DAN ROSENBERG, LIONEL GROUSSIN, ERIC JULLIAN, KARINE PERLEMOINE, SAMIR MEDJANE, ALBERT LOUVEL, XAVIER BERTAGNA, AND JÉRÔME BERTHERAT

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1 X/03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(8): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /jc Transcription Factor 3,5 -Cyclic Adenosine 5 - Monophosphate-Responsive Element-Binding Protein (CREB) Is Decreased during Human Adrenal Cortex Tumorigenesis and Fetal Development DAN ROSENBERG, LIONEL GROUSSIN, ERIC JULLIAN, KARINE PERLEMOINE, SAMIR MEDJANE, ALBERT LOUVEL, XAVIER BERTAGNA, AND JÉRÔME BERTHERAT Department of Endocrinology (D.R., L.G., E.J., K.P., S.M., X.B., J.B.), Institut Cochin, Institut National de la Santé et de la Recherche Médicale U576, René Descartes-Paris V University, Paris, France; Internal Medicine Unit (D.R.), École Nationale Vétérinaire d Alfort, Maisons Alfort; Pathology Laboratory (A.L.), CHU Cochin, Paris, France; and COMETE Network (L.G., J.B., A.L., X.B., J.B.), France Various cellular and molecular alterations of the camp pathway have been observed in adrenal Cushing syndrome. We recently reported the loss of camp-responsive element-binding protein (CREB) expression in the adrenocortical cancer cell line H295R. CREB is the major nuclear target of the camp pathway. This study therefore aimed to analyze the status of the CREB protein in various types of human adrenocortical tumors and normal fetal adrenal cortex. CREB protein status was studied by Western blotting in adrenocortical adenomas (AAs, n 27) and adrenocortical carcinomas (ACs, n 24). A decrease of CREB protein was noticed in the majority of the adrenocortical tumors. The dramatic decrease in CREB protein levels was more pronounced in ACs than in AAs. Levels of the phosphorylated form of CREB were also low in adrenocortical tumors, with a greater decrease in ACs than in AAs. EMSAs also showed decreases in the amounts of CREBcontaining complexes in nuclear extracts from adrenocortical THE camp SIGNALING pathway plays a key role in various physiological functions of the adrenal cortex. The pituitary peptide hormone ACTH is the main regulator of adrenocortical function. This hormone is thought to control the proliferation, differentiation, and secretory properties of the adrenal cortex during fetal development and adulthood by stimulating intracellular camp synthesis (1 3). The stimulation by ACTH of camp production is also required for the expression of steroidogenic enzymes (4). Some causes of hypercortisolism (Cushing syndrome) are associated with overactivation of the camp pathway. Such overactivation may occur in normal adrenal glands if an extracellular signal stimulates the camp pathway, as in ACTH-dependent Cushing syndrome (Cushing disease or ectopic ACTH secretion). It may also occur in diseased adrenal glands, as in ACTH-independent macronodular adrenal hyperplasia, in which illicit hormone receptor expression, particularly for the gastric inhibitory peptide receptor, results in ACTH-independent Cushing syndrome (5, 6). The Abbreviations: AA, Adrenocortical adenoma; Ab, antibody; AC, adrenocortical carcinoma; AU, arbitrary unit; CD, Cushing disease; CREB, camp-responsive element-binding protein; HRP, horseradish peroxidase; NAC, normal adrenal cortex; P, pheocromocytoma; P-CREB, phosphorylated CREB; SF-1, steroidogenic factor 1. tumors. The secretory status of adenomas was strongly correlated with CREB levels, significantly lower in nonfunctioning AAs (n 9) than in functioning AAs (n 9). CREB levels, determined by Western blotting and immunohistochemistry, were very low in the fetal zone of human fetal adrenal cortex, whereas they were normal in the definitive zone. In tumors, adrenocortical cells in several zones were weakly immunohistochemically stained for CREB, whereas CREB was uniformly detected in nonendocrine cell nuclei (e.g. vascular cells, fibroblasts). These results suggest that the absence of CREB may be linked to the development of a highly aggressive tumor with a dedifferentiated benign (nonfunctioning AA) or malignant (AC) phenotype. These findings highlight the similarities between the normal human fetal adrenal gland and adrenal cancers