Osteopontin stimulates invasion of NCI-h295 cells but is not associated with survival in adrenocortical carcinoma
Dirk Weismann,1* * Juliane Briese,2-41 Joscha Niemann,5 Matthias Grüneberger,! Patrick Adam,6
Stefanie Hahner,! Sarah Johanssen,’ Wei Liu,2 Shereen Ezzat,7 Wolfgang Saeger,8 Ana-Maria Bamberger,5 Martin Fassnacht,’ Heinrich M Schulte,9 Sylvia L Asa,2 Bruno Allolio! and Christoph M Bamberger5
! University Hospital of Würzburg, Department of Internal Medicine I, Endocrine and Diabetes Unit, Germany
2 Department of Laboratory Medicine and Pathobiology, University of Toronto and Department of Pathology, University Health Network and Toronto Medical Laboratories, Canada
3 Institute of Molecular Medicine, University of Leeds, UK
4 Department of Obstetrics and Gynaecology, University of Greifswald, Germany
5 Laboratory of Endocrinology and Metabolism of Ageing, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
6 University of Würzburg, Institute of Pathology, Germany
7 The Endocrine Oncology Site Group, Mount Sinai and Princess Margaret Hospitals, Toronto, Ontario, Canada
8 Institute for Pathology, Marienkrankenhaus Hamburg, Germany
9 Endokrinologikum Hamburg, Germany
*Correspondence to:
Dr. Dirk Weismann, University Hospital Würzburg, Department of Internal Medicine I, Endocrine and Diabetes Unit, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. E-mail: weismann_d@klinik.uni- wuerzburg.de
+ These authors contributed equally to this study.
No conflicts of interest were declared.
Received: 4 September 2008
Revised: 14 January 2009 Accepted: 16 January 2009
Abstract
Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin ævß3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin avß3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI-h295 cells (>six-fold increase, p < 0.001). Transfection with integrin avß3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti-integrin 3 antibody, indicating a functional relationship of OPN and integrin avß3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan-Meier analysis including 111 patients with primary tumours graded for OPN staining and follow-up data available. In conclusion, our in vitro data indicate that OPN and integrin avß3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.
Copyright @ 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: adrenocortical cancer; osteopontin; OPN; proliferation; invasion; tissue microarray
Introduction
Adrenocortical carcinoma (ACC) is a rare but highly malignant neoplasm with a poor prognosis. The inci- dence is approximately 1-2 per million population per year [1-6], with a bimodal age distribution peaking in childhood and the fourth decade [7]. The patho- genesis of ACC is poorly understood [8]. Investiga- tion of hereditary tumour syndromes implicates p53 mutations (Li-Fraumeni syndrome) and uniparental paternal isodisomy for IGF II (Beckwith-Wiedemann syndrome) as culprit lesions. The molecular events fol- lowing these primary defects are, however, not well
understood and those of sporadic adrenal tumours are even more obscure. Although a growing number of molecular alterations have been described [2,9-11], a conclusive pathogenetic concept is lacking.
Using gene array analysis, Giordano et al [12] have shown osteopontin (OPN) mRNA expression to be 16- to 20-fold elevated in ACC, being the second highest transcribed mRNA after IGF II . OPN mRNA and/or protein expression levels are increased in many human tumours, including breast [13,14], lung [15], prostate [16], colon [17], ovarian [18], and gastric [19] cancer, and it has been suggested that OPN plays an important role in tumourigenesis, tumour progression,
and metastasis formation [20,21]. Accordingly, high OPN expression was associated with reduced sur- vival in some malignancies [15,22]. OPN enhanced tumourigenicity and metastatis [23,24] potentially in an autocrine fashion [25]. Paracrine effects may pro- vide protection from cytotoxic macrophages, possi- bly through inhibition of nitric acid production [26]. Reported receptors for OPN are CD44, CEACAM1 [27,28], and a variety of integrins [29]. Binding to integrin avß3 heterodimers via its RGD sequence has generally been accepted to be the primary receptor for OPN [30,31].
In this study, we investigated for the first time the expression and localization of OPN in adrenocortical tissue. We examined the role of OPN in invasiveness in NCI-h295 cells in vitro and investigated a link to integrin avB3. Furthermore, we correlated OPN expression with survival of patients registered in the German ACC Registry.
