and function in neoplastic adrenal tissue is of interest for several reasons. First, ACTH has recently been shown to stimulate the expression of its own receptor in the NCI h295 cell line and adrenal fasciculata-reticularis cells (Mountjoy et al., 1994; Penhoat et al., 1994; Lebrethon et al., 1994) in a time- and dose-dependent manner. Therefore, one would expect low ACTH-R mRNA expression in cortisol- producing adrenal tumours because these are associated with suppressed plasma ACTH concentrations. On the other hand, in ACTH-dependent bilateral adrenocortical hyperplasia, ACTH-R expression should be high due to the ACTH drive on the adrenal cortex. Second, ACTH can stimulate cell growth and proliferation through the ACTH receptor, possibly acting on the expression of adrenal oncogenes like IGF-II (Ilvesmäki et al., 1993a; Gicquel et al., 1994b), similar to the effects of ACTH on IGF-II expression in the foetal adrenal (Ilvesmäki et al., 1993b). Therefore, ACTH suppressive therapy in patients with adrenal carcinomas might improve survival, provided that the ACTH-R is expressed in these tumours. Third, the separation between benign and malignant adrenal tumours with conventional histology can be very difficult (Weiss, 1984). The expression and distribution of ACTH-R mRNA transcripts might be a new useful means for the differentiation of these entities.
We, therefore, analysed ACTH-R mRNA expression in a variety of neoplastic adrenal tissues and compared the results with the expression of P450 side-chain cleavage enzyme
(P450scc), the initial and rate-limiting step of adrenal steroidogenesis.
Patients and methods
Adrenal tissue and cell lines
Adrenal tissue from 42 patients with adrenal disease and cells of two adrenocortical cancer cell lines were studied. Clinical data from the patients are shown in Table 1. The clinical and pathological diagnosis was made according to established criteria (Weiss 1984; Baxter & Tyrrel, 1986; Orth et al., 1992). Patients with ACTH-dependent Cushing’s syndrome who underwent bilateral adrenalectomy either had Cushing’s disease refractory to trans-sphenoidal surgery or pituitary irradiation (n = 3) or ectopic ACTH syndrome (n = 3). Patients with non- functional adrenal adenomas had no signs or symptoms of hormonal excess, normal serum potassium levels and a normal suppression of serum cortisol (below 83 nmol/l) after low-dose dexamethasone (2 mg). Patients with the preclinical Cushing’s syndrome had no signs and symptoms of Cushing’s syndrome but had serum cortisol concentrations nonsuppressible by 2 and 8 mg of dexamethasone (Reincke et al., 1992). Plasma ACTH concentrations were in the low normal range or suppressed. All patients with adrenal adenomas causing classical Cushing’s syndrome had suppressed plasma ACTH concentrations. Patients with aldosterone-producing adenomas had hyper- tension, hypokalaemic alkalosis, elevated aldosterone concen- trations and suppressed plasma renin activity. All patients with adrenocortical carcinoma had advanced tumour stages with local invasion of surrounding tissue or distant metastases. Neoplastic adrenal tissue was collected with the approval of the ethical committee of the University Hospital of Würzburg. Normal adult adrenals (n = 7) were obtained after organs were removed from brain-dead patients for transplantation or from
the adjacent normal cortex of a patient with an aldosteronoma (n = 1). Only central parts of the tumours were used, avoiding necrotic areas and contamination with normal adjacent tissue. In addition, the integrity of the tissue was checked by light microscopy. The normal or hyperplastic adrenal cortex was carefully dissected from the medulla with a scalpel using only the adrenal cortex for these experiments. The tissues were snap- frozen and immediately stored at -80℃ until analysed.
The human adrenocortical cancer cell lines studied were SW13 and NCI-h295 (Gazdar et al., 1990).
Northern blot
Total or poly A RNA was isolated from tissue using the guanitidin isocyanate method (Stratagene, Heidelberg, FRG). Degraded mRNA samples on agarose gels stained with ethidium bromide were excluded from the analysis. After electrophoresis of 20 µg total RNA or 3 µg poly A RNA through a 1% agarose/formamide gel hybridization was performed using a @32P-CTP (Amersham, Braunschweig, Germany) labelled (Random Primed Labeling Kit, Boehringer- Mannheim, FRG) full length human ACTH-R cDNA (a 1061 base pair fragment of the human ACTH receptor generated by PCR using human genomic DNA as template and 5’ GAT TTA ACT TAG ATC TCC AGC AAG T 3’ and 5’ CGT TGC CAA GTG CCA GAA TAG TGT 3’ as upstream and downstream primer, respectively (Mountjoy et al., 1992)) and a human cDNA for P450scc (provided by Dr W. L. Miller, University of California, San Francisco (Chung et al., 1986)). The blots were washed twice in 1 x Sodium chloride/sodium citrate buffer (SSC) and 0-5 × SSC (each containing 0-1% SDS) at 60℃. After exposing for autoradiography at -80℃ with intensifying screens, resulting bands were quantified by scanning densito- metry, while the blots were stripped and rehybridized. The large number of tissues used in this study required electrophoresis of the samples on different blots. To facilitate comparison of the relative mRNA expression of ACTH-R and P450scc one ‘index’ tissue (diffuse hyperplastic adrenal of ACTH-dependent Cushing’s syndrome) was run on all blots in parallel for normalization. For standardization the blots were hybridized with a mouse ß-actin cDNA probe. The mRNA steady state concentrations are expressed as % of normal adrenals (=100%) after correcting the signal of each sample for ß-actin expression.
