Expression of Insulin-Like Growth Factor Binding Protein 1-6 Genes in Adrenocortical Tumors and Pheochromocytomas
V. Ilvesmäki1, J. Liu1, P. Heikkilä1, A. I. Kahri1, R. Voutilainen1,2
1 Departments of Pathology and Medicine, University of Helsinki, Helsinki, Finland
2 Department of Pediatrics, Kuopio University Hospital, Kuopio, Finland
The insulin-like growth factor (IGF) system appears to be important in the regulation of adrenal growth and hormone synthesis. As IGF-binding proteins (IGFBPs) modify IGF bioactiv- ity, we investigated the expression of IGFBP 1-6 genes in differ- ent adrenal tumors and hyperplasias to further clarify the role of the IGF system in adrenal pathophysiology. IGFBP-1 mRNA levels were too low to be detected by Northern blot analysis, but could be found by RT-PCR in some tumors and hyperplastic adrenals. Other IGFBPs were detected by Northern blotting. IGFBP-3 mRNA levels were very low in normal adrenals. In adre- nal tumors and hyperplastic adrenals, IGFBP-3 mRNA expres- sion was usually higher than in normal adrenals. In hormonally active adrenocortical carcinomas, IGFBP-2, -4, -5 and -6 mRNA levels were lower than in nonfunctional carcinomas and normal adrenals. The low IGFBP mRNA expression in the hormone-pro- ducing carcinomas was associated with high IGF-II mRNA con- tent. In adrenocortical adenomas from patients with Cushing’s or Conn’s syndrome, mean IGFBP mRNA levels were higher than in normal adrenals or in hormonally inactive adenomas. In nodular and bilateral hyperplasias, IGFBP-2, -3 and -4 mRNA ex- pression was on average higher than in normal adrenals but varied substantially, as did IGFBP mRNA levels in pheochromo- cytomas. In comparison to normal adrenals, pheochromocyto- mas expressed on average higher levels of IGFBP-2 and -4 but less IGFBP-5 and -6 mRNAs. Our data show that the six IGFBPs 1-6 are expressed at variable level in adrenal tumors and hy- perplasias. The low level of IGFBP mRNAs in hormonally active adrenocortical carcinomas was of particular interest.
Key words: Insulin-Like Growth Factor Binding Protein - IGFBP - Adrenal - Adrenocortical - Pheochromocytoma
Introduction
Insulin-like growth factors (IGF-I and IGF-II) are polypeptides structurally related to proinsulin [1,2]. They are produced in multiple tissues and many of their actions are mediated locally via paracrine or autocrine mechanisms. Generally, they can stimulate cellular proliferation and/or differentiation in nor- mal tissues and may also contribute to the autocrine stimula- tion of cell growth in a variety of tumors, including Wilms’ tu-
mor, rhabdomyosarcoma, hepatoblastoma, colon carcinoma, neuroblastoma, leiomyoma, and leiomyosarcoma [3,4]. IGFs are important regulators of adrenocortical steroidogenesis. In experimental conditions, they usually stimulate steroid pro- duction [5,6]. In addition to the regulation of steroidogenesis, IGFs appear to be involved in the regulation of adrenocortical cell growth. IGF-II mRNA expression in human fetal adrenals is very high, and in cell cultures, it is regulated by ACTH, poly- peptide growth factors and protein kinase C dependent mechanisms [8,9,10]. In adult adrenals, IGF-II expression is low compared with that in fetal adrenals [10]. However, in Cushing’s carcinomas, IGF-II is as highly expressed as in fetal adrenals, and it has been postulated that IGF-II may play a role in the pathogenesis of these tumors [11,12,13]. IGF-II is also highly expressed in pheochromocytomas, where it may affect the growth of these tumors [11, 14, 15].
