Deletion of the Adrenocorticotropin Receptor Gene in Human Adrenocortical Tumors: Implications for Tumorigenesis*
MARTIN REINCKE, PATRICIA MORA, FELIX BEUSCHLEIN, WIEBKE ARLT, GEORGE P. CHROUSOS, AND BRUNO ALLOLIO
Department of Internal Medicine, University of Wurzburg, Wurzburg, Germany; and the Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (G.P.C.), Bethesda, Maryland 20892
ABSTRACT
Constitutive activating mutations of G protein-coupled receptors, such as that of TSH, have been implicated in the tumorigenesis of human endocrine neoplasms, such as thyroid adenomas. In a previous study we reported that constitutive activating point mutations of the ACTH receptor (ACTH-R) gene, a member of the G protein-coupled receptor superfamily, were not present in hormone-secreting and nonsecretory adrenocortical neoplasms. In this study, we investigated whether allelic loss of the ACTH-R gene is present in sporadic adrenal tumors. We identified a PstI polymorphism in the promoter region 3 kilobases upstream of the coding region of the ACTH-R gene. The rate of heterozygosity for this polymorphism in 99 unrelated Caucasian individuals was 53.5%. Using this polymorphism, we analyzed loss of heterozygosity (LOH) of the ACTH-R gene in 20 informative cases with benign and malignant adrenocortical tumors. Of 16 patients
with benign lesions, LOH was present in 1 oncocytic nonfunctional adenoma, but not in 15 hyperfunctioning adenomas. Of 4 informative patients with adrenocortical carcinomas, LOH was present in 2 cases. Both patients had advanced tumor stages and showed a more rapid course than carcinoma patients without LOH. Analysis of the flank- ing region of the ACTH-R using the polymorphic microsatelite marker D18S37 and D18S40 showed that this deletion was confined to the ACTH-R gene. Northern blot experiments demonstrated reduced ex- pression of ACTH-R messenger ribonucleic acid in the tumors with LOH of the ACTH-R gene, suggesting functional significance of this finding at the transcriptional level. We conclude that LOH of the ACTH-R gene is possibly involved in adrenal tumorigenesis, contrib- uting to cellular dedifferentiation in adenomas and carcinomas. (J Clin Endocrinol Metab 82: 3054-3058, 1997)
A DRENOCORTICAL carcinoma is a rare, highly malig- nant tumor with an incidence of 1.7/million. In con- trast, benign adrenocortical lesions are very frequent (prev- alence, 1/100). Most of these tumors are detected incidentally by ultrasound or computed tomography (so-called inciden- talomas) (1). Histopathological differentiation of benign from malignant adrenocortical lesions is crucial for treatment and follow-up. However, histopathological classification of adrenocortical carcinomas can be difficult if metastases or infiltration of surrounding tissues are absent, especially in small, well differentiated carcinomas (2, 3).
The human ACTH receptor (ACTH-R) is a member of the G protein-coupled seven-transmembrane domain superfam- ily of receptors and belongs, together with several MSH receptors, to the melanocortin receptor family (4, 5). The ACTH-R gene was recently cloned (4) and mapped on chro- mosome 18p11.2 (6, 7). In a previous study, we reported that no constitutive activating point mutations of the ACTH-R gene were present in adrenocortical neoplasms, in contrast to earlier findings of activating mutations of the TSH receptor
in thyroid adenomas (8). These data and evidence from in vitro experiments (9) suggested that ACTH was a differen- tiating factor of the adrenal cortex, with a low potential of stimulating cell proliferation and tumorigenesis. Thus, in- activation of the ACTH-R signal transduction cascade could result in loss of differentiation and enhanced clonal expan- sion of adrenal tumors.
We recently identified a PstI polymorphism located ap- proximately 3 kilobases (kb) upstream of the ACTH-R-cod- ing region (10). Using this polymorphism, we investigated whether allelic loss of the ACTH receptor gene occurs in adrenocortical neoplasms. We herein report that deletion of the ACTH-R gene at 18p11.2 is present in a subset of adre- nocortical neoplasms characterized by loss of differentiation and/or aggressive growth.