Reverse transcriptase-PCR
Tumour samples with low or undetectable ACTH-R mRNA transcripts were subjected to reverse-transcriptase-PCR (RT-PCR) to investigate low abundance mRNA expression. For first-strand cDNA synthesis, 250 ng total RNA or 40 ng mRNA, 2 ul 10 xrTth buffer, 2 ul 10mM MnCl2 and @ 1997 Blackwell Science Ltd, Clinical Endocrinology, 46, 619-626
1.6 ul 10 mM NTPs and 5’ GTC ATC TGG ACG TTC TGC AC 3’ for ACTH-R and 5’ CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG 3’ for ß-actin downstream primer, respectively were incubated with 2 ul rTth polymerase (Perkin Elmer Cetus) at 75° for 15 min. For polymerase chain reaction the product was incubated with 64 ul H2O, 8 ul 10 x chelating buffer, 6 ul MgCl2, 5’ GAA GAG AGA CAT GTA GCA GGC 3’ and 5’ TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA 3’ as ACTH-R and actin upstream primer, respectively, at 95° for 2 min, 55° for 1 min, 72° for 1 min, 95° for 1 min (35 cycles) followed by a final cycle at 72° for 7 min. The ACTH-R mRNA amplification product had an expected length of 318 bp, the B-actin amplification product a length of 661 bp. The specificity of the amplification products was confirmed by restriction enzyme analysis. The PCR products increased exponentially up to 40 cycles for both ACTH-R and B-actin, so 35 cycles were chosen for the final protocol. Negative control samples such as RNA from other tissues (liver, kidney, fat tissue) and genomic DNA digested with DNAse (Boehringer-Mannheim, FRG) were included in all experi- ments. The sensitivity of the RT-PCR experiment was at least 102 times greater than that of the Northern blot.
Statistical analysis
All values are expressed as mean ± SEM. Significance of differences between group means was assessed using a non- parametric one-way ANOVA (Kruskal-Wallis test) and Mann-Whitney U-test, as appropriate. Correlation between ACTH mRNA and steroidogenic enzyme mRNA concentra- tions was determined by linear regression analysis and expressed as Pearson’s correlation coefficient. A P ≤ 0-05 was considered statistically significant.
Results
Northern blot
Normal and neoplastic adrenocortical tissue expressed five ACTH-R mRNA transcripts, two major bands of 2.0 and 3.8 kb, one intermediate band of 4.2 kb, and two minor bands of 6-8 and 10 kb which were only visible after prolonged exposure. Little variation was observed in the ratio of these transcripts in different adrenocortical tissues (data not shown). For statistical analysis, the 3.8 kb band was selected, although the 2-0 kb band gave similar results. In the tissues analysed, mean ACTH-R mRNA expression ranged from 0 to 287% (normal adrenals, 100 ± 12%) and were significantly different between groups (P= 0.0001) (Figs 1 and 2).
The highest mean ACTH-R mRNA steady state concentra- tions were observed in aldosteronomas (175 ± 29%; P = 0-03
3911 bp
ACTH-R
2800 bp
2800 bp
P450scc
2800 bp
ß-actin
1
2
3
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vs normal adrenals). Low ACTH mRNA levels were present in non-functioning adenomas (23 ± 11%; P = 0-007 vs normal adrenals) and carcinomas (19 ± 12%; P = 0-002 vs normal adrenals). The majority of adrenal carcinomas did not express ACTH-R mRNA, however, in two of the six carcinomas ACTH-R mRNA transcripts were clearly present.