IGFs are bound to specific binding proteins (IGFBP) in serum and other biological fluids. Until now, six high-affinity IGFBPs have been characterized [16], and there are at least four addi- tional novel IGFBP-related proteins [17,18]. IGFBPs are express- ed in multiple tissues including normal human fetal and adult adrenals [19]. IGFBPs may inhibit or stimulate the effects of IGFs, and they provide a storage pool for IGFs in biological fluids [16]. The local IGF/IGFBP production ratio may be critical for the autocrine/paracrine IGF effects. As such, it is important to study IGFBP expression in the tumors where high IGF ex- pression has been linked to a pathogenetic role. In the present work, we have evaluated the expression of the six affinity IGFBP genes (IGFBP-1-6) in adrenal tumors and hyperplasias to further clarify the significance of the IGF system in neoplas- tic adrenal growth.
Materials and Methods
Tissue specimens
Tumor samples were obtained from patients operated at the Helsinki University Central Hospital, and they were sent to the Department of Pathology aseptically. Fresh tumor speci- mens were dissected by the pathologist and representative non-necrotic tissue samples were taken. The histological diag-
noses was made by the pathologist, and the clinical diagnoses were confirmed from the patient records of the hospital. The samples were frozen in liquid nitrogen within one hour after surgery, and then stored at - 70℃ until further use. The tu- mors included in the study were Cushing’s carcinoma (n=3), Conn’s carcinoma (n= 1), a virilizing carcinoma (n=1), hor- monally inactive adrenocortical carcinoma (on clinical basis) (n=2), Cushing’s adenoma (n=4), Conn’s adenoma (n=6), non-functional adrenocortical adenoma (on clinical basis) (n=5), and pheochromocytoma (n=9). One case of bilateral adrenal hyperplasia due to pituitary Cushing’s disease and sev- en cases of nodular hyperplasia without clinical evidence of steroid overproduction were also studied. The adjacent adre- nals from tumor patients were investigated where available. One case of renal cell carcinoma with adjacent normal kidney was used as a control. Normal adrenals and normal liver tissue were obtained as described previously [13].
RNA preparation and analysis
Total RNA was isolated from deep-frozen tissues by guanidine thiocyanate-cesium chloride method [20]. RNA was quantitat- ed spectrophotometrically and transferred onto Hybond N fil- ters (Amersham International, Amersham, Buckinghamshire, UK) by Northern or dot blotting [21]. The filters were hybrid- ized with synthetic oligonucleotide probes for IGFBP-1, -2, -3, -4, -5, and -6 [19]. The oligonucleotide probes were 3’-end-la- beled with terminal transferase (Boehringer Mannheim, Mannheim, Germany) and [o .- 32P]dCTP (6000 Ci/mmol, Amer- sham). Mouse 28S ribosomal RNA cDNA (used as a control probe, [22]) and IGF-II cDNA [23] inserts were labeled by ran- dom priming. The hybridization conditions, autoradiographic detection of hybridization signals and densitometric scanning were performed as described previously [21]. IGFBP-1 mRNA
was also studied by reverse transcription-polymerase chain reaction (RT-PCR) amplification, as previously described [19].
Results
IGFBP-1 mRNA was not detected by Northern blotting in any of the adrenal specimens, but it was found in liver samples, which were used as controls (data not shown). RT-PCR was re- quired for the detection of IGFBP-1 mRNA in adrenal samples (Fig. 1). In normal adrenals, IGFBP-2, -4, -5 and -6 mRNAs were readily detectable via Northern blotting, and were of approxi- mately equal abundance, based on the required exposure times and signal intensities in Northern blots (Figs. 2,3). The relative IGFBP and IGF-II mRNA expression levels in all the spe- cimens studied are summarized in Table 1. As described before [11], IGF-II mRNA was strongly expressed in hormonally active adrenocortical carcinomas and pheochromocytomas, and was also somewhat increased in nonfunctional adrenocortical car- cinomas in comparison to normal adrenals (Table 1). IGF-I mRNA levels in these tumors have previously been shown to be low [11].
IGFBP-1
Although IGFBP-1 mRNA was not detected by Northern blot analysis in adrenal samples, it was detected by using a more sensitive RT-PCR technique. IGFBP-1 was expressed in two Cushing’s carcinomas, one non-functioning adrenocortical car- cinoma, three Conn’s adenomas, two Cushing’s adenomas, one non-functioning adenoma, four nodular hyperplasias, one bi- lateral hyperplasia, three pheochromocytomas, and in some of the tumor adjacent adrenals (part of these samples are shown in Fig. 1).
| Tissue | n | IGF-II | IGFBP-2 | IGFBP-3 | IGFBP-4 | IGFBP-5 | IGFBP-6 |
|---|---|---|---|---|---|---|---|
| Normal adult adrenal | 3 | 100 | 100 | 100 | 100 | 100 | 100 |
| (91-106) | (86-118) | (87-117) | (92-112) | (93-110) | (94-106) | ||
| Adrenocortical carcinoma | 2 | 261 | 143 | 2075 | 176 | 71 | 77 |
| (non-functional) | (186-335) | (88-197) | (303-3847) | (123-228) | (56-86) | (51-102) | |
| Adrenocortical carcinoma | 5 | 1516 | 29 | 562 | 50 | 31 | 15 |
| (hormone-producing) | (873-3014) | (16-50) | (202-1237) | (24-88) | (6-72) | (3-29) | |
| Adrenocortical hyperplasia | 1 | 74 | 193 | 1000 | 1031 | 570 | 135 |
| (Cushing's disease) | |||||||
| Nodular hyperplasia | 7 | 182 | 225 | 313 | 190 | 121 | 80 |
| (43-412) | (61-397) | (31-773) | (76-300) | (48-265) | (17-181) | ||
| Adenoma | 5 | 69 | 86 | 224 | 112 | 74 | 58 |
| (non-functional) | (43-88) | (47-115) | (92-544) | (92-140) | (43-114) | (20-97) | |
| Adenoma | 4 | 77 | 210 | 917 | 363 | 830 | 115 |
| (Cushing) | (43-156) | (24-376) | (533-1167) | (268-489) | (510-1380) | (15-283) | |
| Adenoma | 6 | 134 | 568 | 1864 | 259 | 849 | 202 |
| (Conn) | (54-230) | (227-910) | (106-7533) | (105-389) | (97-2200) | (122-263) | |
| Pheochromocytoma | 9 | 605 | 306 | 627 | 156 | 40 | 26 |
| (202-952) | (68-803) | (427-1073) | (53-308) | (4-112) | (11-48) | ||
| Normal fetal adrenal | 1 | 1428 | 221 | 817 | 89 | 42 | 24 |
| Normal kidney | 1 | ND | 191 | 364 | 304 | 191 | 30 |
| Renal cell carcinoma | 1 | ND | 34 | 9348 | 53 | 23 | 344 |
The values were calculated from scanned autoradiographic signals of Northern and dot blots, as described in Materials and Methods. The filters were sequentially hybridized with cDNA probes for IGF-II and 28S and oligonucleotide probes for IGFBP-2, 3-, -4, -5 and -6. All of the IGF-Il and IGFBP signals were normalized with the respective 28S values. The means and ranges (in parentheses) are shown. The means of normal adult adrenals were adjusted to 100. ND = not determined.
Cushing’s carcinoma 1 Cushing’s adenoma 1
nonfunctioning carcinoma
Cushing’s carcinoma 2 Conn’s adenoma 1
adjacent adrenal 1
Conn’s adenoma 2
adjacent adrenal 2
Conn’s adenoma 3
adjacent adrenal 3
Cushing’s adenoma 2
bilateral hyperplasia
nodular hyperplasia
adjacent adrenal
pheochromocytoma
adjacent adrenal normal liver
negative control
marker
empty lane
603 bp- 310 bp-
-445 bp
-445 bp
nonfunctioning carcinoma Cushing’s carcinoma
empty lane
normal adult adrenal
pheochromocytoma 1
adjacent adrenal 1
pheochromocytoma 2
pheochromocytoma 3
adjacent adrenal 3
kb
IGF-11
6.0
+ 4.8
42.2
IGFBP-2
4-1.5
IGFBP-3
4-2.5
IGFBP-4
4-2.6
GFBP-5
4-6.0
28 S
Cushing’s adenoma 1
Cushing’s adenoma 2
adjacent adrenal 2
Cushing’s adenoma 3
Cushing’s adenoma 4 Conn’s adenoma 1
adjacent adrenal 1
Conn’s adenoma 2
Conn’s adenoma 3
adjacent adrenal 3
Conn’s adenoma 4
adjacent adrenal 4
bilateral hyperplasia
normal adult adrenal
fetal adrenal
kb
IGFBP-2
1.5
IGFBP-3
2.5
IGFBP-4
4-2.6
IGFBP-5
4-6.0
IGFBP-6
4- 1.3
28 S
IGFBP-2
IGFBP-2 mRNA levels were low in hormone-producing carci- nomas (Cushing’s carcinomas, a Conn’s carcinoma, a virilizing carcinoma) (Fig. 2, Table 1). In non-functional carcinomas and adenomas, mean IGFBP-2 mRNA content was approximately the same as in normal adrenals. In most cases of bilaterally or nodularly hyperplastic adrenals, Cushing’s and Conn’s adeno- mas, and pheochromocytomas IGFBP-2 mRNA levels were higher than in normal adrenals (Fig. 3, Table1). However, re- markable variations in IGFBP-2 expression were observed among individual tumors, even within the same clinical and histological conditions (Table 1).
IGFBP-3
IGFBP-3 gene expression was low in normal adrenals. In nor- mal kidney, it was 3.6-fold higher. In both non-functional and hormone-producing carcinomas, IGFBP-3 expression was higher than in normal adrenals (Fig. 2, Table 1). In pheochro-
mocytomas, IGFBP-3 mRNA was expressed consistently (Fig. 2). In hyperplastic adrenals and in adenomas, IGFBP-3 ex- pression was variable and on average somewhat higher than in normal adrenals (Fig. 3, Table 1). In renal cell carcinoma, IGFBP-3 mRNA was very high (Table 1) as described previously [24].
IGFBP-4
IGFBP-4 mRNA level was lower in the hormonally active carci- nomas than in normal adrenals. In Cushing’s adenomas, the mean IGFBP-4 mRNA level was 3.6- and in Conn’s adenomas 2.6-fold compared with normal adrenals. The most abundant IGFBP-4 mRNA expression was observed in bilaterally hyper- plastic adrenals from a patient with Cushing’s disease (10-fold compared with normal adrenals) (Fig. 3, Table 1). In pheochro- mocytomas IGFBP-4 mRNA content was variable with a mean level of 1.6-times that of normal adrenals (Table 1).
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IGFBP-5
IGFBP-5 mRNA content was lower in adrenocortical carcino- mas and pheochromocytomas than in normal adrenals. As in the cases of IGFBP-2, -3, and -4, IGFBP-5 mRNA level was lower in the hormonally active than in the inactive carcinomas (Table 1). In Cushing’s and Conn’s adenomas, IGFBP-5 mRNA content was on average 8.3 and 8.5-fold, respectively, in comparison to levels in normal adrenals (Table 1). In bilaterally hyperplastic adrenals, IGFBP-5 mRNA level was about 6-fold and in nodu- larly hyperplastic adrenals about equal compared with normal adrenals.
IGFBP-6
IGFBP-6 mRNA, expression was very low in hormonally active adrenocortical carcinomas and pheochromocytomas (Table 1). In hormonally inactive adrenocortical carcinomas and differ- ent adenomas, IGFBP-6 mRNA was at the same level as in nor- mal adrenals or lower.
