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¹ University of Colorado Health Sciences, Department of Medicine, 4200 East Ninth Avenue, Denver, Colorado 80262, USA
² Veterans Affairs Medical Center, Denver, Colorado 80220, USA
³ Colorado State University, Department of Physiology, Animal Reproduction and Biotechnology Laboratory, Fort Collins, Colorado 80523, USA

(Requests for offprints should be addressed to L M Glode; Email: mike.glode{at}uchsc.edu)

	Abstract

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic–pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.

	Introduction

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence (Wierman 1996, Neill 2002). Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (see Fig. 1). LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Continuous stimulation of pituitary GnRH receptors (GnRHR) by exogenously administered GnRH agonists, rather than pulsatile stimulation, desensitizes and down-regulates GnRHRs (Neill 2002). The ultimate effect of this chronic stimulation of the pituitary GnRHRs is to decrease LH and FSH production, with subsequent decreases in circulating sex steroid levels (Wierman 1996, Neill 2002).

View larger version (30K): [in this window] [in a new window]   Figure 1 The hypothalamic–pituitary–gonadal axis. Secretion of GnRH from the hypothalamus occurs in a pulsatile fashion and is partially responsible for controlling the number of GnRHRs in the pituitary gland and controls synthesis and secretion of FSH and LH. These gonadotropins in turn regulate function of the testes and ovaries. E, estrogen; P, progesterone; T, testosterone.

The detection of GnRH and its receptor in other tissues including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper will review the current data concerning differential control of GnRH and GnRHR expression and signaling in the hypothalamic–pituitary axis and extrapituitary tissues. Using these data as a backdrop, we will then review the literature about GnRH action in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRHR in cancer cells to promote anti-proliferative effects and control of tumor burden.

	GnRH expression and control

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
GnRH expression profile

At least two isoforms of GnRH have been identified in the mammalian central nervous system (CNS), GnRH-I and GnRH-II. GnRH-I is the hypothalamic decapeptide responsible for LH and FSH secretion from the anterior pituitary originally isolated by Guillemin and Schally (Guillemin 1967). GnRH-II was initially discovered as chicken GnRH-II and displays a diffuse pattern of localization in most tissues (Densmore & Urbanski 2003, Pawson et al. 2003). In the CNS, GnRH-II has been hypothesized to play a role in the behavioral components of reproduction (Pawson et al. 2003). The genes for human GnRH-I and GnRH-II are on chromosomes 8 and 20 respectively (Wierman 1996, Limonta et al. 2003). Both isoforms of GnRH are decapeptides that are characterized by post-translational modifications, including the pyro-glutamic acid at the amino termini and amidated glycine at the carboxy termini. GnRH-I is conserved throughout evolution and has been identified in both vertebrates and invertebrates (Wierman 1996, Limonta et al. 2003). GnRH-I shares a 60% identity between mammals and tunicates, whereas GnRH-II is even more highly conserved with 100% identity between birds and mammals (Wierman 1996, Limonta et al. 2003). GnRH-I is synthesized as a prohormone, human prepro-GnRH that contains 92 amino acids. A signal peptide is contained in the first 23 amino acids, followed by the GnRH decapeptide, a 3 amino acid cleavage site and a 56 amino acid product known as GnRH-associated peptide (GAP). Post-translational processing of GnRH involves cleaving by a prohormone convertase, removal of basic amino acids by a carboxypeptidase, amino-terminus modification through the action of glutaminyl cyclase and carboxy-terminus modification by peptidylglycine -amidating mono-oxygenase (Wierman 1996, Limonta et al. 2003). This enzymatic processing produces mature GnRH and GAP. A physiologic role for GAP has not been determined, although it has been postulated to act as a prolactin-inhibitory factor.

Control of GnRH expression

There are approximately 1500–2000 GnRH neurons in the human (in contrast to the 800 neurons in the rodent) that are diffusely arranged in a neuronal network in the basal hypothalamus after migration (Tobet et al. 2001). The small number and dispersed population make in vivo studies of the control of GnRH expression difficult. Immortalized GnRH neuronal cell culture models as well as isolated GnRH neurons demonstrate that the GnRH ‘pulse generator’ is intrinsic to the neuronal cells, and have served as model systems to define the many membrane and nuclear factors important in GnRH expression across development (Wierman et al. 2004). During human development, the GnRH pulse generator and subsequent gonadotropin release is active in the early neonate, but decreases by 3–4 years of age (Terasawa & Fernandez 2001, MacColl et al. 2002). Until puberty, the GnRH pulse generator is repressed. The exact mechanisms of repression are not known and may involve central -amino butyric acid neuronal activity (Terasawa & Fernandez 2001). Similarly, the factors that reactivate the GnRH pulse generator during puberty are not completely understood, but the process involves activation of glutamatergic pathways (Terasawa & Fernandez 2001).

	GnRHR expression and regulation

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
Mammals, including humans, produce two isoforms of GnRHRs, GnRHR-I and GnRHR-II (previously called chicken GnRH-II receptor) (Clayton & Catt 1981, Grundker et al. 2002a, McArdle et al. 2002, Kang et al. 2003, Ruf et al. 2003). Both isoforms are members of the G-protein coupled receptor (GPCR) family of proteins, couple with Gq and function in the inositol phosphate signaling pathway (see Fig. 2). In functional studies that measured inositol phosphate production, primate GnRH-I receptors demonstrated an approximate 48-fold selectivity for GnRH-I versus GnRH-II (Gault et al. 2003, Pawson et al. 2003, Terasawa 2003). Conversely, GnRH-II receptors demonstrated a 421-fold preference for GnRH-II versus GnRH-I (Gault et al. 2003, Pawson et al. 2003, Terasawa 2003). Unlike the unique GnRH-II receptor and most other GPCRs, the GnRH-I receptor contains no large cytoplasmic C-terminal tail (Clayton & Catt 1981, Grundker et al. 2002a, McArdle et al. 2002, Kang et al. 2003, Ruf et al. 2003). The C-terminus of both types of receptors are phosphorylated in response to GnRH, leading to receptor desensitization. The GnRHR-I is on chromosome 4 and the GnRHR-II is on 1q (Pawson et al. 2003). Although the mRNA for GnRHR-II has been cloned from monkey as well as rodent and fish species, these investigators have suggested that it is not expressed into a functional protein in the mouse or human, making the physiologic relevance of GnRHR-II in human biology a question for further study (Pawson et al. 2003).

