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Endocrine Unit (A.P., P.L., B.C., F.C., C.C., P.F., M.S., M.M.), Medical Genetics Unit (S.B.-P.), and Clinical Biochemistry Unit (S.G.), Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy; Department of Human Pathology and Oncology (G.N.), University of Florence, 50139 Florence, Italy; Department of Internal Medicine (G.A.), University of Ancona, 60131 Ancona, Italy; and Department of Medical and Surgical Sciences (F.M.), University of Padova, 35131 Padova, Italy
Abstract
The molecular mechanisms leading to adrenocortical tumorigenesis
have been only partially elucidated so far. Because the pituitary
hormone ACTH, via activation of the cAMP pathway, regulates both cell
proliferation/differentiation and steroid synthesis in the adrenal
cortex, in this study we focused on the cAMP-dependent transcription
factors cAMP responsive element modulator (CREM) and cAMP responsive
element binding protein (CREB). We studied CREM and CREB expression by
RT-PCR in human normal adrenal cortex (n = 3), adrenocortical
adenomas (n = 8), and carcinomas (n = 8). We found
transcripts corresponding to the isoforms 
, ß, 
, and 
2 of
the CREM gene in all of the normal adrenal tissues, in the adenomas,
and in seven of eight carcinomas. On the other hand, mRNA for the
inducible cAMP early repressor isoforms, which derive from an internal
promoter of CREM gene, was detected in the normal adrenal and in seven
of eight adenomas, but in only three of eight carcinomas. Similarly,
CREB transcripts were readily detectable in all normal adrenals and
adenomas, whereas they were not found in four of eight adrenal
carcinomas. To further characterize the carcinomas, telomerase activity
and the expression of the ACTH receptor gene were determined.
Telomerase activity in the carcinomas resulted in levels
significantly higher than in the adenomas, whereas the levels of ACTH
receptor mRNA were lower in the carcinomas. No correlation was found in
the carcinomas between the levels of the ACTH receptor transcript and
the loss of expression of CREB/inducible cAMP early repressor,
suggesting that this alteration is not secondary to an upstream
disregulation at the receptor level. In conclusion, our results suggest
that an alteration in cAMP signaling may be associated with
malignancies of the adrenal cortex.
IN THE ADRENAL cortex, the pituitary hormone ACTH acts as the major activator of the cAMP-dependent pathway. ACTH regulates cell differentiation, steroid synthesis, and, to a lesser extent, cell proliferation (1). However, the regulation of adrenocortical cells by ACTH-driven cAMP signaling appears to be complex, because an inhibitory role of ACTH on cell proliferation in vitro has also been observed (2, 3). Accordingly, complex multiple molecular mechanisms are likely to be related to adrenocortical tumorigenesis, as it is suggested by different reports. LOH of the ACTH receptor (ACTH-R) gene and reduced levels of mRNA (4) have been observed, for instance, in adrenal cancer and have been associated with cellular dedifferentiation. The observation that approximately one third of patients with MEN type 1 have adrenocortical tumors (5) prompted investigation of the association between mutations of the MEN 1 gene and adrenal neoplasia. LOH at 11q13 has been described in adrenal tumors, although it is not completely clear whether it involves the menin locus or not (6, 7). Furthermore, the activation of the proto-oncogene K-ras (8), p53 mutations (9), or overexpression of IGF II and IGF-binding protein-2 (10) or epidermal growth factor receptor (11) have been observed with variable frequency. However, the molecular mechanisms leading to adrenocortical tumorigenesis appear to be only partially known and are yet to be clearly elucidated.
Most of the intracellular effects of cAMP are mediated by the
activation of PKA. PKA activates, by phosphorylation at specific serine
sites, the nuclear transcription factors cAMP response element
modulator (CREM) and cAMP response element binding protein (CREB)
(12). Phosphorylated CREB and CREM bind as dimers to
palindromic cAMP response element sequences, thus modulating the
expression of cAMP-dependent genes. A classical cAMP response element
has been observed, for instance, in the promoter of 11ß-hydroxylase
(CYP11B1) or aldosterone synthase (CYP11B2) genes (13).
