Nephroblastoma Overexpressed/Cysteine-Rich Protein 61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed Gene-3 (NOV/CCN3), a Selective Adrenocortical Cell Proapoptotic Factor, Is Down- Regulated in Childhood Adrenocortical Tumors
Mabrouka Doghman, Malika Arhatte, Hélène Thibout, Giovanna Rodrigues, Juliana De Moura, Sébastien Grosso, Alina Nico West, Maryvonne Laurent, Jean-Christophe Mas, André Bongain, Gerard P. Zambetti, Bonald C. Figueiredo, Patrick Auberger, Cécile Martinerie, and Enzo Lalli
Institut de Pharmacologie Moléculaire et Cellulaire Centre National de la Recherche Scientifique Unité Mixte de Recherche 6097 and Université de Nice Sophia Antipolis (M.D., M.A., G.R., J.D.M., E.L.), 06560 Valbonne, France; Institut National de la Santé et de la Recherche Médicale U515 (H.T., M.L., C.M.), Hôpital St. Antoine, UPMC, Paris 6, 75012 Paris, France; Institut National de la Santé et de la Recherche Médicale U526 (S.G., P.A.), Faculté de Médecine, 06107 Nice, France; Department of Biochemistry (A.N.W., G.P.Z.), St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; Service de Pédiatrie (J .- C.M.) and Service de Gynécologie Obstétrique (A.B.), Hôpital de l’Archet, Centre Hospitalier Universitaire, 06200 Nice, France; and Instituto de Pesquisa Pelé Pequeno Principe and Centro de Genética Molecular e Pesquisa do Câncer em Crianças (B.C.F.), Curitiba, 80.060-110 Paraná, Brazil
Context: Childhood adrenocortical tumors (ACTs) have a fetal ad- renal phenotype and overexpress steroidogenic factor-1 (SF-1). Nephroblastoma overexpressed (NOV)/cysteine-rich protein 61/con- nective tissue growth factor/nephroblastoma overexpressed gene-3 mRNA is significantly down-regulated in childhood ACTs.
Objective: The objective of the study was to measure NOV protein levels in childhood ACTs and characterize NOV expression regulation and biological function in human adrenocortical cells.
Design and Setting: Protein extracts from ACT and normal adrenal cortex samples, human adrenocortical carcinoma H295R, primary adrenocortical tumors and fetal adrenal cultures, tissue culture su- pernatants, and cell lysates from H295R cells overexpressing SF-1 in an inducible fashion were used.
Main Outcome Measures: NOV protein levels were measured by enzyme-linked immunoassay and immunoblot. Transient transfec- tion assays were used to study the activity of NOV promoter. Terminal
deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, caspase assays, and flow cytometry were used to assess the proapoptotic activity of NOV on cells in culture.
Results: NOV mRNA and protein expression is lower in childhood ACTs than in normal adrenal cortex. No significant difference was observed between adenomas and carcinomas. SF-1 overexpression down-regulates NOV at the transcriptional level. NOV has a selective proapoptotic activity toward human adrenocortical cells. The C-ter- minal domain of NOV is responsible for its proapoptotic effect. NOV protein is expressed in DAX-1-positive human fetal adrenal cells.
Conclusions: NOV is a selective proapoptotic factor for human ad- renocortical cells. Reduced expression of NOV in ACTs may play an important role in the process of childhood ACT tumorigenesis, ac- counting at least in part for the defect of apoptotic regression of the fetal adrenal that has been proposed to be responsible for tumor formation. (J Clin Endocrinol Metab 92: 3253-3260, 2007)
T HE STRUCTURE OF the human adrenal cortex is dif- ferent during fetal life and after birth. In fact, during intrauterine life, most of the inner part of the adrenal cortex (fetal zone) is occupied by large, steroid-secreting cells,
First Published Online June 19, 2007
Abbreviations: ACT, Adrenocortical tumors; CCN, connective tissue growth factor; DAPI, 4’,6’-diamidino-2-phenylindole; DAX-1, dosage- sensitive sex reversal-adrenal hypoplasia congenita critical region on X chromosome-gene 1; FITC, fluorescein isothiocyanate; IGFBP, IGF bind- ing protein; NOV, nephroblastoma overexpressed; P450scc, P450 side- chain cleavage; SF-1, steroidogenic factor-1; TUNEL, terminal deoxy- nucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the en- docrine community.