Materials and methods
Patients and tissues
Clinical data including survival and follow-up data of ACC patients were collected by the German ACC Reg- istry (www.nebennierenkarzinom.de) following writ- ten informed consent from patients and with the approval of the ethics committee of the University of Würzburg (Germany). Tissues were collected fol- lowing written informed consent from patients and with the approval of the ethics committees of the University of Würzburg (Germany), Hamburg (Ger- many) and the University Health Network (Toronto, Canada), respectively. Paraffin-embedded adrenal tis- sue was used for immunohistochemistry and tis- sues snap-frozen in liquid nitrogen were used for immunoblotting. In all cases, the clinical diagnosis was confirmed by histopathology. Paraffin blocks of 15 adrenocortical nodular hyperplasias, 16 adrenocor- tical adenomas, and 17 ACCs were available. Nor- mal adrenal gland specimens (n = 20) were obtained from patients who underwent adrenalectomy during nephrectomy for sporadic renal cell carcinoma.
Immunohistochemistry
Immunohistochemistry was performed with specific monoclonal anti-OPN antibody (1:200, Akm2A1; Santa Cruz, Heidelberg, Germany), anti-integrin avß3 antibody (1: 800, MAB1976; Chemicon, MA, USA), and anti-CD34 and anti-CD68 antibodies (both Dako, Glostrup, Denmark; 1:25 and 1:500, respectively). Serial sections of 4-6 um were cut from the paraffin blocks and mounted on 3-aminopropyl-triethoxysilane- coated slides, deparaffinized in xylene, and rehydrated in graded alcohol and Tris-buffered saline (50 mmol/l Tris, 150 mmol/l NaCl, pH 7.4). After washing with phosphate-buffered saline (PBS), slides were blocked for 10 min at room temperature with the avidin-biotin
blocking system (Dako) and incubated with anti-OPN antibody for 30 min or with anti-integrin avB3 anti- body overnight. Slides were reacted with biotiny- lated secondary antibody (Dako) for 10 min. Staining was performed using peroxidase-labelled streptavidin (Dako) and 3,3’-diaminobenzidine containing 0.03% H2O2. Sections were counterstained with haema- toxylin (Meyer’s Hemalaun; Merck, Germany). Two pathologists performed the immunohistochemical eval- uation independently. Staining was graded as follows: no staining = 0; low staining = 1; medium staining = 2; strong staining = 3.
Protein isolation and western blotting
Western blotting using specific anti-OPN monoclonal antibody (1:200, RB9097-P; Neomarkers), anti- integrin avB3 antibody (1:500, MAB1976; Chemi- con), and anti-actin antibody (1: 500, A3853; Sigma, St Louis, MO, USA) was performed on 40 nor- mal and 40 tumour samples of fresh-frozen mate- rial and on NCI-h295 cells. Tissues and cells were lysed [0.5% sodium deoxycholate, 0.1% sodium dode- cyl sulphate (SDS), 1% Nonidet P-40 and 1× PBS, 100 µg/ml phenyl-methylsulphonyl fluoride (PMSF), 69 µg/ml aprotinin (Sigma), and 1 mM sodium ortho- vanadate] and incubated on ice for 30 min, followed by micro-centrifugation at 10000 g for 10 min at 4℃. Fifty micrograms of protein was transferred onto nitrocellulose membranes (Bio-Rad Laborato- ries). Five per cent non-fat milk in 1x TBST was used to block non-specific binding. Detection was carried out with HRP-coupled secondary antibodies applying the ECL chemiluminescence detection system (Amer- sham, Buckinghamshire, UK). Densitometry for quan- tification of band intensities was performed. Cell lysate from osteosarcoma (Lab Vision) was used as a positive control for western blot analysis.
Tissue arrays
Adrenal tissue microarrays (TMAs) consisting of 167 ACC samples were constructed. Of these 167 ACC samples, 134 derived from surgery at the time of pri- mary diagnosis, 19 from local recurrence, and 14 from distant metastases. Briefly, haematoxylin and eosin (H&E)-stained sections of formalin-fixed and paraffin- embedded tissue blocks were re-evaluated to identify representative areas of well-preserved morphology and marked for tissue punching; TMAs were assembled according to the manufacturer’s instructions (Beecher Instruments, Silver Spring, MD, USA). Briefly, five cores with a diameter of 0.6 mm were punched from each adrenal block and arrayed into a recipient paraf- fin block at a distance of 1 mm between each core. Sections (2 um) were cut on silanized slides also used for conventional immunohistochemical stains. Slides were dried at room temperature for 7 days in order to minimize tissue loss during staining. H&E-stained slides were cut to verify tumour cell content. Tissue
arrays were stained with a monoclonal anti-OPN anti- body (Akm2A1; Santa Cruz). A case was considered evaluable if at least two of the five cores contained tumour.
