REVIEW Gonadotropin-releasing hormone and its receptor in normal and malignant cells

Abstract

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.

Full text

REVIEW
Gonadotropin-releasing hormone and its
receptor in normal and malignant cells
G S Harrison
1
, M E Wierman
1,2
, T M Nett
3
and L M Glode
1
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@uchsc.edu)
Abstract
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.
Endocrine-Related Cancer (2004) 11 725–748
Introduction
Gonadotropin-releasing hormone (GnRH) is the
hypothalamic factor that mediates reproductive compe-
tence (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).
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 (Pitteloud et al. 2002). On the other
hand, agonists of GnRH are delivered in a continuous
mode to turn off reproductive function by inhibiting
gonadotropin production (Labrie et al. 1981), thus
Endocrine-Related Cancer (2004) 11 725–748
Endocrine-Related Cancer (2004) 11 725–748 DOI:10.1677/erc.1.00777
1351-0088/04/011–725 # 2004 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

lowering sex steroid production, resulting in medical
castration (Labrie 1991, Labrie et al. 1993, Huirne &
Lambalk 2001). They have been used in endocrine
disorders such as precocious puberty, endometriosis and
leiomyomata, but are also studied extensively in hormone-
dependent malignancies (Labrie et al. 1980, 1996, Huirne
& Lambalk 2001). More recently, pure GnRH antagonists
have been devised to directly block GnRHRs on the
pituitary and inhibit LH and FSH production (Huirne &
Lambalk 2001). Currently, they are indicated for the
treatment of female infertility as adjunct therapy during
ovarian hyperstimulation for in vitro fertilization and are
under active investigation in various malignancies (Labrie
et al. 1996, Huirne & Lambalk 2001).
The detection of GnRH and its receptor in other
tissues including the breast, ovary, endometrium, placenta
and prostate suggested that GnRH agonists and antago-
nists 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
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 chromo-
somes 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
Hypothalamus
Gonad
Pituitary
FSH
LH
E, P, T
Gonadal
peptides
(-)
(-)
GnRH
GnRHR
Sex Steroids
Gametogenesis
Secondar
y
Sex Characteristics
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.
Harrison et al.: GnRH and its receptor
726
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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 a-
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 mechan-
isms of repression are not known and may involve central
g-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 activa-
tion of glutamatergic pathways (Terasawa & Fernandez
2001).
GnRHR expression and regulation
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 Gqa 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 selec-
tivity 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).
GnRHR expression is regulated in the pituitary across
sexual maturation and in response to GnRH, sex steroids
and gonadal peptides (Norwitz et al. 2002a,b, Ellsworth et
al. 2003a,b, Sadie et al. 2003). Activin A augments GnRH
activation of the GnRHR promoter in aT3 pituitary cells
(Norwitz et al. 2002a,b). Differential control of LHb
versus FSHb gene expression by GnRH is partially
mediated by an up-regulation of GnRHR (Bedecarrats
& Kaiser 2003). An increase in GnRHR number in the
immortalized gonadotrope cell line LbT2 cells resulted in
a disruption of the response of the FSHb but not the LHb
promoter to GnRH (Bedecarrats & Kaiser 2003).
Together, these cell systems suggest that, at the level of
the pituitary, GnRHR-I expression is tightly regulated.
Signaling downstream of GnRHR in the
pituitary
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) path-
way and IP3 stimulates release of intracellular calcium. In
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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,
a,LHb and FSHb, in the anterior pituitary, selectively
modulating gonadotropin synthesis and/or release from
pituitary cells.
In LbT2 immortalized pituitary gonadotrope cells,
studies suggest the importance of G proteins as well as
Gq/11 (shown to be critical in aT3 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 aT3 and LbT2 are related to their
immortalization by SV40 T antigen, remains to be
determined. Desensitization in response to chronic
GnRH administration in LbT2 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 LHb 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 sug-
gested that cross-talk with the EGFR may occur at the
level of the pituitary (Shah et al. 2003). In LbT2 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
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)).
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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 path-
ways.
GnRHR effects on proliferation
Recent data suggest GnRH may also modulate prolifera-
tion 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 LbT2 cells. The cells accumu-
lated 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 observa-
tions are pertinent to the possibility of direct targeting of
peripheral tissues including tumors (see below).
Further support for GnRHR influence on prolifera-
tion 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
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
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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 a and b 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
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 stron-
ger 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.
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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 ai, 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
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 infu-
sions 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).
GnRH analogs in prostate cancer
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 leupro-
lide, 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 Administra-
tion (FDA) approval of leuprolide as a safer means of
achieving medical castration (Anonymous 1984).
Although orchiectomy remains a safe, effective alterna-
tive, 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
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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 ‘Com-
bined androgen blockade confers a statistically significant
but questionable clinical improvement in survival over
orchiectomy or LHRH monotherapy’ and that ‘. . . . treat-
ment 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 administra-
tion 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
Table 1 GnRH agonists and antagonists. Reprinted with permission from van Loenen et al. 2000.
Amino acid sequence
Name
1
pGlu
2
His
3
Trp
4
Ser
5
Tyr
6
Gly
7
Leu
8
Arg
9
Pro
10
Gly-NH
2
Human GnRH
GnRH-I 1 2 3 4 5 6 7 8 9 10
GnRH-II 1 2 3 4 His 6 Trp Tyr 9 10
GnRH-IIII 1 2 3 4 5 6 Trp Leu 9 10
GnRH agonist
Nonapeptides
Leuprorelin 1 2 3 4 5
D-Leu 7 8 9 N-Et-NH
2
Buserelin 1 2 3 4 5 D-SER(Bu
1
)7 8 9 N-Et-NH
2
Goserelin 1 2 3 4 5 D-SER(Bu
1
)7 8 9 AzaGly-NH
2
Histrelin 1 2 3 4 5 D-His(Imbzl) 7 8 9 N-Et-NH
2
Deslorelin 1 2 3 4 5 D-Trp 7 8 9 N-Et-NH
2
Decapeptides
Nafarelin 1 2 3 4 5 (d-Nal)
2
7 8 9 Gly-NH
2
Triptorelin 1 2 3 4 5 D-Trp 7 8 9 Gly-NH
2
GnRH antagonist
Abarelix
D-Ala D-Phe D-Ala 4 5 D-Asp 7 Lys(iPr) 9 D-Ala
Antarelix D-Nal D-Phe D-Pal 4 Phe D-Hcit 7 Lys(iPr) 9 D-Ala
Cetrorelix
D-Nal D-Phe D-Pal 4 5 D-Cit 7 8 9 D-Ala
Ganirelix
D-Nal D-Phe D-Pal 4 5 D-hArg 7 HArg 9 D-Ala
Iturelix (Antide)
D-Nal D-Phe D-Pal 4 NicLys D-NicLys 7 Lys(iPr) 9 D-Ala
Nal-Glu D-Nal D-Phe D-Pal 4 D-Glu D-Glu 7 8 9 D-Ala
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with significant neurologic or urinary obstructive symp-
toms.
The duration of response and survival of patients
treated with GnRH analogs or other means of androgen
deprivation therapy (ADT) depends largely on their
clinical status and volume of disease. Table 2 lists some
of the important factors that have been found to influence
responses (Oefelein & Resnick 2003). The timing of
GnRH agonist ADT (i.e. when to initiate therapy and
for how long to continue it) has generated considerable
discussion and a variety of experimental approaches
simply because, unlike surgical castration, GnRH agonist
therapy is reversible.
With the advent of PSA testing, it has become
possible to detect residual, advancing disease months to
years before any symptoms develop. One of the few
studies on the natural history of patients who go
untreated after a PSA rise is detected after successful
prostatectomy revealed that radionuclide scans and other
body imaging studies do not detect metastases for a
median of 8 years from the time of biochemical
progression (Pound et al. 1999). Predictors of more
rapid development of clinical metastases are the initial
Gleason score, the time-interval between prostatectomy
and initial PSA detection and the doubling time of the
PSA once detectable. In this study, the median time from
radiographic metastases to death was 5 years. These data
suggest that men can wait until radiographic progression
is detected before initiation of ADT. Early treatment of
prostate cancer is reviewed by Labrie et al. (2002).
Against this approach are studies showing that early
use of GnRH agonist or other ADT for prostate cancer
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)).
Table 2 Clinical factors predictive of response and overall survival in patients with prostate cancer treated with androgen
suppression therapy. Reprinted with permission from Oefelein & Resnick 2003.
Variable Good prognostic factors Reference
Nadir PSA Undectable Oefelein et al. 2002
Time to nadir PSA <3 months Oefelein et al. 2002
Baseline PSA Low Sabbatini et al. 1999
Tumor grade Low Albertsen et al. 1995
Bone pain No pain de Voogt et al. 1989
Extent of metastases Minimal Sabbatini et al. 1999
Performance status Low
de Voogt et al. 1989,
Sabbatini et al. 1999
Alkaline phosphatase Low
de Voogt et al. 1989,
Sabbatini et al. 1999
Tumor stage Low de Voogt et al. 1989
Hemoglobin High Sabbatini et al. 1999
Body mass index Low Oefelein et al. 2002
Pretreatment testosterone Low Chodak et al. 1991
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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 prostatect-
omy, 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).
However, the GnRH agonists produce considerable
side-effects including hot flashes, accelerated bone resorp-
tion, impotence, loss of libido, loss of muscle mass and, in
some instances, profound psychologic effects. One
approach to avoid these toxicities would be better
selection of patients for early hormonal ablation. Various
algorithms and neural networks have been developed to
try to better predict which patients would benefit the most
from early ADT (Crawford 2003). Further pharmacologic
intervention can be used as well, adding additional drugs
(and expense) to the patient’s treatment regimen. For
example, calcium and vitamin D supplementation with or
without bisphosphonates can significantly reduce bone
loss (Smith 2003). Hot flashes can be ameliorated in
approximately half of the patients by prescribing venla-
faxine (Quella et al. 1999), estrogens or progestins. A
variety of partially effective measures can be tried to
alleviate sexual dysfunction (Higano 2003).
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 under-
stood; 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, hypophysect-
omy 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)-posi-
tive tumors (Sunderland & Osborne 1991). As with
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)).
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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 (Cas-
tiglione-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 compara-
tively well tolerated, there have been few studies compar-
ing 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
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
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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 mono-
clonal 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).
Studies with hormonotoxins
More recently, a similar approach has been the use of
hormones rather than antibodies to target toxins to
specific cells in the body (Schwartz et al. 1987, Singh &
Curtiss 1991, 1994, Marcil et al. 1993). With this
approach, a toxin is conjugated to a hormone that has
specific receptors in a select population of target cells. The
‘hormonotoxin’ then binds to those receptors, is taken
into the cell by receptor-mediated endocytosis and, when
delivered to the cytoplasm, results in inhibition of protein
synthesis leading to cell death.
Among peptide hormones, cytotoxic analogs of
somatostatin, bombesin and GnRH have been synthe-
sized 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
90
Yor
111
In somatostatin
analogs have been used for both imaging and therapy
2. Receptor mediated
= Toxin
endocytosis
= Ligand
1. Binding
to cell surface
= Receptor
receptor
3. Delivery to
Acidified
cytoplasmic
EF-2
endosome
DT
compartment
PE RTA
DT RIP
PE
Trans-Golgi/ER
4. Inhibition
of Protein
RTA
Synthesis
RIP
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)).
Harrison et al.: GnRH and its receptor
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(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, Wein-
bauer & 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 con-
jugate 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 una-
bated 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
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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 secre-
tion 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-pyrroli-
nodoxorubicin 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 chemother-
apeutic 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–cytotox-
ins 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
Although GnRH analog therapy will suppress gonado-
tropin (and therefore ovarian/testicular steroid hormone)
production and thereby slow progression of breast or
prostate cancers, many such tumors also appear to have
receptors for GnRH (as discussed above). Thus, the
potential exists for these cells to respond directly to
hormonotoxin treatment using GnRH as the targeting
ligand. Ovarian cancer cells, although not responsive to
steroid hormones, express the GnRHR and their growth
is inhibited by GnRH analog treatment.
Comparison of fusion and conjugate GnRH–
toxins
Fusion proteins consisting of GnRH and PAP or PE have
been produced and shown to inhibit growth of cultured
tumor cells expressing GnRHRs (Nechushtan et al. 1997,
Schlick et al. 2000); however, the activity of the fusion
proteins were not directly compared with the activities of
hormonotoxins produced by conjugation of a GnRH
analog to the toxic protein moiety. Since recombinant
fusion proteins are not chemically synthesized, they
should always have one and only one GnRH associated
with each PAP molecule, and it is always at the same site.
For this reason, the uniformity of GnRH–PAP fusion
preparations should be much better than for conjugates
prepared by chemical means, thus making the generation
of large amounts of GnRH–PAP for clinical studies more
feasible. It is likely, however, that recombinant fusion
proteins will have inferior activity to hormontoxins
produced via chemical conjugation for two reasons: (1)
the ends of the GnRH molecule are known to be
important for receptor binding (Karten & Rivier 1986)
and are not likely to be as easily accessible to the receptor
in a fusion protein and (2) the incorporation of a
D-amino
acid in position 6 that is known to enhance receptor
binding affinity approximately 30-fold is impossible in
fusion proteins.
We recently compared GnRH–PAP fusion toxin
(single polypeptide chain) with GnRH–PAP chemical
conjugate for binding and cytotoxic activity in Chinese
hamster ovary (CHO) control cells, and CHO cells
expressing a transfected GnRHR (CHO-GnRHR) (Qi et
al. 2004). In support of the hypothesis that the conjugate
would be more cytotoxic than the fusion protein, we
showed that while the conjugate bound and caused
specific toxicity to GnRHR-positive cells, the GnRH–
PAP fusion proteins were much less active in both of these
regards. We tested two different versions of fusion
protein, corresponding to either full (f, containing post-
translationally modified sequences) or mature (m, without
these sequences). GnRH–fPAP was tested because a
previous study (Schlick et al. 2000) demonstrated that a
GnRH–PAP fusion protein containing these post-trans-
lationally modified sequences was cytotoxic to Ishikawa
cells (an endometrial cell line). While the GnRH–PAP
conjugate bound to GnRHR, albeit at somewhat higher
Harrison et al.: GnRH and its receptor
738
www.endocrinology-journals.org

concentrations compared with control GnRH alone (D-
Lys
6
-GnRH), neither of the fusion proteins (GnRH–fPAP
or GnRH–mPAP) were able to inhibit binding of the
radioligand to GnRHR. However, both the GnRH–PAP
conjugate and GnRH–mPAP fusion proteins inhibited
translation to a similar extent as PAP alone in a cell-free
rabbit reticulocyte translation system, demonstrating that
the PAP molecule in the conjugate and fusion proteins
retained toxicity. Thus, any difference in their cytotoxicity
when tested in cell survival/clonogenic assays could not be
attributed to disruption of PAP function.
To evaluate the ability of GnRH–PAP to inhibit
growth of cells expressing GnRHR on their surface, cells
were treated with increasing amounts of PAP or GnRH–
PAP conjugate or fusion protein. We showed that
GnRH–PAP was able to prevent the growth of cells
expressing GnRHRs including several prostate and breast
cancer cell lines (Qi et al. 2003, Yang et al. 2003). Results
from a clonogenic assay (consisting of counting the
number of colonies formed at the end of a 5-day
incubation period) are shown in Fig. 6 in control CHO
and CHO-GnRHR cells. As seen in the figure, PAP and
GnRH–mPAP fusion showed only non-specific toxicity,
with similar results in both CHO and CHO-GnRHR cells
at all concentrations (P > 0:1, comparing GnRH–mPAP
to PAP alone at 110
8
M). Fifty percent inhibition in
both cell types with these proteins was observed only at
concentrations > 100 nM. In contrast, GnRH–PAP con-
jugate protein showed specific toxicity in CHO-GnRHR
cells (P < 0:002 at 110
8
M compared with PAP alone),
with 50% inhibition seen at approximately 2 nM; in CHO
controls cells, toxicity from the conjugate protein was
within the same concentration range as the fusion protein
and PAP (P > 0:09 at 110
8
M ). These results showed
that the conjugate protein was approximately 50-fold
more cytotoxic to CHO-GnRHR cells compared with
CHO control cells. The fusion protein, however, did not
show any specific toxicity to CHO-GnRHR cells. This
finding is consistent with the lack of fusion protein
binding to GnRHR.
Although the native GnRH sequence was used in the
fusion proteins while the analog GnRH (with
D-amino
acid in position 6, see above) was used in the conjugate
protein, the difference in binding affinity (30-fold) by itself
was not sufficient to explain the three to four log
difference in binding between conjugate and fusion
GnRH–PAP proteins, nor the approximate two log
difference in cytotoxicity.
GnRH–toxin conjugates in cancer cell lines
With this information in hand, we decided to evaluate
GnRH–PAP prepared by chemical conjugation both in
vivo and in vitro. Two sets of studies were performed to
evaluate this conjugate in vitro. First, ovine anterior
pituitary glands were dissociated into single cells and
placed in cell culture. Cultured cells served as controls or
were treated with PAP alone, or with GnRH–PAP
overnight. Following the overnight culture, treatments
were removed by washing the cells with media and then
the cells were incubated for an additional 24 h. At this
time, the cells were collected, homogenized and amounts
of LH (to reflect gonadotrope activity) and prolactin (to
assess activity in a cell type not expressing GnRHRs) were
measured. PAP alone did not alter the amounts of either
LH or prolactin in the pituitary cells compared with
controls. However, GnRH-PAP decreased the amount of
LH in the cells by approximately 80% compared with the
controls, but did not alter the amount of prolactin in the
cells (Fig. 7). Secondly, we tested the chemical GnRH–
PAP conjugate against selected cancer cell lines for direct
toxicity. As shown in Fig. 8, both CHO cells expressing
the GnRHR and aT3-1 cell lines were specifically killed by
the conjugate at concentrations greater than 10 nM (Yang
et al. 2003). We also demonstrated direct GnRH–PAP
cytotoxicity in prostate cancer cell lines (Qi et al. 2003).
0
20
40
60
80
PAP
A
100
120
% Control
GnRH-mPAP
GnRH-PAP Conjugate
0.1 1 10 100 1000
Concentration (nM)
% Control
120
B
100
80
60
40
20
0
0.1 1 10 100 1000
Concentration (nM)
PAP
GnRH-mPAP
Conjugate
Figure 6 Clonogenic assay in (A) CHO-GnRHR or (B) CHO
control cells exposed to varying concentrations of PAP, GnRH–
mPAP fusion protein or GnRH–PAP conjugate. Values are
means
S.E.M. (Reprinted with permission from Qi et al. 2004).
Endocrine-Related Cancer (2004) 11 725–748
www.endocrinology-journals.org 739

These data demonstrate the ability of this conjugate to
specifically inhibit the function of cells expressing
GnRHRs without altering the function of cells lacking
GnRHRs. Moreover, the pituitary cells that were affected
are the cells responsible for stimulating the activity of
gonads. Thus, inhibiting the function of these cells would
eliminate the gonadal steroids on which many prostate
and mammary tumors are dependent for their growth. As
GnRH agonists alone will not result in sterilization, the
ability to achieve this is an important avenue of
investigation. The potential for use of this conjugate in
clinical trials will clearly depend on the level of expression
of the GnRHR in vivo, quantification of which is a current
focus of our laboratories.
GnRH–toxin conjugates in animal studies
Encouraged by the results obtained in the experiments
discussed above, the GnRH–PAP conjugate was next
tested in vivo. In this study, the effect of GnRH–PAP on
reproductive function was evaluated in adult, male dogs.
Four dogs received GnRH–PAP hourly for 36 h (hourly)
and four other dogs received GnRH–PAP as one bolus
injection daily for 3 consecutive days (bolus). One dog
received a single bolus (single). Three adult male dogs
received GnRH without the PAP conjugate and served
as controls. Twenty-five weeks after the initial treatment,
all treated dogs received a second treatment with
GnRH–PAP as a single bolus while control dogs
received GnRH. Serum concentrations of testosterone
and LH were determined by radioimmunoassay, and
testis size was measured for 9 months after treatment.
Stimulation tests (5 mg/kg GnRH) were used to evaluate
the ability of the pituitary gland to release LH. Serum
testosterone concentrations were measured to evaluate
testis function during the study. Concentrations of
testosterone were significantly lower ðP < 0:05Þ in all
animals treated with GnRH–PAP than in controls after
treatment (Fig. 9A). Basal LH was lower ðP < 0: 05Þ in
all treated animals than in the control group between
weeks 0 and 33 post-treatment. Likewise, treatment with
GnRH–PAP reduced ðP < 0:05Þ LH release after GnRH
stimulation in treated animals compared with the
control area under the curve (AUC) (Fig. 9B). Testis
volume was lower ðP < 0:05Þ in all treated versus control
dogs (Fig. 9C). In conclusion, administration of the
GnRH–PAP conjugate at a 25-week interval resulted in
a major disruption of reproductive function in male
dogs which was maintained for at least 11–12 weeks
after the second GnRH–PAP injection (Sabeur et al.
2003).
In all animal and human studies to date from our
group and others, there has been no evidence for
01/gn
5
sllec
300
700
600
500
400
200
100
0
Control Toxin GnRH-Toxin
Figure 7 Effects (meansS.E.M.) of GnRH–toxin or toxin alone
on the ability of cultured anterior pituitary cells to synthesize LH
(solid bars) or prolactin (shaded bars). Toxin alone did not alter
LH synthesis compared with untreated cells, but GnRH–toxin
substantially decreased ðP < 0:01Þ synthesis of LH. In contrast,
GnRH–toxin did not influence the ability of cultured pituitary cells
to synthesize prolactin ðP > 0:05Þ.
Figure 8 Cytotoxicity of GnRH–PAP conjugate against (A)
CHO-GnRHR and (B) aT3-1 cells in the cell proliferation assay,
with various concentrations of GnRH–PAP (*),
D-Lys
6
-GnRH
(~) or PAP (&). Values are means
S.E.M. (Reprinted with
pemission from Yang et al. (2003)).
Harrison et al.: GnRH and its receptor
740
www.endocrinology-journals.org

non-specific toxicity, nor any indication that removal of
GnRH inhibited the function of a variety of organs in
which mRNA for GnRH receptor has been identified (i.e.
gut). The fact that administration of GnRH–PAP or
immunization to eliminate active GnRH from the
circulation only affects reproductive tissues supports the
premise that GnRHR can be safely employed as the target
for hormonotoxins.
Conclusions and future prospects
Over three decades have passed since the elucidation of
the GnRH molecule. Analogs are now widely employed in
the treatment of prostate cancer, eliminating the need for
surgical castration, and ongoing studies suggest similar
benefits in selected breast cancer patients. The expression
of GnRHR-I and GnRHR-II in peripheral tissues,
0 1 3 4 6-7 8-9 9-10 11-12 14-15 18-19 23-24 27-28 29-30 32-33 35-36
1500
2000
2500
3000
3500
4000
4500
mm( emuloV sitseT
3
)
5
10
15
20
25
30
35
40
45
1000
2000
3000
4000
5000
6000
7000
8000
)lm/gp( enoretsotseT mureS
Weeks Post-Treatment
3 day bolus 36 h infusionControl Single bolus
Control Single Bolus 3 day Bolus 36 h infusion
GnRH-PAP
Control
)CUA( HL mureS
0 1 3 4 6-7 8-9 9-10 11-12 14-15 18-19 23-24 27-28 29-30 32-33 35-36
1500
2000
2500
3000
3500
4000
4500
mm( emuloV sitseT
3
)
5
10
15
20
25
30
35
40
45
1000
2000
3000
4000
5000
6000
7000
8000
)lm/gp( enoretsotseT mureS
3 day bolus3 day bolus 36 h infusion36 h infusionControlControl Single bolusSingle bolus
Control Single Bolus 3 day Bolus 36 h infusionControl Single Bolus 3 day Bolus 36 h infusion
GnRH-PAP
Control
GnRH-PAP
Control
)CUA( HL mureS
A
B
C
Figure 9 Effects (meansS.E.M.) of GnRH–PAP on reproductive parameters in male dogs. Treatment regimens are described in the
text. Arrows in (A) indicate the times that treatments were administered. (A) Changes in serum concentrations of testosterone at various
times after treatments. The ability of the anterior pituitary to respond to a GnRH challenge is shown in (B); data shown represent the total
amount of LH released (AUC) following the challenge. Changes in testicular volume with time after treatments is depicted in (C). Since
each of the treatment regimens resulted in decreased testicular volume, data from all the treatment groups were combined. (Reprinted
with pemission from Sabeur et al. 2003)).
Endocrine-Related Cancer (2004) 11 725–748
www.endocrinology-journals.org 741

especially neoplastic tissues, may provide a new target for
GnRH–toxin-directed therapy. This treatment could
potentially achieve both permanent castration by elimina-
tion of pituitary gonadotropes as well as direct cytotoxi-
city to tumors. Animal studies to date support such
development, revealing little toxicity to normal intact
tissues, while clinical studies will depend on the develop-
ment and testing of good manufacturing practices (GMP)
materials. The elucidation of the differences in function of
the GnRH-I and GnRH-II receptors and ligands remains
an area of intense investigation.
Acknowledgements
L M G and T M N are co-founders of Gonex (http://
www.gonex.com), a corporation attempting to commer-
cialize GnRH–toxin conjugates for cancer therapy and
other applications. G S H is a minor stockholder in
Gonex.
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