View larger version (31K): [in this window] [in a new window]   Figure 2 Effects of GnRH activation of its receptor in stimulating diverse signaling pathways in the anterior pituitary. (Reprinted with permission from Ruf et al. (2003)).

	Signaling downstream of GnRHR in the pituitary

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
Effects on gonadotropin gene expression and secretion

GnRH activation of its receptor results in stimulation of diverse signaling pathways in the anterior pituitary (Fig. 2; see Ruf et al. (2003) for detailed discussion of components of the pathway). The GnRHR is coupled to Gq/11 proteins to activate phospholipase C which transmits its signal to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (Kraus et al. 2001, McArdle et al. 2002, Krsmanovic et al. 2003, Ruf et al. 2003). DAG activates the intracellular protein kinase C (PKC) pathway and IP3 stimulates release of intracellular calcium. In addition to the classical Gq/11, coupling of Gs is occasionally observed in a cell-specific fashion. PKC activation in response to GnRH also leads to increases in the mitogen-activated protein kinase (MAPK) pathways including ERK1,2, ERK5, p38MAPK and JNK in pituitary cells (Kraus et al. 2001, McArdle et al. 2002, Ruf et al. 2003). The active MAPKs move to the nucleus where they activate a variety of transcription factors, such as the Ets and/or AP1 families to modulate gene expression. These pathways then differentially regulate the synthesis and secretion of the gonadotropin subunits, , LHß and FSHß, in the anterior pituitary, selectively modulating gonadotropin synthesis and/or release from pituitary cells.

In LßT2 immortalized pituitary gonadotrope cells, studies suggest the importance of G proteins as well as Gq/11 (shown to be critical in T3 gonadotrope cells) to regulate gonadotropin subunit gene expression (Liu et al. 2002, 2003, Vasilyev et al. 2002, Krsmanovic et al. 2003). Whether signaling downstream from the GnRHR changes across pituitary development or the differences between results in the T3 and LßT2 are related to their immortalization by SV40 T antigen, remains to be determined. Desensitization in response to chronic GnRH administration in LßT2 cells results in decreased GnRHR and Gq/11 expression, and down-regulation of PKC, cAMP and calcium-dependent signaling (Liu et al. 2002, 2003). In addition, stimulation of ERK and p38MAPK, as well as c-Fos and LHß protein expression, were blocked (Liu et al. 2002, 2003). These studies suggested that chronic GnRH may also result in desensitization of other Gq-coupled receptors such as the epidermal growth factor (EGF) receptor (EGFR; see Fig. 2) at the level of the pituitary (Shah et al. 2003).

GnRHR cross-talk with other growth factor receptors

In addition to the direct effects of the GnRHR in activating intracellular signaling, recent work has suggested that cross-talk with the EGFR may occur at the level of the pituitary (Shah et al. 2003). In LßT2 pituitary cells, Roelle et al. (2003) showed that EGFR can be activated via GnRHR based upon proteolytic release of local EGF-like ligands from transmembrane precursors. In this system, matrix metalloproteinase (MMP) 2 and 9 allow shedding of the growth factors to activate EGFR. GnRH stimulation of the cells induced Src, Ras and ERK that were dependent on the action of the MMPs, whereas activation of c-Jun N-terminal kinase and p38MAPK by GnRH was unaffected by the inhibition of EGFR or MMPs. GnRH activation of c-Jun and c-Fos was, however, blocked by interference with the MMPs or EGFR signaling. Together, these data underscore the complexities of direct and indirect effects of GnRH/GnRHR action on diverse intracellular signaling pathways.

GnRHR effects on proliferation

Recent data suggest GnRH may also modulate proliferation of gonadotropes via activation of its receptor. Miles et al. (2004) showed that continuous exposure to a GnRH signal or with the GnRH agonist leuprolide resulted in an anti-proliferative effect in LßT2 cells. The cells accumulated in G0/G1. The effects were receptor dependent in that they were blocked with the GnRH antagonist antide. Of interest, the agonist was maximally effective at nanomolar concentrations, yet little effect of native GnRH was observed until concentrations exceeded physiologic levels (Miles et al. 2004). This suggests that the mechanisms of GnRH agonist action at peripheral targets are not through normal physiologic pathways, but instead related to the potency of the agonist to bind to the receptor, the long receptor occupancy and subsequent desensitization of GnRHR, and potential cross-talk with other growth factor signaling cascades. These observations are pertinent to the possibility of direct targeting of peripheral tissues including tumors (see below).

Further support for GnRHR influence on proliferation is provided in a study by Davidson et al. (2004). They demonstrated that activation of the GnRHR results in both cell adhesion and cytoskeletal remodeling. GnRH (10^–7 M) increased adhesiveness of HEK293 kidney cells overexpressing GnRHR. Cytoskeletal remodeling was dependent on focal adhesion kinase I, c-Src, ERK and Rac and independent from the classic phospholipase C signaling pathway (Davidson et al. 2004). These studies are compromised in that they utilized pharmacologic levels of GnRH which may act more like a GnRH agonist at the receptor in contrast to nanomolar concentrations normally effective in activating GnRHR downstream signaling.