previously observed in terms of parallelism in IGF-II production. (J Clin Endocrinol Metab 88: , 2003) intracellular camp pathway may also be constitutively overactivated because of the activating mutation of G s protein in McCune-Albright syndrome (7, 8). Finally, an inactivating heterozygous germline mutation in the protein kinase A regulatory subunit R1A (PRKAR1A) was recently identified in patients with Carney complex, an autosomal dominant disease in which Cushing syndrome may occur secondarily to primary pigmented nodular adrenocortical disease (9, 10). Somatic inactivating mutations of PRKAR1A have also been observed in cases of isolated primary pigmented nodular adrenocortical disease and in sporadic adrenocortical adenomas (AAs) (11, 12). Interestingly, all these diseases are generally benign, malignancy rarely being associated with camp pathway activation in the adrenal cortex. The transcription factor camp-responsive element-binding protein (CREB) plays a central role in the regulation of transcription by camp. CREB is important for cellular differentiation in various cell types. For example, the expression of a constitutively active CREB in preadipocytes in vitro induces adipogenesis, whereas the expression of a dominantnegative mutant of CREB blocks this process (13). In vivo, the expression of a dominant-negative CREB mutant reduces the ability of thymocytes to produce cytokine in response to immunization (14). CREB has also been shown to play a role 3958

2 Rosenberg et al. CREB in Adrenocortical Tumors J Clin Endocrinol Metab, August 2003, 88(8): in differentiation in endocrine tissues, such as thyroid and pituitary (12, 15, 16). Development of malignancy is characterized by progressive dedifferentiation. Very few of the molecular mechanisms involved in adrenocortical tumorigenesis have been identified to date. IGF-II has been shown to be overexpressed in adrenal carcinomas (17, 18), and the overexpression of IGF-II (resulting from maternal 11p15 loss of heterozygosity and duplication of the active paternal IGF-II allele) is very frequent in adrenocortical cancers (19). High levels of IGF-II expression have also been observed in fetal adrenal glands (20). The combination of these observations suggests that malignant tumors may have dedifferentiated to a fetal state. The loss of CREB expression recently reported in the human adrenocortical cancer cell line H295R (21) suggests that changes in the camp signaling pathway within the nucleus may be involved in malignant transformation in the adrenal cortex. In this study, we investigated whether the cellular dedifferentiation occurring during adrenocortical tumorigenesis was associated with a decrease in CREB levels. We found that CREB protein levels were lower in adrenocortical tumors than in nontumoral adrenal cortex (adrenocortical tissue from patients with Cushing disease) or adrenal tumors originating from the medulla (pheochromocytomas). This difference was more marked in malignant adrenocortical tumors and in nonfunctioning benign adrenocortical tumors than in cortisol-secreting adenomas. The very low levels of CREB protein levels in malignant and nonfunctioning adrenocortical tumors are reminiscent of the situation in the transitory fetal zone of the adrenal fetal cortex, in which no CREB signal was detected. This study suggests that changes in the camp pathway within the nucleus, involving a decrease in CREB protein levels, occur during the dedifferentiation process leading to malignant transformation of the adrenal cortex. Materials and Methods Tissue collection Adrenal tumors and tissues were obtained during surgery and immediately dissected by the pathologist, frozen, and stored in liquid nitrogen until use. Adrenal tumors were diagnosed on the basis of classical pathological criteria and with molecular genetics markers, as previously reported by the COMETE network (19). Normal adrenal cortex tissue was obtained from normal glands (adjacent to nonadrenal tumors) and the adjacent cortex of nonsecreting adenomas. Informed consent was given for adrenal tissue collection as part of a protocol approved by the Institutional Review Board of the Cochin Hospital. Fetal adrenal glands (from fetuses aged