which are differentiated to synthesize large amounts of de- hydroepiandrosterone and its sulfate derivative. Conversely, a few layers of cells lying in an outer, subcapsular position (definitive zone) are nonsteroidogenic and constitute a pro- liferative compartment in which most probably the precur- sors of fetal zone cells are generated (1-3). An additional zone (transitional zone) develops between the definitive and the fetal zone late in pregnancy and synthesizes cortisol. The fetal adrenal undergoes a process of rapid involution and remodeling after birth (1, 4). The fetal zone regresses by apoptosis, whereas cells of the definitive zone proliferate and differentiate to form the glomerulosa, fasciculata, and re- ticularis zones of the adult adrenal cortex. Childhood adre- nocortical tumors (ACTs) are thought to be derived from the fetal adrenal, probably through defective apoptosis because of their age distribution, their pattern of hormone secretion,
and their molecular phenotype (5, 6). These tumors can arise isolated or in the context of genetic syndromes (for a review see Ref. 7) and most commonly present with virilization due to production of androgenic steroids, which can be associ- ated with Cushing syndrome. A peculiar epidemiological feature of childhood ACTs is their high incidence in the states of Paraná and São Paulo in southern Brazil, where they are almost invariably found associated with a specific germline TP53 mutation (R337H) (8). In addition, a role for the tran- scription factor steroidogenic factor-1 (SF-1) in childhood ACT pathogenesis has been suggested based on its ampli- fication and overexpression in this type of cancer (9, 10). It is remarkable that an increase in SF-1 dosage is by itself sufficient to increase proliferation of human tumor adreno- cortical cells and to cause adrenocortical tumors in mice (Doghman, M., T. Karpova, G. Rodrigues, M. Arhatte, J. De Moura, L. R. Cavalli,, V. Virolle, P. Barby, G. P. Zambetti, B. C. Figueiredo, L. L. Heckert, E. Lalli, submitted for publication).
A gene expression profiling study performed in childhood ACTs has recently shown that distinct patterns of gene ex- pression distinguish tumors from normal adrenal cortex. Nephroblastoma overexpressed (NOV)/cysteine-rich pro- tein 61/connective tissue growth factor/nephroblastoma overexpressed gene (CCN)-3 is one of the transcripts that is most powerfully down-regulated in childhood ACTs, inde- pendently from their degree of malignancy (11). Conversely, it was previously shown that loss of NOV expression is associated with a poor prognosis in adult adrenocortical tumors (12). NOV is a multidomain-secreted protein, mem- ber of the CCN family, whose expression was described to be mostly restricted to the definitive zone of the fetal adrenal cortex (13). Its known functions are related to cell adhesion and angiogenesis, and its expression is negatively correlated to proliferation and associated with differentiation in many cellular systems (for review see Ref. 14). Here we show that NOV is strikingly down-regulated at the protein level in childhood ACTs and in human adrenocortical cells in a man- ner dependent on SF-1 dosage and characterize its selective proapoptotic activity for human adrenocortical cells.
Subjects and Methods
Tumor samples
Twenty-four childhood ACTs (five adenomas, 18 carcinomas, and one of undetermined histological type) and seven normal adrenal cortex samples were used for mRNA studies. These were the same samples that were used for gene expression profiling using Affymetrix microarrays (11). For protein expression studies, tumor samples were obtained at the time of surgery from eight children with ACTs (three boys and five girls). Five patients presented with virilization and three with virilization associated with Cushing’s syndrome. Histologically, four tumors were classified as adenomas and four as carcinomas. Eight normal adrenal glands resected from age-matched children undergoing surgery for Wilms’ tumor were used as controls. Samples were immediately snap frozen in liquid nitrogen before processing for protein extraction. In one case of adrenocortical adenoma in a 5-yr-old child, tumor cells were dissociated with collagenase and cultured in OptiMEM (Invitrogen, Carlsbad, CA) containing 2% fetal calf serum, following an established protocol (15). All patients and control subjects were included in the study after one of the parents or legal representatives signed an informed consent form approved by the Ethics Committee of the Hospital de Clínicas of the Federal University of Paraná.
