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Amplification of the Steroidogenic Factor 1 Gene in Childhood Adrenocortical Tumors

Department of Pediatrics, Center for Molecular Genetics and Cancer Research in Children (CEGEMPAC) (B.C.F., M.A.D.P., G.A.R.), Department of Pediatrics, Division of Pediatric Hematology and Oncology (M.A.D.P.), and Department of Pediatrics, Division of Pediatric Endocrinology (R.S., L.D.), Federal University of Paraná, Curitiba PR 80.030-110, Brazil; Department of Oncology and Institute for Molecular and Human Genetics/Lombardi Comprehensive Cancer Center (L.R.C., B.R.H.), Georgetown University, Washington, DC 20007; Institut de Génétique et de Biologie Moléculaire et Cellulaire Centre National de Ciencia y Tecnologia–Institut National de la Santé et de la Recherche Médicale (E.L.), Université Louis Pasteur, 67404 Illkirch, France; Departments of Hematology-Oncology (R.C.R.) and Biochemistry (G.Z.) and the International Outreach Program (R.C.R.), St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; and Department of Pediatrics (R.C.R.), University of Tennessee, College of Medicine, Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: Bassem R. Haddad, M.D., Institute for Molecular and Human Genetics, Georgetown University Medical Center, 3800 Reservoir Road NW, Main 4000, Washington, DC 20007. E-mail: haddadb1{at}georgetown.edu.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Southern Brazil has one of the highest incidences of childhood adrenocortical tumors (ACTs), occurring 10–15 times more frequently than worldwide estimates. The reasons for this increase remain elusive. In an attempt to further characterize the genetic changes in childhood ACTs, we recently detected a consistent gain of 9q (or a portion of it) in eight of nine cases of pediatric ACTs and amplification of 9q34 in the majority of these cases using comparative genomic hybridization. Other studies involving both childhood and adult ACTs have corroborated these findings. To follow up on these results, we examined whether the steroidogenic factor 1 (SF-1) gene, which is located in this chromosomal region and plays an important role in the development and function of the adrenal cortex is amplified in these ACT cases. We detected increased copy number of the SF-1 gene in all eight cases with 9q gain, suggesting an association between an increased copy number of the SF-1 gene and adrenocortical tumorigenesis.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
THE INCIDENCE OF childhood adrenocortical tumors (ACTs) is 10–15 times greater in Southern Brazil, particularly in the state of Paraná, compared with the rest of the world (3.4–4.2 cases/million in southern Brazilian states, compared with 0.3 cases/million worldwide) (1). The molecular basis of this disease remains poorly understood, despite several studies investigating the role of particular genes in adrenal tumorigenesis (2). Using comparative genomic hybridization (CGH), we recently evaluated a group of nine cases of pediatric ACTs from southern Brazil and identified a recurrent pattern of chromosomal aberrations. Gain of 9q or a portion of it emerged as the most consistent finding: eight of nine cases showed this gain, and a high level copy number increase (amplification) of 9q34 was detected in five of the eight cases (3). Interestingly, a similar study conducted in the United Kingdom showed gain/amplification of 9q34 in 10 of 11 childhood ACTs (4). Other studies of adult ACTs have also shown frequent gains involving that same chromosomal region (5). Chromosomal segments showing gain/amplification by CGH are generally sites of amplified genes, and gene amplification is one of the ways in which oncogene activation is achieved. Clearly identification of such genes and characterization of their mechanism of action and its timing is critical to better understand the process of tumorigenesis for a given tumor.

Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family of transcription factors and plays an important role in endocrine function, including the regulation of steroid hydroxylases and the transcription of an array of genes involved in reproduction, male sexual differentiation, and the development and function of the adrenal cortex (6, 7, 8, 9). SF-1 maps to 9q33.3, a chromosomal region that we and others (3, 4) identified as showing a gain/amplification in a high percentage of cases with childhood ACTs. Studies in SF-1 knockout mice have shown that the adrenal cortex became hypoplastic sometime after embryonic development (10, 11), and SF-1 was also shown to be essential for the residual adrenal gland growth after unilateral adrenalectomy (12). In humans, the adrenal cortex becomes hypoplastic after birth. Volume of the fetal zone of the human adrenal cortex rapidly involutes after birth decreasing from 70 to 3% of the total adrenal volume (13). The finding of 9q gain in both our previous work and in the study of James et al. (4) in childhood ACTs as well as in other studies of adult ACTs, prompted us to conduct the present investigation to identify potential candidate genes involved in childhood adrenocortical tumorigenesis. Because of the intense steroidogenic activity in most childhood cases of ACTs, we investigated SF-1 on 9q33.3 as one of the amplified candidate genes. We used fluorescence in situ hybridization (FISH) analysis to identify SF-1 amplification in the group of nine childhood ACTs previously analyzed using CGH.

Childhood ACTs, in contrast to ACTs from adults and many other types of cancer, usually occur in individuals with germline TP53 mutations (14). We previously reported a germline point mutation of TP53 encoding an R337H amino acid substitution combined with somatic loss of the second TP53 allele occurring in 35 of 36 childhood ACTs studied from southern Brazil (15). In addition, we evaluated 432 normal individuals from southern Brazil for the presence of the R337H TP53 mutation and none found to be a carrier (data not shown). TP53 maps to chromosome 17p13.1, and its protein consists of four distinct domains: an N-terminal transactivation domain, a central DNA-binding domain, a tetramerization domain (in which arginine 337 is located) and a C-terminal regulatory domain. Arginine 337 forms a salt bridge with aspartic acid 352 across the helix-helix interface to form the tetramer. For this reason, we also evaluated our samples for the presence of that common germline TP53 point mutation and the loss of the wild-type TP53 allele.

This is a follow-up study to our previous work and other studies showing that 9q is a hotspot for amplification in ACT. Because SF-1, which is localized to this region, plays an important role in the development and function of the adrenal cortex, here we investigate its amplification in ACTs.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patient selection

Nine patients with childhood ACTs previously evaluated by CGH (2) were selected for this study. All patients, six girls (aged 9–82 months) and three boys (aged 13–35 months), were referred to the Pediatric Endocrinology Unit of Hospital de Clínicas from Federal University of Paraná, Curitiba, PR, Brazil. A written informed consent, as approved by the Ethics Committee of the Hospital of Clinicas from the Federal University of Paraná, Curitiba, Brazil, was signed by one of the parents of each patient. Patient characteristics are described in Table 1. The tumors were classified as adenomas or carcinomas according to conventional pathological criteria, as previously described (1). Eight tumors were primary and one was a secondary local recurrent tumor (diagnosed almost 2 yr after the resection of the primary tumor). All patients presented with signs and symptoms due to elevated serum levels of androgens and/or glucocorticoids and were described as having functional tumors. Severity of the clinical manifestations was generally proportional to hormone levels and the time between the day of diagnosis and the first documentation of any sign of the disease. Five patients presented with only virilization, whereas the other four presented with virilization plus Cushing’s syndrome. Six of the tumors were classified as carcinoma, whereas three were adenomas (Table 1).

FISH

To evaluate the copy number of the SF-1 gene, we designed a FISH probe consisting of a bacterial artificial chromosome (BAC) clone containing sequences of the SF-1 gene: RP11–91G7 (BACPAC Resources, Oakland, CA). BAC clone DNA was prepared and labeled with biotin-11-deoxyuridine 5-triphosphate (Roche, Indianapolis, IN) using nick translation as previously described (16). Biotin-labeled DNA was detected with fluorescein-avidin DCS (Vector Laboratories Inc., Burlingame, CA). Map location of the BAC clone and the hybridization efficiency of the FISH probe as well as the number of FISH signals detected in normal diploid interphase nuclei were assessed in normal lymphocytes according to a standard FISH mapping protocol (17). Correct chromosomal location of the probe to chromosome 9q33.3 and the presence of two signals per diploid nucleus in more than 98% of the 200 interphase nuclei scored were confirmed.