from 12 to 36 wk) were obtained after autopsy, with the informed consent of the mother. Western immunoblotting Nuclear extracts were prepared from frozen tissue samples as previously described except that okadaic acid (1 mmol/liter) was added to the buffers (22). Protein assays were performed in duplicate, with a kit from Bio-Rad Laboratories, Inc. (Hercules, CA). Proteins were resolved by SDS-PAGE in a 10% polyacrylamide gel and transferred to nitrocellulose sheets. We loaded the equivalent of 20 g protein onto the gel for each nuclear extract. The membranes were probed with antibody (Ab) 9192, a commercially available antiserum directed against a synthetic fragment of the protein (New England Biolabs, Beverly, MA). The binding of this antibody was detected by incubating the membrane with a horseradish peroxidase (HRP)-conjugated secondary antibody. Alternatively, we used Ab 253 directed against the full-length CREB protein (23) as the primary antibody and the Immun-Star goat antirabbit IgG detection kit (Bio-Rad Laboratories, Inc.). Assessment of the phosphorylated form of CREB on same extracts were performed using the commercial antiserum Ab 9191, directed against a phosphorylated synthetic fragment containing Ser 133 (New England Biolabs) or Ab 5322, directed against a phosphorylated CREB peptide containing residues (23). These antibodies were detected in a similar manner to the antibodies directed against nonphosphorylated CREB. For Western blots performed on fetal material, steroidogenic factor 1 (SF-1) was detected by incubation with a commercial polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) followed by a HRP-coupled secondary antibody. Immunoreactive proteins were visualized with the Amersham enhanced chemiluminescence system (Amersham, Ailsbury, UK). The blots were then exposed to x-ray films (BioMax MR, Eastman Kodak Co., Rochester, NY), and signal intensity was measured by scanning densitometry and expressed in arbitrary units (AUs), using the public domain NIH Image program ( (Macintosh system 9.1, Capertino, CA). To facilitate comparisons between experiments, we loaded equivalent amounts of recombinant CREB (4 ng) as an internal control for each gel. For each extract, signal intensity was adjusted according to the signal for recombinant CREB. Bacterial recombinant CREB, Ab 253, and Ab 5322 were generously provided by M. Montminy (La Jolla, CA). EMSAs Gel mobility shift assays were carried with nuclear extracts from H295R and DMS79 from normal adrenal cortex adjacent to nonsecreting tumors and adrenocortical tumors with high and low levels of CREB protein. The nuclear extracts were incubated in binding buffer [20 mmol/liter HEPES, 0.2 mmol/liter ethyl-enediaminetetraacetate, 0.1 mmol/liter NaCl, 0.1 mmol/liter KCl, 10 mmol/liter MgCl 2, 200 mg/ml BSA, 35% glycerol, and 5 mg poly(di-dc)] as previously described (21). We added labeled probe (5 fmol) to this mixture, which contained an amount of nuclear extract equivalent to 5 mg of protein, and incubated the mixture at room temperature for 30 min. Complexes were resolved by electrophoresis in a 6% nondenaturing polyacrylamide gel using an electrophoresis Tris-borate EDTA 0.53 buffer. The gel was dried, and protein-dna complexes were visualized by autoradiography, using BioMax MR film (Eastman Kodak Co.). The synthetic annealed oligonucleotide was end labeled with T4 polynucleotide kinase and [ - 32 P]ATP. The sequence of the human CG CRE oligonucleotide probe was as follows: 59-GAT CCG GCT GAC GTC ATC AAG CTA-39 (24). Immunohistochemistry Paraffin-embedded sections (7 m) were dewaxed in xylene and rehydrated by incubation in a graduated series of ethanol solutions, ending with water. Endogenous peroxidase was inhibited by incubating sections for 30 min with 1% hydrogen peroxide. Antigen retrieval was performed by microwaving sections in 0.01 m citrate buffer, ph 6.0, for 20 min at 800 W. Tissue sections were incubated for 1hatroom temperature with 5% newborn calf serum in PBS. Sections were then incubated overnight at 4Cinahumidified chamber with CREB antibody (Ab 9192, New England Biolabs) diluted 1/100 in PBS containing 5% newborn calf serum. For negative controls, the primary antibody