	GnRH and GnRHR in extra-hypothalamic tissues

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
In addition to the hypothalamus, GnRH-I has also been localized to the endometrium, placenta, breast, ovary, testis and prostate (Clayton & Catt 1981, Wierman 1996, Huirne & Lambalk 2001, Limonta et al. 2003). The exact function of GnRH-I in these tissues is under active investigation. The recent production of a transgenic mouse targeting alkaline phosphatase expression with the rat GnRHR promoter (Granger et al. 2004) and luciferase expression with the mouse GnRHR promoter (McCue et al. 1997) may provide additional model systems to identify and map developmental expression of GnRHR in extrapituitary sites. Several lines of evidence suggest that the GnRHR-I is also expressed in the brain in GnRH neurons to contribute to an ultrashort loop feedback mechanism (Xu et al. 2004).

In the endometrium and myometrium, GnRHR has been detected by radioligand binding experiments and immunohistochemistry (Clayton & Catt 1981). In the placenta, GnRH-I and II are expressed in human placenta and are key regulators of urokinase-type plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor (PAI-1) (Chou et al. 2003a). In endometrial stromal cultures from first trimester decidual tissues, GnRH-I and GnRH-II increased expression of uPA mRNA and protein (Chou et al. 2003a). In contrast, GnRH-I increased but GnRH-II decreased PAI-1 mRNA and protein expression. A GnRH receptor antagonist inhibited the effects of GnRH-I but not GnRH-II.

GnRH-I also increased mRNA for MMP 2 and 9 in decidual stromal cultures with no effect on tissue inhibitors of metalloproteinases (TIMP)-1 (Chou et al. 2003b). In the placenta, GnRH-I and GnRH-II increased the production of MMPs and decreased the expression of TIMPs in trophoblasts. The effects of GnRH-I but not GnRH-II were blocked by a GnRH antagonist (Chou et al. 2003c). These results suggest the complex interaction of GnRH action both in the endometrium and placenta impacting on implantation. The studies also support the hypothesis that a functional GnRH-II may not be expressed in humans, explaining the divergent results with GnRH-II versus GnRH-I. Together, these data suggest that, in the endometrium and placenta, the effects of native GnRH and GnRH agonists may be similar. The down-regulation of GnRHR that occurs at the level of the pituitary in response to tonic stimulation by GnRH may not occur in these peripheral sites. Further studies are needed to clarify the control of GnRH and its receptor in these tissues.

In the ovary, in situ studies have shown the presence of the GnRH-I mRNA in granulosa cells of primary, secondary and tertiary follicles (Kang et al. 2003). Recently, investigators have shown the presence of GnRH-II in human granulosa-luteal cells (hGLCs), immortalized ovarian surface epithelial (OSE) cells and in ovarian cancer cells (Kang et al. 2003). Some studies suggest a physiologic role of the GnRH system in the control of atresia (Kang et al. 2003). GnRH can inhibit DNA synthesis, induce apoptosis and activate genes important for follicular rupture and oocyte maturation such as plasminogen activator, prostaglandin endoper-oxide synthase type 2 and progesterone receptor, and those important for matrix remodeling such as the MMPs (Kang et al. 2003).

GnRHR expression changes in the ovary correlate with the degree of follicular development across the estrous cycle (Kang et al. 2003). GnRH-I induced a biphasic effect on GnRH and GnRHR expression in hGLCs and OSE cells. Estrogen treatment resulted in an initial up- and then down-regulation of GnRH and GnRHR expression. GnRH agonist administration can also down-regulate estrogen receptors and ß in ovarian cells (Kang et al. 2003). Recent work has suggested that the GnRHR promoter is controlled by a unique upstream regulatory sequence in human ovarian granulosa-luteal cells which was not critical in ovarian cancer cells or pituitary cells (Cheng et al. 2002). Thus, there may be tissue-specific regulation of GnRH/GnRHR pathways.

In the breast, both GnRH and GnRHR have been detected by RT-PCR (Kottler et al. 1997). Most studies involved testing of breast cancer cell lines in the absence of normal breast samples (Kakar et al. 1994). GnRH agonists inhibit malignant breast epithelial cells (Kakar et al. 1994). In the absence of studies on normal human breast tissue or normal breast cell lines, the direct action of GnRH via its receptor in the breast in the absence of malignancy remains to be elucidated.

In the testes, GnRH mRNA is expressed in Sertoli cells while the GnRHR is expressed in the Leydig cells by RT-PCR (Bahk et al. 1995, Botte et al. 1998). In early fetal development in the rat, GnRH mRNA expression in the testis precedes that in the ovary, followed by GnRHR expression. In late fetal development, levels of GnRH and its receptor increased first in females in the ovary and then in males in the testes (Dufau et al. 1984, Botte et al. 1999). In cultured testes, GnRH increased GnRHR, and GnRH agonists blocked steroidogenesis, supporting a direct action of GnRH in the testis (Dufau et al. 1984, Botte et al. 1999). These effects of GnRH or GnRH agonists may not be physiologic. No studies are available concerning the regulation of GnRH or GnRHR in the human testis.

In the prostate, GnRHR has been detected in human samples of benign prostatic hypertrophy (BPH) (Bono et al. 2002). GnRHR has been detected by RT-PCR in prostate biopsies with levels lower in normal prostate than in prostate cancer specimens (Bono et al. 2002). No normal human prostate cell lines exist to clarify the role of GnRH signaling in normal prostate.