Human fetal adrenal tissue preparation
Adrenal glands were obtained from human fetuses aged 10-14 wk after elective termination of pregnancy. The research protocol was pre- viously submitted to the French national Agence de la Biomédecine and local Ethical Committee of the Nice University Hospital. Fetal adrenal cells were isolated and cultured as described (15).
Immunofluorescence
It was performed as described (16) using the following antibodies: rabbit anti-NOV (K19M); mouse anti-dosage-sensitive sex reversal- adrenal hypoplasia congenita critical region on X chromosome-gene 1 (DAX-1) (2F4); rabbit anti-P450 side-chain cleavage (P450scc; gift of Dr. W. L. Miller) (17); rabbit anti-SF-1 (Upstate, Lake Placid, NY). As negative control for primary antibodies, species-matched IgG was used. Cell nuclei were counterstained with 4’,6’-diamidino-2-phe- nylindole (DAPI).
Cell culture
H295R cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM/F-12 supplemented with 2% NuSerum (Becton Dick- inson, Lincoln Park, NJ), 1% ITS Plus (Becton Dickinson), and antibiotics. H295R cell clones overexpressing SF-1 in a doxycycline-inducible fash- ion (H295R TR/SF-1) were produced and maintained as described (Doghman, M., T. Karpova, G. Rodrigues, M. Arhatte, J. De Moura, L. R. Cavalli, V. Virolle, P. Barby, G. P. Zambetti, B. C. Figueiredo, L. L. Heckert, E. Lalli, submitted for publication). Proliferation assays were performed in the absence of NuSerum. Hela cells were cultured in DMEM (1 g/liter glucose) supplemented with 10% fetal calf serum (Invitrogen) and antibiotics. COS cells were cultured in DMEM (4.5 g/liter glucose) supplemented with 5% fetal calf serum (Invitrogen) and antibiotics. Jurkat cells were cultured in RPMI 1640 supplemented with 5% fetal calf serum (Invitrogen) and antibiotics.
cDNA amplification and real-time PCR analysis for NOV mRNA expression
This was performed as described (11). Results were normalized to ubiquitin expression levels using the 44C, method (18).
A
150
Relative expression
125
100
75
**
50
25
0
Normal
ACT
B
kDa
N1 N2 N3
N4
T1
T2
T3
T4
N5 N
N7 N8 18
T5 T6 T7 T8
66 -
45 -
NOV
35 -
25
₿-tubulin
A
30
NOV (ng/ml)
20
**
*
10
0
NOV
IGFBP2
+
+
Dox
48 h
72 h
B
Dox
Parental clone
+
luc
-492 NOV promoter
H295R TR/SF-1
+
Parental clone
+
luc
-625 NOV promoter
H295R TR/SF-1
+
Parental clone
+
luc
-2540 NOV promoter
H295R TR/SF-1
+
0
5000
10000
15000
RLU
NOV assays
Secreted NOV protein was quantified by an enzyme linked immu- noassay, as described (19) or extracted from culture supernatants by heparin-Sepharose chromatography, as described (20), and detected by immunoblot.
Immunoblots
The immunoblots were performed as described (10). Normal and tumor adrenal samples were homogenized in Laemmli buffer, and pro-
FIG. 3. NOV reduces H295R cell number in a dose-de- pendent manner. A, H295R cells were cultured in me- dium without NuSerum in the presence of protein buffer only (Ctrl) or with increasing doses [0.01 (0.25 nM), 0.05 (1.25 nM), 0.1 (2.5 nM), and 0.5 (12.5 nM) µg/ml] of re- combinant NOV protein added. Cell numbers were counted after 4 d. Data are normalized by the number of cells present in buffer-treated cultures. Data shown are the average of two experiments performed in duplicate. B, Cell cycle distribution of untreated H295R cells (left) and cells treated with 0.5 µg/ml NOV protein for 4 d (right).
teins were separated on SDS-PAGE and blotted on a polyvinyl difluoride membrane (GE Healthcare, Indianapolis, IN). Antibodies used for im- munoblot were: rabbit anti-NOV (K19M), mouse anti-ß-tubulin (Sigma, St. Louis, MO), and goat anti-IGF binding protein (IGFBP)-2 (Santa Cruz Biotechnology, Santa Cruz, CA).
Transient transfection and luciferase assays
H295R cells were transfected with NOV promoter reporters (20) and pCH110 as a ß-galactosidase expression vector using the jetPEI reagent
A
% of untreated cells
100
T
75
50
T
25
0
Ctrl
NOV
B
0 NOV
0.5 NOV
#
8
2
2
8
B
-
2
&
.
.
-
0
Q
0
200
400
600
1000
FLDE
FLZH
at a nitrogen to phosphate ratio of 5, following the manufacturer’s (Polyplus Transfection, Illkirch, France) instructions. Luciferase assays were realized using a Luminoskan Ascent (Thermo Labsystems, San Jose, CA) luminometer. Results were normalized by ß-galactosidase activities.
Recombinant NOV proteins
The recombinant NOV proteins were produced and purified as de- scribed (19).
Flow cytometry
H295R cells were fixed in 70% ethanol and then treated with RNase A (1 µg/ml) for 30 min at 37 C. DNA was stained with propidium iodide (50 µg/ml), and cells were analyzed for cell-cycle distribution with a FACScan instrument (Becton Dickinson). Results are expressed as pro- liferating index (sum of percentages of cells in S + G2/M phases).
Apoptosis and caspase activity assays
Apoptosis was assayed using the TdT-based in situ cell death detec- tion kit (Roche, Indianapolis, IN). For caspase activity assays, H295R cells were lysed for 30 min at 4 C in lysis buffer [50 mM HEPES (pH 8), 150 mM NaCl, 20 mM EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.2% Triton X-100], and lysates were cleared at 10,000 × g for 15 min at 4 C. Each assay (in triplicate) was performed using 20 µg of protein lysate prepared from control or NOV-stimulated cells and complemented with 5 mm dithio- threitol. Briefly, cellular extracts were incubated in a 96-well plate with 0.2 mM of Ac-DEVD-AMC (caspase-3), Ac-LEHD-AMC (caspase-9), or
Ac-IETD-AMC (caspase-8) as substrates at 37 C. Caspase activity was measured after absorbance at 460 nm (excitation at 390 nm) in the absence or presence of 1 µM of Ac-DEVD-CHO, Ac-LEHD-CHO, and Ac-IETD-CHO, for caspase-3,-9, and -8, respectively. To assay for active caspase-3 levels in Jurkat T cells, cells were treated for 8 or 24 h in the presence of either NOV (2.5 µg/ml) or CH11 anti-Fas antibody (50 ng/ml), as a control of induction of apoptosis. Cells were then washed with PBS, fixed, and permeabilized. Subsequently cells were incubated for 30 min with antiactive-caspase-3-fluorescein isothiocyanate (FITC) monoclonal antibody (BD Biosciences, Franklin Lakes, NJ). After wash- ing, samples were immediately analyzed with a FACScan (Becton Dickinson).
Results
NOV protein is down-regulated in childhood ACT
By microarray analysis we have shown that NOV is one of the transcripts that is more significantly down-regulated in a series of 24 childhood ACTs, compared with normal ad- renal cortex (11). NOV mRNA levels measured by quanti- tative RT-PCR are significantly lower in childhood ACTs than normal adrenal cortex (Fig. 1A). To verify whether NOV protein levels correlate with mRNA levels, we compared NOV protein expression in a series of eight tumors (four adenomas and four carcinomas) and eight samples of age- matched normal adrenal cortex. NOV protein levels are in- variably reduced in all tumor samples, compared with nor- mal tissue, for both adenomas and carcinomas (Fig. 1B).
A
SP IGFBP
VWC
TSP1
CT
N-ter
C-ter
B
30
**
% apoptotic cells
DAPI
**
20
10
TUNEL
0
Ctrl
NOV FL
N-ter
C-ter
Ctrl
NOV
C
20
% apoptotic cells
15
10
5
0
Ctrl
NOV
NOV
Ctrl
NOV
H295R
HeLa
COS
B-Tubulin staining shows similar protein loading in each sample (Fig. 1B).
Increase of SF-1 dosage in H295R cells represses NOV expression at the transcriptional level
H295R cells are the only differentiated human adrenocor- tical cell line with a fetal adrenal phenotype currently avail- able (21). They express SF-1 endogenously (16). We estab- lished cellular clones derived from H295R cells that overexpress SF-1 in a doxycycline-inducible fashion (H295R TR/SF-1) to produce a cellular model for childhood ACT (Doghman, M., T. Karpova, G. Rodrigues, M. Arhatte, J. De Moura, L. R. Cavalli, V. Virolle, P. Barby, G. P. Zambetti, B. C. Figueiredo, L. L. Heckert, E. Lalli, submitted for publica- tion). Gene expression profiling of H295R TR/SF-1 cells re- vealed that NOV is one of the transcripts that is more pow- erfully down-regulated when SF-1 dosage is increased. Microarray results were confirmed by quantitative RT-PCR in two different H295R TR/SF-1 clones (Doghman, M., T. Karpova, G. Rodrigues, M. Arhatte, J. De Moura, L. R. Cav- alli, V. Virolle, P. Barby, G. P. Zambetti, B. C. Figueiredo, L. L. Heckert, E. Lalli, submitted for publication). NOV protein levels are significantly reduced in the supernatant of H295R TR/SF-1 cells treated with doxycycline for either 48 or 72 h to induce SF-1 overexpression (Fig. 2A). The IGFBP2-se- creted protein harbors an IGF-binding domain similar to the one present in the N-terminal half of NOV and is readily detectable in the tissue culture supernatant of H295R cells (22). Remarkably, doxycycline treatment of H295R TR/SF-1 cells does not appreciably modulate IGFBP2 levels in their culture supernatant (Fig. 2A), showing the specificity of the effect of increased SF-1 levels to negatively regulate NOV expression in H295R human adrenocortical cells.
Increased SF-1 dosage regulates NOV expression at the transcriptional level because doxycycline treatment reduced activity of NOV promoter in H295R TR/SF-1 cells, whereas it had no effect on NOV promoter activity in the parental H295R TR clone (Fig. 2B). Deletion analysis showed that the activity of the shortest NOV promoter fragment analyzed (-492) is still repressed by elevated SF-1 levels in H295R TR/SF-1 cells. In the minimal NOV promoter sequences sen- sitive to inhibition by increased SF-1 levels, no putative SF-1 response element can be found by MatInspector (Genomatix, Munich, Germany) analysis. Furthermore, NOV promoter reporters are not sensitive to inhibition by SF-1 after transient transfection in COS cells (data not shown). This data suggest that the specific effect of increased SF-1 levels on NOV pro- moter activity in adrenocortical cells is mediated by other transcription factors whose levels, or activities, are regulated by SF-1 dosage.