The peculiar aspect of CREB and CREM genes resides in the fact that
they can encode different isoforms, either activating or inhibiting
gene expression, by mechanisms of alternative exon splicing,
alternative promoter usage, and autoregulation of promoters
(12). In particular, an internal promoter of CREM gene
directs the expression of the repressor isoforms named inducible cAMP
early repressors (ICERs) in response to cAMP activation
(12). Recently, in the human adrenocortical cancer cell
line H295R, loss of expression of CREB gene and overexpression of the
activator CREM , compared with the normal adrenal cortex, have been
observed (14). It has been hypothesized that this pattern
of expression could be linked to cellular transformation. To better
define whether cAMP-dependent transcription factors play a role in
adrenocortical tumorigenesis, we studied the expression of CREB, CREM,
and ICER in normal human adrenal cortex (n = 3), in
cortisol-secreting adrenocortical adenomas (n = 8), and in
adrenocortical carcinomas (n = 8) by RT-PCR.
Materials and Methods
Patients
Nineteen patients were included in the study after obtaining
informed consent. Three human normal adrenal glands (from patients
undergoing nephrectomy), eight adrenocortical adenomas, and eight
adrenal carcinomas were studied. The
clinical characteristics of the patients are reported in Table 1
. Tissue specimens, obtained at surgery, were immediately
frozen in liquid nitrogen and stored at -80 C until RNA extraction.
Hematoxylin and eosin-stained sections were prepared, reviewed, and
classified according to the criteria of Weiss (15).
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The gene expression of the ACTH-R was evaluated by quantitative/competitive RT-PCR, as described previously (16). Briefly, the following primers were used: 5'-ACTGTCCTCGTGTGGTTTTG-3' and 5'-AGATGAAGACCCCGAGCAG-3'. A nonhomologous RNA competitor was constructed and used in RT-PCR experiments. There was a 66-bp difference between the length of the competitor and the normal ACTH-R transcript. Five increasing amounts of competitor were mixed with fixed amounts of tumoral RNA after RNase-free DNase digestion. The bands corresponding to the competitor and the transcript products were resolved by gel electrophoresis. The densitometric ratios were determined and were then plotted against the number of RNA competitor molecules added to each RT-PCR. The number of ACTH-R mRNA copies was extrapolated considering the value 1 of the competitor/target ratio as the point in which the number of competitor molecules is equal to that of the target (tumoral ACTH-R).
Telomerase assay
Telomerase activity was measured as described previously
(17). Briefly, tissue samples (
100 mg) were homogenized
and centrifuged. The supernatants were frozen and stored at -80 C.
Protein concentration was measured by the Bio-Rad Protein Assay
(Bio-Rad Laboratories, Inc., Hercules, CA). Protein
(6 µg ) was used for telomerase assay. RNase (Roche
Diagnostics, Monza, Italy; 0.5 µg) was used for each assay for 30
min at 37 C to inactivate telomerase. Each extract was assayed
in 47.2 µl reaction mixture containing 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 4.5 mM
MgCl2, 1 mM dNTP,
20 pM TAG-U primer, and 0.5 µM LT4
gene 32 protein (Roche Diagnostics). After 60 min at 30 C
for telomerase-mediated extension of TAG-U primer, the reaction mixture
was subjected to PCR. A 10 min at 72 C step followed PCR cycles after
the addition of a second reaction mixture containing 20 pM
CTA-R primer and 1.5 U Taq Gold (Perkin-Elmer Corp., Norwalk, CT). We diluted 10 µl each PCR product with
490 µl 10 mM Tris-HCl, 1
mM EDTA (pH 7.5), and then 500 µl
ultrasensitive fluorescent dye PicoGreen (Molecular Probes, Inc., Eugene, OR). Fluorescence was measured in a
spectrofluorometer RF-540 (Shimadzu) using standard wavelengths. DNA
concentration was calculated for each sample on a calibration curve
generated by dilutions of a control DNA (0–100 µg/liter). The final
DNA concentration of each sample was determined by subtracting the DNA
amount obtained in the same specimen after RNase treatment. Telomerase
activity was calculated as the mean of duplicates for each sample and
expressed as nanograms of DNA per microgram of protein. A negative
control, obtained after pretreatment of the sample with RNase, was also
assayed for each specimen.
CREB, CREM, and ICER RT-PCR
RT-PCR was performed on total RNAs (0.5 µg for each reaction)
using CREB-, CREM-, and ICER-specific primers. In particular, CREB
primers spanned sequences at the 5'- and 3'-end of CREB mRNA
(18). CREM primers were able to generate two different
amplified products (243 and 390 bp), corresponding to the isoforms ,
ß, (repressors), and 2 (activator) and to the activators ,
, and 1, respectively, as described previously
(19). ICER primers were designed to detect ICER I and ICER
II isoforms, as described previously (20). The primers
were synthesized by Roche Diagnostics. Preliminary
experiments were performed to determine the PCR cycles corresponding to
the exponential phase of amplification. Thereafter, the PCR were always
stopped in the exponential phase (35 cycles). Only in those cases in
which no amplified signal was detectable did we further assess the
negativity by extending the number of cycles. Each experiment
was repeated three times to confirm the results. The quality of RNAs
was assessed by performing additional RT-PCR using primers specific for
the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, as described
previously (21). Finally, in each RT-PCR experiment, a no
RNA reaction was added as a negative control. RT-PCR products were
subjected to agarose gel electrophoresis, Southern blot, hybridization
to CREB-, CREM-, and ICER-specific probes and immunochemiluminescent
detection, as described previously (19, 20). The membranes
were exposed to x-ray films. The time of exposure of the film was kept
constant in each experiment (5 min).