Cell lines
The human adrenocortical cancer cell line NCI-h295 [32] was obtained from the American Type Culture Collection (Manassas, VA, USA). Suspension (NCI- h295: CRL-10296) and adherent (monolayer; NCI- h295R CRL-2128) cells were maintained as described previously [33]. The cells were grown at 37 ℃ in 5% CO2-95% air. For experiments, fetal calf serum (FCS) concentration was reduced to a maximum of 2% or as otherwise specified.
RNA extraction, PCR, and RT-PCR
Total RNA from NCI-h295 cells was isolated using a modification of the one-step phenol/guanidinium thiocyanate method (TRIzol; Invitrogen, Karlsruhe, Germany).
Three micrograms of total RNA was used to synthe- size single-stranded cDNA using the superscript first- strand synthesis system (BioRad, München, Germany) according to the manufacturer’s instructions. PCR was performed on a Mastercycler gradient (Eppendorf, Hamburg, Germany) with HotStarTaq DNA Poly- merase (Qiagen, Hilden, Germany) using OPN primers (5’-GCA GTG ATT TGC TTT TGC CT and 3’-GAT TCT GCT TCT GAG ATG GG) with the following protocol: 15 min of 94 ℃ activation and 37 cycles of 94 ℃/30 s, 65 °C/30 s, and 72°C/1 min. PCR prod- ucts were analysed on 2% agarose gels stained with ethidium bromide.
Transient transfection of NCI-h295 cells and in vitro invasion assay
Transfection of NCI-h295 cells was performed with LipofectAMINE PLUS reagent (Life Technologies, Karlsruhe, Germany). Cells were plated in six-well culture plates (500 000 cells per well). After 24 h, the medium was replaced by 0.8 ml of FCS-free medium and the cells were transfected with 1 ug of the plasmid avB3 integrin with PLUS reagent and lipofectAMINE. After 3 h incubation, aliquots of 1 ml of medium containing 20% FCS were added. Cells were harvested after 24 h. The construction of the expression vectors containing regions of the genes encoding both æv and ₿3 chains has been described [34]. As a negative control, the mock expression vector pcDNA3.1(+) (Invitrogen) was used.
Invasiveness was assayed in a membrane inva- sion culture system (BD BioCoat Matrigel Invasion Chamber; BD Biosciences). Cells were harvested in trypsin-EDTA buffer, washed with RPMI 1640, and subsequently seeded at 2.5 x 104 cells per 500 ul of RPMI 1640 on the Matrigel-coated PET membrane. Two micrograms of human OPN was added directly
to the medium and the plates were incubated at 37 ℃ for 48 h. Migrated cells were fixed and stained with Diff-Quick (Dade Behring AG, Düdingen, Switzer- land) and quantified by counting seven high-power fields (460 um × 700 um) using a Zeiss microscope. Each measurement was performed in triplicate and experiments were repeated five times.
Dye exclusion assay
Cells were seeded (106 cells per ml) in 12-well cell culture plates. OPN was added to a final concentra- tion of 0.1-10 µg/ml. After 96 h, viability was deter- mined by tryptan blue staining (Invitrogen, Eggen- stein, Germany). Each experiment was performed in triplicate.
Hormonal analysis
NCI-h295 cells are a well-characterized model to study steroid hormone synthesis [35,36]. NCI-h295 cells were incubated for 6, 24, and 96 h with OPN (0.1-10 µg/ml). Each experiment was performed in triplicate. At the end of the incubation period, cor- tisol and dehydroepiandrosterone sulphate (DHEA-S) were determined in the cell supernatants by commer- cially available RIAs (DPC Biermann, Bad Nauheim, Germany).
Statistical analysis
The Kruskal-Wallis test or chi-square test and Stu- dent’s t-test or ANOVA were used as appropriate. Survival probabilities were estimated by the univariate Kaplan-Meier method and survival curves were com- pared by the log-rank test. Statistical differences with p values less than 0.05 were considered significant. Analyses were performed with R [37].