NOV induces apoptosis of human tumor adrenocortical cells through caspase activation
To study the biological effects of NOV on human adre- nocortical cells, we treated H295R cells with increasing doses of recombinant full-length NOV protein and monitored their growth over a 4-d period. NOV decreases cell number in a dose-dependent manner (Fig. 3A). This effect of NOV cannot be ascribed to block of cell-cycle progression because the
proliferating index of H295R cells treated with the highest dose of NOV (0.5 µg/ml) was not decreased, compared with untreated cells (36.7 vs. 34.8, respectively; Fig. 3B). On the other hand, full-length NOV in nanomolar concentrations induces a significant increase in the percentage of apoptotic H295R cells, as detected by terminal deoxynucleotidyl trans- ferase-mediated deoxyuridine triphosphate nick end label- ing (TUNEL) staining, after a 24-h treatment (Fig. 4B). The C-terminal NOV fragment, encompassing the sequence con-
A
30-
% apoptotic cells
20
10
0
Ctrl
Z-VAD
Z-DEVD
Z-IETD
Z-LEHD
NOV
B
Caspase 3
Caspase 8
Caspase 9
12.5
A Fluo/min/mg protein
10.0
A Fluo/min/mg protein
1.5
A Fluo/min/mg protein
1.5
7.5
1.0
1.0
5.0
0.5
0.5
2.5
0
0
0
Ctrl
NOV
Ctrl
NOV
Ctrl
NOV
C
100
1.88%
Ctrl
0
100
102
182
30.20%
CH11 24h
Number of events
Number of events
100
0-
103
27.44%
CH11 6h
100
1000
101
102
103
104
2.17%
NOV 24h
100
0
191
182
a
.
1.80%
NOV 6h
Active caspase 3
0
I 4
Active caspase 3
taining the thrombospondin-type I repeat and the CT (Fig. 4A), induces apoptosis of H295R cells nearly as efficiently as the full-length protein, whereas the N-terminal fragment, encompassing the IGFBP and von Willebrand factor type C domain domains, is inactive (Fig. 4B). Remarkably, NOV effect appears to be relatively selective for adrenocortical cells because it has no proapoptotic activity on other cell lines tested (Hela, COS; Fig. 4C). NOV-induced apoptosis is sen- sitive to the pan-caspase inhibitor Z-VAD and is also reduced by treatment with the selective caspase-3 (Z-DEVD), caspase-8 (Z-IETD), and caspase-9 (Z-LEHD) inhibitors (Fig. 5A). Consistent with these results, NOV induces a significant increase of caspase-3, caspase-8, and caspase-9 activities in H295R cells (Fig. 5B). Remarkably, NOV was unable to ac- tivate caspase-3 in Jurkat T leukemic cells (Fig. 5C), whereas this caspase was activated by anti-Fas treatment, as previ- ously described (23). Furthermore, NOV effect on apoptosis of primary tumor adrenocortical cells was measured. Adre- nocortical tumor cells in culture keep their differentiated phenotype, with the majority of cells expressing transcrip- tion factors SF-1 and DAX-1 (Fig. 6A) and steroidogenic enzymes P450scc and P450C17 (not shown). Similarly to the H295R cell line, apoptosis of primary adrenocortical tumor cells could be significantly elicited by recombinant NOV protein in SF-1-positive cells (Fig. 6B and Table 1).
NOV protein is expressed in human fetal adrenal cells
Previous studies have shown that NOV mRNA is detect- able in the human fetal adrenal, mostly in the definitive zone (13). Adrenocortical fetal adrenal cells in culture isolated from fetuses aged 11-14 wk after conception express the DAX-1 transcription factor (24) and the first enzyme in the steroidogenic cascade, P450scc (Fig. 7A). NOV protein is detected in the cytoplasm of the same cells that express
| SF-1-positive apoptotic cells | Nonapoptotic SF-1- positive cells | |
|---|---|---|
| 14 | 468 | Control cells |
| 46 | 419 | NOV-treated cells |
Primary tumor adrenocortical cells isolated from one childhood ACT were isolated by collagenase treatment and cultured in eight- well chamber slides. Equivalent volumes of protein buffer (control) or recombinant full-length NOV protein (10 µg/ml) were added. Num- bers of apoptotic (TUNEL staining) SF-1-positive cells in the cultures were counted after an 18-h incubation. The difference in the number of apoptotic cells between control and NOV-treated samples is sig- nificant (P < 0.0001, Fisher’s exact test).
DAX-1 (Fig. 7B). Remarkably, DAX-1-negative cells, which are not steroidogenic (Fig. 7A), do not express NOV.