In summary, GnRH and GnRHR are expressed in many peripheral tissues. The functional physiologic role of the ligand and its receptor in these sites is under active investigation. Problems with the current literature include the detection of GnRH and GnRHR mRNA often with RT-PCR techniques without functional assays of protein expression and ligand-binding assays. Studies of potential physiologic signaling via GnRHR in extrapituitary tissues are flawed by the sole use of GnRH agonists and/or antagonists with long half-lives, often at pharmacologic levels. These agents may trigger signaling via local GnRHR that is different from potential physiologic paracrine signaling from local GnRH/GnRHR activity. Cell systems serve as models for GnRHR signaling. However, many are not physiologic models. This may explain the divergent pathways detected and confuse the complexities of functional pathways in vivo. Finally, few cutting-edge techniques have been used to date to prove that the effects of GnRH agonists or antagonists in peripheral target tissues are via the GnRHR, such as the use of siRNA, antisense technology or tissue-specific knockouts. Further research is needed to differentiate between GnRH/GnRHR signaling in normal pituitary and extrapituitary sites and show how it differs from that observed in cancer cells.

	Aberrant expression of GnRHR in cancer

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
Various disease and/or transformed epithelial cells are known to express the GnRHR (Friess et al. 1991, Kakar & Jennes 1995, Chatzaki et al. 1996, Kottler et al. 1997, Barbieri 1998, Yin et al. 1998, Ortmann & Diedrich 1999, Borroni et al. 2000, Halmos et al. 2000, Kang et al. 2000, Lee et al. 2000, Noci et al. 2000), and the GnRHRs present on hormonally responsive tumor cells appear to be identical to the high-affinity pituitary receptor. Recently, GnRHR-II, which transmits significantly stronger anti-proliferative effects than GnRHR-I, has been identified in ovarian and endometrial cancers (Kang et al. 2000, Bedecarrats & Kaiser 2003); the significance of this remains to be determined as the role of GnRH-II has not been elucidated.

Cancer types with cells expressing the GnRHR include breast, prostate, endometrial cells in endometrio-sis and endometrial cancer, ovarian, pancreatic and hepatoma (reviewed by Imai & Tamaya 2000). Analysis shows that the GnRHR-I sequences in these cancer cell types are identical to those in pituitary gonadotropes (Chatzaki et al. 1996, Yin et al. 1998), and binding studies have demonstrated the functionality of these receptors. High-affinity and low-affinity GnRH-binding sites have been found in 90% and 50% of ovarian cancer biopsies respectively (Emons et al. 1993, Emons & Schally 1994, Imai & Tamaya 2000). While ovarian cancer is not hormone responsive, the presence of GnRH-binding sites, as well as the intraperitoneal distribution of the tumor, makes this disease an especially attractive model for GnRH–toxin therapy.

About 80% of endometrial cancers and 50% of breast cancers express both GnRH and GnRHR as part of an autocrine system (Emons et al. 2003). Similarly, it has been reported that 86% of human prostate adenocarci-nomas express high-affinity binding sites for GnRH and express mRNA for the receptor; higher Gleason score tumors show reduced receptor numbers, but higher affinity receptors (Halmos et al. 2000). Proliferation of ovarian cancer cells was shown to increase after treatment with an antiserum to GnRH, supporting the idea that GnRH produced by the tumor cells acts as a negative autocrine regulator (Emons et al. 2000). Native GnRH, as well as GnRH agonists and antagonists, inhibited proliferation of cancer cells in a dose- and time-dependent manner (reviewed by Grundker et al. 2002b).

The anti-proliferative effect of GnRH analogs is mediated, at least in part, by changes in signal transduc-tion (recently reviewed by Emons et al. 2003, Limonta et al. 2003). When bound to its ligand in cancer cells, the GnRHR couples to G protein i, activating a phospho-tyrosine phosphatase which dephosphorylates EGFRs (Grundker et al. 2001). This in turn suppresses EGF-induced activation of MAPK and c-fos, thus inhibiting proliferation (Emons et al. 1996, Grundker et al. 2000a). This mechanism of cell signaling in cancer cells is different from that observed in pituitary cells, as described in the section on ‘Effects on gonadotropin gene expression and secretion’. There are also data supporting the role of GnRH-I in apoptosis in tumor cells, but this remains controversial (Grundker et al. 2000b, Wang et al. 2002).

Fewer GnRH-binding sites have been found in sex steroid-independent gynecologic tumors such as cervical carcinoma (Imai et al. 1994). Using RT-PCR to detect message, there was significantly lower expression of the receptor in BPH and normal prostate tissues (Straub et al. 2001). However, GnRH-I behaves as a negative regulator of growth in some tumors outside of the reproductive tract such as melanoma (Limonta et al. 2003). In these cancer cells, the biochemical and pharmacological profiles of GnRHR-I correspond to those in pituitary cells (Limonta et al. 2003). GnRH-I-binding sites have also been described in glioblastoma (van Groeninghen et al. 1998) and in leukemic T cells (Chen et al. 2002).

	Current use of GnRH analogs in cancer

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
With the elucidation of the structure of GnRH-I by Guillemin and Schally (Guillemin 1967), it became possible to synthesize thousands of different analogs of the primary decapeptide. Although agonists were recognized early on (Labrie et al. 1980, 1981, Faure et al. 1982) and have been employed in clinical medicine for more than 25 years, they act as delayed inhibitors of LH and FSH secretion following the initial agonistic activity. The mechanisms underlying this desensitization simulate continuous infusions of GnRH which down-regulate GnRHR and Gq/11 expression resulting in decreased PKC-, cAMP- and Ca-dependent signaling, possibly resulting in decreased bioactive and immunoreactive gonadotropin secretion (Labrie et al. 1996, Labrie 2004). The development of safe and effective GnRH antagonists required considerably longer, necessitating the substitution of three or more amino acids to achieve the desired pharmacologic profiles (van Loenen et al. 2002) (Table 1).