Sequence analysis of ICER RT-PCR products
RT-PCR for the determination of ICER transcripts originated
signals of different length. The specificity of the putative signals
corresponding to ICER I and II isoforms was validated by additional
sequence analysis, as described previously, using
32[P]-ATP-labeled primers (20).
Amplified products were electrophoresed on acrylamide gel (6%)
in the presence of 7 M urea. Then, the gels were blotted on
Whatman 3 M filters (Whatman International
Ltd., Maidstone, UK). After drying, the filters were transferred to an
x-ray cassette and exposed to x-ray films for 12 h.
Statistical analysis
Statistical comparison between groups was performed using the t test. Differences were considered as statistically significant at the 0.05 level.
Results
Histology, ACTH receptor expression, and telomerase activity
Total RNA from normal adrenal glands (n = 3), adrenocortical
adenomas (n = 8), and adrenocortical carcinomas (n = 8) was
subjected to RT-PCR. The histological features of the adrenal
carcinomas, according to the criteria of Weiss (15), are
shown in Table 2.
|
). Telomerase activity, which has been shown to be a
potential marker for differentiating benign from malignant adrenal
tumors (22, 23), was also determined, and it was found to
be higher in the carcinomas (adenomas, 5.41 ± 1.54,
vs. carcinomas, 17.21 ± 1.72 ng DNA/µg protein;
mean ± SE; P = 0.00023)
(Table 1). Analysis of CREB transcripts
RT-PCR analysis of CREB transcripts revealed the presence of a
specific signal in all of the samples from normal adrenal gland and
adrenal adenoma. The specificity of the 1026-bp signal, corresponding
to the full-length transcript of CREB gene, was confirmed by Southern
blotting and hybridization to a CREB-specific probe. The results, as
obtained by chemiluminescent detection, are shown in Fig. 1A (no. 1–3, normal adrenal; 4–11,
adenoma). Conversely, signals corresponding to CREB transcripts were
detectable in only four of eight adrenocortical carcinomas (no.
12–15), whereas no amplified signal was in any case obtained
(experiments were repeated three times) in the remaining samples (no.
16–19). In these cases, no signal was detectable, even after extending
the number of PCR cycles (data not shown). The quality of RNAs was
assessed by the analysis of GAPDH transcripts. A GAPDH-specific signal
was readily detectable in all samples (Fig. 1B), thus excluding that
the absence of CREB transcripts in some samples was due to RNA
degradation. No significant difference in ACTH-R mRNA levels was
observed between the carcinomas expressing and those not expressing
CREB (5.38 ± 0.17 vs. 5.35 ± 0.15 log(no.
copies)/100 ng RNA; mean ± SE,
P = 0.88).
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CREM-specific primers, spanning sequences from exon B (sense
primer) and from exon D (antisense primer) of CREM gene, were designed
for RT-PCR experiments. These primers have been designed and used
previously in our laboratory (19) and are able to generate
two specific signals corresponding to different CREM isoforms (see
Materials and Methods). The expected signal of 243 bp,
corresponding to the CREM repressors , ß, and and to the
activator 2, was detected in all RNAs from normal adrenals (no.
1–3), adenomas (no. 4–11), and carcinomas (no. 12–18), with the
exception of one case (no. 19). The specificity of the signal was
validated by hybridization of RT-PCR products to a CREM-specific probe,
and the results are shown in Fig. 2A.
However, in no case was the presence of the 390-bp signal corresponding
to the activators , , and 1, which was readily detectable
in other cell systems such as germ cells (19), observed in
the adrenal cortex.
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For the detection of ICER transcripts by RT-PCR, primers were
selected from the internal promoter and exon (sense primer) and
from exon Ib (antisense primer) of CREM gene. These primers were
designed to detect ICER I (657 bp) and ICER II (257 bp) isoforms.
Amplified products were hybridized to a specific oligonucleotide probe.
