ADRENOCORTICAL carcinoma is a rare, highly malignant

Transcription

1 X/97/$03.00/0 Vol. 82, No. 9 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1997 by The Endocrine Society 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 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 ADRENOCORTICAL carcinoma is a rare, highly malignant tumor with an incidence of 1.7/million. In contrast, benign adrenocortical lesions are very frequent (prevalence, 1/100). Most of these tumors are detected incidentally by ultrasound or computed tomography (so-called incidentalomas) (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 superfamily 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 chromosome 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 Received February 26, Revision received May 13, Accepted May 21, Address all correspondence and requests for reprints to: PD Dr. Martin Reincke, Schwerpunkt Endokrinologie, Medizinische Universitätsklinik Würzburg, Josef-Schneider-Strasse 2, Wurzburg, Germany. * This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Re 752/5 1). 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 flanking 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 expression 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, contributing to cellular dedifferentiation in adenomas and carcinomas. (J Clin Endocrinol Metab 82: , 1997) in thyroid adenomas (8). These data and evidence from in vitro experiments (9) suggested that ACTH was a differentiating factor of the adrenal cortex, with a low potential of stimulating cell proliferation and tumorigenesis. Thus, inactivation of the ACTH-R signal transduction cascade could result in loss of differentiation and enhanced clonal expansion of adrenal tumors. We recently identified a PstI polymorphism located approximately 3 kilobases (kb) upstream of the ACTH-R-coding 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 adrenocortical 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 according 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- 3054

2 DEPLETION OF ACTH-R GENE IN ADRENOCORTICAL TUMORS 3055 TABLE 1. Clinical data of the 16 adenoma patients informative for the PstI polymorphism in the ACTH receptor gene Patient no. Age (yr) Sex Clinical presentation Max. tumor size (cm) Histology 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 M APA 3.0 Adenoma M APA 3.2 Adenoma F APA 4.0 Compact cell adenoma F CPA 3.0 Adenoma F CPA 4.0 Adenoma F CPA 6.5 Adenoma Incidentaloma, 7.0 Oncocytic adenoma F nonfunctional F, Female; M, male; APA, aldosterone-producing adenoma; CPA, cortisol-producing adenoma. TABLE 2. Clinical data of the patients with adrenal carcinomas informative for the PstI polymorphism in the ACTH receptor gene Patient no. Age (yr) Sex Clinical presentation Max. tumor size (cm) Tumor stage Disease-free survival LOH of ACTH-R M Virilization 9 T2N0M0 15 months a 2 22 F Virilization 12 T2N0M0 15 months a 3 46 M Cushing s syndrome 8 T4N1M1 b 0 month c 4 79 F Conn s syndrome 9 T4N0M0 d 3 months e F, Female; M, male. a Follow-up with radiological imaging and endocrine evaluation every 3 months. b Tumor invasion of the vena cava, pulmonary and liver metastases. c Deceased after 5 months. d Tumor invasion of the vena cava. e Local tumor recurrence after 3 months, thereafter lost to follow-up. LOH of ACTH-R FIG. 1. Restriction map of the ACTH-R gene. 1, Nontranslated exon approximately 10 kb upstream of the ACTH-R coding region (19); 2, ACTH-R-coding region. plantation. After removing adjacent fat tissue, the tissues were snapfrozen 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 manufacturer (Boehringer Mannheim, Mannheim, Germany), the DNA was electrophoresed through a 0.8% agarose gel and blotted onto a nylon membrane (Amersham, Braunschweig, Germany). Hybridization was performed using an [ - 32 P]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 TAG 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 individuals 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 [ - 32 P]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 [ - 35 S]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 -actin cdna probe. The steady state

