The role of the insulin-like growth factor system in adrenocortical tumourigenesis
M. M. Weber, C. Fottner and E. Wolf*
Medical Department II, Laboratory of Endocrine Research, Klinikum Grosshadern, Germany; * Institute for Molecular Animal Breeding/Gene Center, Ludwig-Maximilian University, Munich, Germany
Abstract
Background The insulin-like growth factor (IGF) system plays a central role in the mechanism of transformation and tumourigenesis. Elevated levels of IGF-II and IGF-I have been found in adrenocortical carcinomas.
Material and methods We examined binding characteristics and concentrations of both IGF-receptors in normal adult human adrenocortical glands, and compared them with the IGF-I receptor binding in adrenocortical tumours of various origins. The human IGF-I receptor was overexpressed in the mouse adrenocortical tumour cell line Y1, and growth studied in response to IGF stimulation. The influence of IGF-II on adrenal morphology and function was assessed in transgenic mice that postnatally overexpress IGF-II.
Results While the abundance of the IGF-I receptor in adrenocortical hyperplasias and adenomas was similar to normal tissue, a strong overexpression of the intact IGF-I recep- tor was found in three out of four adrenocortical carcinomas. Y1 cells overexpressing the human IGF-I receptor respond to IGF-I with an increase in thymidine incorporation by 140%. Furthermore, the antiproliferative effect of ACTH is blunted. In transgenic mice postnatally overexpressing IGF-II, adrenal weight is increased, mainly due to a 50% increase in the number of zona fasciculata cells. Plasma corticosterone levels in these mice are twofold higher than in controls, in contrast to similar plasma ACTH levels, thus indi- cating a direct effect of IGF-II on adrenal cell hyperplasia and function.
Conclusion There is substantial evidence that the IGF-system is involved in adrenal growth and tumourigenesis. High local levels of IGF-II in combination with elevated IGF-I receptor concentrations would represent a significant growth advantage of the adrenocortical carcinoma cell and could contribute to a highly malignant phenotype. IGF-II overexpression alone seems not to be sufficient for malignant transformation.
Keywords IGF-I, IGF-II, adrenal tumours. Eur J Clin Invest 2000; 30 (Suppl. 3): 69-75
Introduction
The insulin-like growth factor (IGF) system plays a cen- tral role in the mechanism of transformation and tumourigenesis [1-4]. IGFs inhibit apoptosis, promote tumour growth and induce transformation and metastasis
Medical Department II, Laboratory of Endocrine Research, Klinikum Grosshadern, Germany M.M. Weber, C. Fottner); Institute for Molecular Animal Breeding/Gene Center, Ludwig- Maximilian University, Munich, Germany (E. Wolf).
Correspondence to: Matthias M Weber, PhD, Medizinische Klinik II, Klinikum Großhadern, Marchioninistr. 15, 81377 Munich, Germany. Fax: +49-89-7004418
in many types of malignancies. Elevated levels of IGF-II and - to a lesser extent - of IGF-I have been found in many tumours including adrenocortical carcinomas. Most, if not all mitogenic effects of IGF-I and IGF-II are mediated through the IGF-I receptor, and the majority of tumours express intact IGF-I receptor. Increased levels of the IGF-I receptor have been described in lung tumours, breast carcinomas and Wilm’s tumours [5,6]. Over- expression of the human IGF-I receptor promotes ligand- dependent neoplastic transformation and IGF-induced mitogenesis can be inhibited by interruption of the IGF-I receptor signaling [1-4,6-12].
IGFs are involved in the regulation of growth and dif- ferentiation of the adrenal gland. IGF peptides, receptors and binding proteins are synthesized by the adrenal
glands of various species, and both IGF-I and IGF-II have been found to induce steroidogenesis and mitogenesis in adrenocortical cells in vitro [13-20]. Accumulating data indicate that IGF-II not only is an important fetal adreno- cortical growth factor, but also is involved in the regu- lation of adult adrenal growth and function. In the adult bovine and human adrenal gland IGF-II enhances the steroidogenic effect of ACTH more potently than IGF-I. This effect is mediated through interaction with the IGF-I receptor, and modulated by locally produced IGF binding proteins [14,20]. In the actively growing fetal human adrenal gland, high levels of IGF-II are expressed, whereas in adult adrenal tissue only low IGF-II levels are found. It is assumed that IGF-II mediates the ACTH- induced fetal adrenal growth since ACTH induces IGF-II gene expression in human fetal adrenocortical cells, and since IGF-II is mitogenic in these cells [16,21-23]. Overexpression of IGF-II, which might contribute to neoplastic cell proliferation, has been found in func- tional human adrenocortical carcinomas and in pheo- chromocytomas [24-32]. Furthermore, elevated levels of IGFBP-2 have been described in adrenocortical carci- nomas [33].
