ELSEVIER
MCE
Molecular and Cellular Endocrinology
Review
Adrenocortical tumorigenesis, luteinizing hormone receptor and transcription factors GATA-4 and GATA-6
Susanna Vuorenoja ª, Adolfo Rivero-Mullerª, Sanne Kiiveri b, Malgorzata Bielinska℃, Markku Heikinheimob, David B Wilson℃, Ilpo T Huhtaniemi a,d, Nafis A Rahman ª,*
a Department of Physiology, University of Turku, FIN-20520 Turku, Finland
b Children’s Hospital and Program for Developmental and Reproductive Biology, University of Helsinki, 00029 Helsinki, Finland
” Department of Pediatrics, Washington University, St. Louis, MO, USA
d Institute of Reproductive and Developmental Biology, Imperial College London, Faculty of Medicine, London W12 0NN, UK
Received 14 September 2006; received in revised form 12 November 2006; accepted 12 November 2006
Abstract
Luteinizing hormone (LH/hCG) responsiveness of normal and pathological human adrenal glands as well as the possibility of constitutive expressions of luteinizing hormone receptor (LHR) in adrenal cortex has been reported. Some recent studies showed a correlation between the LHR and abundant GATA-4 expression in both metastasizing and non-metastasizing human adrenocortical tumors, but not in normal adrenals, implicating the putative relevance of LHR and GATA-4 for adrenocortical pathophysiology. However, the physio- and pathophysiological sig- nificance of LHR and GATA-4 in the mechanism of adrenocortical tumorigenesis remains unclear. The paucity of suitable models for adrenal tumorigenesis makes the establishment of proper animal models highly important. LHR expression in the murine adrenal gland is an excep- tion and not found in wild-type (WT) animal. We have previously shown that ectopic LHR expression in the murine adrenal gland can be induced by chronically elevated LH levels. We have generated a gonadotropin-responsive adrenal tumor model in gonadectomized transgenic (TG) mice expressing the inhibin & promoter/Simian Virus 40 T antigen transgene (inha/Tag). Given the induction of expression and regulation of GATA-4 and GATA-6 zinc finger transcription factors in the gonads by gonadotropins, this review will explore their relationship to LHR expression and their role in adrenocortical tumorigenesis. A functional link between LHR and GATA-4 actions in the adrenal pathophysiology is proposed.
@ 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adrenal tumors; Transgenic mice; Gonadotropins; LHR; Transcription factors GATA-4 and GATA-6
Contents
1. Adrenocortical tumors and the luteinizing hormone receptors (LHR)
39
2. Transcription factors GATA-4 and GATA-6
39
2.1. GATA-4 and GATA-6 in human adrenocortical tumors 40
3. Animal models for adrenocortical tumorigenesis 40
3.1. Transgenic (TG) murine models for adrenocortical tumorigenesis
41
3.2. Adrenocortical phenotype of the inha/Tag mice
42
4. Future directions
43
Acknowledgments
43
References 43
Abbreviations: LHR, luteinizing hormone receptor; hCG, human chorionic gonadotropin; ACC, adrenocortical carcinoma; ACT, adrenocortical tumor
* Corresponding author. Fax: +358 2 2502610.
E-mail address: nafis.rahman@utu.fi (N.A. Rahman).
1. Adrenocortical tumors and the luteinizing hormone receptors (LHR)
Adrenocortical carcinoma (ACC) is a rare type of malig- nancy. In the United States, its incidence is approximately 3 per million children under 16 years of age (Wagner et al., 1994), and 0.5-2 per million adults (Schulick and Brennan, 1999). It tends to occur either before the age of five or in the first, fourth or fifth decades of life, and is relatively more common in women (Michalkiewicz et al., 1997; Sandrini et al., 1997; Schulick and Brennan, 1999; Wajchenberg et al., 2000). Only a small proportion of adrenocortical tumors (ACT) cause endocrine diseases in humans. It has been shown in 602 cases of ACC, that 62% of the carcinomas were functional and 38% non-functional (Ng and Libertino, 2003). The func- tional tumors presented with Cushing’s syndrome (40%) and only rarely with pure virilization in adults (Ng and Libertino, 2003). In children the proportion of endocrine disease is even higher (such as primary hyperaldosteronism, hypercortisolism, hyperandrogenism, or hyperestrogenism), and less than 1% are malignant (Bornstein et al., 1999). However, in the group of operated incidentalomas, i.e. incidentally found adrenocortical tumor masses, even 12% can be malignant adrenocortical car- cinomas (Arnaldi et al., 2000; Mantero et al., 2000a,b). The tumors are classified as non-functional or functional, depend- ing on their production of corticosteroid, androgen, estrogen, or mineralocorticoid. Most patients present with large tumor masses and with stage IV disease, which normally means the presence of the metastases (Schulick and Brennan, 1999). There are still no efficient forms of therapy for adrenal tumors, with the exception of tumor removal. Even in patients undergoing com- plete resection, recurrent and metastatic disease is quite common (Reincke et al., 1994; Bornstein et al., 1999). The survival rate
of ACC according to some studies is 15-35% (Ahlman et al., 2001). In a series of 139 patients from the last 20 decades the 5-year survival rate was shown to be improved than in the older reports to 60% (Vassilopoulou-Sellin and Schultz, 2001). In the group of patients with metastases, the 5-year survival rate is reported to range between 15 and 25% (Latronico and Chrousos, 1997a,b).