Subjects and Methods
Normal subjects and patients
Blood was collected from 99 unrelated Caucasian individuals (57 females and 42 males) after giving written informed consent and stored at -80 C until DNA extraction.
Forty-one patients with a variety of adrenal diseases were studied. Twenty of these (49%) were heterozygous for the PstI polymorphism of the ACTH-R gene. The clinical data for these patients are shown in Tables 1 and 2. The clinical and pathological diagnosis was made ac- cording to established criteria (2, 3, 11, 12). Blood and neoplastic adrenal tissue was collected with the approval of the ethical committee of the University Hospital of Wurzburg. Normal adult adrenals (n = 4) were obtained after organs were removed from brain-dead patients for trans-
Received February 26, 1997. Revision received May 13, 1997. Ac- cepted May 21, 1997.
Address all correspondence and requests for reprints to: PD Dr. Martin Reincke, Schwerpunkt Endokrinologie, Medizinische Univer- sitätsklinik Würzburg, Josef-Schneider-Strasse 2, 97080 Wurzburg, Germany.
* This work was supported by a grant from the Deutsche Forschungs- gemeinschaft (Re 752/5-1).
| Patient no. | Age (yr) | Sex | Clinical presentation | Max. tumor size (cm) | Histology | LOH of ACTH-R |
|---|---|---|---|---|---|---|
| 1 | 46 | M | APA | 1.0 | Adenoma | - |
| 2 | 50 | M | APA | 1.1 | Adenoma | - |
| 3 | 48 | F | APA | 1.6 | Adenoma | |
| 4 | 43 | F | APA | 1.7 | Spongiocytic adenoma | |
| 5 | 64 | F | APA | 1.8 | Adenoma | |
| 6 | 42 | F | APA | 2.0 | Adenoma | |
| 7 | 41 | F | APA | 2.0 | Adenoma | |
| 8 | 67 | M | APA | 2.0 | Adenoma | |
| 9 | 53 | F | APA | 2.1 | Adenoma | |
| 10 | 51 | M | APA | 3.0 | Adenoma | |
| 11 | 47 | M | APA | 3.2 | Adenoma | |
| 12 | 66 | F | APA | 4.0 | Compact cell adenoma | |
| 13 | 36 | F | CPA | 3.0 | Adenoma | |
| 14 | 39 | F | CPA | 4.0 | Adenoma | |
| 15 | 46 | F | CPA | 6.5 | Adenoma | |
| 16 | 68 | F | Incidentaloma, nonfunctional | 7.0 | Oncocytic adenoma | + |
F, Female; M, male; APA, aldosterone-producing adenoma; CPA, cortisol-producing adenoma.
| Patient no. | Age (yr) | Sex | Clinical presentation | Max. tumor size (cm) | Tumor stage | Disease-free survival | LOH of ACTH-R |
|---|---|---|---|---|---|---|---|
| 1 | 1.2 | M | Virilization | 9 | T2N0M0 | >15 monthsª | - |
| 2 | 22 | F | Virilization | 12 | T2N0M0 | >15 monthsª | ☐ - |
| 3 | 46 | M | Cushing's syndrome | 8 | T4N1M1b | 0 monthe | + |
| 4 | 79 | F | Conn's syndrome | 9 | T4NOM0ª | 3 monthse | + |
F, Female; M, male.
” Follow-up with radiological imaging and endocrine evaluation every 3 months.
b Tumor invasion of the vena cava, pulmonary and liver metastases.
” Deceased after 5 months.
d Tumor invasion of the vena cava.
e Local tumor recurrence after 3 months, thereafter lost to follow-up.
1
2
0.5 kb
3.1 kb
0.9 kb
Pst I
Pst I
Pst I
Gene
mRNA
296 AA
Protein
plantation. After removing adjacent fat tissue, the tissues were snap- frozen and immediately stored at -80 C until analyzed.