For each tumor, a 4-µm tissue section was hematoxylin and eosin stained and histologically examined to confirm the presence of tumor tissue, and a consecutive 4-µm paraffin section was evaluated by FISH using the SF-1 FISH probe as described earlier (16). In brief, the tissue slide was deparaffinized with Xylene, rehydrated with decreasing ethanol concentrations, pepsin digested for 90 min, and then denatured in 70% formamide/saline sodium citrate (SSC) two times for 4 min at 80 C. After overnight hybridization with the probe at 37 C, the slide was washed three times in formamide/SSCx 2 (1:1) at 42 C, and three times in SSCx 1 at 42 C. The biotin-labeled probe was detected with avidin conjugated to fluorescein isothiocyanate (Vector Laboratories), and the cells were counterstained with 4',6-diamidino-2-phenylindole, and embedded in antifade solution [200 mM 1,4-diazabicyclo[2.2.2]octane, 90% vol/vol glycerol, 20 mM Tris-HCl (pH 8)] to reduce photo-bleaching. Scoring of cells and digital image acquisition were performed using a x63 objective mounted on a DMRBE microscope (Leica, Wetzlar, Germany) equipped with optical filters for 4',6-diamidino-2-phenylindole and Cy3 (Chroma Technologies, Brattleboro, VT) and a cooled charge-coupled device camera (Photometrics, Tucson, AZ). The IPLab software package (Scanalytics Inc., Fairfax, VA) was used for the initial gray-scale image acquisition and consecutive color processing. Considering artifacts and loss of genomic contents in partially cut nuclei, the following scoring system was used: FISH signals in 50 cells for each specimen were counted. Detection of three or more signals in at least 30% of the nuclei with detectable signals was considered as increased copy number per gain. Furthermore, the gene was considered to be amplified in those cases in which 30% or more of the cells showed four or more copies by FISH analysis. In the absence of copy number gain, the presence of two FISH signals per cell in at least 50% of the nuclei was considered as normal diploid.

Estimation of R337H TP53 mutation

Four of the nine patients had the entire TP53 gene sequenced using DNA prepared from their lymphocytes and were found to have the R337H amino acid substitution (15). The presence of the same mutation was evaluated in the remaining five patients using a previously described PCR/HhaI-based assay (15). Loss of heterozygosity (LOH), described as loss of the wild-type TP53 allele and presence of the R337H TP53 mutation in the tumor genome, was studied in eight cases as described (15).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
SF-1 FISH analysis

FISH analysis using the SF-1-specific probe showed increased copy number in eight of the nine specimens, whereas one specimen failed to exhibit copy number gain (majority of cells showing two copies of the gene). Of the eight patients with increased copy number of the SF-1 gene, six showed four or more copies of the gene in at least 30% of the cells and therefore were considered to have SF-1 amplification by FISH. There was an excellent correlation between the CGH findings from our earlier study (3) and the FISH analysis: all eight patients with an increased copy number of the SF-1 gene by FISH had a gain of 9q (or a portion of it) by CGH, whereas the patient with two copies of SF-1 by FISH did not show 9q gain by CGH. In addition, five of the six patients with four or more copies of the SF-1 gene (i.e. SF-1 amplification as detected by FISH) were previously found to have an amplification of the 9q34 chromosomal segment by CGH. Amplification of the gene was detected both in the adenomas (two of three cases) and the carcinomas (three of six cases); it was also detected in cases presenting with virilization only (four of five cases) or both virilization and Cushing’s syndrome (one of four cases). Of note, we did not observe copy number gain in the normal areas of the tissue sections. Table 1 summarizes the FISH and CGH data. Examples of SF-1 FISH studies are shown in Fig. 1 (Fig. 1A shows a case with increased copy number of the SF-1 gene, and Fig. 1B shows case 5 with mostly two copies of the gene).

View larger version (22K): [in this window] [in a new window]   FIG. 1. A, Representative image of SF-1 FISH evaluation of a tumor sample (patient 6) showing mostly amplification. B, Tumor sample with two copies of the gene (patient 5).