3 3056 REINCKE ET AL. JCE&M 1997 Vol 82 No 9 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 computer-based image analysis system (Stemmer, Puchheim, Germany) using the IMAGE program (NIMH, NIH, Bethesda, MD). Results Rate of heterozygosity in normal subjects Fifty-three of the 99 normal subjects (53.5%) were heterozygous 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 adenoma demonstrated LOH of the ACTH-R gene in the tumor tissue (Table 1 and Fig. 3). This tumor was incidentally detected by computed tomography and measured 7 cm in maximum diameter. The patient was clinically asymptomatic and had normal serum potassium levels, normal PRA, and normal suppression of serum cortisol by 2 mg dexamethasone. Surgery was suggested because of its size to exclude adrenocortical carcinoma, and the patient underwent adrenalectomy 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 carcinomas 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 microsatelite 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 FIG. 2.PstI polymorphism of the ACTH-R gene in six normal subjects. Heterozygote individuals (lanes 1, 4, and 5) are informative for LOH analysis. the D18537 or D18540 markers, demonstrating that the deletion 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 available 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 variety of endocrine tissues. Oncogenic transformation by constitutive 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). Adrenocortical tumorigenesis differs from pituitary and thyroid tumorigenesis, 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 innervation by the splanchnic nerve. ACTH in physiological concentrations does not stimulate cell proliferation of adrenocortical 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 -chain of the G s have been identified in benign or malignant adrenocortical tumors (8, 16). On the contrary, activating mutations of the G i2, one of the adenylyl cyclase inhibitory G proteins, were found in very few adrenocortical tumors, but not in a variety of other endocrine and nonendocrine tumors (16, 18). These data suggest that in the adrenal cortex the ACTH/G s /protein kinase A signaling pathway is preferentially important for steroid hormone secretion 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 associated 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 implications 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

4 DEPLETION OF ACTH-R GENE IN ADRENOCORTICAL TUMORS 3057 FIG. 3. LOH of the ACTH-R gene. Leukocytic (L) and tumor (Tu) DNA were digested, electrophorised, and hybridized, as described in Subjects and Methods. Center and right, Two tumors with LOH at the PstI restriction site. Left, Adrenal adenoma without LOH. FIG. 4. ACTH-R mrna expression in adrenocortical tumors assessed by dot blot. Polyadenylated mrna was hybridized with a 32 P-labeled full-length ACTH-R cdna (top) and a mouse b-actin cdna (bottom). Row 1, Two normal adrenals; row 2, two tumors with LOH at the PstI polymorphism; row 3, tumors without LOH (aldosterone-producing adenomas). 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 aldosterone- or cortisol-producing adenomas. Histopathology demonstrated an oncocytic adenoma. Oncocytic adrenal cortical neoplasms are a rare variant of adrenocortical tumors characterized by large tumor cells with abundant finely granular eosinophilic 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 recurrence after adrenalectomy, and an unfavorable outcome. This indicates that deletions of the ACTH-R gene in adrenocortical carcinomas are associated with clonal expansion of undifferentiated and/or highly malignant tumor clones. LOH and microsatellite instability are important characteristics of many tumor types. These DNA deletions affect chromosomal areas of known or supposed tumor suppressor genes. Functional inactivation of the other allele of a tumor suppressor gene occurs generally by missense point mutations 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 tumorigenesis. 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 mutations 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 steroidogenesis 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