Characterization of IGF-I receptor binding in adrenocortical tumours
Although the expression of both types of IGF receptors has been identified in normal and tumourous human adrenal tissue by RT-PCR and immunohistochemistry [24,34], until recently no data were available on the bind- ing characteristics and concentrations of the IGF-I
receptor in human adrenal tumours. In order to further clarify the significance of the IGF-I receptor in tumouri- genesis of the human adrenal gland, we examined the binding characteristics and concentrations of both IGF- receptors in normal adult human adrenocortical glands, and compared them with the IGF-I receptor binding in adrenocortical tumours of various origin [17]. Using membrane preparations of 14 normal adrenocortical glands, we identified and characterized the IGF-I receptor with its typical binding characteristics. The Scatchard analysis revealed a single class of high-affinity binding sites with a dissociation constant (Ka) of 0.16 ± 0.03 nmol L-1, and a receptor concentration (RC) of 19.2 ± 2.5 nmol kg1 protein (Fig. 1). Affinity cross- linking experiments with normal and tumourous adreno- cortical tissue displayed a band at an apparent molecular weight of 135 kDa, corresponding to the size of the nor- mal d-subunit of the IGF-I receptor. When 125I-IGF-I binding in adrenocortical hyperplasias (RC 19-6 ± 2.0 nmol kg-1 protein, Ka 0.19 ± 0.04 nmol L-1, n = 4) and adenomas (RC 17.5 ± 3.1 nmol kg1 protein, Ka 0.21 ± 0.04 nmol L-1, n = 4) was compared with the 125I- IGF-I binding in normal adrenocortical tissue, similar IGF-I receptor concentration and binding kinetics were found (Table 1). In contrast, three out of four hormonally active adrenocortical carcinomas showed a strongly ele- vated specific 125I-IGF-I binding with a three- to fourfold increase in IGF-I receptor concentration, as compared with normal adrenocortical tissue (Table 2). This resulted in a significantly higher mean specific binding and recep- tor concentration in adrenocortical carcinomas, while the binding kinetics and the size of the a-subunit of the IGF-I receptor remained unaltered (n = 4, RC 72.2 ± 21.3 nmol kg-1 protein, K3 0.17 ± 0.02 nmol L-1).
spezifische Bindung (%)
15
12
10
7.5
5
2.5
0
0
0.06
0.25
1
4
16
IGF-I (nM)
☒ normale NNR
0.20
gebunden / frei
0.15
0.10
0.05
0
0
0.01 0.02 0.03 0.04
gebundenes IGF-I (nM)
☐ NNR-Carcinom
analysis was calculated from the binding data using a computer- based analysis device (Ligand). Each point represents the mean of two duplicate determinations and the data are representative for the findings in 14 normal and three out of four carcinomatous adrenocortical tissues (from [17]).
| n | Specific binding (%) | ED50 (nmol L-1) | Ka (nmol L-1) | Receptor concentration (nmol kg-1 protein) | |
|---|---|---|---|---|---|
| Normal adrenal cortex | 14 | 5.0 ± 0.5 | 0.17 ± 0.02 | 0·16 ± 0.03 | 19.2 ± 2.5 |
| Adrenocortical hyperplasia | 4 | 4·1 ± 0.4 | 0.23 ± 0.08 | 0·19 ± 0·04 | 196 ± 2.0 |
| Adrenocortical adenoma | 4 | 4.0 ± 1.1 | 0-28 ± 0.15 | 0.21 ± 0.04 | 17.5 + 3.1 |
| Adrenocortical carcinoma | 4 | 13.8 ± 4.2 | 0.23 ± 0.04 | 0·17 ± 0.02 | 72.2 ± 21.3 |
Values are means ± SEM (from [17]).
| Control (n = 4) | PEPCK-IGF-II (n = 3) | Relative change (%) | |
|---|---|---|---|
| Body weight (g) | 36.6 ± 1.5 | 39.3 ± 4.5 | 107 |
| Adrenal weight (mg) | 1.9 ± 0.46 | 2.6 ± 0.22 | 137 |
| Adrenal volume (mm3) | 1.83 ± 0.44 | 2.5 ± 0.21 | 137 |
| Adrenal cortex volume (mm3) | 1.38 ± 0.36 | 1.96 ± 0.19 | 142 |
| (a) capsule: volume (mm3) | 0.12 ± 0.05 | 0·15 ± 0·01 | 125 |
| (b) zona glomerulosa (mm3) | 0.31 ± 0.09 | 0·36 ± 0.04 | 116 |
| (c) zona fasciculata (mm3) | 0.94 ± 0.25 | 1·44 ± 0.13 | 153 |
| Adrenal medulla (mm3) | 0·45 ± 0·11 | 0·55 ± 0.07 | 122 |
From [36].