Human adrenal LH/hCG responsive tumors have been described (Leinonen et al., 1991; Lacroix et al., 1999). LHR expression has also been reported at low levels in normal human adrenal cortex (Pabon et al., 1996) and hCG has been shown to stimulate the dehydroepiandosterone sulphate synthesis of human fetal adrenals (Jaffe et al., 1981). It would be important to explore further, as suggested in recent studies (Rao Ch et al., 2004; Alevizaki et al., 2006), the possibility whether nor- mal human adrenal glands might contain low levels of LHR and whether they become activated whenever LH levels are ele- vated as a part of physiological or pathological process. Several cases of LH/hCG dependent and ACTH-independent Cushing’s syndrome (CS) have been reported in postmenopausal women (Lacroix et al., 1999; Feelders et al., 2003) and pregnancy asso- ciated CS is a known rare condition (Sheeler, 1994). In a very recent study, aberrant expression of human LHR by adrenocor- tical cells was shown to be sufficient to provoke hyperplasia,
benign adrenocortical tumorigenesis and Cushing’s syndrome features (Mazzuco et al., 2006). Ectopic expression of LHR in the adrenal gland has been shown not only to be associ- ated with ACTH-independent Cushing’s syndrome (CPA), but also with the benign aldosterone-producing adenomas (APA) (Saner-Amigh et al., 2006). The potential role of LH and aber- rantly expressed LHR in the regulation of steroidogenesis in both Conn’s and Cushing’s disease has been shown to have clinical implications (Saner-Amigh et al., 2006). LH has been shown to increase the transcription of aldosterone synthase in an adrenocortical cell line transfected with LHR, which is required for the final reaction in aldosterone synthesis, empha- sizing further the potential role of LHR in primary aldosteronism (Saner-Amigh et al., 2006). There are also reports about malig- nant androgen producing LH-dependent adrenal tumors (Givens et al., 1975; de Lange et al., 1980; Leinonen et al., 1991). Hence, LH/hCG dependent mechanisms do exist among human adrenal pathologies, LH/hCG dependent mechanisms exist and they may be clinically important in certain circumstances, e. g. in connection with polycystic ovarian syndrome (PCOS) and after menopause. It would be interesting to know whether the increased levels of gonadotropins in postmenopausal women or in PCOS patients could affect adrenal function. It is already known that in PCOS patients the elevated levels of LH cor- relate with the overproduction of androgens (Franks, 1995). Only a few animal models exist for studying the initiation, progression, and metastasis of adrenal tumors or for exam- ining new treatment strategies. Thus, there is a great need for novel experimental models for research in adrenocortical cancer.
2. Transcription factors GATA-4 and GATA-6
GATA-4 and GATA-6 belong to the family of zinc finger transcription factors termed the GATA-binding proteins, and they regulate gene expression, cell differentiation and prolif- eration in a variety of tissues (Orkin, 1992; Heikinheimo et al., 1994, 1997). However, recent studies on GATA transcription factors showed a role for these factors also in tumor formation, at least in gonads and adrenals (Kero et al., 2000; Ketola et al., 2000; Kiiveri et al., 2002a,b, 2004; Barbosa et al., 2004; Rahman et al., 2004). GATA-4 is expressed in heart, gonads, gastrointestinal tract and pancreas and in limited number of other tissues (Orkin, 1992; Arceci et al., 1993; Narita et al., 1996; Heikinheimo et al., 1997; Ketola et al., 1999). The phe- notypic characterization of homozygous GATA-4 deficient mice showed its expression and importance in several tissues during the embryonic period (Arceci et al., 1993; Heikinheimo et al., 1997; Ketola et al., 1999, 2004). The GATA-4 deficient mice die in utero by E9.5 mainly due to abnormal cardiac morpho- genesis caused by a disturbance in the ventral folding (Kuo et al., 1997; Molkentin et al., 1997). In humans, GATA-4 has been shown to be linked to congenital heart defects and carcinomas of ovary and esophagus (Pehlivan et al., 1999; Lin et al., 2000; Lassus et al., 2001). GATA-6 was primarily recognized as a separate GATA family member from a chicken cDNA library (Laverriere et al., 1994). GATA-6 resembles GATA-4 in many
ways and besides the organs mentioned above it is also expressed in the lungs and vascular smooth muscle cells (Narita et al., 1996; Morrisey et al., 1997). GATA-6 is required during embryogene- sis and GATA-6 deficient homozygous mice die at E7.5 due to a defect in the development of visceral endoderm (Morrisey et al., 1998). Besides being expressed in the male and female gonads and adrenals, GATA-4 and GATA-6 are expressed at the levels of hypothalamus-pituitary-gonadal/adrenal axis (Kiiveri et al., 1999). There are differences in the expression of GATA factors in normal and neoplastic adrenal tissues. GATA-6 is found in fetal and adult adrenocortical tissue (Kiiveri et al., 1999, 2002a,b, 2004, 2005). GATA-4 expression, instead, is found in fetal but not in wild type murine or human adult adrenal tissue (Kiiveri et al., 2002a). However, while we investigated the adrenal tumors of inha/Tag mice, we found abundant GATA-4 expression in adrenal tumors, while GATA-6 could not be detected (also see below) (Kiiveri et al., 1999). These results implicate a role for GATA-4 in adrenal tumorigenesis and that GATA-4 and GATA-6 have different roles in the adrenal gland. GATA-4 has been shown to act in synergy with steroidogenic factor 1 (SF- 1) in reproduction, steroidogenesis and sexual differentiation (Kiiveri et al., 2005). There is also co-operation between GATA- 4 and SF-1 to regulate the expression of AMH (anti-Müllerian hormone) (Kiiveri et al., 2005). The lack of co-operation of these two transcription factors could be the reason for some cases of abnormal sex differentiation in humans (Tremblay and Viger, 2003). Recently, it has also been found that the human HSD3B2 promoter is activated by GATA transcription factors (Martin et al., 2005), presumably by GATA-6 (Bassett et al., 2005). GATA-6 has been suggested to have a role in androgen synthesis. It is mainly detected in areas, such as zona reticu- laris, which produce androgens and it appears to co-express with SF-1 and P450c17 which are markers for steroidogene- sis (Jimenez et al., 2003; Fluck and Miller, 2004; Kiiveri et al., 2005).
2.1. GATA-4 and GATA-6 in human adrenocortical tumors
Recently, GATA transcription factors have also been studied in normal human adrenals and adrenocortical tumors. In ear- lier studies, it has been postulated that human adrenals express GATA-6 but not GATA-4, which was in harmony with the find- ings from mice (Kiiveri et al., 1999; Jimenez et al., 2003). Later studies showed that GATA-4 could also be detected in normal (fetal) human adrenals as well as at high level in tumor tissues (Pabon et al., 1996; Kiiveri et al., 2002a,b, 2005; Barbosa et al., 2004). However, mRNA expression of GATA-4 was low or hardly detectable in normal adrenal or in adenoma, whereas GATA-6 was significantly increased. These findings support other results that GATA-4 is mainly expressed in adrenal car- cinomas, but not in healthy adrenals or adenomas (Barbosa et al., 2004; Kiiveri et al., 2004; Bassett et al., 2005). However, upon comparison at different types of adrenocortical adenomas, some studies showed that GATA-4 expression was mainly asso- ciated with cortisol rather than aldosterone production (Kiiveri et al., 2004, 2005), whereas in some cases elevated levels in aldos- terone producing adenomas (Bassett et al., 2005). It has also
been demonstrated that GATA-4 is expressed both in metasta- sizing and non-metastasizing tumors, with higher expression in the latter (Barbosa et al., 2004). In contrast to findings in mouse adrenal tumors, expression of GATA-6 has also been found in human adrenal tumors (Kiiveri et al., 2002b, 2004). GATA-6 has been reported to be found highly expressed in both aldos- terone producing adenomas and ACTH-independent Cushing’s syndrome, compared to the normal human adrenal gland (Bassett et al., 2005). This could implicate that GATA-6 is little affected by the tumorigenic process (Barbosa et al., 2004). GATA-6 is apparently more widely found in cortisol producing adenomas, which supports the finding of the role of GATA-6 in androgen and cortisol production (Kiiveri et al., 2004). The same was found in other studies where human adrenocortical cells lines were investigated (Kiiveri et al., 2004). GATA-6 has been found to be more abundantly present in adenomas than carcinomas except in the virilizing tumor subgroup (Kiiveri et al., 2004). This finding was supported when comparing the Weiss score to GATA-6 expression; if Weiss score was low, i.e. the tumor was more differentiated, there was more GATA-6 expression than in the cases of a high Weiss score (Kiiveri et al., 2005). These findings could be interpreted to support the role of GATA-6 in the maintenance of benign form of adrenocortical cells and that the absence of GATA-6 could ease the formation of car- cinomas. However, GATA-6 has been shown to be a regulator of androgen synthesis together with SF-1 and P450c17 (Fluck and Miller, 2004). GATA-6 expression also positively correlates with those of SF-1 and P450c17 (Jimenez et al., 2003; Fluck and Miller, 2004; Kiiveri et al., 2005). When non-steroidogenic cells were co-transfected with GATA-6 and SF-1, it was found that these two synergistically activated steroidogenesis (Jimenez et al., 2003). In human adrenocortical cells, GATA-6 was thought to regulate the P450c17, which also ties the production of cor- tisol and androgens with GATA-6 expression (Jimenez et al., 2003) (Table 1).
3. Animal models for adrenocortical tumorigenesis
There are few early reports from the 1940s about rare “spontaneous” adrenocortical hyperplasia and carcinomas of mouse, showing a strict inbred strain-dependency. The first report on nodular adrenocortical hyperplasia was reported in 1939 for a dilute female brown strain (DBA/2J) of mice after prepubertal gonadectomy (Wooley et al., 1939). A similar phe- nomenon was reported for gonadectomized DBA male mice in 1941 (Wooley et al., 1941). It has recently been verified that prepubertally gonadectomized DBA/2J mice develop adrenal tumors (Bielinska et al., 2003). Other mouse strains like C3H, BALB/c, after prepubertal gonadectomy have been shown to develop adrenal adenomas and in the case of CE (extreme dilu- tion) strains even adrenal adenocarcinomas, whereas the C57Bl mouse strain failed to form any visible adrenocortical neoplasias (Feteke et al., 1941; Wooley and Little, 1945). It has been shown [as early as in the 1940-60s (Feteke et al., 1941; Murthy et al., 1968; Murphy and Rao, 1970), and recently revisited and recon- firmed (Bielinska et al., 2004, 2005)] that at first small groups of densely populated small cells appear below the adrenal capsule
| Animal model | Promoter gene | Gonadectomy needed | Age of the tumor formation (month) | Histopathology | LHR | GATA-4 | Metastases | Cell line established | Reference |
|---|---|---|---|---|---|---|---|---|---|
| Inha/Tag | Inhibin & | Yes | 6-8 | Tumors from X-zone (innermost layers) | In the tumor area | In the tumor area | Liver occasionally | C& 1 | Kananen et al., 1996, Rilianawati et al., 1998, Rahman et al., 2004 |
| DBA/2J | Yes | 6 | A and B cells in the subcapsular region | In the tumor area | In A and B cells | ? | None | Bielinska et al., 2003 | |
| Inh-/- | Yes | 5-6 | Tumors derived from zona fasciculata and reticularis and/or X-zone | In the tumor area | ? | Lung | Matzuk et al., 1994, 1996 | ||
| Inh-/ -- LH-CTP | Yes | 3 | Tumors from X-zone | In the tumor area? | ? | ? | Beuschlein et al., 2003 | ||
| NU/J nude mice | Yes or xenograft | 1-2 | A and B cells in the subcapsular region (resembles DBA/2J mice) | In B cells | In A and B cells | ? | Bielinska et al., 2004, 2005 | ||
| Ferret | Yes | A and B cells in the subcapsular region (resembles DBA/2J mice), myxoid differentiation | In the tumor area | In the tumor area | Liver | Peterson et al., 2004 |
(A cells), which further extend as becoming more spindle shaped in between the zona fasciculata cell columns. Later on these A cells presumably further forms the so called B cells, larger round cells which are believed to secrete hormones as estro- gens (DBA/2J and CE mice) or androgens (C3H mice) (Rosner et al., 1966; Murphy and Rao, 1970). These B cells express several steroidogenic enzymes on the contrary to A cells. This phenomenon has recently been shown in certain inbred mouse strains, like DBA/2J, where pre-pubertal gonadectomy induced sex steroid-producing adrenocortical neoplasm (Bielinska et al., 2003). In this mouse model the adrenal contains first the spin- dle shaped A which further probably forms B cells, which may implicate that gonadectomy caused undifferentiated stem or pro- genitor cells in the subcapsular region to transform into steroid producing B cells. Markers for steroidogenesis, for example P450c17, are only found in B cells (Bielinska et al., 2003). The coexpression of GATA-4 and LHR has also been shown in this adrenocortical tumor mouse model (Bielinska et al., 2003).
Adrenocortical neoplasms are found to be a common cause of morbidity in neutered ferrets. A recent study by screening archival specimens of adrenocortical neoplasms from such ani- mals determined that these adrenal tumors expressed nuclear immunoreactivity for GATA-4 in 19/22 (86%) specimens of ferret adrenocortical carcinomas and was prominent in areas exhibiting myxoid differentiation (Peterson et al., 2004), which
thus could be used as a tumor marker in this species, as the normal ferret adrenocortical cells lacked GATA-4 expression. Two other markers of adrenocortical tumors in gonadectomized mice (Kiiveri et al., 1999; Rahman et al., 2004), inhibin & and luteinizing hormone receptor (LHR), were coexpressed with GATA-4 in some of the ferret tumors. Thus, neutered ferrets can therefore be used as a model for studying adrenocortical tumorigenesis.
3.1. Transgenic (TG) murine models for adrenocortical
tumorigenesis
Until now, among the existing TG murine models the most suitable candidates for adrenal tumors are inh &/Tag (mouse bearing Simian Virus 40 T-antigen under the inhibin & pro- moter) (Kananen et al., 1996; Rilianawati et al., 1998; Rahman et al., 2004), NU/J (Bielinska et al., 2004, 2005), inh-/- (inhibin knockout mice) (Matzuk et al., 1994, 1996) and inh-/ — LH- CTP (Beuschlein et al., 2003) (inhibin knockout mice crossbreed with C-terminal peptide of the human chorionic gonadotropin ß subunit TG mice) (Risma et al., 1995) (see below).
One of the models for adrenal tumors, is the female NU/J nude mouse (Bielinska et al., 2004, 2005). These mice devel- oped adrenocortical neoplasms in response to either prepubertal gonadectomy or gonadotropin elevation from xenografts of hCG-secreting CHO cells (Bielinska et al., 2004, 2005). The CHO cells used stably express a biologically active single chain of hCG variant. Tumors formed in nude mice resemble the ones in DBA/2J strain with spindle shaped A cells expressing GATA- 4 and steroid producing B cells expressing GATA-4 and also the steroidogenic factors LHR and P450c17 (Bielinska et al.,
2003, 2004, 2005). Also in this model the chronic elevation of gonadotropins allows the adrenocortical cells in the subcapsu- lar region to differentiate into the gonadal-like stroma. GATA-4, which is expressed in both of the cell types has been hypothe- sized to be a key factor of the transformation (Bielinska et al., 2005).
Inhibin and activin belong to the TGFB family of growth factors. Inhibin is known as a tumor suppressor in the mouse gonad (Matzuk et al., 1994). It has been shown that inhibin « knockout mice develop spontaneous gonadal tumors and if gonadectomized prepubertally (<6 weeks of age), they develop adrenal tumors with 99% penetrance (Matzuk et al., 1994). Based on the inh-/- observations, it was initially suggested that either the overexpression of gonadotropins or the lack of some gonadal factor might be the cause of tumor formation after gonadectomy (Matzuk et al., 1994; Kananen et al., 1996, 1997). Later studies have indicated that the development of tumors requires a high level of LH (Kero et al., 2000) and a drop in the levels of gonadal activin, which normally inhibits the adrenal tumor growth by apoptosis (Beuschlein et al., 2003). Smad 3, the intracellular signal transducer for TGFß, has shown to be upreg- ulated in inh-/ — mice and its expression has also been found in the stem cell compartment indicating that tumors might arise from dys-regulated stem cells. It is also possible that GATA-4 is an important factor inducing adrenal stem cells to transform to tumor cells. This could provide additional proof for the fact that GATA-4 expression is detected in all the tumors developed after gonadectomy (Bielinska et al., 2003). The adrenocortical stem cells may also be unable to downregulate TGFß signalling, which could result in tumor formation (Looyenga et al., 2004). Of interest, GATA-4 has recently been shown to cooperate with Smad3 to mediate TGFß signalling in the regulation of inhibin &. (Anttonen et al., 2006). Thus, TGFß, Smad3 and GATA-4 might act in the common pathways to promote adrenal tumor forma- tion. Supporting this connection, TGFß-Smad3 signalling has been linked to GATA-4 upregulation in the adrenals (Looyenga et al., 2004). Recent work on inh --- LH-CTP mice (Risma et al., 1995; Beuschlein et al., 2003, 2004) showed that when inh-/- mice are crossbreed with LH-overexpressing LH-CTP mice, they produce large activin-secreting ovarian tumors (Beuschlein et al., 2003). While gonadectomized, they produce large, steroid- producing adrenal tumors putatively arising from the X-zone (Beuschlein et al., 2003). However, in the presence of activin, apoptosis in the X-zone has been shown to prevent the LH- dependent adrenocortical tumor formation (Beuschlein et al., 2003). Activin is thus considered an important regulator of the X-zone together with inhibin, which again is known to regulate the growth of the adrenal X-zone (Beuschlein et al., 2003, 2004). Hence, elevated LH levels alone might not be sufficient to induce adrenal tumor formation in the presence of activin (Beuschlein et al., 2004).
3.2. Adrenocortical phenotype of the inha/Tag mice
We have studied the molecular mechanisms of adrenal tumorigenesis extensively in the inh &/Tag TG mouse model, where the mice bearing the Simian Virus 40 T-antigen (Tag)
under a 6-kb fragment of the murine inhibin «-subunit pro- moter (inha), develop gonadal tumors with 100% penetrance by the age of 5-6 mo (Kananen et al., 1995). When prepubertally gonadectomized, these mice develop adrenocortical tumors by the same age (Kananen et al., 1996; Rilianawati et al., 1998; Rahman et al., 2004). No adrenal tumors could be detected either in any intact TG or in gonadectomized, intact or control non-TG (C57Bl) littermates (Kananen et al., 1996; Rilianawati et al., 1998; Rahman et al., 2004). If functional gonadectomy was induced by administration of a GnRH antagonist or by cross-breeding the transgenic mice into the hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumors appeared (Kananen et al., 1997). This finding led to the hypoth- esis, that tumor development is related to elevated gonadotropin secretion, which is the distinct difference between the surgi- cal and functional gonadectomy models (Kananen et al., 1997). This high secretion of gonadotropins was thus proposed to stimulate the expression of LHR in tumors upon the malig- nization process (Rahman et al., 1998, 2004; Rilianawati et al., 1998).
Recently, it has been shown that GATA-4 and LHR have a direct interrelationship during the adrenal tumorigenesis (Rahman et al., 2004). The onset of adrenal mRNA and pro- tein expression of GATA-4 and LHR coincides and colocalizes in the same tumor areas at 4 mo of age after gonadectomy, which preceded the appearance of discernible adrenocortical tumors at 6 mo of age (Rahman et al., 2004). GATA expres- sion might thus be well connected to the LHR expression in adrenocortical tumorigenesis. These results demonstrated an apparent direct and positive feed-forward amplification link between these two factors, LHR action up-regulating the GATA- 4 message and perhaps also vice versa. These two factors together with Tag expression are thought to cause the forma- tion of LH-dependent adrenocortical tumors in this TG mouse model (Rahman et al., 2004). The adrenal tumor derived cell line (Ca1) from inha/Tag mice was found to express high lev- els of functional LHR as assessed by Northern hybridization, immunocytochemistry, ligand binding, and human CG (hCG)- stimulated cAMP and steroid production (Rilianawati et al., 1998). We were unable to find any follicle stimulating hor- mone receptors (FSHR) in the adrenal tumors or in the Ca1 cells (Rilianawati et al., 1998). While we stimulated the Ca1 cells by hCG, we achieved dose dependent cell proliferation and steroid production (Rilianawati et al., 1998). This helped us additionally to conclude that the expression of the potent oncogene, Tag, in adrenocortical cells allows a tropic hormone (LH) to function as a tumor promoter (Rilianawati et al., 1998). While there are differences between these TG mouse adrenal tumors and human adrenocortical tumors, certain similarities are noteworthy. Even if rare, the human adrenocortical tumors are gonadotropin responsive (Givens et al., 1975; de Lange et al., 1980; Leinonen et al., 1991) (also see above). They may also express some common neuroectodermal and steroidogenic enzymes like in the case of transgenic mice (Kananen et al., 1996, 1997; Rilianawati et al., 1998). These conserved fea- tures makes the ina/Tag TG mouse model potentially useful for identifying factors critical for adrenocortical tumorigenesis in
humans and a very useful model for human adrenocortical cancer research.
4. Future directions
In the future, it is estimated that somatic gene therapy might be a possible way to treat patients with adrenocortical tumors. A potential probe in gene therapy trials could be the Her- pes Simplex Virus Thymidine Kinase (HSV-TK) gene, which alone is non-toxic but induces the killing of dividing cells if exposed to a “prodrug” (Camper et al., 1995). HSV-TK phosphorylates nucleoside analogs such as ganciclovir and acy- clovir into monophosphorylated molecules, which are converted into triphosphate substrates by eukaryotic cells’ own enzymes. The cytotoxic monophosphorylated product is incorporated into elongating DNA of proliferating cells, causing chain termination and cell death (Moolten and Wells, 1990). HSV-TK has already been used in experimental and clinical gene therapy trials as a conditional suicide gene to ablate tumor cells (Moolten and Wells, 1990; Moolten et al., 1990; Mikola et al., 2001). We have produced another TG mouse line, expressing the HSV-TK gene under the inha promoter and crossbred these mice with the orig- inal inha/Tag tumor mice, to obtain double TG mice, which are susceptible to tumor ablation by treatment with antiherpes drugs (e.g. gancyclovir) (Mikola et al., 2001). We have already suc- cessfully treated double TG and single TG mice of both sexes, possessing gonadal tumors, with gancyclovir, and found that the double TG tumors decreased significantly in volume during the antiherpes treatment, whereas controls inha/Tag tumors contin- ued growing at the same time (Mikola et al., 2001). Thus, we hypothesize that targeted expression of the HSV-TK gene would potentially be useful for gene therapy of adrenocortical tumors in this double TG model. Possibly a novel treatment strategy with siRNA (small interfering RNA) technology in vivo could serve also as a potential and novel therapeutic approach. We would hypothesize that LHR and GATA-4 could be targeted by siRNA in vivo strategy simultaneously, which could increase the suc- cess rate. Recently, it has also been found that the lytic peptide hecate-conjugate, a fusion protein of a 23-amino acid (hecate) and a 15-amino acid (81-95) fragment of the human CGB chain could be used as a form of novel anticancer drug that could selec- tively induce the destruction of cancer cells expressing LHR. The cytotoxic effect strongly correlates with the number of LHR. There are already studies done with Hecate conjugate and its positive treatment effects in prostate (Hansel et al., 2001), and breast (Bodek et al., 2003) cancer, and in gonadal tumors (Bodek et al., 2005). The results have already proven the principle that the Hecate-CGB conjugate may provide a novel specific lead into LHR expressing cancer cell therapy by targeted destruc- tion. Our preliminary data on adrenocortical tumor treatment with Hecate conjugate in inha/Tag TG mice showed promising results (Vuorenoja et al., unpublished data). Based on the avail- able data (Kiiveri et al., 2002a,b, 2004; Barbosa et al., 2004), we also believe that in the future it will be possible to use the expres- sion levels of GATA-4, as well as GATA-4 associated markers like LHR as a predictor of the prognostic marker for monitoring adrenocortical tumorigenesis.
Acknowledgments
This work was supported by grants from the Academy of Finland and from Moikoisten Syöväntutkimussäätiö.
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