was replaced with PBS. The sections were rinsed three times in PBS and incubated for 10 min with a HRP-conjugated secondary polymer antirabbit antibody (PicTure Rabbit, Zymed Laboratories, San Francisco, CA). Sections were rinsed in PBS, and the HRP label was detected with a 1/50 dilution of biotinyl tyramide in 1 amplification diluent (TSA biotin system, NEN Life Science Products, Boston, MA). The deposited biotin was detected with a streptavidin-hrp complex diluted 1/100 in TNB blocking buffer (0.1M Tris HCL, PH7.5, 0.15 m NaCl, 0.5% blocking reagent) (TSA biotin system, NEN Life Science Products). To visualize the HRP label, samples were incubated with 0.5 g/liter diaminobenzidine, 1/6000 hydrogen peroxide, and 1/6000 nickel chloride (Amersham). Contiguous sections of the specimens were stained with hematoxylin and eosin for morphological assessment of the adrenal tissue. Immunostained and hematoxylin and eosin-stained sections were examined under an Eclipse E800 microscope (Nikon), and digital images were obtained with a Photometrics, cooled CCD camera (digital camera

3 3960 J Clin Endocrinol Metab, August 2003, 88(8): Rosenberg et al. CREB in Adrenocortical Tumors DXM1200, Nikon, Inc., Melville, NY), driven by ACT-1 software, version 2.10 (Nikon, Inc.). Statistical analysis Western blot data are expressed as means sem. The significance of differences between groups of tumors was determined by ANOVA, with Statview (Abacus Concepts, Berkeley, CA) 5.0 software (Macintosh system 9.1). Values of P 0.05 were considered significant. Results Levels of CREB protein are low in dedifferentiated adrenocortical tumors Twenty-seven AAs and 24 adrenocortical carcinomas (ACs) obtained from 51 patients in surgery were studied by Western blot analysis. The clinical, pathological, and hormonal data for the patients are summarized in Table 1. Two different Abs, Ab 253 (directed against the whole CREB protein) and Ab 9192 (directed against a synthetic CREB peptide), gave similar results (Fig. 1A): Although variable, the signal obtained was stronger in adenomas than in carcinomas. For comparison, CREB signal in cortical tissue samples from 16 patients with Cushing disease (CD) and 18 patients with pheocromocytomas (P) vas evaluated. Strong signals, similar to the higher signals for adrenocortical adenomas, were obtained with all these tissue samples observed in all the CD and P samples (Fig. 1A). The intensity of the CREB signal was quantified for analysis with Ab 253, and significant differences were found between AA (n 15) and AC (n 14). The mean normalized CREB signal in tumors was AU in AA and AU in AC (P 0.01). CREB signal in AAs was comparable to CREB signal in nine normal adrenal cortex used as control ( AU) (Fig. 1B). We investigated whether the variation of CREB protein levels within malignant and benign adrenocortical tumors was associated with parallel changes in the active, phosphorylated form by carrying out immunodetection of phosphorylated CREB (P-CREB) in the same extracts with Ab 5322, an antibody directed against a phosphorylated CREB peptide containing residues This antibody reacts specifically with the protein kinase A phosphorylated form of CREB on Ser 133. The mean ratio of P-CREB signal to recombinant CREB protein signal was AU in AA and AU in AC (P 0.02) (Fig. 1B). We investigated the presence of CRE-binding proteins by EMSAs with a probe containing the CRE palindromic consensus sequence of the CG promoter ( CG CRE probe, Fig. 2). A CREB-containing complex (indicated by the arrow) was retrieved in nuclear extracts from the CREB-expressing cell line DMS79, whereas no such complex was detected with the CREB-deficient H295R adrenocortical cell line (Fig. 2). The CREB-containing complex was also present in nuclear extracts corresponding to two normal adrenal cortex samples (NACa, NACb). In four adrenocortical tumors with low levels (adrenal carcinomas AC1, AC2) or high levels (adrenal adenomas AA1, AA2) of CREB protein, as shown by Western blotting, we observed variation of patterns of CREB complexes parallel to CREB protein status. TABLE 1. Histological, clinical, and hormonal data from patients with adrenocortical tumors Patient no. Diagnosis Sex Age (yr) Secretion Tumor weight (g) 1 AA F 42 G 11 2 AA F 36 G 13 3 AA F 69 G 25 4 AA F 50 G 45 5 AA F 30 G 16 6 AA F 51 G 46 7 AA F 44 G 15 8 AA F 55 G 10 9 AA F 36 G 8 10 AA F 36 G AA F 57 G AA F 48 G AA F 34 G AA F 45 G AA F 42 G AA F 52 NF AA M 56 NF AA M 41 NF AA F 68 NF AA F 38 NF AA F 25 NF AA F 38 NF AA F 64 NF AA F 70 NF AA F 64 NF AA F 37 NF AA M 68 NF AC F 27 A AC F 63 A AC F 77 A AC F 26 A AC F 29 A AC F 15 A AC M 53 G AC F 80 G AC F 52 G AC M 30 G AC M 63 G AC F 28 G AC M 39 G AC F 31 G/A AC F 51 G/A AC M 72 G/A AC F 53 G/A AC F 44 G/A AC F 30 G/A AC F 40 G/A AC F 26 G/A AC F 15 G/A 50 a 50 AC F 53 G/A/M AC F 68 NF 230 F, Female; M, male; G, glucocorticoid secretion; A, androgen secretion; G/A, glucocorticoid and androgen secretion; G/A/M, glucocorticoid, androgen, and mineralocorticoid secretion; NF, nonfunctioning tumor. a Tumor weight (of local recurrence). Loss of secretory activity in AAs is associated with a decrease in CREB content We investigated whether the variability of CREB levels in AAs was related to cortisol secretion by comparing Western blots for nine hypersecreting adenomas with those for nine nonfunctioning adenomas (Fig. 3). The CREB signal was significantly weaker in nonsecreting adenomas than in hypersecreting adenomas ( AU vs AU,

4 Rosenberg et al. CREB in Adrenocortical Tumors J Clin Endocrinol Metab, August 2003, 88(8): FIG. 1. CREB protein in adrenocortical tumors. A, Western immunoblot analysis of CREB in adrenocortical tumors (ACs and AAs) using CREB Abs 253 or 9192 as described in Materials and Methods. CREB recombinant protein (4 ng*) was used as a control. CREB levels were lower in most ACs and a subset of AAs than in adrenal tissues from patients with CD and medulloadrenal tumors (P), in which CREB levels were constantly high. B, Quantification of CREB levels in adrenocortical tumors, as assayed by Western blot analysis. Western blots for CREB were performed with Ab 253 (open circles) and those for Ser133- phosphorylated CREB were performed with antibody 5322 (closed circles). The autoradiographs were quantified as described in Materials and Methods. The results are expressed in arbitrary units. Quantification of CREB levels in nine normal cortex extracts (five specimens adjacent to nonadrenal tumors and four specimens adjacent to nonsecreting adenomas signaled by an arrowhead) is presented as control. The mean CREB and Ser133-phosphorylated CREB protein content for each group is indicated by a dashed horizontal line. P 0.02). CREB signal in hypersecreting adenomas was comparable to CREB signal in cortical tissue samples from 16 patients from CD used as control ( AU) (Fig. 3B). We carried out P-CREB immunoblot on the same extracts, using the commercially available anti-p-creb antibody Ab The results followed a similar pattern, but the difference was not significant: The mean ratio of P-CREB signal to recombinant CREB recombinant protein signal was AU for nonfunctioning adenomas and AU in functioning adenomas (Fig. 3B). CREB is absent from the fetal zone of human fetal adrenal cortex We evaluated CREB protein levels in normal human adrenal cortex by CREB immunoblot analysis of adult adrenal cortex (adjacent to nonsecreting adrenocortical tumors) and fetal adrenal cortex (without dissection of fetal and definitive zones) samples. The CREB signal was strong and of similar intensity for all samples from the adult adrenal cortex (Fig. 4A). By contrast, the CREB signal was weak in fetal adrenal cortex (intermediate between the signal observed in normal adrenal cortex and the absence of signal noted in the CREBdeficient cell line H295R). We investigated whether the fetal and definitive zones contained different amounts of CREB by carrying out Western blot analysis with nuclear extracts of fetal zone and definitive zone, macroscopically dissected from adrenal glands from human fetuses 12 wk and 36 wk into gestation (Fig. 4B). The CREB signal was strong in the definitive zones of both fetuses, whereas it was barely detectable in the fetal zones. By contrast, SF-1 protein content was similar in the various zones of the fetal adrenal cortex. A strong difference in the intensity of the CREB signal between the fetal and definitive zones was