The germline R337H mutation was detected in seven of the nine patients, and the corresponding tumors showed LOH at the TP53 locus, similar to our findings in an earlier study (15). In one patient (patient 9), only tumor DNA was available, which also revealed the presence of the R337H mutation. No DNA was available from normal tissues from this patient, and we were therefore unable to determine whether the mutation was present in the germline, nor could we confirm LOH at the TP53 locus in the tumor. Patient 4, a 23-month-old boy with virilization and Cushing’s syndrome, did not harbor the germline R337H TP53 mutation as was found in the other patients. However, we cannot rule out the possibility of a different genetic variant in the TP53 gene.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Studies of ACTs in both adults and children have shown that these tumors are associated with a large variety of chromosomal alterations, high-level amplifications, and losses and gains of DNA material from different chromosomal regions (18, 19, 20, 21). However, despite this genetic heterogeneity, these studies have also shown that certain regions of the genome show consistent alterations. 9q34 is among the regions that have been shown to be gained/amplified in both childhood and adult ACTs, thus justifying further investigation (3, 4, 5). The orphan nuclear receptor SF-1, which plays a key role in endocrine function, maps to 9q33.3. This region is in very close proximity to the common 9q34 amplicon in ACTs. The multiple studies that have described this amplicon, including ours (3, 4, 5), have all used conventional chromosomal CGH. However, because of the lower resolution of conventional chromosomal CGH, this method is incapable of accurately delineating the exact boundaries of amplicons; hence, it is very possible that the previously described 9q34 amplicon may include the 9q33.3 region and thus the SF-1 gene (22).

In the present study, we investigated the possible gain/amplification of the SF-1 gene in childhood ACTs. We used FISH to detect copy number changes of the gene in a cohort of nine tumors that we had previously evaluated using CGH. These cases included six carcinomas and three adenomas. CGH showed chromosomal aberrations in all nine cases. But despite the high number of alterations, we observed a recurrent pattern; the most striking finding was a consistent copy number gain of chromosomal region 9q34 in eight of the nine tumors (3). A similar finding of 9q34 gain was independently observed in other studies of both childhood (4) and adult ACTs (5). For instance, James et al. (4) reported a gain of 9q34 in 10 of 11 childhood tumors (nine adenomas and two carcinomas), and Dohna et al. (5) observed a gain involving the 9q34 region in 12 of 25 samples (eight adenomas, 14 carcinomas, one metastatic lymph node, and two cell lines). Our FISH analysis was in full agreement with our prior CGH findings. Specifically, all eight patients who showed copy number gain of 9q or a portion of it including 9q34 showed increased copy number of the SF-1 gene by FISH. The only subject that showed no gain of that region by CGH (patient 5) showed no gain of the SF-1 gene by FISH (Table 1). Our study does not address the question regarding which portion of the SF-1 gene is duplicated. However, we are in the process of collecting fresh-frozen ACT tissues to investigate this point in a future study.

Unraveling the complexity behind 9q34 amplification may provide a role that the expression of an amplified gene could play in the pathogenesis of ACTs. We originally hypothesized that the 9q34 amplification detected in our study (3) could indicate the site of an oncogene(s) involved in ACT formation, and thus we proposed that amplification of 9q34 could involve aberrant transcripts of the ABL1 oncogene; this possibility was ruled out using Southern blot analysis (data not shown). However the SF-1 gene may not be the only amplified gene in the 9q34 amplicon. Other genes mapped to 9q34 that may play a role in ACT tumorigenesis include the VAV2 oncogene (23); the TGFß receptor-1 gene, which is an activin A receptor type II-like kinase (24); the TNF receptor-associated factor-2 gene (25); the oncogene 24p3 or lipocalin 2 gene (26); and the retinoid X receptor-.