Phase I trials in men with clinical stage D prostate cancer with the agonist leuprolide acetate demonstrated that doses of 1–10 mg daily effectively reduced testosterone to the castrate range (Warner et al. 1983). A small phase III trial was then completed in similar stage D metastatic prostate cancer patients in which the efficacy of leuprolide, administered as a single daily 1 mg injection, was compared with a single oral 3 mg dose of diethylstilbestrol (DES). Comparable numbers of patients in both groups had suppression of testosterone, dihydrotestosterone and the only tumor marker available at that time, acid phosphatase. Tumor responses were equivalent, but the DES side-effects of gynecomastia, nausea/vomiting and thromboembolism led to the Federal Drug Administration (FDA) approval of leuprolide as a safer means of achieving medical castration (Anonymous 1984). Although orchiectomy remains a safe, effective alternative, it is often rejected by patients because of the psychologic side-effects (Samdal et al. 1991). The excess costs (compared with surgical castration) of these psychologic decisions by individual patients are estimated to be approximately $386/month (Chon et al. 2000), while the overall costs to the USA Medicare healthcare system are about $1.2 billion dollars annually for the two available GnRH agonists, leuprolide and goserelin (Anonymous 2003).

Treatment with GnRH agonists results in transient elevations in testosterone and dihydrotestosterone. In a fraction of patients with metastatic disease, this can result in ‘tumor flare’ characterized by worsening of disease symptoms. These include worsening of bone pain, urethral or ureteral obstruction and, of gravest concern, spinal cord compression or even death in patients with advanced metastatic disease. These effects can be blocked (Labrie et al. 1984, 1987, Kuhn et al. 1989) with the short-term administration of an anti-androgen.

A much more controversial issue is the longer term use of either steroidal (flutamide, bicalutamide or nilutamide) or non-steroidal (megestrol or cyproterone) anti-androgens in combination with the GnRH agonists in the treatment of prostate cancer. While these agents can prevent some of the physiologic effects of the androgen-induced tumor flare, they also have the theoretic advantage of blocking adrenal androgens. In addition, they would be expected to enhance tumor control in the occasional patient who experiences ‘escape’ from chronic GnRH agonist administration. A thorough meta-analysis of 8275 men who participated in 27 randomized trials of medical/surgical castration with or without the addition of anti-androgen therapy concluded that the addition of anti-androgens improved the 5-year survival by only 2 or 3% (Anonymous 2000). As there is some variability in the definition of ‘castrate levels’ of testosterone (traditionally <50 ng/dl) and the fact that men who are surgically castrated rarely have total testosterone levels exceeding 20 ng/dl, the National Comprehensive Cancer Network has recommended using the latter level as a lower threshold (Millikan & Logothetics 1997). A more recent review (Loblaw et al. 2004) of hormonal management for androgen-sensitive prostate cancer concludes that ‘Combined androgen blockade confers a statistically significant but questionable clinical improvement in survival over orchiectomy or LHRH monotherapy’ and that ‘. . . . treatment with antiandrogen monotherapy appears unlikely to lead to a survival benefit in men with localized disease managed with non-definitive therapy (watchful waiting)’. As this active debate on the effects of steroidal versus non-steroidal anti-androgens is beyond the scope of this review article, the reader is referred to Loblaw et al. (2004) for current guidelines and recommendations.

The first GnRH antagonist to be approved for clinical use in the USA was abarelix (Table 1). A randomized phase III multicenter study comparing its efficacy with the combination of leuprolide and bicalutamide revealed that over half of abarelix-treated patients achieved castrate (<50 ng/dl) levels of testosterone by day 4 while 79% of the combination-treated patients had still not achieved castrate levels by day 15 (Trachtenberg et al. 2002) (Fig. 3). Although the rate of fall in prostate specific antigen (PSA) levels among the two groups was similar, the potential avoidance of tumor flare via the use of abarelix in patients with impending cord or ureteral/urethral urinary tract obstruction or severe bone pain persisting on narcotic analgesics led to FDA approval for use in these limited circumstances. Unfortunately, the clinical trials leading to this approval also revealed a low incidence of immediate-onset allergic reactions that included hypotension and syncope. These phenomena led to a black box warning that patients who are administered abarelix need to be observed in the physician’s office for 30 min following each administration and physicians must participate in a training program to ensure their competence to manage these side-effects. Nevertheless, abarelix offers an important alternative to surgical castration, the use of ketoconazole or GnRH agonist plus anti-androgen in the patient who presents with significant neurologic or urinary obstructive symptoms.

View larger version (12K): [in this window] [in a new window]   Figure 3 Median (±S.E.M.) serum testosterone level in patients treated with abarelix depot (broken line) and those treated with leuprolide acetate and bicalutamide (solid line) on study days 1 through 169. (Reprinted with permission from Trachtenberg et al. (2002)).

Against this approach are studies showing that early use of GnRH agonist or other ADT for prostate cancer can improve overall survival. When combined with radiation therapy for advanced local disease, two large randomized trials showed either disease-free or overall survival advantages compared with radiation alone (Pilepich et al. 1995, Bolla et al. 1997). Similarly, in patients with nodal metastases at the time of prostatectomy, immediate medical or surgical castration improved the 5-year survival when compared with waiting until metastases (or rising PSA) were evident (Messing et al. 1999) (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Figure 4 Eastern Cooperative Oncology Group: disease-free and overall survival data. Kaplan–Meier estimates of overall survival and disease-free survival in patients receiving immediate versus deferred hormonal therapy. (From Crawford (2003), adapted with permission from Messing et al.(1999)).