Southern blot
The PstI polymorphism used in this study is located upstream of the ACTH-R gene (Fig. 1). It was detected when DNA was double digested with PstI and MspI/HpaII to study the methylation pattern of the ACTH-R gene in adrenocortical tumors. Digestion with other restriction enzymes and hybridization with different ACTH-R complementary DNA (cDNA) fragments showed that the polymorphism is located 3.1 kb upstream of the ACTH receptor-coding region, within the ACTH-R promoter (data not shown). Leukocytic or tumor DNA was extracted by means of proteinase K digestion and phenol/chloroform extraction. After digestion with PstI according to the instructions of the manufac-
turer (Boehringer Mannheim, Mannheim, Germany), the DNA was elec- trophoresed through a 0.8% agarose gel and blotted onto a nylon mem- brane (Amersham, Braunschweig, Germany). Hybridization was performed using an [@-32P]CTP (Amersham)-labeled (Random Primed Labeling Kit, Boehringer Mannheim) full-length human ACTH-R cDNA (a 1061-bp fragment of the human ACTH-R generated by PCR using human genomic DNA as template and 5’-GAT TTA ACT TÁG ATC TCC AGC AAG T-3’ and 5’-CGT TGC CAA GTG CCA GAA TAG TGT-3’ as upstream and downstream primers, respectively (4). Heterozygous in- dividuals showed two bands of 4.5 and 4 kb.
Using the polymorphic microsatelite markers D18S37 and D18S40 (13, 14) on the short arm of chromosome 18 close to the ACTH-R gene (14), we delineated the extent of the deletion of the ACTH-R gene. One of the primers was end labeled with [ y-32P]ATP, and PCR of leukocytic and tumor DNA was performed as described previously (13).
PCR and direct sequencing of the ACTH-R gene
In all patients informative for the PstI polymorphism, the ACTH-R gene-coding region was amplified using the PCR and directly sequenced by the dideoxy nucleotide chain termination method, using modified T7-DNA polymerase (Sequenase, U.S. Biochemical Corp., Cleveland, OH) in the presence of [@-35S]deoxy-ATP, as described previously (8).
Northern blot
Total or polyadenylated ribonucleic acid (RNA) was isolated from tissue using the guanitidin isocyanate method (Stratagene, Heidelberg, Germany). The RNA integrity was checked by ethidium bromide stain, and degraded RNA samples were excluded. The RNA was directly dot blotted on a nylon membrane. Hybridization was performed using the same probe as that for a Southern blot (15). For standardization, the blots were hybridized with a mouse B-actin cDNA probe. The steady state
messenger RNA (mRNA) concentrations are expressed as a percentage of that in normal adrenals (=100%). Autoradiographic images were digitalized with a video camera and a Macintosh PowerMac 7100 com- puter-based image analysis system (Stemmer, Puchheim, Germany) us- ing the IMAGE program (NIMH, NIH, Bethesda, MD).
Results
Rate of heterozygosity in normal subjects
Fifty-three of the 99 normal subjects (53.5%) were het- erozygous for the PstI polymorphism (Fig. 2).
Loss of heterozygosity (LOH) in adrenocortical adenomas
Of 16 patients with adrenocortical adenomas informative for the PstI polymorphism (15 functional and 1 nonfunctional adenoma), only the patient with a large nonfunctional ade- noma demonstrated LOH of the ACTH-R gene in the tumor tissue (Table 1 and Fig. 3). This tumor was incidentally de- tected by computed tomography and measured 7 cm in max- imum diameter. The patient was clinically asymptomatic and had normal serum potassium levels, normal PRA, and nor- mal suppression of serum cortisol by 2 mg dexamethasone. Surgery was suggested because of its size to exclude adre- nocortical carcinoma, and the patient underwent adrenalec- tomy with uneventful recovery. Histopathology showed an oncocytic (0 cell) adrenal adenoma composed of large tumor cells with abundant eosinophilic cytoplasm. The patient has remained in remission, and follow-up studies have been negative for tumor recurrence.