Discussion
Members of the CCN family of proteins are multimodular factors associated with the extracellular matrix endowed with diverse physiological functions. Their biological activ- ities are often dependent on the specific cellular context, and they may even have opposing effect in different cell types (reviewed in Ref. 25). Here we have shown that NOV/CCN3 has a potent proapoptotic effect on human adrenocortical cells. Remarkably, NOV proapoptotic activity appears to be relatively selective for adrenocortical cells because other cell types are not sensitive to NOV. This is the first time that direct evidence is presented showing a proapoptotic effect of NOV through caspase activation. However, NOV has been associated with the inhibition of proliferation of several other cancer cell types (26-28). Together with data shown here, those reports indicate that the proapoptotic effect of NOV may target a restricted spectrum of tissues. Other members
A
O
DAPI
SF-1
DAX-1
SF-1/DAX-1
B
DAPI
TUNEL
Ctrl
NOV
A
DAPI
P450scc/DAX-1
B
DAPI
NOV/DAX-1
of the CCN family may function as growth factors or induce apoptosis, depending on the cell type (29-31). The molecular bases of those differential effects are still unknown, but they might be accounted for by differential expression of NOV receptors and/ or coupling to different signaling pathways in different cell types.
It is remarkable that in childhood ACTs, NOV expression is invariably reduced at the transcript and protein level (Fig. 1). Reduced expression of an endogenous proapoptotic factor may play an important role in the process of childhood ACT tumorigenesis, accounting at least in part for the defect of apoptotic regression of the fetal adrenal that has been pro- posed to be responsible for tumor formation (6). Signifi- cantly, in childhood ACTs, NOV levels are down-regulated in both the adenoma and carcinoma groups (Ref. 11 and our data). Importantly, increase of SF-1 dosage in human tumor adrenocortical cells is sufficient to repress NOV expression at the transcriptional level (Fig. 2). SF-1 overexpression in childhood ACTs is also a widespread finding, independent from clinical outcome (10). This is at variance with data reported in adult ACT, in which NOV expression is lower in carcinomas, compared with adenomas, and associated with a more severe prognosis (12). Our results suggest that in childhood ACTs, SF-1 overexpression and NOV down-reg- ulation may be essential for tumor formation but not for
progression to a malignant phenotype. Conversely, loss of NOV expression in adult adrenocortical carcinomas may reflect their loss of the differentiated adrenal cortex pheno- type, similarly to what has been shown for other transcripts that are expressed at high levels in normal adrenal cortex and down-regulated in carcinomas (32, 33).
In cultured human fetal adrenal cells, NOV is exclusively expressed by cells that are also positive for DAX-1 staining. Previous studies have shown that in the human fetal adrenal NOV mRNA is mainly expressed in the outer definitive zone (13). This zone represents the adrenal cortex proliferative compartment, in which the precursors of steroidogenic cells are generated. Because of its proapoptotic effect on adreno- cortical cells, it is tempting to speculate that in the physio- logical setting, NOV may play an important role in the dy- namic interplay of diffusible factors that regulate (1, 2, 34, 35) the proliferation and differentiation of fetal adrenal cells.
Acknowledgments
We thank Dr. Nicole Gallo-Payet for advice about fetal adrenal cell culture, Dr. Walter L. Miller for gift of the anti-P450scc antibody, and the personnel of the Service d’Orthogénie of the Archet Hospital for collaboration.
Received February 14, 2007. Accepted May 31, 2007.
Address all correspondence and requests for reprints to: Enzo Lalli, M.D., Institut de Pharmacologie Moléculaire et Cellulaire, Centre Na- tional de la Recherche Scientifique Unité Mixte de Recherche 6097, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail: ninino@ipmc.cnrs.fr.
This work was supported by a Centre National de la Recherche Scientifique ATIP grant, contrat d’interface Institut National de la Santé et de la Recherche Médicale-Centre Hospitalier Universitaire de Nice, CAPES-COFECUB (419/03), Fondation Recherche Médicale, Associa- tion Recherche sur le Cancer (3142), and Princess Grace Foundation (to E.L.); and National Institutes of Health Grant CA63230 (to G.P.Z.). M.D. is a recipient of a Fondation Recherche Médicale fellowship.
Disclosure Information: All authors have nothing to declare.
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