In summary, we show that intact IGF-I and IGF-II recep- tors are present in normal adult human adrenocortical tis- sue. While the abundance of the IGF-I receptor in adrenocortical hyperplasias and adenomas was similar to normal tissue, a strong overexpression of the intact IGF-I receptor was found in three out of four adrenocortical carcinomas [17].
1,5
spezifische Bindung (%)
1,0
0,5
0
0
0.25
1
4
16
32
IGF-I (nM)
☒ untransfizierte Y1
Overexpression of IGF-I receptors in Y1 mouse adrenocortical tumour cells
The combined overexpression of the fetal growth factor IGF-II and the IGF-I-receptor in adrenocortical tumour cells is likely to represent an important growth advantage of the carcinomatous cell and thereby might contribute to
0.02
gebunden / frei
0.01
0
0
2,5
5
10
20
gebundenes IGF-I (pM)
☐ IGF-IR-Y1
from the binding data using a computer-based analysis device (Ligand). Each point represents the mean of two duplicate deter- minations.
the highly malignant phenotype of adrenocortical carci- nomas. In order to evaluate this hypothesis in vitro, we stably transfected the cDNA of the intact human IGF-I- receptor into the mouse adrenocortical tumour cell line Y1. Using reverse transcriptase polymerase chain reaction (PT-PCR), we identified the human IGF-I receptor- mRNA expression. When wild-type Y1 cells were sub- jected to an IGF-I receptor binding assay, only a low specific 125I-IGF-I binding of 0.5% was detected, corre- sponding to a low number of high-affinity IGF-I receptors (1500 receptors cell-1) with typical binding characteristics (Ka 1.6 ± 0-4 nM) as evaluated by Scatchard analysis (Fig. 2). In contrast, transfected Y1-cells exhibited a threefold higher specific binding with a significant increase in the IGF-I receptor number (18.350 receptors cell-1) while the Ka remained comparable (Ka 1.9 ± 1.1 nM). These results confirm the stable integration and expression of the intact human IGF-I receptor-cDNA in the transfected IGF-IR-Y1 cells. While the morphological appearance of IGF-IR-Y1 cells is identical to the parental cells (WT-Y1), the overexpression of IGF-I receptors results in an increased mitogenic response to IGF-I in vitro. When WT-Y1 cells are subjected to proliferation assays under serum reduced conditions, only a small and nonsignificant increase in thymidine incorporation was observed under the influence of IGF-I (6.5 nM), which could be antagonized by the well-known antimitogenic effect of ACTH (10-8 M) in vitro (Fig. 3). In contrast, IGF-IR-Y1 cells respond to IGF-I with an increase in thymidine incorporation by 140%. Furthermore, in these cells the antiproliferative effect of ACTH is blunted and can be further antagonized by exogenous IGF-I (Fig. 3). In summary, these results indicate that the absolute amount of IGF-I receptors cell-1 plays an important role in the regulation of cell growth, and that an overexpres- sion of intact IGF-I receptors in carcinomatous adreno- cortical cells can further enhance the mitogenic reaction of these cells.