LOH in adrenocortical carcinomas
Two of four patients with adrenocortical carcinomas had LOH of the ACTH-R gene. Clinical presentation, tumor stage, and disease-free survival of these patients are shown in Table 2. Compared to patients with adrenocortical carci- nomas without LOH, patients with LOH of the ACTH-R gene had advanced tumor stages, early recurrence, and/or a more rapid course.
Polymorphic microsatelite markers D18S37 and D18S40
All patients were informative for at least one of the mic- rosatelite markers, D18S37 and D18S40. Neither the 3 tumors with LOH of the ACTH-R gene locus nor the 17 tumors without LOH of the ACTH-R gene locus showed LOH using
1
2
3
4
5
6
the D18537 or D18540 markers, demonstrating that the de- letion was confined to the ACTH-R gene locus.
PCR amplification and sequencing of the ACTH-R gene
Using PCR, we amplified the coding region of the ACTH-R gene of DNA from all tumor tissues. Direct sequencing of the PCR products revealed no point mutations or small deletions in the entire ACTH receptor sequence.
ACTH-R mRNA expression
Expression of ACTH-R mRNA was analyzed by Northern and dot blot experiments in 17 of the 20 tumor tissues avail- able for RNA extraction. Compared to normal adrenals (100 ± 12%) and adrenocortical tumors without LOH of the ACTH-R gene (102 + 20%), tumors with LOH showed greatly reduced ACTH-R mRNA steady state concentrations (21 ± 4%; Figs. 4 and 5).
Discussion
CAMP is a key second messenger involved in hormone hypersecretion and/or increased cell proliferation in a va- riety of endocrine tissues. Oncogenic transformation by con- stitutive activation of key regulatory proteins of cAMP, such as G protein-coupled receptors and GTP-binding proteins, have been implicated in the pathogenesis of such diseases as acromegaly and toxic thyroid adenomas (16, 17). Adreno- cortical tumorigenesis differs from pituitary and thyroid tu- morigenesis, as activation of the cAMP/protein kinase A pathway seems to be of little importance in the development of adrenocortical neoplasms. ACTH is the main hormone regulating steroid hormone secretion; however, it fails to cause adrenocortical hypertrophy in the absence of inner- vation by the splanchnic nerve. ACTH in physiological con- centrations does not stimulate cell proliferation of adreno- cortical cells in vitro, and even pharmacological doses of ACTH induce only moderate cell growth (9). In keeping with these findings, activating mutations of neither the ACTH receptor nor the a-chain of the Gs have been identified in benign or malignant adrenocortical tumors (8, 16). On the contrary, activating mutations of the Gi2, one of the adenylyl cyclase inhibitory G proteins, were found in very few adre- nocortical tumors, but not in a variety of other endocrine and nonendocrine tumors (16, 18). These data suggest that in the adrenal cortex the ACTH/Gs/protein kinase A signaling pathway is preferentially important for steroid hormone se- cretion and, hence, for maintenance of a highly differentiated cellular phenotype, but is of relatively little importance for cellular proliferation. Mutational loss of the ACTH-R gene by deletion, therefore, could result in loss of differentiation, a characteristic feature of human tumorigenesis that is asso- ciated with clonal expansion of a malignant cell clone. We herein demonstrate for the first time that allelic loss of a gene for a G protein-coupled receptor, that of the ACTH-R, is present in a subset of adrenocortical tumors, suggesting im- plications for the pathogenesis of these tumors. Three of 20 tumors in our series showed LOH for a PstI polymorphism in the promoter of the ACTH-R gene, suggesting a deletion within the promoter and/or the ACTH-R gene itself. The
L
Tu
L
Tu
L
Tu
1
2
3
ACTH-R
ß-Actin
specificity of the ACTH-R deletion is supported by the data generated using the microsatelite markers D18S37 and D18S40, located 9.4 and 3.2 centimorgans upstream of the ACTH-R (13, 19), respectively, which did not reveal LOH at these loci. The functional significance of our findings at the transcriptional level is supported by reduced steady state concentrations of ACTH-R mRNA found in these tumors compared to those in normal adrenals and adrenocortical tumors without LOH of the ACTH-R gene. One of 16 benign lesions in this study demonstrated LOH of the ACTH-R gene locus. This tumor differed from the other 15 adenomas in size, steroid activity, and histopathology. It was clinically and bio-
chemically nonfunctional, in contrast to adenomas without LOH of the ACTH-R, which were all hyperfunctioning aldo- sterone- or cortisol-producing adenomas. Histopathology dem- onstrated an oncocytic adenoma. Oncocytic adrenal cortical neoplasms are a rare variant of adrenocortical tumors charac- terized by large tumor cells with abundant finely granular eosi- nophilic cytoplasm filled with mitochondria (20, 21). Oncocytic changes can also be found in adrenocortical carcinomas (22), and close postoperative follow-up is required in patients with oncocytic tumors because of their potentially malignant behavior (20).