also observed in experiments involving immunohistochemical staining (Fig. 5A). Most of the adrenocortical cells in the definitive zone were strongly labeled for CREB. Little or no CREB immunostaining was observed in any of the endocrine cells of the fetal zone, whereas a strong signal was observed for non endocrine cells within the fetal zone. CREB immunohistochemical signal is heterogeneous in adrenocortical tumors We then investigated whether the decrease in intensity of the CREB signal observed in most of the ACs and silent adenomas concerned part of, or the totality of, the tumor. We carried out immunohistochemical staining for the CREB protein in six AAs, five ACs, and two normal adrenal glands (adjacent to nonadrenal tumors). Almost all of the cells in the normal adrenal glands displayed strong nuclear immunostaining for CREB (Fig. 5A). In adrenocortical tumors, the CREB signal intensity was low (four adenomas, one carcinoma) or almost undetectable for the whole specimen (three carcinomas, one adenoma) and highly variable (three carcinomas, two adenomas), with very little or no CREB immunostaining, in numerous zones (Fig. 5B). No morphological difference was observed between labeled and unlabeled territories within the same specimens. The CREB immunolabeling decrease contrasted with the positive staining of nonendocrine cells (vascular and hematopoietic) within the same

5 3962 J Clin Endocrinol Metab, August 2003, 88(8): Rosenberg et al. CREB in Adrenocortical Tumors FIG. 2. EMSA of CREBs in adrenocortical tumors with high and low levels of CREB. An oligonucleotide corresponding to the CG CRE was used to study CRE binding to proteins in nuclear extracts from ACs (AC1, AC2) with low levels of CREB and AAs (AA1, AA2) with high levels of CREB and in nuclear extracts from normal adrenal cortex adjacent to nonsecreting tumors (NACa, NACb). Nuclear extracts from CREB-deficient (H295R) and CREB-expressing (DMS79) cell lines were used as controls. Unlabeled CG CRE probe ( CG) was added (300-fold molar excess) to DMS79 nuclear extract for competition. The arrow shows a complex previously demonstrated by supershift in DMS79 to contain CREB (21). zones, and the strong nuclear labeling of the majority of cells from adjacent positive zones. Discussion The molecular changes leading to the development of adrenocortical tumors have not yet been described in detail. Allelic losses at 11p15 and 17p13, associated with the overproduction of IGF-II, have been observed specifically in malignant adrenocortical tumors. Various cellular or molecular changes leading to activation of the camp pathway have been observed in benign adrenocortical tumors and hyperplasia associated with cortisol oversecretion, but such changes are rare in malignant adrenocortical tumors. We previously demonstrated the loss of CREB expression in the H295R cell line, which was derived from a malignant adrenocortical tumor (21). This finding was later confirmed by others (25). Because CREB is the most distal (i.e. nuclear) actor of the camp pathway, we studied this transcription factor in various adrenocortical tumors. We observed a strong decrease in CREB protein levels in two types of adrenocortical tumor: nonsecreting adenomas and carcinomas. Both these types of tumor display a partial loss of differentiation characteristics. In contrast, high/normal CREB levels were observed in well-differentiated tissues such as cortisol-hypersecreting adenomas and nontumoral adrenocortical tissues (normal adrenocortical tissues and hyperplastic cortex from CD patients). Clinicians and pathologists have long suspected that there are pathological links between dedifferentiated (nonsecreting) adenomas and malignant adrenocortical tumors. Indeed, it is often difficult to distinguish these two groups of tumors clearly on the basis of pathological criteria (19, 26). It has been suggested that a sequential multistep process of tumorigenesis occurs, in which adenomatous lesions may precede carcinoma formation (27 29). Our data, showing similarly low levels of CREB protein in malignant adrenocortical tumors and nonsecreting adrenocortical adenomas, provide further evidence to support this notion. Our results are consistent