Results
Characterization of OPN in adrenocortical tissue
Adrenocortical tissue stained positive for OPN and a cytoplasmatic OPN staining pattern was seen. In the normal adrenal gland, OPN was found to be expressed exclusively in the cortex, with the staining intensity increasing in a centripetal fashion (Figure 1A). The adrenal cortex also stained positive for integrin avB3 (Figure 1B). Moderate expression of OPN and integrin avB3 was found in nodular hyperplasia, with OPN being expressed homogeneously and integrin ævß3 showing a more focal punctate pattern (Figures 1C and 1D). Expression of OPN was heterogeneous in adrenocortical adenomas (Figure 1E) and carcinomas (Figure 1G), with staining intensity often being more accentuated in single cells or cell clusters. Between positive tumour nests there were also some posi- tive inflammatory stromal cells, like macrophages and plasma cells. Immunoreactivity against OPN was most
OPN
Integrin
normal adrenal gland
A
B
D
nodular hyperplasia
C
D
Adenoma
E
F
ACC
G
H
pronounced in adrenocortical tumour cells and not in CD68-positive macrophages (data not shown). Within adrenocortical carcinomas, extensive fibrous bands as well as CD34-positive sinusoidal structures showed no OPN expression (data not shown). No significant impact of OPN expression was found on Weiss score criteria (data not shown). Integrin avB3 showed a sim- ilar staining pattern in adrenal adenomas (Figure 1F) and in carcinomas (Figure 1H). Accumulation of inte- grin avß3 in single tumour cells or cell groups was found, yielding a focal punctate membranous pattern or staining of the cytoplasm with perinuclear accumu- lation.
Immunoblot analysis of OPN on human primary adrenal tissue
Western blot analysis confirmed OPN expression in all adrenocortical tissues (Figures 2A-2C), with normal tissue staining less intensively than carcinomas. The
size of the detected protein was identical in all tissues and corresponded to a molecular weight of ~65 kD. Figure 2D represents the intensity of the OPN expres- sion relative to actin levels and significantly higher expression of OPN in adrenocortical carcinomas than in normal adrenal tissue (p < 0.01). Additionally, inte- grin avß3 immunoblotting was performed on normal adrenal tissue, adrenal adenomas, and carcinomas. All samples showed strong expression for integrin avB3 (Figure 2E). There was no difference in the expression level between normal samples and adrenal carcino- mas. The results are the average of seven independent experiments.
Assessment of OPN effects in NCI-h295 cells
Invasion was studied using the Matrigel in vitro invasion assay. A potential functional link between OPN and integrin avB3 was investigated using
J Pathol 2009; 218: 232-240 DOI: 10.1002/path Copyright @ 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
A
1
2
3
4
5
6
OPN
ß actin
B
1
2
3
4
5
6
OPN
ß actin
C
1
2
3
4
5
6
OPN
ß actin
D
**
2.0
Arbitrary Units
1.5
T
T
1.0
Normal
Adenoma
Carcinoma
E
1
2
3
4
5
6
7
8
9
Integrin avß3
ß actin
adrenocortical carcinoma NCI-h295 cells transfected with integrin avB3 (Figure 3A). Stimulation of mock- transfected NCI-h295 cells with 2 ug of OPN strongly enhanced invasiveness (p < 0.001), which was abol- ished by the addition of an anti-OPN antibody. Trans- fection with integrin avB3 also increased cellular inva- siveness (p < 0.001 versus control), which was again abolished by the addition of an anti-integrin ævß3 anti- body. An additive effect was seen after stimulation of integrin avB3-transfected cells with OPN (p < 0.001). This was reversed by inhibition of the integrin receptor by anti-integrin §3 antibodies in transfected cells,
decreasing invasiveness to a level comparable to OPN- stimulated control cells. PCR transcription (data not shown) and transfection efficiency were confirmed by western blotting (Figures 3B and 3C).
No significant effect on proliferation was observed after 96 h of stimulation using cell count analysis (Figure 3D). As a control, co-stimulation of 10 µg/ml OPN with EGF (20 ng/ml) increased the proliferation rate after 96 h significantly (p < 0.01) (Figure 3E).
Cortisol and DHEAS synthesis of NCI cells was not significantly affected by OPN, although cortisol levels were slightly decreased after 96 h stimulation
A
**
**
*
Arbitrary Units
6
4
*
2
0
OPN
+
+
+
+
anti-OPN
+
Integrin avß3
+
+
+
+
anti-integrin ß3
+
+
B
1
2
3
C
1 2 3 4 5
OPN
Integrin avß3
ß actin
ß actin
*
D
E
1.2
1.0
1.0
Arbitrary Units
0.8
Arbitrary Units
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0
0.1
0.3
1
3
10
0.0
Control
EGF
OPN
OPN+EGF
OPN [ug/ml]
with 10 μg/ml OPN (168±9 versus 147±4 μg/dl, p = 0.09).
Tissue array and correlation of OPN with survival in ACC
Using tissue arrays, OPN expression was determined in 167 ACC samples. Due to loss of individual spots during the staining process, 147 samples were avail- able for immunohistochemical analysis. We confirmed high OPN expression in these ACC samples [median 2 (0-3)]. No differences were seen in OPN staining stratified for WHO stage (Table 1) and no differences were seen in the staining pattern of metastases (12/14; positive/total) and relapses (16/18; Figure 4A). For 111 patients (male/female 40/71) with primary tumour
samples represented on the TMA, complete clinical follow-up data were available and OPN staining was evaluable. Survival time was calculated from the date of surgery to the date of death or to the date of last follow-up. No association was seen between the grade of OPN expression and survival (Figure 4B) with or without adjusting for WHO stage (data not shown). Furthermore, no correlation was seen for OPN expres- sion and hormonal excess in ACC (data not shown).
Discussion
Based on gene array analysis, OPN mRNA has pre- viously been reported as overexpressed in ACC, sug- gesting a role in the pathogenesis of this disease. In
J Pathol 2009; 218: 232-240 DOI: 10.1002/path Copyright @ 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
| WHO stage | |||||
|---|---|---|---|---|---|
| I | II | III | IV | p value | |
| n* | 5 | 45 | 24 | 35 | |
| Age | 54 ± 24 | 50 ± 15 | 53 ± 14 | 49 ± 18 | nst |
| Sex (M/F) | 2/3 | 16/29 | 10/14 | 11/24 | ns* |
| Tumour size | 4.7±0.3 | 11.8 ±4.5 | 11.5 ± 3.5 | 13.6±4.3 | <0.001+ |
| OPN expression11 | 2 (1-3) | 3 (1-3) | 2.5 (0-3) | 2 (1-3) | ns|| |
Data are presented as mean ± SD, unless otherwise stated.
* For two patients, WHO stage was not available because the initial tumour size was unknown. This makes a total of 109 patients in this table instead of the 111 patients included in the survival analysis.
p values are based on * ANOVA, # the chi-square test, and | the Kruskal-Wallis test. 1 Median and range.
ns = not significant.
A
Immunohistochemical Grading
Cumulative Proportion Surviving 00
1.0
3
OPN
-0
0.8
- 1
— 2
2
0.6
·- 3
0.4
1
0.2
0
o
0.0
Primary
Relapse
Metastasis
0
50
100
150
Survival Time [months]
this study, we have shown that the normal human adrenal gland physiologically expresses OPN. Stain- ing was found exclusively in the cortex, with the intensity being highest in the zona reticularis. Expres- sion of OPN was heterogeneous in ACCs, with high expression in single tumour cells or cell nests. Western blot analysis confirmed higher expression of OPN in adrenal carcinomas.
In recent publications, OPN expression has been associated with a more aggressive clinical course [38]; linked to matrix metalloproteinase-9 pathway activation [39]; and furthermore, lentiviral-mediated miRNA against OPN suppressed tumour growth and metastasis in vitro [40]. Binding to integrin avB3, OPN has been shown to stimulate lung cancer cell migration by activation of the AKT-NFKB path- way [41]. Integrin avB3 has been widely described as the mediating receptor for OPN in many malig- nancies and models [30,31,42,43]. Accordingly, we proved the expression of integrin avB3 in the same tissues analysed for OPN expression. To investigate a functional link, we studied the invasiveness of OPN- stimulated NCI-h295 cells with and without overex- pression of integrin avB3. We have shown that OPN greatly increases the invasiveness of mock-transfected NCI-h295 cells. Overexpression of integrin avB3
increased invasiveness by itself and OPN stimulation further enhanced invasiveness in an additive fashion. However, inhibition of the integrin receptor reversed only this additional effect. This proves that the effects of OPN on invasiveness are not exclusively transduced by the integrin avB3 receptor in NCI-h295 cells.
In prostate cancer cells (LNCaP cells), co- stimulation with OPN and EGF further enhanced proliferation [44]. Co-stimulation with EGF and OPN also increased proliferation in this study (p < 0.01), whereas stimulation with either EGF or OPN alone did not show a significant effect. As OPN may act as an adhesion factor important for proliferation [45], we performed all tests with soluble as well as with adherent NCI-h295 cells, but no differences in proliferation were observed.
Steroid hormones enhance OPN expression in vari- ous cells [44,46,47]. As important effects of OPN are mediated by autocrine and paracrine actions in some tissues [26], we tested whether OPN has an EGF- like effect on hormone production [48] and might therefore participate in maintaining hormone secre- tion during states of hormone excess, for example, in adenomas or carcinomas. As shown in our results, different concentrations of OPN did not significantly alter cortisol or DHEA levels in NCI-h295 cells, but
testing other models for steroidogenesis may provide additional information [49].
It was suggested that OPN secreted from trans- formed cells is post-translationally modified differ- ently to OPN from normal cells [50]. The expression of OPN is induced by various signalling proteins that are often constitutively active in tumours: growth fac- tors such as the hepatocyte growth factor [51]; onco- genes such as RAS [52]; and tumour promoters such as 12-O-tetradecanoyl phorbol 13-acetate [53]. Recently, it was shown that OPN suppresses NO production by macrophages, which in turn enhances the proliferation of murine colorectal cancer cells (CT 26) in co-culture experiments [54]. We could not exclude such a mech- anism for ACCs.
Tissue array analysis confirmed high expression of OPN in ACC in a large number of patients, but sur- vival analysis with and without adjustment for WHO stage did not indicate a relationship between OPN expression and survival in ACC. One can speculate that such an effect may be seen in cases with concomi- tant overexpression of integrin æv3. However, as our western blot analysis has shown, there is no evidence of varying expression of integrin avß3 in ACCs. It seems likely that adrenocortical tumours behave dif- ferently to other entities, but we cannot rule out that inhibition of this system might have an effect in vivo by reducing invasiveness and therefore tumour growth.
The present study describes the first systematic anal- ysis of OPN expression in the adrenal gland and in adrenocortical tumours. OPN expression was not found to be associated with patient survival in ACC. However, we have found increased cellular inva- siveness of integrin avB3 overexpressing NCI-h295 cells after OPN treatment, indicating a functional link between OPN and integrin avB3. This relationship remains of relevance to our understanding of carcino- genesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.
Acknowledgements
The following pathologists provided tumour material of two or more patients for the tissue array: Professor Gerhard Seitz (Klinikum Bamberg); Professor Harald Stein, Professor Manfred Dietel (Charite University Berlin); Professor Gerhard Mall (Klinikum Darmstadt); Professor Helmut Erich Gabbert (University of Düsseldorf); Professor Werner Schmid (Univer- sity of Essen); Professor Steffen Hauptmann (Martin-Luther University of Halle); Professor Peter Schirmmacher (Univer- sity of Heidelberg); Professor Alfred C Feller (University of Lübeck); Professor C James Kirkpatrick (University of Mainz); Professor Roland Moll (University of Marburg); Professor Cyrus Tschahargane (Lukaskrankenhaus, Neuss); Professor Rainer Horst Krech (Klinikum Osnabrueck); Professor Fer- dinand Hofstaedter (University of Regensburg); Dr Andrea Maria Gassel (Leopoldina Hospital Schweinfurt); and Profes- sor Konrad Mueller-Hermelink (University of Würzburg). The following hospitals/clinicians contributed clinical data from three or more patients to the German Adrenocortical Can- cer Registry: Marcus Quinkler, Wolfgang Oelkers (University
Hospital Charite Berlin); Holger Willenberg (University Hospi- tal Düsseldorf); Michael Morcos (University Hospital Heidel- berg); Peter Langer (University Hospital of Marburg); Detlef Meyer (Leopoldina Krankenhaus Schweinfurt); Christian Fot- tner (University Hospital Mainz); Michael Brauckhoff (Univer- sity Hospital Halle); Felix Beuschlein (University of Freiburg); Horst L Fehm (University Hospital Lübeck); Dagmar Führer (University Hospital Leipzig); and Stefan Petersenn (Univer- sity Hospital Essen). We appreciate the support of Uwe Maeder (Tumor Center, University Hospital Würzburg) in establishing the German ACC Registry database. This work was supported by the Deutsche Krebshilfe (grant No 107313 to CMB and grant No 106080 to MF and BA).
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