Signals corresponding to ICER I and ICER II transcripts were detected
in all normal adrenal glands (Fig. 2B). In adrenocortical adenomas,
ICER I and II transcripts were observed in all samples, with the
exception of one case (no. 7). Conversely, in adrenal carcinomas, the
presence of ICER I and II mRNA was detected in only three of eight
cases (no. 12–14), whereas no detectable levels of expression were
repeatedly found in the remaining cases (no. 15–19) (Fig. 2B). In
those cases in which no signal was detected, the negativity was
confirmed even after extension of the number of PCR cycles (data not
shown). Sequence analysis of the different amplified products, which
was performed in samples from normal adrenals as well as from adenomas
and carcinomas, confirmed that the 657- and 257-bp signals correspond
to ICER I and ICER II, respectively (data not shown), as described
previously in pituitary adenomas (20). The middle two
amplified fragments, previously detected also in pituitary adenomas
(20), contain partial sequences of ICER I (data not shown)
and might correspond to different, so far undescribed, ICER isoforms,
which will need further characterization.
Discussion
ACTH, by activating the cAMP-dependent pathway, has a moderate
effect on adrenal cell proliferation or even a mild anti-proliferative
effect in vitro (2, 3), whereas it plays a
pivotal role in regulating steroid hormone synthesis (1)
and, hence, in maintaining a differentiated phenotype. LOH of the ACTH
receptor gene and reduced expression of ACTH receptor mRNA have been
detected in a subset of adrenocortical carcinomas (4).
Because LOH is a characteristic of many tumor types, it has been
suggested that the ACTH-R gene may act as a tumor suppressor gene and
that the LOH of this gene may result in loss of differentiation and in
growth advantage. The possible involvement of the cAMP-dependent
pathway in neoplastic transformation of adrenocortical cells has been
recently highlighted by the identification of the gene for Carney
complex, a disease characterized by different clinical features,
including the presence of pigmented adrenocortical tumors. In fact,
mutations of the gene encoding the protein kinase type I- regulatory
subunit, an apparent tumor suppressor gene, have been detected in a
subset of patients with this disease (24).
In this report, we focused on two downstream targets of the
cAMP-dependent pathway, i.e. CREM and CREB, and we
investigated their expression in the normal adrenal gland as well as in
adrenocortical adenomas and carcinomas. We detected CREM-specific
transcripts corresponding to the transcriptional activator 2 and to
the inhibitors , ß, and in RNAs from all normal adrenals,
adenomas, and carcinomas, with only one exception in the last group. In
no case were transcripts corresponding to CREM activators , ,
and 1 detectable. Therefore, an altered pattern of CREM expression
does not appear to be a molecular feature associated with adrenal
tumorigenesis. On the other hand, different patterns of expression of
ICER isoforms, which derive from an internal promoter of the CREM gene,
were observed. In particular, whereas ICER isoforms were readily
detectable in all normal adrenal glands and adenomas (with the
exception of one case), lack of expression was found in five of eight
carcinomas. Similarly, CREB transcripts were consistently observed in
normal adrenal cortex and in adrenal adenomas, whereas detectable mRNA
levels were not found in half of the adrenal cancers. The CREB
transcript that we detected corresponds to the full-length transcript
of CREB gene, which originates a transcriptional activator, upon
phosphorylation at Ser-133. Conversely, CREB repressors originate by
mechanisms of alternative exon splicing or by alternative start sites
of translation of CREB gene. As a consequence, lack of transcription of
one or more exons occurs, and the resulting proteins cannot undergo
phosphorylation-mediated activation. In keeping with our results, in a
recent report the absence of CREB expression was observed in the human
adrenocortical cancer cell line H295R (14). However, at
variance with our findings, in that study a compensatory overexpression
of CREM , which is usually absent or expressed at a very low level
in the normal adrenal cortex, was detected in H295 cells
(14). This apparent discrepancy might be due to the
different cell system (i.e. adrenal tumoral tissues obtained
at surgery vs. a single cultured cell line). In view of our
results, it is noteworthy that both the promoter of CREB gene and the
internal promoter of CREM gene that directs the expression of ICER
isoforms, but not the upstream promoter, are autoregulated by cAMP. In
fact, three CREs are present in the promoter region of CREB gene
(25); two pairs of closely spaced CREs are contained in
the internal promoter of CREM gene (26). As a result,
activation of the cAMP pathway, by enhancing the levels of
phosphorylated, hence activated, CREB, stimulates CREB and ICER
expression. These considerations, in conjunction with our experimental
data, support the hypothesis that adrenal malignancies can be
associated to an alteration of the cAMP-dependent signaling. In this
scenario, the lack of expression of CREB (and/or ICER) in a consistent
percentage of cases of adrenal carcinoma may be regarded as a marker of
loss of cell differentiation. To clarify whether or not the absence of
CREB/ICER mRNA was a consequence of a different molecular alteration
such as reduced ACTH-R expression, which may be a feature of adrenal
cancer (4), the levels of transcript of the ACTH-R gene
were determined. The expression levels of this gene did not differ
between the carcinomas expressing CREB/ICER and those showing loss of
CREB/ICER expression, thus suggesting that the absence of CREB/ICER
mRNA in a subset of adrenal carcinomas does not appear to be secondary
to an upstream disregulation at the receptor level. Interestingly, CREB
activation via Ser133-phosphorylation has been
related to cell differentiation in different tissues, such as the brain
(27) and the adipose tissue (28). In the
blood system, phosphorylated CREB has been shown to induce the
differentiation of megakaryocytes (29). A CREB
gain-of-function mutant, which induced high levels of constitutive
Ser133-phosphorylation, has been found to promote
cell differentiation in vitro (30). On the
other hand, mice lacking CREB expression have been generated; the
mutant mice invariably die from respiratory distress, associated to
surfactant deficiency, immediately after birth (31).