5 3058 REINCKE ET AL. JCE&M 1997 Vol 82 No 9 FIG. 5. ACTH-R mrna expression (mean SEM) in normal adrenals, tumors without LOH of the ACTH-R gene, and 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 sufficient 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 expression of ACTH-R mrna are present in a subset of adrenocortical tumors that were either nonfunctional or highly malignant. These data suggest that deletion of a G-coupled receptor may give tumors a growth advantage. Under physiological 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 stimulation of a growth path. References 1. Ross NS, Aron DC Hormonal evaluation of the patient with an incidentally discovered adrenal mass. N Engl J Med. 323: Hough AJ, Hollifield JW, Page DL, et al Prognosis factors in adrenocortical tumors. Am J Clin Pathol. 72: Weiss LM Comparative histologic study of 43 metastasing and nonmetastasing adrenocortical tumors. Am J Pathol. 8: Mountjoy KG, Robbins LS, Mortrud MT, Cone RD The cloning of a family of genes that encode the melanocortin receptors. Science. 275: Siegrist W, Eberle AN Melanocortins and their implication in melanoma. Trends Endocrinol Metab. 6: Gantz I, Tashiro T, Barcroft C, et al Localization of the genes encoding the melanocortin-2 (ACTH hormone) and melanocortin-3 receptors to chromosome 18p11.2 and 20.q by fluorecent in situ hybridization. Genomics. 18: Vamvakopoulos NC, Durkin S, Nierman W, Chrousos GP Mapping the human adrenocorticotropin receptor gene to the small arm of chromosome 18 (18p pter). Genomics. 18: Latronico AC, Reincke M, Mendonca BB, et al No evidence for oncogenic mutations in the adrenocorticotropin receptor gene in human adrenocortical neoplasms. J Clin Endocrinol Metab. 80: Estivariz FE, Iturriza F, Mclean C, Hope J, Lowry PJ Stimulation of adrenal mitogenesis by N-terminal proopiomelanocortin. Nature. 297: Reincke M, Mora P, Beuschlein B, Lehmann R, Karl M, Allolio B No evidence for oncogenic mutations in the adrenocorticotropin receptor gene in human adrenocortical neoplasms. Exp Clin Endocrinol. 103(Suppl 1): Baxter JD, Tyrrel JB The adrenal cortex. In: Felig P, Baxter JD, Broadus AE, Frohman LA, eds. Endocrinology and metabolism, 2nd ed. New York: McGraw-Hill; Orth DN, Kovacs WJ, DeBold CR The adrenal cortex. In: Wilson JD, Foster DW, eds. Williams textbook of endocrinology, 8th ed. Philadelphia: Saunders; Berrettini WH, Ferraro TN, Goldin LR, et al Chromosome 18 DNA markers and manic-depressive illness: evidence for a susceptibility gene. Proc Natl Acad Sci USA. 91: Detera-Wadleigh SD, Yoon SW, Berrettini WH, et al ACTH receptor/ melanocortin receptor-2 maps within a reported susceptibility for bipolar illness on chromosome 18. Am J Med Genet. 60: Reincke M, Beuschlein F, Menig G, et al. ACTH receptor mrna expression in adrenocortical tumors: comparison with P450 steroidogenic enzyme expression. Clin Endocrinol (Oxf). 46: Lyons J, Landis CA, Harsh G, et al Two G protein oncogenes in human endocrine tumors. Science. 249: Parma J, Duprez L, Van Sande J, et al Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature. 365: Reincke M, Karl M, Travis W, Chrousos GP No evidence for oncogenic mutations in guanine nucleotide binding proteins of human adrenocortical neoplasms. J Clin Endocrinol Metab. 77: Naville D, Barjhoux L, Jaillard C, Lebrethon MC, Saez JM, Begeot M Characterization of the transcription start site of the ACTH receptor gene: presence of an intronic sequence in the 5 -flanking region. Mol Cell Endocrinol. 106: Erlandson RA, Reuter VE Oncocytic adrenal cortical adenoma. Ultrastruct Pathol. 15: Mitchell A, Scheithauer BW, Sasano H, Hubbard EW, Ebersold MJ Symptomatic intradural adrenal adenoma of the spinal nerve root: report of two cases. Neurosurgery. 32: Mackay B, el-naggar A, Ordonez NG Ultrastructure of adrenal cortical carcinoma. Ultrastruct. Pathol. 18: Schimmer BP, Tsao J, Knapp M Isolation of mutant adrenocortical tumor cells resistant to cyclic nucleotides. Mol Cell Endocrinol. 8: Schimmer BP, Kwan WK, Tsao J, Qiu R Adrenocorticotropin-resistant mutants of the Y1 adrenal cell line fail to express the ACTH receptor. J Cell Physiol. 163: Van der Riet P, Karp D, Farmer E, et al Progression of basal cell carcinoma through loss of chromosome 9q and inactivation of a single p53 allel. Cancer Res. 54: Lin SR, Lee YJ, Tsai JH Mutations of the p53 gene in human functional neoplasms. J Clin Endocrinol Metab. 78: