Ch. Fottner1 A. Hoeflich2 E. Wolf2 M. M. Weber1
Role of the Insulin-like Growth Factor System in Adrenocortical Growth Control and Carcinogenesis
Abstract
Clinically silent adrenocortical adenomas are the most frequent abnormalities in the adrenal gland. In contrast, adrenocortical carcinoma is a rare tumor with an extremely poor prognosis. The factors responsible for the frequent occurrence of benign adrenocortical tumors on one hand and the rare malignant trans- formation on the other are not known. Several genetic alterations such as loss of imprinting or loss of heterozygosity of the 11p15 gene locus causing a strong IGF-II overexpression have been demonstrated in the majority of adrenocortical carcinomas. In addition to IGF-II overexpression, increased levels of the IGF-I- receptor and IGFBP-2 have been found in advanced human adre- nocortical carcinomas, suggesting an important role for the IGF- system in adrenocortical carcinogenesis. IGFs are potent mito- gens regulating growth and apoptosis through interaction with the IGF-I-receptor, and overexpression of the human IGF-I-recep- tor promotes ligand-dependent neoplastic transformation in a
variety of different cell systems. It is evident, therefore, that high levels of IGF-II in combination with overexpression of the IGF-I-receptor can provide a significant growth advantage for adrenocortical carcinoma cells and thus contribute to the highly malignant phenotype of this rare type of cancer. Additionally, it has been shown that overexpression of IGFBP-2 can promote ma- lignant transformation of Y1 mouse adrenocortical tumor cells through unknown IGF-independent mechanisms. As one possi- ble mechanism, we have recently found altered expression of catalase in IGFBP-2-overexpressing tumor cells, thus implicating IGFBP-2 in influencing intracellular peroxide levels. However, since transgenic mice with IGF-II or IGFBP-2 overexpression in the adrenal gland do not show an increased frequency of adrenal tumors, IGF-II or IGFBP-2 may act as progression factors but not as initiation factors in adrenocortical tumorigenesis.
Key words
IGF-I . IGF-II . IGF-I-receptor . IGFBP . Adrenal . Tumorigenesis
Introduction
Primary adrenocortical neoplasms can be divided into benign adenomas and malignant carcinomas. Clinically silent adreno- cortical adenomas are frequent and can be found in approxi- mately 3 to 7% in adults over 50 years of age as shown by autopsy studies and cross-sectional studies by abdominal CT [1]. In con- trast, adrenocortical carcinoma is a rare but highly malignant tu- mor with an approximate incidence of one new case per million
per year. More than two-thirds of the patients suffer from an ad- vanced tumor stage with local invasion or distant metastasis at the time of diagnosis, and can rarely be cured. Recent progress has been achieved in the understanding of adrenal tumorigen- esis by mapping and identification of genes responsible for sev- eral hereditary tumor syndromes associated with the formation of benign and malignant adrenocortical tumors such as Li-Frau- meni syndrome and Beckwith-Wiedemann syndrome. In Li- Fraumeni syndrome, the molecular basis of the familial suscep-
Affiliation
1 Schwerpunkt Endokrinologie und Stoffwechselerkrankungen, I. Medizinische Klinik und Poliklinik, Johannes Gutenberg University, Mainz, Germany
2 Institute of Molecular Animal Breeding/Gene Center, Ludwig Maximilians University of Munich, Germany
Correspondence
Prof. Dr. med. M. M. Weber · Schwerpunkt Endokrinologie und Stoffwechselerkrankungen · I. Medizinische
Klinik und Poliklinik . Johannes Gutenberg Universität Mainz . Langenbeckstraße 1 . 55131 Mainz . Germany . Phone: +49 (6131) 177260 · Fax: +49 (6131) 175608 . E-Mail: MMWeber@uni-mainz.de
Received 8 December 2003 . Accepted after revision 9 March 2004
Bibliography
DOI 10.1055/s-2004-814563 . ISSN 0018-5043
The IGF-system in normal adrenocortical cells
Alterations of the IGF-system in adrenocortical cancer
BP-4
IGF-II
IGF-I
IGF-I
BP-2
BP-5
IGF-II
BP-2
IGF-II/MP6 -receptor
BP-1
IGF-I-receptor
IGF-I-receptor
BP-3
Differentiated function
Growth
time and dose -dependent stimulation of steroid- biosynthesis by IGF-I and -|| [3,9-11,12,85 -88,23,90,91]
Increased mitogenic effect of IGF-I and -Il in IGF-I-receptor overexpressing adrenocortical tumor cells [33]
predominant stimulation of androgen -secretion more potently by IGF-II than by IGF-| [4,6,8,23]
Stimulation of IGFBP -3, -4 and -5 expression and secretion by IGF-I and -|| [6-8]
Hyperplasia of adrenocortical cells in IGF-II- and GH-transgenic mice [48-50]
Differential modulation of IGF-induced steroid - biosynthesis by IGFBP-1 [89]
Inhibitory effect of IGFBP-2 on adrenocortical growth in IGFBP -2 transgenic mice [79,80]
IGFBP-2 overexpression enhances malignant phenotype of Y1-adrenocortical tumor cells in- vitro [81]
tibility to a variety of cancers including adrenocortical carcino- mas has recently been elucidated by identification of germline point mutations in the p53 tumor-suppressor gene. Changes in the gene locus 11p15.5 with subsequent overexpression of IGF-II have been demonstrated in Beckwith-Wiedemann syndrome, a rare condition characterized by macroglossia, gigantism, earlobe pits, abdominal wall defects and an increased risk for Wilms tu- mor, rhabdomyosarcoma, hepatoblastoma and adrenal carcino- ma, pointing to a possible role of the IGF-system in adrenocorti- cal tumorigenesis [2]. Beckwith-Wiedemann syndrome (BWS) is a sporadic or autosomal-dominant inherited genetic disorder associated with neonatal gigantism, omphalocele and various childhood tumors including Wilm’s tumor, hepatoblastoma, rhabdomyosarcoma, neuroblastoma and adrenocortical carcino- ma. BWS has also been assigned to genetic alterations in the chromosomal region 11p15.5 and the p57KIP2 gene [2]. Overex- pression of the IGF-II gene as observed in many of these tumors and the predisposition of BWS towards adrenocortical cancer have prompted many studies on the role of IGF-II and the 11p15 chromosomal region in sporadic adrenal malignancies.
We and others have previously shown that the IGF system plays an important role in the regulation of the differentiated function of the adrenal gland. IGF peptides, receptors and binding pro- teins are synthesized and secreted by the adrenal glands of var- ious species, and both IGF-I and IGF-II have been found to induce steroidogenesis in adrenocortical cells in vitro and in vivo. This effect is mediated through interaction with the IGF-I-receptor and modulated by locally produced IGF-binding proteins regulated in a specific manner by ACTH and IGFs [3-9]. In addi- tion to its steroidogenic effect, accumulating data indicate that the IGF-system is involved in adrenocortical growth and devel- opment regulation (see Fig. 1). IGF-II seems to be an important adrenocortical growth factor in the fetal adrenal gland, which ex- presses high levels of IGF-II; IGF-II is assumed to mediate ACTH-
induced fetal adrenal growth during development [10-13]. Fur- thermore, characteristic changes in IGF-serum levels during puberty and adrenopause are associated with relevant changes in adrenocortical function [13 - 14].
The Insulin-like Growth Factor System in the Normal Adrenal Gland
Insulin-like growth factors
High levels of IGF-II mRNA and protein but almost no IGF-I are found in the primate fetal adrenal gland whereas IGF-I and IGF- II are expressed at similar levels in the adult adrenal gland. Therefore, it has been postulated that IGF-II promotes growth predominantly during embryogenesis in the human adrenal gland, whereas IGF-I acts mainly after birth [9]. This hypothesis is supported by the strong decline in IGF-II serum and tissue lev- els in the postnatal period, and since systemic IGF-II is a poor promoter of whole body growth compared to IGF-I [15-19]. In the fetal adrenal gland, IGF-II is assumed to mediate ACTH-in- duced adrenal growth since ACTH induces IGF-II gene expression in human fetal adrenocortical cells and since IGF-II is highly mi- togenic in these cells [20]. Furthermore, IGF-II has been implicat- ed in the regulation of fetal adrenal steroidogenesis due to a co- ordinate expression of IGF-II and steroidogenic enzyme mRNA [21,22].
In the normal adult human adrenal gland, IGF-receptors and a variety of specific IGF-binding proteins are expressed in addition to IGF-I and IGF-II, which are assumed to play an important role in the regulation of adrenal growth and function at a paracrine/ autocrine level [4-5,23]. In adult human adrenocortical cells, both IGFs stimulate basal and ACTH-induced steroid biosyn- thesis by upregulating steroidogenic key enzymes and ACTH-re- ceptor expression [5,23-24]. In analogy to the finding in fetal
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| Gene/ protein | Alteration | Prevalence (tumors) | Reference |
|---|---|---|---|
| IGF-I | mRNA overexpression | 0/18 adenomas 0/23 carcinomas | De Fraipont et al. [37] |
| mRNA overexpression | 0/15 adenomas 0/4 carcinomas | Ilvesmäki et al. [41] | |
| > 50% cells positive on immunohistochemical staining | 1/23 adenomas 21/64 carcinomas | Kamio et al. [27] | |
| IGF-II | IGF-II mRNA-over- expression | 0/15 adenomas 5/6 carcinomas | Ilvesmäki et al. [41] |
| IGF-II mRNA-over- expression IGF-II mRNA-over- expression/ LOH 11p15 | 2/17 adenomas 5/6 carcinomas 0/35 adenomas 25/29 carcinomas 2/35 adenomas 24/29 carcinomas | Giquel et al. [54] | |
| Giquel et al. [45,55] Boulle et al. [44] Giordano et al. [42] | |||
| Increased IGF-II protein content I GF-II mRNA over- expression | 1/9 adenomas 9/9 carcinomas 10/11 carcinomas | ||
| Stronger immuno- histochemical staining | 4/64 adenomas 33/67 carcinomas | Erickson et al. [43] | |
| IGF-I- receptor | Increased receptor concentration Stronger immuno- histochemical staining | 0/8 adenomas 3/4 carcinomas 9/23 adenomas 40/64 carcinomas | Weber et al. [26] Kamio et al. [27] |
| IGF-II/ Mannose- 6-Phos- phate- receptor | M6P/Insulin-like growth LOH factor-Il receptor | 2/25 adenomas 11/29 carcinomas | Lebouloux et al. [77] |
| IGFBP-2 | increased protein concentration in tumor tissue | 0/9 adenomas 8/9 carcinomas | Boulle et al. [44] |
| increased serum concentration | 0/36 healthy controls 2/13 compl. remission 23/28 metast. disease | Boulle et al. [78] |
cells, IGF-I and IGF-II preferentially stimulate adrenal androgen secretion by interacting with the IGF-I-receptor. the steroido- genic effect of IGF-II is significantly more potent than the effect of IGF-I due to a modulatory effect of locally produced IGF-bind- ing proteins [5-7].
Insulin-like growth factor receptors
Normal adult adrenocortical cells express both types of IGF re- ceptors as demonstrated by RT-PCR, immunohistochemistry, af- finity cross-linking and Scatchard plot blot analysis. Both IGF-re- ceptors and the insulin-receptor are expressed at a similar level in all zones of the adrenal cortex [25 - 27]. So far, all known mito- genic and differentiating effects of IGF-I and IGF-II in adrenocor- tical cells have been shown to be mediated through interaction with the IGF-I-receptor to which both ligands bind with similar affinity [5,22,26]. The physiological role of the IGF-II/mannose 6-phosphate (IGFII/M6P) receptor - which only binds IGF-II with high affinity - remains unclear. However, recent data show that the IGFII/M6P receptor mediates the clearance and inactiva- tion of IGF-II and facilitates the activation of the growth inhibitor transforming growth factor ß (TGFB). Therefore, the IGFII/M6P
receptor gene has been implicated as a tumor suppressor gene [28].