Significantly, a severe impairment in brain and T cell development has
been observed. Furthermore, transgenic mice, in which expression of a
dominant negative CREB isoform was targeted to the thyroid gland,
exhibited severe growth retardation and primary hypothyroidism;
histologically, the thyroid glands were characterized by poorly
developed follicles (32).
CREB-mediated activation of transcription is a multifactorial process that includes the involvement of different coactivators interacting with the transcriptional apparatus. CREB binding protein (CBP) and p300 are two factors connecting with CREB only in its phosphorylated form (33, 34), thus participating in the molecular events resulting in the stimulation of gene expression. CBP and p300 take part in a variety of cellular processes, such as cell growth, differentiation, and apoptosis (35). It is noteworthy that alterations of the human CBP gene have been implicated in hematological malignancies, such as acute or chronic myeloid leukemia (36, 37). Similarly, inactivation of p300 gene has been related to leukemia (38) as well as to gastric and colorectal carcinomas (39), suggesting that these coactivators of CREB function may serve as tumor suppressor proteins. These data confirm that cAMP signaling is strongly involved in the control of cell growth and differentiation and that alterations disrupting the integrity of this pathway may result in neoplastic proliferation.
Because the number of cases of adrenocortical cancer that we examined so far is limited, it is not possible to conclusively establish whether there is a relationship between the presence or the absence of expression of CREB or ICER and the clinical features of the patients. In a recent report we observed that the high levels of telomerase activity in adrenal carcinomas were positively correlated with the tumor size (23). However, in the present study it may be noteworthy to observe that, among the eight patients with adrenal carcinoma, the two patients who died from metastatic disease did not show detectable levels of CREB and ICER expression. Therefore, although any conclusive statement will be possible only after extending the number of cases examined, preliminary observations seem to suggest that the lack of expression of CREB/ICER in cases of adrenal cancer might be linked to a more severe outcome.
In conclusion, in the present study we have demonstrated for the first time that, whereas cAMP-dependent transcription factors are consistently expressed in the normal adrenal gland and in adrenocortical adenomas, loss of expression of CREB and/or ICER may be a feature of adrenal cancer. Because cAMP signaling is involved in the processes leading to cell differentiation, it can be hypothesized that an alteration of the cAMP-dependent transcription factor machinery may be associated with impaired cell differentiation and with a transformed phenotype in the adrenal cortex.
Acknowledgments
We thank Dr. Marina Scarpelli (Department of Pathology, University of Ancona, Italy); Prof. Andrea Amorosi (Department of Pathology, University of Catanzaro, Italy); Prof. Marco Carini (Urology Unit, Ospedale Santa Maria Annunziata, Florence, Italy); and Prof. Domenico Borrelli and Dr. Andrea Valeri (General Surgery Unit, Ospedale Careggi, Florence, Italy) for valuable contributions.
Footnotes
Address all correspondence and requests for reprints to: Dr. Massimo Mannelli, Department of Clinical Physiopathology, Endocrine Unit, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy.
Abbreviations: ACTH-R, ACTH receptor; CBP, CREB binding protein; CREB, cAMP responsive element binding protein; CREM, cAMP responsive element modulator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICER, inducible cAMP early repressor.
Received December 19, 2000.
Accepted August 13, 2001.
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