Two of four adrenocortical carcinomas showed LOH of the ACTH-R gene. The patients with carcinomas with LOH had advanced tumor stages, aggressive tumor growth, early re- currence after adrenalectomy, and an unfavorable outcome. This indicates that deletions of the ACTH-R gene in adre- nocortical carcinomas are associated with clonal expansion of undifferentiated and/or highly malignant tumor clones. LOH and microsatellite instability are important character- istics of many tumor types. These DNA deletions affect chro- mosomal areas of known or supposed tumor suppressor genes. Functional inactivation of the other allele of a tumor suppressor gene occurs generally by missense point muta- tions eliminating all wild-type tumor suppressor activity and enhancing clonal expansion of a malignant cell clone. LOH of the ACTH-R gene at 18p11.2 suggests that the ACTH-R may act as a tumor suppressor gene in adrenocortical tu- morigenesis. The clinical features of tumors with LOH in our series (loss of steroidogenesis in the oncocytoma, aggressive growth in adrenal carcinomas) is in accordance with this idea. We were not able to detect inactivating point mutations in the remaining ACTH-R allele. However, this does not necessarily exclude inactivation of the other allele, as mu- tations outside of the coding region, such as in the ACTH-R promoter, may have been missed by our approach. Evidence for functional inactivation of the ACTH-R by means other than mutations comes from the mouse adrenocortical tumor cell line Y1. In this cell line, ACTH and compounds such as the long-acting cAMP analog 8-bromo-cAMP stimulate ste- roidogenesis but inhibit cell proliferation (23). Schimmer et al. (24) reported two mutant subclones, Y6 and OS3, that do not express functional ACTH receptors, in contrast to the ACTH-sensitive parental cell line Y1. The ACTH-R gene transcription in these subclones is completely silenced by mechanisms not involving deletions or altered methylation of the ACTH-R gene. These data show that inactivation of the
ACTH-R mRNA expression (%)
140
120
100
mean+SEM
80
60
40
20
0
Normal Adrenals
Tumors without LOH
Tumors with LOH
ACTH receptor can also be caused by as yet unidentified transcription factors and cis-acting DNA promoter elements. Alternatively, deletion of one ACTH-R allele could be suf- ficient for oncogenic transformation, as has been suggested for other tumor suppressor genes. For example, mutations of the p53 tumor suppressor gene located at chromosome 17p affect only one allele in certain tumor types, such as basal cell carcinoma (25) and adrenocortical tumors (26). This can be explained by a dominant negative effect or a gain of function of the mutant p53 protein.
In summary, LOH of the ACTH-R gene and low expres- sion of ACTH-R mRNA are present in a subset of adreno- cortical tumors that were either nonfunctional or highly ma- lignant. These data suggest that deletion of a G-coupled receptor may give tumors a growth advantage. Under phys- iological circumstances, the ACTH-R-cAMP-protein kinase A signaling cascade maintains a differentiated adrenocortical cell phenotype, whereas proliferation of adrenocortical cells is stimulated mainly by peptides and receptors other than ACTH and its receptor. Partial deletion of the ACTH-R gene could, therefore, result in loss of differentiation and stimu- lation of a growth path.
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