Postnatal overexpression of IGF-II in transgenic mice
IGF-II is mitogenic for adrenocortical cells [15,16] and it is assumed to play an important role during fetal adrenal development [22,23]. Furthermore, a strong overexpres- sion of IGF-II in adrenocortical tumours seems to be associated with a highly malignant phenotype [24-27]. In order to evaluate its role in the regulation of adult adrenal growth and steroidogenesis in vivo, we assessed the influ- ence of IGF-II on adrenal morphology and function in transgenic mice, which postnatally overexpress IGF-II. These PEPCK-IGF-II-transgenic mice, in which the human IGF-II gene has been placed under the control of the phosphoenolpyruvate carboxykinase (PEPCK) pro- moter, are characterized by four- to sixfold elevated post- natal IGF-II serum levels, elevated serum IGFBP-2 levels and subtle changes in organ growth, while their total body weight and morphological appearance remain unchanged [35]. However, PEPCK-IGF-II transgenic mice exhibit twofold higher basal corticosterone levels than age- and sex-matched controls (Fig. 4), both in the morning (7.4 ± 1.5 vs. 17.8 ± 3.9 ng mL-1, P < 0.01) as well as in the evening (33.3 ± 6.5 vs. 65.3 ± 12 ng mL-1, P < 0.01). Upon stimulation with ACTH, corticosterone levels were stimulated sixfold to 396 + 17 ng mL-1 in PEPCK-IGF-II transgenic mice, as compared with 230 ± 24 ng mL-1 in the control group (Fig. 4). In contrast to corticosterone, plasma ACTH levels were similar in transgenic and con- trol mice, excluding an indirect effect of IGF-II at the hypothalamic or pituitary level. In vitro, the basal and ACTH-induced corticosterone production of adrenal glands from transgenic mice was higher (twofold and 1.8- fold, respectively) than that of control organs. However, when normalized for adrenal weight, the in vitro corticos- terone secretion was similar in both groups. At autopsy, adrenal weights of transgenic mice were significantly greater than those of control adrenal glands (3.3 ± 0.2 vs.
160
untransfizierte Y1
IGF-IR-Y1
140
Thymidineinbau (% Kontrolle)
120
100
80
60
0
Kontrolle ACTH IGF-I ACTH + Kontrolle ACTH IGF- I ACTH + IGF- I
IGF- I
@ 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30 (Suppl. 3), 69-75
Figure 3 Effect of ACTH and IGF-I on thymidine incorporation in wild-type and in IGF-I receptor overexpressing Y1 cells (IGF-IR-Y1). A total of 104 cells were incubated for 36 h under serumfree con- ditions with ACTH (10-8 M) and/or IGF-I (6-5 nM). In the following, the cells were incubated for another 12 h in the presence of 3H-thymidine, washed, lysed and the incorporated activity was assessed. The bars represent the mean + SEM of six incubations, and the data are representative for three independent incubation experiments.
**
400
T
corticosterone (ng/ml)
350
300
250
200
150
100
**
50
0
basal
ACTH-induced
☐ control mice
☐ PEPCK-IGF-II-mice
adrenal weight in transgenic mice was mainly due to a 50% increase in the number of zona fasciculata cells, while cell volume and zonation of transgenic adrenal glands remained unchanged (Fig. 5, Table 2). In sum- mary, these data indicate that postnatal overexpression of IGF-II induces an increased adrenal weight and elevated corticosterone serum levels, presumably by a direct mito- genic effect of IGF-II on adrenocortical fasciculata cells [36].
Discussion
There is substantial evidence that the IGF system is involved in adrenal growth and tumourigenesis. Over- expression of IGF-II and IGFBP-2 has been found in human adrenocortical carcinomas [33], and we have demonstrated an overexpression of IGF-I receptors in a subgroup of malignant adrenocortical tumours [17]. The mechanism and functional significance of the overexpres- sion of IGF-II and the IGF-I-receptor in adrenocortical carcinomas remains unknown at present. The mitogenic effect of IGF-II is dependent on the presence of the IGF-I receptor, and we have recently shown that in the adult bovine and human adrenal gland the steroidogenic effect of IGF-II is mediated through interaction with the IGF-I receptor [14,20]. In fibroblasts, overexpression of the human iIGF-I receptor promotes ligand-dependent neo- plastic transformation and the absence of IGF-I receptors prevents malignant growth and transformation in vitro
C
T.
C
G
F
M
M
5
and in vivo [6,10,11]. Here we report that in analogy to fibroblasts, the overexpression of IGF-I receptors in Y1 adrenocortical tumour cells is able to induce IGF-depen- dent cell growth and to antagonize the antiproliferative effect of ACTH in vitro. Furthermore, we were able to demonstrate a direct mitogenic effect of IGF-II on adult adrenocortical cells in vivo. It is evident therefore, that high local levels of IGF-II in combination with elevated IGF-I receptor concentrations would represent a signifi- cant growth advantage of the adrenocortical carcinoma cell and could contribute to the highly malignant pheno- type, at least in a subgroup of this rare type of cancer. However, the fact that the described PEPCK-IGF-II mice did not develop macroscopically obvious tumours over a 18-month period [37], suggests that IGF-II overpro- duction by itself is not sufficient for malignant transform- ation, and that additional factors are required for tumourigenesis. Further studies will have to elucidate whether the increase of IGF-I receptor and IGF-II pep- tide concentrations in malignant adrenocortical tumours is merely an epiphenomenon or represents a specific step in tumourigenesis.
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