Since the GnRH analogs are reversible, another less well-studied approach to their use is intermittent therapy. Prostate cancer cells deprived of androgen stimulation eventually become androgen independent, leading to clinical progression of metastases. Resistance to combined androgen blockade in localized disease is not yet understood; its occurrence in metastatic disease is discussed by Labrie et al. (2002). The molecular events accompanying this phenomenon include mutations in or overexpression of the androgen receptor and alterations in other signaling pathways that lead to loss of the capacity for the cells to undergo apoptosis (Avila et al. 2001). In theory, allowing patients to recover from GnRH analog-induced ADT could delay or prevent the emergence of such cells, and prolong the duration of the hormone-sensitive state. Early experiments with this approach in animals demonstrated a 3-fold prolongation of the hormone-sensitive state (Sato et al. 1996). These observations led to a phase II clinical trial that found improved quality of life in men treated with intermittent ADT without obvious compromise in duration of responsiveness or survival (Goldenberg et al. 1999). Although the quality of life in intermittently treated patients is improved, the overall impact on survival and duration of hormone responsiveness is unknown. Two large randomized trials in the USA and Canada are being performed to answer these questions (Pether et al. 2003).

GnRH analogs in breast cancer

It was first recognized that surgical ovarian ablation could produce clinical responses in breast cancer in the late 19^th century by Albert Schinzinger and, later, George Thomas Beatson (Love & Philips 2002). Additional endocrine ablative options including adrenalectomy, hypophysectomy and radiation ablation of the ovaries all received attention in the 20^th century, but all are irreversible and invasive. Response rates to oophorectomy in premeno-pausal metastatic breast cancer patients vary from 30 to 75% with the highest responses in patients with estrogen receptor (ER)- and/or progesterone receptor (PR)-positive tumors (Sunderland & Osborne 1991). As with prostate cancer, gonadal ablation with a GnRH analog was shown to be equivalent to surgical oophorectomy in premenopausal patients with receptor-positive disease although, surprisingly, there was an approximate 15-year delay in such trials, possibly due to the availability of effective anti-estrogens like tamoxifen (Taylor et al. 1998).

With the advent of adjuvant chemotherapy, it was widely recognized that at least some of the benefit of treatment was due to chemically induced menopause. Multiple studies did not resolve the controversy over which effect was more important, the cytotoxic effect of chemotherapy agents on the cancer cells, which could extend to the ER/PR-negative populations, or the chemotherapy-induced ovarian failure (Pritchard 2002). The value of permanent ovarian ablation by surgery or radiotherapy in premenopausal women under 50 years of age with both node-positive and node-negative disease was established by several randomized trials performed by the Early Breast Cancer Trialists’ Collaborative Group (Anonymous 1996). More recently, this group reported on a randomized trial of adjuvant chemotherapy followed by goserelin versus either modality alone in premenopausal node-negative patients. The study accrued 1063 patients from 1990 to 1999 and stratified participants according to ER status. ER-negative patients fared best if they received chemotherapy with some additional improvement by the GnRH agonist treatment. In contrast, ER-positive patients had similar outcomes regardless of the treatment arm, suggesting equivalence of the therapies. A possible exception was noted for those women under 39 years of age where the combination of chemotherapy followed by goserelin resulted in superior disease-free survival (Castiglione-Gertsch et al. 2003). There are many nuances in considering these outcomes, such as reserving the addition of a GnRH agonist only for those patients who are not rendered menopausal by the chemotherapy and the side-effects of chemotherapy versus those of ovarian ablation. Further, newer regimens of chemotherapy which are superior to the ‘classic CMF’ (cytoxan, methotrexate, 5-fluorouracil) regimen used in this study complicate the interpretation of results such that some experts feel ovarian ablation should not be recommended routinely to premenopausal patients otherwise being treated with newer regimens (Pater & Parulekar 2003).

For premenopausal node-positive patients similar findings hold for adjuvant therapy. A large randomized study sponsored by the manufacturer of goserelin compared treatment with ‘classic CMF’ versus goserelin, accruing approximately 800 patients in each group between 1990 and 1996. The disease-free survival was equivalent in those patients (~60%) with ER-positive disease, while those with ER-negative tumors again fared best with chemotherapy. The quality of life was superior in the goserelin-treated group (de Haes et al. 2003). This study did not include a group treated with both modalities, but did document amenorrhea rates which were approximately 65% during the first 6 months of chemotherapy. Again, the practicing clinician must weigh these findings in the light of superior chemotherapy regimens compared with the CMF regimen employed in the trial (Jonat et al. 2002).

Premenopausal patients with metastatic disease have often been treated with the anti-estrogen tamoxifen because of numerous studies showing equivalence with ovarian ablation (Crump et al. 1997). As tamoxifen is relatively inexpensive, easily administered and comparatively well tolerated, there have been few studies comparing its use with the GnRH agonists alone or in combination with tamoxifen. A meta-analysis of four small studies suggested there might be a benefit from the combination, but no large trials have been performed (Boccardo et al. 1999, Pritchard 2000). Most attention in this area as well as in the treatment of postmenopausal patients is now directed at the use of third generation aromatase inhibitors (Buzdar 2004).

	Clinical studies with targeted toxins

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

 
The approach of coupling cytotoxic agents to GnRH has been used in model systems to test therapies for a number of tumors possessing GnRHRs (see section on ‘Aberrant expression of GnRHR in cancer’). To achieve specificity in a GnRH–toxin approach, the GnRHR levels on cell types other than the targeted tumor must be considerably lower than on the tumor cells.

In animal studies, passive immunization was achieved with anti-GnRH antibodies in nude mice with human breast cancer cell xenografts (Jacobs et al. 1999). GnRH–Pseudomonas exotoxin (PE) conjugates were also shown to reduce adenocarcinoma tumor size when injected in a nude mouse xenograft model (Ben-Yehudah et al. 1999). In a human clinical study (Simms et al. 2000), GnRH decapeptide conjugated to diphtheria toxoid was injected into patients with locally advanced prostate cancer. In all patients, antibodies to GnRH and castrate levels of testosterone (which appeared to be reversible) were produced. In short, the presence of elevated levels of functional GnRHR in certain tumor types supports the notion that the GnRHR is a good candidate for targeted therapies.

Many chemotherapeutic agents used to treat cancer require interaction with DNA, transcriptional machinery or microtubules to disrupt cellular function. Therefore, potentially thousands of molecules may have to be delivered to a cell to inhibit its function completely. In the past two decades a novel approach, the use of targeted cytotoxins, has been developed to destroy specific tissue or cell types in the body. In the initial studies, antibodies specific for a particular cell or tissue were generated and conjugated to bacterial or plant toxins (Collier & Kaplan 1984). Following binding of the antibody–toxin conjugate to a molecular target on the cell surface, it is internalized into endosomes. In the case of many plant and bacterial toxins, acidification results in conformational changes in the toxin that permit it to exit the endosome and enter the cytoplasm. Once in the cytoplasm, most toxins inhibit protein synthesis, eventually leading to cell death, reviewed by Thrush et al. (1996) (Fig. 5). Many immunotoxins have been developed to target specific tumors based on unique cell-surface targets discovered through the use of monoclonal antibodies. Whereas cytotoxic small molecules can be delivered by such antibodies, the advantage of toxic proteins such as diphtheria toxin (DT), pokeweed antiviral protein (PAP) and Pseudomonas exotoxin (PE) lies in their enzymatic activity such that a single molecule specifically delivered to a target cell may have a much greater intracellular influence, potentially acting on thousands of substrate molecules within a cell. Thus, compared with traditional chemotherapeutic agents, only a small fraction of the number of molecules needs be delivered to cause cytotoxicity. In the past decade, targeted toxin therapy has been employed in numerous human clinical and animal studies (Brinkmann & Pastan 1994, Bast et al. 1996, Nichols et al. 1997, Frankel et al. 2000, Olsen et al. 2001, Allen 2002, Abou-Jawde et al. 2003) and some agents have been approved by the FDA for use in patients (Gunther et al. 1993, Bast et al. 1996, Nichols et al. 1997, Uckun et al. 1999, Olsen et al. 2001, Abou-Jawde et al. 2003).

View larger version (20K): [in this window] [in a new window]   Figure 5 Mechanism by which hormonotoxins may lead to cell death. The ligand–toxin conjugate binds to specific receptors on the surface of target cells (1) and is then internalized via receptor-mediated endocytosis (2). Once inside the cell, the conjugate (or the toxin alone) enters the cytoplasm by an unknown mechanism (3) and inhibits protein synthesis; the mechanism by which this occurs depends on the type of toxin. Pseudomonas exotoxin (PE) and diphtheria toxin (DT) inactivate elongation factor-2 (EF-2), whereas plant toxins (ricin toxin A (RTA); ribosome-inactivating protein (RIP)) inactivate 28S RNA. In either case, the intoxicated cell is unable to synthesize proteins which results in cell death. (Adapted with permission from Thrush et al. (1996)).

	Studies with hormonotoxins

Top Abstract Introduction GnRH expression and control GnRHR expression and regulation Signaling downstream of GnRHR... GnRH and GnRHR in... Aberrant expression of GnRHR... Current use of GnRH... Clinical studies with targeted... Studies with hormonotoxins Potential use of GnRH-toxins... Conclusions and future prospects References

Among peptide hormones, cytotoxic analogs of somatostatin, bombesin and GnRH have been synthesized in a program headed by Andrew Schally (Letsch et al. 2003). These compounds have shown efficacy in ovarian, breast and renal cell carcinoma cell lines, and xenograft models. Receptors for somatostatin have been demonstrated on breast, kidney, brain and non-small cell lung cancers, and radiolabeled ⁹⁰Y or ¹¹¹In somatostatin analogs have been used for both imaging and therapy (Virgolini et al. 2002). Additionally, a hormonotoxin utilizing corticotropin-releasing hormone (CRH) as the targeting agent was reported to decrease the ability of pituitary cells to respond to CRH (Schwartz et al. 1987, Schwartz & Vale 1988). Thus, this approach appears to have utility for numerous hormone receptors.

As discussed previously and reviewed elsewhere, elimination of gonadal function is an effective therapy for prostate and breast cancers (Miller et al. 1985, Vickery 1986, Nicholson & Walker 1989, Hoffken 1992, Weinbauer & Nieschlag 1992), and for other hormone-dependent abnormalities including fibroid tumors and endometriosis. GnRH analogs have successfully replaced surgery in a number of settings such as metastatic breast or prostate cancers, and their reversibility is particularly useful in settings where return of gonadal function is desirable. On the other hand, in many instances, for example, the ongoing maintenance of the castrate state after a prostate cancer patient becomes ‘hormone refractory’, permanent castration would be preferable. Development of a non-surgical agent that resulted in permanent suppression of gonadal activity after a single administration would thus constitute a major advance in the treatment of hormone-dependent tumors.