with those of a previous study, involving a nonquantitative RT-PCR approach, which reported a decrease in CREB transcript levels in a small series of malignant adrenocortical tumors (30). CREB mrna is subject to various posttranscriptional processing and degradation processes (31, 32). Differences between observations focusing on levels of transcript and protein were therefore anticipated. Indeed, the authors of these previous studies suspected that no CREB transcript was present in four of eight adrenocortical carcinomas, whereas in our study, CREB was detected in all of the 59 adrenocortical tumor specimens tested, although the signal was close to the limits of detection for some carcinomas. The evaluation of CREB protein levels was further validated in our study by the close correlation of protein levels with the tumor content of P-CREB, the functionally active form of CREB, and CREB-DNA complexes, as determined by EMSA. The presence of a variable nonendocrine component (e.g. of vascular and hematopoietic origin) within these tumors may result in differences in CREB levels. We therefore used immunohistochemical techniques to evaluate CREB levels in various adrenocortical tumors. This in situ approach confirmed that little CREB was present in large groups of endocrine cells in most of the tumor specimens. Our observations suggest that CREB protein levels may decrease late in the process of adrenocortical tumor development because some tumor cells had normal CREB signal. The mechanisms leading to changes in CREB protein levels in adrenocortical tumors were not evaluated in this study. Recent studies have provided evidence for regulation of the CREB and P-CREB contents of various tissues by hypoxia or oxidative stress (32 35). Both phenomena are thought to play a major role in the development of various types of malignant tumors (36, 37) and may occur during adrenocortical tumorigenesis because dedifferentiated adrenocortical tumors are associated with profound changes in architecture, including vascular damage, necrosis (26), and hypoxia. The dedifferentiation of adrenocortical tissue is associated with molecular events reminiscent of what is observed in the fetal zone of the fetal adrenal cortex (18, 38 40). The finding that CREB protein levels are very low in dedifferentiated adrenocortical tumors led us to investigate CREB levels in normal fetal adrenal tissue. The CREB was barely detectable by immunoblotting and immunohistochemistry in the fetal

6 Rosenberg et al. CREB in Adrenocortical Tumors J Clin Endocrinol Metab, August 2003, 88(8): FIG. 3. CREB levels in secreting (S) and nonfunctioning (NF) AAs. A, CREB immunoblot, using the polyclonal antibody Ab 9192 as described in Materials and Methods. CREB recombinant protein was used as a control. B, Quantitative representation of CREB and P-CREB (Ser133-phosphoryalted form) levels in secreting vs. nonfunctioning AAs. Western blots for CREB were performed with Ab 9192 (open circles) and Ser133-phosphorylated CREB with antibody 9191 (closed circles). Autoradiographs were quantified as described in Materials and Methods. The results are expressed in arbitrary units. Quantification of CREB levels in 16 cortical tissue samples from CD patients is presented as control. The mean CREB and Ser133-phosphorylated CREB content for each group is indicated by a dashed horizontal line. FIG. 4. CREB levels in the human fetal adrenal cortex. A, Western immunoblot analysis of CREB with the antibody 253 in the CREBdeficient cell line H295R, in fetal adrenal cortex specimen (without dissection of fetal and definitive zone) (F) and in three normal adrenal cortex samples (adjacent to tumor NAC). B, Up: CREB immunoblotting, using the polyclonal Ab 253 with the definitive (Dz) and fetal (Fz) zones of the adrenal cortex at 12 and 36 wk of gestation. CREB recombinant protein (*) was used as a control. Below: Western blot for the adrenocortical nuclear protein SF-1, using the same nuclear extracts as an internal control. zone, whereas it was clearly detected in the definitive zone, which persists after birth. To our knowledge, this is the first report to describe similarity between fetal and tumoral adrenocortical tissue on the basis