Insulin-like growth factor-binding proteins
The biological effects of IGF-I and IGF-II are modulated in vivo by at least six high-affinity IGF-binding proteins (IGFBP-1 to -6) which, depending on the cellular context, are able to stimulate or inhibit IGF-signaling through a variety of mechanisms and have also been shown to exert IGF-independent actions [29]. In adult and fetal human adrenocortical cells and tissue the expres- sion of all six high-affinity IGF-binding proteins (IGFBP) has been shown [6-8,30]. Additionally, conventional and two-dimen- sional Western-ligand blotting and immunoblotting have re- vealed the secretion of IGFBP-1 to -5 by human adrenocortical cells in culture [8,31]. In adult adrenocortical cells, the expres- sion and secretion of IGFBPs is differentially regulated by ACTH, steroids and IGFs [7-8,31]. While ACTH specifically stimulates the abundance of IGFBP-1 and IGFBP-4, IGF-I and IGF-II preferen- tially induce the expression of adrenocortical IGFBP-3 and IGFBP-5. No specific IGFBP-2 and IGFBP-4 regulation has been observed up to now [6-8]. Although the functional role of locally produced IGFBPs in the adrenal gland remains unknown, in vitro data indicate that they modulate the steroidogenic response of IGFs. In adult human adrenocortical cells, interaction between IGFs and locally produced IGFBPs results in a stronger steroido- genic potency of IGF-II compared to IGF-I [6,8]. Also, IGFBP-1 has been shown to inhibit the steroidogenic action of IGF-II and potentiate the effect of IGF-I in primary bovine adrenocortical cell cultures [32].
The Role of the Insulin-like Growth Factor System in Adreno- cortical Tumorigenesis
Insulin-like growth factor I
Many studies have shown that in vitro IGF-I is mitogenic in fetal and adult adrenocortical cells from different species [33]. In vivo, infusion of IGF-I into guinea pigs caused an increase in the frac- tional weight of the adrenal glands, and this effect was more pro- nounced with long R3 IGF-I, an analogue with reduced affinity for IGFBPs [34]. Enlargement of the adrenal glands and increased corticosterone secretion has been observed in growth hormone (GH)-overexpressing transgenic mice characterized by elevated IGF-I serum levels, clearly indicating an important role of the GH/IGF axis for adrenal growth and function. Furthermore, under conditions of elevated GH/IGF-I expression, an increase in zona fasciculata cell number and size has been found [35].
IGF-I expression is present in adrenocortical carcinomas as shown by microarray analysis and immunohistochemistry. How- ever, no IGF-I overexpression could be demonstrated in adrenal tumors in several different studies [36-38] and similar IGF-I protein concentrations were measured by RIA in specimen of normal, adenomatous and malignant adrenal tissue [39] (see Ta- ble 1). Only one report demonstrated statistically insignificantly stronger immunohistochemical staining for IGF-I in adrenocorti- cal adenomas and carcinomas compared to normal adrenocorti- cal tissue. In this study, most (75%) normal adrenocortical tissue exhibited only 10-50% IGF-I-positive cells, whereas in 64% of
adrenal adenomas and in 83% of the carcinomas investigated, more than 50% IGF-I positive cells were found [40].
Insulin-like growth factor II
A strong overexpression of IGF-II is a dominant finding in adre- nocortical cancer, occurring in approximately 90% of malignant adrenal tumors but in almost none of the benign adenomas (see Table 1). Usually more than 100-fold higher levels of IGF-II mRNA are found in malignant tumors [37 -39,41 - 43]. The highest IGF- II levels are associated with a more malignant phenotype of these tumors [44], and overexpression of IGF-II is associated with a 5-fold increased risk for the recurrence of sporadic adre- nocortical carcinomas [45]. From Northern blotting analysis, which showed similar IGF-II mRNA patterns in normal and tu- morous adrenal tissue, concluded that the normal promoters (P3 and P4) of the IGF-II gene are used in adrenocortical tumors and that the IGF-II gene transcription is not altered qualitatively [46,47]. IGF-II mRNA seems to be translated efficiently into IGF- II protein since significantly stronger immunostaining for IGF-II and tenfold concentrations of mature IGF-II protein and - to a lesser degree - IGF-II itself has been found in adrenocortical can- cer [39,45]. In contrast to IGFBP-2, no elevated serum IGF-II lev- els were seen in patients with malignant or metastatic adreno- cortical tumors [44].
In vitro, IGF-II has been shown to be an important autocrine fac- tor in the proliferation of human adrenocortical carcinoma cell line NCI H295R, which secretes large amounts of IGF-II and IGFBP-2 [27]. In order to evaluate the role of IGF-II in the regula- tion of the adult adrenal growth and function in vivo, adrenal morphology and steroid secretion were investigated in PEPCK- IGF-II transgenic mice that overexpress IGF-II postnatally. These IGF-II-transgenic mice are characterized by four to six times the postnatal IGF-II serum levels, elevated serum IGFBP-2 levels and subtle changes in organ growth, while their total body weight and morphological appearance remains unchanged [48]. How- ever, the predominant finding at autopsy was a significantly in- creased adrenal weight of the transgenic mice compared to con- trol adrenal glands. Morphological investigation of adrenal glands from IGF-II transgenic mice and control animals demon- strated that the increase in adrenal weight in transgenic mice was mainly due to a 50% increase in the number of zona fascicu- lata cells, while cell volume and zonation of transgenic adrenal glands remained unchanged. The increase in zona fasciculata volume was paralleled by an enhanced steroidogenesis. PEPCK- IGF-II transgenic mice exhibited twice the basal and ACTH-in- duced corticosterone levels of age- and sex-matched controls both in vivo and in vitro. However, when normalized for adrenal weight, the corticosterone secretion was similar in both groups [49]. PEPCK-IGF-II-transgenic mice did not show any increased frequency of adrenal tumors, indicating that IGF-II-overproduc- tion by itself is not sufficient for malignant transformation of adrenocortical cells, and that additional factors are required for adrenal tumorigenesis [50].
Mechanisms Leading to IGF-II Overexpression
Loss of imprinting (LOI) and loss of heterozygosity (LOH) in the 11p15 gene locus
The mechanism for IGF-II overexpression in adrenal tumors is related, in part, to pathological genomic imprinting. The IGF-II gene maps to the 11p15 chromosomal region and is maternally imprinted; therefore, IGF-II is exclusively expressed from the pa- ternal allele in the adult. Abnormalities in the imprinted 11p15 region (involving the maternally imprinted IGF-II gene and the paternally imprinted p57KIP2 and H19 genes) are highly specific for malignant tumors and are the main finding in adrenal carci- nomas. These alterations at the 11p15 gene locus include loss of heterozygosity (LOH) with paternal duplication of the allele and, less frequently, loss of imprinting (LOI), excessive transcriptional activation or loss of transcriptional suppression. Usually, these genetic alterations result in a strong IGF-II overexpression [51]. In adult patients with non-metastatic sporadic adrenocortical cancer, 11p15 LOH is a strong predictor for a shorter disease-free interval (relative risk ratio: 9) [45]. Although the exact mecha- nism for the dysregulation of the imprinted 11p15 region in adre- nocortical cancer remains unclear, this data suggests that over- expression of IGF-II plays an important role in the transition from benign to malignant adrenal tumors.
H19 gene and p57KIP2 gene
The fact that the predictive value of 11p15 LOH is stronger than that of IGF-II overexpression indicates that additional molecular changes in this region such as loss of maternally expressed tu- mor suppressor genes contribute to adrenal tumorigenesis [45]. Two candidate genes, namely p57KIP2 and H19, also map to the 11p15.5 region. Unlike IGF-II, these two genes are paternally im- printed so that they are normally expressed only from the mater- nal allele. The exact function of H19 is not clear; it encodes an mRNA which is not translated into a protein and has been postu- lated a tumor suppressor gene. p57KIP2 is a known suppressor of the cell cycle progression and is thought to play an important role in Beckwith-Wiedemann syndrome [51]. Thus, genetic al- terations with loss of the maternal and duplication of the pater- nal 11p15 allele can result in not only the overexpression of the paternally expressed IGF-II-gene but also the functional loss of the maternally expressed p57KIP2 and H19 genes [52-56]. In- deed, decreased H19 gene expression and the p57KIP2 gene is a frequent and specific finding in adrenocortical carcinomas [55- 57], and loss of function in these putative tumor-suppressor genes might contribute to the highly malignant phenotype in these tumors. Furthermore, these genes might be involved in the regulation of IGF-II gene expression or mRNA stability, since a strong negative correlation between p57KIP2 and IGF-II mRNA has been found [56] and H19 mRNA has been shown to decrease steady state IGF-II mRNA levels in vitro[53].
Methylation status of the IGF-II gene
Apart from gene dosage effects due to biallelic IGF-II expression, numerous other factors might contribute to IGF-II overexpres- sion in adrenocortical malignancies. Since demethylation of the IGF-II gene could be demonstrated in pediatric adrenocortical cancers, increased IGF-II expression might be correlated with de- methylation at this locus as demonstrated in other malignancies [38,53,58].
Insulin 5’ variable number of tandem repeats
The expression of IGF-II has been found to be associated with the minisatellite DNA polymorphism of a variable number of tandem repeats (VNTR) in the 5’-flanking region of the human insulin gene, another gene located in the 11p15 region [59,60]. This hy- pothesis is supported by the fact that only the short class I Insu- lin 5’ VNTR alleles were found in three out of four pediatric adre- nocortical cancers associated with an increased IGF-II expression [53].
Nephroblastoma overexpressed gene expression
Nephroblastoma overexpressed (novH) gene expression in adre- nocortical carcinomas is significantly and selectively decreased, and there is an inverse correlation with the levels of IGF-II mRNA in these tumors [61,62]. NovH belongs to the CCN (CNTF/ CYR61/NOV) family of proteins which are part of the IGFBP su- perfamily. The function of novH in adrenocortical cancer is un- known so far, but it might play a role as a negative regulator of growth and seems to be involved in cell adhesion.
Insulin-like growth factor-I-receptor
Strong overexpression of the IGF-I-receptor has been described in adrenocortical carcinomas but not in adrenocortical hyperpla- sias or adenomas [26,27]. Competition binding studies and Scatchard plot analysis revealed comparable IGF-I-receptor con- centrations and binding kinetics in normal adrenocortical tissue, adrenal hyperplasias and adenomas, with a single class of high affinity binding sites and a dissociation constant (Kd) of 0.16 ±0.03 nmol/l. In contrast, three out of four hormonally ac- tive adrenocortical carcinomas showed a significantly elevated specific IGF-I binding with a three- to fourfold increase in IGF-I- receptor concentration compared to normal adrenocortical tis- sue. However, the binding affinity and the electrophoretic mobil- ity of the IGF-I-receptor were unaltered in adrenocortical carci- nomas, suggesting overexpression of the functionally intact IGF- I-receptor in these carcinomas [26]. In accordance with the above study, a strong overexpression of the IGF-I-receptor has been found by immunohistochemistry in 8/21 adrenocortical carcinomas but none of the adenomas investigated [27]. In an- other study, the presence of IGF-I-receptor mRNA in adrenocorti- cal carcinomas was demonstrated by RT-PCR [13].
Overexpression of the IGF-I-receptor has been demonstrated in a variety of malignant tumors like colon, breast and lung cancer [29,66] and strong evidence indicates that the IGF-I-receptor plays a pivotal role in tumorigenesis. Increased expression of the human IGF-I-receptor promotes ligand-dependent neoplas- tic transformation in a variety of different cell systems [29,30]. In contrast, absence or decreased levels of the IGF-I-receptor pre- vent malignant growth and transformation in vitro and in vivo and have been demonstrated to confer resistance to oxidative stress and even extend patient lifespan [63-66]. Adrenocortical tumor cells in the Y1 mouse have been used to show that the overexpression of the human IGF-I-receptor results in an in- creased IGF-dependent cell proliferation and antagonizes the an- tiproliferative effect of ACTH in vitro [33].
The functional significance of the strong and specific overexpres- sion of IGF-II and the IGF-I-receptor in the majority of adrenocor- tical carcinomas remains unknown at present. However, the mi-
togenic effect of IGF-II is dependent on the presence of the intact IGF-I-receptor [29,66], and interaction with the IGF-I-receptor has been shown to mediate the steroidogenic and mitogenic ef- fect of IGF-I and IGF-II in the adult human adrenal gland [6,8]. Therefore, high local levels of IGF-II in combination with elevated IGF-I-receptor concentrations possibly represent an autocrine stimulatory loop, thus contributing to adrenocortical tumorigen- esis.
The mechanisms responsible for enhanced IGF-I-receptor ex- pression in adrenal cancer and other malignancies are still un- clear. However, expression of the IGF-I-receptor is regulated by a variety of factors including growth factors, oncogenes, and tu- mor suppressor genes such as p53 [67-70]. In normal cells, ex- pression of wild-type p53 was shown to inhibit IGF-I-receptor gene expression, whereas mutant p53 upregulates IGF-I-recep- tor gene expression in several different tumors [71,72]. In adre- nocortical carcinoma, mutations within the conserved regions of p53 have been found in approximately 30% of malignant adreno- cortical tumors, whereas mutations are rarely found in benign adrenocortical adenomas, suggesting a role for p53 later in adre- nocortical carcinogenesis [73]. Thus, p53 mutations may repre- sent one possible mechanism for the overexpression of IGF-I-re- ceptors in a late-stage adrenocortical tumorigenesis. Further- more, the elevated IGF-II concentration in adrenal malignancies might contribute to the overexpression of IGF-I-receptors in these tumors. It has been shown in CaCo-2 human colon carcino- ma cells that stable overexpression of IGF-II result in an in- creased IGF-I-receptor expression with increased proliferation and anchorage-independent growth [74]; a positive correlation between the expression of IGF-II and IGF-I receptors has been re- ported in colorectal carcinomas [63].
IGF-II/Mannose-6-Phosphat (IGF-II/M6P) receptor
Although the signaling function of the IGFII/M6P receptor re- mains unclear, it seems to play an important role in regulating cell growth by ensuring the clearance and degradation of IGF-II. An antiproliferative effect of the IGFII/M6P receptor has been demonstrated in choriocarcinoma- and in colorectal cells in vitro [75,76]. Furthermore, a high frequency of LOH with concomitant mutations in the remaining IGFII/M6P allele has been found in liver and breast tumors [29]. Recently, it has been shown that the IGFII/M6P receptor might also play an important role in the pathogenesis of adrenocortical malignancies. Loss of heterozy- gosity at the M6P/IGF-II-R locus seems to be a frequent event in adrenocortical tumors, and may thus contribute to increased IGF-II bioavailability in these tumors [77]. The fact that LOH at the M6P/IGF-II-R locus was detected in 58% of all informative malignant but only in 9% of benign adrenocortical tumors indi- cates that molecular changes involving the M6P/IGF-II-R gene lo- cus are a late step in adrenocortical tumorigenesis. Although in- activation of the remaining allele by mutations or abnormal fetal imprinting has not been investigated in adrenocortical tumors this data supports the hypothesis that the M6P/IGF-II receptor may function as a tumor suppressor gene in the adrenal gland [77].
Insulin-like growth factor-binding protein-2
A characteristic change in IGFBP-pattern has been reported in adrenocortical carcinomas, with significantly higher IGFBP-2
levels in malignant than in benign adrenocortical tumors. De- spite elevated levels of IGFBP-2 protein in malignant tumor tis- sue, no increase in IGFBP-2 mRNA levels was detected, suggest- ing a post-transcriptional mechanism in the regulation of IGFBP- 2 abundance in adrenocortical carcinoma [44]. In contrast to IGFBP-2, no change in the secretion of other IGFBPs was found in human adrenocortical tumors [44,78].
Furthermore, significantly higher plasma levels of IGFBP-2 were found in more than 80% of patients with metastasized adreno- cortical carcinoma compared to a normal control group. In these patients, plasma IGFBP-2 levels correlate positively with tumor burden and are inversely correlated with survival. In contrast, there was no significant difference in plasma IGFBP-2 concentra- tion between patients with complete remission, localized dis- ease and healthy controls.
Although high concentrations of IGFBP-2 are a frequent finding in a variety of malignant tumors [30], the functional significance of elevated IGFBP-2 levels in adrenocortical carcinoma tissue re- mains unclear. Until recently, IGFBP-2 has been believed to be mainly a negative regulator of normal somatic growth, most probably by sequestering IGFs from their receptors [79]. In trans- genic mice, overexpression of IGFBP-2 leads to a strong inhibi- tion of GH/IGF-I-induced adrenocortical hypertrophy but has no effect on adrenal hyperplasia. This selective inhibition of cell size increase is an interesting new facet in the spectrum of mecha- nisms by which IGFBP-2 may affect cell growth and differentia- tion [35,79]. However, increasing evidence indicates that IGFBP- 2 also can play an important role in promoting tumor growth [79]. In vitro, overexpression of murine IGFBP-2 in Y1 mouse adrenocortical tumor was associated with significant morpho- logical alterations, enhanced cell proliferation, and increased colony formation as compared to control transfected cells. The enhanced proliferation of IGFBP-2-secreting clones was inde- pendent of exogenous IGFs and the presence of a blocking IGF-I- receptor antibody «IR-3 [81]. These data suggest that elevated levels of IGFBP-2 in the tumor may contribute to the highly ma- lignant phenotype of adrenocortical cancer by an IGF-indepen- dent mechanism. Although the exact mechanism of this tumor- promoting effect of IGFBP-2 is still unknown, recent data indi- cate that IGFBP-2 overexpression results in elevated catalase ac- tivities in different tumor cells [85]. Since catalase controls intra- cellular hydrogen peroxide levels, which represents an impor- tant intracellular signaling molecule, a new link between IGFBP- 2 and intracellular signaling processes has been established.
IGFBP-2 expression and secretion regulation is highly complex and can be influenced by many hormones and growth factors in- cluding IGFs and steroid hormones. The factors responsible for the IGFBP-2 oversecretion observed in advanced adrenocortical cancer are not known. However, IGFBP-2 is the main binding protein secreted by malignant adrenocortical cells [44], whereas IGFBP-2 accounts for only 12% of the IGFBP activity in normal adrenocortical cells, which is not regulated by ACTH or IGFs [8]. This makes it unlikely that the overexpression of IGFBP-2 in adrenocortical carcinomas is merely an epiphenomenon, and suggests that IGFBP-2 plays a specific role in the progression of advanced adrenocortical malignancies.
Discussion
Although the molecular pathogenesis of adrenal tumorigenesis is still poorly understood, accumulating data indicate that adre- nocortical carcinogenesis is a multistep process with the pro- gression of normal adrenal cells to adenoma cells and finally to malignant adrenocortical cells. Based upon clonal analysis and comparative genomic hybridization, the transition from adeno- ma to adrenocortical cancer may take place when a particular clone of cells gains a growth advantage due to accumulated ge- netic changes, as it has been shown by example in the colorectal cancer model. This hypothesis is supported by the investigation of a mixed adrenocortical tumor with histomorphologically be- nign and malignant parts. While the malignant part did show 17p13 loss of heterozygosity, 11p15 uniparental disomy and over- expression of the IGF-II gene, no abnormalities were found in the surrounding benign adenomatous tissue [83]. In adrenocortical tumors, a variety of genetic abnormalities associated with a ma- lignant phenotype have been described. Dysregulation of fetal imprinting or rearrangement of 11p15.5 is one of the dominant changes that are highly specific for malignant adrenocortical tu- mors that lead to significant IGF-II upregulation in the tumor. To- gether with an increased expression of IGF-I-receptors, this re- presents an autocrine stimulatory loop for the adrenocortical tu- mor cell, which may result in a significant growth advantage and thus contribute to the highly malignant phenotype of this tumor. Additionally, IGF-independent effects of IGFBP-2 via mecha- nisms so far unknown could further promote malignant transfor- mation and facilitate metastatic disease. In comparison to the adult human adrenal gland, the fetal adrenal shows higher con- centrations of IGFBP-2 and IGF-I-receptors and more than 25- fold higher IGF-II mRNA levels that drop dramatically after birth [19]. Therefore, adrenocortical carcinomas with strong overex- pression of IGF-II and high IGFBP-2 and IGF-I-receptor levels may be regarded as a regression of the adrenocortical tissue to the highly proliferative fetal state where IGF-II is believed to be involved in the regulation of fetal adrenal growth through inter- action with the IGF-I-receptor. However, the fact that transgenic mice with a strong local overexpression of IGFs or IGFBP-2 did not develop adrenal tumors despite significant effects on adrenal growth and steroidogenesis indicates that IGF-II overproduction alone is not sufficient for malignant transformation, and that ad- ditional factors are required for adrenal tumorigenesis [84]. This is supported by the clinical observation that changes in the IGF- system seem to be rather a late event in the transition of the fre- quent benign adrenal adenomas to the rare but highly malignant adrenocortical cancer, and that these changes are associated with an advanced stage of the disease, a poor clinical prognosis and a high risk of recurrence [44, 45, 78].
Acknowledgements
Matthias Weber and Eckhard Wolf were supported by Deutsche Forschungsgesellschaft Grant WE 1356/4-2.
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