One consequence of the SF-1 gene amplification, particularly if it is associated with high expression of the SF-1 protein, could be an increased steroid hydroxylase activity and steroid production. A recent review of 254 children enrolled in the International Pediatric Adrenocortical Tumor Registry (27) showed that signs of virilization were found in 84.2% of patients, consistent with those of other reports from other continents (27, 28, 29, 30, 31). Virilization was observed either alone or accompanied by clinical manifestations of the overproduction of other adrenocortical hormones, including glucocorticoids, androgens, aldosterone, or estrogens. In our study, all patients presented with virilization, and four of the nine had an associated increase in cortisol levels and Cushing’s syndrome.

The pivotal role of the SF-1 gene in controlling the development of the adrenal cortex is well known from studies of the phenotype of SF-1 null mice (10) and human subjects harboring heterozygote SF-1 mutations (32). A direct role of SF-1 in controlling adrenal cell growth is suggested by recent data showing that liver receptor homolog-1, an orphan nuclear hormone receptor closely related to SF-1, is involved in cell cycle control in the intestine through regulation of G₁ cyclin genes (33). In the adrenal cortex, the R337H p53 mutant protein may exert a tissue-specific effect inducing genomic instability in the adrenal cortex. In this context, cells harboring amplification of the SF-1 gene may acquire selective growth advantage, thus escaping from normal mechanisms of growth control. This, together with the accumulation of other genetic lesions, may lead to tumor formation. In the present study, the germline R337H TP53 mutation and the somatic loss of the wild-type TP53 allele were found in seven of the nine patients studied. The most striking association between germline TP53 mutations and cancer is found in cases of childhood ACTs (14). We believe that the germline R337H TP53 mutation combined with LOH (15) under certain conditions of pH and temperature (34) may occur during the development of the adrenal cortex and thus generate ACTs. Of the eight tumors with SF-1-increased copy number by FISH, six had the germline R337H TP53 mutation and somatic loss at the TP53 locus (wild-type allele) in the tumor, and one (patient 9) had the R337H TP53 mutation in the tumor (this subject was not tested for germline mutation). The only subject with normal copy number of the SF-1 gene also showed the TP53 germline mutation and LOH. Chromosomal instability caused by TP53 dysfunction is well documented; in addition, the loss of the wild-type TP53 was shown to predispose to gene amplification and altered cell cycle arrest (35). We believe that the germline R337H TP53 mutation and the somatic loss of the wild-type allele can contribute to genomic alterations and increased SF-1 copy numbers. Further studies are needed to clarify whether the TP53 mutation and 9q34 amplification, including SF-1 amplification, are part of a cascade of events leading to ACT oncogenesis, similar to what was reported in other tumors, such as in the multistep progression of colorectal cancer (36).

This is the first study showing an association between an increased copy number of the SF-1 gene and ACTs. The significance of this observation needs to be further investigated using a larger cohort of both pediatric and adult subjects. Because this gene is detected at the earliest stages of development of the urogenital ridge in which both the gonads and the adrenal cortex are derived and because this expression is maintained throughout development up until adult life (8), further studies examining the mechanisms and consequences of the amplification of this gene in ACTs are critical to a better understanding of the onset and progression of this disease.

	Acknowledgments

 
We thank Drs. Monica Nunes Lima Cat and Janice D. Rone for their scientific input and valuable discussions while preparing the manuscript.

	Footnotes

 
First Published Online November 16, 2004

Abbreviations: ACT, Adrenocortical tumor; BAC, bacterial artificial chromosome; CGH, comparative genomic hybridization; FISH, fluorescence in situ hybridization; LOH, loss of heterozygosity; SF-1, steroidogenic factor 1; SSC, saline sodium citrate.

This work was supported by Cancer Center Support Grants CA-21765 and CA-71907 from the National Institutes of Health (Department of Health and Human Services); a Center of Excellence grant from the State of Tennessee; the Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil; Fundacao Araucaria, Curitiba, Parana, Brazil (01/2001); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Comité Français d’Evaluation de la Coopération Universitaire avec le Brésil Grant 419/03; and the American Lebanese Syrian Associated Charities.

Received May 24, 2004.

Accepted November 8, 2004.

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