of observations concerning a system other than the genes of the 11p15 region and the IGF family. The low levels of CREB in the fetal zone of the fetal adrenal cortex and in dedifferentiated adrenocortical tumors, both of which undergo rapid increases in size (2, 19), suggest that CREB may be involved in the control of proliferation associated with these physiological and pathological situations. This hypothesis is supported by several lines of experimental evidence in favor of an antiproliferative role for CREB in various endocrine and nonendocrine cell types (33, 41, 42). These results suggest that the camp pathway may display two-way involvement in tumorigenesis in the adrenal cortex. Molecular changes leading to overactivation of the camp pathway may lead to adrenocortical dysregulation, with the persistence of well-differentiated adrenocortical tissue generally displaying hypersecretion. By contrast, molecular changes disrupting the camp pathway are likely to cause defects associated with dedifferentiation of the adrenal cortex (carcinomas and nonfunctional adenomas). Several example of overactivation of the camp pathway associated with adrenocortical hyperplasia or small functioning adenomas are known (5, 7 11, 43), but the results presented here provide evidence for an association between camp pathway deactivation and dedifferentiation of the adrenal cortex. This observation, at the nuclear level, should be considered together with the loss of ACTH receptor gene expression observed in some adrenal cancers (30, 44). Inactivation of the camp pathway from the cell surface to the nucleus would lead to the development of a highly aggressive tumor with a malignant phenotype. Acknowledgments We thank J. F. Massias and F. René-Corail for excellent technical assistance, the surgeons (Profs. Y. Chapuis and B. Dousset), and the medical and paramedical staff of the Surgery and Endocrinology Departments of Cochin Hospital, who managed the patients; Dr. F. Tissier for assistance with pathological examinations; Dr. M. A. Dugue and the staff of the Laudat Hormone Laboratory for hormone assays; and the members of our laboratory for helpful discussions. We also thank Prof. M. Montminy (Salk Institute, La Jolla, CA) for generously providing us with recombinant CREB and CREB antibodies. Received January 14, Accepted May 6, Address all correspondence and requests for reprints to: Prof. Jérôme Bertherat, Service des Maladies Endocriniennes et Métaboliques, Hô-

7 3964 J Clin Endocrinol Metab, August 2003, 88(8): Rosenberg et al. CREB in Adrenocortical Tumors FIG. 5. Immunohistochemical detection of CREB in normal adult and fetal adrenal cortex and in adrenocortical cancers. A, Immunohistochemical detection of CREB in normal adult (A1, A2, and A3) and fetal adrenal cortex (A4, A5, and A6). Dz, Definitive zone; Fz, fetal zone. One representative 20 field (left column) and two 200 fields (middle and right columns) are shown for each tissue. The location of the 200 fields is indicated on the 20 field by dashed rectangles (middle column) and regular rectangles (right column). Well-labeled nonendocrine cells (hematopoietic) in the zone of unlabeled endocrine cells are indicated with arrowheads. B, Immunohistochemical detection of CREB in adrenocortical tumors: AA (B1, B2, and B3) and AC (B7, B8, and B9) showing a homogenous CREB immunolabeling in the adrenocortical adenoma and a demonstrative heterogeneous pattern of CREB immunolabeling in the adrenocortical carcinoma. One representative 20 field (left column) and two 200 fields (middle and right columns) are shown for each tissue. The location of the 200 fields is indicated on the 20 field by dashed rectangles (middle column) and regular rectangles (right column). Well-labeled nonendocrine cells (vascular and hematopoietic) in the zone of unlabeled endocrine cells are indicated with arrowheads. Histological (H/E) evaluation of adjacent sections of the tumors (AA: B4, B5, and B6; AC: B10, B11, and B12) shows no cell morphological differences between immunolabeled and nonimmunolabeled areas. pital Cochin, 27 rue du Fg-St-Jacques, Paris, France. jerome.bertherat@cch.ap-hop-paris.fr. 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