Discussion
We reported previously that IGFBP genes are expressed in hu- man fetal and adult adrenals and that IGFBP peptides are se- creted by adrenocortical cells [19]. In the present study, we have investigated IGFBP gene expression in adrenocortical tu- mors, hyperplastic adrenals, and pheochromocytomas. The most interesting finding was the low expression of all IGFBPs, except IGFBP-3, in hormonally active adrenocortical carcino- mas where IGF-II expression is simultaneously very high.
Of the different IGFBPs, IGFBP-1 mRNA level was the lowest and it was detectable only by RT-PCR. Due to the low IGFBP-1 expression in adrenal samples, IGFBP-1 is likely to have no ma- jor role in the pathophysiology of adrenal tumors. IGFBP-2 mRNA levels were variable, and low values were detected in malignant adrenal tumors. Previously it has been shown that IGFBP-2 expression is increased in prostate and ovarian cancer [25,26]. Elevated serum IGFBP-2 level is also a very common finding in different types of malignant tumors [27,28].
IGFBP-3 gene expression was higher in some adrenal tumors than in normal adrenals, where it was very low and near the detection limit. In breast cancer, IGFBP-3 mRNA levels corre- late with IGF peptide levels, and they are elevated in tumors with poor prognosis [29]. In uterine leiomyosarcomas, IGFBP- 3 protein levels are low, whereas in leiomyomas and normal myometrium, they are clearly higher [30]. IGFBP-3 serum lev- els in malignancies are often decreased together with elevated IGFBP-2 levels [31,32].
IGFBP-4 usually inhibits IGF actions [16], and thus its low lev- els in malignant adrenocortical tumors suggest increased IGF bioactivity in these tumors. High IGFBP-4 level in adrenocorti- cal hyperplasia raises the possibility that it might be regulated by ACTH in vivo.
Previously, it was shown that, in Cushing’s syndrome, the se- rum levels of IGF-I, IGFBP-2 and-3 were elevated, but IGF-II and IGFBP-1 concentrations were normal [33]. Most of these patients had pituitary Cushing’s disease and adrenocortical hyperplasia, whereas our Cushing patients had either adeno-
ma or carcinoma, only one patient had pituitary disease. In this patient, we found high IGFBP-3 and slightly elevated IGFBP-2 mRNA levels. Although these results seem to agree with those of Bang et al. [33], it must be emphasized that serum IGFBP protein levels may depend on various factors such as nutri- tional status or posttranslational modifications during syn- thesis. Clearly more studies comparing tissue mRNA and pro- tein levels as well as serum concentrations are needed.
In conclusion, our results show that IGFBPs 1 - 6 are expressed at varying levels in most adrenal tumors and adrenocortical hyperplasias. The pathogenetic or prognostic significance of altered IGFBP expression in different tumor types is not clear. However, low expression of most IGFBPs in hormonally active adrenocortical carcinomas with abundant IGF-II expression may be significant, as low IGFBP level may lead to high IGF-II bioactivity, which may be involved in tumor progression. In the future, it would also be important to study IGFBP expres- sion in these tumors at the protein level. These levels should also be correlated with serum IGFBP levels in the different pa- tient groups.
Acknowledgements
Ms. Merja Haukka and Ms. Eija Heilio are thanked for technical assistance. This study was financially supported by the Ida Montin Foundation, the Cancer Society of Finland (to JL), the Sigrid Juselius Foundation, and the Nordisk Insulin Foundation (to RV).
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Requests for reprints should be addressed to:
Vesa Ilvesmäki, MD University of Helsinki Haartman Institute Department of Pathology PO Box 21 Haartmaninkatu 3 FIN-00014 Helsinki Finland
Phone:
+ 358 (9) 4346424
Fax: +358 (9) 4346700
E-mail: vesa.ilvesmaki@helsinki.fi
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