Plasma ACTH concentrations did not have a major influence on tumour ACTH-R mRNA levels. In patients with high plasma ACTH concentrations due to ACTH-dependent Cushing’s syndrome, the hyperplastic adrenal tissue did not have increased ACTH-R mRNA levels (88 ± 6%). In patients with suppressed plasma ACTH concentrations as a result of adrenal Cushing’s syndrome, the ACTH mRNA levels in the tumour samples were not low (81 ± 15%). Mean ACTH-R mRNA expression paralleled P450scc mRNA expression in all tissues analysed with the exception of adrenal carcinomas which had low mean ACTH-R mRNA levels but high mean P450scc levels (Fig. 2). A close positive correlation between ACTH-R mRNA and P450scc mRNA was present in all adrenal adenomas independently of endocrine activity (n = 22, r = 0.8, P = 0.0001; Fig. 3) but was not found in carcinomas and cancer cell lines (n = 8, r = - 0-44, P = 0-3). After exclusion of the two carcinoma cell lines there remained no correlation in the carcinomas (n = 6, r = - 0-45, P = 0.33).
Reverse transcriptase-PCR
Tumour tissue with undetectable ACTH-R mRNA levels at Northern blot analysis were subjected to reverse transcriptase- PCR. Low abundance ACTH-R mRNA transcripts were detected in three functional carcinomas and in the NCI h295 cancer cell line, but not in a non-functional carcinoma and the SW13 cell line (Fig. 4).
Discussion
ACTH is the major hormone that regulates glucocorticoid secretion, adrenal androgen secretion and - together with angiotensin II - mineralocorticoid secretion in the adrenal cortex (Baxter & Tyrrel, 1986; Orth et al., 1992). In addition, ACTH regulates the adaptational processes of hypertrophy and hyperplasia, during both physiological and pathophysiological conditions such as major stress. ACTH acts through its specific receptor, the ACTH-R, a member of the superfamily of seven transmembrane domain, G-protein coupled receptors (Mountjoy et al., 1992). The ACTH-R belongs to the melanocortin receptor family which consists of four MSH receptors (MC-R 1, 3 to 5) and the ACTH (or MC-R 2) receptor (Siegrist & Eberle, 1995). The human ACTH-R is mainly expressed in the adrenal cortex but has been recently identified in extra-adrenal tissues
(a)
300
ACTH-R mRNA (%)
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100
0
(b)
120
P450scc mRNA (%)
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including human skin (Slominski et al., 1996) and mouse adipose tissue (Boston & Cone, 1996).
There are three main findings to our report. First, the expression of ACTH-R mRNA transcripts in neoplastic adrenal tissue seems to be largely independent of circulating ACTH concentrations. Patiens with suppressed plasma ACTH because of cortisol-producing adrenal tumours did not have low ACTH-R mRNA levels. Also, patients with ACTH-dependent Cushing’s syndrome with high plasma ACTH concentrations did not have an increased expression of ACTH-R mRNA in this study. This is in contrast to the stimulatory effects of ACTH (and angiotensin II) on ACTH-R mRNA and ACTH-R binding sites in bovine/human fasciculata-reticularis cells in vitro and
ACTH-R mRNA (arbitrary units)
6
4
2
0
0
2
4
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P450 SCC mRNA (arbitrary units)
human tumour cell lines (Lebrethon et al., 1994; Mountjoy et al., 1994; Penhoat et al., 1994).
However, the absence of any difference in ACTH-R mRNA expression between normal adrenal tissue and hyperplastic adrenals of patients with ACTH-dependent Cushing’s syn- drome may be in part explained by the fact that the ‘normal’ adrenals were collected from brain-dead patients in this study. Major stress following trauma or severe illness in these patients may have been present for hours to several days leading to ACTH-mediated stimulation of the adrenal cortex with a consequent increase in expression of ACTH-R mRNA.
Our data support the concept that ACTH-R mRNA expression correlates with the steroidogenic activity of the tumour. For example, non-functional adenomas had very low ACTH-R mRNA levels, patients with ‘preclinical’ Cushing’s syndrome, i.e. adenomas which secrete glucocorticoids in amounts insufficient to cause clinically apparent Cushing’s syndrome (Reincke et al., 1992), had intermediate ACTH-R mRNA levels, whereas classical cortisol-producing adenomas and aldosteronomas had high ACTH-R mRNA concentrations. P450scc is the initial and rate-limiting step of steroidogenesis which converts cholesterol to pregnenolone (Miller, 1988). ACTH-R mRNA levels and P450scc mRNA concentrations showed parallel changes in most of the tissues analysed and a close positive correlation was observed between these two parameters in the adenomas studied. Although our data do not give direct experimental evidence, they support the hypothesis that ACTH-R and P450 enzyme mRNA expression may be regulated by similar transcription factors in neoplastic tissues.
1
2
3
= 661 bp
= 318 bp
ACTH-R
B-ACTIN
B-ACTIN
ACTH-R
ACTH-R
B-ACTIN
CONTR.