Potentially, hormonotoxins could be developed to attack the gonadal cells directly by attaching toxins to gonadotropins (Marcil et al. 1993, Singh & Curtiss 1994). If attached to LH, the toxin could potentially destroy Leydig cells in males, and theca, granulosa and luteal cells in females. Since theca and granulosa cells do not acquire LH receptors until development of a follicle is relatively advanced (i.e. follicles in early stages of development would not be affected), and since the corpus luteum is a transient endocrine organ, an LH–toxin conjugate would not lead to permanent castration in females. Likewise, in males there appears to be a stem cell population that will replace Leydig cells if they are destroyed (Keeney et al. 1990). Thus, an LH–toxin conjugate would only lead to a transient decrease in testicular function. If attached to FSH, again the toxin may lead to destruction of granulosa cells in follicles thus preventing follicular development. However, primordial and primary follicles do not have FSH receptors (Fortune et al. 1991) so as soon as the FSH–toxin conjugate is cleared from the blood stream, follicle maturation would resume. An FSH–toxin conjugate in males may result in destruction of Sertoli cells and lead to aspermia. However, the number of Sertoli cells in the adult testis is not static, at least in some species of animals (Johnson & Thompson 1983). Since Sertoli cells appear capable of dividing in adult males, it seems unlikely that the effects of an FSH–toxin in males would be permanent. Further, Leydig cells would be unaffected. Therefore, testosterone secretion would continue unabated and androgenic stimulation of hormone-dependent tumors would continue. Thus, an FSH–toxin conjugate also would not be useful for inducing permanent gonadal inactivity. For these reasons, ‘chemical castration’ using gonadotropin–toxin conjugates does not appear feasible.

To circumvent problems inherent with the use of LH and/or FSH, a hormontoxin using GnRH as the targeting agent to achieve chemical castration would appear preferable. The GnRH–toxin would not target the gonads directly, but rather destroy gonadotropes in the anterior pituitary gland, the cells that produce the hormones (FSH and LH) responsible for stimulating gonadal activity. Gonadotropes appear to be a terminally differentiated cell line, so destruction of gonadotropes would potentially lead to a permanent loss of gonadal function. Some of the GnRH–toxin approaches used to date have not been permanently toxic to the pituitary gonadotropes. This may be due to the relatively resting G0 state of these cells and the high amounts of toxin moieties such as the doxorubicin analogs required when compared with the enzymatic toxins. On the other hand, use of PE as the toxin in conjugates has been associated with dose-limiting CNS toxicity (Pai et al. 1992).

There are several additional reasons why GnRH is a superior choice to the use of gonadotropins to eliminate gonadal activity: (1) GnRH is a relatively small peptide (10 amino acids) that can be prepared by chemical synthesis in large quantities for nominal cost, (2) GnRH functions similarly in both males and females so a single compound could be used in either sex, (3) numerous analogs of GnRH are available that have much higher affinity for receptors than native GnRH thus enhancing the probability that the hormone–toxin conjugate will bind to receptor, (4) the GnRH molecule can be easily modified to produce analogs that can be readily linked to toxins and (5) there is little evidence that gonadotropes actively divide once puberty has occurred; thus, if existing gonadotropes are destroyed, it does not seem likely that they will be replaced. Gonadotropins are essential for gonadal function, so this would lead to permanent gonadal inactivity in both males and females. In addition, tumor cells that aberrantly express GnRHRs could be directly affected by such a conjugate as discussed above. Therefore, it is possible that for some hormone-sensitive tumors, both direct (via GnRHRs expressed in tumors) and indirect (via reduction in sex steroids by elimination of gonadotropin secretion) effects on the tumor could be achieved.

Several investigators have attempted to develop GnRH–toxin conjugates that destroy gonadotropes. Myers & Villemez (1989) conjugated GnRH to DT toxin, but they did not report its bioactivity. Szoke et al. (1994) reported that a GnRH agonist conjugated to glutaryl-2-(hydroxymethyl)-anthraquinone, a cytotoxic agent, was internalized by rat pituitary cells in vitro, a necessary step for causing cell death. More recently, it was reported (Kovacs et al. 1997) that a GnRH agonist conjugated to 2-pyrrolinodoxorubicin, a highly potent cytotoxic agent, decreased GnRH-stimulated LH secretion in rats by 63% whereas thyrotropin-releasing hormone-induced thyrotropin secretion and GHRH-induced growth hormone secretion were not significantly affected. However, their study lasted for less than 60 days so whether the reduction in gonadotrope function induced by the GnRH–toxin was permanent or temporary was not ascertained. Moreover, chemotherapeutic agents like glutaryl-2-(hydroxymethyl)anthraquinone and 2-pyrrolinodoxorubicin have two disadvantages compared with agents such as DT or similar compounds. First, even though GnRH will facilitate localization to gonadotropes, there is a significant potential for non-specific toxicity because any cell in the body could still take up the conjugate in small amounts due to the chemical nature of the chemotherapeutic agents. Secondly, many chemotherapeutic agents such as the anthracyclines must intercalate into DNA to disrupt cellular function. Potentially thousands of molecules may therefore have to be delivered to a cell to inhibit its function completely. In contrast, the enzymatic activity of some plant and bacterial toxins renders them much more efficacious once internalized. In fact, one molecule of DT has been reported to inactivate as many as five million molecules of elongation factor 6 in cells (Collier & Kaplan 1984).

If GnRH–cytotoxins actually destroy gonadotropes as they should do theoretically, then it is possible that a single dose could lead to permanent suppression of gonadal function in an individual with steroid-responsive prostate or breast cancer. Thus, these compounds would alleviate the need for repeated and continual treatment as is necessary with the GnRH agonists and antagonists in use today. Therefore, GnRH–cytotoxins have the potential to significantly decrease the number of times an individual will need to travel to a clinic to obtain treatment and decrease the lifetime cost of such treatments. In addition to these advantages, many breast and prostate cancers actually express receptors for GnRH and GnRH–cytotoxins have the potential to specifically bind to those receptors and destroy cancer cells, something that GnRH agonists or antagonists cannot do. This is discussed in greater detail in the next section of this review.

	Potential use of GnRH–toxins for prostate, breast and ovarian cancers

Top Abstract