MARKER
However, these results need to be confirmed by other approaches such as cell culture experiments and Western blot investigating directly the causal relationship between ACTH-R expression and steroid biosynthesis. However, to date we and others have been unable to generate an antibody against the ACTH-R, probably due to its short extracellular domains and low antigenity.
Second, adrenocortical carcinomas did not show the positive correlation between ACTH-R mRNA and P450scc mRNA observed in adenomas. Mean ACTH-R mRNA expression in carcinomas was low, as in non-functioning adenomas, but mean P450scc expression in carcinomas was high, similar to cortisol- and aldosterone-producing adenomas. This may be explained as a result of tumour dedifferentiation associated with the malignant phenotype of these neoplasms. However, two of six adrenocortical carcinomas expressed ACTH-R mRNA transcripts to a degree similar to functioning adrenal adenomas. Although the number of tumours studied is limited, this may argue for the expression of functional ACTH-R in up to 30% of these tumours. From human or bovine fasciculata-reticularis cells we know that the mRNA expression of ACTH-R transcripts correlates well with the number of functional ACTH-R (Lebrethon et al., 1994; Penhoat et al., 1994). The finding of ACTH-R mRNA expression in sporadic carcinomas, therefore, raises the question whether ACTH-suppressive therapy with dexamethasone could be an adjunctive therapy for adreno- cortical carcinomas by suppression of the ACTH-related adreno- cortical cell proliferation. This regimen would be similar to the TSH suppressive therapy with thyroxine in thyroid carcinoma. Demonstration of ACTH-R binding sites in adrenal cancer tissue by receptor autoradiography and cell culture experiments showing ACTH-induced carcinoma cell proliferation could give further evidence in vitro to support investigation into the anti-tumour effects of ACTH suppression in a clinical trial.
Third, the histological differentiation between large adenomas and small adrenocortical carcinomas confined to the adrenal bed may be very difficult (Weiss, 1984; Gandour & Grizzle, 1986). Since therapy and follow-up of adrenocortical carcinomas is entirely different from those of adenomas, the distinction between these two entities is important for the clinician. Several histological criteria have been developed to enable the histolo- gical diagnosis of adrenal cancer, if clear-cut signs of malignancy, such as distant metastases, and capsular or blood vessel invasion are absent. These criteria include diffuse growth pattern, tumour cell necrosis, broad fibrous bands, a high mitotic rate and atypical mitoses, and pleomorphism (Hough et al., 1979; Weiss, 1984). Nevertheless, the diagnosis of adrenal carcinoma remains difficult, and additional criteria like carcinoma-specific molecular markers may be of value for the pathologist. The quantitative expression of ACTH-R mRNA and P450scc evaluated by Northern blot in our experiments showed differentiation between adenomas and carcinomas to some degree. However, the observed overlap, especially between non-functional adenomas and adrenocortical carcinomas, limits its usefulness in the histopathological workup of adrenal tumours of unknown malignant potential.
The highest ACTH-R mRNA expression in this series was found in aldosteronomas. Biochemically it is well recognized that aldosterone secretion in aldosteronomas is dependent on plasma ACTH (Orth et al., 1992). Aldosterone secretion is typically unresponsive to orthostasis in these tumours but shows an ACTH dependent diurnal rhythm, which is used as a diagnostic criterion in the differential diagnosis from idiopathic bilateral adrenal hyperplasia. In situ hybridization experiments of ACTH-R mRNA in the normal adrenal gland showed the highest expression in the mineralocorticoid- producing zona glomerulosa (unpublished observation). In this context, the finding of high ACTH-R mRNA expression in aldosteronomas is not surprising.
@ 1997 Blackwell Science Ltd, Clinical Endocrinology, 46, 619-626
In one non-functioning adrenal adenoma and four adrenal carcinomas ACTH-R mRNA transcripts were not detectable by Northern blot. To investigate whether these tumours expressed ACTH-R mRNA in low abundance we used RT-PCR and amplified a 318 bp fragment of the ACTH-R mRNA. In our experience the technique employed is at least 100-fold more sensitive than a conventional Northern blot. With this approach we demonstrated that only one non-functional carcinoma did not express ACTH-R message. The co-amplification of a ß- actin mRNA fragment of 661 bp in all samples showed that the missing amplification was not due to RNA degradation. Our experiments therefore show that most adrenocortical tumour tissues express ACTH-R mRNA, although occasionally to a degree which is undetectable by Northern blot.
Acknowledgements
We would like to thank Dr W. Miller, UCSF, California for the generous gift of the P450scc cDNA. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Re 752/5-1).
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